The specific aim of this study is to characterize at the molecular level the temporal expression of the intermediate filament keratins during fetal stratified squamous epithelial (SSE) differentiation. The long term objective of our research is to understand the functional role of keratins in epithelial cells and the effects of modulations in their gene expression on differentiation. The fetal-primary SSE differentiation presents a unique opportunity to observe several levels of keratin molecular regulation at the point of their initiation. Three SSE that are closely approximated during fetal development, yet have distinct patterns of differentiation will be examined. Cranial skin, tongue, and palate mucosa will be isolated from rat embryos of known gestational age and postnatal and adult animals. The keratins will be examined with respect to their protein synthesis, posttranslational modifications, gene transcription,and genomic organization. The programmed sequence of keratin protein synthesis during fetal development will be defind by SDN-PAGE, IEF and two dimensional gel electrophoresis. Keratin posttranslational modification will be examined by analyzing the incorporation of (32-P) orthophosphate or (3-H) glucosamine by SDS-PAGE, autoradiography and liquid scintillation counting. Modified keratins will be further analyzed by SDS-PAGE and HPLC peptide mapping following enzymatic proteolysis. The fragments with and without modification will be collected in a pure form by HPLC for both amino acid and antigenicity analyses. Keratin gene transcription will be examined by dot-blot and "northern" hybridizations with mRNA immobilized on membranes, in situ hybridizations with mRNA immobilized in tissue sections, and by in vitro translation of the isolated mRNA. 8 keratin cDNA probes, 2 type specific, and 6 gene specific will be used. The mRNA will be translated in a rabbit reticulocyte lysate and products resolved by SDS-PAGE. The genomic organization of the keratin genes in the epithelium and connective tissue at different stages of development will be examined by restriction digestion of the DNA, electrophoretic fractionation, transfer to nitrocellulose and hybridization with the keratin cDNAs. Hpa II and Msp I restriction digests will determine tissue-specific keratin gene methylation patterns. With these results we may begin to understand the functional role of keratins during fetal SSE differentiation and develop a new appreciation of the congenital effects resulting from a derangement in keratin gene expression.