Dynamically regulated focal adhesions coordinate endothelial cell remodelling in developing vasculature.

The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including the process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we have generated a zebrafish mutant that is deficient for the integrin adaptor protein Talin 1 (Tln1). Using a new focal adhesion (FA) marker line expressing endothelial Vinculinb-eGFP, we demonstrate that EC FAs function dynamically and are lost in our tln1 mutants, allowing us to uncouple the primary roles of FAs in EC morphogenesis from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Tln1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored actin dynamics and EC elongation during vascular morphogenesis. Together, we identify that FAs are essential for EC elongation and junction linearisation in flow-pressured vessels and that they influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling. This article has an associated 'The people behind the papers' interview.

Endothelial cells are not productively infected by SARS-CoV-2.

OBJECTIVES: Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious. METHODS: Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier. RESULTS: We show that primary human endothelial cells express very low levels of the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS-CoV-2. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines. CONCLUSIONS: Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

The RNA helicase Ddx21 controls Vegfc-driven developmental lymphangiogenesis by balancing endothelial cell ribosome biogenesis and p53 function.

The development of a functional vasculature requires the coordinated control of cell fate, lineage differentiation and network growth. Cellular proliferation is spatiotemporally regulated in developing vessels, but how this is orchestrated in different lineages is unknown. Here, using a zebrafish genetic screen for lymphatic-deficient mutants, we uncover a mutant for the RNA helicase Ddx21. Ddx21 cell-autonomously regulates lymphatic vessel development. An established regulator of ribosomal RNA synthesis and ribosome biogenesis, Ddx21 is enriched in sprouting venous endothelial cells in response to Vegfc-Flt4 signalling. Ddx21 function is essential for Vegfc-Flt4-driven endothelial cell proliferation. In the absence of Ddx21, endothelial cells show reduced ribosome biogenesis, p53 and p21 upregulation and cell cycle arrest that blocks lymphangiogenesis. Thus, Ddx21 coordinates the lymphatic endothelial cell response to Vegfc-Flt4 signalling by balancing ribosome biogenesis and p53 function. This mechanism may be targetable in diseases of excessive lymphangiogenesis such as cancer metastasis or lymphatic malformation.

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.

Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish.

Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.

VE-cadherin in vascular development: a coordinator of cell signaling and tissue morphogenesis.

Blood and lymphatic vessels make up the vascular system of vertebrates and are lined by specialized endothelial cells. The connections between endothelial cells are formed by adhesion molecules and are essential to maintain cell-cell adhesion, cell-cell communication, and the integrity of our vascular tubes. One key adhesion molecule is the adherens junctional protein vascular endothelial cadherin (VE-cadherin). In addition to its role in endothelial adhesion, it is emerging that this protein is actively involved in modulating key cellular signaling cascades within endothelial cells and can control the behavior of endothelial cells during development and morphogenesis. We describe key studies that highlight VE-cadherin as a regulatory hub in endothelial cell signaling during angiogenesis, vessel morphogenesis, and vascular development.

A Nodal-independent and tissue-intrinsic mechanism controls heart-looping chirality.

Breaking left-right symmetry in bilateria is a major event during embryo development that is required for asymmetric organ position, directional organ looping and lateralized organ function in the adult. Asymmetric expression of Nodal-related genes is hypothesized to be the driving force behind regulation of organ laterality. Here we identify a Nodal-independent mechanism that drives asymmetric heart looping in zebrafish embryos. In a unique mutant defective for the Nodal-related southpaw gene, preferential dextral looping in the heart is maintained, whereas gut and brain asymmetries are randomized. As genetic and pharmacological inhibition of Nodal signalling does not abolish heart asymmetry, a yet undiscovered mechanism controls heart chirality. This mechanism is tissue intrinsic, as explanted hearts maintain ex vivo retain chiral looping behaviour and require actin polymerization and myosin II activity. We find that Nodal signalling regulates actin gene expression, supporting a model in which Nodal signalling amplifies this tissue-intrinsic mechanism of heart looping.

Hyaluronan: a critical regulator of endothelial-to-mesenchymal transition during cardiac valve formation.

During embryonic development, cardiac valves arise at specific regions in the cardiac endothelium that swell up due to enhanced extracellular matrix production (so-called endocardial cushions). An important extracellular matrix component that is produced by the endocardial cells is the glycosaminoglycan hyaluronan. A deficiency in hyaluronan synthesis results in a failure to form endocardial cushions and a loss of their cellularization by a process called endothelial-to-mesenchymal transformation. Expression of the major hyaluronan synthase Has2 is under the influence of both positive and negative regulators. MicroRNA-dependent degradation of Has2 is required to control extracellular hyaluronan levels and thereby the size of the endocardial cushions. In this article, we review the current literature on hyaluronan synthesis during cardiac valve formation and propose that a balanced activity of both positive and negative regulators is required to maintain the critical homeostasis of hyaluronan levels in the extracellular matrix and thereby the size of the endocardial cushions. The activating and inhibitory interactions between microRNA-23, Has2, and hyaluronan are reminiscent of a reaction-diffusion system. Using a mathematical modeling approach we show that the system can produce a confined expression of hyaluronan, but only if the inhibitory signal is transferred to adjacent cells in exosomes.

Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues.

Non-coding microRNA (miRNA) molecules bind their target mRNAs and thereby modulate the amount of protein produced. To understand the significance of a potential miRNA-mRNA interaction, temporal and spatial information on miRNA and mRNA expression is essential. Here, we provide a detailed protocol for miRNA whole mount in situ hybridization. We introduce the use of Morpholino based oligos as antisense probes for miRNA detection, in addition to the current "gold standard" locked nucleic acid (LNA) probes. Furthermore we have modified existing miRNA in situ protocols thereby improving both sensitivity and resolution of miRNA visualization in whole zebrafish embryos and adult tissues.

Bmp signaling exerts opposite effects on cardiac differentiation.

RATIONALE: The importance for Bmp signaling during embryonic stem cell differentiation into myocardial cells has been recognized. The question when and where Bmp signaling in vivo regulates myocardial differentiation has remained largely unanswered. OBJECTIVE: To identify when and where Bmp signaling regulates cardiogenic differentiation. METHODS AND RESULTS: Here we have observed that in zebrafish embryos, Bmp signaling is active in cardiac progenitor cells prior to their differentiation into cardiomyocytes. Bmp signaling is continuously required during somitogenesis within the anterior lateral plate mesoderm to induce myocardial differentiation. Surprisingly, Bmp signaling is actively repressed in differentiating myocardial cells. We identified the inhibitory Smad6a, which is expressed in the cardiac tissue, to be required to inhibit Bmp signaling and thereby promote expansion of the ventricular myocardium. CONCLUSION: Bmp signaling exerts opposing effects on myocardial differentiation in the embryo by promoting as well as inhibiting cardiac growth.

MicroRNA-23 restricts cardiac valve formation by inhibiting Has2 and extracellular hyaluronic acid production.

RATIONALE: Since their discovery almost 20 years ago, microRNAs have been shown to perform essential roles during tissue development and disease. Although roles for microRNAs in the myocardium during embryo development and cardiac disease have been demonstrated, very little is know about their role in the endocardium or during cardiac valve formation. OBJECTIVE: To study the role of microRNAs in cardiac valve formation. METHODS AND RESULTS: We show that zebrafish dicer mutant embryos, lacking mature miRNAs, form excessive endocardial cushions. By screening miRNAs expressed in the heart, we found that miR-23 is both necessary and sufficient for restricting the number of endocardial cells that differentiate into endocardial cushion cells. In addition, in mouse endothelial cells, miR-23 inhibited a transforming growth factor-ß-induced endothelial-to-mesenchymal transition. By in silico screening of expression data with predicted miR-23 target sites combined with in vivo testing, we identified hyaluronic acid synthase 2 (Has2), Icat, and Tmem2 as novel direct targets of miR-23. Finally, we demonstrate that the upregulation of Has2, an extracellular remodeling enzyme required for endocardial cushion and valve formation, is responsible for the excessive endocardial cushion cell differentiation in dicer mutants. CONCLUSIONS: MiR-23 in the embryonic heart is required to restrict endocardial cushion formation by inhibiting Has2 expression and extracellular hyaluronic acid production.

Genetics of congenital heart defects: a candidate gene approach.

By using a candidate gene approach, we have identified novel single-nucleotide polymorphisms specific to patients diagnosed with atrioventricular valve and septum defects. Here we discuss how the gene products, in which these polymorphisms were found, functionally interact to regulate endocardial cushion formation during embryo development. These findings support a model in which mutations in different genes but regulating the same process can cause or make one more susceptible to developing atrioventricular valve and septum defects.

Missorting of the Aquaporin-2 mutant E258K to multivesicular bodies/lysosomes in dominant NDI is associated with its monoubiquitination and increased phosphorylation by PKC but is due to the loss of E258.

To stimulate renal water reabsorption, vasopressin induces phosphorylation of Aquaporin-2 (AQP2) water channels at S256 and their redistribution from vesicles to the apical membrane, whereas vasopressin removal results in AQP2 ubiquitination at K270 and its internalization to multivesicular bodies (MVB). AQP2-E258K causes dominant nephrogenic diabetes insipidus (NDI), but its subcellular location is unclear, and the molecular reason for its involvement in dominant NDI is unknown. To unravel these, AQP2-E258K was studied in transfected polarized Madin-Darby canine kidney (MDCK) cells. In MDCK cells, AQP2-E258K mainly localized to MVB/lysosomes (Lys). Upon coexpression, wild-type (wt) AQP2 and AQP2-E258K formed multimers, which also localized to MVB/Lys, independent of forskolin stimulation. Orthophosphate labeling revealed that forskolin increased phosphorylation of wt-AQP2 and AQP2-E258K but not AQP2-S256A, indicating that the E258K mutation does not interfere with the AQP2 phosphorylation at S256. In contrast to wt-AQP2 but consistent with the introduced protein kinase C (PKC) consensus site, AQP2-E258K was phosphorylated by phorbol esters. Besides the 29-kDa band, however, an additional band of about 35 kDa was observed for AQP2-E258K only, which represented AQP2-E258K uniquely monoubiquitinated at K228 only. Analysis of several mutants interfering with AQP2-E258K phosphorylation, and/or ubiquitination, however, revealed that the MVB/lysosomal sorting of AQP2-E258K occurred independent of its monoubiquitination or phosphorylation by PKC. Instead, our data reveal that the loss of the E258 in AQP2-E258K is fundamental to its missorting to MVB/Lys and indicate that this amino acid has an important role in the proper structure formation of the C-terminal tail of AQP2.