Figure S2.

Apical Rab11 vesicles are not derived from endocytosis. (A) Endocytosed positive vesicles labeled by membrane dye FM4-64 extensively colocalize with YFP-Rab5 vesicles (85%, 72 vesicles from three embryos) but not YFP-Rab11 vesicles (3%, 98 vesicles from three embryos) during apical constriction. Green arrows mark Rab5 vesicles that do not colocalize with FM4-64 vesicles and yellow arrows mark Rab5 vesicles that colocalize with FM4-64 vesicles. Scale bar, 5 μm. (B) Quantification of colocalization between endocytosed FM4-64 vesicles and corresponding Rab vesicles. (C) YFP-Rab5 and mCherry-Rab11 vesicles do not show colocalization. Scale bar, 5 μm. (D and D’) Top panels (D): Montage showing Sqh-mCherry in the ventral cross-section view of wild type and shi^ts embryos during ventral furrow formation. Bottom panels (D’): En face view images showing myosin accumulation at the indicated time points. En face view images were Gaussian filtered with a radius of 0.5 pixel. Contrasts were adjusted to make the cytoplasmic background comparable. Scale bars, 10 μm. Embryos were placed at restrictive temperature (32°C) during cellularization. shi^ts mutant embryos (N = 17 embryos) exhibit ventral furrow formation defects with different level of severity compared to wildtype controls (N = 5 embryos). shi^ts (mild) embryos (N = 10 embryos) progress slower in ventral furrow formation while shi^ts (severe) embryos (N = 7 embryos) fail to form ventral furrow as tissue relaxes after initial constriction. In both group of mutant embryos, apical myosin intensity eventually rises to a level comparable or higher than that in the wildtype controls. Interestingly, a previous study has shown that blocking endocytosis using shi^ts in snail mutant embryos rescued the accumulation of Fog in the apical cytoplasm and partially restored apical Myosin II accumulation (Pouille et al., 2009). It is unclear whether the Myosin II phenotype observed in our study was associated with the role of dynamin in regulating Fog. (E and E’) Disruption of dynamin function does not prevent apical accumulation of Rab11 vesicles. (E) En face view (maximum projection over 2 μm from apical surface) of the ventral region of embryos at the onset of gastrulation (T = 0) or during gastrulation when ventral furrow shows similar morphology between different groups of embryos. (E’) Zoom-in view of the highlighted regions (red boxes) are shown at the bottom. shi^ts (mild) embryos show similar apical Rab11 vesicle accumulation compared to the controls. In shi^ts (severe) embryos, Rab11 vesicles are already present in the broad apical region of the embryo before apical constriction starts. Nevertheless, the amount of apical Rab11 vesicles substantially increases in the constricting cells during ventral furrow formation. Contrasts were adjusted to make the cytoplasmic background comparable. Images were Gaussian filtered with a radius of 0.7 pixel. Scale bars, 10 μm. Scale bars for zoom-in panel, 2 μm.