Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.
Throughout life, blood vessels are constantly remodelled to ensure that oxygen and nutrients reach every part of the body where they are needed. If a tissue is not receiving an adequate blood flow, existing blood vessels may widen or new blood vessels may sprout from their walls. In certain diseases, such as cancer, blood vessels may grow excessively to form disorganized networks, and preventing this growth may help to treat these conditions. However, we do not fully understand how the body controls the size, shape and branching pattern of blood vessels.
For a new blood vessel to sprout out of an existing vessel, the tip of the new branch must first develop. The tip forms when the endothelial cells that line the blood vessel are activated by a protein called vascular endothelial growth factor A (Vegfa), which is produced by the surrounding tissue. The activated endothelial cells respond to Vegfa stimulation by producing the protein Dll4, which talks to neighboring endothelial cells to prevent them from also forming new tips. In a way, this process bears all the signs of a competition between cells, as they fight for which one is allowed to take the lead. The losers of this competition, when forced into subordination by the tips, also serve an important function, as they will help to form and elongate the base of the new sprout.
Although it is known that changes in the levels of Vegfa in tissues can cause blood vessel branching to alter dramatically, the mechanisms that enable this to occur are not well understood. Computer simulations of the process predicted that an unexpected synchronization of Dll4 dynamics would be triggered when Vegfa levels increased; however, this remained to be observed in real cells.
Ubezio, Blanco et al. have now used fluorescent markers to observe the Dll4 production in lab-grown mouse endothelial cells as they formed new vessel sprouts in response to Vegfa. This revealed that the levels of Dll4 fluctuate widely in individual cells. Time-lapse movies of the cells showed that as a new sprout forms, the levels of Dll4 in neighbouring cells fluctuate in an uncoordinated manner. However, increasing the amount of Vegfa in the cells indeed synchronizes these fluctuations. This causes the new sprout to retract and allows the original blood vessel to widen. Increasing the levels of Dll4 had the same effect.
Further experiments confirmed that increasing the amount of Vegfa also reduces blood vessel branching in tumours in mice by synchronizing the fluctuations in the levels of Dll4 in neighbouring endothelial cells. In the future, these results could help refine anti-cancer treatments that work by blocking the activity of Vegfa and Dll4.