its association with microtubules is not promiscuous (Supplementary Figure S3C). Basal-to-apical nuclear migration is correlated with Tpx2-dependent microtubule organization in the apical process in G2-phase We next examined whether Tpx2 is involved in basal-to- apical nuclear migration by reducing its activity using a vector-based RNA interference (RNAi) approach. The protein level of Tpx2 was signiﬁcantly decreased by the RNAi treat- ment; the treatment eventually induced mitotic arrest 24 h after electroporation (Supplementary Figure S4A) and abolished mitotic spindles (Supplementary Figure S4B), as in the case of HeLa cells (Gruss et al, 2002). To examine the effect of TPX2 RNAi treatment on INM, we ﬁrst examined the positions of BrdU-containing cells in the VZ. After BrdU incorporation in S-phase, BrdU-labelled nuclei migrated toward the apical surface during incubation for 90 min (Figure 3F, cont. EP). We observed that this nuclear migration activity was signiﬁcantly decreased with a knockdown of TPX2 (Figure 3F, RNAi EP). Non-electroporated cells in the same area did not show a decrease in this migration activity (Figure 3F, RNAi noEP), suggesting that this aspect of nuclear migration is perturbed cell autonomously. We conﬁrmed that TPX2-RNAi did not result in an increase of apoptotic cells with the 18-h time course of the experiment (Supplementary Figure S4C), and that the perturbed basal-to-apical nuclear migration by TPX2-RNAi was recovered by introduction of the human TPX2 gene (Supplementary Figure S4D). Similar perturbed nuclear movement was also observed using RNAi with a different target sequence (Supplementary Figure S5A), indicating that the perturbation in nuclear migration by TPX2 knockdown was not due to an off-target effect of RNAi. More directly, we analysed the dynamics of basal-to- apical nuclear migration in brain slices treated with TPX2 RNAi and observed (1) a decrease in velocity of migration and (2) a non-linear, stepwise movement during migration (Figure 3G; Supplementary Movies S4 and S5). When TPX2 is knocked down, the average velocity of nuclear movement from basal-to-apical region (50–15 mm from the apical surface, respectively) showed a statistically signiﬁcant de- crease (Figure 3G, from 1.0±0.11 (control) to 0.5±0.07 Mechanisms of interkinetic nuclear migration Y Kosodo et al (TPX2 RNAi) mm/min, P-value o0.001, t-test). In addition, the number of subapical mitotic cells was increased in the TPX2 RNAi-treated brain, suggesting that the nuclei had entered M-phase before reaching the apical surface (Supplementary Figure S5B). This can be attributed to slower nuclear movement in the apical direction. Taken together, these results suggest that the expression of Tpx2 during G2-phase is necessary to regulate proper nuclear migration from the basal-to-apical region of the VZ. We next sought to elucidate how Tpx2 affects G2-phase nuclear migration. TPX2 RNAi might affect epithelial polarity or centrosomal activity; however, we did not ﬁnd any aber- rant pattern using markers of epithelial characteristics such as ZO-1, Par-3 and b-catenin or using a marker for TACC3, a centrosomal protein involved in INM (Xie et al, 2007), in the TPX2 knocked-down tissue (Supplementary Figure S5C–E). We then tested the possibility that microtubule organization in the apical processes of neural progenitor cells is regulated by Tpx2 in a cell cycle-dependent manner. To visualize microtubule organization at speciﬁc cell-cycle stages, we introduced ﬂuorescence-tagged EB3, a microtubule plus- end-binding protein (Mimori-Kiyosue and Tsukita, 2003) and CCPM to neural progenitor cells in the developing brain (Figure 4A). Live imaging of EB3-tagRFP characterized ‘comet-like’ movement of the protein (Supplementary Movie S6), as reported in a previous study of migrating neurons (Tsai et al, 2007), indicating that the expression level of EB3- tagRFP is moderate in our system. Because we noticed that chemical ﬁxation with paraformaldehyde or methanol dis- rupted EB3 localization in tissue (data not shown), we observed signals of EB3-tagRFP and CCPM in living brain slices. In controls, we found that EB3 signals were more sharply distributed inside the apical process in G2-phase than in G1-phase (Figure 4Bab and Cb). By contrast, in the TPX2 knocked-down cells, the proﬁle of EB3 signals during both G1- and G2-phases was essentially indistinguishable from that in normal G1-phase cells (Figure 4Cb), indicating that the sharpened microtubule distribution in the G2-phase apical process requires Tpx2. Given that Tpx2 functions to bundle puriﬁed microtubules (Schatz et al, 2003), Tpx2 is likely to alter microtubule organization in the apical process of G2-phase cells during the S to G2 progression. While Tpx2 Figure 3 Tpx2 shows temporal expression and association with microtubules in neural progenitor cells, and loss of Tpx2 function perturbs basal-to-apical nuclear migration. (A) Immunostaining for Tpx2 (a), incorporation of BrdU followed by a 2-h incubation (a0), and the merged view (a00; Tpx2, green; BrdU, magenta) in a cryosection of E14.5 mouse brain tissue. Open arrowheads indicate the Tpx2 and BrdU double- positive cells. Arrows indicate Tpx2 signals outside the nucleus. Note that dividing cells showed strong expression of Tpx2 on their mitotic spindles (white arrowheads, apical mitotic cell; red arrowhead, basal progenitor cell). Bar ¼ 10 mm. (B) (a) Schematic showing the cell cycle- dependent translocation of GFP in CCPM-electroporated cells. (b) Co-labelling of E13.5 mouse brain tissue using Tpx2 antibody, CCPM and the merged view (b00; Tpx2, magenta; CCPM, green). The white arrowhead indicates the nucleus of a G1-phase neural progenitor cell, whereas the white arrow indicates the apical process of a G2-phase cell identiﬁed by CCPM localization. Bar ¼ 10 mm. (C) Expression of GFP (a) or GFP- Tpx2 (b) in neural progenitor cells in E13.5 mouse brain tissue. Note that GFP-Tpx2 localizes to nuclei and apical processes extended in the VZ but not to basal processes. Bar ¼ 50 mm. (D) Co-expression of CCPM (a), 6myc-TPX2 (a0) and the merged view (a00; CCPM, green; 6myc-Tpx2, magenta). Red arrowheads in (a0) indicate 6myc-Tpx2 localization at apical processes. Bar ¼ 10 mm. (E) HVEM image of GFP-Tpx2 in neural progenitor cells. (a, b) A plasmid encoding GFP-Tpx2 was electroporated into E12.5 mouse brain tissue and incubated for 24 h before dissection. Immunostaining using gold particles was performed on vibratome sections, followed by specimen preparation for HVEM analysis. Note the gold particles localized within the nucleus (a) and on several ﬁbre-like structures in the apical processes (a, b). N, nucleus. Bars ¼ 1 mm. (F) Nuclear positions after BrdU incorporation in S-phase followed by a 1-h or 30-min incubation with LacZ miR RNAi as a control (cont., grey dots) or Tpx2 miR RNAi (RNAi, green dots) in E13.5 mouse brain tissue. y-coordinate: distance from apical surface (below 90 mm), EP: NLS-GFP-positive nuclei (electroporated cells), noEP; NLS-GFP-negative nuclei in the same microscopic frame (magenta dots). Black error bars indicate standard error of the mean (s.e.m.). (G) Tracking of basal-to-apical nuclear movement with LacZ miR RNAi as a control (a) or Tpx2 miR RNAi (b) in slice cultures prepared from E13.5 mouse brain tissue. Positions of nuclei relative to the apical surface (y-coordinate) were measured according to their incubation time (x-coordinate). The time point at which nuclei showed the most apical localization was deﬁned as zero. Numbers and colour codes of nuclei are indicated on the right. For (A–E), apical surface is down. & 2011 European Molecular Biology Organization The EMBO Journal VOL 30 | NO 9 | 2011 1695