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Fig. 1 Venous angioblasts become specified as a separate population and at a different time point than arterial angioblasts. (A) Trunk vasculature of a zebrafish embryo. Veins are pseudocolored blue and arteries red. (B-G) Confocal projections from a time-lapse movie of a Tg(fli1a:EGFP)y1 embryo. (B) At the time point of their specification, angioblasts appear to have a round morphology (arrowheads). The angioblast that will form the DA begin to migrate to the midline of the embryo (arrows). (C) While posterior angioblasts forming the DA arrive at the midline at 16 hpf, angioblasts forming the LDA have just started to migrate (arrows). (D) At 17 hpf, most of the LDA-forming angioblasts have arrived at their final position (arrows). At the same time, a second population of angioblasts of round morphology appears in the region where the bilateral cardinal veins will develop (arrowheads). (E) Angioblasts of this second population start to become elongated and form filopodia (arrows). Asterisks indicate the first population forming the LDA. (F) Additional GFP-positive angioblasts become specified (arrowheads). Some angioblasts of the second population have lost their round morphology and the first cells begin to migrate (arrows) to form the CCVs. (G) At 22 hpf, angioblasts with multiple filopodia migrate (arrows) to form the CCVs, whereas other angioblasts remain in a more central position to form the bilateral cardinal veins. (H,I) Merged confocal projections of the green and red channel showing Kaede protein expression in a Tg(fli1ep:GAL4FF)ubs;(UAS:KAEDE)rk8 embryo. (H) Photoconversion of all endothelial cells (ECs) at 17 hpf, before the second population of angioblasts starts to express Kaede. Note that all ECs are photoconverted (red). (I) Arterial ECs in the LDA can be detected by both photoconverted (red) and new synthesized Kaede (green), whereas venous ECs in the bilateral cardinal veins and the CCVs only express newly synthesized Kaede (green). Note non-endothelial expression of Kaede by blood cells. All images represent dorsal views. Arrowheads, newly specified angioblasts; arrows, migrating angioblasts; DA, dorsal aorta; LDA, lateral dorsal aortae; CCV, common cardinal vein.