New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell-derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gainof-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis.
IntroductionBlood vessels form and expand in both development and disease, via processes that include vasculogenesis, angiogenesis, and intussusception (reviewed in Risau, 1 Eichmann et al, 2 Coultas et al 3 ). Sprouting angiogenesis is the coordinated migration of groups of endothelial cells from vessels and their subsequent fusion to form new interconnections. In this way, simple vascular tubes are ramified and extended to form a primitive vascular plexus. This vessel plexus forms at numerous sites in the embryo, including the yolk sac, the head mesenchyme, and surrounding the neural tube. The primitive vascular plexus is then remodeled under the influence of blood flow and interactions with mural cells. Thus, the initial pattern of vessels serves as a template for remodeling that leads to a mature vasculature.During formation of the primitive vascular plexus, several cellular processes must be regulated and integrated. Specifically, endothelial cells respond to some morphogenetic cues by sprouting, while actively dividing to expand the pool of endothelial cells. One level of integration occurs via the signaling pathways that promote angiogenesis, because many affect both endothelial cell division and morphogenesis. The VEGF signaling pathway is an example of this mode of integration, because it regulates both cell division and branching morphogenesis (reviewed in Rousseau et al, 4 Kliche and Waltenberger, 5 Ferrara et al,6 Nagy and Senger, 7 and Shibuya and Claesson-Welsh 8 ). VEGF-A (VEGF) binds 2 highaffinity receptors on endothelial cells, flk-1 (VEGFR-2) and flt-1 (VEGFR-1), and perturbation of VEGF signaling by genetic deletion of either receptor affects both endothelial cell division and morphogenesis. ...