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.

Preimplantation Development

Trophectoderm (TE) Primitive endoderm (PE)

Inner cell mass (ICM)

Zona pelucidaBlastocoel cavity

Blastomere

Primitive ectoderm (PrEct)

Day 3.0 Day 3.5 Day 4.0

Morula BlastocystCleavage

Confusing nomenclature!

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

Epigenesis vs Preformation

Nicolas Hartsoeker, 1695

Bicoid mRNA in cytoplasm

nanos mRNA in cytoplasm

AnteriorPosterior

Nucleus

Animal

Vegetal

Animal

VegetalDorsal (Spemanns Organiser)

Ventral

P1 AB

AnteriorPosterior

D. melanogaster

C.elegans

X laevis

Predetermined axes in animal developmentMosaic 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’.

Mosaic and Regulated development

Tarkowski, (1959)Nature 184, p1286-7

2-cellembryo

Donor

Recipient

Regulated development in mouse embryos

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 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

Preimplantation Development

Trophectoderm (TE) Primitive endoderm (PE)

Inner cell mass (ICM)

Zona pelucidaBlastocoel cavity

Blastomere

Primitive ectoderm (PrEct)

Day 3.0 Day 3.5 Day 4.0

Morula BlastocystCleavage

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

12 24

Replicationinitiation

M-phase

hr post fertilization0

Second polar bodyZona pelucida

• Maternal and paternal haploid genome remains separate (pronuclei) until first metaphase.

Male pronucleus. Female pronucleus.

Syngamy

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

Non-equivalent contribution of maternal and paternal genomes

?Recipient zygote

Donor zygote

Barton, Surani , Norris (1984)Nature 311, p374-6McGrath 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

Trophectoderm (TE) Primitive endoderm (PE)

Inner cell mass (ICM)

Zona pelucidaBlastocoel cavity

Blastomere

Primitive ectoderm (PrEct)

Day 3.0 Day 3.5 Day 4.0

Morula BlastocystCleavage

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 pepperpattern 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 determinationin preimplantation development

Trophectoderm (TE) Primitive endoderm (PE)

Inner cell mass (ICM)

Zona pelucidaBlastocoel cavity

Blastomere

Primitive ectoderm (PrEct)

Day 3.0 Day 3.5 Day 4.0

Morula BlastocystCleavage

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

Testing the inside outside hypothesis

4-cellembryo

Hillman, Sherman, Graham (1972) J. Embryol. Exp. Morphol. 28, 263-278

• 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

8-cell compaction 16-cell morula

Apical determinantsBasolateraldeterminants

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

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; Hirate et al (2013) Curr Biol 23, p1181-94

• Angiomotin (Amot) sequestration by apical domains underlies Hippo inactivation.

AJ= adherens junctionMer = merlin

Maintenance of TE/ICM specification

• Double negative feedback loop with Oct4/Nanog confines Cdx2 expression to TE cells.

Formation of the blastocel cavity - cavitation

16-32 cell morula Early blastocyst

Physical forces merge fluid filled spaces to form blastocoel cavity

End