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. 2019 Oct 21;29(20):3370–3385.e7. doi: 10.1016/j.cub.2019.08.017

Figure 1.

Figure 1

RhoGEF2 Activates Medial-Apical, but Not Junctional, Rho1 in the Ectoderm

(A) Apical (0 μm) and junctional (1.5 μm) confocal z sections of ventro-lateral ectodermal cells from embryos expressing Myo-II::mCherry and Ani-RBD::GFP, 8 min after the onset of cephalic furrow formation. White arrowheads show planar-polarized Myo-II and Rho1-GTP at vertical junctions.

(B) 7 μm projections of confocal acquisitions in both control and RhoGEF2 shRNA embryos expressing Ani-RBD::GFP.

(C and D) Quantifications of mean medial-apical Rho1-GTP and mean junctional Rho1-GTP intensities in control and RhoGEF2 shRNA embryos. Medial Rho1-GTP is decreased in the RhoGEF2 knockdown condition while junctional Rho1-GTP intensity is unchanged compared to control.

(E) Top panels: apical (0 μm), junctional (2 μm), and lateral (8 and 12 μm) confocal z sections of ectodermal cells in control and RhoGEF2 germline clone embryos expressing Myo-II::mCherry and α-catenin::YFP, a junctional marker. Medial-apical Myo-II is lost in mutant embryos, and Myo-II is still detected at junctions in this condition (white arrowheads). Although half of the RhoGEF2 germline clone embryos express RhoGEF2 zygotically, no rescue has been observed for Myo-II apical levels, suggesting that maternally loaded RhoGEF2 mainly controls the process in a wild-type embryo at this stage. Bottom panel: schematic view of Myo-II and adherens junction distribution in both control and RhoGEF2 mutant ectodermal cells is shown.

(F) Stack-focused image of control embryos (from 2 to 6 μm below apical membrane) and RhoGEF2 germline clone embryos (from 8 to 12 μm below apical membrane) expressing Myo-II::GFP. Note that the planar-polarized Myo-II cables are still detected in RhoGEF2 germline clone.

(G) Myo-II amplitude of polarity at junctions in control and RhoGEF2 germline clone. No difference in junctional Myo-II planar polarity is observed between the two conditions. The weak values of junctional Myo-II polarity measured in this experiment are due to an absence background subtraction during the post-processing of the image.

Scale bars represent 5 μm. Means ± SEM between images are shown. Statistical significance has been calculated using Mann-Whitney U test. Not significant (ns), p > 0.05; ∗p < 0.05; ∗∗p < 0.01. All the panels have the same orientation: dorsal at the top and anterior to the left.

See also Figures S1, S4, and S7 and Video S1.