Figure 1.

Dysregulation of Nwd1 Disturbs the Radial Migration of Neurons and Directs NSPCs to Reside in the VZ/SVZ

(A–K) Nwd1 overexpression in the embryonic neocortex. (A–C) In utero electroporation of control EGFP (A) or Nwd1 together with EGFP (B) was performed on E14.5, and the neocortex was analyzed at E16.5. (C) Distribution of electroporated EGFP⁺ cells in the indicated areas. ∗p < 0.05, ∗∗∗p < 0.001, Welch's t test followed by Holm-Bonferroni correction. (D–H) EGFP-Nwd1 (E) or control EGFP (D) was electroporated at E14.5, and brains were immunostained for Nestin (red) at E16.5. (F and G) Higher magnification of the VZ cells expressing EGFP-Nwd1 (F) and Nestin (G). (H) Ratio of EGFP⁺ or EGFP-Nwd1⁺ cells to the total number of Nestin⁺ cells in the VZ. ∗∗∗p < 0.001, Welch's t test. (I–K) Nwd1 or control EGFP was electroporated at E14.5, and the brains were collected on E18.5. (K) Distribution of EGFP⁺ cells in the indicated layers. ∗p < 0.05, ∗∗p < 0.01, Welch's t test followed by Holm-Bonferroni correction.

(L–S) Nwd1 knockdown represses neuronal migration. An Nwd1 shRNA (shRNA #1 or shRNA #2) was co-electroporated with EGFP at E14.5 and cortices were analyzed at E16.5 (L–O) or E18.5 (P–S). (O) Distribution of EGFP⁺ cells in the indicated areas at E16.5. ∗p < 0.05, ∗∗p < 0.01, Welch's t test followed by Holm-Bonferroni correction. (R) Nwd1 shRNA was co-electroporated with the full-length human NWD1 cDNA at E14.5, and the cortex was analyzed at E18.5. (S) Distribution of EGFP⁺ cells in the indicated areas at E18.5. NS, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, Welch's t test followed by Holm-Bonferroni correction.

All data are presented as means ± SEM. Scale bars, 50 μm in (A), (B), (D), €, and (L)–(N); 5 μm in (F) and (G); 100 μm in (I), (J), and (P)–(R).