The proper development of skin during embryogenesis, and its maintenance throughout life are essential for the protective and mechanical functions it serves. A variety of birth defects including blistering and keratinization disorders and dysplasias, are manifested as skin malformations. Undoubtedly many more epidermal abnormalities are lethal to the developing embryo. Defining the elements involved in the morphogenesis of a stratified epithelium will enable us to better understand how normal functions are perturbed and perhaps increase our ability to diagnose and ultimately prevent such defects. The goal of this proposal is to investigate the structure and expression of two human desmosomal proteins involved in the development and differentiation of skin. Desmoplakins (DPs) I and II are closely related molecules localized in the cytoplasmic plaque of the desmosome, a junction thought to play a crucial role in cell-cell adhesion. Desmosomes also appear to act as specific attachment sites for intermediate filaments (IF). Because of their extreme insolubility their structure and sequence have not been determined and it has been difficult to define their functional relationships to other desmosomal components and IF. DPI appears to be present in all desmosomes, whereas DPII appears to be present only in stratified tissues. Understanding the relationship between DPII expression and stratification has been hampered by the lack of a DPII specific probe. Generation of such a probe is a primary goal of this research. I have isolated several cDNA clones from human foreskin and bovine tongue lambda gt11 libraries. My results suggest that in bovine tongue, there are two messages for the DPs, and these messages are transcribed from a single gene. This possibility will be tested for the human system by Northern and Southern hybridization experiments. The structure of the human DP cDNAs (mRNAs) will be determined by restriction mapping, cross- hybridization experiments and DNA sequence analysis. This may allow identification of sequences which could serve as a DPII specific probe. Genomic clones will also be isolated and the initial characterization of gene structure and flanking regions will be carried out to identify possible mechanisms of transcriptional and post-transcriptional regulation. The spatial distribution and expression of the DPs at the message and protein levels will be studied in human embryonic, fetal and adult skin by in situ hybridization, Northern analysis, immunolocalization and immunoblotting to identify differences in DPI and DPII expression. Finally, the expression of DPI and II will be studied during differentiation and stratification in cultured human keratinocytes. This work will surely increase our understanding of the elements involved in the successful morphogenesis and differentiation of human skin.
