Lecture 2

• Overview of preimplantation development• Specification of the trophectoderm• Specification of primitive endoderm• Stem cell lines from early mouse embryos

You should understand

• Key transcription factors and signalling pathways in preimplantation embryos• Mechanisms governing specification of the trophectoderm lineage• Mechanisms governing specification of the primitive endoderm lineage• Stem cell lines from early mouse embryos and their relationship to earlylineages.

• Roux (1888) shows ‘mosaic development’ of frog embryo following ablation of one cell in two-cell embryo – formation of ‘half’ embryo.

• Driesch (1895) finds opposite is true for sea urchin, normal albeit smaller embryo develops from one of two cells – ‘regulated development’.

Mosaic and Regulated development

Tarkowski, (1959)Nature 184, p1286-7

2-cellembryo

Donor

Recipient

Regulated development in mouse embryos

Chimeras from aggregaton of 8-cell stage embryos

8-cell embryos

Remove zona pellucida

Aggregate in dish

Culture in vitro

Chimeric blastocyst

Transfer to foster mother

Chimeric progeny

Tarkowski (1961) Nature 190, 857-860

Chimeras from transfer of ICM cells

• Gardner later showed fate of TE and PE is determined by blastocyst stage

Gardner (1968), Nature 220, p596-7

Preimplantation Development

Trophectoderm (TE) Primitive endoderm (PE)

Inner cell mass (ICM)

Zona pelucidaBlastocoel cavity

Blastomere

Primitive ectoderm (PrEct)

Day 3.0 Day 3.5 Day 4.0

Morula BlastocystCleavage

1. Oct4/Pou5f1; uniformly expressed in cleavage stages. Switched off in trophectoderm of blastocyst. Knockout fails to develop ICM.

2. Cdx2; stochastically expressed from 8-cell stage. Progressively restricted to outer TE cells of blastocyst. Knockout fails to develop trophectoderm.

3. Nanog; stochastically expressed from 8-cell stage. Switched off in TE. Expressed in salt and pepperpattern in ICM eventually restricted to primitive ectoderm at d4. Knockout fails to develop ICM.

4. Gata6 (+Gata4); stochastically expressed from 8-cell stage. Switched off in TE. Expressed in salt and pepper pattern in ICM eventually restricted to primitive endoderm at d4. Double knockout fails to develop PE.

Four master transcription factors for early lineage determinationin preimplantation development

Trophectoderm (TE) Primitive endoderm (PE)

Inner cell mass (ICM)

Zona pelucidaBlastocoel cavity

Blastomere

Primitive ectoderm (PrEct)

Day 3.0 Day 3.5 Day 4.0

Morula BlastocystCleavage

Inside-Outside Hypothesis

Outside cell

Inside cell

8-cell embryo 16-cell compacted morula

Tarkowski and Wroblewska, (1967) J Embryol Exp Morphol. 18, p155-80

Testing the inside outside hypothesis

4-cellembryo

Hillman, Sherman, Graham (1972) J. Embryol. Exp. Morphol. 28, 263-278

• Cell polarity model posits that divisions at 8-cell stage produce 2 polar or 1 polar and one apolar cell, depending on the plane of division (stochastic).

The role of compaction and the cell polarity model

• Compaction; at 8-cell stage cells flatten along basolateral surfaces (those with cell-cell contacts). Apical (outside facing) surfaces develop distinct features, eg microvilli.

Johnson and Ziomek (1981), Cell 21, p935-942

8-cell compaction 16-cell morula

Apical determinantsBasolateraldeterminants

Polar outside cell

Non-polar Inside cell

• Only outside cells express apical determinants – provides potential mechanism for the differentiated fate decision.

Cell polarity at compaction discriminates outer and inner cells of the morula

Molecular mechanism linking polarity to TE specification?

• Proteins of the apical-basal polarity pathway localise assymetrically in the morula

Inhibition of Hippo signalling in polarised cells induces Cdx2

• Tead4, the downstream effector of Hippo pathway is required for Cdx2 expression in outer cells.

• Tead4 co-activator, dephosphorylated YAP is present in the nucleus only in inner cells of 32 cell morula.

Nishioka et al (2009) Dev Cell 16, p398-410

Maintenance of TE/ICM specification

• Double negative feedback loop with Oct4/Nanog confines Cdx2 expression to TE cells.

Day 3.0 Day 3.5 Day 4.0

High NanogLow GATA6

Low NanogHigh GATA6

• Reciprocal salt and pepper pattern of Nanog and GATA6 in ICM cells of mid-stage blastocysts

Trophectoderm (TE) Primitive endoderm (PE)

Inner cell mass (ICM)

Blastocoel cavity

Primitive ectoderm (PrEct)

Specification of primitive endoderm lineage

Chazaud et al (2006) Dev Cell 10 p615-24.

Fibroblast growth factor (FGF) signalling transducedby MAPK

• Grb2 mutant embryos fail to specify primitive endoderm

Chazaud et al (2006) Dev Cell 10 p615-24.

Grb2

Fgf2r

Gata6NanogGata6

Nanog

Fgf4

Fgf4

Mapk

Fgf4 high Fgfr2 high

• Only Nanog expressing ICM cells seen in Grb2 knockout or with disruption of FGF signalling

• Negative feedback by Gata6 on Nanog and vice versa?

Fibroblast growth factor (FGF) signalling regulates primitive endoderm to primitive ectoderm switching

Primitive ectoderm(PrEct) cell

Primitive endoderm(PE) cell

• Cell sorting mechanism?

• FGF4 gene is activated by Oct4

Chazaud et al (2006) Dev Cell 10 p615-24.

Cell sorting

Embryonic Stem (ES) CellsStem cells and progenitors;

Terminology for differentiative capacity of stem cells/progenitors;• Totipotent; capable of giving rise to all differentiated cell types of the organism, including extraembryonic lineages e.g. morula cells

• Pluripotent; capable of giving rise to cell types of the three germ layers, ectoderm, mesodermand endoderm eg primitive ectoderm cells of the blastocyst.

• Multipotent – capable of giving rise to a limited number of differentiated cell types, e.g.adult stem cells and progenitors

Stem cell; unlimited capacity to self-renew and produce differentiated derivatives

Progenitor cell; limited capacity to self-renew and produce differentiated derivatives

Terminally differentiated cell

ES cells

• Contribute to the germ-line of chimeric animals (blastocyst injection) and can therefore be transmitted to subsequent generations.

• Derived from blastocyst stage embryos

• Grow as ‘clumps’ or ‘colonies’ by culturing with fetal calf-serum (FCS) on layer of inactivated primary embryonic fibroblast cells (PEFs).

• Have stable karyotype and contribute to all three germ layers (but not trophectoderm) when transferred to recipient blastocyst – pluripotent.

• Can be differentiated into embryoid bodies or defined lineages in vitro

Alkaline phosphatase positive

Core transcription factors Oct4, Nanog and Sox2.

Evans and Kaufman (1984) Nature 292, p154-6

What is an ES cell?

• No self-renewing pool of embryonic precursors in ICM or epiblast – ES cells are ‘synthetic’.

• Single cell transcriptomics suggest closest to primitive ectoderm cells of the blastocyst.

Signalling pathways regulating self-renewal and differentiation of mouse ES cells

• Recent evidence suggests LIF +BMP blocks autostimulation of differentiation by FGF4

FGFsVia ERK1/2 pathway

LIF/STAT3 (JAK/STAT)and BMP/Smad/Id

LIF/STAT3 andBMP/Smad/Id

2i - Small moleculeinhibitors of ERK

GSK inhibition(wnt?)

GSK inhibition(wnt?)

Ying et al (2008) Nature 453, p:519-23

Stem cell types isolated from early mouse embryos

Day 4.0 Day 5.5Day 3.5

+LIF +BMP

Polar Trophectoderm

Mural Trophectoderm

Primitive ectodermPrimitive endodermICM

Polar Trophectoderm

Mural Trophectoderm

+FGF4 -LIF+ feeders +FGF

+Activin

Extraembryonicectoderm

Epiblast

Visceralendoderm

Parietalendoderm

ES cell TS cell XEN cell EpiSC(Trophoblaststem cell)

(Extraembryonicendoderm cell)

(Epiblast stem cell)

ChimeraContribution

Germ layersGerm line

TrophectodermPrimitive endoderm

Germ layersGerm line

TrophectodermPrimitive endoderm

Germ layersGerm line

TrophectodermPrimitive endoderm

Germ layersGerm line

TrophectodermPrimitive endoderm

In vitrodifferentiation

(-LIF/-BMP)Germ layersGerm cells

Primitive endoderm

(-FGF)Trophoblast giant

cells)

(-FGF)Parietal endoderm like

(-FGF/Activin)Germ layers

+FGF4 +LIF + feeders

Tanaka et al (1998) Science 282, p2072-5; Brons et al (2007) Nature 448, p191-5;Kunath et al (2005), Development, 132, p1649-61

Interconversion of embryo stem cell types

TSES

EpiSC

XEN

+GATA6 and/or+OCT4+FGF4+LIF

+CDX2and/or-OCT4

+FGF4- LIF

+LIF+2iOr+KLF4

+FGF2+Activin

+serum freemedium

Niwa (2007) Development 134, p635-46

End lecture 2