Figure 4

Defects in head and trunk vascular development result in reduced circulatory function. (A-D) Hemoglobin staining revealed a reduction in the amount of blood cells in 3 dpf WAF-treated embryos, notably in the vasculature of the pharyngeal arches (A, B, brackets, lateral view; C, D, arrowheads, ventral view), with staining abruptly ending in the heart or bulbus artery prior to filling the aortic arches (arrow). (E-H') Microangiography analysis with QTracker 655 fluorescent quantum dots (red) injected into 3 dpf tg[fli:eGfp] transgenic larvae to visualize endothelial cells associated with the vasculature (green). Endothelial vasculature in moderately affected WAF-treated embryos (G) was comparable to controls (E), however in severe cases posterior arch vasculature was lost and circulation was reduced (H, brackets, arrowhead, H', arrowheads). (E', F', G', H') Arrowheads and arrows denote the specific blood vessels associated with the pharyngeal arches. Accumulation of quantum dots in the heart atrium suggests reduced flow into the ventricle (H', dashed line). (I-K) Real time analysis of the flow speed of individual blood cells (I-J', arrowheads) over a 7-somite distance in the dorsal aorta. WAF-treated embryos have reduced blood circulation (K, right half of graph). (L-O') Intersegmental blood vessels had reduced circulation of quantum dots as demonstrated by either a complete absence of flow (M-O, arrowheads) or truncated flow (M-O arrows). Ectopic branching and vascular remodeling was evident in some segments devoid of circulation (N', O'). Abbreviations: BuA, bulbus arteriosus; H, heart; HA, hypobranchial artery; ORA, opercular artery; PHS, primary head sinus. Numbers and affiliated arrowheads in E' and F' represent the first through sixth aortic arch. Scale bars = 200 μm, A-D; 100 μm, E-H'; 50 μm, I, J, N', O'; 20 μm, I', J'; 50 μm, L-O.