Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 15 der Johann Wolfgang Goethe-Universität in Frankfurt am Main Von Suchit Ahuja aus Neu Delhi, Indien Frankfurt 2016 (D30) Vom Fachbereich der Goethe Universität als Dissertation angenommen. Dekan: Gutachter: Datum der Disputation: SUPERVISED BY Dr. Sven Reischauer, Ph.D. Department of Developmental Genetics Max Planck Institute for Heart and Lung Research Bad Nauheim, Germany REVIEWER Prof. Dr. Didier Stainier, Ph.D. Department of Developmental Genetics Max Planck Institute for Heart and Lung Research Bad Nauheim, Germany and Prof. Dr. Anna Starzinski-Powitz, Ph.D. Department of Molecular Cell Biology and Human Genetics Institute of Cell Biology and Neuroscience Johann Wolfgang Goethe University Frankfurt am Main, Germany ERKLÄRUNG Ich erkläre hiermit, dass ich mich bisher keiner Doktorprüfung im Mathematisch- Naturwissenschaftlichen Bereich unterzogen habe. Frankfurt am Main, den ............................................ (Unterschrift) Versicherung Ich erkläre hiermit, dass ich die vorgelegte Dissertation über Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development selbständig angefertigt und mich anderer Hilfsmittel als der in ihr angegebenen nicht bedient habe, insbesondere, dass alle Entlehnungen aus anderen Schriften mit Angabe der betreffenden Schrift gekennzeichnet sind. Ich versichere, die Grundsätze der guten wissenschaftlichen Praxis beachtet, und nicht die Hilfe einer kommerziellen Promotionsvermittlung in Anspruch genommen zu haben. Vorliegende Ergebnisse der Arbeit sind in folgendem Publikationsorgan veröffentlicht: Ahuja S, et al. Dev. Biol. 2016; 412(1): 71-82. Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development. Frankfurt am Main, den ............................................ (Unterschrift) You have to dream before your dreams can come true. Man needs his difficulties because they are necessary to enjoy success. Great dreams of great dreamers are always transcended. A.P.J. Abdul Kalam Aerospace Scientist 1931-2015 Contents Table of Contents Abbreviations ............................................................................................. 11 1. Introduction ........................................................................................... 14 1.1 Congenital Heart Disease .......................................................................... 14 1.2 Heart Valve Development ......................................................................... 16 1.2.1 Origin Of Valvular Cells: Contribution From Different Heart Layers .......................... 17 1.2.2 Endocardial Cushion Formation: Signaling Molecules Involved .................................. 17 1.3 Zebrafish Heart Development .................................................................... 22 1.4 Zebrafish Atrioventricular Canal/Valve Development................................. 25 1.5 Inhibitor of DNA Binding 4 (Id4) .............................................................. 27 1.6 AIM of the Project .................................................................................... 29 2. Materials ................................................................................................... 30 2.1 Antibiotics ................................................................................................ 30 2.2 Antibodies ................................................................................................ 30 2.3 Bacterial Culture medium .......................................................................... 31 2.4 Bacterial Strains ........................................................................................ 31 2.5 Buffers/Solutions ...................................................................................... 31 2.6 Centrifuges ............................................................................................... 33 2.7 Chemicals ................................................................................................. 34 2.8 Enzymes ................................................................................................... 36 2.9 Kits .......................................................................................................... 37 2.10 Microscopes ............................................................................................. 37 2.11 Miscellaneous Equipment .......................................................................... 38 2.12 Oligonucleotides ....................................................................................... 40 2.13 PCR enzymes and mastermix .................................................................... 41 2.14 Plasmids ................................................................................................... 42 2.15 Softwares .................................................................................................. 43 Contents 2.16 Tubes and containers ................................................................................. 43 2.17 Zebrafish Food .......................................................................................... 44 2.18 Zebrafish Lines ......................................................................................... 44 3. Methods ..................................................................................................... 45 3.1 Zebrafish Husbandry ................................................................................. 45 3.1.1 Zebrafish Maintenance .................................................................................................. 45 3.1.2 Zebrafish Breeding ........................................................................................................ 45 3.2 Zebrafish Microinjection ........................................................................... 45 3.2.1 Preparation of Injection plates ....................................................................................... 45 3.2.2 Preparation of Injection Needles ................................................................................... 46 3.2.3 Microinjection ............................................................................................................... 46 3.3 RNA Isolation ........................................................................................... 46 3.4 Reverse Transcription or cDNA Synthesis ................................................. 46 3.5 PCR amplification from cDNA .................................................................. 47 3.6 Agarose Gel Electrophoresis ...................................................................... 47 3.7 PCR Product Elution From Agarose Gel .................................................... 48 3.8 E.Coli Competent Cells Preparation ........................................................... 48 3.9 Transformation of Competent E.Coli ......................................................... 48 3.10 DNA Restriction Digestion ........................................................................ 48 3.11 Cloning .................................................................................................... 49 3.11.1 TA Cloning .................................................................................................................... 49 3.11.2 Cold Fusion ................................................................................................................... 49 3.11.3 Cloning id4 Overexpression Constructs ........................................................................ 50 3.12 Plasmid DNA Isolation ............................................................................. 50 3.13 In situ Hybridisation .................................................................................. 50 3.13.1 Probe Synthesis ............................................................................................................. 50 3.13.2 Embryo/Adult Heart preparation for in situ hybridization ............................................ 51 3.13.3 In situ Hybidization Day 1: Probe Hybridization .......................................................... 51 3.13.4 In situ Hybidization Day 2: Unbound Probe Removal .................................................. 52 3.13.5 In situ Hybidization Day 3: Unbound Antibody Removal and Staining ....................... 53 3.13.6 In situ Hybidization: Microscopy and Imaging ............................................................. 53 Contents 3.14 TALEN (Transcription Activator-Like Effector Nucleases) Induced Mutagenesis ....................................................................................................... 53 3.14.1 TALEN Designing......................................................................................................... 53 3.14.2 TALEN Assembly Day 1 .............................................................................................. 54 3.14.3 TALEN Assembly Day 2 .............................................................................................. 54 3.14.4 TALEN Assembly Day 3 .............................................................................................. 55 3.14.5 TALEN Assembly Day 4 .............................................................................................