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.
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