In electron micrographs, the apical process of G2-phase cells, which had been electroporated with CCPM, was recognized by the dark precipitation of DAB when immunostaining of GFP was detected by the DAB-peroxidase reaction (Figure 4D). Microtubules, which were clearly detected by a heavy DAB precipitation, were highly packed in the apical process of G2-phase cells in the control situation (Figure 4Da). When Tpx2 was knocked down (Figure 4Db), the number of microtubules in the cross-section of a DAB-posi- tive (i.e. G2-phase) apical process was nearly equal to that in the control (Figure 4Dc, 43.0±3.8 (control) or 45.0±2.1 2 (TPX2 RNAi)), but their density in the unit area (0.1 mm ) of apical process was drastically decreased (Figure 4Dd, 18.1±18 (control) or 7.1±0.5 (TPX2 RNAi)), indicating that Tpx2 facilitates microtubule packing in the apical pro- cess of G2-phase cells. Evaluated together, our results imply that basal-to-apical nuclear migration in the G2-phase re- quires Tpx2-dependent reorganization of apical microtubules during the G2-phase. Incorporated microbeads exhibit unidirectional translocation toward the basal region of the VZ The basal accumulation of G1-arrested nuclei (Figure 2) suggests that neural progenitor cells overexpressing p18Ink4c migrate basally after mitosis at the apical surface under conditions in which surrounding cells undergo normal INM. This raised the possibility of a speciﬁc mechanism that conveys nuclei from the apical to the basal region. To test whether the translocation system in the VZ is cell autono- mous or dependent on the activity of surrounding cells, we observed the behaviour of ﬂuorescent microbeads in cultures of brain slices. Magnetic ﬂuorescent beads were incorporated into the VZ from its apical surface by applying a magnet on the pial side, followed by brain slice culture and time-lapse imaging (Figure 5A). Incorporated ﬂuorescent beads were initially aligned on the apical surface. Some beads were translocated to the basal region over the course of brain slice culture (Figure 5B). HVEM imaging conﬁrmed that this treatment did not impair normal cell–cell contact or disrupt normal tissue architecture (Supplementary Figure S6). Once introduced, the beads tended to stay at the apical surface for a short time before beginning basally directed translocation (Figure 5C, see Supplementary Movie S7). During the translocation, beads showed ‘ratcheting,’ back- and-forth movement as observed in the migration of G1-phase nuclei (Figure 1B). Upon reaching the basal region of the VZ, the ﬂuorescent beads terminated their transloca- tion and never entered the neuronal layer (Figure 5D). In addition and importantly, once the beads reached the basal end of the VZ, they remained stationary and did not show the return, basal-to-apical movement (Figure 5C). We then examined whether the basal translocation of beads was due to their association with nuclei migrating in the apical-to- basal direction. A comparison of the trajectories of NLS-GFP- labelled nuclei and ﬂuorescent beads in the same brain slices (Supplementary Movie S8) clearly indicates that nuclei underwent radial migration within the elongated cell shape, whereas beads took more variable orientations (Figure 5E). This result implies that beads move not by adhesion to migrating nuclei. We thus conclude that there is a mechanism by which beads translocate from the apical surface to the basal region through intercellular space in the VZ. & 2011 European Molecular Biology Organization Mechanisms of interkinetic nuclear migration Y Kosodo et al S-phase arrest of neural progenitor cells perturbs apical-to-basal nuclear migration in G1-phase Microbead translocation in the brain slice suggests that G1-nuclei are driven in the apical-to-basal direction by a non-autonomous mechanism. However, such a mechanism is not immediately apparent. One possibility is that active basal-to-apical migration of nuclei increases the nuclear density high on the apical side of the VZ. The resultant close packing of nuclei may crowd out free nuclei that have completed mitosis, so that they are pushed further away from the ventricular surface. If this is the case, apical-to-basal migration should be affected by the acute perturbation of basal-to-apical migration of nuclei in G2-phase. To test this hypothesis, we arrested the cell cycle at S-phase by drug treatment and performed time-lapse observations of migrat- ing nuclei. We ﬁrst searched for an appropriate inhibitor to arrest the cell cycle of neural progenitor cells in the mouse brain slice culture in S-phase. Hydroxyurea (HU), which selectively inhibits ribonucleoside diphosphate reductase (Wright et al, 1990), leads to a dose-dependent reduction in the number of mitotic cells in the VZ (Figure 6A) without inducing apoptosis below 1 mM (Supplementary Figure S7A). As previously reported in chick neuroepithelium (Murciano et al, 2002), we conﬁrmed that HU treatment results in an accumulation of S-phase neural progenitor cells by measuring the rate of BrdU incorporation in nuclei immediately after washing out the HU (Supplementary Figure S7B). The results indicated that the G1–S transition was not impaired by HU treatment. We tracked nuclear movement under these conditions in slice cultures to see the effect of HU administration (Figure 6B; Supplementary Movies S9 and S10). In the presence of HU, the number of apically migrating nuclei was dramatically decreased and S-phase nuclei accu- mulated in the basal region of the VZ. This result was expected, as HU arrests progenitor cells at S-phase, prevent- ing entry into G2-phase. Time-lapse observations revealed that apical-to-basal migration was also quickly perturbed by HU treatment (Figure 6B and C). Under this condition, the average velocity of nuclei moving in the basal direction was signiﬁcantly decreased (Figure 6Cd). We did note a few rapidly moving unidentiﬁed nuclei (3/42 cases with HU, Figure 6C); however, the majority were impeded. This acute delay in apical-to-basal migration is most likely due to the lack of a decrease in basal nuclear density, and of an increase in apical nuclear density, both of which are simultaneously caused by individual nuclei migrating api- cally in the normal situation. To conﬁrm that the perturbation of basally directed nuclear movement is not due to unex- pected effects of HU on G1-phase cells, but instead due to the physical displacement effect, we tested whether microbead translocation (Figure 5) is also perturbed by the same drug treatment. Indeed, most ﬂuorescent beads incorporated from the apical surface translocated shorter distances after treat- ment with 1 mM HU than in the control (Figure 6D). To further corroborate the nuclear displacement effects, we arrested the cell cycle of a population of cells at G1-phase by introducing p18Ink4c plasmid (see Figure 2), and examined the distribution of pulse-labelled nuclei (by two thymidine analogues: CldU and IdU, see the ﬁgure legend), that have not incorporated p18Ink4c plasmid (i.e. not affected by the cell-cycle arrest). When p18Ink4c plasmid was intro- duced to the surrounding cells, the distribution of CldU The EMBO Journal VOL 30 | NO 9 | 2011 1697