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Fig. 3. GSK-3 activity leading to polarised axonal outgrowth is regulated independently of phosphorylation at Ser9 or Ser21. (A) Western blot of lysates form cortical neurons showing total and phosphorylated forms of GSK-3 and GSK-3ß of mice in which the Ser21 and Ser9 phosphorylation sites of GSK3 and GSK3ß had been replaced with Ala (GSK-3/ß^{21A/21A/9A/9A}) compared with wild-type littermates (GSK-3/ß^+/+/+/+). (B,C) Hippocampal neurons from wild-type and double knock-in mice were grown to low density and fixed at 48 hours. Bars, 10 µm. (D) Coronal sections of embryonal (E18) wild-type and double knock-in mice brains. Axonal projections are visualised with antibody against Tau-1, dendritic projections with antibody against Map-2. Single confocal sections are shown. In the developing cortex, the upper cortical plate (cp) contains more dendrites and, thus, more Map-2; the intermediate zone (iz) contains more Tau-1-positive axons but few dendrites. As shown here, this polarity is not disturbed in developing double knock-in brains. Also, staining the axons in the iz for another marker, synaptobrevin, resulted in the same pattern as shown here for the double knock-in mice (right panels). Bars, 25 µm. (E,F) Hippocampal neurons from the same cultures as shown in B and C incubated with the GSK-3 inhibitor SB216763. Bars, 10 µm. (G) Quantification of the experiments. At least 300 neurons from each condition were analysed and the number with one or multiple axons counted based on the length of Tau-1-positive neurites (mean ± s.e.m.). (H) Total length of axons and minor neurites after 70 hours in culture in wild-type and double knock-in neurons (mean ± s.e.m.).

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