1. 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.

2. 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. Four master transcription factors for early lineage determination in preimplantation development Trophectoderm (TE) Primitive endoderm (PE) Inner cell mass (ICM) Zona pelucida Blastocoel cavity Blastomere Primitive ectoderm (PrEct) Day 3.0Day 3.5Day 4.0 MorulaBlastocystCleavage

3. Day 3.0Day 3.5Day 4.0 High Nanog Low GATA6 Low Nanog High 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.

4. 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. Inhibition of FGF signalling also causes failure to specify primitive endoderm

5. Grb2 Fgf2r Gata6 Nanog Gata6 Nanog Fgf4 Mapk Fgf4 high Fgf2r 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

6. Embryonic Stem (ES) Cells Stem 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, 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 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

7. Embryonal carcinoma (EC) cells 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 Teratoma Martin and Evans (1974), Cell 2, p163-172

8. 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 normal karyotype Alkaline phosphatase positive Evans and Kaufman (1984) Nature 292, p154-6 Contribute to all three germ layers (but not trophectoderm) when differentiated in vitro or when transferred to recipient blastocyst – pluripotent. Efficient at homologous recombination allowing development of gene knockout technology.

9. Transcription factor circuitry in ES cells Availability of unlimited quantity of ES cells grown in vitro has facillitated genome wide analysis. Key findings include; Oct, Nanog and Sox2 participate in negative regulatory loops to block expression of core transcription factors of trophectoderm and primitive endoderm lineages. Core transcription factors Oct4, Nanog and Sox2 co-occupy a large proportion of target genes 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). Oct4, Nanog and Sox2 participate in positive feedback loops with themselves and one another to stably maintain the pluripotent state Boyer et al (2005) Cell 122, p947-56

10. 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 ICM (primitive ectoderm) cells of the blastocyst.

11. Homogeneous expression of Oct4 but not Nanog Oct4Nanog Immunostaining of ES cell colonies

12. DNA methylation in ES cells Somatic cellsES cells % meCpG70-80 Santos et al (2002) Dev Biol 241, 172-182.

13. Signalling pathways regulating self-renewal and differentiation of mouse ES cells Recent evidence suggests LIF +BMP blocks autostimulation of differentiation by FGF4 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?) GSK inhibition (wnt?) Ying et al (2008) Nature 453, p519-23

14. Without 2iWith 2i Somatic cellsES cells2i ES cells % meCpG70-80 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. LIF+ serum 2i Ground state pluripotency 2i ES cells more closely resemble embryo precursors of the blastocyst Leitch et al (2013) Nat Struct Mol Biol 20, p311-6

15. Stem cell types isolated from early mouse embryos Day 4.0Day 5.5 Day 3.5 +LIF +BMP Polar Trophectoderm Mural Trophectoderm Primitive ectoderm Primitive endoderm ICM Polar Trophectoderm Mural Trophectoderm +FGF4 -LIF + feeders +FGF +Activin Extraembryonic ectoderm Epiblast Visceral endoderm Parietal endoderm 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 +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 Epigenome (X inactivation Pre-X inactivation Imprinted- X inactivation Imprinted- X inactivation Random- X inactivation

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

17. 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).

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

19. Development of the egg cylinder

20. 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, p2259-69

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