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Lecture 2 Preimplantation development I

Lecture 2 Preimplantation development I. You should understand; E vidence that maternal and paternal genomes are non-equivalent What is meant by mosaic and regulated development and evidence that mouse embryos are highly regulated.

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Lecture 2 Preimplantation development I

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  1. Lecture 2 Preimplantation development I • You should understand; • Evidence that maternal and paternal genomes are non-equivalent • What is meant by mosaic and regulated development and evidence that mouse • embryos are highly regulated. • The inside/outside mechanism regulating the first lineage allocation in mouse • preimplantation development.

  2. Preimplantation Development Cleavage Morula Blastocyst Day 3.0 Day 3.5 Day 4.0 Primitive ectoderm (PrEct) Inner cell mass (ICM) Blastomere Zona pelucida Primitive endoderm (PE) Blastocoel cavity Trophectoderm (TE)

  3. Confusing nomenclature! A ‘derm’ is a cell layer – not a cell type!

  4. Epigenesis vs Preformation Nicolas Hartsoeker, 1695

  5. Predetermined axes in animal development Mosaic development Animal Anterior Posterior D. melanogaster Vegetal Nucleus Bicoid mRNA in cytoplasm nanos mRNA in cytoplasm Anterior Posterior C.elegans P1 AB Ventral Animal X laevis Vegetal Dorsal (Spemanns Organiser)

  6. Mosaic and Regulated development • Roux (1888) shows ‘mosaic development’ of frog embryo following ablation of one cell in • two-cell embryo – formation of ‘half’ embryo. • Driesch (1895) finds opposite is true for sea urchin, normal albeit smaller embryo develops • from one of two cells – ‘regulated development’.

  7. Regulated development in mouse embryos Donor Recipient 2-cell embryo Tarkowski, (1959) Nature 184, p1286-7

  8. 8-cell embryos Remove zona pellucida Aggregate in dish Culture in vitro Transfer to foster mother Chimeric blastocyst Chimeric progeny Chimeras from aggregaton of 8-cell stage embryos Tarkowski (1961) Nature 190, 857-860

  9. Chimeras from transfer of ICM cells Gardner (1968), Nature 220, p596-7 • Gardner later demonstrated this for ICM cells of the blastocyst stage embryo. • In these experiments ICM cells did not contribute to trophectoderm or primitive endoerm lineages

  10. Preimplantation Development Cleavage Morula Blastocyst Day 3.0 Day 3.5 Day 4.0 Primitive ectoderm (PrEct) Inner cell mass (ICM) Blastomere Zona pelucida Primitive endoderm (PE) Blastocoel cavity Trophectoderm (TE)

  11. Non-equivalence of maternal and paternal genomes • Penetration of cumulus cells • Acrosomal reaction penetrates zona pellucida made up of glycoproteins • Sperm and egg plasma membranes fuse and sperm nucleus enters egg. • Fertilization triggers dramatic release of calcium in the egg, setting in train completion of • female meiosis etc.

  12. Pronuclear Maturation Second polar body Zonapelucida Syngamy Male pronucleus. Female pronucleus. 12 24 0 hr post fertilization Replication initiation M-phase • Maternal and paternal haploid genome remains separate (pronuclei) until first metaphase.

  13. Parthenogenesis Parthenogenetic activation - Genetic background - In vitro manipulation - Pronase/hyalouronidase - Heat shock - Ethanol - Strontium chloride • Oocytes can be activated in the absence of fertilization, leading to parthenogenetic development • Parthenogenetic embryos have limited viability, contrasting with other model organisms • Limited viability suggests either that sperm/fertilization confers essential properties for development or • that maternal genome alone is incapable of supporting development

  14. Recipient zygote Donor zygote Non-equivalent contribution of maternal and paternal genomes ? Barton, Surani , Norris (1984) Nature 311, p374-6 McGrath and Solter, (1984) Cell 37, p179-183 • Gynogenetic embryos have retarded growth/development of extraembryonic tissues • Androgenetic embryos have retarded growth/development of embryonic tissues

  15. Preimplantation Development Cleavage Morula Blastocyst Day 3.0 Day 3.5 Day 4.0 Primitive ectoderm (PrEct) Inner cell mass (ICM) Blastomere Zona pelucida Primitive endoderm (PE) Blastocoel cavity Trophectoderm (TE)

  16. 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 Day 4.0 Primitive ectoderm (PrEct) Inner cell mass (ICM) Blastomere Zona pelucida Primitive endoderm (PE) Blastocoel cavity Trophectoderm (TE)

  17. Inside-Outside Hypothesis 16-cell compacted morula 8-cell embryo Outside cell Inside cell Tarkowski and Wroblewska, (1967) J EmbryolExpMorphol. 18, p155-80

  18. Testing the inside outside hypothesis 4-cell embryo Hillman, Sherman, Graham (1972) J. Embryol. Exp. Morphol. 28, 263-278

  19. 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

  20. Cell polarity at compaction discriminates outer and inner cells of the morula 8-cell compaction 16-cell morula Apical determinants Basolateral determinants Non-polar Inside cell Polar outside cell • Only outside cells express apical determinants – provides potential mechanism for • the differentiated fate decision.

  21. Molecular mechanism linking polarity to TE specification? • Proteins of the apical-basal polarity pathway localise assymetrically in the morula

  22. Inhibition of Hippo signalling in polarised cells induces Cdx2 AJ= adherens junction Mer = merlin • 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. • Angiomotin (Amot) sequestration by apical domains underlies Hippo inactivation. Nishioka et al (2009) Dev Cell 16, p398-410; Hirate et al (2013) CurrBiol 23, p1181-94

  23. Maintenance of TE/ICM specification • Double negative feedback loop with Oct4/Nanog confines Cdx2 expression to TE cells.

  24. Formation of the blastocelcavity - cavitation 16-32 cell morula Early blastocyst Physical forces merge fluid filled spaces to form blastocoel cavity

  25. End

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