Mechanisms of interkinetic nuclear migration Y Kosodo et al Figure 1 Quantitative tracking of nuclear movement of cortical neural progenitor cells in embryonic mouse brain slice cultures. (A) Schematic model of INM of neural progenitor cells. Nuclei in the VZ, the closest tissue layer to the ventral surface of the developing brain, show an oscillatory movement along the apical–basal epithelial axis that is associated with the phase of the cell cycle (see Introduction). The colour code of cell-cycle phases is indicated on the right. (B) Representative movement of a nucleus undergoing INM. Nuclei of neural progenitor cells were labelled by NLS-GFP, and their movements in slice cultures prepared from E13.5 mouse brains were tracked by time-lapse microscopy. Using the tracking software, positions of nuclei from 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. Phases of the cell cycle, estimated from previous reports, are indicated below (see Results). Numbers and colour codes of nuclei are indicated on the right (a or b after the numbers indicate daughter cells derived from cell division at the apical surface). Murciano et al, 2002) or microtubules (Messier, 1978) suggest that proper cytoskeletal organization is either directly or indirectly necessary for INM. Indeed, it has been suggested that INM is perturbed by eliminating the activity of several molecules with putative functions in cytoskeletal regulation, including b-catenin (Machon et al, 2003), Cdc42 (Cappello et al, 2006), gap-junction proteins (Pearson et al, 2005), the serine/threonine kinase CK2 (Carneiro et al, 2008), Rac (Minobe et al, 2009), Myosin II (Norden et al, 2009) and Laminin (Tsuda et al, 2010). Components of microtubule- based dynein motor complex, such as Lis1 (Tsai et al, 2005) and dynactin (Del Bene et al, 2008), and centrosomal activity at the apical surface (Tamai et al, 2007; Xie et al, 2007), have been clearly shown to be involved in basal-to-apical nuclear migration. The connection between dynein and the nuclear envelope is maintained by SUN-domain proteins and KASH- domain proteins; loss of their molecular function impairs INM (Del Bene et al, 2008; Zhang et al, 2009). Thus, it is likely that basal-to-apical nuclear migration is actively driven by microtubule-based motors and is therefore cell autono- mous. However, cell cycle-dependent expression has not been reported for known molecular drivers of basal-to-apical nuclear migration, and it remains unexplained why this movement occurs during G2-phase. In the present study, we sought to address a pair of questions that must be answered before a comprehensive model of INM can be developed. Namely, what mechanisms couple the timing and direction of nuclear movement with cell-cycle progression in neural progenitor cells, and how is & 2011 European Molecular Biology Organization apical-to-basal migration achieved? Recent reports demon- strate that the actomyosin system (Schenk et al, 2009) or kinesin-type microtubule plus-end-directed motors (Tsai et al, 2010) are involved in carrying nucleus from the apical region to the basal region of the VZ, although mechanisms by which such cytoskeletal motors act in G1-phase are not addressed. We here report that the basal-to-apical migration of neural progenitor cells that occurs in INM is regulated by Tpx2, a microtubule-associated protein (Gruss et al, 2001; Schatz et al, 2003). Tpx2 is localized to apical processes during G2-phase and alters microtubule organization in a cell cycle- dependent manner. Moreover, we show by in situ experi- ments and computational modelling that the apical-to-basal migration of nuclei in G1-phase occurs mainly through passive, non-autonomous displacement, which we ascribed to the autonomous nuclear movement of G2-phase nuclei moving in the opposite direction. The resulting model of INM describes a mechanism for VZ tissue homeostasis coordi- nated with progenitor cell proliferation. Results Nuclei of neural progenitor cells show characteristic movement depending on the phase of the cell cycle To analyse nuclear movement during INM, we established a system that enabled us to quantitatively track the motion of individual nuclei in living tissue. Nuclei in the dorsal cortex of an E13.5 mouse brain were labelled by green ﬂuorescent protein (GFP) containing a nuclear localization signal (NLS) The EMBO Journal VOL 30 | NO 9 | 2011 1691