The basic helix–loop–helix transcription factor TCF4 recruits the Mediator complex to activate gonadal genes and drive ovarian development

The bipotential gonad is the precursor to the ovary and testis. In mice, gonad differentiation is initiated by commitment of presupporting cells into testicular Sertoli cells or ovarian granulosa cells. While Sry drives testis differentiation, upstream drivers of ovary fate are unclear. We identified binding sites for basic helix–loop–helix (bHLH) transcription factors (TFs) upstream of granulosa genes and sought to investigate whether bHLH TFs regulate granulosa specification. bHLH transcription factor 4 (TCF4) was expressed in presupporting cells before sex determination. As development progressed, TCF4 was maintained in granulosa cells but lost in Sertoli cells. In Tcf4 STOP/STOP mutant mice lacking the TCF4 DNA-binding domain, FOXL2 was reduced in granulosa cells and nuclear morphology was altered. Mutant ovaries failed to undergo morphological changes similar to wild-type littermates. Meanwhile, Tcf4 STOP/STOP mutant testes appeared normal. We found that TCF4 binds the Mediator complex to regulate expression of gonadal genes ( Wt1 , Nr2f2 ) and granulosa-enriched genes including Jun and Fos . In ex vivo gonad culture, inhibition of JUN activation led to decreased Wnt4 expression. These results support the hypothesis that TCF4 regulates an underlying gonadal program that primes the gonad toward a female fate and is silenced in Sertoli cells downstream from Sry .