Fig. 4

Altered cilia affect different aspects of human LUHMES neuron differentiation: axon outgrowth and branching pattern. A The percentage of ciliation in differentiating human LUHMES neurons increases and declines slightly earlier in RFX2 -/- mutants as compared to WT neurons. The percentage of neurons reaching stage 3 of differentiation is delayed in RFX2 -/- mutants as compared to WT neurons throughout the entire differentiation and maturation process (WT n = 134–394; RFX2 -/- n = 162–492). B Altered cilia of LUHMES RFX2 -/- neurons do not render axon outgrowth more efficient (n = 29–90), as compared to non-ciliated LUHMES RFX2 -/- neurons (n = 24–122) (d1-d3 summarized and individual days). C-D Axons of ciliated neurons (n = 69–80) are more branched and arborized as compared to non-ciliated neurons n = 48–107 in WT, whereas in RFX2 -/- mutants there is no difference between ciliated (n = 43–84) and non-ciliated neurons n = 34–103). E Detection by immunocytochemistry demonstrates the effect the cilium imparts on the complexity of axon branching in WT neurons: top – ciliated neuron, bottom – non-ciliated neuron (circles indicate centrosome with or without a cilium; arrowheads indicate axon branch points); Hoechst stain – nucleus, PCNT – centrosome/basal body marker, ARL13B – ciliary marker, Phalloidin – cytoskeleton/F-actin marker. Mean values are shown ± s.d. (A, C-D) and ± s.e.m. (B). The results are from a minimum of three independent experiments with a minimum of two technical replicates each. We conducted regular two-way ANOVA analyses (not repeated measures) with multiple comparisons (Bonferroni’s test) between groups. *p < 0.05; **p < 0.005; ***p < 0.0005; ****p < 0.0001