GLI3 resides at the intersection of hedgehog and androgen action to promote male sex differentiation

Urogenital tract abnormalities are among the most common congenital defects in humans. Male urogenital development requires Hedgehog-GLI signaling and testicular hormones, but how these pathways interact is unclear. We found that Gli3(XtJ) mutant mice exhibit cryptorchidism and hypospadias due to local effects of GLI3 loss and systemic effects of testicular hormone deficiency. Fetal Leydig cells, the sole source of these hormones in developing testis, were reduced in numbers in Gli3(XtJ) testes, and their functional identity diminished over time. Androgen supplementation partially rescued testicular descent but not hypospadias in Gli3(XtJ) mutants, decoupling local effects of GLI3 loss from systemic effects of androgen insufficiency. Reintroduction of GLI3 activator (GLI3A) into Gli3(XtJ) testes restored expression of Hedgehog pathway and steroidogenic genes. Together, our results show a novel function for the activated form of GLI3 that translates Hedgehog signals to reinforce fetal Leydig cell identity and stimulate timely INSL3 and testosterone synthesis in the developing testis. In turn, exquisite timing and concentrations of testosterone are required to work alongside local GLI3 activity to control development of a functionally integrated male urogenital tract.