Junctional remodeling during angiogenesis : the role of Claudin5 during ISV formation in Zebrafish

During sprouting angiogenesis several cellular activities are required such as cell shape changes, cell migration, cell proliferation and cell rearrangement. These processes have to be tightly regulated in order to allow cellular dynamics on one side and maintain tissue integrity on the other side. Cell-cell contacts are the main mediators of dynamic cell behaviors during morphogenesis. These contacts are composed of adherens and tight junctions. To better understand the role of tight junctions during the morphogenetic events of angiogenesis, I have analyzed the function of the endothelial specific Cldn5b during the process of intersegmental vessel (ISV) formation. Although MO-mediated knock-down of cldn5b does not alter the initial outgrowth of endothelial cells (ECs) from the dorsal aorta (DA), it does lead to a halt of EC migration at the level of the horizontal myoseptum (HMS). Furthermore, neighboring ISVs do not connect within the dorsal-longitudinal-anastomotic vessel (DLAV) and detach from the DA. In contrast, the adherens junction specific protein VE-cadherin rather exerts a cell rearrangement function. The different expression pattern of Cldn5b and VE-cadherin support these finding. While VE-cadherin is expressed in every EC of the ISV, Cldn5b is temporally downregulated in early sprouting ECs. Furthermore, a clear reduction of cell number in the ISVs was observed in cldn5b morphants. The latter observation, together with a possible reduction in adhesion between ECs are suggested to cause the phenotypes observed in cldn5b knock-down embryos. Together, this study suggests for the first time a possible role of a Cldn during a morphogenetic event. Additionally, I generated mutant fish lacking cldn5b gene function by using Zinc-Finger Nucleases. The analysis of these mutant fish will help to understand the functional role of a core component of tight junctions during vertebrate blood vessel development. However, the preliminary analysis show only a very weak ISV phenotype compared to cldn5b morphants. I will discuss several ideas explaining the discrepancy between cldn5b morphants and mutants

Oscillatory Flow Modulates Mechanosensitive klf2a Expression through trpv4 and trpp2 during Heart Valve Development

In vertebrates, heart pumping is required for cardiac morphogenesis and altering myocardial contractility leads to abnormal intracardiac flow forces and valve defects [1-3]. Among the different mechanical cues generated in the developing heart, oscillatory flow has been proposed to be an essential factor in instructing endocardial cell fate toward valvulogenesis and leads to the expression of klf2a [4], a known atheroprotective transcription factor [5]. To date, the mechanism by which flow forces are sensed by endocardial cells is not well understood. At the onset of valve formation, oscillatory flows alter the spectrum of the generated wall shear stress (WSS), a key mechanical input sensed by endothelial cells. Here, we establish that mechanosensitive channels are activated in response to oscillatory flow and directly affect valvulogenesis by modulating the endocardial cell response. By combining live imaging and mathematical modeling, we quantify the oscillatory content of the WSS during valve development and demonstrate it sets the endocardial cell response to flow. Furthermore, we show that an endocardial calcium response and the flow-responsive klf2a promoter are modulated by the oscillatory flow through Trpv4, a mechanosensitive ion channel specifically expressed in the endocardium during heart valve development. We made similar observations for Trpp2, a known Trpv4 partner, and show that both the absence of Trpv4 or Trpp2 leads to valve defects. This work identifies a major mechanotransduction pathway involved during valve formation in vertebrates

Cdh5/VE-cadherin Promotes Endothelial Cell Interface Elongation via Cortical Actin Polymerization during Angiogenic Sprouting

SummaryOrgan morphogenesis requires the coordination of cell behaviors. Here, we have analyzed dynamic endothelial cell behaviors underlying sprouting angiogenesis in vivo. Two different mechanisms contribute to sprout outgrowth: tip cells show strong migratory behavior, whereas extension of the stalk is dependent upon cell elongation. To investigate the function of Cdh5 in sprout outgrowth, we generated null mutations in the zebrafish cdh5 gene, and we found that junctional remodeling and cell elongation are impaired in mutant embryos. The defects are associated with a disorganization of the actin cytoskeleton and cannot be rescued by expression of a truncated version of Cdh5. Finally, the defects in junctional remodeling can be phenocopied by pharmacological inhibition of actin polymerization, but not by inhibiting actin-myosin contractility. Taken together, our results support a model in which Cdh5 organizes junctional and cortical actin cytoskeletons, as well as provides structural support for polymerizing F-actin cables during endothelial cell elongation

The tight junctions protein Claudin-5 limits endothelial cell motility

Steinberg's differential adhesion hypothesis suggests that adhesive mechanisms are important for sorting of cells and tissues during morphogenesis (Steinberg, 2007). During zebrafish vasculogenesis, endothelial cells sort into arterial and venous vessel beds but it is unknown whether this involves adhesive mechanisms. Claudins are tight junction proteins regulating the permeability of epithelial and endothelial tissue barriers. Previously, the roles of Claudins during organ development have exclusively been related to their canonical functions in determining paracellular permeability. Here, we use atomic force microscopy to quantify Claudin-5-dependent adhesion and find that this strongly contributes to the adhesive forces between arterial endothelial cells. Based on genetic manipulations, we reveal a non-canonical role of Claudin-5a during zebrafish vasculogenesis, which involves the regulation of adhesive forces between adjacent dorsal aortic endothelial cells.; In vitro; and; in vivo; studies demonstrate that loss of Claudin-5 results in increased motility of dorsal aorta endothelial cells and in a failure of the dorsal aorta to lumenize. Our findings uncover a novel role of Claudin-5 in limiting arterial endothelial cell motility, which goes beyond its traditional sealing function during embryonic development

The novel transmembrane protein Tmem2 is essential for coordination of myocardial and endocardial morphogenesis

Coordination between adjacent tissues plays a crucial role during the morphogenesis of developing organs. In the embryonic heart, two tissues - the myocardium and the endocardium - are closely juxtaposed throughout their development. Myocardial and endocardial cells originate in neighboring regions of the lateral mesoderm, migrate medially in a synchronized fashion, collaborate to create concentric layers of the heart tube, and communicate during formation of the atrioventricular canal. Here, we identify a novel transmembrane protein, Tmem2, that has important functions during both myocardial and endocardial morphogenesis. We find that the zebrafish mutation frozen ventricle (frv) causes ectopic atrioventricular canal characteristics in the ventricular myocardium and endocardium, indicating a role of frv in the regional restriction of atrioventricular canal differentiation. Furthermore, in maternal-zygotic frv mutants, both myocardial and endocardial cells fail to move to the midline normally, indicating that frv facilitates cardiac fusion. Positional cloning reveals that the frv locus encodes Tmem2, a predicted type II single-pass transmembrane protein. Homologs of Tmem2 are present in all examined vertebrate genomes, but nothing is known about its molecular or cellular function in any context. By employing transgenes to drive tissue-specific expression of tmem2, we find that Tmem2 can function in the endocardium to repress atrioventricular differentiation within the ventricle. Additionally, Tmem2 can function in the myocardium to promote the medial movement of both myocardial and endocardial cells. Together, our data reveal that Tmem2 is an essential mediator of myocardium-endocardium coordination during cardiac morphogenesis

Semaphorin-PlexinD1 Signaling Limits Angiogenic potential via the VEGF Decoy Receptor sFlt1

Sprouting angiogenesis expands the embryonic vasculature enabling survival and homeostasis. Yet how the angiogenic capacity to form sprouts is allocated among endothelial cells (ECs) to guarantee the reproducible anatomy of stereotypical vascular beds remains unclear. Here we show that Sema-PlxnD1 signaling, previously implicated in sprout guidance, represses angiogenic potential to ensure the proper abundance and stereotypical distribution of the trunk`s segmental arteries (SeAs). We find that Sema-PlxnD1 signaling exerts this effect by antagonizing the proangiogenic activity of vascular endothelial growth factor (VEGF). Specifically, Sema-PlxnD1 signaling ensures the proper endothelial abundance of soluble flt1 (sflt1), an alternatively spliced form of the VEGF receptor Flt1 encoding a potent secreted decoy. Hence, Sema-PlxnD1 signaling regulates distinct but related aspects of angiogenesis: the spatial allocation of angiogenic capacity within a primary vessel and sprout guidance

Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo

Although many of the cellular and molecular mechanisms of angiogenesis have been intensely studied [ 1], little is known about the processes that underlie vascular anastomosis. We have generated transgenic fish lines expressing an EGFP- tagged version of the junctional protein zona occludens 1 (ZO1) to visualize individual cell behaviors that occur during vessel fusion and lumen formation in vivo. These live observations show that endothelial cells (ECs) use two distinct morphogenetic mechanisms, cell membrane invagination and cord hollowing to generate different types of vascular tubes. During initial steps of anastomosis, cell junctions that have formed at the initial site of cell contacts expand into rings, generating a cellular interface of apical membrane compartments, as defined by the localization of the apical marker podocalyxin-2 (Pdxl2). During the cord hollowing process, these apical membrane compartments are brought together via cell rearrangements and extensive junctional remodeling, resulting in lumen coalescence and formation of a multicellular tube. Vessel fusion by membrane invagination occurs adjacent to a preexisting lumen in a proximal to distal direction and is blood-flow dependent. Here, the invaginating inner cell membrane undergoes concomitant apicobasal polarization and the vascular lumen is formed by the extension of a transcellular lumen through the EC, which forms a unicellular or seamless tube

In Vivo Analysis Reveals a Highly Stereotypic Morphogenetic Pathway of Vascular Anastomosis

Organ formation and growth requires cells to organize into properly patterned three-dimensional architectures. Network formation within the vertebrate vascular system is driven by fusion events between nascent sprouts or between sprouts and pre-existing blood vessels. Here, we describe the cellular activities that occur during blood vessel anastomosis in the cranial vasculature of the zebrafish embryo. We show that the early steps of the fusion process involve endothelial cell recognition, de novo polarization of endothelial cells, and apical membrane invagination and fusion. These processes generate a unicellular tube, which is then transformed into a multicellular tube via cell rearrangements and cell splitting. This stereotypic series of morphogenetic events is typical for anastomosis in perfused sprouts. Vascular endothelial-cadherin plays an important role early in the anastomosis process and is required for filopodial tip cell interactions and efficient formation of a single contact site