Hypospadias is one of the most common birth defects in the world affecting nearly 1% of newborn boys. Hypospadias is the result of disrupted urethral closure where the urethra exits ventrally along the shaft of the penis. The etiology of 70% of urethral closure defects remains unexplained. Androgen signaling in the penis drives cell-to-cell communication between the urethra and the adjacent mesenchyme, which then influences closure of the urethra. Normal penis development requires testosterone. In the absence of testosterone produced by the testis, the urethra fails to close, leading to severe hypospadias. The testis-derived testosterone eventually reaches the presumptive penis, and is metabolized into the more potent dihydrotestosterone by 5-α reductase. Dihydrotestosterone then binds androgen receptor that are localized within the penis, which transcribes a suite of genes that induce penis development. Surprisingly the regulation of 5-α reductase and the diversity of cell populations involved in urethral closure are not well understood. Recently, I discovered a unique cell population in the penis that expresses the steroidogenic enzyme master regulator, Steroidogenic Factor 1 (Sf1). These cells are localized in the ventral-proximal aspect of the penis, express both androgen receptor and 5-α reductase, and appear to be required for urethral closure. My central hypothesis is that these SF1+ cells contribute to proper urethral closure by both producing and responding to androgens. In the two phases of my K99/R00, I will investigate the role of SF1+ cells in penis development and urethral closure. In the K99 phase, I will define the role of SF1+ cells and Sf1 the gene in urethral closure. I will use a combination of the Diptheria toxin, cell ablation mouse model and a penis specific Sf1 gene knockout mouse model. I hypothesize that these cells are essential for urethral closure and are responsible for the penises steroidogenic capacity to convert testosterone to dihydrotestosterone. In the R00 phase, I will reveal how androgen signaling influences SF1+ cell differentiation and urethral closure. Using SF1+ cell conditional androgen receptor knockouts and chromatin binding assays, I will test the hypothesis that androgens are directly involved in SF1+ cell function and differentiation. The results from this study will establish the role of a novel cell population, the function of the gene, Sf1, and androgen responsiveness in penis urethral closure. This new knowledge will provide a better understanding of the mechanisms involved in the occurrence of hypospadias and will further human investigations on the etiology of hypospadias. Ultimately if there is a more comprehensive understanding of penis development, we can begin to develop strategies to prevent this defect that impacts millions of boys. With the technical skills I already have, the data generated in this proposal, and the skills I will acquire during the fellowship, I will be well-poised to ...