Role of Ovol Genes in Epidermal Development

描述(申请人提供)：皮肤表皮由多能外胚层衍生而来。在外胚层细胞成为毛囊间表皮的过程中，它至少面临着三个关键决定：1)不成为神经细胞，2)不成为附属物细胞，3)自我更新(像干细胞或祖细胞通常所做的那样)，或者停止增殖，最终分化为高度特化的细胞，对有机体的生存具有必要的保护功能。这些选择是如何做出的？是什么基因或遗传途径控制或调节了这些选择？如果这些基因/途径出错了，会发生什么？我的研究的长期目标是用多学科的方法来解决这些重要的问题，这些问题不仅将影响对皮肤干细胞增殖和分化的理解，而且还将影响对一般上皮发育和分化机制的理解。这项建议侧重于研究编码锌指转录因子的两个小鼠Ovo基因Ovol1和Ovol2在表皮特化、增殖和分化中的作用。主要目的有三：1)研究Ovol2在胚胎干细胞增殖、外胚层分化和表皮特化中的作用。我们将产生Ovol2缺陷的ES细胞，并在体外分析它们的增殖和存活。我们将采用一种允许ES细胞分化为表皮细胞的体外培养系统，并监测野生型和Ovol2缺陷的ES细胞的表皮分化。我们将进行嵌合小鼠分析，以检验Ovol2缺陷的ES细胞在促进皮肤表皮方面的内在能力。2)研究Ovol2在表皮增殖和分化中的作用。我们将通过建立和分析表皮特异的Ovol2基因敲除小鼠，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，来研究Ovol2在表皮中的功能。3)探讨Ovol1在基因表达和生长停滞中的作用机制。我们将进行生化研究来验证Ovol1蛋白通过与c-Myb竞争结合并通过招募HDAC到其启动子从而促进生长停滞而直接抑制c-Myc和Id2表达的假设。此外，我们将使用转基因“显性阳性”的方法来进一步研究Ovoll在体内对发育中的表皮增殖/分化的调节作用。

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.