Mechanisms of interkinetic nuclear migration Y Kosodo et al using in utero electroporation. Labelled nuclei in cultures of brain slices were tracked using a video imaging system (Supplementary Movie S1), and their location at each time frame was plotted (Figure 1B). After mitosis at the apical surface (time point ¼ 0 in Figure 1B), nuclei migrate basally within the VZ. Before mitosis, nuclei migrate apically. Using previous studies that measured the length of the cell cycle in neural progenitor cells (Takahashi et al, 1995), we were able to correlate the position of tracked nuclei during INM with phases of the cell cycle (Figure 1B). In this way, we conﬁrmed the basic scheme of INM described previously (Sauer, 1935; Sauer and Walker, 1959; Fujita, 1960), suggesting that our experimental set-up faithfully represented in vivo neural progenitor dynamics. Analysis of the kinetics of INM identi- ﬁed three novel features of nuclear movement. First, nuclear ‘ratcheting’, a forward and backward motion of nuclei, occurs while the nuclei migrate toward the basal side during G1-phase (after 0 min in Figure 1B). Second, during the basal- to-apical migration before mitosis (G2-phase, around 120 to 0 min in Figure 1B), the nuclei show linear movements and faster kinetics than nuclei that are moving in the opposite direction. Third, the individual positions of nuclei within the population differ remarkably before they begin basal-to-api- cal migration (during S-phase, before 120 min in Figure 1B). Furthermore, some nuclei show slow basal-to-apical migra- tion during S-phase, although most remain stationary. These features of S-phase nuclei have been indicated by other reports using histological methods (Takahashi et al, 1993; Hayes and Nowakowski, 2000), suggesting that they are not artifacts of our experimental system. Molecular evidence for the cell cycle dependence of INM Individual phases of INM are tightly correlated with phases of the cell cycle, but it has not been determined how migration depends on cell-cycle progression. To address this question, we ﬁrst examined whether INM depends on G1- to S-phase progression. The cell cycle of neural progenitors was arrested at G1-phase by overexpression of p18Ink4c, a cyclin-dependent kinase inhibitor (Guan et al, 1994; Sherr and Roberts, 1999). Introduction of p18Ink4c by in utero electroporation resulted in a decrease in the number of cells expressing Ki67, a marker for the proliferative state (Figure 2A). The electroporated cells were neither labelled by BrdU, which is incorporated into DNA during S-phase (Supplementary Figure S1A), nor observed histologically to be in M-phase. These cells, there- fore, had passed through M-phase and were arrested in G1-phase by the time of analysis (18 h after electroporation). Interestingly, at E10.5, when proliferative cells are dominant, the cell bodies of the p18Ink4c-electroporated cells accumu- lated in the basal region of the VZ, with their long apical processes extended toward the apical surface (Figure 2B). This phenomenon is not speciﬁc to this developmental stage, as statistical measurements showed basal accumulation of G1-arrested nuclei in the VZ at E14.5 as well (Figure 2C and D). The basal nuclear localization of p18Ink4c-expressing cells may be due to differentiation of G1-arrested progenitor cells into neurons that do not migrate to the apical surface. However, we conﬁrmed that the progenitor state is not affected in p18Ink4c-expressing cells based on expression of Sox2 (Supplementary Figure S1B) and Pax6 (Supplementary Figure S1C), markers for apical neural progenitor cells (Go¨ tz et al, 1998; Graham et al, 2003). Furthermore, we did not 1692 The EMBO Journal VOL 30 | NO 9 | 2011 observe any signiﬁcant changes in the pattern of Tuj1 stain- ing, a marker for neurons, nor any increase in expression of Tbr2, a marker for differentiating intermediate progenitor cells (Kowalczyk et al, 2009), 24 h after electroporation (Supplementary Figure S1D and E). These results indicate that the nuclei of neural progenitor cells do not migrate in the apical direction when they are arrested in G1-phase and suggest that entry into S-phase is a prerequisite for basal- to-apical nuclear migration. Tpx2, a microtubule-associated protein, regulates basal-to-apical nuclear migration during G2-phase The dependence of basal-to-apical migration on both cell- cycle progression (Figure 2) and microtubules (Supple- mentary Figure S2; Supplementary Movie S2; Tsai et al, 2005; Xie et al, 2007) indicates that microtubule regulation coupled with the cell cycle might control the timing of nuclear migration. Based on reports from other studies (Gruss et al, 2002; Schatz et al, 2003; Gruss and Vernos, 2004), one possible candidate molecule fulﬁlling this role is Tpx2. Tpx2 is a microtubule-nucleating/bundling protein involved in spindle pole formation around chromosomes dependent on Ran-GTP activity (Gruss and Vernos, 2004). In HeLa cells, Tpx2 protein expression is regulated by the cell cycle; it accumulates in nuclei during S/G2-phase, localizes at the spindle pole during M-phase, and is degraded in early G1-phase (Gruss et al, 2002). We ﬁrst characterized the protein expression pattern of Tpx2 in neural progenitor cells in the embryonic mouse brain. BrdU labelling to identify the phase of the cell cycle (Takahashi et al, 1992) showed that Tpx2 is expressed in neural progenitor cells in S-, G2- and M- phases (Figure 3A, open arrowheads). We noted that, in addition to the nuclear staining, Tpx2 was found in a ﬁbre- like pattern within VZ cells (Figure 3Aa, arrows). Introduc- tion of cell cycle phase marker (CCPM; Figure 3Ba; Supple- mentary Movie S3) into neural progenitor cells showed that these ﬁbre-like structures were located in the apical processes only during G2-phase, and were absent in G1-phase (Figure 3Bb; Supplementary Figure S3A), suggesting that Tpx2 func- tions in the apical processes of the G2-phase neural progeni- tor cells. When GFP-tagged Tpx2 was expressed in neural progenitor cells, GFP-Tpx2 speciﬁcally localized to the nuclei and apical processes of cells in the VZ (Figure 3C). During interphase, GFP-Tpx2 in the apical processes extended toward centrosomes on the apical surface, whereas during M-phase, it was present on mitotic spindles (Supplementary Figure S3B). Co-electroporation of CCPM and 6myc-Tpx2 revealed that the exogenous Tpx2 protein was distributed within the apical processes but never within the basal processes of G2-phase neural progenitor cells (Figure 3D). Ultrastructural analysis using high-voltage electron micro- scopy (HVEM) showed bundled ﬁbre-like patterns of GFP- Tpx2 reminiscent of microtubule structure within the apical processes (Figure 3E). The microtubule bundling activity of Tpx2 had been previously shown in a puriﬁed in vitro system (Schatz et al, 2003), and when taken together, our results strongly suggest that Tpx2 is associated with microtubules in the apical processes of neural progenitor cells. Furthermore, even though both the basal and apical processes of these cells contain large amount of microtubules, the localization of Tpx2 is restricted to the apical processes, suggesting that & 2011 European Molecular Biology Organization