Characterization of the Popeye domain containing gene family in zebrafish Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Bettina Carmen Kirchmaier aus Frankfurt am Main Würzburg, 2010 Eingereicht am:……………………………… Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Thomas Dandekar 1. Gutachter: Prof. Dr. Thomas Brand 2. Gutachter: Prof. Dr. Christoph Winkler Tag des Promotionskolloquiums:……………………………. Doktorurkunde ausgehändigt am:…………………………… Für meine Familie, in Liebe Index 4 CONTENTS Contents .................................................................................................................................................................. 4 Figures .................................................................................................................................................................... 9 Tables .................................................................................................................................................................... 10 1 Summary ......................................................................................................................................... 11 2 Zusammenfassung.......................................................................................................................... 12 3 Introduction ..................................................................................................................................... 13 3.1 The vertebrate heart........................................................................................................................ 13 3.1.1 Formation of the heart field .............................................................................................................. 13 3.1.2 Heart tube assembly and development of polarity ........................................................................... 14 3.1.3 Chamber formation and cardiac conduction development ............................................................... 15 3.1.4 Heart remodeling ............................................................................................................................. 17 3.2 Electrical activity of the adult heart .................................................................................................. 17 3.2.1 The nodes and the fast conduction Purkinje fiber networks ............................................................ 19 3.2.2 Gap junctions ................................................................................................................................... 19 3.2.3 Cardiac currents involved in generation of myocardial action potentials ......................................... 20 3.3 Danio rerio ...................................................................................................................................... 23 3.3.1 The zebrafish as model system for cardiovascular research ........................................................... 23 3.3.2 Zebrafish heart development ........................................................................................................... 24 3.3.3 Development of the cardiac conduction system in zebrafish ........................................................... 25 3.4 The Popeye domain containing (Popdc) gene family ...................................................................... 27 3.4.1 Expression pattern ........................................................................................................................... 27 3.4.2 Protein structure and distribution ..................................................................................................... 28 3.4.3 Interaction partners .......................................................................................................................... 29 3.4.4 Function ........................................................................................................................................... 29 3.5 Aim of the study .............................................................................................................................. 31 Index 5 4 Material ........................................................................................................................................... 32 4.1 Biological material ........................................................................................................................... 32 4.1.1 Bacterial strains ............................................................................................................................... 32 4.1.2 Vectors ............................................................................................................................................ 32 4.1.3 Fish lines ......................................................................................................................................... 33 4.1.4 Primary and secondary antibodies .................................................................................................. 33 4.2 Molecularbiological material ............................................................................................................ 33 4.2.1 DNA and protein ladders ................................................................................................................. 33 4.2.2 Kits ................................................................................................................................................... 34 4.3 Software .......................................................................................................................................... 34 4.4 Equipment ....................................................................................................................................... 35 4.5 Reagents ......................................................................................................................................... 36 4.6 Enzymes ......................................................................................................................................... 37 4.7 Primers ............................................................................................................................................ 38 4.8 Morpholinos .................................................................................................................................... 38 5 Methods .......................................................................................................................................... 39 5.1 Fish care ......................................................................................................................................... 39 5.1.1 Setting up pair crosses .................................................................................................................... 39 5.1.2 PTU treatment to prevent melanization of embryos ......................................................................... 40 5.2 Genexpression manipulation in Danio rerio .................................................................................... 40 5.2.1 Agarose plates for holding embryos ................................................................................................ 40 5.2.2 Microinjections ................................................................................................................................. 40 5.2.3 Morpholinos ..................................................................................................................................... 41 5.2.4 Chemical treatment with cyclopamine ............................................................................................. 41 5.3 Physiological methods/Imaging methods ........................................................................................ 42 5.3.1 Recording of cardiac activity ............................................................................................................ 42 5.3.2 Isolated calcium transient recordings by selective plane illumination microscopy (SPIM) ............... 42 5.4 Microbiological methods .................................................................................................................. 43 5.4.1 Ligation ............................................................................................................................................ 43 5.4.2 Transformation................................................................................................................................. 43 Index 6 5.4.3 Cloning into plasmid vectors ............................................................................................................ 45 5.4.4 Screening bacterial colonies using X-Gal and IPTG ........................................................................ 45 5.4.5 Glycerol stocks ................................................................................................................................ 45 5.5 Molecularbiological methods ........................................................................................................... 46 5.5.1 Preabsorption of antibody for whole mount in situ hybridization ...................................................... 46 5.5.2 Whole mount RNA in situ probe generation ..................................................................................... 46 5.5.3 Whole mount in situ hybridization