REGULATORY ROLE OF RA GENE NETWORKS IN HUMAN FETAL SKIN

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Hormones and growth factors are involved in generating and maintaining cell- and tissue-specific patterns of gene expression during development. Retinoic acid (RA) plays a central role in the maintenance of epithelia, including the epidermis. We propose that RA also functions earlier in skin development by participating in the establishment of keratinocyte lineage through regulation of gene expression during. We hypothesize that RA, acting through its receptors, initiates developmentally-specific gene networks in human fetal skin. Further, the activities of these genes may be modulated by interaction with other cellular factors, such as transforming growth factor beta (TGFbeta). The proposed studies are designed to determine the regulatory roles of genes which belong to the RA gene network and to characterize the functional consequences of their expression in developing human skin. Such functional analysis requires a complex cellular microenvironment, the human fetal skin equivalent (SE), for determining physiologic relevance. In studies detailed in Specific Aim #1, the SE, containing both fetal epidermal keratinocytes and fetal dermal fibroblasts, will serve as a culture model in which to experimentally test the effects of RA and TGFbeta in a fetal skin environment and to determine which receptors and binding proteins mediate their activities. Specific Aim #2 is designed to identify genes, other than the RA receptors, which belong to a fetal keratinocyte RA network. We will characterize genes present in subtractive libraries, enriched for sequences induced early during the response of fetal keratinocytes to RA, to identify genes with regulatory function. Fetal keratinocyte-specific sequences directly regulated by RA will be identified by the presence of the consensus response elements. Finally, the focus of Specific Aim #3 is assignment of function to genes in the RA network. This will be accomplished by demonstration of RA-regulated and cell-type specific gene activity in transient expression assays, stable expression in the fetal SE with subsequent modulation of the morphogenesis and differentiation of human fetal skin cells, as well as characterization of expression during normal human fetal skin development.