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How Visual Asymmetry Starts in Pigeons - Characterizing Melanopsin As a Potential Inducer

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How Visual Asymmetry Starts in Pigeons - Characterizing Melanopsin As a Potential Inducer How visual asymmetry starts in pigeons - Characterizing melanopsin as a potential inducer Inaugural - Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften in der Fakultät für Psychologie der RUHR - UNIVERSITÄT BOCHUM vorgelegt von Rena Klose aus Remscheid Bochum, Mai 2016 Referent: Prof. Dr. Onur Güntürkün Korreferent: Prof. Dr. Jörg T. Epplen Tag der mündlichen Prüfung: 24.08.2016 SINÄ OLET KEMIA! TABLE OF CONTENTS CHAPTER 1: GENERAL INTRODUCTION .............................................................................. 1 1.1 THE PIGEON AS A MODEL SYSTEM FOR VISUAL LATERALIZATION .............................. 2 1.1.1 The eye of the pigeon: Retinal structure and composition ............................. 3 1.1.1.1 Special features of pigeon retina ............................................................ 5 1.1.2 Two visual pathways in pigeons ....................................................................... 6 1.1.2.1 Lateralization of the visual system in pigeons ........................................ 8 1.1.3 Ontogeny of visual lateralization in pigeons .................................................... 9 1.2 A THIRD CLASS OF PHOTOCEPTOR SYSTEMS – MELANOPSIN-CONTAINING INTRINSICALLY PHOTOSENSITIVE RETINAL GANGLION CELLS ................................... 12 1.2.1 Discovery of melanopsin as a third photoreceptive instance inside the retina .............................................................................................................. 13 1.2.2 Melanopsin: diverse functions inside the retina ............................................ 13 1.2.3 Two types of genes: Opn4x vs. Opn4m .......................................................... 15 1.2.3.1 Two types of melanopsin isoforms were found in Opn4x and Opn4m ................................................................................................... 16 1.2.4 Melanopsin signaling ...................................................................................... 16 1.3 MELANOPSIN – A POSSIBLE TRIGGER OF LATERALIZATION IN PIGEONS? ................. 19 1.4 AIMS OF THIS STUDY .................................................................................................. 21 CHAPTER 2: SEQUENCING OF THE PIGEON MELANOPSIN GENE ........................................ 23 2.1 INTRODUCTION .......................................................................................................... 23 2.2 MATERIAL AND METHODS ......................................................................................... 24 2.2.1 General sequencing strategy .......................................................................... 24 2.2.2 Laboratory animals ......................................................................................... 26 2.2.3 RNA preparation ............................................................................................. 26 2.2.3.1 cDNA transcription ................................................................................ 27 2.2.4 Sequencing pigeon melanopsin ..................................................................... 28 i 2.2.5 Further analysis of pigeon specific melanopsin insert ................................... 29 2.2.6 Data analysis ................................................................................................... 30 2.2.6.1 Sequencing data analysis ...................................................................... 30 2.2.6.2 Theoretical structural and functional analysis...................................... 30 2.2.6.3 Phylogenetic analyses ........................................................................... 31 2.3 RESULTS ...................................................................................................................... 31 2.3.1 Identification of pigeons melanopsin gene sequence ................................... 31 2.3.1.1 Key features of the two pigeons melanopsin isoforms ........................ 33 2.3.1.2 A pigeon unique sequence: a more detailed analysis .......................... 34 2.3.2 Functional analysis of pigeon melanopsin ..................................................... 35 2.3.3 Phylogenetic relationship of melanopsin in pigeons and other species ........ 37 2.4 DISCUSSION ................................................................................................................ 41 2.4.1 Two different melanopsin isoforms are expressed in pigeons ...................... 42 2.4.1.1 Structural and potential functional differences of Opn4xS and Opn4xL in pigeons ................................................................................. 43 2.4.2 Melanopsin evolution – melanopsin is highly conserved within the Aves .... 44 2.4.3 Summary and conclusion ............................................................................... 47 CHAPTER 3: IS MELANOPSIN EXPRESSED IN ALL DEVELOPMENTAL STAGES IN PIGEONS? – A qualitative analysis of melanopsin expression pattern in the pigeon retina .......................................................................................................... 48 3.1 INTRODUCTION .......................................................................................................... 48 3.2 MATERIAL AND METHODS ......................................................................................... 48 3.2.1 Laboratory animals ......................................................................................... 48 3.2.2 Retinal tissue preparation for immunohistochemistry - adult animals ......... 49 3.2.3 Retinal tissue preparation for immunohistochemistry - embryos and hatchlings ....................................................................................................... 50 3.2.4 Retinal immunohistochemistry of melanopsin over three developmental stages .............................................................................................................. 51 3.2.5 Tectal tracing .................................................................................................. 52 ii 3.2.5.1 Tracer injection ..................................................................................... 52 3.2.5.2 Tissue preparation ................................................................................ 53 3.2.5.3 Immunohistochemistry of brain tissue ................................................. 54 3.2.5.4 Double labeling of CTB and melanopsin ............................................... 54 3.2.6 Data analysis ................................................................................................... 55 3.3 RESULTS ...................................................................................................................... 55 3.3.1 Melanopsin immunohistochemistry over three developmental stages ........ 55 3.3.2 Tectal tracing .................................................................................................. 57 3.4 DISCUSSION ................................................................................................................ 58 3.4.1 Melanopsin is mainly expressed in retinal ganglion cells in pigeons ............. 59 3.4.2 Melanopsin is expressed in the pigeon retina during the critical period of asymmetry formation .................................................................................... 61 3.4.3 Summary and conclusion ............................................................................... 62 CHAPTER 4: IS MELANOPSIN EXPRESSION ASYMMETRICALLY REGULATED IN THE DEVELOPMENT OF PIGEONS? – A qPCR approach to quantify melanopsin expression in the pigeon retina ................................................................... 63 4.1 INTRODUCTION .......................................................................................................... 63 4.2 MATERIAL AND METHODS ......................................................................................... 63 4.2.1 Laboratory animals ......................................................................................... 64 4.2.2 Tissue preparation .......................................................................................... 65 4.2.3 RNA preparation and quality control ............................................................. 66 4.2.4 cDNA transcription ......................................................................................... 67 4.2.5 Sequencing of putative reference genes ....................................................... 67 4.2.6 Evaluation of putative reference genes for qPCR .......................................... 70 4.2.7 Determining limit of detection (LOD) and limit of quantification (LOQ) ....... 73 4.2.8 Quantitative melanopsin expression analysis ................................................ 74 4.3 RESULTS ...................................................................................................................... 76 4.3.1 Quantification of melanopsin isoforms S and L in adult pigeons .................. 77 4.3.2 Melanopsin expression over development .................................................... 78 4.3.3 Melanopsin expression in dark-incubated embryos ...................................... 79 iii 4.4 DISCUSSION ................................................................................................................ 81 4.4.1 Analyzing relative gene expression: establishment and validation of qPCR in the pigeon retina ........................................................................................ 82 4.4.2 Isoform-related melanopsin
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