Additional Supporting Information may be found in the online version of this article.

SC_11-1232_sm_S1.mov3861KSupplemental Movie 1. Time-lapse imaging of control (red) and Vangl2 KD (green) NPCs migrating in the RMS, recorded at 5-minute intervals for 250 minutes. Time-lapse video recordings were obtained using an inverted Zeiss confocal microscope, LSM5 PASCAL or LSM710, equipped with a stage-top microscope incubator, INU-ZI-F1 (5% CO2 at 37°C; Tokai Hit, Shizuoka, Japan), at low magnification, with a 10× dry objective. Optical Z sections (7–12 in 10-μm Z-steps) were acquired every 5 min over a period of 6 h, and all of the focal planes (70–120 μm depth) were merged. See also Supplemental Fig. 4.
SC_11-1232_sm_supplFigure1.pdf242KSupplemental Fig. 1. Immunostaining for Vangl2 and Dvl2. Coronal sections through the SVZ (A, D), RMS (B, E), and OB (C, F) stained with antibodies for Vangl2 (red in A-C) or Dvl2 (red in D-F), in combination with Dcx (green) or GFAP (blue), in the SVZ (A, D) and RMS (B, E), or with NeuN (green) in the OB (C, F). CC, corpus callosum. (A′, B′, D′, E′) Higher magnification of the boxed regions in (A, B, D, E). Scale bar: A, B, D, E, 50 μm; C, F, 10 μm.
SC_11-1232_sm_supplFigure2.tif55KSupplemental Fig. 2. Schematic representations of the domain structure of Dvl2 and Vangl2 and the deletion constructs used in this study.
SC_11-1232_sm_supplFigure3.pdf67KSupplemental Fig. 3. Knockdown verification. HEK293T cells were cotransfected with vectors expressing both Flag-tagged Vangl2 and an miRNA against either luciferase (as a negative control, CT) or Vangl2 (KD), and the cell extracts were subjected to Western blotting analysis with antibodies to Flag or β-actin (a loading control).
SC_11-1232_sm_supplFigure4.tif124KSupplemental Fig. 4. Supplemental information related to Supplemental Movie 1. (A) Migration speed (control, 139.8 ± 7.2 μm/h, n=28 cells from 3 mice; Vangl2 KD, 112.5 ± 5.9 μm/h, n=23 cells from 3 mice; P = 0.0067). (B-C) Directionality. (B) The NPCs were classified into rostrally migrating (arrow with circle) or non-rostrally migrating (arrows) groups based on the positions of the cell body before and after 1 h in culture. (C) The percentage of rostrally migrating Vangl2 KD NPCs was significantly lower than that of control NPCs (control, 61.0 ± 3.6%, n=95 cells from 3 mice; Vangl2 KD, 42.4 ± 5.0%, n=133 cells from 3 mice; P = 0.0398).
SC_11-1232_sm_supplFigure5.pdf236KSupplemental Fig. 5. Inhibition of Dvl2 did not affect NPC differentiation or proliferation in the SVZ. (A-D) Immunostaining of coronal sections of the SVZ, stained for GFP (green) and Mash1 (red in A, B) or Dcx (red in C, D). (E) The percentages of GFP-Mash1 double-positive transit-amplifying cells (control, 11.2 ± 2.7%, n= 155 cells from 3 mice; Dvl2-C, 16.4 ± 3.1%, n= 128 cells from 3 mice; P = 0.824) and GFP-Dcx double-positive NPCs (control, 72.6 ± 3.7%, n=3; Dvl2-C, 65.1 ± 4.8%, n=3; P = 0.782) were not altered by Dvl2ΔPDZ. (F) The percentage of mitotic cells was not altered by Dvl2ΔPDZ (control, 2.4 ± 0.7%, n= 122 cells from 3 mice; Dvl2-C, 2.1 ± 1.1%, n= 92 cells from 3 mice; P = 0.651). Scale bar: A-D, 10 μm.
SC_11-1232_sm_supplFigure6.tif2386KSupplemental Fig. 6. Inhibition of Dvl2 or Vangl2 did not affect the differentiation of NPCs in the OB. (A-F) Immunostaining of coronal sections through the SVZ infected with control (A, C, E) or Dvl2-C (B, D, F) lentiviral vector, stained for GFP (green), Dcx (red), and NeuN (blue). (G) The percentages of GFP+Dcx+NeuN- (A, B), GFP+Dcx+NeuN+ (C, D), and GFP+Dcx-NeuN- (E, F) cells were not altered by Dvl2-C, Dvl2ΔPDZ, Vangl2ΔC, or full length Vangl2, compared with control. Scale bar: A-F, 10μm.

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