Figure 7.

Wnt5a mediates branching via protein kinase C. A, Canonical Wnt/β-catenin signaling in TOPGAL reporter transgenic mouse line is not detected in E16.5 SCG (outlined with dashed line); the asterisk (*) identifies a neighboring blood vessel. Scale bar, 100 μm. B, C, TCF transcriptional activity is not required for Wnt5a-mediated axon outgrowth. SCG explants infected with GFP (B) or dominant-negative-TCF (dn-TCF) (C) adenovirus show robust neurite outgrowth in response to Wnt5a. Ganglia were immunostained for β-III-tubulin. Scale bar, 320 μm. D, Quantification of neurite outgrowth from SCG explants infected with either GFP or dn-TCF in the presence or absence of Wnt5a-containing media. Values are the mean ± SEM for four explants. No significant difference in neurite outgrowth was observed between Wnt5a-treated explants infected with either GFP or dn-TCF adenovirus. Wnt5a-treated explants infected with GFP or dn-TCF were significantly different from control conditioned media-treated explants; **p < 0.01 and ***p < 0.001, as determined by one-way ANOVA followed by Tukey's multiple-comparisons test. E, Cultured sympathetic neurons stimulated with recombinant Wnt5a (200 ng/ml; 30 min) show increased levels of phosphorylated PKC (p-PKC). In contrast, levels of p-GSK3β, p-c-Jun, p-CaMKII, were unaffected. All Western blots were stripped and reprobed for the p85 subunit of PI3-K as a loading control. F, G, Wnt5a-dependent axonal branching in sympathetic neurons requires PKC activity. Sympathetic neurons cultured for 24 h in the presence of Wnt5a show extensive branching (F) that is abrogated by treatment with a myristoylated PKCα pseudosubstrate (G). Neurons were immunostained for β-III-tubulin. Scale bar, 50 μm. H, I, Quantification of branch points per neuron (H) and neurite length (I) in low-density dissociated cultures treated with control or Wnt5a conditioned media, in the presence or absence of PKCα-pseudosubstrate. Values are the mean ± SEM from three independent experiments. ***p < 0.001 significantly different from control conditioned media-treated neurons, as determined by one-way ANOVA followed by Tukey's multiple-comparisons test. J, Quantification of new branch points per projection per 24 h in low-density compartmentalized cultures treated with control or Wnt5a-containing media exclusively on distal axons (DA), in the absence or presence of PKCα-pseudosubstrate added to DA or cell bodies compartments (CB). Values are the mean ± SEM from three independent experiments. **p < 0.01, significantly different from control conditioned media-treated neurons, as determined by one-way ANOVA followed by Tukey's multiple-comparisons test. PKCα (DA) is significantly different from PKCα (CB), p < 0.05.