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      Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis

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          Abstract

          Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function.

          DOI: http://dx.doi.org/10.7554/eLife.06489.001

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          Organs develop alongside the network of blood vessels that supply them with oxygen and nutrients. One way that new blood vessels grow is by sprouting out of the side of an existing vessel, via a process called angiogenesis. This process relies on signals that are received by the endothelial cells that line the inner wall of blood vessels, and that direct the cells to form a new ‘sprout’, consisting of tip and stalk cells.

          In the developing brain, the Wnt/β-catenin signaling pathway helps direct the formation of blood vessels. In this pathway, a member of a protein family called Wnt signals to specific proteins on the surface of the cells lining the blood vessels. Much effort has gone into uncovering the identity of these proteins, with many studies looking at blood vessel development in the brain of mouse embryos.

          In this study, Vanhollebeke et al. turned to zebrafish embryos to uncover new regulators of angiogenesis and define their roles during the multi-step process of blood vessel development in the brain. A variety of experimental techniques were used to alter and study the activity of different Wnt signaling pathway components. These experiments revealed that the Wnt7a and Wnt7b proteins signal to an endothelial cell membrane protein complex containing the proteins Gpr124 and Reck.

          Vanhollebeke et al. then created ‘mosaic’ zebrafish embryos, which contained two genetically distinct types of cells—cells that were missing one of the components of Wnt/β-catenin signaling pathway, and wild-type cells. Visualizing the growth of the vessels showed that all the new blood vessels that sprouted had normal tip cells. However, the cells in the stalk of the sprout could be either normal or missing a signaling protein.

          These findings demonstrate that Wnt/β-catenin signaling controls the pattern of blood vessel development in the brain by acting specifically on the invasive behaviors of the tip cells of new sprouts, a cellular mechanism that allows efficient organ-specific control of vascularization.

          DOI: http://dx.doi.org/10.7554/eLife.06489.002

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          Most cited references47

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          Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

          TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
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            Delta-like ligand 4 (Dll4) is induced by VEGF as a negative regulator of angiogenic sprouting.

            Genetic deletion studies have shown that haploinsufficiency of Delta-like ligand (Dll) 4, a transmembrane ligand for the Notch family of receptors, results in major vascular defects and embryonic lethality. To better define the role of Dll4 during vascular growth and differentiation, we selected the postnatal retina as a model because its vasculature develops shortly after birth in a highly stereotypic manner, during which time it is accessible to experimental manipulation. We report that Dll4 expression is dynamically regulated by VEGF in the retinal vasculature, where it is most prominently expressed at the leading front of actively growing vessels. Deletion of a single Dll4 allele or pharmacologic inhibition of Dll4/Notch signaling by intraocular administration of either soluble Dll4-Fc or a blocking antibody against Dll4 all produced the same set of characteristic abnormalities in the developing retinal vasculature, most notably enhanced angiogenic sprouting and increased endothelial cell proliferation, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. In a model of ischemic retinopathy, Dll4 blockade also enhanced angiogenic sprouting and regrowth of lost retinal vessels while suppressing ectopic pathological neovascularization. Our data demonstrate that Dll4 is induced by VEGF as a negative feedback regulator and acts to prevent overexuberant angiogenic sprouting, promoting the timely formation of a well differentiated vascular network.
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              Dynamics of endothelial cell behavior in sprouting angiogenesis.

              The vertebrate body contains an extensive blood vessel network that forms, with a few exceptions, by endothelial sprouting from the existing vasculature. This process, termed angiogenesis, involves complex and highly dynamic interactions between endothelial cells and their environment. Pro-angiogenic signals, such as VEGF, promote endothelial motility, filopodia extension and proliferation, and, together with Notch signaling, controls whether specific endothelial cells become lead tip cells or trailing stalk cells. Sprouts then convert into endothelial tubules and form connections with other vessels, which requires the local suppression of motility and the formation of new cell-cell junctions. We here review the dynamics of angiogenesis in the context of key molecules and pathways controlling tip cell selection, sprouting and the formation of new vessels. Copyright © 2010 Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                2050-084X
                08 June 2015
                2015
                : 4
                : e06489
                Affiliations
                [1 ]deptDepartment of Biochemistry and Biophysics , University of California, San Francisco , San Francisco, United States
                [2 ]deptDepartment of Developmental Genetics , Max Planck Institute for Heart and Lung Research , Bad Nauheim, Germany
                [3 ]deptInstitut de Biologie et de Médecine Moléculaires , Université Libre de Bruxelles , Gosselies, Belgium
                [4 ]deptDepartment of Molecular Biology and Genetics , Johns Hopkins University School of Medicine , Baltimore, United States
                [5 ]deptDepartment of Cell Biology , National Cerebral and Cardiovascular Center Research Institute , Osaka, Japan
                [6 ]deptDepartment of Neuroscience , Howard Hughes Medical Institute, Johns Hopkins University School of Medicine , Baltimore, United States
                [7 ]deptDepartment of Ophthalmology , Johns Hopkins University School of Medicine , Baltimore, United States
                University of Toronto , Canada
                University of Toronto , Canada
                Author notes
                Article
                06489
                10.7554/eLife.06489
                4456509
                26051822
                a27c0f1c-c62f-4edc-925c-ac5aa3d4573d
                © 2015, Vanhollebeke et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 15 January 2015
                : 07 May 2015
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100002661, Fonds De La Recherche Scientifique—FNRS;
                Award ID: F.4543.15
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100004410, European Molecular Biology Organization (EMBO);
                Award ID: ALTF 609-2008
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100000854, Human Frontier Science Program;
                Award ID: LT000334/2009-L
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000053, National Eye Institute (NEI);
                Award ID: R01EY081637
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000863, Ellison Medical Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000011, universityHoward Hughes Medical Institute (HHMI);
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: SFB 834
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, universityNational Institutes of Health (NIH);
                Award ID: 5R03 NS074218-02
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000008, David and Lucile Packard Foundation;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100004189, Max-Planck-Gesellschaft;
                Award Recipient :
                Funded by: Fondation ULB;
                Award Recipient :
                Funded by: Action de Recherche Concertée de la Fédération Wallonie-Bruxelles;
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, universityNational Institutes of Health (NIH);
                Award ID: Medical Scientist Training Program
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100003134, Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture;
                Award Recipient :
                The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
                Categories
                Research Article
                Developmental Biology and Stem Cells
                Custom metadata
                2.3
                The membrane proteins Reck and Gpr124 are integral components of a novel Wnt7a/Wnt7b-specific signaling complex, and there is a distinctive requirement for Wnt/β-catenin signaling in tip cells during angiogenesis in the central nervous system.

                Life sciences
                brain vascular development,tip cell,angiogenesis,reck,gpr124,wnt7a/wnt7b,zebrafish
                Life sciences
                brain vascular development, tip cell, angiogenesis, reck, gpr124, wnt7a/wnt7b, zebrafish

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