Classical and genetic approaches to vertebrate development using amphibians
Gerald Thomsen
State University of New York–Stony Brook
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