The O/E family of vertebrate rHLH transcription factors has been implicated in neurogenesis and axonal pathfinding. The mammalian O/E proteins are expressed transiently in the developing CNS and PNS during times of axonogenesis. A Xenopus O/E homologue, Xcoe2, functions downstream of neurogenin and upstream of neuroD in neurogenesis. The murine O/E members are also expressed in olfactory neurons throughout development and may regulate components of the odorant signal transduction cascade. A C. elegans O/E (CeO/E) homologue has been identified in our laboratory and shown to be encoded by the
unc-3 locus, mutations which result in an uncoordinated phenotype. Previous electron microscopic reconstruction of
unc-3 mutants revealed that the axons of the ventral cord motor neurons are severely defasiculated, the neuromuscular junctions occur at ectopic sites and the motor neurons receive input from inappropriate interneurons as a result of the defasiculation. Using a GFP fusion to the
unc-3 promoter and specific antibodies, we observed that CeO/E is expressed in a large subset of the ventral cord motor neurons and in a pair of chemosensory neurons (ASI amphid neurons). The CeO/E protein is expressed transiently, during times of axonogenesis in the ventral nerve cord and throughout development in the ASI neurons. Several observations suggest that the protein may have distinct roles in the two cell types. Specifically, the axonal and dendritic projections of the ASI appear to be normal when labeled with DiI and the
unc-3 mutants enter the dauer pathway under inappropriate conditions. There is also evidence for autoregulation of the gene in ASI neurons - the expression of CeO/E is greatly reduced in
unc-3 mutants. CeO/E lacks a conserved second repeat helix found that is highly conserved in the mammalian protein. Experiments suggest that CeO/E protein is able to homodimerize in vitro, although the DNA binding specificity of CeO/E is distinct from the mammalian O/E proteins. We are currently identifying the binding site for CeO/E by analysis of sequences essential for regulation of expression in the ASI neurons as well as by specific binding site selection methods (Selex). We are using PCR and differential expression methods to isolate downstream targets of CeO/E that might participate in signaling in the ASI neurons and mediate the axonal pathfinding activities in the ventral nerve cord. The isolation of putative cofactors and downstream targets of CeO/E should provide insight into the function of this transcription factor in neuronal differentiation in C. elegans and vertebrates.