A candidate genetic trigger of female sex determination in Marsabit clawed frogs ( Xenopus borealis )

A central question in biology asks why the genetic basis of sex determination evolves quickly in some groups even though disruption of this process carries severe risks such as sterility. A challenge to understanding mechanisms of this rapid evolution is a lack of functionally verified genetic triggers of sex determination. To begin to remedy this, we set out to identify, characterize, and functionally test the genetic trigger of female sex determination in Marsabit clawed frogs (Xenopus borealis), which has a species-specific and probably newly evolved W chromosome. Genomic and transcriptomic data identify a candidate that was expressed specifically in female gonads during sex determination. We knocked out this gene, which is a female-specific duplicate of wnt3.L - hereafter wnt3-W - and induced complete female-to-male sex reversal in 35 of 36 individuals. These sex-reversed individuals had male-typical external and internal morphology, mating behavior, mating calls, testis histology, and are fertile. This demonstrates that wnt3-W is generally necessary for feminization of X. borealis, and adds to only one other functionally verified candidate trigger of sex determination in amphibians. Phylogenetic and cytogenetic analysis indicates that wnt3-W originated recently on the tip of the X. borealis W chromosome, after this species diverged from all other extant Xenopus. Wnt3-W is the first known candidate genetic trigger of sex determination in the Wnt gene family, which is surprising because wnt3 is more generally involved with vital, but not sex-specific functions. Similarities in protein structure are consistent with the possibility that Wnt3-W is a genetic surrogate that utilizes the feminizing genetic network of Wnt4. The discovery of wnt3-W adds to a handful of other secreted peptides that trigger sex determination in animals; its proposed function as a positively acting female-determining gene - if confirmed - would further be distinguished from other known genetic triggers in vertebrates.