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.
Preimplantation Development
Cleavage
Morula
Blastocyst
Day 3.0
Day 3.5
Inner cell mass (ICM)
Zona pelucida
Day 4.0
Primitive ectoderm (PrEct)
Blastomere
Blastocoel cavity
Trophectoderm (TE)
Primitive endoderm (PE)
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.
Cleavage
Morula
Blastocyst
Day 3.0
Day 3.5
Inner cell mass (ICM)
Zona pelucida
Day 4.0
Primitive ectoderm (PrEct)
Blastomere
Blastocoel cavity
Trophectoderm (TE)
Primitive endoderm (PE)
Inside-Outside Hypothesis
8-cell embryo
16-cell compacted morula
Outside cell
Inside cell
Tarkowski and Wroblewska, (1967) J Embryol Exp Morphol. 18, p155-80
Testing the inside outside hypothesis
4-cell
embryo
Hillman, Sherman, Graham (1972) J. Embryol. Exp. Morphol. 28, 263-278
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
Cell polarity at compaction discriminates outer
and inner cells of the morula
8-cell
compaction
Basolateral
determinants
16-cell morula
Apical determinants
Non-polar
Inside cell
Polar outside cell
• Only outside cells express apical determinants – provides potential mechanism for
the differentiated fate decision.
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 outer cells of 16-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.
Specification of primitive endoderm lineage
Day 3.0
Day 3.5
Inner cell mass (ICM)
Blastocoel cavity
Trophectoderm (TE)
Day 4.0
Primitive ectoderm (PrEct)
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 p615-24.
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 p615-24.
Fibroblast growth factor (FGF) signalling regulates
primitive endoderm to primitive ectoderm switching
Fgf4
Nanog
Grb2
Fgf2r
Fgf4
Mapk
Gata6
Nanog
Gata6
Fgf4 high
Fgfr2 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 p615-24.
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
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 differentiatiation in vitro and
in vivo, following transfer into recipient blastocysts. Cannot contribute to germline
Martin and Evans (1974), Cell 2, p163-172
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
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’.
Signalling pathways regulating self-renewal
and differentiation of mouse ES cells
LIF/STAT3 (JAK/STAT)
and BMP/Smad/Id
GSK inhibition
(wnt?)
FGFs
Via ERK1/2 pathway
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, p:519-23
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 corepressors).
Repressed target genes are associated with differentiation into different lineages and are held
in a‘poised’ configuration by epigenetic mechanisms (Polycomb).
•
Boyer et al (2005) Cell 122, p947-56
Stem cell types isolated from early mouse embryos
Day 4.0
Day 3.5
Polar Trophectoderm
ICM
Mural Trophectoderm
+FGF4
-LIF
+ feeders
+LIF
+BMP
ES cell
Day 5.5
Extraembryonic
ectoderm
Visceral
endoderm
Polar Trophectoderm
Primitive ectoderm
Primitive endoderm
Mural Trophectoderm
Parietal
endoderm
Epiblast
+FGF4
+LIF
+ feeders
+FGF
+Activin
TS cell
XEN cell
EpiSC
(Trophoblast
stem cell)
(Extraembryonic
endoderm cell)
(Epiblast stem cell)
Germ layers
Germ line
Trophectoderm
Primitive endoderm
Germ layers
Germ line
Trophectoderm
Primitive endoderm
Chimera
Contribution
Germ layers
Germ line
Trophectoderm
Primitive 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
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
XEN
+GATA6
and/or
+OCT4
+FGF4
+LIF
ES
+CDX2
and/or
-OCT4
TS
+FGF4
- LIF
+FGF2
+Activin
+serum free
medium
+LIF
+2i
Or
+KLF4
EpiSC
Niwa (2007) Development 134, p635-46
End lecture 2
Development of the egg cylinder
FGF4 signals to polar trophectoderm
Day 4.0 blastocyst
Polar trophectoderm
Fgf4
Mural trophectoderm
Fgf4
Fgf4
Fgf2r
•
FGF4 signalling maintains a diploid stem cell population in the polar trophectoderm
Rappolee et al (1994) Development 120, p2259-69
Probably need to drop this
Formation of the blastocel cavity
16-32 cell morula
Early blastocyst
Physical forces merge fluid filled spaces to form blastocoel cavity
Hatching
Four days after fertilization the blastocyst hatches from the zona pellucida
as a precursor to implantation in the uterine wall.