wnt4a Promotes Female Development and Reproductive Duct Elongation in Zebrafish

In laboratory strains of zebrafish, sex determination occurs in the absence of a typical sex chromosome and it is not known what regulates the proportion of animals that develop as male or female. Many sex determination and differentiation genes that act downstream of a sex chromosome are well conserved among vertebrates, but studies that test their contribution to this process have mostly been limited to mammalian models. In mammals, WNT4 is a signaling ligand that is essential for ovary and Mullerian duct development, where it function, in part, to antagonize the male-promoting FGF9 signal. Wnt4 is highly conserved in non-mammalian vertebrates, but it is not known if Wnt4 plays a role in sex determination and/or the differentiation of sex organs outside of mammals. This is an especially interesting question in teleost, such as zebrafish, because they lack an Fgf9 ortholog. Here we show that wnt4a is the ortholog of mammalian Wnt4 , and that wnt4b was present in the last common ancestor of humans and zebrafish, but was lost in mammals. We found that wnt4a is expressed in the somatic cells of juvenile gonads during the time sex determination likely occurs. We show that wnt4a loss-of-function mutants develop predominantly as males and conclude that wnt4a activity promotes female sex determination in zebrafish. Additionally, both male and female wnt4a mutants are sterile because their reproductive ducts do not connect to the vent, where wnt4a is normally expressed. Yet when dissected from homozygous wnt4a mutant gonads, both sperm and eggs can produce fertile offspring. Together these results strongly argue that Wnt4a is a conserved regulator of female sex determination and reproductive duct development in non-mammalian vertebrates.