In the L3 male tail, the nine bilaterally-symmetric Rn cells each begin the execution of a sublineage that generates the components of a ray (two neurons and a structural cell), a hypodermal cell and a programmed cell death. We would like to understand how Rn cells are selected to execute the ray sublineage, and how this program of division and differentiation is coordinated. The bHLH gene
lin-32, similar to the proneural genes of Drosophila, is required for the execution of the ray sublineage. In a
lin-32 hypomorph, many ray neuroblasts (Rn.a cells) are transformed into Rn-like hypodermal cells, and the frequency of ray formation is reduced. To better understand the function and regulation of
lin-32, we performed a screen for modifiers of the hypomorphic allele
lin-32(
e1926). Among the mutants isolated in this screen, we recovered two alleles (
bx108 and
bx115) that semi-dominantly enhance the ray-loss phenotype of several
lin-32 alleles. However, neither
bx108 nor
bx115 animals have any noticeable defects in a wild-type background. Mapping experiments placed
bx108 and
bx115 in the vicinity of
hlh-2, the worm homolog of the E/daughterless bHLH genes described recently by Krause et al. (Development 124:2179). Proteins of the E/daughterless family have been shown to serve as general heterodimerization partners for cell-type-specific bHLH proteins, making
hlh-2 an excellent candidate for our enhancers. Though no mutants had been isolated,
hlh-2(RNAi) had been shown to cause embryonic lethality. Expression studies suggested that
hlh-2 function is restricted primarily to neurogenesis, and may be regulated during development. We sequenced the
hlh-2 gene in
bx108 and
bx115 mutants, and found that both contain missense mutations in the bHLH domain. As alleles of
hlh-2, these enhancer mutations are therefore likely to potentiate the defects of
lin-32 mutants by further weakening the DNA-binding or dimerization activities of mutant LIN-32 proteins. Neither of these subliminal
hlh-2 alleles is likely to be null, and any
lin-32-independent functions of
hlh-2 are probably not revealed by our mutants. In vitro experiments are in progress to test the predictions that LIN-32 and HLH-2 can heterodimerize, and that our mutations disrupt this property. We are currently examining the LIN-32 and HLH-2 expression patterns in the larval male tail in both wild-type and mutant animals, to determine whether
hlh-2 expression is regulated, and to understand potential regulatory dependencies between
hlh-2 and
lin-32. We are also characterizing the lineage alterations in
hlh-2;
lin-32 double mutants, to explore the possibility that the ray sublineage defect differs from that in
lin-32 single mutants. We are interested in understanding whether these two factors simply function together once, to effect the activation of target genes required for neurogenesis, or whether they have more complex roles in the implementation of Rn cell fate.