LIN-5 controls cleavage furrow induction, independent of its role in aster separation. ( A and B) Schematics of the method used for measuring aster separation w.r.t. embryo length (d/l) in embryos coexpressing GFP-tagged PLCδ1-PH (membrane marker), GFP-TBB-2 (microtubules marker), mCherry-H2B (chromatin marker), and mCherry-γ-tubulin (centrosomal marker), referred to as GFP^Mem./Tub.;mCherry^Chr./Cent.. Time is w.r.t. NEBD, which was monitored by the appearance of the GFP-tubulin signal in the pronuclei. In B, dotted straight lines mark the average time for the anaphase onset and furrow initiation for control embryos, as indicated. n represents the number of embryos analyzed. The respective shaded color shows error bars in SEM. (C–H) Schematics on the left depict the effect of different RNAi conditions for the aster separation (in green) and chromosome behavior (in pink). On the right are the representative pseudokymographs of the division plane acquired by time-lapse confocal microscopy of embryos expressing GFP^Mem./Tub.;mCherry^Chr./Cent., which are either untreated (C), treated with various RNAi, or mutated for spd-1 (D–H), as indicated. For comparative analysis, only GFP^Mem./Tub. localization is shown in the pseudokymographs panel. Please note the re-use of the lin-5(RNAi) pseudokymograph in Fig. S1 D. Also, see Fig. S1, H–M. Time is w.r.t. NEBD; the scale bar is 5 µm. (I) Schematic of a one-cell embryo highlighting the selected equatorial cortical region to refer to furrow involution: the appearance of a furrow composed of two adhered back-to-back plasma membranes in a side view. (J) Quantification of the time interval between NEBD and furrow involution in embryos coexpressing GFP^Mem./Tub.;mCherry^Chr./Cent. that are either left untreated, treated with various RNAi, or mutated for spd-1 (in different colors), as indicated. Each dot represents one embryo, and n represents the number of embryos analyzed. In this and other similar graphs used to measure furrow involution timings, 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 using two-tailed Student’s t test.