1. Recap

2. Confusing nomenclature!
A ‘derm’ is a cell layer – not a cell type!

3. Lecture 3
Specification of the second lineage, primitive endoderm and stem cell lines from early mouse embryos
You should understand
Mechanisms governing specification of the primitive endoderm lineage
What is an ES cell
Stem cell lines from early mouse embryos and their relationship to early
lineages.

4. 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.
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 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.
Trophectoderm (TE)
Primitive endoderm (PE)
Inner cell mass (ICM)
Zona pelucida
Blastocoel cavity
Blastomere
Primitive ectoderm (PrEct)
Day 3.0
Day 3.5
Day 4.0
Morula
Blastocyst
Cleavage

5. Specification of primitive endoderm lineage
Day 3.0
Day 3.5
Day 4.0
Inner cell mass (ICM)
Primitive ectoderm (PrEct)
Blastocoel cavity
Trophectoderm (TE)
Primitive endoderm (PE)
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 p

6. Grb2 mutant embryos fail to specify primitive endoderm
Fibroblast growth factor (FGF) signalling transduced by MAPK
Inhibition of FGF signalling also causes failure to specify primitive endoderm
Chazaud et al (2006) Dev Cell 10 p

7. Fibroblast growth factor (FGF) signalling regulates
primitive endoderm to primitive ectoderm switching
Grb2
Fgf2r
Gata6
Nanog
Fgf4
Mapk
Fgf4 high
Fgf2r high
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 p

8. 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

9. Embryonal carcinoma (EC) cells
Teratoma
Teratocarinomas are malignant tumours derived from germ cells and comprising multiple cell types from all three germ layers, indicating the presence of a pluripotent stem cell population.
Occur at high frequency in 129 strain of mouse or can be produced by injecting early
embryo cells into testis or kidney capsule of syngeneic host.
Pluripotent stem cell tissue culture cell lines derived from teratocarcinomas are
termed embryonal carcinoma (EC) cells. They have an abnormal karyotype and express high
levels of alkaline phosphatase.
EC cells can self-renew indefinitely and can undergo lineage differentiation in vitro and
in vivo, following transfer into recipient blastocysts. Cannot contribute to germline
Martin and Evans (1974), Cell 2, p

10. 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
Contribute to all three germ layers (but not trophectoderm) when differentiated in vitro or when transferred to recipient blastocyst – pluripotent.
Have stable normal karyotype
Contribute to the germ-line of chimeric animals (blastocyst injection) and can therefore be
transmitted to subsequent generations.
Efficient at homologous recombination allowing development of gene knockout technology.
Evans and Kaufman (1984) Nature 292, p154-6

11. Transcription factor circuitry in ES cells
Availability of unlimited quantity of ES cells grown in vitro has facillitated genome wide
analysis. Key findings include;
Core transcription factors Oct4, Nanog and Sox2 co-occupy a large proportion of target genes
Oct4, Nanog and Sox2 participate in positive feedback loops with themselves and one another to stably maintain the pluripotent state
Oct, Nanog and Sox2 participate in negative regulatory loops to block expression of core transcription factors of trophectoderm and primitive endoderm lineages.
Other target genes can be either activated or repressed (recruitment of co-activators or co-repressors).
Repressed target genes are associated with differentiation into different lineages and are held in a‘poised’ configuration by epigenetic mechanisms (e.g. Polycomb).
Boyer et al (2005) Cell 122, p947-56

12. What is an ES cell?
Single cell transcriptomics suggest closest to ICM (primitive ectoderm) cells of the blastocyst.
No self-renewing pool of embryonic precursors in ICM or epiblast – ES cells are ‘synthetic’.

13. Homogeneous expression of Oct4 but not Nanog
Immunostaining of ES cell colonies

14. DNA methylation in ES cells
Santos et al (2002) Dev Biol 241,
Somatic cells
ES cells
% meCpG
70-80

15. Signalling pathways regulating self-renewal
and differentiation of mouse ES cells
FGFs
Via ERK1/2 pathway
LIF/STAT3 (JAK/STAT)
and BMP/Smad/Id
LIF/STAT3 and
BMP/Smad/Id
2i - Small molecule
inhibitors of ERK
GSK inhibition
(wnt?)
Recent evidence suggests LIF +BMP blocks autostimulation of differentiation by FGF4
Ying et al (2008) Nature 453, p519-23

16. Ground state pluripotency
2i ES cells more closely resemble embryo precursors of the blastocyst
Without 2i
With 2i
LIF+
serum 2i
Somatic cells
ES cells
2i ES cells
% meCpG
70-80
25-30%
Due to reduced levels of de novo DNA methyltransferases (Dnmt3a, Dnmt3b), and
enhanced levels of enzymes that actively remove methyl groups from CpG.
Leitch et al (2013) Nat Struct Mol Biol 20, p311-6

17. Parietal endoderm like
Stem cell types isolated from early mouse embryos
Day 3.5
Day 4.0
Day 5.5
Polar Trophectoderm
Extraembryonic
ectoderm
Polar Trophectoderm
Primitive ectoderm
Primitive endoderm
Visceral
endoderm
ICM
Mural Trophectoderm
Mural Trophectoderm
Parietal
endoderm
+FGF4
-LIF
+ feeders
+FGF4
+LIF
+ feeders
Epiblast
+LIF
+BMP
+FGF
+Activin
ES cell
TS cell
XEN cell
EpiSC
(Trophoblast
stem cell)
(Extraembryonic
endoderm cell)
(Epiblast stem cell)
Chimera
Contribution
Germ layers
Germ line
Trophectoderm
Primitive endoderm
Germ layers
Germ line
Trophectoderm
Primitive endoderm
Germ layers
Germ line
Trophectoderm
P endoderm
Germ layers
Germ line
Trophectoderm
Primitive endoderm
In vitro
differentiation
(-LIF/-BMP)
Germ layers
Germ cells
Primitive endoderm
(-FGF)
Trophoblast giant
cells)
(-FGF)
Parietal endoderm like
(-FGF/Activin)
Germ layers
Epigenome
(X inactivation
Imprinted-
X inactivation
Imprinted-
X inactivation
Random-
X inactivation
Pre-X inactivation
Tanaka et al (1998) Science 282, p2072-5; Brons et al (2007) Nature 448, p191-5;Kunath et al (2005), Development, 132, p

18. Interconversion of embryo stem cell types
+GATA6
and/or
+OCT4
+CDX2
and/or
-OCT4 TS ES
EpiSC
XEN
+FGF4
+LIF
+FGF4
- LIF
+FGF2
+Activin
+serum free
medium
+LIF
+2i Or
+KLF4
Niwa (2007) Development 134, p635-46

19. ES cells from other species.
Pluripotent human ESCs (Thomson et al, 1998, Science 282, p ) derived from blastocysts
explanted onto mouse feeder cells. Addition of bFGF improves maintenance.
Human ESCs resemble mouse EpiSCs e.g. post X inactivation and dependent on Fgf4 and nodal
signals from feeder cells. Not ground state.
Considered to have significant potential in regenerative therapies.
2i method has opened up the possibility of obtaining ESCs from any mouse strain and from other
species, notable success being rat (Buehr et al, 2008, Cell 135, p ).

20. Hatching
Four days after fertilization the blastocyst hatches from the zona pellucida
as a precursor to implantation in the uterine wall.

21. Development of the egg cylinder)

22. FGF4 signals to polar trophectoderm
Day 4.0 blastocyst
Mural trophectoderm
Polar trophectoderm
Fgf4
Fgf2r
FGF4 signalling maintains a diploid stem cell population in the polar trophectoderm
Rappolee et al (1994) Development 120, p

23. Overview of gastrulation
Brachyury expression marks
the primitive streak
What determines the site of initiation of the primitive streak?

24. Major signalling pathways; BMP and Nodal/Activin
BMPs and Nodal signal by binding type I/II receptor and activating ser/thr kinase to phosphorylate Smads
BMP signal transduced by phosphorylating Smad 1,5, or 8 and Nodal through Smad 2 or 3
Phospho Smads bind Smad4, translocate to the nucleus and activate target genes

25. Major signalling pathways; Nodal fine tuning
Nodal can be regulated at the level of conversion of pro-nodal to nodal by Furin/PACE4
Cer1 and Lefty1 are diffusible antagonists of nodal.

26. Major signalling pathways; canonical Wnt
Binding of Wnt ligand to frizzled/LRP stabilises b-catenin by blocking activity of the destruction
complex comprising Axin, Dvl, and the kinases CK1 gamma and GSK beta.
Stabilised b-catenin translocates to the nucleus, binds to TCF family proteins and activates expression
In the absence of b-catenin TCF proteins repress target genes.
In the absence of wnt ligand, b-catenin is phosphorylated by CK1 and GSK3 and degraded
DKK1 anagonises Wnt signalling by sequestering and internalising LRP
WIF1 and sFRP are frizzled related proteins that bind and sequester Wnt ligands