Vascular lumens are essential for the dispersal of nutrients, oxygen, and immune cells throughout the body. De novo lumen formation necessitates the precise segregation of junctional proteins from apical surfaces. Direct visualization of this process and a comprehensive understanding on how the apical and junctional compartments are segregated and established remain elusive, due to the lack of visualizable systems, proper reporters and manipulative tools. Using zebrafish as an experimental model, we have established a wealth of molecular reporters, photo-convertible and optogenetic tools and combined them with genetic analyses to study the establishment of apical domains from pre-apical junctional complexes. Our study identified Rasip1 as one of the earliest apical proteins recruited, peripheralizing cortical contractility at the junctional patches by inhibiting NMII at the center, thereby initiating the outward flow of junctional complexes. After the establishment of the apical compartments, Rasip1 shuttles between junctions and the apical compartments to the high-tension sites and tunes down the local hyper contractility, restricting Cdh5 to junctions by suppressing apical contractility. Above all, the formation of de novo lumens and maintenance of junctional integrity depend on the precise control of contractility within the apical compartments and junctions, orchestrated by the dynamic recruitment of Rasip1.### Competing Interest StatementThe authors have declared no competing interest.

The vertebrate cardiovascular system is made up by a hierarchically structured network of highly specialised blood vessels. This network emerges during early embryogenesis and evolves in size and complexity concomitant with embryonic growth and organ formation. Underlying this plasticity are actin-driven endothelial cell behaviours, which allow endothelial cells to change their shape and move within the vascular network. In this review, we discuss the cellular and molecular mechanisms involved in vascular network formation and how these intrinsic mechanisms are influenced by haemodynamic forces provided by pressurized blood flow. While most of this review focusses on in vivo evidence from zebrafish embryos, we also mention complementary findings obtained in other experimental systems.

Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.

Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.

Actin filaments are instrumental in driving processes such as migration, cytokinesis and endocytosis and provide cells with mechanical support. During angiogenesis, actin-rich filopodia protrusions have been proposed to drive endothelial tip cell functions by translating guidance cues into directional migration and mediating new contacts during anastomosis. To investigate the structural organisation, dynamics and functional importance of F-actin in endothelial cells (ECs) during angiogenesis in vivo, we generated a transgenic zebrafish line expressing Lifeact-EGFP in ECs. Live imaging identifies dynamic and transient F-actin-based structures, such as filopodia, contractile ring and cell cortex, and more persistent F-actin-based structures, such as cell junctions. For functional analysis, we used low concentrations of Latrunculin B that preferentially inhibited F-actin polymerisation in filopodia. In the absence of filopodia, ECs continued to migrate, albeit at reduced velocity. Detailed morphological analysis reveals that ECs generate lamellipodia that are sufficient to drive EC migration when filopodia formation is inhibited. Vessel guidance continues unperturbed during intersegmental vessel development in the absence of filopodia. Additionally, hypersprouting induced by loss of Dll4 and attraction of aberrant vessels towards ectopic sources of Vegfa165 can occur in the absence of endothelial filopodia protrusion. These results reveal that the induction of tip cells and the integration of endothelial guidance cues do not require filopodia. Anastomosis, however, shows regional variations in filopodia requirement, suggesting that ECs might rely on different protrusive structures depending on the nature of the environment or of angiogenic cues.

Notch signalling coordinates angiogenesis by controlling the specification of endothelial cells into tip cells that lead the way in growing blood vessels and the stalk cells that follow. Michael Potente and colleagues have identified a previously unknown component in Notch signalling regulation in endothelial cells that may provide a mechanism for fine-tuning angiogenesis in response to metabolic requirements and angiogenic stress. They show that the metabolism- and redox-sensing deacetylase SIRT1 deacetylates the Notch1 intracellular domain directly, thereby controlling its stability and turnover and negatively modulating Notch signalling. Inactivation of SIRT1 impairs angiogenesis in zebrafish and mice. Notch signalling is a key intercellular communication mechanism that is essential for cell specification and tissue patterning, and which coordinates critical steps of blood vessel growth1,2,3 . Although subtle alterations in Notch activity suffice to elicit profound differences in endothelial behaviour and blood vessel formation2,3 , little is known about the regulation and adaptation of endothelial Notch responses. Here we report that the NAD+-dependent deacetylase SIRT1 acts as an intrinsic negative modulator of Notch signalling in endothelial cells. We show that acetylation of the Notch1 intracellular domain (NICD) on conserved lysines controls the amplitude and duration of Notch responses by altering NICD protein turnover. SIRT1 associates with NICD and functions as a NICD deacetylase, which opposes the acetylation-induced NICD stabilization. Consequently, endothelial cells lacking SIRT1 activity are sensitized to Notch signalling, resulting in impaired growth, sprout elongation and enhanced Notch target gene expression in response to DLL4 stimulation, thereby promoting a non-sprouting, stalk-cell-like phenotype. In vivo, inactivation of Sirt1 in zebrafish and mice causes reduced vascular branching and density as a consequence of enhanced Notch signalling. Our findings identify reversible acetylation of the NICD as a molecular mechanism to adapt the dynamics of Notch signalling, and indicate that SIRT1 acts as rheostat to fine-tune endothelial Notch responses.