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

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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

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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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