Functional Analysis of HLH-3, a bHLH Achaete/Scute Protein, in HSN Maturation

The basic helix loop helix protein, HLH-3, is a member of the Achaete/Scute family in C. elegans that is required for normal egg-laying behavior. hlh-3 null mutants have an egg-laying defective (Egl) phenotype. A pair of hermaphrodite specific motor neurons (HSNs) are the command neurons regulating egg-laying behavior in the hermaphrodites. Work by others had shown that loss of hlh-3 function results in individuals where only 35% of the HSNs are detected with anti 5HT staining, and among those detected 60% of them have axonal pathfinding defects. When this project was started the fate of the remaining 65% HSNs was unknown. My work shows that the reason they are undetectable is not because they undergo cell death or fate transformation to their sister cells. Instead this work shows that undetected HSNs are arrested in their lamellipodia/filopodia stage in the null mutant, hlh-3(tm1688), and do not progress further. I call these HSNs as immature as they do not progress to express the later markers of terminal differentiation. In the process of analysis of another deletion mutant, hlh-3 (bc277), where the hermaphrodites are not Egl, we were surprised to find mature HSNs (5HT positive) that were nevertheless abnormal in their axonal pathfinding. This work provides evidence that these animals are not Egl because their HSN axons have branches that form varicosities on the vulva muscles. Since hlh-3 (bc277), is a deletion in the region of the 1st putative exon of hlh-3, and mutant hermaphrodites shows normal egg-laying behavior and their HSNs have no maturation defects I tested whether the 1st exon can be transcribed and translated. My work suggests that the gene encodes two distinct functions, which I have called isoforms A (longer) and B (shorter). I show that a transgene containing the 1’ exon, present in HLH-3A, or exon 1, present in HLH-3B is expressed in the embryonic HSNs and not post hatching, which is consistent with the expression pattern seen with the fosmid reporter (HLH-3::YFP). This expression pattern suggests that hlh-3 expression is needed in the embryonic stages but its function is needed in the later stages of development. Based on the phenotypes displayed by hlh-3 (tm1688) (lacks both isoforms) and hlh-3 (bc277) (lacks isoform A) hermaphrodites, I hypothesize that HLH-3A seems to be important for pathfinding and HLH-3B for maturation.