stem cells from the early embryo prof. janet rossant · nishioka et al., dev cell, 2009 20 lats...
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Stem Cells from the Early Embryo Prof. Janet Rossant
1The screen versions of these slides have full details of copyright and acknowledgements
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Stem Cells from the Early Embryo
Prof. Janet RossantHospital for Sick Children
University of Toronto, Ontario, Canada
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Preimplantation mouse development
Zygote 2-cell 4-cell 8-cellCompact morula
Early blastocyst
Mid blastocyst
Lateblastocyst
Trophectoderm
Inner cell mass (ICM)
Primitive endoderm
Epiblast
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Preimplantation embryo development
OOFGO Zyg 2C
4C 8C Mor Blast
Genome activation, compaction, differentiation
Maturation, fertilization, reprogramming
Stem Cells from the Early Embryo Prof. Janet Rossant
2The screen versions of these slides have full details of copyright and acknowledgements
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EpiblastTrophectoderm
Primitive endoderm
Three cell types in the mouse blastocyst
• What do they give rise to?• How are they established?• Can we derive lineage-specific stem cell lines?
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Tarkowski, Nature, 1961Mintz, J. Exp Zool, 1964
The first chimeras8-cell aggregation Blastocyst injection of ICM cells
Gardner, Nature, 1968
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Blastocyst reconstitution experiment
Stem Cells from the Early Embryo Prof. Janet Rossant
3The screen versions of these slides have full details of copyright and acknowledgements
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E 7.5 reconstituted blastocyst chimera
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Visceral and parietal endoderm only
All definitive germ layers including endoderm, plus mesoderm of yolk sac
Injection of rough and smooth cells from E4.5 ICM
Gardner and Rossant, 1979
9Kwon et al., Dev Cell 2008
Some primitive endoderm contribution to gut progenitors
Stem Cells from the Early Embryo Prof. Janet Rossant
4The screen versions of these slides have full details of copyright and acknowledgements
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Epiblast derivatives
Ectoplacental cone
Trophoblastgiant cells
Parietal endoderm
Epiblast
Day 4.5
Mural TE
Polar TEPrimitive endoderm
Trophectoderm derivatives
Primitive endoderm derivatives
Mesoderm
Day 6.0 Day 7.0
Extraembryonicectoderm
Mesoderm
Visceralendoderm
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Extraembryonic lineages pattern the developing body axis
Courtesy Liz Robertson
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Lineage-specific transcription factors expressed at E3.5
Cdx2
Gata6
Nanog
Stem Cells from the Early Embryo Prof. Janet Rossant
5The screen versions of these slides have full details of copyright and acknowledgements
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Cdx2 is restricted to outside cellsbefore pluripotency markers restricted to ICM
Cdx2 Oct4 Merge
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Cdx2 mutant blastocyst forms, but fates are mis-specified
Wild type Cdx2 mutant
Outside markers (Eomesodermin, Troma1)
Inside cell markers (Oct4, Nanog)
E4.5
Strumpf et al., 2005 DevRalston & Rossant, 2008 Dev Biol
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Oct4
Nanog
Blastocyst formation begins but Oct4 and Nanog are not downregulated
Stem Cells from the Early Embryo Prof. Janet Rossant
6The screen versions of these slides have full details of copyright and acknowledgements
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Cdx2
Trophoblasttargets
Oct4/Sox2/Nanog
ICM/ES Trophectoderm/TS
Pluripotency targets
Localized Cdx2 expression initiates lineage segregation
What restricts Cdx2 to outside cells?
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Compaction and polarization promote lineage segregation
Are there signaling pathways differentially activein inside and outside cells?
Ralston and Rossant, 2008also Dietrich and Hiiragi, 2007
Cdx2 expn
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14-3-3Yki/Yap
P Wts/Lats
P
P
Hippo complex
Hippo/Wrts growth control pathway
Hippo activating signal
Wrts/Lats inactive:Scalloped/Tead active
Wrts/Lats active:Scalloped/Tead inactive
Pan, Guan, Irvine and others
14-3-3Wts/Lats
Scalloped/TEAD
Yki/YapScalloped/
TEADX
Stem Cells from the Early Embryo Prof. Janet Rossant
7The screen versions of these slides have full details of copyright and acknowledgements
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Yap nuclear localization correlates with Cdx2 expression
Nishioka et al., Dev Cell, 2009
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Lats kinase activity regulates localization of Cdx2
Nishioka et al., Dev Cell, 2009
Lats2 Yap-S112ALats2-KD
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Inside Outside
Yap
Lats1/2
Cdx2Tead4
YapP
Lats1/2 and Yap control Cdx2 expression in the preimplantation embryo
Cdx2
Trophectoderm (TE)
Inner cell mass (ICM)
Cdx2Tead4Yap
Lats1/2
X
Stem Cells from the Early Embryo Prof. Janet Rossant
8The screen versions of these slides have full details of copyright and acknowledgements
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Cdx2
Trophoblast fatePluripotency
Tead4/Yap
Oct4/Nanog
Oct4/Nanogtargets
Current pathway of ICM/trophectoderm fate
Gata3
Elf5 EomesAscl2
BMP4Ppar
Outside polar cells
Lats kinase active
Inside apolar cells
Tead4/Yap
Lats kinase inactive
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How is epiblast or primitive endoderm decision made?
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Oct4PemOct4
Primitive endoderm segregated to ICM surface by E4.5
E3.5 E4.5
Stem Cells from the Early Embryo Prof. Janet Rossant
9The screen versions of these slides have full details of copyright and acknowledgements
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Epiblast(Primitive ectoderm)
Primitive endoderm
ICM cells
Epiblast(primitive ectoderm)
Primitive endoderm
How does primitive endoderm form?
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‘Salt and pepper’ expression of Nanog and Gata6 in the E3.5 blastocyst
Gata6 = PE marker; Nanog = Epi marker
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ICM cellsNanog positive
Gata6 positive
Epiblast(primitive ectoderm)
Primitive endodermSorting out
How does primitive endoderm form?
Chazaud et al., Dev Cell, 2006Plusa et al., Development, 2008Meilhac et al., Dev Biol, 2009
Stem Cells from the Early Embryo Prof. Janet Rossant
10The screen versions of these slides have full details of copyright and acknowledgements
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Mosaicism of Nanog and GATA6 expression is lost in Grb2-/- E3.5 embryos
Chazaud et al., Dev Cell, 2006
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All ICM cells transform to Nanog positive Epi cells in Grb2-/- mutants lacking PE
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Levels of FGF influence EPI versus PE fate
Yamanaka et al.,2010, DevelopmentAlso see Nichols et al., 2009, Development
Embryos treated with FGFfrom 8-cell stage
Cdx2Gata6Nanog
Control FGF4 (500ng/ml)
Gata6
Embryos treated with FGFR inhibitor from 8-cell stage
MergeNanog
Stem Cells from the Early Embryo Prof. Janet Rossant
11The screen versions of these slides have full details of copyright and acknowledgements
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Graded levels of FGF signals determine EPI vs. PE fate
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Model of primitive endoderm formation
Chazaud et al., Dev Cell, 2006
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Three cell types in the mouse blastocyst
Can we derive lineage-specific stem cell lines?
Epiblast
Trophectoderm
Primitive endoderm
Stem Cells from the Early Embryo Prof. Janet Rossant
12The screen versions of these slides have full details of copyright and acknowledgements
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XEN ES TS
Three lineage-specific progenitor cell lines from the mouse blastocyst
Oct4+Nanog+
Gata6+Sox7+
Cdx2+Eomes+
Trophectoderm
Epiblast progenitor
Primitive endoderm progenitor
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Block FGF/GSK3 (Ying et al., Nature 2008)
Add FGF4 + activin(Tanaka et al., Science, 1998)
TS cellsES cells
Culture conditions determine stem cell development from mouse blastocyst
Add FGF4XEN cells
+ Cond medium(Kunath et al., Development 2007)
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Inner cell, produces FGF4, EPI differentiation
Inner cell, expresses FGFRResponds to FGF by PE differentiation
Outside TE progenitor, expresses FGFRResponds to FGF by proliferation
The same signal from the same cells is usedin two different ways in the blastocyst
FGF4 signal
Stem Cells from the Early Embryo Prof. Janet Rossant
13The screen versions of these slides have full details of copyright and acknowledgements
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• ICM/TE segregation requires position-dependent activation of Hippo signaling for establishing cell fate and FGF signaling for maintenance
• EPI/PE segregation requires stochastic (?) activation of FGF signaling followed by cell sorting
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The mouse blastocyst –a model for understanding lineage formation and its relevance to stem cell development
Epiblast
Trophectoderm
Primitive endoderm
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