Mechanisms of interkinetic nuclear migration Y Kosodo et al Figure 5 Unidirectional translocation of microbeads from the apical surface toward the basal region of the VZ. (A) Schematic of the method used to incorporate ﬂuorescent microbeads (diameters are B2 mm) into embryonic mouse brain tissue using magnetic activity, followed by tracking of their movement in slice cultures using time-lapse microscopy. (B) Images of ﬂuorescent beads in slice cultures (a, b; red) incorporated into E13.5 mouse brain tissue. The white dashed line indicates the apical surface. Beads were aligned at the apical surface at the starting time point (a). Several beads had detached from their original position after 24 h of incubation (b). Bar ¼ 50 mm. (C) Tracking of microbeads in brain slice cultures. Positions of ﬂuorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from the time-lapse images. Movements of nine microbeads acquired from three independent brain slices are displayed (slice numbers (1–3) and beads (A–D) are indicated in the graph). (D) Immunostaining of mouse brain sections from cultured tissue slices after microbead incorporation (a, b; red). Antibody for proliferating cell nuclear antigen (PCNA) stains proliferative cells (a0; green), whereas Tuj1 0 stains post-mitotic neurons (a, a ; magenta). Note that ﬂuorescent microbeads were observed only in the proliferative zone, the VZ (white arrowheads). Yellow dashed lines indicate the apical surface. Bar ¼ 10 mm. (E) Comparison of nuclear and microbead tracks in brain tissue slices. Plane positions of nuclei marked with NLS-GFP (a) or incorporated microbeads (b) were measured along the apical surface (x-coordinate) and apical–basal axis (y-coordinate). Slice numbers (1–3), nuclei and beads (A–D) are indicated in each graph. single-labelled nuclei of p18Ink4c plasmid-negative cells showed a notable accumulation close to the apical surface (Figure 6E), indicating that apical-to-basal nuclear migration is perturbed. Based on the results above, we conclude that the apical-to-basal nuclear migration that occurs in G1-phase is subject to a displacement or crowding-out effect of incoming nuclei migrating in the opposite direction after exiting S-phase. 1698 The EMBO Journal VOL 30 | NO 9 | 2011 & 2011 European Molecular Biology Organization