title: Sequential Ligand-Dependent Notch Signaling Activation Regulates Valve Primordium Formation and Morphogenesis.
abstract: The Notch signaling pathway is crucial for primitive cardiac valve formation by epithelial-mesenchymal transition, and NOTCH1 mutations cause bicuspid aortic valve; however, the temporal requirement for the various Notch ligands and receptors during valve ontogeny is poorly understood.
  
  
  
  The aim of this study is to determine the functional specificity of Notch in valve development.
  
  
  
  Using cardiac-specific conditional targeted mutant mice, we find that endothelial/endocardial deletion of Mib1-Dll4-Notch1 signaling, possibly favored by Manic-Fringe, is specifically required for cardiac epithelial-mesenchymal transition. Mice lacking endocardial Jag1, Notch1, or RBPJ displayed enlarged valve cusps, bicuspid aortic valve, and septal defects, indicating that endocardial Jag1 to Notch1 signaling is required for post-epithelial-mesenchymal transition valvulogenesis. Valve dysmorphology was associated with increased mesenchyme proliferation, indicating that Jag1-Notch1 signaling restricts mesenchyme cell proliferation non-cell autonomously. Gene profiling revealed upregulated Bmp signaling in Jag1-mutant valves, providing a molecular basis for the hyperproliferative phenotype. Significantly, the negative regulator of mesenchyme proliferation, Hbegf, was markedly reduced in Jag1-mutant valves. Hbegf expression in embryonic endocardial cells could be readily activated through a RBPJ-binding site, identifying Hbegf as an endocardial Notch target. Accordingly, addition of soluble heparin-binding EGF-like growth factor to Jag1-mutant outflow tract explant cultures rescued the hyperproliferative phenotype.
  
  
  
  During cardiac valve formation, Dll4-Notch1 signaling leads to epithelial-mesenchymal transition and cushion formation. Jag1-Notch1 signaling subsequently restrains Bmp-mediated valve mesenchyme proliferation by sustaining Hbegf-EGF receptor signaling. Our studies identify a mechanism of signaling cross talk during valve morphogenesis involved in the origin of congenital heart defects associated with reduced NOTCH function.
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Donal
authors.initials: D
authors.lastname: MacGrogan
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Gaetano
authors.initials: G
authors.lastname: D'Amato
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Stanislao
authors.initials: S
authors.lastname: Travisano
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Beatriz
authors.initials: B
authors.lastname: Martinez-Poveda
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Guillermo
authors.initials: G
authors.lastname: Luxán
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Del Monte-Nieto
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Tania
authors.initials: T
authors.lastname: Papoutsi
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Mauro
authors.initials: M
authors.lastname: Sbroggio
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Vanesa
authors.initials: V
authors.lastname: Bou
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Pablo
authors.initials: P
authors.lastname: Gomez-Del Arco
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Manuel Jose
authors.initials: MJ
authors.lastname: Gómez
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Juan Miguel
authors.initials: JM
authors.lastname: Redondo
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.).
authors.forename: Luis J
authors.initials: LJ
authors.lastname: Jiménez-Borreguero
authors.affiliation: From the Intercellular Signaling in Cardiovascular Development and Disease Laboratory (D.M., G.D., S.T., B.M.-P., G.L., G.d.M.-N., T.P., M.S., V.B., J.L.d.l.P.), Regulation of Gene Expression in Vascular Endothelium Laboratory (P.G.-d. A., J.M.R.), Bioinformatics Unit (M.J.G.), and Cardiovascular Imaging Laboratory (L.J.J.-B.), Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain; Department of Molecular Biology, Universidad Autónoma de Madrid, Madrid, Spain (P.G.-d. A.); Department of Genetics, Pediatrics, and Medicine, Albert Einstein College of Medicine, New York, NY (B.Z.); and Instituto de Investigación Sanitaria Hospital, Universitario La Princesa, Madrid, Spain (L.J.J.-B.). jlpompa@cnic.es.
authors.forename: José Luis
authors.initials: JL
authors.lastname: de la Pompa
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002135:Calcium-Binding Proteins; D058750:Epithelial-Mesenchymal Transition; D066246:ErbB Receptors / Q000378:metabolism; D066257:Heparin-binding EGF-like Growth Factor / Q000378:metabolism; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D000072100:Jagged-1 Protein / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008943:Mitral Valve / Q000002:abnormalities / Q000196:embryology / Q000378:metabolism*; D009024:Morphogenesis; D051881:Receptor, Notch1 / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C519665:DLL4 protein, mouse; C587884:Hbegf protein, mouse; D066257:Heparin-binding EGF-like Growth Factor; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D047908:Intracellular Signaling Peptides and Proteins; C000606274:Jag1 protein, mouse; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; C497762:Notch1 protein, mouse; C497571:Rbpj protein, mouse; D051881:Receptor, Notch1; D066246:ErbB Receptors
keywords: endocardial cushions; endocardium; epithelial–mesenchymal transition; heparin-binding EGF-like growth factor; valve morphogenesis