Fig. 9. Model for transcriptional regulatory events in the AIY interneurons. We propose that CEH-10 activates the expression of the TTX-3 protein, which then – either in conjunction with CEH-10 or other as yet unknown transcription factors – activates the expression of ceh-23 and other target genes. TTX-3 and CEH-23 presumably both contribute to the regulation of sra-11 expression. Pan-neuronal features are determined either upstream or in parallel to the ceh-10ttx-3ceh-23 cascade. Regulatory events that determine pan-neuronal and cell-type specific features of AIY (via ceh-10/ttx-3/ceh-23) are unknown and are denoted by a question mark. Based on their prominent roles in neuronal patterning in several systems, we tested whether the C. elegans Pax-6/Eyeless homolog vab-3 (Chisholm and Horvitz, 1995), the C. elegans homolog of MATH1/Atonal, lin-32 (Zhao and Emmons, 1995) and the C. elegans NeuroD homolog cnd-1 (Hallam et al., 2000) couple early embryonic patterning events to the transcriptional regulatory cascade of ceh-10, ttx-3 and ceh-23 in AIY and found this not to be the case (data not shown). It is conceivable that some of the TTX-3 target genes shown here (ser-2, sra-11, C36B7.7, kal-1) are directly involved in axonal defects observed in ttx-3(ks5) mutants; this possibility is denoted by a broken arrow. Isolation of mutations in these genes will address this issue. Although unc-17 hypomorphic mutants do not show axonal defects, it is possible that cholinergic transmission contributes together with other genes to axogenesis of AIY (Table 3). At this point it is not clear whether any of the regulatory events that involve CEH-10, TTX-3 and CEH-23 depend on direct interaction of the respective homeodomain protein with the promoter of the presumptive target gene. Within the ttx-3 promoter we have defined two motifs of 5bp and 7bp length, spaced by 20 nucleotides, which are both necessary for the activity of the promoter; both elements contain a variant of the core homeodomain binding site (A. Wenick and O. H., unpublished). Similar clustered motifs can be found in other presumptive TTX-3 target gene promoters. We speculate that these motifs may represent binding sites for TTX-3 and possible co-factor(s). ceh-10 autoregulation has been demonstrated by Forrester et al., 1998.