Genetic analysis of rab7 mutants in zebrafish

Vascular network formation requires the fusion of newly formed blood vessels and the emergence of a patent lumen between the newly established connections so that blood flow can start. Lumen formation has been shown to depend on the late endosomal/lysosomal pathway in various organs of animal tubular systems. Here, we identified a late endosomal/lysosomal vesicular fraction (Rab7/Lamp2) in early zebrafish angiogenic sprouts, which appears to contribute to apical membrane growth during lumen formation. To study the effect of the late endocytic pathway on vascular development, we generated mutant alleles for all three rab7 genes in zebrafish ( rab7a, rab7ba, rab7bb ). All rab7 genes are expressed in wild-type zebrafish and we did not detect any compensatory effects by the other rab7 isoforms in single knockout mutants, which were all viable. Only the triple mutant was lethal suggesting some functional redundancy. However, the different rab7 isoforms fulfil also at least partially independent functions because eggs laid from mothers lacking two rab7 ( rab7a and/or rab7bb ). showed reduced survival and contained enlarged yolk granules, suggesting maternal contribution of these two rab7 . Finally, we observed minor effects on lumen formation in embryos which still express one copy of rab7 . Our results support the notion that the late endocytic/lysosomal compartment contributes to lumen expansion

Live imaging molecular changes in junctional tension upon VE-cadherin in zebrafish

Forces play diverse roles in vascular development, homeostasis and disease. VE-cadherin at endothelial cell-cell junctions links the contractile acto-myosin cytoskeletons of adjacent cells, serving as a tension-transducer. To explore tensile changes across VE-cadherin in live zebrafish, we tailored an optical biosensor approach, originally established in vitro. We validate localization and function of a VE-cadherin tension sensor (TS) in vivo. Changes in tension across VE-cadherin observed using ratio-metric or lifetime FRET measurements reflect acto-myosin contractility within endothelial cells. Furthermore, we apply the TS to reveal biologically relevant changes in VE-cadherin tension that occur as the dorsal aorta matures and upon genetic and chemical perturbations during embryonic development

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis

Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin-F-actin based oscillatory cell-cell interaction

Angiogenesis and vascular remodeling are driven by extensive endothelial cell movements. Here, we present in vivo evidence that endothelial cell movements are associated with oscillating lamellipodia-like structures, which emerge from cell junctions in the direction of cell movements. High-resolution time-lapse imaging of these junction-based lamellipodia (JBL) shows dynamic and distinct deployment of junctional proteins, such as F-actin, VE-cadherin and ZO1, during JBL oscillations. Upon initiation, F-actin and VE-cadherin are broadly distributed within JBL, whereas ZO1 remains at cell junctions. Subsequently, a new junction is formed at the front of the JBL, which then merges with the proximal junction. Rac1 inhibition interferes with JBL oscillations and disrupts cell elongation-similar to a truncation in ve-cadherin preventing VE-cad/F-actin interaction. Taken together, our observations suggest an oscillating ratchet-like mechanism, which is used by endothelial cells to move over each other and thus provides the physical means for cell rearrangements

Endothelial cell division in angiogenic sprouts of differing cellular architecture

The vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a chord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic sprouting has so far not been investigated at detailed cellular level. Reasoning that different tubular architectures may impose discrete mechanistic constraints on endothelial cell division, we analyzed and compared the sequential steps of cell division, namely mitotic rounding, cytokinesis, actin re-distribution and adherence junction formation, in different blood vessels. In particular, we characterized the interplay between cell rearrangement, mitosis and lumen dynamics within unicellular and multicellular tubes. The lumen of unicellular tubes becomes constricted and is ultimately displaced from the plane of cell division, where a de novo junction forms through the recruitment of junctional proteins at the site of abscission. By contrast, the new junctions separating the daughter cells within multicellular tubes form through the alteration of pre-existing junctions, and the lumen is retained throughout mitosis. We also describe variations in the progression of cytokinesis: while membrane furrowing between daughter cells is symmetric in unicellular tubes, we found that it is asymmetric in those multicellular tubes that contained a taut intercellular junction close to the plane of division. Our findings illustrate that during the course of normal development, the cell division machinery can accommodate multiple tube architectures, thereby avoiding disruptions to the vascular network

Distinct and redundant functions of Esam and VE-cadherin during vascular morphogenesis

The cardiovascular system forms during early embryogenesis and adapts to embryonic growth by sprouting angiogenesis and vascular remodeling. These processes require fine-tuning of cell-cell adhesion to maintain and reestablish endothelial contacts, while allowing cell motility. We have compared the contribution of two endothelial cell specific adhesion proteins - VE-cadherin (VE-cad/Cdh5) and Esama (Endothelial cell-selective adhesion molecule a) - during angiogenic sprouting and blood vessel fusion (anastomosis) in the zebrafish embryo by genetic analyses. Different combinations of mutant alleles can be placed into a phenotypic series with increasing defects in filopodial contact formation. Contact formation in esama mutants appear wild-type like, while esama(-/-); ve-cad(+/-)and ve-cad single mutants exhibit intermediate phenotypes. The lack of both proteins interrupts filopodial interaction completely. Furthermore, double mutants do not form a stable endothelial monolayer, display intrajunctional gaps, dislocalization of Zo-1 and defects in apical-basal polarization. In summary, VE-cadherin and Esama have distinct and redundant functions during blood vessel morphogenesis and both adhesion proteins are central to endothelial cell recognition during anastomosis

Mapping the molecular steps of secretory-lysosome-driven tracheal tube fusion

During development, tubular networks form through the joining of lumenized branches. Further insights into tracheal tube fusion in Drosophila melanogaster now reveal the molecular steps that promote the connection of two apical membrane compartments within a single cell through secretory lysosomes

The Tip Cell Concept Ten Years After : New Players Tune in for a Common Theme

Embryonic growth, organ formation and regeneration rely on properly patterned vascular networks. During these processes, tissue growth is accompanied by an expansion of the respective vascular beds by angiogenesis. Angiogenesis involves the sprouting of new vessels from pre-existing ones and the eventual fusion of these sprouts with other sprouts or blood vessels to form new vascular loops. Sprouting blood vessels are made up of different cell populations: leading tip cells, which guide the sprout, and following stalk cells, which make up the base of the sprout and maintain the connection to the parental vessel. The functional and molecular differences between tip and stalk cells and how such differences ensure proper angiogenesis has drawn great attention within the last decade. In this review, we present recent progress in our understanding of tip cell specification and function during sprouting angiogenesis and anastomosis. We furthermore discuss similarities and variations in tip cell biology in different vascular beds and how they might be important for the formation of structurally and functionally distinct vascular networks

Cell behaviors and dynamics during angiogenesis

Vascular networks are formed and maintained through a multitude of angiogenic processes, such as sprouting, anastomosis and pruning. Only recently has it become possible to study the behavior of the endothelial cells that contribute to these networks at a single-cell level in vivo This Review summarizes what is known about endothelial cell behavior during developmental angiogenesis, focusing on the morphogenetic changes that these cells undergo

Building the complex architectures of vascular networks: Where to branch, where to connect and where to remodel?

The cardiovascular system is the first organ to become functional during vertebrate embryogenesis and is responsible for the distribution of oxygen and nutrients to all cells of the body. The cardiovascular system constitutes a circulatory loop in which blood flows from the heart through arteries into the microvasculature and back through veins to the heart. The vasculature is characterized by the heterogeneity of blood vessels with respect to size, cellular architecture and function, including both larger vessels that are found at defined positions within the body and smaller vessels or vascular beds that are organized in a less stereotyped manner. Recent studies have shed light on how the vascular tree is formed and how the interconnection of all branches is elaborated and maintained. In contrast to many other branched organs such as the lung or the kidney, vessel connection (also called anastomosis) is a key process underlying the formation of vascular networks; each outgrowing angiogenic sprout must anastomose in order to allow blood flow in the newly formed vessel segment. It turns out that during this "sprouting and anastomosis" process, too many vessels are generated, and that blood flow is subsequently optimized through the removal (pruning) of low flow segments. Here, we reflect on the cellular and molecular mechanisms involved in forming the complex architecture of the vasculature through sprouting, anastomosis and pruning, and raise some questions that remain to be addressed in future studies