For all heatshock experiments, three biological replicates were performed. The student t-test is used for statistical comparisons, *p<0.05, **p<0.01, ***p<0.001, n.s.= p>0.05. (A) A schematic representation of the neurons in the RVG, VNC, and PAG of the worm. In heatshock experiments represented in B, C, and D, the number of ASE marker expressing cells in the RVG, VNC, and PAG were scored. In B and D, unc-47, a marker of GABAergic identity (is used in the background and only unc-47(-) neurons are scored. This strategy ensures proper scoring of the ~50 unc-3 dependent cholinergic MNs . (B) Images of wildtype and unc-3 worms with and without CHE-1hs and a quantification of gcy-5prom induction at various stages of development. Every dot in this plot represents an individual worm. The entire RVG, VNC, and PAG of the worm is scored. A significantly higher number of gcy-5prom::gfp neurons are seen in unc-3 mutants as compared to wildtype in response to CHE-1hs induction. No gcy-5prom::gfp neurons are seen in unc-3 mutant worms that get heatshocked but do not contain the heatshockprom::che-1 array (0 cells in >20 worms). (C) gcy-5prom transgene (green bar) and gcy-5 mRNA (smFISH; pink bar) induction in heatshocked worms carrying a single copy insertion of heatshockprom::che-1. Induction of gcy-5 endogenous mRNA (smFISH) is similar to the induction of the gcy-5prom reporter. (D) Expression of various other ASE markers is seen in a significantly higher number of unc-47(-) RVG, VNC, and PAG neurons in unc-3 mutants as compared to wildtype after CHE-1hs induction. The scored markers and their wildtype (without CHE-1hs) expression patterns are listed above each plot. Every dot in these plots represents an individual worm. Markers are scored only in the anterior and posterior regions of the worm, as represented in A. These worms were heatshocked at the L4 stage.