In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY genes of the teleost fish medaka have been characterized as sex-determining genes that trigger formation of the testis. However, the molecular mechanism of the ZZ/ZW-type system in vertebrates, including the clawed frog Xenopus laevis, is unknown. Here, we isolated an X. laevis female genome-specific DM-domain gene, DM-W, and obtained molecular evidence of a W-chromosome in this species. The DNA-binding domain of DM-W showed a strikingly high identity (89%) with that of DMRT1, but it had no significant sequence similarity with the transactivation domain of DMRT1. In nonmammalian vertebrates, DMRT1 expression is connected to testis formation. We found DMRT1 or DM-W to be expressed exclusively in the primordial gonads of both ZZ and ZW or ZW tadpoles, respectively. Although DMRT1 showed continued expression after sex determination, DM-W was expressed transiently during sex determination. Interestingly, DM-W mRNA was more abundant than DMRT1 mRNA in the primordial gonads of ZW tadpoles early in sex determination. To assess the role of DM-W, we produced transgenic tadpoles carrying a DM-W expression vector driven by Ϸ3 kb of the 5-flanking sequence of DM-W or by the cytomegalovirus promoter. Importantly, some developing gonads of ZZ transgenic tadpoles showed ovarian cavities and primary oocytes with both drivers, suggesting that DM-W is crucial for primary ovary formation. Taken together, these results suggest that DM-W is a likely sex (ovary)-determining gene in X. laevis.T he sexual fate of metazoans is determined genetically or by environmental factors, such as temperature. In the former case, heterogametic sex chromosomes determine the male (XY() or female (ZW&) fate in many species of vertebrates. In the XX/XY sex-determining system, the Y-linked SRY genes of most mammals and the DMY/Dmrt1bY gene of the teleost fish medaka have been characterized as sex-determining genes that initiate testis formation, leading to male sexual development (1-5). In contrast, the molecular mechanism for the ZZ/ZW sex-determining system remains unclear, because no sexdetermining genes have been isolated.The Drosophila melanogaster doublesex (dsx) and Caenorhabditis elegans male abnormal (mab)-3 genes are known to control sexual development in these animals (6, 7). The two genes encode proteins containing a zinc finger-like DNA-binding motif called the DM domain. In vertebrates, the DM-domain gene DMRT1 is implicated in sexual development. In the mouse, DMRT1 is essential for postnatal testis differentiation (8, 9). In some other vertebrates, such as the chicken and turtle, DMRT1 expression is connected to testis formation in undifferentiated gonads (10-12). As mentioned above, the medaka fish gene DMY/Dmrt1bY, which is a coorthologue of DMRT1, causes testis formation as a sex-determining gene (3-5). In the chicken, which has the ZZ/ZW system, DMRT1 is located on the Z chromosome, suggesting that gene dosage may induce male development (...

The doublesex and mab-3-related transcription factor 1 (DMRT1) is involved in testis formation in a variety of vertebrates. In the teleost fish, Medaka, DMY/DMRT1Y on the Y chromosome, a duplicate of the autosomal DMRT1 gene, is characterized as a sex-determining gene. We report here the characterization of the Xenopus DMRT1 genes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that X. laevis DMRT1 was expressed throughout the embryo during early development and was restricted to the primordial gonads after embryogenesis. Whole-mount in situ hybridization analysis of the gene confirmed its specific expression in the primordial gonads. To study the transcriptional control of DMRT1 gene expression, we isolated the predicted promoter region of X. tropicalis DMRT1 using databases for this species. Analysis of transgenic tadpoles with a green fluorescence protein (GFP) reporter showed that approximately 3 kb of the 5′-flanking sequence of the DMRT1 gene was implicated in DMRT1 expression in the primordial gonads. We also showed that the C-terminal region of DMRT1 functioned as a transactivation domain in cultured cells, by a luciferase reporter assay using fusion proteins with the DNA-binding domain of GAL4. These findings suggest that DMRT1 functions as an activator of one or more genes involved in sex determination or gonadal differentiation.

The molecular mechanisms of vertebrate ZZ/ZW-type sex-determining systems remain unclear. We recently indicated that a W-linked gene, DM-W is a likely ovary-determining gene in Xenopus laevis. We first examined whether Cyp19 for estrogen-synthesizing enzyme P450 aromatase and Foxl2 showed female-specific expression in developing gonads. Both genes showed much higher expression in ZW than in ZZ gonads during and after sex determination. Importantly, transgenic ZZ gonads expressing exogenous DM-W at the sex-determining stage showed a ZW-type pattern of Cyp19 and Foxl2 expression. These results suggest that DM-W up-regulates Cyp19 and Foxl2 expression to guide primary ovary development in X. laevis.