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

4. Epigenesis vs Preformation Nicolas Hartsoeker, 1695

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

6. 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’. Mosaic and Regulated development

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

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

9. Chimeras from transfer of ICM cells 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 Gardner (1968), Nature 220, p596-7

10. 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

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

13. Parthenogenesis Limited viability suggests either that sperm/fertilization confers essential properties for development or that maternal genome alone is incapable of supporting development 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

14. Non-equivalent contribution of maternal and paternal genomes ? Recipient zygote Donor zygote 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 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

16. 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

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

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

19. 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). 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. Johnson and Ziomek (1981), Cell 21, p935-942

20. 8-cellcompaction16-cell morula Apical determinants Basolateral determinants Polar outside cell Non-polar Inside cell Only outside cells express apical determinants – provides potential mechanism for the differentiated fate decision. Cell polarity at compaction discriminates outer and inner cells of the morula

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 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; Hirate et al (2013) Curr Biol 23, p1181-94 Angiomotin (Amot) sequestration by apical domains underlies Hippo inactivation. AJ= adherens junction Mer = merlin

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

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

25. End