Figure 1. unc-43 functions downstream of lin-44 in proper PDB development. (A-F) Representative images of PDB at the L4 stage. Red arrowheads denote posterior neurites, and dotted arrowheads represent the absence of posterior neurites. Asterisks denote the PDB cell bodies. (G) Quantification of animals with ectopic posterior neurites. Sample numbers are shown in each bar. ***p < 0.001; **p < 0.002; ns, not significant (Fisher’s exact test). (H, I) Representative images of LIN-17::GFP localization (top panels), PDB neurite structure labeled with cytoplasmic BFP (middle panels) and merged images (bottom panels) in wildtype (H) and unc-43(n498n1186) (I) animals. Quantification of the normalized GFP/BFP ratio at the anterior and posterior growth cones for each genotype is shown on the right. Error bars indicate mean ± SEM. ***p < 0.001 (Ratio paired t-test). (J) Quantification of the animals with ectopic posterior neurites. Two independent lines of each rescue experiment are labeled with #1 and #2. Note the quantification of unc-43(n498n1186) is from panel (G). Sample numbers are shown in each bar. ***p < 0.001; *p < 0.033 (Fisher’s e2xact test). Scale bars: 10 μm.
Abstract
During development, many neurons prune their neurites. While many pruning events are activity-dependent, some neurons undergo stereotyped and developmentally regulated neurite pruning, and our understanding of the signaling pathways that mediate this form of pruning remains limited. In this study, using the PDB motor neuron in Caenorhabditis elegans, we show that the Wnt-calcium signaling pathway is required for stereotyped neurite pruning during development. We found that mutants of itr-1/IP3 receptor and two calcium-dependent kinases, unc-43/CaMKII and pkc-2/PKC, exhibit neurite pruning defects. Genetic analysis suggested that they function downstream of lin-44/Wnt in neurite pruning. Human CaMKIIA can induce neurite pruning in C. elegans, and mutations in CaMKII genes in patients with intellectual disabilities affect its pruning function. In vivo calcium imaging revealed that PDB neurites exhibit calcium transients during neurite pruning, which are regulated at least in part by lin-44 and itr-1. Furthermore, we demonstrate that pkc-2 regulates neurite pruning through clathrin-mediated endocytosis. Together, our work reveals the critical functions of Wnt–calcium signaling in neurite pruning.