Establishing sexual dimorphism in the developing nervous system requires the coordinated activities of sex-determining genes and neural regulators. Development of C. elegans male-specific tail neurons depends on a conserved sexual regulator, the DM domain protein MAB-3, and a conserved neural regulator, the proneural bHLH protein LIN-32. Males lacking
lin-32 or
mab-3 activity lack V rays, sensory structures required for mating. Previous studies have indicated that LIN-32 acts downstream of sex-specific Hox gene activity to specify V ray neuroblasts. MAB-3 is expressed with LIN-32 in the ray neuroblasts and potentiates LIN-32 activity in these cells. We have proposed a model in which MAB-3 potentiates LIN-32 activity by repressing target genes that would otherwise prevent LIN-32-directed V ray specification. This model predicts that MAB-3 targets are ectopically expressed in
mab-3 mutants; mutations in these targets should restore V ray development in
mab-3 mutants. The bHLH protein REF-1 is such a target. We identified
ref-1 mutations in a genetic screen for suppressors of
mab-3 V ray defects;
ref-1(lf) mutations fully restore V ray development in
mab-3 mutants. REF-1 is distantly related to the Hairy/Enhancer of Split (Hes) family of antineural regulators. We find that, like Hes proteins, REF-1 opposes proneural gene activity:
lin-32::gfp expression is dramatically reduced in
mab-3 mutant V ray precursors, but is restored by
ref-1 mutations. This suggests that REF-1 represses
lin-32 expression in
mab-3 mutant males; the antineural activity of REF-1 must be prevented by MAB-3 in wild-type males to allow LIN-32-directed V ray specification. Consistent with this, MAB-3 negatively regulates expression of
ref-1::gfp reporters in the V ray lineage. Repression of
ref-1 by MAB-3 is likely direct, as both wild-type MAB-3 and conserved MAB-3 binding sites are required to prevent
ref-1::gfp expression in sensory ray lineages. Furthermore, ectopic expression of
ref-1::gfp in wild-type males phenocopies Mab-3, suggesting that
ref-1 is the primary target of MAB-3 in the V ray lineage. MAB-3 thus promotes sex-specific neurogenesis by regulating two conserved bHLH factors, the proneural protein LIN-32, and the antineural protein REF-1. DM domain proteins control sexual differentiation in diverse metazoans. It is therefore possible that regulation of bHLH expression by these conserved sexual regulators is a general mechanism for specifying sex-specific neurons.