REGULATORY ROLE OF RA GENE NETWORKS IN HUMAN FETAL SKIN

激素和生长因子参与生成和维持 细胞和组织的特定模式的基因表达在发展过程中。 视黄酸（RA）在上皮细胞的维持中起核心作用， 包括表皮。 我们建议RA也在早期发挥作用， 通过参与角质形成细胞的建立来促进皮肤发育 通过调控基因表达的谱系。 我们假设 RA通过其受体启动发育特异性 人类胎儿皮肤中的基因网络。 此外，这些活动 基因可以通过与其他细胞因子的相互作用来调节， 转化生长因子β（TGF β）。 拟议的研究是 旨在确定基因的调控作用，这些基因属于 RA基因网络，并表征其功能后果 在发育中的人类皮肤中表达。 这种功能分析需要一个 复杂的细胞微环境，人胎儿皮肤等效物（SE）， 用于确定生理相关性。 在具体的研究中， 目的#1，SE，含有胎儿表皮角质形成细胞和胎儿表皮角质形成细胞两者， 真皮成纤维细胞将作为培养模型， 实验测试RA和TGF β在胎儿皮肤中的作用 并确定哪些受体和结合蛋白介导 他们的活动。 具体目标#2旨在识别基因，其他 而RA受体属于胎儿角化细胞RA网络。 我们将表征消减文库中存在的基因， 在胎儿角质形成细胞对RA的反应早期诱导的序列， 来鉴定具有调节功能的基因。 胎儿角质形成细胞特异性 由RA直接调节的序列将通过以下物质的存在来鉴定： 共识响应元素。 最后，具体目标#3 是RA网络中基因的功能分配。 这将是 通过证明RA调节和细胞类型特异性基因来完成 在瞬时表达测定中的活性，在胎儿中的稳定表达 SE与随后的形态发生和分化的调制 人胎儿皮肤细胞，以及表征表达过程中， 正常的人类胎儿皮肤发育。

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.