Investigating the role of FOXL2 during urethra closure - Project Summary Hypospadias is one of the most common birth defects in the world, affecting nearly 1% of newborn boys. Hypospadias is abnormal urethra closure, where the urethra exits ventrally along the shaft of the penis, and not the tip. Although hypospadias is common, the origin of 70% of urethral closure defects remains unknown. During urethra closure there are extensive changes in the extracellular matrix composition. Prior to urethra closure, basement membrane surrounding the urethral epithelium is one continuous sheet. As the urethra closes, the basement membrane becomes fragmented. This allows the surrounding mesenchymal cells to invade and support urethra closure. This process is dependent on normal androgen signaling. In the absence of testis-derived testosterone, there is no basement membrane breakdown, no mesenchymal invasion, and the urethra remains open along the ventral aspect of the penis. Surprisingly, the genetic regulation of extracellular matrix modification and the diversity of cell populations involved in urethral closure are not well understood. Recently, my lab discovered a unique cell population in the penis that expresses the gene, Forkhead Box L2 (Foxl2). These cells are located immediately next to the urethral epithelium, express androgen receptor and enzymes that modify extracellular matrix, and are closely associated with urethra closure. My central hypothesis is that the Foxl2 gene, and the cell population that expresses FOXL2, are essential for urethra formation by remodeling the urethral epithelium and responding to androgens. Guided by strong preliminary data and pre-existing publications, we will address this hypothesis in three main aims. In aim 1, well define the role of Foxl2, the gene, in urethra closure. We will investigate the hypothesis that Foxl2 expression is essential for regulating genes required for urethra closure. Hypospadias defects will be investigated in Foxl2-/- mice and the molecular action of FOXL2 will be determined with transcription factor binding assays. Aim 2 will determine the role of FOXL2+ cells during urethra closure, by using a cell ablation mouse model. Aim 3 will reveal how androgen signaling influences FOXL2+ cell differentiation and urethral closure. To test the hypothesis that androgens are directly involved in FOXL2+ cell function and differentiation, we will use FOXL2 conditional androgen receptor knockouts, chromatin accessibility assay, and chromatin binding assays. Results from these studies will establish the role of the gene, Foxl2; the role of a novel group cells; and the role androgen signaling in FOXL2+ cells in penile urethra closure. Our lab has the conceptual and technical expertise to successfully complete the proposed aims. This attained knowledge will provide new avenues in understanding the origins of hypospadias within the human populations. With a more comprehensive understanding of urethra closure, we can begin to develop surgical alternatives, or preventatives for this common birth defects.
