Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics

Significance Here, we show that DMRT1 dosage is the key sex determination factor in birds and is essential for testis development. Furthermore, we provide additional evidence that birds, in contrast to mammals, have acquired cell-autonomous sex identity (CASI) and that the sex hormone environment does not significantly influence avian secondary sexual characteristics. This finding highlights an evolutionary divide between mammals and nonmammalian vertebrates. In mammals, the sex chromosomes determine the type of gonad formed, and sex hormones largely define the secondary sexual phenotype. In birds, the sexual phenotype is directly determined by the sex chromosome content of individual cells in different tissues. Our findings help advance our understanding of the evolution of sex determination systems and the nature of sex identity. In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3–Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9–based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.