Peroxin3, a newly identified regulator of melanocyte development and melanosome biogenesis in zebrafish Danio rerio Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Mirco Brondolin aus San Dona’ di Piave - Italien Bonn 2016 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter Prof. Dr. rer. nat. Michael Hoch 2. Gutachter Prof. Dr. phil. nat. Christoph Thiele Tag der Promotion: 20. März 2017 Erscheinungsjahr: 2017 Per aspera sic itur ad astra “Through hardships to the stars” (Lucius Annaeus Seneca, Hercules furens, act II, v. 437 Table of contents I Table of contents 1 Introduction ...........................................................................................................................1 1.1 Peroxisomes.................................................................................................................. 1 1.1.1 Peroxisome structure and features........................................................................1 1.1.2 Peroxisomal metabolic activity ..............................................................................2 1.1.3 Peroxisome biogenesis...........................................................................................7 1.1.4 Pex3, key component of peroxisome biogenesis................................................ 11 1.1.5 Peroxisome related pathologies ......................................................................... 13 1.2 Zebrafish (Danio rerio) ................................................................................................ 16 1.2.1 Zebrafish in biomedical research ........................................................................ 16 1.2.2 Zebrafish genome features ................................................................................. 17 1.2.3 Zebrafish life cycle............................................................................................... 18 1.2.4 Advantages of zebrafish as biomedical model.................................................... 20 1.2.5 Gene manipulation in zebrafish .......................................................................... 21 1.2.6 Study of metabolic disorders in zebrafish........................................................... 26 1.2.7 Development of therapeutical approaches ........................................................ 27 1.3 Neural crest and its derived tissues ............................................................................ 28 1.3.1 Neural crest during embryogenesis .................................................................... 28 1.3.2 Pigment cell populations..................................................................................... 30 1.3.3 Pigmentation pattern formation in zebrafish ..................................................... 30 1.3.4 Melanocytes........................................................................................................ 33 1.3.5 Melanosomes...................................................................................................... 34 2 Aim of this thesis................................................................................................................. 39 3 Materials and Methods....................................................................................................... 41 3.1 Materials ..................................................................................................................... 41 3.1.1 Common materials.............................................................................................. 41 3.1.2 Equipment........................................................................................................... 41 3.1.3 Standards, Kits and Enzymes............................................................................... 42 3.1.4 Buffers and solutions .......................................................................................... 43 3.1.5 Fish lines.............................................................................................................. 45 3.1.6 Standard fish food............................................................................................... 45 3.1.7 Oligonucleotides ................................................................................................. 46 3.1.8 Antisense Morpholino Oligonucleotides (AMOs) ............................................... 47 3.1.9 Vectors ................................................................................................................ 48 3.1.10 Antibodies ........................................................................................................... 48 3.1.11 Micrororganisms ................................................................................................. 49 II Table of contents 3.1.12 Cell lines............................................................................................................... 49 3.1.13 Bacterial culture media ....................................................................................... 49 3.1.14 Cell culture media................................................................................................ 49 3.1.15 Softwares............................................................................................................. 49 3.2 Methods ...................................................................................................................... 50 3.2.1 Fish work ............................................................................................................. 50 3.2.2 Histology.............................................................................................................. 53 3.2.3 Molecular work ................................................................................................... 54 3.2.4 Biochemical work ................................................................................................ 58 3.2.5 Microbiological work........................................................................................... 59 3.2.6 Bioinformatics ..................................................................................................... 59 3.2.7 Statistics............................................................................................................... 60 4 Results ................................................................................................................................. 61 4.1 Identification of the zebrafish homolog of the human PEX3...................................... 61 4.2 Pex3 gene structural analysi........................................................................................ 64 4.3 Prediction of pex3 functional domains ....................................................................... 66 4.4 pex3 expression in zebrafish ....................................................................................... 69 4.5 Pex3 morphants do not show abnormal development .............................................. 74 4.6 Generation and validation of a pex3 loss of function zebrafish model ...................... 75 4.6.1 TALENs-guided mutation generation .................................................................. 76 4.6.2 CRISPR/Cas9-guided mutation generation.......................................................... 78 4.6.3 Commercial ENU-induced mutagenesis alleles................................................... 79 4.6.4 Establishing High Resolution Melting Analysis (HRMA) to validate mutant alleles80 4.7 In pex3 mutants peroxisomal and mitochondrial metabolism is impaired, resulting in increased oxidative stress ....................................................................................................... 84 4.7.1 In pex3CRISPR/ZMP zebrafish peroxisomal metabolism is impaired ......................... 84 4.8 Pex3 mutation affects neural crest-derived tissues developmen............................... 93 4.8.1 Pex3 mutation affects proper melanophores migration and development ....... 93 5 Discussion.......................................................................................................................... 123 5.1 In zebrafish developing embryos, pex3 is expressed also in developing sensory organs, gill filaments and melanophores .............................................................................. 124 5.2 Pex3 mutant alleles are responsible for mild forms of Peroxisomal Biogenesis Disorders ............................................................................................................................... 126 5.3 Metabolic differences between zebrafish and human account for dissimilarities in phenotypes............................................................................................................................ 127 5.4 pex3 facilitates peroxisome interaction with other organelles ................................ 128 5.5 Melanosomes and melanin synthesis are affected by cellular metabolism ............. 130 5.6 pex3 is targeted also to melanosomes and exert its function on their membrane.. 132 Table of contents III 5.7 pex3 influences melanin synthesis sensing mechanism in melanophores............... 133 5.8 Defective pex3 localization impairs proliferation and migration of specific cell populations ..........................................................................................................................