Effects of Pk2 depletion on AJ orientation in the NE. (A) Still images from time-lapse imaging of the control (myrRFP) and Pk2-KD (myrGFP) NEs from Video 3 with a duration of 1 h 12 min and 3-min intervals. Imaging was initiated at stages 15–17. The green lines represent the cell orientation. A: anterior. P: posterior. The white arrowhead indicates the presumed midline. (B) Representative frequency distribution histogram of cell orientation in the control (blue) and Pk2 KD (red) NEs from Video 3. The darker color represents NEs at earlier stages. Circular angles 0 and 90 correspond to the AP and ML axes, respectively. (C) Scheme of the AJ quantification method used in Fig. 2, F–H and Fig. 10, C–E. The orientation and length of individual AJs were quantified frame by frame. (D and E) Representative images of AJ tracking in the wild-type control (D) and Pk2-KD (E) NEs. (F and G) Representative 2D plot of AJ length and orientation changes in the control (F) and Pk2 KD (G) NEs, which were tracked for 1 h and 28 min. X axis: AJ orientation. Y axis: AJ length. Large red (F) or blue (G) dots indicate AJs before tracking. Large green dots indicate AJs at the end of tracking. The plots include seven control AJs (F) and six Pk2 MO AJs from one embryo (G). (H) Quantification of changes in AJ length in the control and Pk2 MO NEs. 17 AJs (control) and 15 AJs (Pk2 KD) were randomly selected from time-lapse images of one embryo. Changes in AJ length were categorized as follows: elongation (blue, ≥0.8 μm/3 min), shrinkage (red, less than or equal to −0.8 μm/3 min), and no change (green, −0.8< and <0.8 μm/3 min). Based on orientation, AJs were categorized into AP (≤30° from the AP axis), ML (≤30° from the ML axis), and mid (30–45° from either the AP or ML axes). The two-sample Kolmogorov‒Smirnov test was used to compare distributions of AJ length changes between AP and ML junctions. The original numerical data were used for statistical analysis. The total number of AJ length changes was 353 (control) and 595 (Pk2 KD). The same analysis was conducted for two other embryos (Fig. S1, K and L). **P < 0.01, n.s., not significant. Bars: 20 μm.