ABSTRACT The short-lived African killifish Nothobranchius furzeri lives in seasonal freshwater ponds and has evolved remarkable traits to survive in this limited environment. One of those traits is a genetic XX/XY sex-determination system, which ensures an equal distribution of both sexes. Comparisons of female and male genomic sequences identified the Y-chromosomal copy of the TGF-β family member gdf6 as the candidate male sex-determining (SD) gene, which was named gdf6Y in contrast to the X-chromosomal allele gdf6X . CRISPR/Cas9-mediated inactivation of gdf6Y in N. furzeri led to a complete male-to-female sex reversal in XY animals. The homozygous inactivation of gdf6X on the other hand led to a detrimental phenotype post-hatching. This phenotype was compensated by gdf6Y , revealing that the latter became the SD gene while retaining at least some of its original gdf6 function. Gdf6Y is expressed in testicular somatic cells already prior to hatching, where it represses the germ cell-intrinsic feminizing gene foxl2l . We have identified components of the TGF-β signaling pathway, especially the inhibitor of DNA binding genes id1/2/3 , and the mRNA decay activator zfp36l2 , as Gdf6Y targets. We conclude that Gdf6Y exerts its function as the male sex-determining gene by suppressing female-specific genes in the developing gonad of male N. furzeri .

The Tgf-β family member Gdf6Y determines the male sex inNothobranchius furzeriby suppressing oogenesis-inducing genes