Fig. 7

WNT signaling is essential for promoting human LUHMES neuron differentiation. A-B Inhibiting WNT signaling by treating LUHMES neurons with the antagonist Wnt-C59 negatively affects axon outgrowth (how efficiently neurons reach differentiation stage 3) in (A) WT (vehicle n = 190; Wnt-C59 n = 301) but not in (B) an RFX2 -/- background (vehicle n = 214; Wnt-C59 n = 242). Control (vehicle): the same treatment, but without antagonist. C-D WT ciliated neurons (n = 99) reach differentiation stage 3 more efficiently than non-ciliated neurons (n = 91) in the control experiment (vehicle), while no difference was observed between ciliated (n = 129) and non-ciliated neurons (n = 172) after treatment with the antagonist Wnt-C59 (C). No differences were observed in RFX2 -/- neurons (D) under the same experimental conditions (ciliated (n = 114) versus non-ciliated (n = 100); with (n = 129) versus without (n = 113) antagonist Wnt-C59). E–F Wnt-C59 treatment deregulates axon branching. In the control experiment (vehicle), axon branching is promoted in WT ciliated neurons (n = 37) as compared to non-ciliated neurons (n = 38). Upon treatment with the antagonist Wnt-C59 this difference is alleviated (ciliated n = 34; non-ciliated n = 50) (E). In RFX2 -/- neurons with altered cilia there are no differences in axon branching between ciliated (n = 43) and non-ciliated neurons (n = 22) in the control experiment (vehicle); likewise, for the treatment with the antagonist Wnt-C59 (ciliated n = 38; non-ciliated n = 43) (F). Mean values are shown ± s.e.m. (A-D) and ± s.d. (E–F). The results are from two independent experiments with a minimum of three technical replicates each. We conducted an unpaired two-sided t-test (95% confidence level) (A-B), and regular two-way ANOVA analyses (not repeated measures) with multiple comparisons (Bonferroni’s test) between conditions (C-F). *p < 0.05; ***p < 0.0005