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First published online 1 September 2005
doi: 10.1242/dev.02016

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Agrin regulates growth cone turning of Xenopus spinal motoneurons

¹ Institute of Neuroscience, Chinese Academy of Sciences, Shanghai 200031, China
² Department of Biochemistry and Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China
³ Division of Neurobiology, Department of Cell and Molecular Biology, University of California, Berkeley, CA 94720, USA

View larger version (41K): [in a new window]   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.

View larger version (61K): [in a new window]   Fig. 2. Agrin inhibits the neurite extension of cultured Xenopus spinal neurons. (A) Microscopic time-lapse images of a neurite at various time points after the addition of agrin. Top panel, treated with BSA (50 µg/ml in bath) as control; bottom panel, treated with agrin (20 ng/ml in bath). Broken lines indicate the duration of treatment. (B) Normalized neurite extension rate of neurons treated with agrin compared with that of control, BSA. The neurite extension rate was measured and normalized by comparing the neurite extension rate before and after agrin treatment. Each value represents the average±s.e.m.; *P<0.01 (Student's t-test). (C) A dose-response curve of the effect of agrin on neurite extension. Negative values represent the average length of neurite retraction. Approximately 20-40 neurons were assayed for each concentration of agrin tested (0.1-50 ng/ml). (D) The inhibitory effect of agrin on neurite extension was abolished by an agrin-neutralizing antibody (Agrin+Ab), or by overexpressing a MuSK mutant that comprised the extracellular domain of MuSK fused with the Fc region of an Ig (EC-MuSK). Microscopic time-lapse images of a neurite extending from a neuron under different conditions. Cultured spinal neurons prepared from Xenopus embryos were injected with empty vector (Mock) or an MuSK mutant (EC-MuSK), and treated with agrin (20 ng/ml). For treatment with agrin+Ab, agrin was preincubated with its neutralizing antibody (40 ng/ml) at 4°C overnight. (E) Histogram showing the normalized average neurite extension rate under different conditions, as in D. The neurite extension rate was normalized by comparing neurite extension rate before and after the application of agrin. Each value represents the average±s.e.m.; *P<0.01 (Student's t-test).

View larger version (42K): [in a new window]   Fig. 3. Calcium is involved in agrin-induced growth-cone turning. (A) Microscopic images of cultured Xenopus spinal neurons at the beginning (0 min) and the end (60 min) of a 1-hour exposure to bovine serum albumin (BSA, as control, upper panel) and an agrin gradient created by the pulsatile application (arrows) of agrin (lower panel) in the presence of calcium-free solution (CFS). (B) Microscopic images of cultured Xenopus spinal neurons at the beginning (0 min) and the end (60 min) of a 1-hr exposure to an agrin gradient created by the pulsatile application (arrows) of agrin in the presence of DMSO, served as control (upper panel), or thapsigargin (TG; lower panel). (C) Left: histogram showing the average turning angles of growth cones from neurons treated with an agrin gradient in the presence of CFS, or pretreated with DMSO and TG, respectively. Scatter plots show the distribution of the turning angles of each growth cone. Right: histogram showing the average neurite extension rate during the turning assay. Scatter plots show the extension rate of each neurite. Each value represents the average±s.e.m.; *P<0.01 (one-way ANOVA).

View larger version (32K): [in a new window]   Fig. 4. PI3-kinase is involved in agrin-induced growth-cone turning of Xenopus neurons. (A) Microscopic images of cultured Xenopus spinal neurons (preincubated with different PI3-kinase inhibitors for 20 minutes) at the beginning (0 min) and the end (60 min) of a 1-hour exposure to an agrin gradient applied by a micropipette (100 µg/ml in the micropipette, arrows). Agrin (DMSO), Agrin (Wort) or Agrin (LY) depict neurons pretreated with DMSO as control, with wortmannin (100 nM) or with LY294002 (30 µM), respectively. (B) Left: histogram showing the average turning angles of growth cones from neurons. Each value represents the average±s.e.m.; *P<0.01 (Student's t-test). Scatter plots showing the distribution of turning angles of each growth cone. Right: histogram showing the average neurite extension rates during the 1-hour growth cone turning assay. Scatter plots show the distribution of the neurite extension rate of each neuron.

View larger version (36K): [in a new window]   Fig. 5. Rac1 is involved in agrin-induced growth-cone turning of Xenopus neurons. (A) Agrin downregulates Rac1 activity in developing cerebellar granule neurons. Western blots show the time course of activation of Rac1 and Rho GTPase in cultured cerebellar granule cells following treatment with agrin (100 ng/ml) for 1-30 minutes. Left, Rac1 activity; right, Rho activity. Total Rac1 and Rho expression served as loading control (bottom panels). Results are representative of at least three experiments and the fold change compared with the untreated control is presented in the histograms. Each value represents the average±s.e.m. (B) Left: microscopic time-lapse images of cultured neurons from embryonic Xenopus spinal cord expressing GFP (top) and DN-Rac1-GFP (bottom) at the beginning (0 min) and the end (top, 60 min; bottom, 120 min) of exposure to an agrin gradient created by the pulsatile application (arrows) of agrin. Right: superimposed traces depict the trajectory of the neurite extension during the 1-hour (top, expression of GFP) and 2-hour (bottom, expression of DN-Rac1-GFP) turning assay. (C) Left: histogram showing the average turning angles of growth cones from neurons expressing different constructs in the presence of an agrin gradient (100 µg/ml in the micropipette). Scatter plots show the distribution of the turning angles of each growth cone. Right: histogram showing the average neurite extension rate during the turning assay. Scatter plots show the extension rate of each neurite. Each value represents the average±s.e.m.; *P<0.01 (Student's t-test).