REGULATORY ROLE OF RA GENE NETWORKS IN FETAL SKIN

荷尔蒙和生长因子参与生成和维持 发育过程中特定细胞和组织的基因表达模式。 维甲酸(RA)在维持上皮细胞中起着核心作用。 包括表皮。我们建议RA在早期也起作用 皮肤发育通过参与角质形成细胞的建立 血统是通过基因表达的调控来实现的。我们假设 RA通过其受体作用，启动发育特异性 人类胎儿皮肤中的基因网络。此外，这些组织的活动 基因可能通过与其他细胞因子的相互作用来调节，如 即转化生长因子β(TGFbeta)。建议进行的研究包括 被设计用来确定属于 RA基因网络及其功能结果的特征 在人类皮肤发育过程中的表达。这样的功能分析需要 复杂的细胞微环境，相当于人类胎儿皮肤(SE)， 用于确定生理相关性。在具体的研究中 目的#1，SE，包含胎儿表皮角质形成细胞和胎儿 皮肤成纤维细胞，将作为一种培养模型，在其中 维甲酸和转化生长因子β在胎儿皮肤中的实验研究 并确定哪些受体和结合蛋白介导 他们的活动。特定目标2旨在识别基因，其他 而不是属于胎儿角质形成细胞RA网络的RA受体。 我们将对存在于消减文库中的基因进行表征，为 在胎儿角质形成细胞对RA的反应早期诱导的序列， 鉴定具有调控功能的基因。胎儿角质形成细胞特异性 由RA直接调控的序列将通过存在 协商一致的回应要素。最后，具体目标3的重点 是将功能分配给RA网络中的基因。这将是 通过展示RA调节的和细胞类型特异性基因来实现 在瞬时表达分析中的活性，在胎儿中稳定表达 Se与随后的形态发生和分化的调节 人胎儿皮肤细胞，以及在 人胎儿皮肤发育正常。

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.