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Wnt4信号在哺乳动物雌性性别决定中的分子机制。Wnt4与部分性逆转有关。Wnt4基因重复的男性患者表现出从隐睾到XY性反转的性表型。携带Wnt4突变的女性患者缺乏女性生殖道结构。显然，确定Wnt4的作用不仅会增加我们对性别决定的基本理解，而且还会改善对人类性疾病的管理。迄今为止，仅鉴定出两个可能的Wnt4靶基因，并且尚未完全表征。特异性Aim 1基于体外数据验证了Wnt4通过一种新的信号通路起作用的假设，该数据显示在Wnt4刺激后β -连环蛋白重新定位到细胞质膜上。我们将利用原位杂交和免疫组织化学（IHC）和新发现的细胞标记物，确定Wnt4在发育中的卵巢中的详细表达谱；我们将在野生型和Wnt4突变型胚胎中研究β -连环蛋白（Wnt4功能的中介）的定位；最后用Western blot法检测β -catenin的磷酸化状态。特异性目的2验证了Wnt4调控两个基因，血管运动素和小血管蛋白-1的假设，这两个基因都参与了Wnt4表型的过程：细胞迁移和血管生成。这一目标将通过使用转染wnt4的细胞来检测靶基因表达、细胞聚集、迁移和小管形成；利用免疫组化技术评估Wnt4野生型和突变型胚中靶基因的表达；并通过检测小泡蛋白-1缺失性腺在发育过程中的表型。

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.