Role of DAX1 in Testis Determination and Function

在过去的8年里，我们鉴定并描述了具有天然突变的患者， 孤儿核受体DAX 1和SF 1，并证明DAX 1部分通过抑制SF 1- 介导的转录使用靶向诱变，我们开发了鼠Daxl敲除（KO）模型， Daxl在性腺决定、睾丸发育和成年睾丸功能中的作用。在这份补助金中， 我们建议扩展这些研究，这些研究为性腺激素的机制提供了意想不到的见解。 发展我们提出了3个相互关联的目标，重点是Daxl的结构和功能，作为一个转录 阻遏物，确定由Daxl调控的遗传途径，并开发动物模型来解开相互作用， Daxl与其他参与睾丸发育和功能的基因的同源性。具体而言，在目标1中，我们将确定 介导DAX 1转录抑制的分子伴侣。自然发生的DAX 1突变 将被鉴定和表征，以阐明DAX 1在体内功能所需的关键结构域。 在酵母双杂交筛选胚胎性腺文库中鉴定的候选蛋白质将进一步用于 表征了目的2是利用微阵列技术鉴定Daxl调控的遗传通路 在12 dpc，一个时间点，野生型与Daxl缺陷型胚胎性腺中表达的基因分析 当Daxl表达在男性和女性中分化时。目标3旨在探索以下因素之间的相互作用： Daxl与体内其他遗传途径。使用缺乏Daxl的小鼠，我们将探索性腺 在各种细胞类型中选择性拯救Daxl的小鼠的表型。此外，Daxl缺陷小鼠将 可以与其他具有与Daxl交叉的遗传途径改变的菌株杂交（例如，SFL， Sry，Ptc）。这些目标的成功应该提供DAX 1和转录抑制之间的关键联系 与性腺发育相关的多种其他转录因子的连接途径。

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.