Pk2 KD inhibits AP cell orientation in the NE. (A–D) Snapshot images of F-actin distribution during cell intercalation and apical constriction in control NEs expressing Scarlet-UtrCH (purple) and Pk2-KD NEs expressing Lifeact-GFP (green). The white rectangular areas in A and B are enlarged in C and D. White circles indicate T1 junctions. The double arrows show AP elongation of newly formed AJs in the control NE. The white arrowheads indicate short-lived F-actin meshwork at the apical cortex. The purple arrowhead indicates stable F-actin enrichment at the apical cortex in apically constricted cells. Note the presence of short-lived apical F-actin but the absence of cells with stable apical F-actin in the Pk2 KD NE (D). (E) 2D plots representing F-actin abundance at the apical cortex in the control (blue) and Pk2-KD (red) NEs. The x axis represents the apical domain size of individual cells. The y axis represents the integrated fluorescence intensity of phalloidin staining. Dots represent individual cells. 234 cells (control) and 331 cells (Pk2 MO) from three embryos per group were used. Statistical analysis was conducted on two categorized cell populations: cells with small apical domains (<100 μm²) and cells with large apical domains (>100 μm²). The Mann‒Whitney test was used to compare ranks between two groups. (F) Lengths of anteroposteriorly or mediolaterally oriented AJs in the control (left) and Pk2 KD (right) NEs. AJs were categorized into AP (≤30° from the AP axis) and ML (≤30° from the ML axis). The bold and thin lines indicate medians and quartiles, respectively. The Mann‒Whitney test was used to compare mean ranks. n = 339 (control, AP); n = 209 (control, ML); n = 233 (Pk2 KD, AP); n = 402 (Pk2 KD, ML). Three embryos per group were used. (G–G‴) Representative images of phalloidin-stained NE. The control is on the left. Pk2 KD is on the right. Phalloidin staining only (G), overlaid with color coding on the basis of the apical domain size (G′), with the green bars indicating cell orientation (G″), and with GFP indicating Pk2 MO cells (G‴). (H) Snapshot images of the NE expressing Scarlet-UtrCH only (left) and Lifeact-GFP coinjected with Pk1 MO (right) from time-lapse imaging. The green lines represent the orientation of individual cells. (I and J) Frequency distribution histogram of cell orientation in the control (blue), Pk2 KD (red in J), and Pk1 KD (red in K) NEs. Averages and ±S.Ds. are shown. The Kolmogorov‒Smirnov test was used to compare distributions. Three embryos per group were used. The total number of cells was as follows: n = 920 (control in I), n = 1,093 (Pk2 KD in I), n = 120 (control in J), and n = 130 (Pk1 KD in J). (K and L) Quantification of AJ length change and orientation in the control NEs (left) and Pk2 MO NEs (right). The statistical analyses were conducted as described in Fig. 2 H. 15 AJs per embryo were used. The total number of AJ length changes was as follows: n = 353 (control in K), n = 595 (Pk2 KD in K), n = 171 (control in L), and n = 195 (Pk2 KD in L). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Scale bars: 20 μm.