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Lecture 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

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Lecture 3

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  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. 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)

  3. 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.

  4. 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.

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

  6. 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

  7. 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

  8. 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

  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; • 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

  10. 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’.

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

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

  13. 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, p519-23

  14. Ground state pluripotency 2i ES cells more closely resemble embryo precursors of the blastocyst Without 2i With 2i LIF+ serum 2i • 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 StructMolBiol 20, p311-6

  15. Stem cell types isolated from early mouse embryos Day 5.5 Extraembryonic ectoderm Polar Trophectoderm Primitive ectoderm Polar Trophectoderm Visceral endoderm Primitive endoderm ICM Mural Trophectoderm Mural Trophectoderm Parietal endoderm Day 4.0 Day 3.5 +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 P 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 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, p1649-61

  16. 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

  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. Day 4.0 blastocyst Polar trophectoderm Mural trophectoderm Fgf4 Fgf4 Fgf4 Fgf2r Fgf2r • FGF4 signalling maintains a diploid stem cell population in the polar trophectoderm FGF4 signals to polar trophectoderm Rappolee et al (1994) Development 120, p2259-69

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

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