To test this idea, we generated a double mutant of ahr-1;mec-3 an

To test this idea, we generated a double mutant of ahr-1;mec-3 and determined that cAVM neurons now resemble the simple, unbranched morphology Selleck Talazoparib of mec-3 mutant PVD neurons ( Figure 6F). This finding confirms that mec-3 function is necessary for cAVM branching in the ahr-1 mutant. Two alternative

models are consistent with this result: (1) AHR-1 normally limits MEC-3 expression in the touch neurons to prevent branching, and (2) AHR-1 functions downstream to block expression of MEC-3-dependent targets that drive the creation of PVD-like branches. To distinguish between these models, we first asked if AHR-1 regulates mec-3. In wild-type animals, mec-3::GFP is normally expressed in the six light touch neurons and in the FLP and PVD neurons ( Figure 6G) ( Way and Chalfie, 1989). We noted that a mec-3::GFP reporter was strongly expressed in the touch neurons and in FLP but showed a consistently weaker signal in PVD (data not shown). In the ahr-1 mutant, mec-3::GFP expression

was substantially reduced in cAVM in comparison to the wild-type AVM neuron ( Figures 6G and 6H). We used fluorescence in situ hybridization (FISH) to confirm that mec-3 mRNA is expressed at a lower level in PVD and in cAVM than in wild-type AVM ( Figure S4). These findings argued against the idea that AHR-1 inhibits mec-3 expression and favored the alternative possibility that AHR-1 activates mec-3 to specify touch neuron traits. We tested this hypothesis by examining the touch-neuron-specific marker mec-4::mCherry, which check details normally depends on mec-3 function for expression ( Zhang

et al., 2002). mec-4::mCherry is rarely detected in cAVM neurons ( Figure 6I) but is restored by overexpression of MEC-3 in an ahr-1 mutant ( Figure 6J; Table S1). It is also important to note that overexpression of MEC-3 did not prevent the formation of ectopic PVD-like branches or inhibit expression of the PVD-specific marker gene, F49H12.4::GFP in cAVM ( Figure 6J). 3-mercaptopyruvate sulfurtransferase These results are consistent with a model in which MEC-3 must exceed a high threshold to activate expression of light touch neuron genes (e.g., mec-4) but also in which low levels of MEC-3 are sufficient to drive expression of transcripts that specify PVD-like traits (e.g., lateral branching). We therefore considered the hypothesis that AHR-1 negatively regulates PVD-like branching in AVM by inhibiting MEC-3 transcriptional targets ( Figure 6K) and set out to identify these downstream genes. We hypothesized that MEC-3-regulated targets in PVD should include genes that promote branching since PVD neurons show a branchless phenotype in mec-3 mutants ( Smith et al., 2010 and Tsalik et al., 2003). To identify these genes, we used the mRNA tagging method to isolate PVD-specific transcripts from L2 stage larvae immediately prior to the period in which PVD lateral branching is first observed ( Smith et al.

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