The endosomal RIN2/Rab5C machinery prevents VEGFR2 degradation to control gene expression and tip cell identity during angiogenesis.

Sprouting angiogenesis is key to many pathophysiological conditions, and is strongly regulated by vascular endothelial growth factor (VEGF) signaling through VEGF receptor 2 (VEGFR2). Here we report that the early endosomal GTPase Rab5C and its activator RIN2 prevent lysosomal routing and degradation of VEGF-bound, internalized VEGFR2 in human endothelial cells. Stabilization of endosomal VEGFR2 levels by RIN2/Rab5C is crucial for VEGF signaling through the ERK and PI3-K pathways, the expression of immediate VEGF target genes, as well as specification of angiogenic 'tip' and 'stalk' cell phenotypes and cell sprouting. Using overexpression of Rab mutants, knockdown and CRISPR/Cas9-mediated gene editing, and live-cell imaging in zebrafish, we further show that endosomal stabilization of VEGFR2 levels is required for developmental angiogenesis in vivo. In contrast, the premature degradation of internalized VEGFR2 disrupts VEGF signaling, gene expression, and tip cell formation and migration. Thus, an endosomal feedforward mechanism maintains receptor signaling by preventing lysosomal degradation, which is directly linked to the induction of target genes and cell fate in collectively migrating cells during morphogenesis.

Live imaging of angiogenesis during cutaneous wound healing in adult zebrafish.

Angiogenesis, the growth of new blood vessels from pre-existing vessels, is critical for cutaneous wound healing. However, it remains elusive how endothelial cells (ECs) and pericytes (PCs) establish new blood vessels during cutaneous angiogenesis. We set up a live-imaging system to analyze cutaneous angiogenesis in adult zebrafish. First, we characterized basic structures of cutaneous vasculature. In normal skin tissues, ECs and PCs remained dormant to maintain quiescent blood vessels, whereas cutaneous injury immediately induced angiogenesis through the vascular endothelial growth factor signaling pathway. Tortuous and disorganized vessel networks formed within a few weeks after the injury and subsequently normalized through vessel regression in a few months. Analyses of the repair process of injured single blood vessels revealed that severed vessels elongated upon injury and anastomosed with each other. Thereafter, repaired vessels and adjacent uninjured vessels became tortuous by increasing the number of ECs. In parallel, PCs divided and migrated to cover the tortuous blood vessels. ECs sprouted from the PC-covered tortuous vessels, suggesting that EC sprouting does not require PC detachment from the vessel wall. Thus, live imaging of cutaneous angiogenesis in adult zebrafish enables us to clarify how ECs and PCs develop new blood vessels during cutaneous angiogenesis.

ß-Catenin-dependent transcription is central to Bmp-mediated formation of venous vessels.

ß-catenin regulates the transcription of genes involved in diverse biological processes, including embryogenesis, tissue homeostasis and regeneration. Endothelial cell (EC)-specific gene-targeting analyses in mice have revealed that ß-catenin is required for vascular development. However, the precise function of ß-catenin-mediated gene regulation in vascular development is not well understood, since ß-catenin regulates not only gene expression but also the formation of cell-cell junctions. To address this question, we have developed a novel transgenic zebrafish line that allows the visualization of ß-catenin transcriptional activity specifically in ECs and discovered that ß-catenin-dependent transcription is central to the bone morphogenetic protein (Bmp)-mediated formation of venous vessels. During caudal vein (CV) formation, Bmp induces the expression of aggf1, a putative causative gene for Klippel-Trenaunay syndrome, which is characterized by venous malformation and hypertrophy of bones and soft tissues. Subsequently, Aggf1 potentiates ß-catenin transcriptional activity by acting as a transcriptional co-factor, suggesting that Bmp evokes ß-catenin-mediated gene expression through Aggf1 expression. Bmp-mediated activation of ß-catenin induces the expression of Nr2f2 (also known as Coup-TFII), a member of the nuclear receptor superfamily, to promote the differentiation of venous ECs, thereby contributing to CV formation. Furthermore, ß-catenin stimulated by Bmp promotes the survival of venous ECs, but not that of arterial ECs. Collectively, these results indicate that Bmp-induced activation of ß-catenin through Aggf1 regulates CV development by promoting the Nr2f2-dependent differentiation of venous ECs and their survival. This study demonstrates, for the first time, a crucial role of ß-catenin-mediated gene expression in the development of venous vessels.

Visualizing the cell-cycle progression of endothelial cells in zebrafish.

The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.

Looking back and moving forward: recent advances in understanding of cardiovascular development by imaging of zebrafish.

Development requires cell proliferation, migration, differentiation, apoptosis, and many kinds of cell responses. Cells prepare intracellular conditions to respond to extracellular cues from neighboring cells. We have studied the development of the cardiovascular system (CVS) by visualizing morphology and signaling simultaneously using zebrafish, which express probes for both. Endodermal sheet is required for the bilateral cardiac precursor cell (CPC) migration toward the midline. Endothelial cells (ECs) proliferate specifically in the certain regions of blood vessels. Bone morphogenetic proteins (BMP) induce the remodeling of the caudal vein plexus (CVP) to form the caudal vein (CV). Our findings point to the pre-existing neighboring cells as the cells exhibiting certain responses during the development of CVS. In this review, we introduce recent results of our research on angiogenesis and cardiogenesis by spotlighting the mechanism by which ECs and CPCs are regulated by the cells next to themselves. In addition, we discuss the unanswered questions that should be clarified in the future in the field of CVS development.

Vascular and Neuronal Network Formation Regulated by Growth Factors and Guidance Cues.

Blood vessels and nerves are distributed throughout the body and show a high degree of anatomical parallelism and functional crosstalk. These networks transport oxygen, nutrients, and information to maintain homeostasis. Thus, disruption of network formation can cause diseases. Nervous system development requires the navigation of the axons of neurons to their correct destination. Blood vessel formation occurs via vasculogenesis and angiogenesis. Vasculogenesis is the process of de novo blood vessel formation, and angiogenesis is the process whereby endothelial cells sprout from pre-existing vessels. Both developmental processes require guidance molecules to establish precise branching patterns of these systems in the vertebrate body. These network formations are regulated by growth factors, such as vascular endothelial growth factor; and guidance cues, such as ephrin, netrin, semaphorin, and slit. Neuronal and vascular structures extend lamellipodia and filopodia, which sense guidance cues that are mediated by the Rho family and actin cytosol rearrangement, to migrate to the goal during development. Furthermore, endothelial cells regulate neuronal development and vice versa. In this review, we describe the guidance molecules that regulate neuronal and vascular network formation.

EphrinA1-EphA2 signal induces compaction and polarization of Madin-Darby canine kidney cells by inactivating Ezrin through negative regulation of RhoA.

The epithelial cells exhibit either a columnar or a flat shape dependent on extracellular stimuli or the cell-cell adhesion. Membrane-anchored ephrinA stimulates EphA receptor tyrosine kinases as a ligand in a cell-cell contact-dependent manner. The mechanism through which ephrinA1/EphA2 signal regulates the cell morphology remains elusive. We demonstrate here that ephrinA1/EphA2 signal induces compaction and enhanced polarization (columnar change) of Madin-Darby canine kidney epithelial cells by regulating Ezrin, a linker that connects plasma membrane and actin cytoskeleton. Activation of EphA2 resulted in RhoA inactivation through p190RhoGAP-A and subsequent dephosphorylation of Ezrin on Thr-567 phosphorylated by Rho kinase. Consistently, the cells expressing an active mutant of Ezrin in which Thr-567 was replaced with Asp did not change their shape in response to ephrinA1. Furthermore, depletion of Ezrin led to compaction and enhanced polarization without ephrinA1 stimulation, suggesting the role for active Ezrin in keeping the flat cell shape. Ezrin localized to apical domain irrespective of ephrinA1 stimulation, whereas phosphorylated Ezrin on the apical domain was reduced by ephrinA1 stimulation. Collectively, ephrinA1/EphA2 signal negatively regulates Ezrin and promotes the alteration of cell shape, from flat to columnar shape.

Mechanical loading of intraluminal pressure mediates wound angiogenesis by regulating the TOCA family of F-BAR proteins.

Angiogenesis is regulated in coordinated fashion by chemical and mechanical cues acting on endothelial cells (ECs). However, the mechanobiological mechanisms of angiogenesis remain unknown. Herein, we demonstrate a crucial role of blood flow-driven intraluminal pressure (IP) in regulating wound angiogenesis. During wound angiogenesis, blood flow-driven IP loading inhibits elongation of injured blood vessels located at sites upstream from blood flow, while downstream injured vessels actively elongate. In downstream injured vessels, F-BAR proteins, TOCA1 and CIP4, localize at leading edge of ECs to promote N-WASP-dependent Arp2/3 complex-mediated actin polymerization and front-rear polarization for vessel elongation. In contrast, IP loading expands upstream injured vessels and stretches ECs, preventing leading edge localization of TOCA1 and CIP4 to inhibit directed EC migration and vessel elongation. These data indicate that the TOCA family of F-BAR proteins are key actin regulatory proteins required for directed EC migration and sense mechanical cell stretching to regulate wound angiogenesis.

Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits.

Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter gene activities ensure control of the cell with synthetic genetic circuits, as achieved using a combination of in silico and in vivo experiments. However, unexpected effects of downstream reporter genes on upstream regulatory genes may interfere with in vivo observations. This phenomenon is termed as retroactivity. Using in silico and in vivo experiments, we found that a different copy number of regulatory protein-binding sites in a downstream gene altered the upstream dynamics, suggesting retroactivity of reporters in this synthetic genetic oscillator. Furthermore, by separating the two sources of retroactivity (titration of the component and competition for degradation), we showed that, in the dual-feedback oscillator, the level of the fluorescent protein reporter competing for degradation with the circuits' components is important for the stability of the oscillations. Altogether, our results indicate that the selection of reporter promoters using a combination of in silico and in vivo experiments is essential for the advanced design of genetic circuits.

Wnt/ß-catenin signaling regulates VE-cadherin-mediated anastomosis of brain capillaries by counteracting S1pr1 signaling.

Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis.

Cdc42 mediates Bmp-induced sprouting angiogenesis through Fmnl3-driven assembly of endothelial filopodia in zebrafish.

During angiogenesis in vivo, endothelial cells (ECs) at the tips of vascular sprouts actively extend filopodia that are filled with bundles of linear actin filaments. To date, signaling pathways involved in the formation of endothelial filopodia have been studied using in-vitro-cultured ECs that behave differently from those in vivo. Herein, we have delineated a signaling pathway that governs the assembly of endothelial filopodia during angiogenic sprouting of the caudal vein plexus (CVP) in zebrafish. During CVP formation, bone morphogenetic protein induces the extension of endothelial filopodia and their migration via Arhgef9b-mediated activation of Cdc42. Active Cdc42 binds to and stimulates Formin-like 3, an actin-regulatory protein of the formin family, which, in turn, promotes the extension of endothelial filopodia to facilitate angiogenic sprouting of the CVP. Thus, this study has elucidated molecular mechanisms underlying the formation of endothelial filopodia and their role in angiogenesis in vivo.

Endothelial Ca 2+ oscillations reflect VEGFR signaling-regulated angiogenic capacity in vivo.

Sprouting angiogenesis is a well-coordinated process controlled by multiple extracellular inputs, including vascular endothelial growth factor (VEGF). However, little is known about when and how individual endothelial cell (EC) responds to angiogenic inputs in vivo. Here, we visualized endothelial Ca(2+) dynamics in zebrafish and found that intracellular Ca(2+) oscillations occurred in ECs exhibiting angiogenic behavior. Ca(2+) oscillations depended upon VEGF receptor-2 (Vegfr2) and Vegfr3 in ECs budding from the dorsal aorta (DA) and posterior cardinal vein, respectively. Thus, visualizing Ca(2+) oscillations allowed us to monitor EC responses to angiogenic cues. Vegfr-dependent Ca(2+) oscillations occurred in migrating tip cells as well as stalk cells budding from the DA. We investigated how Dll4/Notch signaling regulates endothelial Ca(2+) oscillations and found that it was required for the selection of single stalk cell as well as tip cell. Thus, we captured spatio-temporal Ca(2+) dynamics during sprouting angiogenesis, as a result of cellular responses to angiogenic inputs.

EphrinA/EphA signal facilitates insulin-like growth factor-I-induced myogenic differentiation through suppression of the Ras/extracellular signal-regulated kinase 1/2 cascade in myoblast cell lines.

Insulin-like growth factor-I (IGF-I) activates not only the phosphatidylinositol 3-kinase (PI3K)-AKT cascade that is essential for myogenic differentiation but also the extracellular signal-regulated kinase (ERK) 1/2 cascade that inhibits myogenesis. We hypothesized that there must be a signal that inhibits ERK1/2 upon cell-cell contact required for skeletal myogenesis. Cell-cell contact-induced engagement of ephrin ligands and Eph receptors leads to downregulation of the Ras-ERK1/2 pathway through p120 Ras GTPase-activating protein (p120RasGAP). We therefore investigated the significance of the ephrin/Eph signal in IGF-I-induced myogenesis. EphrinA1-Fc suppressed IGF-I-induced activation of Ras and ERK1/2, but not that of AKT, in C2C12 myoblasts, whereas ephrinB1-Fc affected neither ERK1/2 nor AKT activated by IGF-I. IGF-I-dependent myogenic differentiation of C2C12 myoblasts was potentiated by ephrinA1-Fc. In p120RasGAP-depleted cells, ephrinA1-Fc failed to suppress the Ras-ERK1/2 cascade by IGF-I and to promote IGF-I-mediated myogenesis. EphrinA1-Fc did not promote IGF-I-dependent myogenesis when the ERK1/2 was constitutively activated. Furthermore, a dominant-negative EphA receptor blunted IGF-I-induced myogenesis in C2C12 and L6 myoblasts. However, the inhibition of IGF-I-mediated myogenesis by down-regulation of ephrinA/EphA signal was canceled by inactivation of the ERK1/2 pathway. Collectively, these findings demonstrate that the ephrinA/EphA signal facilitates IGF-I-induced myogenesis by suppressing the Ras-ERK1/2 cascade through p120RasGAP in myoblast cell lines.