title: Context-Dependent Sensitivity to Mutations Disrupting the Structural Integrity of Individual EGF Repeats in the Mouse Notch Ligand DLL1.
abstract: The highly conserved Notch-signaling pathway mediates cell-to-cell communication and is pivotal for multiple developmental processes and tissue homeostasis in adult organisms. Notch receptors and their ligands are transmembrane proteins with multiple epidermal-growth-factor-like (EGF) repeats in their extracellular domains. In vitro the EGF repeats of mammalian ligands that are essential for Notch activation have been defined. However, in vivo the significance of the structural integrity of each EGF repeat in the ligand ectodomain for ligand function is still unclear. Here, we analyzed the mouse Notch ligand DLL1. We expressed DLL1 proteins with mutations disrupting disulfide bridges in each individual EGF repeat from single-copy transgenes in the HPRT locus of embryonic stem cells. In Notch transactivation assays all mutations impinged on DLL1 function and affected both NOTCH1 and NOTCH2 receptors similarly. An allelic series in mice that carried the same point mutations in endogenous Dll1, generated using a mini-gene strategy, showed that early developmental processes depending on DLL1-mediated NOTCH activation were differently sensitive to mutation of individual EGF repeats in DLL1. Notably, some mutations affected only somite patterning and resulted in vertebral column defects resembling spondylocostal dysostosis. In conclusion, the structural integrity of each individual EGF repeat in the extracellular domain of DLL1 is necessary for full DLL1 activity, and certain mutations in Dll1 might contribute to spondylocostal dysostosis in humans.
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Schuster-Gossler
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Cordes
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Müller
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors.forename: Insa
authors.initials: I
authors.lastname: Geffers
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors.forename: Patricia
authors.initials: P
authors.lastname: Delany-Heiken
authors.affiliation: Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, 30625 Hannover, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Taft
authors.affiliation: Institut für Biophysikalische Chemie, Medizinische Hochschule Hannover, 30625 Hannover, Germany Centre for Structural Systems Biology, Deutsches Elektronen-Synchrotron, 22607 Hamburg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Preller
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany gossler.achim@mh-hannover.de.
authors.forename: Achim
authors.initials: A
authors.lastname: Gossler
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins; D053595:Embryonic Stem Cells / Q000378:metabolism; D017930:Genes, Reporter; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D057507:Mice, 129 Strain; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D000072417:Protein Domains; D051881:Receptor, Notch1 / Q000378:metabolism*; D051883:Receptor, Notch2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D002135:Calcium-Binding Proteins; C512925:Dlk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; C497762:Notch1 protein, mouse; C497765:Notch2 protein, mouse; D051881:Receptor, Notch1; D051883:Receptor, Notch2; D011993:Recombinant Fusion Proteins
keywords: Notch activation; Notch signaling; Notch-ligand interaction; mouse DLL1; targeted mutagenesis/allelic series