Figure 7.
Figure 7. Rho activity, Rho-kinase, and F-actin binding are required for Shroom planar polarity. (A–F) Shroom, Baz/Par-3, and F-actin (phalloidin) in embryos expressing wild-type Rho (Rho WT; A and D), embryos expressing dominant-negative RhoN19 (B and E), and Rok2 maternal (C and F) mutants. Anterior is left, and ventral is down. (G and H) Planar polarized enrichment of Shroom (G) and F-actin (H) at AP cell boundaries (75–90° with respect to the AP axis) relative to DV cell boundaries (0–15°). (G) Shroom planar polarity (visualized with the anti-Shroom antibody) was reduced in embryos expressing RhoN19 compared with embryos expressing wild-type Rho (P < 0.0001) and in Rok2 mutants compared with wild type (P < 0.0001). (H) F-actin planar polarity was strongly reduced in embryos expressing RhoN19 compared with embryos expressing wild-type Rho (P < 0.005) and was slightly reduced (but did not reach statistical significance) in Rok2 mutants compared with wild type (P = 0.08). (I) Model. (J–M) Venus:Shroom proteins in stage 7 wild-type embryos visualized with anti-GFP. Wild-type Shroom (J), Shroom ΔRok–binding domain (BD; K), F-actin–binding domain alone (L), and Shroom ΔF-actin–binding domain (M) are shown. Anterior is left, and ventral is down. (N) Shroom protein (the ShrmA isoform aa 1–1,576) contains an F-actin–binding domain (aa 445–920) and a Rho-kinase–binding domain (Rok BD, aa 1,324–1,576). (O) Planar polarized enrichment of Venus:Shroom proteins at AP cell boundaries relative to DV cell boundaries visualized with anti-GFP antibody. Shroom ΔF-actin–binding domain planar polarity was strongly reduced (P < 0.0001) and Shroom F-actin–binding domain and ΔRok–binding domain planar polarity were moderately reduced (P < 0.05) compared with full-length Shroom. Shroom planar polarity was slightly lower than in Fig. 3 C and Fig. 7 G because different antibodies were used to detect endogenous and ectopic Shroom protein (Materials and methods). A single value was obtained for each image by averaging 100–200 edges/image; 12–26 images in 6–18 embryos were analyzed/genotype. Means ± SEM between images are shown. Bars, 10 µm.

Rho activity, Rho-kinase, and F-actin binding are required for Shroom planar polarity. (A–F) Shroom, Baz/Par-3, and F-actin (phalloidin) in embryos expressing wild-type Rho (Rho WT; A and D), embryos expressing dominant-negative RhoN19 (B and E), and Rok2 maternal (C and F) mutants. Anterior is left, and ventral is down. (G and H) Planar polarized enrichment of Shroom (G) and F-actin (H) at AP cell boundaries (75–90° with respect to the AP axis) relative to DV cell boundaries (0–15°). (G) Shroom planar polarity (visualized with the anti-Shroom antibody) was reduced in embryos expressing RhoN19 compared with embryos expressing wild-type Rho (P < 0.0001) and in Rok2 mutants compared with wild type (P < 0.0001). (H) F-actin planar polarity was strongly reduced in embryos expressing RhoN19 compared with embryos expressing wild-type Rho (P < 0.005) and was slightly reduced (but did not reach statistical significance) in Rok2 mutants compared with wild type (P = 0.08). (I) Model. (J–M) Venus:Shroom proteins in stage 7 wild-type embryos visualized with anti-GFP. Wild-type Shroom (J), Shroom ΔRok–binding domain (BD; K), F-actin–binding domain alone (L), and Shroom ΔF-actin–binding domain (M) are shown. Anterior is left, and ventral is down. (N) Shroom protein (the ShrmA isoform aa 1–1,576) contains an F-actin–binding domain (aa 445–920) and a Rho-kinase–binding domain (Rok BD, aa 1,324–1,576). (O) Planar polarized enrichment of Venus:Shroom proteins at AP cell boundaries relative to DV cell boundaries visualized with anti-GFP antibody. Shroom ΔF-actin–binding domain planar polarity was strongly reduced (P < 0.0001) and Shroom F-actin–binding domain and ΔRok–binding domain planar polarity were moderately reduced (P < 0.05) compared with full-length Shroom. Shroom planar polarity was slightly lower than in Fig. 3 C and Fig. 7 G because different antibodies were used to detect endogenous and ectopic Shroom protein (Materials and methods). A single value was obtained for each image by averaging 100–200 edges/image; 12–26 images in 6–18 embryos were analyzed/genotype. Means ± SEM between images are shown. Bars, 10 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal