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Knack, Brent Andrew (2011) Cell adhesion factors in cnidarians. PhD thesis, James Cook University.

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ResearchOnline@JCU

Cell Adhesion Factors in Cnidarians

Thesis Submitted by

Brent Andrew KNACK BSc (Hons)

in August, 2011

for the degree of Doctor of Philosophy

in the School of Pharmacy and Molecular Biology

James Cook University

i

Statement of Sources I declare that this thesis is my own work and has not been submitted in any form for another

degree or diploma at any university or other institution of tertiary education. Information

derived from the published or unpublished work of others has been acknowledged in the

text and a list of references is given.

I declare that the electronic copy of this thesis provided to the James Cook University

Library is, within the limits of the technology available, an accurate copy of the print thesis

submitted.

6th August 2011 Brent Andrew Knack

ii

Statement of Access I, the undersigned, author of this work, understand that James Cook University will make

this thesis available for use within the University Library and, via the Australian Digital

Theses Network, for use elsewhere.

I understand that, as an unpublished work, a thesis has significant protection under the

Copyright Act and I do not wish to place any further restriction on access to this work.


iii

Contribution of Others Nature of Assistance Contribution Names

Core research funding Australian Research

Council Centre of

Excellence for Coral Reef

Studies

Stipend James Cook University,

Graduate Research School

Travel Assistance Faculty of Medicine,

Health and Molecular

Sciences Graduate

Research Scheme

Financial Support

Accommodation Network for Genes and

Evironment in

Development

Database programming

(See page iv)

Dr. Wayne Mallett, JCU

High Performance

Computing

Collaboration and

Technical assistance with

Drosophila Cell Culture

Dr. Tom Bunch, University

of Arizona Data Collection & Support

Collaboration on

Expression of Wnt

signalling component

Chuya Shinzato and

Svetlana Ukolova

iv

Collaborator contributions to development of JCUSMART:

The concepts and programming behind JCUSMART grew from a relatively simplistic installation

of the annotation programs into a large database with a comprehensive search capability. I manaed

and directed the entire development process. I accessed support from JCU High Performance

Computing (HPC) through personal contacts. The contribution of the HPC team members was

primarily programming support. I actively contributed to the design and testing of all system

components developed, as well as working closely with computing staff to correct errors.

During the initial phases of development Dr. Wayne Mallet (System administrator for the JCU

High Performance Computing Department) and myself worked closely on the design of the

computational pipeline, the handling of character conflicts, and the design of the data storage

system. Decisions on what analyses and results were required in order to be informative, how

character conflicts were resolved, and how data should be linked were primarily my responsibility

as Dr. Mallett had very limited knowledge of biology and bioinformatics.

Dr. Mallet was responsible for programming the scripts that initiate analyses, pre-process input

data, and parse key results into the database. As the system administrator, Dr. Mallett was ideally

positioned to install and troubleshoot programs on the computing cluster. Dr. Mallett’s knowledge

of Perl and PostgreSQL allowed the development of a stable analysis platform, which was

compatible with the JCU HPC system.

Throughout the development process, I tested analysis initialisation; data capture & storage; and

database queries. This was a necessary approach, as each of these steps requires interpretation of

results, which was beyond the knowledge of the HPC staff involved. I then directed the changes in

consultation with Dr. Mallett.

During the development process I actively improved my own programming ability by working

with Dr. Mallet to understand Perl and Prostgres databases in more detail. I also worked with

Wade Tattersall (an undergraduate programmer working on the HPC ARCHER Project) and other

personal contacts to understand Python programming which has a PostgreSQL component

allowing greater control of queries and results. With this knowledge I was able to create more

complex database queries and return more specific results than was previously possible. The

current version of the command line interface was primarily programmed by me, using the

programming skills I developed whilst conducting my PhD.

v

Wade Tattersall assisted me to write the initial scripts for commencing BLAST analyses, parsing

BLAST output for relevant data, and entering the results into the JCUSMART database. I took

over the modification and testing of these scripts and further refined these independently.

The web-server interface was programmed and implemented by Wayne Mallet early in the

development of the JCUSMART system. It was designed to give basic reports on up to ~300

sequences. The size of the datasets being analysed quickly outgrew the web-server functionality.

The web-server interface was not progressed beyond its initial incarnation, as HPC staff could not

spare time to update, nor could I take on writing HTML code. The more flexible & capable

command line interface was adopted as it required less external support.

Collaborator contribution to Figure 6.2: Maximum likelihood phylogenetic analysis of

representative β-Integrins

I selected protein sequences for analysis and collected sequences from NCBI protein database.

Integrin phylogenetic analyses are based on extensively edited protein sequence alignments.

Editing of the full length protein sequence alignment (output of ClustalW) was primarily

conducted by Prof. David Miller. Phylogenetic Analysis conducted using MolPhy was performed

by Prof. Miller. I was responsible for editing of raw analysis output into the published figure

presented in Knack et al, 2008 and Figure 6.2.

Every reasonable effort has been made to gain permission and acknowledge the owners of

copyright material. I would be pleased to hear from any copyright owner who has been omitted or

incorrectly acknowledged.


vi

Acknowledgements The greatest thanks must go to the girl who will very soon become my wife, Ally. In my

work, as in the rest of my life you have been my one source of unwavering support and my

inspiration for perserverance. Your faith in me has been nothing short of amazing, even at

the hardest times and with the distance between us, you kept me going. No words can

explain your true contribution to this work.

Secondly I would like to acknowledge the contribution my supervisor, Prof. David Miller, to

faciliating the initiation of this project. I would also like to thank Dr. Eldon Ball for helping

me to appreciate some of the finer aspects of Acropora development and Dr. David Hayward

for preparing and mailing EST clones as soon as he was able. Thanks must also go to Dr. Tom

Bunch, whose hospitality and patience made every aspect of my time in Arizona worthwhile.

Thanks must also go to Dr. Akira Iguchi for the words he shared during my Honours degree,

“Step by step we will do”. This is a phrase that I have reflected upon many times throughout

the past few years.

My fellow lab members, with whom I have shared the highlights and disappointments of my

work, you have made this experience truly enriched. Your friendship and advice will

continue to be invaluable to me as will the colourful language of 5 foreign tongues. I wish

you all the best in what I am confident will be a long and successful life beyond your studies.

Finally, I must thank my parents who supported and encouraged me to embark on on this

study and helped me to remain focused. Without you, I would not have had the ability to

succeed in my studies.

vii

Table of Contents

Statement of Sources.................................................................................................... i

Statement of Access......................................................................................................ii

Contribution of Others.................................................................................................iii

Acknowledgements .....................................................................................................vi

Abstract xv

Chapter 1: Introduction............................................................................................. 1

1.1 Cell adhesion molecules in development and evolution................................................................1 1.2 Acropora millepora -‐ a model organism for evolution and development ...............................6 1.3 Project objectives ......................................................................................................................................... 10

Chapter 2: Methods .................................................................................................12

2.1 Standard PCR Protocol............................................................................................................................... 12 2.2 Amplification of Gene Fragments from A.millepora cDNA libraries....................................... 12 2.3 Recovery of cDNA from Expressed Sequence Tag libraries ...................................................... 13 2.4 In situ Hybridisation ................................................................................................................................... 13 2.4.1 Riboprobe Synthesis ...................................................................................................................................... 13 2.4.2 Fixation of coral developmental stages ................................................................................................ 13 2.4.3 Hybridisation.................................................................................................................................................... 14 2.5 Cloning of pHS S2 cell expression constructs .................................................................................. 15 2.5.1 Generation of native Acropora integrin expression constructs ................................................. 15 2.5.2 PCR amplification from template pBSII constructs......................................................................... 16 2.5.3 Generation of chimeric Acropora integrin expression constructs ............................................ 17 2.5.4 Generation of chimeric mutant expression construct .................................................................... 19 2.5.5 Insertion of HA and MYC tags to ItgβCN1 and AmItgβ2 ............................................................... 20 2.6 Maintenance of Drosophila S2 cells in culture ................................................................................ 23 2.7 Transfection of Drosophila S2 Cells in culture ................................................................................ 23 2.8 Cell spreading assays.................................................................................................................................. 24 2.9 Antibody staining and flow cytometry of integrin expressing cells....................................... 25

viii

Chapter 3: JCUSMART – A simple tool for automated annotation and exploration of

large protein sequence datasets ...........................................................26

3.1 Introduction.................................................................................................................................................... 26 3.2 Methods (development of JCUSMART)............................................................................................... 31 3.3 Results (Testing and low intensity applications)........................................................................... 34 3.4 Discussion........................................................................................................................................................ 34 3.5 Conclusions..................................................................................................................................................... 37

Chapter 4: Diversity of cell adhesion molecules in cnidarians ...................................38

4.1 Introduction.................................................................................................................................................... 38 4.2 Methods............................................................................................................................................................ 41 4.3 Results ............................................................................................................................................................. 42 4.3.1 Cadherins ........................................................................................................................................................... 42 4.3.2 Integrins ............................................................................................................................................................. 43 4.3.3 Lectins ............................................................................................................................................................... 45 4.3.4 Adhesion LRR.................................................................................................................................................... 48 4.3.5 Class B adhesion G-protein coupled receptors................................................................................... 49 4.3.6 Immunoglobulin superfamily.................................................................................................................... 50 4.3.7 Extra-cellular matrix .................................................................................................................................... 51 4.3.8 Novel cnidarian sequences ......................................................................................................................... 52 4.4 Discussion........................................................................................................................................................ 54 4.4.1 The ancestral adhesion repertoire.......................................................................................................... 54 4.4.2 Novel cnidarian sequences ......................................................................................................................... 60 4.4.3 Differences between cnidarian adhesion systems ............................................................................ 61 4.4.4 Interesting absences...................................................................................................................................... 62 4.5 Concluding comments................................................................................................................................ 63

Chapter 5: Developmental roles of cadherins from Acropora millepora....................65

5.1 Introduction.................................................................................................................................................... 65 5.2 Methods............................................................................................................................................................ 71 5.2.1 Sequence identification................................................................................................................................ 71 5.2.2 Cadherin phylogenetics................................................................................................................................ 71 5.2.3 Isolation of riboprobe template cDNA .................................................................................................. 71 5.3 Results ............................................................................................................................................................. 73 5.3.1 Identification of catenin binding cadherins and planar cell polarity components from

Acropora millepora ...................................................................................................................... 73

ix

5.3.2 Phylogenetic analysis of catenin binding cadherins ....................................................................... 75 5.3.3 In situ hybridisation of catenin binding cadherins and planar cell polarity pathway

components ...................................................................................................................................... 77 5.4 Discussion........................................................................................................................................................ 80 5.4.1 Cadherins involved in epithelial cohesion and migration evolved early in metazoan

evolution............................................................................................................................................ 80 5.4.2 Planar cell polarity but not Am_ACadherin is implicated in gastrulation of Acropora

millepora ........................................................................................................................................... 82 5.4.3 Am_ACadherin and planar cell polarity are implicated in development of the Acropora

larval oral pore............................................................................................................................... 84 5.5 Conclusion ....................................................................................................................................................... 86

Chapter 6: Integrins of Acropora millepora ..............................................................87

6.1 Introduction.................................................................................................................................................... 87 6.2 Methods............................................................................................................................................................ 91 6.2.1 Phylogenetic analyses................................................................................................................................... 91 6.2.2 Preliminary ligand binding assay ........................................................................................................... 91 6.2.3 Optimisation of cell spreading conditions ........................................................................................... 92 6.2.4 Analysis of integrin surface expression................................................................................................. 93 6.3 Results ............................................................................................................................................................. 94 6.3.1 Integrin identification and phylogenetic analysis ........................................................................... 94 6.3.2 Ligand Binding of coral integrins ........................................................................................................... 97 6.4 Discussion......................................................................................................................................................102 6.4.1 Novel Coral integrins may interact with 2 distinct ligand types ............................................ 102 6.4.2 Integrins containing AmItgα1 interact with RGD tripeptide ligands................................... 103 6.5 Conclusions...................................................................................................................................................105

Chapter 7: General Discussion................................................................................106

7.1 Modes of gastrulation in cnidarians...................................................................................................106 7.2 Genetic determinants of cnidarian gastrulation...........................................................................110 7.3 Acropora millepora exhibits an inside-‐out mode of gastrulation..........................................113 7.3.1 The mobile presumptive ectoderm and un-coupling of β-catenin from mesendoderm

development.................................................................................................................................. 113 7.3.2 Maintaining stability in the presumptive endoderm ................................................................... 115 7.3.3 Co-ordinating expansion of the presumptive ectoderm ............................................................. 116

Chapter 8: General Conclusions..............................................................................120

x

Reference List ...........................................................................................................123

Appendix A: Supplementary Material .......................................................................135

Chapter 4 ...........................................................................................................................................................135 Chapter 5 ...........................................................................................................................................................150 Chapter 6 ...........................................................................................................................................................170

Appendix B: JCUSMART Survey of the Cnidarian Adhesome......................................177

Cadherins ...........................................................................................................................................................177 Integrins ...........................................................................................................................................................179 Lectins ...........................................................................................................................................................186 LRR Adhesion ..........................................................................................................................................................192 Class B GPCR 193 Immunoglobulin .....................................................................................................................................................198 Extracellular Matrix ..............................................................................................................................................201 Planar Cell Polarity Signalling ..........................................................................................................................213

xi

List of Figures

Chapter 1: Introduction............................................................................................. 1

Figure 1.1 Evolution of metazoan cellular junctions................................................................................3

Figure 1.2 Lifecycle of Acropora millepora ...................................................................................................8

Figure 1.3 Differing gastrulation strategies of Acropora millepora and Nematostella

vectensis ..............................................................................................................................................8



Figure 3.1 Upward approach to identifying genes of interest in a custom dataset ................ 28

Figure 3.2 Downward approach to identifying genes of interest in a custom dataset using

JCUSMART ...................................................................................................................................... 30

Figure 3.3 Computational Workflow of JCUSMART pipeline............................................................. 33

Chapter 4: Diversity of cell adhesion molecules in cnidarians ..................................38

Figure 4.1 Maximum likelihood analysis of Talin proteins from representative

metazoans ....................................................................................................................................... 44

Figure 4.2 Maximum Likelihood analysis of metazoan haemolytic lectins ................................ 47

Figure 4.3 Domain structure of selected cnidarian innovations ...................................................... 53

Chapter 5: Developmental roles of cadherins from Acropora millepora....................65

Figure 5.1 Generalised protein architecture of catenin binding cadherins................................. 67

Figure 5.2 Milestones in cadherin evolution ........................................................................................... 67

Figure 5.3 Protein conservation and architecture of Am_ACadherin ........................................... 74

Figure 5.4 Maximum likelihood analysis of catenin binding Cadherin cytoplasmic

domains............................................................................................................................................ 76

Figure 5.5 Spatial mRNA expression patterns of Am_ACadherin and PCP components in

embryo’s of Acropora millepora ............................................................................................ 78

Figure 5.6 Spatial mRNA expression patterns of Am_ACadherin and PCP & Wnt components

in larvae of Acropora millepora ............................................................................................. 79


Figure 6.1 Maximum likelihood analysis of representative α-‐Integrins ...................................... 95

Figure 6.2 Maximum likelihood analysis of representative β-‐Integrins....................................... 96

xii

Chapter 7: General Discussion ...............................................................................106

Figure 7.1 Modes of Gastrulation in cnidarians and bilaterians ....................................................107

Figure 7.2 Phylogeny of cnidarian classes ...............................................................................................108

Figure 7.3 Differing gastrulation strategies of Acropora millepora and Nematostella

vectensis .........................................................................................................................................109

Appendix A: Supplementary Material .......................................................................135

Chapter 4

Supplementary Figure 4.1 JCUSMART search terms used for identification of adhesion

genes ............................................................................................................135

Supplementary Figure 4.2 Multiple sequence alignment of Talin proteins used for

maximum likelihood analysis ...........................................................139

Supplementary Figure 4.3 Multiple sequence alignment of meatzoan haemolytic lectins

used for maximum likelihood analysis .........................................141

Supplementary Figure 4.4 Spatial mRNA expression pattern of haemolytic lectin

A036-‐E7 in Acropora millepora larva, polyp & adult ..............142

Supplementary Figure 4.5 Boxshade alignment of full length Am_LRIG3 protein with

representative metazoan LRIG3 orthologues .............................143

Supplementary Figure 4.6 Boxshade alignment of full length Am_PTPRD protein with

representative metazoan PTPRD orthologues ...........................146

Chapter 5

Supplementary Figure 5.1 Aligned nucleic acid and protein sequence of Am_ACadherin 150

Supplementary Figure 5.2 Boxshade alignment of representative Dachsous C-‐terminal

domains .......................................................................................................157

Supplementary Figure 5.3 Multiple sequence alignment of cadherin cytoplasmic domains

used for maximum likelihood analysis .........................................158

Supplementary Figure 5.4 Boxshade alignment of AmFlamingo protein with

representative metazoan Flamingo orthologues ......................160

Supplementary Figure 5.5 Boxshade alignment of full length Am_Van Gogh Like protein

with representative metazoan Van Gogh orthologues............166

Supplementary Figure 5.6 Boxshade alignment of full length Am_Dishevelled protein with

representative metazoan Dishevelled orthologues...................168

xiii

Chapter 6

Supplementary Figure 6.1 Multiple sequence alignment of α-‐Integrin proteins used for

maximum likelihood analysis ............................................................170

Supplementary Figure 6.1 Multiple sequence alignment of β-‐Integrin proteins used for

maximum likelihood analysis ............................................................173

xiv

List of Tables



Table 3.1 Programs used by JCUSMART in the annotation of protein sequences .................. 32

Table 3.2 Conditions under which sequences may not be identifiable using JCUSMART... 36

Table 3.3 Contribution of JCUSMART to publication........................................................................... 37

Chapter 4: Diversity of cell adhesion molecules in cnidarians ..................................38

Table 4.1 Datasets used to explore the adhesome of basal metazoans....................................... 41

Table 4.2 Distribution of cadherin family proteins in basal metazoans ..................................... 42

Table 4.3 Distribution of integrin and associated proteins in basal metazoans ..................... 43

Table 4.4 Distribution of lectin family proteins in basal metazoans ............................................ 45

Table 4.5 Distribution of adhesion -‐LRR proteins in basal metazoans........................................ 48

Table 4.6 Distribution of class B G-‐Protein Coupled Receptors in basal metazoans............. 49

Table 4.7 Distribution of immunoglobulin superfamily proteins in basal metazoans ......... 50

Table 4.8 Distribution of extracellular matrix proteins in basal metazoans............................. 51

Table 4.9 Presence and absence of selected bilaterian adhesion genes with

developmental and immunological significance............................................................ 55

Chapter 5: Developmental roles of cadherins from Acropora millepora ...................65

Table 5.1 Distribution of catenin binding cadherins and planar cell polarity components

in cnidarians .................................................................................................................................. 75


Table 6.1 Concentrations of Mg2+ (MgCl2) and Ca2+ (CaCl2) added to Robb’s Saline for

optimisation of cell spreading conditions......................................................................... 92

Table 6.2 Percentage of cells expressing detectable α and β-‐Integrins on the cell surface

following transient transfection..........................................................................................100

Table 6.3 Percentage of cells expressing epitope tagged β-‐Integrins on the cell surface

following transient transfection..........................................................................................101

Chapter 7: General Discussion ...............................................................................106

Table 7.1 Function of adhesion protein families with demonstrated involvement in

bilaterian gastrulation.............................................................................................................112

xv

Abstract Cell adhesion is central to metazoan evolution and development, facilitating multicellularity,

intercellular communication and co-‐ordinated cell movements. Morphological studies

indicate that cellular and developmental processes involving dynamic changes in adhesive

state, such as cell migration and gastrulation, were established early in metazoan evolution.

However, much of our current understanding surrounding the involvement of cell adhesion

molecules in these processes has been obtained from comparative analyses of bilaterian

development. Cnidarians present an exciting opportunity to investigate cell adhesion and

developmental processes in the simplest extant animals to possess a tissue layer level of

organisation. As the nearest out-‐group to the Bilateria, cnidarians are ideally positioned for

comparative studies between diploblastic and triploblastic development whilst being highly

informative regarding ancestral gene function and protein family evolution.

The diversity of adhesion proteins in cnidarians has remained largely unexplored, with no

single analysis offering an overview of the cnidarian adhesion complement or “adhesome”.

To better understand the complexity of the cnidarian adhesome, sequence data from four

model cnidarians (Acropora millepora – coral, Nematostella vectensis – Sea Anemone, Hyrda

magnipapillata – Hydra, Clytia hemispherica – Hydrozoan Jelly-‐fish) was annotated and all

potential adhesion proteins categorised according to similarity to described protein families.

The cnidarian adhesome shows overall similarity with that of invertebrate bilaterians,

containing a substantial array of recognisable cadherins, integrins, lectins and extracellular

matrix proteins comparable to Drosophila and sea urchin. Such conservation suggests that

most recognised adhesion proteins involved in bilaterian development and innate immunity

were already established in the ureumetazoan ancestor. All four species also demonstrated

an expanded set of lectin domain containing putative pattern recognition receptors which

may act in opsonisation of microbial pathogens. In contrast to this, each species lacked the

large immunoglobulin complement observed in deuterostomes suggesting the predominate

mechanism of microbial recognition may be facilitated by lectins rather than

immunoglobulins.

Catenin binding cadherins and components of the planar cell polarity (PCP) pathway were

among the developmentally significant proteins conserved between cnidarians and

bilaterians. Expression of cadherins, such as E-Cadherin and N-Cadherin, which bind

cytoplasmic β-catenin, strongly influence the ability of cells to undergo epithelial to

mesenchymal transtion, a central event in developmental processes such as gastrulation.

The assymetrical distribution of planar cell polarity cadherins and other PCP proteins at

xvi

both the cell and tissue levels, also affects tissue morphology by co-‐ordinating cellular

structures and providing positional cues during morphogenesis. To identify the potential for

conserved catenin binding cadherins and PCP proteins to participate in cnidarian

development, patterns of mRNA expression were assessed in embryos and larvae of the

coral Acropora millepora. Surprisingly, the only known catenin binding Cadherin from coral

and the first identified outside the Bilateria, Am_ACadherin, does not appear to participate in

gastrulation, which is inconsistent with bilaterian modes of gastrulation. Planar cell polarity,

however may be active during Acropora gastrulation, with AmVan Gogh (AmVangl) and

AmDachsous (AmDs) expressed assymetrically during gastrulation. Am_ACadherin was

instead implicated in oral pore development following gastrulation as indicated by a highly

restricted pattern of expression in the oral ectoderm. In situ mRNA hybridisation also

strongly suggests the involvement of non-‐canonical Wnt/PCP in oral pore development,

however none of the PCP adhesion proteins exhibited oral pore restricted patterns of

expression during larval stages. These results suggest both Cadherin-‐catenin signalling and

PCP are signficant during cnidarian development and may facilitate co-‐ordinated tissue

mobility such as involution of the oral ectoderm.

Survey of the cnidarian adhesome also identified α and β integrins, which, like cadherins,

play signifcant roles in the early development of bilaterians. The expression patterns for 3 (1

α-‐Integrin & 2 β-‐Integrins) of the 5 (3 α-‐Integrins & 2 β-‐Integrins) integrins identified in

Acropora have previously been reported, showing restiction to the presumptive endoderm

throughout gastrulation. The ligand binding properties of basal integrins have not been

reported, obscuring their developmental function. To identify the ligand binding properties

of AmItgα1-ItgβCN1 and AmItgα1-AmItgβ2 integrin heterodimers, transgenic cell spreading

assays were performed on a number of Drosophila ligands. These experiments suggested

coral AmItgα1 containing integrins may bind to arginine-‐glycine-‐aspartate (RGD) sequence

containing proteins. RGD specific integrins are abundant throughout the Bilateria, and along

with Laminin binding integrins, have been suggested to be present in the Urbilaterian

ancestor (the last common ancestor of the Bilateria). Phylogenetic analysis of α-‐Integrins

including the more recently identified AmItgα3 (Acropora) and NvItgα2 (Nematostella)

suggest that these proteins may bind to a second distinct ligand type. The ligand diversity of

cnidarian integrins may therefore be comparable with basal bilaterians.

xvii

Invovlement of cadherins and integrins in gastrulation appears to be conserved between

cnidarians and bilaterians as suggested by mRNA expression during gastrulation of

Acropora millepora. The mode of gastrulation exhibited by Acropora millepora is among the

more peculiar strategies of gastric cavity formation, where by a flat cellular bilayer folds to

produce a spherical bilayer at the end of gastrulation. Expression patterns of the coral

integrins and cadherins, along with protein localisation data for β-catenin, allow the

description of a new model for gastrulation in Acropora. These data suggest the presumptive

ectoderm of the coral embryo is mobile, with expansion of this tissue layer co-‐ordinated by

planar cell polarity. Stability in the presumptive endoderm is suggested to be mediated by

integrin ligand binding, providing the basis for mesoglea (the connective tissue layer that

separates cnidarian ectoderm and endoderm) development. This model is inside-‐out with

respect to most modes of gastrulation which rely upon co-‐ordinated migration of the

presumptive endoderm and represents a unique arrangement of conserved adhesion

proteins and cellular signalling processes.

Chapter One Introduction

1

Chapter 1: Introduction

1.1 Cell adhesion molecules in development and

evolution Cell adhesion and changes in adhesive state are essential for the normal development and

function of metazoans. Developmental processes including fertilisation, gastrulation, and

neurogenesis along with immunity and allorecognition are all dependant on co-‐ordinated

changes in the adhesive capability of cells (Halbleib & Nelson 2006; Kinashi 2005). The

ability of a cell to adhere to other cells, to maintain contact with extracellular matrix, or to

actively migrate to another location, is governed by the expression of a broad group of cell

surface proteins collectively known as cell adhesion molecules. Although this name may

suggest a simple function in making cells “stick”, cell adhesion molecules have far more

important roles than simply securing cellular structures.

Each biological process involving adhesion, such as gastrulation, requires multiple cells to

have structural organisation and behave in a co-‐ordinated manner (Gumbiner 2005). Cell

adhesion molecules perform two fundamental cellular functions, cohesion and intracellular

signalling, which together contribute to tissue organisation and co-‐ordination in

multicellular organisms. The cohesive function is a consequence of extracellular domain

(ecto-‐domain) structure and determines ligand specificity, whereas the cytoplasmic domain

facilitates cell signalling and cytoskeletal attachment by interacting with cytoplasmic

proteins. Unlike most cell surface receptors, which only transduce signals from the

environment into the cell, a number of cell adhesion molecule families frequently respond to

internal cellular stimuli, changing affinity, expression level or signalling ability through

intrinsic structural response, transcriptional regulation and protein regulatory mechanisms

(eg. phosphorylation, domain cleavage, and endocytosis). The range of cytoplasmic

interactions, multiplicity of fast and slow regulatory mechanisms and the ability to

specifically determine cellular cohesion, make adhesion molecules particularly versatile

morphogenic effectors, capable of influencing the structure, organisation and gene

expression profile of both cells and tissues.


2

The influence of cell adhesion molecules on cellular organisation and co-‐ordination is

common to all multicellular organisms. Whereas unicellular organisms are thought to

primarily utilise adhesive processes for anchoring to their environment, the maintenance of

multicellular form immediately dictates an added role for adhesion in generating continual

cell-‐cell contact (Abedin & Nicole King 2010). The link between multicellularity and

increased adhesive capability is well established (Abedin & King, 2010; King, 2004; Nichols

et al, 2006)and none more evident than in the most complex incarnation of multicellularity,

the Metazoa. An overview of the metazoan tree of life is presented in Figure 1.1A.

Enrichment of cell adhesion genes is among the features that distinguish metazoans from

other eukaryotes, along with enrichment of signal transduction and cell differentiation genes

(Rokas 2008). Even the most morphologically simple metazoan, Trichoplax adhaerens

(essentially a flat epithelial bilayer sandwiching multinucleated fibre cells), contains a

complement of adhesion genes, which is beyond that of the closest unicellular relatives of

metazoans, the Choanoflagellata (King et al., 2008; Srivastava et al., 2008). Expansion of

genetic adhesive capability during metazoan evolution is reflected in the diversity of cellular

junctions observed in different animal lineages. Figure 1.1B&C illustrates the expansion of

adhesive junction types from simple anchorage junctions in choanoflagellates to the

communication, barrier and dynamic cohesive junctions of vertebrates. Although this

increased complexity of adhesive systems was a significant step in the transition to

multicellularity, most of what is known about the function of cell adhesion molecules

originates from studies of bilaterians.

Investigation of adhesion mechanisms within bilaterian animals has demonstrated that

many adhesion and associated signalling mechanisms are conserved from flies to

vertebrates. This is particularly evident in systems affecting early morphogenesis such as

Cadherin-‐β-‐catenin, integrin signalling and planar cell polarity (Klein & Mlodzik, 2004;

Nichols et al., 2006). Despite widely variegated gastrulation strategies in flies, fish, frogs and

mammals, these conserved adhesion systems maintain influence over the same tissue level

mechanisms during the formation of three germ layers (See Chapter 7). Through

conservation of ligand specificity and intracellular signalling properties, adhesion proteins

with similar structure in each taxon govern tissue morphogenic mechanisms such as tissue

flexibility, migration and directional division. Whilst this level of functional conservation has

clearly been demonstrated in higher animals, the morphogenesis of basal phyla, cnidarians

and sponges, is strikingly different.

3

Figure 1.1 Evolution of metazoan cellular junctions. A Phylogeny of the Metazoa (adapted from Technau et al., 2005). A wide range of evidence suggests a common (monophyletic) origin for metazoans from a unicellular ancestor. Cnidarians are basal among animals with a tissue layer level of organisation (Eumetazoa) and are informative about the last common ancestor of cnidarians and bilaterians. B & C Distribution of adhesive cell junctions in metazoans (adapted from Abedin and King, 2010). The diversity of cell junctions expanded multiple times during animal evolution. The presence (grey) or absence (white) of junction types is evidenced by genetic surveys (Gene), electron microscopy (Morph), and experimental (Exp) data. Although experimental data is lacking for non-‐bilaterians, the presence of genes that function in adherens and septate junctions distinguish metazoans from their unicellular ancestors and subsequent evolution of intercellular communication in the eumetazoan ancestor may have been critical to tissue layer formation. Enrichment of communicating, barrier and cohesive junctions is a feature of chordate evolution and particularly evident in vertebrates. Some caveats apply to the data presented: 1 α and β integrins are absent in choanoflagellates but present in C.owczarzaki, a model unicellular organism used to infer the genetic content of Opisthokonts, which are basal to holozoans. 2 Only some sponges exhibit basal lamina like structures and ECM comprised of Collagen IV, a major component of basal lamina.


4

The methods of gastrulation observed in basal animals are even more diverse than in

bilaterians. This is particularly clear in cnidarians where all strategies present in higher

animals are represented as well as a number of unusual gastrulation methods (Byrum and

Martindale, 2004). Despite broad resemblances to bilaterian gastrulation, formation of the

gastric cavity in cnidarians and the gastrulation-‐like events of sponge embryonic

development result in only two embryonic germ layers. These animals are therefore

referred to as diploblasts. Furthermore, larvae and primary polyps of cnidarians exhibit

radial symmetry, only possessing distinction of oral and aboral structures and not a dorsal-‐

ventral axis or bilateral symmetry, characteristic of higher animals. Given the great impact of

adhesive systems on morphogenesis, these clear developmental differences between

bilaterians and basal phyla are suggestive of changes in adhesion system specificity,

regulation and downstream signalling events.

Evolution of other milestone developmental processes associated with differential adhesion

is also believed to have originated in sponges and cnidarians. The Porifera (sponges)

represent the most basal metazoan phylum to exhibit continual cell-‐cell contact during

development and show specificity in cohesion that allows sorting of dissociated cells

(Burger et al., 1975) . They are also the most basal animal to establish cell layers during

embryonic development, although they lack a single dedicated gastric cavity, which is

defining of true gastrulation. The novelties found in cnidarians are even more significant to

animal evolution. Cnidarians do develop a single dedicated gastric cavity, and possess a

primitive nervous system, which is proposed to facilitate sensory responses to the

environment. They also have added complexity of life cycles that include a medusa jellyfish

stage, which commonly have dedicated defensive structures in the form of

cnidoctye/nematocyte (stinging cell) covered tentacles. The development of comparable

dedicated tissues in higher animals has been shown to require cell adhesion molecules to

direct tissue development.

Despite the multitude of novelties that arose before the Bilateria and the significant

differences between cnidarian development and that of higher animals, very little

information exists about the function of adhesion in cnidarians and sponges. Simply

considering differences among bilaterians provides a highly limited view of the evolution of

adhesion molecules and adhesion-‐signalling systems. This is due to both the large amount of

time between cnidarian and bilaterian divergence and the genetically derived nature of

some common laboratory model organisms such as Drosophila melanogaster. Knowledge of

adhesion in basal animals is limited to sporadic identifications of differentially regulated


5

adhesion molecules during coral bleaching and metamorphosis, investigation of a single

coral lectin (Millectin), and limited surveys of two complete genomes.

The complete genomes of only 1 sponge (Amphimedon queenslandica) and 1 cnidarian (the

starlet sea anemone, Nematostella vectensis) are currently published (Putnam et al., 2007;

Srivastava et al., 2010). To date, the Nematostella genome has been the only representative

of non-‐bilaterian animals regularly included in comparative analyses of cell adhesion

molecules. Surveys of the available genomes have been targeted towards identification of

genes or conserved functional protein domains associated with developmentally important

cell adhesion molecules. These targeted investigations fail to report many novel or expanded

components of the adhesion complement and are instead focused on highlighting the

ancestral nature of components from a minimal set of developmental adhesion systems.

However, the great diversity of cnidarian developmental processes and near stochastic

patterns of gene loss within the Cnidaria (Forêt et al. 2010; Miller et al. 2007) indicates that

investigation of a single cnidarian genus is often insufficient to accurately infer ancestral

relationships. It is important to be mindful of this when information regarding the cnidarian

adhesion complement is inferred from a single species. Additionally, detailed evolutionary

relationships have been explored for only three families of adhesion genes (integrins,

cadherins and G-‐Protein Coupled Receptors) leaving the evolutionary features of most

adhesion families enigmatic.

Understanding the origins of developmentally significant cell adhesion molecules is

important, although sequence based analyses provide no information as to the function of

these basal cognates. The specificity, regulation, and spatial and temporal expression of

cnidarian adhesion molecules are expected to be substantially different to that of higher

animals and are more informative of ancestral function than identifying the presence or

absence of a homologue. However, no investigation into these aspects of cnidarian adhesion

systems has previously been performed for any family of cell adhesion molecule.


6

The overall lack of information regarding the ancestral function of cell adhesion molecules

and the evolution of adhesion molecule families is best addressed by investigation of

cnidarian adhesion systems. As the most basal organisms with a tissue layer level of

organisation, cnidarians are ideally positioned to be informative about ancestral

developmental processes while illuminating aspects of molecular evolution. Understanding

the adhesion complement or “adhesome” of more than a single representative species, and

performing investigations into the specificity and expression patterns of key developmental

cell adhesion molecules from cnidarians is the most efficient way to fill critical gaps in our

knowledge of diploblastic development and evolution.

1.2 Acropora millepora - a model organism for

evolution and development Cnidarians are a large and successful phylum of predominantly marine animals, which

diverged from bilaterians approximately 500-‐600 million years ago and have expanded to

over 9000 described species. They are characterised by the presence of cnidocytes (stinging

cells) that are used for defence and prey capture. The Cnidaria are divided into four classes:

Anthozoa, Scyphozoa, Cubozoa and Hydrozoa, each with defining life cycle features.

Acropora are a genus of reef building scleractinian corals abundant throughout the Indo-‐

Pacific region. Phylogenetic analyses have revealed that along with Nematostella (sea

anemones), Acropora are members of the basal class of cnidarians, the Anthozoa (see Miller

& Ball, 2000 for review). Investigations into anthozoan genomes have revealed that despite

a simple, radially symmetrical body plan, these basal animals possess surprisingly

sophisticated genetics, containing a similar gene number and signalling complement to

vertebrates (Miller & Ball, 2000). The sophisticated genetic complement and basal position

of Acropora both within the Cnidaria and among the Eumetazoa (“True metazoans” –

Animals with a tissue layer level of organisation), positions this genus ideally for

investigations of evolution and development.

Acropora millepora is among the best described species of cnidarian in terms of both

genetics and development, and exhibits several features that are likely to involve differential

expression of cell adhesion molecules, such as nervous system development and

metamorphosis (Ball et al., 2004; Putnam et al., 2007). The gastrulation strategy of Acropora

millepora is particularly peculiar, involving the early formation of a flat cellular bilayer and

folding of the presumptive ectoderm around the presumptive endoderm (Figure

1.2)(Hayward et al., 2004). This is in contrast to that of Nematostella vectenisis, which has a

typical blastula stage and gastrulation occurs via invagination (Hayward et al., 2004) (Figure


7

1.3). Whereas movements similar to those seen in Nematostella gastrulation have been

highly investigated in bilaterian models, the factors facilitating tissue re-‐arrangements

during coral gastrulation are obscured.

Despite the gross morphological differences, Nematostella and Acropora are closely related

on an evolutionary timescale (both are Sub-‐Class Hexcorallia) and commonly have a similar

complement of many signalling pathway components (Lee et al., 2007). The biological

differences and genetic similarity of these two cnidarians provide a unique opportunity for

comparative studies of cell adhesion in closely related animals, whilst elucidating

mechanisms of an unusual developmental strategy.

In addition to the developmental and evolutionary implications of studying cell adhesion in

Acropora, there are coral specific processes reported to involve cell adhesive changes.

Acropora live in obligatory symbiosis with unicellular algae called Zooxanthellae. The

uptake and maintenance of symbionts in the gastroderm of adult Acropora millepora

requires expression of Millectin, a cell adhesion factor of the lectin family (Kvennefors et al.,

2008; Kvennefors et al., 2010). Whether other adhesion factors play a role in the recognition,

uptake and translocation of algal symbionts from ectodermal cells to the gastrodermis

remains unresolved.

The wide spread loss of Zooxanthellae from coral tissue is known as coral bleaching and is

also suggested to involve changes in cell adhesion. Although bleaching involves both

apoptotic and necrotic processes, the sloughing of gastrodermal tissue has also been

reported and is assumed to be due to a change in cellular cohesive state (Kvennefors et al.,

2008; Kvennefors et al., 2010). Separate experiments have found changes in the expression

level of undescribed adhesion molecules (eg. Lectins and cadherins) during thermal stress

(Seneca et al., unpublished; (Gates et al., 1992), which further implicates adhesion molecules

as central players in coral bleaching. The precise adhesion genes that are responsible for

sloughing of zooxanthellae containing gastrodermal cells are equivocal and could be

resolved through direct analysis of adhesion protein function, although such an undertaking

is not straightforward in sensitive marine invertebrates like coral.


8

Figure 1.2 Lifecycle of Acropora millepora. Electron micrographs of each major stage in the embryonic development of Acropora millepora (Image courtesy of E.Ball, Unpublished). The anthozoan lifecycle lacks the medusa stage observed in other cnidarian classes and asexual reproduction in coral results in colonial expansion rather than budding of a separate individual. Sexual reproduction occurs annually when mature Acropora colonies release sperm and ova into the water column where fertilisation and embryonic development takes place. Developmental times from fertilisation are temperature dependant and times indicated are within normal ranges for embryos collected in late September at Magnetic Island, Queensland.

Figure 1.3 Differing gastrulation strategies of two representative cnidarians, Acropora millepora and Nematostella vectensis. Gastrulation in Acropora (A; Hayward et al., 2004) is preceded by formation of a flat cellular bilayer, which reduces in circumference and thickens at the onset of gastrulation. The edges then begin to fold upward producing a concavity on the side of the presumptive endoderm. As the concavity deepens (gastrula) the blastopore becomes apparent and eventually closes to make a sphere. By contrast, Nematostella gastrulation (B; Lee et al., 2007) is preceded by blastula (a hollow spherical monolayer of cells) formation and occurs by involution of presumptive endoderm from one side of the blastula. Unlike Acropora, the blastopore does not close and becomes the larval oral pore.


9

There are a number of limitations to using Acropora millepora as a model organism.

Maintaining coral in a marine aquarium is a difficult task due to fastidious temperature,

light, pH and nutritional requirements and those kept in large reef aquariums do not

reproduce sexually in a predictable manner, which makes them unsuitable for

developmental studies. Sexual reproduction of Acropora millepora on the reef flat is an

annual event and occurs by broadcast spawning of sperm and ovum bundles. The annual

reproductive cycle and generation times of 5-‐10 years have inhibited the development of

gene knockdown, protein over expression and cell tracking methods in this model.

Application of these techniques, which have become the standard tools of developmental

research and functional analyses, has been further constrained by the fragility of coral

embryonic stages and requirement of a high salt and high pH (pH 7.8 – 8.0) environment.

The study of adhesion in coral morphogenesis has therefore required some exploration and

extension of the techniques established in other model metazoans.


10

1.3 Project objectives Developmental roles of adhesion molecules are poorly comprehended in basal metazoans,

which provide a fundamental view of ancestral gene function and the evolutionary history of

gene families. The objective of this project is to address the deficit in understanding of

ancestral adhesion gene complement and function using a combination of bioinformatic and

molecular techniques. Acropora millepora is the primary model for these investigations,

which focus on a number of specific aspects of cnidarian adhesion systems.

A representative view of adhesion genes in cnidarians has not yet been achieved from

previous studies exploring the genome of Nematostella vectensis. Through the investigation

of four large cnidarian datasets (genome, transcriptome and expressed-‐sequence tag

projects), the first component of this project aims to establish an overview of the cnidarian

adhesome and provide insight into the evolution of adhesion genes originating in the

eumetazoan ancestor. Accurate annotation of coding sequences is central to identifying the

gamut of adhesion proteins within cnidarians and to discerning their evolutionary

relationships. The datasets analysed from Acropora millepora, Nematostella vectensis, Hydra

magnipapillata and Clytia hemispherica contain over 100,000 predicted peptides that must

be considered for adhesive potential. No single, publically available tool is able to provide

the level of annotation required and permit sufficient data exploration for gene discovery.

Therefore, a secondary aim of the above survey component was to assemble a tool capable

of high throughput annotation and exploration of protein sequences which would allow

analysis of private data for genes with specific protein features (eg. conserved domains or

signal peptides). Development of such software has potential to contribute to projects

beyond simply identifying adhesion related genes and is applicable to all experiments

requiring discrimination of uncharacterised sequences.

The second aim of this project focuses on elucidating the functional roles of specific families

of adhesion proteins found during the survey described above. Members of the Cadherin and

integrin families are of particular interest due to their critical function in cell sorting,

directed cell migration and neuronal pathfinding in bilaterians. The diversity and

developmental impact of these proteins has not been functionally assessed in cnidarians,

although the expression patterns for some of the coral integrins during development of

Acropora millepora have been previously reported (Knack et al. 2008). This work on coral

integrins will be extended as a part of the second project component by determining the

ligand specificities of 3 Acropora integrins using transgenic cell spreading assays. Whereas


11

some preliminary data on cnidarian integrins has been published, the expression and

function of Cadherin mediated cell signalling systems from cnidarians are recondite. Chapter

5 explores the phylogenetic relationships between cnidarian catenin binding cadherins,

which have been demonstrated to be central to bilaterian gastrulation mechanisms. The

expression of coral cadherins and planar cell polarity components are also investigated in

Chapter 5, revealing potential roles in both embryonic and larval development.

Chapter Two Methods

12

Chapter 2: Methods

2.1 Standard PCR Protocol The standard protocol used to perform amplification from plasmids is as follows:

H20 to 20.0ul Buffer (10x) 2.0µl dNTPs (2mM ea) 1.5µl MgCl2 (25mM) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Taq Polymerase 0.6µl Template (variable)

2.2 Amplification of Gene Fragments from A.millepora

cDNA libraries Gene targets were amplified by PCR from an equal mix of cDNA libraries

representing embryonic, larval and adult tissues of A.millepora. cDNA libraries were

generated by Dr. David Hayward (Australian National University) with RNA isolated

from multiple individuals at adult, planula and prawnchip stages of development.

PCRs used to amplify partial sequences of target genes (Chapters 5 and 6) were

performed using the standard PCR protocol (Chapter 2.1) with the following

alterations:

• Primer concentration 2.5µl Primer (20µM ea)

• MgCl2 concentration 2.5µl MgCl2 (25mM)

• Template volume 0.5µl

• Final volume 25µl

1 95oC 2min 2 95oC 30sec 3 52-‐56oC 30sec 4 72oC 1min/Kb 5 Repeat steps

2-‐4 25x

6 72oC 2x extension

time 7 4oC Hold

Chapter Two Methods

13

2.3 Recovery of cDNA from Expressed Sequence Tag

libraries cDNA isolated from EST libraries were donated by Eldon Ball and David Hayward

(Australian National University, Canberra) and shipped to James Cook University on

Filter Paper. Recovery of cDNA was performed by placing the filter paper in a 1.5ml

microfuge tube and adding 50µl of ddH20 before overnight incubation at 4oC. 5µl of

resuspended cDNA was then used to transform competent E.coli NM522.

2.4 In situ Hybridisation 2.4.1 Riboprobe Synthesis

Anti-‐sense riboprobes were synthesised from partial or full-‐length cDNA cloned in

pGEM-‐T or pBluescript II KS-‐ vectors. Vectors were linearised by restriction

digestion to produce a 5’ end, 5’ overhang. Reverse Transcription was performed

using T7 or SP6 RNA polymerase (Promega) in the presence of digoxygenin (DIG)

labelled UTP. A sample of the resulting RNA was visualised by electrophoresis

through a 1% agarose gel. Labelled RNA was purified by ethanol precipitation and

resuspension in RNase-‐free water before being hydrolysed using carbonate buffer to

produce a theoretical fragment length of 250bp. Finally, riboprobes were purified by

ethanol precipitation and resuspended in resuspension buffer (50% formamide,

50% TE, 0.1% Tween-‐20).

2.4.2 Fixation of coral developmental stages

Developmentally staged embryos and larvae from Acropora millepora were fixed for

10-‐12minutes in 4% (v/v) formaldehyde in HEPES buffered Millipore Filtered Sea

Water pH8.0 (0.2µm filter; HEPES-‐MPFSW). Embryos and larvae were then washed

repeatedly in MPFSW before dehydration through a methanol/H20 series (20%,

50%, 70%, 90%, 100%x2) and stored in absolute methanol at -‐20°C.

Chapter Two Methods

14

2.4.3 Hybridisation

In situ hybridisation was carried out by methods similar to that previously described

(Hayward et al., 2001). Staged embryos were removed from storage and allowed to

come to room temperature before rehydration to 50% through a methanol series

(90%, 70%, 50%), followed by washing with PBS then PBT (once each). Overnight

incubation (4°C) in RIPA was performed to remove the large amount of lipid present

in the coral embryos & larvae. Animals were then dehydrated through an ethanol

series (25%, 50%, 70%, 90%, 100% x2) and treated with Xylene for 3-‐4hrs. Xylene

was removed by multiple washes with absolute ethanol followed by rehydration

through ethanol series and washing with PBT.

Prehybridisation was performed by replacing PBT with hybridisation solution and

incubating at hybridisation temperature (50°C-‐57°C) for 2hrs. Riboprobe was added

at a final dilution of 1/125 and allowed to incubate for 72hrs at hybridisation

temperature. Unbound probe was removed by extensive washing in several volumes

of hybridisation wash solution at 55°C-‐60°C. Hybridisation wash solution was

gradually replaced by PBT before addition of Alkaline Phosphatase conjugated anti-‐

DIG antibodies (Roche) at a 1:1600 dilution by PBT. The animals were incubated in

antibody suspension for 2 hours with gentle agitation and washed with PBT.

Detection was performed using NBT/BCIP (Vector Laboratories) diluted in PBT as

per the manufacturer’s instructions. Colour development was stopped by washing

with PBT and background colour was removed with absolute ethanol where

necessary.

Chapter Two Methods

15

2.5 Cloning of pHS S2 cell expression constructs Expression constructs were generated from pBluescript II KS-‐ plasmids containing complete

coding sequence of AmItgα1 (EU239371), ItgβCN1 (AF005356) or AmItgβ2 (EU239372) as

described in Knack et al. 2008. For each integrin subunit, both native and chimeric genes

were cloned into the pHS vector, which has previously been described for the expression of

integrins in Schneider’s line 2 (S2) cells ( Bunch & Brower, 1992; Jannuzi et al., 2002; Bunch

et al., 2004). Chimeric genes were then used as a template to incorporate integrin

heterodimer activating mutations by overlap extension PCR. Primer sequences and

amplification conditions follow.

2.5.1 Generation of native Acropora integrin expression

constructs

Native constructs included complete coding sequence amplified using the below

primers and conditions. PCR products of the expected size (~3Kb) were cleaned

using a PCR cleanup kit (MoBio) before direct restriction digestion (NEB restriction

enzymes) of PCR products and pHS vector. PCR clean up was again performed prior

to ligation of 200ng insert at 6:1 ratio insert:vector using T4 ligase (Promega).

Transformation was carried out by heat shock of 50µl competent NM522 cells. DNA

was isolated from overnight cultures of selected colonies (Axygen Miniprep kit) and

sequenced (Macrogen).

Note: Ext (Brown) is random sequence which exhibits very low specificity with

sequences in the Acropora transcriptome and pHS vector. This sequence was

included in primers to improve the specificity of restriction digests used during

cloning. 5’ and 3’ Ext are not complementary to each other or internal primer

sequences to minimise primer dimer formation.

Chapter Two Methods

16

Ext HndIII-‐Alpha1 5' Tm =60oC 5’-TGTCAACACGCGCTGTACGAAAGCTTATGCTCTTCACTTCAATAACTTG-3’ Ext EcoRI-‐Alpha1 3' Tm=58oC 5’-TGTCAACACGCGCTGTACGAGAATTCCTACAGGGCTGTCGTTTCT-3’ Ext KpnI-‐BetaCN1 5' Tm=60oC 5’-TGTCTTCACGCGCATCCGCAGGTACCATGAAGCGAAGGCTTTGCTT-3’ Ext SacI-‐BetaCN1 3' Tm=58oC 5’-TGTCAACACGGGCTTCAGCAGAGCTCACTACTGTCTTCCACCAGC-3’ Ext HindIII-‐Beta2 5' Tm=58oC 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ Ext SacI-‐Beta2 3' Tm=58oC 5’-TGTCTTCACGCGCTACGGCAGAGCTCTTATTTCCCACCATACGTAGG-3’ 2.5.2 PCR amplification from template pBSII constructs

H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Pfu Polymerase 0.6µl pBSII Template 50ng

1 95oC 2min 2 95oC 45sec 3 55oC 30sec 4 72oC 7min 5 Repeat steps

2-‐4 30x

6 72oC 8min 7 4oC Hold

Chapter Two Methods

17

2.5.3 Generation of chimeric Acropora integrin expression

constructs

Chimeric constructs included 5’ sequence from Acropora integrins coding the

extracellular region of the integrin protein and 3’ coding sequence from Drosophila

malanogaster integrin-‐αPS2C or integrin-‐βPS. 5’ and 3’ sequences were amplified

using the conditions and primers described in PCR1. Chimeras were generated from

PCR1 products using overlap extension PCR (PCR2) before direct digestion, cleanup

and ligation to pHS vector. Transformation was carried out by heat shock of 50µl

competent NM522 cells. DNA was isolated from overnight cultures of selected

colonies (Axygen Miniprep kit) and sequenced (Macrogen). Chimeric genes are

referred to as AmItgα1-‐ItgαPS2, AmItgβ1-‐PS and AmItgβ2-‐PS.

5’ Fragment Primers Ext HndIII-‐Alpha1 5' Tm =60oC 5’-TGTCAACACGCGCTGTACGAAAGCTTATGCTCTTCACTTCAATAACTTG-3’ Alpha1Δ 3' Tm=57oC 5’-CCACCACGGCGTCTTCT-3’ Ext KpnI-‐BetaCN1 5' Tm=60oC 5’-TGTCTTCACGCGCATCCGCAGGTACCATGAAGCGAAGGCTTTGCTT-3’ BetaCN1Δ 3' Tm=60oC 5’-GGGAGCCTCTGTCGGGC-3’ Ext HindIII-‐Beta2 5' Tm=58oC 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ Beta2Δ 3' Tm=57oC 5’-TTCAGCTTCCTTTGGGCA-3’ 3’ Fragment Primers Ext EcoRI-‐AlphaPS2 3' 5’-TGTCAACACGCGCTGTACGAGAATTCCTACAGGTGCTCGTCGCC-3’ Alpha1-‐AlphaPS2 adaptor 5’-GAAGAAGACGCCGTGGTGGGTCGTCGTACTGGCCGC-3’ Ext SacI-‐BetaPS 3' 5’-TGTCAACACGCGCTGTACGAGAGCTCCTATTTGCCCGCATACATG-3’ BetaCN1-‐BetaPS adapt 5’-TGCCCGACAGAGGCTCCCATGTTGGGCATCGTTATGG-3’ Beta2-‐BetaPS adapt 5’-GTTTGCCCAAAGGAAGCTGAAATGTTGGGCATCGTTATGG-3’

Chapter Two Methods

18

PCR1 – Amplification of 5’ and 3’ Sequences H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Pfu Polymerase 0.5µl pBSII Template 50ng PCR1- 5’ Amplification Cycle 3’ Amplification Cycles

1 95oC 2min 1 95oC 2min 2 95oC 45sec 2 95oC 45sec 3 59oC 30sec 3 59oC 30sec 4 72oC 6min 4 72oC 2min 5 Repeat

steps 2-‐4 30x

5 Repeat steps 2-‐4

30x

6 72oC 4min 6 72oC 4min 7 4oC Hold 7 4oC Hold

PCR2 – Overlap Extension PCR H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl PCR1 5’ product 50ng PCR1 3’ product 50ng Pfu Polymerase 0.5µl pBSII Template 50ng

1 95oC 2min 2 95oC 45sec 3 60oC 30sec 4 72oC 6min 5 Repeat

steps 2-‐4 10x

7 4oC Hold Add Outer

primers

8 95oC 45sec 9 59oC 30sec 10 72oC 7min 11 4oC Hold

Chapter Two Methods

19

2.5.4 Generation of chimeric mutant expression construct

A single amino acid mutation was introduced into the chimeric expression vector

pHSItgβ2-‐βPS pHS by overlap extension PCR. 5’ and 3’ sequences were amplified

using the conditions and primers described in PCR1. Chimeras were generated from

PCR1 products using overlap extension PCR (PCR2) before direct digestion, cleanup

and ligation to pHS vector. Transformation was carried out by heat shock of 50µl

competent NM522 cells. DNA was isolated from overnight cultures of selected

colonies (Axygen Miniprep kit) and sequenced (Macrogen). Chimeric AmItgβ2

containing an activating mutation is referred to as AmItgβ2L>D-‐PS.

5’ Fragment Primers

Ext HindIII-‐Beta2 5' 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ AmItgb2L>D S-‐ 5’1065-GGCTTGTCTGATAAGTTGGTCAAGGTTAGATGAGTCCTCTCTCAACG-1019 3’

3’ Fragment Primers Ext SacI-‐BetaPS 3' 5’-TGTCAACACGCGCTGTACGAGAGCTCCTATTTGCCCGCATACATG-3’ AmItgb2L>D S+ 5’1019-CGTTGAGAGAGGACTCATCTAACCTTGACCAACTTATCAGACAAGCC-1065 3’

PCR1 – Amplification of 5’ and 3’ sequences

H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl Fwd and Rev Primers (20µM ea) 1.0µl Pfu Polymerase 0.5µl pBSII Template 50ng


steps 2-‐4 10x

7 4oC Hold

Chapter Two Methods

20

PCR2 – Overlap Extension PCR H20 to 20.0ul Buffer (10x) (including MgCl2) 2.0µl dNTPs (2mM ea) 2.0µl PCR1 5’ product 50ng PCR1 3’ product 50ng Pfu Polymerase 0.5µl pBSII Template 50ng


steps 2-‐4 10x

7 4oC Hold Add outer

primers

8 95oC 45sec 9 59oC 30sec 10 72oC 7min 11 4oC Hold

2.5.5 Insertion of HA and MYC tags to ItgβCN1 and AmItgβ2

HA and MYC epitope tags were cloned into expression constructs for ItgβCN1 and

AmItgβ2 respectively. Epitope tag sequences were inserted into the loop encoding

region between bases 258 and 259 of ItgβCN1 and bases 248 and 249 of AmItgβ2.

This loop region was extended using sequence from the corresponding loop in the

Drosophila integrin ItgβPS. Sequences 5’ and 3’ of the insertion site were amplified

in separate PCRs from the coral template using the following primer combinations

before restriction digestion.

Template 5’ Sequence 3’ Sequence

5’ Primer 3’ Primer 5’ Primer 3’ Primer

ItgβCN1 Ext-‐KpnI βCN1

5’

ItgβCN1

insert tag 5’ S-‐

βCN1 insert tag

3’ S+

βCN1 NgoMIV

3’ S-‐

AmItgβ2 Ext-‐HindIII β2

5’

AmItgβ2

insert tag 5’ S-‐

AmItgβ2 insert

tag 3’ S+

AmItgβ2 NcoI

3’S-‐

Chapter Two Methods

21

Sense and anti-‐sense oligonucleotides encoding the HA and MYC epitopes flanked by

SacI and BamHI restriction sites were annealed by immersing a microfuge tube

containing equal portions of sense and antisense oligonucleotides in boiling water

for 5 minutes before allowing to cool slowly to room temperature. Three way

ligation was performed to combine the 5’ and 3’ coral sequences with the HA or MYC

tag insert. The resulting DNA containing the epitope tag corresponded

approximately 1/3 of original coral template in the 5’ end. This sequence was

inserted into the ItgβCN1 or AmItgβ2 expression vector by restriction digestion and

ligation. Success of the cloning process was determined by restriction digestion and

sequencing. Only 2 vectors, ItgβCN1-‐HA and AmItgβ2-‐MYC, were completed in the

timeframe required to perform cell spreading experiments. HA and MYC tagged

ItgβPS were kindly donated by Dr. Tom Bunch (University of Arizona) for

experimental control.

5’ and 3’ Fragment Primers

coral adaptor sequence SacI restriction site BamHI restriction site Ext KpnI-‐BetaCN1 5' 5’-TGTCTTCACGCGCATCCGCAGGTACCATGAAGCGAAGGCTTTGCTT-3’ ItgbCN1 insert tag 5’S-‐ 5’-ctcgacgagctcgaactcgacgagtaggagctgctgccggacatggcggagccgccgccaccagccgccagttcagcTTTTGCACCTACATTACTGT-3’ ItgbCN1 insert tag 3’S+ 5’-gatctgggatccagttccgccagcggatacgaagagtactctgccggcgaaattGTGCAAGTTCAGCCAAAG-3’ Ext HindIII-‐Beta2 5' 5’-TGTCTTCACGGGCCACGGCAAAGCTTATGCGGATATTTTGGGTTACA-3’ AmItgb2 insert tag 5’S-‐ 5’-

ctcgacgagctcgaactcgacgagtaggagctgctgccggacatggcggagccgccgccaccagc

cgccagttcagcTGTATCAAGTGGCTTGTTC-3’

AmItgb2 insert tag 3’S+ 5’-gatctgggatccagttccgccagcggatacgaagagtactctgccggcgaaattAATGTGAAAGTGAAACCACA-3’

Chapter Two Methods

22

HA and MYC epitope tag oligonucleotides

SacI restriction site BamHI restriction site HA or MYC Tag sequence Myc loop middle S+ 5’-cgtcgagcttctactcgcagagctcctcggagcagaagctgatctccgaagaggatctgg-3’ Myc loop middle S-‐ 5’-gatcccagatcctcttcggagatcagcttctgctccgaggagctctgcgagtagaagctcgacgagct-3’ HA1 loop middle S+ 5’-cgtcgagcttctactcgcagagctcctcgTACCCGTACGATGTGCCGGATTACGCCg-3’ HA1 loop middle S-‐ 5’-gatccGGCGTAATCCGGCACATCGTACGGGTAcgaggagctctgcgagtagaagctcgacgagct-3’

Chapter Two Methods

23

2.6 Maintenance of Drosophila S2 cells in culture All experiments were performed using Drosophila S2/M3 cell line (Schneider’s line 2 cells

adapted for growth in M3 medium (Schneider 1972); referred to as S2 Cells). S2 cells were

maintained at a density of 2x106 – 10x106 cells/ml in Shields and Sang M3 medium (M3;

Sigma) supplemented with 12.5% heat inactivated foetal calf serum (FCS), 100U/mL

Penicillin and 100μg/mL Streptomycin (PS). Cells were cultured under controlled

temperature conditions at room temperature (25OC) with an air gas phase. Transformed cell

lines were maintained in M3 + FCS + PS with 2x10-‐7 Molar Methotrexate (MTX).

2.7 Transfection of Drosophila S2 Cells in culture Unless otherwise stated, transfections were performed in 6 well tissue culture plates

(2ml/well) 1 day after resuspension of cells at 3x106 cells/ml in M3 +FCS +PS medium to

allow cells to reach the exponential growth phase and recover from stress. Immediately

prior to transfection, DNA mix was prepared by adding serum free M3 medium and the

following components to achieve a final volume of 100µl:

• Transient transfection only -‐ 1µl 250ng/µl GFP expression plasmid (under

Actin promotor)

• Transient transfection only -‐ 2µl 250ng/µl Mys (ItgβPS) RNAi

• Equal parts of:

o 250ng/µl pH8CO (methotrexate resistance; Rebay et al., 1991)

o 250ng/µl α integrin pHS expression construct

o 250ng/µl β integrin pHS expression construct

Negative controls contained empty pHSMCS and pH8CO vectors only. 100µl of diluted

Cellfectin (Invitrogen) was also prepared immediately prior to transfection by addition of

10µl Cellfectin to 90µl serum free M3 medium. The diluted Cellfectin and DNA mix were

gently combined before incubation at room temperature for 15minutes. During incubation,

growth medium (M3 + FCS +PS) on the cells to be transfected was replaced with 2ml serum

free M3 medium. After incubation and working no more than 2 wells at a time, M3 (serum

free) medium was removed from the cells, 800µl M3 (serum free) medium was added to the

200µl Cellfectin-‐DNA mix before being gently added to cells. Cells were incubated in the

transfection mix for 5hrs at room temperature. After incubation, the transfection mix was

replaced with 2ml M3+FCS+PS and incubated overnight. Following this, medium was

replaced with supplemented M3 medium containing MTX.

Chapter Two Methods

24

2.8 Cell spreading assays Potential ligands for coral integrins (Rbb-‐Tiggrin, Vitronectin, Fibronectin, Tennectin, Twow,

PacI, Drosophila Trimeric Laminin, Sucrose fractionated Drosophila Laminin) were diluted

in Phosphate Buffered Saline (PBS) and 200µl added to the wells of a 96well tissue culture

plate. The plate was sealed with parafilm and incubated overnight at 4OC. Prior to assay,

liquid was removed from the wells by decanting and replaced with 200µl of spreading

medium (optimal: Robb’s Saline + 1mM Mg2+ + 0.2mM Ca2+; Alternatives: BES-‐Tyrodes (1mM

Mg2+, 10µM Ca2+, 1% BSA), PBS (1mM Mg2+, 10µM Ca2+, 1% BSA), serum free M3 medium (no

additional cations)).

Cells at 3 days post-‐transfection were counted and collected by centrifugation at 1000g for 2

minutes. Cells were resuspended at 4x105cells/ml in spreading medium (Optimal: Robb’s

Saline with 1mM Mg2+ and 0.2mM Ca2+). Spreading medium was removed from the wells and

replaced with 100µl of cells in spreading medium (4x104cells/well). Attachment and

spreading was allowed to proceed for 2hrs before fixation with 2% paraformaldehyde (PFA)

by PBS. Images were captured on a Nikon X5000 attached to a Zeiss Axiovision 200

microscope.

Chapter Two Methods

25

2.9 Antibody staining and flow cytometry of integrin

expressing cells Duplicate cultures of S2 cells were sampled 2 days after transfection with coral and/or fly

integrin subunits. Samples of 0.5x106 -‐ 1x106 cells were resuspended in 50µl of primary

antibody diluted as follows:

• Rabbit anti-‐AmItgα1 1:500 dilution (1:100 and 1:1000 also trialled)

• Rabbit anti-‐ItgβCN1 1:500 dilution (1:100 and 1:1000 also trialled)

• Rabbit anti-‐AmItgβ2 1:500 dilution (1:100 and 1:1000 also trialled)

• Mouse anti-‐ItgαPS1 1:500 dilution

• Mouse anti-‐ItgαPS2C 1:1000 dilution

• Rabbit anti-‐HA-‐488 1:1000 dilution

• Rabbit anti-‐MYC 1:1000 dilution

Samples were incubated in primary antibody for 20-‐25minutes before washing with 500µl

M3 serum free medium and resuspension in 50µl secondary antibody diluted as follows:

• Goat anti-‐Rabbit 546 1:1000 dilution – Coral integrins

• Goat anti-‐mouse-‐546 1:1000 dilution – Fly integrins

Samples were incubated in secondary antibody for 20-‐25minutes before washing with

500µl serum free M3 medium and fixation in 500µl 2% PFA by PBS. Surface antigen

detection was then performed by flow cytometry. To sort antigen expressing cells for cell

spreading assays (Section 6.3.2), cells were washed 2x in 500µl Robb’s Saline after

incubation with secondary antibody and maintained on ice prior to cell sorting.

Chapter Three JCUSMART

26

Chapter 3: JCUSMART – A simple tool for automated annotation and exploration of large protein sequence datasets

3.1 Introduction The traditional view of many evolutionary and developmental biologists has been that the

simple morphology of basal invertebrates such as cnidarians is a reflection of low genetic

complexity. The genomic complexity of cnidarians has often been underestimated through

the reasonable expectation that simple morphology is correlated with low complexity.

However, studies in the early 2000’s examined this expectation and demonstrated specific

examples where aspects of cnidarian genetics were considerably more complex than

expected (Schmitt & Brower, 2001; reviewed in Ball et al., 2004; Martindale et al., 2002).

Compelling evidence for broad genetic complexity within basal metazoans arose from

analysis of the moderately sized expressed sequence tag (EST) projects of Acropora

millepora and Nematostella vectensis. Sequence analysis of these EST data showed that

Acropora possesses a higher proportion of sequences with greater similarity to humans than

traditional invertebrate models (ie. Drosophila and Caenorhabditis) (Kortschak et al., 2003).

Both Acropora and Nematostella datasets contain a significantly higher than expected

representation of signalling pathways previously assumed to be vertebrate specific, and

exhibit a degree of ancestral gene maintenance not observed in bilaterians (Technau et al.

2005). Evidence for ancestral complexity within the Cnidaria was consolidated with the

publication of Nematostella genome, which supported the above findings and revealed that

the number of coding genes is more similar to that of Deuterostomes than Ecydosozoans

(Ball et al., 2004; Martindale et al., 2002; Schmitt & Brower, 2001). The demonstrated lack of

correlation between genetic and phenotypic complexity has lead to a new appreciation for

the complexity that arose prior to bilaterian evolution.

The dramatic change in understanding of cnidarian genetics and its implications for

metzoan evolution exemplifies the importance of sequence analysis as a research tool. The

power of sequence analysis to yield results capable of making a significant impact on any

field of study is heavily reliant on both the size and quality of the sequence data available.

Development of high throughput sequencing technologies including Illumina and 454

sequencing has made generation of high quality, large scale datasets (such as transcriptome


27

and genome sequencing) feasible for a much wider range of non-‐traditional model

organisms. Some publically available examples available at the Ensembl genome browser

(http://www.ensembl.org/index.html) include Dolphin, Alpaca, Wallaby, Turkey and Shrew.

Thee hundred and twenty three (323) whole genome projects are currently (July 2011) in

the public domain (available through Ensembl) and the decreasing cost of sequencing is

stimulating the occurrence of private large scale datasets. Despite the increasing ease of

generating data, the ability to navigate large datasets in order to explore genetic

relationships and genetic novelties remains out of reach for most individual molecular

biologists and requires the assistance of a dedicated bioinformatician. As a result, the huge

potential of large scale sequencing for furthering our understanding of genetic diversity is

underutilised.

Increasing the accessibility of large datasets for analysis by a greater proportion of the

scientific community hinges on accurate annotation of sequence data, principally of coding

sequences and predicted peptides. This is particularly important in organisms such as

cnidarians where current data indicates a significant proportion of the detectable coding

sequences are dissimilar to all sequences described in the primary public databases

(Swissprot and NCBI). However, annotation of these datasets by molecular biologists is often

limited by the procedural aspects of operating commonly accepted annotation software,

including installation on an appropriate computing environment, operation of the

computing environment and the annotation tools using a command line interface, and

management of the output data.

Two of the most powerful and common annotation tools are BLAST, to explore pairwise

similarity, and HMMER to explore conservation of functional domains within protein

sequences. Like most common annotation tools, these can be utilised for individual

sequences by virtue of online servers accessible through a graphical interface. These public

servers carry heavy limitations to compute time and the number of sequences that can be

processed and are therefore not suitable for large scale processing.

Limitations to the capacity of graphical and easy to manage annotation tools have

contributed to the development of an upward or “gene by gene” approach to annotation and

large dataset exploration (Figure 3.1). The “gene by gene” approach starts with a small

number or narrow range of sequences to be annotated and often yields a narrow range of

results. For example, in the case of identifying a single or small number unknown sequences,


28

BLAST can be performed online against public databases and similarity to reported

sequences can be identified. For this application, the “gene by gene” approach is ideal.

However, this approach is less suitable for identifying sequences of interest from a pool of

unknowns (eg. a transcriptome or genome sequence dataset). This task requires use of a

known homologue of the sequence of interest, which is used to query a custom BLAST

database and may require some verification of results, such as BLAST of the best hits for

each gene of interest against NCBI non-‐redundant (nr) or SwissProt databases. The analysis

process quickly becomes time consuming as the number of sequences increases.

Figure 3.1 Upward approach to identifying genes of interest in a custom dataset. The process undertaken to analyse the data is shown in A, whilst the impact on the size of the gene’s of interest group is displayed in B As the identification process proceeds, the number of genes that need to be considered increases with each step. Steps marked with “*” have potential to introduce identification / annotation error to the system, through false matches which may not be easily detectable if only BLAST is considered.

A B


29

The time required to processes data by the gene by gene approach can reduce the number of

annotative tools that are used, which can have adverse affects on the accuracy of the final

annotation. Often the only tool employed by molecular biologists in sequence identification

is BLAST. As with all bioinformatics programs, BLAST has implicit limitations. When

considering large proteins with common or repeated domains, for example those of the

Cadherin family, which can contain more than 30 Cadherin domains, the BLAST analysis fails

to accurately distinguish between members of the family, even if repeat masking is applied.

In these cases, and cases where the query sequence is potentially highly derived as expected

in cnidarian data, the protein architecture (domain structure, presence of signal peptides

and transmembrane regions etc) must also be considered. The Simple Modular Architecture

Research Tool (SMART) (Letunic et al. 2009; Schultz et al. 1998) served from Heidelberg

University (http://smart.embl.de) is one online tool that allows for more accurate peptide

annotation through identification of protein architecture, however the same limits to input

size as with other online serves apply.

In order to more effectively explore large datasets from potentially divergent organisms, an

approach that asks “What genes are present?” must be used instead of one that asks “Is this

specific gene present?”. This more permissive, downward approach (Figure 3.2) is better

suited to surveying large or multiple datasets for whole families of genes. Using a downward

approach, genes of interest are identified by searching pre-‐assigned annotations for specific

combinations of features that are consistent with the genes of interest. Through control of

search terms, this approach allows greater flexibility and control over the scope of results

obtained.

For public genome project data, the level of exploration offered by a downward approach is

primarily accessed through genome browsers (eg. JGI genomes and Ensembl), which usually

offer results for BLAST against SwissProt, HMMER analysis against Pfam and SMART, and

organisation of sequences into Eukaryotic Cluster of Orthologous Groups (KOG) or Kyoto

Encyclopaedia of Genes and Genomes (KEGG) pathways. Using a genome browser to survey

public genomes for genes with specific features often begins with exploring the contents of

the appropriate KOG and/or KEGG annotations. The results can then be narrowed on the

basis of protein architecture (structural features of a protein including signal peptides,

conserved domains and transmembrane regions) or BLAST annotations. Unfortunately,

accurate mapping of private data to the KOG and KEGG hierarchies is not always possible

and is particularly complicated for lower organisms such as cnidarians due to small dataset

size and the strong vertebrate bias in publically available genomes on which KOG and KEGG


30

are based. Instead exploration of large private datasets must be based on the remaining

annotative tools, protein architecture and BLAST. Fortuitously, software to perform these

analyses are freely available for academic purposes and can be installed on multiple

computing platforms.

Figure 3.2 Downward approach to identifying genes of interest in a custom dataset using JCUSMART. The process undertaken to analyse the data is shown in A, whilst the impact on the size of the gene’s of interest group is displayed in B. As the process proceeds, the number of sequences to consider is reduced in a manner reflecting the stringency of search criteria. Error is not introduced into the system, however some sequences may not be identifiable due to the nature of the custom dataset. These sequences are more easily detected using the downward approach rather than falsely annotated.

In order for large scale sequencing data to be effectively utilised by a greater proportion of

molecular biologists, a single access point for data annotation and exploration using a

downward approach must be available. Currently, no single package is publically available

to perform both of these functions. To address this deficit, I have developed a tool named

JCUSMART that aims to act as a single platform for automated annotation of large protein

datasets and for exploration of the resulting annotations using a downward approach.

Adoption of this tool will facilitate exploration of large datasets by a larger number of

molecular biologists who have only a basic background in bioinformatic techniques.

A B


31

3.2 Methods (development of JCUSMART) In order to provide a more accessible single platform for employing a downward approach

to large scale data exploration I developed a system named JCUSMART that allows

assessment of both BLAST results and protein architecture. JCUSMART was designed to

address both analytical and user interface requirements.

The analytical requirements of JCUSMART were:

• To perform timely analysis of large private datasets by BLAST against public

datasets and determination of protein architecture

• To extract key information from raw results for storage and display

• To store key information in a searchable format

Analytical requirements were addressed using a computational pipeline, which was capable

of thin deployment over a local computing cluster thereby reducing analysis time. Outputs

from annotative programs (Table 3.1) were parsed for key information, which was passed

into a PostgreSQL database. The computational workflow is detailed in Figure 3.3. BLAST

annotation was not included in the automated analysis in order to give greater control over

the BLAST parameters. A separate script was developed to enter BLAST data into the

database.

To make this tool accessible the user interface was required to:

• Be presented for use in a single, intuitive, graphical environment

• Have simple operation for initiation of analyses and exploration of results

• Present results clearly

JCUSMART is accessible through either a web interface

(https://kanga.hpc.jcu.edu.au/jcusmart/index.html) or command line interface. Both offer

the ability to launch new JCUSMART analyses and search the database for sequences with

common annotations. Both interfaces have simple operation and display results in an easily

interpreted table.


32

Program Function Origin Link HMMER2: HMMPFAM

Predict Conserved Domains using HMMs. PFAM and SMART databases utilised

Howard Hughes Medical Institute and Dept. of Genetics, Washington University

http://hmmer.janelia.org/

TMHMM Predict Transmembrane Helicies

Center for biological sequence analysis – Technical University of Denmark (CBS-‐DTU)

http://www.cbs.dtu.dk/services/TMHMM (Krogh et al., 2001)

SignalP Predict signal peptides

CBS-‐DTU http://www.cbs.dtu.dk/services/SignalP (Bendtsen et al., 2004)

TargetP Predict cell

membrane/ mitchondreal/ Chloroplastic signal peptides

CBS-‐DTU http://www.cbs.dtu.dk/services/TargetP (Krogh et al., 2001)

SEG Identify low complexity segments

CBS-‐DTU (Wootton, 1994)

NCOILS Predict coiled-‐coil regions

CBS-‐DTU (Lupas et al., 1991)

Prospero Identify internal repeats

Welcome Trust Centre for Huan Genetics

http://www.well.ox.ac.uk/ariadne/intro.shtml (Emanuelsson et al., 2000)

Netphos Predict Phosphorylation sites

CBS-‐DTU http://www.cbs.dtu.dk/services/NetPhos (Blom et al., 1999)

Table 3.1 Programs used by JCUSMART in the annotation of protein sequences, showing the information gathered and the program origin. Conserved Functional domains were detected using HMMPFAM in conjunction with PFAM and SMART Hidden Markov Model Libraries. TMHMM was used to predict the presence of transmembrane helices. Membrane targeting signal sequences were identified using SignalP and TargetP. Low complexity segments and coiled-‐coil predictions were predicted by SEG and NCOILS respectively. Prospero and Netphos were used to identify internal repeats and putative phosphorylation sites. All programs are freely available for academic use. No citation is available for HMMER2 as per the program manual.


33

Figure 3.3 Computational Workflow of JCUSMART pipeline. JCUSMART accepts protein sequences in FASTA format. The input file is parsed for characters that raise errors in the analysis programs, removing or replacing characters where required (this does not affect the protein sequence). JCUSMART then loads the cleaned input file to each program for analysis. Each program runs on a separate node of the James Cook University High Performance Computing Cluster (HMMPFAM on 2 nodes) and is assigned the maximum number of CPUs allowed by the program. Raw output files are parsed for results informative to most molecular biologists, which are then entered into the JCUSMART database. The JCUSMART graphic user interface and command line interface allow text based searching of database results for identification of sequences containing specific combinations of domains and protein architectural features (eg. Singal peptide, specific combinations of domains, transmembrane helix, and phosphorylation sites). Refining search terms allows narrowing of the number of sequences of interest. BLAST results are also stored in the JCUSMART database, however this analysis is run independently. Results of BLAST can also be searched for key words allowing identification of a greater number of potential sequences of interest (dependant on similarity limits imposed on BLAST analysis), whilst providing enough architectural information to accurately assign orthology even in cases where BLAST alone is insufficient.


34

3.3 Results (Testing and low intensity applications) Initial testing and development was centred around the sequences of the three integrins

from Acropora millepora which had previously been characterised in terms of protein

architecture (Knack et al., 2008). For each annotation program, a comparison was made

between the known protein architecture, the raw program output and the results in the

database. These results were consistent for the three sequences used. The same comparison

was made for a 10 sequence analysis and also found to be 100% consistent, demonstrating

annotations were not lost or re-‐assigned before entry into the database. Analysis of 100 and

1000 sequence datasets were performed in order to check for errors in the database.

SignalP, HMMER, TMHMM and TargetP programs produce a database entry for every

sequence analysed. Errors could therefore be identified by comparing the number of input

sequences to the number of entries for these programs. Re-‐testing using these datasets was

performed after each adjustment to the system. Testing and development of database

queries were also perform using these initial datasets.

3.4 Discussion The increasing ease of generating large amounts of sequence data is broadening the scope of

what can be achieved through sequence analysis. However, a large proportion of molecular

biologists who could benefit from exploring large private datasets for proteins containing

specific features of interest are not utilising the data for their maximum benefit. In many

cases this is simply due to inexperience with the procedural aspects of bioinformatic

analysis on large datasets. JCUSMART provides a single tool for running basic automated

annotation of large datasets and exploring the results using a downward approach.

Currently the JCUSMART pipeline can analyse protein data from a whole genome containing

30,000 sequences in less than 3 days. After analysis, multiple large datasets that may consist

of over 200,000 sequences in total can then be searched for specified features in a matter of

minutes. Common queries used to explore data from one or more datasets in the JCUSMART

database include:

• Searching for proteins with specified conserved domains

• Searching for proteins with specified architectural features (eg. signal peptide or

number of transmembrane domains)

• Searching for sequences that have BLAST hits with descriptions matching specified

key words using Boolean operators (AND, OR, NOT)


35

For many of the data types (eg. conserved domains and BLAST hits) E-‐value cutoffs can be

specified, allowing control over the specificity of results returned by a query, and any chosen

fields can be retrieved using a list of sequence names generated using JCUSMART or any

external method. The rapid cross dataset search times and ability to control search

stringency makes this tool ideal for comparative studies and identification of novel or

divergent sequences of interest from a large pool of data. A number of limitations currently

exist in JCUSMART. The current web based interface lacks features such as downloadable

results file and fetching sequences from the database in FastA format. These issues have

been resolved in the command line interface. The web interface could also be improved by a

graphical display of results with links to external information (eg. conserved domain

descriptions), which would make interpretation of results more straightforward than the

current tabular format.

JCUSMART is primarily designed to facilitate identification of sequences of interest from a

pool of unknown sequences. It is not designed to identify orthologous sequences or group

sequences automatically. This is due to the difficulties of automating the integration of

BLAST (which is potentially misleading for divergent or basal animals) and protein

architecture information to reach an accurate result. Protein grouping is better performed

manually using the JCUSMART information as it allows the user to consider their prior

knowledge of the proteins of interest. Protein identification therefore involves refining

search criteria to suit a specific question. Some sequences may not be identifiable using

JCUSMART for a number of reasons (Table 3.2). It should be noted that the inability to

identify a homologue of a particular gene of interest is not a definitive indication of its

absence from an organism, it may simply be absent from the dataset.


36

Reason for Absence Description

Fragmentation of genes

into multiple models

This is an inherent limitation of the contiguous sequence assembly

process (unrelated to JCUSMART). In the case of genome sequencing

this is aided by increased sequence coverage and EST support.

Incomplete models may

not represent the gene

sufficiently to be

identified using the

search criteria

Gene models and therefore predicted peptides, may represent

regions of a gene that are specific to a particular biological function

or to a specific gene. These sequences cannot be resolved without

laboratory investigation (ie. cloning and sequencing).

Incomplete models more

closely resemble other

sequences

Limitations in BLAST and HMMER can produce misleading results.

Often in this case the results of BLAST against NCBI nr will not agree

with the domain structure of the predicted peptide.

Inappropriate search

criteria

Narrowing search criteria to focus on a diagnostic region of the gene

of interest can sometimes correct this. Search criteria involving

keywords commonly require refinement.

Table 3.2 Conditions under which sequences may not be identifiable using the JCUSMART method. Both errors in gene or protein prediction, such as fragmentation of genes into multiple models or truncated gene models, and errors in the search approach (ie. inappropriate search criteria) can result in unidentifiable sequences.

Currently the JCUSMART database holds annotations for a total of 112,868 sequences

(~35Gb) from five basal animal datasets:

• Nematostella vectensis genome filtered models – 27273 sequences

• Hydra magnipapillata EST project protein predictions – 19845 sequences

• Acropora millepora Transcriptome protein predictions – 48 335 sequences

• Clytia hemispherica EST project protein predictions – 8219 sequences

• Monosiga brevicolis Genome predicted peptides – 9196 sequences

The application of JCUSMART in identification of specific gene families across several genera

has already made a significant contribution to a number of research projects. The projects

and the application of JCUSMART is outlined in Table 3.3. One further documented

application for JCUSMART has been in the identification of adhesion genes throughout the

Cnidaria. This survey of the cnidarian adhesion complement is presented in Chapter 4 and

was the model use case for JCUSMART.


37

Research Contribution using JCUSMART Foret et al (2010), New tricks

with old genes: the genetic

bases of novel cnidarian traits.

Trends in Genetics, 26 (4), 154-‐

158

Identification of CEL-III like lectins in Clytia hemispherica but

not in Nematostella vectensis or Hydra magnipapilata. This

distribution of CEL-III demonstrates the stochastic nature of

gene loss in cnidarians

Detection of Selenium related

proteins in Acropora millepora

(In preparation)

Identification of GPx (1), TR (2), Other Seleno-‐proteins (8),

selenium binding proteins(2), other related factors (1) from

Acropora millepora EST data. These sequences formed the

basis of protein characterisation and coral stress experiments

to meet PhD project requirements.

Detection of caspase and NOD

related genes in Acropora

millepora, Nematostella

vectensis, Hydra magnipapillata,

Clytia hemispherica

Identification of NOD and caspase proteins from each of 4

cnidarian datasets prior to cloning and laboratory

investigation used to meet PhD project requirements.

Table 3.3 JCUSMART has already contributed to published and unpublished work though the identification of genes of interest and sequence annotation. The project and the contribution of JCUSMART are outlined in the table.

3.5 Conclusions Through the use of a distributed computational pipeline and a relational PostgreSQL

database, JCUSMART delivers accurate and highly searchable automated protein annotation

in a timely manner. Having already demonstrated its usefulness in a number of projects,

JCUSMART provides an efficient tool for employing a downward approach to identifying

sequences of interest, and facilitates the exploration of large protein datasets by molecular

biologists with limited bioinformatic knowledge.

Acknowledgements Initial development and implementation of pre-‐processing, job dispatch and data storage

systems were performed in conjunction with Dr. Wayne Mallet of the James Cook University

High Performance Computing Department. Testing and development of BLAST dispatch,

BLAST data storage and command line query scripts were performed with the assistance of

Wade Tattersall of the JCU ARCHER project. Thanks also to Russell Sim and David Lang for

guidance on programming and query design.

Chapter Four Diversity of Adhesion Molecules in cnidarians

38

Chapter 4: Diversity of cell adhesion molecules in cnidarians

4.1 Introduction Cellular contacts with the extracellular matrix (ECM) and surrounding cells play a critical

role in the development and survival of animals, as demonstrated by the multitude of severe

and often lethal phenotypes resulting from misregulation of cell cohesion and cell migration

(Hogg & Bates, 2000). A combination of genome analyses and functional studies in

representative bilaterians has led to an appreciation that cell adhesion molecules, the

proteins facilitating cellular cohesion and migration, form a complex network capable of

regulating diverse biological processes in both protostomes and deuterostomes (Klein &

Mlodzik, 2004; Schambony, Kunz, & Gradl, 2004; Tan et al., 2001). The importance of cell

adhesion molecules to fundamental processes such as morphogenesis and immunity has

been described in detail. Examples include integrins, which function in leukocyte trafficking

and formation of the gastric cavity (Kinashi, 2005; Serini, Valdembri, & Bussolino, 2006;

Yang et al., 1999), cadherins, which influence cell polarity and directed cell movements

(Kimura-‐yoshida et al., 2005; Rodríguez, 2004), and immunoglobulins, which are the

predominant immune mediators in bilaterians, facilitating vertebrate adaptive immunity.

Comparative analyses have also identified that many families of adhesion genes, such as

integrins, catenin binding cadherins, and several components of the basement membrane

are conserved throughout the Bilateria, playing analogous roles in a variety of model

organisms. However, the complement and function of cell adhesion molecules from outside

the Bilateria is poorly investigated.

Biological processes governed by cell adhesion in bilaterian animals are also observed in

lower metazoans such as cnidarians and sponges. Cnidarians, the most basal phylum to

contain a tissue layer level of organisation (Technau et al., 2005), undergo a broad variety of

processes that centre around adhesion molecule expression and dynamic changes in

adhesive state including complex morphogenic cell re-‐arrangements during gastrulation and

metamorphosis (Grasso et al., 2008; Hayward et al., 2004), allorecognition between

individuals and colonies (Nicotra et al., 2009), innate immunity (Miller et al., 2007), nervous

system development (de Jong et al., 2006) and the uptake/loss of zooxanthellae in symbiotic

cnidarians such as coral (Gates et al., 1992; Kvennefors et al., 2008, 2010). This diverse

phylum is also positioned at the base of the Eumetazoa (‘true animals’), owing to possession

of a nervous system and dedicated tissues. Furthermore, gastrulation results in development

of only two germ layers rather than three as observed in bilateral animals. These attributes


39

make cnidarians a significant comparator to bilaterian systems and particularly informative

for studies of evolution and development, allowing the genetics of the last common

eumetazoan ancestor to be inferred. Despite the importance of cnidarians to understanding

the fundamental roles of adhesion molecules in tissue development and the molecular

evolution of adhesion genes, the diversity and function of adhesion genes in cnidarians are

largely unexplored.

Investigations encompassing aspects of cnidarian adhesion have previously been focused on

elucidating the distribution of a single gene family (defined by the presence of specific

functional domains) throughout the whole of the Metazoa. The evolution of only 3 families of

adhesion molecules (integrins, cadherins and G-‐Protein Coupled Receptors) has been

investigated in this manner with each analysis suggesting several family members

originated prior to the eumetazoan ancestor (Hulpiau & van Roy, 2011; Nordström et al.,

2009; Sebé-‐pedrós et al., 2010). No common evolutionary trend could be determined from

these proteins families, as each family has a broad variety of functions and therefore

selective pressures, however, expansion of protein families is generally evident at the base

of the bilaterian and vertebrate lineages. Although these surveys provide perspective on the

origin of specific adhesion families, they provide a limited view of the cnidarian adhesion

gene complement or ‘adhesome’ as a whole. Such targeted studies also have a restricted

capacity to consider the importance of novel or structurally distinct family members in a

biological context, which is central to understanding the significance of ancestral adhesion

molecules.

In order to establish an accurate overview of the cnidarian adhesome, the present analysis

considered 7 families of adhesion molecules with described roles in bilaterian development

or immunity. In addition to investigation of the integrin, cadherin and immunoglobulin

families mentioned above, members of the lectin, class B G-‐protein coupled receptors

(GPCR), Adhesion leucine rich repeat (LRR) and extracellular matrix families were also

identified. Analysis of these families attempted to identify all possible members with

relevance to cell adhesion processes, although additional members of these families may be

detectable using less stringent criteria.


40

Previous studies into the evolution of adhesion molecules often considered only a single

representative, the sea anemone Nematostella vectensis, as representative of cnidarians as

whole. However, the genetic diversity and near stochastic nature of gene loss observed in

the Cnidaria suggest the use of a single species may not be suitable to gain insight into the

evolution of adhesion genes originating in the eumetazoan ancestor. To overcome this

limitation the present analysis examines EST, transcriptome and genomic data from 4 model

cnidarians, Acropora millepora, Nematostella vectensis, Hydra magnipapillata and Clytia

hemispherica. This selection of cnidarians includes representatives from 2 taxonomic

classes, Hydrozoa (Hydra and Clytia) and Anthozoa (basal among cnidarians; Acropora and

Nematostella), and 2 habitats, marine (Acropora and Clytia) and estuarine (Hydra and

Nematostella). Developmental strategies also vary between the 4 species, with Hydra and

Nematostella developing directly from larvae to adult, whilst Acropora and Clytia (which

also exhibits a medusa stage) undergoing metamorphosis to a sedentary polyp. The variety

of the taxonomic classes, habitats and developmental strategies are typical of cnidarians and

allows this analysis to more accurately elucidate conservation, novelty, and differences in

cnidarian adhesion systems, whilst developing a strong basis for inferring the adhesion

repertoire of the common metazoan ancestor.


41

4.2 Methods The adhesion complement of four cnidarians (Acropora millepora, Nematostella vectensis,

Hydra magnipapillata and Clytia hemispherica) and one choanoflagellate (Monosiga

brevicolis) was assessed using the automated annotation and data exploration capacities of

JCUSMART (described in Chapter 3). Predicted peptides with potential cell adhesion

capabilities were identified from 5 large sequencing datasets (Table 4.1) by searching

JCUSMART annotations with the terms presented in Supplementary Figure 4.1. These

searches focused on the discovery of peptides associated with 7 broad families of cell

adhesion molecules (cadherins, integrins, lectins, Adhesion-‐Leucine Rich Repeat (Adhesion-‐

LRR), Adhesion G-‐protein coupled receptors (Adhesion-‐GPCRs), immunoglobulin

superfamily, and extracellular matrix (ECM)), which are central to developmental,

immunological and defensive processes in metazoans. Many of the predicted peptides could

be further categorised into sub-‐families according to the presence of key architectural

features used in the classification of bilaterian proteins. Orthology of a prediction to any

previously described sequence was assigned only after manual consideration of both the

predicted protein architecture and results of BLASTp against the NCBI non-‐redundant (nr)

database. As detailed in chapter 3, genes were more easily detected in anthozoans, owing to

the larger size of the available data and longer predicted peptides. Therefore the absence of

some genes from this analysis (particularly from Clytia) may reflect limitations of the

predicted peptide collection rather than gene losses.

Animal Data type Number of sequences

Reference

Acropora millepora Transcriptome (2010) 48 335 Miller et al., unpublished

Nematostella vectensis Genomic predicted peptides

(version 1)

27 273 Putnam et al., 2007

Hydra magnipapillata Unigenes (EST contigs) 19 845 Bosch et al.,

unpublished

Clytia hemispherica Unigenes (EST contigs) 8 219 Houliston et al., 2010

Monosiga brevicolis Genome predicted peptides

(version 1)

9 196 King et al., 2008

TOTAL 112 868

Table 4.1 Predicted peptide datasets used to assess the adhesion complement of four cnidarians with diverse lifecycles and developmental features. Each dataset was annotated and explored using JCUSMART as described in Chapter 3. The Number of Sequences presented in the table represents an unadjusted total of the sequences analysed and does not take into account occasions where models were later combined to provide complete coding sequences.


42

4.3 Results 4.3.1 Cadherins

Hydra magnipapillata

Clytia hemispherica

Nematostella vectensis

Acropora millepora

Monosiga brevicolis

Catenin binding 1 -‐ 4 1 -‐ Flamingo -‐ -‐ 1 1 -‐ Calsyntenin -‐ -‐ 1 2 -‐ FAT -‐ -‐ 1 -‐ -‐ FAT-Like -‐ -‐ 2 -‐ -‐ Dachsous like -‐ -‐ 10 1 6 Unique -‐ -‐ 2 -‐ 4

Table 4.2 Distribution of Cadherin family proteins in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.

A modest number of predicted peptides from each cnidarian contained extracellular

Cadherin (EC) domains (maximum of 56 distinct sequences in the complete dataset

available, the Nematostella genome) and very few peptides contained other conserved

domains. Although the number of cadherins in each animal was limited there was a

surprising richness of cadherins with recognised developmental and signalling roles

including orthologues of Type-‐III Catenin Binding cadherins (Figure 5.3), Flamingo (CELSR)

(Supplementary Figure 5.4), FAT and FAT-like (in Nematostella), Dachsous (Supplementary

Figure 5.2), Cadherin23 and Calsyntenin as well as a limited number of Protocadherins.

The number of genes in each sub-‐family is comparable between anthozoans, however, large

cadherins are difficult to detect and may obscure understanding of the complete hydrozoan

Cadherin complement. It is expected that representatives of the developmentally significant

cadherins (above) are in fact present in each cnidarian, supporting previous suggestions by

Hulpiau and van Roy (2011) that their origin predates the cnidarian divergence. Monosiga

peptides containing EC domains are unlike those of the Metazoa, consisting of novel

combinations of domains or chains of EC domains up to 58 repeats long.

Investigation of mRNA expression patterns during coral development and discussion of

putative protein function of some members of the Cadherin superfamily are presented in

Chapter 5.


43

4.3.2 Integrins Hydra

magnipapillata Clytia

hemispherica Nematostella vectensis

Acropora millepora

Monosiga brevicolis

integrin Alpha 1 1 2 3 -‐ integrin Beta 2 1 4 2 -‐ integrin Linked Kinase

1 1 1 1 -‐

ILK associated Ser/Thr Kinase

1 -‐ 1 -‐ 1

Talin 2 2 1 2 1 PINCH 1 -‐ 1 1 -‐ Parvin 1 -‐ 1 1 -‐ Paxillin 1 1 1 1 1 FAK -‐ 1 1 1 -‐ c-Src 1 -‐ -‐ 1 -‐

Table 4.3 Distribution of integrin and integrin associated proteins in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.

All components of the integrin signalling system reported in vertebrates were identified in

the cnidarians except Nematostella c-Src, Clytia PINCH & Parvin, and Hydra Focal adhesion

Kinase, although these too are expected to be present. Cytoplasmic signalling proteins of the

integrin pathway such as Talin, integrin Linked Kinase (ILK), PINCH and α-‐Parvin are

represented by a single peptide prediction in each cnidarian, however the number of α and β

integrin models is variable. As previously reported in Knack et al (2008), Acropora contains

only 2 integrin β-‐subunits (confirmed here by exhaustive transcriptome searches) compared

to Nematostella 4, Hydra 2 and Clytia 1. The distribution of α-‐Integrins is similarly fluctuant

between cnidarians, with Acropora possessing 3, Nematostella 2, Clytia 1, and Hydra 1. This

distribution combined with phylogenetic analyses (Figures 6.1 & 6.2) suggests that the

eumetazoan ancestor possessed a complement of at least 2 β and 1 α integrins and

expansion in each cnidarian is the result of independent duplications. The heterodimer

combinations and ligand binding properties of the cnidarian integrins are unknown, but may

hold clues as to their biological function and relationship with bilaterian integrins.

Evolutionary relationships and ligand binding properties of Acropora integrins are

presented in Chapter 6. Patterns of mRNA expression for Acropora integrins have previously

been reported by Knack et al (2008).


44

Figure 4.1 Maximum likelihood analysis of Talin proteins from representative metazoans. Clytia Talin1 and Clytia Talin2 are closely associated, grouping together with Nematostella with strong bootstrap support (91%). Cnidarian sequences form an independent clade basal to the bilaterian and vertebrate clade, suggesting that duplication in Clytia is genus specific and does not support an ancestral origin of the duplication observed in vertebrates. Phylogenetic analysis is based on alignment of Talin proteins in the region of overlap between Clytia talin1 and Clytia talin2 (Supplementary Figure 4.2). Branch numbers represent percentage bootstrap support based on 100 bootstrap replicates.

In contrast to the complete conservation of integrin signalling components in basal

metazoans, the complement of Monosiga lacks critical components. Protein domains

resembling the α-‐integrin repeat domain were detected however no complete α or β

integrins could be identified. ILK, PINCH and α-‐Parvin were also absent, however Talin was

found to be present, which suggests an alternative function for FERM domain proteins,

which are enriched in choanoflagellates (Nicole King et al., 2008). Phylogenetic analysis of

Talin genes (Figure 4.1) places Monosiga and cnidarian Talin genes independent of

metazoan counterparts showing that vertebrate Talin1 and Talin2 arose from a lineage

specific independent duplication.


45

4.3.3 Lectins Hydra



Acropora millepora

Monosiga brevicolis

C-‐Type Soluble

12 4 42 3

C-‐Type Secreted 3 5 13 6 C-‐Type Transmembrane

4 2 10 2

Galectin 19 4 42 2 Fucolectin 1 -‐ 25 21 -‐ Mannose Binding (Legume Like)

2 -‐ 2 3 2

Haemolytic -‐ 2 -‐ 3 -‐ Table 4.4 Distribution of lectin family proteins in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.

lectin families known to be important to metazoan processes including cell-‐cell adhesion

and immunity were represented in cnidarians with unexpected diversity. The most

expansive lectin family of higher animals, the C-‐type lectins, were also particularly well

represented in all four of the cnidarians investigated and comprised the greatest proportion

of the lectins identified from each species. Galactose binding lectins were the next most

abundant being found in similar numbers to C-‐type lectins. Despite the number of C-‐type

lectins (42 from Nematostella), there was only a limited selection of predicted peptides that

are likely to be secreted (13 -‐ Nematostella) as determined by the presence of a signal

peptide. Even fewer models (9 -‐ Nematostella) contained a transmembrane helix and could

be considered potential cell surface antigens. The C-‐type lectin family did however contain a

surprising diversity of associated domains, which more closely resembles vertebrates than

protostome invertebrates (Wood-‐Charlson & Weis, 2009). One model of C-type lectin 16A

(CLEC16A), which is highly expressed in circulating immune cells and associated with a

range of auto-‐immune diseases in man, was found in each cnidarian. The CLEC16A models

were the only predicted lectins to demonstrate significant homology to known lectins (based

on BLAST) and have not previously been reported outside the Bilateria.

lectins containing a collagen domain, followed by a lectin domain were conserved

throughout the cnidarians. This structure is consistent with the Collectin family (Figure 4.3),

which has only been reported once in lower animals during a microarray analysis of

Acropora metamorphosis (Grasso et al., 2008). Unlike the bilaterian proteins, which consist

of Collagen -‐ C-‐Type lectin combination, 14 of the Collectins from cnidarians contain a

galactose binding domain (Gal_lectin), and only 2 (1 Nematostella and 1 Clytia) have a C-‐


46

type lectin domain. Nematostella, Acropora and Clytia each contain three models with

predicted signal peptides and none of the sequences have transmembrane helices,

suggesting they are secreted.

Fuctose binding lectins (Fucolectins/F-‐type lectins) are also not reported in pre-‐bilaterian

animals and have limited abundance in basal deuterostomes such as the purple sea urchin

(Strongylocentrotus purpuratus – 2), yet a surprising number were identified in the

Nematostella genome (26) and Acropora transcriptome (23). More modest numbers were

identified in Hydra and Clytia, which contained 2 and 1 predicted peptides respectively,

suggesting an anthozoan specific expansion of the Fucolectin repertoire.

Acropora and Clytia each contain 2 members of the haemolytic lectin family, which has a

restricted distribution in metazoans being first reported in the sea cucumber, Cucumeria

echinata (Hatakeyama et al., 1994) then in Acropora millepora (Grasso et al., 2008).

Exhaustive searches of Hydra and Nemtatostella data failed to identify homologues of this

family suggesting selective gene losses have occurred (Forêt et al., 2010). Maximum

likelihood analysis (Figure 4.2) shows the Acropora haemolytic lectins clade independently

of the Clytia sequences, suggesting lineage specific duplications occurred from a single

ancestral gene. In situ hybridisation of coral haemolytic lectins demonstrate expression in

cells thought to be nematoblasts, the precursors to nematocysts (“stinging” cells)

(Supplementary Figure 4.4).

The lectin complement of Monosiga is much smaller than that of the cnidarians with only 15

peptides matching the search criteria. The majority of these (9) contain C-‐type lectin

domains and no representatives of the collectin, fucolectin or haemolytic lectin families are

present.


47

Figure 4.2 Maximum likelihood analysis of metazoan haemolytic lectins shows sequences from Clytia and Acropora form separate clades with 100% bootstrap support, suggesting duplication of the ancestral haemolytic lectin occurred independently in each genus. The limited distribution of CEL-III like haemolytic lectins implies a number of independent gene losses occurred in the Cnidaria and elsewhere during bilaterian evolution (Foret et al., 2010). Phylogenetic analysis is based on alignment of haemolytic lectins shown in Supplementary Figure 4.3. Branch numbers represent percentage bootstrap support based on 100 bootstrap replicates. Acropora sequences are labelled according to their EST of origin (Grasso et al., 2008) and Clytia sequences correspond to Contigs IL0ABA2YE22RM1 (1) and IL0ABA8YL09RM1 (2).


48

4.3.4 Adhesion LRR Hydra



Acropora millepora

Monosiga brevicolis

LRR-‐Dsl -‐ -‐ 1 -‐ -‐ LRR-‐EGF -‐ 2 4 1 -‐ LRR-‐FN3 -‐ -‐ 1 -‐ 1 LRR-‐IG -‐ -‐ 2 4 -‐ LRR-‐LDLa -‐ -‐ -‐ -‐ 12 LRR-‐Sushi -‐ -‐ 1 -‐ 1 LRR-‐VWA -‐ 1 -‐ -‐ -‐ Scribble -‐ -‐ 1 1 -‐

Table 4.5 Distribution of proteins containing a leucine rich repeats (LRR) and an adhesion domain in basal metaoans. For each species investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.

Overall the adhesion-‐LRR complement of cnidarians is much smaller and shows less

diversity than that of bilaterians. More than 230 Leucine Rich Repeat containing proteins

were identified across the 5 species investigated and only 26 of them had potential adhesive

capability based on the presence of one or more adhesion domains. LRR containing models

proposed to have adhesive functions were sorted into dsl, EGF, FN3, IG, LDLa, SUSHI, and

VWA groups based on domain content and none of these groups were identified in Hydra.

Cnidarians appear to lack the LRR and LDLa domain combination, characteristic of the

relAxin hormone receptor family (LGR 7 and LGR 8) consistent with previous suggestions

that these proteins arose early in the vertebrate lineage (Wilkinson et al., 2007). This

domain combination was however dominant in Monosiga, which contained 12 LRR-‐LDLa

proteins compared to 1 LRR-‐Sushi and 1 LRR-‐FN3.

The number of models that are likely orthologues of known bilaterian proteins was limited

to 4. Each of the anthozoans contained 1 model of Leucine-Rich Repeats and Immunoglobulin-

Like Domains 3 (LRIG3) and 1 of Scribble. LRIG3 is suggested to play a role in tumour

suppression and neural tube closure (Abraira et al., 2010; Zhao et al., 2008), whilst Scribble

is crucial for signalling from the Cadherin FAT-1 and may be a cross-‐over point between

Planar Cell Polarity and Hippo signalling (Abraira et al., 2010; Zhao et al., 2008).


49

4.3.5 Class B adhesion G-protein coupled receptors Hydra



Acropora millepora

Monosiga brevicolis

7tm_2 Only 4 4 28 23 1 GPS-‐7tm_2 1 2 13 8 3 VLGR1 -‐ -‐ 1 1 -‐ CLECT-‐GPS-‐7tm_2

-‐ -‐ 3 -‐ 1

NvX Group -‐ -‐ 6 -‐ -‐ GPCR125 -‐ -‐ 1 1 -‐ Novel -‐ -‐ 3 2 1

Table 4.6 Distribution of Class B Adhesion G-‐Protein Coupled Receptors in basal metaoans. For each speacies investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.

The Class B G-‐Protein Coupled Receptors (characterised by a 7tm_2 domain) comprise the

most interesting group of GPCRs due to their roles in developmental signalling processes.

Like the Adhesion-‐LRR family, proteins containing a 7tm_2 domain were rare in all the

organisms investigated and a restricted distribution among cnidarians was observed for 3

protein subgroups. The majority of 7tm_2 proteins from each species have no detectable

extracellular domains and adhesion domains are extremely rare, consistent with reports

that adhesion-‐GPCRs expanded after the divergence of vertebrates (Nordström et al., 2009).

Orthologues of Frizzled, Smoothened and Very Large G-protein Couple Receptor1 (VLGR1)

were identified in each cnidarian but absent from Monosiga. C-‐type lectin and 7tm_2 domain

proteins were only identified in Nematostella as were proteins containing a Somatomedin B

(SO) domain. SO-‐7tm_2 proteins have previously been reported from Nematostella

(Nordström et al., 2009) and the present investigation confirms that these proteins are not

shared with other cnidarians. The third group with a restricted distribution, the GPCR125

orthologues, were found only in the anthozoans. Novel structures were also found in 6 genes

(3 Nematostella, 2 Acropora, 1 Monosiga).


50

4.3.6 Immunoglobulin superfamily Hydra



Acropora millepora

Monosiga brevicolis

DCC 1 -‐ 1 -‐ -‐ DS-‐CAM 1 1 9 -‐ -‐ F11/Contactin 1 -‐ 1 -‐ -‐ L1-‐like -‐ -‐ 2 1 -‐ N-‐CAM -‐ -‐ 3 4 -‐ IgLON 2 2 13 2 Ig and FN3 Containing

4 1 23 20 -‐

Ig and Adhesion

2 9 11 -‐ -‐

COLIG -‐ -‐ 5 9 -‐ TIR (Toll Pathway)

4 -‐ 12 10 2

RPTP -‐ -‐ 2 1 -‐ MALT-1 -‐ 1 2 2 -‐

Table 4.7 Distribution of immunoglobulin Superfamily proteins in basal metaoans. For each speacies investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.

The abundance of immunoglobulins containing any other associated domain is much smaller

in cnidarians than higher animals, with the largest representation consisting of 87 individual

predicted peptides (Nematostella). The majority of sequences found in Nematostella (55)

were associated with Fibronectin Type3 (FN3) domains and could be further categorised

into 6 sub-‐groups (N-‐CAM, L1-‐like, Contactin, DS-‐CAM, Repeat Protein Tyrosine Phosphatase

(RPTP), and Other Ig and FN3 containing), whilst most of the remaining sequences were

specified as part of the DCC, or Toll like families. Interestingly no Toll Like Receptors (TLR)

possessing the canonical structure known from higher animals were identified during this

survey. One LRR containing Toll receptor (NvTLR-1) has previously been identified in

Nematostella (Miller et al., 2007) and remains the only example from a cnidarian despite

exhaustive searches.

A number of models unrelated to the above sub-‐groups were also identified including

members of the FGF receptor family, Bystin proteins, and DCLK2/3 proteins. Mucosa

Associated Lymphoid Tissue 1 (MALT1) was also found in Nematostella, Clytia and Acropora,

and although these are not adhesion related genes they have previously only been reported

in vertebrates, C.elegans and Dictostellium. Partial putative models of extremely large

adhesion molecules were also identified in cnidarians including Titin, Roundabout and

VEGFR, however genes similar to those functioning in the adaptive immunity of vertebrates,

such as I-CAM and V-CAM were not identified in either cnidarians or choanoflagellates.


51

One novel group of proteins, consisting of a Collagen domain followed by 2-‐5 Ig domains was

identified in Nematostella and Acropora, but are absent from the hydrozoan data. The

structure (Figure 4.3) is somewhat reminiscent of collectins, however the function has not

been described.

4.3.7 Extra-cellular matrix Hydra



Acropora millepora

Monosiga brevicolis

Collagen4 2 1 6 -‐ Fibrillar Collagen 12 12 10 2 Collagen 6 4 12 1 Minicollagen 20 2 3 -‐ Fibrinogen Domain Containing

2 44 92 3

Fibrillin 4 5 15 -‐ FN2 Domain Containing

1 2 11 -‐

FN3 Domain Containing

-‐ -‐ 1 2

Laminin Alpha 2 -‐ 9 1 Laminin Beta -‐ -‐ 1 -‐ Laminin Gamma 4 -‐ 4 1 Thrombospondin 4 6 3 -‐ Rhamnospondin -‐ 5 6 10 -‐ HSPG2 1 1 1 -‐ SPOCK 1 1 1 -‐

Table 4.8 Distribution of Extracellular matrix proteins in basal metaoans. For each speacies investigated, the number of protein models assigned to each sub-‐family (left hand column of table) is given. Numbers shown only include sequences that could be confidently assigned on the basis of JCUSMART analysis.

The major components of the invertebrate extra-‐cellular matrix (ECM) such as type 4

Collagen, Laminins, Fibrillin and Heparin Sulphate Proteoglycan 2 (HSPG2) were identified

throughout the Cnidaria and a similar number of proteins in each family were detected

across Nematostella, Hydra, Cyltia and Acropora. All of these classes of ECM proteins occur

in the Bilateria with the exception of the mini-‐collagens, a family of proteins containing

simplified collagen like domains which were identified as a structural component of the

nematocyst cell wall in Hydra. Their existence in Acropora has been acknowledged in the

literature (Srivastava et al., 2010) however the relative number of mini-‐collagen domain

proteins in Nematostella (2), and Clytia (2) has not been reported. The laminin complement

of Nematostella is similar to that of vertebrates consisting of 3 or more alpha chains, 3 or

more gamma chains and only 1 beta chain. Alpha and Gamma chains are also predicted in

Hydra, whereas no laminins were detected in Clytia.


52

A few of the large multidomain matrix proteins and proteoglycans were also represented

including Usherin, Sidekick, Nidogen and HSPG2, Titan, Polydom, SPARC and SPOCK, Slit and

notch. A surprising discovery was the presence in Nematostella of a model similar to

periostin, a matrix protein involved in regulation of bone mass in response to load in

vertebrate models. Fibronectin, Vitronectin, Tenascin’s and von Willebrand Factor were

absent from cnidarians further supporting their designation as vertebrate specific genes.

4.3.8 Novel cnidarian sequences Rhamnospondins are a family of thrombospondin and lectin domain containing proteins,

which have immune functions in Hydractina. This survey has identified homologues of

Rhamnospondins in the wider cnidarian community but not in Monosiga suggesting this

family originated in cnidarians. Similarly, minicollagen proteins, previously known from

Hydra, were found in each of the cnidarians, although this is perhaps unsurprising given

their role in cnidocyte cell wall formation. A novel protein family consisting of a signal

peptide followed by a Collagen domain and 2-‐4 Ig domains (Figure 4.3) was also found in

Nematostella and Acropora. The only other novel cnidarian adhesion family are GPCR

proteins containing a Somatomedin-‐B domain in extracellular region. This family are

described as the NvX GPCR family and through this analysis have been confirmed as

Nematostella specific. A few examples of novel protein architectures not shared between the

cnidarians were also identified in the adhesion GPCR family, although these protein

predictions need to be experimentally confirmed.


53

Figure 4.3 Domain structure of selected cnidarian innovations. Collectin like proteins, containing a Galactose binding lectin domain rather than the C-‐type lectin domain of Collectins, were identified as novel sequences in the Cnidaria. A Family of proteins, which I named COLIG’s, containing a Collagen domain followed by 2-‐4 immungoglobulin domains was also identified for the first time in this analysis. COLIG’s are restricted to the anthozoan data and appear to be absent from hydrozoans, however it is unclear whether this is an anthozoan innovation or an example of hydrozoan gene loss. Rhamnospondins, which are characterised by a series of thrombospondin domains followed by a rhamnose binding lectin domain, also appear to be an innovation of the Cnidaria or ureumetazoan ancestor. Each of these families hold a structure similar to pattern recognition receptors of bilaterian humoral immune systems. The presence of a predicted signal peptide suggests these proteins are secreted, which may allow them to function in opsonisation of bacteria or other invasive micro-‐organisms.


54

4.4 Discussion 4.4.1 The ancestral adhesion repertoire Cnidarian adhesion genes or gene families possessing features of known bilaterian proteins

are likely to have been retained from the Ureumetazoan ancestor (the last common ancestor

of cnidarians and bilaterians). The presence and absence of known gene families is

summarised in Table 4.9. Genes from each of the 7 adhesion families (cadherins, integrins,

lectins, Adhesion-‐LRR, Adhesion-‐GPCRs, immunoglobulin superfamily, and ECM) were

identified in all 4 cnidarians investigated and many showed homology to described

bilaterian protein architectures, although clear orthologues of higher animal proteins were

rarely apparent. Comparison of the cnidarian cell adhesion molecule complement with the

published bilaterian adhesion gene repertoire, has revealed a number of significant

functional and evolutionary aspects of cell adhesion.

The cnidarian extracellular matrix, cadherin, and immunoglobulin families all showed

characteristics more consistent with invertebrates than chordates. The extracellular matrix

proteins were remarkably well conserved with all major components of invertebrate ECM

(eg. Type-‐IV and fibrillar collagens, laminins, fibrilin, fibulin and Thrombospondin) present

in each cnidarian. The most surprising aspect of the cnidarian ECM was the diversity of

proteins associated with human disorders, which were identified primarily from the

Nematostella genome. These proteins include Titin, Usherin, Heparin Sulphate Proteoglycan 2

(HSPG2), MEGF, Polydom, and SPOCK. Although the precise roles of these proteins in

cnidarian development are unclear, their presence suggests the capacity to produce complex

cell-‐ECM interactions that facilitate cell migration, cilia formation and regulate matrix

assembly, is an ancestral trait.


55

Family Genes present Genes absent Novel Genes Genes not previously Identified outside Bilateria

cadherins • Type-‐III Cadherin (catenin binding)

• FAT • FAT-‐Like • CELSR/Flamingo • Dachsous • Protocadherins

• Type I/II/IV Cadherin (Catenin Binding)

• Desmocolins • Desmogliens • CDH26 • 7D family

• 2 unique -‐ Nematostella

• Type-‐III Cadherin

• Dachsous • FAT • FAT-‐Like

integrins • α-‐Integrin • β-‐Integrin • integrin Linked Kinase (ILK)

• ILK associated phosphatase

• PINCH • Parvin • Talin • Kindlin • ADAM • ADAM_TS • GON family

-‐ -‐ -‐

lectins • C-‐type • Galactose Binding • Fucolectins • Haemolytic lectins

• Selectins • Collectin-‐Like • Rhamnospondins

• Collectins • Fucolectins

Immunoglobulin • N-‐CAM • L1-‐like • F11/Contactin • IgLON • RPTP • DCC • DS-‐CAM • Ig-‐FN3 • TIR containing • NvTLR-1 • MALT-‐1

• Canonical Toll Receptors

• Adaptive Immune Components

• COLIGs • RPTP • MALT-1

Adhesion LRR • LRIG3 • Scribble • LRR-‐EGF • LRR-‐dsl • LRR-‐IG • LRR-‐Sushi

-‐ -‐ • LRIG family • Scribble

Class B GPCRs • CLECT-‐7tm_2 • GPCR125 • VLGR

• Group II • Group VI • Group VII • Group VIII • Methuselah family

• Somatomedin B-‐GPCRs

• 2 Unique Acropora

• 3 Unique Nematostella

-‐


56

Extracellular Matrix

• Fibrillar Collagens • Collagen 4 family • Contactin • Collagen Triple Helix containing

• Fibrinogen containing

• Fibrillin • Polydom • Fibulin • Thrombospondins • Periostin • Titan • Perlecan/HSPG2 • MEGF • Netrin • Fibropellin • Notch • Nidogen • Slit • Agrin • Usherin • Fras1 • Rabconnectin • Sortilin • Sorting nexin • Sidekick • SPOCK

• Fibronectin • Vitronectin • Osteonectin • Von Willebrand Factor

• Tenascin

• Minicollagens -‐

Table 4.9 Presence and absence of selected bilaterian adhesion genes with developmental and immunological roles. The present analysis has identified a number gene families previously thought to be bilaterian restricted including Type-‐II cadherins, FAT cadhrins and Fucolectins. The abundance of known bilaterian adhesion genes demonstrates that many of the adhesion systems recognised from higher animals were already established in the last common eumetazoan ancestor. Further diversity in mammalian systems is therefore likely to be the result of lineage specific expansions occurring after the cnidarian divergence. Genes involved in vertebrate sight and hearing are also present in cnidarians and are likely to have been sequestered from other biological functions, which are yet to be elucidated in lower animals. Gene families absent from the cnidarian data are largely associated with evolution of closed circulatory systems and circulating cellular immune systems. Hormone receptors associated with long range cellular signalling are also absent from cnidarians.


57

cadherins that influence the early embryogenesis of higher animals have only recently been

reported in one cnidarian, Nematostella vectensis (Fung et al., 2008; Hulpiau & van Roy,

2009; Whittaker et al., 2006), and no expression or functional data has been published from

this model. Conservation of Type-‐III Cadherin, Flamingo, and Dachsous among the

cnidarians (as well as FAT and FAT-‐like protein in Nematostella) combined with their

absence from choanoflagellates suggest the critical roles these proteins play in β-‐catenin

signalling and planar cell polarity originated prior to the cnidarian divergence (See Chapter

5 for discussion). Reports from the genome of Amphimedon queenslandica (Porifera) have

also yielded a sequence for a Type-‐III Cadherin, but do not indicate the distribution of other

cadherins in sponges (Srivastava et al., 2010). The presence of these cadherins in both

cnidarians and sponges implies that Cadherin based morphogenic systems date back to the

metazoan common ancestor.

Despite the richness of morphologically crucial cadherins in cnidarians, the structure and

low total number of Cadherin proteins is most consistent with the invertebrate complement.

Type-‐III cadherins possess a characteristic domain structure and are not found in chordates

(with few exceptions). Using the JCUSMART method, the total number of genes with 3 or

more Cadherin domains in Nematostella (the most complete dataset used here) was found

to be 20 -‐ 4 more than found by Hulpiau et al (2011). In either case, the number of cnidarian

cadherins is similar to that of fly (17) and sea urchin (14), compared to over 110 sequences

in mouse and human (Fung et al., 2008; Hulpiau & van Roy, 2009; Whittaker et al., 2006),

supporting previous suggestions that diversification of cadherins occurred specifically in

chordates (Hulpiau & van Roy, 2011).

Immunoglobulin proteins lacked the complexity observed in higher animals. Whereas the

sea urchin genome yielded over 20 adhesion-‐LRR proteins with a wide variety of associated

extracellular domains, only 13 sequences were found across the 4 cnidarians (Nematostella -‐

9, Acropora -‐1, Clytia -‐3). The range of associated domains was also limited with only

EGF(7), Ig(3), FN3(1), Sushi (1), VWA(1), represented. The immunoglobulin family

contained representatives of all Ig-‐FN3 sub-‐families and the IgLON family, however, the total

number of immunoglobulin proteins falls far short of what is observed in sea urchin (~400;

Whittaker et al., 2006). Such an explosive expansion of the immunoglobulin superfamily in

deuterostomes most likely facilitated the occurrence of adaptive immunity, the components

of which are absent from cnidarians. Both the adhesion-‐LRR and immunoglobulin families

have recognised roles in innate immunity, serving largely as pattern recognition receptors

(PRRs) in other model organisms (Pancer & Cooper, 2006). This function is apparently


58

conserved in cnidarians, with a member of the IgLON family implicated in allorecognition of

Hydractinia (Hydrozoa) colonies (Nicotra et al., 2009). The relative simplicity of these

adhesion families, as identified during this survey, suggest cnidarian pattern recognition

mediated by immunoglobulin and adhesion-‐LRR proteins is primitive when compared to

bilaterian systems.

The predominant PRR system of cnidarians may instead centre around the lectin family.

Cnidarian lectins were found to be surprisingly diverse and include a variety of C-‐type and

galactose binding lectins (Gal-‐lectins) with a range of associated domains. This level of

complexity in cnidarian C-‐type lectins more closely resembles the vertebrate lectin diversity

than that of model protostomes (Cambi et al., 2005; Dam & Brewer, 2010), which is

consistent with previous reports of genetic simplification among Ecdysozoans (eg.

Drosophila and Caenorhabditis) (Lin et al., 2000; Wood-‐Charlson & Weis, 2009). Both C-‐type

and Gal-‐lectins have been suggested to play roles in pattern recognition (Cambi et al., 2005;

Dam & Brewer, 2010; Weis et al., 1998). Pattern recognition is particularly important to

cnidarians that exist in obligatory symbiosis with intracellular algae (Zooxanthellae) and

lectins have been shown to facilitate the uptake of symbionts into host cells through specific

interaction with glycoproteins of the algal cell wall (Lin et al., 2000; Wood-‐Charlson & Weis,

2009). The most prominent example of this interaction comes from the only characterised

PRR lectin from a coral, Millectin, a C-‐type lectin isolated from Acropora millepora,

(Kvennefors et al., 2008; Kvennefors et al., 2010).

The lectin family also yielded some of the most interesting and unexpected cnidarian

innovations, all of which have implications for innate immunity. The first unexpected find

was the presence of fucolectins (F-‐type/Fucose binding), secreted innate immune effectors

first identified in eels (Honda et al., 2000) that have not previously been described in lower

animals. The anthozoans, Nematostella and Acropora, each contain an expanded set of more

than 20 (26 and 23 respectively) fucolectins compared to between 2 and 8 representatives

in bilaterians, suggesting that fucolectins form a central part of the anthozoan immune

system. Other unexpected finds included orthologues of CLEC16A (a disease associated

protein highly expressed in mammalian B cells and dendritic cells) and CEL-III (haemolytic

activity), further adding to the secreted innate immune repertoire of cnidarians.


59

Comparison of the cnidarian adhesome with that of Monosiga was consistent with previous

indications that adhesion systems sustaining unicellular life are considerably simpler than

what is required in multicellular animals (Abedin & Nicole King, 2010). This was particularly

evident in the ECM, which contained limited examples of fibrillar collagens, a limited set of

laminin α and γ chains, and lacked all components of functional basement membranes (eg.

Type-‐IV collagens and laminin-β). Immunoglobulin domains were almost completely absent

from Monosiga, as were adhesion GPCR proteins, and whilst many of the domains known

from other adhesion families (eg. Cadherins and integrins) were identifiable in low

abundance (eg. Cadherin & integrin α domains), the overall protein architecture was unlike

that of animal adhesion molecules.

In contrast to systems such as innate immunity, characterised by genes absent in pre-‐

metazoans and likely to have originated in the ureumetazoan or urmetazoan ancestor,

integrin signalling is suggested to have originated well before the evolution of

multicellularity despite its absence from Monosiga. All components of the integrin signalling

complex were identified in cnidarians and only the terminal signalling molecules (α and β

integrins) varied in number. This finding is consistent with investigations into the pre-‐

metazoan ancestry of integrins, suggesting the integrin heterodimer, ILK, Talin, PINCH and

α-‐Parvin originated in the Unikont ancestor with canonical integrin signalling (involving FAK

and c-Src) appearing in the Opsithokont ancestor (Sebé-‐pedrós et al., 2010) (Although the

later is based on data from a single species). The function of integrin signalling components

in the primitive model organisms investigated by Sebé-‐Pedrós et al (2010), and therefore

the pre-‐metazoan ancestral function, is likely to be distinct from that of the metazoan

integrin system. Instead, these proteins are likely to have been co-‐opted from other

biological processes into morphogenic roles concurrent with the evolution of animal

multicellularity.


60

4.4.2 Novel cnidarian sequences Novelty in the cnidarian adhesome is primarily centred around expansion of the immune

system with most phylum specific protein families exhibiting strong similarity to secreted

pattern recognition proteins. Models containing an N-‐terminal collagen domain followed by

a C-‐terminal Gal_lectin domain were identified in each of the cnidarians, an architecture

which closely resembles that of Collectins (Collagen – C-‐type lectin). Vertebrate Collectins

function in humeral immunity through opsonisation, neutralisation, agglutination,

phagocytosis and activation of the complement system in addition to modulating apoptotic

cell clearance (Gupta & Surolia, 2007; van de Wetering et al, 2004). Each of these immune

responses are expected to also occur in cnidarians as apoptotic processes and components

of the complement system (eg. C3, MASP, Ficolins (also identified by the present analysis),

and Factor B) have both been identified (Ainsworth et al., 2007; Kimuraet al., 2009; Lasi et

al., 2010). However, the range of proteins able to elicit these responses awaits clarification.

Two other taxon specific families with a similar general architecture, consisting of a

conserved homophilic matrix protein domain (TSP1 or Collagen) in conjunction with an

antigen recognition domain (lectin or Immunoglobulin) were also identified. The

Rhamnospondin family (TSP1 – Rhamnose binding lectin) have previously been identified in

Hydractinia, where they are suggested to mediate bacterial defence in the oral region

(Schwarz et al., 2007). The present analysis detected Rhamnospondins in the wider

cnidarian community, suggesting they are not a hydrozoan innovation. Rhamnospondins

have also been reported from the tunicate Botyllus schlosseri (Oren et al, 2007), however

their identification in this urochordate is unconvincing. The final family of novel proteins

with the matrix-‐recognition architecture, which have here been given the name “COLIGs”, is

previously undescribed anywhere in the Metazoa, consisting of a collagen domain followed

by 2 Ig domains. This structure again suggests a role in resisting colonisation by micro-‐

organisms.

Whereas the Collectin-‐like proteins, Rhamnospondins and COLIGs provide secreted defence

by opsonisation, the minicollagens, which are also unique to cnidarians, are reported to be

critical to physical defence. Minicollagens are demonstrated components of the cnidocyte

cell wall, which is a defining defensive structure in all cnidarians. It is therefore unsurprising

that minicollagen proteins were identified in both anthozoans and hydrozoans.


61

4.4.3 Differences between cnidarian adhesion systems Although many of the major components of bilaterian adhesion could be identified in all of

the cnidarians, a number of proteins with adhesion domains had a restricted distribution.

These proteins showed no bias towards any one biological process or adhesion family. The

Anthozoans were found to each possess a single LRR & Ig domain containing protein, which

showed clear orthology to vertebrate LRIG3. The LRIG family was thought to be vertebrate

specific and LRIG3 has been experimentally determined to influence neural crest formation

and complex tissue morphogenesis (eg. inner ear morphogenesis), possibly through

modulation of the FGF and Wnt pathways (Abraira et al., 2010; Zhao et al., 2008).

Orthologues of XPTP-D, a Xenopus receptor Protein Tyrosine Phosphatase involved in

retinal axon extension and neurite outgrowth (Johnson & Holt, 2000; Johnson et al., 2001),

were also restricted to Anthozoans. The prospect of ancient roles for these genes in

embryogenesis and neurogenesis of anthozoans is exciting and warrants further

investigation, however a reason for their absence from hydrozoans cannot yet be suggested.

The genome of Nematostella vectensis revealed a number of proteins that are not found in

other cnidarians such as NvX group and C-‐type lectin domain containing (Group V) GPCRs.

Extensive targeted searches of the available cnidarian data have confirmed that these

families are species specific innovations. Nematostella also possesses the only known

canonical Toll receptor in cnidarians (Miller et al., 2007). The canonical structure of the Toll

receptor is expected to be ancestral and has apparently been lost from other model

cnidarians.

The most peculiar distribution among cnidarians is that of the haemolytic lectins, which

bind to target cells via the lectin domain and cause osmotic rupture by pore formation.

Haemolytic lectins were identified in only 1 of the anthozoans (Acropora; Grasso et al.,

2008) and 1 of the hydrozoans (Clytia), with the 2 sequences in each species resulting from

independent duplications (Figure 4.2). These cnidarian lectins are orthologous to the CEL-III

haemolytic lectin from sea cucumber (Cucumaria echinata) and together make up the entire

known metazoan complement of haemolytic lectins. The distribution of haemolytic lectins in

echinoderms and cnidarians suggest these genes have been lost in a number of independent

lineages, with parallel losses occurring in anthozoans and hydrozoans.


62

The remarkable distribution of haemolytic lectins, along with the restriction of canonical

Toll receptors, highlights both the ubiquity and stochastic nature of gene loss in cnidarians

and throughout the Metazoa (Forêt et al., 2010). Furthermore, cnidarian haemolytic lectins

are retained only in species that inhabit a marine environment, suggesting estuarine and

marine cnidarians have distinct defensive strategies and that adaptation to an estuarine

habitat has impacted on gene loss in Nematostella and Hydra.

4.4.4 Interesting absences The cnidarian adhesome lacks a number of genes present in bilaterians, particularly those

recognised to be chordate specific expansions such as some classes of cadherins and

integrins. The missing Cadherin family members include Type I and II cadherins (eg. E-

Cadherin and N-Cadherin) as well as the recently evolved desmocolins and desmoglians. The

absence of β-catenin binding Type I and II cadherins was predictable due to a demonstrated

degree of functional analogy with invertebrate Type-‐III cadherins, which are considered the

ancestral ‘classical’ Cadherin (Hulpiau & van Roy, 2009; 2011). Other predictable absences

include proteins associated with adaptive immunity and matrix proteins such as Fibronectin

and Von Willebrand Factor, which function in maintenance of vascular systems, a structure

lacking from cnidarians.

integrins with VWA domains involved in binding of collagens and those functioning in

leukocyte trafficking were also absent from all the cnidarians investigated. Cnidarian

integrins are not directly related to mammalian types and have unknown binding

capabilities, although the ability to bind collagen is unlikely (See Chapter 6). Reelin, a cell

surface receptor, which functions in positioning neuroblastic cells, is another significant

absence from the cnidarian adhesome. This exclusion may at first seem surprising given the

presence of other large neural adhesion proteins in Nematostella such as Slit, Netrin, and

Roundabout, however, Reelin binds to a soluble ligand, acting in the neurocrine system.

Hormone receptors are completely absent from cnidarians, however they are prevalent in

basal deuterostomes such as sea urchin.


63

4.5 Concluding comments The overall composition of the 7 adhesion families making up the cnidarian adhesome is

remarkably similar to adhesion systems of bilaterian invertebrates, with the exception of the

lectin family, which has undergone significant simplification in Ecdysozoans. This degree of

conservation suggests that most of the recognised bilaterian adhesion components affecting

developmental, innate immune and defensive processes were already established in the

ureumetazoan ancestor. Determining which of these adhesion systems are linked to the

origin of metazoan multicellularity and the earliest forms of morphogenesis will require

similar surveys of the recently released sponge genome (Amphimedon queenslandica). Such

studies could also clarify the influence of adhesion proteins on the evolution of dedicated

tissues, which are a eumetazoan feature absent from sponges.

The abundance of secreted innate immune proteins observed in all 4 species investigated

here, highlights the importance of pattern recognition to cnidarian survival. The aqueous

environment inhabited by cnidarians could easily facilitate colonisation by pathogenic

micro-‐organisms, which may go some way towards explaining the development of a

predominantly secretory cnidarian immune system in preference to forms of cell-‐contact

mediated immunity. The secretion of mucus as a general stress response in cnidarians may

also increase the efficacy of these immune mediators in resisting colonisation by reducing

loss of secreted proteins to the water column. Alternatively, a proportion of the pattern

recognition receptors may be associated with nematocysts (‘stinging’ cells) that form the

primary mechanism for cnidarian defence and prey capture. In situ RNA hybridisation of

both haemolytic lectins and Collectin-‐like proteins in Acropora polyps demonstrate

expression in putative nematoblasts, the precursors to nematocysts (Grasso et al., 2008;

Supplementary Figure 4.4). Further investigation of pattern recognition protein expression

is required to determine whether the secreted innate immune proteins of cnidarians

constitute a form of humoral immunity or if the innovation in cnidarian pattern recognition

is primarily associated with cnidarian specific structures.

Whether the cnidarian pattern recognition complement represents a phylum specific

expansion or the higher animal repertoire is a simplification of the ancestral state is unclear.

However, surveys of other lower metazoan phyla will resolve these issues as complete data

becomes available for a broader range of basal animals. The evolutionary and functional

impacts of cnidarian innate immunity are therefore an area of interest for future

investigations and may also contribute to our knowledge of symbiont uptake and coral

bleaching mechanisms, which will aid in reef conservation efforts.


64

Many of the cell adhesion molecules directing morphogenic cell movements during

gastrulation and neural development were identified in each of the cnidarians. The effect of

these proteins in the development of diploblastic animals however has not been explored,

with most studies instead focusing on the expression and regulation of transcription factors

expressed in tissue restricted patterns. The insight into cnidarian cell adhesion presented

here establishes a strong foundation for future analyses aimed at understanding the

expression and function of genes that ultimately determine tissue differentiation and

morphogenesis in lower animals.

Chapter Five Developmental roles for cadherins from Acropora millepora

65

Chapter 5: Developmental roles of cadherins from Acropora millepora

5.1 Introduction Developmental processes in metazoans require regulated adhesion and communication

between cells in order to properly co-‐ordinate morphological change and ensure correct

functional associations are established. Members of the Cadherin family are critical to these

processes and whilst their primary capability may appear to be adhesion between cells

achieved through calcium dependant homophilic interactions, cadherins have a far more

influential part to play than simply mediating cell-‐cell contact (Hulpiau & van Roy, 2009;

Magie & Martindale, 2008; Nollet et al., 2000; Yagi & Takeichi, 2000). Differential regulation

and a variety of cytoplasmic connections allow cadherins to take an active role in regulating

the cytoskeleton, cell shape and tissue polarisation as well as playing roles in cell sorting and

the intracellular signalling that determines morphogenesis (Gumbiner, 2005; Halbleib &

Nelson, 2006).

The earliest recognised cadherins were isolated because of their clear roles in gastrulation

and are now considered “classical” or Type-‐I cadherins (Yoshida and Takeichi, 1982; Kemler

1992). Members of this group, which include E-Cadherin (Cdh1) and N-Cadherin (Cdh2), have

5 extracellular Cadherin domains, followed by a transmembrane helix and a characteristic

cytoplasmic region. There are now more than 15 recognisable groups of cadherins based on

the arrangement of conserved domains and motifs along the protein (referred to herein as

the protein architecture). Phylogenetic analysis of first true Cadherin repeat in the

ectodomain has identified 6 major clades and revealed substantially more familial

associations (Hulpiau & Frans van Roy, 2009; Nollet et al., 2000; F van Roy & Berx, 2008).

Although there are many sub-‐families of cadherins, some genes stand out as particularly

significant for their functions in development, such as Type-‐I cadherins, CELSR

(Flamingo;Fmi), Dachsous (Ds) and FAT (Ft) cadherins (Halbleib & Nelson, 2006). Like Type-‐

I cadherins, those from the Type-‐III and Type-‐IV groups have been shown in different

animals to be critical to the cellular re-‐arrangements of germ layer formation (Miller &

Mcclay, 1997; Oda et al., 1998; Choi and Gumbiner, 1989; Hatta et al., 1987; Takeichi et al

1986). Each of these 3 families, which vary in overall protein architecture (Figure 5.1), share

a common feature -‐ the ability to make functional connections to the cytoskeleton through

binding cytoplasmic catenins (Yagi & Takeichi, 2000). The Cadherin cytoplasmic domain

(CCD) is a defining feature of these groups and contains 2 highly conserved motifs, the juxta-‐


66

membrane domain (JMD), responsible for binding δ-catenin (p120-catenin), and the catenin

binding domain (CBD), responsible for binding β-catenin (van Roy & Berx, 2008). The ability

of these proteins to influence the cytoplasmic pool of β-catenin sets them apart and conveys

the influence over morphogenic events for which these cadherins are known (Gumbiner,

2005).

Two functions of catenin binding cadherins are particularly well conserved in the Bilateria,

cell cohesion and cell migration. In vertebrates, expression of E-Cadherin is detectable at

adherens junctions even before gastrulation has occurred. Like in adult tissues, the role of E-

Cadherin at this stage of development is to maintain stable cellular contacts and limit the

movement of cells within the cell layer. As gastrulation commences, cells fated to become

mesoderm and endoderm begin to express N-Cadherin. As these cells begin to migrate by

ingression or involution, they cease to express E-Cadherin at the cell surface (Takeichi,

1988). This occurs by both endocytosis of membrane associated E-Cadherin and

transcriptional inactivation (Le et al., 1999; Hatta et al., 1987). The loss of epithelial

character associated with the migratory or invasive phenotype has been demonstrated to be

a direct result of N-Cadherin expression. Moreover, co-‐expression of N-Cadherin and E-

Cadherin produces cells that have the same invasiveness as those expressing N-Cadherin

alone (Nieman et al., 1999).

In protostomes, Type-‐IV Cadherin (DE-Cadherin/Shotgun in Drosophila) is functionally

analogous to vertebrate E-Cadherin, showing a similar protein localisation and undergoing

the same down-‐regulation during mesoderm development (Oda et al., 1998). Like in

vertebrates, DN-Cadherin is up-‐regulated in migrating mesodermal cells, although the

protein architecture of DN-Cadherin (a Type-‐III Cadherin) is significantly different from that

of DE-Cadherin (type-‐IV) and vertebrate N-Cadherin (type-‐I). In sea urchin, an invertebrate

deuterostome, no functional analogue of N-Cadherin has been described. Instead, the

flexibility of cell junctions that allows invagination of presumptive mesendoderm has been

suggested to be facilitated by de-‐coupling of LvG-Cadherin from the cytoskeleton. Protein

localisation studies indicated that whilst LvG-Cadherin expression at the cell surface is

maintained, cell surface expression of α-catenin and β-catenin is greatly reduced compared

to the surrounding ectoderm (Miller & Mcclay, 1997). Together, these observations suggest

both protostomes and deuterostomes require at least 1 catenin binding cadherin that

facilitates tissue stability and in most cases a 2nd to promote a migratory or invasive

phenotype.


67

Figure 5.1 Generalised protein architecture of catenin binding cadherins. Catenin binding cadherins possess distinct ectodomain structures allowing classification as Type-‐I/II, Type-‐III or Type-‐IV. Type-‐I & Type-‐II cadherins (classical cadherins) contain 5 Cadherin domains (ectodomain repeats; EC) followed by a transmembrane domain and Cadherin cytoplasmic domain. The highly conserved Cadherin cytoplasmic domain (CCD) is responsible for functional connections to the cytoskeleton through interaction with δ-‐catenin via the JMD motif, and β-catenin via the CBD motif. Unlike Type-‐I & Type-‐II cadherins, which are chordate specific, Type-‐III & Type-‐IV cadherins possess longer extracellular domains consisting of 8 or more EC repeats and a membrane proximal “Primitive Classic Cadherin Domain” (PCCD). The PCCD consists of a single “non-‐chordate” domain, followed by alternating EGF-‐like and Laminin G (LamG) domains. Cadherins of Type-‐IV structure have 8 EC domains and their PCCD contains only 1 EGF-‐like and 1 LamG domain. By contrast, the number of EC domains in Type-‐III cadherins varies, with some proteins containing as many as 30 EC domains. The Type-‐III EC domains are also preceded by N-‐Terminal EC-‐like domains, which hold a similar structure but are not capable of homophilic binding. The PCCD of Type-‐III cadherins contains a 3 EGF-‐like domains and 2 LamG domains, more than other types of catenin binding cadherins.

Figure 5.2 Milestones in cadherin evolution (adapted from Hulpiau and Van Roy, 2009). The proposed appearance of features in the Cadherin domain structure throughout evolution are marked in blue with losses marked in red. Genomic surveys suggest early cadherins contained a wide variety of associated domains (eg. Nhh, vWA and IgCAM domains) and simplification of these associations occurred in the bilaterian lineage. Catenin binding cadherins have been suggested to be a bilaterian invention, first occurring as Type-‐III cadherins in the Urbilateria (common ancestor of bilaterians). From the Type-‐III structure, these cadherins are believed to have diversified through a serries of domain simplifications into Type-‐IV and Type-‐I/II cadherins in protostomes and chordates respectively.


68

In addition to roles in making cytoskeletal connections at the cell membrane, β-catenin is an

important cytoplasmic signalling molecule. If not active at the cell membrane, ‘free’

cytoplasmic β-catenin is rapidly degraded following phosphorylation by the GSK3β complex,

consisting of Glycogen Synthase Kinase-3β (GSK3β), Adenomatous Polyposis Coli (APC) and

Axis Inhibition Protein (Axin). Alternatively, β-catenin can function as an activating co-‐factor

to T-‐Cell Factor (TCF/LEF) transcription factors, which lay downstream of the canonical Wnt

Pathway. Canonical Wnt signalling through the Frizzled group of cell surface receptors

inhibits GSK3β mediated degradation of β-‐catenin, thereby stabilising the cytoplasmic pool

and effecting its nuclear translocation (Nelson & Nusse, 2004; Schambony et al., 2004). In

the nucleus, TCF/LEF is activated by replacement of the repressing co-‐factor, Groucho, with

the activating co-‐factor β-‐catenin (Chen & Courey, 2000; Range et al., 2005). Through this

mechanism, β-‐catenin is critical to canonical Wnt signalling, which is commonly regarded as

one of the most important signalling cascades in bilaterian development, governing diverse

processes such as axis specification and limb development.

Canonical Wnt signalling is recognised to activate epithelial to mesechymal transitions

(EMT), which is a critical step during gastrulation of bilaterians (Chuai & Weijer, 2009).

Among the main features of a full EMT is the loss of epithelial adhesions (adherens

junctions) and increase in cell motility (Shook & Keller, 2003). It is therefore perhaps

unsurprising that Canonical Wnt has a reciprocal inhibitory relationship with the expression

and function of CCD containing cadherins, as both compete for β-catenin activity to facilitate

opposite adhesive states (Vincan & Barker, 2008; Wang et al., 2010). Canonical Wnt

signalling has been demonstrated to stabilise and up-‐regulate the E-Cadherin repressor,

Snail, which is required for EMT (Yook et al., 2006; Katoh and Katoh, 2006; Yook et al.,

2005). Furthermore β-catenin has been suggested to directly repress transcription of E-

Cadherin (van Roy & Berx, 2008). Conversely, E-Cadherin has been demonstrated to inhibit

the translocation of β-catenin to the nucleus in over-‐expression experiments (Logan et al.,

1999; Wikramanayake et al., 2003), thereby acting in a positive feedback loop to maintain

cellular adhesions and highlighting the importance of β-catenin regulation in determining a

cell’s adhesive state.

The developmental roles of CELSR, Ds and Ft cadherins are also influenced by Wnt signalling,

although here signalling through the non-‐canonical pathway is upstream of their expression

(Strutt, 2003). These cadherins are all components of systems that determine the

polarisation of cells within an epithelial cell sheet or planar cell polarity (PCP). Two main

systems of planar cell polarity have been described, the Fmi-Fz (‘core’) pathway and the Ds-

Ft pathway. In most well described examples, these systems function within the same tissue


69

and both are required for correct morphogenesis (for review see Fanto and McNeill, 2004),

however regulatory mechanisms between these systems remain enigmatic. In each system,

cell polarity is effected by the asymmetrical localisation of membrane bound components to

opposing ends of the cell.

Interactions between the membrane bound components have been shown to occur through

direct binding of opposing extracellular domains. The Fmi-‐Fz system has opposing

localisation of Fz-‐Dgo-‐Dsh-‐Fmi and Vangl-‐Pk-‐Fmi complexes, whereas the Ds-‐Ft system relies

on opposing localisation of Ds and Ft at the membrane as well as asymmetrical cellular

localisation of four-‐jointed (fj), a golgi protein that modifies the Ds protein (Wu & Mlodzik,

2009). Components of each system are expressed in a graduated pattern across the

epithelium and polarisation of tissues is determined by the relative expression and activity

of each system. The relationship between their relative expression and morphological

events is not simple. Two classical examples are the Drosophila Wing and Eye, where both

the Fmi-‐Fz and Ds-‐Ft systems are expressed. In the Wing, graduated activity of Ds-‐Ft along

the proximal-‐distal axis opposes that of Fmi-‐Fz, whereas in the eye, the activity gradient of

both systems are concurrent (Wu & Mlodzik, 2009). Although there are a number of

unresolved aspects of planar cell polarity function, the influence of appropriate PCP on

morphogenesis has been clearly demonstrated in two distinct processes. Convergent

extension, the process of elongating an epithelial sheet through intercalation of existing

cells, is seen in vertebrate gastrulation and is a critical process for neuralation (Shindo et al.,

2008; Wang et al., 2006). The second process is polarised cell division, whereby

establishment of tissue polarity determines the direction of mitotic spindle alignment

through constraint of the cells shape (Gong et al., 2004).

The involvement of PCP in both convergent extension and polarised cell division has been

shown in Zebrafish, Drosophila and Caenorhabditis, suggesting these are evolutionary

conserved morphogenetic events (Carreira-‐Barbosa et al., 2009; Gong et al., 2004; Seifert &

Mlodzik, 2007). Similarly, the role of β-catenin in both transcriptional and membrane

associated roles has been highly investigated in protostomes and deuterostomes. Very few

investigations however, have described homologues of β-catenin and PCP adhesion systems

in ‘lower’ metazoans.


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Consideration of the distribution and arrangement of adhesion domains within Cadherin

proteins from Nematostella and choanoflagellates has revealed the ancestral nature of the

Cadherin extracellular domain and describes a variety of domain associations that have

since been lost in the Bilateria (Abedin & King, 2009). The presence of the catenin binding

Cadherin cytoplasmic domain is reportedly unique to metazoans however, in a number of

basal cognates, the overall architecture of proteins containing this domain has not been

reported. Hulpiau and Van Roy (2009) proposed the model of Cadherin evolutionary

milestones described in Figure 5.2, suggesting that a number of developmentally significant

cadherins, including those that bind β-Catenin are restricted to the Bilateria, despite the

parallels in early development of selected cnidarians and bilaterians.

Genomic surveys of the sea anemone Nematostella vectensis have previously revealed the

existence of Canonical and Non-‐canonical Wnt components including β-‐catenin, the GSK3β

complex, Frizzled and Van Gogh, however no direct analysis of the Cadherin complement of

cnidarians has been made (Kusserow et al., 2005; Putnam et al., 2007). Furthermore, the

expression patterns of the Nematostella Wnt genes and β-catenin have been described and

discussed in terms of their signalling during gastrulation (Lee et al., 2007; Wikramanayake

et al., 2003), however the localisation and putative roles of planar cell polarity genes and

catenin binding cadherins have not been reported outside the Bilateria.

Investigation of cadherin-‐catenin and planar cell polarity signalling components in

cnidarians and sponges will resolve the importance of these systems to metazoan evolution

and reveal their ancestral significance in development. Here, I describe the distribution of

genes significant to Cadherin-‐catenin signalling and planar cell polarity in 4 representative

cnidarians, with different developmental features. The expression of catenin binding

cadherins and a number of planar cell polarity components are investigated by in situ

hybridisation of coral embryos and larvae and the functional and evolutionary implications

of these systems are considered.


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5.2 Methods 5.2.1 Sequence identification Sequences for “classical” cadherins and other key components of the Wnt -‐ Planar Cell

Polarity pathway were identified using JCUSMART (See Chapter 3). Sequences from 4 large

datasets representing Acropora millepora, Nematostella vectensis, Hydra magnipapilata and

Clytia hemispherica were assessed for the presence of key architectural features and overall

sequence similarity (BLASTp) to known representatives of target genes already in Genbank.

5.2.2 Cadherin phylogenetics Maximum likelihood analysis was performed on 28 cnidarian and bilaterian cadherins

possessing a classical Cadherin cytoplasmic domain which consists of both a JMD motif and a

CBD motif. Due to the highly repetitive nature of the Cadherin extracellular domain, this

analysis is based on truncated sequences corresponding to 148 positions within the

cytoplasmic region and encompassing both the JMD and CBD motifs. Maximum likelihood

analysis was performed using PhyML3.0 (Guindon & Gascuel, 2003) and supported by 100

bootstrap replicates.

5.2.3 Isolation of riboprobe template cDNA Partial sequences of selected genes identified in the Acropora millepora transcriptome were

amplified from Acropora cDNA libraries and cloned into pGEM-‐T vector (Promega) as

described in Chapter 2.2. Amplified regions correspond to sections of the coding sequence

that exhibit the least homology to other genes and do not encompass internal repeats of

functional protein domains. These strategies were utilised to increase the specificity of the

resulting probe. Although a number of primer combinations were assessed, the sequences of

only the most successful pair for each gene are shown below. Full or partial sequences for

the remaining genes were recovered from the Acropora millepora Expressed Sequence Tag

(EST) project. In situ hybridisation was performed as described in Chapter 2.4.


72

Am_ACadherin F3 5’-‐GGGCAACATCATCTCAAGC-3’

Am_ACadherin R5 5’-‐ATGCGATTGAGGTTATCGAA-3’

AmFlamingo F4 5’-‐GGTCCACGTACCACCAAG-3’

AmFlamingo R4 5’-‐AGAGAATCAAGGAATATCAAG-3’

Am_Van_Gogh like F3 5’-‐GTGATCGCAATATGGGCTT-3’

Am_Van_Gogh like R3 5’-‐TCTGCTTCTTCATAGAAACG-3’

Am_Wnt16 Forward 5'-‐GTACAGTGGTTGGGGAATGG-‐3'

Am_Wnt16 Reverse 5' -‐ CAGTGCAAGAGCCAGAAACA-‐3'

Am_beta-‐Catenin Unigene: B025-‐E11

Am_Frizzled4 Unigene: C007-‐H4

AmDachsous Unigene: D041-‐C8

Am_WIF Unigene: C014-‐E11

Am_Axin Unigene: D027-‐D10

Am_Dishevelled Unigene: B036-‐C4


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5.3 Results 5.3.1 Identification of catenin binding cadherins and planar cell

polarity components from Acropora millepora Two sequences with homology to the catenin-‐binding Cadherin cytoplasmic domain were

identified in the Acropora millepora transcriptome using JCUSMART. One contiguous

sequence (Contig6389 + Contig5439; Sequence presented in Supplementary Figure 5.1) of

8,933bp, which I named Am_ACadherin, showed significant homology to Type-‐III cadherins

in both protein architecture and cytoplasmic domain sequence alignment (Figure 5.3).

Although the 5’ sequence of Am_ACadherin could not be resolved with the available data, the

cytoplasmic region contains both a p120/δ catenin binding juxta-‐membrane domain (JMD)

and β-catenin binding domain (CBD) (Figure 5.3 A). The second sequence (Contig10872),

7,786bp, was identified as an orthologue of Dachsous (Ds) (see Supplementary Figure 5.2 for

cytoplasmic region boxshade alignment), an atypical protoCadherin that participates in

planar cell polarity in bilaterians. AmDachsous (AmDs), the first reported Ds orthologue

outside the Bilateria, contains only a CBD in the cytoplasmic region as is consistent with

bilaterian Dachsous genes.

Two partial sequences (Contig8737 and Contig3665) corresponding to the central and C-‐

terminal regions (3,073bp and 3,898bp respectively) of the core planar cell polarity

Cadherin CELSR/Flamingo (Fmi) were also identified using JCUSMART. The presence of coral

orthologues of core (Fmi) and secondary (Ds) planar cell polarity pathway cadherins

prompted investigation of other components of this pathway. Almost all investigated genes

associated with Cadherin-‐catenin signalling, PCP signalling and Wnt/PCP pathway are

represented throughout the Cnidaria (Table 5.1). The range of genes apparently absent from

Hydra and Clytia is likely due to data limitations rather than gene losses. These signalling

pathways can therefore be considered evenly represented among species with different

developmental mechanisms, suggesting they are of fundamental importance to survival.


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Figure 5.3 Protein conservation and architecture of Am_ACadherin

A) Alignment of Am_ACadherin cytoplasmic domain to representative sequences from bilaterians. Shading demonstrates >50% consensus to Am_ACadherin (Back –conservation, Grey – conservative substitution). Am_ACadherin contains both a JMD (Yellow box) and CBD (blue box), which are required for interaction with β-‐catenin and the cytoskeleton.

B) Domain structure of Am_ACadherin. Sequences gained from the 2010 assembly of Acropora millepora show only an N-‐terminal truncated model. The total number of EC repeats is therefore unknown. The assembly of 2 overlapping contigs (Contig 6389 + Contig5394) demonstrates Am_ACadherin contains at least 13 EC domains followed by the canonical structure of PCCD, TMH, CCD consistent with Type-‐III cadherins. This is the first reported type-‐III Cadherin outside of the Bilateria suggesting the developmentally critical function of cadherins in facilitating cell adhesion and influencing β-‐catenin balance originated earlier than previously believed.

(Hs) Homo sapiens; (Mm) Mus Musculus; (Bm) Bombyx mori; (Gb) Gryllus bimaculatus; (Dm) Drosophila melanogaster; (Af) Artemia franciscana; (Lv) Lytechinus variegates; (Sp) Strongylocentrotus pupuratus; (Ap) Asterina pectinifera; (Se) Sexostrea echinata; (BS) Botryllus schlosseri; (Ci) Ciona intestinalis; (Cj) Cardina japonica; (Le) Ligia exotica; (At) Achaearanea tepidariorum


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Gene Acropora Nematostella Hydra Clytia

Type-‐III Cadherin 1 3 -‐ -‐ Beta-‐Catenin 1 1 1 1 Axin 1 1 1 -‐ APC 1 1 1 -‐ GSK3b 1 1 1 1 Dishevelled 1 1 1 1 CELSR/Flamingo 1 1 -‐ -‐ Frizzled 4 4 2 1 Van Gogh/Strabismus 1 1 1 1 Diego -‐ -‐ -‐ -‐ LRP5/6 1 1 -‐ -‐ Dachsous 1 1 -‐ -‐ FAT -‐ 3 -‐ -‐ Four-‐jointed -‐ -‐ -‐ -‐ Wnt16 1 1 1 1 Groucho 1 1 1 1 Inversin 3 (partial) 1 -‐ -‐

Table 5.1. Distribution of catenin binding cadherins and planar cell polarity pathway components in cnidarians. For each protein, the number of homologues that were positively identified by the JCUSMART method are shown for each cnidarian species investigated. This value may vary from previously published values, for example, 2 Frizzled proteins have been reported in Clytia (Momose & Houliston, 2007), however only 1 protein could be confirmed as Frizzled on the basis of EST data used in JCUSMART analysis, despite the presence of 4 protein models with BLAST hits to Frizzled. A “-‐“ indicates no sequences were identified but may not be suggestive of gene loss, due to inherent limitations of the initial data and sequence assembly and analysis. Representatives of all components of the Cadherin-‐catenin (Green) and planar cell polarity pathways (blue) are present in cnidarians with the exception of the cytoplasmic-‐side regulatory proteins, Diego and Four-‐jointed. Genes involved in Wnt/PCP signalling are coloured yellow. Conservation of a single homologue of most genes across cnidarian species with minimal to no expansion of gene repertoire suggests that systems influencing β-catenin distribution and cell polarity are highly constrained despite varying developmental methods in each species. Identifiers for models included in table are presented in Appendx B.

5.3.2 Phylogenetic analysis of catenin binding cadherins Maximum likelihood analysis of catenin binding cadherins (Figure 5.4) shows that

sequences from protostomes and deuterostomes form two distinct major clades. Cadherins

with similar overall protein architecture (ie. each Cadherin type) group together within

these major clades. Consistent with this, Type-‐III cadherins from protostomes and

deuterostomes clade separately from each other and from the cnidarian Cadherin

sequences, which also possess Type-‐III architecture. The position of the cnidarian

sequences, as a third major clade is well supported albeit on a long branch, which most

likely reflects the large amount of time since cnidarian divergence from other metazoans

rather than a lack of evolutionary constraint on the cytoplasmic region of cnidarian

cadherins.


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Figure 5.4 Maximum likelihood analysis of Cadherin Cytoplasmic Domains of Type-‐I, Type-‐II, Type-‐III & Type-‐IV cadherins from representative metazoans. Cadherin major clades reflect the taxonomy of each animal as Protostome (Green), Deuterostome (yellow) or Cnidarian (blue). Bilaterian Type-‐III cadherins occur in 2 well supported, lineage specific clades (Deuterostome & Protostome), which reflect their function as epithelial stabilising or invasion promoting as well as reflecting their evolutionary history. The position of Type-‐I & Type-‐II cadherins within the Deuterostome clade, and Type-‐IV cadherins within the Protostome clade is consistent with previous suggestions that these families arose from independent lineage specific diversification of ancestral Type-‐III cadherins. The position of cnidarian Type-‐III cadherins in an independent clade is likely due to the early divergence of the cnidarians from bilaterian evolution and is not informative as to the function of cnidarian or ancestral catenin-‐binding cadherins. Branch numbers are bootstrap values (100 replicates). Values of less than 50 are not marked. The edited alignment of sequences used for maximum likelihood analysis and Genbank accession numbers are presented in Supplementary Figure 5.3. (Hs) Homo sapiens; (Mm) Mus Musculus; (Bm) Bombyx mori; (Gb) Gryllus bimaculatus; (Dm) Drosophila melanogaster; (Af) Artemia franciscana; (Lv) Lytechinus variegates; (Sp) Strongylocentrotus pupuratus; (Ap) Asterina pectinifera; (Se) Sexostrea echinata; (BS) Botryllus schlosseri; (Ci) Ciona intestinalis; (Cj) Cardina japonica; (Le) Ligia exotica; (At) Achaearanea tepidariorum


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5.3.3 In situ hybridisation of catenin binding cadherins and planar

cell polarity pathway components In situ RNA hybridisation of Am_ACadherin shows that detectable levels of expression are

not present until after gastrulation, at the sphere stage (Figure 5.6). Expression in spheres is

restricted to a small region of ectoderm on one side of the larva, likely to correspond to the

site of the future oral pore. Expression in the involuted ectodermal cells of the oral pore is

apparent in pear and planula larvae, however no expression could be detected after

metamorphosis.

Coral orthologues of planar cell polarity (PCP) components exhibit a variety of spatial

expression patterns throughout development. Two components of the Fz-Fmi PCP system,

Am Van Gogh like (AmVangl) and AmDishevelled (AmDsh), along with AmDs of the FAT-Ds

PCP system, are first expressed at the prawnchip stage, prior to gastrulation (Figure 5.5).

Each of these three genes are expressed only in the presumptive ectoderm of the prawnchip,

however, whilst AmDsh is expressed ubiquitously throughout this presumptive germ layer,

AmVangl and AmDs are expressed in a gradient from one side of the embryo. It is not clear

whether these graded expression patterns are overlapping or opposing. In contrast to these

early patterns, only one of the three cloned Acropora Frizzled genes are expressed prior to

gastrulation (AmFz4), however its expression is restricted to the presumptive endoderm

(Ukolova et al., unpublished). AmFmi expression is also not detectable until after

gastrulation.

The expression of PCP related genes in larval stages occurs in two main regions, the aboral

ectoderm and the oral region. AmFmi, AmDsh, AmVangl and AmDs are all expressed

throughout the aboral ectoderm of planula larvae. In the oral region, AmFmi and AmDsh are

expressed in the oral endoderm, whilst genes involved in maintaining non-‐canonical Wnt

signalling (Wnt16, Axin, Wnt inhibitory factor (WIF) and Groucho) are expressed either in a

ring of ectodermal cells surrounding the oral pore (Wnt16, Axin) or in the oral ectoderm

itself (WIF, Groucho) (Figure 5.6). Although its role in canonical or non-‐canonical Wnt

signalling is unclear, AmFz4 is also strongly expressed in the oral ectoderm and potentially

overlaps the area of Wnt16 expression.


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Figure 5.5. In situ hybridisation of prawnchip (left) and early donut (right) stage Acropora embryos with genes from the Cadherin-‐catenin and planar cell polarity pathways. Embryos are shown with presumptive blastopore in the centre and the presumptive endoderm visible. Unlike catenin binding cadherins from other species, Am_ACadherin (A & B) is not expressed during gastrulation of Acropora millepora, suggesting this protein is not required to maintain cellular stability during embryonic morphogenesis. Am_β-catenin (C & D) and Am_Dishevelled (E & F) are both expressed in the presumptive ectoderm, which is in contrast to blastula forming animals where embryonic expression of both proteins occurs in the presumptive mesendoderm. Planar cell polarity genes Am_Van_Gogh (G & H) and AmDachsous (I & J) demonstrate a lateral gradient of expression in the presumptive ectoderm during embryonic development (the relative direction of these gradients could not be confirmed), which may indicate an active PCP. However, the apparent absence of AmFlamingo expression (K &l) during this stage would suggest otherwise. Am_Dishevelled and Am_β-catenin insitu hybridisations (C-‐F) were performed by C.Shinzato, Unpublished)


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Figure 5.6. In situ hybridisation of Acropora larvae with genes from the Cadherin-‐catenin and planar cell polarity pathways. Larvae are shown with oral pore at top except in B where oral pore is centred. Am_ACadherin is expressed in a restricted pattern at the oral pore commencing at sphere stage (A & B) and is maintained during the pear (C & D) and planula (E) larval stages. This pattern overlaps that of β-catenin (F-‐H), consistent with canonical interactions of β-catenin and CCD containing cadherins and a role in oral pore development. Expression patterns of non-‐canonical Wnt signalling components Wnt16 & Frizzled 4 (M & P) suggest Planar Cell Polarity may also function in the oral pore. This is supported by repressors of canonical Wnt signalling WIF (N) and Axin (O) in the oral pore, however the absence of PCP effector (Dachsous, Van Gogh, Flamingo and Dishevelled) expression from the oral ectoderm is contradictory. In situ hybridisation of Am_β-catenin (F-‐H) performed by E.Ball, Unpublished; In situ hybridisation of Wnt components (M-‐P) performed by S.Ukolova, unpublished; In-‐situ hybridisation of AmGroucho (Q) performed by L.Grasso, Unpublished.


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5.4 Discussion 5.4.1 Cadherins involved in epithelial cohesion and migration

evolved early in metazoan evolution cadherins capable of binding cytoplasmic β-catenin have long been recognised to have roles

in embryonic development where their expression correlates to both stable cohesive and

invasive cellular phenotypes. Using JCUSMART, I have identified 4 cnidarian cadherins (3

complete from Nematostella vectensis and 1 5’ truncated from Acropora millepora) with

conserved catenin binding motifs in their cytoplasmic domain. Genomic surveys have

revealed the presence of CCDs in representatives of all metazoan taxa, including a cnidarian

(Nematostella vectensis) and sponge (Amphimedon queenslandica – preliminary sequences)

(Abedin & King, 2009; King et al., 2008; Sakarya et al., 2007). These domains are notably

absent from the genome of Monosiga brevicolis, a representative of the choanoflagellata,

which are considered the closest extant metazoan outgroup (Abedin & King, 2009; King et

al., 2008). The absence of CCDs from choanoflagellates suggests the CCD arose only after the

animal transition to multicellularity in the common metazoan ancestor (King et al., 2008).

Although the CCD is recognised as metazoan specific, the complete coding sequence of CCD

containing proteins from non-‐bilaterian representatives has not previously been

investigated. The protein architecture of each cnidarian sequence is consistent with that of

type-‐III cadherins (Figure 5.1), found primarily in non-‐chordates (with the exception of Hz-‐

cadherins) and have not previously been reported outside the Bilateria. Cadherins of this

type are distinct from typical chordate (type-‐I and II) catenin binding cadherins due to the

presence of a variable number of N-‐terminal Cadherin (EC) repeats followed by a

characteristic combination of NC, CE and LamG domains (the PCCD complex), which may

play a role in translocation to the plasma membrane (Oda & Tsukita, 1999). The sponge CCD

containing protein, first reported by Sakarya et al (2007), is also a Cadherin however is

somewhat divergent in its ecto-‐domain architecture. Following the N-‐terminal EC repeats

(>12), are 12 EGF domains and 1 LamG domain. The domain organisation and the absence of

a PCCD are dissimilar to type-‐III cadherins and all other CCD containing proteins. The

sponge protein has instead been considered a FAT-‐like Cadherin (King et al., 2008) despite

its unknown functional significance. Although this one reported sponge catenin binding

Cadherin does not exhibit an architecture characteristic of any described ‘classic’ Cadherin

types, future analyses of the recently completed A.queenslandica genome (Srivastava et al.,

2010) may reveal a more complex complement of CCD containing cadherins in sponges.


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Type-‐III cadherins have previously been suggested to be a bilaterian ‘invention’ due to their

presence in both protostomes and deuterostomes (Hulpiau & van Roy, 2009). The

identification of CCD containing proteins from basal metazoans as cadherins with type-‐III

structure contradicts this notion. Instead, their presence suggests that type-‐III cadherins

arose early in metazoan evolution and diversified in the bilaterian lineage, giving rise to

functionally similar type-‐IV and type-‐I cadherins in protostomes and chordates respectively.

Type-‐III cadherins have subsequently been lost from the majority of chordates with only

three representatives (Hz-‐cadherins) remaining in vertebrates.

Phylogenetic analysis of the catenin binding region (Figure 5.4) was unable to identify any

clear relationship between cnidarian and bilaterian CCD containing cadherins. Protostome

and deuterostome sequences produce 2 discrete clades as is consistent with previously

published analyses (Hulpiau & van Roy, 2009). Type-‐III cadherins also form 2 clades

consistent with the functional distinction between type-‐III cadherins from different

bilaterian lineages. Grouping of cnidarian sequences with either clade would suggest a

common evolutionary history, however the cnidarian sequences form a third discrete clade

and are not clearly associated with either of the bilaterian type-‐III Cadherin groups. The

functional significance of the cnidarian CCD containing cadherins therefore remains unclear

as they are not more closely related to either invasive or stabilising type cadherins. It may be

that cadherins fulfilling both of these roles are present in cnidarians and that the distinction

between invasiveness and stability is more closely associated with differential regulation

than with the interaction between the cytoplasmic domain and catenins. Differential

regulation however, only partially explains this distinction in bilaterians, as expression of E-

Cadherin on N-Cadherin expressing cells does not diminish the invasive phenotype (Nieman

et al., 1999). To further the explanation, a mechanism of matrix-‐metalloprotease mediated

cleavage of the E-Cadherin ectodomain has been proposed (Xian et al., 2005), although the

possibility of similar protein level regulation in cnidarians would not be reflected in the

present analysis.

The apparent tight association of the CCD with cadherins in even the most primitive

metazoan phylum, the sponges, and the high degree of conservation in the catenin binding

motifs of the CCD, indicates the presence of a functional constraint on the Cadherin-‐catenin

relationship. The critical nature of roles for β-catenin and Cadherin described during

gastrulation and normal function of bilaterian animals is the most likely reason for this

constraint. It therefore stands to reason that similar vital roles for the conserved Cadherin-‐

catenin signalling axis in germ layer specification and morphogenesis could be evident

during development of more primitive metazoans such as cnidarians.


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5.4.2 Planar cell polarity but not Am_ACadherin is implicated in

gastrulation of Acropora millepora The presence of catenin binding cadherins at adherens junctions of the presumptive

ectoderm prior to gastrulation is conserved throughout the Bilateria. A similar distribution

in the presumptive ectoderm was expected for Am_ACadherin prior to gastrulation of

Acropora millepora. However, in situ hybridisation of Acropora embryos revealed no

detectable expression of Am_ACadherin until after gastrulation was complete (Figures 5.5 &

5.6). In each branch of the Bilateria for which catenin binding cadherins have been

described, a role analogous to that of E-Cadherin in stabilising the structure of the

presumptive endoderm has been demonstrated. The unexpected absence of Am_ACadherin

from gastrulation stages indicates it does not function in tissue stability prior to

gastrulation. Only a single Cadherin capable of binding β-catenin has been identified from

A.millepora, suggesting that the most highly conserved role for the Cadherin-‐catenin axis is

not represented in this species.

The most direct evolutionary interpretation of the absence of cadherins effecting tissue

stability is that the tissue stabilising function arose after the divergence of cnidarians. This

prospect is however highly unlikely. Cnidarians demonstrate a considerable range of

gastrulation processes including invagination, ingression, delamination and epiboly (Byrum

and Martindale, 2004). Such diversity is even reflected among coral and the bi-‐layer folding

exhibited by Acropora is a particularly peculiar mode of gastrulation. In contrast to

Acropora, the gastrulation of Nematostella occurs by invagination following blastula

formation (Kraus & Technau, 2006; Lee et al., 2007; Magie, Daly, & Martindale, 2007)

variations of which are reasonably common in bilaterians. The clear evolutionary

relationship between Nematostella and bilaterian gastrulation suggests that stabilisation of

the presumptive ectoderm by a catenin-‐binding Cadherin is likely to be represented in some

parts of the cnidarian lineage. As such, the tissue stabilising function can still be considered

ancestral. The absence of this function in Acropora is therefore attributable to a genus

specific diversification related to the mode of gastrulation and is not reflective of the

ancestral state.

Simplification of Acropora catenin binding Cadherin function is also supported by the results

of cnidarian genomic surveys. Whereas a single Type-‐III Cadherin was identified in coral, the

genome of Nematostella vectensis has revealed 3 predicted Cadherin genes with Type-‐III

architecture. Two of the Nematostella genes appear to be tandem repeats and may produce

only a single functional protein (based on protein alignment – not shown), however the

third gene resides on a separate scaffold of the genome assembly and is likely to be a distinct


83

and functional Cadherin. The presence of only a single Cadherin in coral compared to 2 in

Nematostella could indicate that functional simplification is paralleled by gene loss. This

then raises the question of how epithelial associations are maintained when the major

adhesive component of adherens junctions has been lost. When considered along with the

incomplete nature of the Acropora transcriptome, there is a reasonable possibility that

further catenin binding cadherins may be found in the upcoming Acropora millepora genome

(Miller et al, unpublished).

Planar cell polarity has also been implicated in gastrulation, however most studies have

focused on roles during convergent extension processes such as neuralation. In Acropora,

expression of AmDsh, AmVangl and AmDs is restricted to the presumptive ectoderm (Figure

5.5). Unlike AmDsh, expression of AmVangl and AmDs is observed as a gradient across the

embryo. The relative orientation of the gradients could not be confirmed although the

presence of graded expression is consistent with classical descriptions of these genes in

polarised epithelial tissues (Wu & Mlodzik, 2009). These patterns of expression suggest that

planar cell polarity has a role in morphogenesis of the coral ectoderm during gastrulation.

Like most forms of gastrulation, that of Acropora requires progressive changes in cellular

arrangement. The best described methods of achieving the necessary re-‐arrangements are

directed cell movements (eg. convergent extension, integrin mediated migration, ingression,

and involution) and polarised cell division. Directed cell movements are expected to play a

minimal role in Acropora gastrulation with no clear morphological evidence for the common

forms. Despite this, the process by which convergent extension is regulated is worth

considering. Experiments in Xenopus have demonstrated that convergent extension

requires active planar cell polarity controlled by Dsh and that non-‐canonical Wnt is only

permissive in this process and not required (Wallingford et al., 2000). Of more direct

importance is the demonstration that planar cell polarity, through Dishevelled, is

responsible for directing polarised cell division along the AV axis in Zebrafish. Similarly the

expression of AmDsh in the presumptive ectoderm of coral (Figure 5.5) could play roles in

permitting cell movement and directing cell polarity. This would also be consistent with a

lack of catenin binding Cadherin in this tissue. Asymmetric distribution of AmVangl and

AmDs may therefore act in establishing cell polarity and the direction of cell division,

allowing extension of the ectodermal cell sheet to occur in an ordered manner where a

single plane of ectodermal cells is maintained.


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Contradictory to this suggestion is the absence of detectable AmFmi and Fz expression in the

ectoderm prior to gastrulation. Frizzled and Fmi are the major cell surface components of

the core (Fz-‐Fmi) planar cell polarity pathway and are expected to be present in polarised

epithelium. Whilst it is likely that multiple Frizzled proteins remain undiscovered in

Acropora, some of which may be expressed in the presumptive ectoderm, the same is less

likely to be true for Flamingo. Genome surveys from a range of representative metazoans

indicate diversification of CELSR occurred only in the vertebrate lineage, so alternatively

expressed coral cognates are not expected. The roles and organisation of non-‐bilaterian

planar cell polarity systems are yet to be defined, however the asymmetrical distribution of

AmDs and AmVangl and their potential involvement in directing cell division are a starting

point for future investigations which would fill a gap in both our knowledge of cnidarian

gastrulation and the evolution of a major metazoan patterning system.

5.4.3 Am_ACadherin and planar cell polarity are implicated in

development of the Acropora larval oral pore. The possibility of developmental roles for Am_ACadherin and components of the planar cell

polarity pathway were investigated by in situ hybridisation of Acropora millepora

developmental stages. Spatial patterns of expression in and around the oral pore (larval

mouth structure) for a number of the genes investigated were consistent with roles in oral

pore development, a process which is poorly described in Acropora. Morphological analyses

have demonstrated that following Acropora gastrulation the blastopore closes completely

(sphere stage) prior to establishment of the oral pore (pear stage). This is in contrast to the

development of other cnidarians, such as Nematostella, where the blastopore does not close

instead becoming the oral pore. Morphological data from Acropora also show that cells of

the oral pore are ciliated and more closely resemble the epithelial organisation of

ectodermal tissue (Ball et al., 2002), suggesting they are of ectodermal origin. The possibility

that oral pore formation occurs through involution of ectodermal tissue is therefore not

unreasonable.

Expression of Am_ACadherin was first detectable in a restricted region of ectoderm in sphere

stages and later expressed specifically in the oral pore, during pear and planula stages

(Figure 5.6). Expression both prior to and following establishment of the larval mouth,

suggests a role in oral pore development. The restricted pattern of larval expression also

coincides with that described for Amβ-catenin, supporting the proposition of an ancestral

Cadherin-‐catenin signalling axis. The association of Amβ-catenin with Am_ACadherin in these

cells is further supported by expression of AmWIF and AmGroucho in the same cell

population. In bilaterian models, WIF has been demonstrated to compete with Wnts for Fz

binding, thereby antagonising the canonical Wnt system and subsequent stabilisation and


85

nuclear translocation of β-catenin. Groucho, on the other hand, represses the transcriptional

targets of Wnt signalling by binding TCF/LEF transcription factors in the absence of nuclear

β-catenin. Expression of WIF and Groucho together suggests that β-catenin is not

transcriptionally active in the oral pore and is more likely to be associated with Cadherin at

the plasma membrane. Evidence for a Type-‐III Cadherin-‐β-catenin interaction in cells of the

oral pore, which are potentially undergoing involution, is consistent with an

invasive/migratory type function for Am_ACadherin.

Genes up stream of the planar cell polarity pathway were also found to be expressed in the

oral region of Acropora larvae. AmWnt16 is expressed in a restricted pattern in a ring of cells

at the edge of the oral pore. Bilaterian orthologues of Wnt16 have been demonstrated to

activate the planar cell polarity pathway whilst inhibiting canonical Wnt signalling in

bilaterians. These effects constitute non-‐canonical Wnt signalling. The functional

significance of AmWnt16 in coral development has not yet been clearly demonstrated,

however gene expression is abolished by the presence of nuclear beta-‐catenin following

alsterpaullone treatment (Ukolova et al., unpublished), which is consistent with previously

described reciprocal antagonism between the canonical and non-‐canonical Wnt systems

(Bryja et al., 2009; Topol et al., 2003). AmAxin is also expressed in the ectodermal cells

surrounding the mouth of coral larvae. In bilaterians, Axin is a constituent of the GSK3β

complex responsible for the degradation of cytoplasmic β-catenin and is capable of binding

β-catenin directly. Expression coinciding with Wnt16 is consistent with the expected

negative regulation of the canonical Wnt system. The role of Axin as part of the GSK3β

complex outside the Bilateria is however currently under scrutiny due to the absence of the

beta-‐catenin binding motif in Amphimedon, Nematostella and Acropora Axin orthologues

(Srivastava et al., 2010; Ukolova et al unplublished).

The presence of a functional planar cell polarity system at the outer lip of the oral pore may

have roles in orientating cilia appropriately for directing food to the gastric cavity or simply

in defining the structure of the oral pore from surrounding ectodermal tissue. The

expression patterns of planar cell polarity effectors, however, contradict the existence of a

non-‐canonical Wnt mediated planar cell polarity in this area. In situ hybridisation of AmFmi,

AmDsh, AmVangl, and AmDs failed to identify expression of components from either the Fmi-‐

Fz or FAT-Ds PCP systems at the outer lip of the oral pore. The only possible PCP component

expressed in the oral pore was AmFz4. Although AmFz4 expression may overlap that of

AmWnt16, co-‐expression is not necessarily suggestive of a role in PCP signalling. Frizzled

proteins are recognized to hold diverse functions so no conclusion as to the implication of

AmFz4 in the oral pore can be drawn without the support of functional data.


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Despite the apparent lack of PCP component expression, the suggested role of Wnt16

expression in oral pore development cannot be ignored. Protein localisation studies are the

only conclusive indicator of planar cell polarity and provide added sensitivity over in situ

hybridisation. These experiments may uncover asymmetric distribution of protein within a

limited cell population thereby re-‐enforcing non-‐canonical Wnt signalling as a factor in oral

pore development. Likewise, protein localisation and further analysis of the morphological

movements during oral pore formation could clarify the role of Am_ACadherin. Further

investigation involving both protein localisation and in vivo functional studies are also

required to clarify the roles of Cadherin-‐catenin signalling and planar cell polarity in

cnidarian gastrulation. Of particular interest is the influence of these cadherins on the

nuclear translocation of β-catenin. It has already been demonstrated in Nematostella that

over expression of the CCD can inhibit gastrulation, however no direct studies into the

function of ‘classic’ cadherins have been undertaken outside the Bilateria.

5.5 Conclusion Investigation into the Cadherin complement of cnidarians has revealed an ancestral origin

for two adhesion systems that are important in bilaterian development. Here I have

described that the conserved Cadherin-‐catenin axis, active during bilaterian development, is

conserved in cnidarians and likely to have originated in the eumetazoan ancestor. Whilst the

function of this system in cnidarians is yet to be tested, early investigations into expression

in Acropora suggest roles in involution rather than the tissue stabilising functions seen in

bilaterians. The possibility that the function of the Cadherin-‐catenin axis is simplified in

Acropora is also evidenced. Components of the planar cell polarity pathway, from signalling

to membrane bound effectors, are also conserved in cnidarians. Expression in both

gastrulation and oral pore development in Acropora implies planar cell polarity is not

limited to previously described functions in bilaterian development and is of broader

morphogenic significance than currently recognised.

Chapter Six Integrins of Acropora millepora

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Chapter 6: Integrins of Acropora millepora

6.1 Introduction Cohesion of cells to the extracellular matrix (ECM) is critical to the evolution of animal

multicellularity and morphogenesis of complex metazoan body structures (Abedin & King,

2010). The integrin family of transmembrane cell surface receptors are the primary

mediators of cell-‐ECM interactions in bilaterians (De Arcangelis & Geogres-‐Labouesse, 2000)

and have been implicated in a broad array of biological processes including gastrulation,

neuron outgrowth and leukocyte trafficking. Integrin genes are present in all metazoans

investigated to date, although the number of α and β-‐subunits varies greatly between taxa

(Sebé-‐pedrós et al., 2010). The diversity of integrin αβ heterodimers and the ability to

rapidly switch between high and low affinity states in response to intracellular stimuli

(inside-‐out) or extracellular ligand binding (outside-‐in) (Ginsberg et al., 2005; Hynes, 2002;

Takagi et al., 2002) is central to the versatility of integrin function. This integration of

dynamic cell adhesion with bi-‐directional signalling allows cellular behaviours such as

migration, growth, differentiation and deposition of basement membrane components

(Brown, 2000) to be guided by positional cues (Ramos et al., 1996; Streuli, 2009), providing

a basis for many of the key events in animal development.

The role of integrins in facilitating morphogenesis and tissue development is conserved

throughout the Bilateria (Brower, 2003; Knack et al., 2008; Reber-‐Müller, et al., 2001;

Whittaker & Desimone, 1993) and functional investigations have demonstrated that the

integrin signalling system is highly specific in terms of protein expression, ligand binding

and heterodimer combination (Humphries et al., 2006; Hynes, 2002). Whereas protein

expression is governed by transcriptional regulation, integrin heterodimer combination and

ligand binding are inextricably linked. The α and β integrin subunits make an unequal

contribution to ligand interactions, with the α-‐subunit holding the greatest influence over

ligand specificity and the β-‐subunit being primarily responsible for signal transduction

through cytoplasmic interactions and formation of cytoskeletal connections (Berman et al.,

2003; Legate, et al., 2006; Martin-‐Bermudo et al., 1997; Nishiuchi et al., 2003). As a single β-‐

subunit may associate with many α-‐subunits (Kinashi, 2005), different αβ integrin

combinations are capable of eliciting similar cellular behaviours on a range of substrates

through specific ligand binding. This is particularly significant to developmental processes

as diversity in the ligand specificity can facilitate behaviours such as cohesion, polarisation


88

and migration on basement membranes with various physical properties, thereby

contributing to the co-‐ordination of many distinct tissues during development.

The most diverse integrin complement belongs to mammals, containing 18α and 8β

subunits and although only 24 combinations are expressed as functional integrin receptors

(Hynes, 2002), the complexity of this complement is far greater than other bilaterians such

as sea urchin (3α & 4β), fly (5α & 2β), and worm (2α & 1β) (Brower, 2003; Putnam et al.,

2007; Schmitt & Brower, 2001). Although there are differences in the number of integrin

subunits between bilaterians, ligand specificities from different model systems are often

similar, allowing all described integrin receptors to be broadly categorised as a Laminin,

RGD tripeptide, Collagen or Leukocyte-‐specific type according to their ligand binding

properties (Huhtala et al., 2005; Hynes, 2002). Mammals, flies, worms and sea urchin all

contain at least 1 integrin receptor that binds Laminin and 1 that binds RGD tripeptide

containing proteins (eg. Fibronectin, Tiggrin), suggesting the last common ancestor of

bilaterians exhibited similar ligand specificities. Collagen binding and leukocyte-‐specific

integrin receptors are found only in chordates and appear to be independent diversifications

linked to the evolution of circulating adaptive immune cells and complex collagen structures.

In contrast to the wealth of information regarding integrin function in higher animals, the

expression, heterodimer formation and ligand binding properties of lower animal integrins

have received little attention. Recent genomic surveys of basal metazoan phyla have

discovered that the cnidarian and sponge integrin complement is similar to that of other

invertebrates, possessing up to 2α & 4β, and 5α & 7β respectively (Sebé-‐pedrós et al., 2010);

Chapter 4). Despite similarity in gene numbers between lower animals and bilaterian

invertebrates, phylogenetic analyses including sequences from Nematostella have failed to

determine a clear relationship between the integrins of cnidarians and bilaterians (Knack et

al., 2008; Magie & Martindale, 2008) offering little insight as to the function of cnidarian

integrins.

Although there are differences in integrin receptors between phyla, a number of

publications suggest integrin cytoplasmic signalling is remarkably similar to that of higher

animals, in even the simplest metazoans. Investigations into integrin systems of

representative cnidarians, sponges and lower metazoans (eg. Trichoplax adhaerens) have

identified the presence of complete integrin subunits and all of the major cytoplasmic

components required for canonical integrin signalling in every animal model considered.

Investigations extending beyond the animal kingdom also identified a complete signalling

complement in Capsaspora owczarzaki, a filose amoeboid nucleariid serving as an out group


89

to the Metazoa (Nichols et al., 2006; Sebé-‐pedrós et al., 2010). Despite the integrin system

traditionally being considered an animal novelty (Rokas, 2008), the presence of canonical

integrin signalling in such a distant Metazoan relative suggests that the cellular signalling

events following integrin ligand binding are highly constrained and have been conserved

since before the first metazoans evolved.

In addition to maintenance of integrin signalling from a basal Opsithokont ancestor, the

distribution of integrin gene expression is also conserved throughout the animal kingdom.

The jelly-‐fish Podocoryne carnea (Reber-‐Müller et al., 2001), the coral Acropora millepora

(Knack et al., 2008), and all bilaterian models investigated to date each express α and β

integrins in the presumptive mesendoderm prior to gastrulation (Bökel & Brown, 2002;

Davidson, Hoffstrom, Keller, & DeSimone, 2002; Marsden & Burke, 1998; Ramos, Whittaker,

& Desimone, 1996). Despite diverse modes of gastrulation (See Chapter 7.1), expression of

integrins in mesendodermal cells crosses the diploblast – triploblast divide, demonstrating

an unusually high degree of conservation in the pattern of expression consistent with

integrins playing an indispensible role during animal development.

Whereas evidence is available for the conservation of cytoplasmic signalling and expression

of integrins in at least one aspect of metazoan development, gastrulation, there is no

evidence to suggest ligand specificity is equally conserved between cnidarian and bilaterian

integrins. Investigation of cnidarian integrin ligand specificity aimed to address the lack of

functional data from non-‐bilaterian models and clarify the roles of integrins in the

ureumetazoan ancestor. As phylogenetics is demonstrated to be of limited predictive value

(Knack et al., 2008), the ligand binding specificities of cnidarian integrins must be

determined empirically using an ancestrally informative model. Anthozoan cnidarians such

as the coral Acropora millepora offer a unique perspective on the ancestral role of integrins

during development, having retained much of the genetic complexity from the common

eumetazoan ancestor (Technau et al., 2005). They are also the most basal phylum to possess

true tissues and are therefore likely to be highly informative regarding the genetic origins of

tissue morphogenesis, a process which is strongly influenced by integrin mediated adhesion.


90

Investigation of integrin-‐extracellular matrix interaction in higher animals, such as flies, is

routinely performed by assessing structural changes of cultured cells in response to contact

with potential ECM ligands (Jannuzi et al., 2002; Nieves et al., 2010). The maintenance of

coral cells in culture has not yet been established and developing such a system is beyond

the scope of the present investigation. To overcome this obstacle, it was necessary to turn to

an established experimental system already proven to be useful in assessing the structure

and function of integrins. Cultured Drosophila S2 cells have often been employed to conduct

such investigations and hold several advantages over other model systems including access

to a wide variety of genetic markers, allowing exploration of cellular affects, availability of

inducible expression vectors and convenient & efficient knockdown of endogenous

integrins. Investigation of coral integrin ligand specificity was therefore conducted using

transgenic expression of coral αβ subunit combinations in Drosophila S2 cells and exposing

transfected cells to a variety of known Drosophila integrin ligands. These studies provide a

basis for direct comparison between higher animal ligand specificity and that of

developmentally significant coral integrins. The phylogenetic distribution of integrin α and β

subunits from a wider range of cnidarians was also considered in order to determine the

broader relevance of coral ligand specificity in an evolutionary context.


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6.2 Methods 6.2.1 Phylogenetic analyses integrins from Acropora millepora and Nematostella vectensis were identified using the

method described in Chapter 3. Maximum likelihood analysis of β-‐Integrin subunits was

performed using MolPhy with 1000 bootstrap replicates (Knack et al., 2008). Maximum

likelihood analysis of α-‐Integrin subunits was performed using PhyML (1000 bootstrap

replicates) and includes cnidarian sequences (AmItgα2, AmItgα3, NvItgα2) not present in

published phylogenetic analyses. integrin phylogenies are based on extensively edited

alignments presented in Supplementary Figure 6.1 (α-‐Integrin alignment) & 6.2 (β-‐Integrin

alignment).

6.2.2 Preliminary ligand binding assay Clones of AmItgα1, ItgβCN1 and AmItgβ2 were isolated from the Acropora millepora EST

project and RNA isolates as described in Chapter 2.2 & 2.3. Cloning of the 9 expression

constructs used throughout the following experiments was performed using a series of

overlap PCRs as detailed in Chapter 2.5. Procedural aspects of the following experiments,

including the maintenance of Drosophila S2 cell cultures, are presented in Chapter 2.6-‐2.9.

Preliminary assessment of the ligand binding properties of coral integrins was performed

using cell spreading on Rbb-‐Tiggrin, Vitronectin, Fibronectin, Tennectin, Twow, PacI,

Trimeric Laminin (Drosophila), and sucrose fractionated Laminin. Wells pre-‐incubated with

PBS were used as negative controls for each cell types. Cells expressing ItgαPS1 ItgβPS or

ItgαPS2 ItgβPS were used as positive controls for interaction with Laminin and RGD type

ligands respectively. Combinations of coral integrins (excluding tagged β constructs) were

co-‐transfected (Cellfectin – Invitrogen) with a constitutively expressing eGFP construct,

pH8CO vector (Rebay et al., 1991) and 500ng Mysopheroid (ItgβPS) RNAi. Cell spreading on

Laminin type substrates was performed using 5x 1:5 serial dilutions of stock laminin. Cell

spreading was assessed by phase contrast microscopy under an inverted light microscope

(Zeiss).


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6.2.3 Optimisation of cell spreading conditions Serum free-‐ M3 medium (no added cations), BES-‐Tyrodes (1mM Mg2+, 10µM Ca2+, 1% BSA),

Robb’s Saline (1mM Mg2+, 10µM Ca2+, 1% BSA) and PBS (1mM Mg2+, 10µM Ca2+, 1% BSA)

were compared for their ability to support specific integrin mediated cell spreading on Rbb-‐

Tiggrin. Cells expressing AmItgα1 AmItgβ2 or ItgαPS2 ItgβPS (positive control) were

included in the cell spreading assay, conducted in replicate as described in Chapter 2.8.

The impact of Mg2+ and Ca2+ concentration on the percentage of cells exhibiting integrin

mediated cell spreading and the morphology of spreading cells was assessed using the

following concentrations of cations in Robb’s saline with 1% BSA (Table 6.1). Cells

expressing AmItgα1 AmItgβ2 or ItgαPS2 ItgβPS (positive control) were included in the cell

spreading assay, which was conducted in replicate as described in Chapter 2.8.

Combination Mg2+ (MgCl2)

Concentration

(mM)

Ca2+ (CaCl2)

Concentration

(mM)

1 50 10

2 5 0.2

3 0.1 0.2

4 0.01 0.2

5 0 (EDTA) 0 (EDTA)

6 1 0.002

7 1 0.02

8 1 0.2

Table 6.1 Concentrations of Mg2+ (MgCl2) and Ca2+ (CaCl2) added to Robb’s Saline for optimisation of cell spreading conditions. Integrin activation and ligand interaction is dependant on the availability of divalent cations in the surrounding medium. The divalent cation concentration in the extracellular fluid surrounding coral integrins (sea water) is expected to differ from that for Drosophila integrins (hemolymph), therefore the concentration of divalent cations required for optimum integrin activation may also differ. Comparison of S2 cell supporting media identified Robb’s Saline to give the highest degree of integrin mediated cell spreading. The divalent cation concentration included in preparation of Robb’s Saline was altered according to the 8 combinations shown in the table before use as a supporting medium for cell spreading assays.


93

6.2.4 Analysis of integrin surface expression integrin expressing cells were prepared for flow cytometry as described in Chapter 2.9.

Secondary detection at 546nm was used for cells transiently transfected with integrin (α

and β) and eGFP expression constructs. Cells exhibiting detectable levels in the GFP range

were considered successfully transfected, whilst cells exhibiting detectable emission in both

the GFP and 546nm ranges were considered integrin+. Integrins tagged with the HA epitope

were detected by direct immuno-‐fluorescence at 488nm, therefore cells were not

transfected with the eGFP expression vector.


94

6.3 Results 6.3.1 Integrin identification and phylogenetic analysis Investigation of integrin diversity in four representative cnidarians conducted using

JCUSMART (See Chapter 3) identified similar numbers of α and β subunits in Hydra, Clytia,

Nematostella and Acropora. Whilst the complete complement of Nematostella has

previously been reported (Putnam et al., 2007; Srivastava et al., 2010), the present analysis

identified 2 novel α-‐subunits from Acropora millepora and previously undescribed integrins

from Hydra (1α & 2β) and Clytia (1α & 1β). Due to limitations of the available data, the

number of integrins identified in Hydra and Clytia are likely to represent only a portion of

the total complement, however the Acropora (3α and 2β) and Nematostella (2α & 4β)

integrin complement is expected to encompass all distinct genes present in each genome.

Maxmium Likelihood analysis performed on a selection of integrin sequences was broadly

consistent with previous investigations (Ewan et al., 2005; Hughes, 2001; Huhtala et al.,

2005; Knack et al., 2008). Sequences from Clytia and Hydra, which are potentially

informative, were excluded from the analysis as gene models were not of sufficient length to

allow accurate phylogenetics. Integrin phylogenetics is complicated by homoplasy and poor

conservation of primary sequence, making unambiguous alignment difficult, therefore the

trees presented here are based upon extensively edited alignments (Supplementary Figure

6.1).

Analysis of integrin α-‐subunits shows grouping of sequences with RGD and Laminin binding

properties into 2 distinct clades, with the Cnidarian sequences forming a separate clade.

Deuterostome and protostome sequences were present in both the RGD and Laminin clades

suggesting the capacity to bind both ligand types was already established in the common

bilaterian ancestor. The position of the cnidarian α sequences in an indpendent clade is not

informative as to the ligand binding specificities of basal metazoans. This analysis also

revealed that NvItgα2 and AmItgα3 are closely related, forming the Cnidarian Minor Clade

however, association with the other cnidarian sequences (Cnidarian Major Clade) is poorly

supported.

Phylogenetics of β-‐Integrins is also consistent with published data, showing clustering of

chordate β1, β4 and β3 families and independent grouping of non-‐chordate sequences

(urchin, Fly, Cnidarian, Sponge) according to taxonomy. This pattern demonstrates the

independent expansion of β-‐Integrins in several lineages, including the Cnidaria where

Nematostella encodes 2 sequences orthologous to each of the Acropora β-‐Integrins.


95

Figure 6.1 Maximum likelihood phylogenetic analysis of representative α-‐Integrins. Numbers at branch points indicate the percentage of 1000 bootstrap replicates supporting the topology resolved using PhyML. Integrin sequences α7, α6, α3, α5, αV, α8 and αIIb taken from human. Bilaterian sequences are resolved into Laminin, RGD and Alpha 4/9 clades consistent with previous analyses. The ambiguous position of ItgαPS3 is likely due to the highly derived nature of this protein when compared with other Drosophila integrins. The position of GcItgα away from the distinct clades is suggested to result from early divergence. This position would be further resolved by inclusion of other sponge sequences. In contrast to the bilaterian sequences, which group in a ligand specific manner, the cnidarian sequences form an independent clade. This grouping suggests functional divergence had already occurred in the Urbilateria. Two novel cnidarian sequences (NvItga2 and AmItga3) are only weakly associated with the other cnidarian sequences, reflective of their sequence divergence. The presence of two distinct cnidarian clades suggests that cnidarian integrins may interact with multiple ligands.


96

Figure 6.2 (Excerpt from Knack et al., 2008) Maximum likelihood phylogenetic analysis of representative β-‐Integrins. Numbers at branch points indicate the percentage of 1000 bootstrap replicates supporting the topology resolved using MolPhy. In contrast to the α-‐integrin phylogeny, the grouping of the β-‐subunits in lineage specific clades suggests beta integrins have evolved through a number of lineage specific expansions, as is consistent with previous publications. Cnidarian sequences clade together with strong bootstrap support. Within the Cnidarian clade, Acropora and Nematostella sequences show 2 sub-‐clades consisting of ItgβCn1, NvItgβ3, NvItgβ4, and AmItgβ2, NvItgβ1,NvItgβ2, PcItgb. The position of Acropora and Nematostella sequences into two sub-‐clades suggests the sea anemone possess 2 β-‐Integrins for each coral β-‐Integrin, which may be the result of a recent duplication in Nematostella.


97

6.3.2 Ligand Binding of coral integrins

Preliminary ligand binding Assay The preliminary cell spreading assay to assess the ligand binding properties of coral

integrins was performed under non-‐optimised conditions using the following ligands:

• Rbb-‐Tiggrin

• Vitronectin

• Fibronectin

• Tennectin

• Twow

• PacI

• Drosophila Trimeric Laminin

• Sucrose fractionated Drosophila Laminin

Assessment of transfection efficiency by visualisation of eGFP expression prior to cell

spreading showed that ~30% of the total cell population expressed GFP 2 days after

transformation. Accordingly, ~30% of control cells for RGD tripeptide ligands (transformed

with αPS2 + βPS) spread on Rbb-‐Tiggrin, Vitronectin, Fibronectin, Tennectin, Twow, and

PacI. Positive control cells for Laminin interaction (transfected with αPS1 + βPS), spread

weakly on the trimeric Laminin and sucrose fractionated Laminins, indicating the quality of

the available Drosophila Laminin substrates was less than ideal. Cells transfected with coral

integrins showed both a low percentage of spread cells (~ 2-‐5% above empty vector

transfected controls) and a small degree of spreading on all substrates, suggesting

interactions with the tested substrates may only be weak. The highest degree of spreading

was observed in AmItgα1β2 and AmItgα1β1 transformed cells on Rbb-‐Tiggrin, a 53 amino

acid recombinant portion of the RGD containing Drosophila Tiggrin protein (Jannuzi et al.,

2002), consisting of 25 bases up and down stream of the RGD sequence. Rbb-‐Tiggrin also

facilitates a particularly strong interaction with the primary Drosophila RGD-‐binding

integrin (αPS2βPS), resulting in a highly spread morphology. Rbb-‐Tiggrin was therefore

used to assess RGD-‐ligand binding in all subsequent experiments.


98

0ptimisation of Cell spreading conditions In order to determine the optimal buffer for cell spreading assays, 4 buffers, Serum free-‐ M3

medium (no added cations), BES-‐Tyrodes (1mM Mg2+, 10µM Ca2+, 1% BSA), Robb’s Saline

(1mM Mg2+, 10µM Ca2+, 1% BSA) and PBS (1mM Mg2+, 10µM Ca2+, 1% BSA), were compared

for their impact on cell morphology and the capacity to allow coral integrin mediated

spreading on Rbb-‐Tiggrin. More than 95% cells stably transfected with αPS2βPS spread to a

diameter of no less than 3x that of the nucleus in all media. By comparison, very few of the

coral expressing cells demonstrated notable integrin mediated cell spreading. In all

transfections, cells appeared to have a more regular morphology (consistent with healthy

cells) in M3 media than saline based media, with PBS causing the highest degree of cell

death. Although the most protective media for S2 cells was M3, cells in this media showed

the smallest degree of spreading on Rbb-‐Tiggrin. The highest degree of cell spreading and

the most acceptable level of negative impact on cell health was observed in cells spread in

Robb’s saline, therefore subsequent experiments were conducted using only this medium.

Investigations into the optimal divalent cation concentration for integrin activation were

also assessed morphologically. High concentrations of divalent cations (5mM Mg2+ + 1mM

Ca2+ and above) resulted in formation of numerous cell processes, which were inconsistent

with integrin mediated adhesion. Concentrations of less than 1mM Mg2+ or 0.2mM Ca2+

facilitated cell spreading in only a small proportion of the cell population, whereas 1mM

Mg2+ and 0.2mM Ca2+ allowed optimal activation of coral integrins with a maximum of 7% of

cells in the AmItgα1 + AmItgβ2 transfected sample spreading beyond background levels on

Rbb-‐Tiggrin. Spreading in all other coral samples was observed to be between 2% and 5% of

the population under the same conditions compared to over 30% in αPS2βPS control cells

(transient transfection).


99

Surface expression of integrins following transient transfection Expression of integrins on the cell surface assessed by flow cytometry of duplicate cell

transfections showed that for each of the 8 transfections, 30-‐35% of the total cell population

expressed GFP (Table 6.2), indicating transfection was successful in all samples. The number

of transfected (GFP+) cells staining with anti-‐PS1 and anti-‐PS2 antibodies was higher in

ItgαPS1 (9.8%) and ItgαPS2 (10.0%) expressing cells than pHSMCS transfected negative

controls (1.5% and 0.4% respectively), indicating that approximately 10% of the total

population expresses detectible levels of Drosophila integrins 2 days after stress induction

of the heat shock promoter (ie. during transfection). Immunostaining of coral integrin

transfected cells with antibodies against AmItgα1, AmItgβ1, and AmItgβ2 showed no

detectable levels of surface expression (secondary detection).

Flow cytometry 1 day after transfection and in the presence of Mn2+ performed on a replica

set of transfections demonstrated 5-‐10% of the population was GFP+, however also failed to

detect coral integrins using the anti-‐ AmItgα1, AmItgβ1, and AmItgβ2 antibodies (data not

shown).

Expression of Acropora β-‐Integrins containing epitope (HA or c-‐MYC) tagged Drosophila βPS

serine loops inserted into the extracellular hybrid domain was assessed by flow cytometry 2

days after transfection (Table 6.3). Cells transfected with αPS2 + βPS-‐HA showed expression

in ~24% of the total population above empty vector (pHSMCS) transfected controls.

Similarly, cells transfected with αPS2 + βPS-‐MYC exceeded expression in controls by ~16%.

In contrast to the clear expression of Drosophila integrins, the percentage of the total cell

population expressing AmItgα1AmItgβ1-‐HA was only 1.8% above empty vector transfected

controls and AmItgα1AmItgβ2-‐MYC was detected in only ~1% of the total population above

the control level. Together, this suggests that whilst coral integrins are expressed on the

surface of transfected cells, they are only present in a minimal proportion of the transfected

cell population


100

Cell Line % of Total GFP+

Antibody % of Total GFP+/integrin+

pHSMCS 34.5 α-‐ItgαPS1 α-‐Mouse 546 1.2

ItgαPS1 ItgβPS 31.3 α-‐ItgαPS1 α-‐Mouse 546 9.8

pHSMCS 35.2 α-‐ItgαPS2 α-‐Mouse 546 0.4

ItgαPS2 ItgβPS 23.3 α-‐ItgαPS2 α-‐Mouse 546 10.0

pHSMCS 35.4 α-‐AmItgα1 α-‐Rabbit 546 0.0

AmItgα1 AmItgβ1 34.9 α-‐AmItgα1 α-‐Rabbit 546 0.1

AmItgα1 AmItgβ2 30.8 α-‐AmItgα1 α-‐Rabbit 546 0.1

AmItgα1-‐PS2 AmItgβ1-‐PS 34.6 α-‐AmItgα1 α-‐Rabbit 546 0.0

AmItgα1-‐PS2 AmItgβ2-‐PS 31.7 α-‐AmItgα1 α-‐Rabbit 546 0.0

AmItgα1-‐PS2 AmItgβ2L>D-‐PS 35.7 α-‐AmItgα1 α-‐Rabbit 546 0.1

pHSMCS 34.4 α-‐AmItgβ1 α-‐Rabbit 546 0.1

AmItgα1 AmItgβ1 34.6 α-‐AmItgβ1 α-‐Rabbit 546 0.0

AmItgα1-‐PS2 AmItgβ1-‐PS 35.1 α-‐AmItgβ1 α-‐Rabbit 546 0.0

pHSMCS 34.7 α-‐AmItgβ2 α-‐Rabbit 546 0.1

AmItgα1 AmItgβ2 31.0 α-‐AmItgβ2 α-‐Rabbit 546 0.1

AmItgα1-‐PS2 AmItgβ2-‐PS 31.9 α-‐AmItgβ2 α-‐Rabbit 546 0.1

AmItgα1-‐PS2 AmItgβ2L>D-‐PS 36.9 α-‐AmItgβ2 α-‐Rabbit 546 0.1

Table 6.2 Percentage of cells expressing detectable α and β integrins on the cell surface following transient transfection (as detected by subunit specific antibodies and anti-‐rabbit Alexa546 secondary antibodies). The Antibody column shows the specificity of the primary (top) and secondary (bottom) antibodies used for detection of integrins. Secondary antibodies were conjugated with the Alexa546 fluorophore. The percentage of GFP+ cells in each transfection indicates 30-‐35% of the total population were transfected effectively. Cells transfected with Drosophila integrins show integrin expression in 8-‐10% of the total cell population above empty vector controls. Integrin expression was not detected in cells transfected with coral integrins, suggesting primary anti-‐bodies against coral integrins did not bind to target epitopes.


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.


Antibody % of Total integrin+

% of Total Expressing integrin

pHSMCS -‐ α-‐HA 488 3.17 ItgαPS2 ItgβPS-‐HA -‐ α-‐HA 488 29.46 26.29 AmItgα1 AmItgβ1-‐HA -‐ α-‐HA 488 4.97 1.8


Antibody % of Total GFP+/integrin+

% of Total Expressing integrin

pHSMCS 19.13 α-‐MYC α-‐Rabbit 546

0.21

ItgαPS2 ItgβPS-‐MYC 35.01 α-‐MYC α-‐Rabbit 546 18.14 17.84

ItgαPS2 ItgβPS-‐MYC 34.15 α-‐Rabbit 546 0.27

AmItgα1 AmItgβ2-‐MYC 27.17 α-‐MYC α-‐Rabbit 546

1.23 1.02

AmItgα1 AmItgβ2-‐MYC 27.8 α-‐Rabbit 546 0.18

Table 6.3 Percentage of cells expressing epitope tagged β integrins on the cell surface following transient transfection (as detected by anti-‐HA or anti-‐MYC antibodies). The percentage of GFP+ cells in each transfection indicates 20-‐35% of each population were transfected effectively. Anti-‐HA antibodies were tagged with Alexa488, which possesses the same peak emission wavelength as GFP, as such cells transfected with HA tagged integrins were not co-‐transfected with GFP. The percentage of the total cell population expressing Drosophila integrins (26% for HA-‐tagged integrins and 18% for MYC tagged integrins) was much higher than that of coral (1.8% for HA tagged integrins and 1% for MYC tagged integrins), indicating that whilst expressed in a low percentage of the population, coral integrins were present on the cell surface.

Cell spreading of GFP+/AmItgβ+ cells The ~1% of cells expressing both GFP and surface β-‐Integrins spread poorly on all

substrates follow cell sorting. All samples exhibited between 20% and 25% dead or

damaged cells which is likely due to extended periods in divalent cation free Robb’s saline

during cell sorting and the physical stress of the sorting process. Only 70% of healthy

αPS2βPS expressing cells spread on Rbb-‐Tiggrin, 25% less than expected. Spreading of coral

integrin expressing cells was similarly poor with ~2% of healthy cells spreading in each

sample, indicating that the cell sorting processes had a substantial negative impact on cell

viability and spreading.


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6.4 Discussion The range of substrates that stimulate integrin mediated cellular behaviours such as

migration, differentiation and matrix deposition, is strongly influenced by the diversity of

integrin subunits expressed by the cell. These cellular behaviours govern a wide variety of

developmental processes. As such, understanding the diversity of integrins throughout the

Metazoa provides insight into the developmental roles, ancestral function and evolution of

the integrin family.

6.4.1 Novel Coral integrins may interact with 2 distinct ligand

types Examination of large protein datasets for Acropora, Nematostella, Hydra, and Clytia has

demonstrated that cnidarians possess similar numbers of α and β subunits to higher

invertebrates, which may appear somewhat surprising given the relative simplicity of the

cnidarian body plan. However, the relationship between morphological complexity and

genetic complexity is not linear (Ball et al., 2004) and phylogenetic analysis of integrins

provides another example of this disparity. Maximum-‐Likelihood analyses (Figures 6.1 and

6.2) show that cnidarian sequences form a phylum specific clade in both α and β integrin

phylogenies, indicating that integrin diversity among the Cnidarian integrin complement has

resulted from a number of independent duplications occurring after the divergence of

cnidarians from bilaterian evolution. Several lineage specific expansions have also occurred

in the Bilateria as evidenced by the existence of 3 clades of vertebrate β-‐Integrins and

grouping of α-‐subunits from each bilaterian phyla according to their ligand binding

specificity. Despite the clear evolutionary path of bilaterian integrins, a high degree of

primary sequence divergence and homoplasy effects have obscured the evolutionary

relationship between cnidarian and bilaterian sequences, leaving little clue as to the function

of basal cognates.

Inclusion of 2 novel coral sequences in the α-‐subunit phylogenetic analysis has

demonstrated that NvItga2 and AmItga3 form a clade only weakly associated with the other

cnidarian α-‐Integrins, suggesting they share a common origin independent of other

described cnidarian α-‐subunits. By contrast, the 2 coral β-‐subunits are closely associated

with each other and have a clear relationship to the integrins of Nematostella (2 β-‐Integrins

orthologous to each Acropora sequence), which could suggest 2 duplication events within

the cnidarian lineage or maintenance of 2 ancestral genes. The number of sequences in

Nematostella (2α & 4β) and Acropora (3α & 2β) combined with the phylogenetic

distributions suggest the ureumetazoan ancestor contained 2α integrins and 1 or 2β


103

sequences. As the ligand binding specificity of integrin receptors is largely dependant on the

α-‐subunit, the independent origin of the 2 cnidarian α-‐subunits clades implies the cnidarian

integrin complement facilitates interaction with 2 distinct ligand types. Given cnidarians are

considered informative as to the genetic complement of the Eumetazoan ancestor, this

animal is also likely to have possesed the capacity to bind 2 distinct ligand types. Whether

these ligands bare any relation to bilaterian ligands was the focus of the functional studies

presented here.

6.4.2 Integrins containing AmItgα1 interact with RGD tripeptide

ligands Preliminary investigation of ligand binding showed that none of the 5 αβ combinations of

coral integrins spread significantly on RGD or Laminin based substrates compared to cells

expressing Drosophila integrins. This result could have been observed for a number of

reasons including failure of transfection and failure of integrins to bind efficiently to

substrates resulting from sub-‐optimal activation or vastly different ligand specificity. Co-‐

transfection with constitutively expressed GFP demonstrated the transfection efficiency to

be consistently 30-‐35% of the total population, a figure which was confirmed by flow

cytometry and supported by the proportion of Drosophila ItgαPS2 expressing cells that

spread on RGD peptides. Poor cell spreading mediated by coral integrins is therefore not

likely due to failure of transfection.

The capacity for integrins to switch between low and high affinity conformations is highly

influenced by the availability of divalent cations, which interact directly with amino acid side

chains to facilitate activation (Takagi et al., 2002). The cell media (M3 media) used to carry

out previous investigations of integrin structure-‐function using the Drosophila S2 cell

system is specifically designed to mimic the extracellular fluid contacting Drosophila cells in

vivo. This media contains 18mM Mg2+ and 6.8mM Ca2+ and allows efficient activation of

Drosophila integrin receptors, however, the extracellular fluid encountered by coral

integrins is seawater, which is vastly different in composition and contains 50mM Mg2+ and

10mM Ca2+ in a common artificial preparation. Performing cell spreading experiments on

Rbb-‐Tiggrin using range cation concentrations and a simplified saline media allowed up to

8% of cells transfected with AmItgα1 + AmItgβ2 to spread on Rbb-‐Tiggrin. This figure was

7% above spreading of empty vector control cells and provided an indication that coral

integrin receptors containing the AmItgα1 subunit bind to RGD tripeptide ligands.


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In order for the observed spreading on RGD ligands to be attributed to interaction with coral

integrins, the presence of coral integrins on the cell surface had to be confirmed. The

complete lack of GFP+/integrin+ cells in all samples detected with coral specific antibodies

(Table 6.2) strongly suggests the antibodies do not bind to target epitopes. These polyclonal,

peptide derived antibodies have confirmed specificity for their target peptides (data not

shown), however appear unable to recognise the folded integrin structure despite the

location of the target epitope on an exposed surface of the protein structure in both high and

low affinity conformations. Detection of epitope tagged β-‐subunits using anti-‐HA or anti-‐

MYC antibodies was able to detect expression in 1% of the cells transfected with AmItgα1-‐

AmItgβ1-‐HA and AmItgα1-‐AmItgβ2-‐MYC constructs compared to 26% or 18% of cells

transfected with ItgαPS2-‐ItgβPS-‐HA/MYC respectively (Table 6.3). Although expression only

occurs in a small percentage of the total population, the detection of epitope tagged β-‐

subunits confirms that coral integrins were successfully expressed on cell surface.

The percentage of coral integrin transfected cells that spread on Rbb-‐Tiggrin (2-‐7%) was

higher than that of cells expressing detectable levels of epitope tagged integrins on the cell

surface (1%), which is consistent with control transfections using the same epitope (18%

surface expression, 30% spreading on Rbb-‐Tiggrin). The presence of a higher percentage of

cells spreading than exhibiting detectable surface expression in both coral integrin and

control transfected cells suggests that spreading in coral integrin transfected populations

was in fact mediated by integrin-‐ligand interaction.

Although only a small percentage of cells expressed coral integrins successfully, exploration

of the ligand binding specificity of AmItgα1 containing heterodimers has provided the first

indications that like bilaterians, cnidarians also possess integrins which demonstrate affinity

for RGD tripeptide containing ligands. RGD sequences are common in bilaterian extracellular

matrix proteins, occurring in a range of developmentally expressed proteins including

Fibronectin, Vitronectin, Osteopontin, Easter, Tiggrin Fibrinogen, Fibrillin, and

Thrombospondin (Barczyk et al., 2010; Humphries et al., 2006). Recent data has revealed

that many of these proteins known to act as integrin ligands are not present in cnidarians

(see chapter 4), which limits the range of conserved potential ligands. Cnidarians possess

multiple representatives of Fibrinogen, Fibrillin and Thrombospondin containing xGD

sequences that may facilitate integrin binding in place of RGD sequences. Such tolerance to

changes in the first position of the recognition motif has previously been demonstrated in

the case of Drosophila thrombospondin where a KGD sequence facilitates binding of αPS2

integrins (Bentley & Adams, 2010). Identifying which of the matrix proteins conserved in

cnidarians and higher animals are capable of mediating integrin adhesion requires


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identification of xGD motifs in a wider range of proteins and comprehensive functional

assays encompassing the recently identified AmItgα2 and AmItgα3 subunits.

6.5 Conclusions Whilst the number of α and β integrins present in some representatives of lower animals

has recently been acknowledged, the true complexity of the basal metazoan integrin

repertoire is only now coming to light. The relationship of integrin genes from 2 anthozoans

within the context of the broader metazoan complement has for the first time suggested that

integrins from outside the Bilateria also demonstrate affinity for 2 types of ligand. The

distinct ligand specificities found in anthozoans are likely to reflect the binding capacity of

integrins from the Ureumetazoan ancestor, although the depth of their origin in metazoan

evolution is unclear, requiring further data from the Porifera. Furthermore, the indication

that AmItgα1 specifically binds RGD tripeptide sequences, combined with the ubiquity of

xGD sequences in conserved matrix proteins, such as thrombospondin, implies that RGD

tripeptide proteins represent one of the two predicted ancestral integrin ligand types. The

developmental roles of the full range of cnidarian integrins is yet to be explored, however,

understanding the ligand specificity of one cnidarian α-‐subunit expressed during

gastrulation has provided a solid basis for future investigations into potentially conserved

roles for integrins in diploblastic and triploblastic development.

Chapter Seven General Discussion

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Chapter 7: General Discussion

7.1 Modes of gastrulation in cnidarians Cnidarians are considered the earliest branching (“lowest”) members of the animal kingdom

to display true gastrulation owing to their formation of a single gastric cavity (archenteron)

via ordered re-‐arrangement of cellular structure. Despite these re-‐arrangements resulting in

only two embryonic germ layers and a relatively simple body plan, morphological evidence

has revealed that all methods of gastrulation known from studies of triploblastic

development are also displayed within the phylum Cnidaria (Byrum and Martindale, 2004;

(Kraus & Technau, 2006). These methods include invagination, immigration/ingression,

delamination and epiboly as well as mixed modes of gastrulation (Figure 7.1 ).

Acropora millepora and Nematostella vectensis are the best studied examples of cnidarian

gastrulation in terms of morphology and genetic control. Both species are anthozoans, the

basal class of cnidarian (Figure 7.2) and have shared phylogeny to the level of sub-‐class

(Hexcorallia). As such, Acropora and Nematostella are considered closely related on an

evolutionary time-‐scale relative to classical models of comparative embryology (eg.

Drosophila, Xenopus, Gallus), and are ideally positioned for investigations of evolution and

development (Ball et al., 2002; Ball et al., 2004). However, in spite of their close evolutionary

relationship A.millepora and N.vectensis exhibit significant differences in gastrulation

strategy.


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Figure 7.1 Cnidarians exhibit a wide variety of modes of gastrulation including delamination, invagination, epiboly and ingression (a). The diversity of cnidarian modes of gastrulation encompass all observed bilaterian modes of gastrulation. Images of gastrulation in selected bilaterian models are shown (b-‐f). Mixed modes of gastrulation are common in bilaterians as demonstrated in X.Laevis, which combines epiboly of presumptive ectodermal cells with invagination of the presumptive mesoderm (e). Invagination and ingression are combined in amniotes such as chick (f) and human, where the primitive streak is formed by invagination of the presumptive ectoderm. As cell division drives ectodermal cells towards the primitive streak, they begin to lose epithelial character, with completion of the epithelial to mesenchymal transition marked by ingression / immigration into the blastocoel. Adapted from Byrum & Martindale, 2004 (a); Fujimoto et al., 2004 (b); Ball et al., 2004 (c); Wu & McClay, 2007 (d); Gilbert, 2003 (e); Wolpet et al., 2010 (f)


108

Figure 7.2 Anthozoans (eg. Acropora millepora and Nematostella vectensis) are accepted to be basal among the Cnidaria (Ball et al., 2004; Technau et al., 2005). Unlike other classes of cnidarians, anthozoans possess a circular mitochondrial genome, which is consistent with the mitochondrial genome of bilaterians (Bridge et al., 1992). The basal position of anthozoans is also supported by phylogenetic studies of rRNA sequences (Odorico & Miller, 1997). As the basal class, anthozoans are most likely to reflect developmental aspects of the Eumatezoan ancestor, making them useful comparators for investigating evolution and development. The diversity of anthozoan developmental strategies complicates this comparison, however, consideration of data from developmentally distinct anthozoans that are closely related on an evolutionary time scale, such as Nematostella vectensis and Acropora millepora, assists in resolving many potential ambiguities of anthozoan development.

Gastrulation in Nematostella vectensis primarily occurs by invagination, with limited

evidence for a coinciding immigration of a minor cell population (See Kraus and Technau,

2006; Magie and Martindale, 2007 for discussion). Prior to gastrulation, the 128 cell stage

embryo somewhat resembles a prawn-‐chip stage embryo of A.millepora (Figure 7.3; Figure

1.2), which develops further into a spherical pre-‐gastrulation blastula. At the

commencement of gastrulation, the blastula invaginates, creating a blastopore. As the

invagination deepens, cells of the presumptive endoderm migrate towards the aboral pole,

whilst the cells adjacent to the presumptive ectoderm maintain contact with the inner

surface of the presumptive ectoderm and exhibit a polarised morphology typical of

migrating cell types. At the conclusion of gastrulation, the presumptive endoderm is in

proximity to the interior of the presumptive ectoderm, separated by an acellular connective

tissue layer known as the mesoglea. By this stage, the blastopore has developed pharyngeal

structure derived of ectodermal tissue and is now considered an oral pore.


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Whereas invagination is common in the Bilateria (eg. fly, frog and sea urchin) and the

dominant mode of gastrulation in anthozoans (Kraus & Technau, 2006), the mode of

gastrulation exhibited by A.millepora is remarkable and peculiar even among cnidarians. In

contrast to Nematostella and other animals that form a spherical blastula prior to

gastrulation, cells of the A.millepora embryo are organised into a flat bilayer (prawn-‐chip

stage) prior to gastrulation that persists beyond the 128 cell stage. At the onset of

gastrulation, the cells thicken leaving a concavity on the side of the presumptive endoderm.

As the concavity deepens, the blastopore becomes apparent and eventually closes to make a

spherical bi-‐layer embryo (Hayward et al., 2004). Opening of the oral pore and elongation of

the body column then marks the commencement of the planula larva stage, which is

morphologically similar to that of other cnidarians in which the mesoglea is apparent

(Figure 7.3)

Figure 7.3 Gastrulation strategies of Acropora millepora and Nematostella vectensis are distinct despite a close evolutionary relationship. Gastrulation in Acropora (A) is preceded by formation of a flat cellular bilayer, which reduces in circumference and thickens at the onset of gastrulation. The edges then begin to fold upward producing a concavity on the side of the presumptive endoderm. As the concavity deepens (gastrula) the blastopore becomes apparent and eventually closes to make a sphere (Hayward et al., 2004). By contrast, Nematostella gastrulation (B) is preceded by blastula formation and occurs by involution of presumptive endoderm from one side of the blastula (Lee et al., 2007). Unlike Acropora, the blastopore does not close and becomes the larval oral pore. Involvement of a conserved set of developmental genes in both modes of gastrulation implies the existence of differences in gene expression and the involvement of downstream effectors of morphological change, such as cell adhesion molecules.


110

7.2 Genetic determinants of cnidarian gastrulation Most studies of cnidarian early development have focussed on assessing the expression of

transcription factors such as Snail, Twist, Brachyury, Otx, Pax, Sox genes and the Wnt system

(Hayward et al., 2004; Martindale et al., 2004; Momose & Schmid, 2006; Shinzato et al.,

2008; Smith et al., 1999). Transcription factors represent the classical targets of

developmental genetic investigation largely owing to the broad scale

developmental/morphological affects following expression of some transcription factors.

For example, Pax-6 (Eyeless) knockdown in Drosophila produces an eyeless phenotype and

over-‐expression results in ectopic eyes (Halder et al., 1995). Although the effects of other

transcription factors may be more subtle, the ability to influence the activity of whole gene

networks make transcription factors convenient targets for investigating the development of

atypical model systems such as cnidarians.

Comparative analyses between cnidarians and bilaterians into the diversity and expression

of transcription factors have identified two important points: (1) many transcription factors

involved in bilaterian gastrulation were already established at the time of cnidarian

divergence (although examples of specification and diversification are evident within

specific gene families) (Nichols, W. Dirks, Pearse, & King, 2006; Putnam et al., 2007;

Srivastava et al., 2010; Technau et al., 2005) and (2) many of the transcription factors that

function in bilaterian gastrulation also contribute to cnidarian gastrulation (de Jong et al.,

2006; Martindale, 2005; Martindale et al., 2004; Matus et al., 2006). Although these

principles are well evidenced, the diversity of gastrulation strategies observed among

cnidarians implies that genes responsible for cellular re-‐organisation during gastrulation are

dissimilar in expression and/or function between species -‐ even in closely related and

genetically similar species such as Acropora millepora and Nematostella vectenesis. This

complex relationship between genetic complement and morphological events is highlighted

by the substantial differences observed in the mode of gastrulation utilised by Acropora and

Nematostella (Figure 7.3). To further understand how networks of similar genes function to

produce vastly different phenotypes, investigations must consider not only the expression &

function of transcription factors, but also the function of downstream genes that facilitate

cellular behaviours such as division, apoptosis and migration.


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Cell adhesion molecules are the primary effectors of a number of cellular behaviours

including cell migration and cell & tissue polarisation, which are likely to contribute to re-‐

organisation in all modes of gastrulation. The most comprehensive survey into the diversity

of cnidarian adhesion molecules, presented in Chapter 4, revealed that cnidarians contain at

least 8 of the major adhesion genes demonstrated to function during bilaterian gastrulation

(Table 7.1). Members of the Cadherin and integrin families are of particular importance

among the genes identified as they have well defined roles in facilitating cell movements

during bilaterian gastrulation (Bökel & Brown, 2002; Halbleib & Nelson, 2006; Shimizu et al.,

2005; Yagi & Takeichi, 2000). The identification of cnidarian orthologues to so many critical

gastrulation genes supports the early evolution of not only the transcriptional genes

implicated in gastrulation, but also a wide variety of the terminal effectors, more than might

be expected from morphological comparison.

Although identifying the conserved adhesion proteins with potential roles in gastrulation is

an important first step, the simple presence of these genes in cnidarians provides little

insight into their function and significance to cnidarian gastrulation. In addition to protein

identification, the JCUSMART analysis also allowed the domain structure of proteins with

potential roles in cnidarian gastrulation to be assessed. Where complete models were

available, it was found that the domain structure of cnidarian adhesion proteins are

consistent with those of higher animal homologues, suggesting a similar capacity for protein

level interactions, particularly of regulatory mechanisms. This implies some degree of

functional similarity between cnidarian and bilaterian orthologous adhesion proteins (Table

7.1), which is further evidenced by motif conservation including the CCD & PCCD complex in

type-‐III cadherins (Chapter 5.4.1) and the MIDAS, ADMIDAS & NPxY/F motifs in integrins

(Knack et al., 2008).

In determining the influence of conserved cnidarian adhesion proteins on gastrulation, the

temporal and spatial patterns of gene expression must also be considered. In situ RNA

hybridisation of Acropora millepora embryos has demonstrated that integrins, planar cell

polarity implicated cadherins, and components of the Wnt system (eg. β-‐catenin) are all

expressed during gastrulation (Knack et al., 2008; Chapter 5). Whilst these genes are also

expressed during bilaterian gastrulation, several aspects of their expression in Acropora are

not consistent with bilaterian modes of gastrulation and allow a new model to be proposed

for adhesion protein function during gastrulation of Acropora millepora.


112

Gastrulation related adhesion Gene

Maximum Number of Models Identified

in cnidarians

Pathway Functions Molecular Function Reference

Flamingo (Starry Night / CELSR)

1 • Homophilic cell adhesion • Selective Recruitment of Fz and Vangl

Chen et al., 2008;

Usui et al., 1999; Wu & Mlodzik, 2009

Frizzled 4

• Receptor for Wnt Ligands • Recruits DSH to plasma membrane during PCP

• Binds cytoplasmic DSH • ligand binding of Vangl prior to cell-‐autonomous PCP

Wu & Mlodzik, 2008; Guo et al., 2004; Wang et al., 2006;

Van Gogh (Strabismus

) 1

• Planar cell polarity (Fmi-Fz group)

• Invagination • Convergent extension • Directional cell division

• Interaction with FAT-Ds group

• ligand binding of Fz prior to cell-‐autonomous PCP

Bastock et al., 2003; Wu & Mlodzik, 2008; Jessen et al.,

2002

FAT 3 • Heterophilic ligand binding of Ds

Seifert & Mlodzik, 2007

Dachsous 1

• Planar cell polarity (FAT-Ds group)

• Invagination • Convergent extension • Directional cell division Interaction with Fmi-Fz group

• Heterophilic ligand binding of Ft

Seifert & Mlodzik, 2007

Type –III cadherins 2-‐3

• Adherins junction component

• Invagination • Mobility of presumptive mesoderm

• Mesoderm specification

• Neural development

• Homophilic ligand interactions Stabilise tissue (DE/G Cadherin)

• Increase tissue mobility (DN-Cadherin)

• Binds β-catenin at plasma membrane

Wu & McClay, 2007; Byrum et al 2009; Iwai et al., 1997

α-‐Integrins 3

• Heterodimer formation with β-‐Integrins

• Primary influence over ligand binding

• Major Ligand types: RGD, Laminin, Collagen, Leukocyte specific

Oloumi et al., 2004; Schmidt & Friedl, 2010; Hynes et al.,

2002; Marsden and Burke, 1998

β-‐Integrins 4

• Cell motility • Mesoderm migration • Stable adhesion to basement membranes

• Extracellular matrix deposition

• Influence proliferation, differentiation & apoptosis

• Heterodimer formation with α-‐Integrins

• Cytoskeletal connections • Binds ILK

Oloumi et al., 2004; Schmidt & Friedl, 2010; Hynes et al.,

2002; Marsden and Burke, 1998

Table 7.1 Adhesion gene families demonstrated to function during bilaterian gastrulation are conserved in cnidarians. Examination of the cnidarian adhesome has revealed at least 8 of the gene families that have conserved function throughout the Bilateria are also present in cnidarians. These families include the cell adhesion components of the 2 planar cell polarity pathways (Fmi-Fz, Ft-Ds), integrins and Type-‐III cadherins. The high degree of conservation of protein architecture and functional motifs between cnidarian models and bilaterian proteins suggest that cnidarians are capable of a similar range of cellular re-‐arrangements such as convergent extension, directional cell division, planar cell polarity and integrin mediated motility. Involvement of these proteins during cnidarian gastrulation is likely, however the variety of cnidarian modes of gastrulation suggest these processes are demonstrated selectively across a range of cnidarian taxa.


113

7.3 Acropora millepora exhibits an inside-out mode of

gastrulation 7.3.1 The mobile presumptive ectoderm and un-coupling of β-

catenin from mesendoderm development

In most bilaterians, the regions of an embryo fated to become endoderm, mesoderm and

ectoderm are determined well before morphological changes of gastrulation have

commenced. Asymmetrical distribution of maternal mRNA, proteins, organelles and

cytoskeletal elements define the anterior-‐posterior (AP) axis prior to fertilisation and

subsequent signalling cascades then guide cell fate (Lee et al., 2007). Although the site of

gastrulation is variable among bilaterians with respect to the AP and DV axes it is consistently

influenced by a conserved network of signalling genes including β-catenin, bmp2/4/dpp,

Brachyury, Forkhead, Otx, Snail, and Twist (Martindale, 2005). Among these influential genes,

the role of β-catenin appears to be particularly well conserved with the accumulation of

nuclear β-catenin protein marking the site of cell migration and the future blastopore (Lee et

al., 2007; Logan et al., 1999; Range et al., 2005).

The influence of nuclear β-catenin accumulation appears to be conserved from prior to the

cnidarian-‐bilaterian divergence, as both cnidarians and bilaterians that gastrulate by

invagination exhibit a similar domain of nuclear β-catenin localisation prior to gastrulation.

Blastula stage embryos of Nematostella vectensis (Sea anemone) (Matus et al., 2006) and

Clytia hemispherica (Hydrozoan -‐ jelly fish) (Momose & Houliston, 2007) exhibit high levels of

nuclear β-catenin in the presumptive mesendoderm, at the site of gastrulation. This finding is

consistent with protein localisation analyses in bilaterians including Lytechinus variegates

which shows nuclear β-catenin in mesendodermal cells (Weitzel et al., 2004; Wu & McClay,

2007). Like β-‐catenin, the domain of Dishevelled (Dsh) expression is also indicative of the site

of mesendoderm formation in Nematostella and Lytechinus, as well as the scleractinian corals

Pocillopora meandrina, and Fungia scutaria (Marlow & Martindale, 2007). Inhibition of Dsh

expression is sufficient to inhibit the onset of gastrulation in both Nematostella and

Lytechinus embryos (Lee et al., 2007), as is β-catenin inhibition. The domains of expression

for Dsh and β-catenin overlap and the similarity in phenotypes produced upon knockdown are

explained by their cellular activity and protein level interactions.


114

In Acropora millepora however, the domain of β-catenin expression (as indicated by in situ

RNA hybridisation) and nuclear localisation of β-catenin (and Dishevelled) is restricted to the

presumptive ectoderm. This domain of expression is opposite to what might be expected from

investigation of blastula forming animals, where expression occurs in the presumptive

meso/endoderm. Furthermore, the expression centres around the pole furthest the site of

blastopore development. To understand the implications of these expression patterns, the

cellular function of β-catenin must be considered.

Translocation of β-catenin to the nucleus is a key component of the canonical Wnt signalling

pathway in which binding of soluble Wnt protein to Frizzled receptors results in decreased

ubiqutination of β-catenin (mediated by Dsh). Subsequently, free (unbound) β-catenin

accumulates in the cytoplasm and nuclear translocation occurs. In the nucleus β-catenin is an

obligatory co-‐factor to the TCF/LEF-‐1 transcription complex, which activates a network of

genes including the Snail family. The net result of this activation is down-‐regulation of

epithelial characteristics such as cell adhesion and proliferation, accompanied by a marked

increase in cell mobility, survival and matrix deposition consistent with a mesenchymal

phenotype. Restriction of β-catenin and Dsh expression to the presumptive ectoderm of

Acropora embryos is therefore suggestive of an increased mobility of the ectoderm rather

than meso/endoderm prior to gastrulation.

Up-‐regulation of the Snail family of transcription factors downstream of nuclear β-‐catenin-‐

TCF/LEF activation is conserved among bilaterians and is widely considered a necessary step

in the formation of the archenteron and mesenchymal development. In Acropora however, the

domain of β-catenin expression in the presumptive ectoderm (Shinzato unpublished; Chapter

5.3.3) opposes the expression Am_Snail (Hayward et al., 2004), demonstrating a clear

uncoupling of this conserved mechanism, which would otherwise be expected to play a major

role in facilitating the tissue mobility required for gastrulation. A similar uncoupling of

mesendoderm specification from archenteron formation has also been suggested in

Nematostella. In Nematostella, the domains of β-catenin and snail expression overlap,

however, Kumburegama et al (2011) have demonstrated embryos inhibited in β-‐catenin

signalling still undergo invagination but fail to express AmSnail and specify endoderm.

Whether such an uncoupling is common among the Cnidaria remains to be determined, as do

the factors that drive endoderm specification both up and down-‐stream of Snail expression.


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7.3.2 Maintaining stability in the presumptive endoderm In addition to its transcriptional role down-‐stream of canonical Wnt signalling, β-catenin is

also implicated in the regulation of stable cell adhesion and planar cell polarity. In chordates,

E-Cadherin is the predominant epithelial marker responsible for cell-‐cell adhesion and

maintaining the stability of tissues/ cell sheets through lateral homophilic interaction. An

analogous role is played by DE-Cadherin in Drosophila and G-‐Cadherin in sea urchin, despite

significantly varied structures. At the plasma membrane, β-catenin binds directly to the

cytoplasmic region of active catenin binding Cadherin (E/DE/G), linking it to cytoskeletal

Actin. The tissue stabilising activity of catenin binding cadherins is therefore linked to the β-

catenin balance. Additionally, nuclear translocation of β-catenin in response to canonical Wnt

signalling results in down-‐regulation of E-Cadherin expression via TCF and Snail mediated

repression, as well as protein level Cadherin degradation. Conversely, stabilisation of

adherens junctions (AJ ; the junction type where E-Cadherin is most prevalent) by over

expression of E-Cadherin is sufficient to inhibit β-catenin translocation and gastrulation.

The change in epithelial phenotype from stable epithelium to migratory (Epithelial to

mesenchymal transition) is also influenced by the expression of another catenin-‐binding

Cadherin, N-Cadherin. During gastrulation, down-‐regulation of E-Cadherin occurs

concurrently with up-‐regulation of N-Cadherin (DN-Cadherin in Drosophila), which is both

necessary & sufficient for producing a migratory cell phenotype. Expression of catenin

binding cadherins can therefore produce either stable or invasive phenotypes in chordates

and protostomes although an analogous Cadherin has not been identified in sea urchins.

Given the domain of nuclear β-catenin in Acropora prawnchip stage embryos, it would be

expected that a catenin-‐binding Cadherin functionally analogous to E/DE/G-‐Cadherin (ie.

Stabilises the epithelium) is expressed in the presumptive mesendoderm. This pattern would

be inverse to what is observed in bilaterians where E/DE/G-‐Cadherin expression is restricted

to the presumptive ectoderm, outside the nuclear β-catenin domain. Alternatively, a migratory

type catenin binding Cadherin functionally analogous to N-Cadherin would have an expected

expression restricted to the presumptive ectoderm. However, only a single catenin-‐binding

Cadherin, Am_ACadherin, was identified in Acropora (Chapter 4.3.1) and no mRNA expression

could be detected during gastrulation (Chapter 5.3.3). This result suggests the unique absence

of a tissue stabilising catenin binding Cadherin during Acropora gastrulation and raises the

question of how presumptive endoderm stability is maintained throughout morphogenesis.


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The Acropora integrins, AmItgα1, ItgβCN1, and AmItgβ2, are each expressed specifically in the

presumptive endoderm throughout gastrulation (Knack et al., 2008). In bilaterians, integrins

have been shown to interact with a range of cell surface and matrix proteins to produce both

stable interactions and cell motility. Integrin signalling through integrin Linked Kinase (ILK) is

also critical to matrix deposition. Acropora contains all of the genes required for integrin

signalling through ILK including ILK, PINCH, Parvin, and FAK (Chapter 4.3.2) and although the

precise mechanism is unknown, the production of the mesoglea, a thin connective tissue layer

between the ectoderm and endoderm, is apparent towards the end of gastrulation, suggesting

matrix accumulation occurs during gastrulation.

The prospect that Acropora integrins stabilise the endodermal tissue through interaction with

and deposition of extracellular matrix proteins in a positive feedback loop (as observed

higher animals) is therefore not unreasonable. Identifying which matrix proteins present in

the mesoglea are likely candidates for integrin interaction is an area for further study,

although this task is greatly aided by results in Chapter 6, which indicate AmItgα1 containing

integrin heterodimers are likely to interact with RGD type ligands.

7.3.3 Co-ordinating expansion of the presumptive ectoderm The folding of presumptive ectoderm around the presumptive endoderm, the hallmark

feature of Acropora millepora’s unique mode of gastrulation, is accompanied by 2 important

observations: 1) The cells of each germ layer continue to divide throughout gastrulation (as

detectable by examination with a light microscope), and 2) the ectodermal tissue maintains a

relatively constant depth of 1-‐2cells (Ball et al., unpublished). Together these observations

imply expansion of the ectodermal tissue occurs by a co-‐ordinated process that maintains a

single cohesive epithelial sheet.

Maintenance and expansion of an epithelium in a single plane commonly results from planar

cell polarity (PCP), in which the orientation of individual cells are directionally co-‐ordinated

(polarised) throughout a tissue. Cellular polarisation is characterised by asymmetrical

localisation of PCP adhesion proteins and associated regulatory proteins. These proteins

function in 2 parallel groups; the core group consisting of Frizzled (Fz), Dishevelled (Dsh), Van

Gogh (Vang; also known as Strabismus -‐Stbm), Prickle (Pk), Flamingo (Fmi), Diego (Dgo); and

the Dachsous group consisting of FAT (Ft), Dachsous (Ds) and Four-‐jointed (Fj). Both groups

are conserved throughout the Bilateria (with lineage specific expansion of some members),

however their distribution among the Cnidaria has not previously been investigated. In

Chapter 5.3.1, JCUSMART analysis allowed orthologues of all major planar cell polarity


117

components to be identified in cnidarians, with the exception of the cytoplasmic regulation

proteins Diego and Four-jointed.

In addition to local polarisation of individual cells, broad gradients of expression and protein

activity result in global polarity across the tissue. The Drosophila wing is among the classic

examples of both global and cellular polarity. Cellular polarity is detected by localisation of Fz-

Dsh-Dgo-Fmi to the distal plasma membrane and Vang-Pk-Fmi at the proximal plasma

membrane, whilst global polarity is demonstrated by clear gradients of Dachsous expression

(high proximal, low distal) and Four-jointed expression (low proximal and high distal) across

the wing. (Wu and Mlodzik, 2009).

Expression of Am_Daschous and Am_Van_Gogh in gradients across the presumptive ectoderm

of gastrulating Acropora embryos (Chapter 5.3.3) is consistent with the global expression

gradients observed in polarised epithelia such as the Drosophila wing (Dachsous) and mouse

phalangeal limb buds (Van Gogh; (Gao et al., 2011). The expression patterns of these Acropora

genes suggest PCP is involved in co-‐ordinating the expansion of the presumptive ectoderm

during gastrulation. PCP is commonly implicated during bilaterian gastrulation in facilitating

convergent extension (Shindo et al, 2008; Wang et al., 2006) and has recently been implicated

in Nematostella gastrulation, where expression of Vang at the animal pole is required for

archenteron invagination as demonstrated by morpholino knockdown (Kumburegama et al.,

2011). In both Acropora and Nematostella, Vang expression is restricted to the germ layer

exhibiting nuclear β-catenin localisation (the proposed mobile germ layer), which supports a

role for PCP in directing cell mobility across different modes of cnidarian gastrulation.

However, in contrast to Acropora where AmVangl is expressed in a gradient along a potential

second axis, Nv_Vang expression centres around the animal pole and forms a gradient along

the oral-‐aboral axis. This inconsistency in expression implies that whilst PCP is involved in the

morphogenic movements of both Acropora and Nematostella, mobility in the Acropora

presumptive ectoderm is not a simple translocation of the gene expression and cell

movements leading to archenteron invagination in blastula forming animals.

The precise role of planar cell polarity during Acropora gastrulation is not yet clear, although

one common function of PCP stands out as a favourable possibility. The prospect of

convergent extension (CE; the PCP mediated extension of a tissue along a single axis by

cellular intercalation) occurring in the presumptive ectoderm seems unlikely, as intercalation

of cells has not been reported. Furthermore, the region of PCP gene expression is quite broad,

encompassing the entire tissue, which is in contrast to expression patterns during bilaterian

CE events. The existence of a PCP in order to orientate auxiliary structures (eg. hairs, cilia) as


118

observed in both the Drosophila wing and Mouse cochlea, is another possibility, although cilia

are not evident on the ectodermal surface until after gastrulation in Acropora. Whilst

expression of PCP genes prior to gastrulation may aid in co-‐ordinating ciliary beat in later

development, the delay in assembly of cilia implies a more fundamental function for PCP in

early development.

PCP has been demonstrated to influence the axis of cell division in a range of model systems,

including Drosophila and Sea Urchin. In Drosophila wing development and during sea urchin

gastrulation, PCP gene activity restricts cell division to a single direction, allowing the cell

sheet to expand in a single plane. Restriction of division to a single plane is consistent with

the morphological observations of Acropora development, hence the expression of AmVangl

and AmDachsous throughout the presumptive ectoderm and across a potential second axis,

may function to maintain the direction of cell division. This rationale also offers a mechanism

for maintaining flexible intercellular connections in the proposed mobile germ layer, which

shows no sign of classical stabilising proteins such as N-Cadherin and integrin. Whilst an

active PCP directing the axis of cell division and maintaining the stability of the presumptive

ectoderm is at present the most attractive prospect, understanding of PCP in cnidarians is in

its infancy. Further investigations into the Acropora PCP system, its influence on the direction

of cell division and potential roles in cilia mediated motility are required. Focusing on protein

localisation and functional inhibition analyses would provide the most direct and informative

evidence into the presence and function of planar cell polarity during Acropora development.

Investigating these aspects in Nematostella and Clytia as well as Acropora would provide a

solid basis for drawing more general conclusions regarding the role of PCP in cnidarians.

The inside-‐out model of coral gastrulation presented here represents a consolidation of much

of the available molecular and morphological evidence obtained from Acropora millepora,

however, each aspect of the model holds potential for further investigation. Questions remain

as to the transcriptional targets of nuclear β-catenin in conjunction with TCF/LEF such as the

manner in which nuclear β-catenin influences the profile of adhesion molecule expression,

and the consequences of this relationship for ectodermal mobility. The uncoupling of nuclear

β-‐catenin signalling from Snail expression and mesendoderm specification also requires

exploration in Acropora and may offer a new perspective on mechanisms of tissue re-‐

organisation involving differential adhesion in cnidarians. Other questions surround the

purpose of Dachsous and Van Gogh expression in the presumptive ectoderm. Are other PCP

genes expressed? Are the patterns of protein localisation as expected for PCP or do they have

additional roles in the development basal cnidarians not seen in higher animals? The

influence of canonical and non-‐canonical Wnt signalling on β-catenin nuclearisation and


119

establishment of PCP also remains to be explored in Acropora. Investigating these aspects of

Acropora development require substantial methodological development to allow creation of

gene knockdowns, detailed exploration of sub-‐cellular protein localisation and profiling of

changes in gene expression during development and in response to genetic manipulation.

Chapter Eight General Conclusion

120

Chapter 8: General Conclusions

The importance of cell adhesion and dynamic changes in adhesive state, which are accepted to

be essential aspects of normal bilaterian development and function, have received little

attention in the context of cnidarian development. This project has addressed some of the

major deficits surrounding understanding of cnidarian adhesion systems, including the nature

of the cnidarian adhesion complement or “adhesome”, the involvement of cadherins in early

coral development, and the identification of the major integrin ligand type. Consideration of

these aspects of cnidarian adhesion along with previously published data has also facilitated a

new model of Acropora gastrulation to be proposed.

Investigating the nature of the cnidarian adhesome in a manner that would incorporate data

from multiple sources required development of a single platform for identification and

analysis of adhesion molecules. Chapter 3 details the development of such a platform, called

JCUMSART, which allows multiple large protein datasets to be assessed for protein models

with specific features. Although the analysis and data storage pipeline was originally designed

for identification of adhesion molecules, it has a demonstrated capacity for broader

application, being used for identification of selenium processing proteins as well as NOD and

Caspase related proteins.

The survey of cnidarian adhesomes (Chapter 4) encompassed data from 4 model cnidarians

(Acropora millepora, Hydra magnipapillata, Clytia hemispherica and Nematostella vectensis),

exhibiting different developmental strategies and life cycles. The differences between these

species allowed both an overview of the capacity of adhesion in cnidarians and comparison of

specific aspects of their adhesomes. A high degree of similarity between cnidarian and

bilatarian adhesomes suggested many of the adhesion components affecting developmental,

innate immune and defensive processes were already established in the ureumetazoan

ancestor. All 4 cnidarian species possessed an abundance of secreted pattern recognition

proteins with possible immune function, which is a clear advantage in an aquatic environment

where microbial transfer is easily facilitated.


121

Novelty among the cnidarians also centred around secreted pattern recognition proteins with

2 families (Collectin and Coll-‐IG) of PRR’s being identified for the first time in cnidarians. It is

unclear, whether the generalised expansion of pattern recognition receptors in the Cnidaria is

primarily associated with humoral immunity or symbiont uptake, which would hold

significant implications for studies assessing the genetic component of coral bleaching.

Type-‐III cadherins were among the proteins identified for the first time in cnidarians using

the JCUSMART. Cadherins with type-‐III structure include DE and G Cadherin which stabilise

embryonic germ layers during embryogenesis of invertebrates. An analogous role is played by

Type-‐I cadherins in vertebrates (eg. E-Cadherin). These proteins are of particular interest

during development due to conserved roles in β-catenin regulation, central to germ layer

specification. Sequence analysis shows that a single type-‐III Cadherin, Am_ACadherin, is

present in Acropora millepora, providing an opportunity to assess conservation of the

adhesion-‐germ layer specification mechanism in a model organism with a peculiar mode of

gastrulation (Chapter 5). Surprisingly, Am_ACadherin is not expressed during gastrulation of

Acropora millepora, demonstrating a significant departure from an otherwise conserved

specification system (Chapter 5.4.2). Another member of the Cadherin family, Dachsous, was

found to be expressed during coral gastrulation (Chapter 5.4.2), implying a role for planar cell

polarity in coral morphogenesis. This prospect is supported by the expression of another PCP

gene, Van Gogh, which is expressed in a similar pattern.

The cell adhesion survey also identified that the integrin pathway is strongly conserved in all

cnidarians considered. The complement of cnidarian α and β integrins is in alignment with

the complexity predicted by previous studies. In both Nematostella and Acropora, JCUSMART

allowed identification of novel α-‐Integrin subunits. Phylogenetic analysis demonstrates that

these sequences are divergent with respect to other coral integrins. The α-‐Integrin is accepted

to be the major contributor to integrin heterodimer ligand specificity and as such, the

divergence of the novel α subunits suggests cnidarians are capable of binding two distinct

ligands (Chapter 6.4.1). Ligand binding experiments conducted by transgenic expression of

coral integrins in a drosophila S2 cell system suggested that one of the ligands is an RGD

sequence (Chapter 6.4.2). The RGD sequence is common among extracellular matrix proteins

and the evidence provided here provides a solid basis for future investigation into the role of

integrins during coral development though manipulation of matrix proteins.


122

Consideration of results from each chapter of the project allowed a novel model of Acropora

gastrulation to be developed (Chapter 7.3). This model is inside-‐out with respect to blastula

forming animals, largely owing to the expression of β-catenin in the presumptive ectoderm,

rather than presumptive endoderm. The model suggests an increased mobility of the

presumptive ectoderm with respect to the presumptive endoderm. In the absence of a tissue

stabilising Type-‐III Cadherin, the presumptive endoderm is proposed to be stabilised through

integrin mediated interaction with and lay-‐down of extra-‐cellular matrix proteins. These

proteins, which are likely to possess accessible RGD sequences, ultimately form a component

of the mesoglea. Mobile interactions between cells in the presumptive ectoderm are proposed

to be facilitated by components of the planar cell polarity pathway, which may also act to co-‐

ordinate the direction of ectodermal cell division as the presumptive ectoderm folds around

the presumptive endoderm. Whilst each aspect of this model warrants future investigation,

this is the first such model to be proposed for what is a distinctive and unique mode of

gastrulation, which may hold broader implications for our understanding of endoderm

specification and the roles of cell adhesion in cnidarians.

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Appendix A Supplementary Material

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Appendix A: Supplementary Material

Chapter 4 Supplementary Figure 4.1

Protein Family HMMPFAM

Search Terms

BLAST

Search Terms

Cadherin 1. Cad 2. cad NOT dict NOT scad 3. CA NOT EGF NOT egf

NOT CAP NOT CAD_1 NOT CARD NOT ICAR NOT Dicty NOT ICAT NOT CAS NOT Pro_CA NOT SCAMP NOT SCA7

4. Protocad 5. cadg

6. Cadherin 7. Cadherin 8. Protocad 9. protocad 10. ProtoCad 11. Proto-cad 12. Proto-Cad 13. Proto-CAD 14. Dachsous 15. FAT NOT Fatty 16. Desmoglein 17. Desmocollin 18. CELR 19. CELSR 20. Selectin 21. selectin 22. falmingo 23. Flamingo 24. Calsyntenin 25. Calsyntenin 26. Starry 27. starry 28. E_value < 1E-25

integrin 29. ILK 30. ntegrin 31. INB 32. PSI 33. psinew 34. Talin 35. Talin 36. ICAP-1 37. FG-GAP 38. fg-gap 39. DISIN 40. LWEQ (Talin

diagnostic) 41. Int_alpha 42. int_alpha 43. ADAM

44. ntegrin 45. isintegrin 46. Talin 47. Talin 48. ntergrin AND inked 49. E_value < 1E-15

lectin 50. lectin 51. lectin 52. PA-I 53. Intimin 54. intimin 55. APT

68. CELIII 69. ndosialin (Endosialin) 70. lectin 71. lectin 72. c-type 73. Attractin


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56. Agglutinin 57. LECT 58. Knot1 59. PLEC 60. TECPR 61. ftp 62. ricin 63. Ricin 64. CRD 65. F5_F8 66. blect 67. B_lectin

74. attractin 75. Polycystin 76. polycystin 77. PKD 78. Pkd

Adhesion LRR 79. LRR 80. LRR, 7tm_1

Class B Adhesion GPCR 81. 7tm_2 82. Frizzled

83. Frizzled 84. Frizzled 85. FRIZZLED 86. Smoothened 87. smoothened 88. SMOOTHENED 89. E_value < 1E-10

Immunoglobulin

Superfamily

90. titin 91. -set 92. ig NOT Lig NOT signal

NOT align NOT lig 93. Ig 94. IG NOT PIG NOT AIG 95. Adhes 96. adhes 97. CAM NOT SCAMP

NOT calmodulin NOT Calmodulin

98. cell AND adhesion 99. CAM NOT |CAM (ie.

"pipe"CAM - this occurs in some identifiers) NOT almodul

100. cam NOT camp NOT came

101. mmunoglobulin 102. robo 103. Robo 104. oundabout 105. Neogenin 106. neogenin 107. DCC 108. Netrin AND eceptor 109. olorectal AND

cancer 110. ontactin 111. BOC 112. rother AND of 113. CDO 114. ysteine AND

ioxygen 115. pinin 116. Pinin 117. 1E-20 118. Toll NOT olloid AND

recept 119. toll NOT olloid AND

recept 120. 1E-10

Extracellular Matrix 121. LY NOT zf-LYAR 162. ollagen


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NOT LYR NOT CAP_GLY - LDL RECEPTOR

122. NtA - AGRIN 123. Reeler 124. reeler 125. Reelin 126. Reelin 127. NIDO 128. REJ 129. PLAT / LH2 130. MANEC 131. manec 132. FAP 133. fn3 134. FN 135. LamN 136. aminin_N 137. aminin_I 138. collagen 139. Collagen 140. C4 NOT HC4 NOT

C48 NOT PC4 NOT -C4 141. fn1 142. TB NOT PTB NOT

BTB NOT TBP NOT TBC NOT TB2 NOT PNTB NOT TBPIP

143. Fib_ 144. fib_ 145. fibrinogen 146. FBG 147. G2F 148. COLFI 149. C8 NOT PLAC8 150. steopontin 151. OSTEO 152. LINK 153. COLFI 154. ECM1 155. Col_cut 156. FReD 157. FRED 158. fred 159. Fred 160. TSP 161. tsp

163. aminin 164. ibulin 165. ibrillin 166. ibrin 167. nectin 168. steopontin 169. enascin 170. yaluron 171. xtracellular AND

atrix 172. thrombospondin

Other 173. Dicty (Dicty_CAD) 174. dicty 175. C4 NOT HC4 NOT

DC4 NOT C4d 176. CD36 177. Spondin 178. AMOP 179. VWA

194. ZO1 195. mucin binding 196. collagen binding 197. osteonectin 198. fibrillin binding 199. notch 200. Titan 201. Nidogen


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180. Sushi 181. notch 182. zu 183. MucBP 184. Collagen_bind 185. fn_bind 186. FBP 187. FAP 188. Fb_signal 189. amelogenin 190. ZP 191. FOLN 192. Kazal 193. LamNT

202. Usherin 203. Tensin 204. Slit 205. CSMD 206. Sidekick 207. sema 208. NL 209. cell AND adhesion 210. Cell AND adhesion 211. cell AND Adhesion 212. Cell AND Adheison 213. CAM NOT |CAM

NOT almodulin 214. cam NOT camp

NOT came 215. mmunoglobin 216. Robo 217. robo 218. oundabout 219. eogenin 220. DCC 221. Netrin AND receptor 222. olorectal AND

cancer ontactin 223. BOC 224. rother AND of 225. CDO 226. ysteine AND

ioxygen 227. pinin 228. Pinin

Supplementary Figure 4.1 Terms used to identify adhesion molecules during searches of JCUSMART annotation of Nematostella, Acropora, Clytia and Hydra datasets. Independent searches and search terms are used for HMMPFAM and BLAST. Boolean search terms were used in the following hierarchy: “OR” > “AND” > “NOT”. Each bullet point in the Figure constitutes an “OR” statement. E-‐value limits (where present) apply to all terms in the search statement.


139

Supplementary Figure 4.2


140

Supplementary Figure 4.2 Multiple sequence alignment of Talin proteins from representative metazoans used for maximum likelihood analysis (Figure 4.1). Proteins alignment only includes the region of overlap between Clytia talin1 and Clytia talin2. Alignment is coloured by percentage identity between all sequences included in the alignment.

Genbank Accessions: Human_Talin1 NP_006280.3; Human_Talin2 NP_055874.2; Ciona_Talin (Blom, Gammeltoft, & S Brunak, 1999); Urchin_Talin XP_001193631.1; Danio_Talin1 NP_001009560.1;Danio_Talin2 NP_957487.2; Drosophila_Rhea NP_648238.1; Apis_Rhea XP_391944.4


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Supplementary Figure 4.3 Multiple sequence alignment of metazoan haemolytic lectins used for maximum likelihood analysis (Figure 4.2). Alignment is coloured by percentage identity between all sequences included in the alignment. Acropora sequences are labelled according to their EST of origin (Grasso et al., 2008) and Clytia sequences correspond to Contigs IL0ABA2YE22RM1 (1) and IL0ABA8YL09RM1 (2).

Genbank Accessions: CEL-III BAC75827.1


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Supplementary Figure 4.4 In situ hybridization of haemolytic lectin A036-‐E7 from Acropora millepora. Expression commences in planula stage larvae and continues into adult life as demonstrated by cell specific staining (A & B). In larvae (A) and polyp stages (B) haemolytic lectin is expressed in the oral half of the animal, which is exposed to the environment after settlement. Cell specific staining suggests expression is restricted to nematoblasts, the precursor cells to nemtaocysts. In situ hybridization of sectioned adult tissue (C) demonstrates expression in cells basal to nematocysts, which hold a similar shape to nematocysts and are also expected to be nematoblasts. Expression of haemolytic lectin in nematoblasts, suggests a role in cellular defenses in coral. Image C courtesy of T..Ainsworth (unpublished).


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Supplementary Figure 4.5 Am_LRIG3 1 ------MPRSNMASVNIVTAWIVMCLVLFG-------------AALR-KDPNAKACAFAK Nv_LRIG3 1 -----------MASWFSTFSWLVLYIWILC-------------VFVSSQQTEGTACLEIK A_mel_LRIG3 1 MSNTEQIFFDHRFVWKYKWAIVILPILFLT--------SSNFIKANKISYGNIKDNQCPV Dr_LRIG3 1 ------------MPQPARAVSSVLLLLLLS----------GFTAVTGDSRRT-EHEPCPS Xl_LRIG3 1 -----------MCTMPLSWRRIIYLLLLFT------PLVLGFKSLERRVRRD--RGPCPS Mm_LRIG3 1 -----------MGAPGLRAATAALGLLLCAGLGRAGPAGSGGHGAPGQLLDDDAQRPCPA Am_LRIG3 41 PCICFGTKVDCKNRSLRMIPEGIPRNTTHLDLSVNQLTTLDMTKLSRLTKLRVLL----- Nv_LRIG3 37 PCYCLGNRVNCEGKHLTAIPVGIPSDITALNLGYNNIVDLDPKQLLKFTALKELFNPIPG A_mel_LRIG3 53 ECDCLGNVVDCINLQLIGAPSGLPPWTEILGLKGNNIASLEPDVLLHLTKLKELDLSGNK Dr_LRIG3 38 VCSCTGNLADCSRLKAGRTVERLPARITRLDLSHNKLRVFPEALFSSLPQLSEIKLSNN- Xl_LRIG3 42 PCRCLGDLLDCSRRKLTVVPSNLPEWLVQLDLSHNKLSSIKASSMNHLHNLRELRLNNN- Mm_LRIG3 50 ACHCLGDLLDCSRRRLVRLPDPLPAWVTRLDLSHNRLSFIQTSSLSHLQSLQEVKLNNN- Am_LRIG3 96 ------------------------------------------LHKNQLREVPPWQALPSS Nv_LRIG3 97 SFNPIPGSFNPIPGGFNPIPGGFNPILVASIPFLVASILSCHLHHNNIRGIPPWDSFPST A_mel_LRIG3 113 FGDDFKIILSEGTHLQMLKVNKNQLTQVPDMFFVKNITHLALAHNSITDINGTALLNLQR Dr_LRIG3 97 ---EFESIPDLGP-------------------NAGNLSSLILASNRIGRVSSERLSPLLT Xl_LRIG3 101 ---ELQIIPDLGP-------------------LSANITLFSLTNNKIEVILPEHLTPYQS Mm_LRIG3 109 ---ELETIPNLGS-------------------ISANIRQLSLAGNAIDKILPEQLEAFQS Am_LRIG3 114 LQRLWLHQNLISVVPSQRLTKKMSLAMLSLNNNNISVIEPYAFANLTSIWSL-------- Nv_LRIG3 157 LMTLTLHHNKIVAIPSLNTTKQHALRNLYLNSNKISSIGHHAFTNLSNLQNLYSIAYLRY A_mel_LRIG3 173 LQNLDLSXNKISVIRNGSFLAPNCLHNRNLNKNQIKVIENGSLDNLTS-LEELRLN---- Dr_LRIG3 135 LETLDLSNNNIVDVYAGAFPPIP-LKNLFMNNNRISTLEHGCFSNLSSSLLVLKLN---- Xl_LRIG3 139 LETLDLSNNLLAELKAGSFPTLQ-LKYLYINNNRISTMQSGAFDNLSATLQVLTLN---- Mm_LRIG3 147 LETLDLSNNNISELRT-AFPPLQ-LKYLYINNNRVSSMEPGYFDNLASTLLVLKLN---- Am_LRIG3 166 -------------KLGRNKLEAIPVAALSQVTGLEILDLTKNSIREIRVRLSHSIKSVIK Nv_LRIG3 217 TSLTEYKKEDCIKKLGKNRLTSVPSDALSQLQSLKRLDLSRNFFTSILASAFNRLSSLEV A_mel_LRIG3 228 ----------------KNYLTQLK-DLFTNLKKLRILEINRNELQTIQGLSLRGLKNLKE Dr_LRIG3 190 ----------------KNRLNSIP-AKIFSLPHLQHLELSRNRLRRVEGLTFQGLHGLRS Xl_LRIG3 194 ----------------KNRISHIP-SKMFKLSNLQHLELNRNRIKEILGLTFQGLDSLKS Mm_LRIG3 201 ----------------RNRISAIP-PKMFKLPQLQHLELNRNKIKNVDGLTFQGLGALKS Am_LRIG3 213 LKLSKNKIYNISAFAFWEMEKMQELHLDNNNLTSISKTWFFGVPMLRILNFDNNRIRVIE Nv_LRIG3 277 LKLSKNRISTIRG-AFWGQNKLQQLYLDRNNFTAIQTSSFFGLRDLQNLYLQSNQISTII A_mel_LRIG3 271 LHLKKNKIETLDDGAFWPLENLTILELDFNLLTMVRKGGLFGLEHLQKLTLSHNRIRTIE Dr_LRIG3 233 LKMQRNGISRLMDGAFWGLNNMEVLQLEFNNLTEVSKGWLYGLLTLQQLHLSHNSISRIK Xl_LRIG3 237 LRIQRNSIARLMDGAFWGLSTMEVLQLDHNRLTEITKGWLYGLLMLQKLHLSQNAISSIS Mm_LRIG3 244 LKMQRNGVTKLMDGAFWGLSNMEVLQLDHNNLTEITKGWLYGLLMLRELHLSQNAINRIS Am_LRIG3 273 ESKMPGVNWKLAFRLQILILANNEFTHIYSTTFAHLKYLRRLILNTNRIHYLADEAFSDL Nv_LRIG3 336 TTKFA--DWRSFPVLRKLNLERNRLSHIQDTTFKHLLALKVLNLANNRIYHISQGSFSDL A_mel_LRIG3 331 -----IQAWDRCKEIIELDLSYNEISTIERDTFEFLEKLKKLKLDHNQITYIADGAFSST Dr_LRIG3 293 -----PDAWEFCQKLAELDLSWNQLSRLEEGSFVGLSVLEQLHIGNNRISFIADGAFRGL Xl_LRIG3 297 -----PDAWEFCQKLSELDVSFNQLTRLEESSFGGLGLLSGLHIGNNKINFIADGAFRGL Mm_LRIG3 304 -----PDAWEFCQKLSELDLTFNHLSRLDDSSFLGLSLLNALHIGNNKVSYIADCAFRGL Am_LRIG3 333 LSLQTLDLRYNALPSTALEG-GVFANLNSVWLVRLDGNRITRISASSFHGLTSAISMNLS Nv_LRIG3 394 RSLEGLDLSNNDISWTVEEMNGPFRGLTNLASLRLDGNRITAIAATAFLGLENIKYLNLS A_mel_LRIG3 386 PNLQILELKFNKISYMVEDINGAFDPLGQLWKLGLAHNRIKSINKNAFTGLSNVTELDLS Dr_LRIG3 348 TNLQTLDLKFNEISWTIEDMNGPFSALDNLRKLFLQGNRIRSVTRKSFTGLEMLEQLDLS Xl_LRIG3 352 SSLNSLDLKSNDISWTIEDMNGTFSGLERLQRLTLQDNRITSITKKAFSWLDALEYLDLS Mm_LRIG3 359 TSLKTLDLRNNEISWTIEDMSGAFSGLDRLRQLILQGNRIRSITKKAFAGLDTLEHLDLS Am_LRIG3 392 ANAISSIDRFAFYEMTSLQYLYFDTEKLICNCEIKWLPTWLREK-NIQDDVRGLCSYPEN Nv_LRIG3 454 ANIITSIQENSFQGMDKLQKLWLNTSQLMCDCKIKWFGSWLRSRPSTRHTVRARCLHPQT A_mel_LRIG3 446 GNNITSIQENAFVSMTRLTKLRMNSSVLVCDCGLQWLSMWLREH-SYTD-AEVYCGFPHW Dr_LRIG3 408 NNAIMSLQANAFSQMKKLSELHLNTSSLLCDCQLKWFSLWVAEQ-AFLALLNASCAHPHL Xl_LRIG3 412 DNAITSMQTNAFSQMKSLQQLYLNTTSLLCDCQLKWLPKWLAEN-NFQTFVNASCGHPQI Mm_LRIG3 419 GNAIMSLQSNAFSQMKKLQQLHLNTSSLLCDCQLRWLPQWVAEN-NFQSFVNASCAHPQL


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Am_LRIG3 451 LAGRSILNISLSNLECDNSGSSPEVIKHPQGKKVLLGQNVTLRC---VVSWKNYSITVNW Nv_LRIG3 514 LYMKSVFNITPDAFVCDTSNPIPQISVHPPSQKAMRGDNVTLRCRVQVVQRNSSSVRLQW A_mel_LRIG3 504 LQGMSLTQLHHKNFTCDEYPK-PRIIEEPKSQMGIKGDNVTLVCR--ATSTAIARLHFTW Dr_LRIG3 467 LKGRSVFSIAQDEFVCDDFPK-PQITVQPETQSAIKGNNVTFVCS--AASSSDSPMTFAW Xl_LRIG3 471 LKGKIIFAVSPDDFVCDDFPK-PQITVQPETQSAIKGSNVTFICS--AASSSESPMTFAW Mm_LRIG3 478 LKGRSIFTVSPDGFVCDDFPK-PQITVQPETQSAIKGSDVSFTCS--AASSSDSPMTFAW Am_LRIG3 508 TKNGRPLKGAHLKIHTQSKDDY-----RGAYTSELHLRSVSRRDSGKYQCVAYSTEFVPV Nv_LRIG3 574 RLGYEVISERDITDFVRTDDDD-----IVTYNSDLFLISVDFSDAGLYQCIASN-KYGSQ A_mel_LRIG3 561 KHDNIEINDDNLQINTDSTENG-----VTEATSILHLTNVTHANAGKYQCMVTN-TYGTT Dr_LRIG3 524 KKDNELLSEPEIQNQAHVRAAAGETGELTEYTTTLQLRQVEFTSEGRYQCVISN-HFGSS Xl_LRIG3 528 KKDNELLHDSEIENFAHLRAQG---GDVMEYTTILRLRNVEFINEGKFQCVISN-HFGPT Mm_LRIG3 535 KKDNEALQDAEMENYAHLRAQG---GELMEYTTILRLRNVEFTSEGKYQCVISN-HFGSS Am_LRIG3 563 RSHWAQLSVLGFPKLTQTPTDVLVEPGKTFKLYCKAQGHPMPTLLWQKDGGRS-FPAADD Nv_LRIG3 628 FSKKARLEVLEFPVLTRKPHDVSVHVGGLIKLPCAATGYPVPVISWRMGDGKSKFPAAEE A_mel_LRIG3 615 YSAKAKLSILIYPSFSKIPHDIRVIAGSTARLECSAEGQPSPQIAWQKDGGND-FPAARE Dr_LRIG3 583 YSNKAKLTVNMLPFFTKTPMDLTIRAGATARLECAASGHPSPQIAWQKDGGTD-FPAARE Xl_LRIG3 584 YSVKAKLTVNMLPLFTKKPMDLTIRAGSTARLECAAVGHPTPQIAWQKNGGTD-FPAARE Mm_LRIG3 591 YSVKAKLTINMLPSFTKTPMDLTIRAGAMARLECAAVGHPAPQIAWQKDGGTD-FPAARE Am_LRIG3 622 RRIKYAEGLEVCEIRNAQYKDSGKYTCFAKNVAGSANASATVTVLEPPGFTGPWRKKVTV Nv_LRIG3 688 KRIEHLPSEHLFIIRNARGVDTGAYTCTATNGAGSINATAYVTVLEVPRFMQSMVSKR-V A_mel_LRIG3 674 RRMHMMPTDDVLFIVDVKTADSGVYSCTAQNLAGLIVANATLTILETPSFVKPMENKE-V Dr_LRIG3 642 RRMHVMPRDDVFFIVDVKTEDIGVYSCTAQNTAGAISANATLTVLETPSFLRPLLDRA-V Xl_LRIG3 643 RRMHVMPEDDVFFIVNVKTEDIGVYSCTAQNSAGSISANATLTVLETPSFLRPLMDRT-A Mm_LRIG3 650 RRMHVMPEDDVFFIVDVKIEDIGVYSCTAQNSAGSVSANATLTVLETPSFLRPLLDRT-V Am_LRIG3 682 SAGDSLVLECYVRGAPQPLVIWFKDGIKIQMNDRVVLTESRQLLVITKATDDDGGRYDCE Nv_LRIG3 747 RTGESAVLECKASGSPMPRFTWYKDDKKVTLSARVVAHG--QLLVFVTVLRDDEGTYTCQ A_mel_LRIG3 733 TVGGSIVLECMASGMPRPKLSWRKNGNPLLATERHFFTAEDQLLIIVDTRISDAGSYECE Dr_LRIG3 701 AKGETAVLQCIAGGSPPPRLNWTKDDSPLQATERHFFAAGNQLLIIVDAAEGDAGTYTCE Xl_LRIG3 702 SKGETTVLQCIVGGSPTPRVNWTKDDSPLVVTERHFFAAGNQHLIIVDTDLEDAGIYTCE Mm_LRIG3 709 TKGETAVLQCIAGGSPPPRLNWTKDDSPLVVTERHFFAAGNQLLIIVDSDVSDAGKYTCE Am_LRIG3 742 VANSQGNDTR-TMMVAIEPEKCTGVV----KKSNSNNSNYVKYDKKTFLGIIVVVVVACI Nv_LRIG3 805 VSNSLGTARQNTRLTVVEGSELQTCQ----DKES-------RYDKKTFLGIIVISVVTCV A_mel_LRIG3 793 MSNSLGSVVGASHLTVKPAPISTP-----------SPGSVNEDD---ILGLIIITVVCCA Dr_LRIG3 761 MSNPLGTERGNLRLSVLPNPNCDQGPAGGGAVGAAGSGRGPEDDGWTTVGIVIIAVVCCV Xl_LRIG3 762 VSNILGTERGNIHLTVLPNPTCDS---------PVNAIQTAEDDGWATAGIVIIAVVCCV Mm_LRIG3 769 MSNTLGTERGNVRLSVIPTPTCDS---------PHMTAPSLDGDGWATVGVVIIAVVCCV Am_LRIG3 797 VVTSMVWLFIQYNTLSCGKRRAPRRSLHTGPFGVDYSDESNSKALN-QEISYIPLKLSSS Nv_LRIG3 854 VGTSLVWLLVIYCARRGSHRR--RHKLRARPFQADGTDTTNSKLTHRDELSYIPLKSSSS A_mel_LRIG3 839 VGTSIVWVVIIYQTRRR-----LNNVAQGRPHAQPTPTLT--GTVADTQ----------- Dr_LRIG3 821 VGTSLVWVVIIYHTRRRNEDCSVTNTDETNLPADIPSYLSSQGTLAERQDGYMLPSESGS Xl_LRIG3 813 VGTSLVWVVIIYHTRRKNEDCSVTNTDETNLPVDTPSYLSSQGTLAERQDGYGS-SETGS Mm_LRIG3 820 VGTSLVWVVIIYHTRRRNEDCSITNTDETNLPADIPSYLSSQGTLADRQDGYIS-SESGS Am_LRIG3 856 SSTQTSRESPRSTATFLTASEAAQGRCAVATLAGCSSENASGSEEKSSGNDNISSENSLK Nv_LRIG3 912 GSTQTSRDSPRSTATFLASSDSHG----AAFPVSASIDTHSGSE-KTTG--NISSENSLK A_mel_LRIG3 881 THIYLETS------------SQHSKDSGTGDSTNPSSDQLQLCLP--------------- Dr_LRIG3 881 SHQFISSSIGGFYMPPKDMNSLCQLDTGSEADLEAAIDPLLCHYQGPVGSLLTPGAHYSA Xl_LRIG3 872 -HQFIASSMSGYFLQQRD-NGACNLDNGSEADLEVATDPLLFNYTGVPGPLYLRGNPYDP Mm_LRIG3 879 HHQFVTSSGGGFFLPQHDGAGTCHFDDSSEADVEAASDPFLCPFVGSTGPVYLQGNLYSP Am_LRIG3 916 ESRSSLASFCRSESSLPCCQEVVTSAQVHGSDSDSEKSRPTLAMFARNTS---------- Nv_LRIG3 965 GSHCSLTSSCPSVESEPVRQVVH--VQIHSSDSDSEKCKETSPTRRKETANL-------- A_mel_LRIG3 914 -----------------------------EEIVTCSVNNE-------------------- Dr_LRIG3 941 ELPDTYTVCVSEPR------LLSDSYSRKRDFYTCSSSLDPCDHMMLPHDIP-------- Xl_LRIG3 930 DAYEIFHAGYSMDRRTPNANFYESEYLKQKELGLFGHQHDDCYKIACSHGVQSLAGRIVG Mm_LRIG3 939 DPFEVYLPGCSSDPRTALMDHCESSYVKQDRFSCARPSEEPCERSLKSIPWP-HSRKLTD Am_LRIG3 966 --------------------------CSD---HDPGEHKLTFPDCIPYNKKTCTDHEKFS Nv_LRIG3 1015 -------------------------ACSQSDCEDASEHCGSAAQHCGTNAGYCGIPSNHC A_mel_LRIG3 925 --------------------------EEPSAVVNVGAPLLRYTNHERIVHENKDCAV--- Dr_LRIG3 987 -------------SCVDD------CETDQCVLPRSGSYMGTFGKAAWRPTQDHSAVILHE Xl_LRIG3 990 PTCSHKEDIEIK-MSLDTDILGLKHTVDQGILTSCSTYLGTFGKPVWRPQLDSPCGYVQP Mm_LRIG3 998 STYPPNEGHTVQTLCLNKSSVDFSTGPEPGSATSSNSFMGTFGKPLRRPHLDAFSSSAQP


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Am_LRIG3 997 SK--DVTPDVCVTFYPPSEKETSCDTDVVESTLILT------------------------ Nv_LRIG3 1050 GTPLETSGTSSGHYRTFRGLGPEYDSNDVGSVLIKSKEMIVRTPSSEHFSTTHV------ A_mel_LRIG3 ------------------------------------------------------------ Dr_LRIG3 1028 N-----------------PYAALHAEEELTHTPSHTHSSVYEQPFDSRTIDSNPTTPLGS Xl_LRIG3 1049 SFSQLT-SHTIPQTKLNMQLENGKDESQRTNIPDENATFFPKITSDYQRTSGFQCYELDT Mm_LRIG3 1058 PDCQPRPCHGKSLSSPELDSESEENDKERTDFREENHRCTYQQIFHTYRTPDCQPCDSDT Am_LRIG3 1031 SK--DVTPDVCVTFYPPSEKETSCDTDVVESTLILT------------------------ Nv_LRIG3 1104 GTPLETSGTSSGHYRTFRGLGPEYDSNDVGSVLIKSKEMIVRTPSSEHFSTTHV------ A_mel_LRIG3 ------------------------------------------------------------ Dr_LRIG3 1071 N-----------------PYAALHAEEELTHTPSHTHSSVYEQPFDSRTIDSNPTTPLGS Xl_LRIG3 1108 SFSQLT-SHTIPQTKLNMQLENGKDESQRTNIPDENATFFPKITSDYQRTSGFQCYELDT Mm_LRIG3 1118 PDCQPRPCHGKSLSSPELDSESEENDKERTDFREENHRCTYQQIFHTYRTPDCQPCDSDT

Supplementary Figure 4.5 Boxshade alignment of full length Am_LRIG3 protein with representative metazoan LRIG3 orthologues demonstrates a reasonable degree sequence conservation throughout the protein. Shading demonstrates >50% consensus to Am_LRIG3 (Black –conservation, Grey – conservative substitution). The degree of conservation supports the assignment of Acropora protein model Contig16713 as the Acropora orthologue of LRIG3, which was initially established by JCUSMART analysis.

Genbank Accesions: Mm_LRIG3 NP_796126.4; Dr_LRIG3 NP_001103817.1; Xl_LRIG3 NP_001103840.1 A_mel_LRIG3 XP_001121890.2


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Supplementary Figure 4.6 Am_PTPRD 1 MLFFKVAWLLALATIGVAVRAFRLTAIKNVITVNVRENVTLRWTYEVSRGERYTITWGTR Nv_PTPRD 1 ------------------------------------PTVTLTPKDFAGVTRGFVYMICNV Sp_PTPRD 1 --------------------------------ITQEQGEFMVTFPFGYHSGFNHGYNCAE Dr_PTPRD 1 ------------------------------------------------------------ Xl_XPTP-D 1 ------MQVPSCQTMNIARPVVVLLCFLLHAGAETPPKLTRTPVDQIGVSGGVASFICQA Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 61 ADLLFTQESWQKTAQPSPKMPTKYANRVKIVGKASLFIQRVDLSDDGFYNCHIQGDFVST Nv_PTPRD 25 TGSPMPTLTWYKNGSVIRNSR--IQIFTMPYGGLIRIGPLRAKIHKATYECEAD-NGVGP Sp_PTPRD 29 STNFASLRWIN---------------------------------YGKRASCLER------ Dr_PTPRD 1 ------------------------------------------------------------ Xl_XPTP-D 55 TGDPRPKIVRNKKGKKVSNQRFEVIEFDDGSGSVLRIQPLRTPRDEAIYECVASNSVG-- Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 121 SK----------DINLTVIDPPRILTQLPLELTWIEGNRQEINCDADGKPKPRVTWHKDG Nv_PTPRD 82 P-LRDSSTLIVHTASTKPAGFPRIAKHP-SFKQRRSGDDVTFTCAAVGTPTPNITWFKDR Sp_PTPRD 50 -VVDDGRKMVWVEADSLPAGYPEITSNPRRYMVVENNRPATIYCGATGNPEPEILWFKEF Dr_PTPRD 1 --------------DQLPAGFPTIDMGP-QLKVVERTRTATMLCAASGNPDPDISWFKDF Xl_XPTP-D 113 -EVATTTRLTVLREDQIPRGFPTIDMGP-QLKVVERTRTATMLCAASGNPDPEITWFKDY Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 171 SVVKSGHRR-----------------ATLEFKSVSYKDEGSYKCVAKN-IGGKKEKQ--- Nv_PTPRD 140 LPIVLNDP--RVSVLSD--------GRKLRITDLRESDNGKYSCVARNALGMVFSSQAMP Sp_PTPRD 109 VPIEANDR---ISMTDS----------GLQFSRAQLSDQGRYECAAKNSLGT-RYS--VE Dr_PTPRD 46 LPVNTSNN-GRIKQLRSESFGGTPIRGALQIEQSEESDQGKYECVATNNDGT-RYS--AP Xl_XPTP-D 171 LPVDTSNNNGRIKQLRS---------GALQIEQSEESDQGKYECVATNSAGT-RYS--AP Mm_PTPRD 1 ------------------------------------------------------------ Am_PTPRD 210 ----VKVNVLYAPKETNITTNLPQDTVDDGSIITITCEALGNPPPSYKFFINGKPIQDKK Nv_PTPRD 190 ARLFIYLTLSVVRSKPEFTLRPQDKTVDPDTDVELKCSARGSPTPSIVWEVDGSRISGQN Sp_PTPRD 153 AQVYVKD----RQVEPRFTILPENQEVVPGGSVNLTCAAYGSPMPRVRWMKAGMDLDDMD Dr_PTPRD 102 ANLYVRELREVRRVPPRFSIPPTDNEIMPGGSVNITCVAVGSPMPYVKWMLGSEDLTPED Xl_XPTP-D 219 ANLYV----RVRRIAPRFSIPPTNHEIMPGGSVNITCVAVGSPMPYVKWMLGSEDLTPED Mm_PTPRD 1 ---------------------------MPGGSVNITCVAVGSPMPYVKWMLGAEDLTPED Am_PTPRD 266 FERSGILSITAIGFKQRGIYSC-------------------------------------- Nv_PTPRD 250 ----G--ELIIRGIQRSGNYSCIADSNMGRVQAYAYVTVRLLPFAPSRPNAASITSDAIK Sp_PTPRD 209 DLPIGRNVLQLRDIYESANYTCVATSLLGTIDTSARVTVRTRPSVPNSPVVTGYTSSSLT Dr_PTPRD 162 DMPIGRNVLELTDVRQSANYTCVAMSTLGVIEAVAQITVKALPKPPGVPQVTERTATSIT Xl_XPTP-D 275 DMPIGRNVLELTDVRQSANYTCVAMSTLGVIEAIAQINVKALPKPPGTPMVTESTATSIT Mm_PTPRD 34 DMPIGRNVLELNDVRQSANYTCVAMSTLGVIEAIAQITVKALPKPPGTPVVTESTATSIT Am_PTPRD 288 ----------------------------------------------RPENDRGTGPISEV Nv_PTPRD 304 ITWHPRGAHVVTSYEVQYRLKG-KTEWQEITN-VRGMQSLVVSGLR----PFSSYEFRVF Sp_PTPRD 269 VEWSTLSSDTVTSHVLEYKRSRSGGSFTEMEIPSPQDTSFTIEGLL----PYTEYAVRLL Dr_PTPRD 222 LTWDSGNPEPVSYYIIQHKPKNSEDSFKEIDG--VATTRYSVGGLS----PYSDYQFRVV Xl_XPTP-D 335 LTWDSGNPEPVSYYIIQHKPKSSEEQYKEIDG--VATTRYSVAGLS----PYSEYEFRVV Mm_PTPRD 94 LTWDSGNPEPVSYYIIQHKPKNSEEPYKEIDG--IATTRYSVAGLS----PYSDYEFRVV Am_PTPRD 302 AV-YVRYA--PRITHPPGNKIVDENNPVGVRCEAEGYPPPLIKWVKLLGNKNVANGTNWV Nv_PTPRD 358 AVNSLGRSRPSLMAEYTTSETKPGSAPRNIEAMGVSDTSFKAGWLPPISANGRIRGYRLY Sp_PTPRD 325 AVNSIGRSNPSDEVTAMTGEDVPSQ-PEQFEGVAISASQIRLTWMMR-DTEPQIIRYELY Dr_PTPRD 276 AVNNIGRGPPSEDIEAKTAEQAPSTAPRQVRGRMLSATTAIIHWDEPEEANGQITGYRVY Xl_XPTP-D 389 AVNNIGRGPPSEPVMTRTSEQAPSSPPRNVQARMLSSTTILVQWEEPEEPNGQIQGYRVY Mm_PTPRD 148 AVNNIGRGPASEPVLTQTSEQAPSSAPRDVQARMLSSTTILVQWKEPEEPNGQIQGYRVY Am_PTPRD 359 LRSAQRTDQGRYRCIATNGFGRDAFAEFEINVYFAPVINRTASSQDVPA--WAGIPTNLS Nv_PTPRD 418 YSLDLLEDISQWRFMASS-TNSTTVTGLTRQTTHFFRILAYNIAGDGPLSEVVAVKTQKG Sp_PTPRD 383 YNVSTNDND---MHKTITPTTDYVLDNLRPNTLYHIRLAARSETGEGASSPVITVRTQQS Dr_PTPRD 336 YTTDPSQHVNQWEKQIVRTSNFLTIPGLTPNKTYYIKVLAFTSVGDGPLSSDLQIIAKTG Xl_XPTP-D 449 YTMDPTQHINSWTKHNVADSQITTIGNLEPQKTYSVKVLAFTSVGDGPLSNDIQVITQTG Mm_PTPRD 208 YTMDPTQHVNNWMKHNVADSQITTIGNLVPQKTYSVKVLAFTSIGDGPLSSDIQVITQTG Am_PTPRD 417 CV-ATANPAPRYE---------WTKASGVVATSEKSGWLQVTPQEG------------DG Nv_PTPRD 477 VP-GQPRSVQLSVLSSTAIRVTWSPP--RYPGDGIFGY-DVYYNKS-----KQDMDTVIG Sp_PTPRD 440 APGAPPQEVSGTVLSSTSIEVRWSPPPLEDQNGDITGY-KIIYRKMSLVSTNNPEMNVPV Dr_PTPRD 396 VP-SQPTDFKGEAKSETSILLSWNPPTQTGQDNQIIGY-ELLYKKG----DDKEEKRVSF Xl_XPTP-D 509 VP-SQPLNFKAEPESETSILLSWTPP----RSDTISSY-DLYYKDG----DHAEEV-ITI Mm_PTPRD 268 VP-GQPLNFKAEPESETSILLSWTPP----RSDTIASY-ELVYRDG----DQGEEQRITI


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Am_PTPRD 455 FE--PYTCTVENNKGSDSMVIRLVKVGIPVVQLKVVRRQSDSLFLEWLLLSDGATNSYI- Nv_PTPRD 528 AFQMNTKEIMGLTPYTVYKVAVAAKSDKGVGPMSFVLTARTDEDRPSDAPQNIQGRSRDS Sp_PTPRD 499 EADVRSYILEALRKYTLYDIRVVACTAIGDGPPSDSLSIRTAEDAPEGPPRKVRVRVHNS Dr_PTPRD 450 EPTT-TYLLKELKPFTTYMFQLAARSKHGIGAYTNEISAETPQTQPSAPPQEVKCTSHSS Xl_XPTP-D 558 DPAT-SYRLQGLKPNSLYYFRLAARASVGLGASTTEISARTMQSKPSAPPQDIRCNSQSS Mm_PTPRD 318 EPGT-SYRLQGLKPNSLYYFRLSARSPQGLGASTAEISARTMQS---------------- Am_PTPRD 512 -----SHYTLQYHTDHSNGGIERTIRISSEKTN--------------------------- Nv_PTPRD 588 TTLEISWDPPRPEHRNGLITHFTIKYRAKGQRG---AKYLTVDATKTSVVLHNLDKFTNY Sp_PTPRD 559 TTIRVQWQAPDPDLQNGEIRGYRIDYQTVTEE---------------------------- Dr_PTPRD 509 TSILVSWKPPPVELQNGIMTKYTIQYAATEGDDTSMRQVSDIPPEKYHYLLENLEKWTEY Xl_XPTP-D 617 TSILVSWLPPPVEKQNGIIIGYSLKYAGVDADDIKPHEILGISSETRQYLLEQLEKWTQY Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 540 ----------------YQLHDLQPYTKYTIELFATN------------------------ Nv_PTPRD 645 FIWVQASTKQGDGPFSDKHTVSTAEDVPNGAPRDVRIRVHNSTTMTVKWN-PPTTKQDGK Sp_PTPRD 591 ------------------------------------------------------------ Dr_PTPRD 569 RVTVNAHTEAGEGPESLPQLIRTEEDVPSGPPRKVEVEAVNSTSVKVLWRSPVPSRQHGQ Xl_XPTP-D 677 RIIVIAYTDVGPGPESSPILIRTDEDVPSGPPRKVEVEAVNSTSVKVSWRSPVANKQHGQ Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 560 ------------------------------------------------------------ Nv_PTPRD 704 ILGYMVFYTRVDDQGNQLRPPAQPESKDSMSEE-----NHKVHLTGLAPETTYQIEVAAY Sp_PTPRD 591 -------------GDPVGSPQVLFINQPDLR---------VAVLSDLLPKTFYSIEIAAF Dr_PTPRD 629 IRGYQVHYVRMVNGEPVGHPVIKDILMDDAQWEYDDSAEHELVLSDLHAETTYSVTVAAY Xl_XPTP-D 737 IRGYQVLYVRMENGEPKGQPMLKDVMLADA---------QEMIISGLQPETAYSITVTAY Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 560 -------KHFSSETSKVEAMTTEAAPSPPRNVKVKIINGTAVNVKWEAPLRPNGPLLFYT Nv_PTPRD 759 TIKGDGARSVTRLAKTMAQSPDPPFVYLERASGDPISDVTLAWRTSATGVIEYKVRYAKS Sp_PTPRD 629 TVRGDGIRSTTEIQQTPGEVPTTPVAFRVENNLDDTYTATWSPPVETHGDLV-------- Dr_PTPRD 689 TTKGDGARSKPKLVTTTGAVPEKPRLM-VSPTNMGTALLQWHPPPNTFGPLQ-------- Xl_XPTP-D 788 NTKGDGARSKPKIVSTSAAVPGKPRLV-ISHTQMNTALIQWYPPVETFGPVI-------- Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 613 VSYDKKEYIP-NGGTKILSVLPNITEVVIGALTPFSNYSIKVKVRNSLTVNESKPVTIST Nv_PTPRD 819 VRRLRGRDQGNLKMKEIKFKSHTTKQKFQSLANGIWYLFNVSLRTKAGWSPETSRWIEIP Sp_PTPRD 681 TYRLSYGPESGRPLYLDLRPSEFQSYTFDDLRLGTRYEFKLTASNEEGNSNEAIFYYTTH Dr_PTPRD 740 GYRLRFGRKDVEPLTVIEFSERENHYTTKEIHKGASYTFRLSARNKVGFGEETVKEISTP Xl_XPTP-D 839 GYRLKFGRKDLDMLTTFEFFEKEEHFTATDIHKGALYIFKLSARNKVGYGEEIVKELSIP Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 672 KMAAPSEPLAFEANMTGAHSATLWWRKPRQLNGKILLFKIRMRWRYKNVKGTY---RFIT Nv_PTPRD 879 PGPPSGPPLNVRAVAVSSTSVEVTWSAPDIWNRGGPILGYSVMYNPANKRGDVTVKNVTS Sp_PTPRD 741 EGTPSGSPMNVTASPLSSSSISVSWDPPLEEERNGLILKYTIRYYS----SAMPTELTNR Dr_PTPRD 800 EDTPAGYPQGIVAESSTTTTIQVSWQPVALAERNGAVVKYALQYKDINS-PRSPSELFIT Xl_XPTP-D 899 EEPPSGFPQSIHCDSTSSTSVLITWQHPNLEERNGLLTKYTLMYRDINL-PHYPIEVPIV Mm_PTPRD 361 ------------------------------------------------------------ Am_PTPRD 729 KKIPAKVTPRERRRLRRYTMDYRVLRLEPLREIQLTRIQPFAFISVHVSEGTGITENEVF Nv_PTPRD 939 PNIFKVVLKRLKKFREYEVRVRAIGMLGMGPASEPFIDRT-KEDGKCHCRDVRPRTVGSD Sp_PTPRD 797 TTETDIVLTSLVPNTAYSVEVRAHTSVGPGPYSTKDIATTPREAPASAPLDLRCAAPHGT Dr_PTPRD 859 APESTVTLDGLKADTTYDIKMCAFTSKGPGPYSPSVQFRT-QPVNQVFAKNFHVRAAMKT Xl_XPTP-D 958 PADTTVTLTGLKPDTTYDVKLRAHTSKGPGPFSPSVQFRT-LPVDQVFAKNFHVKAVMKT Mm_PTPRD 361 ----------------------------------------------MFAKNFHVKAVMKT Am_PTPRD 789 WSP--WSNNYTLQTLEG----APSAPRNVELKQNKG--ASVKVTWLPPEYPNGIIQK--- Nv_PTPRD 998 WVILEWLPPRIDAGIDT----PLTHADEVRVDALT--------ADVQTFKVQGLVPH-MK Sp_PTPRD 857 QLEVKWSHPSNIQRSGPGYNYLVYSTNSADNTDVSTWGRPVSAGEFTKLELRGL-QEETT Dr_PTPRD 918 SVLLTWEIPENYNPAQP---FTILYDNG-QSVEVD--------GKLTQKLIVNL-QPETQ Xl_XPTP-D 1017 SVLLSWEIPENYNSALP---FKILYDDGKMAVEVD--------GRATQKLITNL-KPETS Mm_PTPRD 375 SVLLSWEIPENYNSAMP---FKILYDDGKMVEEVD--------GRATQKLIVNL-KPEKS Am_PTPRD 838 ----------------YRIIYSNNSIKNYTAEVTK--GLKNTSLFYILSGLHKDSTYQ-- Nv_PTPRD 1045 YTVRLSASNAMGEG--PRAELSISTKKGKPPALAKPTMLRDEMNNGLIPVEL-HRAS--- Sp_PTPRD 916 YYVAVKLDTPEGNSPLSQIITCLTGTSAPSEPRSFDAHIASDNTINLVWSEP-SDIPGRL Dr_PTPRD 965 YSFLL---TNRGNS-AGGLQHRVSTMTAPDILRTKPFLISKTNADGMVTVEL-PGVQ--- Xl_XPTP-D 1065 YSFVL---TNRGNS-AGGLQHRVAAKTAPDVLKTKPVFIGKTNSDGMITVEL-PEVL--- Mm_PTPRD 423 YSFVL---TNRGNS-AGGLQHRVTAKTAPDVLRTKPAFIGKTNLDGMITVQL-PDVP---


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Am_PTPRD 878 -----------------------------INVQAFTSFPG-------EVSPTVRRDIKTP Nv_PTPRD 1099 --------EKNGPISYY----------LVVVVPLDKDG----------KYPQGSNPDEYF Sp_PTPRD 975 KDYVIKYHTSDDFRREYPDYYDGGGEIEILASEQRVSLSGQLFKPN-MEYEFNITARTEA Dr_PTPRD 1017 --------TAERVRAYY-----------IVVVPLKKQRPGKFIKLWENPDEMNLEELLRE Xl_XPTP-D 1117 --------VNDKIKGYY-----------IVIVPLKKSR-GKFIKPWESPDEMEFDELLKD Mm_PTPRD 475 --------ANENIKGYY-----------IIIVPLKKSR-GKFIKPWESPDEMELDELLKE Am_PTPRD 902 GDVLEQSKVD-------------------------------------------------- Nv_PTPRD 1131 KKVRRRS--AREAGQVKPYIAAKFLADQFPKKFLVGDEGFKNQYGYYNKRLVANSYYTVF Sp_PTPRD 1034 IRGPPASILVRTRIQALEHIPK----PTIQNSTMNGQS--IKIALPTITQNAALLDYLYV Dr_PTPRD 1058 INRTSVSLRFRRHLELKPYIAACF--HELPNEFTLGDA--KMYGEFQNKQLLNGQEYVFF Xl_XPTP-D 1157 ISRKRRSLRFRREAEQKPYIAAHF--DLLPTEFTLGDQ--KQYGGFENKQLQNGQEYVFF Mm_PTPRD 515 ISRKRRSIRYGREVELKPYIAAHF--DVLPTEFTLGDD--KHYGGFTNKQLQSGQEYVFF Am_PTPRD 912 --------------------------------------------TASLYGGVFAGIIAFV Nv_PTPRD 1189 SRAYVQTDDGDFVYTSSPFTDPVLITPPSAAG----LSGGGKGGLNLAYVIIPVVLLLVI Sp_PTPRD 1088 IVIGLRNKNG-EPVALTISPNDIKTEDLEVQSRGRRSAQHPRSRRAATGQDTEPYIAAKL Dr_PTPRD 1114 VLAVLEISDS-MLYAASPYSDPVVFADIDPQP---------IIDEEEGLIWVVGPVLAVI Xl_XPTP-D 1213 VLAVIEHSES-AMFATSPYSDPVVSMEIDPQP---------MTDEEEGLIWVVGPVLAVV Mm_PTPRD 571 VLAVMDHAES-KMYATSPYSDPVVSMDLDPQP---------ITDEEEGLIWVVGPVLAVV Am_PTPRD 928 FVIIIVALVIRNH-RKRRDGTDLSGKQYAVANSNDSGIGDKESLDRVPGVKCVQGV---- Nv_PTPRD 1245 VTVIVLAVLYVMRMRRRRR------------------SKKESKCKDEEKSEPSDPVEM-- Sp_PTPRD 1147 NGEEMPASFVI-------------------------GDGSEVNGYRNKPLKEGEYYTLFT Dr_PTPRD 1164 FIICIVIAILLY--KRKRAESEARK-------------GSLPSGKEMPSHHPTDPVEL-- Xl_XPTP-D 1263 FIICIVIAILLY--KRKRTESDSRK-------------SSLPNSKEIPSHNPTDPVEL-- Mm_PTPRD 621 FIICIVIAILLY--KRKRAESESRK-------------SSLPNSKEVPSHHPTDPVEL-- Am_PTPRD 983 RRGSEDVIGPGKFVTIKPIPIQRLPEYCAINHADRNKGFREEFLSIRVPGS--FTWENSQ Nv_PTPRD 1285 RRLNFQ--TPAMID--HP-PISVLDLPKHIAILKADNNSQFTQEYESIEPGQTFTADSSQ Sp_PTPRD 1182 RAVILSENGTAMMS--HP-PIPVSQFQEHIERLKGNDAMLFSQEYESIEPGQQFTWDHSN Dr_PTPRD 1207 RRLNFQ--TPGMAS--HP-PIPVMELADHIERLKANDNLKFSQEYESIDPGQQFTWEHSN Xl_XPTP-D 1306 RRLNFQ--TPGMAN--HP-PIPILELEDHIERLKANDNLKFSQEYESIDPGQQFTWEHSN Mm_PTPRD 664 RRLNFQ--TPGMAS--HP-PIPILELADHIERLKANDNLKFSQEYESIDPGQQFTWEHSN Am_PTPRD 1041 KPENKAKNRFNSIIPYDHTRVTLTELEGSPGSDYINANFIDGYDHPFKFIATQGPVANTF Nv_PTPRD 1340 MECNKGKNRYPNIHAYDHSRVRLSYINGIEGSDYINANFCDGYRKERAYIATQGPMQHTA Sp_PTPRD 1239 LETNKPKNRYANVIAYDHSRVILSPMEGLPGSDYTNANYCDGYRKQNAYIATQGPLLETM Dr_PTPRD 1262 LEVNKPKNRYANVIAYDHSRVLLSAIDGIPGSDYINSNYIDGYRKQNAYIATQGALPETF Xl_XPTP-D 1361 LEVNKPKNRYANVIAYDHSRVLLSAIDGIPGSDYINSNYIDGYRKQNAYIATQGPLPETF Mm_PTPRD 719 LEVNKPKNRYANVIAYDHSRVLLSAIEGIPGSDYVNANYIDGYRKQNAYIATQGSLPETF Am_PTPRD 1101 GDFWRTVWEQGVCVVIMVTNIVEGGRIKCLKYWPSASPEMYGLVLVSPQGEEELADYVVR Nv_PTPRD 1400 ADFWRMVWEQRTFTIVMLTREEERGRVKCDQYWPTDGTDVYEGIEVSLVDWVELANYTIS Sp_PTPRD 1299 ADFWRMVWEQRTNTIVMMTKLEERNRVKCDQYWPTRDQEKYGFIQVTLLDTTELATYTVR Dr_PTPRD 1322 GDFWRMIWEQRSANIVMMTRLEERSRVKCDQYWPNRGTETYGLIQVTLLDTVELATYCVR Xl_XPTP-D 1421 GDFWRMMWEQRSATVVMMTKMEERSRIKCDQYWPSRGTETYGLIQVTLLDTVELATYTVR Mm_PTPRD 779 GDFWRMIWEQRSATVVMMTKLEERSRVKCDQYWPSRGTETHGLVQVTLLDTVELATYCVR Am_PTPRD 1161 KFSIQMISKSAEHSAREVTQYHFTAWPDQGVPTHATSLLAFLRKVRTSVPEDSGPILIHC Nv_PTPRD 1460 TL---QICKEGASQPREVKHFQFTGWPDHGVPAHPTPFLAFLRRVKFYNPPDAGPIVVHC Sp_PTPRD 1359 SF---ALVKNRSMEKREVKQFQFTAWPDHGVPEHATSVLAFISRVKSCNPPDAGPIVVHC Dr_PTPRD 1382 TF---ALYKNGSSEKREVRQFQFTAWPDHGVPEHPTPFLAFLRRVKSCNPPDAGPMVVHC Xl_XPTP-D 1481 TF---ALYKNGSSEKREVRQFQFTAWPDHGVPEHPTPFLAFLRRVKTCNPPDAGPMVVHC Mm_PTPRD 839 TF---ALYKNGSSEKREVRQFQFTAWPDHGVPEHPTPFLAFLRRVKTCNPPDAGPMVVHC Am_PTPRD 1221 SAGVGRTGTYIVLDAMLDQIAAEGVVDIYGFISHIRQQRSFMVQTEGQYVFVHNALEEYV Nv_PTPRD 1517 SAGVGRTGCFIVIDSMLERLRHEETVDIYGHVTVLRTQRNYMVQTQEQYIFSHDAILEAV Sp_PTPRD 1416 SAGVGRTGAYIVIDSMLERIKHEKTVDIYGHVTCLRAQRNYMVQTEEQYIFIHEALYEAV Dr_PTPRD 1439 ------------------------------------------------------------ Xl_XPTP-D 1538 SAGVGRTGCFNVIDAMLERIRHEKTVDIYGHVTLMRAQRNYMVQTEDQYIFIHDALLEAV Mm_PTPRD 896 SAGVGRTGCFIVIDAMLERIKHEKTVDIYGHVTLMRAQRNYMVQTEDQYIFIHDALLEAV Am_PTPRD 1281 TCGSTEFPVSDLPERLRILNTVDPDSGDSLVVAEFKNLGLGASDGDSFIVASRQENKSKN Nv_PTPRD 1577 SCGNTEVHARNLLHHIKKLTELGKGE-VTGLEEEFKVVDPSQAKKHKYGAATLAINRPKN Sp_PTPRD 1476 ASGTTEVHARNLYGHIQKLTALEPGETITGMENEFKRLASQKAQPSRFVSANIPANKFKN Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1598 TCGNTEVPARNLYAYIQKLTQIEPGENVTGMELEFKRLASFKAHTSRFISANLPCNKFKN Mm_PTPRD 956 TCGNTEVPARNLYAYIQKLTQIETGENVTGMELEFKRLASSKAHTSRFISANLPCNKFKN


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Am_PTPRD 1341 RYA-ILPFDRTRVTLWPYTGMVGSDYINASFVDSFQQREAFIATQAPLENTVVDFWRMTW Nv_PTPRD 1636 RLANILPYETTRVHISPVRGVEGSDYINASFIDGYRQRGLFIATQGPLQDTVDDFWRMLM Sp_PTPRD 1536 RLLNIQPYEGNRVCLQPIRGVEGSDYINASHIDGYRQRNAYIATQGPLAETTEDFWRMLW Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1658 RLVNIMPYESTRVCLQPIRGVEGSDYINASFIDGYRQQKAYIATQGPLAETTEDFWRMLW Mm_PTPRD 1016 RLVNIMPYESTRVCLQPIRGVEGSDYINASFLDGYRQQKAYIATQGPLAETTEDFWRMLW Am_PTPRD 1400 EYEAYSIVMLTTMNELQEGQCA-YYLPRREEFIVYGLLMVEVETEDQRNGFCRRRLRVTN Nv_PTPRD 1696 EQNSNIIVMLTQLHEGEWEKCYKYWPTDRSARHQYYIVDPIAEHEYPQFVIRDFKVKDA- Sp_PTPRD 1596 EQNSTIIVMLSKLREMGREKCHQYWPAERSARYQYFVVDPMSEYNMPQYILREFKVTDA- Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1718 EHNSTIVVMLTKLREMGREKCHQYWPAERSARYQYFVVDPMAEYNMPQYILREFKVTDA- Mm_PTPRD 1076 EHNSTIVVMLTKLREMGREKCHQYWPAERSARYQYFVVDPMAEYNMPQYILREFKVTDA- Am_PTPRD 1459 TKSGEIRSIYHFHFKEWAERSIPLNGMGLLDMMKCITRVQQQTGN-GPIVIHCSDGSGRT Nv_PTPRD 1755 -RSDTVRNIKQFHFLGWPETGVPKSGEGIIDLIGQVQRAYEQQEEEGPITVHCSDGVGRT Sp_PTPRD 1655 -RDGQSRTIRQFQFTDWPEQGVPKSGEGFIDFIGQVHKTKEQFGQEGPISVHCSAGVGRT Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1777 -RDGQSRTVRQFQFTDWPEQGVPKSGEGFIDFIGQVHKTKEQFGQDGPISVHCSAGVGRT Mm_PTPRD 1135 -RDGQSRTVRQFQFTDWPEQGVPKSGEGFIDFIGQVHKTKEQFGQDGPISVHCSAGVGRT Am_PTPRD 1518 GTFCAINVALERVKLDGTIDIFQTVRRLRTQRPLMVQTEEQYKLCYEITRLFVESFNDYS Nv_PTPRD 1814 GVFTALFIVLERMRSEGVVDLFQTVKLLRTQRPAMVQSQEQYLFCYKTALEYLGSFDHYA Sp_PTPRD 1714 GVFITLSVVLERMRYEGIVDMFQTVKMLRTQRPAMVQTEDQYQFCYHAALEYLGSFDHYT Dr_PTPRD ------------------------------------------------------------ Xl_XPTP-D 1836 GVFITLSIVLERMRYEGVVDIFQTVKMLRTQRPAVVQTEDQYQFCYRAGLEYLGSFDHYA Mm_PTPRD 1194 GVFITLSIVLERMRYEGVVDIFQTVKMLRTQRPAMVQTEDQYQFCYRAALEYLGSFDHYA Am_PTPRD 1578 DLK Nv_PTPRD 1874 I-- Sp_PTPRD 1774 --- Dr_PTPRD --- Xl_XPTP-D 1896 T-- Mm_PTPRD 1254 T--

Supplementary Figure 4.6 Boxshade alignment of full length Am_PTPRD protein with representative metazoan PTPRD orthologues demonstrates a high degree of sequence conservation C-‐terminal to the transmembrane region. Shading demonstrates >50% consensus to Am_PTPRD (Black –conservation, Grey – conservative substitution). The degree of conservation in the C-‐terminal region supports the assignment of Acropora protein model Contig12034 as the Acropora orthologue of PTPRD, which was initially established by JCUSMART analysis.

Genbank Accesions: Dr_PTPRD XP_002663075.2; Sp_PTPRD XP_001184195.1; Mm_PTPRD NP_001014310.1; Xl_XPTP-D NP_001083850.1


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Supplementary Figure 5.1 Aligned Nucleic Acid and Protein sequence of Am_ACadherin (Type-‐III Cadherin) supercontig (Contig6389 + Contig5439 in the 2010 Acropora millepora transcriptome assembly).

Forward Frame 3: 1 D C G T T S K C E A G L L K F S V E G T 3 GACTGTGGTACAACTAGCAAATGCGAGGCTGGTTTGTTAAAGTTCTCAGTTGAAGGAACA 21 S L F K I D P R T G V V S V G S T P L D 63 TCCCTGTTTAAAATTGATCCGCGCACTGGTGTTGTAAGTGTTGGCTCCACTCCTCTCGAT 41 Y E K Q R E H V F T V V V E D F G E K I 123 TATGAAAAGCAGAGAGAGCATGTGTTTACTGTCGTTGTGGAAGATTTTGGCGAGAAGATA 61 Y K S R G F V T I D V R N T D D E K P Q 183 TACAAATCCAGGGGATTTGTTACGATTGACGTGCGAAATACGGATGACGAGAAGCCACAG 81 F L E S D Y T I S I A E D A E T G R P L 243 TTCCTAGAGAGTGATTACACGATCAGTATTGCTGAAGACGCCGAGACGGGGAGGCCTCTA 101 A A I L A R D A D G D P V K Y S I T S G 303 GCAGCTATACTTGCCAGAGATGCAGATGGGGATCCTGTTAAGTACTCTATAACTAGCGGT 121 D S G G I F Q I N P T T G V L S L K S S 363 GATAGTGGTGGCATTTTCCAGATTAACCCAACAACTGGAGTTCTTTCTTTGAAATCAAGC 141 I K G N P R T Q Y T L Q V K A S N S A Q 423 ATCAAAGGAAATCCTCGGACACAGTACACGCTTCAAGTGAAGGCGTCGAATTCTGCGCAG 161 D S R F D E V R V V V N I E D S N D N R 483 GATTCTCGTTTCGATGAAGTGCGTGTTGTGGTCAACATCGAGGACAGCAATGATAATAGG 181 P V F T D C P P E V P V E E N K P R G H 543 CCAGTGTTCACTGATTGCCCGCCTGAGGTCCCAGTAGAGGAGAACAAGCCAAGAGGACAC 201 R V Y Q V V A Q D T K D R G R N K E I E 603 AGGGTCTATCAGGTCGTCGCTCAGGATACTAAGGACAGGGGAAGAAACAAAGAGATTGAA 221 Y F L V T G G E R L F E I D N T T G V I 663 TATTTTCTCGTTACCGGTGGAGAAAGGTTGTTTGAGATTGATAACACTACCGGAGTTATA 241 K T L T S L D R E T K D Q H T L I I M A 723 AAAACCTTAACCAGCTTGGACAGGGAAACCAAAGATCAGCATACATTAATTATAATGGCT 261 E D G G H G R N S A E R L L S Y C I L D 783 GAGGATGGAGGGCATGGAAGAAATTCGGCGGAGAGACTTCTTTCATATTGTATTCTTGAC 281 V K V V D Q N D N F P F F L T R T Y Y A 843 GTCAAAGTTGTAGACCAGAACGACAATTTCCCCTTCTTCTTGACCAGAACTTACTATGCC 301 S V W Q G A P V N T E V L T V R A A D M 903 AGCGTCTGGCAAGGAGCGCCTGTGAATACAGAGGTTTTAACCGTAAGGGCAGCGGACATG 321 D T R V N A N I D N S E V Q Y Q L V N A 963 GACACGAGAGTGAATGCCAATATTGACAATAGCGAAGTGCAGTATCAGTTGGTCAATGCT 341 D D K F Q V E L A T G V I K T K A T L V 1023 GATGACAAATTTCAAGTTGAACTGGCAACTGGAGTGATTAAAACCAAAGCCACGTTGGTC 361 S F V G K V Q L Q I R A I N K Q P M A I


151

1083 AGCTTTGTGGGAAAAGTACAGTTGCAGATAAGAGCCATTAACAAACAACCCATGGCTATA 381 S E E R P R T S T T T V E I N V E K D K 1143 AGTGAGGAAAGACCACGCACGTCAACGACTACTGTGGAAATCAACGTCGAAAAAGATAAA 401 P P A F A P S A V Y K S A I N E D V K I 1203 CCACCCGCGTTTGCACCGTCGGCGGTTTATAAAAGTGCAATAAACGAAGACGTCAAAATA 421 G K E V Q Q I L A I S Q V D R K N K I V 1263 GGAAAGGAGGTTCAACAGATCTTAGCGATCAGCCAGGTAGACAGGAAAAACAAAATAGTG 441 Y S F V K S N P E G E Q K F Q I D P A T 1323 TACAGTTTTGTGAAGTCCAATCCAGAGGGAGAACAAAAATTTCAAATTGACCCGGCGACT 461 G N I T T A S T L D Y E Q V K E Y R L Q 1383 GGCAACATCACAACTGCTAGCACGCTGGATTATGAACAAGTGAAGGAGTACAGGCTTCAG 481 F R A T D I A T N L Y A T C V V I I S L 1443 TTCCGAGCTACTGATATTGCGACGAACCTTTACGCGACTTGCGTGGTCATCATTTCGCTG 501 I D V N D D T P T F K L E E Y T A R V P 1503 ATCGATGTCAACGACGACACGCCAACGTTCAAGCTAGAAGAATACACTGCTAGAGTTCCG 521 E N A A V D F N V I T I E A D D R D T A 1563 GAAAATGCAGCAGTAGATTTTAATGTGATCACTATAGAAGCCGACGACAGGGATACAGCT 541 L S G Q V G Y T L E V S D S S E G Q F F 1623 TTGTCTGGTCAAGTTGGTTACACCCTAGAAGTGTCCGACAGTTCCGAAGGACAATTCTTC 561 A I N G Q T G V M I T K N S F D R E D P 1683 GCCATCAATGGTCAAACCGGAGTGATGATTACGAAAAATTCGTTTGATCGAGAGGATCCG 581 K H I P K Y N V F A V A T D K G V P P L 1743 AAACACATTCCGAAGTACAATGTGTTTGCCGTGGCAACAGACAAGGGCGTTCCTCCTCTC 601 A G K L D F E T K K T Y N I S I T A T D 1803 GCTGGAAAACTTGACTTTGAAACGAAGAAGACGTACAACATCTCCATCACCGCAACAGAT 621 R K D S A T V P V V V N V L D T N D N I 1863 AGAAAAGACAGTGCTACAGTTCCAGTGGTGGTCAATGTTCTGGACACCAACGATAACATT 641 P Q F R N L I Y E A I I P E N S P P G Q 1923 CCTCAATTTCGCAATCTAATTTACGAGGCCATTATTCCAGAAAACAGCCCTCCTGGCCAA 661 R V A T V F A T D L D S P K I Q N D L R 1983 AGGGTGGCGACAGTCTTTGCCACTGACTTGGACAGTCCGAAAATCCAAAACGATCTCCGA 681 Y S L D A D G Q K N F A V D A V S G L I 2043 TATTCCCTCGACGCAGACGGACAGAAGAACTTTGCAGTTGATGCTGTGAGTGGTCTGATC 701 T T A N Q R L D R E V N P V V T F T A F 2103 ACCACTGCCAATCAAAGGCTGGATCGAGAGGTGAATCCAGTGGTTACCTTCACTGCTTTT 721 A F D G K H R G E A L I R V T L R D V N 2163 GCTTTTGATGGCAAACACAGAGGGGAAGCTTTGATTCGAGTCACGCTCCGAGATGTCAAC 741 D N S P Y F P N P P Y V G Y V E E N L D 2223 GACAACAGTCCATACTTTCCCAACCCTCCCTATGTCGGCTATGTAGAGGAAAACCTAGAT 761 P G A S V M V I Q A F D L D S G I D G E 2283 CCGGGGGCAAGTGTCATGGTTATTCAAGCGTTCGATCTGGATTCCGGCATTGACGGAGAA 781 I V Y S L D D S S N N K F K I D R N S G 2343 ATCGTTTACTCTTTAGATGACAGCTCCAACAACAAGTTTAAGATCGATCGTAATTCCGGC


152

801 L V T T V E T M E K E T A V N S F T I V 2403 CTTGTAACAACTGTGGAAACTATGGAAAAAGAGACTGCTGTGAATTCGTTCACAATCGTT 821 V K A T D K G S P A L S G T V T A T I R 2463 GTGAAAGCAACCGACAAAGGAAGTCCAGCTCTGAGTGGAACTGTAACGGCTACCATCCGA 841 V S D G N D Q A P V F N P R E Y R Q K V 2523 GTATCTGATGGAAACGATCAAGCGCCAGTATTCAACCCCAGGGAATACAGGCAAAAAGTG 861 P E D S P P G F L V T Q V K A T D Q D E 2583 CCGGAGGATTCTCCCCCAGGATTCCTCGTCACGCAAGTGAAAGCTACCGACCAGGACGAG 881 G Y N A E L E F T I T A G N D P Y Q F Y 2643 GGATATAACGCAGAGCTCGAGTTCACCATTACGGCGGGCAATGACCCTTACCAGTTCTAC 901 I D P K T G E I L V S G M L D F D H G K 2703 ATCGACCCGAAAACAGGCGAAATACTTGTATCGGGTATGCTGGATTTTGACCATGGAAAG 921 K S Y N L T V M V S D R G V P P K Q A A 2763 AAGTCCTATAACCTGACAGTTATGGTTAGTGATCGCGGTGTACCACCTAAACAAGCTGCG 941 K P A F V Y I T I V D S N D N P P V F V 2823 AAACCAGCTTTTGTTTACATAACTATCGTGGATTCCAATGATAATCCGCCGGTCTTTGTG 961 P A E Y S I K V T E G T K P G D T V Q L 2883 CCTGCTGAATACAGCATTAAAGTCACCGAGGGTACAAAACCTGGAGACACTGTGCAACTG 981 V T A V D Q D T G T N A L F T F G I A D 2943 GTAACAGCGGTTGATCAGGACACGGGTACTAATGCTCTTTTTACGTTTGGCATCGCTGAC 1001 G D D A D M F G I R P D P K N S S I G F 3003 GGTGATGATGCTGATATGTTTGGTATAAGACCCGATCCCAAGAACAGCAGTATTGGGTTC 1021 I Y T V L Q L D R E T V P Q Y N L T V T 3063 ATTTACACGGTGCTGCAGTTGGATCGCGAAACCGTTCCTCAGTACAATCTCACCGTCACC 1041 A T D T G G L Q G V A V V R I T V L D T 3123 GCGACCGATACTGGCGGGCTCCAAGGCGTTGCTGTGGTGCGTATTACTGTCTTAGACACC 1061 N D N G P W F Q P R Y Y E G S I T V T S 3183 AACGATAACGGCCCGTGGTTCCAACCTCGCTACTATGAAGGTTCAATTACGGTGACTAGT 1081 D S N S Q Q E I T T V K V F D P D E P S 3243 GATTCGAACTCACAACAAGAAATTACCACAGTGAAAGTCTTTGACCCAGATGAGCCAAGC 1101 N G P P F S F S I E S T K P A T D A T R 3303 AATGGACCTCCGTTCAGCTTTTCGATTGAAAGTACAAAGCCTGCCACTGATGCAACTCGC 1121 F G L R K D P K E P Q T A N E V Y S I G 3363 TTTGGGTTGCGAAAAGATCCCAAAGAACCCCAAACCGCAAACGAGGTGTACTCAATCGGA 1141 A F T R Q V P E W E L T I K A I D S G K 3423 GCTTTCACGCGTCAAGTTCCTGAATGGGAACTGACAATCAAGGCTATTGATAGTGGAAAG 1161 P V A M F N S T L V F V W V V D D K N L 3483 CCGGTGGCCATGTTCAACTCGACGCTTGTGTTCGTTTGGGTTGTTGATGACAAGAACTTG 1181 N E P F D G A L T I I V N A Y D D K F A 3543 AACGAACCATTTGACGGAGCATTGACCATCATAGTAAACGCCTACGATGACAAATTTGCC 1201 G G I I G K A Y Y Q D V D Y M G D E N T 3603 GGCGGTATCATCGGAAAGGCCTACTACCAAGACGTGGACTACATGGGAGACGAGAACACA 1221 Y S M S E Q E Y F T L G E L T G D I S A 3663 TACTCTATGAGTGAGCAAGAGTATTTCACCTTGGGCGAACTTACTGGTGACATAAGTGCT


153

1241 A A N I P V G L Y K F E I E V L E Q R L 3723 GCGGCAAACATTCCTGTGGGACTATACAAGTTCGAGATAGAGGTGCTTGAGCAACGGCTT 1261 R P N T N T F K T V T S S V S V I V Q S 3783 CGGCCAAACACCAACACTTTTAAAACCGTAACGTCTTCAGTATCGGTCATCGTGCAAAGT 1281 V P R A A I L Q S V A V Q I L A Y R R P 3843 GTGCCCAGGGCTGCGATTCTGCAAAGCGTGGCTGTGCAGATTCTCGCGTATCGCAGGCCG 1301 A L F V A D I Y T N F R Q K L A G I F G 3903 GCCCTCTTTGTGGCTGACATTTATACCAACTTCCGACAAAAGTTAGCTGGAATCTTCGGC 1321 V Q E A D I L I F S V Q R A P S K R V P 3963 GTTCAGGAAGCTGACATTTTGATCTTTAGCGTTCAAAGGGCTCCAAGCAAGCGTGTTCCT 1341 L A D V F G V E I Q L A V R S S G S S F 4023 CTCGCGGACGTGTTTGGAGTCGAGATTCAACTGGCTGTTCGTTCGTCGGGAAGTTCTTTC 1361 M D K M D V V R G I V E G K A E L E A L 4083 ATGGATAAGATGGATGTTGTCAGGGGAATAGTAGAAGGAAAGGCAGAACTAGAGGCATTG 1381 S L K I G D I G I D V C A A E R Q D V G 4143 AGCTTGAAGATCGGAGATATTGGTATTGATGTGTGTGCTGCGGAGCGACAAGATGTTGGC 1401 V C N N K V E A S S A F T V V S G D I G 4203 GTTTGCAACAATAAAGTGGAGGCTTCGTCAGCGTTCACTGTAGTCAGCGGTGACATTGGC 1421 Q I E P S K S S L T V V S I D I T L K A 4263 CAAATAGAACCGAGCAAGTCTAGTTTAACAGTAGTATCCATTGACATCACACTGAAAGCC 1441 V Y T S I L P P D I N C T T G T P C L H 4323 GTCTACACATCAATCCTTCCACCAGATATAAACTGCACAACAGGCACTCCCTGTCTACAT 1461 G G T C H N A V P K G I I C E C G R D Y 4383 GGGGGCACGTGTCACAACGCAGTTCCCAAGGGGATCATTTGCGAATGTGGACGAGATTAC 1481 L G P E C Q S T T R T F R G N S Y L W L 4443 CTTGGACCAGAGTGTCAGAGCACTACGAGAACGTTCAGAGGAAATTCATACTTGTGGCTT 1501 D K L A S Y E R S S I S L Q F M T G S A 4503 GATAAGCTCGCATCCTATGAACGGAGCTCGATTTCGCTACAGTTTATGACTGGCTCTGCC 1521 N G L L L Y Q G P L Y N G A N N G L P D 4563 AATGGCTTGTTGCTTTACCAAGGACCTCTGTACAATGGGGCCAACAATGGTCTTCCAGAT 1541 S I A L Y L V D G F A K L V I A L G P H 4623 TCGATTGCATTGTATTTGGTGGACGGATTTGCCAAACTGGTGATCGCCCTTGGGCCACAC 1561 P M T P L E L Y M N K G D R L D D R T W 4683 CCTATGACACCATTAGAGCTGTACATGAACAAGGGAGATCGTTTGGATGACAGGACGTGG 1581 H T V E V I R E R K K V V L R I D K C S 4743 CACACGGTTGAGGTCATTCGAGAACGCAAGAAAGTTGTGCTGAGGATCGACAAGTGTTCC 1601 Y S K I V E D Y G Q I V E D R S S C E I 4803 TATTCGAAAATTGTAGAGGATTATGGTCAAATTGTCGAGGACAGGTCCTCATGCGAAATC 1621 K G E I W G S A I Y L N G F G P L Q I G 4863 AAGGGAGAAATCTGGGGCTCCGCGATCTACTTGAATGGTTTTGGTCCTCTTCAAATTGGT 1641 G V E N S I S D M K I N F T G F S G C I 4923 GGCGTGGAAAACTCGATCAGCGATATGAAGATAAACTTCACTGGTTTCTCCGGCTGCATT 1661 R N I Y N N G R M Y D L F N P L K E V N


154

4983 CGAAATATTTACAACAATGGCAGGATGTACGATTTGTTTAATCCATTGAAAGAAGTCAAC 1681 T E L G C R L N N Q C P N C N G R G Y C 5043 ACGGAGTTGGGATGCCGGCTCAATAACCAGTGTCCAAACTGCAATGGGCGTGGCTACTGC 1701 E P F W N Y A I C V C D L G F G G A N C 5103 GAACCCTTCTGGAATTATGCTATCTGTGTGTGTGATCTTGGTTTCGGTGGAGCTAATTGT 1721 D S R T Q A N W Y R A N S F T Q Y R V K 5163 GACTCAAGGACACAAGCCAACTGGTATCGTGCAAATAGCTTTACCCAGTACCGCGTAAAA 1741 Q V K R K R R E L V P A P V S M A N E F 5223 CAAGTTAAGAGAAAGCGTCGTGAACTTGTTCCAGCGCCGGTGTCGATGGCAAATGAATTC 1761 Y T N I A L Q V R V S P N S S N V V I F 5283 TACACAAACATTGCCTTGCAAGTCAGGGTGTCTCCGAATTCATCTAATGTTGTCATTTTC 1781 L A S N S L G T E F N R I D V K N H V L 5343 TTAGCGTCCAACAGTTTGGGAACTGAATTTAACAGGATTGATGTCAAGAATCATGTGTTG 1801 R Y A F R L G D R M K I L K I P Q L N V 5403 CGTTACGCGTTCCGATTGGGTGACCGTATGAAGATCCTCAAGATCCCTCAACTGAACGTG 1821 T D D K Y H T V I V K R E G N R A S L Q 5463 ACGGACGATAAGTATCACACTGTGATCGTGAAGAGGGAGGGTAACCGCGCCAGCCTTCAG 1841 I D Y R G K V E G T T G G L H K L L N M 5523 ATCGATTATAGGGGCAAAGTGGAAGGAACAACAGGAGGACTTCACAAGCTGCTAAACATG 1861 G G G S F F T G G L P N I T E V R V V E 5583 GGTGGAGGAAGTTTCTTTACTGGGGGCTTGCCTAATATTACTGAGGTTAGGGTAGTGGAA 1881 A F V N S G G N A V L R T A E G N I I S 5643 GCGTTTGTTAATAGCGGAGGCAACGCTGTCCTGCGCACAGCTGAGGGCAACATCATCTCA 1901 S G M G S A Y T G I G S Y M S N V I T V 5703 AGCGGGATGGGATCCGCATACACTGGCATCGGTAGTTACATGAGCAATGTAATTACTGTC 1921 N N F G G V D V S Y G V S G A P H V Q R 5763 AATAACTTTGGAGGCGTTGATGTATCGTACGGAGTTAGCGGTGCGCCACACGTTCAGCGG 1941 T I K T K S S I F T G S S G V I T R I S 5823 ACCATCAAAACAAAAAGCAGCATCTTTACAGGAAGCAGCGGTGTGATAACCAGAATCAGT 1961 V S R G H S V E F M N S R F F T R K T K 5883 GTCAGTAGGGGTCATAGTGTGGAATTCATGAACAGTCGTTTCTTTACACGCAAGACCAAG 1981 Q K Q K V I I S S S G G S V S G G S G G 5943 CAGAAGCAAAAGGTAATCATCAGTTCATCTGGAGGTTCAGTGTCCGGCGGAAGTGGAGGT 2001 A S G G S G G A S G S G G S V G V S G G 6003 GCCTCGGGAGGAAGTGGTGGTGCTTCAGGCTCGGGCGGAAGTGTTGGTGTATCTGGAGGA 2021 G G A S V G G S I L G S S A S M D T K G 6063 GGTGGAGCTTCCGTCGGAGGCAGTATATTGGGAAGTAGCGCATCCATGGATACAAAAGGC 2041 N L R S Y G S G F G T W T I A G A G P N 6123 AACCTTAGATCCTATGGCAGTGGTTTCGGGACTTGGACCATCGCGGGCGCAGGCCCTAAT 2061 E A G D V Q V I G D F G G C T A S N S Y 6183 GAAGCTGGAGATGTTCAAGTTATAGGTGATTTTGGAGGGTGCACTGCATCAAACAGTTAC 2081 N G L D L D S H P T I E A R R Q N V E F 6243 AATGGTCTGGATTTGGACAGCCATCCCACTATAGAAGCGCGCCGTCAGAATGTTGAGTTC


155

2101 P C P C G S N F C R H G G T C V S A D P 6303 CCCTGTCCGTGTGGCTCAAACTTCTGTCGTCACGGAGGCACTTGTGTAAGCGCCGACCCG 2121 P Y C L C P V G W S G P V C E S I V K D 6363 CCTTATTGTTTGTGCCCAGTGGGCTGGAGCGGTCCAGTATGCGAGAGTATCGTCAAAGAT 2141 P R P G Q R P S S R W A N P A V I A C I 6423 CCAAGGCCAGGCCAACGGCCGTCAAGCAGATGGGCAAATCCCGCAGTCATCGCTTGCATT 2161 L V I L L A I L V I I G A V L L K R R P 6483 CTCGTCATTCTCCTGGCAATTCTCGTCATAATTGGCGCCGTACTTCTAAAGCGCCGTCCC 2181 Q P A V V A V V E D G H V H D N V R P Y 6543 CAGCCAGCCGTTGTAGCCGTCGTCGAAGACGGACATGTACACGACAACGTGCGTCCTTAC 2201 H D E G A G E E D N F G Y D I S T L M K 6603 CACGACGAAGGCGCAGGCGAGGAGGACAACTTTGGCTACGACATTTCAACGCTTATGAAG 2221 Y T Y V E N G V A G T G G V G H G K F K 6663 TACACTTACGTGGAAAACGGTGTGGCAGGCACGGGCGGTGTGGGCCATGGAAAATTCAAG 2241 N G G S S G E E E F T A T E T K P L L Q 6723 AACGGTGGTAGTAGCGGTGAAGAAGAGTTTACTGCGACCGAGACTAAGCCGTTGTTACAA 2261 G A M P D D D L H F K T T T I T K R K V 6783 GGTGCAATGCCCGACGATGACTTGCATTTCAAGACGACGACCATCACTAAGCGAAAGGTG 2281 V H P D S I D V K Q F I D T R V S E A D 6843 GTACATCCCGATAGCATCGACGTTAAGCAGTTTATTGATACACGTGTTTCTGAGGCGGAT 2301 G E Y I L S I D E L H I Y R Y E G D D S 6903 GGCGAGTACATCCTGTCCATCGACGAACTACATATTTATCGTTATGAGGGAGATGATTCG 2321 D V D D L S E L G D S D E E P D E E E E 6963 GATGTGGATGATTTGAGTGAATTGGGAGACAGCGACGAAGAACCCGACGAGGAAGAAGAG 2341 Q E F A F L Q D W G K K F D N L N R I F 7023 CAAGAATTTGCTTTCTTGCAAGATTGGGGCAAGAAATTCGATAACCTCAATCGCATTTTC 2361 N E D E - F M F S D E L C F V H N V C A 7083 AACGAGGACGAGTAGTTCATGTTCAGCGATGAACTATGTTTTGTACATAATGTTTGTGCT 2381 F - N H F - P Q I S T P G A N T A F A Y 7143 TTCTAGAATCATTTCTAGCCACAGATTAGTACACCGGGTGCAAATACGGCCTTCGCATAC 2401 K K N F F - V N S S R K W T - N N R C K 7203 AAGAAGAACTTTTTCTAGGTAAATTCTTCTCGTAAGTGGACTTAGAATAACCGTTGTAAA 2421 F F L K R K A T L V L C G L H H L V - M 7263 TTTTTTTTAAAACGGAAGGCTACACTTGTCTTGTGCGGCTTACACCACCTCGTGTAAATG 2441 P R I S Y L T S F G T V D V I G - T - A 7323 CCTAGAATTTCATATTTGACATCATTTGGAACGGTGGACGTGATTGGTTAAACTTAAGCC 2461 K H F V - H K A F A K R L S G T L W - H 7383 AAACATTTCGTTTAGCATAAAGCATTTGCAAAAAGATTAAGTGGAACACTCTGGTAACAT 2481 E I K N F Y F V C G A F D N T L K E N - 7443 GAAATTAAGAACTTTTATTTTGTGTGTGGAGCATTTGATAACACTCTCAAAGAGAACTAG 2501 L S S Y Y T G L V L F I A M Y M Q S L V 7503 CTGTCTAGTTATTACACAGGGTTGGTTCTTTTTATCGCAATGTACATGCAGTCTTTGGTT 2521 F R V D N V I S L E I F Q W Q F I L A - 7563 TTTCGTGTTGATAACGTAATTTCCCTAGAAATATTCCAATGGCAGTTTATTCTTGCATAA


156

2541 W K C I L L - M R F S H C C R I Y F V W 7623 TGGAAATGCATTCTGCTATAAATGCGATTTTCTCACTGTTGTAGAATATATTTTGTATGG 2561 T L L I A F - Y R Y F Y S G E L Y F A S 7683 ACATTGTTAATAGCCTTTTAGTACCGTTATTTCTATTCTGGTGAGTTGTATTTCGCCAGC 2581 F K C - A V V N - M H I A C D P L P S W 7743 TTCAAATGTTAAGCCGTAGTTAACTGAATGCATATTGCATGTGATCCCTTGCCATCATGG 2601 R T - S T Q - A E H C A R S Q S F F G V 7803 CGGACCTAAAGTACTCAATAGGCAGAACACTGCGCTCGTTCACAGTCCTTTTTCGGCGTC 2621 I V S K K R - S R A - E S D V F E A Q T 7863 ATCGTCTCCAAGAAGAGATAATCGAGAGCTTAAGAAAGTGACGTTTTTGAGGCACAGACG 2641 E T R I E H F G H Q D S G P S Q I F K P 7923 GAGACCAGAATTGAACATTTCGGACACCAGGACAGTGGTCCCTCCCAGATTTTCAAACCA 2661 V V P R I F Y N I N V V V K R Q L K E E 7983 GTTGTCCCTAGGATATTTTACAATATAAATGTGGTCGTTAAAAGACAACTTAAAGAGGAA 2681 N S S L P L S V R G S K T S - A Q A R - 8043 AACAGCTCACTTCCGCTTTCCGTTCGTGGCTCAAAAACGTCGTGAGCTCAAGCTCGCTAA 2701 L Y Q C V A N I K P E I K C F H - F - S 8103 TTATACCAGTGCGTCGCAAACATCAAACCTGAAATTAAATGTTTTCATTGATTTTAAAGC 2721 Q E Y L L I L I K I S R - D L S R - P S 8163 CAAGAATATTTACTTATCCTAATTAAAATTTCGCGTTAAGATCTTTCTCGATAGCCGTCT 2741 N K - - T S - D S I N F S N F - R T R A 8223 AACAAGTGATGAACGAGTTAAGATTCAATTAATTTCAGTAATTTTTGACGAACGAGAGCT 2761 K I - F V V F S V S - Y P F L I V F H - 8283 AAGATTTGATTCGTCGTTTTCTCCGTATCTTGATATCCTTTTTTAATAGTTTTCCACTGA 2781 C R I Y T V F W K F K S A V - R E K P A 8343 TGTAGAATTTATACTGTTTTCTGGAAATTCAAATCTGCAGTTTGAAGGGAAAAACCGGCA 2801 L G R Q D A F Y T Q Y A F H C S T A C - 8403 CTCGGAAGGCAAGATGCATTTTATACCCAGTATGCATTCCATTGTTCTACAGCTTGTTGA 2821 A S E I V I A C L D G N V H - L G L I C 8463 GCAAGTGAAATAGTTATTGCATGTTTAGATGGAAATGTACACTAGTTGGGGCTAATATGC 2841 R H L H S L T Y C C S V V R Y F S M F L 8523 CGCCACCTTCATTCGTTGACGTATTGTTGCTCGGTCGTTAGATATTTTTCTATGTTCCTT 2861 L L C V I I - K Y M K P R L L V K I L C 8583 CTTTTATGTGTAATAATTTAAAAGTACATGAAACCTAGATTGCTAGTCAAGATACTTTGC 2881 - A F - N N Y E K G F - I 8643 TGAGCTTTTTAGAATAACTATGAAAAAGGCTTTTGAATA


157


Supplementary Figure 5.2 Boxshade Alignment of Representative Dachsous C-‐terminal domains. Conservation of the Catenin Binding Domain (CBD; Blue) but not the Juxta-‐Membrane Domain (JMD) distinguishes the Dachsous cytoplasmic region from that of TypeI-‐IV / “classical” cadherins. The Extracellular region in each of the proteins included in the above alignment contains only Cadherin repeat (EC) domains consistent with the canonical structure of Dachsous. Shading demonstrates >50% consensus to AmDachsous (Black –conservation, Grey – conservative substitution).

Genbank Accesions: Dm_Ds NP_523446.2; Mm_Ds1 NP_001156415.1; Dr_Ds XP_001921284.2


158


Supplementary Figure 5.3 Multiple sequence alignment of cadherin cytoplasmic domains used for maximum likelihood analysis presented in Figure 5.4. Analysis is based on the highly conserved Juxta-‐Membrane Domain (JMD) and Catenin Binding Domain (CBD). Shading corresponds to consensus of 30% or more. Analysis is based on 148 positions of 29 sequences.


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(Hs) Homo sapiens; (Mm) Mus Musculus; (Bm) Bombyx mori; (Gb) Gryllus bimaculatus; (Dm) Drosophila melanogaster; (Af) Artemia franciscana; (Lv) Lytechinus variegates; (Sp) Strongylocentrotus pupuratus; (Ap) Asterina pectinifera; (Se) Sexostrea echinata; (BS) Botryllus schlosseri; (Ci) Ciona intestinalis; (Cj) Cardina japonica; (Le) Ligia exotica; (At) Achaearanea tepidariorum

Genbank accession numbers: Hs_CDH1 AAI46663.1; Mm_Cdh1 AAH98501.1; Hs_CDH9 AAI09385.1; Mm_Cdh9 NP_033999.1; Bm1-‐Cadherin BAD91049.1; Gb1-‐Cadherin BAD91050.1; Dm_shg NP_476722.1 ; Af1-‐Cadherin BAD91054.1; Fc1-‐Cadherin BAD91052.1; Bm2-‐Cadherin BAD91048.1; Gb2-‐Cadherin BAD91051.1 ; Dm_CadN AAZ66476.1; Af2-‐Cadherin BAD91055.1; LvG-Cadherin AAC06341.1; SpG-‐Cadherin XP_001175592.1; Ap-‐Cadherin BAC06834.1; SE-Cadherin BAC06836.1; Bs-‐Cadherin AAB88396.1; 1_Ci0100151854 NP_001121583.1; Cj-‐Cadherin BAD91056.1; LE-Cadherin BAD91057.1; At-‐Cadherin BAD91058.1; Dm_cadN2 ABI31322.2


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Supplementary Figure 5.4 Boxshade alignment of AmFlamingo protein with representative metazoan Flamingo (CELSR) orthologues demonstrates the degree of conservation. Highly conserved regions correlate with functional domains (coloured boxes). Although the current model of AmFlamingo is truncated at the N-‐terminus, all diagnostic C-‐terminal domains are present supporting the designation of this model as the Acropora orthologue of Flamingo, which was initially established by JCUSMART annotation. Shading demonstrates >50% consensus to AmFlamingo (Black –conservation, Grey – conservative substitution). Signal sequences have been removed from the N-‐terminus of each protein.

Genbank Accesions: Mm_CELSR2 NP_001004177.2; Dr_CELSR1a XP_002661517.2; Dm_Flamingo NP_724962; Nv_Flamingo JGI proteinID 84228


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Supplementary Figure 5.5 AmVangl 1 -----MPSHRSDRGHRDRDHDH------------------------NDKRERQRLADDTG Nv_Vangl 1 ------------------------------------------------------------ Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 1 ------------------------------------------------------------ Dm_Vang 1 MENESVKSEHSGRSRRSRNHNNNGGGGGGGGGGGGGSVNNGYHRERDRSRHSHRSTHSSK Mm_Vangl1 1 ---MDTESTYSGYSYYSSHSKKSHRQGER-TRERHKSPRNKDGRGSEKSVTIQAPAGEPL Dr_Vangl1 1 ---MDTDSIHSGYSHHSNRSRGSNKQSERSSRDRHKPHSRDSSSRSDKNVTISATSVQPQ Xl_Vangl1 1 ---MDTESNHSGYSHHS--------------RGRQQRERRHDGQKS-----VNVTVGQPL AmVangl 32 SQEILDEG---------------------IIQVQVIPQDDNWGETATSITETATSIVT-L Nv_Vangl 1 -------------------------------------QDDNWGETATTITETATSIVT-L Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 1 -MDDFDDG---------------------VIEVQVIPQDDNWGETATTITETATSVYSDL Dm_Vang 61 SAKGFQRGDMAPYQTSVNMTGDGSHDGQEVIEVQILPQDENWGENTTAVTGNTSEQSISM Mm_Vangl1 57 LANDSAR------------------------TGAEEVQDDNWGETTTAITGTSEHSIS-Q Dr_Vangl1 58 QPDQSS--------------------------APTDAQDDNWGETTTAVTGTSDHSLS-Q Xl_Vangl1 39 LGEDSGRG-----------------------EEDEEGQDDRWGETTTAVT--SERSAS-F AmVangl 70 SETSLSEVGKDKRSFSCSFLR-HIKLVPAAILSICAVVSPILFVVFPLV----------- Nv_Vangl 23 SEGDLSEIGDEKKGLAFGVCR-HFKLIPAAIISLIALASPILFLVIPVV----------- Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 39 DDTDPFGEEIEKHRFQCPNSKQALNILLSVILALMAVVSPIAFVIIPNV----------- Dm_Vang 121 EDINNMWHRESDKGFSFACRR-YVESSFYFLLGCGAFFSPVAMVVMPYVGFFPSAFDHPE Mm_Vangl1 92 EDIARISKD-MEDSVG-LDCKRYLGLTVASFLGLLVFLTPIAFILLPQILWR-------- Dr_Vangl1 91 EDLAGFGKD-TEGPVEKLNCIRFLPLALTLLLGLLVLVTPLSFLILPQLMWP-------- Xl_Vangl1 73 EDLAPVGTS-RIPDTVKPNTWIMIGFYLSCALGLFAILTPPAFIVLPQVFWG-------- AmVangl 118 -TWKP----EPCGYDCDGVFISFAVKELLLVIAIWALYFRRSRATMPRIFVLRVGMMLLG Nv_Vangl 71 -LWKP----PMCGIECDGLFLSLAVKELILVVGIWALYFRKSRVTMPRVFVLRVGVLVLA Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 88 -SSSLKVTGEECGSVCEGLYITIAIKEIVLIFGLWALYIRPNKVDLPRLNVFKVGMMVLV Dm_Vang 180 ITQTVRTQLLACSEQCKGQLVSLAARLLLLAIGLWAVFMRRTSATMPRIFLYRALVLLLV Mm_Vangl1 142 ------EELKPCGAICEGLLISVSFKLLILLIGTWALFFRKQRADVPRVFVFRALLLVLI Dr_Vangl1 142 ------ERLQTCGTACEGLFLSLAFKLLILLLAGWALFMRPTRASLPGIAVFRALLGLLT Xl_Vangl1 124 ------SQLEPCGVVCEGLYISLAFKLLLLFLASWAVFLRPPRCDLPRLVEFHALLIVLL AmVangl 173 FVVIVCFWLFYGLRIIA---------KQTSQFSDILRFAVNFTDTMLFLHYASLVVLWVR Nv_Vangl 126 FLVLVCYWLFYGLRIIA---------KRGQDFGEILKFAVTLVDSLLFLHYLSVIVLWLR Hm_Vangl 1 ------------------------------------------------------------ Ch_Vangl 147 YVSIICFWLFYSIRMIG---------NGTNQY-LTVSFASSFLDAMLFLHYMALVLMWIR Dm_Vang 240 TICTFAYWLFYIVQVTNGAKIVVETGGDAVDYKSLVGYATNFVDTLLFIHYVAVVLLELR Mm_Vangl1 196 FLFVVSYWLFYGVRILD---------SRDQNYKDIVQYAVSLVDALLFIHYLAIVLLELR Dr_Vangl1 196 LLLLLSYWLFFGVRILD---------SQDDNYQGIVQFAVSLVDALLFIHYLAVVLLEIR Xl_Vangl1 178 FFFLSSYWLFYGVRVLG---------PQEKNLLGVVEYAVSLVDALIFIHYLALILLELR AmVangl 224 QTDPLFTLKVIRSTDGESKFYNIGPLSIQRTAAFVLEQYYKDFPEYNPFLMQT---PART Nv_Vangl 177 QSEPMFTLKVIRSTDGVNKFYNVGLLSIQRAAIYVLEQYYKDFPEYNPYMMSV---PARS Hm_Vangl 1 -HENIYNVSVIRNVDGSRKHYMIGQCSIQKAAVNVLEKYYIDFNEYNPYLPRP---TSRS Ch_Vangl 197 PMEKVYTVSIIRNVDGMRRYYNIGQSSIQKAAVFCLERYYIDFTEYNPYMPRP---QSRT Dm_Vang 300 HQQPCYYIKIIRSPDGVSRSYMLGQLSIQRAAVWVLQHYYVDFPIFNPYLERIPISVSKS Mm_Vangl1 247 QLQPMFTLQVVRSTDGESRFYSLGHLSIQRAALVVLENYYKDFTIYNP-NLLT---ASKF Dr_Vangl1 247 HLQPCFSLCVVRSTDGETHHYNMGQLSIQRAALVTLEHYYKDFTVHNP-ALLT---AAKS Xl_Vangl1 229 QLQPFFYLKVMRSSDGEMRFYSLGTLSIQRAAMFVLENYYKDFPVFRP-DPPV---VRKR AmVangl 281 SHKQFS-SLTFYDIDGK---QND--KVNPRARAILTAASTRRRDPARNDRFYEEAEFERK Nv_Vangl 234 SAKQFS-TLKFYDIDGK---LQDNSKVNPRTRAIITASSQGRRNPGRNDRFYEEAEFERK Hm_Vangl 57 KINKFS-NIKFYDLDNKMDMGNGKNFSQQASKAVIAAAALGRRKEGRNDRFYEELEIDRR Ch_Vangl 254 KINKLA-GLKIYDLDGK---GDG-TLTQQASKAFIAAAAAGRRKEGRNDRFYEEQELDRR Dm_Vang 360 QRNKISNSFKYYEVDGV-----SNSQQQSQSRAVLAANAR-RRDSSHNERFYEEHEYERR Mm_Vangl1 303 RAAKHMAGLKVYNVDGP-------SNNATGQSRAMIAAAARRRDSSHNELYYEEAEHERR Dr_Vangl1 303 RAAKHLAGLKVYNVDGAG------SDAATAQSRAKMAAAARQRDTSHNELYYEEAEHDRR


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Xl_Vangl1 285 --TRHNNHPQVYSVDGP-------NSSTVSQSQTLISTSTN-----YKERYYEEAEHARK AmVangl 335 IKRRKARLFNACEEAFGHIKRVQLE---RGPCVPMDPEETAQSLFPSFARPLQKYLRTVR Nv_Vangl 290 LKRRKARLVATCEEAFGHIKRVQLE---RGPSVPMDPEETAQSLFPSFARPLQKYLRTVK Hm_Vangl 116 VRKRKARLVAAAEEAFGHIARLNAFDTSKKANGSMNPDEAAQAIFPTLARPLQKYLRTTR Ch_Vangl 309 IRKRKARLVAAAEEAFGHVARLNAFDSSKKADGSMDPEEAAQAVFPTLARPLQKYLRTTR Dm_Vang 414 VKKRRARLITAAEEAFTHIKRIHNEP---APALPLDPQEAASAVFPSMARALQKYLRVTR Mm_Vangl1 356 VKKRRARLVVAVEEAFIHIQRLQAEEQQKSPGEVMDPREAAQAIFPSMARALQKYLRTTR Dr_Vangl1 357 VRKRKARLVVAVEEAFTHVRRLQDEEKKKPPGDEMDPREAAQAIFPSMARALQKYLRTTR Xl_Vangl1 331 VRRRKARLVMSVHDAFSQLKRLVQQDEEWKLPNTLHPREAAQSIFPLIAQSLQRYLRSTQ AmVangl 392 LHHRYTMDGIIKHLAHCLTFDMTPKAFLERYLKDQPCGEFT-TKRRCQSWSLVCEEQVTK Nv_Vangl 347 QHHRHNMDAILKHLAHCLTFDMSPKAFLERYLNDQPCIEYTGASVGPQSWSLVCEEQVTS Hm_Vangl 176 QQLHYPLESIMKHLAHCIMFDMSAR----------------------------------- Ch_Vangl 369 QQLYYPLESILKHLAHCISYELSSKAFIERYTCDQPCISYV-GYDGRQEWTLVTDSSPTR Dm_Vang 471 QQPRHTFESILKHLAHCLKHDLSPRAFLEPYLTESPVMQSEKERRWVQSWSLICDEIVSR Mm_Vangl1 416 QQHYHSMESILQHLAFCITNSMTPKAFLERYLSAGPTLQYDKDRWLSTQWRLISEEAVTN Dr_Vangl1 417 QQHCHSMDSIQAHLAFCITNNMTPKAFLESYLTAGPTLQYGRESSQTRHWTLVSEASVTS Xl_Vangl1 391 QAHLHSMEGIIQHLTLCLTHRMSPQAFLEQYLHPGPPVQYPSNPYG--VWTLVSEESVTS AmVangl 451 TLSSSTVFQLKGDDLSLVVTVRRLPYLDISEDEFDFESNKFVLRLNSETSV Nv_Vangl 407 GLSNSTVFQLKTEVLSLVVTVNNIPHFVVDEDEFDFENNKFVLKLNSETSV Hm_Vangl --------------------------------------------------- Ch_Vangl 428 QLNEGTVFQLKHEDISLVIAVSKLPVFAMSELPFNGDSNRFVFRLNSETSV Dm_Vang 531 PIGNECTFQLIQNDVSLMVTVHKLPHFNLAEEVVDPKSNKFVLKLNSETSV Mm_Vangl1 476 GLRDGIVFVLKCLDFSLVVNVKKIPFIVLSEEFIDPKSHKFVLRLQSETSV Dr_Vangl1 477 PLRNGSEFQLKSSDFSLVVTSKTIPHLKLSEEYVHPKSHKFVLQLQSETSV Xl_Vangl1 449 PLRSDLTFCLQCSDTQLLVTVCGIPFLKLSETFISPNSHRLIVSSKPETNL

Supplementary Figure 5.5 Boxshade alignment of full length Am_Van Gogh Like (AmVangl) protein with representative metazoan Van Gogh orthologues demonstrates a reasonable degree of sequence conservation. Shading demonstrates >50% consensus to AmVangl (Black –conservation, Grey – conservative substitution). The degree of conservation supports the assignment of Acropora protein model Contig17842 as the Acropora orthologue of Van Gogh, which was initially established by BLASTp analysis (E-‐value = 1E-‐100).

Genbank Accesions: Mm_Vangl1 NP_808213.2; Dr_Vangl1 NP_991313.1; Xl_Vangl1 NP_001089844.1; Dm_Vang NP_477177.1


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Supplementary Figure 5.6 Am_Dsh 1 -------MEETKVIYHIDDEDTPYLVKLPKSPTDVTLSDFKNVL-NRPS--YKFFFKSMD Nv_Dsh 1 -------MDETKVIYHIDDEDTPYLVKLGKTPDIVTLGDFKNVL-NRPN--YKFFFKSMD Xt_Dsh1 1 -------MAETRIIYHIDEEETPYLVKLPVPPEKVTLADFKNVLSNRPVHHYKFFFKSMD Mm_Dsh1 1 -------MAETKIIYHMDEEETPYLVKLPVAPERVTLADFKNVLSNRPVHAYKFFFKSMD Dr_Dsh1 1 -------MAETKIIYHIDEEETPYLVKLSVAPEKVTLADFKNVLSNRPVNSYKFFFKSMD Dm_Dsh 1 MDADRGGGQETKVIYHIDDETTPYLVKIPIPSAQVTLRDFKLVL-NKQNNNYKYFFKSMD Ch_Dsh 1 -----MAEKETKIIYHVDDEETPYLVKIPKPPDQVTLGDFKSVI-NRPN--FKFFFKSMD Hv_Dsh 1 ----MSVPDETKIIYHVDDEETPYLVKIPKSPSLVTLGDFKNVI-NRPN--YRFFFKSMD Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 51 DDFGVVKEEISDEEMNLPCFNGRVVAWLVTSDTSSVSDTG--RDDQQSVISG-TSDLHPP Nv_Dsh 51 DDFGVVKEEISDDETHLPCFNGRVVAWLVTSDTSSVSGGGDLASDQQSIISASPSDLHVP Xt_Dsh1 54 QDFGVVKEEISDDNAKLPCFNGRVVSWLVLAE-SSHSDGG-------SQSTESRTDLPLP Mm_Dsh1 54 QDFGVVKEEIFDDNAKLPCFNGRVVSWLVLAE-GAHSDAG-------SQGTDSHTDLPPP Dr_Dsh1 54 QDFGVVKEEVSDDNAKLPCFNGRVVSWLVLAE-SSHTDGM-------SVCTDSHTEHPPP Dm_Dsh 60 ADFGVVKEEIADDSTILPCFNGRVVSWLVSADGTNQSDNC-------SELPTSECELGMG Ch_Dsh 53 DDFGVVKEEIIDDDAPLPCFNGRVVSWVVPPE-DGSCDGQ-------SQHSGDGIFVPVQ Hv_Dsh 54 DDFGVVKEEIIDDDTILPCFNGRVVSWVVPPEEGSNDCNS-------------------T Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 108 MPRTVGIGDSRPPSFHHGGQSTAGDFDSEAESTVSSRRG--SRRREKHSIN-RNEVIHG- Nv_Dsh 111 VQRTGGIGDSRPPSFHHGGHSTAGDFDSEVDSTISSRRGGSSRRKERHSSRGIGHRSHR- Xt_Dsh1 106 IERTGGIGDSRPPSFHPNASSSRDGLDNETGTDSVVSHRRDRHRRKNRETHDDVPRINGH Mm_Dsh1 106 LERTGGIGDSRPPSFHPNVASSRDGMDNETGTESMVSHRRERARRRNR---DEAARTNGH Dr_Dsh1 106 LERTGGIGDSRPPSFHANAVNSRDGLDTETGSEPVLRHRRERERERTRRRRDDSERV--- Dm_Dsh 113 LTNRKLQQQQQQHQQQQQQQQQQHQQQQQQQQQQVQPVQLAQQQQQQVLHHQKMMGNP-- Ch_Dsh 105 SSNSNISRSSTMRSKERVQDSDAESIVSRRSSRSRSSRKYESDADRRSRRSHREHRN--- Hv_Dsh 95 NSGESALKQGRSNVSKKDRFPDNESVCSHKSVRSNRSRRLDTENENRSKVRNHRDHRSD- Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 164 ----RRRHESDRYESAS-VMSSDIETTSYQDSSDDQSSVSRFSSTTEGSQ--LLRGRHRR Nv_Dsh 170 ----SGVRQGERFDTTS-TMSSDIETTSYMDSSDDQ---SRFSSTTEGSH--LLSGRHRR Xt_Dsh1 166 PKLDRIRDPGG-YDSASTVMSSELESSSFVD-SDEDENTSRLSSSTEQSTSSRLIRKHKR Mm_Dsh1 163 PRGDRRRDLGLPPDSASTVLSSELESSSFID-SDEEDNTSRLSSSTEQSTSSRLVRKHKC Dr_Dsh1 163 -----VRDSAMGCDSGS-IMSSELESSSFID-SEDEEDASRLSSSTEQSSSFQLMKRHKR Dm_Dsh 171 ----LLQPPPLTYQSAS-VLSSDLDSTSLFG-TESELTLDRDMTDYSSVQ--RLQVRKKP Ch_Dsh 162 ---------YDQYDSAS-MMSSDLETTSFVD-SEEESQMS---SATESSR--YVGGNKRR Hv_Dsh 154 ------------FDSSS-IMSSDLETTSFVD-SDEESRISSTTENSKYGG-----ARQKR Hm_Dsh 1 ------------------------------------------------------------ Am_Dsh 217 RRRRPRMPRVQRCSSFSTITESTMSLNIITVTLNMEKVNFLGISIVGQSNKRGDGGIYVG Nv_Dsh 220 RRRRPRMPRVQRCSSFSTITESTMSLNIITITLNMDKVNFLGISIVGQSNKKGDGGIYVG Xt_Dsh1 224 RRRKQKMRQIDRSSSFSSITDSTMSLNIITVTLNMEKYNFLGISIVGQSNDRGDGGIYIG Mm_Dsh1 222 RRRKQRLRQTDRASSFSSITDSTMSLNIITVTLNMERHHFLGISIVGQSNDRGDGGIYIG Dr_Dsh1 216 RRRRHKVAKIDRSSSFSSITDSTMSLNIITVTLNMEKYNFLGISIVGQSNDRGDGGIYIG Dm_Dsh 223 QRRKKRAPSMSRTSSYSSITDSTMSLNIITVSINMEAVNFLGISIVGQSNRGGDGGIYVG Ch_Dsh 206 RRRRQRMPRVERCSS--------------------------------------------- Hv_Dsh 195 RRRKQRMPRVERCSSFSTITESTMSLNIITVVLNMDKINFLGISIVGQANKKGDGGIYVG Hm_Dsh 1 ------------------IYE----------------------------KKK-------- Am_Dsh 277 SIMKGGAVDLDGRIEPGDMLLQVNDVNFENMSNDDAVRVLREMVHKPGPITLTVAKCWDP Nv_Dsh 280 SVMKGGAVDLDGRVEPGDMLLQVNDVNFENMSNDDAVRVLREMVHKPGPITLTVAKCWDP Xt_Dsh1 284 SIMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAVRVLREIVSKPGPISLTVAKCWDP Mm_Dsh1 282 SIMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAVRVLREIVSQTGPISLTVAKCWDP Dr_Dsh1 276 SIMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAVRILREIVSKNGPISLTVAKCWDP Dm_Dsh 283 SIMKGGAVALDGRIEPGDMILQVNDVNFENMTNDEAVRVLREVVQKPGPIKLVVAKCWDP Ch_Dsh ------------------------------------------------------------ Hv_Dsh 255 SVMKGGAVDADGRIEPGDMILAVGDVNFENMSNDDAVRVLRECVHKPGPIMLTVAKCWDP Hm_Dsh 7 SVLQS-------------TIPGVGDVNFENMSNDDAVRXPQRVRTQTWSHHADGAKCWDP Am_Dsh 337 TPKGYFTLPPSEPVRPIDTSAWVQHTTAMNQFGN---PVVQYGKQPNSQSITTMTSTSSS Nv_Dsh 340 TPKGYFTLPHSDPVRPIDTSAWVQHTTAMNQFAAQQGQFVPPAKPGNSQSLSTMTSTSSS Xt_Dsh1 344 TPRSYFTIPRAEPVRPIDPAAWITHTSALTGAYPRY------------------------ Mm_Dsh1 342 TPRSYFTIPRADPVRPIDPAAWLSHTAALTGALPRYGTSPCS-------------SAITR Dr_Dsh1 336 SPRSYFTIPRAEPVRPIDPAAWISHTTALTGSYPQN------------------------ Dm_Dsh 343 NPKGYFTIPRTEPVRPIDPGAWVAHTQALTSHDSIIAD---------------------- Ch_Dsh ------------------------------------------------------------ Hv_Dsh 315 NPKGYFTVPRNDVTRPIDPAAWMQHSEAVRASGGLLGGRTGS---------PSMSTMTST Hm_Dsh 54 NPKGYFTVPRNDVTRPIDPQLGCNIPEAVRASGGLLGGRTGS---------PSMSTMTST


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Am_Dsh 394 MTSSLPESDIPGYTDIPDGEGLSLSTDMTTVVKAMAAPDSGLDVRDRMWLKITIPNAFIG Nv_Dsh 400 MTSSLPESDR--YTEIPEGEALTINTDMSTVVKAMAAPDSGLDIRDRMWLKITIPNAFIG Xt_Dsh1 380 --------------EQEDSPLSV-KSDMATIVKVMQLPDSGLEIRDRMWLKITISNAVIG Mm_Dsh1 389 TSSSSLTSSVPGAPQLEEAPLTV-KSDMSAIVRVMQLPDSGLEIRDRMWLKITIANAVIG Dr_Dsh1 372 --------------EFDELPLTMGKTDMATIVKVMQLPDSGLEIRDRMWLKITIANAVIG Dm_Dsh 381 --------------IAEPIKERLDQNNLEEIVKAMTKPDSGLEIRDRMWLKITIPNAFIG Ch_Dsh ------------------------------------------------------------ Hv_Dsh 366 SDSLTSSIPESDRYIDHSDLGLSIKTDMNTVIKVMASPDSGLVVRDRMWLKITIPNAFIG Hm_Dsh 105 SDSLTSSIPESDRYIDHSDLGLSIKTDMNTVIKVMASPDSGLVVRDRMWLKITIPNAFIG Am_Dsh 454 SDVVDWLYSRVDGFQDRREARKYACNLLKSGFIRHTVNKITFSEQCYYVFGDLCGNMAAL Nv_Dsh 458 SDVVDWLYTHVEGFMDRREARKYACNLLKAGYIRHTVNKITFSEQCYYVFGDLCGNLAAL Xt_Dsh1 425 ADVVDWLYTHVEGFKERREARKYASSMLKHGYLRHTVNKITFSEQCYYVFGDLCGNVAAL Mm_Dsh1 448 ADVVDWLYTHVEGFKERREARKYASSMLKHGFLRHTVNKITFSEQCYYVFGDLCSNLASL Dr_Dsh1 418 GDVVDWLYSRVEGFKDRRDARKYASSLLKHGYLRHTVNKITFSEQCYYTFGDLCQNMATL Dm_Dsh 427 ADAVNWVLENVEDVQDRREARRIVSAMLRSNYIKHTVNKLTFSEQCYYVV-NEERNPNLL Ch_Dsh ------------------------------------------------------------ Hv_Dsh 426 SDLVDWLFAHVDGFQDRRDARKYASKLLKAELIKHTVKKVTFSEQCYYVFGDLNSSMKGL Hm_Dsh 165 SDLVDWLFAHVDGFQDRRDARKYA------------------------------------ Am_Dsh 514 SIAEHEGDG--DTDTLGPLPVHPHGTPWMAG-PPSYGYVQMPPYPTAPPPVMVDGQTPGY Nv_Dsh 518 SLNENEGD----QDTLGPLPPQQQAIPWMTGGPPAYGYHQMPSYPPSMAP--SDAKTPGY Xt_Dsh1 485 NLNEGSSGT-SDQDTLAPLPHPAA-PWPLG-QGYSYQYPLAPPCFPPTYQEPGFSYGSGS Mm_Dsh1 508 NLNSGSSGA-SDQDTLAPLPHPSV-PWPLG-QGYPYQYPGPPPCFPPAYQDPGFSCGSGS Dr_Dsh1 478 NLNEGSSGAGSEQDTLAPLPPPSTNPWPMGGQPFPYPPFTAPPAFPPGYSDPCHSFHSGS Dm_Dsh 486 GRGHLHPHQLPHGHGGHALSHADTESITSDIGPLPNPPIYMPYSATYNPSHGYQPIQYGI Ch_Dsh ------------------------------------------------------------ Hv_Dsh 486 ILDDFESNELPLPHSSHCGSWVGNNQSSQYVMQMGGMPLIHPGMIVRGSPSITSSSEMGV Hm_Dsh ------------------------------------------------------------ Am_Dsh 571 GQSLY------------------------------------------------------- Nv_Dsh 572 AQSFYSGTSQHSGSQSPQSASG-------------------------------------- Xt_Dsh1 542 AGSQHSEGK----STYSGVFLP-------------------------------------- Mm_Dsh1 565 AGSQQSEGSKSSGSTRSSHRTP------------GREERRATG--------AGG-----S Dr_Dsh1 538 AGSHHSEGSRSSSSNPSIGRIQRAVQ--------REKERKSTGSESDSGKRAGGRRVERS Dm_Dsh 546 AERHISSGSSSSDVLTSKDISA-------------------------------------- Ch_Dsh ------------------------------------------------------------ Hv_Dsh 546 APSASNYSNVPPNYMDMYSQIYQPTFNPYFAHNSGSQGSQHTGSNSGSGGSFRGHQLNQQ Hm_Dsh ------------------------------------------------------------ Am_Dsh ------------------------------------------------------------ Nv_Dsh 594 SSRKGSERDRGAGDDHKSSGGSSSERSGS------------------------------- Xt_Dsh1 ------------------------------------------------------------ Mm_Dsh1 600 GSESDHTVPSGSGSTGWWERPVSQLSRGSSPRSQA-------------------SAVAPG Dr_Dsh1 590 ASQLSHRSHALSSRSHTHSRVPSQHSRTSFSYSHAPFTKYGHTSCALSERSHASSYGPPG Dm_Dsh 568 -SQSDITSVIHQANQLTIAAHGSNKSSGS------------------------------- Ch_Dsh ------------------------------------------------------------ Hv_Dsh 606 QQLVMQQQHYDYDQATIRSSSSSDKSRGSKSSLESKRVLLATTDHLASDKIISPDTVSLN Hm_Dsh ------------------------------------------------------------ Am_Dsh ----------------------------------------------------------- Nv_Dsh ----------------------------------------------------------- Xt_Dsh1 ----------------------------------------------------------- Mm_Dsh1 641 LPP---LHPLTKAYAVVGGPPGGPPVRELAAVPPELTGSRQSFQKAMGNPCEFFVDIM- Dr_Dsh1 650 LPPPYSLARLTPKGAVCSGPPGAPPVREMGAIPPELTASRQSFQHAMGNPCEFFVDIM- Dm_Dsh 596 ----------SNRGGGGGGGGGGNNTNDQDVSVFNYVL--------------------- Ch_Dsh ----------------------------------------------------------- Hv_Dsh 666 GIRESNISNNNYEKGENARNNTSREFGRLDTIPREISASKQSFRMAMGNSSNEFFVDVM Hm_Dsh -----------------------------------------------------------

Supplementary Figure 5.6 Boxshade alignment of full length Am_Dishevelled (Am_Dsh) protein with representative metazoan Dishevelled orthologues demonstrates a high degree of sequence conservation. Shading demonstrates >50% consensus to Am_Dishevelled (Black –conservation, Grey – conservative substitution). The degree of conservation supports the assignment of Acropora protein model Contig1475 as the Acropora orthologue of Dishevelled, which was initially established by BLASTp analysis (E-‐value = 1E-‐136). This Acropora sequence has since been verified by sequencing of cloned DNA (S.Ukolova, unpublished).

Genbank Accesions: Hm_Dsh XP_002162745; Hv_Dsh AAG13667.1; Dm_Dsh NP_511118.2; Mm_Dsh1 NP_034221.3; Dr_Dsh1 XP_698367.5; Xt_Dsh1 NP_001116886.1


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172

Supplementary Figure 6.1 Multiple sequence alignment of α-‐Integrin proteins used for maximum likelihood analysis presented in Figure 6.1. Analysis is based on extensively edited alignment of 843 positions. Shading corresponds to consensus of 30% or more between all sequences included in the alignment.

integrin sequences α7, α6, α3, α5, αV, α8 and αIIb -‐Homo sapiens; integrin αPS1-‐3 -‐Drosophila melanogaster; (Sp) Strongylocentrotus pupuratus; integrin-‐α Pat2 and integrin-‐α Ina1 -‐Caenorhabditis elegans; (Am) Acropora millepora; (Nv) Nematostella vectensis; (Pc) Podocoryne carnea; (Gc) Geodium cydonium; (Lv) Lytechinus verigatus

Genbank accession numbers: LvSU2 AAC23572; SpαP AAD55724; DmPS2 P12080; Itgα5 P08648; ItgαV P06756; Itgα8 P53708; Itgαllb P08514; Itgα6 P23229; Itgα7 Q13683; Itgα3 P26006; ItgαPS3 O44386; AmItgα1 EU239371; Itgα4 P13612; Itgα9 Q13797; NvItgα1 XP_001641435; ItgαPat2 P34446; ItgαPS1; Q24247; PcItgα AAG25993; ItgαIna1 Q03600; GcItgα CAA65943


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176

Supplementary Figure 6.2 Multiple sequence alignment of β-‐Integrin proteins used for maximum likelihood analysis presented in Figure 6.2. Analysis is based on extensively edited alignment of 991 positions. Shading corresponds to consensus of 30% or more between all sequences included in the alignment

(Hs) Homo sapiens; (Dm) Drosophila melanogaster; (Ce) Caenorhabditis elegans; (Sp) Strongylocentrotus pupuratus; (Am; ItgbCn1) Acropora millepora; (Nv) Nematostella vectensis; (Sd) Suberites domuncula; (Gc) Geodium cydonium; (ItgβPo1) Ophlitaspongia tenuis;

Genbank accession numbers: HsItgβ3 P05106; HsItgβ5 P18084; HsItgβ6 P18564 ; HsItgβ2 P05107 ; HsItgβ7 P26010 ; HsItgβ1 P05556 ; SpItgβG AAB39739 ; SpItgβL AAC28382 ; SpItgβC AAB39740 ; DmβPS P11584 ; CePat3 Q27874 ; AmItgβ2 EU239372 ; NvItgβ1 XP_001641468 ; NvItgβ2 XP_001627336 ; PcIntB AAG25994 ; ItgbCN1 AAB66910 ; NvItgβ3 XP_001637894 ; NvItgβ4 XP_001621822 ; OtItgβ1 AAB66911 ; SdItgβ CAB38100 ; GcItgβ CAA77071 ; HsItgβ4 P16144 ; Dmβ-‐nu Q27591 ; HsItgβ8 P26012

Appendix B JCUSMART survey of the cnidarian adhesome

177

Sub-Family Organism Sequence Id BLAST e_value HMMPFAM

Signal Peptide TM Conclusion Notes

Flamingo Nvec jgi|Nemve1|84228|e_gw.9.5.1 CELSR 0CA (8x), EGF (2-3), LamG, EGF, LamG, EGF (3x), HormR_1, GPS, 7tm_2 No 1 Flamingo start and stop codons present

Flamingo Acropora Contig3665cadherin EGF LAG seven-pass G-type receptor 1 [... 2.00E-98 HormR, GPS, 7tm_2 No 7 flamingo c-term best hit to Nvec flamingo; overlaps Contig8737

Flamingo Acropora Contig8737cadherin, EGF LAG seven-pass G-type receptor 2 (f... 1.00E-171 CA, EGF (2x), LamG, EGF,LamG, EGF(2x) No No flamingo middle best hit to Nvec flamingo; overlaps Contig3665

FAT Nvec jgi|Nemve1|197466|fgenesh1_pg.scaffold_5000272 FAT3 SP, CA (29x), EGF, LamG, EGF, LamG, EGF Yes No FAT 4 like start and stop codons presentFAT Nvec jgi|Nemve1|20161|gw.67.8.1 FAT1 CA (34x), LamG, EGF (3x), TM No 1 FAT 1 No start and no stop codonFAT Nvec jgi|Nemve1|20073|gw.193.26.1 FAT4 0 CA (11x), EGF (3x), LamG, EGF, LamG, EGF No No FAT 4, partial FAT4 has CA (25x) but the rest is identicial in structure. No start no stopCalsynetenin Nvec jgi|Nemve1|167342|estExt_gwp.C_860192 Calsyntenin 2 1.00E-84 CA (2x), CA (wobbly) LamG (wobbly) Yes 1 Calsyntenin 2 start and stop codons presentCalsynetenin Acropora Amil_rep_c147915 calsyntenin 1 [Danio rerio] 8.00E-79 CA (2x), LamG, TM No 1 CalsynteninCalsynetenin Acropora Contig31873 calsyntenin 1 [Danio rerio] 8.00E-76 CA, LamG Yes No Calsyntenin partialCatenin Binding Acropora Contig 6389 + Contig5394

Cj-cadherin [Caridina japonica] 1.00E-129

CA (6x) + CA (7x), EGF, LamG, EGF, LamG, EGF, TM, Cadherin_C No 1 Type III cadherin these two contigs overlap on the basis of DNA assembly

Catenin Binding Nvec jgi|Nemve1|239897|estExt_fgenesh1_pg.C_210083 FAT3 0 SP, CA (24x), EGF, LamG, LamG, EGF, TM, Cadherin_C Yes 1 Type III cadherin start and stop codons presentCatenin Binding Nvec jgi|Nemve1|244010|estExt_fgenesh1_pg.C_1040031 FAT3 0 SP, CA (30x), EGF, LamG, EGF, LamG,EGF, TM, Cadherin_C Yes 1

Type III cadherin Uli Technau's Endothelial Cadherin start and stop codons present

Catenin Binding Nvec jgi|Nemve1|239899|estExt_fgenesh1_pg.C_210088 No good hits EGF, TM, Cadherin_C (wobbly) No 1 Type III cadherin start and stop codons present combines with 239898 Extracellular region!Catenin Binding Nvec jgi|Nemve1|223294|fgenesh1_pg.scaffold_2546000001 Cadherin_C No No

Unknown - Classical Cadherin (partial) No start codon, stop codon present

Catenin Binding Hydra CL3352Contig1 No good hits Cadherin_C No No

Unknown - Classical Cadherin (partial) has a M but not likely to be the real start

Cad23/Dachsous Monosiga jgi|Monbr1|27065|fgenesh2_pg.scaffold_17000053 No good hits CA (2x) No No start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|206603|fgenesh1_pg.scaffold_71000072 No good hits CA (4x) Yes 1 Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|129879|e_gw.270.98.1 CELSR 3 1.00E-78 CA (4x) No No Cadherin start and stop codons presentCad23/Dachsous Acropora Contig12092

FAT tumor suppressor homolog 3 [Gallus gallus] 2.00E-49 CA (6x),TM Yes 1

Cad23/Dachsous Nvec jgi|Nemve1|243040|estExt_fgenesh1_pg.C_770089 FAT1 1.00E-59 CA (7x) Yes 1 Cadherin 17 start and stop codons presentCad23/Dachsous Acropora Contig8381 CA (7x), TM Yes 1Cad23/Dachsous Nvec jgi|Nemve1|129882|e_gw.270.7.1 CELSR 1.00E-30 CA (8x) No No Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|27265|fgenesh2_pg.scaffold_18000084 cadherin23 1.00E-35 CA (8x) Yes no Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|214866|fgenesh1_pg.scaffold_212000008 FAT4 1.00E-80 CA (9x) Yes No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|221587|fgenesh1_pg.scaffold_851000001 dachsous 1.00E-99 CA (9x) No No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|207189|fgenesh1_pg.scaffold_77000086 Hedgling 1.00E-178 CA (14x) Yes No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|248422|estExt_fgenesh1_pg.C_5570001 Hedgling 0 CA (17x) No No Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|29591|fgenesh2_pg.scaffold_36000032 fat 1.00E-51 Ca (20x) Yes No Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|27264|fgenesh2_pg.scaffold_18000083 FAT4 1.00E-57 CA (24x) No 2 Cadherin start and stop codons presentCad23/Dachsous Monosiga jgi|Monbr1|31013|estExt_fgenesh2_pg.C_20691 FAT4/cadherin23 1.00E-173 CA (24x) Yes No Cadherin start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|239898|estExt_fgenesh1_pg.C_210085 FAT1 0 CA (30x) Yes No Cadherin

start and stop codons present probably should be combined with 239899 to make one model duplicate of technau's

Cad23/Dachsous Monosiga jgi|Monbr1|37884|estExt_fgenesh1_pg.C_170117 FAT4 1.00E-160 CA (30x) Yes 2 Cadherin 16 like start and stop codons presentCad23/Dachsous Nvec jgi|Nemve1|207195|fgenesh1_pg.scaffold_77000092 dachsous 1.00E-153 CA (36x) Yes 1 dachsous start and stop codons present

Cad23/Dachsous Acropora Contig10872

dachsous 1 [Rattus norvegicus] >gi|149068442... 0 CA (19x), TM cadherin_C No 1 Dachsous Matches Unigene D041-C8. C-terminal region alignment places with Dachsous.

MBCDH1 Monosiga jgi|Monbr1|14707|e_gw1.3.170.1gb|AAP78679.1| MBCDH1 [Monosiga brevicollis] 0 EGF, CA (2x) No No

gb|AAP78679.1| MBCDH1 [Monosiga brevicollis] start, no stop

unidentified Hydra gb|DT613897.1 No good hits CA No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DT617585.1 No good hits CA No No Unknown - Cadherin (partial) No start no stopunidentified Clytia IL0ABA14YL05RM1 protocadherin gamma A2 1.00E-12 CA No No No start No stopunidentified Nvec jgi|Nemve1|110347|e_gw.100.165.1 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|127187|e_gw.236.58.1 Hydra Homolgue only CA No No No start, no stop ref|XP_002122245.1| PREDICTED: similar to Sushi, von Willebrand ... 4.00E-91unidentified Nvec jgi|Nemve1|127202|e_gw.236.61.1 No good hits CA No No Start codon, no stop codonunidentified Nvec jgi|Nemve1|18675|gw.236.62.1 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|219496|fgenesh1_pg.scaffold_416000004 No good hits CA No No start and stopunidentified Nvec jgi|Nemve1|223469|fgenesh1_pg.scaffold_2837000001 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|225510|fgenesh1_pg.scaffold_9093000001 No good hits CA No No Start codon, no stop codonunidentified Nvec jgi|Nemve1|225923|fgenesh1_pg.scaffold_14475000001 No good hits CA No No No start, stop presentunidentified Nvec jgi|Nemve1|225928|fgenesh1_pg.scaffold_14604000001 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|238641|estExt_fgenesh1_pg.C_70089 No good hits CA No No start and stopunidentified Nvec jgi|Nemve1|4597|gw.4224.3.1 starry night 1.00E-31 CA No No No start, no stopunidentified Nvec jgi|Nemve1|67148|gw.100.168.1 No good hits CA No No No start, no stopunidentified Nvec jgi|Nemve1|219501|fgenesh1_pg.scaffold_416000009 No good hits CA (wobbly), F5_F8_type_C No No start and stop

Appendix B: JCUSMART Survey of the Cnidarian Adhesome

Cadherins


178

Sub-Family Organism Sequence Id BLAST e_value HMMPFAM

Signal Peptide TM Conclusion Notes

unidentified Nvec jgi|Nemve1|244693|estExt_fgenesh1_pg.C_1310026 No good hits CA Yes No start and stopunidentified Hydra CL1102Contig1 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|CO538479.1 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN603165.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN811522.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN812369.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DN815486.2 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Hydra gb|DT619671.1 No good hits CA (2x) No No Unknown - Cadherin (partial) No start no stopunidentified Nvec jgi|Nemve1|148827|e_gw.2646.1.1 No good hits CA (2x) No No Start codon, no stop codonunidentified Nvec jgi|Nemve1|52838|gw.77.119.1 starry night 1.00E-30 CA (2x) No No No start, no stopunidentified Clytia SA0AAB45YD15CTG FAT tumor suppressor 1.00E-16 CA (2x) Yes No Start codon, no stop codonunidentified Hydra CL5439Contig1 No good hits CA (3x) No 1 Unknown - Cadherin (partial) No start no stopunidentified Hydra CL5681Contig1 No good hits CA (3x) No 2 Unknown - Cadherin (partial) No start no stopunidentified Nvec jgi|Nemve1|219493|fgenesh1_pg.scaffold_416000001 No good hits EGF, CA (3x) No No start and stopunidentified Nvec jgi|Nemve1|219500|fgenesh1_pg.scaffold_416000008 protocadherin 1.00E-24 CA (3x) No No start and stopunidentified Nvec jgi|Nemve1|220674|fgenesh1_pg.scaffold_557000003 CELSR 1.00E-39 CA (3x) No No No start, stop presentunidentified Nvec jgi|Nemve1|40605|gw.212.44.1 starry night 1.00E-59 CA (3x) No No No start, no stopunidentified Nvec jgi|Nemve1|98014|e_gw.48.27.1 No good hits CA (3x) No No No start, stop presentunidentified Nvec jgi|Nemve1|153449|e_gw.6181.1.1 CELSR 1.00E-47 CA (3x) No No No start, stop presentunidentified Nvec jgi|Nemve1|48709|gw.12488.2.1 fat (dros) 1.00E-60 CA (4x) No No No start, no stopunidentified Nvec jgi|Nemve1|70250|gw.100.177.1 CELSR 1.00E-47 CA (4x) No No No start, no stop

unidentified Nvec jgi|Nemve1|91648|e_gw.27.37.1gb|AAI01037.1| PCDHGC4 protein [Homo sapiens] 1.00E-70 CA (5x) Yes No

cadherin partial ( or PCDH-Gamma-C4) No stop codon

unidentified Nvec jgi|Nemve1|124570|e_gw.212.50.1gb|ABX84114.1| hedgling [Nematostella vectensis] 1.00E-67 CA (5x) No No No start no stop

unidentified Nvec jgi|Nemve1|41182|gw.270.64.1 CELSR 1.00E-59 CA (5x) No No No start no stop

unidentified Nvec jgi|Nemve1|118913|e_gw.157.87.1

ref|NP_059088.2| cadherin EGF LAG seven-pass G-type receptor 2 i… 4.00E-77 CA (5x) No No No start no stop

unidentified Clytia IL0ABA4YB04RM1cadherin EGF LAG seven-pass G-type receptor 3 [Ra... 1.00E-27 CA (6x) No 1 No start No stop

unidentified Nvec jgi|Nemve1|10520|gw.100.7.1 FAT 4 or Cadherin 23 1.00E-98 CA (7x) No No No start no stopunidentified Nvec jgi|Nemve1|40082|gw.212.41.1 CELSR 1.00E-120 CA (8x) No No No start no stopunidentified Monosiga jgi|Monbr1|13666|e_gw1.2.131.1 FAT4 1.00E-154 CA(12x) No No No start, no stopunidentified Nvec jgi|Nemve1|122785|e_gw.193.27.1 FAT 4 0.00E+00 CA (16x) No No start codon, No stop codonunidentified Nvec jgi|Nemve1|89517|e_gw.21.14.1 FAT1 0 CA (17x) No No No start no stopunidentified Nvec jgi|Nemve1|30053|gw.23.10.1 FAT 4 0 CA (18x) No No No start no stopunidentified Nvec jgi|Nemve1|107177|e_gw.85.10.1 dachsous/FAT4 0.00E+00 CA (24x) No No No M and no stopNovel Architecture Nvec jgi|Nemve1|216996|fgenesh1_pg.scaffold_280000006 No good hits CUB, EGF (3x), CA (2x) No No start and stop codons present. Scaffold 280. Ab inito model with no EST support.Novel Architecture Nvec jgi|Nemve1|247587|estExt_fgenesh1_pg.C_3180027 No good hits TSP1, CA (7x), EGF Yes No start and stop codons present. Scaffold 318. Ab inito with partial EST support.Novel Architecture Monosiga jgi|Monbr1|11672|fgenesh1_pg.scaffold_31000029 gb|ABX84114.1| hedgling 1.00E-40

EGF (3x), CCP, EGF, CCP (wobbly), EGF, CA (15x), EGF (2x), CCP (wobbly), EGF (2x) Yes 5 start and stop codons present

Novel Architecture Monosiga jgi|Monbr1|30335|fgenesh2_pg.scaffold_48000016 gb|ABX84114.1| hedgling 1.00E-180

Lam_NT (wobbly), LamEGF (4x), furin_3, LamG (wobbly), CA (46x), FN3 (wobbly), Y_Phosphatase Yes Yes start and stop codons present

Novel Architecture Monosiga jgi|Monbr1|11339|fgenesh1_pg.scaffold_28000057 No good hits

VWD, EGF, iptmega(wobbly), CA (6x wobbly), PKD (wobbly) SH) Yes 2 start and stop codons present

Novel Architecture Monosiga jgi|Monbr1|12200|fgenesh1_pg.scaffold_36000025 FAT4 0.00E+00 CA (58x) No 1 Cadherin novel or error start and stop codons present


179

Integrins

Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes Integrin Beta Hydra gb|DT609127.1 integrin beta chain [Podocoryne carnea] 1.00E-43 INB (wobbly) No Integrin beta - AmItgb2 homologue second best hit was AmItgb2 1E-43

Integrin Beta Nvec jgi|Nemve1|80868|e_gw.3.432.1 integrin beta 2 [Acropora millepora] 0INB, EGF (4x wobbly), Integrin_B_tail, Integrin_b_cyt 1 Integrin beta - AmItgb2 homologue

Integrin Beta Nvec jgi|Nemve1|123281|e_gw.198.1.1 integrin beta 2 [Acropora millepora] 0INB,EGF (4xwobbly), Integrin_B_tail,Integrin_b_cyt 1 Integrin beta - AmItgb2 homologue

Integrin Beta Clytia SA0AAB34YK24RM1 integrin subunit betaCn1 [Acropora millepora] 1.00E-33EGF (4x wobbly), Integrin_B_tail (wobbly) 1 Integrin beta - ItgbCN1 homologue

Integrin Beta Hydra CL4502Contig1 integrin subunit betaCn1 [Acropora millepora] 6.00E-86 INB No Integrin beta - ItgbCN1 homologue

Integrin Beta Nvec jgi|Nemve1|91193|e_gw.26.1.1 integrin subunit betaCn1 [Acropora millepora] 0INB, EGF (4x wobbly), Integrin_B_tail, Integrin_b_cyt 1 Integrin beta - ItgbCN1 homologue

Integrin Beta Nvec jgi|Nemve1|90281|e_gw.23.1.1 integrin subunit betaCn1 [Acropora millepora] 1.00E-168INB,EGF (4x wobbly), Integrin_B_tail Integrin beta - ItgbCN1 homologue This one is thought to be a splice varient

Integrin Beta Nvec jgi|Nemve1|143492|e_gw.826.1.1 integrin subunit betaCn1 [Acropora millepora] 1.00E-177INB,EGF (4xwobbly), Integrin_B_tail,Integrin_b_cyt 1 Integrin beta - ItgbCN1 homologue

Integrin Beta Acropora Contig12118 integrin subunit betaCn1 [Acropora millepora] 0 No AmItgB1Integrin Beta Acropora Contig17193 integrin beta chain [Podocoryne carnea] 1.00E-174 1 AmItgB2Integrin Alpha Clytia SA0AAB95YG15RM1 integrin alpha chain [Podocoryne carnea] 2.00E-74 int_alpha_rpt (4x) No Integrin alpha - partial

Integrin Alpha Nvecjgi|Nemve1|238305|estExt_fgenesh1_pg.C_50011

integrin, alpha 4 (antigen CD49D, alpha 4 subun... 2.00E-54

int_alpha_rpt (3x), Integrin_alpha2 1 Integrin alpha

Integrin Alpha Nvecjgi|Nemve1|196726|fgenesh1_pg.scaffold_3000047 integrin alpha 1 [Acropora millepora] 1.00E-149

int_alpha_rpt (4x), Integrin_alpha2,Integrin_alpha 2 Integrin alpha - AmItga1 homologue TMH 1 is in N-term therefore only 1 real TMH.

Integrin Alpha Hydra CL3123Contig1integrin, alpha 8 [Gallus gallus] >gi|124950|sp… 2.00E-35

int_alpha_rpt (2x), Integrin_alpha2 No Integrin alpha - partial

Integrin Alpha Acropora assembled Contigs integrin alpha 1 [Acropora millepora] 0 AmItga1Integrin Alpha Acropora assembled Contigs integrin alpha 1 [Acropora millepora] 0 AmItga2 -NEWIntegrin Alpha Acropora assembled Contigs integrin alpha 1 [Acropora millepora] 0.00E+00 AmItga3 -NEW

Integrin Alpha Repeat Containing Clytia SA0AAB7YH09RM1 AmItga1 1.00E-05Integrin_alpha2 (wobbly), Integrin_alpha (wobbly) integrin alpha related

Integrin Alpha Repeat Containing Hydra gb|DN813198.2 integrin alpha 1 [Acropora millepora] 6.00E-12 int_alpha_rpt integrin alpha relatedIntegrin Alpha Repeat Containing Hydra gb|CA302076.1 integrin alpha 2 [Pseudoplusia includens] 1.00E-10 No domains integrin alpha relatedIntegrin Alpha Repeat Containing Clytia IL0ABA2YN22RM1 Integrin alpha 4 [Mus musculus] 2.00E-14 int_alpha_rpt (2x wobbly) integrin alpha related

Integrin Alpha Repeat Containing Hydra CL6512Contig1integrin alpha 4 [Rattus norvegicus] >gi|149... 3.00E-14 int_alpha_rpt (wobbly) integrin alpha related

Integrin Alpha Repeat Containing Clytia SA0AAB95YG15NM1 integrin alpha chain [Podocoryne carnea] 2.00E-29 No Domains integrin alpha relatedIntegrin Alpha Repeat Containing Hydra CL6142Contig1 integrin alpha chain [Podocoryne carnea] 2.00E-20 int_alpha_rpt integrin alpha relatedIntegrin Alpha Repeat Containing Hydra gb|CV181210.1 integrin alpha chain [Podocoryne carnea] 3.00E-12 No domains integrin alpha related

Integrin Alpha Repeat Containing Hydra gb|DN244145.2integrin alpha-PS2 [Culex quinquefasciatus] ... 5.00E-15 int_alpha_rpt integrin alpha related

FG-GAP repeats occur in other proteins but int_alpha repeats are more restricted.

Integrin Alpha Repeat Containing Hydra gb|DT620726.1Integrin, alpha 2b (platelet glycoprotein IIb of ... 2.00E-08 No domains integrin alpha related

Integrin Alpha Repeat Containing Hydra CL10025Contig1 ITGA7 variant protein [Homo sapiens] 9.00E-13 int_alpha_rpt (wobbly) integrin alpha relatedIntegrin Alpha Repeat Containing Hydra CL10282Contig1 No good hits int_alpha_rpt (wobbly) integrin alpha related

Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|13072|fgenesh1_pg.scaffold_55000008 No good hits int_alpha_rpt (2x wobbly) integrin alpha related

Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|9496|fgenesh1_pg.scaffold_15000186 No good hits int_alpha_rpt (2x wobbly) integrin alpha related

Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|10253|fgenesh1_pg.scaffold_20000037 quinoprotein (ISS) [Ostreococcus tauri] 1.00E-29 int_alpha_rpt (2-4x) integrin alpha related

Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|25795|fgenesh2_pg.scaffold_11000195 quinoprotein (ISS) [Ostreococcus tauri] 9.00E-15 int_alpha_rpt (2-4x) integrin alpha related

Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|25557|fgenesh2_pg.scaffold_10000207 quinoprotein (ISS) [Ostreococcus tauri] 8.00E-07 int_alpha_rpt (2x wobbly) integrin alpha related

Integrin Alpha Repeat Containing Mbrevjgi|Monbr1|25858|fgenesh2_pg.scaffold_12000007 quinoprotein (ISS) [Ostreococcus tauri] 3.00E-05 int_alpha_rpt (2x wobbly) integrin alpha related

Integrin Alpha Repeat Containing Nvec jgi|Nemve1|115107|e_gw.130.30.1glycosylphosphatidylinositol phospholipase D [Mus... 1.00E-160 int_alpha_rpt (6x) No Integrin alpha related BLAST hit has only 2 int_alpha_rpts. Start and stop codons present.

Integrin Alpha Repeat Containing Nvecjgi|Nemve1|224736|fgenesh1_pg.scaffold_5705000001

ref|NP_777241.1| glycosylphosphatidylinositol specific phospholi... 2.00E-45 1.00E-45 int_alpha_rpt (4x) No Integrin alpha - partial BLAST hit has only 2 int_alpha_rpts. No start and No stop codons.

Integrin Alpha Repeat Containing Acropora Contig19889 integrin alpha 1 [Acropora millepora] 1.00E-150 int_alpha_rpt (2) NoIntegrin Alpha Repeat Containing Acropora Contig33553 integrin alpha 1 [Acropora millepora] 1.00E-141 int_alpha_rpt (2) NoIntegrin Alpha Repeat Containing Acropora Contig7700 integrin alpha 1 [Acropora millepora] 4.00E-61 int_alpha_rpt (4) No

Talin Clytia SA0AAA26YF22RM1talin-1 [Culex quinquefasciatus] >gi|1678730... 2.00E-20

Talin_middle(wobbly), MA_2 (wobbly), ILWEQ Talin - middle

Talin Clytia IL0ABA5YH08RM1 talin [Podocoryne carnea] 1.00E-134 B41_5, PTBI_2 (wobbly) Talin - N-termTalin Hydra CL1Contig29 talin [Podocoryne carnea] 1.00E-118 B41/FERM_N Talin - N-term This may be the N-terminus of one of the other partial talins.

Talin Mbrevjgi|Monbr1|23461|fgenesh2_pg.scaffold_4000204 talin 2 [Mus musculus] 1.00E-127

B41, PTBI (wobbly), Talin_middle (wobbly), ILWEQ (wobbly) Talin 2 - N-term

Talin Clytia IL0ABA4YJ15RM1talin 1 [Danio rerio] >gi|55139380|gb|AAV413... 1.00E-155

MA (wobbly), VBS, ILWEQ (wobbly) Talin 1 - C-term

Talin Mbrevjgi|Monbr1|6317|fgenesh1_pg.scaffold_4000199 talin 1, isoform CRA_b [Homo sapiens] 1.00E-180 VBS, MA (wobbly), ILWEQ Talin 1 - C-term

Talin Hydra CL5745Contig1 talin 1, isoform CRA_b [Homo sapiens] 9.00E-72 ILWEQ Talin 1 - C-term

INB_2, EGF_2 (4), INB_2, EGF_2 (4),


180

Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes

Talin Nvecjgi|Nemve1|159935|estExt_gwp.C_110123 Tln2 protein [Mus musculus] 0

B41, PTBI (wobbly), Talin_middle, 5bar (wobbly), MA (wobbly), VBS, ILWEQ Talin 2

Talin Hydra CL9211Contig1 talin 2 [Mus musculus] 3.00E-79 MA (wobbly), ILWEQ (wobbly) Talin 2 - C-term

Talin Clytia SA0AAB5YM11RM1 talin 2, isoform CRA_a [Homo sapiens] 1.00E-149VBS, MA (wobbly), ILWEQ (wobbly) Talin 2 - C-term

Talin Acropora Contig10699 talin 1, isoform CRA_b [Homo sapiens] 0 VBS, MA (wobbly), ILWEQ Talin1 - C-termTalin Acropora Amil_rep_c147660 talin 1, isoform CRA_b [Homo sapiens] 1.00E-65 ILWEQ Talin1 - C-termTalin-related Hydra gb|DT615285.1 Tln2 protein [Mus musculus] 2.00E-54 MA (wobbly) Talin 2 - partial

Talin-related Acropora Contig943talin 1 [Danio rerio] >gi|55139380|gb|AAV413 1.00E-111 MA (wobbly), ILWEQ (wobbly)

Talin-related Nvec jgi|Nemve1|123513|e_gw.200.72.1huntingtin interacting protein 1 [Homo sapiens] >... 6.00E-70 ILWEQ Talin related

Talin-related Mbrevjgi|Monbr1|36287|estExt_fgenesh1_pg.C_40453

huntingtin interacting protein 1 related, isoform... 9.00E-17 SH3, ILWEQ Talin related

Talin-related Mbrev jgi|Monbr1|14137|e_gw1.2.850.1 huntingtin-interacting protein 1 [Homo ... 2.00E-24 ILWEQ Talin related

Talin-related Hydra gb|CV659942.1talin 1 [Danio rerio] >gi|55139380|gb|AAV413... 9.00E-49 ILWEQ (wobbly) Talin related

Talin-related Clytia IL0ABA22YH03RM1talin 1 [Gallus gallus] >gi|81175199|sp|P54939.... 4.00E-58 ILWEQ (wobbly) Talin related

Talin-related Clytia SA0AAB72YJ22RM1 Talin 1 [Homo sapiens] 4.00E-30 ILWEQ Talin relatedTalin-related Hydra gb|DR435962.1 talin 2 [Homo sapiens] 1.00E-31 No domains Talin related

Talin-related Hydra CL1594Contig1 talin 2 [Xenopus (Silurana) tropicalis] >gi|... 1.00E-29 ILWEQ Talin related

I/LWEQ domains bind to actin. It has been shown that the I/LWEQ domains from mouse talin and yeast Sla2p interact with F-actin PUBMED:9159132. The domain has four conserved blocks, the name of the domain is derived from the initial conserved amino acid of each of the four blocks PUBMED:9159132. I/LWEQ domains can be placed into four major groups based on sequence similarity:1. Metazoan talin.2. Dictyostelium discoideum (Slime mould) TalA/TalB and SLA110.3. Metazoan Hip1p .4. Saccharomyces cerevisiae Sla2p .

Talin-related Clytia SA0AAB23YM10RM1 talin 2 [Xenopus (Silurana) tropicalis] >gi|... 4.00E-17 ILWEQ Talin relatedTalin-related Clytia IL0ABA6YH10RM1 Tln2 protein [Mus musculus] 4.00E-65 No Domains Talin relatedTalin-related Hydra CL3431Contig1 vinculin [Drosophila melanogaster] 1.00E-29 ILWEQ (wobbly) Talin related vinculin has its own domain called "vinculin". Integrin Linked Kinase Hydra CL2782Contig1 Integrin-linked protein kinase [Salmo salar]... 1.00E-124 Ank (3x), tyrkin Integrin-linked kinase

Integrin Linked Kinase Clytia SA0AAA16YD20RM1integrin-linked kinase [Gallus gallus] >gi|1050... 1.00E-145 Ank (3x), tyrkin Integrin-linked kinase

Integrin Linked Kinase Nvec jgi|Nemve1|31638|gw.2.85.1integrin-linked kinase [Danio rerio] >gi|339918... 1.00E-135 Ank (3x), trykin Integrin-linked kinase

Integrin Linked Kinase Acropora Contig5477integrin-linked kinase [Gallus gallus] >gi|1050... 1.00E-148 Ank (3x), trykin Integrin-linked kinase

FAK Acropora Contig1043 FAK 0FAK Nvec jgi|Nemve1|60034|gw.13.293.1 FAK 0FAK Clytia CL7784Contig1 FAK 1.00E-51Parvin Hydra CL698Contig1 Alpha-parvin [Osmerus mordax] 1.00E-85Parvin Acropora Contig28157 Alpha-parvin [Osmerus mordax] 1.00E-77

Parvin Nvecjgi|Nemve1|207272|fgenesh1_pg.scaffold_78000074 Alpha-parvin [Osmerus mordax] 1.00E-55

Paxillin Hydra CL1132Contig1dbj|BAA18998.1|,paxillin gamma [Homo sapiens] 1.00E-120

Paxillin Acropora Contig8199ref|NP_963882.1|,paxillin [Danio rerio] >gi|41350255|gb|AAS00452 1.00E-125

Paxillin Mbrevjgi|Monbr1|19246|estExt_Genewise1.C_20560

ref|NP_002850.2|,paxillin isoform 2 [Homo sapiens] 1.00E-42

Paxillin Nvec jgi|Nemve1|104924|e_gw.75.150.1 paxillin [Culex quinquefasciatus] 1.00E-122Paxillin Clytia SA0AAB113YJ11CTG paxillin, isoform B [Drosophila melanogaster] 1.00E-129

c-Src Acropora Contig2876dbj|BAG70102.1|,c-src tyrosine kinase [Homo sapiens] 1.00E-140

c-Src Clytia SA0AAB19YJ19RM1dbj|BAG70102.1|,c-src tyrosine kinase [Homo sapiens] 1.00E-78

PINCH Acropora Contig17667 gb|ABS17667.1|,PINCH-1 [Xenopus laevis], 1.00E-117PINCH Hydra CL5059Contig1 PINCH 1.00E-81

PINCH Nvecjgi|Nemve1|160257|estExt_gwp.C_120473

gb|EDL42131.1|,"LIM and senescent cell antigen-like domains 1 1.00E-122

ILKAK Hydra CL1236Contig1integrin-linked kinase-associated protein phosp... 7.00E-75 PP2C

Integrin-linked kinase associated Phosphatase

ILKAK Nvec jgi|Nemve1|110119|e_gw.98.154.1ref|NP_075832.1| integrin-linked kinase-associated serine/threon… 2.00E-88 PP2C

integrin-linked kinase-associated serine/threonin...

ILKAK Mbrevjgi|Monbr1|36991|estExt_fgenesh1_pg.C_90170

Integrin-linked kinase-associated serine/threonin... 6.00E-53 PP2C

Integrin-linked kinase-associated serine/threonin...

Disintegrin Containing Hydra gb|DN813922.2 No good hits DISIN Disintegrin containingDisintegrin Containing Hydra gb|DN811041.2 hemicentin1 DISIN, TSP1 Disintegrin containing


181


Disintegrin Containing Nvec jgi|Nemve1|5599|gw.3050.2.1kinesin [Leishmania infantum JPCM5] >gi|1340... 2.00E-16 DISIN (wobbly) DISIN containing

ADAM Nvec jgi|Nemve1|20904|gw.66.19.1a disintegrin and metalloprotease domain 10b [D... 1.00E-167 Reprolysin, DISIN ADAM

ADAM Nvecjgi|Nemve1|175837|estExt_gwp.C_4720015

a disintegrin and metalloprotease domain 10b [D... 1.00E-124

pep_M12B_propep, Reprolysin, DISIN ADAM

ADAM Nvec jgi|Nemve1|5671|gw.3057.2.1a disintegrin and metalloprotease domain 10b [D... 2.00E-56 No domains ADAM

ADAM Clytia SA0AAA13YF04RM1A disintegrin and metalloprotease domain 13 [Xeno... 7.00E-65 reprolysin, DISIN, acr ADAM

ADAM Mbrevjgi|Monbr1|22131|fgenesh2_pg.scaffold_2000338

a disintegrin and metalloprotease domain 17 pre... 8.00E-46 Reprolysin, DISIN ADAM

ADAM Hydra gb|CN552903.1a disintegrin and metalloprotease domain 17 pre... 2.00E-37 reprolysin ADAM

reprolysin: The members of this family are enzymes that cleave peptides. These proteases require zinc for catalysis. Members of this family are also known as adamalysins. Most members of this family are snake venom endopeptidases, but there are also some mammalian proteins such as P78325 and fertilin Q28472. Fertilin and closely related proteins appear to not have some active site residues and may not be active enzymes.

ADAM Mbrevjgi|Monbr1|37642|estExt_fgenesh1_pg.C_150079

a disintegrin and metalloproteinase domain 17a ... 2.00E-08 DISIN (wobbly), GRAN_2 (wobbly) ADAM

ADAM Hydra CL6661Contig1a disintegrin and metalloproteinase domain 24 (... 6.00E-14 reprolysin, DISIN ADAM

ADAM Clytia SA0AAB61YH16CTGadam (a disintegrin and metalloprotease [Aed... 1.00E-64 reprolysin, DISIN, acr ADAM

ADAM Hydra CL970Contig1ADAM metallopeptidase domain 10 [Gallus gallus]... 3.00E-77 reprolysin, DISIN ADAM


ADAM metallopeptidase domain 10 [Pongo abeli... 6.00E-50



ADAM metallopeptidase domain 10 [Pongo abeli... 2.00E-46


ADAM Nvec jgi|Nemve1|91360|e_gw.27.264.1ADAM metallopeptidase domain 10, isoform CRA_a [H... 4.00E-45 Reprolysin, DISIN, acr ADAM

ADAM Hydra gb|DN814600.2ADAM metallopeptidase domain 12 [Bos taurus]... 4.00E-36 DISIN, acr ADAM acr = ADAM_CR = ADAM cystein rich region

ADAM Clytia SA0AAA4YP11CTGADAM metallopeptidase domain 12 [Xenopus (Si... 3.00E-56 DISIN, acr ADAM

ADAM Nvecjgi|Nemve1|212032|fgenesh1_pg.scaffold_148000031

ADAM metallopeptidase domain 8 precursor [Homo ... 4.00E-45 DISIN, acr ADAM

ADAM Clytia SA0AAB70YK05CTGADAM metallopeptidase domain 9 [Rattus norve... 6.00E-37 reprolysin, DISIN ADAM

ADAM Nvec jgi|Nemve1|126434|e_gw.232.25.1ADAM metallopeptidase with thrombospondin type 1 ... 4.00E-39 Reprolysin ADAM

ADAM Hydra gb|CV659930.1ADAM metallopeptidase with thrombospondin type 1 ... 2.00E-29 reprolysin, acr (wobbly) ADAM

ADAM Nvec jgi|Nemve1|16735|gw.148.22.1ADAM33 [Mus musculus] >gi|123229989|emb|CAM18043.... 9.00E-38 DISIN, acr ADAM

ADAM Nvecjgi|Nemve1|200723|fgenesh1_pg.scaffold_23000088 ADAM33 protein [Homo sapiens] 3.00E-15 Reprolysin, DISIN, acr ADAM

ADAM Mbrevjgi|Monbr1|23347|fgenesh2_pg.scaffold_4000090 No good hits Reprolysin, DISIN, SH3 ADAM Novel SH3 domain?


tace, isoform A [Drosophila melanogaster] >gi|4... 2.00E-54



tumor necrosis factor alpha converting enzyme [Ga... 1.00E-34

Ribosomal_L9_N, Reprolysin, DISIN ADAM Tace/ADAM17


tumor necrosis factor-alpha-converting enzyme [Su... 1.00E-16

DISIN (wobbly), DEFSN_4 (wobbly) ADAM Tace/ADAM17


tumor necrosis factor-alpha-converting enzyme mut... 7.00E-18 No domains ADAM Tace/ADAM17

ADAM Hydra gb|DN240294.2a disintegrin and metallopeptidase domain 34 [M... 4.00E-19 DISIN ADAM - putative

ADAM Hydra CL2583Contig1ADAM metallopeptidase domain 10 [Sus scrofa]... 8.00E-27 DISIN (wobbly) ADAM - putative

ADAM Hydra gb|CV985877.1ADAM metallopeptidase domain 33 isoform beta pr... 2.00E-11 DISIN ADAM - putative

ADAM Hydra gb|CN631642.1disintegrin and metalloprotease domain 33 [Mus mu... 1.00E-09 DISIN, TSP1 ADAM - putative

ADAM Hydra CL8785Contig1MIND-MELD [Drosophila melanogaster] or ADAM 7.00E-12 DISIN ADAM - putative mind meld is an ADAM

ADAM Nvec jgi|Nemve1|40207|gw.95.86.1tace, isoform A [Drosophila melanogaster] >gi|4... 2.33E-156 Reprolysin, DISIN ADAM17 homologue tace is ADAM17

ADAM Nvec jgi|Nemve1|6627|gw.3466.2.1a disintegrin and metalloprotease domain 10b [D... 7.00E-22 Pep_M12B_propep (wobbly) M12B peptidase family


182


ADAM Nvecjgi|Nemve1|198740|fgenesh1_pg.scaffold_11000152

a disintegrin and metalloproteinase domain 9... 6.00E-15 Pep_M12B_propep M12B peptidase family

ADAM_TS Nvec jgi|Nemve1|127746|e_gw.242.21.1metalloprotease disintegrin 16 with thrombospond... 7.00E-34 acr (wobbly), TSP1 (wobbly) ADAM_TS

ADAM_TS Nvecjgi|Nemve1|212944|fgenesh1_pg.scaffold_166000015

ref|NP_001074489.1| a disintegrin-like and metalloprotease (repr… 2.00E-80

acr, TSP1, ADAM_spacer(wobbly), TSP1 (4x wobbly), SLG (wobbly) ADAM_TS

ADAM_TS Hydra gb|CX054604.1 ADAMTS20 A short isoform [Mus musculus] 1.00E-42 ADAM_spacer ADAM_TSADAM_TS Nvec jgi|Nemve1|139926|e_gw.497.1.1 ADAMTS6 variant 2 [Homo sapiens] 2.00E-66 ADAM_spacer, TSP1 (4x), PLAC ADAM_TSADAM_TS Nvec jgi|Nemve1|99266|e_gw.52.22.1 ADAMTS6 variant 2 [Homo sapiens] 1.00E-102 ADAM_spacer, TSP1 (4x), PLAC ADAM_TS

ADAM_TS Nvecjgi|Nemve1|240559|estExt_fgenesh1_pg.C_310043

ADAM metallopeptidase with thrombospondin type ... 8.00E-51

Pep_M12B_propep, ADAM_spacer, TSP1 (3x) ADAM_TS


ADAM metallopeptidase with thrombospondin type ... 1.00E-63

pep_M12B_propep, Reprolysin, acr (wobbly), TSP1 (3x), FN3 ADAM_TS FN3 domain is 1E-05… interesting

ADAM_TS Nvec jgi|Nemve1|87270|e_gw.15.9.1ADAM metallopeptidase with thrombospondin type ... 1.00E-157

pep_M12B_propep, Reprolysin, acr (wobbly), TSP1 (4x) ADAM_TS


gb|EAW95600.1| ADAM metallopeptidase with thrombospondin type 1 ... 1.00E-75

pep_M12B_propep, Reprolysin, acr (wobbly), TSP1, ADAM_spacer (wobbly), TSP1 (3x) ADAM_TS



pep_M12B_propep, Reprolysin, acr (wobbly), TSP1, ADAM_spacer (wobbly), TSP1 (4x), SLG (wobbly) ADAM_TS


ref|NP_922932.2| ADAM metallopeptidase with thrombospondin type … 1.00E-104

pep_M12B_propep, Reprolysin, TSP1, acr (wobbly), ADAM_spacer (wobbly), TSP1 (3x) ADAM_TS


gb|EAW68907.1| ADAM metallopeptidase with thrombospondin type 1 … 1.00E-51 Reprolysin, acr, TSP1 ADAM_TS


gb|EAW99151.1| ADAM metallopeptidase with thrombospondin type 1 … 9.00E-58 Reprolysin, acr, TSP1 ADAM_TS

ADAM_TS Nvec jgi|Nemve1|101427|e_gw.60.18.1ref|NP_112217.2| ADAM metallopeptidase with thrombospondin type … 1.00E-125 Reprolysin, acr, TSP1 ADAM_TS


gb|EDL11544.1| a disintegrin-like and metallopeptidase (reprolys… 1.00E-54 Reprolysin, acr, TSP1 (2x) ADAM_TS

ADAM_TS Nvec jgi|Nemve1|30434|gw.314.48.1gb|AAQ94616.1| COMPase precursor [Homo sapiens] 0 Reprolysin, acr, TSP1 (4x) ADAM_TS



Reprolysin, acr, TSP1, ADAM_spacer, TSP1 (2x) ADAM_TS

ADAM_TS Clytia IL0ABA21YG19RM1A disintegrin-like and metallopeptidase (reprolys... 4.00E-42 Reprolysin, TSP1 (wobbly), MAM ADAM_TS


ref|NP_922932.2| ADAM metallopeptidase with thrombospondin type … 3.00E-76 Reprolysin, TSP1, ADAM_spacer ADAM_TS

ADAM_TS Nvec jgi|Nemve1|101342|e_gw.60.83.1ref|NP_001029049.2| ADAM metallopeptidase with thrombospondin ty… 6.00E-21 TSP (3x) ADAM_TS - putative

ADAM_TS Nvec jgi|Nemve1|115857|e_gw.134.16.1 ADAMTS3 protein [Homo sapiens] 1.00E-20 TSP1 ADAM_TS - putative

ADAM_TS Clytia SA0AAB75YP11RM1ADAM metallopeptidase with thrombospondin type 1 ... 1.00E-15 TSP1 (2x), TSP1 (wobbly) ADAM_TS - putative

ADAM_TS Clytia SA0AAB108YD12CTG ADAMTS-like 3 [Homo sapiens] 1.00E-19 TSP1 (3x) ADAM_TS - putativeADAM_TS SA0AAB75YH20RM1 ADAMTS9 1.00E-15 TSP1 (3x) ADAM_TS - putative

ADAM_TS Nvec jgi|Nemve1|5633|gw.2032.1.1ref|NP_001074489.1| a disintegrin-like and metalloprotease (repr… 1.00E-16 TSP1 (wobbly) ADAM_TS - putative

ADAM_TS Hydra CL1752Contig1ADAM metallopeptidase with thrombospondin type ... 4.00E-10 TSP1, MAM ADAM_TS - putative


ref|NP_780523.2| a disintegrin-like and metalloprotease (reproly… 3.00E-27 GON ADAM_TS GON

ADAM_TS Nvec jgi|Nemve1|95408|e_gw.39.117.1ref|NP_891550.1| ADAM metallopeptidase with thrombospondin type … 0

Reprolysin, acr, TSP1, ADAM_spacer, TSP1 (13x), GON ADAM_TS GON

ADAM_TS Hydra gb|CN632985.1 ADAMTS9 protein [Homo sapiens] 7.00E-23 TSP1, GON ADAM_TS GON ref|XP_002166940.1| PREDICTED: similar to abnormal GONad develop... 1.00E-81

FG-GAP containing Nvec jgi|Nemve1|103100|e_gw.67.19.1 Bardet-Biedl syndrome 7 [Mus musculus] 0 FG-GAP (wobbly), BB1(wobbly)Bardet-Biedl syndrome 7 [Mus musculus]

FG-GAP containing Hydra CL337Contig1 embryonic-1 [Hydra vulgaris] 1.00E-126 FG-GAP (3x wobbly) Embryonic-1

Embryonic-1 = SP, one wobbly FG-GAP repeat in its C-terminal. BLAST is hitting an almost perfect match along the full length of the contig (267aa). Embryonic is 340aa.

FG-GAP containing Hydra CL1Contig798 embryonic-1 [Hydra vulgaris] 1.00E-133 FG-GAP (4x wobbly) Embryonic-1

FG-GAP containing Hydra CL8462Contig1ASPIC/UnbV domain-containing protein [Opitut... 2.00E-16 FG-GAP (3x wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|235159|estExt_fgenesh1_pm.C_1000002

Bardet-Biedl syndrome 2 [Mus musculus] >gi|2045... 0 FG-GAP (3x wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|161101|estExt_gwp.C_180226

DEX1 (DEFECTIVE IN EXINE FORMATION 1) [Arabidop... 2.00E-46 FG-GAP (5x wobbly) FG-GAP containing

FG-GAP containing Clytia SA0AAA22YK19RM1FG-GAP repeat protein [bacterium Ellin514] >g... 4.00E-11 FG-GAP (5x wobbly) FG-GAP containing


183


FG-GAP containing Nvecjgi|Nemve1|245375|estExt_fgenesh1_pg.C_1570033

FG-GAP repeat-containing protein [Arabidopsis t... 9.00E-44 FG-GAP (2x wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|200317|fgenesh1_pg.scaffold_21000002

integrin alpha FG-GAP repeat containing 1 [P... 1.00E-103 FG-GAP (5x wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|191884|estExt_GenewiseH_1.C_2190096

integrin alpha FG-GAP repeat containing 2 [D... 2.00E-82 FG-GAP (3x wobbly) FG-GAP containing

FG-GAP containing Hydra CL4337Contig1integrin alpha FG-GAP repeat containing 2 [D... 2.00E-27 No domains FG-GAP containing

FG-GAP containing Clytia IL0ABA24YM21RM1integrin alpha FG-GAP repeat containing 2 [Homo... 3.00E-60 FG-GAP (2x wobbly) FG-GAP containing

FG-GAP containing Hydra CL6840Contig1integrin alpha FG-GAP repeat containing 2 [R... 2.00E-17 FG-GAP (wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|242072|estExt_fgenesh1_pg.C_560046 ITFG3 [Salmo salar] 2.00E-08 FG-GAP (2x wobbly) FG-GAP containing

FG-GAP containing Hydra CL7613Contig1 No good hits FG-GAP (2x wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|203954|fgenesh1_pg.scaffold_47000013 No good hits FG-GAP (2x wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|225852|fgenesh1_pg.scaffold_13181000001 predicted proteins only FG-GAP (2x wobbly) FG-GAP containing

FG-GAP containing Nvec jgi|Nemve1|52083|gw.92.151.1 predicted proteins only FG-GAP (3x wobbly) FG-GAP containing

FG-GAP containing Nvecjgi|Nemve1|225927|fgenesh1_pg.scaffold_14603000001

Rhs family protein [Vibrio vulnificus CMCP6] >g... 5.00E-22 FG-GAP (2x wobbly) FG-GAP containing

FG-GAP containing Clytia SA0AAB75YF22CTGFG-GAP repeat domain protein [Verrucomicrobi... 5.00E-47 FG-GAP (7x wobbly) Integrin alpha/FG-GAP containing

FG-GAP containing Clytia SA0AAB109YA16RM1integrin alpha FG-GAP repeat containing 1 [P... 2.00E-68 FG-GAP (5x wobbly) Integrin alpha/FG-GAP containing Often there are missing or weak FG-GAPs in alpha subunits

FG-GAP containing Mbrevjgi|Monbr1|30147|fgenesh2_pg.scaffold_44000012

FG-GAP repeat-containing protein [Nostoc pun... 1.00E-25 FG-GAP (14x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|12516|fgenesh1_pg.scaffold_40000040 Fibronectin type III domain protein [Chloroh... 2.00E-69 FG-GAP (7x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|34687|estExt_fgenesh2_pg.C_460013 Fibronectin type III domain protein [Chloroh... 2.00E-65 FG-GAP (7x wobbly)


Integrins alpha chain [Anabaena variabilis ATCC... 1.00E-19 FG-GAP (13x wobbly)


integrins alpha chain [Stigmatella aurantiaca... 7.00E-15 FG-GAP (19x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|25716|fgenesh2_pg.scaffold_11000116 No good hits FG-GAP

FG-GAP containing Mbrevjgi|Monbr1|30879|estExt_fgenesh2_pg.C_20417 No good hits FG-GAP (2x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|6487|fgenesh1_pg.scaffold_4000369 No good hits FG-GAP (2x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|11330|fgenesh1_pg.scaffold_28000048 No good hits FG-GAP (3x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|12825|fgenesh1_pg.scaffold_46000035 predicted peptides only FG-GAP (13x wobbly)


FG-GAP containing Mbrevjgi|Monbr1|39187|estExt_fgenesh1_pg.C_410059 predicted peptides only FG-GAP (13x wobbly), trykin

FG-GAP containing Mbrevjgi|Monbr1|11861|fgenesh1_pg.scaffold_32000092 predicted peptides only

FG-GAP (13x wobbly), tyrkin, tSNARE/MA (wobbly), CCP (wobbly), EGF



FG-GAP containing Mbrevjgi|Monbr1|11774|fgenesh1_pg.scaffold_32000005 predicted peptides only FG-GAP (14x wobbly), Tyrkin




FG-GAP containing Mbrevjgi|Monbr1|31515|estExt_fgenesh2_pg.C_40272 predicted peptides only FG-GAP (20x wobbly)





184







































185





FG-GAP containing Mbrevjgi|Monbr1|10795|fgenesh1_pg.scaffold_24000009 predicted peptides only FG-GAP, FG-GAP (6x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|11321|fgenesh1_pg.scaffold_28000039 predicted peptides only FG-GAP, FG-GAP (6x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|39388|estExt_fgenesh1_pg.C_1080001 predicted peptides only FG-GAP(11x wobbly)

FG-GAP containing Mbrevjgi|Monbr1|37922|estExt_fgenesh1_pg.C_180022 quinoprotein (ISS) [Ostreococcus tauri] 9.00E-16 FG-GAP (5x wobbly)


vcbs [Stigmatella aurantiaca DW4/3-1] >gi|115... 6.00E-17 FG-GAP (7x wobbly)


186

Lectins

Sub Family Organism Sequence Id BLAST e_value HMMPFAM SP TM Conclusion Notes C-type Secreted Clytia SA0AAA6YN21RM1

gb|ACN91267.1| mannose receptor C1-like protein [Danio rerio] e-06 CLECT (2x) Yes No C-lectin - Secreted

C-type Secreted Clytia SA0AAB8YD20RM1 hydra homolgue only CLECT (2x) Yes No C-lectin - Secreted

ref|XP_002155423.1| PREDICTED: similar to type II transmembrane ... 263 2.00E-68ref|XP_002153863.1| PREDICTED: type II transmembrane C-type lect... 253 1.00E-65ref|XP_002159076.1| PREDICTED: similar to type II transmembrane ... 252 4.00E-65

C-type Secreted Clytia IL0ABA28YD22RM1 three hydra homolgues only e-66 CLECT (2x) Yes No C-lectin - Secreted

ref|XP_002153863.1| PREDICTED: type II transmembrane C-type lect... 254 6.00E-66ref|XP_002155423.1| PREDICTED: similar to type II transmembrane ... 254 8.00E-66ref|XP_002159076.1| PREDICTED: similar to type II transmembrane ... 246 2.00E-63

C-type Secreted Clytia SA0AAA21YL24RM1 No good hits EGF, CLECT Yes No C-lectin - SecretedC-type Secreted Clytia SA0AAB66YG22RM1 mannose receptor, C type 1 e-43 TSP1, CLECT (4x) Yes No C-lectin - Secreted

blast hits all have multiple c-lectin domains but no TSP1 domain. The TSP1 domain hits at e-11 -pfam and e-17 SMART.

C-type Secreted Hydra CL5624Contig1 No good hits CLECT Yes No C-lectin - SecretedC-type Secreted Hydra gb|CO370876.1 tyrosine kinase receptor [Hydra vulgaris] 3.00E-35 CLECT Yes No C-lectin - SecretedC-type Secreted Hydra gb|DN813687.2 tyrosine kinase receptor [Hydra vulgaris] 3.00E-23 CLECT Yes No C-lectin - SecretedC-type Secreted Mbrev

jgi|Monbr1|10838|fgenesh1_pg.scaffold_24000052 No good hits Candida_ALS, CLECT (2x) Yes No C-lectin - Secreted

C-type Secreted Mbrev

jgi|Monbr1|26977|fgenesh2_pg.scaffold_16000165 No good hits CLECT Yes No C-lectin - Secreted


jgi|Monbr1|27090|fgenesh2_pg.scaffold_17000078 No good hits CLECT (2x) Yes No C-lectin - Secreted


jgi|Monbr1|33819|estExt_fgenesh2_pg.C_250040 No good hits CLECT (3x) Yes No C-lectin - Secreted


jgi|Monbr1|7558|fgenesh1_pg.scaffold_7000290 No good hits CLECT (4x), LamG (2x) Yes No C-lectin - Secreted


jgi|Monbr1|30149|fgenesh2_pg.scaffold_44000014 No good hits

CLECT, int_alpha_rpt(2x very wobbly) Yes No C-lectin - Secreted

C-type Secreted Nvec

jgi|Nemve1|196946|fgenesh1_pg.scaffold_4000026 No good hits CLECT Yes No C-lectin - Secreted













C-type Secreted Nvec jgi|Nemve1|83039|e_gw.7.243.1 No good hits CLECT Yes No C-lectin - SecretedC-type Secreted Nvec

jgi|Nemve1|212709|fgenesh1_pg.scaffold_161000025 matrilin 2 [Xenopus (Silurana) tropicalis] >gi|... 2.00E-22 CLECT (2x), VWA Yes No C-lectin - Secreted


jgi|Nemve1|196785|fgenesh1_pg.scaffold_3000106

gb|AAR24388.1| mannose receptor C1 [Sus scrofa] 1.00E-82

CLECT (3x), F5_F8_type_C, CLECT, LamG (wobbly), CLECT, Gal_lectin, CLECT (5x), disc_4, F5_F8_type_C, disc_4, F5_F8_type_C, SEA Yes No C-lectin - Secreted Unique Archtecture with SEA F5_F8_type_C and CLECT



CUB and Sushi multiple domains 3 isoform 2 [Hom... 1.00E-79

F5_F8_type_C, CCP (12x), CLECT Yes No C-lectin - Secreted CUB and Sushi has lots of (CUB,CCP) repeated


jgi|Nemve1|208381|fgenesh1_pg.scaffold_91000082 No good hits ntp (wobbly, CLECT (wobbly) Yes No C-lectin - Secreted



brevican [Danio rerio] >gi|134054440|emb|CAM... 6.00E-10

ntp (wobbly), EGF (wobbly), LINK_2, CLECT Yes No C-lectin - Secreted

C-type Soluble Clytia IL0ABA9YE18RM1 chondroitin sulfate proteoglycan 2 [Xenopus ... e-22 CLECT No No C-lectin - Soluble chondroitin sulfate proteoglycan 2 is big with multiple domainsC-type Soluble Clytia SA0AAB101YI01CTG hydra homolgue only e-21 CLECT No No C-lectin - Soluble ref|XP_002168680.1| PREDICTED: similar to predicted protein, par…C-type Soluble Clytia SA0AAB12YJ20RM1

DC-SIGN protein isoform B [Canis lupus familiaris] e-12 CLECT, egf No No C-lectin - Soluble ref|XP_002168680.1| PREDICTED: similar to predicted protein, par... 253 4.00E-65 hydra homologue

C-type Soluble Clytia SA0AAB3YK03CTG CLEC16A protein [Homo sapiens] e-92 No domains No No CLEC16A family - Soluble ref|XP_393990.2| PREDICTED: similar to CG12753-PA, isoform A [Ap... 369 1.00E-100C-type Soluble Hydra CL123Contig2 Cnidarian only CLECT No No C-lectin - Soluble ref|XP_001639701.1| predicted protein [Nematostella vectensis] >... 8.00E-50C-type Soluble Hydra CL9574Contig1 collectin-43 [Bos taurus] 3.00E-08 CLECT No No C-lectin - SolubleC-type Soluble Hydra CL8505Contig1

gb|AAA29218.2| tyrosine kinase receptor [Hydra vulgaris] 1.00E-20 CLECT No No C-lectin - Soluble

C-type Soluble Hydra gb|CV659831.1 tyrosine kinase receptor [Hydra vulgaris] 6.00E-48 CLECT No No C-lectin - SolubleC-type Soluble Hydra gb|CV985524.1 tyrosine kinase receptor [Hydra vulgaris] 1.00E-18 CLECT No No C-lectin - SolubleC-type Soluble Hydra gb|DN816073.2 tyrosine kinase receptor [Hydra vulgaris] 1.00E-23 CLECT No No C-lectin - Soluble


187


C-type Soluble Hydra gb|DT611934.1 tyrosine kinase receptor [Hydra vulgaris] 1.00E-71 CLECT No No C-lectin - SolubleC-type Soluble Hydra gb|DT616206.1 tyrosine kinase receptor [Hydra vulgaris] 2.00E-56 CLECT No No C-lectin - SolubleC-type Soluble Hydra CL5450Contig1 No good hits CLECT (2x) No No C-lectin - SolubleC-type Soluble Hydra CL648Contig1 No good hits CLECT (2x) No No C-lectin - SolubleC-type Soluble Hydra gb|DN812128.2 No good hits CLECT, F5_F8_type_C No No C-lectin - SolubleC-type Soluble Hydra CL5387Contig1

C-type lectin domain family 16, member A [Mus m... 1.00E-41 No Domains No No CLEC16A family - Soluble

C-type Soluble Mbrev

jgi|Monbr1|35486|estExt_fgenesh1_pg.C_20250 No good hits CLECT No No C-lectin - Soluble


jgi|Monbr1|24761|fgenesh2_pg.scaffold_7000309 No good hits

CLECT (wobbly), Recep_L_domain (wobbly) No No C-lectin - Soluble


jgi|Monbr1|17036|estExt_gwp_gw1.C_20126 gb|AAP78681.1| MBCTL2 [Monosiga brevicollis] 0 CCP (3x very wobbly), CLECT No No

gb|AAP78681.1| MBCTL2 [Monosiga brevicollis] same domain structure also

C-type Soluble Nvec jgi|Nemve1|17962|gw.655.3.1 ACAN protein [Homo sapiens] 2.00E-20 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|45368|gw.367.41.1 ACAN protein [Homo sapiens] 2.00E-20 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|149561|e_gw.3035.1.1 aggrecan isoform 1 precursor [Homo sapiens] 9.00E-17 CLECT No No C-lectin - SolubleC-type Soluble Nvec

jgi|Nemve1|239025|estExt_fgenesh1_pg.C_110046 aggrecan isoform 2 precursor [Homo sapiens] 5.00E-15 CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|7634|gw.4914.1.1

Brevican [Homo sapiens] >gi|20380804|gb|AAH27971.... 3.00E-15 CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|154303|e_gw.7191.2.1 brevican core protein 8.00E-22 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|108202|e_gw.89.172.1 brevican isoform 1 [Rattus norvegicus] >gi|1... 3.00E-19 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|83478|e_gw.7.7.1

Brevican soluble core protein precursor [Xenopus ... 5.00E-12 CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|88150|e_gw.17.229.1 C-type lectin [Pocillopora damicornis] 9.00E-24 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|87968|e_gw.17.235.1 C-type lectin [Pocillopora damicornis] 1.00E-22 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|6023|gw.37.6.1 C-type lectin 1 [Anguilla japonica] 3.00E-13 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|103399|e_gw.68.85.1

chondroitin sulfate proteoglycan 2 [Xenopus laevis] 5.00E-15 CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|138199|e_gw.425.7.1

dbj|BAA95671.1| C-type lectin [Cyprinus carpio] 1.00E-12 CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|85784|e_gw.12.21.1 endocytic receptor Endo180 [Homo sapiens] 8.00E-10 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|129425|e_gw.266.73.1 gb|AAA87847.1| brevican core protein 1.00E-21 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|137796|e_gw.413.1.1 gb|AAA87847.1| brevican core protein 1.00E-21 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|118816|e_gw.156.96.1 gb|ABD16187.1| c-type lectin [Danio rerio] 1.00E-10 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|102786|e_gw.66.179.1

gb|ACJ64661.1| hypothetical protein A043-D8 [Acropora millepora] 1.00E-18 CLECT No No C-lectin - Soluble


gb|ACN53515.1| C-type lectin [Pocillopora damicornis] 1.00E-09 CLECT No No C-lectin - Soluble


mannose receptor C type 1 precursor [Homo sapie... 6.00E-13 CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|66344|gw.3.677.1 mannose receptor C1-like protein [Danio rerio] 3.00E-15 CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|149329|e_gw.2934.3.1

mannose receptor, C type 2, isoform CRA_a [Mus mu... 2.00E-09 CLECT No No C-lectin - Soluble


neurocan [Mus musculus] >gi|40675766|gb|AAH6511... 8.00E-19 CLECT No No C-lectin - Soluble


neurocan [Rattus norvegicus] >gi|1709256|sp|P55... 7.00E-16 CLECT No No C-lectin - Soluble

C-type Soluble Nvec

jgi|Nemve1|207088|fgenesh1_pg.scaffold_76000072 No good hits CLECT No No C-lectin - Soluble

C-type Soluble Nvec


C-type Soluble Nvec


C-type Soluble Nvec


C-type Soluble Nvec

jgi|Nemve1|229557|fgenesh1_pm.scaffold_85000002 No good hits CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|29214|gw.76.113.1 No good hits CLECT No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|64326|gw.15.244.1 No good hits CLECT No No C-lectin - Soluble


188


C-type Soluble Nvec


ref|NP_001075276.1| Fc fragment of IgE, low affinity II, recepto… 1.00E-12 CLECT No No C-lectin - Soluble


ref|NP_001138689.1| Fc fragment of IgE, low affinity II, recepto… 1.00E-13 CLECT No No C-lectin - Soluble


regenerating islet-derived 2 [Mus musculus] >gi... 5.00E-11 CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|145810|e_gw.1517.2.1 No good hits CLECT (wobbly) No No C-lectin - SolubleC-type Soluble Nvec jgi|Nemve1|78993|e_gw.1.634.1 No good hits CLECT (wobbly) No No C-lectin - SolubleC-type Soluble Nvec

jgi|Nemve1|203353|fgenesh1_pg.scaffold_42000068 No good hits CLECT, I-set, ntp No No C-lectin - Soluble

C-type Soluble Nvec

jgi|Nemve1|203358|fgenesh1_pg.scaffold_42000073 No good hits CLECT, ntp No No C-lectin - Soluble

C-type Soluble Nvec

jgi|Nemve1|210423|fgenesh1_pg.scaffold_121000042 No good hits EGF, CLECT No No C-lectin - Soluble

C-type Soluble Nvec

jgi|Nemve1|210418|fgenesh1_pg.scaffold_121000037 No good hits EGF, LINK, CLECT No No C-lectin - Soluble

C-type Soluble Nvec


chondroitin sulfate proteoglycan 2 variant V3 [Xe... 3.00E-13 LINK, CLECT No No C-lectin - Soluble

C-type Soluble Nvec jgi|Nemve1|84933|e_gw.10.237.1 protein CLEC16A, putative [Ixodes scapularis] 0 No domains No No CLEC16A family - SolubleCollectin Clytia SA0AAA11YD17CTG No good hits Collagen, CLECT No No Collectin - putative

Collectin Nvecjgi|Nemve1|199302|fgenesh1_pg.scaffold_14000067

hypothetical protein A044-C2 [Acropora millepora] 5.00E-36 Collagen, CLECT Yes No Collectin - putative Collectins have a collagen and CLECT domain. Start and stop codons present

Collectin-Like Hydra CL1Contig283hypothetical protein A044-C2 [Acropora millepora] 1.00E-69 Collagen, Gal_lectin No No Collectin like gb|ACJ64658.1| hypothetical protein A044-C2 [Acropora millepora]

Collectin-Like Hydra CL5890Contig1hypothetical protein A044-C2 [Acropora millepora] 1.00E-33 Collagen, Gal_lectin No No Collectin like gb|ACJ64658.1| hypothetical protein A044-C2 [Acropora millepora]

Collectin-Like Hydra CL684Contig1hypothetical protein A044-C2 [Acropora millepora] 8.00E-51 Collagen, Gal_lectin No No Collectin like collagen, Gal_lectin is not found in other organisms

Collectin-Like Nvecjgi|Nemve1|232015|fgsh_est.C_scaffold_14000009

hypothetical protein A044-C2 [Acropora millepora] 7.00E-81 Collagen, Gal_lectin Yes No Collectin like start and stop codons present

Collectin-Like Clytia IL0ABA6YM11RM1hypothetical protein A044-C2 [Acropora millepora] 3.00E-40 Collagen, Gal_lectin Yes No Collectin like

IL0ABA6YM11RM1 gb|ACJ64658.1| hypothetical protein A044-C2 [Acropora millepora] 169 3.00E-40IL0ABA6YM11RM1 ref|XP_002160660.1| PREDICTED: similar to predicted protein isof... 167 9.00E-40IL0ABA6YM11RM1 ref|XP_001639343.1| predicted protein [Nematostella vectensis] >... 160 1.00E-37. Starts with an M.

Collectin-Like Clytia IL0ABA19YN17RM1Hypothetical protein A044-C2 [Acropora millepora] 3.00E-28 Collagen, Gal_lectin Yes No Collectin like

A044-C2 is part of contig C_mge-A048-G3-post22-T which has no good blast hits and domains: SP, Collagen, Gal_lectin and 1 predicted TM in the N-terminal overlapping the SP - this is not likely to be a true TM.

Collectin-Like Nvecjgi|Nemve1|220309|fgenesh1_pg.scaffold_499000008

hypothetical protein A044-C2 [Acropora millepora] 4.00E-11

Collagen (2x), Gal_lectin (wobbly) No No Collectin like - putative start and stop present

Collectin-Like Nvecjgi|Nemve1|232014|fgsh_est.C_scaffold_14000008

hypothetical protein A032-H1 [Acropora millepora] 5.00E-62 Collagen, Gal_lectin Yes 1 Collectin like

start and stop present predicted TM in the N-terminal overlapping the SP - this is not likely to be a true TM

Collectin-Like Clytia SA0AAB24YI18RM1hypothetical protein A032-H1 [Acropora millepora] 9.00E-64 Collagen, Gal_lectin Yes 1 Collectin like

ref|XP_002157988.1| PREDICTED: similar to predicted protein isof... 262 3.00E-68ref|XP_002158019.1| PREDICTED: similar to predicted protein isof... 262 3.00E-68gb|ACJ64659.1| hypothetical protein A032-H1 [Acropora millepora] 247 9.00E-64ref|XP_001639420.1| predicted protein [Nematostella vectensis] >... 242 2.00E-62 predicted TM in the N-terminal overlapping the SP - this is not likely to be a true TM

Collectin-Like Acropora Contig31112hypothetical protein A044-C2 [Acropora millepora] 2.00E-93 Collagen, Gal_lectin Yes No Collectin like

Collectin-Like Acropora Contig12324hypothetical protein A032-H1 [Acropora millepora] 3.00E-93 Collagen, Gal_lectin Yes No Collectin like

Collectin-Like Acropora Contig8056hypothetical protein A044-C2 [Acropora millepora] 5.00E-37 Collagen, Gal_lectin No No Collectin like

Collectin-Like Acropora Contig4833hypothetical protein A044-C2 [Acropora millepora] 5.00E-11 Collagen, Gal_lectin Yes No Collectin like

Collectin-Like Acropora Contig4828hypothetical protein A043-H7 [Acropora millepora] 1.00E-86 Collagen, Gal_lectin No No Collectin like

C-type Transmembrane Clytia SA0AAA8YD10CTG

ref|NP_055545.1| malectin [Homo sapiens] >gi|2495712|sp|Q14165.1… e-64 No domains Yes 1 Malectin

SA0AAA8YD10CTG ref|XP_002168415.1| PREDICTED: similar to predicted protein [Hyd... 301 4.00E-80SA0AAA8YD10CTG ref|XP_001639476.1| predicted protein [Nematostella vectensis] >... 268 4.00E-70. No domains is consistent with the blast hit. Malectin is a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation

C-type Transmembrane Clytia SA0AAB55YA06CTG hydra homolgue only Ig, CLECT (2x) Yes 1 C-type Lectin - lectin/Ig

ref|XP_002160934.1| PREDICTED: similar to predicted protein [Hyd... 315 5.00E-84 this hit contains 2 CLEC, TM.

C-type Transmembrane Hydra gb|CN774438.1 No good hits CLECT No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III C-type Transmembrane Hydra gb|DN812863.2 tyrosine kinase receptor [Hydra vulgaris] 5.00E-27 CLECT No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III C-type Transmembrane Hydra CL8965Contig1 No good hits CLECT No 3 C-type Lectin - TM Can not be confirmed that these are Type II or Type III C-type Transmembrane Hydra CL1143Contig1

malectin [Homo sapiens] >gi|2495712|sp|Q14165.1... 2.00E-57 No Domains No No Malectin

Malectin is a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation


189


C-type Transmembrane Mbrev

jgi|Monbr1|33817|estExt_fgenesh2_pg.C_250037 MBCTL1 [Monosiga brevicollis] 3.00E-14 CLECT No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III. Start and stop codons present

C-type Transmembrane Mbrev

jgi|Monbr1|34706|estExt_fgenesh2_pg.C_460051 MBCTL1 [Monosiga brevicollis] 1.00E-177 CLECT (2x) Yes 1 AAP78680 MBCTL1 MBCTL1 has 3 lectin domains however this may have been an edited model

C-type Transmembrane Nvec

jgi|Nemve1|210164|fgenesh1_pg.scaffold_117000056 aggrecan isoform 1 precursor [Homo sapiens] 2.00E-16 ntp (wobbly, CLECT (wobbly) No 1 C-type Lectin - TM Can not be confirmed that these are Type II or Type III. Start and stop codons present



gb|ABW80963.1| acidic repeat protein [Treponema paraluiscuniculi] 1.00E-55

Astacin, TSP1, CLECT, TSP1 (2x) No 2 C-type Lectin - TM Can not be confirmed that these are Type II or Type III. Start and stop codons present


jgi|Nemve1|221181|fgenesh1_pg.scaffold_685000004 cyclase family protein [Roseovarius sp. 217] ... 1.00E-15 CLECT (wobbly), cyclase No 2 C-type Lectin - TM

Can not be confirmed that these are Type II or Type III. Start and stop codons present Unique architecture CLECT, TM, Cyclase, TM


jgi|Nemve1|248367|estExt_fgenesh1_pg.C_5290004

gb|ABK78718.1| dendritic cell immunoactivating receptor [Anas pl… 1.00E-07 CLECT Yes 1 C-type Lectin - TM Type I


jgi|Nemve1|197952|fgenesh1_pg.scaffold_7000235 No good hits CLECT Yes 1 C-type Lectin - TM Type I



gb|AAR24388.1| mannose receptor C1 [Sus scrofa] 1.00E-72

CLECT (3x), F5_F8_type_C, CLECT, LamG (wobbly), CLECT (5x), disc4, F5_F8_type_C (2x), SEA (wobbly) Yes 1 C-type Lectin - TM Type I Unique Architecture


jgi|Nemve1|199676|fgenesh1_pg.scaffold_16000126 Pod-EPPT [Podocoryne carnea] 8.00E-11 CLECT (wobbly), ntp (wobbly) Yes 1 C-type Lectin - TM Type I



PTX (wobbly), CLECT, ntp (wobbly) Yes 1 C-type Lectin - TM Type I


jgi|Nemve1|198924|fgenesh1_pg.scaffold_12000147 No good hits CLECT (2x) Yes 2 C-type Lectin - TM Type I probably only single pass


jgi|Nemve1|179566|estExt_GenewiseH_1.C_130133

ref|NP_055545.1| malectin [Homo sapiens] >gi|2495712|sp|Q14165.1… 1.00E-53 No domains Yes No Malectin

malectin has no domains. Malectin is a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation

Fucolectin Nvecjgi|Nemve1|216294|fgenesh1_pg.scaffold_256000002 Fucolectin-4 precursor [Esox lucius] 2.00E-06 CLECT, ftp No 2 Fucolectin - Transmembrane

There are only 2 transmembrane fucolectins… Drosophila- furrowed (CCP repeats, one CLECT, 1 ftp) and Tachylectin-4 (SP, ftp) which recognizes bacterial lipopolysaccharide, probably through binding to fucose-like sugars. Start and stop codons are presnt. Probable artifact due to lack of EST support and SP, highly repetative TM region, short scaffold.

Fucolectin Nvecjgi|Nemve1|214591|fgenesh1_pg.scaffold_204000015 FBP32 precursor [Morone chrysops] 2.00E-31 ftp Yes No Fucolectin - Secreted (FBP32 like)

Fucolectin Nvecjgi|Nemve1|221173|fgenesh1_pg.scaffold_683000006 Fucolectin-4 precursor [Esox lucius] 8.00E-28 ftp Yes No Fucolectin - Secreted

Fucolectin Nvecjgi|Nemve1|221640|fgenesh1_pg.scaffold_887000005 No good hits F5_F8_type_C, ftp (wobbly) Yes No Fucolectin - Secreted

Fucolectin Nvec jgi|Nemve1|144857|e_gw.1185.2.1 bryohealin precursor [Bryopsis plumosa] 8.00E-18 ftp No No Fucolectin - Soluble

Fucolectin Nvec jgi|Nemve1|100075|e_gw.55.26.1 FBP32 precursor [Morone saxatilis] 1.00E-41 ftp No No Fucolectin - Solubleeel-Fucolectin Tachylectin-4 Pentaxrin-1 Domain, ftp domain is the SMART version of F5_F8_type_C/FA58C

Fucolectin Nvec jgi|Nemve1|123063|e_gw.196.12.1 FBP32II precursor [Morone chrysops] 3.00E-51 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|11782|gw.196.4.1 FBP32II precursor [Morone chrysops] 9.00E-38 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|106318|e_gw.81.40.1 fucolectin [Fundulus heteroclitus] 2.00E-27 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|8429|gw.9884.1.1 Fucolectin-4 precursor [Esox lucius] 7.00E-32 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|130395|e_gw.279.36.1 Fucolectin-4 precursor [Esox lucius] 5.00E-31 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|17374|gw.224.24.1 Fucolectin-4 precursor [Esox lucius] 3.00E-30 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|113390|e_gw.117.83.1 Fucolectin-4 precursor [Esox lucius] 3.00E-29 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|101196|e_gw.60.139.1 Fucolectin-4 precursor [Esox lucius] 2.00E-26 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|16092|gw.174.11.1 Fucolectin-4 precursor [Esox lucius] 4.00E-24 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|28280|gw.196.56.1 Fucolectin-4 precursor [Esox lucius] 6.00E-23 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|101150|e_gw.59.65.1 Fucolectin-4 precursor [Esox lucius] 5.00E-18 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|155896|e_gw.9311.2.1 Fucolectin-4 precursor [Esox lucius] 4.00E-17 ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|6868|gw.741.1.1 Fucolectin-4 precursor [Esox lucius] 2.00E-13 ftp No No Fucolectin - Soluble

Fucolectin Nvecjgi|Nemve1|217467|fgenesh1_pg.scaffold_298000014 Fucolectin-4 precursor [Esox lucius] 7.00E-07 ftp No No Fucolectin - Soluble

Fucolectin Nvec jgi|Nemve1|9509|gw.10015.2.1 No good hits ftp No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|123139|e_gw.196.46.1 FBP32 precursor [Morone saxatilis] 3.00E-47 ftp (2x) No No Fucolectin - SolubleFucolectin Nvec jgi|Nemve1|112201|e_gw.110.50.1 fucose binding lectin [Dicentrarchus labrax] 5.00E-43 ftp (2x) No No Fucolectin - Soluble

Fucolectin Nvecjgi|Nemve1|239599|estExt_fgenesh1_pg.C_170022

furrowed [Aedes aegypti] >gi|108875029|gb|EA... 3.00E-12 Ricin_B_lectin (wobbly), ftp No No Fucolectin - Soluble

Fucolectin Nvec jgi|Nemve1|65775|gw.61.259.1 No good hits ftp No No Fucolectin - SolubleFucolectin Hydra CL3322Contig1 No good hits ftp Yes No Fucolectin - SecretedFucolectin Acropora Contig2805 BTB, ftp FucolectinFucolectin Acropora Contig8409 ftp FucolectinFucolectin Acropora run002_427609 ftp FucolectinFucolectin Acropora Contig11806 ftp FucolectinFucolectin Acropora Amil_c11587 ftp FucolectinFucolectin Acropora run002_434733 ftp FucolectinFucolectin Acropora run001daytona_1706338 ftp (2x) Fucolectin


190


Fucolectin Acropora run001daytona_1732288 ftp FucolectinFucolectin Acropora Amil_c56240 ftp FucolectinFucolectin Acropora run001daytona_1712753 ftp FucolectinFucolectin Acropora run002_436148 ftp FucolectinFucolectin Acropora Contig18100 ftp, disc4 FucolectinFucolectin Acropora run001daytona_1711122 ftp FucolectinFucolectin Acropora run001daytona_1719357 ftp FucolectinFucolectin Acropora run001daytona_1723531 ftp FucolectinFucolectin Acropora run001daytona_1714209 ftp FucolectinFucolectin Acropora run001daytona_1704584 ftp FucolectinFucolectin Acropora Contig2564 ricin_B_lectin(wobbly),ftp FucolectinFucolectin Acropora Amil_c24802 ftp FucolectinFucolectin Acropora Contig25156 ftp FucolectinFucolectin Acropora Amil_c83456 ftp Fucolectin

legume like Hydra CL4270Contig1mannose-binding endoplasmic reticulum-golgi inter... 3.00E-75 Lectin_leg-like Yes 1

mannose-binding endoplasmic reticulum-golgi inter...

legume like Hydra CL641Contig1Vesicular integral-membrane protein VIP36 precurs... 1.00E-84 Lectin_leg-like No No

Vesicular integral-membrane protein VIP36 precurs...

legume like Mbrevjgi|Monbr1|33100|estExt_fgenesh2_pg.C_160036

ref|XP_001654670.1| vesicular mannose-binding lectin [Aedes aegy… 1.00E-66 Lectin_leg-like Yes 1 vesicular mannose-binding lectin

legume like Mbrevjgi|Monbr1|34633|estExt_fgenesh2_pg.C_430019 vesicular mannose-binding lectin [Aedes aegy... 1.00E-52 Lectin_leg-like Yes 1 vesicular mannose-binding lectin

legume like Nvec jgi|Nemve1|129670|e_gw.268.1.1gb|EDM14702.1| lectin, mannose-binding, 1, isoform CRA_a [Rattus… 1.00E-102 Lectin_leg-like No 1 lectin, mannose-binding

legume like Nvec jgi|Nemve1|82673|e_gw.6.366.1 vesicular mannose-binding lectin [Aedes aegy... 2.00E-90 Lectin_leg-like Yes 1 vesicular mannose-binding lectinGal-lectin Hydra CL1084Contig1 No good hits Gal_lectin Yes 1Gal-lectin Hydra CL1096Contig1 No good hits Gal_lectin No NoGal-lectin Hydra CL2279Contig1 No good hits Gal_lectin Yes 1Gal-lectin Hydra CL3738Contig1 No good hits Gal_lectin No NoGal-lectin Hydra gb|CO537930.1 No good hits Gal_lectin No NoGal-lectin Hydra gb|DN245375.2 No good hits Gal_lectin No NoGal-lectin Hydra gb|DT614072.1 No good hits Gal_lectin Yes NoGal-lectin Hydra CL1172Contig1 No good hits Gal_lectin (2x) No No

Gal-lectin Hydra CL320Contig1rhamnose-binding lectin OLL [Spirinchus lanceola... 5.00E-29 Gal_lectin (2x) Yes No

Gal-lectin Hydra CL3363Contig1rhamnose-binding lectin WCL3 [Salvelinus leucoma... 3.00E-27 Gal_lectin (2x) Yes 1

Gal-lectin Hydra CL385Contig2 24 kDa egg lectin [Oncorhynchus tshawytscha] 4.00E-31 Gal_lectin (2x) No NoGal-lectin Hydra CL385Contig3 24 kDa egg lectin [Oncorhynchus tshawytscha] 2.00E-30 Gal_lectin (2x) No NoGal-lectin Hydra CL385Contig1 rhamnose binding lectin [Tribolodon brandtii] 3.00E-34 Gal_lectin (3x) No No

Gal-lectin Hydra CL9067Contig1rhamnose-binding lectin WCL3 [Salvelinus leucoma... 1.00E-31 Gal_lectin (3x) No No

Gal-lectin Hydra CL1523Contig1rhamnose-binding lectin WCL3 [Salvelinus leucoma... 3.00E-30 Gal_lectin (4x) No No

Gal-lectin Hydra CL2416Contig1rhamnose binding lectin STL3 [Oncorhynchus m... 4.00E-26 Gal_lectin (4x) No No

Gal-lectin Hydra CL55Contig1rhamnose binding lectin STL3 [Oncorhynchus m... 7.00E-30 Gal_lectin (4x) No No

Gal-lectin Hydra CL5403Contig1 No good hits Gal_lectin (wobbly) No No

Gal-lectin Hydra CL6176Contig1nematocyst outer wall antigen precursor [Clytia h... 2.00E-38

Gal_lectin (wobbly), keratin_B2 (wobbly) No No

Gal-lectin Nvecjgi|Nemve1|241976|estExt_fgenesh1_pg.C_540065 No good hits CD20 (wobbly), Gal_lectin

Gal-lectin Nvecjgi|Nemve1|214497|fgenesh1_pg.scaffold_202000001 depsiphilin [Cooperia oncophora] 1.00E-10 Gal_lectin

Gal-lectin Nvecjgi|Nemve1|216053|fgenesh1_pg.scaffold_247000022 depsiphilin [Cooperia oncophora] 4.00E-07 Gal_lectin

Gal-lectin Nvecjgi|Nemve1|232013|fgsh_est.C_scaffold_14000007

hypothetical protein A043-H7 [Acropora millepora] 5.00E-40 Gal_lectin

Gal-lectin Nvec jgi|Nemve1|18799|gw.247.36.1 lectomedin 1 alpha-like [Ciona intestinalis] 1.00E-12 Gal_lectinGal-lectin Nvec jgi|Nemve1|102384|e_gw.64.13.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|106512|e_gw.82.91.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|111827|e_gw.108.32.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|111847|e_gw.108.96.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|111952|e_gw.108.39.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|118227|e_gw.152.81.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|124712|e_gw.213.49.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|124735|e_gw.213.92.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142147|e_gw.645.22.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142154|e_gw.645.34.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142157|e_gw.645.18.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142164|e_gw.645.13.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142165|e_gw.645.31.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|142168|e_gw.645.15.1 No good hits Gal_lectinGal-lectin Nvec jgi|Nemve1|148507|e_gw.2440.4.1 No good hits Gal_lectin

Gal-lectin Nvecjgi|Nemve1|205905|fgenesh1_pg.scaffold_64000028 No good hits Gal_lectin

Gal-lectin Nvecjgi|Nemve1|216054|fgenesh1_pg.scaffold_247000023 No good hits Gal_lectin


191


Gal-lectin Nvecjgi|Nemve1|246049|estExt_fgenesh1_pg.C_1940004 No good hits Gal_lectin

Gal-lectin Nvec jgi|Nemve1|128256|e_gw.247.49.1 novel rhamnose binding lectin [Danio rerio] 9.00E-09 Gal_lectin

Gal-lectin Nvec jgi|Nemve1|87157|e_gw.15.283.1rhamnose-binding lectin precursor [Branchiostoma ... 3.00E-16 Gal_lectin




Gal-lectin Nvec jgi|Nemve1|82364|e_gw.5.561.1 skin mucus lectin [Leiognathus nuchalis] 1.00E-08 Gal_lectin

Gal-lectin Nvecjgi|Nemve1|209268|fgenesh1_pg.scaffold_103000043

rhamnose-binding lectin OLL [Spirinchus lanceola... 3.00E-13 Gal_lectin (2x wobbly)

Gal-lectin Nvec jgi|Nemve1|106614|e_gw.82.111.1 No good hits Gal_lectin (2x)Gal-lectin Nvec jgi|Nemve1|140909|e_gw.552.7.1 No good hits Gal_lectin (2x)


Rhamnose-binding lectin [Salmo salar] >gi|20... 2.00E-15 Gal_lectin (2x)

Gal-lectin Nvec jgi|Nemve1|174671|estExt_gwp.C_3180014rhamnose-binding lectin WCL3 [Salvelinus leucoma... 3.00E-30 Gal_lectin (2x)

Gal-lectin Nvec jgi|Nemve1|122916|e_gw.194.48.1 rhamnose binding lectin [Tribolodon brandtii] 7.00E-37 Gal_lectin (3x)Gal-lectin Nvec jgi|Nemve1|142158|e_gw.645.36.1 rhamnose binding lectin [Tribolodon brandtii] 2.00E-36 Gal_lectin (3x)Gal-lectin Nvec jgi|Nemve1|111951|e_gw.108.118.1 rhamnose binding lectin [Tribolodon brandtii] 2.00E-34 Gal_lectin (3x)Gal-lectin Nvec jgi|Nemve1|131398|e_gw.294.64.1 rhamnose binding lectin [Tribolodon brandtii] 3.00E-21 Gal_lectin (3x)

Gal-lectin Nvecjgi|Nemve1|237832|estExt_fgenesh1_pg.C_10304 rhamnose binding lectin [Tribolodon brandtii] 1.00E-14 Gal_lectin (4x)


hypothetical protein A044-C2 [Acropora millepora] 2.00E-14 Gal_lectin (wobbly)

Gal-lectin Nvecjgi|Nemve1|222353|fgenesh1_pg.scaffold_1557000001 No good hits Gal_lectin (wobbly)


gb|AAX09934.1| ganglioside M2 activator-like protein [Aurelia au… 1.00E-22

Gal_lectin (wobbly), pgtp_13 (wobbly)

Gal-lectin Mbrevjgi|Monbr1|12732|fgenesh1_pg.scaffold_44000010 No good hits EGF (2x), Gal_lectin No 1

Gal-lectin Mbrevjgi|Monbr1|27352|fgenesh2_pg.scaffold_19000006

Beta-galactosidase precursor, putative, expressed... 2.00E-18 Gal_lectin No No

Gal-lectin Clytia IL0ABA5YA20RM1 novel rhamnose binding lectin [Danio rerio] e-10 Gal_lectin No No

ref|XP_002161306.1| PREDICTED: similar to novel rhamnose binding... 198 2.00E-49 this has 2xGal_lec. The Rhaminose binding lectin has one Gal_lec domain and is very short… poor hit indicates this may be something different but length is similar (this starts with an M)

Gal-lectin Clytia SA0AAB36YH22RM1 hydra homolgue only Wobbly Gal_lectin No 1 ref|XP_002169231.1| PREDICTED: similar to LOC733382 protein [Hyd... 151 1.00E-34

Gal-lectin Clytia SA0AAB73YD10CTG Cnidarian homolgues only Gal_lectin Yes No

ref|XP_002169260.1| PREDICTED: similar to predicted protein, par... 218 3.00E-55ref|XP_002159989.1| PREDICTED: similar to predicted protein [Hyd... 218 4.00E-55ref|XP_002160043.1| PREDICTED: similar to predicted protein [Hyd... 153 1.00E-35ref|XP_001639419.1| predicted protein [Nematostella vectensis] >... 130 1.00E-28gb|ACJ64660.1| hypothetical protein A043-H7 [Acropora millepora] 119 3.00E-25

Gal-lectin Clytia IL0ABA4YP02RM1gb|ABY71251.1| nematocyst outer wall antigen precursor [Clytia h... 0 Gal_lectin Yes No gb|ABY71251.1| nematocyst outer wall antigen precursor [Clytia h...

haemolytic Clytia IL0ABA8YL09RM1 CELIII 1.00E-93 2x ricin CELIII

haemolytic Clytia IL0ABA2YE22RM1 CELIII 1.00E-92 2xricin CELIII

has same domain structure and a good blast - heamolytic lectin gb|ACJ64657.1| hypothetical protein A049-E7 [Acropora millepora] 319 4.00E-85gb|ACJ64656.1| hypothetical protein A036-E7 [Acropora millepora] 305 6.00E-81

haemolytic Acropora A036-E7 A036-E7 0 2xricin CELIIIhaemolytic Acropora A049-E7 A049-E7 0 2xricin CELIIIhaemolytic Acropora Contig20384 CELIII 1.00E-131 2xricin CELIII

legume like Acropora Contig6923ref|NP_446338.1| lectin, mannose-binding, 1 [Rattus norvegicus] 1.00E-96 Lectin_leg-like

legume like Acropora run002_224851 vesicular mannose-binding lectin [Aedes aegy... 1.00E-72 Lectin_leg-likelegume like Acropora Contig768 ref|XP_001843193.1| VIP36 1.00E-85 Lectin_leg-like


192

LRR Adhesion

Sub Family Organism Sequence Id BLAST E-value HMMPFAM SP TM Conclusion Notes

LRR-dsl Nematostella jgi|Nemve1|200004|fgenesh1_pg.scaffold_18000128leucine rich repeat containing 15 [Rattus norve... 6.00E-22 lrrnt (wobbly), LRR (8x 7wobbly), GCC2_GCC3, dsl Yes 7 LRR-dsl

LRR-EGF Clytia SA0AAA15YN12CTG noLRR (3x wobbly), EGF (3x wobbly), ringv (wobbly), EGF (wobbly) No 1 LRR-EGF

LRR-EGF Nematostella jgi|Nemve1|203583|fgenesh1_pg.scaffold_44000028 uromodulin 2.00E-14 lrrct, EGF (3x) No 1 LRR-EGF

LRR-EGF Nematostella jgi|Nemve1|199637|fgenesh1_pg.scaffold_16000087slit-like 2 [Homo sapiens] >gi|74748436|sp|Q6EM... 2.00E-22 LRR (9x 8wobbly), lrrct, EGF (4x) Yes 1 LRR-EGF

LRR-EGF Nematostella SA0AAB6YN08RM1 no good hits LRR (6x wobbly), lrrct (wobbly), EGF Yes 1 LRR-EGF

LRR-EGF Nematostella jgi|Nemve1|208146|fgenesh1_pg.scaffold_89000008 microneme protein 4 [Eimeria tenella] 1.00E-40 lrrct (wobbly), EGF (5x) Yes 1 LRR-EGFVILL/gel domains now called GEL by SMART - Gelsolin/severin/villin homology domain

LRR-EGF Clytia jgi|Nemve1|197001|fgenesh1_pg.scaffold_4000081 no good hits lrrnt, LRR (5x), EGF, GCC2_GCC3 (all wobbly) Yes No LRR-EGF

LRR-FN3 Monosiga jgi|Monbr1|31954|estExt_fgenesh2_pg.C_60352Leukocyte-antigen-related-like, isoform B [Dros... 2.00E-22 lrrnt, LRR (10x wobbly), lrrct (wobbly), FN3 (5x) Yes 1 LRR-FN3

LRR-FN3 Nematostella jgi|Nemve1|248399|estExt_fgenesh1_pg.C_5430006ref|NP_001094549.1| protein tyrosine phosphatase, receptor type,… 2.00E-48

F-box (2x wobbly), LRR (3x wobbly), Pentaxin, LamG (wobbly), FBG, PTX, 109ultra, f5_f5_type_C (2x wobbly), MAM (wobbly), LamG (2x wobbly), VirB8 (wobbly), FN3 (9x),TM, Ion_trans_2, Yes 3 LRR-FN3

4260-4282, 4328-4350, 4508-4525 = TM regions. The first 2 overlap with the Ion_trans domain. May not be a surface protein because it has an F-box.

LRR-IG Nematostella jgi|Nemve1|247910|estExt_fgenesh1_pg.C_3760020gb|EDL91410.1| leucine-rich repeats and immunoglobulin-like doma… 6.00E-32

lrrnt, LRR (7x 6wobbly), lrrct, IG, I-set, EGF (wobbly) Yes No LRR-IG

LRR-IG Nematostella jgi|Nemve1|238858|estExt_fgenesh1_pg.C_90051ref|NP_700356.2| leucine-rich repeats and immunoglobulin-like do… 1.00E-131

lrrnt, LRR (10x 6wobbly), lrrct, IG, I-set (2x), ChitinBD_3 (wobbly) Yes 1 LRIG3

LRR-LDLa Monosiga jgi|Monbr1|38718|estExt_fgenesh1_pg.C_300050 no good hits LRR (3x wobbly), LDLa, tyrkin No 1 LRR-LDLa

LRR-LDLa Monosiga jgi|Monbr1|29577|fgenesh2_pg.scaffold_36000018 Epha4a protein [Danio rerio] 5.00E-24 FBG (wobbly), LRR (wobbly), LDLa, tyrkin Yes 1 LRR-LDLadomain structure is very different to Ephrin recept 4a. The fibrinogen C-term may be false hit making this an kinase.

LRR-LDLa Monosiga jgi|Monbr1|37789|estExt_fgenesh1_pg.C_160136 no good hits LRR (2x), LDLa, SKG6 (all wobbly) Yes 1 LRR-LDLa

LRR-LDLa Monosiga jgi|Monbr1|34608|estExt_fgenesh2_pg.C_420022zeta-chain associated protein kinase 70kDa isof... 3.00E-35 LRR (3x wobbly), LDLa (wobbly), tyrkin yes 1 LRR-LDLa

LRR-LDLa Monosiga jgi|Monbr1|28676|fgenesh2_pg.scaffold_28000035 no good hits LRR, LDLa, tyrkin Yes 1 LRR-LDLa

LRR-LDLa Monosiga jgi|Monbr1|33342|estExt_fgenesh2_pg.C_180096Nef associated protein 1 [Homo sapiens] >gi|152... 3.00E-40 lrrnt (wobbly), LDLa, Pkinase_tyr, UPF0066 yes 1 LRR-LDLa TMH is in the SP

LRR-LDLa Monosiga jgi|Monbr1|12385|fgenesh1_pg.scaffold_38000060 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin yes 2 LRR-LDLa only 1 TMH is real.LRR-LDLa Monosiga jgi|Monbr1|32039|estExt_fgenesh2_pg.C_70171 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin yes 2 LRR-LDLa only 1 TMH is real.

LRR-LDLa Monosiga jgi|Monbr1|34526|estExt_fgenesh2_pg.C_390046gb|EEB18061.1| tyrosine-protein kinase transmembrane receptor RO… 2.00E-32 LRR (3x wobbly), LDLa, tyrkin,Pkinase_tyr Yes 2 LRR-LDLa only 1 TMH is real.

LRR-LDLa Monosiga jgi|Monbr1|7318|fgenesh1_pg.scaffold_7000050 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin yes 3 LRR-LDLa one is in the sp

LRR-LDLa Monosiga jgi|Monbr1|33520|estExt_fgenesh2_pg.C_200100ref|ZP_03154554.1| WD-40 repeat protein [Cyanothece sp. PCC 7822… 1.00E-48

WD40 (7x 1wobbly), DUF1042, ADK (wobbly), LRR (wobbly), LDLa, tyrkin, No No LRR-LDLa

LRR-LDLa Monosiga jgi|Monbr1|25751|fgenesh2_pg.scaffold_11000151 no good hits LRR (3x wobbly), LDLa (wobbly), tyrkin (wobbly) Yes No LRR-LDLa

LRR-SUSHI Nematostella jgi|Nemve1|197996|fgenesh1_pg.scaffold_8000037 no good hitsMAM (wobbly), CCP (wobbly), SR, LRR (4x), EGF (2x wobbly) Yes 5 LRR-SUSHI

LRR-SUSHI / LRR-dsl Monosiga jgi|Monbr1|33936|estExt_fgenesh2_pg.C_270059 no good hits LRR (6x 3wobbly), CCP (4x wobbly), dsl, SKG6 Yes 1 LRR-SUSHI / LRR-dsl

dsl- Ligands of the Delta/Serrate/lag-2 (DSL) family and their receptors, members of the lin-12/Notch family, mediate cell-cell interactions that specify cell fate in invertebrates and vertebrates -pfam

Scribbled Nematostella jgi|Nemve1|10117|gw.267.1.1 Scrib protein [Mus musculus] 1.00E-122 LRR (13x wobbly), PDZ (4x) No No scribbledScribbled has a role in cell polarity pathways and is associated with Fat-1.

Scribbled Acropora Contig8961scribbled homolog [Danio rerio] >gi|71000206... 1.00E-150 LRR (15x wobbly), PDZ (4x) No No Scribbled

VWA-LRR Clytia SA0AAB78YB05CTG no good hits VWA (wobbly), lrrnt (wobbly) Yes No VWA-LRRLRR-IG Acropora Amil_c39609 gb|AAI38423.1|,Lrig2 protein [Mus musculus] 1.00E-14 LRRCT, I-set LRR-IGLRR-IG Acropora run002_428190 no good hits LRRCT, I-set LRR-IGLRR-EGF Acropora Amil_c47067 no good hits LRR(3x), EGF (2x) LRR-EGF

LRR-IG Acropora Contig16713sp|Q6P1C6.1|LRIG3_MOUSE RecName: Full=Leucine-rich repeats and i… 1.00E-109 LRRNT, LRR (14x wobbly), lrrct, Ig, I-set (2x) Yes 1 LRIG3

LRR-IG Acropora Contig24535 gb|EDL91410.1| LRIG 1.00E-51 LRR (12x), Ig (2x) LRR-IG


193

Class B GPCR

Sub Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes

7tm_2 Only Acropora Amil_c32588brain-specific angiogenesis inhibitor 3 [Mus mu... 1.00E-11 7tm_2 7tm_2

7tm_2 Only Acropora Amil_c47079class B secretin-like G-protein coupled receptor ... 3.00E-17 7tm_2 7tm_2

7tm_2 Only Acropora Amil_c53870latrophilin-like protein 2 [Haemonchus contortus]... 3.00E-22 7tm_2 7tm_2

7tm_2 Only Acropora Amil_c5902G protein-coupled receptor 64 [Mus musculus] 6.00E-08 7tm_2 7tm_2

7tm_2 Only Acropora Amil_c68305G protein-coupled receptor 133 [Danio rerio]... 1.00E-37 7tm_2 7tm_2

7tm_2 Only Acropora Contig10827 Bai3 protein [Mus musculus] 2.00E-32 7tm_2 7tm_2

7tm_2 Only Acropora Contig11360G protein-coupled receptor 133 [Bos taurus] ... 9.00E-24 7tm_2 7tm_2

7tm_2 Only Acropora Contig13559cadherin EGF LAG seven-pass G-type receptor 1 p... 3.00E-26 7tm_2 7tm_2

7tm_2 Only Acropora Contig15714 GPR133 protein [Homo sapiens] 8.00E-19 7tm_2 7tm_2

7tm_2 Only Acropora Contig1586G protein-coupled receptor 133 [Bos taurus] ... 2.00E-38 7tm_2 7tm_2

7tm_2 Only Acropora Contig17422Latrophilin receptor protein 2 [Brugia malay... 1.00E-13 7tm_2 7tm_2

7tm_2 Only Acropora Contig19559G-protein coupled receptor GPR133 [Homo sapiens] 3.00E-49 7tm_2 7tm_2

7tm_2 Only Acropora Contig19686G protein-coupled receptor 157 [Danio rerio]... 6.00E-23 7tm_2 7tm_2

7tm_2 Only Acropora Contig19849latrophilin 3, isoform CRA_d [Rattus norvegicus] 3.00E-33 7tm_2 7tm_2

7tm_2 Only Acropora Contig5114 latrophilin 3, isoform CRA_d [Homo sapiens] 2.00E-22 7tm_2 7tm_2

7tm_2 Only Acropora Contig7929G protein-coupled receptor 112 [Mus musculus] >g... 6.00E-25 7tm_2 7tm_2

7tm_2 Only Acropora run001daytona_1711571latrophilin-like protein 2 [Haemonchus contortus]... 7.00E-41 7tm_2 7tm_2

7tm_2 Only Acropora Contig22262G protein-coupled receptor 157 [Danio rerio]... 8.00E-43 7tm_2(wobbly) 7tm_2

7tm_2 Only Acropora Contig26829G protein-coupled receptor 157 [Danio rerio]... 2.00E-45 7tm_2(wobbly) 7tm_2

7tm_2 Only Acropora Contig7015cAMP receptor TasA [Polysphondylium pallidum] 5.00E-14 7tm_2(wobbly) 7tm_2

7tm_2 Only Acropora run001daytona_1717054brain-specific angiogenesis inhibitor 2 [Mus mus... 7.00E-13 7tm_2(wobbly) 7tm_2

7tm_2 Only Acropora run001daytona_1725222 latrophilin 3 precursor [Homo sapiens] 7.00E-11 7tm_2(wobbly) 7tm_2

7tm_2 Only Acropora run001daytona_4304G-protein coupled receptor MtH2, putative [Ixodes... 2.00E-10 7tm_2(wobbly) 7tm_2

7tm_2 Only Clytia SA0AAB90YJ23RM1 latrophilin 2, isoform CRA_a [Homo sapiens] 1.00E-30 7tm_2 7tm_2

7tm_2 Only Clytia SA0AAB153YN24CTGlatrophilin 3, isoform CRA_h [Rattus norvegicus] 2.00E-25 7tm_2 7tm_2

7tm_2 Only Clytia SA0AAB37YC22RM1G protein-coupled receptor 157 [Danio rerio]... 4.00E-28 Dicty_CAR (wobbly), 7tm_2 7tm_2

7tm_2 Only Clytia SA0AAB111YC11CTG latrophilin 3 precursor [Homo sapiens] 3.00E-10 Dicty_CAR (wobbly), 7tm_2 7tm_2

7tm_2 Only Hydra gb|DT607625.1G protein-coupled receptor 133 [Bos taurus] ... 3.00E-15 7tm_2 7tm_2

7tm_2 Only Hydra CL2460Contig1latrophilin 3, isoform CRA_h [Rattus norvegicus] 1.00E-14 7tm_2 7tm_2

7tm_2 Only Hydra gb|CX054457.1G protein-coupled receptor 133 [Danio rerio]... 2.00E-12 7tm_2 (wobbly) 7tm_2

7tm_2 Only Hydra CL8507Contig1G-protein coupled receptor, putative [Ixodes scap... 2.00E-10 7tm_2 (wobbly) 7tm_2

7tm_2 Only Mbrevjgi|Monbr1|33227|estExt_fgenesh2_pg.C_170070

cAMP receptor cAR [Dictyostelium aureostipes] 6.00E-19 Dicty_CAR/7tm_2 (wobbly) 7tm_2


194


7tm_2 Only Nvecjgi|Nemve1|202568|fgenesh1_pg.scaffold_36000045

G protein-coupled receptor 157 [Danio rerio]... 2.00E-45 7tm_2 7tm_2

7tm_2 Only Nvecjgi|Nemve1|133486|e_gw.317.3.1

latrophilin 1, isoform CRA_a [Rattus norvegicus] 1.00E-44 7tm_2 7tm_2

7tm_2 Only Nvec jgi|Nemve1|35189|gw.75.111.1G protein-coupled receptor 157 [Danio rerio]... 1.00E-44 7tm_2 7tm_2


G-protein coupled receptor GPR133 [Homo sapiens] 8.00E-41 7tm_2 7tm_2

7tm_2 Only Nvec jgi|Nemve1|36088|gw.29.89.1G protein-coupled receptor 133 [Bos taurus] ... 2.00E-39 7tm_2 7tm_2


latrophilin 3, isoform CRA_d [Rattus norvegicus] 9.00E-34 7tm_2 7tm_2


latrophilin 3, isoform CRA_d [Rattus norvegicus] 1.00E-31 7tm_2 7tm_2


gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 9.00E-30 7tm_2 7tm_2

7tm_2 Only Nvec jgi|Nemve1|53186|gw.299.70.1gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-29 7tm_2 7tm_2



7tm_2 Only Nvecjgi|Nemve1|139162|e_gw.463.5.1 latrophilin 1, isoform CRA_d [Homo sapiens] 1.00E-26 7tm_2 7tm_2







7tm_2 Only Nvecjgi|Nemve1|187681|estExt_GenewiseH_1.C_1050036



G protein-coupled receptor 112 [Mus musculus] >g... 1.00E-17 7tm_2 7tm_2 Group III - Has unseen FA5/8 domains



7tm_2 Only Nvec jgi|Nemve1|43482|gw.166.57.1latrophilin 2 [Homo sapiens] >gi|56204774|emb|CA... 5.00E-17 7tm_2 7tm_2


G protein-coupled receptor 133 [Bos taurus] ... 3.00E-14 7tm_2 7tm_2


7tm_2 Only Nvecjgi|Nemve1|242602|estExt_fgenesh1_pg.C_670060

G protein-coupled receptor 157 [Danio rerio]... 1.00E-42 7tm_2 (wobbly) 7tm_2


gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-19 7tm_2 (wobbly) 7tm_2


G protein-coupled receptor 133 [Bos taurus] ... 3.00E-13 7tm_2 (wobbly) 7tm_2

7tm_2 Only Nvecjgi|Nemve1|131887|e_gw.302.38.1 predicted peptides only 7tm_2 (wobbly) 7tm_2

7tm_2 Only Nvecjgi|Nemve1|198777|fgenesh1_pg.scaffold_11000189 predicted peptides only 7tm_2 (wobbly) 7tm_2

7tm_2 Only Nvecjgi|Nemve1|211774|fgenesh1_pg.scaffold_143000051 No good hits 7tm_2 (wobbly) 7tm_2

CLECT-GPS-7tm_2 Mbrev

jgi|Monbr1|37365|estExt_fgenesh1_pg.C_120196 MB7TM1 [Monosiga brevicollis] 2.00E-08 CLECT (wobbly), GPS, 7tm_2

Group V -Adhesion homologues in C.elegans (2) - PFAM


195

Sub Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes CLECT-GPS-7tm_3 Nvec


gb|EEB17331.1| class B secretin-like G-protein coupled receptor … 9.00E-45 CLECT, GPS, 7tm_2


CLECT-GPS-7tm_4 Nvec

jgi|Nemve1|204814|fgenesh1_pg.scaffold_54000011 latrophilin 3, isoform CRA_b [Homo sapiens] 2.00E-48 CLECT, GPS, 7tm_2


CLECT-GPS-7tm_5 Nvec

jgi|Nemve1|211772|fgenesh1_pg.scaffold_143000049 latrophilin 2, isoform CRA_a [Homo sapiens] 6.00E-29 CLECT, GPS, 7tm_2


Frizzled Clytia SA0AAB17YK01RM1 frizzled 2 [Hydra magnipapillata] 1.00E-122 Frizzled/7tm_2 Frizzled

Frizzled Clytia IL0ABA3YD02RM1frizzled-8 [Xenopus laevis] >gi|3869266|gb|A... 2.00E-22 Fz

Frizzled - putative There are described Frizzled proteins in Hydra.

Frizzled Clytia SA0AAB36YE16RM17-transmembrane receptor frizzled-1 [Xenopus... 8.00E-20 Fz, Gal_lectin(wobbly)

Frizzled - putative

Frizzled Clytia IL0ABA4YO10RM1frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 2.00E-19 Fz, Gal_lectin(wobbly)

Frizzled - putative

Frizzled Hydra CL4401Contig1 frizzled 2 [Hydra magnipapillata] 1.00E-101 Fz Frizzled

Frizzled Hydra CL6396Contig1 frizzled receptor [Hydra vulgaris] 1.00E-127 Fz Frizzlednext best hit frizzled [Aedes aegypti] >gi|108875685|gb|EA... 6.00E-07

Frizzled Hydra CL10411Contig1 frizzled receptor [Hydra vulgaris] 7.00E-60 No domains Frizzled

Frizzled Hydra gb|CX835366.2PREDICTED: Frizzled4/9/10 [Hydra magnipapill... 3.00E-59 Fz (wobbly) Frizzled

Frizzled Hydra CL2039Contig1 Fzd7 protein [Mus musculus] 6.00E-25 FzFrizzled - putative

Frizzled Hydra CL5686Contig1frizzled homolog 8 [Rattus norvegicus] >gi|1... 5.00E-16 Fz, gal_lectin (wobbly)

Frizzled - putative

Frizzled Hydra gb|CN560484.1 frizzled 2 [Hydra magnipapillata] 1.00E-33 No domainsFrizzled - putative

Frizzled Nvecjgi|Nemve1|171640|estExt_gwp.C_1830043

7-transmembrane receptor frizzled-1 [Xenopus... 1.00E-180 Fz, Frizzled Frizzled


frizzled homolog 10 [Xenopus (Silurana) trop... 1.00E-155 Fz, Frizzled Frizzled

Frizzled Nvecjgi|Nemve1|139208|e_gw.466.3.1

frizzled homolog 4 [Gallus gallus] >gi|17433062... 1.00E-137 Fz, Frizzled Frizzled

Frizzled Nvecjgi|Nemve1|183962|estExt_GenewiseH_1.C_530042

frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 0 Fz, Frizzled Frizzled

PREDICTED: similar to More Of MS family memb... 1.00E-107

GPCR125 Acropora Contig5283 Gpr125 protein [Mus musculus] 5.00E-75 LRRCT, Ig, HormR_1, GPS, 7tm_2 GPCR 125

GPCR125 Nvecjgi|Nemve1|242046|estExt_fgenesh1_pg.C_550116 Gpr125 protein [Mus musculus] 3.00E-69

LRR (5x 3wobbly), lrrct1, IG, Horm_R1 (wobbly), 7tm_2 GPCR 125

VLGR1 Acropora Contig33900G protein-coupled receptor 98 [Danio rerio] ... 1.00E-142 Calx-beta, GPS, 7tm_2

VLGR1/GPCR 98 - c-term

GPS-7tm_2 Acropora Contig13413 Bai3 protein [Mus musculus] 2.00E-40 GPS, 7tm_2 GPS-7tm_2GPS-7tm_2 Acropora Contig14869 GPR133 protein [Homo sapiens] 2.00E-55 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Acropora Contig17113latrophilin 1, isoform CRA_a [Rattus norvegicus] 1.00E-24 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Acropora Contig3639G protein-coupled receptor 133 [Bos taurus] ... 7.00E-45 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Acropora Contig5847latrophilin-like protein 2 [Haemonchus contortus]... 3.00E-38 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Acropora Contig8177G protein-coupled receptor 133 [Homo sapiens] >... 1.00E-57 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Acropora run001daytona_697863 Lphn3 protein [Mus musculus] 6.00E-65 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Acropora run001daytona_1134667EGF, latrophilin and seven transmembrane dom... 3.00E-21 GPS, 7tm_2(wobbly) GPS-7tm_2

GPS-7tm_2 Clytia SA0AAA8YB23RM1G-protein coupled receptor 112 [Homo sapiens] >... 3.00E-49 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Clytia SA0AAB111YO10CTGG protein-coupled receptor 133 [Homo sapiens] >... 4.00E-12 GPS, 7tm_2 (wobbly) GPS-7tm_2

GPS-7tm_2 Hydra CL9076Contig1gb|EEB17331.1| class B secretin-like G-protein coupled receptor … 2.00E-18 GPS, 7tm_2 (wobbly) GPS-7tm_2


196


GPS-7tm_2 Mbrev jgi|Monbr1|3267|gw1.2.830.1latrophilin-like protein 2 [Haemonchus contortus]... 3.00E-25 GPS (wobbly), 7tm_2 (wobbly) GPS-7tm_2

GPS-7tm_2 Mbrevjgi|Monbr1|6049|fgenesh1_pg.scaffold_3000547 No good hits GPS (wobbly), 7tm_2 (wobbly) GPS-7tm_2

GPS-7tm_2 Mbrevjgi|Monbr1|38087|estExt_fgenesh1_pg.C_200011 MB7TM1 [Monosiga brevicollis] 0 PMT (wobbly), GPS, 7tm_2 MB7TM1 7tm_2 may be a 7TM_GPCR_str

VLGR1 Nvecjgi|Nemve1|242264|estExt_fgenesh1_pg.C_600022

G protein-coupled receptor 98 [Danio rerio] ... 0

Calx-beta (2x), LamG, calx-beta (8x), IG (wobbly), calx-beta (9x), GPS, 7tm_2 VLGR1/GPCR98 SPU_027371 neural development

GPS-7tm_2 Nvecjgi|Nemve1|125574|e_gw.221.20.1

latrophilin 1 [Bos taurus] >gi|46576871|sp|O... 3.00E-67 GPS (wobbly), 7tm_2 GPS-7tm_2


G-protein coupled receptor GPR133 [Homo sapiens] 4.00E-53 GPS (wobbly), 7tm_2 GPS-7tm_2


latrophilin 2, isoform CRA_a [Rattus norvegicus] ... 5.00E-28 GPS (wobbly), 7tm_2 (wobbly) GPS-7tm_2

GPS-7tm_2 Nvec jgi|Nemve1|13642|gw.297.15.1G protein-coupled receptor 133 [Bos taurus] ... 5.00E-49 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Nvecjgi|Nemve1|146118|e_gw.1586.3.1 flamingo 1 [Gallus gallus] 2.00E-40 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Nvec jgi|Nemve1|20971|gw.105.20.1G protein-coupled receptor 133 [Bos taurus] ... 8.00E-58 GPS, 7tm_2 GPS-7tm_2 7tm_2 may be a 7TM_GPCR_Sru 0.045

GPS-7tm_2 Nvecjgi|Nemve1|211777|fgenesh1_pg.scaffold_143000054 latrophilin 2, isoform CRA_a [Homo sapiens] 9.00E-31 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Nvecjgi|Nemve1|217885|fgenesh1_pg.scaffold_317000008

latrophilin 1 [Rattus norvegicus] >gi|2239297|g... 2.00E-54 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Nvecjgi|Nemve1|237703|estExt_fgenesh1_pg.C_10031

latrophilin-like protein 2 [Haemonchus contortus]... 2.00E-14 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Nvec jgi|Nemve1|24490|gw.39.39.1latrophilin 2 splice variant baabe [Bos taurus] 2.00E-71 GPS, 7tm_2 GPS-7tm_2 Group I on Phylogenetics

GPS-7tm_2 Nvec jgi|Nemve1|25584|gw.39.51.1 latrophilin 3, isoform CRA_c [Homo sapiens] 2.00E-53 GPS, 7tm_2 GPS-7tm_2


latrophilin-like protein 2 [Haemonchus contortus]... 2.00E-35 GPS, 7tm_2 GPS-7tm_2

GPS-7tm_2 Nvecjgi|Nemve1|241064|estExt_fgenesh1_pg.C_390006 latrophilin 2, isoform CRA_a [Homo sapiens] 1.00E-13 GPS, 7tm_2 (with a GPS in one loop) GPS-7tm_2

Novel Acropora Contig18752G-protein coupled receptor 112 [Homo sapiens] >... 5.00E-50

CA (wobbly), IG, SEA (wobbly), HormR1 (wobbly), GPS, 7tm_2 Novel

Novel Acropora Contig9410latrophilin 2 splice variant baaae [Bos taurus] 3.00E-32 SEA, HormR_1, GPS, 7tm_2 Novel

Novel Mbrevjgi|Monbr1|34543|estExt_fgenesh2_pg.C_400022

gb|ABB84827.1| epidermal growth factor domain-containing protein… 3.00E-23

EGF (3x), LamG (wobbly), EGF, TSP1, OPT (wobbly)/ (GPS (wobbly), 7tm_2 (wobbly)) Adhesion Novel

Novel Nvecjgi|Nemve1|217955|fgenesh1_pg.scaffold_319000019 GPR133 protein [Homo sapiens] 2.00E-58 PA14 (2x wobbly), GPS, 7tm_2 Adhesion Novel

Novel Nvecjgi|Nemve1|199785|fgenesh1_pg.scaffold_17000074

G protein-coupled receptor 112 [Mus musculus] >g... 4.00E-59

disc_4 (3x), IG (wobbly), FN3 (wobbly), FN3,GPS, 7tm_2 Novel

Novel Nvecjgi|Nemve1|238736|estExt_fgenesh1_pg.C_80029

G protein-coupled receptor 126 [Gallus gallu... 3.00E-47

DUF885, SLG (wobbly), GCC2_GCC3, igc2_5, HormR_1 (wobbly), GPS, 7tm_2 Novel

NvX Group Nvecjgi|Nemve1|218502|fgenesh1_pg.scaffold_351000013

gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-21 SO_2 (wobbly), 7tm_2 No SO (NvX)


gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 3.00E-26 SO_2, 7tm_2 No SO (NvX)


gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-20 SO_2, 7tm_2 No SO (NvX)


gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 3.00E-27 SO_2, 7tm_2 Yes SO (NvX)


class B secretin-like G-protein coupled receptor ... 6.00E-38

SO_2, c8 (wobbly), doub1_11 (wobbly), 7tm_2 No SO (NvX)


gb|EEB14446.1| class B secretin-like G-protein coupled receptor … 1.00E-32 Collagen, SO_2, 7tm_2 No SO (NvX) Novel


197


Smoothened Acropora Contig8091smoothened homolog [Rattus norvegicus] >gi|6226... 1.00E-133 Fz,Frizzled,7tm_2 smoothened

Like frizzled but operates in the hedgehog pathway

Smoothened Clytia SA0AAB54YE06CTGsmoothened [Xenopus laevis] >gi|13194566|gb|... 2.00E-43 Frizzled Smoothened


Smoothened Nvecjgi|Nemve1|208236|fgenesh1_pg.scaffold_90000004

smoothened homolog (Drosophila) [Mus musculus] 1.00E-135 Fz, Frizzled Smoothened


Smoothened Nvec jgi|Nemve1|92220|e_gw.29.7.1smoothened [Homo sapiens] >gi|6226142|sp|Q99835... 2.00E-92 Fz, Frizzled Smoothened



198

Immunoglobulin

Sub-Family Organism Sequence Id BLAST E_value HMMPFAM Signal PeptideTM Conclusion NotesCOLIG Acropora Contig6942 Collagen, I-setCOLIG Acropora Contig657 Collagen, I-set (2x), IgCOLIG Acropora Contig16723 Collagen, I-setCOLIG Acropora Contig27548 Collagen, I-set (2x), IgCOLIG Acropora Contig24993 Collagen, igc2 (2x)COLIG Acropora Contig11455 Collagen, I-set (2x), SushiCOLIG Acropora Contig21270 Collagen, igc2 (2x)COLIG Acropora Amil_c21796 Collagen, I-setCOLIG Acropora Contig10772 Collagen, I-setCOLIG Nvec jgi|Nemve1|207840|fgenesh1_pg.scaffold_85000017 predicted peptides only Collagen (wobbly), IG (3x) Yes No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|208243|fgenesh1_pg.scaffold_90000011 predicted peptides only Collagen (wobbly), igc2 (2x), I-set, igc2, ultra No No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|221269|fgenesh1_pg.scaffold_709000005 No good hits Collagen, igc2 (2x) No No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|209947|fgenesh1_pg.scaffold_114000019 predicted peptides only Collagen, igc2 (2x), IG Yes No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.COLIG Nvec jgi|Nemve1|200560|fgenesh1_pg.scaffold_22000082 predicted peptides only Collagen, igc2, I-set, igc2, I-set, igc2 No No Collagen-Ig Containing Has 1 TMH in the N-terminal. Probably the signal peptide.

DCC Hydra CL1Contig813neogenin 1 [Danio rerio] >gi|23428357|gb|AAK330... 2.00E-13 igc2_5 (2x) Yes No Neogenin putative partial

DCC Nvec jgi|Nemve1|105427|e_gw.77.120.1 neogenin variant 1b [Xenopus borealis] 3.00E-43 igc2, I-set (2x) Yes No Neogenin partial (Putative)

DS-CAM Clytia SA0AAB54YD20RM1gb|AAZ85125.1| Down Syndrome adhesion molecule splice variant 3.... 9.00E-25 igc2_5, IG, FN3 (3x) No No DS-CAM partial

DS-CAM Hydra CL2309Contig1Down Syndrome adhesion molecule splice variant 3.... 3.00E-20 IG, FN3 (3x) No 2 DS-CAM partial

DS-CAM Nvec jgi|Nemve1|104479|e_gw.73.146.1 down syndrome cell adhesion molecule [Aedes ... 2.00E-33 FN3 (2x) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|101511|e_gw.61.65.1 down syndrome cell adhesion molecule [Aedes ... 2.00E-27 FN3 (2x) No No DS-CAM partial (putative)

DS-CAM Nvec jgi|Nemve1|2043|gw.439.10.1 Down syndrome cell adhesion molecule [Rattus no... 8.00E-27 FN3 (2x) No No DS-CAM partial (putative)

DS-CAM Nvec jgi|Nemve1|108927|e_gw.92.51.1gb|AAL99986.1|AF487348_1 Down syndrome cell adhesion molecule-li… 9.00E-30 FN3 (2x) No No DS-CAM partial (putative)

DS-CAM Nvec jgi|Nemve1|120490|e_gw.171.38.1gb|AAL99986.1|AF487348_1 Down syndrome cell adhesion molecule-li… 1.00E-29 FN3 (2x) No No DS-CAM partial (putative)

DS-CAM Nvec jgi|Nemve1|224185|fgenesh1_pg.scaffold_4113000001 Down syndrome cell adhesion molecule [Xenopu... 4.00E-23 FN3 (3x) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|127554|e_gw.239.11.1 Down syndrome cell adhesion molecule-like... 2.00E-42 FN3 (3x) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|43898|gw.239.46.1 DSCAM splice variant 4.12 [Drosophila yakuba] 2.00E-28 igc2 (wobbly) No No DS-CAM partial (putative)DS-CAM Nvec jgi|Nemve1|224982|fgenesh1_pg.scaffold_6945000001 dscam [Drosophila mojavensis] >gi|193911131|... 1.00E-16 igc2 (wobbly), madsneu2 (wobbly) No No DS-CAM partial (putative)

F11/Contactin Hydra CL2585Contig1 contactin [Drosophila melanogaster] >gi|5597662... 2.00E-31 igc2_5, IG (wobbly), FN3 (2x wobbly) No No contactin putative partialF11/Contactin Nvec 199756|fgenesh1_pg.scaffold_17000045 Contactin 6 [Mus] 3.00E-96 IG (6x), FN3 (4x) Contactin This gene is a very close match to Contactin but was filtered Ig and Adhesion Clytia SA0AAA18YH11RM1 predicted proteins only CCP, CCP (wobbly), HYR, GCC2_GCC3 (2x), IG No No IG-CCPIg and Adhesion Clytia SA0AAB9YG10RM1 Bcam protein [Xenopus tropicalis] 2.00E-09 IG, I-set, IG (2x), EGF No No Ig-EGFIg and Adhesion Clytia SA0AAB66YD22CTG predicted proteins only Wap (wobbly), Kazal_3 (wobbly), IG (wobbly), KU, Kazal_3, FOLN (wobbly), Kazal_3Yes No Ig-KazalIg and Adhesion Clytia IL0ABA11YM18RM1 predicted proteins only I-set, IG, IG (wobbly), LDLa Yes No Ig-LDLaIg and Adhesion Clytia SA0AAB26YD21RM1 No good hits IG, igc2_5, TSP1, igc2_5 Yes 1 IG-TSP1Ig and Adhesion Clytia IL0ABA15YJ23RM1 No good hits igc2_5 (2x), TSP1 No 1 Ig-TSP1Ig and Adhesion Clytia SA0AAA1YF21RM1 roundabout 1 [Aedes aegypti] >gi|108879336|g... 2.00E-14 IG (2x), TSP1 (wobbly) No No Ig-TSP1Ig and Adhesion Clytia SA0AAA8YP06RM1 No good hits IG, igc2_5, TSP1, igc2_5 Yes No Ig-TSP1Ig and Adhesion Clytia SA0AAA8YI05RM1 No good hits IG (2x), GCC2_GCC3, VWA No No IG-VWAIg and Adhesion Hydra CL2877Contig1 No good hits IG (wobbly), TSP1 (wobbly), igc2_5 No No Ig-TSP1Ig and Adhesion Hydra CL1208Contig1 SPEG complex locus [Bos taurus] 5.00E-50 I-set, serkin_6 No 3 SPEG striated preferentially expressed geneIg and Adhesion Nvec jgi|Nemve1|198435|fgenesh1_pg.scaffold_10000027 No good hits KR, IG (wobbly), F5_F8_type_C (wobbly), spt (wobbly) Ig- KRIg and Adhesion Nvec jgi|Nemve1|248172|estExt_fgenesh1_pg.C_4390008 predicted peptides only CUB, igc2 No 2 Ig-CUBIg and Adhesion Nvec jgi|Nemve1|213715|fgenesh1_pg.scaffold_183000001 No good hits IG, CUB Ig-CUBIg and Adhesion Nvec jgi|Nemve1|246222|estExt_fgenesh1_pg.C_2050018 No good hits igv1 (wobbly), igc2 (wobbly), CUB Ig-CUBIg and Adhesion Nvec jgi|Nemve1|209643|fgenesh1_pg.scaffold_109000021 predicted peptides only F5_F8_type_C, EGF (4x), CCP (2x), HYR, EGF, CCP, GCC2_GCC3, IG (wobbly)Yes No Ig-EGF, CCPIg and Adhesion Nvec jgi|Nemve1|202086|fgenesh1_pg.scaffold_32000098 perlecan (heparan sulfate proteoglycan 2) [Mus m... 8.00E-23 F5_F8_type_C, I-set (2x), IG No 1 Ig-F5_F8_type_CIg and Adhesion Nvec jgi|Nemve1|241670|estExt_fgenesh1_pg.C_490046 echinonectin [Lytechinus variegatus] 4.00E-32 F5_F8_type_C, IG, PAN_1 Yes No Ig-F5_F8_type_CIg and Adhesion Nvec jgi|Nemve1|215547|fgenesh1_pg.scaffold_230000039 No good hits IG (3x), LDLa, IG (3x) Yes No Ig-LDLaIg and Adhesion Nvec jgi|Nemve1|220325|fgenesh1_pg.scaffold_502000002 predicted peptides only TPR (8x 5wobbly), I-set No No Ig-TPRIg and Adhesion Nvec jgi|Nemve1|203987|fgenesh1_pg.scaffold_47000046 No good hits IG, TY Yes 2 Ig-TYIg and Adhesion Nvec jgi|Nemve1|197457|fgenesh1_pg.scaffold_5000263 predicted peptides only WAP (3x), IG (2x), zp No 1 Ig-WAPIg and FN3 containing Acropora Amil_c7199 no good hits Ig, I-set, FN3 (2x) No 2 Ig-FN3

Ig and FN3 containing Acropora Contig10504 neuroglian 7.00E-66 Ig (6x), FN3 (wobbly) Yes No Ig-FN3ref|XP_001637406.1| predicted protein [Nematostella vectensis] >… 1E-130

Ig and FN3 containing Acropora Contig10617 no good hits I-set, FN3 No 1 Ig-FN3Ig and FN3 containing Acropora Contig11811 sidekick 2 [Rattus norvegicus] >gi|149054713... 0 I-set (2x), Ig, FN3 (13x) No 1 Ig-FN3Ig and FN3 containing Acropora Contig16551 Ret proto-oncogene [Xenopus laevis] 9.00E-78 Ig, I-set, Ig, FN3, tyrkin Yes No Ig-FN3

Ig and FN3 containing Acropora Contig1699roundabout 1 [Danio rerio] >gi|13509385|gb|AAK2... 2.00E-53 Ig (2x), I-set, Ig (2x), FN3 No 4 Ig-FN3

Ig and FN3 containing Acropora Contig212 neural cell adhesion molecule 1 [Danio rerio] >... 7.00E-19 Ig, I-set, Ig, FN3 (4x wobbly) Yes No Ig-FN3Ig and FN3 containing Acropora Contig22827 retII [Homo sapiens] 1.00E-64 I-set, Ig, FN3 (4x), tyrkin No 1 Ig-FN3Ig and FN3 containing Acropora Contig2812 no good hits Ig (wobbly), FN3 No 1 Ig-FN3Ig and FN3 containing Acropora Contig31261 Ncam1 protein [Danio rerio] 5.00E-37 Ig (5x), FN3 No No Ig-FN3

Ig and FN3 containing Acropora Contig5333gb|AAN32614.1|AF304305_1 Down syndrome cell adhesion molecule li… 4.00E-85 Ig, FN3 (4x), Ig, FN3 No 1 Ig-FN3

Ig and FN3 containing Acropora Contig6272 no good hits Ig (wobbly), FN3 No No Ig-FN3

Ig and FN3 containing Acropora Contig6343ref|NP_523604.2| Leukocyte-antigen-related-like, isoform A [Dros… 1.00E-89 Ig, I-set (2x), FN3 (4x) Yes No Ig-FN3

Ig and FN3 containing Acropora Contig8183 no good hits Ig (wobbly), FN3 No No Ig-FN3Ig and FN3 containing Acropora Contig9170 no good hits I-set Yes No Ig-FN3Ig and FN3 containing Acropora Contig9397 no good hits I-set, FN3 No 1 Ig-FN3Ig and FN3 containing Acropora Contig942 Ncam1 protein [Danio rerio] 1.00E-33 Ig (4x), FN3 Yes No Ig-FN3Ig and FN3 containing Acropora run001daytona_1717233 no good hits Ig (wobbly), FN3 No No Ig-FN3


199

Sub-Family Organism Sequence Id BLAST E_value HMMPFAM Signal PeptideTM Conclusion Notes

Ig and FN3 containing Acropora run002_426847 kalirin, RhoGEF kinase isoform 3 [Homo sapiens]... 5.00E-16 Ig (wobbly), FN3 No No Ig-FN3Ig and FN3 containing Acropora run002_428308 no good hits Ig (3x), FN3 No No Ig-FN3Ig and FN3 containing Clytia SA0AAB49YE16CTG No good hits IG (wobbly), FN3 (2x) No No Ig-FN3Ig and FN3 containing Hydra CL574Contig1 No good hits igc2_5 (wobbly), FN3_2 (wobbly) No 1 Ig-FN3Ig and FN3 containing Hydra CL3251Contig1 No good hits IG (3x 2wobbly), FN3 No No Ig-FN3Ig and FN3 containing Hydra gb|CV464365.1 nephrin [Mus musculus] 6.00E-12 igc2 (wobbly), FN3 No No Ig-FN3Ig and FN3 containing Hydra CL6367Contig1 myosin light chain kinase [synthetic construct] 2.00E-49 Serkin (wobbly), I-set, FN3 (wobbly) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|243882|estExt_fgenesh1_pg.C_1010020 No good hits IG (2x), FN3 (wobbly) Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|213889|fgenesh1_pg.scaffold_186000034 predicted peptides only IG (2x), FN3 (wobbly) Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|241554|estExt_fgenesh1_pg.C_470053 mesoglein variant 1 [Aurelia aurita] 5.00E-30 IG (2x), FN3, zp Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|199622|fgenesh1_pg.scaffold_16000072 No good hits IG, FN3 (wobbly), IG, SEFIR (wobbly) Yes 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|216320|fgenesh1_pg.scaffold_257000003 No good hits igc2 (wobbly), FN3 (wobbly) No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|240082|estExt_fgenesh1_pg.C_240078 neural cell adhesion molecule [Gallus gallus] 1.00E-21 igc2, FN3 No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|201959|fgenesh1_pg.scaffold_31000119 predicted peptides only igc2, FN3 (2x 1wobbly) No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|218534|fgenesh1_pg.scaffold_353000001 predicted peptides only igc2, IG, igc2, FN3 No 1 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|219947|fgenesh1_pg.scaffold_458000005 predicted peptides only IG (2x 1wobbly), FN3 (wobbly) No 2 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|216378|fgenesh1_pg.scaffold_259000002 No good hits IG, FN3 Yes 2 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|241594|estExt_fgenesh1_pg.C_470130 No good hits igc2, FN3 Yes 2 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|201955|fgenesh1_pg.scaffold_31000115 predicted peptides only IG (wobbly), IG (3x), IG (wobbly), IG, madsneu2, FN3No 3 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|202226|fgenesh1_pg.scaffold_33000070 predicted peptides only igc2, FN3 (2x), MFS No 10 Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|225380|fgenesh1_pg.scaffold_8390000001 predicted peptides only I-set, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|204514|fgenesh1_pg.scaffold_51000096 titin, isoform CRA_e [Homo sapiens] 1.00E-28 IG (2x wobbly), FN3 (3x 2wobbly) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|216592|fgenesh1_pg.scaffold_265000021 predicted peptides only IG (2x), FN3 (2x) No No Ig-FN3

Ig and FN3 containing Nvec jgi|Nemve1|85090|e_gw.10.263.1sidekick homolog 2 (chicken), isoform CRA_a [Homo... 3.00E-38 IG, FN3 No No Ig-FN3

Ig and FN3 containing Nvec jgi|Nemve1|217293|fgenesh1_pg.scaffold_291000027 No good hits IG, FN3 (2x 1wobbly) Yes No Ig-FN3

Ig and FN3 containing Nvec jgi|Nemve1|201938|fgenesh1_pg.scaffold_31000098neural cell adhesion molecule 1, isoform CRA_a [M... 9.00E-13 igc2, FN3 No No Ig-FN3

Ig and FN3 containing Nvec jgi|Nemve1|216597|fgenesh1_pg.scaffold_265000026 predicted peptides only igc2, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|218290|fgenesh1_pg.scaffold_340000001 predicted peptides only igc2, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|218823|fgenesh1_pg.scaffold_370000001 predicted peptides only igc2, FN3 (2x) No No Ig-FN3Ig and FN3 containing Nvec jgi|Nemve1|202038|fgenesh1_pg.scaffold_32000050 predicted peptides only V-set (wobbly), IG, FN3 No No Ig-FN3Ig_LON Acropora Amil_c2655 limbic system-associated membrane protein [Ratt… 8.00E-13 Ig (3x) Yes 1Ig_LON Acropora Contig18527 No good hits Ig Yes 1 Ig-LON subgroup likeIg_LON Clytia SA0AAB35YA21RM1 fibroblast growth factor receptor A [Nematostella... 6.00E-20 IG (3x 1wobbly) Yes 0 Ig-LON subgroup Not an FGFR part of IgLON subgroupIg_LON Clytia SA0AAB22YF24RM1 No hits Found IG (wobbly) Yes 0 Ig-LON subgroup like

Ig_LON Hydra CL1538Contig1 neural cell adhesion molecule 1 [Felis catus] 8.00E-10 IG (2x) No 1 Ig-LON subgroupThis can not be an N-CAM as there areno FN3 domains before the TM. Must be an IgLON subgroup

Ig_LON Hydra CL2516Contig1 No good hits IG (wobbly), IG Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|210858|fgenesh1_pg.scaffold_128000042 No good hits IG (3x 2wobbly) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|246644|estExt_fgenesh1_pg.C_2300034 No good hits IG (3x 2wobbly) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|246645|estExt_fgenesh1_pg.C_2300038 No good hits IG (3x) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|246329|estExt_fgenesh1_pg.C_2110042 predicted peptides only IG (3x) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|214858|fgenesh1_pg.scaffold_211000043 predicted peptides only IG (wobbly), igc2, IG (wobbly) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|222990|fgenesh1_pg.scaffold_2139000001 predicted peptides only igc2 (2x) Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|204525|fgenesh1_pg.scaffold_51000107 fibroblast growth factor receptor B [Nematostella... 1.00E-29 igc2 (2x), IG Yes 1 Ig-LON subgroupIg_LON Nvec jgi|Nemve1|183662|estExt_GenewiseH_1.C_500040 No good hits IG (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|220356|fgenesh1_pg.scaffold_506000010 No good hits igc2 Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|198407|fgenesh1_pg.scaffold_9000196 No good hits igc2 (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|205973|fgenesh1_pg.scaffold_65000008 No good hits igc2 (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|242464|estExt_fgenesh1_pg.C_640060 predicted peptides only igc2 (wobbly), IG (wobbly) Yes 1 Ig-LON subgroup likeIg_LON Nvec jgi|Nemve1|207612|fgenesh1_pg.scaffold_82000061 No good hits IG (wobbly), igc2 No 2 Ig-LON subgroup likeL1-Like Acropora Contig7988 neuroglian 7.00E-77 Ig (4x), FN3 (5x) No No L1L1-Like Acropora Contig20212 neuroglian 1.00E-69 Ig (3x), FN3 (5x) No 1 L1L1-Like Nvec jgi|Nemve1|240518|estExt_fgenesh1_pg.C_300161 neurofascin [Danio rerio] >gi|224383688|gb|A... 5.00E-82 IG (2x), igc2 (2x), I-set (wobbly), FN3 (5x 3 wobbly)No 2 neurofascinL1-Like Nvec jgi|Nemve1|87913|e_gw.17.200.1 hBRAVO/Nr-CAM precursor [Homo sapiens] 9.00E-29 FN3 (2x) No No Nr-CAM like (putative) fragmented contactin 6 fgenesh1_pg.scaffold_17000045

MALT-1 Clytia SA0AAA20YJ09CTGref|NP_694508.1| mucosa associated lymphoid tissue lymphoma tran… 3.00E-49 Peptidase_C14 No No MALT1

MALT 1 appears to be only in vertebrates, C.elegans, Dictostellium.

MALT-1 Nvec jgi|Nemve1|244962|estExt_fgenesh1_pg.C_1400022mucosa associated lymphoid tissue lymphoma transl… gb|EAW63079.1| 2.00E-52 igc2 (2x), Peptidase_C14 No No MALT1


MALT-1 Nvec jgi|Nemve1|211581|fgenesh1_pg.scaffold_140000023mucosa associated lymphoid tissue lymphoma transl… gb|EDM14684.1| 2.00E-68 igc2, IG, Peptidase_C14 No No MALT1


MALT-1 Acropora Contig6524ref|NP_694508.1|,mucosa associated lymphoid tissue lymphoma tran 1.00E-43 MALT1

MALT-1 Acropora Contig26161ref|NP_694508.1|,mucosa associated lymphoid tissue lymphoma tran 1.00E-48 MALT1

N-CAM Acropora Contig259immunoglobulin superfamily, member 9 [Mus muscu... 6.00E-43 Ig, (4x), FN3 (2x) Yes 1 N-CAM (putative)

N-CAM Acropora Contig3409 Ncam1 protein [Danio rerio] 2.00E-36 Ig (4x), FN3 No 1 N-CAM (putative)N-CAM Acropora Contig2418 neural cell adhesion molecule 1 [Bos taurus] 1.00E-42 Ig (5x), FN3 (2x) Yes 1 N-CAM (putative)N-CAM Nvec jgi|Nemve1|241555|estExt_fgenesh1_pg.C_470054 neural cell adhesion molecule 1 [Danio rerio] >... 9.00E-31 IG (2x), igc2 (wobbly), FN3 (2x 1wobbly) No 1 N-CAM (putative)N-CAM Nvec jgi|Nemve1|236748|estExt_fgenesh1_kg.C_470007 neural cell adhesion molecule 2 [Danio rerio] >... 2.00E-36 IG (3x), igc2 (wobbly), FN3 (2x) Yes 1 N-CAM (putative) This is N-CAM according to the KOG based approachN-CAM Nvec jgi|Nemve1|241557|estExt_fgenesh1_pg.C_470057 ncam2 [Danio rerio] 4.00E-34 IG, igc2, IG, igc2, igv1_8 (wobbly), FN3 (2x) Yes No N-CAM (putative)N-CAM Nvec jgi|Nemve1|185528|estExt_GenewiseH_1.C_710203 protogenin homolog a (Gallus gallus) [Danio ... 1.00E-75 igc2, IG (2x), igc2, FN3 (2x) Yes No protogeninRepeat PTP Acropora Contig12034 XPTP-D protein [Xenopus laevis] 1.00E-154 Ig, I-set, Ig (2x), FN3 (4x), PTP (2x) Yes 1 PTPRepeat PTP Nvec jgi|Nemve1|210515|fgenesh1_pg.scaffold_123000015 predicted peptides only MAM, IG (3x), igc2 (wobbly) Yes No PTP N-term putative partialRepeat PTP Nvec jgi|Nemve1|88020|e_gw.17.7.1 XPTP-D protein [Xenopus laevis] 0 igc2, I-set, igc2, FN3 (8x), Y_phosphatase, PTPc_3 No 1 PTP-D proteinToll Like TIR Hydra CL6112Contig1 Toll-receptor-related 1 [Hydra magnipapillata] 1.00E-102 TIR HyTRR1Toll Like TIR Hydra CL6921Contig1 Toll-receptor-related 2 [Hydra magnipapillata] 1.00E-58 TIR HyTRR2Toll Like TIR Hydra CL9285Contig1 myeloid differentiation primary response factor 8... 1.00E-16 Death (wobbly), TIR (wobbly) MyD88


200

Sub-Family Organism Sequence Id BLAST E_value HMMPFAM Signal PeptideTM Conclusion Notes

Toll Like TIR Hydra CL9986Contig1 myeloid differentiation primary response gene 88... 4.00E-07 Death (wobbly), TIR (wobbly) MyD88Toll Like TIR Mbrev jgi|Monbr1|30936|estExt_fgenesh2_pg.C_20536 No good hits PfkB, TIR (wobbly) PfkB family includes carbohyrdate and pyriamidine kinases.Toll Like TIR Mbrev jgi|Monbr1|27923|fgenesh2_pg.scaffold_22000108 predicted proteins only TUDOR_7 (wobbly), TIR (wobbly), TUDOR_7(wobbly)Toll Like TIR Nvec jgi|Nemve1|82163|e_gw.5.187.1 myeloid differentiation factor 88 [Larimichthys c... 5.00E-37 Death, TIR MyD88Toll Like TIR Nvec jgi|Nemve1|196737|fgenesh1_pg.scaffold_3000058 TIR1 [Acropora millepora] 2.00E-25 IG, igc2, IG (2x), TIR NvIL-1R

Toll Like TIR Nvec jgi|Nemve1|116780|e_gw.141.61.1 TIR1 [Acropora millepora] 3.00E-20 TIR NvIL-1RThis sequence is N-term truncated and also contains Ig domains (Miller et al 2007 Genome biology)

Toll Like TIR Nvec jgi|Nemve1|204009|fgenesh1_pg.scaffold_47000068 Toll9 [Culex quinquefasciatus] >gi|167864481... 6.00E-18 igc2, IG, TIR NvIL-1RToll Like TIR Nvec jgi|Nemve1|211916|fgenesh1_pg.scaffold_146000015 predicted proteins only TIR (wobbly) TRRToll Like TIR Nvec jgi|Nemve1|16632|gw.367.25.1 TIR1 [Acropora millepora] 2.00E-19 TIR TRRToll Like TIR Nvec jgi|Nemve1|91199|e_gw.26.221.1 Toll-like receptor (AGAP012385-PA) [Anopheles g... 3.00E-35 TIR TRRToll Like TIR Nvec jgi|Nemve1|57985|gw.141.125.1 Toll-receptor-related 2 [Hydra magnipapillata] 7.00E-17 TIR (wobbly) TRRToll Like TIR Nvec jgi|Nemve1|56601|gw.152.108.1 Toll-receptor-related 2 [Hydra magnipapillata] 4.00E-12 TIR (wobbly) TRRToll Like TIR Nvec jgi|Nemve1|217512|fgenesh1_pg.scaffold_300000025 predicted proteins only SAM (wobbly), TIR (wobbly)Toll Like TIR Nvec jgi|Nemve1|223246|fgenesh1_pg.scaffold_2476000001 predicted proteins only SAM (wobbly), TIR (wobbly)Toll Like TIR Nvec jgi|Nemve1|240728|estExt_fgenesh1_pg.C_330073 predicted proteins only Arm (3x 2wobbly), TIR(wobbly)Toll Like TIR Acropora Contig6496 LRR (2x), lrrct, TIRToll Like TIR Acropora run002_428851 TIRToll Like TIR Acropora Contig1450 TIRToll Like TIR Acropora run001daytona_1723038 TIRToll Like TIR Acropora Amil_c81406 TIRToll Like TIR Acropora Contig18494 Death, TIR MyD88Toll Like TIR Acropora Contig13468 TIR (wobbly)Toll Like TIR Acropora Contig33214 TIR (wobbly)Toll Like TIR Acropora Contig5811 TIRToll Like TIR Acropora Contig7691 TIR


201

Extracellular Matrix

Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion NotesCollagen Cltyia SA0AAB17YN15CTG collagen-like protein [Bacillus cereus Q1] >... 2.00E-28 Collagen CollagenCollagen Cltyia SA0AAA20YH02CTG collagen [Hydra vulgaris] 1.00E-23 VWA, Mucin (wobbly), Fasciclin (wobbly) Collagen -Hydra

Collagen Cltyia SA0AAA15YD01RM1gb|EDL92062.1| procollagen, type VI, alpha 3 (predicted), isofor… 8.00E-19 Collagen (5x) Collagen 6A3

Collagen Cltyia IL0ABA3YP01RM1 collagen [Hydra vulgaris] 8.00E-87 WAP, VWA Collagen - HydraCollagen Hydra CL2056Contig1 collagen, type XII, alpha 1 [Gallus gallus] >gi... 1.00E-23 VWA CollagenCollagen Hydra CL4008Contig1 collagen [Hydra vulgaris] 1.00E-22 VWA Collagen -HydraCollagen Hydra CL6422Contig1 gb|ABG80452.1| collagen [Hydra vulgaris] 0 Collagen (wobbly), VWA, Collagen, VWA Collagen - HydraCollagen Hydra CL3504Contig1 gb|ABG80452.1| collagen [Hydra vulgaris] 1.00E-158 Collagen (4x) Collagen - HydraCollagen Hydra CL602Contig2 gb|ABG80452.1| collagen [Hydra vulgaris] 1.00E-87 Collagen (3x) Collagen - HydraCollagen Hydra CL9498Contig1 gb|ABG80452.1| collagen [Hydra vulgaris] 5.00E-50 Collagen (3x) Collagen - Hydra

Collagen Monosigajgi|Monbr1|11132|fgenesh1_pg.scaffold_26000095 collagen [Hydra vulgaris] 8.00E-39

Collagen (6x), VWA, Collagen (3x), VWA, Collagen (3x), VWA, Collagen (19x) Collagen - Hydra

Collagen Nvec jgi|Nemve1|137069|e_gw.389.18.1 collagen alpha 2(I) chain precursor [Sorangi... 4.00E-22 Collagen (wobbly) CollagenCollagen Nvec jgi|Nemve1|79543|e_gw.1.96.1 collagen type VI alpha 5 [Homo sapiens] 3.00E-30 VWA (2x) Collagen

Collagen Nvecjgi|Nemve1|215413|fgenesh1_pg.scaffold_227000024 collagen type VI alpha 6 [Homo sapiens] >gi|... 5.00E-25 WAP (wobbly), VWA (4x) Collagen

Collagen Nvec jgi|Nemve1|106478|e_gw.82.69.1 collagen type XII alpha-1 precursor 1.00E-25 VWA CollagenCollagen Nvec jgi|Nemve1|828|gw.658.2.1 collagen-like protein [Hydra vulgaris] 1.00E-33 VWA CollagenCollagen Nvec jgi|Nemve1|133516|e_gw.318.15.1 collagen, type XII, alpha 1 [Gallus gallus] >gi... 2.00E-31 VWA Collagen

Collagen Nvecjgi|Nemve1|198700|fgenesh1_pg.scaffold_11000112

collagen, type XII, alpha 1, isoform CRA_c [Homo ... 4.00E-21 VWA Collagen

Collagen Nvec jgi|Nemve1|105672|e_gw.78.76.1 collagen, type XXII, alpha 1 [Gallus gallus] 2.00E-22 VWA CollagenCollagen Nvec jgi|Nemve1|88931|e_gw.19.298.1 Col protein [Suberites domuncula] 3.00E-35 Collagen Collagen

Collagen Nvecjgi|Nemve1|188642|estExt_GenewiseH_1.C_1270035 Col protein [Suberites domuncula] 5.00E-23 Collagen (2x) Collagen

Collagen Nvec jgi|Nemve1|128410|e_gw.250.80.1 Col3a1 protein [Bacillus cereus G9241] >gi|47... 4.00E-39 Collagen (2x) Collagen

Collagen Nvecjgi|Nemve1|205490|fgenesh1_pg.scaffold_60000013 collagen type XI alpha-2 [Danio rerio] >gi|1... 3.00E-23 TSPN, Collagen (5x) Collagen 11A2

Collagen 4 Cltyia IL0ABA11YI22RM1 collagen [Hydra vulgaris] 2.00E-70 VWA, Collagen (6x), C4 (2x) C4 - Hydra

Collagen 4 Hydra CL136Contig1type IV collagen alpha 1 chain precursor [Hydra v... 0 Collagen (22x), C4 (2x) C4 - Col4a1

Collagen 4 Hydra CL602Contig1 Col4a6 protein [Mus musculus] 5.00E-80 Collagen (2x), C4 (2x) C4 - Col4a6

Collagen 4 Nvec jgi|Nemve1|127140|e_gw.235.32.1alpha-1 type IV collagen >gi|119629514|gb|EAX0910... 1.00E-94 C4 (2x) C4 - Col4a1

Collagen 4 Nvec jgi|Nemve1|127157|e_gw.235.48.1 alpha2(IV)-like collagen [Strongylocentrotus pu... 2.00E-20 C4 C4

Collagen 4 Nvec jgi|Nemve1|16003|gw.235.37.1sp|P27393.1|CO4A2_ASCSU RecName: Full=Collagen alpha-2(IV) chain… 6.00E-76 C4 (2x) C4 - Col4a2

Collagen 4 Nvec jgi|Nemve1|148660|e_gw.2548.1.1 3 alpha procollagen [Strongylocentrotus purpura... 2.00E-28 Collagen (3x), C4 C4 - Col3a

Collagen 4 Nvecjgi|Nemve1|221793|fgenesh1_pg.scaffold_1002000001

sp|P27393.1|CO4A2_ASCSU RecName: Full=Collagen alpha-2(IV) chain… 2.00E-28 Collagen (4x), C4 C4 - Col4a2

Collagen 4 Nvecjgi|Nemve1|157742|estExt_gwp.C_10237

sp|P27393.1|CO4A2_ASCSU RecName: Full=Collagen alpha-2(IV) chain… 6.00E-76 EGF (10x) C4 - Col4a2

Collagen-triple Helix Cltyia SA0AAA1YC09RM1 collagen triple helix repeat protein [Clostri... 3.00E-14 Collagen (5x) CTHDCCollagen-triple Helix Hydra gb|DN811540.2

ref|YP_142550.1| collagen triple helix repeat containing protein… 8.00E-18 Collagen (3x) CTHDC

Contactin-like Nvec jgi|Nemve1|60863|gw.3.462.1 UNCoordinated family member (unc-89) [Caenor... 2.00E-35 I-set, igc2, IG, I-set (wobbly) contactin?

Contactin-like Nvec jgi|Nemve1|80685|e_gw.3.464.1 UNCoordinated family member (unc-89) [Caenor... 4.00E-34 IG, I-set, IG contactin?Fibrilin Cltyia SA0AAB91YK06RM1 Fibrillin -1 6.00E-31 EGF (6x 1wobbly) FibrillinFibrilin Cltyia SA0AAB39YH05CTG Fibrillin -1 1.00E-24 EGF (5x), SEA (wobbly), EGF (wobbly) FibrillinFibrilin Cltyia SA0AAB122YL04RM1 Fibrillin -2 5.00E-44 EGF (9x) Fibrillin BLAST vs swissprot: Full=Fibrillin-2 [human] 5E-44

Fibrilin Cltyia IL0ABA1YF08RM1 fibrillin [Podocoryna carnea] 0EGF, TB, EGF (7x 1wobbly), TB, EGF (5x), TB, EGF (6x) Fibrillin

TB- This cysteine rich repeat is found in TGF binding protein and fibrillin - PFAM

Fibrilin Cltyia SA0AAB4YB12RM1sp|O08746.1|MATN2_MOUSE RecName: Full=Matrilin-2; Flags: Prec... 4.00E-58 CUB, LDLa (3x), EGF (9x), trypsin Fibrillin hits to fribrillin at 1E-58

Fibrilin Hydra CL5183Contig1 fibrillin [Oikopleura dioica] >gi|18029271|g... 4.00E-36 EGF (6x) FibrillinFibrilin Hydra CL1329Contig1 fibrillin 1 [Rattus norvegicus] >gi|4959650|gb|... 5.00E-29 EGF (10x 5wobbly) Fibrillin?Fibrilin Hydra gb|DN603738.2 fibrillin 1 [Bos taurus] >gi|1706768|sp|P98133.... 6.00E-23 EGF (3x)Fibrilin Hydra CL4709Contig1 fibrillin 4 [Danio rerio] 9.00E-22 EGF (5x)Fibrilin Nvec jgi|Nemve1|212|gw.113.1.1 fibrillin [Podocoryna carnea] 0 EGF (14x) FibrillinFibrilin Nvec jgi|Nemve1|22881|gw.113.27.1 fibrillin 2 [Mus musculus] 9.00E-89 EGF (8x) FibrillinFibrilin Nvec jgi|Nemve1|11770|gw.113.6.1 fibrillin 2 [Rattus norvegicus] >gi|4959652|gb|... 5.00E-82 EGF (8x) FibrillinFibrilin Nvec jgi|Nemve1|70073|gw.28.267.1 fibrillin 3, isoform CRA_a [Homo sapiens] 2.00E-83 EGF (12x) Fibrillin

Fibrilin Nvecjgi|Nemve1|221487|fgenesh1_pg.scaffold_791000001 Fibrillin-1 4.00E-82 EGF (16x) Fibrillin

Fibrilin Nvec jgi|Nemve1|32913|gw.1.126.1 Fibrillin-1 4.00E-69 EGF (10x) FibrillinFibrilin Nvec jgi|Nemve1|61301|gw.1.495.1 Fibrillin-1 6.00E-68 EGF (10x) FibrillinFibrilin Nvec jgi|Nemve1|80370|e_gw.3.48.1 Fibrillin-2 1.00E-82 EGF (16x) FibrillinFibrilin Nvec jgi|Nemve1|108241|e_gw.89.14.1 Fibrillin-2 [mus] 6.00E-35 Laminin_EGF (5x) Fibrillin


202

Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion Notes

Fibrilin Nvecjgi|Nemve1|223762|fgenesh1_pg.scaffold_3290000001 Fibrillin-3 5.00E-68 EGF (12x) Fibrillin

Fibrilin Nvecjgi|Nemve1|163197|estExt_gwp.C_380029 Fibrillin-3 precursor, putative [Pediculus humanu... 6.00E-89 Cub (2x), EGF (15x), vwc (wobbly) Fibrillin

Fibrilin Nvec jgi|Nemve1|24372|gw.113.33.1 fibrillin 1 [Sus scrofa] >gi|13626617|sp|Q9T... 6.00E-39 EGF (3x) Fibrillin?

Fibrilin Nvecjgi|Nemve1|224395|fgenesh1_pg.scaffold_4621000001 fibrillin 2b [Danio rerio] >gi|184198736|gb|... 7.00E-39 EGF (3x) Fibrillin?

Fibrilin Nvec jgi|Nemve1|18668|gw.113.18.1 fibrillin 1 [Bos taurus] >gi|1706768|sp|P98133.... 3.00E-21 EGF (3x)Fibrilin Nvec jgi|Nemve1|112718|e_gw.113.12.1 fibrillin 1 precursor [Homo sapiens] 1.00E-31 EGF (2x)Fibrillar Collagens Cltyia SA0AAB139YM20RM1 No good hits COLFI (wobbly) COLFIFibrillar Collagens Cltyia SA0AAB3YD15CTG

gb|AAA48707.1| alpha-1 type XI collagen/pro-collagen alpha1 type V 2.00E-37 Collagen (3x), COLFI COLFI - Col11A1

Fibrillar Collagens Cltyia IL0ABA5YB21RM1 alpha 1 type I collagen [Oncorhynchus mykiss] 1.00E-64 Collagen(7x), COLFI COLFI - COl1A1Fibrillar Collagens Cltyia IL0ABA5YM21RM1 fibrillar collagen [Hydra vulgaris] 2.00E-85 Collagen(7x), COLFI COLFI - HydraFibrillar Collagens Cltyia IL0ABA25YC24RM1 fibrillar collagen [Hydra vulgaris] 7.00E-83 Collagen(7x), COLFI COLFI - HydraFibrillar Collagens Cltyia IL0ABA1YB18RM1 fibrillar collagen [Hydra vulgaris] 1.00E-73 Collagen (16x), COLFI COLFI - hydraFibrillar Collagens Cltyia SA0AAB109YA10RM1 fibrillar collagen [Hydra vulgaris] 2.00E-46 Collagen, COLFI COLFI - HydraFibrillar Collagens Cltyia SA0AAB121YB12RM1 fibrillar collagen [Hydra vulgaris] 7.00E-22 Collagen, COLFI COLFI - HydraFibrillar Collagens Cltyia IL0ABA16YI03RM1 fibrillar collagen [Hydra vulgaris] 5.00E-14 Collagen (3x) COLFI - Hydra putativeFibrillar Collagens Cltyia SA0AAA9YB21RM1 fibrillar collagen [Hydra vulgaris] 9.00E-14 VWA COLFI - Hydra putativeFibrillar Collagens Cltyia IL0ABA1YF15RM1

gb|AAM77398.1|AF525468_1 fibrillar collagen precursor [Hydra vul... 6.00E-75 Collagen(7x), COLFI COLFI - Hydra

Fibrillar Collagens Hydra CL8418Contig1 alpha 1 type V collagen [Gallus gallus] >gi|675... 7.00E-17 Collagen (wobbly), COLFI COLFI - Col5a1Fibrillar Collagens Hydra CL208Contig1

gb|AAM77398.1|AF525468_1 fibrillar collagen precursor [Hydra vul... 0 Collagen (17x), COLFI COLFI - Hydra

Fibrillar Collagens Hydra CL16Contig3 gb|ABG80449.1| fibrillar collagen [Hydra vulgaris] 0 Collagen (17x), COLFI COLFI - HydraFibrillar Collagens Hydra gb|DN815934.2 gb|ABG80449.1| fibrillar collagen [Hydra vulgaris] 3.00E-06 Collagen (wobbly) COLFI - Hydra putativeFibrillar Collagens Hydra gb|DN811440.2 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 1.00E-108 VWA COLFI - HydraFibrillar Collagens Hydra gb|DN636048.2 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 2.00E-51 Collagen (2x) COLFI - HydraFibrillar Collagens Hydra gb|CN554418.1 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 1.00E-21 Collagen COLFI putativeFibrillar Collagens Hydra gb|CX832276.2 gb|ABG80450.1| fibrillar collagen [Hydra vulgaris] 2.00E-18 Collagen (wobbly) COLFI putativeFibrillar Collagens Hydra CL2403Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 0 Collagen (10x) COLFI - HydraFibrillar Collagens Hydra CL2038Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 6.00E-85 COLFI COLFI - HydraFibrillar Collagens Hydra CL7128Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 3.00E-78 Collagen COLFI - HydraFibrillar Collagens Hydra CL4453Contig1 gb|ABG80451.1| fibrillar collagen [Hydra vulgaris] 4.00E-74 Collagen (3x) COLFI - HydraFibrillar Collagens Monosiga

jgi|Monbr1|31893|estExt_fgenesh2_pg.C_60234 No good hits COLFI COLFI

Fibrillar Collagens Monosiga

jgi|Monbr1|31892|estExt_fgenesh2_pg.C_60233 No good hits COLFI, MIT (wobbly) COLFI

Fibrillar Collagens Nvec

jgi|Nemve1|184625|estExt_GenewiseH_1.C_600080 COL11A1 protein [Homo sapiens] 2.00E-41 COLFI COLFI

Fibrillar Collagens Nvec jgi|Nemve1|80481|e_gw.3.268.1 collagen, type II, alpha 1 [Xenopus laevis] ... 5.00E-55 COLFI COLFIFibrillar Collagens Nvec

jgi|Nemve1|177989|estExt_GenewiseH_1.C_50144 fibrillar collagen [Lethenteron japonicum] 4.00E-47 COLFI COLFI


jgi|Nemve1|226013|fgenesh1_pg.scaffold_16382000001 GCN1 general control of amino-acid synthesis 1-... 1.00E-12 COLFI (wobbly) COLFI



procollagen, type XXVII, alpha 1, isoform CRA_a [... 4.00E-52 Collagen (12x), COLFI COLFI - Col17A1

Fibrillar Collagens Nvec jgi|Nemve1|80945|e_gw.3.433.1 alpha 2 type I collagen [Canis lupus familia... 7.00E-43 Collagen (6x), COLFI COLFI - Col2a1A


203



jgi|Nemve1|205652|fgenesh1_pg.scaffold_61000084 Col2a1a protein [Danio rerio] 4.00E-24 Collagen (16x), COLFI COLFI - Col2a1A


jgi|Nemve1|240903|estExt_fgenesh1_pg.C_360100 collagen, type V, alpha 3 [Mus musculus] >gi|73... 8.00E-30 Collagen (15x), COLFI COLFI - Col5A3

Fibrillar Collagens Nvec jgi|Nemve1|182|gw.3.2.1 fibrillar collagen [Podocoryne carnea] 2.00E-08 Collagen (6x) COLFI putativeFibrillar Collagens Nvec

jgi|Nemve1|204742|fgenesh1_pg.scaffold_53000081 collagen, type I, alpha 2 [Rattus norvegicus] >... 1.00E-53 WAP, Collagen (16x), COLFI COLFI -Col1A2

Fibrinogen Domain Cltyia IL0ABA19YP18RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 2.00E-39 Conotoxin (wobbly), EGF (wobbly), FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB16YA15RM1 microfibrillar-associated protein 4 type I [Perca... 2.00E-26 FBG FBGFibrinogen Domain Cltyia SA0AAB31YC18RM1

angiopoietin 1 [Danio rerio] >gi|148725450|emb|C... 4.00E-24 FBG FBG - angiopoietin

Fibrinogen Domain Cltyia SA0AAB30YI23RM1

Angiopoietin 1 [Danio rerio] >gi|190339926|gb|AAI... 1.00E-22 FBG FBG - angiopoietin

Fibrinogen Domain Cltyia SA0AAB6YB01CTG

angiopoietin 1, isoform CRA_a [Rattus norvegicus]... 3.00E-24 FBG FBG - angiopoietin

Fibrinogen Domain Cltyia SA0AAB1YF10RM1

angiopoietin 2 [Danio rerio] >gi|15077796|gb|AA... 3.00E-33 FBG FBG - angiopoietin

Fibrinogen Domain Cltyia SA0AAB2YN22RM1

angiopoietin 2 [Danio rerio] >gi|15077796|gb|AA... 8.00E-30 FBG FBG - angiopoietin

Fibrinogen Domain Cltyia SA0AAB44YF22CTG

angiopoietin 4 [Homo sapiens] >gi|17433288|sp|Q... 5.00E-32 FBG FBG - angiopoietin

Fibrinogen Domain Cltyia SA0AAB30YC22RM1 angiopoietin-1 [Bos taurus] 2.00E-21 FBG FBG - angiopoietinFibrinogen Domain Cltyia SA0AAB14YK05RM1

angiopoietin-like protein 4 [Bos taurus] >gi|1884... 4.00E-23 FBG FBG - angiopoietin

Fibrinogen Domain Cltyia SA0AAB40YC16RM1

fibrinogen C domain containing 1 [Mus musculus]... 5.00E-35 FBG FBG - FCDC

Fibrinogen Domain Cltyia SA0AAB121YJ19CTG fibrinogen C domain containing 1 [Rattus nor... 9.00E-37 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAB25YB20CTG fibrinogen C domain containing 1 [Rattus nor... 3.00E-36 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAB151YO02CTG fibrinogen C domain containing 1 [Rattus nor... 1.00E-33 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAB10YA11RM1 fibrinogen C domain containing 1 [Xenopus (S... 2.00E-34 FBG FBG - FCDCFibrinogen Domain Cltyia SA0AAA22YK09CTG fibrinogen-like 1 [Bos taurus] >gi|122140308... 2.00E-33 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB108YC15CTG fibrinogen-like 1 precursor [Homo sapiens] >gi|... 9.00E-33 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB6YL01RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 6.00E-43 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAA5YL04RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 2.00E-41 gth (wobbly), FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB3YK12RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 3.00E-41 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB19YK18CTG fibrinogen-like 2 [Gallus gallus] >gi|600989... 1.00E-39 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB16YN22RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 1.00E-38 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB2YG24RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 1.00E-31 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB41YK15RM1 fibrinogen-like 2 [Gallus gallus] >gi|600989... 3.00E-30 FBG FBG - fibrinogenFibrinogen Domain Cltyia SA0AAB18YN18RM1

Fcn3-A protein [Xenopus laevis] >gi|213625378|gb|... 7.00E-41 FBG FBG - Ficolin

Fibrinogen Domain Cltyia SA0AAB103YH02RM1

ficolin 1 precursor [Homo sapiens] >gi|20455484... 7.00E-30 FBG FBG - Ficolin

Fibrinogen Domain Cltyia SA0AAB119YM21RM1

ficolin B [Mus musculus] >gi|119370492|sp|O7049... 4.00E-38 FBG FBG - Ficolin

Fibrinogen Domain Cltyia SA0AAB87YN23CTG ficolin-2 [Xenopus laevis] >gi|55154000|gb|A... 9.00E-32 FBG FBG - FicolinFibrinogen Domain Cltyia SA0AAB28YI15RM1

gb|EAW88140.1| ficolin (collagen/fibrinogen domain containing) 1… 4.00E-39 FBG FBG - Ficolin

Fibrinogen Domain Cltyia SA0AAA19YE16RM1

ref|NP_112638.2| ficolin (collagen/fibrinogen domain containing)… 9.00E-48 FBG FBG - Ficolin

Fibrinogen Domain Cltyia SA0AAB36YJ19RM1

ref|NP_112638.2| ficolin (collagen/fibrinogen domain containing)… 9.00E-19 FBG FBG - Ficolin

Fibrinogen Domain Cltyia SA0AAB40YO07CTG

sp|Q9WTS8.2|FCN1_RAT RecName: Full=Ficolin-1; AltName: Full=Fico… 7.00E-24 FBG FBG - Ficolin


204


Fibrinogen Domain Cltyia SA0AAB20YN11RM1

tenascin C [Rattus norvegicus] >gi|183013175|gb... 1.00E-42 FBG FBG - Tenascin C

Fibrinogen Domain Cltyia SA0AAB19YC15RM1

tenascin R [Gallus gallus] >gi|61216379|sp|Q005... 5.00E-44 EGF (wobbly), FBG FBG - Tenascin R

Fibrinogen Domain Cltyia SA0AAA17YC03RM1

tenascin R [Rattus norvegicus] >gi|61216102|sp|... 3.00E-33 FBG FBG - Tenascin R

Fibrinogen Domain Cltyia SA0AAB71YF03RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 3.00E-44 EGF (wobbly), FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAA24YI07RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 2.00E-36 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB36YN13RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 1.00E-35 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAA5YE04RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 7.00E-33 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAA3YN21RM1 tenascin R, isoform CRA_a [Rattus norvegicus] 1.00E-28 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB6YH05CTG tenascin R, isoform CRA_b [Rattus norvegicus] 2.00E-34 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB127YB20CTG TNR protein [Homo sapiens] 4.00E-30 FBG FBG - Tenascin RFibrinogen Domain Cltyia SA0AAB57YG08CTG tenascin-W [Gallus gallus] 4.00E-29 FBG FBG - Tenascin WFibrinogen Domain Cltyia SA0AAB9YJ17RM1 FIBCD1 protein [Homo sapiens] 1.00E-33 FBG FBG DCFibrinogen Domain Hydra CL3068Contig1 No good hits FBG (wobbly) FBGFibrinogen Domain Hydra CL4995Contig1 predicted peptides only FBG (wobbly) FBGFibrinogen Domain Monosiga

jgi|Monbr1|37116|estExt_fgenesh1_pg.C_100171 No good hits EGF, FBG, EGF (2x) (all wobbly) FBG

Fibrinogen Domain Monosiga

jgi|Monbr1|25354|fgenesh2_pg.scaffold_10000004 No good hits FBG FBG

Fibrinogen Domain Monosiga

jgi|Monbr1|24183|fgenesh2_pg.scaffold_6000092 predicted peptides only FBG FBG

Fibrinogen Domain Nvec

jgi|Nemve1|213673|fgenesh1_pg.scaffold_181000014 adaptor-related protein complex 3, mu 1 subu... 6.00E-10 FBG FBG

Fibrinogen Domain Nvec jgi|Nemve1|104480|e_gw.73.98.1 angiopoietin-1 [Culex quinquefasciatus] >gi|... 6.00E-19 FBG FBGFibrinogen Domain Nvec

jgi|Nemve1|210726|fgenesh1_pg.scaffold_126000052 egg protein [Galaxea fascicularis] 2.00E-36 FBG (wobbly) FBG

Fibrinogen Domain Nvec jgi|Nemve1|124495|e_gw.211.54.1

gb|EAW88140.1| ficolin (collagen/fibrinogen domain containing) 1... 4.00E-19 FBG FBG


jgi|Nemve1|200589|fgenesh1_pg.scaffold_22000111 No good hits EGF (wobbly), FBG FBG

Fibrinogen Domain Nvec jgi|Nemve1|65269|gw.16.345.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|66974|gw.38.279.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|84873|e_gw.10.325.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|92024|e_gw.29.228.1 No good hits FBG FBGFibrinogen Domain Nvec jgi|Nemve1|95763|e_gw.40.258.1 No good hits FBG FBGFibrinogen Domain Nvec

jgi|Nemve1|201999|fgenesh1_pg.scaffold_32000011 No good hits FBG (wobbly) FBG












jgi|Nemve1|239078|estExt_fgenesh1_pg.C_110150 No good hits FBG (wobbly) FBG


jgi|Nemve1|248548|estExt_fgenesh1_pg.C_6550005 No good hits FBG (wobbly) FBG

Fibrinogen Domain Nvec jgi|Nemve1|85018|e_gw.10.356.1 No good hits FBG (wobbly) FBG


205


Fibrinogen Domain Nvec jgi|Nemve1|9022|gw.9208.1.1 No good hits FBG (wobbly) FBGFibrinogen Domain Nvec

jgi|Nemve1|218031|fgenesh1_pg.scaffold_325000006 No good hits FBG (wobbly), TSP1 FBG


jgi|Nemve1|229259|fgenesh1_pm.scaffold_63000016 No hits found FBG FBG

Fibrinogen Domain Nvec jgi|Nemve1|150226|e_gw.3344.3.1 predicted peptides only FBG FBGFibrinogen Domain Nvec

jgi|Nemve1|200421|fgenesh1_pg.scaffold_21000106 predicted peptides only FBG FBG







ref|YP_001803317.1| putative pathogenesis related protein [Cyano… 5.00E-43 FBG FBG

Fibrinogen Domain Nvec jgi|Nemve1|110903|e_gw.103.133.1 techylectin-5B [Culex quinquefasciatus] >gi|... 9.00E-25 FBG FBGFibrinogen Domain Nvec jgi|Nemve1|154536|e_gw.7411.1.1

tenascin R [Danio rerio] >gi|30909302|gb|AAP370... 1.00E-18 FBG FBG - Tenascin R

Fibrinogen Domain Nvec jgi|Nemve1|152370|e_gw.4892.4.1 tenascin XB [Homo sapiens] 1.00E-20 FBG FBG - Tenascin XBFibrinogen Domain Nvec jgi|Nemve1|101484|e_gw.61.218.1 tenascin Y variant [Gallus gallus] 9.00E-18 FBG FBG - Tenascin YFibrinogen Domain Nvec jgi|Nemve1|134301|e_gw.334.16.1 angiopoietin-like 1 [Xenopus (Silurana) trop... 3.00E-37 FBG FBG - angiopoietinFibrinogen Domain Nvec jgi|Nemve1|109792|e_gw.96.16.1

angiopoietin-like protein 4 [Bos taurus] >gi|1884... 3.00E-27 FBG FBG - angiopoietin


sp|O43827.1|ANGL7_HUMAN RecName: Full=Angiopoietin-related prote... 1.00E-44 FBG FBG - angiopoietin


sp|Q24K15.1|ANGP4_BOVIN RecName: Full=Angiopoietin-4; Short=ANG-.. 1.00E-33 FBG FBG - angiopoietin

Fibrinogen Domain Nvec jgi|Nemve1|53126|gw.155.41.1

sp|Q24K15.1|ANGP4_BOVIN RecName: Full=Angiopoietin-4; Short=ANG-... 4.00E-34 FBG FBG - angiopoietin


sp|Q5EA66.1|ANGL7_BOVIN RecName: Full=Angiopoietin-related prote... 7.00E-54 FBG FBG - angiopoietin


sp|Q9UKU9.1|ANGL2_HUMAN RecName: Full=Angiopoietin-related prote.. 9.00E-30 FBG FBG - angiopoietin


sp|Q9UKU9.1|ANGL2_HUMAN RecName: Full=Angiopoietin-related prote... 2.00E-32 FBG FBG - angiopoietin


sp|Q9Y264.1|ANGP4_HUMAN RecName: Full=Angiopoietin-4; Short=A... 3.00E-25 FBG FBG - angiopoietin


jgi|Nemve1|218034|fgenesh1_pg.scaffold_325000009 collagen, type XI, alpha 2 [Xenopus (Siluran... 1.00E-40 FBG FBG - Collagen

Fibrinogen Domain Nvec jgi|Nemve1|123220|e_gw.197.32.1 fibrinogen C domain containing 1 [Xenopus (S... 3.00E-60 FBG FBG - FCDCFibrinogen Domain Nvec

jgi|Nemve1|216157|fgenesh1_pg.scaffold_250000030 fibrinogen C domain containing 1 [Xenopus (S... 3.00E-57 FBG FBG - FCDC

Fibrinogen Domain Nvec jgi|Nemve1|153808|e_gw.6595.1.1 fibrinogen C domain containing 1 [Xenopus (S... 2.00E-26 FBG FBG - FCDCFibrinogen Domain Nvec jgi|Nemve1|35431|gw.12.187.1

sp|A2AV25.1|FBCD1_MOUSE RecName: Full=Fibrinogen C domain-contai... 4.00E-33 FBG FBG - FCDC


sp|A2AV25.1|FBCD1_MOUSE RecName: Full=Fibrinogen C domain-contai... 1.00E-20 FBG FBG - FCDC


sp|Q6AX44.1|FBCDA_XENLA RecName: Full=Fibrinogen C domain-con... 2.00E-54 FBG FBG - FCDC


jgi|Nemve1|228530|fgenesh1_pm.scaffold_22000015

sp|Q6AX44.1|FBCDA_XENLA RecName: Full=Fibrinogen C domain-con... 8.00E-52 FBG FBG - FCDC


jgi|Nemve1|228654|fgenesh1_pm.scaffold_29000006

sp|Q8N539.2|FBCD1_HUMAN RecName: Full=Fibrinogen C domain-con... 1.00E-53 FBG FBG - FCDC



sp|Q8N539.2|FBCD1_HUMAN RecName: Full=Fibrinogen C domain-con... 6.00E-42 FBG FBG - FCDC


sp|Q8N539.2|FBCD1_HUMAN RecName: Full=Fibrinogen C domain-contai... 1.00E-48 FBG FBG - FCDC


sp|Q95LU3.1|FBCD1_MACFA RecName: Full=Fibrinogen C domain-contai... 3.00E-57 FBG FBG - FCDC


sp|Q95LU3.1|FBCD1_MACFA RecName: Full=Fibrinogen C domain-contai... 2.00E-53 FBG FBG - FCDC

Fibrinogen Domain Nvec jgi|Nemve1|118635|e_gw.155.49.1 fibrinogen [Branchiostoma belcheri tsingtaunese] 4.00E-35 FBG FBG - fibrinogenFibrinogen Domain Nvec jgi|Nemve1|113410|e_gw.118.81.1 FCN protein [Xenopus laevis] 3.00E-42 FBG FBG - Ficolin


206


Fibrinogen Domain Nvec jgi|Nemve1|113476|e_gw.118.82.1 FCN protein [Xenopus laevis] 7.00E-42 FBG FBG - FicolinFibrinogen Domain Nvec jgi|Nemve1|90597|e_gw.24.49.1

ficolin (collagen/fibrinogen domain containing)… ref|NP_112638.2| 3.00E-57 Collagen, FBG Ficolin same domain structure as Ficolin


jgi|Nemve1|198298|fgenesh1_pg.scaffold_9000087 ficolin 2 isoform a precursor [Homo sapiens] >g... 3.00E-50 FBG FBG - Ficolin


ficolin A [Mus musculus] >gi|13124179|sp|O70165... 2.00E-53 FBG FBG - Ficolin

Fibrinogen Domain Nvec jgi|Nemve1|125975|e_gw.226.21.1 ficolin B [Bos taurus] >gi|75061130|sp|Q5I2E... 4.00E-39 FBG FBG - FicolinFibrinogen Domain Nvec jgi|Nemve1|95664|e_gw.40.235.1

sp|O00602.2|FCN1_HUMAN RecName: Full=Ficolin-1; AltName: Full=Fi... 1.00E-55 FBG FBG - Ficolin






sp|O75636.2|FCN3_HUMAN RecName: Full=Ficolin-3; AltName: Full=Co... 6.00E-37 FBG FBG - Ficolin


sp|Q5I2E5.1|FCN2_BOVIN RecName: Full=Ficolin-2; AltName: Full=Fi... 4.00E-57 FBG FBG - Ficolin


sp|Q5I2E5.1|FCN2_BOVIN RecName: Full=Ficolin-2; AltName: Full=Fi... 9.00E-51 FBG FBG - Ficolin


sp|Q5I2E5.1|FCN2_BOVIN RecName: Full=Ficolin-2; AltName: Full=Fi… 5.00E-51 FBG FBG - Ficolin


sp|Q9WTS8.2|FCN1_RAT RecName: Full=Ficolin-1; AltName: Full=Fico... 3.00E-42 FBG FBG - Ficolin


sp|Q9WTS8.2|FCN1_RAT RecName: Full=Ficolin-1; AltName: Full=Fico... 3.00E-30 FBG FBG - Ficolin

Fibrinogen Domain Nvec jgi|Nemve1|129798|e_gw.269.68.1 Tenascin-R - BLASTp vs swissprot 2.00E-47 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|147942|e_gw.2188.3.1

gb|EAW90994.1| tenascin R (restrictin, janusin), isoform CRA_a [… 8.00E-36 FBG FBG - Tenascin R



sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; ... 2.00E-55 FBG FBG - Tenascin R


sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; Alt... 3.00E-59 FBG FBG - Tenascin R






sp|Q00546.1|TENR_CHICK RecName: Full=Tenascin-R; Short=TN-R; Alt… 8.00E-24 FBG FBG - Tenascin R


sp|Q8BYI9.1|TENR_MOUSE RecName: Full=Tenascin-R; Short=TN-R; Alt... 1.00E-44 FBG FBG - Tenascin R


sp|Q8BYI9.1|TENR_MOUSE RecName: Full=Tenascin-R; Short=TN-R; Alt… 3.00E-53 FBG FBG - Tenascin R


sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt.. 2.00E-54 FBG FBG - Tenascin R


sp|Q92752.2|TENR_HUMAN RecName: Full=Tenascin-R; Short=TN-R; Alt... 8.00E-58 FBG FBG - Tenascin R













Fibrinogen Domain Nvec jgi|Nemve1|140550|e_gw.527.4.1 tenascin R, isoform CRA_b [Rattus norvegicus] 2.00E-55 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|113867|e_gw.121.24.1 tenascin-R [Homo sapiens] 1.00E-54 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|113913|e_gw.121.63.1 tenascin-R [Homo sapiens] 1.00E-54 FBG FBG - Tenascin RFibrinogen Domain Nvec jgi|Nemve1|108972|e_gw.93.52.1 tenascin-R [Homo sapiens] 4.00E-53 FBG FBG - Tenascin RFibrinogen Domain Nvec

jgi|Nemve1|214077|fgenesh1_pg.scaffold_191000041 tenascin-R [Homo sapiens] 3.00E-51 FBG FBG - Tenascin R


207


Fibrinogen Domain Nvec jgi|Nemve1|107155|e_gw.85.168.1 tenascin-R [Homo sapiens] 2.00E-42 FBG FBG - Tenascin RFibropellin/Fibrosurfin Clytia IL0ABA10YE07RM1 fibrosurfin [Paracentrotus lividus] 3.00E-29 No domainsFibropellin/Fibrosurfin Clytia IL0ABA8YH17RM1 RecName: Full=Adhesive plaque matrix prote... 3.00E-34Fibropellin/Fibrosurfin Clytia SA0AAB50YK15CTG fibropellin Ib 7.00E-58Fibropellin/Fibrosurfin Clytia SA0AAB73YD11RM1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-51Fibropellin/Fibrosurfin Hydra gb|CX831322.2 fibropellin Ia 2.00E-18 No domainsFibropellin/Fibrosurfin Monosiga

jgi|Monbr1|25143|fgenesh2_pg.scaffold_9000077 fibrosurfin [Paracentrotus lividus] 7.00E-57 EGF (7x), FN3 (2x), Tyrkin

Fibropellin/Fibrosurfin Nvec jgi|Nemve1|103841|e_gw.70.1.1 fibropellin Ib 1.00E-114Fibropellin/Fibrosurfin Nvec jgi|Nemve1|104325|e_gw.72.146.1 fibrosurfin [Paracentrotus lividus] 2.00E-97Fibropellin/Fibrosurfin Nvec jgi|Nemve1|1059|gw.947.1.1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-67Fibropellin/Fibrosurfin Nvec jgi|Nemve1|106413|e_gw.81.97.1 fibropellin III 1.00E-41Fibropellin/Fibrosurfin Nvec jgi|Nemve1|110299|e_gw.99.104.1 fibropellin Ib 2.00E-27Fibropellin/Fibrosurfin Nvec jgi|Nemve1|110621|e_gw.101.87.1

Notch homolog 2 (Drosophila), isoform CRA_b [Homo... 5.00E-42

Fibropellin/Fibrosurfin Nvec jgi|Nemve1|114572|e_gw.126.143.1 fibropellin Ia 2.00E-29Fibropellin/Fibrosurfin Nvec jgi|Nemve1|128516|e_gw.251.27.1 RecName: Full=Adhesive plaque matrix prote... 3.00E-67Fibropellin/Fibrosurfin Nvec jgi|Nemve1|130363|e_gw.278.1.1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-82Fibropellin/Fibrosurfin Nvec jgi|Nemve1|136382|e_gw.374.52.1 fibropellin III 1.00E-42Fibropellin/Fibrosurfin Nvec

jgi|Nemve1|223067|fgenesh1_pg.scaffold_2231000001 fibropellin Ia 6.00E-42

Fibropellin/Fibrosurfin Nvec jgi|Nemve1|40287|gw.934.3.1 fibropellin Ia 2.00E-54Fibropellin/Fibrosurfin Nvec jgi|Nemve1|52068|gw.170.90.1 RecName: Full=Fibropellin-1; AltName: Ful... 1.00E-108Fibropellin/Fibrosurfin Nvec jgi|Nemve1|60721|gw.29.181.1

notch homolog 1b [Danio rerio] >gi|60418506|gb|... 7.00E-41

Fibropellin/Fibrosurfin Nvec jgi|Nemve1|61395|gw.24.212.1 RecName: Full=Fibropellin-1; AltName: Ful... 2.00E-49Fibropellin/Fibrosurfin Nvec jgi|Nemve1|84211|e_gw.9.242.1 RecName: Full=Fibropellin-1; AltName: Ful... 3.00E-68Fibropellin/Fibrosurfin Nvec jgi|Nemve1|84597|e_gw.9.264.1 fibropellin Ia 1.00E-58Fibropellin/Fibrosurfin Nvec jgi|Nemve1|87929|e_gw.17.277.1 fibropellin Ia 1.00E-93

Fibulin Cltyia SA0AAB11YG20RM1 Fbln2 protein [Danio rerio] 2.00E-32 EGF (5x) Fibulinref|XP_001625395.1| predicted protein [Nematostella vectensis] >... 2.00E-47

Fibulin Cltyia SA0AAB49YL02RM1 Fibulin 1 2.00E-26 EMI (wobbly), EGF (6x), Gal_lectin FibulinFibulin Cltyia SA0AAB131YO02CTG Fibulin 1 [Homo sapiens] 5.00E-30 EGF (4x) FibulinFibulin Hydra gb|CN774245.1 fibulin-6 [Homo sapiens] 2.00E-25 TSP1 (2x) hemicentin-1

Fibulin Nvecjgi|Nemve1|182469|estExt_GenewiseH_1.C_380136 fibulin 2 [Danio rerio] 1.00E-80 EGF (10x) Fibulin

Fibulin Nvec jgi|Nemve1|919|gw.1034.1.1 fibulin-6 [Homo sapiens] 2.00E-41 TSP1 (4x) fibulin

Fibulin Nvecjgi|Nemve1|216821|fgenesh1_pg.scaffold_273000013 fibulin-6 [Homo sapiens] 5.00E-85 EGF (12x) Fibulin

Fibulin Nvec jgi|Nemve1|110159|e_gw.98.68.1 fibulin-6 [Homo sapiens] 8.00E-87 TSP1 (6x) Fibulin

Fibulin Nvec jgi|Nemve1|12396|gw.273.23.1hemicentin 1, isoform CRA_c [Homo sapiens] / Fibulin-6 [Homo sapiens] 8.00E-56 I-set, TSP1 (3x) hemicentin

Fibulin Nvec jgi|Nemve1|384|gw.329.6.1hemicentin 2 [Mus musculus] >gi|123228129|emb|CA... 5.00E-47 IG, I-set, igc2, I-set, igc2 hemicentin

Fibulin Nvecjgi|Nemve1|224641|fgenesh1_pg.scaffold_5311000001 Hemicentin-1 [human] 4.00E-42 EGF (5x)

Hemicentin-1/Fibrillin-6

Fibulin Nvecjgi|Nemve1|205787|fgenesh1_pg.scaffold_62000106 hemicentin [Homo sapiens] 3.00E-46 IG, igc2 (4x), ig, igc2 hemicentin

Fibulin Nvecjgi|Nemve1|185165|estExt_GenewiseH_1.C_660169 hemicentin 1, isoform CRA_c [Homo sapiens] 3.00E-36 TSP1 (3x)


208


FN2 Domain Containing Cltyia SA0AAB59YM08RM1 coagulation factor XII precursor 1.00E-08 FN2 (3x) FN2

FN2 domains!!! These are supported as E-06 - E-11. there is only one FN2 domain in Nvec (genome paper).

FN2 Domain Containing Hydra CL1445Contig3 predicted peptides only Endotoxin_N (wobbly), FN2 FN2FN2 Domain Containing Hydra CL1445Contig1 predicted peptides only FN2 (wobbly) FN2FN2 Domain Containing Nvec jgi|Nemve1|134613|e_gw.343.31.1 matrix metalloproteinase 9 [Equus caballus] ... 4.00E-24 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|92581|e_gw.30.41.1 vMMP-Lio1 [Liophis poecilogyrus] 1.00E-20 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|28616|gw.30.33.1 vMMP-Lio1 [Liophis poecilogyrus] 8.00E-18 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|92494|e_gw.30.37.1 vMMP-Lio1 [Liophis poecilogyrus] 9.00E-18 FN2 (2x) FN2FN2 Domain Containing Nvec jgi|Nemve1|9233|gw.686.4.1 matrix metalloproteinase 2 isoform a preproprot... 1.00E-11 FN2 FN2FN2 Domain Containing Nvec jgi|Nemve1|142575|e_gw.686.3.1 matrix metallopeptidase 9 (gelatinase B, 92k... 1.00E-09 FN2 FN2FN2 Domain Containing Nvec jgi|Nemve1|130895|e_gw.287.17.1 matrix metallopeptidase 9 (gelatinase B, 92k... 3.00E-09 FN2 FN2FN2 Domain Containing Nvec jgi|Nemve1|86166|e_gw.13.285.1

matrix metalloproteinase 9 [Notophthalmus virides... 3.00E-08 FN2 FN2

FN2 Domain Containing Nvec

jgi|Nemve1|201172|fgenesh1_pg.scaffold_26000101 No good hits PAN (2x), FN2 FN2


jgi|Nemve1|201718|fgenesh1_pg.scaffold_30000069 predicted peptides only EGF, SLG (wobbly), disc (wobbly), FN2 FN2


jgi|Nemve1|198667|fgenesh1_pg.scaffold_11000079 MAM domain containing 4 [Mus musculus] 9.00E-80 FN2, MAM (23x) MAMDC

FN3 Domain Containing Monosiga

jgi|Monbr1|6293|fgenesh1_pg.scaffold_4000175

gb|EEB11871.1| Fibronectin type-III domain-containing protein 3A… 2.00E-41 FN3 (7x) FN3 DC

FN3 Domain Containing Monosiga

jgi|Monbr1|32243|estExt_fgenesh2_pg.C_90007 fibronectin, type III domain-containing protein... 7.00E-75 No domains FN3DC


jgi|Nemve1|243939|estExt_fgenesh1_pg.C_1020048 fibronectin type III domain containing 3B [Homo... 1.00E-118 FN3 (9x) FN3DC

Fras1 Cltyia SA0AAA25YK13CTG Fras1 1.00E-108 No domains Fras1Fras1 Hydra CL917Contig2 FRAS1 protein [Homo sapiens] 6.00E-18 vwc (3x) Fras1

Fras1 Nvec jgi|Nemve1|107201|e_gw.85.18.1FRAS1 related extracellular matrix protein 2, iso… gb|EAX08608.1| 0 NIbeta_1 (3x) Fras1

Fras1 Nvecjgi|Nemve1|207897|fgenesh1_pg.scaffold_85000074

ref|NP_001131133.1| Fras1 related extracellular matrix protein 2… 0 CA, Nibeta_1 (all wobbly)

Fras1 related ECM protein 2

HSPG2 Cltyia SA0AAB41YI05CTG heparan sulfate proteoglycan 2 [Gallus gallu... 1.00E-81 LamG, EGF (2x), LamG, EGF (2x), LamG HSPG2

HSPG2 Clytia SA0AAB56YE16RM1 heparan sulfate proteoglycan 2 [Gallus gallu... 5.00E-34 igc2_5, I-set No Noperlecan puative partial

HSPG2 Hydra CL8208Contig1 heparan sulfate proteoglycan 2 [Danio rerio]... 2.00E-22 I-set, IG No Noperlecan putative partial

HSPG2 Nvec jgi|Nemve1|21768|gw.196.36.1 Hspg2 protein [Mus musculus] 1.00E-136igc2, LamG_3, EGF (2x), LamG_3, EGF, EGF (wobbly), LamG_3 No No Perlecan/HSPG2

Insulin Receptor Like Monosiga

jgi|Monbr1|12183|fgenesh1_pg.scaffold_36000008 insulin receptor, putative [Ixodes scapularis] 7.00E-69 Recep_L_domain, FN3 (2x wobbly), tyrkin insulin receptor

Insulin Receptor Like Monosiga

jgi|Monbr1|7936|fgenesh1_pg.scaffold_9000066

insulin receptor (AGAP012424-PA) [Anopheles gam... 2.00E-46

EGF (6x), Recep_L_domain (wobbly), FN3 (2x 1wobbly), tyrkin insulin receptor like

Insulin Receptor Like Nvec


gb|AAI70430.1| Insulin receptor, beta-subunit [Xenopus laevis] >… 2.00E-75

Recep_L_domain, furin, Recep_L_domain, FN3 (3x) insulin receptor beta

Laminin Alpha Hydra gb|DT619742.1 laminin, alpha 5 [Mus musculus] >gi|12328398... 4.00E-28 EGF (3x) Laminin A putativeLaminin Alpha Hydra CL9494Contig1 laminin alpha-1, 2 chain, putative [Ixodes scapul... 9.00E-05 No domains Laminin A putativeLaminin Alpha Monosiga

jgi|Monbr1|6041|fgenesh1_pg.scaffold_3000539 laminin, alpha 1 precursor [Homo sapiens] >gi|2... 4.00E-14

Laminin_N, EGF (3x 2wobbly), PAN (wobbly) Laminin A putative

Laminin Alpha Nvec jgi|Nemve1|110910|e_gw.103.117.1 laminin A chain, putative [Aedes aegypti] >g... 0

Laminin_N, Laminin_EGF (15x), Laminin_B Laminin A

Laminin Alpha Nvec

jgi|Nemve1|248148|estExt_fgenesh1_pg.C_4300002 laminin subunit alpha [Culex quinquefasciatu... 1.00E-115

Laminin_N, Laminin_EGF (7x), Laminin_B, Laminin_EGF (6x) Laminin A

Laminin Alpha Nvec

jgi|Nemve1|208267|fgenesh1_pg.scaffold_90000035 laminin alpha 3 subunit isoform 3 [Homo sapi... 4.00E-64 Laminin_N, Laminin_EGF (11x) Laminin A

Laminin Alpha Nvec jgi|Nemve1|109158|e_gw.93.3.1 laminin subunit alpha [Culex quinquefasciatu... 1.00E-59 EGF (6x 1wobbly) Laminin ALaminin Alpha Nvec jgi|Nemve1|111045|e_gw.103.86.1 laminin, alpha 1 [Danio rerio] >gi|71370785|... 6.00E-51 Laminin_EGF (4x) Laminin A


209


Laminin Alpha Nvec jgi|Nemve1|108950|e_gw.93.116.1 laminin, alpha 5 [Mus musculus] >gi|12328398... 3.00E-45 Laminin_EGF (5x) Laminin ALaminin Alpha Nvec jgi|Nemve1|155465|e_gw.8623.2.1 laminin alpha-1, 2 chain [Culex quinquefasci... 2.00E-29 Laminin_EGF (5x) Laminin A putativeLaminin Alpha Nvec jgi|Nemve1|34098|gw.27.98.1 laminin, alpha 2, isoform CRA_a [Mus musculus] 3.00E-26 EGF (4x) Laminin A putativeLaminin Alpha Nvec jgi|Nemve1|155008|e_gw.8010.1.1 laminin alpha 1 [Danio rerio] 1.00E-22 EGF (2x 1wobbly) Laminin A putative

Laminin Beta Nvecjgi|Nemve1|187372|estExt_GenewiseH_1.C_1000105 laminin, beta 1 precursor [Homo sapiens] >gi|51... 0

Laminin_N, Laminin_EGF (13x), SPEC (wobbly)/HAMP (wobbly), MA (wobbly) Laminin B

Laminin Gamma Hydra gb|CX831146.2 LAMC1 protein [Homo sapiens] 9.00E-34 Laminin_N Laminin GLaminin Gamma Hydra CL5311Contig1 laminin gamma 1 chain [Aedes aegypti] >gi|10... 1.00E-39 EGF (2x) Laminin G putativeLaminin Gamma Hydra gb|CO538695.1 laminin gamma 1 [Gallus gallus] 1.00E-24 EGF (2x 1wobbly) Laminin G putativeLaminin Gamma Hydra gb|DT616193.1

laminin, beta 2 [Mus musculus] >gi|19913504|gb|... 1.00E-24 EGF (2x) Laminin G putative

Laminin Gamma Monosiga

jgi|Monbr1|24778|fgenesh2_pg.scaffold_8000012 laminin subunit gamma-3 [Culex quinquefascia... 2.00E-73

CUB, Kelch (wobbly), PSI (3x wobbly), EGF (4x), CUB, EGF (2x), Kelch (5x wobbly), PSI (4x wobbly), EGF (3x wobbly) Laminin G putative

Laminin Gamma Nvec jgi|Nemve1|119462|e_gw.162.15.1

laminin, gamma 1 precursor [Homo sapiens] >gi|2... 0

Laminin_N, Laminin_EGF (11x), tSNARE (wobbly), MA (wobbly), hr1 (wobbly) Laminin G

Laminin Gamma Nvec


sp|Q61292.1|LAMB2_MOUSE RecName: Full=Laminin subunit beta-2; Al… 1.00E-107

Laminin_N, Laminin_EGF (9x), Laminin_B (wobbly), Laminin_EGF, MA (wobbly) Laminin G

Laminin Gamma Nvec

jgi|Nemve1|245480|estExt_fgenesh1_pg.C_1620024 laminin, beta 2 [Gallus gallus] >gi|2708707|gb|... 1.00E-94

Laminin_N, Laminin_EGF (7x), Laminin_B, Laminin_EGF (3x), MA (wobbly) Laminin G

Laminin Gamma Nvec

jgi|Nemve1|221570|fgenesh1_pg.scaffold_839000001 laminin beta-2 chain [Aedes aegypti] >gi|108... 6.00E-50 Laminin_EGF (10x) Laminin G

MEGF Cltyia IL0ABA9YP03RM1 MEGF6 [homo sapien] 1.00E-51 EMI (wobbly), furin (wobbly), EGF (7x) MEGFMEGF Cltyia SA0AAB125YO15CTG MEGF6 [rat] 3.00E-50 EGF (7x), CCP MEGF

MEGF Cltyia SA0AAA2YG05RM1sp|Q7Z7M0.2|MEGF8_HUMAN RecName: Full=Multiple epidermal growth … 8.00E-33 EGF (4x 3wobbly) MEGF

MEGF Monosigajgi|Monbr1|38323|estExt_fgenesh1_pg.C_230054

multiple EGF-like-domains 8 [Homo sapiens] >gi|... 4.00E-54

CUB, EGF (2x), Kelch (5x wobbly), EGF (wobbly), ftp (wobbly), PSI (5x 4wobbly), EGF (18x), CUB, EGF (2x), KELCH (3x 2wobbly), PSI (3x 2wobbly), EGF (4x), lim (wobbly) MEGF

MEGF Nvec jgi|Nemve1|97756|e_gw.47.6.1 MEGF8 [Homo sapiens] 0

CUB, EGF (wobbly), Kelch (5x wobbly), PSI (5x wobbly), EGF (4x 2wobbly), CUB, EGF (3x wobbly), Kelch (7x wobbly), PSI, P_2, PSI (2x), EGF (4x) MEGF

MEGF Nvec jgi|Nemve1|20469|gw.71.15.1sp|Q80T91.3|MEG11_MOUSE RecName: Full=Multiple epidermal growth … 1.00E-120 EGF (14x) MEGF

MEGF Nvec jgi|Nemve1|104003|e_gw.71.71.1 multiple EGF-like-domains 10 [Xenopus (Silur... 2.00E-47 EGF (5x wobbly) MEGF?Minicollagen Cltyia IL0ABA3YI18RM1_p minicollagen 1 [Clytia hemisphaerica] 0.0003 Collagen (wobbly) Minicollagen

Minicollagen Cltyia SA0AAB59YD11RM1ref|XP_002163332.1| PREDICTED: hypothetical protein [Hydra magni… 8.00E-22 Collagen (wobbly), GRP (wobbly) Minicollagen

GRP is a rare domain that is found in plants (not in combination), there are only 4 in metazoa. Only blast hit is PREDICTED: hypothetical protein [Hydra magni... 8.00E-22 ref|XP_002163332.1|

Minicollagen Hydra CL1Contig600 PREDICTED: similar to minicollagen-15 [Hydra... 1.00E-34 Collagen (wobbly) Minicollagen minicollagen similar region is interupted

Minicollagen Hydra CL1Contig103pir||C41132 collagen-related protein 3 precursor - Hydra magnipa...sphaerica] 8.00E-12 GRP (wobbly), Collagen Minicollagen

Minicollagen Hydra CL1Contig299nematoblast-specific protein nb001 [Hydra oligactis] 2.00E-10 Collagen Minicollagen

Minicollagen Hydra CL1Contig531gb|ABR19841.1| minicollagen-15 [Hydra vulgaris] >gi|193792440|gb… 3.00E-10 Collagen (wobbly) Minicollagen

Minicollagen Hydra CL334Contig2 hypothetical protein A032-H1 [Acropora millepora] 4.00E-10 Collagen Minicollagen A032-H1 is a minicollagen containing SP-Collagen-CLECT protein.

Minicollagen Hydra CL1Contig549gb|ABR19841.1| minicollagen-15 [Hydra vulgaris] >gi|193792440|gb… 5.00E-09 Collagen (wobbly) Minicollagen

Minicollagen Hydra CL1Contig295minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 1.00E-08 Collagen (wobbly) Minicollagen

Minicollagen Hydra CL1Contig296collagen-related protein 2 - Hydra magnipapillata (f... 2.00E-08 Collagen (wobbly) Minicollagen


Minicollagen Hydra CL334Contig1 hypothetical protein A032-H1 [Acropora millepora] 9.00E-08 Collagen Minicollagen A032-H1 is a minicollagen containing SP-Collagen-CLECT protein. Minicollagen Hydra CL334Contig3 hypothetical protein A032-H1 [Acropora millepora] 9.00E-08 Collagen Minicollagen A032-H1 is a minicollagen containing SP-Collagen-CLECT protein.


Minicollagen Hydra gb|CD680901.1collagen-related protein 2 - Hydra magnipapillata (f... 8.00E-06 Collagen (wobbly) Minicollagen


210


Minicollagen Hydra CL1Contig495collagen-related protein 2 - Hydra magnipapillata (f... 0.0001 Collagen (wobbly) Minicollagen

Minicollagen Hydra CL1Contig97collagen-related protein 3 precursor - Hydra magnipa... 0.0006 Collagen Minicollagen

Minicollagen Hydra CL1Contig638minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 0.049 Collagen (wobbly) Minicollagen

Minicollagen Hydra CL1Contig500minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 0.093 Collagen (wobbly) Minicollagen

Minicollagen Hydra CL1Contig572minicollagen-15 [Hydra vulgaris] >gi|193792440|gb... 0.17 No domains Minicollagen

Minicollagen Hydra CL1Contig287collagen-related protein 4 - Hydra magnipapillata (f... 0.43 Collagen Minicollagen

Minicollagen Hydra CL1Contig83ref|XP_002163332.1| PREDICTED: hypothetical protein [Hydra magni… 3.00E-88 Collagen (wobbly), GRP Minicollagen

Minicollagen Nvecjgi|Nemve1|238353|estExt_fgenesh1_pg.C_50097 mini-collagen [Acropora donei] 0.029 Collagen Minicollagen

Minicollagen Nvecjgi|Nemve1|211803|fgenesh1_pg.scaffold_144000020 mini-collagen [Acropora donei] 0.096 Collagen Minicollagen

Minicollagen Nvec jgi|Nemve1|81941|e_gw.5.7.1 mini-collagen [Acropora donei] 0.28 Collagen (wobbly) MinicollagenNetrin Hydra gb|DT613962.1 ntn1b [Danio rerio] 4.00E-16 Laminin_N netrin-like

Netrin Nvec jgi|Nemve1|118526|e_gw.154.10.1 netrin [Nematostella vectensis] 1.00E-155 Laminin_N, Laminin_EGF (3x), c345c Netrin [Nvec]c345c - Netrin C-terminal Domainhe domain is also found in cobra venom factor and in complement factors C3, C4 and C5

Notch Nvec jgi|Nemve1|41337|gw.2075.6.1 Notch 2 [Takifugu rubripes] 5.00E-73Other Clytia IL0ABA17YC15RM1 papilin, isoform A [Drosophila melanogaster] >g... 6.00E-48 TSP1, ADAM_spacer (wobbly) Papilin related

Other Hydra CL84Contig2emb|CAJ80765.2| thrombospondin type 1 repeat-containing protein … 0 TSP1 (8x)

thrombospondin type 1 repeat-containing protein 2 precursor [Hydra magnipapillata]

Other Hydra CL1487Contig2gb|AAD43811.1|AF159157_1 head-activator binding protein precurso… 1.00E-130 FN3

head-activator binding protein precursor [Hydra vulgaris]

Other Nvecjgi|Nemve1|193237|estExt_GenewiseH_1.C_2820017 nidogen [Aedes aegypti] >gi|108876881|gb|EAT... 5.00E-72 EGF (12x ?wobbly), LY (4x) Nidogen

Other Nvec jgi|Nemve1|87211|e_gw.15.190.1sp|Q61982.1|NOTC3_MOUSE RecName: Full=Neurogenic locus notch … 8.00E-52 EGF (10x)

Notch3 homolgue_N terminal

Other Nvecjgi|Nemve1|238669|estExt_fgenesh1_pg.C_70144 periostin isoform 2 [Danio rerio] >gi|42627706|... 7.00E-50 Fasciclin (3x) periostin

Other Nvecjgi|Nemve1|199904|fgenesh1_pg.scaffold_18000028 usherin isoform B [Homo sapiens] 0

Laminin_N, Laminin_EGF (10x), FN3 (4x), LamG (2x), FN3 (11x) Usherin

Other Nvecjgi|Nemve1|199903|fgenesh1_pg.scaffold_18000027 usherin isoform B [Homo sapiens] 1.00E-118 LamG, Laminin_N, laminin_EGF (5x) Usherin

Other Nvec jgi|Nemve1|59990|gw.18.225.1 usherin isoform B [Homo sapiens] 0 FN3 (16x) Usherin?

Other Nvec jgi|Nemve1|20140|gw.66.6.1rabconnectin-3 beta isoform 2 [Homo sapiens] >g... 0 WD40 (8x) Rabconnectin synaptic transport scaffold for Rab3 GEP and GAP

Other Nvecjgi|Nemve1|240569|estExt_fgenesh1_pg.C_310074

sortilin-related receptor, LDLR class A repeats... ref|NP_035566.2| 0 vps10, LY (3x), LDLa (12x), FN3 (3x) Sortilin lysosomal trafficking

Other Nvecjgi|Nemve1|206807|fgenesh1_pg.scaffold_74000003 sidekick 1 [Gallus gallus] >gi|82242600|sp|Q8AV... 8.00E-67 FN3 (13x) sidekick?

Other Nvecjgi|Nemve1|209051|fgenesh1_pg.scaffold_100000057

stabilin 2 precursor [Homo sapiens] >gi|1455595... 1.00E-159

EGF (4x wobbly), Fasciclin (2x), EGF (6x), Fasciclin (2x) stabilin engulfment

Other Nvec jgi|Nemve1|134716|e_gw.344.35.1 sorting nexin 2 [Gallus gallus] >gi|60098595... 1.00E-137 PX, vps5 sorting nexin endosomal trafficking

Other Nvecjgi|Nemve1|204357|fgenesh1_pg.scaffold_50000041 sidekick 1 [Gallus gallus] >gi|82242600|sp|Q8AV... 2.00E-55

Pan (wobbly), FN3, SEA (wobbly), FN3 (11x) Sidekick?

Other Nvecjgi|Nemve1|242037|estExt_fgenesh1_pg.C_550105

sidekick homolog 1 (chicken), isoform CRA_a [Homo... 1.00E-42 FN3 (9x), Ion_trans_2 sidekick?

Other Nvec jgi|Nemve1|35615|gw.11.151.1 bone morphogenetic protein gb|AAC41710.1| 4.00E-44 CUB (2x) BMP1 like

Other Nvec jgi|Nemve1|36325|gw.11.185.1bone morphogenetic protein 1 [Branchiostoma flori... 1.00E-31 CUB (2x) BMP1 like

PAN-FBG Nvecjgi|Nemve1|206268|fgenesh1_pg.scaffold_67000113 GP2 THP-like protein [Montipora capitata] 5.00E-17 PAN (wobbly), EGF PAN

PAN-FBG Nvecjgi|Nemve1|239485|estExt_fgenesh1_pg.C_150108 No good hits PAN (wobbly), COLFI (wobbly) PAN-COLFI

PAN-FBG Nvecjgi|Nemve1|220640|fgenesh1_pg.scaffold_549000011 No good hits PAN (wobbly), FBG PAN-FBG

PAN-FBG Nvecjgi|Nemve1|221450|fgenesh1_pg.scaffold_771000004 No good hits PAN (wobbly), FBG (wobbly) PAN-FBG

PAN-FBG Nvecjgi|Nemve1|240565|estExt_fgenesh1_pg.C_310061 No good hits PAN (wobbly), FBG (wobbly) PAN-FBG

PAN-FBG Nvecjgi|Nemve1|210066|fgenesh1_pg.scaffold_116000022 predicted peptides only PAN (wobbly), EGF (4X) PAN-FBG

PAN-FBG Nvecjgi|Nemve1|202154|fgenesh1_pg.scaffold_32000166 predicted peptides only

Pan (wobbly), EGF (Wobbly), FBG (wobbly) PAN-FBG


211


PAN-FBG Nvecjgi|Nemve1|205103|fgenesh1_pg.scaffold_56000069 predicted peptides only Pan (wobbly), EGF, FBG (wobbly) PAN-FBG

PAN-FBG Nvecjgi|Nemve1|199873|fgenesh1_pg.scaffold_17000162 predicted peptides only PAN (wobbly), FBG (wobbly) PAN-FBG




PAN-FBG Nvecjgi|Nemve1|243269|estExt_fgenesh1_pg.C_830083 predicted peptides only PAN (wobbly), FBG (wobbly) PAN-FBG

PAN-FBG Nvecjgi|Nemve1|205658|fgenesh1_pg.scaffold_61000090 predicted peptides only PAN, EGF, FBG (wobbly) PAN-FBG

PAN-FBG Nvecjgi|Nemve1|207720|fgenesh1_pg.scaffold_83000081 predicted peptides only PAN, EGF, FBG (wobbly) PAN-FBG

PAPPA Nvec jgi|Nemve1|10113|gw.217.3.1gb|AAC50543.1| pregnancy-associated plasma protein-A preproform … 0

LamGL, Notch (2x wobbly), Peptidase_M43, CCP (5x), Notch PAPPA

Rhamnospondin Acropora Contig6659

gb|ABD95939.1|,rhamnospondin 1 [Hydractinia symbiolongicarpus] 6.00E-66 Rhanspondin











Rhamnospondin Acropora Amil_c933








Rhamnospondin Cltyia SA0AAB1YK18RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-108 TSP1 (6x)Rhamnospondin Cltyia SA0AAA8YF13RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 8.00E-98 TSP1 (5x), Gal_lectin Gal_lectinRhamnospondin Cltyia SA0AAB114YB09RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 1.00E-86 TSP1 (5x)Rhamnospondin Cltyia SA0AAA22YC16RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 2.00E-63 TSP1 (4x)Rhamnospondin Cltyia IL0ABA24YA14RM1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 6.00E-71 TSP1 (4x)Rhamnospondin Nvec jgi|Nemve1|86049|e_gw.12.133.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 5.00E-92 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|34322|gw.12.157.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 2.00E-88 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|138675|e_gw.439.15.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 5.00E-87 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|2608|gw.2423.1.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 7.00E-85 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|2130|gw.3298.1.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 4.00E-80 TSP1 (6x) RhamnospondinRhamnospondin Nvec jgi|Nemve1|82974|e_gw.6.92.1 rhamnospondin 1 [Hydractinia symbiolongicarpus] 8.00E-56 TSP1 (6x) Rhamnospondin

SPOCK Cltyia SA0AAB33YO03RM1sparc/osteonectin, cwcv and kazal-like domains pr... 4.00E-28 Kazal, efhand (wobbly), TY SPOCK

ref|XP_002155894.1| PREDICTED: similar to predicted protein [Hyd... 2.00E-78ref|XP_001641707.1| predicted protein [Nematostella vectensis] >... 3.00E-43

SPOCK Hydra CL2620Contig1gb|EDL32181.1| sparc/osteonectin, cwcv and kazal-like domains pr… 7.00E-23 Kazal, efhand (wobbly), TY SPOCK

SPOCK Nvecjgi|Nemve1|196524|fgenesh1_pg.scaffold_2000146

sparc/osteonectin, cwcv and kazal-like domains pr... 1.00E-28

FOLN (wobbly), Kazal, efhand (2x wobbly), TY SPOCK

Thrombospondin Cltyia IL0ABA26YN08RM1 HyTSR1 protein [Hydra vulgaris] 0 TSP1 (16x) HyTSRThrombospondin Cltyia IL0ABA4YL14RM1 HyTSR1 protein [Hydra vulgaris] 1.00E-144 TSP1 (8x) HyTSR


212


Thrombospondin Cltyia SA0AAA6YH14RM1 HyTSR1 protein [Hydra vulgaris] 4.00E-84 TSP1 (5x) HyTSRThrombospondin Cltyia IL0ABA18YA16RM1 HyTSR1 protein [Hydra vulgaris] 3.00E-83 TSP1 (5x) HyTSRThrombospondin Cltyia SA0AAB5YL07RM1 HyTSR1 protein [Hydra vulgaris] 8.00E-55 TSP1 (3x) HyTSRThrombospondin Cltyia SA0AAA8YF14CTG HyTSR1 protein [Hydra vulgaris] 6.00E-37 TSP1 (6x), MAM (wobbly) HyTSRThrombospondin Hydra gb|CX055898.1 HyTSR1 protein [Hydra vulgaris] 1.00E-124 TSP1 (4x) HyTSRThrombospondin Hydra gb|CF777333.1 HyTSR1 protein [Hydra vulgaris] 1.00E-113 TSP1 (3x) HyTSRThrombospondin Hydra gb|DR436385.1 HyTSR1 protein [Hydra vulgaris] 4.00E-74 TSP1 (2x) HyTSRThrombospondin Hydra CL954Contig1 HyTSR1 protein [Hydra vulgaris] 6.00E-45 TSP1 (3x) HyTSRThrombospondin Nvec jgi|Nemve1|116985|e_gw.142.42.1 HyTSR1 protein [Hydra vulgaris] 5.00E-78 TSP1 (6x) HyTSRThrombospondin Nvec jgi|Nemve1|35366|gw.12.184.1 HyTSR1 protein [Hydra vulgaris] 6.00E-66 TSP1 (5x) HyTSRThrombospondin Nvec jgi|Nemve1|33857|gw.12.136.1 HyTSR1 protein [Hydra vulgaris] 2.00E-48 TSP1 (6x) HyTSR


213

Planar Cell Polarity Signalling

Sub-Family Organism Sequence Id BLAST E_value HMMPFAM SP TM Conclusion NotesPrickle Acropora Contig10161 Prickle like 2 1.00E-126 PET, 3xLIM PricklePrickle Acropora Contig12413 Prickle 1.00E-86 PET, 3xLIM PricklePrickle Acropora Contig2872 Lim-9 1.00E-69 PET, 3xLIM PricklePrickle Acropora Contig14408 Lim-9 1.00E-92 PET, 6x LIMPrickle Acropora run001daytona_1711137 Prickle 1.00E-86 PET, 3xLIM PricklePrickle Acropora run002_436495 Prickle 1.00E-86 PET, 3xLIM PricklePrickle Clytia SA0AAB127YD03CTG Prickle 1.00E-86 3x LIM Prickle

Prickle Nvecjgi|Nemve1|177334|estExt_GenewiseH_1.C_20501 Lim-9 1.00E-112 PET, 6x LIM

Prickle Nvec jgi|Nemve1|79617|e_gw.2.68.1 Prickle 2 1.00E-129 PET, 3xLIM PricklePrickle Hydra CL9755Contig1 prickle 1.00E-31 PET PrickleDishevelled Acropora Contig1475 Dsh (Lv) 1.00E-135 clonedDishevelled Nvec jgi|Nemve1|50431|gw.98.139.1 Dsh 1.00E-176 clonedDishevelled Clytia IL0ABA16YH15RM1 Dsh 1.00E-44

Dishevelled Hydra gb|DN603381.2 Dsh (Hydra) 1.00E-67cloned -XP_002162745

Van Gogh Hydra CL8947Contig1 Vangl 1.00E-67Van Gogh Acropora Contig17842 Vangl 1.00E-100

Van Gogh Nvecjgi|Nemve1|113928|e_gw.121.14.1 Vangl 1.00E-96

Van Gogh Clytia SA0AAA8YK14RM1 Vangl 1.00E-92Inversin Acropora Contig20726 Vangl 1.00E-56Inversin Acropora Contig28177 Vangl 1.00E-73Inversin Acropora Contig499 Vangl 1.00E-63

Inversin Nvecjgi|Nemve1|94580|e_gw.37.192.1 Vangl 1.00E-63

beta-catenin Acropora !"#$%"%&'()*+,-..beta-catenin Nvec Genbank: AAL49498beta-catenin Hydra Genbank: AAC47137beta-catenin Clytia SA0AAA13YE16CTG 1.00E-170LRP5/6 Acropora Contig26866 LRP6 1.00E-156LRP5/6 Acropora run001daytona_1726367 LRP6 1.00E-126LRP5/6 Acropora run002_425839 LRP6 0 *LRP5/6 Acropora Contig33249 LRP6 0LRP5/6 Acropora Contig14656 LRP6 1.00E-127LRP5/6 Acropora Contig34020 LRP6 1.00E-87LRP5/6 Nvec jgi|Nemve1|32389|gw.3.133.1 LRP6 1.00E-81Kynepk Acropora Contig1784 1.00E-67Kynepk Acropora Amil_c20217 1.00E-74Kynepk Nvec 1.00E-84

Kynepk Hyrda CL2103Contig1Genbank XP_002157574

Atrophin Acropora Contig34266 1.00E-125Atrophin Hydra CL5991Contig1 1.00E-129Atrophin Hydra CL6620Contig1 1.00E-114

Atrophin Nvecjgi|Nemve1|93577|e_gw.33.159.1 1.00E-46

Frizzled Acropora Amil_c46307 + Amil_c11915

Frizzled Acropora Contig20909 + EST_C007-H4 Fz4 Human 1.00E-156Frizzled domain

ref|XP_001622965.1| 3063bp

Frizzled Acroporarun001daytona_628203 + Contig 7411 Fz7 1.00E-172

Frizzled domain XP_001647540.1 0

Frizzled Acropora Contig30897 + EST_D019-E7 Fz2 Xenoppus 1.00E-32 FRI domains 3286bp



214


Frizzled Acropora

EST_D016-E8 + Amil_rep_c193699 + Amil_c4306 + run001daytona_1734729 + Contig24514 893bp

Frizzled Acropora Contig6070 Fz8 Danio 1.00E-175Frizzled domain

ref|XP_001634995.1| 2958bp

Frizzled Acropora run001daytona_1732102 Fz2 Xenoppus 1.00E-32 FRI domainsFrizzled Acropora Contig8892 Fz7 Danio 1.00E-41 FRI domains ref|XP_001647540.1|Frizzled Acropora Contig4001 Frizzled-related 1.00E-38 FRI domains 976bpFrizzled Acropora Contig12602 Fz8 Mus 1.00E-21 FRI domains 954bp

Frizzled Acropora Contig11982 Fz10 Danio 1.00E-147Frizzled domain

ref|XP_001630630.1| 3063bp

Frizzled Acropora Contig8177 latrophillin3Frizzled domain

Frizzled Acropora Contig8091 smoothenedFrizzled domain

Frizzled Clytia SA0AAB17YK01RM1frizzled 2 [Hydra magnipapillata] 1.00E-122

Frizzled/7tm_2 Frizzled

Frizzled Clytia IL0ABA3YD02RM1frizzled-8 [Xenopus laevis] >gi|3869266|gb|A... 2.00E-22 Fz Frizzled - putative There are described Frizzled proteins in Hydra.

Frizzled Clytia SA0AAB36YE16RM17-transmembrane receptor frizzled-1 [Xenopus... 8.00E-20

Fz, Gal_lectin(wobbly) Frizzled - putative

Frizzled Clytia IL0ABA4YO10RM1frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 2.00E-19

Fz, Gal_lectin(wobbly) Frizzled - putative

Frizzled Hydra CL4401Contig1frizzled 2 [Hydra magnipapillata] 1.00E-101 Fz Frizzled

Frizzled Hydra CL6396Contig1frizzled receptor [Hydra vulgaris] 1.00E-127 Fz Frizzled

next best hit frizzled [Aedes aegypti] >gi|108875685|gb|EA... 6.00E-07

Frizzled Hydra CL10411Contig1 frizzled receptor [Hydra vulgaris] 7.00E-60 No domains Frizzled

Frizzled Hydra gb|CX835366.2PREDICTED: Frizzled4/9/10 [Hydra magnipapill... 3.00E-59 Fz (wobbly) Frizzled

Frizzled Hydra CL2039Contig1 Fzd7 protein [Mus musculus] 6.00E-25 Fz Frizzled - putative

Frizzled Hydra CL5686Contig1frizzled homolog 8 [Rattus norvegicus] >gi|1... 5.00E-16

Fz, gal_lectin (wobbly) Frizzled - putative

Frizzled Hydra gb|CN560484.1 frizzled 2 [Hydra magnipapillata] 1.00E-33 No domains Frizzled - putative


7-transmembrane receptor frizzled-1 [Xenopus... 1.00E-180 Fz, Frizzled Frizzled


frizzled homolog 10 [Xenopus (Silurana) trop... 1.00E-155 Fz, Frizzled Frizzled

Frizzled Nvecjgi|Nemve1|139208|e_gw.466.3.1

frizzled homolog 4 [Gallus gallus] >gi|17433062... 1.00E-137 Fz, Frizzled Frizzled

Frizzled Nvecjgi|Nemve1|183962|estExt_GenewiseH_1.C_530042

frizzled-5 [Xenopus laevis] >gi|17432995|sp|... 0 Fz, Frizzled Frizzled

PREDICTED: similar to More Of MS family memb... 1.00E-107

Axin Acropora Contig4409 Axin2 1.00E-24Axin Clytia SA0AAB78YI15RM1 Axin1 1.00E-08Axin Hydra CL7700Contig1 Axin 1.00E-09

Axin Nvecjgi|Nemve1|248853|estExt_fgenesh1_pg.C_40840001 Axin1 1.00E-03

Axin Nvecjgi|Nemve1|182113|estExt_GenewiseH_1.C_340142 Axin1 1.00E-28


215


Flamingo Nvec jgi|Nemve1|84228|e_gw.9.5.1 CELSR 0

CA (8x), EGF (2-3), LamG, EGF, LamG, EGF (3x), HormR_1, GPS, 7tm_2 No 1 Flamingo start and stop codons present

Flamingo Acropora Contig8737 + Contig3665APC Acropora Contig20548

APC Nvecjgi|Nemve1|137471|e_gw.401.17.1

APC Hydra CL4732Contig1Wnt16 Clytia SA0AAB3YI03CTG Hydra Wnt16 1.00E-86Wnt16 Hydra BAH23775.1Wnt16 Nvec ABF48091.1Wnt16 AcroporaGSK3beta Acropora Contig33988 GSK3b 0GSK3beta Clytia SA0AAB127YF01CTG GSK3b 0GSK3beta Hydra gb|CV985547.1 GSK3bGSK3beta Nvec 190252 GSK3bGroucho Acropora Contig21079

Groucho Nematostellajgi|Nemve1|184640|estExt_GenewiseH_1.C_600171

Groucho Clytia SA0AAB51YI19RM1Groucho Hydra CL3019Contig1

cloned from cDNA -Ukolova, unpublished

Documents

Cell adhesion factors in cnidariansresearchonline.jcu.edu.au/36991/1/36991-knack-2011... · 2014-12-23 · as Dr. Mallett had very limited knowledge of biology and bioinformatics