title: An Intronic Flk1 Enhancer Directs Arterial-Specific Expression via RBPJ-Mediated Venous Repression.
abstract: The vascular endothelial growth factor (VEGF) receptor Flk1 is essential for vascular development, but the signaling and transcriptional pathways by which its expression is regulated in endothelial cells remain unclear. Although previous studies have identified 2 Flk1 regulatory enhancers, these are dispensable for Flk1 expression, indicating that additional enhancers contribute to Flk1 regulation in endothelial cells. In the present study, we sought to identify Flk1 enhancers contributing to expression in endothelial cells.
  
  
  
  A region of the 10th intron of the Flk1 gene (Flk1in10) was identified as a putative enhancer and tested in mouse and zebrafish transgenic models. This region robustly directed reporter gene expression in arterial endothelial cells. Using a combination of targeted mutagenesis of transcription factor-binding sites and gene silencing of transcription factors, we found that Gata and Ets factors are required for Flk1in10 enhancer activity in all endothelial cells. Furthermore, we showed that activity of the Flk1in10 enhancer is restricted to arteries through repression of gene expression in venous endothelial cells by the Notch pathway transcriptional regulator Rbpj.
  
  
  
  This study demonstrates a novel mechanism of arterial-venous identity acquisition, indicates a direct link between the Notch and VEGF signaling pathways, and illustrates how cis-regulatory diversity permits differential expression outcomes from a limited repertoire of transcriptional regulators.
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Philipp W
authors.initials: PW
authors.lastname: Becker
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Natalia
authors.initials: N
authors.lastname: Sacilotto
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Svanhild
authors.initials: S
authors.lastname: Nornes
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Alice
authors.initials: A
authors.lastname: Neal
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Max O
authors.initials: MO
authors.lastname: Thomas
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Chris
authors.initials: C
authors.lastname: Preece
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Indrika
authors.initials: I
authors.lastname: Ratnayaka
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: Benjamin
authors.initials: B
authors.lastname: Davies
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.).
authors.forename: George
authors.initials: G
authors.lastname: Bou-Gharios
authors.affiliation: From the Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine (P.W.B., N.S., S.N., A.N., M.O.T., I.R., S.D.V.) and The Wellcome Trust Centre for Human Genetics (C.P., B.D.), University of Oxford, Oxford, United Kingdom; and Institute of Ageing and Chronic Disease, Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom (K.L., G.B.-G.). sarah.deval@ludwig.ox.ac.uk.
authors.forename: Sarah
authors.initials: S
authors.lastname: De Val
mesh_terms: D000818:Animals; D001158:Arteries / Q000196:embryology / Q000378:metabolism*; D001665:Binding Sites; D042783:Endothelial Cells / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D050980:GATA Transcription Factors / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D020868:Gene Silencing; D017930:Genes, Reporter; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism*; D007438:Introns; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018919:Neovascularization, Physiologic; D050783:Proto-Oncogene Proteins c-ets / Q000378:metabolism; D051880:Receptors, Notch / Q000378:metabolism; D055747:SOX Transcription Factors / Q000378:metabolism; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*; D014680:Veins / Q000196:embryology / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D050980:GATA Transcription Factors; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D050783:Proto-Oncogene Proteins c-ets; C497571:Rbpj protein, mouse; D051880:Receptors, Notch; D055747:SOX Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C583604:Vegfaa protein, zebrafish; D029961:Zebrafish Proteins; C517430:rbpja protein, zebrafish; C467480:vascular endothelial growth factor A, mouse; C000592875:Kdr protein, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2; C000595917:kdr protein, zebrafish
keywords: arterial-venous specification; artery; endothelial cells; mice; notch; veins; zebrafish