Download Classical and genetic approaches to vertebrate development using

Classical and genetic approaches to vertebrate development using
amphibians
Gerald Thomsen
State University of New York–Stony Brook
[email protected]
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…
1. Classical Approaches
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Fate maps
Explants - specification
Transplants - induction
Physical/chemical perturbation
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
Classical approaches:
• 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?
Specification test on Xenopus blastula
epidermis
Specification tests led to screens that identified
localized RNAs - some are cell fate determinants
.
Animal pole:
An-1
RNA helicase
An-2
Mitochondrial ATPase subunit
An-3
Zinc finger protein
Smurf1
Ubiquitin ligase = neurectoderm
Ectodermin Ubiquitin ligase = neurectoderm
Vegetal pole
Vg1
Xlsrts
Wnt-11
VegT
TGFß member = mesendoderm
Structural RNA anchors Vg1
Wnt growth factor – dorsal axis, PCP
T-box txn factor = mesendoderm
• dozens isolated, mostly by differential cDNA screening
• localization mechanisms somewhat understood.
Tissue transplantation to test cell commitment
– 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)
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 substance
e.g. growth factor
Animal cap assay to screen for inducers
*
= test substance
e.g. growth factor
* A popular variation is to inject a candidate mRNA
into the animal pole and test its effects on the isolated cap
Mesenchyme & blood induction by BMP-4
Untreated
Treated with BMP
Mesenchyme, blood
untreated
+ Vg1
nodals and activin act similarl
Mesoderm & endoderm induction signals in late blastula
Nodals 1,2,5
Vg1
Dorsal nuclear ß-catenin
(cortical rotation &/or wnt11)
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…
Expression cloning
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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
Chordin gene expression in Spemann Organizer
head mesoderm
notochord
initial gastrulation
(stage 10)
Sasai et al, Cell. 1996
early gastrulation
(stage 10.5)
end of gastrulation
(stage 13)
Noggin gene expression in Spemann
Organizer and notochord
Stage 10
Smith & Harland (1996)
Stage 10
Stage 13
Stage 13
Some key developmental genes isolated by
expression cloning in Xenopus
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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…
Does my favorite gene have a developmental function?
• Gain of function tests
– Overexpress gene of interest in embryo and
observe effects.
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.
BMP-4 downregulated in neural plate
Neural
plate
midgastrulation st 11
St 12
Brivanlou & Thomsen 1996
• 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
RNAseH-mediated depletion of materna ß-catenin
mRNA by antisense DNA oligos
ß-catenin depleted embryos
Rescue with injected ß-catenin mRNA
MO knockdown of maternal ß-catenin
blocks 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…
Gene regulation
• 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)
Also …purification of regulatory proteins
- & use of egg and embryo in vitro extracts
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Cell cycle regulators…e.g MPF = cyclin + cdk
Transcription factors
Replication factors
Translation systems
Protein degradation / ubiquitin-proteasome
Chromatin assembly
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- RFP
Pax6-GFP
Lens gamma crystalline- RFP + Pax6-GFP = yellow eye
Natural recessive mutations of X. tropicalis
curly
bubblehead
Mutants recovered from a gynogentic haploid screen
rough diamond
balloon head
Fly
First mutant: Morgan 1910
Genome:
~ 2001
Xenopus
Harland, Grainger 2005
2005
The starlet
sea anemone,
Nematostella
vectensis
(Anthozoa)
egg masses
Lab of Mark Q. Martindale
Deuterostomia
Bryozoa
Articulata
Phoronida-*
Inarticulata
Pr otostomia
Spiralia
Acoelomorpha-*
Cnidaria-*
Ctenophora-*
Porifera
Choanoflagellata
“Bilateria”
“Radiata”
Metazoa