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.

泌尿生殖道异常是人类最常见的先天性缺陷。男性泌尿生殖系统发育需要Hedgehog-GLI信号传导和睾丸激素，但这些途径如何相互作用尚不清楚。我们发现Gli3 ^XtJ突变小鼠表现出隐睾症和尿道下裂，这是由于GLI3丢失的局部作用和睾丸激素缺乏的全身作用所致。胎儿睾丸间质细胞是发育中的睾丸中这些激素的唯一来源，在Gli3 ^XtJ睾丸中的数量减少，并且其功能同一性随时间而降低。补充雄激素可部分^缓解Gli3 ^XtJ的睾丸^后遗症，但不能^缓解尿道下裂突变体，将GLI3丢失的局部作用与雄激素不足的全身作用解耦。将GLI3激活剂（GLI3A）重新引入Gli3 ^XtJ睾丸可恢复刺猬通路和类固醇生成基因的表达。在一起，我们的结果表明激活形式的GLI3的新功能可以翻译刺猬信号，以增强胎儿Leydig细胞的身份，并在发育中的睾丸中及时刺激INSL3和睾丸激素的合成。反过来，需要精巧的时间和睾丸激素的浓度来与局部GLI3活性同时起作用，以控制功能性整合的男性泌尿生殖道的发育。