Figure 6. unc-3 directly controls the expression of genes required for SAB neuronal signaling.

A–F: Cis-regulatory mutational analysis is shown for genes expressed in SAB neurons [unc-17/VAChT, cho-1/ChT, exp-1(GABAR), unc-8 (DEG/ENaC), glr-4 (GluR), del-1(DEG/ENaC)]. Multiple transgenic lines were analyzed for each construct. See Table S4 for detailed quantification of the promoter analysis data. Lines indicate genomic region fused to gfp (green), tagrfp (red), or yfp (yellow). (+) indicates robust expression in the SAB neurons, or ventral nerve cord MNs, or head (at least 70% of the animals) in at least 2 independent transgenic lines. (+/−) indicates significant reduction in the number of neurons expressing the reporter gene in at least 50% of the animals (in at least 2 independent transgenic lines) when compared to transgenic animals carrying longer genomic fragments of the cis-regulatory region. (−) indicates complete loss or faint expression in the SAB neurons, or ventral nerve cord MNs, or head in at least 50% of the animals (in at least 2 independent transgenic lines) when compared to transgenic animals carrying longer genomic fragments of the cis-regulatory region. (*) indicates that the COE motif (vertical light blue line) is conserved in at least 3 other nematode species. MUT indicates that COE motif has been mutated by always substituting the same 2 nucleotides in the core sequence (for example, COE wild-type site: TCCCNNGGGA >> COE MUT site: TGGCNNGGGA). See also Figure S6 and Table S3.

G: Schematic that summarizes the key findings of this paper. The transcription factor unc-3 (likely together with unknown factors “X”) regulates synaptogenesis by controlling madd-4/Punctin expression, which is a pre-synaptically secreted molecule required for AChR clustering in head muscle. UNC-3 also coregulates the expression of ACh pathway genes (unc-17/cha-1, cho-1), ionotropic neurotransmitter receptor (NT) genes (glr-4, glr-5, exp-1), and ion channel genes (del-1, unc-8). The core sequence (8 nucleotide positions) of the sequence logo of the UNC-3 binding site (COE motif) is shown to indicate direct regulation by UNC-3. The entire sequence logo of the COE motif is presented in Table S3 and was generated by combining 22 COE motifs found in all SAB-expressed genes shown here. The post-synaptic target of SAB MNs is head muscle (depicted on the left). SAB MNs receive input form the AVL interneuron, as well as the command interneurons (AVA, AVD, AVE) depicted on the right.

H: unc-3 likely cooperates with distinct regulatory factors (factor X in SAB neurons, factor Y in VNC motor neurons) to activate the expression of MN type-specific target genes, as well as the expression of shared genes such as cholinergic pathway genes and synaptogenic genes.