The Identification and the Functional Validation o f Eye Development and Regeneration Genes in Schmidtea Mediterranea Beatriz Calvo Lozano A questa tesi doctoral està subjecta a la llicència Reconeixement - NoComercial – SenseObraDerivada 3.0. Espanya de Creat ive Commons . Esta tesis doctoral está sujeta a la licencia Reconocimiento - NoComercial – SinObraDerivada 3.0. España de Creative Commons . Th is doctoral thesis is licensed under the Creative Commons Attribution - NonCommercial - NoDerivs 3.0. Spain Licens e . GENETIC DEPARTMENT GENETIC DOCTORAL PROGRAMME BIOLOGY FACULTY “UNIVERSITAT DE BARCELONA” 2009-2012 THE IDENTIFICATION AND THE FUNCTIONAL VALIDATION OF EYE DEVELOPMENT AND REGENERATION GENES IN SCHMIDTEA MEDITERRANEA A thesis submitted in partial fulfilment of the requirements of the degree of Doctor by the “Universitat de Barcelona” by Beatriz Calvo Lozano Supervisor and Tutor Author Prof. Emili Saló i Boix Beatriz Calvo Lozano Barcelona, May 2015 I. CONTENTS CONTENTS I ABBREVIATIONS IV 1. INTRODUCTION 1 1.1 THE EYES OF SCHMIDTEA MEDITERRANEA......................................................................................... 1 1.2 STRUCTURE AND FUNCTION OF PLANARIAN EYES................................................................................. 2 1.3 REGENERATION AND DEVELOPMENT OF PLANARIAN EYES....................................................................... 8 1.4 EYE SIGNALLING PATHWAYS........................................................................................................... 13 1.4.1 PHOTOTRANSDUCTION PATHWAY....................................................................................... 13 1.4.2 RHODOPSIN TRANSPORT................................................................................................ 14 1.4.3 ABC TRANSPORTER..................................................................................................... 19 1.4.4 PIGMENT BIOSYNTHESIS................................................................................................. 21 1.4.5 ATP-DEPENDENT PROCESSES (PHOSPHORYLATION).............................................................. 25 1.4.6 STRUCTURAL PROTEINS................................................................................................. 28 2. OBJECTIVES 33 3. RESULTS AND DISCUSSION 35 3.1 SELECTION OF CANDIDATE GENES................................................................................................... 35 3.2 SEQUENCE CLONING................................................................................................................... 36 3.3 FUNCTIONAL STUDY OF CANDIDATE GENES: RNA INTERFERENCE AND IN SITU HYBRIDIZATION........................ 37 3.3.1 PHOTOTAXIS ASSAY....................................................................................................... 38 3.3.2 RNAi-PROBES........................................................................................................... 42 3.3.3 IN SITU-PROBES..................................................................................................... 47 3.4 GENE EXPRESSION ANALYSIS AND FUNCTIONAL STUDY OF PUTATIVE OPSIN GENES........................................ 49 3.4.1 RHODOPSINS............................................................................................................... 50 3.4.2 PEROPSINS ................................................................................................................ 53 3.4.3 MELANOPSINS.............................................................................................................. 55 3.5 GENE EXPRESSION ANALYSIS AND FUNCTIONAL STUDY OF PUTATIVE GENES RELATED TO PIGMENT BIOSYNTHESIS.. 62 3.5.1 SMED-WHITE FRAGMENTS............................................................................................... 62 3.5.2 SMED-VER (VERMILION).................................................................................................. 71 3.6 GENE EXPRESSION ANALYSIS AND FUNCTIONAL STUDY OF PUTATIVE TRANSCRIPTION FACTORS......................... 74 3.6.1 SMED-EXD /SMED-PBX (EXTRADENTICLE / PRE-B-CELL LEUKAEMIA HOMEOBOX)........................... 74 3.6.2 SMED-MITF-1, SMED-MITF-2 AND SMED-MITF-3.................................................................. 77 3.6.3 SMED-HOX1 AND SMED-HOX2......................................................................................... 80 3.6.4 SMED-TLX (T-CELL LEUKAEMIA HOMEOBOX GENE)................................................................ 82 3.6.5 SMED-HMT (HISTONE LYSINE METHYLTRANSFERASE).............................................................. 83 3.6.6 SMED-YY1 (YIN YANG 1).............................................................................................. 84 3.6.7 SMED-FOX (FORK HEAD BOX).......................................................................................... 85 CONTENTS I 3.7 GENE EXPRESSION ANALYSIS AND FUNCTIONAL STUDY OF PUTATIVE ENZYMES........................................... 88 3.7.1 SMED-TPR1 AND SMED-TPR2......................................................................................... 88 3.7.2 SMED-KINASE RHODOPSIN KINASE ).................................................................................. 92 3.7.3 SMED-VATP SYNTHASE (VACUOLAR ATP SYNTHASE SUBUNIT C)......................................... 94 3.8 GENE EXPRESSION ANALYSIS AND FUNCTIONAL STUDY OF PUTATIVE SIGNALLING AND TRANSPORT PROTEINS....... 98 3.8.1 SMED-DYE (DEAD EYE).................................................................................................. 98 3.8.2 SMED-CENTRIN............................................................................................................ 101 3.9 GENE EXPRESSION ANALYSIS AND FUNCTIONAL STUDY OF THE SMED-MICROARRAY GENE SELECTION............... 104 3.9.1 SMED-MICROARRAY...................................................................................................... 104 3.9.2 SMED-MICROARRAY SELECTION FOR THIS STUDY.................................................................. 105 3.9.3 SMED-MRP (MULTIDRUG RESISTANCE PROTEIN)..................................................................... 105 3.9.4 SMED-FGFL (FIBROBLAST GROWTH FACTOR RECEPTOR-LIKE 1) SMED-TITIN (TITIN A)..................... 109 3.9.5 SMED-FIB (FIBROPELLIN/JAGGED)..................................................................................... 111 3.9.6 SMED-DUK3, SMED-DUK5, SMED-DUK6, SMED-DUK10........................................................ 111 4. CONCLUSIONS 113 5. MATERIAL AND METHODS 115 5.1 PLANARIAN MAINTENANCE............................................................................................................. 115 5.2 DATABASES FOR SEQUENCE SELECTION............................................................................................ 115 5.2.1 454 PYROSEQUENCING EST DATABASE............................................................................. 116 5.2.2 454 EST AND ILLUMINA GAIIX DATABASE........................................................................ 116 5.2.3 ILLUMINA ESTS, HEAD REGENERATION TRANSCRIPTOME......................................................... 117 5.2.4 Sm454 FLX TITANIUM AND ABI SOLID 3 SEQUENCING....................................................... 117 5.2.5 THE SMED-MICROARRAY................................................................................................ 117 5.2.6 PFAM ANALYSIS............................................................................................................ 117 5.3 TOTAL RNA EXTRACTION............................................................................................................. 118 5.4 CDNA REVERSE TRANSCRIPTION.................................................................................................... 118 5.5 SEQUENCE CLONING.................................................................................................................... 119 5.5.1 TA-CLONING METHOD.................................................................................................... 119 5.5.2 T7-CLONING METHOD.................................................................................................... 123 5.5.3. PRIMER SEQUENCES...................................................................................................... 127 5.6 RNA INTERFERENCE (RNAI)....................................................................................................... 129 5.7 IN SITU HYBRIDIZATION................................................................................................................. 130 5.8 IMMUNOSTAINING......................................................................................................................... 130 5.9 TRANSMISSION ELECTRON MICROSCOPY............................................................................................ 131 5.10 PHOTOTAXIS ASSAY..................................................................................................................... 131 5.11 MICROSCOPY IMAGING AND OTHER INSTRUMENTS................................................................................ 131 6. BIBLIOGRAPHY 133 CONTENTS II 7. APPENDIX 155 7.1 DNA SEQUENCES................................................................................................................................... 155 7.2 STEP-BY-STEP PROTOCOLS ................................................................................................................. 163 PROTOCOL 1 TOTAL RNA ISOLATION