Abstract
The primitive endoderm 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. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. 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. Fgf receptor/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 Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.
Highlights
A key cell fate decision in early mammalian development occurs when cells in the inner cell mass (ICM) decide to follow either the primitive endoderm or epiblast cell fate [1]
Control embryoid bodies were formed from media containing the same DMSO percentage as used with the inhibitors, this ranged from 0.02% to 0.08% (See figure legends for the concentration used in each experiment). 25 ml drops of the cell suspension were pipetted onto the inside of the lid of a 10 cm tissue culture dish. 10 mls of phosphate-buffered saline (PBS) (Invitrogen) was placed in the bottom of the plate
Increasing expression of the primitive endoderm fate markers and loss of the pluripotency marker Nanog suggests that the outer layer of the embryoid bodies is undergoing a developmental program similar to that observed in the embryonic primitive endoderm
Summary
A key cell fate decision in early mammalian development occurs when cells in the inner cell mass (ICM) decide to follow either the primitive endoderm or epiblast cell fate [1]. There has been a rapid increase in our understanding of how the primitive endoderm versus epiblast cell fate decision is regulated (Reviewed in [2,3]). These studies have used mouse embryos, as well as embryoid bodies which can be formed from mouse embryonic stem (mES) cells. The outer cell layer of embryoid bodies is an epithelium with many similarities to the embryonic primitive endoderm, and has been used as a model of its development [4,5]
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