AUS DEM DEPARTMENT INNERE MEDIZIN KLINIK FÜR INNERE MEDIZIN IV: NEPHROLOGIE UND ALLGEMEINMEDIZIN DES UNIVERSITÄTSKLINIKUMS FREIBURG IM BREISGAU Role of the transcription factors hic1, myca and the basement membrane in the zebrafish pronephros regeneration after laser induced injury Inaugural-D issertation zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs -Universität Freiburg im Breisgau Vorgelegt 2016 von Anne Wurditsch, geb. Bunjes geboren in Herrenberg Dekan: Prof. Dr. Norbert Südkamp Erster Gutachter: Prof. Dr. med. Gerd Walz Zweiter Gutachter: PD Dr. med. Ekkehart Lausch Jahr der Promotion: 2019 TABLE OF CONTENTS ABSTRACT ............................................................................................................................................................. VI 1. INTRODUCTION.............................................................................................................................................. 1 1.1. KIDNEY DEVELOPMENT: PRONEPHROS, MESONEPHROS, METANEPHROS ...................................................................... 1 1.2. ZEBRAFISH AS A MODEL ORGANISM ....................................................................................................................... 3 1.3. ZEBRAFISH PRONEPHROS ..................................................................................................................................... 4 1.4. COLLECTIVE CELL MIGRATION ............................................................................................................................... 8 1.5. COLLECTIVE CELL MIGRATION (CCM) IN THE ZEBRAFISH PRONEPHROS ........................................................................ 10 1.6. ACUTE KIDNEY INJURY ....................................................................................................................................... 11 1.7 KIDNEY RECOVERY AFTER LASER INDUCED INJURY IN ZEBRAFISH (A MODEL FOR AKI) ..................................................... 13 1.8 THE CRISPR/CAS9 SYSTEM............................................................................................................................... 16 1.9 THE TRANSCRIPTION FACTORS HIC1 AND MYCA ...................................................................................................... 21 1.10 AIMS OF THIS WORK ......................................................................................................................................... 23 2. MATERIAL AND METHODS ........................................................................................................................... 24 2.1. MATERIALS AND SOLUTIONS .............................................................................................................................. 24 2.2. HOUSING AND MATING CONDITIONS OF ADULT ZEBRAFISH ....................................................................................... 33 2.3 EMBRYO COLLECTION AND MAINTENANCE ............................................................................................................ 34 2.4 ZEBRAFISH EMBRYO INJECTIONS ......................................................................................................................... 34 2.5. ZEBRAFISH LINES ............................................................................................................................................. 34 2.6 LYSIS OF ZEBRAFISH EMBRYOS OR AFTER FIN CLIPPING ............................................................................................. 36 2.7 POLYMERASE CHAIN REACTION .......................................................................................................................... 36 2.8 RESTRICTION ENZYME DIGEST ............................................................................................................................ 37 2.9 CLONING AND LIGATION ................................................................................................................................... 38 2.10 TRANSFORMATION ........................................................................................................................................... 38 2.11 IN VITRO TRANSCRIPTION .................................................................................................................................. 39 2.12 LITHIUM CHLORIDE PRECIPITATION ...................................................................................................................... 39 2.13 MINICULTURE AND –PREPARATION ..................................................................................................................... 39 2.14 PLASMID PREPARATION (MIDI) .......................................................................................................................... 40 2.15 PHENOL-CHLOROFORM EXTRACTION ................................................................................................................... 40 2.16 AGAROSE-GEL ELECTROPHORESIS ....................................................................................................................... 41 2.17 DNA SEQUENCING .......................................................................................................................................... 41 2.18 WHOLE-MOUNT IMMUNOSTAINING .................................................................................................................... 41 2.19 LASER ABLATION ............................................................................................................................................. 42 2.20 STATISTICAL ANALYSIS ...................................................................................................................................... 43 3. RESULTS ....................................................................................................................................................... 44 3.1. HIC1 (SA10158) DOES NOT IMPAIR PRONEPHRIC REGENERATION AFTER LASER INDUCED INJURY ...................................... 44 3.2. CRISPR/CAS9 HIC1......................................................................................................................................... 47 3.3. CRISPR/CAS9 MYCA ....................................................................................................................................... 54 3.4. SHROOM1 KNOCKOUT DOES NOT IMPAIR PRONEPHRIC REPAIR AFTER LASER ABLATION ................................................... 58 3.5. SHROOM3 IS NOT REQUIRED FOR PRONEPHRIC REPAIR AFTER LASER INDUCED INJURY ..................................................... 59 3.6. THE PRONEPHRIC BASEMENT MEMBRANE IS NOT REQUIRED FOR THE REPAIR PROCESS .................................................. 63 4. DISCUSSION ................................................................................................................................................. 69 iii 5. GERMAN SUMMARY / ZUSAMMENFASSUNG .............................................................................................. 73 6. ACKNOWLEDGEMENTS ................................................................................................................................ 74 7. REFERENCES ................................................................................................................................................. 75 8. APPENDIX .................................................................................................................................................... 82 8.1 ABBREVIATIONS ...................................................................................................................................................... 82 8.2 TABLES ................................................................................................................................................................. 84 8.3 FIGURES ................................................................................................................................................................ 85 8.4 EIDESSTATTLICHE VERSICHERUNG………………………………………………………………………………………………………………………….86 8.5 ERKLÄRUNG ZUM EIGENANTEIL DER DISSERTATIONSSCHRIFT………………………………………………………………………………………..87 8.6 PUBLIKATIONEN…………………………………………………………………………………………………………………………………………………88 iv v ABSTRACT ABSTRACT Acute kidney injury (AKI) with the possible progression to chronic kidney disease is still a worldwide public health challenge. Although spontaneous recovery might take place, AKI is generally associated with poor outcome, since many of the affected patients remain dialysis- dependent. Therefore, the need for understanding the underlying pathophysiology and the regeneration process remains urgent. The laser-mediated ablation of collectively migrating tubular epithelial cells of the zebrafish pronephros represents a unique AKI model. In this system, transient injuries of the kidney tubule are repaired within hours via collective cell migration (CCM) of the neighbouring tubular epithelium. Here, I further characterize the molecular factors that connect CCM to tissue repair. Previous work has implicated the transcription factors hypermethylated in cancer 1 (hic1), myca in the regeneration process. Furthermore, the basement membrane is considered to be essential guiding matrix for the regenerating renal epithelia. My results show that the pronephros regeneration is not affected in genetic loss-of-function models of hic1 or its putative targets shroom1 and shroom3. Since