Transcript Germ Layer Formation/Fate Mapping (Slagle)

“It is not birth, marriage or death,
but gastrulation which is truly the
most important time in your life.”
Lewis Wolpert
Germ layer formation: from blastula to animal
onset of zygotic transcription
Germ layer formation: from blastula to animal
Fate-mapping the early Xenopus embryo
animal
vegetal
blastula
Fate-mapping the early Xenopus embryo
animal
ectoderm (epidermis, nervous system)
mesoderm (blood, muscle, kidneys, bones)
endoderm (digestive, respiratory systems)
vegetal
blastula
Origin of mesoderm: Nieuwkoop experiments
ectoderm
ectoderm
What happened to mesoderm?
endoderm
How is endoderm formed?
Origin of endoderm: maternal factors
• maternally supplied transcript
animal
•T-box transcription factor
• activates endoderm cascade
cell-autonomously
maternal vegT
vegetal
• inhibition blocks endoderm
(and mesoderm) formation
Cell autonomous and non-autonomous effects
Cell autonomy: genetically mutant cell/tissue exhibits
phenotype associated with loss of gene product
(gene product required by cell producing it)
Cell non-autonomy: genetically mutant cell/tissue
causes phenotypes in other (genetically wild-type) cells
(gene product required by cells other than those
producing it)
X
X
A
X
X
B
Origin of mesoderm: Nieuwkoop experiments
• mesoderm induced from ectoderm
when apposed to endoderm
• effect has limited range (4-5 cell
diameters)
• origin of “animal cap” assay
Origin of mesoderm: Nieuwkoop experiments
• induction event tolerant of small
physical separation (filter or spacing
between tissues): secreted
molecule(s) responsible
Origin of mesoderm: Nieuwkoop experiments
• type of mesoderm induced depends
on duration of contact with
endoderm/signal
Origin of mesoderm: Nieuwkoop experiments
“young”
• “heterochronic” transplants:
competence of ectoderm to form
mesoderm restricted to gastrula
stages
“old”
What is the mesoderm-inducing signal?
• secreted factor
• act at a distance
• expressed by endoderm at right time (late
blastula/gastrula stages)
• ectoderm competent to respond
Finding the signal:
• “cut and dump” cap assay: expose caps to candidate
secreted factors, assay for response
• functional cap screen: inject eggs with candidate genes,
cut young caps, assay for response
• expression screen: determine endoderm-specific
transcripts
• 30+ years of screening: TGFβ and FGF signaling
What is the mesoderm-inducing signal?
What is the mesoderm-inducing signal?
FGF signaling pathway
What is the mesoderm-inducing signal?
• brachyury: founding member of
T-box transcription factor family
• expressed throughout
mesoderm
• crucial for posterior mesoderm
development (similar to FGFs)
Additional findings
• Brachyury activates Fgf expression, leading to positive
feedback loop
• FGFs do not induce the entire mesoderm spectrum,
only posterior fates
• FGFs do not have unique targets involved in
mesoderm induction
Additional findings
• VegT directly activates expression of Nodal
• Nodals induce expression of their antagonist, Lefty
(mechanism for restricting inductive properties)
Making matters more complex…
animal
Spemann’s
organizer
(dorsal
blastopore lip)
vegetal
ventral
β-catenin
dorsal
• vegetal β-catenin moves
dorsally after fertilization
• induces high levels of Nodal
expression
• Nodal induces expression of
“dorsal determinants”
Discovery of the dorsal organizer
Spemann-Mangold experiment
*Donor organizer will induce/respecify host tissue to more dorsal fates!*
Spemann’s organizer: patterning the DV axis
animal
vegetal
ventral
high
Nodal
dorsal
ventral
dorsal
Spemann’s organizer: patterning the DV axis
Bra
Gsc
Nodal
ventral
dorsal
Spemann’s organizer: patterning the DV axis
Evolutionary conservation of mesoderm induction
yolk
Main differences
• holoblastic vs. meroblastic cleavage
• endoderm formation is cell non-autonomous
• no maternal VegT homologue
(zygotic homologue does not activate Nodal
expression)
Evolutionary conservation of mesoderm induction
ectoderm
neuroectoderm
mesoderm
ventral
lateral
dorsal
endoderm (gut) organizer
yolk
ectoderm
tail somites
and
neuroectoderm
yolk
“no Nodal”
Conserved mechanisms
• Nodal signaling is required
• β-catenin induces high levels of
dorsal nodal
• dorsal organizer (shield) inhibits
ventral signaling for DV patterning
• brachyury expressed throughout
mesoderm
• Wnt, FGF, BMP signaling maintain
and pattern mesoderm fates
Recap: vertebrate germ layer formation
• Endoderm origin varies between species
– Cell autonomous in Xenopus: maternal VegT
– Cell non-autonomous in zebrafish: maternal YSL signals
• Mesoderm is induced by signals (e.g. Nodal) from
endoderm and maintained by feedback loops
(FGF-Wnt-Brachyury)
• Concurrent with dorsal-ventral patterning
– Non-dorsal mesoderm produces FGFs, Wnts, BMPs
– Dorsal organizer inhibits ventral signals to form signaling
gradients and pattern mesoderm
Applying knowledge of embryonic tissue induction
Applying knowledge of embryonic tissue induction
mouse embryonic stem cells induced to cardiac muscle using
FGF, BMP, and low activin; day 7 of induction protocol