Fig. 6

Endothelial cell migration against blood flow is impaired in AA1 in alk1-deficient embryos. (A) 2D maximum projections of selected time points from time-lapse two-photon imaging of proximal cranial vessels in control and alk1-morphant Tg(fli1a:nEGFP)^y7 embryos, ~24-34hpf. Vessels of interest are outlined to improve clarity. AA1 nuclei are color coded according to their behavior over the course of the movie. Green, cells remain in proximal AA1; pink, cells move from AA1 into the OFT/heart; yellow, cells enter proximal AA1 from more distal segments. Scale bar: 50 µm. (B) Quantification of endothelial cells grouped by initial (Time_i, 24hpf) and final (Time_f, 34hpf) positions. n=3 controls, 4 alk1 morphants. Below the graph, names of alk1-positive vessels are shaded gray, and direction of cell migration with respect to blood flow is depicted by black arrows. (C) 2D maximum projections of selected time points from time-lapse confocal/two-photon imaging of the right side of AA1 in Tg(fli1a:nEGFP)^y7;Tg(fli1a.ep:mRFP-CAAX)^pt505 control and alk1-morphant embryos. See also Movies 1,2. Endothelial cell membranes, magenta; endothelial cell nuclei, gray. White arrowheads track a single cell nucleus over time in each embryo. Dorsal views, anterior down. Scale bar: 20µm. (D-F) From these movies, we quantified summed migration track length (D), net nuclear displacement (E) and tortuosity ratio (F). Each data point represents mean values for a single embryo, 6-10 cells per embryo. Only cells that could be tracked for a minimum of four consecutive frames were included in the analysis. Graphs represent mean±s.e.m. for n=3 controls and 3 alk1 morphants. Two-tailed Student′s t-test, *P<0.05, **P<0.01.