Role of Ovol Genes in Epidermal Development

描述（由申请人提供）：皮肤表皮来源于多能性外胚层。在外胚层细胞成为滤泡间表皮的过程中，它面临着至少三个关键的决定：1)不成为神经细胞，2)不成为附属细胞，3)自我更新（就像干细胞或祖细胞通常做的那样），或者停止增殖，最终分化成一个高度特化的细胞，对生物体的生存具有重要的保护功能。这些选择是如何做出的？哪些基因或遗传途径控制或调节这些选择？如果这些基因/途径出错会发生什么？我研究的长期目标是使用多学科方法来解决这些重要问题，这些问题不仅会影响对皮肤干细胞增殖和分化的理解，还会影响对上皮发育和分化机制的理解。本研究的重点是研究编码锌指转录因子的两个小鼠卵形基因Ovol1和Ovol2在表皮规范、增殖和分化中的作用。有三个具体目的：1)表征Ovol2在胚胎干（ES）细胞增殖、外胚层分化和表皮形成中的作用。我们将产生缺乏Ovol2的胚胎干细胞，并分析其体外增殖和存活。我们将采用能使胚胎干细胞向表皮细胞分化的体外培养系统，监测野生型和ovol2缺失型胚胎干细胞的表皮分化情况。我们将进行嵌合小鼠分析，以检验缺乏ovol2的胚胎干细胞对皮肤表皮的内在能力。2)表征Ovol2在表皮增殖和分化中的作用。我们将通过产生和分析表皮特异性Ovol2敲除小鼠来研究Ovol2在表皮中的功能，特别是验证Ovol2是表皮干细胞/祖细胞自我更新和/或增殖所必需的假设。3)研究Ovol1在基因表达和生长阻滞中的作用机制。我们将进行生化研究来验证Ovol1蛋白直接抑制c-Myc和Id2表达的假设，通过与c-Myb竞争结合，并通过将hdac招募到其启动子，从而促进生长停滞。此外，我们将采用转基因“显性阳性”方法来进一步研究Ovoll在体内调节发育中的表皮增殖/分化中的作用。

DESCRIPTION (provided by applicant): Skin epidermis is derived from the pluripotent ectoderm. In an ectodermal cell's path to become interfollicular epidermis, it faces at least three critical decisions: 1) not to become a neural cell, 2) not to become an appendage cell, 3) to self-renew (as a stem or progenitor cell normally does), or to cease proliferation and terminally differentiate into a highly specialized cell that carries an essential protective function for the organism's survival. How are these choices made? What genes or genetic pathways govern or modulate these choices? What happens if these genes/pathways go awry? The long-term goal of my research is to use a multidisciplinary approach to address these important questions that will impact the understanding of not only stem cell proliferation and differentiation in skin, but also the mechanisms of epithelial development and differentiation in general. This proposal focuses on addressing the role of two mouse Ovo genes, Ovol1 and Ovol2, encoding zinc finger transcription factors, in epidermal specification, proliferation, and differentiation. There are three specific aims: 1) Characterize the role of Ovol2 in embryonic stem (ES) cell proliferation, ectodermal differentiation, and epidermal specification. We will generate ES cells deficient in Ovol2, and analyze their proliferation and survival in vitro. We will adapt an in vitro culture system that allows the differentiation of ES cells into epidermal cells, and monitor epidermal differentiation of wild-type and Ovol2-deficient ES cells. We will perform chimeric mouse analysis to examine the intrinsic ability of Ovol2-deficient ES cells in contributing to the skin epidermis. 2) Characterize the role of Ovol2 in epidermal proliferation and differentiation. We will examine the function of Ovol2 in epidermis through the generation and analysis of epidermis-specific Ovol2 knockout mice, particularly testing the hypothesis that Ovol2 is required for self- renewal and/or proliferation of epidermal stem/progenitor cells. 3) Investigate the mechanism of Ovol1 function in gene expression and growth arrest. We will perform biochemical studies to test the hypothesis that Ovol1 protein directly represses c-Myc and Id2 expression by competing with c-Myb for binding and by recruiting HDACs to their promoters, thereby facilitating growth arrest. Furthermore, we will employ a transgenic "dominant positive" approach to further address the in vivo role of Ovoll in the regulation of proliferation/differentiation in developing epidermis.