Rac1 activation is required for the inhibition of cell packing by Pk2. (A–F) Representative images of Xenopus gastrula ectoderms expressing myrGFP only (A), myrGFP with DN-RhoA (B), DN-Rac1 (C), Flag-Pk2 (D), Flag-Pk2+DN-RhoA (E), or Flag-Pk2+DN-Rac1 (F). Imaging was initiated at stage 11.5. Pigmented wild-type embryos were used for image acquisition and quantification. (G–K) Quantification of cell packing on the basis of variations in AJ linearity (G), AJ length (H and J), and the number of neighboring cells (I and K). Statistical analyses in G, H, and J were conducted on the original numerical data. One-way ANOVA was used to compare mean ranks, with the Bonferroni correction for multiple pairwise comparisons in G. The Kolmogorov‒Smirnov test was used to compare distributions in H and J. The chi-square test was used in I and K. Three embryos per group were used. The total number of cells and AJs was as follows: 214 cells and 781 AJs (myrGFP only); 161 cells and 594 AJs (DN-RhoA only); 184 cells and 681 AJs (DN-Rac1 only); 236 cells and 828 AJs (Pk2 only); 177 cells and 656 AJs (Pk2 with DN-RhoA); and 189 cells and 694 AJs (Pk2 with DN-Rac1). *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. Scale bars: 20 μm.