LIN-5 controls furrow initiation timing independent of its function in spindle positioning. (A–C) Representative images from the time-lapse confocal microscopy of two-cell stage embryo coexpressing GFP^Mem./Tub.;mCherry^Chr./Cent. in control (A) and in lin-5(RNAi) (B), and lin-5(RNAi) in spd-1(ts) background (C). 75 s on the top panel represents the time w.r.t. NEBD. Note a delay in furrow involution in the anterior AB cell at the two-cell stage in lin-5(RNAi) and lin-5(RNAi) in spd-1(ts) embryos compared with control embryos. The scale bar is 10 µm. Since a significant percentage (∼40%) of LIN-5–depleted embryos in spd-1(ts) show cytokinesis failure at the late stage after furrow ingression (see Fig. 5), this analysis was conducted in the rest of the embryos that did not show cytokinesis failure and progress to the two-cell-stage embryo. (D and E) Schematic represents the furrow involution in AB cell (D) and the quantification of the time interval between NEBD and furrow involution in embryos coexpressing GFP^Mem./Tub.;mCherry^Chr./Cent. that are either left untreated or depleted for LIN-5, or LIN-5 depletion was performed in spd-1(ts) background (E), as indicated. Each dot represents one embryo, and n is the number of embryos analyzed. The solid black line on the graph represents the mean, whose values and SEM values are also mentioned at the bottom. ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test. (F and G) Representative images from the time-lapse confocal microscopy of the one-cell embryo coexpressing GFP^Mem./Tub.;mCherry^Chr./Cent. in lin-5(RNAi). The spindle was characterized into two categories based on orientation at anaphase: nonskewed spindle (F) and skewed spindle (G). 75 s on the left panel represents the time w.r.t. NEBD. The scale bar is 10 μm. (H and I) Schematic represents the furrow involution in P0 cell (H) and the quantification of the time interval between NEBD and furrow involution in embryos coexpressing GFP^Mem./Tub.;mCherry^Chr./Cent. with nonskewed spindle and skewed spindle (I), as indicated. Each dot represents one embryo, and n is the number of embryos analyzed. The solid black line on the graph represents the mean, whose values and SEM values are also mentioned at the bottom. ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test.