Classical and genetic approaches to vertebrate development using amphibians

Gerald Thomsen

State University of New York–Stony Brook

gerald.h.thomsen@stonybrook.edu

Amphibian Model

Xenopus laevis

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Studying Development with Amphibian Embryos

1. Classical Approaches - “cut and paste”

2. Induction and cell differentiation assays

3. Functional screens for developmental regulators

4. Analysis of candidates by gain and loss of function

5. Cell biology and morphogenesis

6. Gene regulation

7. Genetics - Xenopus tropicalis and the future…

• Fate maps• Explants - specification• Transplants - induction• Physical/chemical perturbation

1. Classical Approaches

Vogt’s method of amphibian fate mapping

Making a fate map of Xenopus

Fluorescent dextran labeling of

Building the frog 32 cell stage fate map:

C1

C4

Dale and Slack 1984

Fate map of the Xenopus blastula

• Specification test– Autonomous execution of a developmental

program.– Test cell’s or tissue’s potential for

autonomous differentiation when removed from the embryo.

– Gives clues about the nature of cell fate determination: determinants or induction?

Classical approaches:

Specification test on Xenopus blastulaepidermis

Animal pole:

An-1 RNA helicaseAn-2 Mitochondrial ATPase subunitAn-3 Zinc finger protein

Smurf1 Ubiquitin ligase = neurectodermEctodermin Ubiquitin ligase = neurectoderm

Vegetal pole

Vg1 TGFß member = mesendodermXlsrts Structural RNA anchors Vg1Wnt-11 Wnt growth factor – dorsal axis, PCPVegT T-box txn factor = mesendoderm

Specification tests led to screens that identified localized RNAs - some are cell fate determinants

.

• dozens isolated, mostly by differential cDNA screening• localization mechanisms somewhat understood.

– Do cells respond to their neighbors or are they committed to their own progranms?

– Induction is where interactions between cells affects fate.

– First evidence that vertebrates use induction to develop was demonstrated with amphibians (newts)

Tissue transplantation to test cell commitment

Pieter Nieuwkoop showed that mesoderm is induced

Grafts with lineage tracing

1. Classical Approaches - “cut and paste”

2. Induction and cell differentiation assays

3. Functional screens for developmental regulators

4. Analysis of candidates by gain and loss of function

5. Cell biology and morphogenesis

6. Gene regulation

7. Genetics - Xenopus tropicalis and the future…

Animal cap assay to screen for inducers

= test substancee.g. growth factor

Animal cap assay to screen for inducers

= test substancee.g. growth factor

* A popular variation is to inject a candidate mRNAinto the animal pole and test its effects on the isolated cap

*

Mesenchyme & blood induction by BMP-4Untreated Treated with BMP

Mesenchyme, blood

untreated + Vg1

nodals and activin act similarl

Nodals 1,2,5Vg1

Dorsal nuclear ß-catenin(cortical rotation &/or wnt11)

Mesoderm & endoderm induction signals in late blastula

1. Classical Approaches - “cut and paste”

2. Induction and cell differentiation assays

3. Functional screens for developmental regulators

4. Analysis of candidates by gain and loss of function

5. Cell biology and morphogenesis

6. Gene regulation

7. Genetics - Xenopus tropicalis and the future…

• Pool of random cDNA clones, grid of ESTs, wells etc• Synthesize mRNA from pooled clones• Inject and score pool activity (e.g axis induction)• split pool and retest fractions• reiterate sib selection• Identify active clone

Expression cloning

Chordin gene expression in Spemann Organizer

notochord

initial gastrulation(stage 10)

early gastrulation(stage 10.5)

end of gastrulation(stage 13)

Sasai et al, Cell. 1996

head mesoderm

Noggin gene expression in SpemannOrganizer and notochord

Smith & Harland (1996)

Stage 10 Stage 10

Stage 13Stage 13

Some key developmental genes isolated by expression cloning in Xenopus

• Noggin• Chordin• Sizzled• Cerberus• Dickkopf• Kremen

Organizer specific genes in X. tropicalis

Kokha et al,

Other ways to find candidates….

•Differential screen– subtractive selection– gene chip assay - e.g. VegT induced genes.

– in silico = NCBI Differential Display (DD)• Protein interaction partners - Y2H, proteomics

• Systems biology Networks• MFCG (my favorite cool gene…)

1. Classical Approaches - “cut and paste”

2. Induction and cell differentiation assays

3. Functional screens for developmental regulators

4. Analysis of candidates by gain and loss of function

5. Cell biology and morphogenesis

6. Gene regulation

7. Genetics - Xenopus tropicalis and the future…

• Gain of function tests– Overexpress gene of interest in embryo and

observe effects.

Does my favorite gene have a developmental function?

Induction of an ectopic dorsal axis by a constitutively active Type I activin receptor (ALK4)

Ventralization by dorsal BMP-4 overexpression

control

treated

BMP-4 and wnt8 expressed in ventral marginal zone mesoderm

S.O.

Brivanlou & Thomsen 1996

BMP-4 downregulated in neural plate

midgastrulation st 11

Neural plate

St 12

• Loss of function tests

– Dominant-negative proteins– Antisense oligonucleotide targeting of mRNAs to

block protein expression.

• RNAse-H mediated destruction via DNA oligo• Translation inhibition with Morpholino oligo• Splicing inhibition with Morpholino

– Gene mutations?

DNA oligo-mediated mRNA destruction by endogenous oocyte RNAseH

Slack 2000

ß-catenin depleted embryos

Rescue with injected ß-catenin mRNA

RNAseH-mediated depletion of materna ß-cateninmRNA by antisense DNA oligos

MO knockdown of maternal ß-cateninblocks spemann organizer formation

MO knockdown of two X. laevis chd alleles

Region-specific D-V patterning gene expression

1. Classical Approaches - “cut and paste”

2. Induction and cell differentiation assays

3. Functional screens for developmental regulators

4. Analysis of candidates by gain and loss of function

5. Cell biology and morphogenesis

6. Gene regulation

7. Genetics - Xenopus tropicalis and the future…

Cell biological observations and perturbations

• cell shape and cytoarchitecture• cytoskeleton and nuclear structure / assembly• adhesion• movement• cell and tissue polarity

A - P axis elongation

1. Classical Approaches - “cut and paste”

2. Induction and cell differentiation assays

3. Functional screens for developmental regulators

4. Analysis of candidates by gain and loss of function

5. Cell biology and morphogenesis

6. Gene regulation

7. Genetics - Xenopus tropicalis and the future…

• promoter analysis - mutate and inject into nucleus

• transgenics- stable, heritable, link tissue-specific promoters to reporters (GFP, RFP, etc)…basis of screens?

• Transcription factor assays/purification with egg/embryo extracts- “get in the cold room”…or modern proteomics

• inducible systems– Stage specific promoters– Heat shock promoter– Protein chimeras - fused regulatory domains (e.g.

GR)

Gene regulation

• Cell cycle regulators…e.g MPF = cyclin + cdk• Transcription factors• Replication factors• Translation systems• Protein degradation / ubiquitin-proteasome• Chromatin assembly

Also …purification of regulatory proteins- & use of egg and embryo in vitro extracts

1. Classical Approaches - “cut and paste”

2. Induction and cell differentiation assays

3. Functional screens for developmental regulators

4. Analysis of candidates by gain and loss of function

5. Cell biology and morphogenesis

6. Gene regulation

7. Genetics - Xenopus tropicalis and the future…

Enter Xenopus tropicalis

X.l X.t.

transgenic X. tropicalis

Cardiac actin- RFPPax6-GFPLens gamma crystalline- RFP + Pax6-GFP = yellow eye

Natural recessive mutations of X. tropicaliscurly

bubblehead

Mutants recovered from a gynogentic haploid screen

rough diamond

balloon head

First mutant: Morgan 1910 Harland, Grainger 2005

Fly Xenopus

Genome: ~ 2001 2005

The starlet sea anemone,

Nematostellavectensis

(Anthozoa)

egg masses

Lab of Mark Q. Martindale

Deuterostomia

“Bilateria”“Radiata”

Metazoa

Pr otostomia

Choanoflagellata

Cnidaria-*

PoriferaCtenophora-*

Acoelomorpha-*

BryozoaArticulataPhoronida-*Inarticulata

Spiralia