Behavior of CD41-GFP^low definitive blood precursors in the zebrafish AGM. Time-lapse confocal fluorescence imaging of live CD41-gfp embryos pre-incubated with Bodipy TR (lateral views of the trunk). (A) 54 hpf, combined fluorescence from 2 focal planes (1 and 2) 6 μm apart, overlaid with transmitted light image (see Videos S4,S5). Rostral is to the top. Three cells (red, blue, and yellow dots) initially all in focal plane 1—in the abluminal mesenchyme dorsal (yellow and blue cells) or lateral (red cell) to the PCV—move to the deeper plane 2 one after another (arrowheads) to enter the PCV lumen and circulation. These successive moves in depth can be directly seen in Video S4. The yellow cell moves to plane 2 at t = 37.5 minutes.; it and the blue cell (still in plane 1) move toward eachother (still in the DP joint) until the yellow one passes below the blue (t = 45 to 47.7 minutes), then moves straighter within the PCV lumen (between 65 and 80 minutes), after which it is taken by the blood flow. The blue cell moves to plane 2 at 115 to 117.5 minutes, then is in the PCV lumen, in which it is last seen at t = 137.5 minutes. Finally the red cell moves to plane 2 at 195 minutes, after which it is in the PCV lumen, where it is last visible at 217.5 minutes. (B) 48 hpf; 2 (likely sister) GFP⁺ cells from the DP joint, initially linked by a tether (arrow), enter one after the other into the PCV lumen and circulation, in a PU.1 Mo injected embryo (Video S6). Scale bars, 20 μm. y, yolk tube.