Fig. 1. Growth-cone turning induced by a gradient of agrin. (A,B) Left: microscopic images of a neuron (cultured for 5-10 hours) at the beginning (0 min) and the end (60 min) of a 1-hour exposure to a drug gradient created by pulsatile application (arrows) with a micropipette. Right: superimposed traces depict the trajectory of neurite extension during the 1-hour turning assay. (A) Top, BSA (50 µg/ml in pipette); bottom, agrin (100 µg/ml in pipette). (B) Top, Ab, agrin-neutralizing antibody (200 µg/ml in pipette); bottom, agrin preincubated with the agrin-neutralizing antibody. (C) Left: histogram showing the average turning angles of Xenopus neuronal growth cones induced by a gradient of BSA, agrin, agrin-neutralizing antibody (Ab) and agrin preincubated with the antibody (Agrin+Ab). Each value represents the average±s.e.m.; ^*P<0.01 (one-way ANOVA). Scatter plots show the distribution of the turning angles for each growth cone of Xenopus neurons examined. Right: histogram showing the average neurite extension rate during the 1-hour exposure to drug gradient. Scatter plots show the distribution of neurite extension rate for the Xenopus neurons examined. (D) Overexpression of a MuSK mutant inhibited agrin-induced growth-cone turning. Left: microscopic images of cultured Xenopus spinal neurons, prepared from embryos injected with either empty vector (Mock) or EC-MuSK, at the beginning (0 min) and the end (60 min) of a 1-hour exposure to an agrin gradient created by the pulsatile application (arrows) of agrin (100 µg/ml in the micropipette). Right: superimposed traces of neurites depicting the trajectory of neurite extension during the 1-hour turning assay. (E) Left: histogram showing the averaged turning angles of growth cones from neurons expressing different constructs in the presence of an agrin gradient applied with a micropipette (agrin, 100 µg/ml in the micropipette). Each value represents the average±s.e.m.; ^*P<0.01 (Student's t-test). Scatter plots show the distribution of the turning angles of each growth cone. Right: histogram showing the average neurite extension rate during the 1-hour growth cone turning assay. Scatter plots show the distribution of neurite extension rate of each neuron.