RGC translocation kinetics. (A) Developing eye of a 34-hpf embryo. ath5:gap-GFP transgene labels RGCs. The dashed box shows the typical area displayed in subsequent montages. Bar, 50 µm. (B) Typical example of RGC translocation in LSFM. Arrowheads, basal process. Bar, 10 µm. (C) Kinetics of RGC translocation in a spinning disk confocal microscope. 0 indicates mitotic position of cells. Eight single trajectories (n = 4 experiments) and a mean trajectory ± SD are shown plus the mean of wild-type trajectories in LSFM. (D) Kinetics of RGC translocation in LSFM. 0 indicates mitotic position of cells. 140 single trajectories and a mean trajectory ± SD are shown. Green phase, directionally persistent movement; gray phase, fine positioning. (E) MSDs of RGCs for directional phase and fine positioning. MSDs are from the first 95 min after mitosis and the first 95 min after reaching the basal side. α value is given with a 95% confidence interval. (F) Directionality ratio of RGCs in directional movement and fine positioning. Data from E. (E and F) Error bars represent SEM. Final directionality ratios: directional = 0.88; fine positioning = 0.28. The scheme defines the directionality ratio between the distance from start to finish of the trajectory (d) and the length of the trajectory (D). (G) Aphidicolin/hydroxyurea stalls cells in S phase. Thus, nuclei do not migrate toward the apical side for mitosis. (H) ath5:gap-GFP embryos treated with 150 µM aphidicolin/20 mM hydroxyurea imaged in a spinning disk microscope from 34 hpf. Imaging started 1 h after drug addition. Bar, 5 µm. (B and H) White dots, RGC followed. Time is shown in hours and minutes. Dashed lines delimit the apical and basal sides. (I) RGC layer still forms upon cell cycle inhibition. Fewer mitotic cells (right) compared with control (left) are seen by pH3 staining (magenta). Dashed lines mark the retinal outline and RGC layer. Bar, 50 µm.