Shroom is required for Rho-kinase localization and force generation during convergent extension. (A–C) Localization of Rho-kinaseK116A and Baz/Par-3 in wild-type (WT; A), ShrmΔ13.6 mutant (B), and ShrmAB (C) RNAi embryos at stage 8. Anterior is left, and ventral is down. (D) Junctional enrichment is the ratio of the mean pixel intensity at adherens junctions divided by the mean pixel intensity in the medial–apical cortex and cytoplasm (pixels ≥1 µm from the cell boundary). Planar polarity is the mean intensity of AP edges (75–90° relative to the AP axis) divided by the mean intensity of DV edges (0–15°). (E) Rho-kinase was less junctionally enriched in Shroom mutants in stage 8 (P < 0.0001). (F) Rho-kinase was less planar polarized in Shroom mutants in stage 8 (P = 0.05). Wild-type Rho-kinase planar polarity was slightly higher than in Fig. 1 C because different Rho-kinaseK116A transgenes and fixation methods were used (Materials and methods). (G and H) Z stack (G) and cross section (H) of Rho-kinaseK116A in a stage 8 wild-type embryo (0 µm is at the level of adherens junctions, and −2 to 8–µm images are at the indicated distance basal to the junctions). (I and J) Z stack (I) and cross section (J) of Rho-kinaseK116A in a stage 8 ShrmΔ11 mutant. (K) Peak retraction velocities after laser ablation of control (flp dsRNA injected) and ShrmAB RNAi embryos in early stage 8. ShrmAB RNAi embryos had slower retraction velocities at AP edges (P = 0.003) and no change at DV edges (P = 0.36) compared with controls (19 AP and 8 DV ablations in flp RNAi; 16 AP and 6 DV ablations in ShrmAB RNAi). ShrmΔ11 and ShrmΔ13.6 were combined for the analysis in E and F. A single value was obtained for each image by analyzing 100–200 edges/image, and 8–11 images in 5–6 embryos were analyzed/genotype. *, P ≤ 0.05. Means ± SEM between images are shown. Bars, 10 µm.
