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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

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slide1

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 early
  • lineages.
slide2

Mosaic and Regulated development

  • 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’.
slide3

Regulated development in mouse embryos

Donor

Recipient

2-cell

embryo

Tarkowski, (1959)

Nature 184, p1286-7

slide4

8-cell embryos

Remove zona pellucida

Aggregate in dish

Culture in vitro

Transfer to foster mother

Chimeric blastocyst

Chimeric progeny

Chimeras from aggregaton of 8-cell stage embryos

Tarkowski (1961) Nature 190, 857-860

slide5

Chimeras from transfer of ICM cells

Gardner (1968), Nature 220, p596-7

  • Gardner later showed fate of TE and PE is determined by blastocyst stage
slide6

Preimplantation Development

Cleavage

Morula

Blastocyst

Day 3.0

Day 3.5

Day 4.0

Primitive ectoderm (PrEct)

Inner cell mass (ICM)

Blastomere

Zona pelucida

Primitive endoderm (PE)

Blastocoel cavity

Trophectoderm (TE)

slide7

Four master transcription factors for early lineage determination

in preimplantation development

1. Oct4/Pou5f1; uniformly expressed in cleavage stages. Switched off in trophectoderm of blastocyst.

Knockout fails to develop ICM.

Cleavage

Morula

Blastocyst

Day 3.0

Day 3.5

Day 4.0

2. Cdx2; stochastically expressed from 8-cell stage. Progressively restricted to outer TE cells of

blastocyst. Knockout fails to develop trophectoderm.

Primitive ectoderm (PrEct)

Inner cell mass (ICM)

3. Nanog; stochastically expressed from 8-cell stage. Switched off in TE. Expressed in salt and pepper

pattern 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.

Blastomere

Zona pelucida

Primitive endoderm (PE)

Blastocoel cavity

Trophectoderm (TE)

slide8

Inside-Outside Hypothesis

16-cell compacted morula

8-cell embryo

Outside cell

Inside cell

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

slide9

Testing the inside outside hypothesis

4-cell

embryo

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

slide10

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.
  • 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).

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

slide11

Cell polarity at compaction discriminates outer

and inner cells of the morula

8-cell

compaction

16-cell morula

Apical determinants

Basolateral

determinants

Non-polar

Inside cell

Polar outside cell

  • Only outside cells express apical determinants – provides potential mechanism for
  • the differentiated fate decision.
slide12

Molecular mechanism linking polarity to TE specification?

  • Proteins of the apical-basal polarity pathway localise assymetrically in the morula
slide13

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

slide14

Maintenance of TE/ICM specification

  • Double negative feedback loop with Oct4/Nanog confines Cdx2 expression to TE cells.
slide15

Specification of primitive endoderm lineage

Day 3.0

Day 3.5

Day 4.0

Primitive ectoderm (PrEct)

Inner cell mass (ICM)

Primitive endoderm (PE)

Blastocoel cavity

Trophectoderm (TE)

High Nanog

Low GATA6

Low Nanog

High GATA6

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

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

slide16

Fibroblast growth factor (FGF) signalling transduced

by MAPK

  • Grb2 mutant embryos fail to specify primitive endoderm

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

slide17

Fgf4

Fgf2r

Mapk

Grb2

Fgf4

Nanog

Gata6

Nanog

Gata6

Fgf4 high

Fgfr2 high

Fibroblast growth factor (FGF) signalling regulates

primitive endoderm to primitive ectoderm switching

Primitive

ectoderm

(PrEct) cell

Primitive

endoderm

(PE) cell

Cell sorting

  • FGF4 gene is activated by Oct4
  • Only Nanog expressing ICM cells seen in Grb2 knockout or with disruption of FGF signalling
  • Negative feedback by Gata6 on Nanog and vice versa?
  • Cell sorting mechanism?

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

slide18

Embryonic Stem (ES) Cells

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

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, mesoderm
  • and 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
slide19

ES cells

  • 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).

Alkaline phosphatase positive

Core transcription factors Oct4, Nanog and Sox2.

  • Have stable karyotype and contribute to all three germ layers (but not trophectoderm)
  • when transferred to recipient blastocyst – pluripotent.
  • Contribute to the germ-line of chimeric animals (blastocyst injection) and can therefore be
  • transmitted to subsequent generations.
  • Can be differentiated into embryoid bodies or defined lineages in vitro

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

slide20

What is an ES cell?

  • Single cell transcriptomics suggest closest to primitive ectoderm cells of the blastocyst.
  • No self-renewing pool of embryonic precursors in ICM or epiblast – ES cells are ‘synthetic’.
slide21

Signalling pathways regulating self-renewal

and differentiation of mouse ES cells

LIF/STAT3 (JAK/STAT)

and BMP/Smad/Id

FGFs

Via ERK1/2 pathway

GSK inhibition

(wnt?)

2i - Small molecule

inhibitors of ERK

LIF/STAT3 and

BMP/Smad/Id

GSK inhibition

(wnt?)

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

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

slide22

Stem cell types isolated from early mouse embryos

Day 5.5

Extraembryonic

ectoderm

Polar Trophectoderm

Primitive ectoderm

Polar Trophectoderm

Visceral

endoderm

Primitive endoderm

Day 4.0

ICM

Day 3.5

Mural Trophectoderm

Mural Trophectoderm

Parietal

endoderm

+FGF4

-LIF

+ feeders

+FGF4

+LIF

+ feeders

Epiblast

+LIF

+BMP

+FGF

+Activin

EpiSC

TS cell

XEN cell

ES cell

(Extraembryonic

endoderm cell)

(Trophoblast

stem cell)

(Epiblast stem cell)

Germ layers

Germ line

Trophectoderm

Primitive endoderm

Germ layers

Germ line

Trophectoderm

Primitive endoderm

Germ layers

Germ line

Trophectoderm

Primitive endoderm

Germ layers

Germ line

Trophectoderm

Primitive endoderm

Chimera

Contribution

(-LIF/-BMP)

Germ layers

Germ cells

Primitive endoderm

(-FGF)

Trophoblast giant

cells)

(-FGF)

Parietal endoderm like

(-FGF/Activin)

Germ layers

In vitro

differentiation

Tanaka et al (1998) Science 282, p2072-5; Brons et al (2007) Nature 448, p191-5;

Kunath et al (2005), Development, 132, p1649-61

slide23

Interconversion of embryo stem cell types

+CDX2

and/or

-OCT4

+GATA6

and/or

+OCT4

XEN

ES

TS

+FGF4

+LIF

+FGF4

- LIF

+FGF2

+Activin

+serum free

medium

+LIF

+2i

Or

+KLF4

EpiSC

Niwa (2007) Development 134, p635-46