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Morphogenesis of hierarchical vascular networks depends on the integration of multiple biomechanical signals by endothelial cells, the cells lining the interior of blood vessels. Expansion of vascular networks arises through sprouting angiogenesis, a process involving extensive cell rearrangements and collective cell migration. Yet, the mechanisms controlling angiogenic collective behavior remain poorly understood. Here, we show this collective cell behavior is regulated by non-canonical Wnt signaling. We identify that Wnt5a specifically activates Cdc42 at cell junctions downstream of ROR2 to reinforce coupling between adherens junctions and the actin cytoskeleton. We show that Wnt5a signaling stabilizes vinculin binding to alpha-catenin, and abrogation of vinculin in vivo and in vitro leads to uncoordinated polarity and deficient sprouting angiogenesis in Mus musculus. Our findings highlight how non-canonical Wnt signaling coordinates collective cell behavior during vascular morphogenesis by fine-tuning junctional mechanocoupling between endothelial cells. When a new blood vessel is created, a leader cell branches out from the lining of an existing vessel before being joined by other cells moving together in the same direction. A protein called Wnt5a regulates this process by helping the cells to orient themselves and finely coordinating their migration, but the exact details of this mechanism are still unclear. One way that cells can communicate is by touching and physically exerting forces on each other. This is made possible by structures called cellular junctions, which are present at the interface between cells. These can transmit forces within a tissue because they are connected with elements that form the cells’ internal skeletons. A protein known as vinculin is involved in these connections. To find out what role Wnt5a plays in cell migration, Carvalho et al. prevented blood vessel cells from creating the protein. The results showed that Wnt5a helps cells to move together by stabilizing vinculin at cell junctions. This strengthens the physical communication between cells and allows them to efficiently coordinate their movements. Indeed, in the mouse retina, deleting vinculin from cells that make blood cells impaired the formation of new blood vessels. Problems in the way that blood vessels grow are very common in the human population. In addition, Wnt5a is linked to cancer progression, which also relies on coordinated movement of cells. A better grasp of the role of this protein could therefore be relevant to understand how blood vessels are formed, but also how certain cancers invade surrounding tissues.

Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus. Blood vessels play an essential role in growth and development as they transport many important molecules that help cells to survive. Throughout life, the forces that act on the blood vessels help to remodel the vessel network to ensure that blood gets to the parts of the body that need it. For example, the movement of blood across the surface of the endothelial cells that line the inside of the blood vessels applies a force called “shear stress” to the cells. The endothelial cells respond and adapt to the stress by altering their shape, patterns of gene activity and internal organization (known as their polarity). It was not fully understood exactly how the forces acting on endothelial cells help to remodel the blood vessel network. Franco et al. have now investigated how a signalling pathway known as non-canonical Wnt signalling affects the remodelling of blood vessels in mice, and found that this pathway stabilizes existing connections between vessels. Disrupting non-canonical Wnt signalling, by genetically engineering mice to lack proteins called Wnt5a and Wnt11, increased the sensitivity of endothelial cells to shear stress. Franco et al. then built a computer model that simulates blood flow and endothelial cell polarity in a network of blood vessels; this enabled them to measure the endothelial cells’ response to blood flow in complex vascular networks. The model was then used to show that endothelial cells lacking non-canonical Wnt signalling are able to reorient and become polarized against the direction of blood flow at lower levels of shear stress. Thus, non-canonical Wnt signalling helps to raise the threshold of shear stress above which endothelial cells change their properties. Further work is now needed to identify how non-canonical Wnt signalling interferes with the ability of the endothelial cells to sense shear stress levels.

Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.

The establishment of a functional patterned vascular network is crucial for development, tissue growth and homeostasis. The mis-patterning or dysfunction of this network is associated with cancer, stroke and arteriovenous malformations. The formation of a functional vascular network requires two distinct processes – formation of primitive vascular plexuses through sprouting angiogenesis; and vascular remodelling, the reorganization of primitive plexuses into a hierarchical network of arteries, capillaries and veins. During sprouting angiogenesis endothelial cells polarise and migrate towards sources of VEGF while upon vascular remodelling, they polarise and migrate against the blood flow direction. Nevertheless, the molecular mechanisms regulating migration and polarisation during sprouting and vascular remodelling are poorly understood.Here, we investigate the mechanisms of polarisation and migration during sprouting angiogenesis by unravelling the role of Arp2/3 complex in vascular morphogenesis, using a combination of in vitroapproaches with the power of mouse genetics. We show that Arp2/3 is crucial for endothelial cell migration, for the establishment of cellular protrusions and that its depletion drastically affects vascular morphogenesis during development and tumour angiogenesis.At the same time, we show that blood flow and VEGF interact to orchestrate patterns of endothelial cell polarity at the network-level. We demonstrate that this interaction defines the transition between two distinct morphogenic events, vascular sprouting and vascular remodelling. Accordingly, manipulation of VEGF gradients or blood flow in vivocompromises normal polarity patterns, resulting in delayed or premature remodelling of blood vessels. At the molecular level, we show that mechanotransduction at adherens junctions is key for VEGF-induced polarisation and negatively regulates flow-dependent collective polarisation. Specifically, we propose that vinculin recruitment is key for mechanotransduction and the establishment of a transition zone. In addition, we generated a polarity reporter mouse with endothelial cell nuclei and Golgi labelled that will enable the study the dynamics of endothelial cell polarisation downstream of VEGF and blood flow in development, homeostasis and disease.Given the physiological relevance of vascular patterning in health and disease, our approach will give insights in defining the cellular and molecular principles involved in vascular patterning.

During spinal cord development, Notch signalling regulates both the maintenance of progenitors and neuronal specification. In most spinal cord domains, a single Notch ligand is expressed however in the V2 domain two ligands (Dll1 and Dll4) are expressed. It is not known how Dll4 expression is regulated and how Dll4-mediated Notch signalling has a role in the specification of V2 interneurons (V2a, V2b and V2c) that are generated from V2 progenitors.To investigate how Dll4 expression is regulated, we have focused on three proneural bHLH genes (Mash1, Ngn1 and Ngn2), expressed in V2 progenitor cells. We characterized Dll4-expressing cells, by double RNA in-situ hybridization with each of the proneural genes. Our results revealed that Dll4+ cells can coexpress all three proneural genes but to different extents. Next, we have overexpressed each proneural gene (singly and in combination) in the chick neural tube, using in ovo electroporation. Embryos were tested in terms of: Dll4expression and V2 cell-type specification.Although electroporation of either Mash1 or Ngn1 leads to downregulation of Dll4 expression in the V2 domain, only NGN1 is capable of inducing ectopic Dll4 expression. Moreover, both double electroporation of Mash1 and Ngn1, or Mash1 and Ngn2, represses Dll4expression in the V2 domain, while leading to ectopic expression in other ventral spinal cord domains.Concerning cell-type specification, electroporation of Mash1 represses both V2a and V2b cell fate. Electroporation of either Ngn1 or Ngn2 increases the number of V2a INs, while repressing V2b INs. Double electroporation of Mash1/Ngn1, Mash1/Ngn2 or Ngn1/Ngn2increases the number of V2a INs and represses V2b fate.Together, the results presented in this thesis show that MASH1 and NGN1 can repress Dll4 expression and that this repression correlates with alterations in V2 IN specification. Our results indicate that NGN2 can affect V2 specification, although it does not regulate Dll4expression.

Blood vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs, sprouting angiogenesis and vascular remodeling, is established by a shift on collective front-rear polarity of endothelial cells. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns. Competing Interest Statement The authors have declared no competing interest.

Abstract Sprouting angiogenesis is fundamental for development and contributes to multiple diseases, including cancer, diabetic retinopathy and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on the mechanisms that endothelial tip cells use to invade into tissues. Here, we prove that endothelial tip cells use long lamellipodia projections (LLPs) as the main cellular protrusion for invasion into non-vascular extracellular matrix. We show that LLPs and filopodia protrusions are balanced by myosin-IIA (MIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of MIIA promotes excessive LLPs formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents LLPs development and leads to excessive filopodia formation. We further show that MIIA inhibits Rac1-dependent activation of Arp2/3, by regulating the maturation state of focal adhesions. Our discoveries establish the first comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way towards new strategies to block invasive tip cells during sprouting angiogenesis. Competing Interest Statement The authors have declared no competing interest.