Although the genetic triggers for gonadal sex differentiation vary across species, the cell biology of gonadal development was long thought to be largely conserved. Here, we present a comprehensive analysis of gonadal sex differentiation, using single-cell sequencing in the embryonic chicken gonad during sexual differentiation. The data show that chicken embryonic-supporting cells do not derive from the coelomic epithelium, in contrast to other vertebrates studied. Instead, they derive from a DMRT1+/PAX2+/WNT4+/OSR1+ mesenchymal cell population. We find a greater complexity of gonadal cell types than previously thought, including the identification of two distinct sub-populations of Sertoli cells in developing testes and derivation of embryonic steroidogenic cells from a differentiated supporting-cell lineage. Altogether, these results indicate that, just as the genetic trigger for sex differs across vertebrate groups, cell lineage specification in the gonad may also vary substantially.

Our understanding of avian sex determination and gonadal development is derived primarily from the studies in the chicken. Analysis of gynandromorphic chickens and experimental chimeras indicate that sexual phenotype is at least partly cell autonomous in the chicken, with sexually dimorphic gene expression occurring in different tissue and different stages. Gonadal sex differentiation is just one of the many manifestations of sexual phenotype. As in other birds, the chicken has a ZZ male: ZW female sex chromosome system, in which the male is the homogametic sex. Most evidence favours a Z chromosome dosage mechanism underling chicken sex determination, with little evidence of a role for the W chromosome. Indeed, the W appears to harbour a small number of genes that are un-related to sexual development, but have been retained because they are dosage sensitive factors. As global Z dosage compensation is absent in birds, Z-linked genes may direct sexual development in different tissues (males having on average 1.5 to 2 times the expression level of females). In the embryonic gonads, the Z-linked DMRT1 gene plays a key role in testis development. Beyond the gonads, other combinations of Z-linked genes may govern sexual development, together with a role for sex steroid hormones. Gonadal DMRT1 is thought to activate other players in testis development, namely SOX9 and AMH, and the recently identified HEMGN gene. DMRT1 also represses ovarian pathway genes, such as FOXL2 and CYP19A1. A lower level of DMRT1 expression in the female gonads is compatible with activation of the ovarian pathway. Some outstanding questions include how the key testis and ovary genes, DMRT1 and FOXL2, are regulated. In addition, confirmation of the central role of these genes awaits genome editing approaches.

The exact genetic mechanism regulating avian gonadal sex differentiation has still not been completely resolved. The most likely scenario involves a dosage mechanism, whereby the Z-linked DMRT1 gene triggers testis development. However, the possibility still exists that the female-specific W chromosome may harbour an ovarian determining factor. In this study, we provide evidence that the universal gene regulating gonadal sex differentiation in birds is Z-linked DMRT1, and not a W-linked (ovarian) factor. Three candidate W-linked ovarian determinants are HINTW, FET1, and FAF. To test the association of these genes with ovarian differentiation in chicken, we examined their expression following experimentally-induced female-to-male sex reversal, using the aromatase inhibitor, fadrozole. Administration of fadrozole on day 3 of embryogenesis induced a significant loss of aromatase enzyme activity in female gonads and masculinisation. However, expression levels of HINTW, FAF and FET1 were unaltered following experimental masculinisation. Furthermore, comparative analysis showed that FAF and FET1 expression could not be detected zebra finch gonads. In addition, an antibody raised against the predicted HINTW protein failed to detect it endogenously. These data do not support a universal role for these genes or indeed the W sex chromosome in ovarian development in birds. We found that DMRT1 (but not the recently identified Z-linked HEMGN gene) is male up-regulated in embryonic zebra finch and emu gonads, as in chicken. As chicken, zebra finch and emu exemplify the major evolutionary clades of birds, we proposed that Z-linked DMRT1, and not the W sex chromosome, regulates gonadal sex differentiation in birds.

Sexual differentiation in birds is controlled genetically as in mammals, although the sex chromosomes are different. Males have a ZZ sex chromosome constitution, while females are ZW. Gene(s) on the sex chromosomes must initiate gonadal sex differentiation during embryonic life, inducing paired testes in ZZ individuals and unilateral ovaries in ZW individuals. The traditional view of avian sexual differentiation aligns with that expounded for other vertebrates; upon sexual differentiation, the gonads secrete sex steroid hormones that masculinise or feminise the rest of the body. However, recent studies on naturally occurring or experimentally induced avian sex reversal suggest a significant role for direct genetic factors, in addition to sex hormones, in regulating sexual differentiation of the soma in birds. This review will provide an overview of sex determination in birds and both naturally and experimentally induced sex reversal, with emphasis on the key role of oestrogen. We then consider how recent studies on sex reversal and gynandromorphic birds (half male:half female) are shaping our understanding of sexual differentiation in avians and in vertebrates more broadly. Current evidence shows that sexual differentiation in birds is a mix of direct genetic and hormonal mechanisms. Perturbation of either of these components may lead to sex reversal.