Role of DAX1 in Testis Determination and Function

超出所提供的空间。 在过去的8年中，我们鉴定和表征了在孤儿核受体DAX1和SF 1中具有天然发生的突变的患者，并证明DAX1部分地通过抑制SF 1介导的转录起作用。使用靶向诱变，我们开发了一种小鼠Daxl基因敲除（KO）模型，并确定了Daxl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Daxl的结构和功能作为一个转录抑制因子，确定由Daxl调控的遗传途径，并开发动物模型，以解开Daxl与其他基因参与睾丸发育和功能的相互作用。具体地，在目标1中，我们将鉴定介导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的目标是使用在12 dpc时野生型与Daxl缺陷的胚胎性腺中表达的基因的微阵列分析来鉴定由Daxl调节的遗传途径，所述12 dpc是Daxl表达在男性和女性中发散的时间点。目的3是探讨Daxl与其他遗传通路的体内相互作用。使用缺乏Daxl的小鼠，我们将探索在各种细胞类型中选择性拯救Daxl的小鼠的性腺表型。此外，Daxl缺陷型小鼠将与其他品系杂交，这些品系具有与Daxl交叉的遗传途径的改变（例如，Sfl、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。性能现场=

EXCEED THE SPACE PROVIDED. Over the last 8 years, we identified and characterized patients with naturally occurring mutations in the orphan nuclear receptors, DAXl and SF1, and demonstrated that DAXl acts in part by inhibiting SF1- mediated transcription. Using targeted mutagenesis, we developed a murine Daxl knockout (KO) model and identified roles for Daxl 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 Daxl structure and function as a transcriptional represser, identify genetic pathways regulated by Daxl, and develop animal models to unravel the interplay of Daxl with other genes involved in testis development and function. Specifically, in Aim 1 we will identify molecular partners that mediate DAXl 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 Daxl using microarray analyses of genes expressed in wild type versus Daxl-deficient embryonic gonads at 12 dpc, a timepoint when Daxl expression diverges in males and females. Aim 3 is designed to explore the interaction of Daxl with other genetic pathways in vivo. Using mice that lack Daxl, we will explore the gonadal phenotypes of mice with selective rescue of Daxl in various cell types. In addition, Daxl-deficient mice will be crossed to other strains with alterations in genetic pathways proposed to intersect with Daxl (e.g., Sfl, 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. PERFORMANCE SITE ========================================Section End===========================================