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.