Sprouting angiogenesis enables the growth of new blood vessels from preexisting ones. This process requires intracellular signaling through vascular endothelial growth factor receptors (VEGFRs) to promote angiogenesis and intercellular signaling through the Notch pathway to oppose angiogenesis. Tip cells (which are thought to determine the direction of growth of a sprout) have higher abundance of the Notch ligand delta-like 4 (Dll4) than stalk cells in the sprout. Because the abundance of Dll4 can be affected by VEGFR activity, Jakobsson et al. investigated the role of VEGFR abundance on tip cell selection. Computational modeling indicated that, when competing with wild-type cells, cells with half the normal amount of VEGFR2 were more likely to become stalk cells, whereas cells with half the normal amount of VEGFR1 were more likely to become tip cells. Embryonic stem cells from wild-type mice were mixed with those from mice heterozygous for Vegfr2 (Vegfr2+/egfp) or from mice heterozygous for Vegfr1 (Vegfr1+/lacz), cultured to form spheroids called embryoid bodies, and treated with VEGF-A to induce sprouting angiogenesis. Compared with wild-type cells, Vegfr2+/egfp cells were less likely and Vegfr1+/lacz cells were more likely to assume the tip cell position in the resulting mosaic sprouts. Embryoid bodies were also formed from wild-type cells and cells from mice heterozygous for Dll4 (Dll4+/lacz) or from mice heterozygous for Nrarp (Nrarp+/lacz) (which have higher Notch activity). Dll4+/lacz or Nrarp+/lacz cells were less likely than wild-type cells to be in the tip cell position, indicating that reduced Dll4 abundance or increased Notch signaling reduces the ability to compete for the tip cell position. Immunofluorescence analysis of sprouts formed from wild-type cells revealed higher abundance of Dll4 and VEGFR2 in tip cells and higher abundance of VEGFR1 in stalk cells, and treatment with the Notch inhibitor DAPT increased the abundance of VEGFR2. In mosaic angiogenic sprouts treated with DAPT, tip cells were no longer predominantly those with higher Vegfr2 or with lower Vegfr1, indicating that competition for the tip cell position based on VEGFR abundance depends on Notch signaling. Time-lapse microscopy of angiogenic sprouts showed that cells constantly changed position and that Vegfr1+/lacz cells stayed in the tip cell position longer than wild-type or Vegfr2+/egfr cells. The authors suggest that constant competition within the endothelial cell population in a vascular sprout for the tip cell position may ensure accurate migration toward a VEGF-A gradient.
L. Jakobsson, C. A. Franco, K. Bentley, R. T. Collins, B. Ponsioen, I. M. Aspalter, I. Rosewell, M. Busse, G. Thurston, A. Medvinsky, S. Schulte-Merker, H. Gerhardt, Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat. Cell. Biol. 12, 943–953 (2010). [PubMed]