Role of DAX1 in Testis Determination and Function

描述（由申请人提供）：在过去的8年中，我们鉴定并表征了孤儿核受体DAX1和SF 1中天然发生突变的患者，并证明DAX1部分通过抑制SF 1介导的转录发挥作用。使用靶向突变，我们开发了一种小鼠Dax1基因敲除（KO）模型，并确定了Dax1在性腺决定，睾丸发育和成年睾丸功能中的作用。在这项资助中，我们建议扩展这些研究，这些研究为性腺发育机制提供了意想不到的见解。我们提出了3个相互关联的目标，专注于Dax1的结构和功能作为一个转录抑制因子，确定由Dax1调控的遗传途径，并开发动物模型，以解开Dax1与其他基因参与睾丸发育和功能的相互作用。具体而言，在目标1中，我们将确定介导DAX1转录抑制的分子伴侣。将鉴定和表征天然存在的DAX1突变，以阐明体内DAX1功能所需的关键结构域。将进一步表征在胚胎性腺文库的酵母双杂交筛选中鉴定的候选蛋白。目的2的目标是确定Dax1调控的遗传途径，使用微阵列分析的基因表达在野生型与Dax1缺陷的胚胎性腺在12 dpc，一个时间点时，Dax1的表达在男性和女性的分歧。目的3：探讨Dax 1与其他遗传通路的相互作用。使用缺乏Dax 1的小鼠，我们将探索在各种细胞类型中选择性拯救Dax 1的小鼠的性腺表型。此外，Dax 1缺陷小鼠将与其他品系杂交，这些品系的遗传途径发生了改变，这些改变被认为与Dax 1相交（例如，Sf1、Sry、Ptc）。这些目标的成功应该提供DAX1和转录抑制途径之间的关键联系，这些途径连接了性腺发育中涉及的各种其他转录因子。

DESCRIPTION (provided by applicant): Over the last 8 years, we identified and characterized patients with naturally occurring mutations in the orphan nuclear receptors, DAX1 and SF1, and demonstrated that DAX1 acts in part by inhibiting SF1- mediated transcription. Using targeted mutagenesis, we developed a murine Dax1 knockout (KO) model and identified roles for Dax1 in gonadal determination, testis development, and adult testis function. In this grant, we propose to extend these studies, which have provided unexpected insight into mechanisms of gonadal development. We propose 3 inter-related aims that focus on Dax1 structure and function as a transcriptional repressor, identify genetic pathways regulated by Dax1, and develop animal models to unravel the interplay of Dax1 with other genes involved in testis development and function. Specifically, in Aim 1 we will identify molecular partners that mediate DAX1 transcriptional repression. Naturally occurring DAX1 mutations will be identified and characterized to elucidate key structural domains required for DAX1 function in vivo. Candidate proteins identified in a yeast two-hybrid screen of an embryonic gonadal library will be further characterized. The goal of Aim 2 is to identify the genetic pathways regulated by Dax1 using microarray analyses of genes expressed in wild type versus Dax1-deficient embryonic gonads at 12 dpc, a timepoint when Dax1 expression diverges in males and females. Aim 3 is designed to explore the interaction of Dax1 with other genetic pathways in vivo. Using mice that lack Dax1, we will explore the gonadal phenotypes of mice with selective rescue of Dax1 in various cell types. In addition, Dax1-deficient mice will be crossed to other strains with alterations in genetic pathways proposed to intersect with Dax1 (e.g., Sf1, Sry, Ptc). Success in these aims should provide a critical link between DAX1 and transcriptional repression pathways that link a variety of other transcription factors involved in gonadal development.