Figure 3. Axin directs axon formation in cultured hippocampal neurons. A–F, Axin is required for axon formation. Dissociated hippocampal neurons were transfected with shAxin construct, together with GFP expression construct, and fixed at 3 DIV (A, B), 4 DIV (C, D), or 5 DIV (E, F) after transfection. Neurons were costained with antibodies against GFP, axonal markers Tau-1 (green), smi-312 (green), or synapsin I (Syn I; green), and dendritic marker MAP2 (red) as indicated. The axon was defined as Tau-1, smi-312, or synapsin I positive, longer than 100 μm, and at least twice as long as the other processes. Arrows indicated the axons; arrowheads indicated the non-axon neurites. Scale bar, 20 μm. B, D, F, Quantitative analyses of axonal phenotypes. The axonal phenotypes of transfected hippocampal neurons were classified into three groups: NA (no-axon; negative for Tau-1, smi-312, or Syn I but positive for MAP2), SA (single-axon), and MA (multiple-axon) neurons. More than 300 neurons were examined and quantified for each condition. Data were presented as mean ± SEM; **p < 0.01, n = 3, shAxin versus control (Student's t test). G–I, Expression of shRNA-resistant Axin restores axon formation in Axin-deficient neurons. G, Expression of shRNA-resistant Axin (AxinRes–WT) in HEK 293T cells. HEK 293T cells were transfected with expression constructs encoding wild-type (Axin–WT) or shRNA-resistant Axin (AxinRes–WT) together with shAxin construct as indicated. H, shRNA-resistant Axin construct restores the expression of Axin protein in shAxin-transfected hippocampal neurons. I, Expression of shRNA-resistant Axin rescues axon formation in Axin-deficient neurons. More than 300 neurons were examined and quantified for each condition. Data were presented as mean ± SEM; **p < 0.01, n = 3, one-way ANOVA. J, Knockdown of Axin does not cause axon degeneration or a transition of axon to dendrite. Cultured neurons were cotransfected with shAxin construct and GFP expressing construct at 3 DIV. The neurons were then fixed at 3 d after transfection and stained with antibodies for Tau-1 and MAP2. Arrows indicated the magnified axonal distant ends, which is positive for Tau-1 (red) but negative for MAP2 (blue). Scale bar, 50 μm.