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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. - PowerPoint PPT Presentation
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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 blastocoel cavity - cavitation
16-32 cell morula Early blastocyst
Physical forces merge fluid filled spaces to form blastocoel cavity
End