UNC-43/CaMKII mediates the sexually dimorphic cholinergic transmission. (A and B) Transcriptome database analysis to identify genes expressed in the cholinergic motor neurons and enriched in males after the L4 stage. (A) Venn diagram analysis acquires genes with sex-differential expression during development. Blue: gene group with lower (<10%) or comparable expression in males compared with hermaphrodite during the L3 stage (Kim et al., 2016); red: gene group with higher expression in males compared with hermaphrodite in both the L4-stage and adult stage (Kim et al., 2016); green: gene group expressed in the cholinergic motor neurons (Taylor et al., 2021). Genes with the criteria of P value <0.05 (analyzed with DESeq2 and Likelihood-Ratio tests) and mean counts ≥ 1 are listed. (B) Gene list of male-enriched genes after the L4 stage and expressed in the cholinergic motor neurons (208). (C–E)rab-3 mutation cannot block the sexually dimorphic cholinergic transmission at C. elegans NMJ. Endogenous acetylcholine transmission was assessed by recording mEPSCs from body-wall muscles of rab-3 mutant hermaphrodites (Herm.) and males. Representative mEPSC traces (C), the mean mEPSC frequencies (D), and the mean mEPSC amplitudes (E) are shown. The data for WT are the same as in Fig. 1, H and I. In D and E, data are presented as mean values ± SEM. Ns represent the number of animals tested. (F–H) Partial knockdown of UNC-43 expression in males reduces cholinergic transmission. Endogenous acetylcholine transmission was assessed by recording mEPSCs from body-wall muscles in males with unc-43 knockdown (unc-43 RNAi male). Representative mEPSC traces (F), the mean mEPSC frequencies (G), and the mean mEPSC amplitudes (H) are shown. (I) Locomotion behavior analysis of unc-43 knockdown males (unc-43 RNAi male). The averaged and individual crawling locomotion velocities were plotted. In D, E, and G–I, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Bonferroni’s multiple comparisons. *P < 0.05, ***P < 0.001, n.s., not significant.
Figure S3.

UNC-43/CaMKII mediates the sexually dimorphic cholinergic transmission. (A and B) Transcriptome database analysis to identify genes expressed in the cholinergic motor neurons and enriched in males after the L4 stage. (A) Venn diagram analysis acquires genes with sex-differential expression during development. Blue: gene group with lower (<10%) or comparable expression in males compared with hermaphrodite during the L3 stage (Kim et al., 2016); red: gene group with higher expression in males compared with hermaphrodite in both the L4-stage and adult stage (Kim et al., 2016); green: gene group expressed in the cholinergic motor neurons (Taylor et al., 2021). Genes with the criteria of P value <0.05 (analyzed with DESeq2 and Likelihood-Ratio tests) and mean counts ≥ 1 are listed. (B) Gene list of male-enriched genes after the L4 stage and expressed in the cholinergic motor neurons (208). (C–E)rab-3 mutation cannot block the sexually dimorphic cholinergic transmission at C. elegans NMJ. Endogenous acetylcholine transmission was assessed by recording mEPSCs from body-wall muscles of rab-3 mutant hermaphrodites (Herm.) and males. Representative mEPSC traces (C), the mean mEPSC frequencies (D), and the mean mEPSC amplitudes (E) are shown. The data for WT are the same as in Fig. 1, H and I. In D and E, data are presented as mean values ± SEM. Ns represent the number of animals tested. (F–H) Partial knockdown of UNC-43 expression in males reduces cholinergic transmission. Endogenous acetylcholine transmission was assessed by recording mEPSCs from body-wall muscles in males with unc-43 knockdown (unc-43 RNAi male). Representative mEPSC traces (F), the mean mEPSC frequencies (G), and the mean mEPSC amplitudes (H) are shown. (I) Locomotion behavior analysis of unc-43 knockdown males (unc-43 RNAi male). The averaged and individual crawling locomotion velocities were plotted. In D, E, and G–I, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Bonferroni’s multiple comparisons. *P < 0.05, ***P < 0.001, n.s., not significant.

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