Figure 10.

GPM6a is involved in the formation of polarized axons in vivo. A, Localization of LN in the IZ of mouse embryonic brain. Left, Distribution of β2-LN (also called S-LN; red) and GFP-positive neurons (green) in a representative E16 coronal brain section. Nuclei were stained with DAPI (blue). Right, Fluorescence intensity profiles of anti-β2-LN Ab in each area in the E16 coronal brain sections in the boxed areas in the photograph are indicated. Average intensities of anti-β2-LN Ab immunofluorescence are shown. B, Immunohistochemical analysis of GPM6a in embryonic brain. GPM6a immunoreactivity (red) was mainly localized in the IMZ at stage E15 and in the cortical plate at stage E16. Nuclei are stained with DAPI (blue). C, D, RNAi of GPM6a using in utero electroporation of shRNA against GPM6a or one of two negative control shRNA plasmids (Bsm) in mice at the developmental stage of E16. C, Left, Low magnifications of representative immunofluorescent staining of morphological transitions in E16 coronal brain sections of mice. Neurons were stained with DAPI (blue) and anti-EGFP (green) to visualize the RNAi-introduced neurons more clearly. Morphological transition was impaired by GPM6a-KD. Middle, Higher magnification of the representative morphological transition focusing on the IZ that was impaired by GPM6a-KD (top, negative control, N.C., Bsm; bottom, shGPM6a#8). Arrows indicate the neurons drawn in D (see below). Right, Drawings of representative neurons in the middle panels. Scale bar, 50 μm. D, Magnified drawing of several typical neurons in the middle panels of C. E, F, Quantitative analysis of the effect of negative controls (Bsm, shNeg) and shGPM6a (#5, #7, and #8) on neuronal morphology (classified as having unipolar/bipolar or multipolar morphology) (E) and the number of neurites in the IZ (F). **p < 0.05, ****p < 0.0001 versus negative control (N.C.; Bsm) using one-way ANOVA. More than 100 EGFP-positive neurons from three to four brains were examined in each group. The data represent means ± SEM. There was rarely an effect of either of the two negative controls, the shNeg (including a nontarget sequence under the U6 promoter) and the Bsm (without shRNA-expression sequence under the U6 promoter, only restriction enzyme-targeting sequence), on the neurons. The shGPM6a#8 induced and the shGPM6a#5 had a tendency to induce multipolar cells or increase the number of neurites. G, Efficiency of each shRNA vector was examined by immunoblotting of the expression of EGFP-GPM6a transfected into COS-7 cells. The arrowhead indicates the position of GPM6a. α-tubulin was blotted as a loading control. H, I, RNAi and rescue experiment of GPM6a in vivo. The shGPM6a#8 and/or rescue-GPM6a expression vectors were electroporated into the cortex at E14, followed by fixation at E16. Quantitative analysis of RNAi and rescue effects on neuronal morphology (classified as having unipolar/bipolar or multipolar morphology) (H) and the number of neurites in the IZ (I) were measured (N.C., Bsm 85.8 ± 2.0 vs shGPM6a#8 45.4 ± 4.5, ***p < 0.001; shGPM6a#8 45.4 ± 4.5 vs shGPM6a#8 + rescue-WT-GPM6a 77.6 ± 3.6, **p < 0.005, one-way ANOVA). More than 100 EGFP-positive neurons from three to four brains were examined in each group. The data represent means ± SEM.