Chapter 9 the Down Syndrome Cell Adhesion Molecule

8/13/2019 Chapter 9 the Down Syndrome Cell Adhesion Molecule

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Chapter 9

The Down Syndrome Cell Adhesion Molecule

Hitesh Kathuria and James C. Clemens

Abstract The Down syndrome cell adhesion molecules (DSCAMs) are a struc-

turally and functionally conserved family of cell surface receptors that playimportant roles in nervous system organization. These receptors are expressed

on both axons and dendrites where they engage in isoform-specific binding

interactions between DSCAM receptors on opposing cell surfaces. Massive

alternative splicing of arthropod DSCAM transcripts greatly expands the

complexity of the DSCAM family by endowing these organisms with the ability

to produce tens of thousands of distinct receptor isoforms that undergo homo-

philic binding. In addition to homophilic binding, DSCAM extracellular

domains serve as receptors for other proteins such as the attractant netrin-1.

These diverse interaction properties allow DSCAMs to control a variety ofnervous system patterning processes including axon path-finding and targeting,

neurite branch segregation, self-recognition, and neurite tiling.

Keywords DSCAM Ig domain Alternative splicing Axon guidance Neuron Synapse Drosophila

9.1 Introduction

In 1998, Julie Korenbergs group at the Cedars-Sinai Research Institute identi-

fied a new member of the immunoglobulin superfamily of cell surface receptors

(Yamakawa et al. 1998). Structurally, this protein was similar to previously

identified proteins that function as cell adhesion molecules (CAMs), but it

possesses a unique arrangement of domains within the extracellular region.

The gene encoding this novel cell surface receptor is located on human chromo-

some 21 in band 21q22. This is significant because an increased expression of

J.C. Clemens (*)

Department of Biochemistry, Purdue University, 175 S. University St., West Lafayette,IN 47907, USA

e-mail: [email protected]

M. Hortsch, H. Umemori (eds.),The Sticky Synapse,

DOI 10.1007/978-0-387-92708-4_9, Springer ScienceBusiness Media, LLC 2009

207


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genes in this chromosomal region correlates with the manifestation of Down

syndrome (Ds) phenotypes, including mental retardation (Korenberg et al.

1992, Delabar et al.1993, Korenberg et al.1994). Northern blot analysis and

in situ hybridization studies revealed that this gene is broadly expressed within

the nervous system (Yamakawa et al.1998). Based on these observations, theprotein was dubbed the Down syndrome cell adhesion molecule (DSCAM,

pronounced [de-es-kam) (Yamakawa et al.1998).

Since this initial report, the DSCAM family has grown with the discovery of

paralogs and homologs in mammals, fish, birds, insects, sea urchins, crusta-

ceans, and mollusks. In chordates DSCAM is generally written in all capital

letters, while non-chordate Dscams are written with only the first letter capita-

lized. DSCAM receptors are expressed throughout the nervous system during

development and have been found to play widespread roles in patterning the

nervous system, including axon targeting, neurite arborization, and branchsegregation (Zipursky et al. 2006, Schmucker 2007). An additional role for

hyper-variable arthropod Dscams has been described in innate immunity, but

this role is beyond the scope of this chapter and will therefore not be described

(for reviews on this subject, see Watson et al.2005, Dong et al.2006).

9.2 Identification of DSCAM Family Members

As mentioned previously, the founding member of the DSCAM family was

identified as a novel member of the immunoglobulin superfamily and as a

putative cell adhesion molecule (Yamakawa et al.1998). Since the gene encod-

ing this membrane receptor localizes to human chromosome band

21q22.222.3, a region that is critical for the neurological phenotypes of Ds, it

was tempting to speculate that duplication of the DSCAM gene might play a

causative role in the production of these phenotypes. The finding that DSCAM

is expressed largely in the developing nervous system is consistent with a

putative role in Ds. However, a subsequent study (Ronan et al. 2007) further

demarcated the Ds critical region on chromosome 21 that is responsible for the

generation of Ds-related phenotypes and determined that the DSCAM gene

resided outside of this region. This suggests that DSCAM is unlikely to be a

causative agent in the development of Down syndrome.

In an effort to further characterize DSCAM function, Yamakawa et al.

(1998) isolated mouse homologs of DSCAM by using the human cDNA

sequence as a probe to screen a mouse brain cDNA library. Tissue in situ

hybridization of mouse DSCAM on mouse tissues revealed that DSCAM

expression is largely localized to the central nervous system. The timing of its

expression suggests that DSCAM may play a role during early nervous system

development.

The third DSCAM family member that was identified came from the class

Insecta. Drosophila Dscam was first characterized as an unknown tyrosine

208 H. Kathuria and J.C. Clemens


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phosphorylated protein that physically associates with the Dock (Dreadlocks)

SH2 domain (Schmucker et al. 2000). Dock is an adapter protein that comprises

three SH3 domains and one SH2 domain and is required for axon guidance in

flies (Clemens et al. 1996, Garrity et al. 1996). Since dock mutants exhibit defects

in axon guidance during the development of the visual system in an adult fly(Garrity et al.1996), as well as in the embryonic nervous system (Desai et al.

1999), it was proposed that Dock serves as a vital link that connects targeting

receptors to downstream regulators of the actin cytoskeleton to control neural

patterning. This hypothesis was further supported by Hing et al. (1999) with the

discovery that a known regulator of the actin cytoskeleton, Pak (p21-activated

kinase), physically and genetically interacts with dock to control axon guidance.

These findings suggest that manipulating Pak signaling is one mechanism that

insect Dscam family members employ to control connectivity within the ner-

vous system. Additionally, a subsequent human DSCAM study found thatDSCAM directly binds to Pak and stimulates Pak phosphorylation and activity

(Li and Guan 2004). This suggests that DSCAM-mediated control of Pak

activity appears to be a general property of all DSCAM receptors.

Following the identification of DSCAM genes in human, mouse, and fruit

flies, DSCAM homologs have been identified primarily by sequence database

comparisons in a number of other species. DSCAMs appear to be present in

most animals that have a nervous system. The notable exception is theCaenor-

habditis elegans genome, which lacks a DSCAM gene product that exhibits clear

sequence conservation. Despite the growing number of DSCAM reports inother species, most of what we know about DSCAM function comes from

Drosophila. Therefore, much of this chapter will center onDrosophila Dscam

function with some additional examples of vertebrate DSCAM functions.

9.3 General Domain Structure

DSCAMs are type I cell surface transmembrane receptors that belong to the

immunoglobulin (Ig) superfamily (Yamakawa et al. 1998). Members of the

DSCAM protein family are made up of approximately 2,000 amino acids

with an average molecular weight of 221 kDa. The amino acid sequence and

domain structure of the extracellular region is conserved and encompasses ten

Ig domains and six fibronectin type III modules (Fig. 9.1). Nine of the Ig

domains are tandemly arrayed in the membrane distal (N-terminal) region of

the extracellular domain. The six fibronectin type III modules are tandemly

arrayed in the membrane proximal region with the tenth Ig domain located

between fibronectin modules four and five. This unique arrangement of Ig and

fibronectin domains is the hallmark that distinguishes DSCAM receptors from

other Ig superfamily members.

The extracellular domain is connected to the cytoplasmic region of DSCAM

by a single membrane-spanning domain. In the case of human DSCAM, it has

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been shown that the transmembrane domain is necessary and sufficient for

association of the DSCAM receptor with the netrin-binding receptor deleted in

colorectal carcinomas (DCC) (Ly et al.2008). It is unclear whether this is a

general property of all DSCAM receptors or whether this is a property that is

specific for human DSCAM.

The cytodomains of DSCAMs range in size from approximately 300 to 400amino acids and contain no previously characterized catalytic domains or

substantial sequence motifs. DSCAM family members within the same phylum

tend to have cytoplasmic domain sequences that are fairly well conserved.

However, comparison of cytoplasmic DSCAM sequences across the phyla

(Arthropoda, Chordata, Platyhelminthes, Echinodermata, and Mollusca) reveals

little sequence conservation. In general, DSCAM cytodomains contain multiple

tyrosines, which are thought to serve as binding sites for SH2 domain-containing

proteins such as Dock, as well as a C-terminal putative PDZ domain-binding

site. PDZ (named after the first three letters of proteins containing this domain:PSD95, DlgA, and ZO-1) domain-containing proteins frequently bind to the C-

terminal sequences of transmembrane receptors and serve as scaffolds that hold

together signaling complexes (Ponting et al. 1997). No proteins have been

reported to interact with this site in DSCAMs and it is currently unknown if

this region functionally interacts with PDZ domain-containing proteins.

9.4 DSCAM Molecular Diversity

DSCAM transcripts usually undergo alternative splicing. Therefore (most or

all),DSCAMgenes typically express multiple protein isoforms. The extent and

complexity ofDSCAMgene transcript alternative splicing varies greatly among

DSCAM Domain Structure

signal peptide Ig domainFibronectin III

domain

Transmembrane

domain

Ectodomain Cytodomain

Fig. 9.1 DSCAM domain structure. The general domain structure of DSCAM receptor family

members is shown. Symbols representing subdomains within DSCAMs are labeled below the

receptor structure. The DSCAM ectodomain is separated from the cytodomain by a single

transmembrane domain segment. The majority of DSCAM receptor sequences are located in

the ectodomain, which is composed of an N-terminal signal peptide, ten Ig domains and sixfibronectin type III domains in the order shown. The C-terminal cytodomain contains no

catalytic domains or substantial sequence motifs



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species. Non-arthropodDSCAM transcripts undergo little if any alternative

splicing (Barlow et al. 2002). In contrast, unprecedented alternative splicing

of some of the arthropodDscam transcripts leads to the production of tens

of thousands of different Dscam protein isoforms (Schmucker et al. 2000,

Graveley et al. 2004, Watson et al. 2005, Brites et al. 2008). Thus far, in allarthropods examined (fruit fly, mosquito, honey bee, beetle and Daphnia) a

Dscam gene has been detected that exhibits hyper-variable alternative splicing.

However, Dscam paralogs that do not exhibit hyper-variable splicing have also

been described in these organisms (Funada et al.2007, Millard et al.2007).

TheDrosophila Dscamgene serves as an example to illustrate hyper-variable

Dscamsplicing and the impact this splicing has on Dscam receptor molecular

diversity. TheDrosophila melanogaster genome contains four Dscam genes. The

first that was identified is called Dscam and the subsequently identified paralogs

are known asDscam2,Dscam3, andDscam4(Millard et al.2007). Of these fourgenes, only the (original)Dscam gene product is hyper-variable through the

process of alternative splicing.Dscam2 has been shown to encode two splice

variants, whileDscam3 and Dscam4 are predicted to encode a single protein

product (Millard et al.2007).

The Drosophila Dscam gene contains 115 exons (Fig.9.2). Twenty of these

are considered as constant exons and are present in allDscamtranscripts, while

95 are variable and their inclusion in the Dscam mRNA is controlled by

alternative splicing (Schmucker et al.2000). Each mature Dscam mRNA com-

prises 24 exons: 20 constant exons and 4 variable exons (Fig.9.2). The variableexons are exons 4, 6, 9, and 17, which are arrayed within theDscam gene in

linear clusters. Exon 4 has 12 alternatives, exon 6 has 48 alternatives, exon 9 has

33 alternatives, and exon 17 has 2 alternatives (Fig.9.2). Alternative exons are

included in the Dscam mRNA in a mutually exclusive fashion such that each

mRNA will contain exactly one of each alternative exon 4, 6, 9, and 17. As a

result, all Dscam mRNAs encode Dscam protein isoforms that share the same

overall domain structure, but differ in the amino acid sequence at four distinct

regions. The first half of Ig domain 2 is encoded by alternative exon 4, the first

half of Ig domain 3 is encoded by alternative exon 6, Ig domain 7 is encoded byalternative exon 9, and the transmembrane domain is encoded by alternative

exon 17.

This extraordinary example of alternative splicing lendsDrosophila Dscama

unique advantage over other cell adhesion genes in that, by itself, theDscam

gene encodes a large family of molecularly diverse cell surface receptors. Math-

ematically, a total of 19,008 (12 48 33) different extracellular domains fused

to one of two different transmembrane domains (resulting in 38,016 isoforms)

can be produced by the flyDscam gene (Schmucker et al.2000). A standard

nomenclature has been adopted by Dscam researchers to designate the usage of

alternative exons. The particular variant within an alternative exon cluster is

represented by a code consisting of two numbers separated by a decimal point.

For example, the second variant within the alternative exon 4 array is repre-

sented as 4.2 in this scheme. To refer to a particular Dscam splice form that



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contains alternative exons 4.2, 6.20, 9.27, and 17.1 the first numbers of the code

are dropped and the alternative exon variant numbers are listed sequentially

and separated by decimal points: 2.20.27.1.

Dscam mRNA expression studies reveal that all alternative exons appear to

be used with the exception of alternative exons 6.11 and 9.33, which have not

been detected in any Dscam cDNA (Neves et al.2004). Splicing of the alter-

native exons appears to be independent of each other such that random combi-

nations of exons 4, 6, and 9 are produced. Individual neurons are predicted to

contain 1450 copies of the Dscam mRNA, and it is known that this population

consists of a mixture of different splice forms (Neves et al.2004).

In general, splicing of the alternative exons appears to be random; however,

developmental and tissue-specific alternative exon trends have been observed.

Within the alternative exon 4 cluster, exon 4.2 is rarely used in Drosophila

embryos but its usage greatly increases in later developmental stages (Celotto

and Graveley2001, Neves et al.2004). Splicing of alternative exon 6 appears to

be random. The greatest splicing biases occur during alternative exon 9 selec-

tion. Alternative exons 9.6, 9.9, 9.13, 9.30, and 9.31 are highly favored in the

embryonic stage, whereas during later stages of development a more even

distribution of exon 9 variants are used (Neves et al.2004). Exon 9 also appears

Exon 4

12 Alternatives

Exon 6

48 Alternatives

Exon 9

33 Alternatives

Exon 17

2 Alternatives

Gene

mRNA

4 6 9 17

Protein

2 3 7

Drosophila Dscam

Fig. 9.2 Drosophila Dscam gene, transcript, and protein structure. The Drosophila Dscam gene,

transcript, and protein are shown. Constant exons are represented as black vertical barsin the

gene structure and dark gray segments in the mRNA structure. Variable exons are represented

as shortergray barsin the gene structure and aslight graysegments in the mRNA structure.

The constant exons are present in all Dscam mRNAs while only one of each variable domain

exon is selected for inclusion in the Dscam mRNA. All Dscam protein isoforms share the

same overall domain structure as shown, but differ in sequence in the gray-coloredregions

comprising the N-terminal halves of Ig domains 2 and 3, all of Ig domain 7, and the

transmembrane domain



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to have a tissue-specific bias. For example, photoreceptor cells selectively use

the same five variants that are prevalent in the embryo, while other neurons use

a broader repertoire (Neves et al. 2004). At present the significance of the

developmental and tissue-specific splicing preferences is not known.

9.5 Homophilic Interactions

The extracellular domain of a DSCAM receptor expressed on one cell surface

can physically interact with the extracellular domain of a DSCAM receptor

located on a neighboring cell membrane (Agarwala et al.2000). These interac-

tions, which span two cellular membranes, are referred to astransinteractions

and only occur between two identical DSCAM receptor proteins (homophilic).For example, there are two DSCAM paralogs in chicken: DSCAM and

DSCAML (Yamagata and Sanes 2008). Non-neuronal cultured cells expressing

DSCAM will aggregate due to the trans homophilic interactions of DSCAM

ectodomains. Similarly, cells expressing DSCAML will also form aggregates

through DSCAML ectodomaintrans homophilic interactions. However, cells

expressing DSCAM do not form heterophilic interaction with cells expressing

DSCAML (Yamagata and Sanes2008).

This homophilic-specific binding property appears to be a conserved attri-

bute shared by all DSCAM family members. Perhaps even more remarkablethan the sheer number of Drosophila Dscam ectodomains (19,008) is the

way these isoforms interact with each other. Dscam isoforms interact intrans

(Fig. 9.3), but generally only do so when they contain identical sequences

(alternative exons) at all three variable Ig domains (Wojtowicz et al. 2004,

2007). If they differ in one or more variable Ig domains (2, 3, and 7), no

interaction is detectable. In some rare cases heterophilic interactions have

been detected, but in each of these instances two of the variable domains are

identical while the non-identical third domains are highly related to each other

(Wojtowicz et al.2007).

Because homophilic interaction relies on sequence identity at Ig domains 2,

3, and 7, it was proposed that these domains are major sites of interaction

between Dscam monomers (Wojtowicz et al. 2004). Recent crystallographic

and mutagenic studies support this hypothesis (Meijers et al.2007, Wojtowicz

et al.2007, Sawaya et al.2008). These structural studies have determined that

specific sequences within the variable regions of Ig domains 2, 3, and 7 partici-

pate in binding interactions between identical Dscam splice forms. The indivi-

dual variable domains of one monomer bind to the corresponding variable

domain of the identical second monomer in an antiparallel configuration such

that Ig 2 binds Ig domain 2, Ig 3 binds Ig 3, and Ig 7 binds Ig 7 (Fig. 9.3).

Overall, the structure adopted by the first seven Ig domains of each interacting

Dscam monomer resembles an S-shape in which Ig domains 2, 3, and 7 are

roughly in a linear arrangement. The S-shape conformation is only observed



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when Dscam isoforms are participating in homophilic binding. When

Dscam is monomeric, the distal Ig domains 14 exist in a horseshoe con-

formation (the top half of the S), while Ig domains 57 are in a flexible

extended conformation (Meijers et al.2007; Sawaya et al.2008). This horse-

shoe conformation has been observed in other Ig superfamily members such

as insect hemolin (Su et al. 1998) and the neural CAM axonin-1 (Freigang

et al. 2000).

Fig. 9.3 Dscam isoform-specific homophilic interaction. A schematic representation of theDscam receptor is shown. (AC) Ig domains are represented as rounded rectangles and

fibronectin domains as ellipses. Ig domains 19 are numbered. A representation of a lipid

bilayer separates the cytodomain from the ectodomain. Ig domains 2, 3, and 7 contain

antiparallel-binding determinants represented by white and black geometric shapes. These

binding determinants interact such thatblackdeterminants bind towhitedeterminants if and

only if they are the same shape. (A) Identical Dscam monomers: Ig domains 14 pack into a

horseshoe-shaped conformation while the other extracellular domains do not participate in

higher order structures. (B)Identical Dscam isoforms engaged in trans homophilic interaction:

Theblackdeterminants in Ig domains 2, 3, and 7 of each receptor bind to the respective white

determinants present in the other receptor in an antiparallel manner. This results in the

formation of an ordered S-shaped conformation involving Ig domains 17 of each mono-mer and stable receptor binding. (C)Non-identical Dscam isoforms: The two Dscam isoforms

shown have identical Ig domains 2 and 7 but differ in Ig domain 3 as indicated by differently

shaped binding determinants (crosses versus circles). This results in an inability to form astable interaction between these different isoforms



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The most well-documented biological response of Dscam homophilic inter-

action is repulsion of neurites expressing identical (interacting) forms. The

molecular mechanism of homophilic repulsion is unknown, but it is hypothe-

sized to function in a stepwise manner. Initially, the identical ectodomains

interact to create an adhesive complex. This in turn activates the Dscamcytodomain to initiate a signaling cascade that ultimately modifies the cytoske-

leton to direct movement away from the sites of interaction (Hughes et al.2007,

Matthews et al.2007, Soba et al.2007). Indeed, mutation of the cytodomain

leads to a stable adhesive interaction instead of repulsion (Hughes et al.2007,

Matthews et al.2007, Soba et al.2007). A second signal must also be generated

to disrupt adhesion between the trans interacting ectodomains. It is thought

that this occurs by one or more of the following mechanisms: a conformational

shift, receptor internalization, or a proteolytic event.

In Drosophila, Dscam-mediated homophilic repulsion has been demon-strated to control neurite branch segregation (Zhan et al. 2004) and self-

avoidance (Hughes et al. 2007, Matthews et al. 2007, Soba et al. 2007),

while Dscam2-mediated homophilic repulsion contributes to neuronal tiling

(Millard et al. 2007). In mice, DSCAM-mediated homophilic repulsion is

involved in aspects of neuronal self-avoidance and tiling (Fuerst et al.2008).

9.6 Branch Segregation and Self-Avoidance

Hyper-variable arthropod Dscam genes provide a molecular mechanism for

neurons to distinguish self from non-self. In the case of theDrosophila Dscam

gene, neurons select a nearly random population of 1450 Dscam isoforms

from a pool of roughly 38,000 possibilities. Because of this, neighboring neu-

rons express a unique collection of Dscam isoforms and are consequently

unlikely to contain any isoforms in common. Therefore, the Dscam isoforms

expressed by an individual neuron serves as a molecular signature that can be

used to differentiate itself from all other neurons (Hattori et al.2007).

This signature is the molecular basis of self-avoidance inDrosophilaand is

used to help pattern the nervous system. Since only identical Dscam ectodo-

mains interact, neighboring neurons do not repel each other because they do

not express identical isoforms (Fig.9.4A). Therefore, individual neurons can

bundle into nerves (fasciculate) and grow along common pathways. If neurons

that normally fasciculate are experimentally forced to express a Dscam isoform

in common using a transgene, the neuron bundle will defasciculate and the

individual neurons will move away from each other (Fig. 9.4B) (Schmucker

et al.2000, Zhan et al.2004).

In the case of branched neurons, neurite branches display isoforms in com-

mon since each branch originates from the same cell body. Homophilic inter-

actions between identical Dscam isoforms on each sister branch leads to the

initiation of repulsion and the dispersion of sister branches. This mechanism



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ensures that neurites emanating from the same cell body segregate properly to

different targets instead of converging on a single target (Fig.9.4C) (Wang et al.

2002, Zhan et al.2004). It also allows dendritic branches of a neuron to fully

disperse for the uniform exploration of a spatial field (Fig.9.4D) (Zhu et al.

2006, Hughes et al.2007, Matthews et al.2007, Soba et al.2007).

Dscam mutant neurons display defects in segregation of sister branches to

their proper target fields. This can be illustrated within the mushroom bodies

(MB), which are multi-lobed structures in the Drosophila brain that are

involved in olfactory learning and memory. Axons extend from the MB cell

bodies down a common pathway called the peduncle. At the distal end of the

peduncle the MB axons branch. One branch targets to the dorsal lobe, while

the second branch targets to the medial lobe. In Dscam mutant MB axons, the

branches form but frequently target to the same lobe (Wang et al.2002, Zhan

et al.2004).

A

+1+1

+1

B

T1

T2

C

D

Fig. 9.4 Dscam-mediated repulsion: self-recognition versus tiling. (AC) Neurons are repre-

sented schematically. Axons (lines) extending from neuron cell bodies (ovals) end in growthcones, which are complex motile structures that elaborate numerous filopodia. (A) Each

neuron expresses and displays different Dscam populations on their plasma membranes as

indicated by different shading patterns. Since these neurons do not express common Dscam

isoforms, they do not recognize each other as self. Their neurites do not repel each other and

are able to fasciculate and grow along common pathways. ( B) In addition to their normal

unique combination of Dscam isoforms expressed from their endogenous Dscamgenes (as in

panel A), each neuron also expresses a Dscam isoform in common due to the presence of a

transgene. Interaction between these common isoforms is interpreted as recognition of self

and leads to neurite repulsion, which prevents them from growth along a common pathway.

(C) In the case of axons that branch, each branch will contain the same Dscam isoforms

allowing for self-recognition. This results in repulsion between the two growth cones and helpsensure that each branch seeks out different targets (T1 and T2) rather than both converging on

the nearest target (T1). (D) The dendritic fields from four hypothetical neurons are shown.

Self-recognition mediated by hyper-variable Dscam causes neurites originating from the same

cell body to spread out and effectively cover a large spatial area. Tiling interactions (gray

double-headed arrows) mediated by non-diverse Dscam receptors such as Dscam2 prevent

overgrowth of the dendrites from one cell body into the area occupied by the dendrites coming

from a different cell body



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Dscam mutant neurons display defects in patterning dendritic arborizations.

Sensory neurons in theDrosophilalarva body wall elaborate complex dendritic

arborizations that spread out to evenly cover the sensory field. The highly

branched dendrites of these neurons, known as dendritic arborization (da)

neurons, never cross over each other in the plane of the body wall, elegantlyillustrating the principle of self-avoidance (Fig.9.4D).Dscam mutant da neu-

rons still elaborate dendritic fields that display complex branch patterns; how-

ever, these branches cross over one another and fail to evenly disperse within the

body wall (Hughes et al.2007, Matthews et al.2007, Soba et al.2007).

Self-avoidance is a general property that likely functions in all species to

properly disperse neurite branches. In arthropods hyper-variable Dscam genes

have arisen to provide one mechanism to recognize and move away from self.

SinceDrosophila Dscam is expressed in most, if not all neurons during devel-

opment, the single Dscam gene may be sufficient to confer self-avoidanceproperties to all neurites. Outside the arthropod lineage, a similar DSCAM

diversity has not been detected; therefore, it is unlikely that DSCAMs are the

only molecules responsible for this process. For example, DSCAM mutant mice

display defects in self-avoidance of neurite arbors in two sub-populations of

retina amacrine cells that normally express DSCAM protein (Fuerst et al.

2008). However, other retina amacrine cell populations exhibited normal neur-

ite patterning in these mice. Therefore self-avoidance in non-arthropods may

require the cell-type-specific expression of different repulsion receptor genes.

Alternatively, a more general self-avoidance mechanism may have evolved inthese organisms, which is yet to be elucidated.

9.7 Tiling

Tiling within the nervous system is the process of completely and evenly filling a

spatial region with multiple synaptic domains of a particular neural class.

Similar to tiles in a floor, the synaptic domains of each neuron are contained

within a distinct region that approaches, but does not overlap with adjacent

domains. The result of this is a uniform distribution of neural processes that are

restricted to each individual domain. One possible mechanism to constrain

neural processes to a particular domain (tile) is to utilize a repulsive receptor

system. Receptors expressed on the processes of adjacent tiling neurons could

prevent overlap of neurite fields by inducing repulsion upon receptor interac-

tion (Fig.9.4D).

Hyper-variable Drosophila Dscam is not a good candidate to perform this

role. While Dscam diversity is well suited for self-avoidance, since individual

neurons make different isoforms, Dscam is incapable of signaling repulsion

between processes from different neurons. Non-variable Dscam receptors, on

the other hand, which are expressed in a cell-type-specific manner, have been

demonstrated to function in neuron tiling. InDrosophila, Dscam2gene function



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is required for tiling the L1 subclass of lamina neurons within the visual system

(Millard et al.2007).Dscam2mutant L1 neurons target the correct layer in the

medulla, but extend processes laterally and invade neighboring columns (tiles).

Presumably this is due to the lack of homophilic interaction-based repulsion

between L1 neuron axons. A similar role for DSCAM-mediated tiling has beenuncovered in mice (Fuerst et al.2008). DSCAM is required in two sub-popula-

tions of retinal amacrine cells for proper dispersion of these neurons in a spatial

field. In the absence of DSCAM, these neurons migrate together and inappro-

priately fasciculate.

In both the preceding examples, DSCAM family members serve as homo-

typic repulsion receptors, whose function is to preserve the spatial patterning

(tiling) between members of the same neuron type. Since tiling is not self-

avoidance, but rather avoidance of members within a particular class, it

would follow that multiple tiling receptors must exist. This is because in somebiological environments, different neuron classes will tile independently of each

other within a shared spatial region (Hughes et al.2007, Matthews et al.2007,

Soba et al. 2007). For example, da class III dendrites exhibit tiling in the

Drosophila body wall. Da class IV dendrites also exhibit tiling in the same

environment. Therefore, since class III dendrites do not repel those of class

IV, the receptors that prevent class III dendrites from overlapping must be

different from those that prevent class IV dendrites from overlapping. It is

known that Dscam and Dscam2 are not responsible for da class IV tiling. An

attractive possibility is that Dscam3 or 4 might play a role in tiling theseneurons.

9.8 Non-repulsive DSCAM Functions

There is a growing body of evidence indicating that the DSCAM family of

receptors can function in roles other than signaling homotypic repulsion. Loss-

of-function studies inDrosophilasuggest that Dscam is involved in aspects of

axon target selection in both the olfactory system (Hummel et al.2003) and

mechanosensory neurons (Chen et al.2006). The phenotypes observed in each

of these studies are inconsistent with merely a loss of Dscam-mediated homo-

philic repulsion and hint at a possible instructive requirement of Dscam splice

forms for target selection (Chen et al.2006). For example, it might be the case

that Dscam receptors signal attraction or even adhesion in response to either

different ligands (non-Dscam) or changes in Dscam receptor molecular

contexts.

Recent studies in chordates have uncovered new functions for DSCAMs.

Yamagata and Sanes (2008) isolated chick Dscam and DscamL, orthologs of

human DSCAM and DSCAML1, respectively, and have determined that these

receptors function in a manner similar to sidekick receptors to pattern laminar

arborizations in the chick retina (see Chapter 10 for a description of sidekick



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receptors). Like other Dscams, the ectodomains of these receptors were found

to participate in homophilic, but not heterophilic, interactions. Instead of

inducing repulsion between neurons, chick Dscam or DscamL homophilic

interactions result in trans-synaptic adhesion between neurons at synaptic

junctions within the inner plexiform layer (IPL) of the chick retina. Dscamprotein is predominantly localized to the S5 sublamina of the IPL, while

DscamL receptors localize to sublamina S1, S2, and S4. Depletion of Dscam

expression disrupts the laminar patterning of S5 by causing the processes of

affected neurons to extend beyond the S5 boundary. The processes of non-

Dscam-expressing neurons in other sublamina were not affected by Dscam

depletion. Moreover, ectopic expression of Dscam rerouted neuronal processes

to the Dscam-positive layer S5. Similarly, ectopic expression of DscamL

rerouted neurites to layers other than S3, a layer that is DscamL negative.

Taken together, these data suggest that Dscam signaling is not limited tohomotypic repulsion, but can also promote adhesion, and hint at putative

roles for Dscams in synaptic specification or maintenance (Yamagata and

Sanes2008).

9.9 Non-DSCAM Interactions

During vertebrate spinal cord development commissural axons are attracted tothe ventral midline due to expression of netrin-1 by the floor plate cells. Deleted

in colorectal cancer (DCC) is a netrin-1 cell surface receptor expressed on

commissural axons that function as a key mediator of the attractive and out-

growth promoting properties of netrin-1 (Keino-Masu et al. 1996). While

impairment of DCC function blocks netrin-1-stimulated outgrowth of commis-

sural axon explants, it does not completely block turning of these axons toward

a netrin-1 source (Keino-Masu et al. 1996). These results suggest that an

additional netrin receptor functions with DCC to induce axon turning toward

the netrin-1 gradient.

In addition to homophilic interactions, the extracellular domain of rat

DSCAM has been shown to engage in heterophilic interactions with netrin-1

(Ly et al.2008). A protein truncation analysis revealed that the netrin-1-binding

site is located within a region containing DSCAM Ig domains 79. Addition-

ally, DSCAM and DCC form a receptor complex in the absence of netrin-1.

This complex requires the transmembrane domain of DSCAM and it dissoci-

ates upon netrin-1 stimulation (Ly et al. 2008). Like knockdown of DCC,

knockdown of DSCAM by siRNA does not completely block the turning

response of rat commissural axons to netrin-1. However, simultaneous knock-

down of both DSCAM and DCC results in a complete blockage of netrin-1-

induced axon turning (Ly et al.2008). Finally, ectopic expression of DSCAM in

Xenopusspinal neurons is sufficient to confer a turning response to netrin-1 in

the absence of DCC activity (Ly et al. 2008). These studies demonstrate that



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vertebrate DSCAM has additional heterophilic binding partners, which enable

it to function as a netrin-1 receptor and mediate axon turning, much in the same

way as DCC functions. A recent study in Drosophila has generated similar

findings and suggests that netrin binding might be general property of all

DSCAMs (Andrews et al. ).

9.10 Concluding Remarks

It has been a decade since the discovery of the founding DSCAM family

member. DSCAM genes have been identified in virtually all organisms that

have a nervous system. During this time our understanding of DSCAM bio-

chemical properties and biological functions has grown, but is far from com-

plete. Thus far, all DSCAMs have been reported to engage in isoform-specifichomophilic interactions. The biological consequences of these interactions

appear to vary between homotypic repulsion and adhesion. This may simply

reflect different species-specific roles that have evolved for DSCAMs since what

we know about DSCAM function is being pieced together from experiments in

a wide variety of organisms. An alternative and attractive hypothesis is that the

signaling output from homotypic interactions is dependent on the signaling

contexts in which these interactions occur. For example, it might be the case

that axonaxon- or dendritedendrite-based DSCAM homotypic interactions

lead to repulsion to segregate neurite branches, disperse arborizations, and tilereceptive fields. On the other hand, axondendrite-based DSCAM homotypic

interactions may be instructive for the selection and stabilization of synaptic

partners as in the chicken IPL. It may even be the case that the hyper-variable

arthropod Dscams play an instructive role in connection specificity as well,

rather than simply serving as a means to distinguish self from non-self. Recent

studies have added a new wrinkle to the DSCAM story with the discovery that

DSCAMs also functions as netrin receptors. Finally the involvement of

DSCAM in human Down syndrome and other nervous system structural dis-

orders awaits critical assessment.

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Chapter 9 the Down Syndrome Cell Adhesion Molecule