UNC-43 functions at cholinergic motor neurons to support cholinergic synaptic transmission and sexually dimorphic locomotion behaviors. (A) Schematic for construction of the unc-43 conditional deletion lines. Two loxP sites were inserted into the genomic loci at the promoter and the first intron of unc-43 (K11E8.1d and K11E8.1g) coding sequence using CRISPR/Cas9, the minigene encoding Cre recombinase under unc-17, unc-25, and myo-3 promoters were used to knockout unc-43 gene in cholinergic motor neurons, GABAergic motor neurons, and muscle cells, respectively. (B–D) Endogenous acetylcholine transmission was assessed by recording mEPSCs from body-wall muscles of unc-43 conditional knockout hermaphrodites. Representative mEPSC traces (B), the mean mEPSC frequency (C), and the mean mEPSC amplitudes (D) are shown. (E–G) Locomotion behavior analysis of unc-43 conditional knockout hermaphrodites and males. The averaged and individual crawling locomotion velocities were plotted. The data used for the analysis of wild-type hermaphrodites and males in G are the same as presented in F. In C–G, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Dunnett multiple comparisons for C and D, one-way ANOVA with post-hoc Bonferroni’s multiple comparisons for E–G. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.