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The Role of Non-Coding Rnas for Skeletal Development and Diseases

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The Role of Non-Coding Rnas for Skeletal Development and Diseases ZENTRUM FÜR KINDER- UND JUGENDMEDIZIN, SEKTION PÄDIATRISCHE GENETIK Universitätsklinikum Freiburg im Breisgau The role of non-coding RNAs for Skeletal development and diseases Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Franziska Friedrich geb. Eckmann geboren in Freiburg im Breisgau Freiburg im Breisgau Dezember 2017 Dekanin der Fakultät für Biologie: Prof. Dr. Bettina Warscheid Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner Betreuer der Arbeit: Prof. Dr. med. Bernhard Zabel Weiterer Betreuer der Arbeit: Tim J.M. Welting, PhD, Associate Professor of Molecular Cartilage Biology, Maastricht UMC+, the Netherlands Betreuer an der Fakultät für Biologie: Prof. Dr. Annette Neubüser Referentin: Prof. Dr. Annette Neubüser Ko-Referent: Dr. Georgios Pyrowolakis Drittprüfer: Prof. Dr. Wolfgang Driever Datum der mündlichen Prüfung: 19.01.2018 Erklärung 1. Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quellen gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs- beziehungsweise Beratungsdiensten (Promotionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. 2. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. 3. Die Bestimmungen der Promotionsordnung der Fakultät für Biologie sind mir bekannt, insbesondere weiß ich, dass ich vor Vollzug der Promotion zur Führung des Doktortitels nicht berechtigt bin. Datum und Unterschrift Table of contents SUMMARY...........................................................................................................................................IV ZUSAMMENFASSUNG .........................................................................................................................VI INDEX OF FIGURES ........................................................................................................................... VIII INDEX OF TABLES ................................................................................................................................ XI ABBREVIATIONS ................................................................................................................................. XII 1 INTRODUCTION ........................................................................................................................... 1 1.1 SKELETAL DEVELOPMENT ................................................................................................................. 1 1.1.1 Development of Bones: Endochondral ossification ............................................................. 1 1.1.2 Wnt signalling is linked to bone development .................................................................... 3 1.1.3 The role of hypoxia during chondrogenesis and bone development .................................. 5 1.2 DISORDERS OF THE SKELETON .......................................................................................................... 7 1.3 CARTILAGE-HAIR HYPOPLASIA .......................................................................................................... 8 1.4 RMRP AND THE RNASE MRP COMPLEX ......................................................................................... 10 2 AIM AND OBJECTIVES OF THIS STUDY ...................................................................................... 14 3 MATERIALS AND METHODS ...................................................................................................... 15 3.1 MATERIALS ................................................................................................................................. 15 3.1.1 Antibiotics ........................................................................................................................... 15 3.1.2 Antibodies and sera ........................................................................................................... 15 3.1.3 Bacterial strains ................................................................................................................. 15 3.1.4 Buffers and solutions ......................................................................................................... 15 3.1.5 Cell lines .............................................................................................................................. 17 3.1.6 Chemicals and reagents ..................................................................................................... 17 3.1.7 Enzymes .............................................................................................................................. 19 3.1.8 Equipment .......................................................................................................................... 19 3.1.9 Kits ...................................................................................................................................... 20 3.1.10 Ladder ............................................................................................................................ 21 3.1.11 Materials for cell culture ............................................................................................... 21 3.1.12 Plasmids ......................................................................................................................... 21 3.1.13 Software and databases ............................................................................................... 22 3.1.14 Supplies .......................................................................................................................... 22 3.1.15 Oligonucleotides ............................................................................................................ 23 3.2 METHODS .................................................................................................................................. 25 3.2.1 Biochemical Methods......................................................................................................... 25 3.2.2 Cell culture methods .......................................................................................................... 27 i 3.2.3 Cloning of DNA fragments ................................................................................................. 30 3.2.4 CRISPR/Cas9 knockout ....................................................................................................... 31 3.2.5 DNA standard methods ..................................................................................................... 31 3.2.6 Ethical statement ............................................................................................................... 32 3.2.7 Histological methods ......................................................................................................... 33 3.2.8 Polymerase chain reaction (PCR) techniques .................................................................... 34 3.2.9 RNA standard methods ...................................................................................................... 37 3.2.10 RNA-Seq ......................................................................................................................... 38 3.2.11 Statistical Analysis ......................................................................................................... 38 3.2.12 Work with Zebrafish ...................................................................................................... 39 4 RESULTS .................................................................................................................................... 42 4.1 NEWLY MUTATIONS IN RMRP CAUSE CARTILAGE-HAIR HYPOPLASIA VARIANT ........................................ 42 4.2 CHH MOUSE MODEL .................................................................................................................... 45 4.2.1 General Considerations ...................................................................................................... 45 4.2.2 Analysis of the mouse model ............................................................................................. 50 4.2.3 Alternative approach ......................................................................................................... 50 4.3 CHH ZEBRAFISH MODEL ................................................................................................................ 54 4.3.1 General consideration ........................................................................................................ 54 4.3.2 Analysis of the zebrafish model ......................................................................................... 57 4.3.3 Planned alternative approach ........................................................................................... 61 4.4 RMRP IS UP-REGULATED AT EARLY STAGES OF HYPERTROPHIC DIFFERENTIATION .................................... 64 4.4.1 Many signalling pathways are altered in CHH patients ................................................... 68 4.5 WNT SIGNALLING HAS AN INFLUENCE ON RMRP EXPRESSION ............................................................
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