Wnt4 expression in the differentiating gonad of the frog Rana rugosa

Wnt4, a member of the Wnt family, is known to influence the sex-determination cascade. In mice having a targeted deletion of Wnt4, masculinization occurs in XX pups. Therefore, in addition to Sry, Wnt4 is also involved in sex determination in mice. In humans, a chromosomal duplication of the WNT4 causes feminization of XY-individuals. Thus, for better understanding of the mechanism of sex determination in vertebrates, it is necessary to examine the expression of Wnt4 at early gonadal development stages in non-mammalians. We first isolated the Wnt4 cDNA from the tetsis of the frog Rana rugosa. R. rugosa Wnt4 had a high similarity (>86%) at the amino acid level with zebra fish, chicken, mouse, and human Wnt4s. We next employed RT-PCR analysis to examine whether Wnt4 was expressed in a sexually dimorphic fashion at early stages of gonadal development in R. rugosa. Wnt4 was transcribed first in the embryos at the late gastrula stage, and its expression was maintained until the indifferent gonad differentiated into a testis or an ovary. Wnt4 expression in the differentiating gonad appeared in a non-sexually dimorphic pattern. The results, taken together, suggest that Wnt4 is highly conserved through evolution, and that its expression in the indifferent gonad takes place with no sexual dimorphism. Thus, Wnt4 is not a key factor to initiate the development of a testis or an ovary from the indifferent gonads in R. rugosa. However, this gene probably forms part of a gonadal-development pathway in this species.