Extension of filopodia toward sources of Slit is required for axon repulsion. (A) Polar histogram plots of filopodium distribution. Data were quantified from DIC images taken over a 10-min period after application of the indicated gradient; orientation of micropipette-generated gradients indicated by black triangle (open, mock; filled, Slit). Data are binned from six independent experiments examining a total of 24 growth cones. (B) Numbers of filopodia on the side of the growth cone facing toward (darker shades, proximal, 0–90°) or away from (lighter shades, distal, 90–360°) the Slit gradient. (C) Lengths of filopodia by relative orientation to mock (orange) or Slit (blue) gradients. Filopodia proximal to the Slit gradient were significantly longer (10.5 ± 5.4 µm) than filopodia exposed to a mock gradient (7.7 ± 3.6 µm) or filopodia distal to the gradient (8.9 ± 4.3 µm). ***, P < 0.001; ****, P < 0.0001; n.s., not significant; one-way ANOVA. (D) Traces of growth cone positions over a 30-min period after application of mock or Slit gradients. Representative DIC images show axons just before gradient application (0’) and after 20 or 40 min; insets show higher magnification views of growth cones at the corresponding time points. A total of 25 nM CD was added to the bath media at the time of the gradient onset in iii. Long filopodia were frequently observed in Slit gradients applied to control (ii, white arrowheads), but not in CD-treated (iii) or mve neurons (iv). (E) Axon turning angles in response to gradients (mean ± SD). n ≥ 18 biological replicates for each condition. ***, P < 0.001; one-way ANOVA. (F) Summary of relationships between filopodia extension toward sources of Slit and axon repulsion. Bars, 10 µm. WT, wild type.