Figure 5. Apical membrane contractility regulates lumen formation during sprouting angiogenesis.

a) Tg(kdr-l:ras-Cherry)^s916 embryos with mosaic expression of Myl9b-EGFP or Myl9bAA-EGFP were analysed at 2 dpf. EGFP-positive ISVs were classified by eye into three categories according to their level of EGFP expression (low, moderate, strong) and screened for the presence of a lumen. A multinomial log-linear model was used to test for association of cell count in the different categories with the mutation status (WT: n=55 ISVs from 24 embryos; AA: n=31 ISVs from 9 embryos; data pooled from three independent experiments; p=0.30 (low), p=0.11 (moderate), p=0.0002 (high), p=0.00024 (total); **, p<0.01). EGFP-positive cells where the presence or absence of a lumen could not be appreciated were referenced as undetermined.

b) Tg(kdr-l:ras-Cherry)^s916 embryos with mosaic expression of Myl9b-EGFP or Myl9bAA-EGFP were imaged at 2 dpf. Arrowhead, disconnected lumen fragment. Arrow, side lumen branch. Scale bars are 10 μm. Images are representative of 6 embryos analysed.

c) Tg(kdr-l:ras-Cherry)^s916 embryos with mosaic expression of Myl9bAA-EGFP were imaged from 35 hpf. Arrowheads, lumen. Time is in hours:minutes:seconds. Scale bar is 10 μm. Images are representative of 3 embryos analysed.

d) Schematic model of lumen formation by inverse membrane blebbing during sprouting angiogenesis in vivo. Haemodynamic forces generate a positive pressure difference between the luminal and the cytoplasmic sides of the apical membrane (1). Consequently, inverse blebs expand along the apical membrane at sites of weak attachment of the cortex to the membrane (2). Following bleb expansion, F-actin polymerises and myosin-II is recruited at the apical membrane of growing blebs (3). Actomyosin contraction leads to bleb retraction (4), and selective bleb retraction ensures unidirectional lumen expansion (5).