Figure 4.
Cortical levels of LIN-5/GPR-1/2 are essential for the timely enrichment of NMY-2 at the equatorial cortex. (A–G) Confocal image of a control embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2) in pseudocolor intensity gradient (A). The selected furrow region of GFP-NMY-2–expressing embryos in control or GFP-NMY-2–expressing embryos in lin-5(ts) background is utilized to build time-lapse pseudokymographs under various RNAi conditions, as indicated in B–G: control (B); lin-5(RNAi) in lin-5 (ts) background (C); gpr-1/2(RNAi) (D); par-2(RNAi) (E); par-5(RNAi) (F); and lin-5(RNAi) and par-5(RNAi) in lin-5(ts) background (G). Pseudokymographs begin at 100 s after NEBD. The scale bar is 10 and 5 µm for the embryo and the pseudokymograph, respectively. A heatmap of fluorescence intensity is given (bottom right) for reference. (H and I) Schematic of the method used to quantify GFP-NMY-2 fluorescence intensity at furrow region over time. Quantification of GFP-NMY-2 intensity at the furrow region in control embryos (black line), as well as in lin-5(RNAi) in lin-5 (ts) background (pink line), gpr-1/2(RNAi) (blue line), par-2(RNAi) (orange line), par-5(RNAi) (cyan line), and par-5(RNAi) and lin-5(RNAi) in lin-5(ts) (asparagus line) embryos. n is the number of embryos analyzed. Time is w.r.t. NEBD. Error bars, SEM. ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test. Please also see Fig. 3 and Fig. S3 for cortical and central plane localization of GFP-NMY-2 in LIN-5–inhibited embryos. (J–M) The representative pseudokymographs of the division plane (depleted on the top right) were acquired by time-lapse confocal microscopy of control embryos expressing GFPMem. and mCherryTub., which were either left untreated (J), or depleted for PAR-5 (K), lin-5(ts) embryos expressing GFPMem. and mCherryTub. that were treated with lin-5(RNAi) (L), or lin-5(ts) embryos expressing GFPMem. and mCherryTub. that were treated with lin-5(RNAi) and par-5(RNAi) (M), as indicated. Please note that only the GFPMem. signal is shown (in green) for easy comparative analysis of furrow involution timing. Control (n = 17); par-5(RNAi) (n = 10); lin-5(RNAi) in lin-5(ts) background (n = 10); and lin-5(RNAi) and par-5(RNAi) in lin-5(ts) background (n = 12). n is the number of embryos analyzed. The pseudokymograph begins at 150 s after NEBD. The scale bar is 5 µm. (N) Quantification of the time interval between NEBD and furrow involution in control, par-5(RNAi) embryos coexpressing GFPMem. and mCherryTub., or lin-5(ts) embryos coexpressing GFPMem. and mCherryTub. that are treated with lin-5(RNAi) or lin-5(RNAi), and par-5(RNAi), as indicated. Each dot represents one embryo, and n is the number of embryos analyzed. ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test.

Cortical levels of LIN-5/GPR-1/2 are essential for the timely enrichment of NMY-2 at the equatorial cortex. (A–G) Confocal image of a control embryo expressing endogenously tagged NMY-2 with GFP (GFP-NMY-2) in pseudocolor intensity gradient (A). The selected furrow region of GFP-NMY-2–expressing embryos in control or GFP-NMY-2–expressing embryos in lin-5(ts) background is utilized to build time-lapse pseudokymographs under various RNAi conditions, as indicated in B–G: control (B); lin-5(RNAi) in lin-5 (ts) background (C); gpr-1/2(RNAi) (D); par-2(RNAi) (E); par-5(RNAi) (F); and lin-5(RNAi) and par-5(RNAi) in lin-5(ts) background (G). Pseudokymographs begin at 100 s after NEBD. The scale bar is 10 and 5 µm for the embryo and the pseudokymograph, respectively. A heatmap of fluorescence intensity is given (bottom right) for reference. (H and I) Schematic of the method used to quantify GFP-NMY-2 fluorescence intensity at furrow region over time. Quantification of GFP-NMY-2 intensity at the furrow region in control embryos (black line), as well as in lin-5(RNAi) in lin-5 (ts) background (pink line), gpr-1/2(RNAi) (blue line), par-2(RNAi) (orange line), par-5(RNAi) (cyan line), and par-5(RNAi) and lin-5(RNAi) in lin-5(ts) (asparagus line) embryos. n is the number of embryos analyzed. Time is w.r.t. NEBD. Error bars, SEM. ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test. Please also see Fig. 3 and Fig. S3 for cortical and central plane localization of GFP-NMY-2 in LIN-5–inhibited embryos. (J–M) The representative pseudokymographs of the division plane (depleted on the top right) were acquired by time-lapse confocal microscopy of control embryos expressing GFPMem. and mCherryTub., which were either left untreated (J), or depleted for PAR-5 (K), lin-5(ts) embryos expressing GFPMem. and mCherryTub. that were treated with lin-5(RNAi) (L), or lin-5(ts) embryos expressing GFPMem. and mCherryTub. that were treated with lin-5(RNAi) and par-5(RNAi) (M), as indicated. Please note that only the GFPMem. signal is shown (in green) for easy comparative analysis of furrow involution timing. Control (n = 17); par-5(RNAi) (n = 10); lin-5(RNAi) in lin-5(ts) background (n = 10); and lin-5(RNAi) and par-5(RNAi) in lin-5(ts) background (n = 12). n is the number of embryos analyzed. The pseudokymograph begins at 150 s after NEBD. The scale bar is 5 µm. (N) Quantification of the time interval between NEBD and furrow involution in control, par-5(RNAi) embryos coexpressing GFPMem. and mCherryTub., or lin-5(ts) embryos coexpressing GFPMem. and mCherryTub. that are treated with lin-5(RNAi) or lin-5(RNAi), and par-5(RNAi), as indicated. Each dot represents one embryo, and n is the number of embryos analyzed. ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test.

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