UNC-43 is enriched in males at the cholinergic motor neuron terminals. (A) Schematic illustration of split GFP complementation experimental design. Seven copies of the split GFP11 were inserted into the N-terminal of unc-43 (K11E8.1d) genomic loci by CRISPR-Cas9 system. The split GFP1-10 was expressed in B-type cholinergic and GABAergic motor neurons by unc-129 and unc-25 promoters, respectively. (B–D) The puncta fluorescence intensities and densities, marked by the cholinergic synaptic UNC-43::split GFP in dorsal nerve cord axons in the L3-staged, L4-staged, and adult hermaphrodites (Herm.) and males (Male). Representative images (B), mean puncta intensity (C), and density (D) are shown. (E–G) The puncta fluorescence intensities and densities, marked by the GABAergic synaptic UNC-43::split GFP in dorsal nerve cord axons in the adult hermaphrodites (Herm.) and males (Male). Representative images (E), mean puncta intensity (F), and density (G) are shown. In B and E, Scale bars, 10 μm. In C, D, F, and G, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Bonferroni’s multiple comparisons for C and D and two-tailed and unpaired Student’s t test for F and G. ***P < 0.001, n.s. not significant.