Vascular Development In Zebrafish Research Articles

Sphingosine-1-Phosphate Receptor-1 Controls Venous Endothelial Barrier Integrity in Zebrafish

Endothelial sphingosine-1-phosphate (S1P) receptor-1 (S1P(1)) affects different vascular functions, including blood vessel maturation and permeability. Here, we characterized the role of the zS1P(1) ortholog in vascular development in zebrafish. zS1P(1) is expressed in dorsal aorta and posterior cardinal vein of zebrafish embryos at 24 to 30 hours postfertilization. zS1P(1) downregulation by antisense morpholino oligonucleotide injection causes early pericardial edema, lack of blood circulation, alterations of posterior cardinal vein structure, and late generalized edema. Also, zS1P(1) morphants are characterized by downregulation of vascular endothelial cadherin (VE-cadherin) and Eph receptor EphB4a expression and by disorganization of zonula occludens 1 junctions in posterior cardinal vein endothelium, with no alterations of dorsal aorta endothelium. VE-cadherin knockdown results in similar vascular alterations, whereas VE-cadherin overexpression is sufficient to rescue venous vascular integrity defects and EphB4a downregulation in zS1P(1) morphants. Finally, S1P(1) small interfering RNA transfection and the S1P(1) antagonist (R)-3-amino-(3-hexylphenylamino)-4-oxobutylphosphonic acid (W146) cause EPHB4 receptor down-modulation in human umbilical vein endothelial cells and the assembly of zonula occludens 1 intercellular contacts is prevented by the EPHB4 antagonist TNYL-RAW peptide in these cells. The data demonstrate a nonredundant role of zS1P(1) in the regulation of venous endothelial barrier in zebrafish and identify a S1P(1)/VE-cadherin/EphB4a genetic pathway that controls venous vascular integrity.

The F-BAR protein NOSTRIN participates in FGF signal transduction and vascular development

F-BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F-BAR proteins in vivo. Here we report, that the F-BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.

The Synaptic Proteins β-Neurexin and Neuroligin Synergize With Extracellular Matrix-Binding Vascular Endothelial Growth Factor A During Zebrafish Vascular Development

The goal of this study was to determine the in vivo functions of the synaptic proteins neurexins and neuroligins in embryonic vascular system development using zebrafish as animal model. In the present study, we show that the knockdown of the α-form of neurexin 1a induces balance defects and reduced locomotory activity, whereas β-neurexin 1a and neuroligin 1 morphants present defects in sprouting angiogenesis and vascular remodeling, in particular in the caudal plexus and subintestinal vessels. Coinjection of low doses of morpholinos for β-neurexin 1a and neuroligin 1 together or in combination with morpholinos targeting the -heparin--binding isoforms of vascular endothelial growth factor A (encoded by the VEGFAb gene) recapitulates the observed abnormalities, suggesting synergistic activity of these molecules. Similar coinjection experiments with morpholinos, targeting the enzyme heparan sulfate 6-O-sulfotransferase 2, confirm the presence of a functional correlation between extracellular matrix maturation and β-neurexin 1a or neuroligin 1. Our data represent the first in vivo evidence of the role of neurexin and neuroligin in embryonic blood vessel formation and provide insights into their mechanism of action.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

141 Tissue factor pathway inhibitor regulates vascular development in zebrafish

Introduction Angiogenesis requires the coordinate regulation of multiple biological pathways, including haemostasis. Tissue factor pathway inhibitor (TFPI) is a potent anticoagulant molecule, inhibiting tissue factor-led coagulation. However, the role of TFPI in vascular development is unknown. Zebrafish (Danio rerio) provide a unique model system to study vascular development in vertebrates. Despite the divergence of jawed fish (teleosts) over 430 million years ago, there is notable conservation of the constituent molecules of the clotting cascade. Multiple features make this vertebrate model unique, including its genetic accessibility, the ability to titrate the degree of genetic knock-down, external embryonic development, and the transparent nature of the embryos. Methods Using in-situ hybridisation techniques, we demonstrate TFPI expression during early vertebrate development. We then utilise transgenic fish with labelled endothelium (Fli1GFP) and erythrocytes (GATA1dsRed), to study in real time, concomitant fluorescent imaging of both structural development and dynamic blood flow observation, in living zebrafish embryos. Results TFPI expression was identified at 24 h post fertilisation (hpf) in the pronephros (Abstract 141 figure 1—dark blue staining denotes TFPI expression; none seen in control embryos). Subsequently, TFPI mRNA became more abundant, localising to developing kidney, gut, and vasculature. Morpholino (MO)-based knockdown of TFPI resulted in coagulopathy and disordered vascular development (Abstract 141 figure 2. left column: green endothelial Fli1GFP, right column: red erythrocytes. AB: uninjected controls. CD: MO controls. E–P: TFPI knockdown). Abnormally targetted (ie, vessels sprouting from normal site but growing in abnormal direction; grey arrows) and extra vessels (ie, superfluous vessels not seen in controls; white arrows in Abstract 141 figure 2EFHIKL white arrows Abstract 141 figure 2GJMP) in 25.2±2.3% (p Conclusion These data represent the first demonstration of TFPI expression in zebrafish and the first description of a unique phenotype following TFPI knockdown. They support a model in which TFPI acts a molecular break to angiogenesis both in vivo, during early vertebrate embryogenesis, and in vitro in mature human endothelial cells, secondary to constitutive regulation of VEGF signalling.

The Transcription Factor HOXC9 Regulates Endothelial Cell Quiescence and Vascular Morphogenesis in Zebrafish via Inhibition of Interleukin 8

The transcription factor HOXC9 belongs to the homeobox gene family acting as developmental morphogen in several species. HOXC9 is EXPRESSED in different vascular beds in vivo. Yet vascular functions of HOXC9 have not been studied. This study was aimed at characterizing HOXC9 functions in human vascular endothelial cells and in zebrafish vascular development. HOXC9 was abundantly expressed in resting human umbilical vein endothelial cells and was downregulated by hypoxia. Overexpression of HOXC9 inhibited endothelial cell proliferation, migration, and tube formation in vitro. Expression profiling and chromatin immunoprecipitation experiments in human umbilical vein endothelial cells identified interleukin 8 as the major HOXC9 target and demonstrated the direct binding of HOXC9 to the interleukin 8 promotor. HOXC9 overexpression led to reduced endothelial interleukin 8 production, whereas HOXC9 silencing increased interleukin 8. The antimigratory and antiangiogenic effect of HOXC9 overexpression could be rescued by external interleukin 8 administration. Corresponding to the cellular experiments, endothelial-specific overexpression of HOXC9 and morpholino-based interleukin 8 loss-of-function experiments inhibited zebrafish vascular development. The data identify HOXC9 as an endothelial cell active transcriptional repressor promoting the resting, antiangiogenic endothelial cell phenotype in an interleukin 8-dependent manner.

CYP Pathway Modulators Alter Development and Angiogenesis in Zebrafish Embryos

Epoxyeicosatrienoic acids (EETs) are CYP metabolites of arachidonic acid that have vascular actions including vasodilation. Arachidonic acid is also metabolized by CYP hydroxylase enzymes to 20‐hydroxyeicosatetraenoic acid (20‐HETE) that causes vasoconstriction and increases vascular reactivity. These vascular properties make CYP metabolites attractive targets for therapeutics. Thus, we determined the actions of chemical compounds that modulate EETs or 20‐HETE on cardiovascular development. A zebrafish embryo model system was utilized to compare heart and caudal vascular plexus development. Tg(kdr:EGFP) zebrafish embryos were exposed to compounds at 10 hours post fertilization (hpf), and incubated until 48hpf when images were taken, quantified, and analyzed using a 2‐tailed t‐test to determine significance. Heart development was affected significantly with a 20‐HETE antagonist (n=19), eliciting a heart width of 176.7 ± 8.2 μm compared with 114.5 ± 17.1 μm in the control fish (n=4). (p<0.05). A soluble epoxide hydrolase inhibitor (sEHI), EET analogs, and combined EET analog/sEHI all caused a narrowing of the caudal vascular plexus. All compounds affecting the caudal vascular plexus caused a 13–21% narrowing compared with control fish (p<0.05). These data demonstrate that chemical compounds that modulate EETs and 20‐HETE have distinct actions on heart and vascular plexus development in the zebrafish embryos. Therefore, we conclude that 20‐HETE antagonism alters heart and vascular development whereas sEHI and EET analogs significantly alter vascular development in zebrafish.

Two Functional MicroRNA-126s Repress a Novel Target Gene p21-Activated Kinase 1 to Regulate Vascular Integrity in Zebrafish

MicroRNAs (miRNAs) are key regulators of vascular development and diseases. The function and underlying mechanism of endothelial miRNAs have not been fully defined. To investigate the role of endothelial miR-126 in zebrafish vascular development. Two homologs of miR-126, miR-126a (namely miR-126 in previous literature) and miR-126b, with only 1 nucleotide difference in their mature sequences, were identified in zebrafish genome. In vitro analysis showed that both precursors could sufficiently produce mature functional miRNAs. Expression analyses by Northern blot and quantitative RT-PCR showed that both miR-126s accumulated significantly 12 hours after fertilization and were specifically expressed in endothelial cells of zebrafish. Inhibition of miR-126a or miR-126b with specific morpholinos caused cranial hemorrhage, and simultaneous inhibition of both miR-126s resulted in a pronounced hemorrhage in higher percentage of embryos. Bioinformatics prediction showed that the targets of miR-126a/b partially overlapped but essentially differed. p21-activated kinase1 (pak1) was identified as a novel target of miR-126a/b, and pak1 3' untranslated region was differently regulated by these 2 miRNAs. Quantitative RT-PCR, in situ hybridization, and Western blot analyses showed that the level of pak1 was reduced when miR-126a/b were overexpressed. Notably, pak1 expression in endothelial cells was increased when miR-126a/b were knocked down. Furthermore, overexpression of the active form of human pak1 caused cranial hemorrhage, and knockdown pak1 effectively rescued the hemorrhage caused by inhibiting miR-126a/b. Two functional endothelial cell-specific miRNAs, miR-126a and miR-126b, synergistically regulate zebrafish vascular integrity, and pak1 is a critical target of miR-126a/b in vascular development.

Angioblast Behavior in Arterial-Venous Segregation

AbstractAbstract SCI-43Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from pre-existing vessels (angiogenesis). Using high resolution imaging of zebrafish vascular development we discovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated in part by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial/venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development. Disclosures:No relevant conflicts of interest to declare.

Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

BackgroundPlatelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization.Methodology/Principal FindingsIn order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels.Significance/ConclusionOur data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.

Arterial-venous segregation by selective cell sprouting: an alternative mode of blood vessel formation.

Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from preexisting vessels (angiogenesis). With high-resolution imaging of zebrafish vascular development, we uncovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial-venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

D008 First evidences of frizzled-7 involvement in vessel formation

Recent evidence links the Wnt/frizzled pathway to proper vascular growth but the molecular players are still unknown. Recently, we evidenced that frizzled7 (Fzd7) interacted with a Wnt pathway modulator sFRP1 and was involved on endothelial cell morphogenesis. Here, we investigated whether Fzd7 may be involved in vessel formation. Knocking down (KD) of Fzd7 by injecting morpholino oligonucleotides impaired intersomitic endothelial cells migration in zebrafish embryo. Phenotypes were rescued by the injection of Fzd7 mRNA. To elucidate a role of Fzd7 in vascular formation, we set up a model of vessel formation using a murine embryonic stem (ES) cell model. Embryoid bodies (EBs) differentiating under VEGF induction in vitro were stained for CD31 to visualize the formation of vascular structures. We showed that Fzd7 expression was modulated during the kinetic of EB differentiation and that Fzd7 was expressed in EC assembled in vascular structures. Fzd7 was highly localized in cell-cell junctions, colocalized with VE-cadherin and beta catenin-positive junctions. In order to analyze the role of Fzd7 in endothelial cell function, effect of Fzd7 knock down by small interfering RNAs was investigated in human umbilical vein endothelial cells (HUVEC). KD of Fzd7 had no effect on cell proliferation whereas a strong inhibition of HUVEC migration and tube formation was observed. Indeed, Fzd7 siRNA decreased cell migration to 59±17 % (n=3, p This finding suggests that Fzd7 may play a crucial role during vascular formation. Fzd7 is essential for zebrafish vascular development in vivo and regulates endothelial cells functions involved in angiogenesis in vitro.

Functional analyses of human and zebrafish 18-amino acid in-frame deletion pave the way for domain mapping of the cerebral cavernous malformation 3 protein

Cerebral cavernous malformations (CCMs) may cause recurrent headaches, seizures, and hemorrhagic stroke and have been associated with loss-of-function mutations in CCM1/KRIT1, CCM2, and CCM3/programmed cell death 10 (PDCD10). The CCM3/PDCD10 amino acid sequence does not reveal significant homologies to protein domains with known structure. With the help of the only published human in-frame deletion of the CCM3 gene (c.97-?_150+?del), CCM3:p.L33_K50del, we have identified the interaction domain of CCM3 with the oxidant stress response serine/threonine kinase 25 (STK25, YSK1, SOK1) and with the mammalian Ste20-like kinase 4 (MST4, MASK). Consistently, nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analyses revealed two STK25 phosphorylation sites at serine 39 and threonine 43. The corresponding in-frame deletion of zebrafish ccm3a, dccm3:p.L31_K48del, also resulted in impaired interaction with STK25 and MST4. In agreement with the observed redundant biochemical functionality of zebrafish ccm3a and its duplicate ccm3b, simultaneous inactivation of both genes resulted in a progressive cardiovascular phenotype in zebrafish indistinguishable from ccm1 and ccm2 mutants. The pronounced cardiovascular dilatations could be recapitulated by morpholino-induced in-frame skipping of the exon encoding the STK25 and MST4 binding site of zebrafish Ccm3a if Ccm3b was repressed in parallel. Using a novel zebrafish model of CCM, we could thus demonstrate that the newly mapped STK25 and MST4 interaction domain within the CCM3 protein plays a crucial role for vascular development in zebrafish.

Meis1 regulates the development of endothelial cells in zebrafish

The differentiation of endothelial cells is tightly connected with the formation of blood vessels during vertebrate development. The signaling pathways mediated by vascular endothelial growth factor ( vegf) are required for these processes. Here we show that a proto-oncogene, meis1, plays important roles in the vascular development in zebrafish. Knockdown of meis1 by anti-sense meis1 morpholino ( meis1 MO) led to the impairment of intersegmental vessel (ISV) formation. In meis1 morphants, the expression of an artery marker was reduced in dorsal aorta (DA), and the expression of vein markers was expanded in DA and posterior cardinal vein (PCV), suggesting the defects on artery development. Furthermore, the expression of vegf receptor, flk1, was significantly decreased in these embryos. Interestingly, flk1 MO-injected embryos exhibited similar defects as meis1 morphants. Thus, these results implicate that meis1 is a novel regulator involved in endothelial cell development, presumably affecting the vegf signaling pathway.

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Multicellular organisms coordinate their growth, development and homeostasis through cell-cell communication mediated by a relatively small number of extracellular signaling molecules, often referred to as growth factors. Nowadays, it is difficult to open a new volume of a developmental biology

A Chemical Genetic Screen Identified Small Molecules Modulating Zebrafish Vascular Development and Angiogenesis.

The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. Abnormal angiogenesis is linked to over 70 health conditions and inhibition of angiogenesis is an excellent target for cancer therapy (including hematopoietic malignancies) as tumor growth requires new blood vessels. In this study, we performed a chemical genetic screen in developing zebrafish embryos to identify compounds that modulate zebrafish vascular development and angiogenesis. A zebrafish transgenic line with the Flk1 promoter controlling the GFP (green fluorescent protein) reporter was used. The entire vascular network of the Flk1:GFP fish was marked by GFP and could be visualized under fluorescence microscopy. Screens were performed in 96 well plates with three embryos/well. The outer membranes of healthy zebrafish embryos expressing green fluorescent vasculature were removed by enzymatic digestion with protease at the 16–18 somite stages. Small molecules/compounds were then added to the water at concentrations of 0.1 μM, 1 μM, and 5 μM for 72 hrs. After 24, 48 and 72 hrs of exposure to the compound, the embryos were visually inspected for viability, gross morphological defects, heartbeating rate and circulation. The known angiogenic inhibitor, PD173074 was used as control. An initial screening of 780 compounds in a small molecule compound library identified four small molecules with potent activities in inhibiting zebrafish vascular development. Notably, 1–5 μM of Hit #1 inhibited the growth of cranial vessels and disrupted vascular patterning, which resulted in uneven spacing between intersegmental vessels. While Hit #2 and #5 also inhibited the growth of cranial vessels, the vascular patterning remained unaffected, however, the drug-treated embryos had weak or missing intersegmental vessels. Fish embryos treated by Hit #3 had an enlarged heart and thinner vessels. Interestingly, this screen also identified one compound (Hit #4) with pro-angiogenic activity. Embryos treated with Hit #4 had increased numbers of intersegmental vessels. Hit #2 is a known inhibitor of c-Jun N-terminal kinase and angiogenesis (SP600125), was recently reported to inhibit the proliferation and migration of human endothelial cells in vitro as well as inhibit solid tumor growth in mice. This observation lends validity to the zebrafish screen and the potential utility of the other hits. Experiments are currently in progress to investigate the detailed time course of the angiogenic inhibition and the potential molecular mechanism. Studies of these angiogenic inhibitors may lead to the development of potent anti-cancer drugs while the pro-angiogenic compound may prove useful in facilitating tissue/organ regeneration. We conclude that the zebrafish model is likely to yield valuable information regarding vasculogenesis and its manipulation.

BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish

For the proper development of vertebrate embryos as well as for survival of the adult organism, it is essential to form a functional vascular system. Molecules involved in this process are members of highly conserved families of proteins that exert conserved functions across species. Bone morphogenetic proteins (BMP) are extracellular factors that are regulated by extracellular modulators and bind to BMP receptors, which in turn activate intracellular signaling cascades. BMPs are necessary not only for induction of endothelial and hematopoietic lineages but also for further endothelial and hematopoietic cell differentiation. Previously, we identified BMPER (BMP endothelial cell precursor derived regulator) and demonstrated its spatiotemporal expression at sites of vasculogenesis and direct modulation of BMP activity. To directly investigate the role of BMPER in vascular development, we cloned the BMPER ortholog in zebrafish ( zbmper). It is expressed at sites of high BMP activity, including vascular precursor cells located in the aortic arches and the intermediate cell mass during zebrafish embryonic development. Knockdown of zbmper results in a dorsalized phenotype, a reduced number of gata1 expressing hematopoietic precursor cells and of circulating blood cells as well as in a vascular phenotype. The generation of the caudal vein is compromised and the pattern guiding of the intersomitic vessels is disturbed, indicating that zbmper is required for early steps in vascular pattern formation and hematopoiesis in zebrafish.

A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP) s843 transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.

BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish

BMPER is a conserved regulator of hematopoietic and vascular development in zebrafish

Cooperative interaction of Angiopoietin-like proteins 1 and 2 in zebrafish vascular development

Angiopoietin-like protein (Angptl) 1 and Angptl2, which are considered orphan ligands, are highly homologous, particularly in the fibrinogen-like domain containing the putative receptor binding site. This similarity suggests potentially cooperative functions between the two proteins. In this report, the function of Angptl1 and Angptl2 is analyzed by using morpholino antisense technology in zebrafish. Knockdown of both Angptl1 and Angptl2 produced severe vascular defects due to increased apoptosis of endothelial cells at the sprouting stage. In vitro studies showed that Angptl1 and Angptl2 have antiapoptotic activities through the phosphatidylinositol 3-kinase/Akt pathway, and coinjection of constitutively active Akt/protein kinase B mRNA rescued impaired vascular development seen in double knockdown embryos. These results provide a physiological demonstration of the cooperative interaction of Angptl1 and Angptl2 in endothelial cells through phosphatidylinositol 3-kinase/Akt mediated antiapoptotic activities.