Buds are directly released as Plts. (A) Mk behavior tracks from E13.5 FL organotypic cultures. Representative examples of changes in cell volume and sphericity of Mks that did not undergo Plt production (i); died (ii); formed proPlts (iii); and released Plts via budding (iv). Black arrow (iii), initiation of proPlt formation; colored arrows (iv) highlight Plt release events. The absolute count and frequency of each Mk behavior is indicated. Vertical line indicates end of track. (B) Stills from a budding Mk movie (see Video 3). Colored arrows highlight Plt release events. Scale bar, 10 µm. (C) Volume of circulating Plts, free FL Plts (o = 738) present at the start of experiment, and nascent Plts (o = 138) released by budding. P = 0.95, one-way ANOVA. (D) Number of Plts released per shedding event. (E) 3D z-projection of E13.5 FL Mks (CD41, gray) and endothelium (Flk1-GFP, green) in vivo. Optical section from boxed region. Distribution of Plt-associated protein VWF indicated in red. Scale bars, 10 µm. n = 4 independent experiments. Blue arrow, trans-endothelial bud.