10 - Early stages of mammalian development

Cleavage 

• Zygote to 8-cell stage 

• Cell division without cell growth

• Overall size of the embryo stays the same 

• Above shows development of a mouse embryo 

Cleavage = cell division without cell growth, so 8-cell stage embryo is the same size as the zygote 

• From 2- to 8- cell stage cells are called blastomeres and are thought to be equivalent 

• Fate of the blastomeres has not been decided 

• Zona pellucida - prevents embryo from implanting prematurely in the fallopian tube 

• If this happens, it is called ectopic pregnancy (life threatening)

Evidence for blastomere equivalence 

• Sometimes, during cleavage, the embryo can split, generating identical (monozygotic) twins 

• A blastomere can be removed from the embryo without affecting development 

• Taken for preimplantation diagnosis 

Compaction 

• At compaction, blastomeres maximise cell to cell contact to form morula 

• Requires the cell adhesion molecule E-cadherin - express high levels of this - calcium dependent

• Following compaction, the first differentiation event occurs

• Cells on the surface differentiate to become trophectoderm cells 

• Blastomeres at 8-cell stage are totipotent 

• Following compaction, cells on the outside become restricted in their developmental potential 

How is the development of the trophectoderm regulated? 

• Prior to compaction, all cells express the transcription factors at equal levels - Oct4 and Cdx2 

• Oct4 required for development of inner cells 

• Cdx2 required for trophectoderm 

• Above is immunofluorescence staining

• Antibodies added (green and red) - shows yellow as a combination of them both

But following compaction, cells on the outside have higher levels of Cdx2 and cells on the inside have higher levels of Oct4 

• Cdx2 switches off Oct4 (Oct4 is required for pluripotency of the ESCs) 

• Oct4 switches off Cdx2 

• Differential expression of these key transcriptional factors drives the cells in the early embryo to become either trophectoderm, or cells of the inner mass 

What happens is Cdx2 is blocked? 

• Trophectoderm does not develop and the blastocyst does not form 

• Cdx2 - knocked off early on = embryo death

Function of the trophectoderm 

1. Pump sodium ions inside the morula to form blastocyst - water follows through osmosis 

2. Hatching from the zona pellucida - if it doesn't hatch, can't implant

3. Implantation 

4. Maintenance of pregnancy (in human; secretion of chorionic gonadotropin - also the basis of most pregnancy tests) 

5. Formation of the placenta 

Blastocyst formation 

• 1st function of the trophectoderm is the pump Na+ ions inside the morula 

• Accumulation if Na+ ions draws in water osmotically

• Fluid filled cavity forms 

• Zona pellucida stops the blastocyst from expanding too much

• The ability of the trophectoderm to pump Na+ ions depends on polarisation of ion channels  and the Na+ pump 

• Basal part - expression of Na/K ATPase

• Na moves down conc gradient 

• E cadherin - expressed on lateral side of cells 

• Apical - Na+ ion channels 

Cell polarisation 

• Cell polarity refers to the intrinsic asymmetry observed in cells 

• Asymmetry can be in the shape of cells ir in the organisation of cellular components 

• Epithelial cells like trophectoderm become polarised along the apical-basal axis 

• This means that protein consumption composition at apical and basal surfaces is different 

• The plane of cell division plays an important role in cell fate - depends on which proteins they are inheriting 

Mural and polar trophectoderm 

After blastocyst formation, 2 types of trophectoderm cells: 

1. Polar trophectoderm cells are in direct contact with the underlying epiblast

2. Mural trophectoderm cells line the blastocoel cavity 

Mural trophectoderm required for hatching from zona 

• Mural trophectoderm (TE) secretes the enzyme strypsin that lyses a hole in the zona, allowing the blastocyst to escape 

• The blastocyst is now ready to implant 

• Mural TE gives rise to trophoblast giant cells which are terminally differentiated and non-invasive (don't play a role in helping the embryo implant)

Polar trophectoderm required for implantation 

• Upon contact with the uterine epithelium, polar TE differentiates into highly proliferating cytotrophoblastic cells 

• Some cells fuse to form syncytiotrophoblastic cells (when nuclei divide too rapidly) with high invasive capacity 

• This syncytium is responsible for implantation of the embryo within the uterine wall 

Epiblast and hypoblast 

• The inner cell mass of mature blastocyst = ESCs + primitive endoderm (aka hypoblast) 

• ESCs are pluripotent and give rise to all call types in the mature organism ‘

Primitive endoderm / hypoblast 

• Cells positioned on the surface of the inner cell mass following blastocyst development, differentiate to become primitive endoderm (aka hypoblast) 

• They are formed prior to implantation 

• Cytoplasm looks darker - more RER for protein production 

Function 

• Deposit basement membrane at basal surface (thin sheet of membrane matrix; important for compartmentalisation etc

• Give rise to some extra-embryonic tissues (do not form part of the embryo); important for supporting growth of embryo 

• Secrete signalling molecules that regulate the fate of the epiblast cells & regulate their development

• Epiblast cells when still in the embryo - when removed they are called ESCs (but same cell type)

Primitive endoderm differentiation 

Why do cells on the surface of the inner cell mass become primitive endoderm? 

FGFs

• Important role in development 

• Secreted from a cell and bind to receptors on the same cell or a different cell type 

• Cytoplasmic domain of the cell becomes phosphorylated when receptor binds 

• Primitive endoderm differentiation depends on fibroblast growth factors (FGF) binding to their receptors (FGFRs) on the surface of the primitive endoderm cells 

• This interaction leads to the activation of several intracellular signalling pathways 

• The signalling causes some genes to be turned on, and others to be turned off 

• This causes the cells to differentiate 

How does FGF signalling cause the surface cells to differentiate? 

• FGF signalling in the surface cells leads to expression of a transcription factor called Gata6 

• Gata6 is required for primitive endoderm differentiation 

• It suppresses expression of Oct-4 and other pluripotency genes 

• Turns on genes that are needed to make basement membranes - includes collagen and laminin 

Gene expression in the primitive endoderm 

• Oct4 - a transcription factor required to prevent premature differentiation of ESCs and maintain pluripotency 

• GATA6 - a transcription factor required for differentiation of primitive endoderm 

Oct4 = blue 

Gata6 = red 

The peri-implantation stage of mouse development 

Primitive endoderm 

Primitive endoderm → parietal and visceral endoderm 

• These call types are all known as ‘extraembryonic endoderm’ because they do not form part of the embryo proper

• Visceral - become more columnar 

Parietal endoderm 

• Squamous epithelial - flat

• Extensive RER - to excrete lots of extracellular membrane proteins 

Function 

• Secrete vast quantities of extracellular matrix proteins, which are incorporated into a specialised basement membrane called Reichert’s membrane (only in rodents)

Role of Reichert’s membrane 

• Prevents maternal cells entering parietal yolk sac and destroying the embryo

• In embryos without this membrane - the embryo dies 

What drives parietal endoderm differentiation?

• Factors derived from the TE induce primitive endoderm cells to differentiate into parietal endoderm 

• A key molecule is PTHrP (parathyroid hormone related peptide) that is expressed by TE but not by ESCs/epiblast cells 

• Acts on primitive endoderm cell and stimulates them to differentiate to visceral cells 

Visceral endoderm 

• Huge apical vacuoles 

• Very abundant microvilli 

Function: 

• Provides essential nutrients for the growing embryo 

• Supports growth of embryo before formation of placenta 

• Secretes signalling molecules that influence the differentiation of epiblast cells; required for correct patterning of the embryo

What drives visceral endoderm differentiation? 

• Factors derived from the embryonic stem cells / epiblast cells induce primitive endoderm cells to differentiate to visceral endoderm 

• BMPs (bone morphogenetic proteins) secreted by the embryonic stem cells play an important role in visceral endoderm differentiation

Polarisation of epiblast to form primitive ectoderm 

• Epiblast cells (round) now became polarised to form an epithelial (pseudostratified epithelium) - looks multilayer but is actually only single - due to nuclei laying at different levels 

What induces the cells to polarise? 

• Signals from basement membrane induce polarisation of epiblast 

• Immunostaining to show where laminin is situated