DESCRIPTION (Taken from the applicant's abstract): The long-term objective is to identify markers whose modulation results in epithelial differentiation. This objective has a direct impact on the application of epithelial grafts as therapy for full thickness skin injury and as a delivery system for specific gene products. Connexin-negative cells are found in the basal cell layers that harbor stem cells. Connexin expression or lack thereof will be correlated with markers thought to be indicative of stem cells. For correlation with label retaining cells, which are putative stem cells, thymidine-labeled mouse skin will be used. For correlation with K19 expressing follicular cells, also purported to be stem cells, human skin will be utilized. To accomplish this aim, FACS analysis and immunohistochemistry combined with fluorescent and confocal microscopy will be employed. The functionality of gap junctions within both mouse skin and human hair follicles will also be evaluated. Cell populations able to carry out dye transfer will be compared to those populations containing label retaining cells in mouse skin, and K19 expressing cells in human hair follicles. These experiments will further indicate whether stem cells lack gap junction-mediated cell-to-cell communication. The connexin-negative cells of the epidermis will be isolated and compared in culture with other cells of the basal layer. For these experiments, antibodies against the extracellular domain of connexin molecule will be used as a negative selection factor in isolating cells by FACS. Individual cell populations will be assayed for culture life-span, and colony forming efficiency at each passage. Finally, retroviral vectors will be used to introduce both Cx43 gene and antisense oligonucleotides into keratinocytes. The effects will be assessed in submerged cultures, organotypic cultures and on organotypic cultures grafted onto athymic mice. The proposed research plan, as well as the activities described in the application, are aimed at achieving the career goals.