Mechanisms of interkinetic nuclear migration Y Kosodo et al Figure 6 Cell-cycle arrest in S-phase perturbs apical-to-basal nuclear movement of neural progenitor cells in G1-phase. (A) Assessment of drug conditions that inhibit S-phase of neural progenitor cells in slice cultures prepared from E13.5 mouse brain tissue. (a–c) Immunostaining of cryosections prepared from brain slices after drug treatment. Concentrations of HU are indicated in the panels. Green, phosphorylated Histone H3 (mitotic cells); red, ZO-1 (apical surface). Bar ¼ 50 mm. (d) M-phase neural progenitor cells in cultured slices after drug treatment. The average number of mitotic cells per 100 mm of apical surface under several conditions are indicated. Note that HU resulted in a concentration- dependent reduction of M-phase neural progenitor cells. FU, 5-ﬂuorouracil. (B) Tracking of nuclear movements in brain slice cultures with HU treatment. Nuclei of neural progenitor cells were marked with NLS-GFP, and their distances from the apical surface (y-coordinate) were plotted versus their incubation time (x-coordinate). Red arrowheads indicate the time point when control vehicle (a) or 1 mM HU (b) was added to the medium (6 h after incubation started). Numbers and colour codes of nuclei are indicated on the right (a or b after the numbers indicates daughter cells derived from cell division at the apical surface). (C) Apical-to-basal nuclear movements following cell division at the apical surface during 8 h of incubation after adding (a) control vehicle, (b) 0.5 mM HU or (c) 1 mM HU. Colour codes are explained above. (d) Average velocities of nuclei are shown in (a–c) (20–22 cases in each condition). **Po0.005, ***Po0.001, t-test. Error bars indicate s.e.m. (D) Effect of HU treatment on microbead movement induced from the apical surface. Positions of ﬂuorescent beads relative to the apical surface (y-coordinate) were measured at each time point (x-coordinate) from time-lapse images. Six hours after adding microbeads to the brain slice cultures, control vehicle (a) or 1 mM HU (b) was added to the medium (indicated as time point 0). Tracked movements of 10 microbeads in each condition are displayed. (E) Effect of co-existing G1-arrested cells on the basally oriented nuclear movement of non-arrested cells. After in utero electroporation of plasmids of p18Ink4c and NLS-GFP into the E13.5 mouse brain, two thymidine analogues (CldU and IdU) were introduced into pregnant mice at different times (5 and 2 h, respectively) before ﬁxing the embryos. With this procedure, CldU single-labelled cells were identiﬁed as having been in S-phase 2–5 h before ﬁxation. During this time window, CldU single-labelled cells reach the apical surface, therefore their distribution pattern indicates nuclear position of late M-phase or G1-phase cells. Nuclei of p18Ink4c plasmid-negative cells were identiﬁed by the absence of NLS-GFP signals, and their position from the apical surface was measured. Histogram shows the comparison of the GFP-negative CldU single-labelled nuclear positions of six electroporated brains from three independent experiments (total number of measured nuclei; control n ¼ 550, p18Ink4c n ¼ 594). Computational modelling displays non-autonomous nuclear migration To evaluate the possibility that apical-to-basal nuclear migra- tion occurs as a result of the displacement of nuclei in the apical VZ, we constructed a computational model for INM, and qualitatively compared the outcomes predicted by the model with the experimental data. To approximate the VZ occupied by nuclei, we designed a simpliﬁed two-dimen- sional model in which nuclei are represented as circles in a planar VZ constrained in the radial, but not lateral, direction. & 2011 European Molecular Biology Organization Basic rules were set for nuclei to move in the VZ to avoid neighbouring nuclei (Figure 7Aa): (1) A searching radius (SR) around each nucleus was assumed. (2) Each nucleus searches the area of the SR and then goes to the centre of gravity of an area within the SR that is unoccupied by nuclei. (3) The cell- cycle phase of each nucleus has a randomly provided initial value (colour codes are indicated in Figure 7Ab); the lengths of the cell-cycle phases were taken from values that have been experimentally measured in the developing mouse brain (Takahashi et al, 1995). If no commands are provided to The EMBO Journal VOL 30 | NO 9 | 2011 1699