Hypospadias is a congenital malformation of the external genitalia that is characterized by a failure of urethral tube closure and incomplete formation of the prepuce and ventral penis. In the US the incidence of hypospadias approximately doubled, without explanation, between 1968 and 1993 and now affects 1 in 250 live male births. Paradoxically, external genital development is only beginning to be studied with tools of modern molecular genetics. External genital development begins with formation of the genital tubercle (GT). Two distinct phases of genital development occur in mammals; the first involves the initial outgrowth and patterning of the genital tubercle in both male and female embryos. The second phase involves either continued growth and differentiation of the penis, or arrest of outgrowth and differentiation of the clitoris. In all species studied to date, this second phase of male phallic development requires embryonic exposure to an androgen, either testosterone or dihydrotestosterone (DHT). Disruption of androgen signaling can induce hypospadias both in mammals and in alligators. As in humans, the PIs have identified widespread hypospadias and microphallus in natural populations of alligators. They recently found that disruption of the Fgfr2iiib pathway results in hypospadias in mice. Their preliminary data suggest that Fgfr2iiib expression in the GT is positively regulated by the Androgen Receptor (AR). This identifies a critical link between sex steroids and the genetic circuitry that regulates genital patterning. In this proposal, the PIs intend to investigate whether androgens interact with the Fgfr2 pathway in external genitalia of mice and alligators. Their aims include (1) To determine whether the spatio-temporal expression patterns and quantitative levels of Fgfr2iiib, Fgf10, AR and ER are conserved in mouse and alligator genital tubercles; (2) To determine whether androgen and estrogen differentially regulate Fgfr2 expression in mouse and alligator genital tubercles; and (3) To determine whether the Fgf10-Fgfr2 pathway is altered in natural populations of alligators exposed to endocrine active compounds. This will test directly the extent to which genital developmental mechanisms are conserved across species.