Figure 3.

Acute DBZ insufficiency leads to impaired radial migration, poor neurite elongation and a loss of centrosome-nucleus coupling. A, The efficiencies of RNA interference with different shRNAs against DBZ (Nos. 1–3) were evaluated. No. 1 markedly suppressed EGFP-DBZ expression and was subsequently used for all DBZ-knockdown experiments. B, shRNAs against DBZ No. 1 suppressed endogenous DBZ expression in N2a cells. GAPDH was used as a loading control. C, The mU6pro scramble vector (mU6 sc) was used as a control (left). DBZ-knockdown radially migrating cells did not migrate far beyond the intermediate zone (right). CP, Cortical plate; IZ, intermediate zone. The neocortex was subdivided evenly into five bins (1–5) from the ventricular zone (VZ) to the CP. The occurrences of GFP-labeled cells (cell bodies) in each bin were counted. D, DBZ-knockdown neurons that were cotransfected with the hDBZ migrated into the cortical plate (left), whereas sole DBZ-knockdown neurons did not migrate (right, the same image of C). Scale bar, 100 μm. E, Neurons in the cortical plate were examined in the brain slices. Neurons, their centrosomes and their nuclei, were labeled with DS-Red2 (shown in red), EGFP-CETN2 (green), and Hoechst (blue), respectively. The nucleus–centrosome distances were greater in neurons with RNAi against DBZ compared with controls. CETN2, Centrin 2. Arrows indicate centrosomes, and arrowheads represent the edges of nuclei. Scale bar, 5 μm. F, At E17.5, DBZ-expressing neurons were collected from the cortices and dissociated for primary culture. The lengths of the primary neurites were measured after culture for 96 h. Acute DBZ-insufficient neurons had shorter primary neurites than controls. Scale bar, 100 μm. The values represent the mean ± SEM; *p < 0.05.