Transcript 2014 lecture 3x

Recap
Confusing nomenclature!
A ‘derm’ is a cell layer – not a cell type!
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.
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)
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
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 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 differentiation 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
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 (e.g. Polycomb).
•
Boyer et al (2005) Cell 122, p947-56
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’.
Homogeneous expression of Oct4 but not Nanog
Oct4
Nanog
Immunostaining of ES cell colonies
DNA methylation in ES cells
Santos et al (2002) Dev Biol 241, 172-182.
% meCpG
Somatic cells
ES cells
70-80
70-80
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, p519-23
Ground state pluripotency
2i ES cells more closely resemble embryo precursors of the blastocyst
Without 2i
With 2i
LIF+
serum
2i
% meCpG
•
Somatic cells
ES cells
2i ES cells
70-80
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
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
+FGF4
+LIF
+ feeders
Epiblast
+FGF
+Activin
TS cell
XEN cell
EpiSC
(Trophoblast
stem cell)
(Extraembryonic
endoderm cell)
(Epiblast stem cell)
Germ layers
Germ line
Trophectoderm
P 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
ImprintedX inactivation
ImprintedX inactivation
RandomX inactivation
Epigenome
(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, 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
ES cells from other species.
• Pluripotent human ESCs (Thomson et al, 1998, Science 282, p1145-47) 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, p1287-98).
Hatching
Four days after fertilization the blastocyst hatches from the zona pellucida
as a precursor to implantation in the uterine wall.
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
Overview of gastrulation
Brachyury expression marks
the primitive streak
What determines the site of initiation of the primitive streak?
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
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.
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