Figure 2.
Cortical accumulation of LIN-5, but not its asymmetric cortical distribution, is vital for timely furrow induction. (A–C) 3D projected confocal images of a one-cell embryo expressing endogenous LIN-5 tagged with mNeonGreen (mNG) (LIN-5-mNG) at the time of furrow formation in control (A), par-2(RNAi) (B), and par-3(RNAi) (C). Here, and in subsequent embryo images, the A is to the left, and the P is to the right. The scale bar is 10 µm. (D) Schematic of the method to quantify the enrichment of LIN-5-mNG at the furrow initiation stage. Here and in subsequent Fig. panels, Mem. represents membrane intensity; A: anterior; P: posterior; Bkgd. represents background intensity, and Cyto. represents cytoplasmic intensity. (E) Quantification of LIN-5-mNG level at the anterior polar cortex (gray circle) and posterior polar cortex (pink circle) in control (n = 9), par-2(RNAi) (n = 12), and par-3 (RNAi) (n = 8) embryos at furrow onset. n is the number of embryos analyzed. Error bars are the SEM; ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test. (F–J) Representative images from the time-lapse confocal microscopy of embryo coexpressing GFPMem./Tub.;mCherryChr./Cent. in control (F) and various RNAi/mutant conditions, as indicated (G–J): control (n = 12); par-2(RNAi) (n = 9); par-3(RNAi) (n = 9); spd-1(ts) (n = 13); par-2(RNAi) in spd-1(ts) (n = 9). n is the number of embryos analyzed. The scale bar is 10 µm. (K–O) Representative pseudokymographs of the division plane (shown on the top right) acquired by time-lapse confocal microscopy of control (K) and various RNAi/mutant embryos coexpressing GFPMem./Tub.;mCherryChr./Cent., as indicated (L–O). Please note that for the easy comparative analysis of furrow involution timing, only the GFPMem./Tub. signal is shown (in gray). Pseudokymograph begins at 100 s w.r.t NEBD. The scale bar is 5 µm. (P) Quantification of the time interval between NEBD and furrow involution in embryos coexpressing GFPMem./Tub.;mCherryChr./Cent., which were either left untreated (control), or treated with various RNAi, in spd-1(ts) background, or GFP-Mem./Tub.;mCherryChr./Cent. embryos in spd-1(ts) background that is treated with par-2(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. (Q) Aster separation w.r.t embryo length (d/l) in embryos GFPMem./Tub.;mCherry-Chr./Cent., as shown for Fig. 1, A and B in control and different RNAi/mutant conditions. Time is w.r.t. NEBD, which was monitored by the appearance of the tubulin signal in the pronuclei. Dotted straight lines mark the average time for the anaphase onset and furrow initiation in control embryos. The respective shaded color shows the error bars in SEM. n is the number of embryos analyzed.

Cortical accumulation of LIN-5, but not its asymmetric cortical distribution, is vital for timely furrow induction. (A–C) 3D projected confocal images of a one-cell embryo expressing endogenous LIN-5 tagged with mNeonGreen (mNG) (LIN-5-mNG) at the time of furrow formation in control (A), par-2(RNAi) (B), and par-3(RNAi) (C). Here, and in subsequent embryo images, the A is to the left, and the P is to the right. The scale bar is 10 µm. (D) Schematic of the method to quantify the enrichment of LIN-5-mNG at the furrow initiation stage. Here and in subsequent Fig. panels, Mem. represents membrane intensity; A: anterior; P: posterior; Bkgd. represents background intensity, and Cyto. represents cytoplasmic intensity. (E) Quantification of LIN-5-mNG level at the anterior polar cortex (gray circle) and posterior polar cortex (pink circle) in control (n = 9), par-2(RNAi) (n = 12), and par-3 (RNAi) (n = 8) embryos at furrow onset. n is the number of embryos analyzed. Error bars are the SEM; ns, P > 0.05; ***P < 0.001, as determined by two-tailed Student’s t test. (F–J) Representative images from the time-lapse confocal microscopy of embryo coexpressing GFPMem./Tub.;mCherryChr./Cent. in control (F) and various RNAi/mutant conditions, as indicated (G–J): control (n = 12); par-2(RNAi) (n = 9); par-3(RNAi) (n = 9); spd-1(ts) (n = 13); par-2(RNAi) in spd-1(ts) (n = 9). n is the number of embryos analyzed. The scale bar is 10 µm. (K–O) Representative pseudokymographs of the division plane (shown on the top right) acquired by time-lapse confocal microscopy of control (K) and various RNAi/mutant embryos coexpressing GFPMem./Tub.;mCherryChr./Cent., as indicated (L–O). Please note that for the easy comparative analysis of furrow involution timing, only the GFPMem./Tub. signal is shown (in gray). Pseudokymograph begins at 100 s w.r.t NEBD. The scale bar is 5 µm. (P) Quantification of the time interval between NEBD and furrow involution in embryos coexpressing GFPMem./Tub.;mCherryChr./Cent., which were either left untreated (control), or treated with various RNAi, in spd-1(ts) background, or GFP-Mem./Tub.;mCherryChr./Cent. embryos in spd-1(ts) background that is treated with par-2(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. (Q) Aster separation w.r.t embryo length (d/l) in embryos GFPMem./Tub.;mCherry-Chr./Cent., as shown for Fig. 1, A and B in control and different RNAi/mutant conditions. Time is w.r.t. NEBD, which was monitored by the appearance of the tubulin signal in the pronuclei. Dotted straight lines mark the average time for the anaphase onset and furrow initiation in control embryos. The respective shaded color shows the error bars in SEM. n is the number of embryos analyzed.

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