Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes.
The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable.
The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers.
Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which
normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.
|Date of Award||24 Jun 2014|
|Supervisor||Andrew Chalmers (Supervisor) & Melanie Welham (Supervisor)|
- stem cells
- preimplantation embryo
- epithelial polarity