Role of DAX1 in Testis Determination and Function

在过去的8年里，我们鉴定和描述了自然发生的基因突变的患者 孤儿核受体DAX1和SF1，并证明DAX1部分通过抑制SF1-1发挥作用。 中介转录。利用定向突变，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、睾丸发育和成人睾丸功能中的作用。在这笔赠款中， 我们建议扩展这些研究，这些研究为性腺的机制提供了意想不到的见解。 发展。我们提出了三个相互关联的目标，重点是Daxl的结构和作为转录因子的功能 抑制，确定受Daxl调控的遗传途径，并开发动物模型来解开相互作用 Daxl基因与其他与睾丸发育和功能有关的基因。具体来说，在目标1中，我们将确定 介导DAX1转录抑制的分子伙伴。自然发生的DAX1突变 将被鉴定和表征，以阐明体内DAX1功能所需的关键结构域。 在胚胎性腺文库的酵母双杂交筛选中发现的候选蛋白将进一步 特色化的。目标2的目标是使用微阵列识别受Dax1调控的遗传途径 12DPC时野生型与Daxl缺陷型胚胎性腺基因表达的分析 当Daxl在男性和女性中的表达分化时。Aim 3旨在探索 Daxl与体内的其他遗传途径。使用缺乏Daxl的小鼠，我们将探索性腺 不同细胞类型Daxl选择性拯救小鼠的表型。此外，Daxl基因缺陷的小鼠将 与遗传途径发生改变的其他菌株杂交，建议与Dax1交叉(例如，SF1， SRY、PTC)。这些目标的成功应该在DAX1和转录抑制之间提供关键的联系 连接多种其他参与性腺发育的转录因子的途径。

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.