(A) Representative images of neurite extension from t = 0 min (cyan asterisk) to t = 45 min (magenta dotted line) at DIV2 in Vangl2 cKO and control neurons cultured on Ncad-Fc-coated substrates. Scale bar, 10 µm. (B) Quantification of the average growth cone velocity in control and Vangl2 cKO neurons; n = 24–28 neurons. (C) Quantification of total time spent in pauses during a 45 min period for Vangl2 cKO and control growth cones. (D) Quantification of the time per pause in Vangl2 cKO neurons and controls. (E) Quantification of growth cone speed (excluding pauses) in Vangl2 cKO and control growth cones. For C, D, E: n = 10–11 neurons. (F) Graph of the cumulative distance covered by four representative individual Vangl2 cKO (purple) and control (green) growth cones over time. A pause was defined as no movement of the centroid of a growth cone for one minute or more (black arrows). (G, H) Average growth cone velocity and total pause duration in control and Vangl2 cKO neurons on laminin substrates. n = 32–40 neurons (for total time paused, n = 16–20 neurons). (I) Bar graph showing the axonal lengths of both control (n = 94) and Vangl2 cKO (n = 84) neurons in the control condition or treated with Netrin-1 or Wnt5a. (J, K) Average growth cone velocity and total pause duration in control and Vangl2 cKO neurons on PLL substrates. PLL at 1 mg*ml−1n = 21–23 neurons; PLL at 10 mg*ml−1: n = 25–27 neurons. Data are presented as box-and-whisker plots (min/max) based on 3 (B–E, I) or 2 (G, H, J, K) independent experiments; *p<0.05, **p<0.01 or ***p<0.001, by Student’s t-test (B, C, G, H, J), the Mann-Whitney test (D, E, K), or 2-way ANOVA followed by Tukey’s multiple-comparisons test (I).