Temporal modulation of collective cell behavior controls vascular network topology

Vascular network density determines the amount of oxygen and nutrients delivered to host tissues, but how the vast diversity of densities is generated is unknown. Reiterations of endothelial-tip-cell selection, sprout extension and anastomosis are the basis for vascular network generation, a process governed by the VEGF/Notch feedback loop. Here, we find that temporal regulation of this feedback loop, a previously unexplored dimension, is the key mechanism to determine vascular density. Iterating between computational modeling and in vivo live imaging, we demonstrate that the rate of tip-cell selection determines the length of linear sprout extension at the expense of branching, dictating network density. We provide the first example of a host tissue-derived signal (Semaphorin3E-Plexin-D1) that accelerates tip cell selection rate, yielding a dense network. We propose that temporal regulation of this critical, iterative aspect of network formation could be a general mechanism, and additional temporal regulators may exist to sculpt vascular topology. , Many animals have a network of blood vessels that supplies oxygen and nutrients to every part of the body. Each organ contains a unique pattern of blood vessels; some have lots of densely packed vessels, while others have fewer vessels that are more widely spaced. New blood vessels typically form by sprouting from the side of pre-existing vessels. This involves the endothelial cells that line the inner wall of blood vessels moving outwards to create a sprout that is made up of ‘tip cells’ and ‘stalk cells’. Tip cells are found at the front of the growing vessels and encourage the formation of new sprouts, while the stalk cells trail behind and elongate the sprout. Two signaling pathways that involve two proteins called VEGF and Notch interact with each other to control which cells become tip cells and which become stalk cells. Cells with higher levels of VEGF signaling will become tip cells. These cells also activate Notch signaling, which in turn blocks VEGF signaling in their neighboring cells. This feedback mechanism enables a new sprout to form by forcing cells present around a newly formed tip cell to become stalk cells. However, it was still not understood how the different organs develop blood vessel networks with different densities. In 2011, researchers revealed that two other proteins, Semaphorin3E and its receptor Plexin-D1, are expressed in tip cells in the back of the eye in mice and control the VEGF/Notch signaling pathway. Now Kur et al. – including some of the researchers involved in the 2011 work – have used a combination of predictive computer simulations and experimental approaches to understand this interaction in more detail. The analysis showed that Semaphorin3E and Plexin-D1 speed up VEGF/Notch signaling, which causes new tip cells to form at a faster rate, and results in a more densely packed network of blood vessels. For example, in mice that lack Semaphorin3E and Plexin-D1, VEGF/Notch signaling was slower and new tip cells formed more slowly, which resulted in the blood vessel network at the back of the mice’s eyes being less dense. Kur et al. propose that different organs have different ‘molecular metronomes’ that control the pace of VEGF/Notch signaling. A fast acting metronome would yield a dense network, while a slower one would form a less dense network. This helps to explain how diverse densities of blood vessel networks are formed in different organs. This work may aid efforts to develop therapeutic approaches for controlling the development of new blood vessels in cancers and other diseases.