Rab11-positive vesicle-like structures accumulate apically in constricting cells during ventral furrow formation. (A) Apical constriction during ventral furrow formation. Scale bar, 10 μm. (B) The imaging setup for acquiring the en face view of constricting cells. (C) Rab11 compartments (endogenous YFP-Rab11) in an embryo at the onset (0 min) and 9 min into ventral furrow formation. Top panel, maximum projection over 2 μm depth from en face view; middle and bottom panels, a single slice and maximum projection of the cross-section view, respectively. Red boxes: Z position of the corresponding en face view. Red arrows: perinuclear Rab11 vesicles; green arrows and blue outlines: perinuclear Rab11 compartments; yellow outline: nucleus. (D) Zoom-in view of a small ROI in the ventral region of the embryo. (E) Montage showing Rab11 and Myosin II distribution over depth during apical constriction. (F and G) 3D reconstruction of Rab11 positive structures in ventral mesoderm constricting cells. 0 min: onset of gastrulation. Rab11 perinuclear compartments and vesicles are segmented separately using ilastik. An individual cell is shown in G as an example. Cell outlines (yellow dotted lines) are determined based on Gap43-mCherry signal. (H) The intensity profile of summed intensity of Rab11 vesicles along Z direction. 0 μm: cell apex. Error bar: SD. N = 13 cells. All scale bars are 5 μm unless mentioned otherwise.