Molecular Mechanisms of Heart Development and Trabeculation Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften vorgelegt beim Fachbereich 15 der Goethe-Universität in Frankfurt am Main von Filomena Ricciardi aus Frankfurt am Main Frankfurt 2015 (D30) Vom Fachbereich der Goethe Universität als Dissertation angenommen. Dekan: Gutachter: Datum der Disputation: SUPERVISED BY Prof. Dr. Felix B. Engel Experimental Renal and Cardiovascular Research Department of Nephropathology Institute of Pathology Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) Erlangen, Germany Prof. Dr. Didier Y. Stainier Department of Developmental Genetics Max Planck Institute for Heart and Lung Research Bad Nauheim, Germany Eidesstattliche Erklärung: Hiermit versichere ich, dass ich die Dissertationsschrift selbstständig verfasst und keine anderen als die angegebenen Quellen und Hilfsmittel benutzt habe, alle Ausführungen, die anderen Schriften wörtlich oder sinngemäß entnommen wurden, kenntlich gemacht sind und die Arbeit in gleicher oder ähnlicher Fassung noch nicht Bestandteil einer Studien- oder Prüfungsleistung war. Filomena Ricciardi “Ein Gelehrter in seinem Laboratorium ist nicht nur ein Techniker; er steht auch vor den Naturgesetzen wie ein Kind vor der Märchenwelt” Marie Curie, Physikerin und Chemikerin 1987-1934 Contents I Abbreviations ........................................................ 10 1 Introduction .......................................................... 12 1.1 Heart Development ......................................................................................................................................... 12 1.2 Trabeculation .................................................................................................................................................... 14 1.3 Cytoskeleton ...................................................................................................................................................... 16 1.4 focal adhesion .................................................................................................................................................... 18 1.4.1 Flightless I ........................................................................................................................................................ 20 1.5 Cell Polarity (GolGi) ......................................................................................................................................... 21 1.6 Cell miGration .................................................................................................................................................... 24 1.6.1 Memo1 ............................................................................................................................................................... 25 1.7 Aim of the study ............................................................................................................................................... 25 2 Materials ............................................................... 27 2.1 Equipment .......................................................................................................................................................... 27 2.1.1 Miscellaneous equipment .......................................................................................................................... 27 2.1.2 PCR cycler ........................................................................................................................................................ 28 2.1.3 Microscopes ..................................................................................................................................................... 28 2.1.4 Centrifuges ....................................................................................................................................................... 29 2.1.5 Protein Separation ....................................................................................................................................... 29 2.2 Miscellaneous materials ............................................................................................................................... 29 2.2.1 Disposables ...................................................................................................................................................... 29 2.2.2 Non disposables ............................................................................................................................................. 30 2.3 Chemicals ............................................................................................................................................................ 31 2.4 Buffers and solutions ..................................................................................................................................... 33 2.5 Growth media .................................................................................................................................................... 37 2.6 Zebrafish feed .................................................................................................................................................... 37 2.7 Kits ......................................................................................................................................................................... 37 2.8 Plasmids ............................................................................................................................................................... 38 2.9 Antibodies ........................................................................................................................................................... 38 2.10 Enzymes ............................................................................................................................................................ 39 2.11 Size markers .................................................................................................................................................... 40 2.12 OliGonucleotides ............................................................................................................................................ 40 2.13 Morpholinos .................................................................................................................................................... 41 2.14 Strains ................................................................................................................................................................ 41 2.14.1 Bacterial strains ......................................................................................................................................... 41 2.14.2 Zebrafish lines ............................................................................................................................................. 42 2.15 Computer software ...................................................................................................................................... 42 3 Methods ................................................................ 43 3.1 Zebrafish husbandry and microinjection .............................................................................................. 43 3.1.1 Maintenance ................................................................................................................................................... 43 3.1.2 Breeding ........................................................................................................................................................... 43 3.1.3 Microinjection ................................................................................................................................................ 44 Contents 3.2 Bacteria-based techniques .......................................................................................................................... 45 3.2.1 Propagation of E. coli .................................................................................................................................. 45 3.2.2 Generation of chemically competent E. coli ...................................................................................... 45 3.2.3 Transformation od competent E. coli .................................................................................................. 46 3.2.4 Isolation of plDNA from E. coli ................................................................................................................ 46 3.3 DNA-related methods .................................................................................................................................... 46 3.3.1 Amplification of DNA .................................................................................................................................. 46 3.3.2 DNA restriction .............................................................................................................................................. 48 3.3.3 Purification of DNA ...................................................................................................................................... 49 3.3.4 Agarose gel electrophoresis ..................................................................................................................... 49 3.3.5 Extraction of DNA from agarose gels .................................................................................................