1. 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.

2. Epigenesis vs Preformation
Nicolas Hartsoeker, 1695

3. 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)

4. 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’.

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

6. 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,

7. 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

8. 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)

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

10. 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)

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
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.

13. 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?

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, p
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
Inner cell mass (ICM)
Primitive ectoderm (PrEct)
Zona pelucida
Blastomere
Blastocoel cavity
Trophectoderm (TE)
Primitive endoderm (PE)

16. 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)

17. 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

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

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, p

20. 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.

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, p ; Hirate et al (2013) Curr Biol 23, p

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

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