Endothelial cells show surprising cell rearrangement behaviour during angiogenic sprouting; however, the underlying mechanisms and functional importance remain unclear. By combining computational modelling with experimentation, we identify that Notch/VEGFR-regulated differential dynamics of VE-cadherin junctions drive functional endothelial cell rearrangements during sprouting. We propose that continual flux in Notch signalling levels in individual cells results in differential VE-cadherin turnover and junctional-cortex protrusions, which powers differential cell movement. In cultured endothelial cells, Notch signalling quantitatively reduced junctional VE-cadherin mobility. In simulations, only differential adhesion dynamics generated long-range position changes, required for tip cell competition and stalk cell intercalation. Simulation and quantitative image analysis on VE-cadherin junctional patterning in vivo identified that differential VE-cadherin mobility is lost under pathological high VEGF conditions, in retinopathy and tumour vessels. Our results provide a mechanistic concept for how cells rearrange during normal sprouting and how rearrangement switches to generate abnormal vessels in pathologies.
23 24How vascular tubes build, maintain and adapt continuously perfused lumens to meet 25 local metabolic needs remains poorly understood. Recent studies showed that blood 26 flow itself plays a critical role in the remodelling of vascular networks 1,2 , and 27 suggested it is also required for lumenisation of new vascular connections 3,4 . 28 However, it is still unknown how haemodynamic forces contribute to the formation of 29 new vascular lumens during blood vessel morphogenesis. 30Here we report that blood flow drives lumen expansion during sprouting angiogenesis 31 in vivo by inducing spherical deformations of the apical membrane of endothelial 32 cells, in a process that we termed inverse blebbing. We show that endothelial cells 33 react to these membrane intrusions by local and transient recruitment and contraction 34 of actomyosin, and that this mechanism is required for single, unidirectional lumen 35 expansion in angiogenic sprouts. 36Our work identifies inverse membrane blebbing as a cellular response to high external 37 pressure. We show that in the case of blood vessels such membrane dynamics can 38 drive local cell shape changes required for global tissue morphogenesis, shedding 39 light on a pressure-driven mechanism of lumen formation in vertebrates. 40 41Blood vessels form a vast but highly structured network that pervades all organs in 42 vertebrates. During development as well as in pathological settings in adults, vascular 43 networks expand through a process known as sprouting angiogenesis. New blood 44 vessels form from the coordinated migration and proliferation of endothelial cells into 45 vascular sprouts. Subsequent fusion of neighbouring sprouts, defined as anastomosis, 46 then leads to the formation of new vascular loops, whose functionality relies on their 47 successful lumenisation and perfusion 5 . During anastomosis, endothelial lumens form 48 both through apical membrane invagination into single anastomosing cells 49 (unicellular lumen formation), and through de novo apical membrane formation at 50 their nascent junction (multicellular lumen formation) 3,4 . Since the tip of endothelial 51 sprouts can be occupied by either one or several cells as they compete for the tip 52 position 6,7 , we asked whether similar mechanisms of lumen formation apply to 53 unicellular and multicellular endothelial sprouts prior to anastomosis. 54Using a zebrafish transgenic line expressing an mCherry-CAAX reporter for 55 endothelial plasma membrane (Tg(kdr-l:ras-Cherry) s916 ), we imaged lumen formation 56 in tip cells as they sprout from the dorsal aorta (DA) to form the intersegmental 57 vessels (ISVs) from 30 hours post-fertilisation (hpf). We found that lumens expand in 58 sprouting ISVs prior to anastomosis, and do so by invagination of the apical 59 membrane either into single tip cells, or along cell junctions when the tip of a 60 sprouting ISV is shared between several cells (Fig. 1a,b). 61To test if this mechanism of lumen formation is conserved in other vertebrates, we 62 perfo...
During blood vessel formation, endothelial cells (ECs) establish cell-cell junctions and rearrange to form multicellular tubes. Here, we show that during lumen formation, the actin nucleator and elongation factor, formin-like 3 (fmnl3), localizes to EC junctions, where filamentous actin (F-actin) cables assemble. Fluorescent actin reporters and fluorescence recovery after photobleaching experiments in zebrafish embryos identified a pool of dynamic F-actin with high turnover at EC junctions in vessels. Knockdown of fmnl3 expression, chemical inhibition of formin function, and expression of dominant-negative fmnl3 revealed that formin activity maintains a stable F-actin content at EC junctions by continual polymerization of F-actin cables. Reduced actin polymerization leads to destabilized endothelial junctions and consequently to failure in blood vessel lumenization and lumen instability. Our findings highlight the importance of formin activity in blood vessel morphogenesis.
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