The decision of an undifferentiated cell to become a neuron is a crucial developmental process, precisely governed by an spatiotemporally orchestrated regulatory network. So far, except bHLH transcription factors, no other regulatory factors that can fulfill proneural activities have been reported. However, the phenotypes of bHLH knockouts in other organisms suggest that bHLH factors are not required for neurogenesis in all parts of the nervous system.In this sturdy we aim first to get a comprehensive and definitive picture on the role of proneural bHLH transcription factors in early neuronal specification in C. elegans. We started by analyzing HLH-2, the only worm orthologue of Da/E proteins, the conserved heterodimeric partner of other lineage-restricted bHLH factors. In order to reveal the spatial, temporal and differential pattern of HLH-2 expression during embryonic neurogenesis, we systematically lineaged the
hlh-2 fosmid reporter, using 4D microscopy and imaging software (Schnabel R, et al, 1997). Our results indicate while
hlh-2 is broadly expressed all through embryogenesis, it is not ubiquitously expressed thoughout the developing nervous system; that is, we could find neuronal lineages that never express HLH-2. Furthermore, we analyze the extent of neurogenesis in the absence of
hlh-2 using pan-neuronal and lineage specific markers. Interestingly, similar to flies mutant for daughterless, we observed that while many neurons fail to generate a substantial number of neurons are still present in in
hlh-2 maternal and zygotic null mutants.Both the mutant and expression analysis indicate that
hlh-2 may not be required for global neurogenesis. Hence we hypothesize that some Asc/Ato-type bHLH factor can act independently of
hlh-2. To get a conclusive picture of bHLHs function in regulating neurogenesis, we are currently analyzing the extend of neurogenesis in animals carrying null mutant alleles of various Asc and Ato-type bHLH genes (incl. Atonal, NeuroD and Neurogenin) in addition to analysis of their expression. So far, we have found relative restricted effects in neurogenesis in
hlh-16,
cnd-1,
ngn-1 and
lin-32 null mutants.Taken together, our preliminary phenotypic analysis of bHLH knockouts, and in line with data from other organisms, suggest the existence of other unidentified proneural genes. We are taking an unbiased forward genetic approach to uncover genes involved in driving neuronal specification in lineages that are bHLH independent.