Figure 1. Blood pressure drives unicellular and multicellular lumen expansion in angiogenic sprouts.

a) Schematic illustration of unicellular and multicellular lumen formation in angiogenic sprouts.

b) Tg(kdr-l:ras-Cherry)^s916 embryos were imaged from 32 hpf. Black arrows, cell junction. Magenta arrows, apical membrane. Time is in hours:minutes:seconds. Scale bar is 10 μm. Images are representative of 10 embryos analysed.

c) Mouse retinas were harvested at P6 and stained for ICAM-2, ZO-1 and Isolectin IB₄. Isolectin IB₄ staining was used to draw the cell outline (white dotted line). White arrow, unicellular membrane invagination. Scale bar is 10 μm.

d) The number of endothelial sprouts with unicellular or multicellular lumens were quantified in P6 mouse retinas stained for ICAM-2 and ZO-1 (n=487 sprouts from 9 retinas).

e) Tg(kdr-l:ras-Cherry)^s916 embryos were imaged from 33 hpf after the addition of 4x tricaine. Blood flow stopped after 20-25 minutes of treatment, leading to a decrease in blood pressure noticeable through the decrease in diameter of the dorsal aorta (double-headed arrow). At 48 hpf, embryos were returned to 1x tricaine and imaged further. Magenta arrows, apical membrane. Magenta filling, lumen. Times are in hours:minutes:seconds and correspond to the times after addition of 4x tricaine (left panels) and after washout (right panels). Scale bar is 20 μm. Images are representative of 7 embryos analysed.