Molecular Mechanisms of Wnt4 Action in Developing Gonad

描述（由申请人提供）：拟议研究的长期目标是了解Wnt 4信号在哺乳动物雌性性别决定中的分子机制。Wnt 4与部分性逆转有关。Wnt 4重复的男性患者表现出从隐睾到XY性反转的性表型。携带Wnt 4突变的女性患者缺乏女性生殖道的结构。显然，定义Wnt 4的作用不仅会增加我们对性别决定的基本理解，而且还会改善人类性病理学的管理。在该领域迄今为止，只有两个可能的Wnt 4靶基因已被确定，他们没有完全表征。特定目的1基于显示Wnt 4刺激后β-连环蛋白重新定位于细胞质膜的体外数据，检验了Wnt 4通过新型信号传导途径起作用的假设。我们将使用原位杂交和免疫组织化学（IHC）与新的可用的细胞标记物，确定详细的Wnt 4在卵巢发育中的表达谱;我们将检查β-连环蛋白的本地化-建议调解Wnt 4功能-在野生型和Wnt 4突变胚胎;最后，我们将通过Western印迹分析测定β-连环蛋白的磷酸化状态。特异性目的2测试了Wnt 4作用调节两个基因，血管动蛋白和小窝蛋白-1的假设，这两个基因都参与了Wnt 4表型中观察到的过程：细胞迁移和血管生成。这一目标将通过以下方式实现：使用Wnt 4转染的细胞检查靶基因表达、细胞聚集、迁移和小管形成;使用IHC评估Wnt 4野生型和突变胚胎中的靶基因表达;以及检查发育期间小窝蛋白-1缺失性腺的表型。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to understand the molecular mechanisms of Wnt4 signaling in mammalian female sex determination. Wnt4 is associated with partial sex reversal. Male patients with duplications in Wnt4 exhibit sexual phenotypes ranging from cryptorchidism to XY sex reversal. A female patient carrying a Wnt4 mutation lacks structures of the female reproductive tract. Clearly, defining Wnt4 action would not only add to our basic understanding of sex determination, but would also improve management of human sexual pathologies. In the field to date, only two possible Wnt4 target genes have been identified, and they are not fully characterized. Specific Aim 1 tests the hypothesis that Wnt4 acts through a novel signaling pathway based on in vitro data showing the relocalization of beta-catenin to the cytoplasmic membrane after Wnt4 stimulation. We will identify the detailed expression profile of Wnt4 in the developing ovary, using in situ hybridization and immunohistochemistry (IHC) with newly available cell markers; we will examine the localization of beta-catenin-the proposed mediator of Wnt4 function-in wild type and Wnt4 mutant embryos; finally we will assay the phosphorylation state of Beta-catenin by Western blot analysis. Specific Aim 2 tests the hypotheses that Wnt4 action regulates two genes, angiomotin and caveolin-1, which are both involved in processes seen in the Wnt4 phenotype: cell migration and angiogenesis. This aim will be accomplished using Wnt4-transfected cells to examine target gene expression, cell aggregation, migration and tubule formation; by assessing target gene expression in Wnt4 wild type and mutant embryos using IHC; and by examining ttie phenotype of caveolin-1 null gonads during development.