The long term objective of this project is to elucidate the role of keratin and vimentin intermediate filaments (IFs) in the differentiation and development of simple epithelium. Although much is known about the gene arrangement, protein sequence and tissue specific expression of IFs, their cellular function remains unclear. The biochemical heterogeneity of the numerously described IF subunits suggests that IFs have functionally diverse roles in different specialized cells. The murine simple epithelial keratins Endo A and Endo B (mouse homologues of the human keratins K8 and K18, respectively) are expressed in all simple epithelia of the adult and are the first IFs expressed in early embryogenesis. Filaments are first observed in the 8-cell embryo and later in trophectoderm and parietal and visceral endoderm. Visceral endoderm cells form a compact, polarized epithelium surrounding the developing embryo and only display IFs composed of Endo A and Endo B while parietal endoderm cells are highly migratory in the developing embryo and appear to coexpress vimentin IFs. Although vimentin expression is normally restricted to cells in vitro and mesenchymal cell types, its expression is also correlated with the metastatic potential of some epithelial neoplasms. Because large numbers of early embryonic cell types are difficult to obtain, the HR9 parietal endoderm cell line and F9 embryonal carcinoma (EC) cells are useful models of early embryonic cell types. F9 Ec cells can be induced to differentiate to visceral or parietal endoderm in monolayer or aggregate cultures. Aggregate differentiation of F9 cells serves as an extremely useful three- dimensional model for visceral endoderm development. As a means of defining the function of keratin and vimentin IFs in these simple epithelial cell types, we propose to examine: 1) the biological and structural effects of IF disruption induced by the expression of mutant Endo Bor vimentin proteins in HR9 cells nad differentiating F9 cultures; 2) the effects of vimentin expression on the phenotype of compact visceral endoderm cells and 3) the molecular and biological aspects of F9 EC cell differentiation in the absence of Endo B protein in cells possessing site- directed mutagenized Endo B alleles. These molecular approaches to study biological behavior will enhance our understanding of simple epithelial cell structure and morphogenesis, early embryonic development, and potentially the metastatic phenotype of transformed epithelial cells.
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