Foxl2 is required for the initiation of the female pathway in a temperature-dependent sex determination system in...

KDM6B-mediated epigenetic modification of the testicular regulator Dmrt1 has previously been identified as the primary switch of the male pathway in a temperature-dependent sex-determination (TSD) system; however, the molecular network of the female pathway has not yet been established. Here, we have functionally characterized for the first time an upstream regulator of the female pathway, the forkhead transcription factor FOXL2, in Trachemys scripta, a turtle species with a TSD system. FOXL2 exhibited temperature-dependent female-specific expression patterns before the onset of gonadal differentiation and was preferentially localized in ovarian somatic cells. Foxl2 responded rapidly to temperature shifts and estrogen. Importantly, forced expression of Foxl2 at the male-producing temperature led to male-to-female sex reversal, as evidenced by the formation of an ovary-like structure, and upregulation of the ovarian regulators Cyp19a1 and R-spondin1. Additionally, knockdown of Foxl2 caused masculinization at the female-producing temperature, which was confirmed by loss of the female phenotype, development of seminiferous tubules, and elevated expression of Dmrt1 and Sox9. Collectively, we demonstrate that Foxl2 expression is necessary and sufficient to drive ovarian determination in T. scripta, suggesting a crucial role of Foxl2 in female sex determination in the TSD system.