Figure 1: General concept of the study.

(a) Schematic of a sprout showing the differential properties of its ECs, which are activated or inhibited. Active ECs inhibit adjacent cells through Dll4/Notch signalling, are migratory and competitive for the tip. They overtake other ECs (cell rearrangement) due to higher VE-cadherin endocytosis rates (which makes them weakly adhesive) and formation of junctional cortical protrusions. (b) Scheme depicting the effectors modified in the memAgent-Spring ATP model (MSM-ATP) and the signalling pathways included in the MSM-ATP. E^FIL (representing filopodial F-actin), E^COR (referring to cortical actin), and E^ADH (denoting intercellular adhesion) determine respectively, the probability of filopodia extension, the formation of polarized junctional protrusions and cellular adhesion levels, determined by VE-cadherin endocytosis. The pink lines indicate the ATP effector links that were investigated. (c) Schematic drawing of the in vitro EC spheroid assay. Cells with different genotypes (and corresponding colours) are cultured in a sphere and allowed to sprout for 24 hours upon growth factor stimulation. Subsequently, the colour of the cell at the tip is counted for each sprout, allowing to quantify the tip cell contribution as shown in the graph for 1:1 WT^GFP:WT^RED and PFKFB3^KD/GFP:WT^RED mosaic spheroids. Data are mean±s.e.m.; n=30 from 3 donors; **P<0.01; Fisher’s exact test; adapted from ref. 5 with permission from Elsevier. (d) Screenshots of MSM-ATP simulations, showing how cell positions change over time and how, similar as in the in vitro assay, the genotype (colour) of the cell at the end of the in silico experiments can be assessed. The red arrows represent the movement of the cell to its next position in the next simulation screenshot. (e) Scheme illustrating how MSM-ATP simulations are performed using vessel sprouts consisting of ten ECs. Each colour represents a different EC. ATP, adenosine triphosphate; DLL4, delta-like 4; NICD, Notch1 intracellular domain; PFKFB3, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3; VE-cadherin, vascular endothelial cadherin; VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor 2.