Endothelial Ca2+ oscillations reflect VEGFR signaling-regulated angiogenic capacity in vivo

Sprouting angiogenesis is a well-coordinated process controlled by multiple extracellular inputs, including vascular endothelial growth factor (VEGF). However, little is known about when and how individual endothelial cell (EC) responds to angiogenic inputs in vivo. Here, we visualized endothelial Ca2+ dynamics in zebrafish and found that intracellular Ca2+ oscillations occurred in ECs exhibiting angiogenic behavior. Ca2+ oscillations depended upon VEGF receptor-2 (Vegfr2) and Vegfr3 in ECs budding from the dorsal aorta (DA) and posterior cardinal vein, respectively. Thus, visualizing Ca2+ oscillations allowed us to monitor EC responses to angiogenic cues. Vegfr-dependent Ca2+ oscillations occurred in migrating tip cells as well as stalk cells budding from the DA. We investigated how Dll4/Notch signaling regulates endothelial Ca2+ oscillations and found that it was required for the selection of single stalk cell as well as tip cell. Thus, we captured spatio-temporal Ca2+ dynamics during sprouting angiogenesis, as a result of cellular responses to angiogenic inputs. Throughout life, new blood vessels grow out like branches from existing vessels in a process called “sprouting angiogenesis”. This involves some of the endothelial cells that line the inner surface of the blood vessel migrating outwards, creating a vessel sprout made up of tip cells and stalk cells. Sprouting is controlled by two opposing signaling systems. One pathway is triggered by a molecule called vascular endothelial growth factor (VEGF). This molecule binds to receptor proteins to activate a range of signaling processes that stimulate endothelial cells to become tip cells, and so encourage the formation of new sprouts. However, it was not known exactly when or how the endothelial cells respond to these signals. By contrast, the Notch signaling pathway inhibits sprouting angiogenesis. The two signaling pathways interact with each other: VEGF signaling in tip cells activates Notch signaling in neighboring cells, which then prevents VEGF signaling in these cells. This feedback mechanism helps a new sprout to form by suppressing tip-like activity in the cells surrounding a new tip cell, forcing these cells to become stalk cells. Activating VEGF receptors also causes brief increases, or oscillations, in the level of calcium ions inside the endothelial cells. Now, Yokota, Nakajima et al. have investigated VEGF activity by genetically engineering zebrafish embryos so that fluorescent proteins in