Preimplantation development I 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.

Epigenesis vs Preformation Nicolas Hartsoeker, 1695

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

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’.

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

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

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

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

Cell lineages A ‘derm’ is a cell layer – not a cell type!

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

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.

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

Parthenogenesis Activate oocyte; genetic background, ethanol, strontium chloride. Inhibit extrusion of second polar body using cytochalasin-b. no Unfertilized oocyte Diploid Parthenogenetic zygote Diploid parthenogenetic Embryo (maternal genome only) Syngamy Mammalian parthenogenetic embryos have limited viability; Requires factor associated with sperm or maternal and paternal genomes non-equivalent?

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

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

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. Cleavage Morula Blastocyst Day 3.0 Day 3.5 Day 4.0 Inner cell mass (ICM) Primitive ectoderm (PrEct) Zona pelucida 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 16-cell morula Basolateral determinants Apical determinants Apolar 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 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) Curr Biol 23, p1181-94

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

Formation of the blastocoel cavity - cavitation 16-32 cell morula Early blastocyst Cl-