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

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

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 expression differences in adult pigeons ...... 82 4.4.3 Exogenous or endogenous regulation of melanopsin expression over the development in pigeons? ...... 84 4.4.4 Indicators of asymmetrical melanopsin expression in pigeons? ...... 87 4.4.5 Summary and conclusion ...... 89

CHAPTER 5: CAN PIGEON MELANOPSIN BE ACTIVATED BY LIGHT? – A calcium imaging approach to visualize light responses of pigeon melanopsin in vitro ...... 91

5.1 INTRODUCTION ...... 91 5.2 MATERIAL AND METHODS ...... 91

5.2.1 General cloning strategy ...... 91 5.2.2 Calcium imaging ...... 93

5.2.2.1 Addition of NheI and SacII restriction sites for molecular cloning by PCR ...... 93 5.2.2.2 TOPO® TA® cloning and transformation ...... 95 5.2.2.3 Plasmid mini-preparation (alkaline lysis method) ...... 95 5.2.2.4 Plasmid midi-preparation ...... 97 5.2.2.5 Cloning of melanopsin in pmCherry-N1 vector ...... 97 5.2.2.6 Transfection HEK cells ...... 98 5.2.2.7 Fluo-4 calcium assay ...... 99 5.2.2.8 Data analysis ...... 99

5.2.3 Fluo-4 calcium assay under the confocal microscope ...... 100

5.2.3.1 Transfection HEK cells ...... 100 5.2.3.2 Fluo-4 calcium assay ...... 100 5.2.3.3 Data analysis ...... 100

5.3 RESULTS ...... 101

5.3.1 In vitro calcium assay ...... 101

5.3.1.1 Comparison of isoform S and L ...... 104 iv

5.3.2 Live calcium assay...... 104

5.4 DISCUSSION ...... 106

5.4.1 Pigeon melanopsin is activated by blue light ...... 107 5.4.2 Activation of pigeon melanopsin: isoform S vs. L ...... 109 5.4.3 Summary and conclusion ...... 111

CHAPTER 6: ELECTROPHYSIOLOGICAL CHARACTERIZATION OF PIGEON MELANOPSIN

ON THE BASIS OF THE Gi/o SIGNALING PATHWAY IN VITRO ...... 112

6.1 INTRODUCTION ...... 112 6.2 MATERIAL AND METHODS ...... 112

6.2.1 Recloning of pigeon melanopsin ...... 113 6.2.2 Transfection HEK cells ...... 114 6.2.3 In vitro patch-clamp ...... 115 6.2.4 Statistical analysis ...... 118

6.3 RESULTS ...... 118

6.3.1 Light-dependent melanopsin activation on the basis of the Gi/o signaling pathway ...... 119 6.3.2 Repetitive activation of pigeon melanopsin ...... 120 6.3.3 Wavelength-dependence of pigeon melanopsin activation ...... 122

6.4 DISCUSSION ...... 123

6.4.1 Pigeon melanopsin is able to activate the Gi/o signaling pathway ...... 124 6.4.2 Electrophysiological properties of pigeon melanopsin in comparison to other melanopsins ...... 125 6.4.3 Electrophysiological properties of melanopsin isoform L and their possible influence on the induction of visual asymmetries ...... 127 6.4.4 Summary and conclusion ...... 128

CHAPTER 7: GENERAL DISCUSSION ...... 129

7.1 Melanopsin is able to influence the ontogeny of visual lateralization in pigeons .. 129

7.1.1 Transducing asymmetrical light information – possible transfer mechanism from the retina to the brain ...... 132

7.2 Melanopsin and cryptochrome: Possible interaction partners? ...... 139 7.3 Conclusion and outlook ...... 143

LIST OF REFERENCES ...... 146 APPENDIX ...... 178

A. CHEMICALS AND EQUIPMENT ...... 178

A.1 List of chemicals and reagents ...... 178 A.2 Enzymes and corresponding buffers ...... 179 A.3 Pharmaceuticals ...... 180 A.4 Antibodies ...... 180 A.5 Buffer and solutions ...... 180 A.6 Kits and manuals ...... 183 A.7 Technical equipment and consumable supplies ...... 185 A.8 Software ...... 186 A.9 Accession numbers for BLAST ...... 187 A.10 Plasmids ...... 188 A.11 Oligonucleotide primers ...... 189

B. LIST OF ABBREVIATIONS ...... 190 C. LIST OF FIGURES ...... 194 D. LIST OF TABLES ...... 196 E. SUPPLEMENTARY DATA ...... 197

E.1 Supplementary data Chapter 2 ...... 197 E.2 Supplementary data CHAPTER 4 ...... 202

CURRICULUM VITAE ...... 223 PUBLICATIONS ...... 225 ACKNOWLEDGEMENTS/DANKSAGUNG ...... 226 ERKLÄRUNG ...... 228

SUMMARY

Asymmetries of the nervous system are a well investigated trait in all kinds of vertebrates and even invertebrates (Vallortigara and Rogers, 2005; Frasnelli, 2013), ranging from lateralization of vocalization (for review see Ocklenburg et al., 2013) to limb preferences (for review see Ströckens et al., 2013b). Favoring one hemisphere over the other for special tasks prevents competition between both brain halves and allows the enhancement of cognitive capacities (Toga and Thompson, 2003; Vallortigara and Rogers, 2005). However, insights about the ontogeny of lateralization are relatively sparse, although advantages, anatomical structure and behavioral performances are well understood. In order to understand the development of asymmetries the well investigated visual system of pigeons provides a perfect model system (Güntürkün et al., 2014). The visual system of pigeons reveals a stable functional and structural asymmetry in the tectofugal pathway (Güntürkün, 2002a, 2005) with a right eye/left brain hemispheric advantage for discrimination performances which are based on anatomical asymmetries (Güntürkün, 1985; Güntürkün, 1997; Yamazaki et al., 2007). This pathway is shaped into a lateralized system as a result of an asymmetrical light stimulation during a critical time period in the late embryonic development (Güntürkün, 2002a, 2002b; Skiba et al., 2002). Due to a genetic predisposition the pigeon embryo takes up an asymmetrical position inside the egg, with the right eye being exposed to the translucent eggshell while the left eye is directed towards the embryos body (Kuo, 1932). The egg is repetitively stimulated by light since the brooding pigeons regularly leave the nest and turn the eggs (Buschmann et al., 2006). This asymmetrical light stimulation is able to induce the development of an asymmetrical tectofugal system in the ascending visual pathway (Güntürkün, 2002a). In addition, dark-incubation of eggs prevents the development of visual asymmetries in pigeons (Skiba et al., 2002; Güntürkün, 2005; Manns et al., 2008). However, the classical retinal photoreceptors, rods and cones, are not functionally developed before hatching (Bagnoli et al., 1985; Rojas et al., 2007). Therefore, a further photosensitive instance is needed. Recent studies identified a third class of photoreceptive molecules, which are expressed in retinal ganglion cells (Berson, 2003). Since retinal ganglion cells are present already at an early developmental stage and possess functional connections to primary visual areas before hatch (Manns and Güntürkün, 1997; Theiss and Güntürkün, 2001), retinal ganglion cell-bound photoreceptors could serve as a potential transducer for visual lateralization. The photopigment that renders these retinal ganglion cells intrinsically photosensitive is called melanopsin (Hattar et al., 2002; Rollag et al., 2003; Berson, 2003).

vii

SUMMARY

This thesis aimed to characterize pigeon melanopsin as a possible inducer of light-mediated visual asymmetries in pigeons. First of all, the melanopsin gene was sequenced using polymerase chain reaction (PCR), demonstrating the occurrence of at least two different isoforms. Furthermore, the expression of melanopsin was qualitatively proven by immunohistochemical staining and quantitatively examined by quantitative real-time PCR across three developmental stages (adult, hatchling, and embryo). The immunohistochemical staining demonstrated melanopsin expression across all stages in the (developing) retinal ganglion cell layer and quantitative analyses revealed that there is a presumably endogenous regulated melanopsin expression in embryos exhibiting no genetically determined left/right differences. Moreover, light-sensitivity was analyzed by calcium imaging and patch-clamp experiments in vitro, demonstrating that pigeon melanopsin forms a functional especially blue light-sensitive photopigment which can be repetitively activated and only a short light pulse is sufficient for a long-lasting activation. Altogether, these data confirm that melanopsin is expressed during the crucial time frame of asymmetry formation in pigeon embryos and forms a functional blue-light sensitive photopigment, which delivers a sustained photo-response once activated. Therefore, these results provide important evidences for melanopsin as a light-sensitive instance inside the pigeon retina before hatch, which might be able to transduce the asymmetrical light stimulus to the brain shaping the tectofugal pathway into a lateralized system.

viii

ZUSAMMENFASSUNG

Asymmetrien des Nervensystems sind eine gut untersuchte Eigenschaft verschiedenster Vertebraten und sogar Invertebraten (Vallortigara and Rogers, 2005; Frasnelli, 2013). Diese Asymmetrien reichen von der Lateralisierung von Vokalisierung (Review: Ocklenburg et al., 2013) bis hin zu Gliedmaßen-Präferenzen (Review: Ströckens et al., 2013b). Das Bevorzugen einer Hemisphäre für spezifische Aufgaben vermeidet die Konkurrenz zwischen beiden Hirnhälften und erlaubt die Steigerung von kognitiven Kapazitäten (Toga and Thompson, 2003; Vallortigara and Rogers, 2005). Allerdings ist über die Entstehung dieser Asymmetrien bisher wenig bekannt, obwohl Vorteile, anatomischer Aufbau und asymmetrische Verhaltensmuster gut verstanden sind. Daher stellt das intensiv untersuchte visuelle System der Taube ein perfektes Modelsystem dar, um die Entwicklung von Asymmetrien zu verstehen (Güntürkün et al., 2014). Das visuelle System der Taube besitzt eine ausgeprägte funktionale und strukturelle Asymmetrie im tektofugalen System (Güntürkün, 2002a, 2005). Dieses visuelle System weist einen Vorteil für das rechte Auge und die linke Hemisphäre in Diskriminationsaufgaben auf. Diese Verhaltensasymmetrien basieren wiederum auf anatomischen Asymmetrien (Güntürkün, 1985; Güntürkün, 1997; Yamazaki et al., 2007). Durch eine asymmetrische Lichtstimulation, während einer kritischen Phase in der späten embryonalen Entwicklung, wird dieser visuelle Weg in ein lateralisiertes System geformt (Güntürkün, 2002a, 2002b; Skiba et al., 2002). Eine genetische Prädisposition führt dabei zu einer asymmetrischen Position des Embryos innerhalb des Eies, dabei wird das rechte Auge zur lichtdurchlässigen Eierschale gerichtet, während das linke Auge in Richtung des Körpers zeigt (Kuo, 1932). Das Ei wird wiederholt mit Licht stimuliert, da die brütenden Elterntiere das Nest regelmäßig verlassen und die Eier drehen (Buschmann et al., 2006). Diese asymmetrische Lichtstimulation reicht aus, um die Entwicklung eines asymmetrischen tektofugalen Systems im aufsteigenden visuellen Pfad zu fördern (Güntürkün, 2002a). Werden die Eier allerdings im Dunklen inkubiert, werden diese visuellen Asymmetrien im tektofugalen System nicht ausgeprägt (Skiba et al., 2002; Güntürkün, 2005; Manns et al., 2008). Allerdings sind zu diesem Zeitpunkt die klassischen Photorezeptoren, Stäbchen und Zapfen, noch nicht funktional entwickelt (Bagnoli et al., 1985; Rojas et al., 2007). Daher muss es vor dem Schlupf bereits eine weitere photosensitive Instanz in der Retina geben. Vor kurzem konnten Studien zeigen, dass es eine dritte Klasse von photorezeptiven Molekülen in der Retina gibt, die in retinalen Ganglienzellen exprimiert werden (Berson, 2003). Photorezeptoren, die in den retinalen Ganglienzellen lokalisiert sind, könnten potentielle Auslöser der visuellen

ZUSAMMENFASSUNG

Lateralisierung sein, da retinale Ganglienzellen bereits in frühen embryonalen Entwicklungsstufen vorhanden sind und diese bereits funktionale Verbindungen zu primären visuellen Arealen vor dem Schlupf haben (Manns and Güntürkün, 1997; Theiss and Güntürkün, 2001). Das Photopigment, dass diese retinalen Ganglienzellen intrinsisch photosensitiv macht, wird Melanopsin genannt (Hattar et al., 2002; Rollag et al., 2003; Berson, 2003). Diese Arbeit zielte darauf ab, Melanopsin als einen möglichen Auslöser von Licht-induzierten visuellen Asymmetrien in der Taube zu charakterisieren. Als erstes wurde das Melanopsin-Gen der Taube mittels Polymerase-Kettenreaktion (PCR) sequenziert. Dies zeigte, dass mindestens zwei Isoformen des Melanopsin-Proteins in der Taube vorkommen. Des Weiteren wurde die Expression von Melanopsin qualitativ mittels immunhistochemischen Färbungen nachgewiesen und quantitativ mit Hilfe der quantitativen real-time PCR über alle Entwicklungsstufen (adulte Taube, Küken und Embryo) bestimmt. Die immunhistochemischen Färbungen bestätigten, dass Melanopsin in allen Stufen in (sich entwickelnden) retinalen Ganglienzellen exprimiert wird und die quantitative Analyse zeigte, dass die Expression von Melanopsin im Taubenembryo vermutlich endogen reguliert wird und dass es keine genetisch-determinierten rechts/links Unterschiede gibt. Außerdem wurde die Licht-Sensitivität mittels Calcium-Imaging und der Patch-Clamp Technik in vitro analysiert. Dadurch konnte nachgewiesen werden, dass Melanopsin ein funktionales Photopigment formt, das hauptsächlich durch blaues Licht und vor allem repetitiv aktiviert werden kann. Dabei reicht bereits ein kurzer Lichtpuls aus, um eine lang-anhaltende Aktivierung zu erreichen. Zusammenfassend bestätigen diese Daten, dass Melanopsin während der kritischen embryonalen Entwicklungsphase der Asymmetrie-Ausprägung exprimiert wird und ein funktionelles sensorisches Photopigment formt, welches mit blauem Licht aktiviert werden kann. Einmal aktiviert geht eine langanhaltende elektrische Antwort von Melanopsin aus. Daher liefern diese Ergebnisse entscheidende Beweise, dass Melanopsin bereits eine Licht-sensitive Instanz innerhalb der Retina der Taube vor dem Schlupf darstellt, die geeignet ist, die asymmetrische Lichtstimulation an das Gehirn weiterzuleiten und daher den tektofugalen Weg in ein lateralisiertes System zu formen.

CHAPTER 1: GENERAL INTRODUCTION

At first glance, we seem to live in a symmetrical world, with the most obvious example for symmetry found in the shape of our body. We humans belong, like a lot of animals, to the clade of bilateria which exhibit a fundamental body plan including a bilateral symmetry (Finnerty, 2003). However, on a closer look our body appears more and more asymmetrical. For instance our liver, gall bladder, inferior vena cava and trilobed lung are positioned on the anatomical right side of the body, whereas the pulmonary venous atrium, stomach, pancreas, spleen, aorta and bilobed lung are orientated on the left side (Lambert et al., 2016). Only a very small percentage (0.001%) of the population shows an inverted arrangement of viscera and arteria (situs inversus, Lambert et al., 2016). However, our viscera are not the only asymmetrical part of or body. Over 150 years ago Broca discovered that our brain is asymmetrically organized, with functional as well as structural differences between the left and right hemispheres (Broca, 1865, Toga and Thompson, 2003). One of the earliest brain asymmetries found was the specialization of the left hemisphere for human language discovered in post mortem studies by Broca (1861) and Wernicke (1874) and subsequently laterality was a trait thought to be uniquely “human” for a long time. Nevertheless, Nottebohm was able to provide one of the first evidences for brain lateralization beyond the human species. He investigated the chaffinch (Fringilla coelebs) song system, showing that left hypoglossal sectioning leads to loss of most song components in contrast to right hypoglossal sectioning which only leads to the loss of a few or even no song components. Another example of lateralization, probably the most familiar one in humans, is handedness (Hopkins et al., 1987), with about 90% of all people being right handed while the remaining 10% are either left manual or ambidextrous (Corballis, 1997). Limb preferences (on population or individual level) can also be found in a lot of vertebrates (for review see Ströckens et al., 2013b) ranging from mammals (dogs: Tan, 1987; Quaranta et al., 2004; chimpanzees: Hopkins et al., 2011, rats: Güven et al., 2003) to amphibians (toads: Bisazza et al., 1996) and birds (parrots: Brown and Magat, 2011).

CHAPTER 1: GENERAL INTRODUCTION

Thus, a multitude of studies has been conducted showing that lateralization is a widespread phenomenon among all vertebrate classes (Vallortigara and Rogers, 2005; e.g. lateralization of vocalization in many vertebrate species: for review see Ocklenburg et al., 2013; lateralization of the visual system in amphibians, reptiles, mammals and birds: Bisazza et al., 1998, Bisazza et al., 2002, Vallortigara and Rogers, 2005, Güntürkün et al., 2000; Güntürkün, 2005). Despite the evidence that brain lateralization is associated with many advantages as it avoids competition between both hemispheres, enhances cognitive capacities and neuronal efficiencies (Toga and Thompson, 2003; Vallortigara and Rogers, 2005), little is known about the precise mechanisms which lead to brain lateralization. Under consideration are genetic (McManus, 2002), epigenetic (Previc, 1991) as well as environmental factors (Manns, 2006). However, it has been evidenced that the induction of visual lateralization is triggered by the environmental factor light during the embryonic development in pigeons and chickens (Rogers, 1982; Güntürkün, 2002b, 2005). Therefore, these species serve as a useful model for understanding the mechanism of the development of lateralization. And the lateralized visual system of pigeons and chickens has been the target in a multitude of studies (for review see: Manns and Ströckens, 2014). But the precise mechanisms of how embryonic light stimulation can be perceived remain unknown, since classic photosensitive instances are still in development and not functional yet. Hence, this thesis aims to unravel the mystery of light-induced induction of visual lateralization in pigeons by characterizing a possible photosensitive candidate that might be able to transduce the asymmetrical light signal during early embryonic development.

1.1 THE PIGEON AS A MODEL SYSTEM FOR VISUAL LATERALIZATION

Pigeons (Columba livia) represent an excellent model organism for the investigation of cerebral lateralization on the behavioral and structural levels. The well-established pigeon model has several advantages: Pigeons show impressive cognitive capacities, an excellent work ethic, longevity, and are easy to handle. In addition, the well-studied visual system exhibits a stable lateralization on the population level (Güntürkün et al., 2014). Additionally, all retinal fibers cross completely in the optic chiasm (Cowan et al., 1961), leaving the

CHAPTER 1: GENERAL INTRODUCTION contralateral hemisphere with input only from one eye. This enables easy testing of the cognitive capacities and functions of each hemisphere separately by just blocking one hemisphere, e.g. by covering one eye with an eye cap (Manns and Güntürkün, 1999a). The following section will be focusing on the visual system of pigeons, its basic composition, characteristic features and the specific lateralization.

1.1.1 The eye of the pigeon: Retinal structure and composition

The visual system is one of the most important senses in the animal kingdom, and birds are one of the most visually dependent class of all vertebrates (Güntürkün, 2002b). The eyes of a bird take up a considerable part of the whole skull volume (Güntürkün, 2000), and as Rochon-Duvigneaud (1943) said “A bird is a wing guided by an eye”. This statement is also reflected by the fact that pigeons possess around 2.4 million axons in the optic nerve (Binggeli and Paule, 1969), around 2-fold more than for example counted in humans (Curcio and Allen, 1990). The retinal structure and the composition of the pigeon retina demonstrates extensive similarities to most vertebrates and therefore to the mammalian retina. The precise structure will be described in the following section. Before a light stimulus can be converted into an electric impulse that can be forwarded to the brain, light approaching to the eye has first to pass through the cornea, the anterior chamber, the lens and the vitreous body before reaching the retina. The retina itself is structured in seven different layers. These layers can be classified as nuclear layers, which are rich in cell bodies, and as plexiform layers, which possess fewer cell bodies but more fibers. First, light has to pass the ganglion cell layer (GCL), then the inner plexiform layer (IPL) and the inner nuclear layer (INL), subsequently the outer plexiform layer (OPL) and the outer nuclear layer (ONL) before reaching the photoreceptors in the photoreceptor layer (PRL) and the retinal pigment epithelium (RPE, cf. Figure 1.1; Wässle and Boycott, 1991; Hankins et al., 2008; Güntürkün, 2000; Wässle, 2004; Swaroop et al., 2010). The RPE is the rearmost layer of the retina, close to the PRL. In the PRL the light signal is converted into an electrical impulse by two well-known types of photoreceptors: the rods and the cones (Wässle and Boycott, 1991; Wässle, 2004). Rods can perceive quite subtle light differences, but are unable to perceive colors. Cones, on the other hand, can mediate the perception of colors, but are much less sensitive in low light conditions (Bowmaker and Knowles, 1977; Bowmaker and Dartnall, 1980; Swaroop et al., 2010). Both photoreceptor types can be divided into two different segments, 3

CHAPTER 1: GENERAL INTRODUCTION an inner (IS) and an outer (OS) segment. Besides connecting the OS and the synapse, biosynthesis of proteins and cell metabolism occurs in the IS. In the OS of rods and cones a group of light sensitive membrane bound G-protein-coupled receptors called opsins, can be found. In short, absorbance of incoming light leads to a conformational change in the opsins and thereupon, the light signal is transduced into a change of membrane potential resulting in a hyperpolarization of rod and cone somata which form the ONL (Swaroop et al., 2010; Baker and Kerov, 2013). Rhodopsin is the rod opsin exhibiting an absorption maximum at 500 to 506 nm in birds. Whereat iodopsin and other related photopigments can be found in cones of avian retina (absorption maxima: 562 nm, 514 nm, 467 nm, 413 nm; Bowmaker and Knowles, 1977; Nalbach et al., 1993). Aside from the specific photopigments, rods and cones can be usually distinguished by the presence of oil droplets in cones (see 1.1.1.1). Subsequently, the signal, evoked by rods and cones, is propagated to bipolar cells, localized in the INL via the fibers of the OPL (Wässle and Boycott, 1991). Next, bipolar cells transduce the signal to retinal ganglion cells (RGCs), located in the GCL. But before reaching the RGCs the signal can be modulated by different cell types: horizontal and amacrine cells. Horizontal cells, found in the INL, build up connections between bipolar cells, photoreceptors and ganglion cells, while amacrine cells, also located in the INL, integrate, modulate and interpose the visual messages presented to ganglion cells. Then, RGCs transfer the information via the nerve fiber layer (NFL) and the optic nerve further to primary visual areas of the brain (Wässle and Boycott, 1991; Wässle, 2004; Lamb et al., 2007; Joselevitch, 2008). Furthermore, a small population of ganglion cells can be found in the INL, called displaced retinal ganglion cells (dRGCs). Displaced retinal ganglion cells are involved in the control of certain eye movements, mediated by a bisynaptic pathway from the retina to the vestibulo-cerebellum and transfer information to the accessory optic system (Karten et al., 1977; Heaton et al., 1979; Fite et al., 1981). Another relatively newly recognized type of ganglion cells found in the GCL is the intrinsically photosensitive ganglion cell (ipRGCs; cf. Figure 1.1). These cells are able to receive and process light stimulation without receiving input from rods and cones (Berson, 2003; Hankins et al., 2008; Foster, 2009) and will be focused in section 1.2. Between the cell layers (GCL, INL, ONL) the fiber layers IPL and OPL can be found, consisting of fibers of adjacent cells of the nuclear layers (Güntürkün, 2000). In addition to this basic structure and composition of the vertebrate retina the pigeon retina exhibits some special features, which will be focused on in the next section.

CHAPTER 1: GENERAL INTRODUCTION

Figure 1.1: Schematic overview of the vertebrate retina. The retina is composed of seven distinct layers: GCL, IPL, INL, OPL, ONL, PRL and RPE. The rearmost layer RPE is close to the PRL. The PRL is formed by the outer and inner segments of rods and cones which perceive and convey the visual information. The somata of rods and cones are located in the ONL and are laterally connected via horizontal cells. Signals are transferred via bipolar cells located in the INL. These cells are in turn laterally connected with amacrine cells and RGCs. The GCL contains regular RGCs as well as ipRGCs, which intrinsically photosensitive. RGCs transfer the signal via the optic nerve to the brain. The yellow arrow indicates the direction of incoming light (abbreviations: A = amacrine cells, B = bipolar cells, C = cones, G = ganglion cells, GCL = ganglion cell layer, H = horizontal cells, INL = inner nuclear layer, IPL = inner plexiform layer, ONL = outer nuclear layer, OPL = outer plexiform layer, PRL = photoreceptor layer, pRGC = photosensitive ganglion cells, R = rods, RPE = retinal pigment epithelium; modified from Hankins et al., 2008).

1.1.1.1 Special features of pigeon retina

The basic structure of the pigeon retina (described in 1.1.1) is rather similar to most mammalian retinae (Güntürkün, 2000). Aside from the similarities to mammals described above there are some unique differences in the composition of the avian eye. The avian retina is completely avascularized which prevents light scattering and shadows and, therefore, facilitates sharper vision. Due to the absence of vascularization another nutritional device in avian retina is needed: the pecten (Bellhorn and Bellhorn, 1975; Güntürkün, 2000). The pecten is a highly vascularized structure mainly involved in supplying nutrients to the retina (Kiama et al., 1998; Wolburg et al., 1999; Kiama et al., 2001) as well as in controlling the intraocular pH

CHAPTER 1: GENERAL INTRODUCTION

(Brach, 1975). The pecten proceeds from the ventral retina above the accession of the optic nerve into the eyeball towards the lens (Güntürkün, 2000). Furthermore, the avian retina possesses oil droplets located inside the inner segments of cones, serving as specific light filters. They collect a significant amount of light and enhance the outer segment photon capture rate, concurrently shielding the retina from detrimental light conditions, providing an ultraviolet (UV) shield. Oil droplets can be found in different colors and are heterogeneously distributed in the red field (red and orange droplets) and the yellow field (greenish-yellow droplets; Emmerton, 1983; Young and Martin, 1984; Güntürkün, 2000). In addition to the pecten and oil droplets, the avian retina shows differences in cell types and cell densities in comparison to typical mammalian retinae. In contrast to trichromatic mammals (Bowmaker et al., 1997; Vorobyev, 2004), pigeons are potential pentachromates, possessing five kinds of cones (three cone types and one double cone) in their retina (Emmerton and Delius, 1980). Additionally, in mammals the highest density of rods and cones is located at the fovea centralis, an area in the central retina responsible for sharp central vision. This holds true for avian species as well, but due to their laterally placed eyes, pigeons and other birds possess two foveae: a central fovea which is responsible for frontal and binocular perception and a lateral fovea for monocular vision (Binggeli and Paule, 1969). Avian vision is one of the most pronounced sensory systems in the animal kingdom, being able to perceive UV wavelengths and considerably more colors compared to mammals (Goldsmith, 1990; Bennett and Cuthill, 1994). However, to recognize a light stimulus the retinal signal has to be transferred onto higher brain structures. The possible pathways retinal information can enter will be discussed in the next section.

1.1.2 Two visual pathways in pigeons

In pigeons visual information which has been processed by the photoreceptors in the retina and transferred towards the brain via RGCs and the optic nerve will be forwarded to visual brain areas. All fibers of RGCs in each retina cross almost completely in the optic chiasm (Cowan et al., 1961; Weidner et al., 1985), propagating information only to the contralateral hemisphere. Subsequent to decussation, most of the visual information enters one of the two prominent visual pathways in pigeons: either the tectofugal pathway or the thalamofugal pathway. Frontal binocular visual field is represented by the tectofugal pathway, while lateral monocular vision is transferred via the thalamofugal pathway (Güntürkün and Hahmann, 6

CHAPTER 1: GENERAL INTRODUCTION

1999; Hellmann and Güntürkün, 1999). It is important to note that most of all visual information is processed via either the thalamofugal or the tectofugal pathway, but in addition to these two pathways a proportion of projections is directed to other brain regions like the nucleus of the basal optic root (nBOR, Wylie et al., 2009). But this side aspect will not be focused on in this thesis. Visual information from the retina entering the thalamofugal pathway is first transmitted to the nucleus geniculatus lateralis pars dorsalis (GLd) in the thalamus which receives input from the contralateral retina. Fibers originating from the GLd arise bilateral to the ipsilateral and contralateral visual wulst of the telencephalic anterior forebrain. This pathway only receives input from a fraction of ganglion cells in lateral retinal areas and is to some extent comparable with the geniculocortical pathway in mammals (cf. Figure 1.2 A and B; Shimizu and Karten, 1993; Luksch, 2003; Wylie et al., 2009). The thalamofugal pathway is responsible for monocular recognition of new complex stimuli before binocular analysis (Güntürkün et al., 1993; Shimizu and Karten, 1993).

Figure 1.2: Two pathways in the avian visual system. A Thalamofugal pathway. Visual information is processed from the retina to the ipsi- and contralateral to the wulst via the GLd. B Schematic overview of the formation of the thalamo- (green) and tectofugal (blue) pathway in the pigeon brain. C Tectofugal pathway. Fibers arising from the retina project bilaterally to the TO, followed by bilateral projections to the RT. Rotundal projections run to the ipsilateral entopallium. Different thickness of arrows indicates differences in projection strength in the pigeon (abbreviations: E = entopallium, GLd = nucleus geniculatis lateralis pars dorsalis, R = retina, RT = nucleus rotundus, TO = optic tectum, W = wulst; modified from Rogers, 1996 and Manns, 2006).

The tectofugal pathway is the most important visual subsystem of the pigeon, and 75-95% of all fibers of RGCs proceed via this pathway (Remy and Güntürkün, 1991). The tectofugal system corresponds to the extrageniculocortical system of mammals. Arising from the retina, fibers cross almost completely in the optic chiasm and proceed to the contralateral 7

CHAPTER 1: GENERAL INTRODUCTION optic tectum (TO, tectum opticum). The TO sends bilateral projections to the nucleus rotundus (RT), which in turn projects to the ipsilateral entopallium in the telencephalon and the associated entopallial belt (cf. Figure 1.2 B and C; Benowitz and Karten, 1976; Hardy et al., 1985; Engelage and Bischof, 1993; Hellmann and Güntürkün, 1999; Manns et al., 2007). The tectofugal pathway is responsible for the recognition of pattern and shape as well as color perception. Lesion studies have shown that lesions in the TO, the RT or the entopallium cause pronounced deficits with respect to pattern, shape and color discrimination and lowered the discrimination efficiency concerning these properties (Hodos and Karten, 1970, 1974; Jarvis, 1974).

1.1.2.1 Lateralization of the visual system in pigeons

Lateralization of brain functions is a feature that is not exclusively found in mammals. As already mentioned, Nottebohm (1971) was able to show that the song system of chaffinches is lateralized. Around 30 years ago, lateralization of visual discrimination was described in chicken hatchlings, which exhibit a better discrimination performance with their right eye than with the left one (Mench and Andrew, 1986). Further studies in chicken hatchlings revealed that there is a transient lateralization in the thalamofugal pathway with more fibers projecting from the left GLd to right wulst than vice versa. This lateralization pattern disappears in adult chickens (Rogers and Sink, 1988; Rogers and Deng, 1999; Koshiba et al., 2003). While in the thalamofugal pathway no comparable asymmetry is present in the pigeon, either in juvenile or in adult birds, the pigeon´s visual system demonstrates a stable lateralization in the tectofugal system (Ströckens et al., 2013a). The tectofugal system of pigeons shows structural as well as functional lateralization with a dominance of the right eye/left brain hemisphere for visual object discrimination tasks (Güntürkün, 1985; Skiba et al., 2002). Additionally, on the behavioral level, for example in grain-grit discrimination or more complex tasks like differentiation between shapes and colors, the right eye exhibits significantly higher performance than the left one (Güntürkün, 1985; Güntürkün and Kesch, 1987; von Fersen and Lea, 1990; Manns and Güntürkün, 1999b). In general, the left hemisphere (right eye) is specialized for the discrimination of local features and a detailed analysis of objects while the right hemisphere relies on spatial configurations (Rashid and Andrew, 1989; Yamazaki et al., 2007). On the anatomical level, previous studies revealed morphological left-right differences in the tectofugal pathway (Güntürkün, 2002a). 8

CHAPTER 1: GENERAL INTRODUCTION

Tectofugal projections of the TO to the RT are lateralized. There are more fibers projecting from the right TO to the left RT than from the left TO to the right RT (cf. Figure 1.2 C and Figure 1.3 B; Güntürkün et al., 1993; Güntürkün et al., 1998). Anatomical studies on the tectal level show that the somata of neurons of the superficial layer 2-12 are larger in the left than in the right hemisphere. In contrast, neurons of the tectal layer 13-15 have larger somata in the right hemisphere (Figure 1.3 B; Güntürkün, 1997). Additionally, stronger bilateral innervations of the left RT correspond with larger rotundal neurons on the same side (Manns and Güntürkün, 1999b). Unilateral lesions in the left RT caused a diminished performance in discrimination tasks on the ipsi- and contralateral eye, while lesions on the right side do not affect the performance (Güntürkün and Hahmann, 1999). Other lesion experiments have demonstrated that lesions of the tractus occipitomesencephalicus (OM) in the left-hemispheric telencephalon had stronger effects on the visual system as lesions in the right hemisphere (Güntürkün and Hoferichter, 1985). Asymmetrical organization within the brain features a specific biological use, hence a distinct asymmetry enhances success in visually guided behaviors such as foraging as shown for example in better performance in grain/grit discrimination (Güntürkün, 2002a). Therefore, the tectofugal system of pigeons exhibits an ideal model for a stable lateralization on functional as well as on structural level. Whereas anatomical structure and functions of lateralization are well understood, its ontogeny remains mainly unknown. Hence, the question arises: How can these asymmetries develop? The mechanisms that affect the development of lateralization will be introduced in the next section.

1.1.3 Ontogeny of visual lateralization in pigeons

It has been well known for a long time that brain asymmetries allow the enhancement of cognitive abilities and efficiency, and these asymmetries are a favorable trait of vertebrates and even invertebrates (Rogers and Andrew, 2002; Vallortigara and Rogers, 2005; Frasnelli, 2013). Asymmetries of the brain seem to be a species-wide selective advantage and many studies had been conducted on behavioral and structural asymmetries (Toga and Thompson, 2003). But the development of asymmetries remains mainly in darkness. To answer the question of how asymmetries can develop, several factors have been discussed in the recent years: genetic (McManus, 2002), epigenetic (Previc, 1991) as well as environmental factors (Manns, 2006). Studies on the well-investigated visual system of pigeons revealed a stable 9

CHAPTER 1: GENERAL INTRODUCTION functional and structural asymmetry in the tectofugal pathway (Güntürkün, 2002a, 2005) which is triggered by the environmental factor light. The same holds true for the transient thalamofugal lateralization in chicken hatchlings (Rogers, 1982). Asymmetries in the brain of the pigeon already develop in the embryo as a result of an asymmetric light stimulation inside the egg. The right eye is turned to the translucent eggshell while the left eye is inclined into the interior of the egg and directed towards the embryo´s body (Kuo, 1932, Figure 1.3 A). Due to this asymmetrical position of the embryo, the right eye is mainly exposed to incident light, while the left eye is occluded by the embryo´s body. During breeding, the birds leave the nest for short periods and regularly turn the eggs. This behavior guarantees that the embryo can be repetitively stimulated by light (Buschmann et al., 2006). These light pulses are likely to have long-term effects, since the light-induced cellular processes can remain active even when the birds return to their nests and sit down on their eggs disrupting the influence of light (Buschmann et al., 2006). This asymmetrical light stimulation leads to the well-known behavioral and anatomical lateralization in the tectofugal pathway (Figure 1.3 B and section 1.1.2.1). In contrast, studies confirmed that in completely dark-incubated pigeon embryos no lateralization of the tectofugal system for example in discrimination tasks is detectable (Skiba et al., 2002; Güntürkün, 2005; Manns et al., 2008). The same effect is found in chicken hatchlings and the transient lateralization of the thalamofugal pathway (Rogers, 1982). A study of Lesley Rogers (1982) also revealed that a short period of light stimulation (2 hours, 400 lux) within the last days before hatch were sufficient to establish thalamofugal lateralization also in previously dark-incubated chicken hatchlings. As altricial birds pigeons exhibit a prolonged phase of developmental plasticity until post-hatch stages. By covering the right eye of the pigeon hatchling directly after hatching for 10 days, developed asymmetries can be reversed. In contrast, a deprivation of the left eye strengthens the right eye superiority (Manns and Güntürkün, 1999a). This plasticity is also found to some extent in precocial chicks allowing a turn of lateralization in the thalamofugal pathway by artificial occluding of the right eye and light stimulation of the left eye (Rogers, 1990). This indicates that a stabilization process is necessary to consolidate the asymmetries in the visual system (Manns and Güntürkün, 2009). Hence, activation and stabilization of brain asymmetries can be divided into two distinct phases: In a first step, already described in detail in the paragraph above, visual asymmetries are induced due to an asymmetrical light

CHAPTER 1: GENERAL INTRODUCTION stimulation during embryonic development. A second step occurs right after hatch, the induced asymmetries are stabilized under binocular light incidence. In this phase the ascending tectofugal pathway is influenced and stabilized by interactions of the telencephalic top-down as well as intra- and interhemispheric projections (Manns, 2006; Manns and Güntürkün, 2009).

Figure 1.3: Ontogeny of lateralization in the tectofugal system. A Position of a pigeon embryo inside the egg. Due to an asymmetrical position, the right eye is exposed to the translucent eggshell while the left eye is directed to the embryos body. B Lateralization of the tectofugal system. Projections from the right TO to the left RT are stronger than vice versa. Light blue dots indicate exemplary cells. Larger dots represent larger cells than smaller dots. Thickness of the arrows indicates projection strength (abbreviations: E = entopallium, RT = nucleus rotundus, TO = optic tectum; modified from Güntürkün and Manns, 2010). These findings described above illustrate the importance of the environmental factor light in inducing lateralization in pigeons. However, the underlying cellular mechanisms, that trigger the light-evoked lateralization cascade which shape the brain into its lateralized circuits, remain still unknown. This mechanism has to involve a photosensitive instance, which is able to process early light stimulation, and such a trigger has to fulfill several requirements: I. The possible inducer has to be photosensitive. II. The possible inducer has to be already functionally present inside the retina during the critical embryonic time frame. III. The possible inducer must be activated by wavelengths that are able to pass through the eggshell. IV. The possible inducer must be able to propagate the sensory input to visual areas of the brain to shape the visual pathway into a lateralized system.

CHAPTER 1: GENERAL INTRODUCTION

However, the classical photoreceptors, rods and cones, are not functionally developed in the crucial embryonic stage (Bagnoli et al., 1985; Rojas et al., 2007), and, therefore, have to be excluded as a trigger of visual lateralization. Hence, another photosensitive instance is needed. One promising candidate is the photosensitive molecule melanopsin. Melanopsin was recently regarded as a “third” photoreceptive system in the eye by Russel G. Foster (2009). Melanopsin will be introduced in detail in the following sections.

1.2 A THIRD CLASS OF PHOTOCEPTOR SYSTEMS – MELANOPSIN-CONTAINING INTRINSICALLY PHOTOSENSITIVE RETINAL GANGLION CELLS

Almost 90 years ago Clyde Keeler incidentally discovered mice with severe inherited retinal degeneration, phenocopying the human disease Retinitis pigmentosa (Keeler, 1927; van Gelder, 2008; Berger et al., 2010). Surprisingly, these apparently visually blind mice exhibited visual responses like pupil constriction. Keeler (1927) reasoned that “We can imagine the possibility of other forms of stimulation by light” and predicted the presence of photoreceptors in addition to rods and cones in the retina. However, for a long period of time, only two photoreceptor systems in the retina of mammals and other vertebrates were conversant: rods for the perception of light and dark and cones for the detection of colors (Bowmaker and Dartnall, 1980). In 1991 Russel G. Foster rediscovered Keeler´s observations and demonstrated that mice with a degeneration of the retina and thus neither possessed rods nor cones, show an unchanged circadian rhythm (Foster et al., 1991). In the last two decades a subset of retinal ganglion cells were identified to be photosensitive (named: intrinsically photosensitive retinal ganglion cells, short ipRGCs), which Clyde Keeler already predicted around 90 years ago. These ipRGCs were able to induce depolarization triggered by light stimulation, although the input of rods and cones was lacking in these ganglion cells (Berson et al., 2002). Therefore, the conventional view on the retina regarding photosensitive instances had to be altered. Rods and cones are not the only photosensitive cells of the eyes and the existence of third photoreceptor system was accepted (Foster, 2009). But how do these ipRGCs gain their photosensitivity?

CHAPTER 1: GENERAL INTRODUCTION

1.2.1 Discovery of melanopsin as a third photoreceptive instance inside the retina

In 1998 Provencio et al. (1998b) isolated and identified a novel photopigment called melanopsin from photosensitive dermal melanophores of Xenopus laevis. Besides melanophores, melanopsin was found in the outer lamina of the INL of the retina (Provencio et al., 1998b). Since that time, a lot of studies had been conducted on this photosensitive molecule, and melanopsin has been postulated as the responsible photopigment for the intrinsic photosensitivity of ipRGCs (Gooley et al., 2001; Hattar et al., 2002; Berson et al., 2002; Bellingham et al., 2006). Several studies of melanopsin knock-out mice showed that the loss of melanopsin resulted in a loss of photosensitivity in ipRGCs (Panda et al., 2002; Panda et al., 2003; Hankins et al., 2008). The next section introduces the photosensitive molecule melanopsin in more detail.

1.2.2 Melanopsin: diverse functions inside the retina

The investigation of melanopsin-controlled behaviors were the target of a multitude of studies. Consequently, melanopsin was discovered as a mediator of non-image forming vision (Panda et al., 2003) and as a contributor to a broad range of physiological responses (Hankins et al., 2008). Studies in mice lacking melanopsin expression revealed a diminished pupillary light reflex (Lucas, 2003) and, thereupon, melanopsin was investigated as a regulator of pupil constriction (Berson, 2003; Lucas, 2003; Hankins et al., 2008). Furthermore, the role of melanopsin in circadian entrainment was investigated with a rod/cone (-/-) knock-out mouse model which revealed normal circadian response, whereas an additional melanopsin deficiency resulted in a complete loss of photoentrainment of the circadian oscillator (Foster et al., 1991; Panda et al., 2003). Another study indicated that mice lacking melanopsin exhibited a loss in clock gene expression rhythms strengthen acceptance for the molecular clock function of melanopsin (Dkhissi-Benyahya et al., 2013). Therefore, it was assumed that circadian rhythm is maintained by these photosensitive melanopsin-containing ganglion cells, with neither rods nor cones being required for such photic entrainment, and further experiments proved this assumption (Lucas and Foster, 1999; Lucas et al., 1999). Admittedly, this fact does not exclude that rods and cones provide a contribution to the circadian rhythm. Retrograde tracing studies showed that ipRGCs are innervating the suprachiasmatic nucleus (SCN), which contains the circadian pacemaker (Moore and Lenn, 1972; Herbert, 1994; Berson

CHAPTER 1: GENERAL INTRODUCTION et al., 2002). The SCN still responded to light stimulation even if when rods and cones were blocked. These SCN innervations of fibers of ipRGCs were found in different species like rats (Hattar et al., 2002), mice (Panda et al., 2002) and chickens (Diaz et al., 2014). In general, retina and pineal gland act as circadian oscillators of biochemical and physiological processes. They are responsible for the circadian entrainment to 24 hours light and dark cycles. Melanopsin expression is found in both, the retina and pineal gland which is regulated on a circadian basis (Bailey and Cassone, 2005). Since light can modulate melanopsin expression, prolonged darkness (18 hours darkness, 6 hours light) led to a three-times higher melanopsin expression in chicken (Lima et al., 2006), and in mice the highest expression is found one hour before the light was switched on and the lowest expression was found a few hours after switching the light on (Gonzalez-Menendez et al., 2009). But no diurnal expression of melanopsin was found in ipRGCs in chicken (Chaurasia et al., 2005). Additionally, an involvement of melanopsin in light-induced sleep was investigated in melanopsin knock-out mice which showed decreased total sleep during a light pulse (Muindi et al., 2013). But in addition to circadian rhythm, this third class of photoreceptors regulates more than the body-clock (Foster, 2009). Although only studies on pupillary light reflexes and circadian entrainment existed in the past, recent studies elucidated a more extensive role of melanopsin in visual perception. These studies revealed that melanopsin-positive cells have more widely spread projections within the brain, and melanopsin seems to support spatial visual perception. Melanopsin plays a role in object localization, discrimination and contrast detection in mice (Ecker et al., 2010; Schmidt et al., 2014; Hankins and Hughes, 2014). Ecker et al. (2010) were able to demonstrate that mice lacking rods and cones (-/-) can discriminate grating stimuli from equiluminant gray und showed a measurable visual acuity. Former studies in mammals postulated that most of ipRGCs project to the SCN with much less projections to the lateral geniculate nucleus (LGN) which is origin of thalamo-cortical projections neurons in mammals (van Gelder, 2003a; Brown et al., 2010). In the last years researchers agreed that these intrinsically photosensitive retinal ganglion cells form more extensive innervations to visual brain areas than only to the SCN. Between 25-40% of all dorsal LGN (dLGN) neurons receive input from melanopsin-positive cells in mice (Brown et al., 2010; Davis et al., 2015). In macaque monkeys melanopsin-containing ipRGCs were responsible for color and irradiance detection via projections to the LGN (Dacey et al., 2005). Altogether, this leads to the assumption that melanopsin-based photoreception contributes significantly to sensory light

CHAPTER 1: GENERAL INTRODUCTION processing (Brown et al., 2010), and ipRGCs are able to detect small changes in irradiance contributing to general visual perception (Hankins and Hughes, 2014; Davis et al., 2015).

1.2.3 Two types of genes: Opn4x vs. Opn4m

At present, the melanopsin gene, commonly termed Opn4, has already been identified in several vertebrates, including the African clawed frog (Xenopus laevis; Provencio et al., 1998b), mouse (Mus musculus; Provencio et al., 2000), rat (Rattus norvegicus; Hattar et al., 2002), human (Provencio et al., 2000), zebrafish (Danio rerio; Bellingham et al., 2002), chicken (Gallus gallus; Chaurasia et al., 2005; Tomonari et al., 2005), macaque monkey (Dacey et al., 2005), ruin lizard (Podacris sicula; Frigato et al., 2006) and also in fresh water turtle

(Trachemys scripta elegans; Dearworth et al., 2011). However, the pigeon melanopsin gene sequence has not been described at this time. In the mammalian retina melanopsin-containing ipRGCs were restricted to 1-2% of all ganglion cells, while the remaining ganglion cells are mainly not photosensitive (Provencio et al., 2000; Hattar et al., 2002). In general, all melanopsin genes share greater homologies and are more common with invertebrate opsins than classical visual pigments (Hankins et al., 2008). However, melanopsin exhibits large differences between mammals and non-mammals. Analysis of melanopsin genes in different kinds of vertebrates revealed that two distinct melanopsin genes exist in the vertebrate lineage (Bellingham et al., 2006; Hankins et al., 2008): I. The mammalian-like melanopsin: Opn4m (m = mammalian) II. The invertebrate-like melanopsin: Opn4x (x = Xenopus) Both melanopsin genes evolved quite separately and are true orthologues (Bellingham et al., 2006). While mammals exclusively possess Opn4m and Opn4x is not found, either in eutherian or marsupial mammals, some non-mammalian vertebrates express both variants (m and x) which can be found on different chromosomes (e.g. chickens; Bellingham et al., 2006; Verra et al., 2011). In addition to in ipRGCs Opn4x is found in pineal gland and the medial preoptic nucleus in chicken (Chaurasia et al., 2005), and in situ hybridization revealed melanopsin expression in areas which were associated with phototransduction and visual system function, like the TO (Bailey and Cassone, 2005). In humans, melanopsin (Opn4m) can only be found in the eye (Provencio et al., 2000). In addition to differences in localization between both types of melanopsin genes, there are also large differences between melanopsin and other visual 15

CHAPTER 1: GENERAL INTRODUCTION opsins. The comparison of the conserved transmembrane domains revealed only 27% of identity of melanopsin with visual opsins. Yet, Opn4m genes among each other were remarkably identical (94-100%), and Opn4x still shows identities of 89-100% (Hankins et al., 2008). In addition to Opn4m and Opn4x both melanopsin genes possess different splice variants, which will be the focus of the next section.

1.2.3.1 Two types of melanopsin isoforms were found in Opn4x and Opn4m

In 2005 Tomonari et al. were able to demonstrate that two different transcripts of Opn4x can be found in chicken: A short (S) and a long (L) isoform. The short isoform is generated due to an 86 bp insertion in exon 8 (of 9 exons), leading to a premature STOP codon after nucleotide 1242, resulting in a shortened protein (S: 425 aa, L: 543 aa; aa = amino acid). This findings were confirmed by Verra et al. (2011) who additionally found three isoforms of the Opn4m melanopsin gene of the chicken. Two isoforms generated by alternative splicing were also known for Opn4m in mice. The short isoform of mice is 466 aa long, whereas the long isoform has 521 aa. Both isoforms differ only in the length of their C-termini. Also, in the Atlantic cod two melanopsin isoforms (S: 561 aa, L: 615 aa) were identified by Drivenes et al. (2003). Different isoforms seem to be a widespread feature of melanopsin. However, the distinct functions of these isoforms remain unclear whereas the general signaling pathway of melanopsin is conversant and will be introduced in the upcoming section.

1.2.4 Melanopsin signaling

Despite all the dissimilarities concerning the sequence and the amino acids composition, the three-dimensional structure of Opn4m and Opn4x is rather similar. As a structural homologue of other opsins, both melanopsin genes exhibit an extracellular N-terminus and seven transmembrane domains (7 TM). As already mentioned, melanopsin shows more similarities to invertebrate opsins, and also melanopsin signaling is more comparable to an invertebrate signaling pathway (Panda, 2005). Furthermore, melanopsins exhibit, like other opsins, a lysine in the seventh transmembrane domain, which acts as retinal attachment site. The chromophore is bound via a protonated Schiff base linkage (Wang et al., 1980; Peirson et al., 2009). Additionally, melanopsin is bistable, since it exhibits an intrinsic

CHAPTER 1: GENERAL INTRODUCTION photoisomerase function (Panda, 2005; Melyan et al., 2005). And the tertiary structure is stabilized by disulfide bonds in the second and third extracellular loop (Karnik et al., 1988). Melanopsin signal transduction proceeds via heterotrimeric guanine-nucleotide binding protein (G-protein)–coupled receptors that use 11-cis retinaldehyde (11-cis retinal) or a close variant as their chromophore (Graham et al., 2008; Georg et al., 2014). Classical vertebrate photoreceptors (e.g. rods and cones) signal through photoreceptor-specific pertussis toxin (PTX)-sensitive G-proteins called transducins. On the contrary invertebrate opsin signal through PTX-insensitive Gq-protein pathways. The same holds true for melanopsin which mainly activates a Gαq/Gα11-protein coupled signaling pathway (Figure 1.4 A; Panda, 2005; Hankins et al., 2008; Sekharan et al., 2012). Melanopsin is a blue-light sensitive photopigment (476-484 nm; Panda, 2005; Torii et al., 2007; Matsuyama et al., 2012) and, therefore, blue light activates melanopsin upon absorption of a photon and the subsequent conformational change due to photoconversion of the bound chromophore 11-cis retinal into all-trans retinal. This leads to the activation of the G-protein and the melanopsin-coupled

G-protein is dissociated into the Gα subunit and the βγ complex. The activated Gα subunit leads to an activation of the phospholipase C-β (PLC-β). PLC-β cleaves phosphatidylinositol-4,5- bisphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG), which contribute as second messengers in the activation of protein kinase C (PKC). PKC is capable of opening transient receptor potential (Trp) channels, which leads to an influx of sodium (Na+) and calcium ions (Ca2+), causing a depolarization of the cell (Figure 1.4 A). This is contrary to rods and cones, whose activation lead to a hyperpolarization of the cell (Panda, 2005; Hankins et al., 2008; Sekharan et al., 2012). Subsequently to the activation melanopsin is capable of photoconverting all-trans retinal back to the active form in a arrestin-dependent manner due to its intrinsic photoisomerase activity (bistability) in contrast to vertebrate opsins, which release the all-trans retinal from the opsin (Panda, 2005; Melyan et al., 2005; Cameron et al., 2014). The signals from these cells are propagated via the retinohypothalamic tract (RHT) to the SCN or LGN (in mammals), the optic tectum in birds or other retino-recipient areas in the brain (Hattar et al., 2002; Panda et al., 2002; van Gelder, 2003a; Brown et al., 2010). As already depicted in 1.2.2, melanopsin-based photoreception contributes significantly to sensory light processing via these projections.

CHAPTER 1: GENERAL INTRODUCTION

Figure 1.4: Melanopsin Gq (A)/Gi/o (B) signaling pathway. A.1/B.1 Melanopsin in absence of light. The seven transmembrane receptor melanopsin is located in the plasma membrane. The extracellular N-terminus protrudes out of the membrane, the intracellular C-terminus is directed to the cytoplasm inside the cell. The G-protein α-subunit is bound to the plasma membrane as well to the βγ subunit. The Trp-channel/GIRK channels in the plasma membrane are closed. A.2 Melanopsin-dependent Gq-coupled signaling cascade upon incidence of a light pulse. Excitation via light leads to a conformational change in the melanopsin structure. This conformational change activates the G-protein and βγ subunit dissociates. The Gα subunit activates the PLC is. Activated PLC cleaves PIP2 to IP3 and DAG. These products lead to an activation of PKC, which is responsible for opening the Trp-channels resulting in an influx of Na+ and Ca2+ which in turn results in a depolarization of the cell. B.2 Schematic overview of a possible melanopsin activated Gi/o signaling pathway upon incidence of a light pulse. Light activates melanopsin and the βγ subunit of the GPCR dissociates from the Gα subunit. The βγ subunit activates GIRK channels and the efflux of K+ leads to a hyperpolarization of the cell (abbreviations: βγ = βγ subunit of the G-protein-coupled receptor (GCPR), Ca2+ = calcium ion, DAG = diacylglycerol, Gα = Gq alpha subunit, GIRK = G-protein-coupled inwardly-rectifying potassium + + channel, IP3 = inositol triphosphate, K = potassium ion, Na = sodium ion, PIP2 = phosphatidylinositol-4,5-bisphosphate, PKC = protein kinase C, PLC = phospholipase C, Trp = transient receptor potential channel; A: modified from Hankins et al., 2008; B: modified from Riven et al., 2006; Xue et al., 2011; Stewart et al., 2012).

CHAPTER 1: GENERAL INTRODUCTION

However, it has to be noted, that recent studies demonstrated that melanopsin is also able to activate the Gi/o signaling pathway (Figure 1.4 B; Riven et al., 2006; Bailes and Lucas, 2013; Lüscher and Slesinger, 2010; Bailes and Lucas, 2013). In 2013 Bailes and Lucas demonstrated a direct Gi/o coupling due to its sensitivity to pertussis toxin in human melanopsin. In this pathway melanopsin is also activated by blue light, which leads to a photoconversion of the bound cis-retinal and a conformational change as already described above. Subsequently, the G-protein-coupled receptor (GPCR), which in this case couples specifically PTX-sensitive heterotrimeric G-proteins, is activated. The βγ subunits released from the PTX-sensitive G-proteins, bind directly to G-protein-coupled inwardly-rectifying (GIRK) potassium channel, causing them to open. This results in an efflux of potassium ions (K+) leading to a hyperpolarization of the cell (Lüscher and Slesinger, 2010). However, in general, the Gq pathway has been proven to be more efficient and common for melanopsin (Bailes and Lucas, 2013). The possible connection between the photosensitive molecule melanopsin and the ontogeny of visual asymmetries will be described in the next section.

1.3 MELANOPSIN – A POSSIBLE TRIGGER OF LATERALIZATION IN PIGEONS?

The structure and function of cerebral asymmetries among all classes of vertebrates and even invertebrates are reasonably well understood (Vallortigara and Rogers, 2005; Frasnelli, 2013). However, the ontogeny of lateralization remains mostly unknown. Previous sections discussed that lateralization of the tectofugal system in pigeons is dependent on asymmetrical light stimulation during late embryonic development, highlighting the pigeon as an ideal model system to investigate the ontogeny of lateralization. However, classical photoreceptors are not functionally developed at the crucial embryonic stage shortly before hatch (Bagnoli et al., 1985; Rojas et al., 2007). Therefore, they have to be excluded as potential mediators of light-induced lateralization. As already mentioned in 1.1.3, a possible inducer has to fulfill at least four requirements. Why is melanopsin a promising candidate as a trigger for lateralization in pigeons? A closer look at the four main requirements elucidates the promising role of melanopsin as an inducer of lateralization:

CHAPTER 1: GENERAL INTRODUCTION

I. The possible inducer has to be photosensitive. Melanopsin belongs to a third class of photoreceptors inside the retina (Provencio et al., 1998b; Berson et al., 2002; Hankins et al., 2008) provoking the intrinsic photosensitivity of this subtype of retinal ganglion cells in different vertebrates (ipRGCs; Do and Yau, 2010). II. The possible inducer has to be already functionally present inside the retina during the critical embryonic time frame. Melanopsin can already be found in the retina from early developmental stages on. Opn4x is available starting from embryonic day 8 (E8) in chicken before any sign of formal vision appears (Verra et al., 2011; Bertolesi et al., 2014; Diaz et al., 2014). Opn4m expression is continuously elevated from E4-6 to postnatal days (Verra et al., 2011). Additionally, mice express Opn4m already before birth with rods and cones being non-functional until P10. Nonetheless, juvenile mice show negative phototaxis, with melanopsin knock-out mice exhibiting no neonatal light avoidance. (Johnson et al., 2010). Altogether, this indicates that melanopsin is able cover the perception of light responses already before hatch/birth and can mediate visual information to the brain prior to the functional development of classical photoreceptors. III. The possible inducer must be activated by wavelengths that are able to pass through the eggshell. Melanopsin is a blue light-sensitive photopigment (Panda, 2005; Torii et al., 2007). This wavelength is able to pass the barrier of the eggshell. Since white eggshells are translucent to light, approximately 8% of the ambient light is able to pass through the eggshell (Rogers, 2006; Maurer et al., 2015). IV. The possible inducer must be able to propagate the sensory input to visual areas of the brain to shape the visual pathway into a lateralized system. It has been shown that ganglion cells possess functional connections to the brain before hatch in pigeons (Theiss and Güntürkün, 2001), and in chicks, retino-tectal projections are forming and arriving at laminae of the optic tectum by E6 to 8 (Crossland et al., 1975; Verra et al., 2011). First synapses of RGC axons on tectal neurons were established during embryonic development starting from E15 in pigeons (Manns and Güntürkün, 1997). Furthermore, the first expression of Opn4x

CHAPTER 1: GENERAL INTRODUCTION

coincides with the formation of retino-tectal projection fibers to the TO at beginning of E8 in chicks (Verra et al., 2011). Additionally, pigeon eggs were repetitively, although only for a short period of time, light exposed when the adult birds leave the nest (Buschmann et al., 2006). However, melanopsin shows remarkably sustained light response (Wong et al., 2005; Zhang et al., 2012; Hannibal et al., 2013) permitting long-lasting activation with only short stimulation frequencies. In summary, due to its properties melanopsin seems to be a promising candidate for the light- triggered induction of visual asymmetries in pigeons.

1.4 AIMS OF THIS STUDY

Melanopsin has been intensively studied in rodents and other vertebrates, but in this thesis a novel impact of melanopsin should be investigated. For a long time it is known that functional and structural asymmetries of the visual system of pigeons develop due to an asymmetrical light stimulation inside the egg. But the precise mechanisms which can transduce the asymmetrical light stimulation into a cascade that shapes the brain into its lateralized system remain unknown, since classical photoreceptors were not functionally developed during the critical embryonic developmental stage. Therefore, ipRGCs provide a light-sensitive system (in addition to rods and cones), which is already present inside the retina before hatch in chicken (and before birth in mice). The photosensitive molecule melanopsin renders these ganglion cells photosensitive. For that purpose, the possible influence of melanopsin on the establishment of visual asymmetries should be investigated by characterizing the pigeon melanopsin in the different studies of the thesis, since this can give deeper insights into the mechanisms that establish cerebral asymmetries. Therefore, the aims of the present thesis are the following: 1. DNA sequencing of the pigeon melanopsin gene: Melanopsin is postulated as a valid candidate of triggering functional and structural light-induced lateralization in pigeons. However, the existence of melanopsin in pigeons has not been proven yet. Therefore, the first part of this thesis deals with sequencing and analyzing the potential melanopsin gene sequence of pigeons by polymerase chain reaction (PCR).

CHAPTER 1: GENERAL INTRODUCTION

2. Qualitative analysis of melanopsin expression pattern in the pigeon retina: To fulfill all requirements of a potential lateralization inducer, the melanopsin protein has to be present inside the retina during the critical embryonic time frame of asymmetry formation. The second part of this thesis comprises the analysis of the qualitative expression pattern of melanopsin in the pigeon retina over the development by immunohistochemistry. 3. Quantitative analysis of melanopsin expression pattern in the pigeon retina: In addition to a qualitative analysis of the expression of melanopsin, the third part of this thesis constitutes a quantitative real-time polymerase chain reaction (qPCR) to investigate the melanopsin expression in more detail. Furthermore, this study also aims to decipher the expression pattern of melanopsin in dark-incubated embryos, since dark-incubated animals do not develop the asymmetries observed in normal-incubated pigeons. 4. In vitro analysis of pigeon melanopsin light responses: Melanopsin is postulated as a blue light sensitive molecule. However, light sensitivities can be modulated due to sequence and conformational changes. This part of the thesis comprises a calcium imaging study to examine if the pigeon melanopsin gene encodes a functional light-sensitive protein in vitro. 5. Electrophysiological characterization of the pigeon melanopsin: The previous studies clarified the fundamental expression pattern und light responses of pigeon melanopsin. Therefore, this study investigates the electrophysiological and photochemical properties of pigeon melanopsin in more

detail, on the basis of the on the basis of the Gi/o signaling pathway by patch-clamp measurements.

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

CHAPTER 2: SEQUENCING OF THE PIGEON MELANOPSIN GENE

Some parts of this chapter were already worked on during my master thesis. These parts include RNA extraction and cDNA transcription (2.2.3), sequencing of pigeon melanopsin (2.2.4), preliminary sequencing data (2.4.1.1) and preliminary phylogenetic analysis (2.4.1.2), all other parts comprising a deeper sequence analysis (functional and structural) were only achieved during this thesis. Master thesis analysis was revised and nomenclature was modified based on additional analyses. All figures shown in this chapter were exclusively prepared for this thesis and not presented in my master thesis before.

2.1 INTRODUCTION

Functional and structural asymmetries of the nervous system are a common feature of vertebrates and even invertebrates (Vallortigara and Rogers, 2005; Frasnelli, 2013). However, it is unknown if lateralization is mainly determined by genetic, epigenetic factors or triggered by environmental factors (genetic: McManus, 2002, epigenetic: Previc, 1991, environmental factors: Manns, 2006). There is strong evidence for an involvement of the environmental factor light in the induction of asymmetries of the visual system in pigeons (Güntürkün, 2002a; Skiba et al., 2002). A right eye/left hemispheric dominance in discrimination tasks is caused by an asymmetrical light stimulation before hatch due to an asymmetrical position of the pigeon embryo inside the egg, with the right eye being exposed to stronger light stimulation than the left one (Kuo, 1932). This lateralized stimulation induces the development of an functional lateralization based on structural asymmetries in the tectofugal system afflicting visual-guided behavior in the adult bird (Skiba et al., 2002). However, it is unclear how such light stimulation is able to induce lateralization, since classical photoreceptors which normally detect light in adult animals are not functionally expressed in the embryonic retina before hatch (Bagnoli et al., 1985; Rojas et al., 2007). In 1998 Provencio et al. identified a novel opsin in Xenopus laevis dermal melanophores belonging to the class of seven transmembrane G-protein-coupled 23

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN receptors - melanopsin. After this first discovery of an additional photoreceptor system many studies were conducted demonstrating melanopsin expression in different tissues but especially in the retina in all kind of different species (African clawed frog: Provencio et al., 1998b; Atlantic cod: Drivenes et al., 2003; chicken: Tomonari et al., 2005, Chaurasia et al., 2005; human: Provencio et al., 2000; mouse: Pires et al., 2009; zebrafish: Bellingham et al., 2002). A subset of retinal ganglion cells (RGCs) were identified as photosensitive and able to induce depolarization triggered by light stimulation, independent of an input of rods and cones (Berson, 2003). These intrinsically photosensitive RGCs achieve their photosensitivity by the molecule melanopsin. Melanopsin is a required component for light responses in these cells with a melanopsin knock-out leading to a loss of their photosensitivity (Hankins et al., 2008). These photosensitive properties prompted me to study melanopsin in the pigeon in respect transduction of visual lateralization due to asymmetrical light stimulation. However, there were no melanopsin-based studies conducted in pigeons before. Therefore, in order to examine possible melanopsin expression in pigeons, polymerase chain reaction (PCR) was performed to amplify the potential melanopsin gene of pigeons which was subsequently sequenced. Furthermore, the pigeon melanopsin structure, function and phylogenetic relations were analyzed. These data provide the first sequence of melanopsin in pigeons and a principal evidence for a possible influence on the development of visual lateralization.

2.2 MATERIAL AND METHODS

The composition of all buffers, substances and manufactures of products used in this study can be found in detail in the appendix (A. Chemicals and equipment).

2.2.1 General sequencing strategy

Melanopsin has already been identified in several bird species, e.g. in chicken (Gallus Gallus; Chaurasia et al., 2005), turkey (Meleagris gallopavo; Kang et al., 2010) and zebra finch (Taeniopygia guttata; West et al., 2007). In these species melanopsin protein is translated from at least nine exons. First, sequencing of the unknown pigeon melanopsin required amplification of the putative melanopsin gene. Therefore, a polymerase chain reaction (PCR) was established. PCR is a technique capable of producing a specific enrichment of desired DNA

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN or cDNA fragments resulting in a high (exponential increasing) copy number. Usually, PCR involves two oligonucleotide primers flanking the fragment to be amplified and repeated steps of temperature changes called cycles. A cycle includes an initial heat denaturation step of the double stranded DNA or cDNA at ~95 °C, subsequently followed by annealing of primers to their complementary sequences. The temperature of this annealing step varies for each primer pair and needs to be determined individually. In the third step, the novel strands of DNA were extended by annealed primers using a thermo-stable DNA polymerase. Extension is always executed in 5’-3’ direction and complementary to the template DNA strand. This step typically occurs at 72 °C, the optimal working temperature for the commonly used Taq DNA polymerase. Elongation times can vary with fragments length: the larger the desired fragment, the more the time is needed for elongation. Generally, extension time for 1 kb takes approximately one minute under optimum conditions for the Taq polymerase. A cycle (denaturation, annealing and elongation) is normally repeated 30 (up to 50) times leading to an exponential amplification of the desired fragment. Before starting the first cycle an initial denaturation step at 95 °C is required to denature hydrogen bonds between both DNA strands and after the last cycle a final elongation at 72 °C for 5-10 minutes is recommended (Mullis et al., 1986; Saiki et al., 1988).

Figure 2.1: Position of the selected primers on the putative pigeon melanopsin gene. A putative melanopsin sequence (e.g. chicken) is shown in this figure comprising nine exons. Green arrow heads show the position of forward primers while blue arrow heads show the position of the reverse primers. Primer pair used for part I (exon 1 to 6) was named I and the primer pair designed for the second part (part II, exon 5 to 9) was marked with II.

All primers for sequencing of pigeon melanopsin were designed according to the known sequences of close pigeon related species deposited in the BLAST (Basic Local Alignment Search Tool; Altschul et al., 1990) database (accession numbers: chicken (Gallus gallus) long isoform = EU124630.1; turkey (Meleagris gallopavo) = XM_003205485.1; zebra finch (Taeniopygia guttata) = XM_002191902.1). Due to the length of those melanopsin genes

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

(e.g. chicken long isoform is 1629 bp long; Torii et al., 2007), pigeon melanopsin was sequenced in two parts (cf. Figure 2.1): I. exon 1 to 6 II. exon 5 to 9 Both parts were estimated to measure around 800 to 1000 bp. Additionally, a special tag was added to the primers to facilitate sequencing.

2.2.2 Laboratory animals

Six pigeons (Columba livia) from local breeders were included in this experiment. Animals were housed in individual cages at a 12 hour dark-/light cycle and had free excess to food and water. Additionally, one chicken (Gallus gallus) from a local breeder was included in this experiment and directly sacrificed upon arrival in the laboratory. All experiments were performed in compliance with the guidelines of the German Animal Welfare Law for the prevention of cruelty to animals as suggested by the European Communities Council Directive of November 24, 1986 (86/609/EEC) and were approved by the animal ethics committee of the Landesamt für Natur, Umwelt und Verbraucherschutz (North Rhine-Westphalia, Germany). All efforts were made to minimize the number of animals used and maintain the highest possible standards of animal welfare.

2.2.3 RNA preparation

Sequencing of pigeon melanopsin required genetic material from the retina. Therefore, retina to extract retinal ribonucleic acid (RNA) had to be collected.

Preparation of retinal RNA All animals were deeply anesthetized with an overdose of Equitisin (0.45 ml/100 g) and decapitated. Eyeballs were dissected from the skull and the vitreous body and the lens were separated from the eye and discarded. Then, the retina was removed and stored in a sterile Eppendorf tube (Eppendorf, Germany). The tube was frozen in liquid nitrogen and stored at -80 °C until further processing. For RNA preparation retinal tissue was frozen in liquid nitrogen and homogenized. An amount of 50 mg frozen tissue was used for preparation. The RNA was prepared using the Nucleo Spin RNA II Kit (Machery & Nagel, Germany) as described

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN in the user’s manual except for the following changes: In the last step after centrifugation, the supernatant was given on the column’s membrane again and centrifuged anew with 13000 rpm (Eppendorf 5417 C, Eppendorf, Germany) for one minute at room temperature. The prepared RNA was frozen in liquid nitrogen and stored at -80 °C. All working materials and used surfaces were “decontaminated” from ribonucleases with RNaseZap® (Ambion, USA).

2.2.3.1 cDNA transcription

To identify the pigeon melanopsin gene retinal RNA (for isolation cf. 2.2.3) was transcribed into complementary DNA (cDNA) initially. The transcription of RNA into cDNA was carried out via a variant of the common polymerase chain reaction, the reverse transcription PCR (RT-PCR). In this approach the enzyme reverse transcriptase (RT) is utilized, which is able to transcribe RNA into its complementary DNA. To define the target region for transcription, two reverse primers (I and II, cf. Figure 2.1) directed against the melanopsin gene (based on the sequence of chicken, turkey and zebra finch) were designed (using Primer3; Koressaar and Remm, 2007; Untergasser et al., 2012, cf. Table 2.1 and Figure 2.1). The following standard reaction mix was used for a 20 µl reverse transcription reaction: Retinal RNA 1 µl Reverse primer 0.8 µl (8 pmol) 10 x RT Buffer 2 µl (1x) dNTPs 2 µl (0.5 mM) Murine RNase Inhibitor 0.25 µl (10 U) Sensiscript RT 0.5 µl (10 U)

PCR-grade H2O 9.45 µl All reagents were obtained from Sensiscript RT Kit (Qiagen, Germany; except for murine RNase Inhibitor, NEB, Germany), the protocol was modified to the standard reaction mix described above. The samples where incubated for 1 h at 37 °C (Thermocycler T-Professional, Biometra, Germany) and subsequently stored at 4 °C until further use.

Chicken cDNA Chicken RNA was obtained with the same procedure as described above for pigeons. For cDNA transcription the reverse primer II for melanopsin part II was used.

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

Table 2.1: Melanopsin reverse primers used for the transcription of retinal RNA into cDNA. Primers were designed according to the chicken melanopsin gene. In italic letters the sequencing tail added to the primer sequences is marked (abbreviations: A = adenine, bp = base pair, C = cytosine, G = guanine, T = thymine). Primer Primer sequence Target direction 5´ 3´ Part I R CAGGAAACAGCTATGACTCTAGAT GGGTGAATTATTGCATATATGATGG

Part II R CAGGAAACAGCTATGACTCTAGATAACTCTGTAGATGAGCTAAGCCC

2.2.4 Sequencing pigeon melanopsin

Sequencing of pigeon melanopsin first required an amplification of the putative melanopsin gene as already mentioned before (cf. 0). Therefore, a PCR was conducted. The following standard reaction mix was used for a 30 µl PCR reaction: Template cDNA 1 µl (100 ng cDNA) 5’ Primer 1.2 µl (0.4 pmol) 3’ Primer 1.2 µl (0.4 pmol) dNTPs 3 µl (0.2 nM)

MgCl2 1.2 µl (2.5 mM)

TM Biotherm without MgCl2 3 µl (1x) Taq DNA polymerase 0.5 µl (0.5 U)

PCR H2O 20.1 µl

The general PCR cycles used for cDNA templates were as follows: 1. Initialization 95 °C, 5 min 2. Denaturation 94 °C, 30 s

3. Annealing 54 °C, 45 s 31 cylces 4. Elongation 72 °C, 45 s 5. Final elongation 72 °C, 10 min 6. Final hold 4 °C, ∞ All primer sequences are found in Table 2.2 (ordered from Metabion, Germany). At least 4 µl of the PCR reaction sample was previously analyzed on a 2.5% agarose (Gibco-BRL, USA)-TBE gel via electrophoresis to ensure PCR efficiency. Prior to loading the gel, 2 µl loading buffer and 4 µl TBE were added to the sample. In addition to the PCR-DNA product, a marker (pUC19,

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

Fermentas, Germany) was applied to the gel in order to estimate the size of the amplified molecules. A voltage of 220 V/cm was applied for 30 min. The negatively charged DNA migrates to the positively charged plus terminal. The covered distance is dependent on the DNA size. Longer molecules migrate more slowly than shorter fragments. The DNA bands were visualized with ethidium bromide (1% EtBr, Sigma, Germany, in TBE) for 10 min. EtBr is a DNA-intercalating molecule which interacts with double strands. Under UV light EtBr is fluorescent, so the bands appear visible on the gel. Accordingly, the PCR product was sequenced by the sequencing service of the Human genetics department (Ruhr-University Bochum). Table 2.2: Melanopsin oligonucleotide primers used for amplification of the putative melanopsin gene in pigeons via PCR. Primers were designed according to the chicken melanopsin gene. In italic letters the sequencing tail added to the primer sequences is marked (A = adenine, bp = base pair, C = cytosine, G = guanine, T = thymine). Primer Primer sequence Target direction 5´ 3´ F GTAAAACGACGGCCAGTGAATTCTCCAGCCACATTCTGTGACCAAG Part I R CAGGAAACAGCTATGACTCTAGAT GGGTGAATTATTGCATATATGATGG

F GTAAAACGACGGCCAGTGAATTCTATTGTGTATGTCTTGTCCTGGT Part II R CAGGAAACAGCTATGACTCTAGATAACTCTGTAGATGAGCTAAGCCC

2.2.5 Further analysis of pigeon specific melanopsin insert

Sequencing pigeon melanopsin revealed a pigeon specific insert of 87 bp found in exon 8 (cf. 2.3.1.1). Since this insert was not found in any database (e.g. BLAST), it was ensured that this insert is found in general in pigeons. Therefore, a PCR with primers specifically amplifying the region around the insert was conducted (cf. Figure 2.2 and Table 2.3).

Table 2.3: Melanopsin oligonucleotide primers used for amplification of the pigeon specific melanopsin DNA insert sequence via PCR. Primers were designed according to the pigeon melanopsin sequence (abbreviations: A = adenine, bp = base pair, C = cytosine, G = guanine, T = thymine). Primer Primer sequence Target direction 5´ 3´ Pigeon F GCTGTTCCCTGCTTAAGGTTCC specific insert R GCTCTACAGGGTCAAGCTCTACATC

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

Chicken RNA (transcribed into cDNA), which usually does not exhibit this insert, was used as a negative control. PCR conditions were identical to the conditions described in 2.2.4. Amplified fragments were analyzed on a 2.5% agarose gel in TBE. A voltage of 220 V/cm was applied for 30 min. In addition to the PCR DNA sample, a marker (pUC19, Fermentas, Germany) was added to the gel in order to estimate the size of the fragments. The gel was stained with EtBr (1%, Sigma, Germany, in TBE) for 10 min. Photos of the gel were documented using Vilber Lourmat Fusion SL (PeqLab, Germany).

Figure 2.2: Position of the selected primers for analysis of the pigeon specific melanopsin insert. The pigeon specific insert (exon 8a, grey striped) is shown with the surrounding exons (7 and 8b, grey). Green arrow head shows the position of forward primer while blue arrow head show the position of the reverse primers.

2.2.6 Data analysis

2.2.6.1 Sequencing data analysis

Sequence of pigeons melanopsin gene was analyzed using SerialCloner (SerialBasics, USA) and the BLAST algorithm (Altschul et al., 1997, http://blast.ncbi.nlm.nih.gov/Blast.cgi).

2.2.6.2 Theoretical structural and functional analysis

Hydrophobicity analysis was performed using TMpred (Hofman and Stoffel, 1992; http://www.ch.embnet.org/software/TMPRED_form.html). The online tool PredictProtein (http://ppopen.informatik.tu-muenchen.de/; Yachdav et al., 2014) was used to predict functional sites of pigeon melanopsin protein isoforms. The program is able to predict secondary structure, families of related proteins, solvent accessibility and transmembrane helix prediction as well as important domains like e.g. myristoylation sites.

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

2.2.6.3 Phylogenetic analyses

To analyze the sequence of the pigeon melanopsin gene, nucleotide sequences of previously characterized melanopsin genes of other vertebrate species were used for comparison using the BLAST algorithm (Altschul et al., 1997; http://blast.ncbi.nlm.nih.gov/Blast.cgi). Accession numbers for gene and protein sequences can be found in the appendix Table A.9. A Minimum-Evolution tree (MEGA6, Tamura et al., 2011; Tamura et al., 2013) was constructed using the nucleotide sequence from the newly determined pigeon melanopsin gene sequence (isoform S and L) and melanopsin sequences reported before. ClustalX (Version 2: http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used to align the nucleotide sequences. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004) and with the Close-Neighbor- Interchange (CNI) algorithm at a search level of 1, the Minimum-Evolution tree was searched. The Neighbor-joining algorithm (Saitou and Nei, 1987) was used to generate the initial tree. The analysis involved 14 nucleotide sequences: African clawed frog (Xenopus laevis), Atlantic cod (Gadus morhua), brown rat (Rattus norvegicus), Carolina anole (Anolis carolinensis), chicken (Gallus gallus) isoform L, human (Homo sapiens), mouse (Mus musculus) isoform S and L, pigeon (Columba livia) isoform S and L, ruin lizard (Podacris sicula), turkey (Meleagris gallopavo), zebra finch (Taeniopygia guttata) and zebrafish (Danio rerio). All positions containing gaps and missing data were eliminated, and in the final dataset a total of 1264 positions were found.

2.3 RESULTS

In the following sections first the results of the DNA sequencing of melanopsin will be presented and afterwards closer analysis on the pigeon specific insert will be introduced. Finally, the results of the comparison of the pigeon melanopsin gene to other vertebrate melanopsin genes will be illustrated.

2.3.1 Identification of pigeons melanopsin gene sequence

Decoding of the pigeons melanopsin DNA sequence by PCR was successful. Pigeons melanopsin was amplified in two parts (I and II, cf. 0), and both parts, I and II, could be 31

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN sequenced. Obtained sequences were merged and closer analyzed. In summary, the whole sequence of pigeon melanopsin can be found in Figure 2.3 A (whole nucleotide sequence can be found in the supplementary data Table E.1.2a and b). In general, the melanopsin gene of pigeons is composed of nine exons, where exon 8 is subdivided according to Tomonari et al. (2005). The results also reveal that pigeon melanopsin has two isoforms, both isoforms will be discussed in detail in the next section. The length of all exons can be found in Table 2.4, however, the lengths of exons 1 and 9 are based on the open reading frame (ORF).

Figure 2.3: Illustration of the entire melanopsin gene sequence (exon 1 to 9) in pigeons without introns. A illustrates the whole melanopsin DNA sequence independent on the isoform. B depicts the short melanopsin isoform, containing an 86 bp insert comprising a premature STOP codon in exon 8c. C resembles the long melanopsin isoform missing exon 8c, which leads to the first STOP codon in exon 9, resulting in a longer protein. Large stripes depict an insert of 87 bp which has not been reported in any database related to melanopsin yet, therefore, this seems to be unique in pigeons.

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

Table 2.4: Exon length of the melanopsin DNA sequence found in pigeons. The number of melanopsin exons found in pigeons, the corresponding length in bp and additionally the special features of exon 8a (a 86 bp pigeon-specific insert) and exon 8c which is only found in the small isoform (containing a STOP codon; bp = base pair) are specified.

Exon number Length Specific feature

1 133 bp /

2 135 bp /

3 204 bp /

4 171 bp /

5 157 bp /

6 108 bp /

7 175 bp /

8a 87 bp pigeon specific insert

8b 151 bp /

8c 86 bp only in isoform S, contains a STOP codon

9 390 bp /

2.3.1.1 Key features of the two pigeons melanopsin isoforms

Tomonari et al. (2005) demonstrated that chicken melanopsin has two different isoforms. Also pigeon melanopsin exhibits two types of transcripts. On the one hand cDNA contains a 1329 bp ORF that predicts a 442 amino acid (aa) protein for a small isoform (now designated as isoform S, Opn4xS) and, on the other hand, cDNA contains a 1713 bp ORF that is translated into a 570 aa protein for the longer isoform (now designated as isoform L, Opn4xL) with the first 440 aa being identical. Sequence similarity searching (using BLAST) between both isoforms reveals that isoform S includes an additional sequence of 86 bp at position 1320 in the ORF which contains a premature STOP codon (cf. Figure 2.3 A, B and C), encoding a deduced 442 aa protein resulting in a shortened C-terminus. Therefore, this sequence insert is designated as exon 8c, while the actual exon 8 is designated as exon 8b (Figure 2.3and Table 2.1). Additionally, the pigeon melanopsin transcript comprises a special feature: an 87 bp nucleotide sequence is inserted in the ORF of both isoforms after position 1083. This finally results in a 29 aa insertion in the protein (cf. Figure 2.3 B and C). This 33

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN sequence is not found in any database (e.g. BLAST) and seems to occur only in the pigeon melanopsin DNA sequence. This sequence is analyzed in the following section in greater detail.

2.3.1.2 A pigeon unique sequence: a more detailed analysis

As already depicted above, pigeons inhibit an 87 bp nucleotide insertion. To prove that this insert occurs in all pigeons, four additional pigeons (additional to the two sequenced ones) were analyzed by PCR. The results (Figure 2.4) revealed that all pigeons tested possess this insert. Gel electrophoretic analysis of the PCR products showed a PCR fragment of 280 bp for all pigeons.

Figure 2.4: PCR analysis of pigeon specific melanopsin insert in different pigeons. The gel electrophoretic analysis revealed that all four pigeons tested (T07, T828, T63, T947) possess the 87 bp pigeon specific insert. This additional nucleotide sequence results in a protein enlarged by 29 aa. The amplified PCR product is 280 bp long. As a control chicken cDNA was tested. Chickens do not have this nucleotide insertion, but the sequence surrounding is highly conserved. Lacking of the insert leads to a shortened 193 bp PCR product. Chicken PCR fragment confirms this data. No DNA (H2O) served as a negative control (NTC = no template control) with no amplified product apparent (puc19 marker, Fermentas, Germany).

Chicken cDNA served as a control, because chickens do not exhibit this insert (Tomonari et al., 2005), but the region around this nucleotide insertion is highly conserved between chickens and pigeon. Oligonucleotide primers used were able to amplify a 193 bp fragment in chicken. The difference between chicken and pigeon PCR fragments exactly resembles the pigeon specific melanopsin insert. The negative control containing water did not show any amplification products.

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

2.3.2 Functional analysis of pigeon melanopsin

Functional analysis was first performed by a transmembrane domains (TM) prediction according to TMpred (transmembrane prediction tool; Hofman and Stoffel, 1992, http://www.ch.embnet.org/software/TMPRED_form.html). This analysis reveals typical opsin characteristics with seven transmembrane domains, an extracellular N-terminal tail and a cytoplasmic C-terminus (Figure 2.5, blue bars and shade; for more details cf. supplementary data Table E.1.1; cf. Bellingham et al., 2002; Drivenes et al., 2003; Bellingham et al., 2006).

Figure 2.5: Deduced amino acid sequence of pigeon melanopsin protein. The seven transmembrane domains (TM) predicted by transmembrane prediction tool TMpred (Hofman and Stoffel, 1992, http://www.ch.embnet.org/software/TMPRED_form.html) are indicated by blue horizontal bars and blue shaded. Green boxes indicate potential Schiff base counterions (Y and E), DRY tripeptide and the retinal attachment site K300 (K), marked in red are the additional aa found in isoform L (abbreviations: aa = amino acid, TM = transmembrane domain).

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

Table 2.5: Pigeon melanopsin protein sequence analysis (PredictProtein, PROSITE). In pigeon melanopsin different interaction sites can be found e.g. N-glycosylation sites, phosphorylation sites (cAMP- and cGMP-dependent PK phosphorylation sites, PKC phosphorylation sites and Casein kinase II phosphorylation sites). Additionally visual pigments retinal binding sites and GPCR family 1 signatures were identified as well as N-myristoylation sites. The amount and the position of these sites are indicated in this table. Bold letters mark additional sites which are only found in the long isoform. Sites which are different between both isoforms were indicated by italic letters. Protein sequence analysis was conducted using the online tool PredictProtein (http://ppopen.informatik.tu-muenchen.de/; Yachdav et al., 2014; abbreviations: cAMP = cyclic adenosine monophosphate, cGMP = cyclic guanosine monophosphate, GPRC = G-protein-coupled receptor, PK = protein kinase, PKC = protein kinase C).

Pigeon melanopsin amino acid sequence

Pattern Amino acid position Isoform S Isoform L I. 180 III. 389 I. 180 III. 389 N-glycosylation site II. 330 II. 330 IV. 480 cAMP- and cGMP- dependent PK I. 132 I. 132 phosphorylation site I. 45 VI. 333 I. 45 VI. 333 II. 130 VII. 418 II. 130 VII. 404 PKC phosphorylation site III. 213 VIII. 438 III. 213 VIII. 419 IV. 224 IV. 224 IX. 439 V. 232 V. 232 X. 469 I. 9 V. 357 I. 9 V. 357 Casein kinase II II. 213 VI. 391 II. 213 VI. 391 phosphorylation site III. 320 III. 320 VII. 419 IV. 328 IV. 328 VII. 557 I. 32 I. 32 IV. 478 N-myristoylation site II. 98 II. 98 V. 504 III. 164 III. 164 VI. 554

GPCR family 1 signature I. 104 I. 104

Visual pigments (opsins) I. 279 I. 279 retinal binding site

A lysine (K) residue is found at position 285 (corresponding to K296 of bovine rod opsin) which is responsible for chromophore (e.g. retinal) binding via Schiff base linkage (Applebury and Hargrave, 1986; Shichida and Imai, 1998; Menon et al., 2001; Bellingham et al., 2006). Additionally, a DRY motif (D = aspartic acid, R = arginine, Y = tyrosine) is located at position D115/R116/Y117 which can regulate conformational changes and activation in G- protein-coupled receptors (Bockaert and Pin, 1999; Bellingham et al., 2006; Rovati et al., 2007) as well as two potential Schiff base counterions (Y55 and E163, Y = tyrosine, E = glutamic acid) which can stabilize a protonated Schiff base were discovered (Shichida and Imai, 1998; 36

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

Terakita et al., 2000; Drivenes et al., 2003; Bellingham et al., 2006). Additionally, a protein sequence analysis was conducted using the online tool PredictProtein (http://ppopen.informatik.tu-muenchen.de/; Yachdav et al., 2014). This tool runs a PROSITE analysis. PROSITE is a method to determine the possible function of a protein by analyzing biologically important patterns and profiles (Bairoch et al., 1997). Since the pigeon melanopsin gene sequence was unknown before now, this analysis offers valuable clues about protein interactions and especially about the possible differences between both isoforms. All data can be found in Table 2.5 and supplementary data can be found in the appendix (Figure E.1.2a and b). Analysis of pigeon melanopsin (Opn4xS and L) reveals different phosphorylation sites. A cAMP- (cyclic adenosine monophosphate) and cGMP- (cyclic guanosine monophosphate) dependent protein kinase (PK) phosphorylation site and several PKC phosphorylation sites (isoform S: 8; isoform L: 10) are identified. Additionally, some Casein kinase II phosphorylation sites are determined (isoform S: 6; isoform L: 8). Three N-glycosylation sites are found in isoform S and four in isoform L. Particularly important are the N-myristoylation sites, whereof isoform S possess three and isoform L twice as much (6). Furthermore, a G-protein-coupled receptor family 1 signature (Venkatakrishnan et al., 2013) and a visual pigment (e.g. retinal) binding site are located in both isoforms (Applebury and Hargrave, 1986). The latter is equivalent to the lysine at position 285 already described.

2.3.3 Phylogenetic relationship of melanopsin in pigeons and other species

Pigeon melanopsin ORF sequences (isoform S and L) were first compared to other species melanopsins using the BLAST algorithm (Altschul et al., 1997, http://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify possible sequence similarities. Sequence similarities of the nucleotide sequences range from 66%-90% for isoform S with a query coverage (QC) varying between 35% and 93%. Data for isoform L appears quite similar with identities ranging from 66%-90% and a query coverage of 27%-95%. QC indicated the percentage of the query sequence that overlaps the subject sequence and identity demonstrates the percentage of similarity between the query and subject sequences over the length of the covered area. The expect value (E value) describes the random background noise: in a database of random sequences it would be likely to find E sequences that matched the

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN query sequence equally well. Therefore, an E value around 0 indicates that the possibility that the found similarities are produced by chance is extremely unlikely and the more “significant” is the match (complete data can be found in Table 2.6).

Table 2.6: Pigeons melanopsin gene ORF sequence compared to other melanopsin genes (BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi). Query coverage indicated the percentage of the query sequence that overlaps the subject sequence and identity shows the percentage of similarity between the query and subject sequences over the length of the coverage area. The E-value represents the significance of the results: in a database of random sequences it would be likely to find E sequences that matched the query sequence equally well. Pigeon melanopsin isoforms share large similarities with chicken and turkey, but also with zebra finch melanopsin. Additionally, similarities can be found especially in comparison to other classes like Carolina anoles, ruin lizards, African clawed frogs and Atlantic cods. In comparison to humans, mice (isoform S and L) and brown rats the query coverage as well as the identity is rather low (abbreviations: S = short, L = long).

Pigeon melanopsin nucleotide sequence

Species Isoform S Isoform L Query Query E value Identity E value Identity coverage coverage S 93% 0.0 90% 94% 0.0 90% Gallus gallus L 93% 0.0 90% 95% 0.0 90% Melagris 93% 0.0 90% 95% 0.0 90% gallopavo Taeniopygia 93% 0.0 90% 83% 0.0 90% guttata Anolis 83% 0.0 78% 76% 0.0 78% carolinensis Podacris sicula 79% 0.0 78% 62% 0.0 78%

Xenopus laevis 68% 0.0 76% 57% 0.0 76%

Danio rerio 48% 2e-93 69% 50% 2e-93 69%

Gadus morhua 65% 3e-95 69% 50% 4e-95 69%

Homo sapiens 50% 6e-35 66% 40% 8e-35 66%

S 43% 1e-31 68% 38% 1e-32 68% Mus musculus L 43% 1e-31 68% 37% 1e-32 68% Rattus 35% 4e-25 66% 27% 5e-25 66% norvegicus

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

Table 2.7: Pigeons melanopsin amino acid sequence compared to other melanopsin genes. Query coverage indicated the percentage of the query sequence that overlaps the subject sequence and identity shows the percentage of similarity between the query and subject sequences over the length of the coverage area. The E-value represents the significance of the results: in a database of random sequences it would be likely to find E sequences that matched the query sequence equally well. Also for the protein sequence pigeon melanopsin isoforms share large similarities with chicken and turkey, but also with zebra finch melanopsin. In general the protein analysis mostly reflects the nucleotide sequence data (BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi; abbreviations: S = short, L = long).

Pigeon melanopsin amino acid sequence

Species Isoform S Isoform L Query Query E value Identity E value Identity coverage coverage S 97% 0.0 86% 76% 0.0 86% Gallus gallus L 99% 0.0 85% 100% 0.0 83% Melagris 99% 0.0 86% 99% 0.0 83% gallopavo Taeniopygia 99% 0.0 85% 93% 0.0 84% guttata Anolis 98% 0.0 71% 99% 0.0 66% carolinensis Podacris sicula 99% 0.0 72% 87% 0.0 68%

Xenopus laevis 81% 0.0 73% 98% 0.0 56%

Danio rerio 75% 2e-148 62% 58% 2e-147 62%

Gadus morhua 94% 2e-163 56% 73% 7e-163 56%

Homo sapiens 76% 6e-124 51% 73% 2e-123 45%

S 78% 6e-126 78% 60% 6e-125 53% Mus musculus L 94% 1e-125 46% 60% 2e-124 53% Rattus 85% 2e-126 48% 66% 3e-125 48% norvegicus

Sequence similarity searching on the chicken (Gallus gallus) genome revealed an identity of 90% independent on the considered isoform, but query coverage ranges from 93% to 95% (QC: pigeon isoform S 93% to chicken isoform S and L, pigeon isoform L 94% to chicken isoform S, 95% to chicken isoform L). Comparison of the pigeon melanopsin gene with

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN genomes of species of different classes in public databases (BLAST) revealed in general a strong similarity to other bird species (chicken (see above), turkey: pigeon isoform S: 90%, QC 93%; isoform L: 90%, QC 95% and zebra finch: pigeon isoform S: 90%, QC 93%; isoform L: 90%, QC 83%). E values for all these comparisons are 0. The amino acid identity shared by melanopsins from different species ranged from 48% - 86% for isoform S and 45% - 86% for isoforms L when the entire sequence was compared (cf. Table 2.7). Highest similarity is found in the comparison of pigeon´s melanopsin protein to chicken and turkey melanopsin aa sequence. These results mostly resemble the sequence identity found for the ORF nucleotide sequence (cf. Table 2.6).

Figure 2.6: A Minimum-Evolution tree regarding evolutionary relationships of melanopsin. The evolutionary relationship was inferred using the Minimum Evolution method (Rzhetsky and Nei, 1994). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004). The Minimum-Evolution tree was searched using the Close-Neighbor-Interchange (CNI) algorithm (Nei and Kumar, 2001) at a search level of 1. The Neighbor-joining algorithm (Saitou and Nei, 1987) was used to generate the initial tree. The analysis involved 14 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1234 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). Closest relation is found between pigeon, chicken, turkey and zebra finch. Melanopsin genes of Carolina anoles and ruin lizards are found on the same branch. Melanopsin of humans, brown rats and mice can be found on a completely separated branch compared to the other species.

To investigate phylogenetic relationships of pigeon melanopsin a Minimum-Evolution tree was calculated using the maximum composite likelihood methods employing MEGA6 (Tamura et al., 2013). Analysis included the ORF nucleotide sequence from the newly

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN investigated pigeon melanopsin gene sequence (isoform S and L) and melanopsin sequences reported before, the results can be found in Figure 2.6. Both pigeon melanopsin sequences (S and L) are the most closely related to each other. According to the data shown in Table 2.6 and Table 2.7 pigeon melanopsin (both isoforms) show the closest grade of relationship with zebra finches. Furthermore, there is also a close relationship to the chicken and turkey sequence. Melanopsin genes of Carolina anoles and ruin lizards are also closely related to each other, demonstrating also a relationship to pigeon melanopsin. Melanopsin of zebrafish and Atlantic cods show relatively close relationships. In contrast to all species described above, melanopsin sequences of humans, mice and brown rats can be found on a completely separated branch. But mammalian genes seem to be closely related to each other. These data reflect the sequence comparison using the BLAST algorithm shown before.

2.4 DISCUSSION

The genetic sequence of melanopsin is well known in several species e.g. amphibians (African clawed frog: Provencio et al., 1998b), birds (chicken: Chaurasia et al., 2005; zebra finch: West et al., 2007; turkey: Kang et al., 2010) and also in humans (Provencio et al., 2000) but until now, the melanopsin gene of pigeons had not been explored. To allow research on a possible role of melanopsin in asymmetry induction in the visual system of pigeons, the sequence of the pigeon melanopsin gene was investigated in this study. The melanopsin gene of the pigeon was successfully deciphered and the results demonstrated at least two isoforms. One comprises a sequence insertion in exon 8 containing a premature STOP codon translating a C-terminally shortened protein. Additionally, both isoforms showed a special characteristic that seems to be unique to pigeons: a sequence insertion in exon 8 of 87 bp which is not present in any other melanopsin sequence known. Functional sequence analysis revealed seven transmembrane domains, further opsin key features and several co- and post-translational protein modification sites (e.g. N-myristoylation, phosphorylation etc.). Extensive similarities were identified in comparison to the melanopsin sequences of chicken, turkey and zebra finch.

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

2.4.1 Two different melanopsin isoforms are expressed in pigeons

The melanopsin sequence of pigeons was deciphered in this study. A 1329 bp ORF is found for the small isoform (isoform S), and a 1713 bp ORF generates the long isoform (isoform L) of melanopsin. A 442 aa protein is translated from the ORF of isoform S that contains a premature STOP codon. This STOP codon occurs due to an 86 bp insertion (exon 8c, cf. Figure 2.3) in exon 8 leading to a C-terminally shortened protein, whereas ORF of isoform L results in a 570 amino acid protein. Therefore, it is likely that alternate splicing occurs to generate two isoforms. Different isoforms are also described in other species, for example chicken (Tomonari et al., 2005; Verra et al., 2011) and mice (Hughes et al., 2012). Furthermore, the two isoforms of pigeon and chicken melanopsin share a highly identical structure, since the short isoform of chicken also contains an 86 bp insert in the smaller isoform after nucleotide 1242 (Tomonari et al., 2005). The comparison of both inserted sequences (using the BLAST algorithm, Altschul et al., 1997) shows that pigeon and chicken inserts share great similarities with an identity in 77 positions of 86 bp (90%), a QC of 100% and an E value of 3e-30. However, the premature STOP codon of chicken melanopsin appears 24 bp later than the premature STOP codon in pigeons. Furthermore, it has been reported in chicken, that there is a differential expression of two types of melanopsin during development in retina (Tomonari et al., 2005, Verra et al., 2011). Therefore, it could be possible that there is also a variable expression of both isoforms during development in pigeons. This will be the examined in more detail in a further study of this thesis (cf. Chapter 4). Additionally, it was revealed that there are at least two isoforms expressed in mice (Pires et al., 2009). A long isoform assembled of 521 aa and a shorter isoform resulting in a 466 aa protein, with the first 454 aa being identical. In mice alternate splicing of exon 9 results in a shortened exon 9 in isoform S and a STOP codon in exon 10. The long isoform has an extended exon 9 containing a STOP codon (Pires et al., 2009). This mechanism could be comparable to pigeon melanopsin, but it has to be considered that in mice (and all other mammals) a distinct melanopsin gene is expressed – Opn4m (mammalian melanopsin). This will be also discussed in more detail in the next section (cf. 2.4.2). However, the concrete functions of these different isoforms and expression pattern in general are not known yet. One of the first studies investigating the different effects of melanopsin isoform S and L in mice was conducted by Jagannath et al. (2015) very recently. They examined the influence of melanopsin (Opn4m, isoform S and L) knockdown by RNA 42

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN interference in retinal degenerate mice (lacking rods and the majority of cones). This study established that an Opn4mS knock-down resulted in a significant attenuation of pupillary light response whereas an Opn4mL knockdown reveals no effects on these responses. However, they demonstrated that Opn4mL regulates negative masking. Negative masking describes a reduction of activity in diurnal animals often observed after a decrease in illumination and in nocturnal animals after an increase in illumination (Mrosovsky, 2009). Additionally, this study demonstrated that both isoforms can contribute to circadian entrainment via SCN projections. Furthermore, a potential shift in the light sensitivity is conceivable, since recent studies discuss the generation of a variety in ipRGC light responses induced by different melanopsin isoforms for example in mice (Pires et al., 2009). Since little is known about the functional differences of melanopsin isoforms in general, the structural and thus possible functional differences of pigeon melanopsin will be discussed in the next section.

2.4.1.1 Structural and potential functional differences of Opn4xS and Opn4xL in pigeons

Both isoforms of pigeon melanopsin (Opn4xS an Opn4xL) encode a seven transmembrane domain protein and contain all the characteristic features (cf. Figure 2.5) of an opsin, like a lysine residue in the 7th transmembrane domain (Applebury and Hargrave, 1986), a DRY motif (Rovati et al., 2007, Bockaert and Pin, 1999) and two potential Schiff base counterions (Drivenes et al., 2003, Bellingham et al., 2006). These findings are in concordance with various studies in fish (Drivenes et al., 2003), chicken (Bellingham et al., 2006), mice (Pires et al., 2009) and turtles (Dearworth et al., 2011) all confirming the occurrence of these specific motifs in different melanopsin genes. Nonetheless, both isoforms differ only in the lengths of their C-terminal tails. The opsin-specific elements (7 transmembrane domains, DRY motif etc.) are located in the first part of the protein up to amino acid 300, therefore, these features do not vary between both isoforms. However, the C-terminus comprises a lot of co- and posttranslational modification sites that can influence structure as well as function of the translated protein. There are N-glycosylation sites, different phosphorylation sites (e.g. PKC phosphorylation sites), N-myristoylation sites and a GPCR family 1 signature site as well as a visual pigment retinal binding site (cf. Table 2.5). Due to the prolonged C-terminus isoform L comprises one additional N-glycosylation site, two more PKC phosphorylation sites, two more Casein kinase II phosphorylation sites and three additional N-myristoylation sites. Isoform S contains four 43

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

N-glycosylation sites in and isoform L one additional site. This co-translational protein modifications are important for both, structure and function of eukaryotic proteins (Imperiali and O'Connor, 1999). Then, both isoforms exhibit several phosphorylation sites, but they only differ in the number of PKC phosphorylation sites and Casein kinase II phosphorylation sites, two more of each are found in Opn4xL. Only one cAMP- and cGMP-dependent protein kinase phosphorylation site is found in both isoforms. These phosphorylation sites can alter the structural conformation by post-translational protein modification via the addition of a phosphate group (Glass et al., 1986, Casteel et al., 2010, Johnson, 2009) and, therefore, four additional phosphorylation sites can influence structure and function of isoform L considerably. Isoform L carries around 21% more phosphorylation sites in the prolonged C-terminal region. Most importantly, twice as much N-myristoylation sites are found in Opn4xL. Myristoylation is an important co-translational modification and plays an essential role in mediating reversible interactions with the cell membrane and other proteins. In addition protein targeting and function can be regulated (Farazi et al., 2001). In particular, the influence on transportation and cell localization might play a crucial role in melanopsin membrane trafficking. Since melanopsin is a seven transmembrane domain protein, it has to be ensured that the protein is transported to the cell membrane to enable its function as a light receptor. Therefore, it is conceivable that isoform L might be more efficient transported to the cell membrane than isoform S. This hypothesis will be closer examined in another study of this thesis (cf. Chapter 5).

2.4.2 Melanopsin evolution – melanopsin is highly conserved within the Aves

The newly identified melanopsin nucleotide sequence of pigeons exhibits very high identity with chicken (90%, QC: 93-95%) and turkey (90%, QC: 93-95%) and zebra finch (90%, QC: 83-93%), while the level of homology is lower compared to the other species. However, considerable levels of conservation compared to reptiles (78%, QC: 62-83%), amphibians (76%, QC: 57-68%) and fish (69%, QC: 48-65%) were evident. Nevertheless, a comparison to mammals revealed much lower concordance with only 66% with a query coverage ranging between 27 and 50% (cf. Table 2.6). Therefore, it seems that the sequence of pigeon melanopsin is to some extend conserved in the fish, amphibian, reptile and bird class with strong disparities to mammals. Protein sequence comparison revealed similar results (Table 2.7) with large identities to birds (83-86%, QC: 76-99%) and less similarities to reptiles 44

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN

(66-72%, QC: 87-99%), amphibians (56-73%, QC: 81-98%), fish (56-62%, QC: 58-94%) and fewest to mammals (45-78%, QC: 60-94%). According to the data shown in Table 2.6 and Table 2.7 pigeon melanopsin (both isoforms) shows a high grade of relationship with chicken, turkey and zebra finch melanopsin genes with all four species belonging to the Aves and more precisely to the Neognathae clade (Hackett et al., 2008), whereat chicken and turkey are members of the Galliformes order, pigeon belong to the Columbiformes and zebra finch to the Passeriformes (Hackett et al., 2008). However, the relation between Passeriformes (zebra finch) and Columbiformes (pigeon) is closer, since both belong to the Neoaves superorder. Chicken and turkey on the contrary belong to the superorder Galloanserae, with both superorders having evolved from the Neognathae (Hackett et al., 2008). Phylogenetic analysis of melanopsin nucleotide sequences (cf. Figure 2.6) is in full agreement with sequence comparisons for the avian class as described above. Pigeon melanopsin shares the highest grade of phylogenetic homology with zebra finch, their closest relatives (of the species compared) with both belonging to the Neoaves. Turkey and chicken (both belonging to the Galloanserae) melanopsin sequences are phylogenetically closest related. Both Galloanserae species, turkey and chicken, were found on the same branch and show the highest homology. Therefore, it seems likely that phylogenetic conserved features of the melanopsin sequence are found within the Neognathae clade. Figure 2.7 shows the phylogenetic tree of the vertebrate subphylum (Maddison and Schulz, 2007; Ocklenburg et al., 2013; Amemiya et al., 2013) for comparison with melanopsin gene phylogenetic tree (cf. Figure 2.6). The vertebrates are divided in several classes (Mammalia, Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes). The melanopsin genes of Carolina anoles and ruin lizards are found on the same branch on the Minimum-Evolution tree, resembling the Reptilia branch found in Figure 2.7. These melanopsin sequences show the closest phylogenetic familiarity to the Aves as expected from the evolution of the vertebrate subphylum. The relationship of zebrafishes and Atlantic cods, which belong to the Actinopterygii a subgroup of bony fishes (Osteichthyes), to Amphibia and Aves is also reflected by the phylogenetic melanopsin relationship. Though, mammals evolved from the same Amniote stem like reptiles and birds, with a Sauropsid branch forming the ancestors of all extant birds and reptiles and a Therapsid branch forming the ancestors of all extant mammals (Jarvis, 2009). However, the phylogenetic position of human, mouse and rat

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN melanopsin (all from the mammalian class) within the Minimum-Evolution tree does not resemble this relationship at all (cf. Figure 2.6 and Figure 2.7). The analysis revealed a position of mammalian melanopsin even outside the Amniote clade suggesting a completely independent gene. The position mammalian melanopsin genes within the Minimum-Evolution tree is in agreement with an older analysis of Drivenes et al. (2003) and Frigato et al. (2006) strengthening the reliability of the current analysis. This extraordinary position is explained by the two distinct melanopsin genes described before as Opn4m (mammalian melanopsin) and Opn4x (non-mammalian/invertebrate-like melanopsin). On the one hand, Drivenes et al. (2003) suggested a hypothesis for the extraordinary position of mammals: The position of mammalian melanopsin could be interpreted with an ancient melanopsin gene. This ancient gene was duplicated into two paralogous genes which adjacent had acquired different characteristics. Even some non-mammalian vertebrates seem to exhibit a second melanopsin gene: Opn4m e.g. chicken and zebrafish (Bellingham et al., 2006).

Figure 2.7: Phylogenetic tree of the Vertebrata subphylum. The tree shows the evolutionary context of vertebrates. Extinct species and Agnathae are not shown (modified from Maddison and Schulz, 2007; Ocklenburg et al., 2013; Amemiya et al., 2013).

On the other hand, it was suggested that these results indicate that the Opn4x gene was lost as a result of chromosomal reorganization relatively early in mammalian evolution, since Opn4x is not found in any eutherian or marsupial mammals (Bellingham et al., 2006; Hankins et al., 2008). However, this strengthens the hypothesis that the melanopsin gene we identified in pigeons is the Opn4x gene belonging to the non-mammalian melanopsin genes and is in accordance with the data from BLAST. This is not excluding that there might be an Opn4m gene in pigeons as well. However, this explains the discrepancy between mammalian 46

CHAPTER 2: SEQUENCING OF PIGEON MELANOPSIN and non-mammalian melanopsin found in the phylogenetic tree of melanopsin sequences. However, BLAST similarity search suggests at least some identities between mammalian and non-mammalian melanopsin genes. Especially the “core” region (until amino acid 357 of pigeon melanopsin and mouse melanopsin isoform L) show a higher grade of concordance than the C-termini. Cytoplasmic C-terminal regions reveal no significant identities, reflecting the differences between both genes (supplementary data Figure E.1.1), whereas the N-terminal region including the seven transmembrane domains shows more similarities. Particularly the opsin characteristics already described for pigeon melanopsin, e.g. a DRY motif and Schiff base counterions (Y and E) are found in mouse melanopsin as well (Bellingham et al., 2006; Pires et al., 2009). Hence, the presence of these motifs and the seven transmembrane structure build the basis of the G-protein-coupled receptor and opsin structure.

2.4.3 Summary and conclusion

Two isoforms of the pigeon melanopsin nucleotide sequence could be identified in this study, resulting in two proteins which only differ in length of the C-termini. However, protein analysis reveals that most of the important motifs for opsins are located in the frame up to amino acid 300, whereas essential motifs for protein structure and transport, like the N-myristoylation sites, are comprised in the C-terminal tail. This might result in differential membrane targeting of the long isoform. Phylogenetic analyses strengthen the assumption of the pigeon melanopsin identified belonging to the non-mammalian melanopsin genes. Taken together, this study confirmed for the first time, the presence of the light-sensitive photopigment melanopsin in pigeons as well as valuable insights into its structure, delivering a crucial basis for the experiments described below.

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

CHAPTER 3: IS MELANOPSIN EXPRESSED IN ALL DEVELOPMENTAL STAGES IN PIGEONS? – A qualitative analysis of melanopsin expression pattern in the pigeon retina

3.1 INTRODUCTION

The first study of this thesis confirmed the occurrence of a melanopsin gene in pigeons (Chapter 2). This leads to the question of whether the previously sequenced photopigment melanopsin is also found in the pigeon retina across all developmental stages and, therefore, may be a possible inducer of visual lateralization in pigeon embryos. Immunohistochemically stainings of melanopsin in embryonic, post-hatch and adult pigeons should be performed in this study to identify the expression pattern of melanopsin and to prove the role of melanopsin during lateralization in pigeons. In addition, tectal tracing should be executed to determine a possible expression of melanopsin in retinal ganglion cells.

3.2 MATERIAL AND METHODS

The composition of all buffers, substances and manufactures of products used in this study can be found in detail in the appendix (A. Chemicals and equipment).

3.2.1 Laboratory animals

Two adult pigeons (Columba livia) of unknown sex were used in this study. Animals were obtained from local breeders. All pigeons were housed in individual cages with a 12 hour dark-/light cycle and free excess to food and water. Embryos and hatchlings were bred by breeding pairs housed in our lab and hatched by their parents. All experiments were performed in compliance with the guidelines of the German Animal Welfare Law for the prevention of cruelty to animals as suggested by the European Communities Council Directive of November 24, 1986 (86/609/EEC) and were approved by the animal ethics committee of 48

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS the Landesamt für Natur, Umwelt und Verbraucherschutz (North Rhine-Westphalia, Germany). All efforts were made to minimize the number of animals used and maintain the highest possible standards of animal welfare.

3.2.2 Retinal tissue preparation for immunohistochemistry - adult animals

Preserving tissue in a “life-like” state is essential for immunohistochemically staining (Gage et al., 2012). Posthumously endogenous enzymes induce autolysis of tissue, cells and proteins (Srinivasan et al., 2002). Hence, transcardial perfusion was performed to fixate the retina of adult animals. For optimal fixation the blood of the whole system is initially exchanged with a physiological saline solution and subsequently rinsed thoroughly with a fixative (e.g. paraformaldehyde).

Transcardial perfusion Prior to transcardial perfusion, animals were incubated in complete darkness for one hour. This dark-incubation allows better dissection of the retinal pigment epithelium, since the outer segments of rods are then retracted from the retinal pigment epithelium (Burnside and Nagle, 1983). Afterwards, also in darkness, Heparin was injected to prevent vascular obliteration during perfusion (1000 Units Heparin, Ratiopharm, Germany; 0.1 ml Heparin and 0.1 ml physiological saline solution, NaCl). After 15 minutes, the animal was deeply anesthetized with an overdose of Equithesin (0.45 ml/100 g body weight). Before starting perfusion, the pigeon had to be fully anaesthetized, which means that it had to be non-responsive to all physical stimulations. The perfusion procedure started with an incision through the thorax to expose the heart. Subsequently, the pericardium was removed without injuring the heart. Once the heart is laid open, it can be punctured, and the cannula was placed into the posterior end of the left atrium. An additional incision was made at the tip of the right ventricle to allow the outflow of solutions that had already passed the vascular system. First, the system was perfused with 0.9% saline solution (pre-warmed to 37 °C, perfusion pressure 2.0, approximately a rate of about 50 ml/minute) until only clear saline was rinsing out the right ventricle. Immediately afterwards the system was perfused with ice-cold 4% paraformaldehyde (PFA) to fix tissue and proteins. Additionally, PFA was injected into the eyes without injuring the retina. Afterwards the animal was decapitated and eyeballs were dissected from the skull. The vitreous body and the lens were separated from the eye and 49

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS discarded. Then the retina was removed carefully and post-fixed in 4% PFA containing 30% sucrose at 4 °C for 2 h. Subsequently the retina was incubated in 30% sucrose in 0.12 M phosphate-buffered saline (PBS) at 4 °C overnight for cryoprotection. Retina was stored at 4 °C until further processing.

Retina bleaching Bleaching of retinal tissue allows a better dissection of the RPE covering rods and cones. The retina was incubated in a bleaching solution containing 1.54 mM NaCl, 12% hydrogen peroxide (H2O2) and some drops of ammonium hydroxide (NH4OH) for 20 min and subsequently rinsed two times with PBS (approx. 30 min). RPE was carefully dissected from the retina. The prepared retina was immersed with Tissue-Tek® (Jung, Germany) and frozen in -37 °C cold isopentane. The retina was stored at -80 °C until further processing.

Sections Sections of 12 µm thickness were cut on a cryostat (Cryostat Leica CM3050 S, Leica, Germany), mounted on microscopy-slides (Menzel-Gläser, Thermo Scientific, Germany) and frozen at -20 °C until further processing.

3.2.3 Retinal tissue preparation for immunohistochemistry - embryos and hatchlings

Instead of a transcardial perfusion embryonic and post-hatch tissues were fixed using an immersion fixation.

Embryonic tissue At embryonic day 16 (E16) the eggs were removed from the parents and the eggshell was carefully opened. The embryo was slowly pulled of the egg and directly decapitated with a scissor. Eyes were dissected, perforated with a scalpel and immersion-fixated in 4% PFA for 24 h at 4 °C. Afterwards the lens and the virtuous body were removed and the prepared retina was again incubated 90 min in 4% PFA, followed by an overnight incubation at 4 °C in 30% sucrose (in PBS). Like retina from adult pigeons, embryonic retinae were embedded in Tissue-Tek®, frozen in -37 °C cold isopentane and stored at -80 °C.

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Post-hatch tissue At post-hatch day 2 hatchlings (PH2) were removed from the nest and immediately decapitated with scissors. Further processing was equivalent to the embryonic tissue described in the section above.

Sections Sections of 16 µm thickness were cut on a cryostat (Cryostat Leica CM3050 S, Leica, Germany), mounted on microscopy-slides (Menzel-Gläser, Thermo Scientific, Germany) and frozen at -20 °C until further processing.

3.2.4 Retinal immunohistochemistry of melanopsin over three developmental stages

Immunohistochemistry serves as a useful method to visualize protein expression patterns via a specific antibody protein interaction. In this case 3,3'-Diaminobenzidine (DAB) was used as a staining reagent. Initially, a primary antibody directed against an amino acid sequence of a certain protein (here: melanopsin long isoform) is applied to the tissue, binding its specific antigen. A biotinylated secondary antibody is directed against the primary antibody (here: primary antibody from rabbit, secondary antibody has to be an anti-rabbit antibody). The biotin anchor of the secondary antibody is capable of binding an avidin molecule. This avidin molecule is already bound to three of four possible biotin molecules which are in turn coupled with horseradish peroxidase (HRP). Binding of avidin and biotin is highly specific and extremely stable (Livnah et al., 1993). HRP catalyzes the reaction of hydrogen peroxide and DAB to a dark brown colorant (Adams, 1981). The hydrogen peroxide results from the reaction of glucose oxidase and glucose, this guarantees a continuous low concentration of hydrogen peroxide and allows a slower and more specific staining reaction in comparison to the direct addition of hydrogen peroxide. To enhance the staining intensity, cobalt and nickel are added to the reaction (Shu et al., 1988).

Experimental procedure

Initially, tissue specimens (3.2.2 and 3.2.3) were pretreated in 0.3% H2O2 (J.T. Baker, Netherlands) for 30 min to saturate all endogenous peroxidases. Then slides were incubated in 10% normal donkey serum in PBS containing 0.1% Triton-X (PBS-T) to block unspecific binding sites. After a washing step in PBS for 5 min, slices were incubated 72 h at 4 °C with the 51

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS primary rabbit anti-melanopsin antibody (α-melanopsin2 from rabbit; immunopurified, Bethyl Laboratories, USA; a gift from Ana M. L. Castrucci, sequence: LSSSTEFLEAVEKFLS; 1:750 in 0.3% PBS-T, containing 1% normal donkey serum). On the next day slides were rinsed for 10 min in PBS three times to remove all unbound antibodies. Subsequently, slides were incubated with a secondary α-rabbit antibody (biotinylated from α-rabbit VECTASTAIN® Elite-ABC-Kit, Vector Laboratories, USA, diluted 1:200 in PBS-T) for one hour at room temperature, followed by three 10 min washing steps in PBS to remove unbound secondary antibody. Then slices were incubated in VECTASTAIN® ABC reagent (reagent A and reagent B, in a 1:100 dilution in PBS-T, pre-incubation time: 30 min). Afterwards slides were washed two times in PBS for 10 min and subsequently incubated for 10 min in 0.1 M sodium acetate buffer. Then slides were pre- incubated in DAB reaction solution. DAB reaction solution contained a combination of three compounds: compound A (5 g ammonium nickel sulfate and 800 mg ß-D-glucose in 100 ml 0.2 M sodium acetate buffer), B (2 DAB pellets (Sigma, USA) in 80 ml in Aqua dest.) and C (80 mg ammonium chloride and 80 mg Cobalt (II)-Chloride + 6 H2O in 20 ml Aqua dest.). The ratio between A, B and C was 5:4:1. To start the reaction 120 µl of Glucose oxidase (1 mg/ml in Aqua dest., Sigma-Aldrich, Germany) was added to the DAB reaction solution. Slides were incubated three times in this solution for 5 min each (renewing the solution every 5 min). The reaction was stopped in 0.1 M sodium acetate buffer. Finally, slides were washed three times 10 min in PBS and stored at 4 °C in PBS overnight. On the net day slides were dehydrated in an ethanol series (70%, 80%, 90%, 100%, 100% ethanol and 2-times Xylol) and mounted in DPX Mountant (Sigma-Aldrich, Germany).

3.2.5 Tectal tracing

Tectal tracing allows the tracking of fiber projections between different structures of the central nervous system (Deller et al., 2000). To reveal whether possible melanopsin-containing cells project to the optic tectum, adult pigeons were injected with the retrograde tracer choleratoxin subunit B (CTB; Luppi et al., 1990).

3.2.5.1 Tracer injection

Surgery was conducted under semi aseptic conditions. Tracer was applied into the optic tectum as follows: Pigeons were deeply anesthetized with an intramuscular injection of

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

Ketamine/Xylazine (Ketavet/Ketamine: 100 mg/ml, Zoetis, Germany; Rompun/Xylazine: 20 mg/ml, Bayer Healthcare, Germany), both pharmaceuticals were mixed in a 7:3 ratio (0.12 ml/100 g body weight). Feathers of the scalp and around the ears were sheared and the pigeon was placed laterally in a stereotaxic frame (according to Karten and Hodos, 1967). To control the body temperature an electric heating pad was placed under the pigeon´s body (around 41 °C). The scalp was opened with a sagittal incision along the left lateral skull. For injection of the tracer into the left TO a small craniotomy was performed using a microdrill. The TO was localized with the help of stereotactic coordinates from the pigeon brain atlas (Halpern et al., 1968). The tracer CTB (recombinant, Sigma, Germany, 1% in deionized water) was injected through a glass micropipette (inner tip diameter 15–20 nm, custom-made) with a mechanic pressure device (WPI Nanoliterinjector 2000, World Precision Instruments, Germany). A total of five injections sites were equally distributed over the whole left TO. Per injection site around 100 nl of tracer were applied (11 injections with a volume of 9.2 nl each, 1 min interval, in a depth of 1 mm). The micropipette remained in place for about 5 min to ensure tracer diffusion into the brain tissue. After all five injections skin was sutured with surgical yarn. After surgery pigeon received an intramuscular injection of 0.6 ml Rimadyl (Zoetis, Germany; 10 mg/ml) two times per day for analgesia. Tracer injection was followed by a survival time of the animal of five days.

3.2.5.2 Tissue preparation

Consequently to five days of survival time the pigeons were perfused with the same procedure described in 3.2.2. The brain and the right retina were removed post-fixed for 2 hours in PFA with a supplement of 30% sucrose. Subsequently, the brain and retina were incubated in a solution of 30% sucrose in PBS overnight for cryoprotection. The retina was treated like described in 3.2.2. Before slicing, brain tissue had to be embedded in gelatine (30% sucrose and 15% gelatine in PBS, neutral red). Following to embedding, brain was again post fixed in 4% PFA with 15% sucrose overnight at 4 °C and afterwards stored in 15% sucrose in PBS at 4 °C overnight for cryoprotection. Subsequently, brain was cut in coronal sections (30 µm) on a freezing microtome (Leica Microsystems, Germany, slice). Brain was sliced in 10 series, collecting every tenth slide for one series. Slices were stored in PBS containing 0.1% sodium azide at 4 °C.

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

3.2.5.3 Immunohistochemistry of brain tissue

The injection site of the tracer had to be validated to ensure that the injections were in the right targeted area and that enough tracer was applied. For the examination of the injection sites an immunohistochemically staining was performed. The general protocol was identical to the protocol described in 3.2.4 except for the following changes: Brain slices were not mounted on microscopy slides, therefore, immunohistochemically staining was performed free-floating. For the detection of the injected tracer CTB a goat anti-CTB antibody (1:5000, 72 h, Calbiochem, Germany; Cat no. 227040) was used as primary antibody. In addition slices were incubated in normal rabbit serum to block unspecific binding sites. A rabbit anti-goat biotinylated antibody (biotinylated from α-rabbit VECTASTAIN® Elite-ABC-Kit, Vector Laboratories, USA, diluted 1:200 in PBS-T) was used as secondary antibody. DAB reaction also contained only little changes, including a prolonged incubation time in DAB reaction solution for 10 min and a reduced Glucose oxidase concentration to 100 µl (1 mg/ml in Aqua dest., Sigma-Aldrich, Germany) per reaction volume. Afterwards slices were mounted on microscopy-slides (Menzel-Gläser, Thermo Scientific, Germany) in mounting solution (0.12 M PBS containing 10% absolute ethanol and 0.1% gelatine), air-dried and dehydrated in an ethanol series (70%, 80%, 90%, 100%, 100% ethanol and 2-times Xylol) and mounted in DPX Mountant (Sigma-Aldrich, Germany).

3.2.5.4 Double labeling of CTB and melanopsin

Melanopsin and CTB were visualized by using a fluorescent immunohistochemically staining. After 10 min pre-treatment in PBS, unspecific binding sites were blocked by 1 h incubation in 10% normal donkey serum in 0.3% PBS-T. A 5 min washing step in PBS was followed by 86 h incubation in both primary antibodies simultaneously (antibodies and concentration cf. Table 3.1). Unbound primary antibody was removed by washing three times for 10 min in PBS. Afterwards slides were incubated fluorescence-conjugated secondary antibody (antibodies and concentration cf. Table 3.1) for one hour at room temperature, followed by three washing steps in PBS (10 min each). Slides were mounted in DAPI Fluoromount-G® (SouthernBiotech, USA) and stored in darkness.

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

Table 3.1: Antibodies used for fluorescent immunohistochemically staining. *in 0.3% PBS-T containing 1% normal donkey serum; +in 0.3% PBS-T Antibody Antibody type Antigen Concentration Manufacturer rabbit anti- Bethyl primary melanopsin 1:750* melanopsin2 Laboratories, USA Calbiochem, goat anti-CTB primary CTB 1:1000* Germany anti-rabbit Alexa secondary rabbit 1:500+ Invitrogen, USA 488 anti-goat Alexa secondary goat 1:500+ Invitrogen, USA 594

3.2.6 Data analysis

DAB stained retinal and brain sections were analyzed using the AXIO Imager M1 microscopes (Zeiss, Germany). Pictures of relevant areas were photographed using the camera AxioCam MRm (Zeiss, Germany) installed on the microscope. Fluorescence labelled retinal slides were analyzed with LSM510 Meta confocal laser scanning microscope (Zeiss, Germany) in the laboratory of Prof. Dr. Carsten Theiss and analyzed using LSM Image Browser (Zeiss, Germany).

3.3 RESULTS

3.3.1 Melanopsin immunohistochemistry over three developmental stages

Immunohistochemically stainings using an anti-melanopsin antibody recognizing the long isoform demonstrated that melanopsin is expressed in the retina from an early developmental stage onwards (Figure 3.1). In adult pigeons, melanopsin is expressed in the ganglion cell layer (GCL, Figure 3.1 A and D). In post-hatch (Figure 3.1 B and E) and embryonic (Figure 3.1 C and F) retinae melanopsin-positive cells can be found in the developing ganglion cell layer confirming the expression of melanopsin already in early developmental stages. Due to the developmental stages the cytoarchitecture differs from adult animals showing a more diffuse and scattered labeling in the developing GCL. The distribution of melanopsin-positive cells over the GCL in general seems to be uniform over the retina. In total a large number of cells in the GCL are stained, though some unstained cells are found distributed across the retina. In all other retinal layers no labelling can be detected at least for sporadic stained cells 55

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS in the INL in adults and the developing INL in hatchlings and embryos. Control stainings without primary antibody over all developmental stages (Figure 3.1 G, H, I) showed no labelling. In A and D the retinal pigment epithelium was removed, in all other cases the RPE shows its naturally black color.

Figure 3.1: Immunolabeling of the pigeon retina with an anti-melanopsin antibody. A High levels of melanopsin are found in the ganglion cell layer of adult pigeons. B and C Staining of melanopsin in post hatch (PH2, B) and embryonic (E16, C) retinae revealed labeling of the developing ganglion cell layer. D-F Higher magnification of melanopsin labelled cells in adult animals (D), hatchlings (E) and embryos (F). G, H and I Negative control without primary antibody prevented staining in adult animals (G), hatchlings (H) and embryos (I). Black labelling covering the (developing) photoreceptors in B, C, E, F, G, H and I is caused by the retinal pigment epithelium which was not removed in these animals and has naturally a black color (scale bars: 50 µm; abbreviations: GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, PRL: photoreceptor layer).

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

3.3.2 Tectal tracing

Tectal injections of CTB and subsequent fluorescent immunohistochemically staining label a large number of cells in the GCL (Figure 3.2 A, red) in adult pigeons. CTB is found in the whole cell bodies sparing the nucleus.

Figure 3.2: Fluorescent immunolabeling of the pigeon’s retina according to tectal CTB injections. A Immunohistochemically staining against the tracer CTB results in strongly labelled ganglion cells. B Immunohistochemically staining of the pigeon retina with an anti-melanopsin antibody results in labelled cells in the ganglion cell layer. C Overlay of A and B showing a co-localization of CTB (red) and melanopsin (green) in the retinal ganglion cells. All retinal slices are counterstained with DAPI (blue) D Exemplary injections sites of CTB in the TO. White arrows indicate double labelled cells in the retina, black arrows indicate exemplary three of the injections sites into the TO (scale bars: A-C 25 µm, D 1000 µm; abbreviations: GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, PRL: photoreceptor layer, TO: optic tectum).

Fluorescent melanopsin staining reveals the same staining pattern already found in Figure 3.1 (A, D) with a large number of cells being stained in ganglion cell layer (Figure 3.2 B, 57

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS green). Furthermore, fluorescent labeling shows more accurate the distribution of melanopsin in the cells. No melanopsin staining is found in the cell core, most intensive staining is found in the cell membrane and the cytoplasm, since a DAPI counterstain (Figure 3.2 A, B, C, blue) allows visualization of the cell core and evaluation of the staining localization. Some unspecific green fluorescence is found in the OPL, but this labeling is not co-localized with nuclei. Double labeling of CTB and melanopsin reveals co-localization of both (Figure 3.2 C) in the ganglion cell layer. However, not every CTB-positive cell was melanopsin-positive and vice versa. Control staining of CTB in the TO validates positions of the injection sites (Figure 3.2 D). No spreading of the tracer in other retino-recipient brain regions was found.

3.4 DISCUSSION

The results of this study give insight for the first time into the protein expression pattern of melanopsin in the retina of pigeons. Melanopsin isoform L is found in a large number of cells in the ganglion cell layer labelled by immunohistochemically staining in adult pigeons. In hatchlings (PH2) and pigeon embryos (E16) the majority of cells in the developing ganglion cell layer were demonstrated to be melanopsin-positive, confirming the appearance of melanopsin already in early developmental stages. Furthermore, retrograde tracer injections into the TO identified the melanopsin-positive cells in the ganglion cell layer as retinal ganglion cells proving that at least a subpopulation of RGCs projects to this primary visual area in adult pigeon. The existence of melanopsin-containing presumably retinal ganglion cells in the crucial developmental stage (E16) strongly supports the hypothesis that melanopsin could be the initial transducer of light-mediated visual lateralization in pigeons. However, it has to be mentioned that the antibody used only detects the long melanopsin isoform due to the reason that no suitable antibody was available detecting either the short or both isoforms in pigeons. Therefore, all conclusions drawn from this study include only the long isoform of pigeon melanopsin (Opn4xL). Hence, it is conceivable that there are even more melanopsin-positive cells expressing isoform S in the retina.

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

3.4.1 Melanopsin is mainly expressed in retinal ganglion cells in pigeons

Immunohistochemically staining of melanopsin isoform L in retinae of adult pigeons revealed a large number of cells labelled in the ganglion cell layer, though some unstained cells are found consistently spread over the retina. Injections of the retrograde tracer CTB into the TO resulted in a large number of stained cells in the ganglion cell layer. A large number of cells labelled with CTB were also melanopsin-positive proving co-localization of tracer and melanopsin. This indicates that melanopsin is mainly expressed in RGCs that project to the TO (Güntürkün, 2000). The TO is the largest primary visual area in the pigeons visual system with 75-95% of all RGCs projecting via this pathway (Remy and Güntürkün, 1991; Güntürkün, 2000). The tectofugal pathway represents the frontal binocular visual field in pigeons (Güntürkün and Hahmann, 1999; Hellmann and Güntürkün, 1999). This connection of the TO to melanopsin-containing RGCs allows that melanopsin is able to contribute to visual perception also in the pigeon. Recent studies highlighted that melanopsin plays a role in object localization, discrimination and contrast detection for example in mice (Ecker et al., 2010; Schmidt et al., 2014; Hankins and Hughes, 2014) in addition to the classical role of melanopsin as regulator of pupil constriction and contributor to the circadian rhythm via projections to the SCN (Lucas, 2003; Berson, 2003; Hankins et al., 2008; Foster, 2009). However, it is extremely likely that more RGCs were both CTB- and melanopsin-positive since the retrograde tracing did not cover the whole TO, not every retino-tectal projections is captured. Furthermore, not every ganglion cell develops projections to the TO, some exhibit projections to other primary visual structures like the GLd in the thalamus being a part of the thalamofugal pathway (Luksch, 2003; Wylie et al., 2009). The expression patterns of melanopsin had been closely analyzed in a number of different species with the most prominent expression found in the retina. Retinal expression has been demonstrated in mammals, fish, amphibians and birds (Provencio et al., 1998b; Provencio et al., 2000; Bellingham et al., 2002; Drivenes et al., 2003; Chaurasia et al., 2005; Tomonari et al., 2005). However, expression patterns could vary among different species, and it has to be distinguished between mammalian (Opn4m) and non-mammalian/invertebrate- like melanopsin (Opn4x). In humans melanopsin (Opn4m) can exclusively be found in the eye, mostly restricted to ganglion and amacrine cells (Provencio et al., 2000). In mammals generally, the ocular expression of melanopsin is restricted to retinal ganglion cells and the RPE (Provencio et al., 2000; Hattar et al., 2002; Hankins et al., 2008). In amphibians like 59

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

Xenopus laevis, melanopsin can be found in a few retinal ganglion cells, the iris, RPE and the outermost lamina of the inner nuclear layer where horizontal cells are typically found (Provencio et al., 1998b). In teleosts, melanopsin expression was detected in horizontal, amacrine and ganglion cells (Bellingham et al., 2002; Drivenes et al., 2003). Atlantic cods exhibit two different melanopsins (Opn4m and Opn4x) in the proximal inner nuclear and ganglion cell layers (Drivenes et al., 2003). In birds, several studies showed melanopsin (Opn4x) expression in retinal ganglion cells, pronounced expression in the outer half of the inner nuclear layer (suggesting localization in bipolar cells as well as horizontal cells) and less abundant expression in the proximal half of the INL (suggesting localization in amacrine cell bodies) in chicken. In addition, expression is found in photoreceptor cells (Chaurasia et al., 2005; Tomonari et al., 2005; Lima et al., 2006). In comparison, the expression of pigeon melanopsin (Opn4xL) seems to be quite restricted to the ganglion cells (in the GCL) except for some sporadic stained cell in the INL. These cells could be melanopsin-positive amacrine, bipolar or horizontal cells as described in chickens or simply displaced retinal ganglion cells. The expression pattern of pigeon melanopsin seems to resemble more the human or mammalian melanopsin expression in general, since in mice 95% of all melanopsin-positive cells found in the RGCs whereas only 5% of melanopsin-positive cells were amacrine cells (in the INL or possibly displaced RGCs; Provencio et al., 2002). Surprisingly, the majority of ganglion cells was identified to contain melanopsin in the pigeon. This large amount of cells stained is astonishing, since a lot of studies postulated that for example melanopsin-containing ganglion cells were restricted to 1-2% of all retinal ganglion cells in mammals (Provencio et al., 2000; Hattar et al., 2002; Foster, 2005), while the remaining ganglion cells are mainly not intrinsically photosensitive. However, more recent studies revealed that there might be also wide-spread expression in mammals, since new reporter cell lines in mice revealed a larger number of melanopsin-positive cells (Brown et al., 2010; Ecker et al., 2010). Additionally, the specificity of the antibody used in this study was already demonstrated by Lima et al. (2006). They stained melanopsin in retinal ganglion cells, bipolar and horizontal cells in chickens. The chicken sequence detected by this antibody is almost identical to pigeon melanopsin except for a single amino acid exchange (Lima et al., 2006). For completion, melanopsin can also be found in other tissues in addition to the eye. Originally isolated from dermal melanophores of Xenopus laevis (Provencio et al., 1998b),

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS melanopsin had been detected for example in the pineal gland and medial preoptic nucleus in chicken (Opn4x; Chaurasia et al., 2005). However, the question of this thesis only incorporates retinal melanopsin expression. Therefore, the analysis was restricted to the retina, and does not exclude the possibility of extra-ocular expression in pigeons as well.

3.4.2 Melanopsin is expressed in the pigeon retina during the critical period of asymmetry formation

If melanopsin indeed functions as a mediator of an asymmetrical light signal needed for the ontogeny of visual lateralization in pigeons, some criteria have to be fulfilled (cf. 1.1.3 and 1.3). In this part of the thesis, the second requirement was evaluated: Melanopsin has to be functionally present inside the retina already during the critical embryonic time frame. Immunohistochemical stainings of melanopsin isoform L revealed in pigeon hatchlings, two days after hatch (PH2) and in pigeons embryos, one day before hatch (E16) distinct melanopsin protein expression in the developing ganglion cells layer. In comparison to adult pigeons, the number of cells stained seems to be quite similar. Since in adult pigeon melanopsin-positive cells have been proven to be retinal ganglion cells, it is highly probable that the stained cells in the developmental stages might differentiate to retinal ganglion cells as well. This melanopsin expression at an early developmental stage is consistent with previous findings in other species. Tomonari et al. demonstrated in 2005 in chicken that at E7 melanopsin (Opn4x) could be detected in subset of RGCs which were identified as such due to their localization. At E15 horizontal cells and at E17 cells of the INL were also identified as melanopsin-positive. Post-hatch, a small subset of ganglion cells, bipolar and horizontal cells expressed melanopsin (Tomonari et al., 2005). Also Diaz et al. (2014) examined the protein and mRNA levels of melanopsin (Opn4x) in primary cell cultures of ganglion and horizontal cells in chicken and they were able to detect melanopsin during early developmental stages (ganglion cells: E8 and horizontal cells: E15). In another study in chicks melanopsin protein (Opn4x) was expressed weakly at E8, followed by high expression after E11 in the whole embryonic retina (Verra et al., 2011). Verra and colleagues also demonstrated Opn4m protein expression in chicken as early as E4, which remained elevated from E6 to the postnatal days. This early developmental expression was also found in different mammals (Opn4m), for example in mice and rats, in prenatal ganglion cells (Fahrenkrug et al., 2004; Foster, 2005). In rats (at E18) prenatal melanopsin was detected in PACAP-positive cells which later became ganglion cells 61

CHAPTER 3: QUALITATIVE MELANOPSIN EXPRESSION ANALYSIS

(Fahrenkrug et al., 2004). In conclusion, early expression of melanopsin isoform L in pigeons is in concordance with early expression patterns of melanopsin found in other species ranging from birds to mammals.

3.4.3 Summary and conclusion

Melanopsin has been postulated as the responsible photopigment for the intrinsic photosensitivity of ipRGCs (Gooley et al., 2001; Hattar et al., 2002; Berson et al., 2002; Bellingham et al., 2006). These cells were able to propagate visual information to primary visual brain areas without input from rods and cones (Hankins et al., 2008). Therefore, melanopsin is discussed in this thesis as a potential candidate of transducing light-dependent visual lateralization in pigeons. However, the intrinsic photosensitivity of melanopsin-containing retinal ganglion cells is dependent upon the presence of melanopsin (Lucas, 2003). This chapter demonstrates the early developmental expression of melanopsin in pigeons. Already at E16, the crucial embryonic stage for the induction of asymmetries in the tectofugal system (Skiba et al., 2002), melanopsin expression is found the developing retinal ganglion cell layer and most likely in developing ganglion cells. This could enable these melanopsin-containing cells to be photosensitive even before rods and cones are functionally developed (Bagnoli et al., 1985; Rojas et al., 2007). Therefore, the second requirement of an inducer of visual asymmetries is confirmed: Melanopsin is present inside the retina already during the critical embryonic time frame. The functionality will be analyzed further in two other studies of this thesis (cf. Chapter 5 and 6). This study provides the first evidence of the existence of melanopsin expressing ganglion cells over the critical time period of asymmetry formation in pigeon embryos, therefore, strongly arguing that melanopsin is a valid candidate for the induction of lateralization of the visual system due to an asymmetrical light input during embryonic development.

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

CHAPTER 4: IS MELANOPSIN EXPRESSION ASYMMETRICALLY REGULATED IN THE DEVELOPMENT OF PIGEONS? – A qPCR approach to quantify melanopsin expression in the pigeon retina

4.1 INTRODUCTION

A previous study (Chapter 2) has already demonstrated the existence of at least two melanopsin isoforms in pigeons which share strong similarities to invertebrate opsins (Tomonari et al., 2005; Kang et al., 2010). Additionally, a second study (Chapter 3) was able to confirm the expression of melanopsin in retinal ganglion cells already in the crucial embryonic developmental stage. Altogether, supporting the hypothesis that melanopsin could be the mediator of asymmetrical light incidence during embryonic development causing functional and structural visual lateralization in pigeons. This raises the questions: How abundant are both melanopsin isoforms across the development of pigeons? And is there a potentially asymmetrical expression of the possible inducer of lateralization, melanopsin, in the retina? Hence, asymmetrical expression could be a second genetically-determined asymmetrical factor in addition to the asymmetrical position inside the egg, which results in a unilateral incidence of light restricted to the right eye. To address these questions, melanopsin expression has to be characterized over all developmental stages. The relative abundance (kinetics of expression) of genes can be sensitively and specifically quantified using a quantitative real-time PCR (qPCR) approach (Taylor et al., 2010). Therefore, in this study, melanopsin isoform S and L were quantified in adult pigeons, hatchlings (PH2) and embryos (E16) using a qPCR approach.

4.2 MATERIAL AND METHODS

Quantitative real-time PCR (qPCR) is a standard analytical method for the quantification of gene expression rates in cells or tissues. In contrast to conventional PCR,

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS qPCR enables absolute or relative quantification of a chosen target gene. A two-step quantification experiment requires, first, a reverse transcription step of RNA into more stable cDNA. Subsequently, in a second step the single-stranded cDNA is amplified following the PCR principle, and a fluorescent dye intercalating double-stranded DNA (Bustin, 2000) is applied. As in a conventional PCR, the number of amplicons shows an exponential increase after each cycle. Specific primers amplify a sequence of the target gene to be analyzed and the intercalating fluorescent dye binds the double-stranded amplification product. The increased fluorescence can be monitored in real-time. The fluorescence signal correlates with the amount of the sample to be analyzed (Bustin, 2000). The quantification cycle (Cq or formerly threshold cycle Ct) is determined by the intersection of the amplification curve and a fixed threshold (Livak and Schmittgen, 2001; Bustin et al., 2009). The experiment in this study was designed to identify the relative expression values providing information about differences in gene expression e.g. over different developmental stages (Pfaffl, 2006). Relative gene expression analysis should be normalized to the expression of at least two stably expressed reference genes (Bustin et al., 2009). This quantitative gene analysis was performed in accordance to the MIQE guidelines (Bustin et al., 2009, Huggett et al., 2013, Johnson et al., 2014). These guidelines provide the minimum information needed for publication of qPCR experiments and ensure reliable and accurate quantification results (cf. supplementary data E.2). The composition of all buffers, substances and manufactures of products used in this study can be found in detail in the appendix (A. Chemicals and equipment).

4.2.1 Laboratory animals

Eight of each adult pigeons (Columba livia), hatchlings, embryos and dark-incubated embryos of unknown sex were analyzed in this study. Adult animals were obtained from local breeders, housed in individual cages with a 12 hour dark-/light cycle and free excess to food and water. Embryos (designated as embryos or normal-incubated embryos, E) and hatchlings

(PH) were bred by their parents housed in the lab. For dark-incubated embryos (ED), fertilized eggs of six breeding pairs within the lab were incubated in complete darkness throughout the entire period of incubation using four still-air incubators (Brutmaschine Typ 3000, Bruja GmbH, Germany) adjusted at the same temperature (37 °C) and humidity (60-75%). All experiments were performed in compliance with the guidelines of the German Animal 64

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Welfare Law for the prevention of cruelty to animals as suggested by the European Communities Council Directive of November 24, 1986 (86/609/EEC) and were approved by the animal ethics committee of the Landesamt für Natur, Umwelt und Verbraucherschutz (North Rhine-Westphalia, Germany). All efforts were made to minimize the number of animals used and maintain the highest possible standards of animal welfare.

4.2.2 Tissue preparation

For quantitative gene expression analysis retinae of adult pigeons, hatchlings, embryos and dark-incubated embryos were used. Procedures of tissue preparations were completed during the light cycle of the housing room. The retinal tissue was immediately preserved in RNAlater® (Sigma-Aldrich, Germany) at 4 °C for at least one day and afterwards stored at -80 °C to prevent RNA degradation. Retinae of the different developmental stages were prepared under RNase-free conditions as follows:

Adult pigeons Adult pigeons were decapitated with a small animal guillotine and the eyes were quickly removed. Lens and vitreous body were discarded and a small incision was made next to the pecten. The retina was carefully dissected out of the eye and the retinal pigment epithelium was detached from the retina. All tissues were collected on virtually similar time points around noon.

Hatchlings and embryos Hatchlings (PH 1-2) were directly decapitated, eyes were dissected and the retinae were quickly removed. For embryonic tissue, the eggshells were carefully removed with a forceps at embryonic day 15-16 and embryos were slowly pulled out of the egg and directly decapitated using a scissor. The retinae were prepared as described for the hatchlings. Tissue collection was conducted at virtually similar time points around noon.

Dark-incubated embryos All dark-incubated eggs were dissected in a darkened room to prevent any light stimulation. Preparation was carried out according to the same procedure as described above.

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4.2.3 RNA preparation and quality control

Total RNA preparation For total RNA preparation up to 20 mg RNAlater® stabilized tissue of adult retina or whole hatchling/embryonic retinae were homogenized (Polytron PT1200, Kinamatica AG, Switzerland) and total RNA was isolated according to the manufacturer's instructions (RNeasy Mini Kit, Qiagen, Germany) except for the following changes: An optional DNase I digestion step after binding of the RNA onto spin column was added as recommended in the user´s manual (RNase-Free DNase Set, Qiagen, Germany) to remove genomic DNA. RNA was eluted with 30 µl RNase-free H2O (NEB, Germany) with a prolonged 2 min incubation time. A second elution step with 20 µl RNase-free H2O was added for a higher final RNA yield. Total RNA was frozen immediately in five aliquots in liquid nitrogen. RNA concentration was determined using spectrophotometric measurement at 260 nm (absorption maxima for nucleic acids) and 280 nm (absorption maxima for proteins; BioTek’s Take3™ and Gen5™, Biotek, Germany). The ratio of 260 nm/280 nm was utilized to evaluate the purity of DNA and RNA preparations and to exclude protein contaminations (Glasel, 1995; Wilfinger et al., 1997). Values around 2.0 indicate highly pure RNA while values around 1.8 reveal pure DNA (Glasel, 1995; Wilfinger et al., 1997). For RNA preparation all working materials and surfaces were decontaminated from ribonucleases with RNaseAWAY® (Thermo Scientific, Germany).

Additional RNA quality control (RIN measurement) Determining the integrity of RNA used for quantification experiments is a necessary step to ensure that all experiments were performed with intact RNA (Bustin et al., 2009). Therefore, determining the RNA integrity number (RIN) is the method of choice. The ratio of 28S/18S RNA is determined via an electrophoretogram. Based on this ratio RINs can be calculated. RIN values range between 1 (degraded) and 10 (intact; Mueller et al., 2004). Measurement was performed using Agilent RNA 6000 Pico Kit (Agilent Technologies, USA). RNA to be analyzed was adjusted to a final concentration of 2 ng/µl. All measurements were conducted according to the manufacturer's recommendation using the 2100 Bioanalyzer Instrument (Agilent Technologies, USA). Threshold for RIN values was set to 7, all RNA with a RIN less than 7 were excluded from upcoming experiments to ensure valid quantification results. 66

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4.2.4 cDNA transcription cDNA transcription for sequencing of potential reference genes Selection of suitable reference genes is an indispensable step to reliable quantify gene expression levels (Bustin et al., 2009). For this purpose total retinal RNA was transcribed into cDNA with iScript™ Select cDNA Synthesis Kit (Bio-Rad, Germany) containing Oligo(dT) (deoxythymidine) primers according to the manufacturer's protocol. All cDNA samples were stored at 4 °C until use. cDNA transcription for qPCR To obtain cDNA which should be included into upcoming quantitative expression analysis, RNA was reverse transcribed using QuantiTect® Reverse Transcription Kit (Qiagen, Germany). 100 ng of retinal total RNA of adult animals (n = 8, for each left and right retina) were reverse transcribed according to the user’s manual. For RNA isolated from embryos (E or ED) and hatchlings 1 µg of retinal total RNA (n = 8 for each left and right retina) was reverse transcribed. All samples were diluted 1:10 with Aqua dest. and stored at -80 °C until use.

4.2.5 Sequencing of putative reference genes

Selection and evaluation of suitable reference genes is an inevitable step for valid gene expression analysis using qPCR (Bustin, 2000; Pfaffl, 2006). Since the pigeon’s genome has not been fully sequenced yet, the initial steps to quantify melanopsin in the pigeon retina include sequencing of putative reference genes and evaluation of suitable combinations by stability measurements. Potential reference genes for pigeon retina were selected based on the current literature. In other species, several reference genes have been successfully used in retinal tissue in the last decades (Pires et al., 2009; Dearworth et al., 2011; Rocha-Martins et al., 2012). Eight commonly used reference genes were included in sequencing process and can be found in Table 4.1. Primers were designed according to the species indicated in the table using BLAST (Altschul et al., 1990) and Primer3 (Koressaar and Remm, 2007; Untergasser et al., 2012). Gapdh sequence has already been identified in pigeons (accession number: AF036934.1) and sequence was confirmed in this experiment. All partial sequences of finally used reference genes can be found in the supplementary data (Table E.2.1)

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

Table 4.1: Overview of potential reference genes for quantitative melanopsin expression analysis in pigeon retina. Primers for sequencing were designed according to sequences of other species, the corresponding sequence accession numbers for NCBI-DDBJ/EMBL/GenBank, as well as the reference, are shown. Genes finally used as reference genes in the quantitative gene expression analysis are marked in green (abbreviations: Actb: β-actin, ARP: acidic ribosomal protein, Cytb: Cytochrome b, Gapdh: Glyceraldehyde 3-phosphate (GAP) dehydrogenase, Hprt1: hypoxanthine phosphoribosyltransferase 1, Mapk1: mitogen activated (MAP) kinase 1, Nad4: Nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4, Ppia: peptidylprolyl isomerase A).

Potential reference Species Accession number Reference gene

Actb Gallus gallus NM_205518.1 Dearworth et al., 2011

ARP Gallus gallus NM_204987.1 Pires et al., 2009

Cytb Gallus gallus AF_102874.1 Dearworth et al., 2011

Gapdh Columba livia AF_036934.1 Rocha-Martins et al., 2012

Hprt1 Gallus gallus NM_204848.1 Rocha-Martins et al., 2012

Mapk1 Gallus gallus NM_204150 Rocha-Martins et al., 2012

Trachemys Nad4 AF_206699.1 Dearworth et al., 2011 scripta elegans

Ppia Gallus gallus NM_001166326.1 Rocha-Martins et al., 2012

Amplification of potential reference genes by polymerase chain reaction (PCR) Amplification of potential reference genes was conducted by PCR. All PCR reactions were performed using the Taq DNA Polymerase Kit (Qiagen, Germany) and T100™ Thermal Cycler (Bio-Rad, Germany). For all exact annealing temperatures and primer pairs for each putative reference gene compare Table 4.2 (principle of PCR cf. 0).

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

Table 4.2: Overview of primer used for amplification of putative reference genes for sequencing. The target genes with the corresponding primer pairs for amplification, suitable annealing temperatures and desired fragment sizes are shown. All primers were obtained from Life Technologies, Germany. Genes finally used as reference genes in the quantitative gene expression analysis are marked in green (abbreviations: A = adenine, bp = base pair, C = cytosine, F = forward primer, G = guanine, T = thymine, R = reverse primer).

Primer Primer sequence Annealing Fragment Target gene direction 5´ 3´ temperature size F GATGATGATATTGCTGCGCTCG Actb 58 °C 1690 bp R CCTCCCTCCCGCACATACT F CGATGCCCAGGGAAGACAG ARP 60 °C 940 bp R CCGAAGCCCATATCCTCGTC F TGGCACCCAACATTCGAAAAT Cytb 56 °C 865 bp R AGTTTGTTGGGGATAGGGCG F GGAACATCCCGAAGAAGCAAAG Gapdh 58 °C 1201 bp R TATCAGCCTCTCCACCTCCC F TCGGCGATGATGAACAAGGT Hprt1 56 °C 1327 bp R GCAATTCCACTGGCTGATGG F AGTCCTTTTGAGCATCAGACGT Mapk1 56 °C 901 bp R CTGGCTGGAATCTAGCAGTTTC F CACCCTCATCAGTTCTCTACCAC Nad4 60 °C 468 bp R GCTCAGTGGGTTTGGATTATGC F CCAACCCCGTCGTGTTCTT Ppia 56 °C 479 bp R GCAGTCGGTGATGGTGATCT

The following standard reaction mix was used for a 30 µl PCR reaction: Template cDNA 1 µl (100 ng cDNA) 5’ Primer 0.5 µl (0.1 µM) 3’ Primer 0.5 µl (0.1 µM) dNTPs 1 µl (0.2 mM) CoralLoad PCR Buffer (10x) 5 µl (1x) Taq DNA polymerase 1 µl (1 U)

PCR-grade H2O 21 µl

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The general PCR cycles used for cDNA templates were as follows: 1. Initialization 94 °C, 3 min 2. Denaturation 94 °C, 30 s 3. Annealing 56-60 °C, 30 s 40 cylces 4. Elongation 72 °C, 60 s 5. Final Elongation 72 °C, 10 min 6. Final hold 4 °C, ∞

Validation of PCR products All amplified PCR products were analyzed on a 1.5% agarose (Sigma-Aldrich, Germany containing 5 µl/100 ml Midori Green Advance, Nippon Genetics GmbH, Germany)-TBE (Tris/Borate/EDTA buffer) gel via electrophoresis. In addition to the fragments to be examined a 100 bp DNA Ladder (New England Biolabs, Germany) was applied, which allows estimation of fragment sizes. Electrophoresis was conducted for 45 min at 100 V. DNA molecules were visualized under UV light (Chemidoc MP, Bio-Rad, Germany). Desired bands of all putative reference genes were excised from the gel and purified using the QIAEX II Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's recommendations. All purified fragments were either submitted for sequencing or diluted to 5000 copies/µl (Online tool "DNA Copy Number and Dilution Calculator", ThermoFischer Scientific, Germany) as a positive control in subsequent qPCR experiments.

Sequencing of PCR products of putative reference gene Purified PCR products were sequenced by the sequencing service of the Faculty of Chemistry and Biochemistry, Ruhr-University Bochum. Data was analyzed using SnapGene Viewer® (GSL Biotech LLC, USA) and BLAST.

4.2.6 Evaluation of putative reference genes for qPCR

Selection and evaluation of proper reference genes for expression normalization is a crucial point when qPCR is performed. Quantification can only produce reliable results if the necessary accuracy of reference genes is given. But accurate quantification of nucleic acids requires suitable primer pairs and a stable expression of the reference genes. Initially, the primer specificity and efficiency were evaluated, followed by gene expression stability 70

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS measurements to determine the best combination of reference genes for expression analysis in the pigeon retina.

Primer specificity and efficiency In accordance to the identified sequences of putative reference genes (cf. 4.2.5) primers for qPCR analysis were designed using Primer3 (Untergasser et al., 2012). These primers have to amplify targeted sequences ranging from 80 to 150 bp and had to fulfill the following criteria to ensure specific and efficient amplification: The length of all primers should only range between 18 – 23 bp and the length of each forward and reverse primer pair should not differ greatly. All melting temperatures (Tm) should be around 59–60 °C and primers were recommended to span an exon-exon junction to avoid amplification of genomic DNA (Taylor et al., 2010). The primers’ sequences used to evaluate the putative reference genes in qPCR can be found in Table 4.3. Specificity and efficiency of all primers had to be determined prior to evaluation of reference gene stability. Therefore, a dilution series of cDNA (of two adult animals) was prepared (four data points: 1:10, 1:100, 1:1000, 1:10000 in Aqua dest.). All qPCR measurements for all data points were performed in triplicates using SsoFast™ EvaGreen® Supermix (Bio-Rad, Germany) and the thermocycler CFX384 Touch Real-Time PCR Detection System (Bio-Rad, Germany). The desirable range for primer efficiency varies between 90 and 110% (Taylor et al., 2010). The following standard reaction mix was used for a 5 µl qPCR reaction: Template cDNA 1 µl (cDNA dilution series) 5’ + 3’ Primer Mix 0.15 µl (300 nM) 1x SsoFast™EvaGreen®Supermix 2.5 µl

PCR-grade H2O 1.35 µl

The general qPCR cycles were as follows: 1. Initial enzyme activation 95 °C, 30 s 2. Denaturation 95 °C, 5 s 3. Annealing + Elongation 60 °C, 15 s 40 cycles 4. Fluorescence intensity detection 5. Melting curve 65 °C to 95 °C, 0.5 °C per 5 s, fluorescence detection after each heating step

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

Table 4.3: Overview of primers used for evaluation of putative reference genes for qPCR. The target genes with the corresponding primer pairs for amplification, suitable annealing temperatures and desired fragment sizes are depicted. All primers were obtained from Life Technologies (Germany). Genes finally used as reference genes in the quantitative gene expression analysis are marked in green (abbreviations: A = adenine, bp = base pair, C = cytosine, F = forward primer, G = guanine, T = thymine, R = reverse primer).

Primer Primer sequence Annealing Fragment Target direction 5´ 3´ temperature size F TGTACCCTGGCATTGCTGAC Actb 60 °C 87 bp R CAGGCGGGGCAATGATCTT F CGGCAGCATTTACAATCCCG ARP 60 °C 82 bp R GACGTTACGAACACCCTCCA F CATACACAGAGGACCAGGTTGT Cytb 60 °C 87 bp R CGTTTGCATGGAGGTTTCGG F CATACACAGAGGACCAGGTTGT Gapdh 60 °C 91 bp R TCATTCAGTGCAATGCCAGC F TCATGGACAGGACGGAGAGG Hprt1 60 °C 86 bp R ACCCTTGAGTACACAGAGAGC F TGTGCTTCATCGTGACCTCA Mapk1 60 °C 81 bp R GCCAGTCCAAAGTCACAAATCTT F CAACTCCTGAACTGGCCTCC Nad4 60 °C 90 bp R GGTAGTCATAGGTGCAGGCC F ATCCTGTCCATGGCCAACG Ppia 60 °C 89 bp R TGCTTGCCATCCAACCACTC

Since primers play a crucial role for reliable quantification of gene expression, primer efficiencies were calculated. Therefore, a standard curve was derived from a dilution series

(as described above) and Cq values were plotted against the logarithm of the cDNA concentration. The slope of the standard curve was used to calculate the efficiency (EPCR, cf. Formula 1, Stahlberg et al., 2003):

1 -slope EPCR = 10 -1 (1)

EPCR = PCR efficiency; EPCR = 1 corresponds to 100% efficiency Additionally, the melting curves of amplified gene products were generated by decreasing the temperature from 65 °C to 95 °C in 0.5 °C steps. The fluorescence was plotted against temperature and differentiation of this curve results in a view of the melting dynamics, with

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS one clear peak indicating good primer specificity amplifying only one specific product and excluding the formation primer dimers.

Selection of reference genes by gene expression stability evaluation Selection of reference genes played an essential role in quantitative expression analysis of genes of interest. Therefore, potential reference genes were tested for their expression stability (cf. Table 4.1), except for one: Mapk1 was excluded beforehand as it already showed insufficient data when primer specificity and PCR efficiency were investigated. Eight retina samples (of either adult pigeons, hatchlings or normal- or dark-incubated embryos) with each left and right retinae were included in the measurements. Every primer pair of a putative reference gene was tested in triplicates and 1 µl of cDNA was used in 1:10 dilution (cDNA transcription cf. 4.2.4). Quantitative PCR was performed as described above, and for analysis the Gene Study tool of the Bio-Rad CFX Manager™ (Bio-Rad, Germany) was utilized to calculate the ideal pair of reference genes via pairwise variation of the candidate reference genes, thereby, calculation of the gene stability measure M was performed according to the algorithm proposed by Vandesompele et al. (2002) and Hellemans et al. (2007).

4.2.7 Determining limit of detection (LOD) and limit of quantification (LOQ)

Since, evaluation of qPCR performance parameters is a necessary step to gain valid quantification results, it is indispensable to determine the limit of detection (LOD) and the limit of quantification (LOQ) before starting gene expression analysis. The LOD describes the lowest concentration at which 95% of a replicate can be detected, and as such is the lowest amount of the gene of interest which is detectable in a single reaction (Bustin et al., 2009; Nutz et al., 2011). The LOQ is the lowest concentration of the gene of interest that still can be reliably quantified. It describes the point when the coefficient of variation (CV) is greater than 20% (deviation of measured and nominal values; Armbruster and Pry, 2008; Bustin et al., 2009; Nutz et al., 2011). Both, LOD and LOQ have to be determined for all reference genes used and for the genes of interests. Therefore, a starting concentration of 2560 copies of all genes analyzed was diluted to 2.5 copies (online tool "DNA Copy Number and Dilution Calculator", ThermoFisher Scientific) in 1:1 dilution steps. All samples were tested in octuplicates. LOD was determined using Formula 2: 73

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Cq = -(2.303m)∙ ln (#copies) + n (2) with #copies = nominal value, m = slope; n = y-intercept 100 %deviation= (3) #copies - #copies' #copies = nominal value; #copies' = calculated value LOQ was calculated using Formula (3), after the copy number for every replicate and the theoretically calculated copy number were determined. Since the cutoff value of the deviation of LOQ was set at 20%, the standard curve was adjusted to the range of LOQ.

4.2.8 Quantitative melanopsin expression analysis

After successful reference gene sequencing, evaluation and determination of LOD and LOQ the expression of the gene of interest, melanopsin, can be analyzed. Primers for melanopsin were designed on basis of the pigeon melanopsin gene sequence (cf. Chapter 2). These primers have to meet the same criteria as the primer designed for the reference genes (cf. 4.2.6). Two different primer pairs (cf. Table 4.5) were used for melanopsin quantification: I. Primers amplifying the short melanopsin isoform (Opn4xS, isoform S) II. Primers amplifying the long melanopsin isoform (Opn4xL, isoform L) For these primers´ specificity and efficiency as well as LOD and LOQ have been determined as described previously. In all measurements no template controls (NTC) and positive controls of all target genes were applied. All samples were measured in triplicates and repeated in three individual runs. An inter-run calibration (IRC) was performed to calibrate the three runs among each other and to exclude technical variations. As IRC a standard curve of known concentration was established, using seven data points of a serial dilutions of purified Nad4 fragments with a starting concentration of 10.000 copies (dilution in 5-fold dilution steps).

Relative gene expression of melanopsin was determined by using the ΔΔCq method (Livak and Schmittgen, 2001, for used reference genes cf. Table 4.4). For analysis the relative quantity of a target gene normalized to the quantities of the reference targets was calculated using Bio-Rad CFX Manager™ (Bio-Rad, Germany). The following standard reaction mix was used for a 5 µl qPCR reaction:

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

Template cDNA 1 µl (adult 1:100 dilution;

PH,E, ED 1:10 dilution) 5’ + 3’ Primer Mix 0.15 µl (300 nM) 1x SsoFast™ EvaGreen® Supermix 2.5 µl

PCR-grade H2O 1.35 µl

Table 4.4: Overview of reference genes used for quantification of melanopsin. Reference genes used for quantification of melanopsin in all developmental stages. The gene stability values M and CV are listed for the used combination of reference genes. According to Hellemans et al., 2007 in homogeneous samples M and CV (coefficient of variation) values less than 0.5 and 25%, respectively, are acceptable for suitable reference genes.

Reference gene Mean CV Developmental stage M value combination [%]

Adult ARP and Ppia 0.1730 6.02

Hatchling (PH) Gapdh and Ppia 0.2526 8.78

Embryo (E) Gapdh and Ppia 0.1942 8.45

Dark-incubated Gapdh and Ppia 0.2208 7.67 embryo (ED)

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Table 4.5: Overview of primers used for quantification of melanopsin. The target gene with the corresponding primer pair for amplification, suitable annealing temperature and desired fragment size are shown. All primer were obtained from Life Technologies (Germany; abbreviations: A = adenine, bp = base pair, C = cytosine, G = guanine, T = thymine, F = forward primer, Opn4xS = short melanopsin isoform, Opn4xL = long melanopsin isoform, R = reverse primer).

Primer Primer sequence Annealing Fragment Target gene direction 5´ 3´ temperature size F CACCCTCATCAGTTCTCTACCAC Opn4xS 60 °C 142 bp R GCTCAGTGGGTTTGGATTATGC F AGTCCTTTTGAGCATCAGACGT Opn4xL 60 °C 86 bp R CTGGCTGGAATCTAGCAGTTTC

Statistical analysis Expression data for all developmental stages were statistically analyzed using SPSS Statistics Software 20 (IBM Corporation, USA). Kolmogorov-Smirnov tests were used to check normal distribution of the data sets. Univariate analysis of variance (ANOVA) and multivariate ANOVA were computed to investigate expression differences between and within (expression left eye versus right eye) all developmental stages. LSD test was used for post‐hoc comparisons between developmental stages. For expression analysis within a developmental stage a paired-sample t-Test had been calculated.

4.3 RESULTS

Quantitative PCR was used to determine the relative abundance of melanopsin in the pigeon retina at different developmental stages. In order to analyze the relative expression of melanopsin in pigeon raw Cq data of both melanopsin isoforms were normalized to the selected reference genes (adult: ARP & Ppia; hatchling, embryo and dark-incubated embryo:

Gapdh & Ppia) using the ΔΔCt method to obtain the relative expression values (Gene Study, CFX Manager™, Bio-Rad, Germany). This is one of the first qPCR studies performed with pigeon retinal tissue and in order to establish valid qPCR analysis in pigeon retina in our laboratory a multitude of factors had to be evaluated. All experiments were performed in accordance to the MIQE guidelines (Bustin et al., 2009). All details and requirements of these guidelines can be found in the supplementary data E.2. Specificity and efficiencies of primers were previously determined by amplification of a dilution series and a melting curve analysis (cf. 76

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS supplementary data Figure E.2.2a and b). Additionally, the linear dynamic range of used reference genes was analyzed to allow reliable quantification results (supplementary data Figure E.2.3). All primers exhibit comparable amplification efficiencies within the appropriate range of 90-110% (Taylor et al., 2010). Reliable quantification ranges were determined by LOD/LOD relations (supplementary data Figure E.2.4 and Table E.2.5).

4.3.1 Quantification of melanopsin isoforms S and L in adult pigeons

The relative expression levels of both melanopsin isoforms in adult pigeon retina were analyzed by qPCR using ARP and Gapdh as reference genes (M value = 0.173, CV = 6.02%) for accurate relative quantification. Data for both melanopsin isoforms S and L are shown in Figure 4.1 (relative expression values of isoform L: 1.034 (± 0.079 SEM) for the left eye and 1.016 (± 0.083 SEM) for the right eye; isoform S: 0.507 (± 0.147 SEM) for the left eye and 0.367 (± 0.075 SEM) for the right eye).

Figure 4.1: Comparison of the relative melanopsin expression levels of isoform S and L in the retina of adult pigeons. Melanopsin isoform S shows significantly less expression in comparison to isoform L (paired t-Test p < 0.05, n = 8). No significant expression difference is found between the left and the right eye (paired t-Test, p > 0.05, n = 8). All expression data (± SEM) is shown normalized to the expression of housekeeping genes ARP and Ppia. Expression of isoform L was significantly higher compared to isoform S in adult pigeons in both eyes (left eye: paired t-Test; t(15) = -2.44; p < 0.05, n = 8 and right eye: paired t-Test; t(15) = -4.74; p < 0.05, n = 8). No expression differences either in isoform S or L were found between the left and the right eye (paired t-Test S: t(15) = 1.22; p > 0.05, n = 8; L: t(15) = 0.34; p > 0.05, n = 8). However, it has to be noticed that the quantification results of isoform S has 77

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to be considered carefully since the mean Cq of isoform S was 35.84 ± 0.70 for the right eye and 35.28 ± 0.62 for the left and, therefore, around two Cq larger than permitted by the limit of quantification (33.98). The limit of quantification for isoform L was set to 33.77 with a mean

Cq of 28.91 ± 0.22 for the right eye and 28.68 ± 0.23 clearly ranging below this cut-off value (cf. supplementary data Figure E.2.4, Table E.2.5 and Table E.2.6 a).

4.3.2 Melanopsin expression over development

Immunohistochemically data already demonstrated melanopsin isoform L is expressed throughout the development of pigeons (cf. 3.3.1) and quantitative analysis by qPCR strongly supports these findings. Melanopsin expression was examined in pigeon embryos and hatchlings using ARP and Ppia as reference genes (E: M value = 0.1942, CV = 8.45%; PH: M value = 0.2526, CV = 8.78%). Melanopsin isoform L is already detectable as early as E16, and expression is traceable from E16 to PH2 to adult pigeons (relative expression values: 0.594 (±0.090 SEM) at E16 for the left eye and 0.683 (±0.097 SEM) for the right eye, 0.872 (±0.152 SEM) at PH2 for the left eye and 1.079 (±0.175 SEM) for the right eye). No expression differences between the left and right eye within any developmental stage (E, PH) were found

(paired t-Test E: t(15) = -0.84; p > 0.05, n = 8; PH: t(15) = -1.80; p > 0.05, n = 8), whereas analysis between both developmental stages revealed significant more expression (ANOVA;

F(3, 28) = 0.60; p < 0.05, n = 8) in the right eye of hatchlings compared to embryos. Comparison of expression values of adult animals (isoform L) showed also no expression differences between these developmental stages in the right eye (ANOVA, p > 0.05, n = 8; Figure 4.1 and Figure 4.2), but significantly more expression was evident in the left eye of adult pigeons as compared to embryos (ANOVA, p < 0.05, n = 8), whereas no expression difference compared to the left eye of hatchlings was detectable. Since melanopsin isoform S was barely detectable, it could not be reliably quantified, although primer efficiencies and specificity tested before for isoform S were in the appropriate range. Therefore, for the comparison of gene expression between left and right retinae, only isoform L was analyzed. All expression data was reliably quantified since all mean Cq values were within the acceptable range determined by LOD and LOQ measurements (PH: Opn4xL right eye = 30.14 ± 0.07 and left eye = 30.20 ± 0.08, E: Opn4xL right eye = 30.83 ± 0.14 and left eye 30.90 ± 0.12, with LOD = 35.63 and LOQ = 33.77, see supplementary data Table E.2.5 and Table E.2.6 b and c) as well reference genes were proven to be appropriate. 78

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Figure 4.2: Relative expression levels of melanopsin isoform L in the retina of pigeon embryos and hatchlings. No significant expression difference is found between the left and the right eye neither in embryos nor in hatchlings (paired t-Test, p > 0.05, n = 8). Additionally, no significant differences in melanopsin expression in the left eye between both developmental stages is obvious. Right retinae of hatchlings express significantly more melanopsin than the right eye of embryos (ANOVA, p < 0.05, n = 8). All expression data (± SEM) is shown normalized to the expression of housekeeping genes Gapdh and Ppia. A power-analysis was conducted to calculate the required sample size to reach significance within the samples using G*Power 3.17 (Faul et al., 2007; Faul et al., 2009). Power analysis for t-Test with dependent samples was conducted using the following parameters for a two-sided test: α = 0.05, power = 0.95, effect size dz (PH: dz = 0.637, E: dz= 0.298), as calculated by the G*Power software from the means and standard deviations of the left and the right eye. The total sample size required for the effect to reach significant differences between the left and right eye was n = 35 for hatchlings and n = 148 for embryos (cf. 4.4.4).

4.3.3 Melanopsin expression in dark-incubated embryos

In order to quantitatively evaluate the melanopsin gene expression in respect to possible lateralization effects normal-incubated and dark-incubated embryonic retinae were analyzed. On average, melanopsin isoform L relative gene expression values found in the retinae of in dark-incubated embryos were 0.580 (± 0.169 SEM) in the left eye and 0.632 (± 0.138 SEM) in the right eye (for normal-incubated embryos compare 4.3.2). No significant expression differences within the group were found (paired t-Test; t(15) = -0.30; p > 0.05, n = 8) as well as no significant differences between normal- and dark-incubated

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS embryos (ANOVA, p > 0.05, n = 8) were found, even though, there is a significant melanopsin isoform L expression difference in the left eye compared to adults and in the right eye compared to adults and hatchlings (ANOVA, p < 0.05, n = 8). In dark-incubated embryonic retinal tissue, isoform S was barely detectable and could not be reliably quantified (cf. embryos and hatchlings 4.3.2). Quantification results of isoform L in dark-incubated embryos lay in the range necessary for valid quantification determined by LOD and LOQ (ED: Opn4xL mean Cq right eye 30.91 ± 0.24 and left eye 31.10 ± 0.24, LOD 35.63 and LOQ 33.77, see supplementary data Table E.2.5 and Table E.2.6 c and d).

Figure 4.3: Comparison of melanopsin isoform expression values between normal-incubated and dark-incubated pigeon embryos. Melanopsin isoform L shows no significant expression differences neither between the left and the right eye within both conditions, nor between the two groups (paired t-Test p > 0.05, n = 8 and unpaired t-Test p > 0.05, n = 8). All expression data (± SEM) is shown normalized to the expression of housekeeping genes Gapdh and Ppia.

A power-analysis was conducted to calculate the required sample size to reach significance in the sample of dark-incubated embryos using G*Power 3.17 (Faul et al., 2007; Faul et al., 2009). The procedure was similar as described above (4.3.2) with the following parameters: α = 0.05, power = 0.95, effect size dz 0.105, as calculated by the G*Power software from the means and standard deviations of isoform L in the left and the right eye. The total sample size required for the effect to reach significant differences between the left and right eye was 1173 dark-incubated embryos (cf. 4.4.4). Furthermore, an analysis of variance of the Delta absolute values of normal- and dark-incubated pigeon embryos (cf.

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

Figure 4.4 and supplementary data Table E.2.7) revealed a higher variance in dark-incubated embryos.

Figure 4.4: Analysis of variance between normal- and dark-incubated pigeon embryos. Boxplot of the Delta absolute values of normal- and dark-incubated embryos illustrating the higher variance in dark- than in normal-incubated embryos.

4.4 DISCUSSION

The results of this study provide the first insight into the precise mRNA expression pattern of melanopsin in the retina of pigeons. Melanopsin isoform L exhibits a significantly stronger expression in adult pigeons than isoform S. Isoform S expression in pigeon embryos and hatchlings could not be quantified due to its very low expression rate indicating that isoform S might, if at all, just have a minor influence on mediating an asymmetrical light signal during embryonic development. Furthermore, no expression differences within a developmental stage were identified for isoform L, illustrating no asymmetrical expression between the left and the right eye. Referring to normal- and dark-incubated embryos no significant expression differences were observed neither in the left nor in the right retina. All aspects, especially concerning the impact of melanopsin on light-induced lateralization, will be discussed in the next sections in more detail.

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

4.4.1 Analyzing relative gene expression: establishment and validation of qPCR in the pigeon retina

This study provides the first quantitative gene expression analysis of melanopsin in the pigeon retina. Therefore, before starting gene expression analysis of a gene of interest qPCR had to be established and validated in pigeon retina to ensure accurate and reliable quantification. The whole study was conducted in accordance to the MIQE guidelines which provide the minimum information required for publication of qPCR-based experiments (Bustin et al., 2009). Therefore, quality of used RNA was controlled by spectrophotometric measurements and RIN determination, qPCR specificity and efficiency of reference genes were validated, as well as the limit of detection (LOD) and the limit of quantification (LOQ; Armbruster and Pry, 2008; Bustin et al., 2009; Nutz et al., 2011). Because, the pigeon´s genome is not fully characterized yet, first of all different potential reference genes for retinal expression analysis that have been successfully used for qPCR studies in retinae of other species had to be sequenced (Pires et al., 2009; Dearworth et al., 2011; Rocha-Martins et al., 2012). Sequencing of eight commonly used reference genes in retinal tissue was performed (Actb, ARP, Cytb, Gapdh, Hprt1, Mapk1, Nad4 and Ppia) and subsequently to stability measurements two appropriate reference genes for each developmental stage were selected for normalization of gene expression. For adult retinal tissue ARP and Ppia were proven to be suitable reference genes, whereas in juvenile and embryonic stages expression should be normalized to Gapdh and Ppia. According to Hellemans et al. (2007) gene expression stability measure (M) values less than 0.5 (CV < 25%) were sufficient for reliable quantification results for homogenous samples (heterogeneous samples: M < 1, CV < 50%), identifying the chosen reference genes as perfectly suitable. Taken together, qPCR performed in this study ensures highly accurate expression data since reference genes used were accurately proven to be good normalizers and qPCR was performed according the MIQE guidelines (Bustin et al., 2009). Furthermore, this study provides a valid basis for further gene expression analysis in the pigeon retina.

4.4.2 Isoform-related melanopsin expression differences in adult pigeons

This study revealed significant expression differences between melanopsin isoform S and L in adult pigeons whereas in embryos and hatchlings isoform S expression was barely

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS detectable and hence could not be quantified. Therefore, the analysis of differential expression patterns between the long and the short isoform will only be focused on adult pigeons. Isoform L expression in comparison to isoform S is around 1.8-fold higher in the left eye and 2.7-fold in the right eye, and overall isoform L expression is around 2.4-fold higher. Differential expression of both melanopsin isoforms was also confirmed for other vertebrate species e.g. chicken (Tomonari et al., 2005) and mice (Pires et al., 2009; Hughes et al., 2012). Upregulation of melanopsin (Opn4x) irrespective of the isoform was published by Verra et al. in 2011, demonstrating increased melanopsin expression from E8 to E11. Tomonari et al. (2009) conducted a qPCR analysis in the chicken retinae, where nearly 10-fold increased expression was demonstrated for the long isoform of chicken melanopsin in comparison the shorter isoform (Opn4xL: 90.8%, Opn4xS: 9.2%) at embryonic day 17. Additionally, they showed a continuous increase of isoform L expression from E10 (expression set to 1) to E14 (expression relative to E10: 11.8-fold) which finally reached a 122.6-fold relative expression at E17. These results were in concordance with the expression data found in pigeons with significantly higher expression of isoform L in adults and barely detectable expression of isoform S in hatchlings and embryos. However, isoform S expression was quantified by Tomonari et al. (2009) in neural retina of chicken (E10-17), whereas quantification of isoform S was not possible in pigeons during the development due to the too low expression rates. These differences might be explained by the degree maturation at hatch. Pigeons are altricial birds, whereas chicken are precocial (Jones and Jones, 2011). Therefore, isoform S might differentially regulated in precocial chickens than in the ontogeny of pigeons. On the contrary qPCR studies in mice indicated a 40x higher expression of isoform S (Opn4mS) in comparison to isoform L (Opn4mL) (Pires et al., 2009). The authors suggested that these expression differences might be a result of a higher de novo expression or increased mRNA stability (Pires et al., 2009). Deeper analyses of mRNA and protein levels revealed constant expression of isoform S mRNA and elevated protein levels starting postnatally at day 1 (P1) during development in mice, whereas a marked increase of isoform L was initially found for mRNA and protein in later developmental stages (P14 and P30; Hughes et al., 2012). This might be contradictory at first glance, but it has to be noted that mice and pigeons express orthologous melanopsin genes. The mammalian (Opn4m) and non-mammalian (Opn4x) melanopsin genes exhibit only a small percentage of sequence concordance whereas the functions appear rather similar (Hankins et al., 2008). And mice and pigeons exhibit

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS completely different circadian rhythms: mice are nocturnal (Hut et al., 2011), and at least some birds are diurnal animals (e.g. pigeons and chickens; Lazareva et al., 2012). The second difference in the circadian rhythm might influence melanopsin expression even more and potentially explain the different expression between isoform S and L. Furthermore, these results of melanopsin expression in pigeons are in accordance to the data obtained in Chapter 2 and Chapter 3. Pigeon melanopsin isoform L exhibits a multiplicity of co- and posttranslational modification sites due to its prolonged C-terminal tail (cf. 2.3.2. and Table 2.5; e.g. one additional N-glycosylation site, two more PKC phosphorylation sites, two more Casein kinase II phosphorylation sites and three additional N-myristoylation sites). Altogether, this predicts intense membrane targeting and higher grade of functionality (Glass et al., 1986; Farazi et al., 2001; Johnson, 2009; Casteel et al., 2010). Additionally, the staining of melanopsin isoform L protein (cf. 3.3.1) demonstrated a large number of stained ganglion cells. In conclusion, these results are also reflected by the significantly higher expression of melanopsin isoform L mRNA in adult pigeons. Therefore, especially melanopsin isoform L might be a promising candidate to contribute to the ontogeny of visual lateralization, since it is expressed at the critical developmental time frame, whereas isoform S exhibits only a marginal, barely detectable expression in embryos and hatchlings perhaps excluding it as a potential inducer.

4.4.3 Exogenous or endogenous regulation of melanopsin expression over the development in pigeons?

Expression analysis of the mRNA of melanopsin in pigeon retina in different developmental stages via qPCR demonstrated that there are no significant expression differences between the left and the right eye within a developmental stage. Additionally, melanopsin isoform L expression of adult pigeons is quite similar to the expression in hatchlings, illustrating an “adult” expression pattern already in two day old hatchlings. Significantly more expression was found the left eye in adults in comparison to the left eye in normal- and dark-incubated embryos. Also the right eye of adults expresses significantly more melanopsin isoform L than the right eye of dark-incubated embryos and additionally the right eye of hatchlings demonstrated a significant higher melanopsin expression than the right eye of normal- and dark-incubated embryos. As explained above the discussion will focus on the expression of isoform L. 84

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

Melanopsin is usually perceived as one of the circadian regulators (Berson, 2003). Therefore, a lot of studies have been conducted to investigate the circadian expression patterns of melanopsin (Gooley et al., 2001; Hattar et al., 2002; Berson, 2003). In chicken pineal gland the highest melanopsin expression was found at night. Importantly, no diurnal expression of melanopsin was found in ipRGCs in chicken (Chaurasia et al., 2005). Bailey and Cassone (2005) postulated that melanopsin mRNA is regulated on a circadian basis in the pineal gland in chicken, but they only detected a slight increase in melanopsin mRNA in the retina overnight. Furthermore, Lima et al. (2006) demonstrated via qPCR analysis that a prolonged period of darkness (18 hours darkness, 6 hours light) leads to a 3-fold higher melanopsin mRNA expression in the chicken retina at the time point when the light is switched on in contrast to prolonged day light (6 hours darkness, 18 hours light). They suggested that melanopsin expression may be modulated by light in chicken via an inhibition of transcription (Lima et al., 2006). In mice, the number of melanopsin-expressing cells did not change between E16 and E19, while a significant increase was observed between the day before birth and the day of birth (E19-P0). The increase of melanopsin-positive cells around birth seems to be light-independent, since Gonzalez-Menendez et al. (2011) reported that retinae collected at night right after birth or those collected some hours later during the light period did not differ statistically (Gonzalez-Menendez et al., 2011). No changes in the number of cells were observed after early postnatal light exposure. Comparison of the number of melanopsin-expressing cells in mice pups in different light conditions (constant light, constant darkness, regular dark/light cycles) revealed no variation (Gonzalez-Menendez et al., 2011). In addition, in another study rats were reared under different light conditions varying between constant darkness to constant bright light from the day of birth (P0) to weaning day (P22). Subsequently, the number of melanopsin-positive cells was determined by immunohistochemistry. This study revealed that there are no circadian alternations in melanopsin-expressing cells and that the number of melanopsin-expressing cells is not dependent on the ambient light conditions in rats (Cambras et al., 2015). However, it has to be noted that a constant number of melanopsin-expressing cells does not necessarily exclude/include an increase/decrease in melanopsin expression in these cells. And on the contrary in adult mice the highest melanopsin expression was found one hour before the light was switched on and the lowest expression was found a few hours after

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS the light was switched on (Gonzalez-Menendez et al., 2009). Another qPCR study on mammalian melanopsin in rats reported that melanopsin expression oscillated with a peak around onset of dark and in the middle of the darkness period, respectively. Additionally, they showed that constant dark and light abolished the melanopsin expression rhythm (Mathes et al., 2007). This led to the conclusion that melanopsin regulation is not relying on a circadian oscillator but is directly illumination-dependent (Mathes et al., 2007). Analysis of the melanopsin mRNA expression by qPCR in albino rats showed an upregulation at dusk and a nadir at dawn (Hannibal et al., 2013). The latter authors suggested that input from retinal networks can regulate the melanopsin mRNA expression and that the regulation of melanopsin is a mechanism of the retina to adapt to environmental changes in light and dark conditions (Hannibal et al., 2013). In pigeons no expression differences are found either between the left and the right eye of normal- and dark-incubated embryos or between both groups. The same holds true for the comparison of adult pigeons and pigeon hatchlings. On the one hand the unchanged amount of melanopsin isoform L mRNA in normal- and dark-incubated pigeon embryos could indicate an endogenous regulation of melanopsin expression independently of light incidence. On the other hand an exogenous (e.g. light-dependent) regulation might be conceivable. If melanopsin expression is regulated exogenously, differential expression between the left and right eye of normal-incubated embryos would be expected. Due to the embryos asymmetrical position inside the egg (Kuo, 1932), only the right eye is stimulated by light, whereas the left eye is light-deprived. However, it is conceivable that the amount of light irradiating the egg is not sufficient or not manifesting a circadian rhythm. Since the brooding pigeons regularly leave the nest and turn their eggs, the eggshell is repeatedly exposed to the ambient light. However, these periods were rather short, with a stimulation of 55 s approximately every 43 min (Buschmann et al., 2006) and are not comparable to a natural continuous dark/light rhythms. Therefore, this light incidence could be too short to actively regulate melanopsin expression in a circadian/illumination-dependent manner. However, Hannibal et al. (2013) suggested that endogenous mechanisms may play a more important role in during development whereas in adult animals regulation of melanopsin expression is exogenously determined and hence illumination-dependent. This suggests the hypothesis of an endogenous regulation during pigeon ontogenesis as well. Admittedly, the regulation in adult pigeons would just be a speculation since all animals analyzed were all scarified at the same

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS time of the day. Experiments as carried out by Gonzalez-Menendez et al. (2011) would be needed to analyze if there is also a illumination-dependent regulation of melanopsin expression found in adult pigeons. But, as soon as the pigeon is hatched (analyzed at PH1/2), the expression is adapted to the adult pattern, possibly indicating an illumination-dependent regulation directly after hatch. However, this thesis focuses on the ontogeny of asymmetries during early light-stimulation in pigeons. Therefore, the focus was set to the critical developmental period (E16). In summary, an endogenous regulation of melanopsin expression is the most likely regulation mechanism during the development inside the egg, whereas an illumination-dependent regulation is possible immediately after hatching and in adult pigeons.

4.4.4 Indicators of asymmetrical melanopsin expression in pigeons?

As already illustrated, an asymmetrical light stimulation during a critical embryonic time frame shortly before hatch leads to stable structural and functional asymmetries of the tectofugal system in pigeons (Güntürkün, 2002a, 2005). Since rods and cones are not functionally developed at that time point (Bagnoli et al., 1985; Rojas et al., 2007), melanopsin is discussed as a potential mediator in this thesis. Chapter 2 and 3, already provided important evidences for the possible involvement of melanopsin as shown by the validation of melanopsin expression during the critical time frame in retinal ganglion cells in pigeons. Therefore, this study aims to unravel more precisely the expression pattern (already discussed above 4.4.2 and 4.4.3). Since this thesis is dealing with the ontogeny of asymmetries, the question arises if there might be secondary asymmetrical component in the induction of lateralization in addition to an asymmetrical light input. Asymmetrical melanopsin expression could influence the induction of asymmetries on a secondary level in addition to the transmission of an asymmetrical light stimulus to the brain. At first glance, no expression differences were found between the left and the right eye within any developmental group. In particular, no left/right differences appeared at the critical developmental stage, neither in normal- nor in dark-incubated pigeon embryos. This could indicate that there is no additional asymmetrical genetically-determined factor in addition to the asymmetrical light stimulation due to the genetic predisposition of asymmetrical position inside the egg. On the one hand, if melanopsin is exogenously regulated by light, more expression in either the left or right eye would be expected due to the 87

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS asymmetrical light stimulation. On the other hand if melanopsin expression is regulated endogenously an asymmetrical expression is possible but not necessary (as already depicted in 4.4.3). In this case an asymmetrical expression would indicate a second genetically predetermined asymmetry in addition to the asymmetrical position inside the egg. However, this seems rather unlikely since no differences between the left and the right eye were found neither in normal- nor in dark-incubated pigeon embryos. A power analysis (cf. 4.3.3) revealed that at least 148 normal-incubated embryos would be needed to find significant differences between the left and the right eye, which may favor the right eye. In dark-incubated embryos 1173 animals would be necessary. This indicates that there might be a more pronounced asymmetric effect in normal-incubated embryos than in dark-incubated embryos. However, both quantities of animals exceed ethically justifiable and feasible amounts of laboratory animals which can be incorporated in a study. Since the effect of left and right eyed expression asymmetries is minimal, it might be concluded that this lateralization is functionally not relevant. However, a variance analysis showed a considerably higher variance in relation to the Delta absolute values of the melanopsin expression in dark-incubated embryos compared to normal-incubated embryos (cf. Figure 4.4). This might be explained with a stabilizing effect of light, even if the expression in general might be endogenously regulated in embryos. Stabilizing effects of light (especially UV light) were conversant in different kind of cells. Studies on the stabilizing effects of light demonstrated that in plant cells light effects the stability of some light-responsive mRNAs (Filipowicz and Hohn, 1996), and in human cells a dose-dependent UV light stabilization of preformed mRNA was found (Hilgers et al., 1991). Bollig et al. (2002) suggested that UV light induces stabilization of mRNA expression and is able to affect a broad spectrum of mRNAs. The same might hold true for the expression of melanopsin in normal-incubated pigeon embryos since UV light is able to penetrate the eggshell (Maurer et al., 2015). This light-induced stabilization process in normal-incubated embryos is missing in dark-incubated ones, therefore, this fact might explain the higher variance and the overall more variable expression pattern. This indicates also a larger variance between the individuals in the group of dark-incubated embryos, whereas the overall means of expression values were quite similar. However, no significant expression differences were found between the left and the right eye within a developmental stage. Values differed between 0.38 (± 0.05 SEM) and 1.00

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS

(± 0.14 SEM) in the left eye and 0.43 (± 0.07 SEM) – 1.13 (± 0.25 SEM) in the right eye (cf. Table E.2.6c) in normal-incubated embryos. This variability within the eyes between different individuals, even if not differing significantly at all, is conceivable to influence the development of the degree of lateralization in the adult animal. This could be in agreement with studies on visual lateralization of pigeons which showed that animals with higher degree of asymmetries were more successful in discrimination tasks (e.g. grain/grit discrimination) under binocular conditions (Güntürkün et al., 2000). Furthermore, a recent study demonstrated that the degree of lateralization strongly varies between individuals at transfer tasks (Letzner et al., 2014). Therefore, a difference in the melanopsin expression rate in the right eye during development might explain different strengths in the degree of lateralization in adult pigeons. Hence, if more melanopsin is expressed in the RGCs, it might be conceivable that light stimulation leads stronger signal in GCL which can be subsequently propagated to the brain. In conclusion, the asymmetrical light stimulation due to the embryos asymmetrical position inside the egg remains the only asymmetrical and genetically-determined parameter during the development, since melanopsin expression is not lateralized. However, the amount of melanopsin expressed varies between individuals, even though not statistically significant. This might influence the different degrees of lateralization observed in adult pigeons.

4.4.5 Summary and conclusion

In summary, this study revealed that melanopsin isoform L is expressed over all developmental stages in pigeon, and adult expression patterns are already reached after hatch. Therefore, this fact strongly supports the hypothesis that melanopsin isoform L could be an inducer of visual lateralization in pigeons due to an asymmetrical light stimulation in a critical embryonic time frame. In contrast, isoform S has to be virtually excluded as a potential trigger due to the barely detectable expression in embryos (normal- and dark-incubated) and hatchlings. The results of the expression in normal- and dark-incubated embryos indicate an endogenous regulation during embryonic development whereas an illumination-dependent regulation is conceivable after hatch. Indeed, the genetically-determined asymmetrical position inside the egg remains the only asymmetrical factor, since no significant expression differences were found between the left and the right eye, neither in normal- nor in dark-incubated pigeon embryos. However, these results strengthen the hypothesis that

CHAPTER 4: QUANTITATIVE MELANOPSIN EXPRESSION ANALYSIS melanopsin is a promising candidate as the transducer of the asymmetrical light stimulation onto higher brain regions inducing visual lateralization in pigeons.

CHAPTER 5: ANALYSIS OF MELANOPSIN LIGHT RESPONSES

CHAPTER 5: CAN PIGEON MELANOPSIN BE ACTIVATED BY LIGHT? – A calcium imaging approach to visualize light responses of pigeon melanopsin in vitro

5.1 INTRODUCTION

Previous studies confirmed that melanopsin is expressed in retinal ganglion cells (Chapter 3), which are already present at an early developmental stage and were able to process information via functional connections to primary visual areas before hatch in chicken and pigeons (Manns and Güntürkün, 1997; Theiss and Güntürkün, 2001; Diaz et al., 2014). Hence, melanopsin-containing retinal ganglion cells could serve as a potential inducer of visual lateralization in pigeons. Our previous studies already demonstrated the existence of two melanopsin isoforms in the pigeon (Chapter 2) and provided the first qualitative and quantitative evidence of melanopsin expression in (developing) retinal ganglion cells in the critical developmental period (Chapter 3 and 4). However, the proof that pigeon melanopsin resembles a functional sensory photopigment is still missing. Therefore, this study was conducted to test the capacity of pigeon melanopsin isoforms to from a functional sensory photopigment. Light responses of melanopsin isoforms heterologous expressed in HEK cells were analyzed via calcium imaging on the basis of the Gq-coupled signaling pathway.

5.2 MATERIAL AND METHODS

The composition of all buffers, substances and manufactures of products used in this study can be found in detail in the appendix (A. Chemicals and equipment).

5.2.1 General cloning strategy

Examining the light sensitivity of pigeon melanopsin is an important step to specify the role of melanopsin in the transduction of visual lateralization in pigeons. 91

CHAPTER 5: ANALYSIS OF MELANOPSIN LIGHT RESPONSES

Figure 5.1: Schematic overview representing the general cloning strategy of pigeon melanopsin into pm-Cherry-N1 vector. Restriction sites added to pigeon melanopsin by fusion PCR allow subsequent cloning into pmCherry-N1 expression vector. Only most important sites are outlined (dotted blue lines = restriction sites; abbreviations: AmpR = ampicillin resistance gene, Col\E1\origin = replication site, CMV-Promotor = cytomegalovirus promoter, mCherry = mCherry gene sequence, Mel S/L = melanopsin isoform S or L).

CHAPTER 5: ANALYSIS OF MELANOPSIN LIGHT RESPONSES

Therefore, the whole open reading frame of pigeons melanopsin (isoform S and L) was obtained from Life Technologies (Germany) based on the sequence identified in Chapter 2. Restriction sites for NheI or SacII were added to the first or to the last melanopsin coding nucleotides (without STOP codon), respectively, using a fusion PCR. Forward primer was flanked with NheI restriction site as well as a Kozak sequence, reverse primer comprised SacII restriction site. Kozak sequence is necessary for translation initiation (Kozak, 1990) and restriction sites for subsequent ligation to pmCherry-N1 vector (also comprising NheI and SacII restriction sites). Before ligation into pmCherry-N1 the fusion PCR product (melanopsin ORF flanked by NheI and SacII) was subcloned into a pCRTM2.1® vector system using TOPO® Cloning® (see Figure 5.1).

5.2.2 Calcium imaging

Calcium is an essential intracellular messenger in neurons. Therefore, calcium imaging provides a useful method to study elevations of intercellular Ca2+ (for review see: Grienberger and Konnerth, 2012). To examine capacity of pigeons melanopsin isoforms to form a functional sensory photopigment the activation of melanopsin upon a light pulse should be investigated on basis of the Gq signaling pathway (Panda, 2005, for an overview see: 1.2.4). Therefore, a Fluo-4 DirectTM calcium assay (Invitrogen, Germany) should be conducted in vitro analyzing the light responses of pigeon melanopsin on the basis of a Ca2+ influx into the cell. Fluo-4 provides a system which allows to detect changes in intracellular Ca2+ concentrations due to a fluorescence increase upon binding Ca2+.

5.2.2.1 Addition of NheI and SacII restriction sites for molecular cloning by PCR

The full length pigeon melanopsin (obtained from Life Technologies, Germany) was amplified using PCR (principle cf. 0). Additionally, PCR fused a NheI restriction site (and an additional Kozak sequence) in front of the START codon and a SacII restriction site at the end of the nucleotide sequence (for PCR cf. 4.2.5). The following standard reaction mix was used for a 50 µl PCR reaction (primer sequences can be found in Table 5.1):

CHAPTER 5: ANALYSIS OF MELANOPSIN LIGHT RESPONSES

Template cDNA 1 µl (10 ng Plasmid) 5’ Primer 2.5 µl (0.5 µM) 3’ Primer 2.5 µl (0.5 µM) dNTPs 1 µl (0.2 mM) Buffer BiothermTM

with MgCl2 (10x) 5 µl (1x) Taq DNA polymerase 1 µl (1 U)

PCR H2O 37 µl

The general PCR cycles used for Plasmid templates were as follows: 1. Initialization 95 °C, 5 min 2. Denaturation 94 °C, 30 s

3. Annealing 60 °C, 45 s 35 cycles 4. Elongation 72 °C, 1:45 min 5. Final Elongation 72 °C, 10 min 6. Final hold 4 °C, ∞

Table 5.1: Overview of primer used for addition of NheI and SacII restriction sites. This table indicates the direction of the primer and the added restriction sites in italic letters (blue: NheI; red: SacII) for both melanopsin isoforms and green letters indicate the Kozak sequence (abbreviations: A = adenine, bp = base pair, C = cytosine, G = guanine, T = thymine).

Melanopsin isoform S (addition of NheI + SacII restriction sites)

Forward primer 5’- GCTAGCCACCATGGGCACCCAGCCCCACG -3’

Reverse primer 5’- CCGCGGTCTTCCCTTTTCTGTGGTTGCTGCG -3’

Melanopsin isoform L (addition of NheI + SacII restriction sites)

Forward primer 5’- GCTAGCCACCATGGGCACCCAGCCCCACG -3’

Reverse primer 5’- CCGCGGTGACAGAAATTTCTCAACAGCTTCA -3’

PCR products were analyzed on a 1.5% agarose (LE, Biozym, Germany) TAE gel containing 0.001% EtBr. The 1 kb marker (Fermentas, Germany) was loaded to the gel in order to estimate the fragment sizes. All PCR products were mixed with 1x loading buffer upon loading onto agarose gel and a voltage of 135 V/cm was applied for 30 min. Afterwards bands 94

CHAPTER 5: ANALYSIS OF MELANOPSIN LIGHT RESPONSES were cut out of the gel and extracted from the gel matrix using QIAquick Extraction Kit (Qiagen, Germany). Extraction was carried out as declared in the user´s manual, though 500 µl QG Buffer and 30 µl elution buffer were used. Incubation time was prolonged to 4 minutes. As a control of an efficient extraction samples were analyzed on agarose gel as described before.

5.2.2.2 TOPO® TA® cloning and transformation

TOPO® TA Cloning® kits (Life Technologies, Germany) provide one-step cloning strategy for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector. This insertion is based on the Taq polymerase-specific property to add a single deoxyadenosine (A) to the 3´ ends of PCR products. The pCR™2.1-TOPO® vector provided with the kit contains single 3´-thymidine (T) overhangs for TA Cloning® and a covalently bound Topoisomerase I. Thymidine overhangs allow the PCR product to ligate with the vector and the Topoisomerase I ligates the fragment permanently into the vector (Shuman, 1991, 1994). TOPO® TA Cloning® reaction was conducted according to the manufactures recommendation and cloning reaction was subsequently transformed into TOP10® chemically competent E. coli cells. TOP10® E. coli cells (50 µl; Invitrogen, Germany) were thawed on ice and 2 µl of TOPO® TA® cloning reaction was added. After 30 min incubation on ice, cells were treated with a heat shock 30 s at 42 °C, followed by 2 min incubation on ice. Subsequently 250 µl S.O.C. medium (super optimal broth containing glucose, Invitrogen, Germany) was added and incubated at 37 °C for one hour. Afterwards cells were plated on pre-warmed Kanamycin plates (150 µl of transformation mixture onto LB (lysogeny broth) plates containing 50 µg/ml Kanamycin and 20 mg/ml (20 mM) X-gal. Plates were incubated overnight at 37 °C. On the next day, bacteria were selected via “blue-white screening” since bacteria containing the desired fragment were not capable of producing functional β-galactosidase. Therefore, the colonies appear white in contrast to colonies lacking the construct which appear blue (Vieira and Messing, 1982).

5.2.2.3 Plasmid mini-preparation (alkaline lysis method)

Single bacteria colonies identified as comprising the construct (appearing white) were picked with a sterile toothpick from the plate and transferred into 4 ml LB medium containing suitable antibiotics. Solitary colonies were incubated at 37 °C overnight at 300 rpm to allow

CHAPTER 5: ANALYSIS OF MELANOPSIN LIGHT RESPONSES bacteria to grow. On the next day 1.5 ml of the overnight cultures was transferred into another tube and centrifuged at 8000 rpm for 2 min. The supernatant was discarded and the pellet was re-suspended in 100 l buffer 1 (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 g/ml RNAse A). Afterwards 100 l of buffer 2 (lysis buffer; 200 mM NaOH, 1% SDS) was added to start lysis of the bacteria. Reaction tubes were inverted carefully and after 10 s pre-chilled buffer 3 (neutralization buffer; 3 M KOAc, pH 5.5) was added to stop the lysis reaction. Reaction mixture was centrifuged at 13.000 rpm for 5 min and the pellet containing the cell debris was discarded. The supernatant was transferred into a new tube containing 500 l of 96% EtOH (ethanol). The mixture was incubated at -20 °C for half an hour, afterwards shortly vortexed and immediately centrifuged at 13.000 rpm for 5 minutes. Again supernatant was discarded and 500 µl 70% EtOH was added to the pellet containing the plasmid DNA. After 3 min centrifugation at 13.000 rpm the supernatant was again discarded and the pellet was air-dried for approximately 30 min and afterwards resolved in in 30 l distilled water.

Analysis of cloning and transformation Plasmid DNA obtained from plasmid mini-preparation was analyzed using restriction digestion and subsequent gel electrophoretic analysis. Restriction sites were generated using fusion PCR (cf. 5.2.2.1). These specific restriction sites were recognized by specific restriction enzymes and these enzymes were able to cleave DNA at their individual recognition sites (Roberts, 1976) resulting in multiple DNA fragments with different length. Restriction endonucleases (NheI and SacII, NEB, Germany) were used according to the manufactures recommendations. The following standard reaction mix was used for a 30 µl digestion reaction: Template DNA 450 ng Buffer B4 10x (NEB) 3 µl (1x) BSA 3 µl (100 µM) NheI + SacII 0.5 µl each (10 U each)

PCR-grade H2O up to 30 µl The mixture was incubated at 37 °C for 30 min at 300 rpm and further analysis was conducted using the gel electrophoresis technique as described before.

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5.2.2.4 Plasmid midi-preparation

Larger amounts of plasmid DNA were required for the cell culture transfection. Therefore, plasmid midi-preparations were conducted instead of plasmid mini-preparations. An overnight culture of previously analyzed mini-preparation was incubated in 50 ml LB medium containing appropriate antibiotics at 37 °C overnight at 300 rpm. Plasmid midi-preparation was performed using Qiagen Plasmid Midi Kit 100 (Qiagen, Germany; according the manufactures recommendations) which is also based on a modified alkaline lysis method.

5.2.2.5 Cloning of melanopsin in pmCherry-N1 vector

The aim of this study is to measure light-dependent activation of melanopsin in a HEK (human embryonic kidney) cell model in vitro. Therefore, melanopsin had to be finally cloned into a pmCherry-N1 expression vector for the transfection of HEK cells. First, melanopsin previously cloned into pCRTM2.1 and amplified in E.coli, was excised from the vector using restriction enzymes. Therefore, melanopsin pCRTM2.1 plasmid (containing either melanopsin isoform S or L) and pmCherry-N1 vector were digested with NheI and SacII. As a result of the digestion, the melanopsin fragment is able to bind the complementary restriction sites on the digested pmCherry-N1 vector. Both, fragments and vector, were then ligated with each other using a T4-Ligase. The following standard reaction mix was used for a 30 µl digestion reaction: Template DNA 450 ng Buffer B4 10x (NEB) 3 µl (1x) BSA 3 µl (100 µM) NheI + SacII 0.5 µl each (10 U each)

PCR-grade H2O up to 30 µl 30 min, 37 °C CIP 1 µl (10 U) 1 h, 37 °C Inactivation of CIP, 15 min, 65 °C

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The following standard reaction mix was used for a 20 µl ligation reaction: Digested Vector DNA 1 µl Ratio 5:1 Digested construct DNA 4 µl BSA 3 µl (100 µM)

T4-Ligase 1 µl (400 U)

T4-Buffer (10x) 2 µl (1x)

PCR-grade H2O up to 20 µl 16 °C, over night

Subsequently to ligation, newly obtained melanopsin pmCherry-N1 plasmid was transformed in TOP10® chemically competent E. coli cells using the same protocol as described above (cf. 5.2.2.2). Afterwards, a plasmid mini-preparation was conducted followed by restriction digestion and subsequent gel electrophoretic analysis (for protocols cf. 5.2.2.3) to examine the transformation results. A plasmid midi-preparation was performed as described above (cf. 5.2.2.4) to gain a higher yield of plasmid DNA for HEK cell transfection.

5.2.2.6 Transfection HEK cells

HEK cells were used as a common expression system in molecular biology for studying heterologous expressed constructs (Thomas and Smart, 2005). This cell line was a transfection product of a human embryonic kidney cell line with DNA fragments of adenovirus type 5 (Graham et al., 1977). A HEK tsA-201 cell line (ATCC, American Type Culture Collection, VA, USA) was used for transient expression of either pigeon melanopsin isoform S or L in this experiment. Cells utilized in this experiment were kept in a humidified incubator under 5% CO2 at 37 °C and grew in Dulbecco’s modified eagle medium (DMEM) including 10% fetal bovine serum and 1000 U Penicillin/Streptomycin. HEK tsA-201 cells were seeded on 24-well plates one day prior to transfection. Each cell well, containing 60-70% confluent cells, was transfected using 1 µl Lipofectamine® 2000 (Invitrogen, Germany) reagent and 1 µg DNA, both were diluted in Opti-MEM (Invitrogen, Germany) for heterologous expression of melanopsin

(according to manufactures instructions). Cells were incubated at 37 °C and 5% CO2 overnight.

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Fixation of cells for microscopy Transfection control was examined under the microscope using fixated cells. Therefore, cells were seeded on poly D-lysine coated coverslips and were allowed to grow until 70% confluence. Transfection was conducted as described above. 24 h post transfection coverslips were washed 2 times with 1x PBS and incubated in 4% PFA for 30 min. Then coverslips were again washed 2 times with 1x PBS and covered with Roti®-Mount FluorCare (Carl Roth, Germany). All steps were performed protected from light.

5.2.2.7 Fluo-4 calcium assay

One day after transfection cells were seeded into poly D-lysine coated, 96-well, black wall, clear bottom microplates (50 µl/per well). On the following day cells could be used for the calcium assay. Measurements of the Ca2+ influx (increase in relative fluorescence) was conducted via Fluo-4 DirectTM Calcium Assay kit (Invitrogen, Germany). Fluo-4 calcium dye was prepared according to the manufactures manual. Cells were loaded with 50 µl of Fluo-4 calcium indicator dye and afterwards incubated for 1 h at 37 °C in 5% CO2 protected from light. In certain cases either 9-cis or all-trans retinal as co-factors were supplemented to the wells (final concentration 10 µM). Calcium assay was performed in a multilabel plate reader (Victor X3, Perkin Elmer) at room temperature. The application protocol for the measurement could be found in Table 5.2. Emission was measured with a filter for 510 nm. The protocol was programmed using Victor 2030 Workstation software (Perkin Elmer, Germany).

Table 5.2: Application protocol for Fluo-4 calcium assay. Cells were stimulated two seconds with a light pulse at 485 nm at 1 s intervals over 60 repeats.

Application protocol: Fluo-4 calcium assay

Duration [s] 2 1 Wavelength [nm] 485 light off Repeats 60

5.2.2.8 Data analysis

For data analysis, fluorescence counts were normalized to the minimum fluorescence signal and data for the same condition (9-cis, all-trans, no retinal) were averaged. This relative fluorescence increase was plotted against the time (Excel, Microsoft, USA).

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5.2.3 Fluo-4 calcium assay under the confocal microscope

Confocal microscopy technology enables live recordings of cells. Therefore, a calcium assay was performed under a confocal microscope to visualize the Ca2+ influx of the melanopsin-transfected HEK cells analyzed before in a multilabel plate reader.

5.2.3.1 Transfection HEK cells

HEK tsA-201 cells were seeded on 35 mm well plates and nurtured until a confluence of 60-70%. Cells were transfected with pigeon melanopsin (either isoform S or L) using FuGENE® HD Transfection Reagent (Promega, Germany). Transfection reagent was prepared according to the manufactures manual and loaded onto the cells in addition with 2 µg DNA.

Cells were incubated at 37 °C and 5% CO2 overnight.

5.2.3.2 Fluo-4 calcium assay

Fluo-4 calcium assay was prepared as described above (see 5.2.2.7) and 10 µM 9-cis retinal was supplemented as a co-factor. Calcium assay was monitored and digital images were acquired from cell culture using a Leica TCS SP5 confocal laser scanning microscope (Leica, Germany) and Leica Application Suite Advanced Fluorescence software (LAS AF 2.6) was used to capture all photomicrographs. Melanopsin was excited at 488 nm (22% laser intensity), calcium assay at 496 nm (27% laser intensity) and mCherry at 561 nm (36% laser intensity). Captured images were transferred into ImageJ (ImageJ, USA, Schneider et al., 2012) for processing and image overlay.

5.2.3.3 Data analysis

Data was analyzed using Test-Time Analyzer (ImageJ, USA; Schneider et al., 2012) and subsequently exported to Excel. Total intensity over time was analyzed in regions of interest (ROI). Data was normalized to the minimum fluorescence signal and relative fluorescence increase was plotted against the time (Excel, Microsoft, USA).

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5.3 RESULTS

The main objective of the present work was to analyze the possible role of melanopsin in the induction of visual asymmetries in pigeons and therefore, this study was conducted to test the capacity of pigeon melanopsin isoforms to from a functional sensory photopigment. Functional expression and light-sensitivity of pigeons melanopsin was analyzed in HEK cells in vitro. According to Kumbalasiri et al. (2007) HEK cells provide the necessary environment for proper trafficking and activation of melanopsin.

5.3.1 In vitro calcium assay

Light sensitivity is one of the key requirements of a possible trigger of light-induced visual lateralization. To validate whether both melanopsin isoforms were capable to be activated by light, calcium imaging was conducted in living HEK cells employing the Ca2+

2+ indicator dye Fluo-4. Light-induced melanopsin activation would lead to Gq-mediated Ca release via downstream activation of PLC (Panda, 2005; Peirson and Foster, 2006; Graham et al., 2008). Microscopic images of transfected HEK cells revealed that pigeon melanopsin was successfully expressed in HEK cells (Figure 5.2, isoform S: A-C, isoform L: D-F). But, isoforms S showed a strongly reduced membrane trafficking compared to isoform L. Isoform L appeared to be properly targeted to plasma membrane while isoform S seems to stick in the endoplasmatic reticulum (cf. Herrick-Davis et al., 2006). Calcium imaging was conducted in a microplate reader (2 s blue light signal, 1 s darkness, 60 repeats) and fluorescent counts were normalized to minimum fluorescence signal. Representative data is shown as a mean of six wells in Figure 5.3 (A isoform S and B isoform L). The short melanopsin isoform showed nearly no light-induced elevation in Ca2+ levels when the calcium assay was performed in the plate reader (Figure 5.3 A). A very small activation peak was detectable if 9-cis retinal (peak 1.040 ± 0.015) was supplied as a co-factor. No activation was detected either with all-trans retinal (peak 1.012 ± 0.011) or without any supplement (peak 1.011 ± 0.008). These results have to be considered carefully since these small peaks could just represent measuring artifacts.

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Figure 5.2: Representative photomicrographs showing the heterologous expression of melanopsin isoform S and L C-terminally tagged with mCherry in HEK cells. HEK cells are transfected with either the melanopsin isoform S or L-pmCherry construct. Red fluorescent pmCherry is an indicator for successfully transfected melanopsin-positive HEK cells. A (isoform S) and D (isoform L) show a transmitted-light microscope picture of the HEK cells. B (isoform S) and E (isoform L) visualize the red fluorescent pmCherry signal of transfected melanopsin-positive cells. C (isoform S) and E (isoform L) represent an overlay of both signals (Scale bars = 100 µm).

However, melanopsin isoform L triggered intracellular Ca2+ release in HEK cells when activated by a two second blue light pulse (485 nm), but the Ca2+ response was extremely dependent on the co-factors supplied (Figure 5.3 B). In comparison to all-trans (peak 1.169 ± 0.040) or no retinal (peak 1.192 ± 0.089) melanopsin isoform L induced the largest Ca2+ influx if 9-cis retinal (peak 1.849 ± 0.071) was supplied as a co-factor. In all cases, the untransfected HEK cells served as a negative control and showed no light-dependent increase in fluorescence based on a Ca2+ influx.

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Figure 5.3: Light-induced changes in intracellular Ca2+ levels by pigeon melanopsin in transfected HEK cells. Light-induced changes in intracellular Ca2+ levels were measured in HEK cells transfected with the melanopsin isoform S (A) or L (B) pmCherry constructs. Fluo-4 was used as a fluorescent Ca2+ indicator dye and measurements were conducted in a microplate reader. Fluorescent counts were normalized to minimum fluorescence signal and representative data are shown as a mean of six wells ± SEM. Two co-factors (either 9-cis or all-trans retinal) were supplemented. This demonstrates that pigeons melanopsin isoform L could be activated by blue light (485 nm) and the strongest signal is reached with 9-cis retinal as a co-factor (black dots, n = 6). All-trans retinal (black rhomb, n = 6) and no supplementation (black/grey triangle, n = 6) result in a lower signal. Isoform S only reveals barely detectable changes in fluorescence signal. Non-transfected HEK cells show no Ca2+-signal and served as a control condition (grey asterisk, n = 6).

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5.3.1.1 Comparison of isoform S and L

Figure 5.4 shows the direct comparison of both melanopsin isoforms under optimal conditions with 9-cis retinal as a supplement. Isoform L induced robust Ca2+ responses, detected by changes in fluorescence, which were considerably larger than the Ca2+ release induced by isoform S which is nearly identical to baseline control conditions of untransfected HEK cells.

Figure 5.4: Direct comparison of light-induced changes in intracellular Ca2+ levels of melanopsin isoform S and L. Light-induced changes in intracellular Ca2+ levels were measured in HEK cells transfected with the melanopsin isoform S (black squares, n = 6) or L (black dots, n = 6) pmCherry constructs. Fluo-4 was used as a fluorescent calcium indicator dye and measurements were conducted in a microplate reader. Fluorescent counts were normalized to minimum fluorescence signal and representative data are shown as a mean of six wells ± SEM. Only 9-cis as a supplement was added. This demonstrates that pigeons melanopsin isoform L showed a high Ca2+ influx when activated by blue light (485 nm), whereas isoform S produced nearly no Ca2+ influx and remained on the baseline level. Untransfected HEK cells show no Ca2+-signal and served as a control condition (grey asterisk, n = 6).

5.3.2 Live calcium assay

In contrast to a microplate reader calcium assay (as conducted in 5.3.1), which analyses a multitude of cells in a well in parallel, a calcium assay under the confocal microscope allows the visualization of Ca2+ elevation in single cells under optical control in real-time. Figure 5.5 shows reprensensitve photomicrographs of HEK cells expressing either isofrom S (A) and isofrom L (B) of example measurements under the confocal microscope.

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Figure 5.5: Representative photomicrographs showing the heterologous expression of either melanopsin isoform S (A) or L (B). Both isoforms C-terminally tagged with mCherry were expressed in HEK cells. The photomicrographs represent either the inactive state without light (light „OFF“, A.1, B.1) or the active state of melanopsin with a blue light pulse (light „ON“, A.2, A.2). Red mCherry fluorescence indicates succesfully transfected HEK cells. Green flourescence indicates Ca2+ influx detected by a calcium-sensitive fluorecent dye (Fluo-4).

Before a blue light pulse was given onto the cells, no Ca2+-dependent increase in fluorecence is visible (no green fluorescence increase). Hence, merely the red fluorecence of pmCherry is detectable in successfully transfected cells expressing one of the two melanopsin isofroms (isofrom S B.1, isofrom L B.2). In general the expression pattern described in 5.3.1 (Figure 5.2) was confirmed, with an efficient membrane transport of isofrom L. Isofrom S showed again a more clustered cytoplasmatic localization (possibly within the endoplasmatic reticulum). During a blue light pulse an increase in green fluorecence (as an indicator of Ca2+ influx due to melanopsin-dependent Gq signaling pathway activaton) was clearly dectable in all successful transfected cells. Fluorescent counts were again normalized to minimum fluorescence signals, 11 successful transfected cells for each isoform were analyzed over one minute (results can be found in Figure 5.6). In this case both isoforms showed a reasonably similar Ca2+ influx (peak isoform S 1.723 ± 0.101, isoform L 1.876 ± 0.078). Solely in cells transfected with the smaller isoform the Ca2+ influx decreases faster over time than observed for isoform L.

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Figure 5.6: Light-induced changes in intracellular Ca2+ levels of melanopsin isoform S and L. Light-induced changes in intracellular Ca2+ levels were measured in HEK cells expressing either melanopsin isoform S (grey squares, n = 6) or L (black dots, n = 6). Fluo-4 was used as a fluorescent calcium indicator dye and measurements were conducted under a confocal microscope. Fluorescent counts were normalized to minimum fluorescence signal and representative data are shown as a mean of 11 cells ± SEM. Only 9-cis as a supplement was added. This data indicates that pigeon melanopsin isoform S and L showed a reasonably identical Ca2+ influx when activated by light (485 nm).

5.4 DISCUSSION

A possible inducer of visual lateralization in pigeons has to be photosensitive and functionally present inside the retina already during the critical embryonic time frame. Using fluorescent calcium imaging, this study investigated the functionality of the recently discovered pigeon melanopsin isoforms S and L (cf. Chapter 2). Pigeon melanopsin was heterologously expressed in HEK tsA-201 cells to test its capacity to form a functional photopigment. This is a very critical point in the evaluation of melanopsin as a potential inducer of lateralization. As already shown in the previous studies of this thesis, melanopsin (isoform L) is expressed in the critical embryonic time frame in pigeons. However, to transfer asymmetrical embryonic light stimulation to the brain to shape the visual pathway into a lateralized system, melanopsin has to be light-sensitive. The induction of Ca2+ currents measured by a fluorescent Ca2+ indicator dye were used to study light-dependent melanopsin activation in vitro. Particularly melanopsin isoform L was proven to form a functional sensory photopigment. Activation of HEK cells expressing melanopsin isoform L with a blue light pulse

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(485 nm) led to a robust increase in intracellular Ca2+ as indicated by a fluorescence increase. Supplemented 9-cis retinal is necessary to reach the maximal signal, either in a multilabel plate reader or in a confocal assay, whether isoform S activation is only detectable when stably transfected cells were manually selected in a calcium assay conducted under the confocal microscope. Untransfected cells exhibited no Ca2+ signal confirming that the obtained Ca2+ signals can be traced back specifically to melanopsin activation.

5.4.1 Pigeon melanopsin is activated by blue light

To analyze if pigeon melanopsin resembles a functional sensory photopigment in the retina, functionality was tested using a calcium assay in vitro. As already explained in detail in the general introduction, in principle, melanopsin is able to activate the Gq signaling pathway (Panda, 2005; Qiu et al., 2005; Melyan et al., 2005). In this signaling cascade Ca2+ displays an essential intracellular messenger (Grienberger and Konnerth, 2012). Therefore, melanopsin was expressed in HEK tsA-201 cells which provide the necessary environment for proper trafficking and activation of melanopsin since HEK cells express protein characteristics of neuronal cells (Kumbalasiri et al., 2007). This section will focus on the functional properties of pigeon melanopsin isoform L, since isoform S only revealed a strong light-dependent activation in a few number of cells. However, differences in the functionality of both isoforms will be discussed in the next section (cf. 5.4.2). First of all, this study revealed that pigeon melanopsin exhibited the strongest Ca2+ signal in the presence of 9-cis retinal as a co-factor and this is in agreement with a bunch of studies revealing that the supplementation of 9-cis retinal or 11-cis retinal is necessary to obtain a proper melanopsin signal (Hartwick et al., 2007; Graham et al., 2008; Georg et al., 2014). In the recent years, several studies had been conducted on melanopsin proving that it forms a functional sensory photopigment which can be activated by blue light in different species (Sekaran et al., 2003; Melyan et al., 2005; Bellingham et al., 2006; Contin et al., 2010). In 2006 Contin et al. demonstrated in primary cultures of chicken retinal ganglion cells (at E8) that light had a direct effect on 3H-melatonin expression. Suppression experiments by different inhibitors of the invertebrate photocascade revealed that an early light detection is principally possible before vision occurs (Contin et al., 2006). The same group showed a connection to melanopsin-based vision and the Gq-coupled pathway. In embryonic chicken RGC cell culture expressing melanopsin a 30 s blue light stimulus was able to induce a 107

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significant and rapid elevation in IP3 levels (1.3-fold) and very rapid and distinct increase in Ca2+ levels (Contin et al., 2010). The same holds true for ipRGC cultures of neonatal rats where a light stimulus leads to an intracellular rise in Ca2+ (Hartwick et al., 2007). In mice lacking rods and cones (-/-) it was revealed that Ca2+ currents can be induced by blue light stimulation (470 nm) and that the light-evoked Ca2+ currents are dependent on intensity and duration of the light stimulus (Sekaran et al., 2003). Heterologous expression of human melanopsin in Neuro-2a cells was proven to be sufficient to render these cells photosensitive which relies on the presence of 9/11-cis retinal (Melyan et al., 2005). A similar experiment was conducted by Kumbalasiri et al. (2007). They demonstrated in a HEK cell line stably expressing human melanopsin that light stimulation leads to an increase in the intracellular Ca2+ levels. Furthermore, they showed that Ca2+ is released from internal stores by depletion experiments with thapsigargin (Kumbalasiri et al., 2007). Also in Xenopus melanophores melanopsin acts via a phosphoinositide signaling pathway which is similar to the invertebrate phototransduction pathway, since supplementation of Ca2+ inhibits melanopsin signaling (Isoldi et al., 2005; Panda, 2005). And even in the chordate amphioxus melanopsin-expressing micorvillar photoreceptors has been proven to release internal Ca2+ stores upon a light stimulus (Peinado et al., 2015). A recent study of Spoida et al. (2016) compared calcium signals of the long isoforms of human and mouse melanopsin as optogenetic tools. They demonstrated that mouse melanopsin showed a sustained Ca2+ response, whereas the human melanopsin signal immediately declined in amplitude (Spoida et al., 2016). The Ca2+ signal of pigeon melanopsin seems to be more comparable to mouse melanopsin isoform L as indicated by only a moderate decline in amplitude. However, the signaling pathway associated with melanopsin can vary depending on the used cell line (Panda, 2005; Qiu et al., 2005; Melyan et al., 2005; Sekaran et al., 2007). This study indicated via calcium imaging that the activation the

Gq-coupled signaling pathway by pigeon melanopsin is possible. However, the signaling pathway proven to be functional in a cell line may not reflect the regular transduction mechanism in retinal ganglion cells. But in RGCs melanopsin signaling seems actually to be coupled to Gq signaling (Graham et al., 2008; Hughes et al., 2015). In conclusion, pigeon melanopsin (isoform L) appears to form a functional light sensitive photopigment which is able to be activated by blue light (485 nm) and able to induce a Gq-coupled signaling pathway in general.

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5.4.2 Activation of pigeon melanopsin: isoform S vs. L

Pigeon melanopsin isoforms S or L were heterologous expressed in a HEK tsA-201 cell line to analyze the functionality of both isoforms. However, extreme differences were found between both isoforms. Isoform L appeared properly targeted to plasma membrane, while isoform S accumulates more in the cytoplasm. The membrane transport of isoform S seems to be considerably lower than of isoform L. On a closer view the short melanopsin isoform appears to stick in the endoplasmatic reticulum (cf. Herrick-Davis et al., 2006). This might be the result of the shortened C-terminus. The premature STOP codon of isoform S leads to shortened protein (442 aa; isoform L: 570 aa, first 440 aa are identical, cf. Chapter 2: 2.3.1.1). The possible implications of this reduced C-terminal tail were already discussed in detail in Chapter 2 (cf. 2.4.1.1) and are briefly summarized in this discussion. Due to the shortened C- terminus of isoform S, many protein interaction/modification sites were lost. Most importantly, isoform L exhibits twice as many N-myristoylation sites. Myristoylation, as an important co-translational modification, plays a crucial role in mediating reversible interactions with the cell membrane and regulates protein targeting and function (Farazi et al., 2001, cf. Chapter 2 2.3.2 and 2.4.1.1). Additionally, phosphorylation sites can alter the structural conformation by post-translational protein modification via the addition of a phosphate group (Glass et al., 1986, Casteel et al., 2010, Johnson, 2009) and therefore, the structure and function of isoform S might be severe damaged or impaired by the absence of four phosphorylation sites in comparison to isoform L. This might be reflected by the weak membrane trafficking demonstrated in this study. However, the general structure like the seven transmembrane domains are not affected by the shortened C-terminus, generally not excluding the formation of a functional sensory photopigment In addition to the obvious differences in membrane trafficking the analysis of functionality revealed differences as well. At first glance, results of the multilabel plate reader are contradictory to the data obtained by the confocal calcium assay. On the one hand, only a marginal fluorescence increase was detectable for isoform S in the calcium assay conducted in the plate reader, whereas a large fluorescence signal was observable in cells transfected with isoform L and 9-cis retinal as a supplement. On the other hand, the confocal calcium assay revealed that isoform S is principally able to induce Ca2+ currents (measured by an increase in fluorescence) comparable to isoform L. However, there are considerable differences between both assays. The plate reader detects a multitude of cells and the fluorescence signal 109

CHAPTER 5: ANALYSIS OF MELANOPSIN LIGHT RESPONSES represents a mean of all cells. In the confocal assay single cells were analyzed and “functionally” transfected cells can be selected. The vast majority of not functionally transfected cells (transfected cells in which isoform S remains in the endoplasmatic reticulum; included in the plate reader assay) can be excluded. These results indicate that isoform S resembles a functional sensory photopigment in principal. But possibly due to the absence of transport motifs, which might result in reduced membrane targeting, it seems to play a minor role in the transduction of light in the pigeon. In addition, a faster decline in signal was found in isoform S (confocal calcium assay) compared to isoform L. These results might be explained by the different C-terminal length as well. Also Spoida et al. (2016) suggested that the different Ca2+ signals of the long isoforms of human and mouse melanopsin might be explained by the differences in the C-terminal domains. The same might hold true for pigeons, since different phosphorylation patterns can strongly influence light-induced Ca2+ responses (Blasic et al., 2012a; Blasic et al., 2012b; Blasic et al., 2014; Fahrenkrug et al., 2014). Only a few studies have been conducted analyzing the functionality of the different melanopsin isoforms. Tomonari et al. (2005) investigated two non-mammalian (Opn4x) isoforms in chicken which are highly comparable to the two isoforms found in pigeons. Unfortunately, they did not include further functional analysis of both isoforms. Another study showed by whole cell patch-clamp recordings that chicken Opn4x and Opn4m expressed in Neuro-2a cells both encode a functional sensory photopigment, but they did not differentiate between different isoforms as well (Bellingham et al., 2006). It has to be mentioned, that Torii et al. (2007) showed results in chicken contrary to the data presented in this study for pigeons. They showed a functionality of Opn4x isoform S and Opn4m isoform L, but they failed to obtain a detectable amount of active opsins capable of binding with 11-cis retinal for Opn4xL. However, the study of Torii et al. had to be considered carefully, since they did not describe why the expression failed. In mice both isoforms of Opn4m expressed in Neuro-2a cells encode a functional sensory photopigment in the presence of 9-cis retinal (Pires et al., 2009). Different isoforms of zebrafish and mouse melanopsin were expressed in HEK293 cells by Blasic et al. (2014). This study revealed different kinetics using a calcium assay due to different C-terminal length of the melanopsin genes (Blasic et al., 2014). Altogether, these results strengths the hypothesis (cf. Chapter 2, 3 and 4) that isoform L plays a more important role in the mediation of light stimulation by melanopsin in pigeons,

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5.4.3 Summary and conclusion

The observations of this study are the first demonstration that pigeon melanopsin is able to render HEK cells in culture photosensitive, and that pigeon melanopsin is capable of sensing photic information from the environment. Blue light (485 nm) is able to induce a melanopsin-based Ca2+ current measurable due to an increase in fluorescence by a Ca2+-sensitive dye. However, this study confirms the more important role of isoform L which showed increased membrane trafficking and a higher Ca2+ signal when activated by blue light. Therefore, this study delivers important evidence for the possible role of melanopsin in the induction of visual asymmetries, since it reveals that pigeon melanopsin forms a functional sensory photopigment. This is an important requirement to be fulfilled by a potential inducer of visual lateralization in pigeons. A closer evaluation of the light-sensitive properties of melanopsin will be conducted in the next study (Chapter 6).

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CHAPTER 6: ELECTROPHYSIOLOGICAL CHARACTERIZATION OF PIGEON

MELANOPSIN ON THE BASIS OF THE Gi/o SIGNALING PATHWAY IN VITRO

6.1 INTRODUCTION

Melanopsin, a photosensitive pigment, first identified from dermal melanophores in Xenopus (Provencio et al., 1998b), is introduced as a potential lateralization inducer in this thesis. Our previous studies, as detailed above, have confirmed the presence of two pigeon melanopsin isoforms in general, and the presence of isoform L during the critical embryonic time frame (Chapter 2, 3 and 4). Additionally, blue light sensitivity and the ability to form a functional sensory photopigment in HEK cells could be evidenced (Chapter 5). However, a closer analysis of the electrophysiological properties had not been conducted yet, and the functional presence inside the retina before hatch is a key element a possible inducer has to fulfill. Therefore, this study aims to unravel the light-mediated performance of pigeon melanopsin. Melanopsin in general is able to activate either the Gq-coupled signaling pathway (Panda, 2005; Qiu et al., 2005; Melyan et al., 2005; already investigated in Chapter 5) or the

Gi/o pathway (Bailes and Lucas, 2013). Hence, the electrophysiological properties of pigeon melanopsin should be investigated on the basis of the Gi/o signaling pathway in vitro by patch-clamp experiments.

6.2 MATERIAL AND METHODS

The composition of all buffers, substances and manufactures of products used in this study can be found in detail in the appendix (A. Chemicals and equipment).

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6.2.1 Recloning of pigeon melanopsin

Before starting the patch-clamp experiments melanopsin had to be recloned into a pEGFP-N1 vector (Addgene, USA) for methodical reasons, since the green fluorescence by the EGFP tag was more easily detected by the microscope than the red fluorescence of the mCherry tag (pmCherry-N1 vector).

Figure 6.1: Schematic overview representing the recloning strategy of pigeon melanopsin. Melanopsin pmCherry-N1 vector was digested with the same restriction enzymes as a pEGFP-N1 expression vector which allowed subsequent cloning of melanopsin into pEGFP-N1 expression vector. Only most important sites are outlined (dotted blue lines = restriction sites, abbreviations: AmpR = ampicillin resistance gene, bp = base pair, CMV-Promotor = cytomegalovirus promoter, EGFP = EGFP gene sequence, mCherry = mCherry gene sequence, Mel S/L = melanopsin isoform S or L).

EGFP-N1 vector and melanopsin cloned into pmCherry-N1 (now called pmCherry- N1_Mel_S: comprising isoform S; pmCherry-N1_Mel_L: comprising isoform L) were both 113

CHAPTER 6: ELECTROPHYSIOLOGICAL MELANOPSIN CHARACTERIZATION digested with NheI and SacII as described before (cf. 5.2.2.5). Fragments were purified using gel electrophoresis and bands were excised from the gel matrix (Wizard SV Gel and PCR Clean- Up System, Promega, Germany; cf. 5.2.2.1). Afterwards fragments were ligated and transformed into competent TOP10® E. coli with the same procedure as described before (cf. 5.2.2.5; Figure 6.1). Transformation efficiency was screened using a plasmid mini-preparation with subsequent restriction digestion and gel electrophoretic analysis (cf. 5.2.2.3). Subsequent to a successful validation of the product, constructs were amplified using a plasmid midi-preparation (cf. 5.2.2.4). All purified constructs (now designated as pEGFP-N1_Mel_S and pEGFP-N1_Mel_L) were sequenced by the sequencing service of the Faculty of Biochemistry, Ruhr-University Bochum.

6.2.2 Transfection HEK cells

HEK 1.2 cells used in this experiment were developed from a HEK293 cell line, which stably expressed G-protein-coupled inwardly-rectifying potassium channel (GIRK) subunits 1 and 2 (courtesy of Dr. A. Tinker, UCL London). Thus, this cell line was an ideal model to investigate the electrophysiological properties of melanopsin isoform L based on the Gi/o signaling pathway.

Cell culture and cell splitting Adherent growing HEK 1.2 cells were cultured in T-25 cell culture flasks containing 5 ml

HEK 1.2 media in incubator with 5% CO2 at 37 °C. HEK 1.2 media comprises the antibiotic G418 (Geneticin®; Gibco, Germany) which allowed a selection cells which stably express GIRK channels. At an optic density of 80% cells were split. Before cell splitting old cell culture medium was discarded and substituted with 3 ml fresh medium per flask. Afterwards cells were rinsed from the bottom of the flask carefully and transferred into a new 15 ml tube. Cells were now singularized and the desired volume (depending on the density) was transferred into a new cell culture flask containing fresh HEK 1.2 medium. Subcultures in 35 mm cell culture wells containing 2 ml HEK 1.2 medium was necessary for cell transfection for patch-clamp experiments. Depending on the cell density between 20 and 70 µl cell suspension was added to the wells. Cells were ready for transfection with plasmid DNA approximately two days after cell seeding of the subcultures and achieving a confluence of 70 to 80%.

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Transfection with FuGENE® HD Transfection Reagent Transfection of cells was conducted using FuGENE® HD Transfection Reagent (Promega, Germany) as declared in the user’s manual. Two µg of plasmid DNA of pEGFP-N1_Mel_S and L were used for transfection of a 35 mm well. Before transfection cell media was exchanged for an antibiotic-free medium. Subsequent to transfection cell were incubated 24 h in the incubator. On the experimental day cell were singularized via cell splitting as described for the main culture above. For a faster and better adhesion, flasks were covered with Poly-L-lysine (Sigma-Aldrich, Germany). Afterwards, cells were incubated at least 2 h in the incubator and since transfection cells were stored in lightproof wells for light protection.

6.2.3 In vitro patch-clamp

There are different possibilities of configurations for patch-clamp experiments like an inside-out patch, an outside-out patch or a whole cell patch (Sakmann and Neher, 1984). In the following experiments a whole cell patch was applied. The cell attached configuration originally developed by Neher and Sakmann (1976) builds the basis for the whole cell patch. A patch micropipette is slowly attached to the cell with a slight overpressure to avoid contaminations of the pipette tip. Then, to obtain a high resistance seal called giga seal, the micropipette is pressed against a cell membrane and a short negative pressure is applied. The electrical resistance across the seal is then in the dimension of one gigaohm or higher and the membrane is firmly attached to the pipette (Neher and Sakmann, 1976, Sakmann and Neher, 1984; see Figure 6.2).

Patch-clamp process All pipettes were pulled on the experimental day and were subsequently fire polished. Patch pipettes should have a resistance between 2 and 7 MΩ and the tip diameter should range between 0.5 and 2 µm. The osmolarity of the intra- and extra cellular medium should be around 295 and 305 mOsm (extra cellular solution: 20 mM NaCl, 120 mM KCl, 2 mM CaCl2, 10 mM HEPES-NaOH, pH 7.3, stored at 4 °C; intra cellular solution: 100 mM K-Aspartate,

40 mM KCl, 5 mM MgATP, 10 mM HEPES-NaOH, 2 mM EGTA, 2 mM MgCl2, 0.01 mM NaGTP, pH 7.3, stored at -20 °C). Patch laboratory was darkened and all experiments were conducted at room temperature (25 °C). All measurements were executed in the whole cell modus. For 115

CHAPTER 6: ELECTROPHYSIOLOGICAL MELANOPSIN CHARACTERIZATION preparation of the cells medium was exchanged against extracellular medium (5 ml) and 9-cis retinal was added to a final concentration of 1 µM. Then, successfully transfected cells were selected via the green fluorescence tag (eGFP) using the polychrome on the patch microscope. The actual patch process was conducted without visual control just on the basis of changes of the electrical resistance. Regulation of the amplifier (EPC-9) was performed via Pulse & PulseFit software (HEKA Elektronik, Germany) and the patch pipette was adjusted via a micromanipulator. The patch-clamp process was started with plunging the pipette into extracellular medium (Figure 6.2 A and B). A rectangular test pulse of +5 mV with a duration of 5 ms was applied with the amplifier. Driving the pipette up to the cell the resistance increased (Figure 2.1 B).

Figure 6.2: Schematic drawing of whole cell patch-clamp measurement configuration. The whole cell patch-clamp configuration was used to measure light-evoked melanopsin currents via the GIRK signaling pathway. First the pipette was plunged into the extracellular medium (A) and directed to close proximity of a transfected cell (B). A short suction led to the cell-attached configuration (C, resistance Rm exceeded 1 GΩ, gigaohm seal). Again a short and strong suction was applied to rip the cell membrane off within the pipette tip. This leads to the occurrence of capacitive currents as a confirmation of the whole cell configuration (D). All measurements conducted were executed in the whole cell modus (modified from Li, 2008; Kodandaramaiah et al., 2012).

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Table 6.1: Application protocols of different patch-clamp experiments in HEK 1.2 cells expressing pigeons melanopsin isoform L. Protocol A shows the simple stimulation protocol. B indicates the long-term activation measurement protocol. Repetitive activation was measured using protocol C. Protocol D measured the dependency on the duration of the activation pulse. Protocol E was run to measure the wavelength dependency. In all protocols cells were recorded for the whole time.

Application protocol A: simple stimulation

Duration [s] 10 1 10 40 Wavelength [nm] light off 470 light off 560 Repeats 1

Application protocol B: long-term activation

Duration [s] 10 0.2 290 50 Wavelength [nm] light off 470 light off 560 Repeats 1

Application protocol C: repetitive activation

Duration [s] 10 0.2 9 40 Wavelength [nm] light off 470 light off 560 Repeats 20

Application protocol D: duration activation pulse

Duration [s] 10 0.1 - 10 10 10 10 30 Wavelength [nm] light off 470 light off 560 light off 560 Repeats 5x: 100 ms, 200 ms, 500 ms, 1000 ms; pseudo-randomized

Application protocol E: wavelength dependency

Duration [s] 10 1 10 40 Wavelength [nm] light off 380380 -650 650 light off 560 Repeats 28 in 10 mm steps; pseudo-randomized

After releasing the gauge pressure and producing negative pressure the occurrence of a giga seal was checked. A giga seal was equated with a cell-attached configuration and had to meet the following criteria (Figure 6.2 C): I. Flat current track II. Capacitive artifacts at the beginning and the end of a pulse

III. Resistance Rm exceeded 1 GΩ

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Capacitive artifacts were manually corrected using the amplifier, and the cell membrane was ripped off under the pipette through a short and strong negative pressure. For a successful whole cell configuration the following criterion had to be met (Figure 6.2 D): occurrence of capacitive currents (larger than in cell attached configuration). Again, these capacitive artifacts were manually corrected via the amplifier (electric charge and discharge of the cell membrane). Then, to measure melanopsin-mediated currents, cell was clamped to a membrane potential of -60 mV. The extracellular solution had a high potassium concentration of 120 mM, which allowed measuring an activation of the GIRK channels coupled to the Gi/o signaling pathway by a potassium influx. These measurements were possible because the equilibrium potential of potassium is 0 mV, which exceeds the normal membrane potential (-60 mV). The exact measurement/application protocols could be found in Table 6.1.

6.2.4 Statistical analysis

Data obtained from electrophysiological recordings were statistically analyzed using SigmaPlot 12.0 software (Systat Software, USA). A Shapiro–Wilk test was used to check the normal distribution of the data sets. A Mann–Whitney–Wilcoxon/Wilcoxon rank-sum test was conducted for post-hoc comparisons. For all results significance threshold was set at p = 0.05 (represented by *, p<0.01 = **, p≤0.001 = ***). All data are expressed as means ± SEM.

6.3 RESULTS

The electrophysiological properties of melanopsin play a crucial importance in the evaluation of melanopsin as a possible inducer of lateralization. The results of Chapter 5 already showed the blue light-dependent activation of melanopsin. Hence, these properties were closer analyzed using a patch-clamp approach. Melanopsin responses to different stimulation protocols were measured on the basis of the Gi/o signaling pathway in HEK cells stably expressing GIRK channel subunits 1 and 2. Previous studies on human and mouse melanopsin already showed that the Gi/o signaling pathway is a suitable way to analyze light-dependent melanopsin activation (Spoida et al., 2016). All measurements were conducted using a whole cell patch-clamp mode. Unfortunately, melanopsin isoform S could

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CHAPTER 6: ELECTROPHYSIOLOGICAL MELANOPSIN CHARACTERIZATION not be electrophysiologically analyzed, due to reduced expression and weak membrane transport as already indicated in Chapter 5.

6.3.1 Light-dependent melanopsin activation on the basis of the Gi/o signaling pathway

Calcium imaging data from Chapter 5 already allowed evidence for light-dependent activation of pigeon melanopsin, especially isoform L. In this study, the light-dependent activation was closer analyzed using a patch-clamp approach. The analysis showed that pigeon melanopsin isoform L induced large GIRK currents after activation with a blue light pulse (470 nm; Figure 6.3 A and B, 1 s light stimulation with 470 nm and 1 nW/mm2). These robust currents occur after an activation by a light pulse with a maximal amplitude of 118 pA ± 6.6 pA (mean of seven cells; Figure 6.3 B). These melanopsin induced GIRK currents could be completely deactivated with a second light pulse of a longer wavelength (green light, 560 nm).

Application protocol A: simple stimulation

Duration [s] 10 1 10 40 Wavelength [nm] light off 470 light off 560 Repeats 1

Figure 6.3: Simple stimulation of pigeon melanopsin isoform L in vitro. A simple stimulation of pigeon melanopsin isoform L (application protocol A). An example measurement track shows the melanopsin- dependent activation of GIRK currents (light pulse of 470 nm). Currents can be deactivated with a second light pulse of 560 nm. B Current amplitude in transfected cells (n = 7). C Kinetics of activation and deactivation time constants of melanopsin-induced GIRK currents (n = 7, application protocol A). All data are shown as means ± SEM (*** p < 0.001; blue boxes: light stimulation at 470 nm; green boxes: light stimulation at 560 nm; grey boxes: darkness; n = number of experiments; pA = pico ampere). Comparison of the kinetics of activation and deactivation time constants of melanopsin-induced GIRK currents revealed a significant faster activation (τON = 1.86 ± 0.02) than deactivation kinetic (τOFF = 5.72 ± 0.003; p < 0.001; Figure 6.3 D). Long-term Stimulation 119

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(Figure 6.4) revealed that GIRK currents were maintained for several minutes after one 0.2 s activation pulse (470 nm), although the currents amplitude declines over time, but remained at approximately 40% of the maximal amplitude. However, these currents, which were maintained over several minutes, were only completely deactivated after the application of a long-wavelength light pulse.

Application protocol B: long-term activation

Duration [s] 10 0.2 290 50 Wavelength [nm] light off 470 light off 560 Repeats 1

Figure 6.4: Long-term stimulation of pigeon melanopsin isoform L in vitro. Long-term stimulation (application protocol B). Example measurement track shows that current sustained over minutes at ~40% of the maximal amplitude. All data are shown as means ± SEM (blue boxes: light stimulation at 470 nm; green boxes: light stimulation at 560 nm; grey boxes: darkness; n = number of experiments; pA = pico ampere).

6.3.2 Repetitive activation of pigeon melanopsin

Possible repetitive activation was analyzed applying a 200 ms blue light pulse followed by darkness (9s) and deactivation (green light, 560 nm, 40s). Pigeon melanopsin isoform L was capable of a repetitive activation of GIRK currents over 20 trails. Relative GIRK currents did not decrease over these trails (Figure 6.5 A and B). Dependency of activation pulse duration was investigated by activating melanopsin with blue light pulses of different duration (100 ms - 10 s), and after each trial melanopsin was deactivated by a green light pulse (560 nm). This experiment revealed that a 200 ms blue light pulse sufficed for an almost complete activation of pigeon melanopsin isoform L (relative GIRK current 0.934 ± 0.022).

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Application protocol C: repetitive activation

Duration [s] 10 0.2 9 40 Wavelength [nm] light off 470 light off 560 Repeats 20

Figure 6.5: Repetitive activation of pigeon melanopsin isoform L in vitro. A Example measurement track shows the repetitive activation of melanopsin (application protocol 3). GIRK currents are induced at 470 nm and deactivated with 560 nm in transfected cells. B Comparison of the maximal amplitude of GIRK currents in transfected cells in the curse of repetitive activation (n = 6). All data is shown as a mean ± SEM (blue boxes: light stimulation at 460 nm; green boxes: light stimulation at 560 nm; grey boxes: darkness; n = number of experiments; pA = pico ampere).

A light pulse of 100 ms induced already around 65% of the maximal GIRK current (relative GIRK current 0.624 ± 0.455). Light pulses between 200 ms and 1 s lead to maximal GIRK current activation (relative GIRK current 500 ms: 0.949 ± 0.029, 1 s: 0.913 ± 0.039), while light pulses over 10 s showed a slightly reduced current amplitude (Figure 6.6; relative GIRK current 0.777 ± 0.039).

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Application protocol D: duration activation pulse

Duration [s] 10 0.1 - 10 10 10 10 30 Wavelength [nm] light off 470 light off 560 light off 560 Repeats 5x: 100 ms, 200 ms, 500 ms, 1000 ms; pseudo-randomized

Figure 6.6: Dependency of activation pulse duration on the relative amplitude of the GIRK currents. Comparison of the maximal melanopsin evoked GIRK currents in transfected cell (application protocol D, n = 7). All data is shown as a mean ± SEM (n = number of experiments; ms = millisecond).

6.3.3 Wavelength-dependence of pigeon melanopsin activation

The previously described experiments revealed that pigeon melanopsin is able to induce robust GIRK currents when activated with one blue light pulse of 470 nm and that deactivation is possible with green light pulses of 560 nm. Hence, the question about a wavelength-dependency arose, since only two different wavelength were tested for pigeon melanopsin before now (470 nm and 560 nm). Therefore, wavelength-dependency of activation was tested with repetitive stimulation with different wavelength ranging from 380 – 650 nm (in a pseudo-randomized manner). The maximal GIRK currents induced by pigeon melanopsin were detected with the application of wavelength ranging from 420 nm to 470 nm. The maximal current was observable at 430 nm (relative GIRK current 0.929 ± 0.023). Half maximal activation was achieved with a wavelength of 520 nm (relative GIRK current 0.482 ± 0.070). Starting from a wavelength of 540 nm no activation of pigeon melanopsin isoform L was detectable (Figure 6.7, relative GIRK current 560 nm: 0.025 ± 0.011, 620 nm: 0.024 ± 0.016).

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Application protocol E: wavelength dependency

Duration [s] 10 1 10 40 Wavelength [nm] light off 380380 -650 650 light off 560 Repeats 28 in 10 mm steps; pseudo-randomized

Figure 6.7: Wavelength-dependency of pigeon melanopsin isoform L. Comparison of the maximal melanopsin evoked GIRK currents in transfected cells in dependency of the applied wavelength (application protocol E, n = 5). All data is shown as a mean ± SEM.

6.4 DISCUSSION

This study characterized pigeon melanopsin isoform L electrophysiologically on the basis of the Gi/o signaling pathway in a HEK cells line stably expressing subunit 1 and 2 of GIRK channels. A previous study conducted by Spoida et al. (2016) already confirmed the practicability of this pathway to characterize the principal actions of melanopsin by light activation. The results of this experimental series illustrated that melanopsin isoform L induces robust GIRK currents when activated with a blue light pulse of 470 nm. These currents can be maintained (at approximately 40% of the maximum current) for several minutes and just a short activation pulse of 200 ms is sufficient for maximal activation. Pigeon melanopsin evoked GIRK currents can be deactivated with a green light pulse at 560 nm, but it can be subsequently repetitively activated without a decrease of the amplitude of the currents. Analysis of the wavelength dependency revealed that isoform L is mainly activated by blue light ranging from wavelength between 420 nm and 470 nm. No activation can be found if wavelengths starting at 540 nm were applied. Overall, these results demonstrate that pigeon melanopsin isoform L forms a functional opsin which is light-sensitive and blue light activation leads to robust and strong currents.

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Unfortunately, pigeon melanopsin isoform S could not be analyzed to due low expression levels and week membrane transport. The shortened C-terminus (already described in detail in Chapter 2, cf. 2.4.1.1) could play a crucial role, since the C-terminus is essential for membrane trafficking, subcellular localization and G-protein binding (Glass et al., 1986, Imperiali and O'Connor, 1999; Farazi et al., 2001; Johnson, 2009; Casteel et al., 2010; Oh et al., 2010). This seems to play a key role for the expression differences of isoform S and L. However, our previous studies (Chapter 3, 4 and 5) already strengthened the hypothesis that isoform L might play the major role in asymmetry formation in pigeons. Possible implication of isoform L will be discussed in the following sections.

6.4.1 Pigeon melanopsin is able to activate the Gi/o signaling pathway

The results of this study confirm that pigeon melanopsin is capable of the activation the Gi/o signaling pathway in addition to the Gq-coupled signaling pathway, which had been proven in Chapter 5. In general, a light activation of the Gi/o pathway results in a cleavage of the βγ subunit of the G-protein-coupled receptor from Gα subunit. The βγ subunit activates the GIRK channel which leads to a K+ flux (Riven et al., 2006; Stewart et al., 2012; Casoni et al., 2012; Bailes and Lucas, 2013). Hence, these K+ currents were detected in HEK cells (comprising GIRK channels) expressing pigeon melanopsin isoform L in the conducted patch-clamp experiments when activated with blue light. This is proof of principle that this expression system in suitable to analyze the photochemical properties of pigeons melanopsin isoform L.

The activation of both the Gq- and Gi/o-coupled signaling pathways was also confirmed in human melanopsin (Bailes and Lucas, 2013). As already mentioned before, the signaling pathway associated with melanopsin can vary depending on the expression system (Panda, 2005; Qiu et al., 2005; Melyan et al., 2005; Sekaran et al., 2007). The signaling pathway proven to be functional in a cell line may not reflect the native transduction mechanism in retinal ganglion cells. However, pigeon melanopsin has been proven to be able to activate the Gq- (cf.

Chapter 5) and Gi/o-coupled signaling pathway if it is expressed in different HEK cells lines. In the retina the Gq-coupled signaling is reported to be the major pathway (Graham et al., 2008;

Schmidt et al., 2011) whereas Gi/o-coupled signaling is conceivable in somata and dendrites of retinal neurons as well (Clark et al., 2009).

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6.4.2 Electrophysiological properties of pigeon melanopsin in comparison to other melanopsins

Analyses of the electrophysiological properties of pigeon melanopsin revealed a lot of similarities to other melanopsins analyzed by other research groups. The fast kinetics of the induced currents in the conducted experiments were comparable to cone opsins which were used for the control of the Gi/o signaling pathway (Masseck et al., 2014). The wavelength dependency revealed a maximal amplitude in a blue-light wavelength ranging between 420 and 470 nm. Blue-light sensitivity of melanopsin was also confirmed for example in chicken (476-484 nm), mouse (467 nm) and human (479 nm; Panda, 2005; Torii et al., 2007; Matsuyama et al., 2012; Bailes and Lucas, 2013). Mouse melanopsin expressed in COS cells presented the maximum activation in the mid blue range (420-440 nm). However, the authors suggested that the range might be shifted due to the expression in COS cells (Newman et al., 2003). The possibility of repetitive activation and the fact that the relative GIRK currents did not decrease over all activation trials strongly argues for a bistability of pigeon melanopsin. Bistable opsins do not need external photoisomerases which otherwise regenerate the retinaldehyde all-trans to 11-cis retinal, due to an intrinsic photoisomerase activity of melanopsin (Shichida and Matsuyama, 2009). It has been demonstrated that mouse melanopsin heterologous expressed in HEK cells is capable of re-isomerization of all-trans retinal to 11-cis retinal (Matsuyama et al., 2012). Additionally, Matsuyama et al. (2012) proposed a bistability of melanopsin. They claimed that melanopsin bound to 11-cis retinal resided in the resting state whereas the active melanopsin (entitled as meta-melanopsin) binds a converted all-trans retinal (Matsuyama et al., 2012). This bistability was also demonstrated for human melanopsin (Melyan et al., 2005; Panda, 2005). In 2015 Emanuel and Do also suggested a tristability of mouse melanopsin including two resting states and one signaling state (Emanuel and Do, 2015). Despite the robust currents induced by pigeon melanopsin upon a blue light pulse an intrinsic desensitization tendency is observable remaining at approximately 40% of the maximal amplitude. After termination of the activating light pulse the induced currents declined. However, the currents remained on the reduced level for several minutes without further reductions. It is conceivable that only some melanopsin pigments remain active after termination of the light pulse whereas another portion returns to the resting state. This might

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CHAPTER 6: ELECTROPHYSIOLOGICAL MELANOPSIN CHARACTERIZATION potentially be explained by bleaching mechanisms or a necessary regeneration of retinal (Do and Yau, 2013; Emanuel and Do, 2015). Additionally, the long isoform of pigeon melanopsin behaves rather similar to human and to some extend to mouse melanopsin (Spoida et al., 2016). Spoida et al. (2016) compared the different biophysical properties of the long isoforms of human and mouse melanopsin heterologous expressed in HEK cells comprising GIRK channel subunits 1 and 2. They were able to demonstrate that that mouse melanopsin was maximally activated by a short light pulse of 100 ms and a repetitive activation was possible without a decline in amplitude. In human melanopsin, a light pulse >500 ms was required for maximal activation and repetitive activation led to a decrease in the amplitude (50% of the primary amplitude). Both melanopsins were activated by a blue light pulse, but the amplitude declined to 20% in human melanopsin over time (Spoida et al., 2016). Pigeon melanopsin seems to resemble properties of both mammalian melanopsins compared to these data obtained from human and mouse. First, pigeon melanopsin shares similarities with human melanopsin. Both amplitudes declined over time, but the amplitude of pigeon melanopsin remains at ~40%, whereas human melanopsin declined to 20%. However, similarities were found to mouse melanopsin as well. In both melanopsins a very short activation pulse was sufficient (pigeon: 200 ms, mouse: 100 ms) and the signal amplitude did not decline by repetitive activation. The time constants as well as the half-maximal activation was comparable to both, human and mouse melanopsin (half maximal activation: pigeon (520 nm), human (505 nm), mouse (525 nm); Spoida et al., 2016). In conclusion, all three opsins were able to induce robust currents mediated via GIRK channels upon an activating light stimulus and were able to control the Gi/o signaling pathway. Solely the amplitude of the induced currents of pigeon melanopsin is smaller compared to human and mouse melanopsin. Differences of pigeon melanopsin compared to human and mouse melanopsin might be explained by sequence differences. Both human and mouse melanopsin belong to the group of mammalian melanopsins (Opn4m) whereas pigeon melanopsin resembles the group of non-mammalian melanopsins (Opn4x). Sequence analysis (cf. Chapter 2) revealed a slightly higher query coverage (QC) between human and pigeon melanopsin in comparison to pigeon and mouse melanopsin (human: QC = 73%, identity = 45%; mouse: QC = 60%, identity = 53%). Sequence variations might explain differences in electrophysiological properties. This is in accordance to the study of Bellingham et al. (2006) which characterized Opn4x and Opn4m of chicken (unfortunately not considering the

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CHAPTER 6: ELECTROPHYSIOLOGICAL MELANOPSIN CHARACTERIZATION different isoforms). Melanopsin-expressing Neuro-2A cells provoked larger currents when expressing Opn4m than Opn4x in patch-clamp recordings. Another study also conducted in Neuro-2A cells expressing mouse melanopsin demonstrated light-dependent inward currents in the presence of 9-cis retinal in a whole cell patch-clamp approach (Pires et al., 2009). Furthermore, no differences in regard to the amplitude, spectral sensitivity and kinetics were found in comparison between mouse isoform S and L (Pires et al., 2009). The same holds true for Neuro-2A cells expressing human melanopsin were a sustained inward current was found upon a light stimulus (Melyan et al., 2005). And a perforate patch-clamp study conducted in ipRGC cultures from neonatal rats demonstrated action potentials in response to light exposure (Hartwick et al., 2007). In conclusion, pigeon melanopsin forms a functional sensory opsin, which is maximally activated with blue light in a range of 420-470 nm.

6.4.3 Electrophysiological properties of melanopsin isoform L and their possible influence on the induction of visual asymmetries

First of all, this study clearly demonstrated that the pigeon melanopsin (isoform L) gene sequence encodes a functional sensory photopigment with a maximal blue light sensitivity ranging between 420 and 470 nm. This is in concordance with the previous results (Chapter 5, melanopsin can be activated by a 485 nm light pulse) and has a strong impact on the role of melanopsin as a potential inducer. A potential inducer has to be activated by a wavelength that is able to pass through the eggshell and this holds true for blue light (Rogers, 2006; Maurer et al., 2015). Additionally, the analysis of the activation pulse dependency revealed that a short blue light pulse (200 ms up to 1 s) sufficed for an almost complete activation of pigeon melanopsin isoform L. This indicates that only short light exposure is sufficient to activate a robust melanopsin-based current. Pigeon eggs were incubated by the adult pigeons which indeed regularly leave the nest, but for a mere period of time (Buschmann et al., 2006). Therefore, it is quite important that a short light pulse is sufficient for melanopsin activation and that the short periods of asymmetrical light stimulation can be transferred onto higher brain regions. Furthermore, melanopsin can be repetitively activated by light and most importantly a short light pulse results in a long lasting activation of the melanopsin-containing cell. This remarkably sustained melanopsin responses were found in different melanopsins in mice and rats (Hannibal et al., 2001; Wong et al., 2005; Hannibal et al., 2013). In a melanopsin 127

CHAPTER 6: ELECTROPHYSIOLOGICAL MELANOPSIN CHARACTERIZATION knock-out mice model, Zhang et al. (2012) were able to demonstrate that retinal dopaminergic neurons showed no sustained light responses whereas an absence of the melanopsin knock-out allowed these sustained responses. Therefore, this sustained activation can be traced back to melanopsin, since the transmission of rods and cons was blocked (Zhang et al., 2012). As already lined out the photochemical properties exhibited by pigeon melanopsin especially strengthen an influence of melanopsin isoform L in the transmission of visual lateralization during embryonic development in pigeons.

6.4.4 Summary and conclusion

The aim of this study was to characterize melanopsin in regard to its electrophysiological properties to function as a potential inducer of lateralization in pigeons. The expression of melanopsin in the critical time period has already been proven in previous studies (Chapter 3 and 4). Additionally, the first evidence that pigeon melanopsin forms a functional blue light-sensitive photopigment was demonstrated in Chapter 5. In this study, the functionality was closer analyzed and revealed a blue light sensitivity, as well as a sustained responses to a short light stimulus. Furthermore, a repetitive activation was possible without a decrease in the signal amplitude. To sum up, the electrophysiological properties of pigeon melanopsin confirmed its ability to form a functional sensory photopigment and strengthen its role as a potential inducer. A short blue light incidence is sufficient to induce long-lasting activation of the retinal cells even after disappearance of the light pulse. This is important to transfer only short but repetitive asymmetrical embryonic light stimulation onto higher brain region to manifest structural and behavioral lateralization.

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CHAPTER 7: GENERAL DISCUSSION

The aim of this thesis was to characterize melanopsin in regard to its properties to function as a potential inducer of lateralization in pigeons. The current data provide the evidence that two isoforms of a non-mammalian/invertebrate-like melanopsin gene are expressed in pigeons. Furthermore, this thesis demonstrates the expression of a long melanopsin isoform during the critical embryonic time frame of asymmetry induction shortly before hatch. In addition, the potential signaling pathways of pigeon melanopsin were investigated, proving that pigeon melanopsin is able to activate either a Gq- or Gi/o-coupled signaling pathway in vitro. Moreover, current data represents the evidence that pigeon melanopsin forms a functional photosensitive pigment inside the retina. Therefore, this thesis provides the first data for an advanced role of melanopsin in the transduction of an asymmetrical light stimulus during embryonic development in pigeons. Even though no direct proof of melanopsin-containing RGC activation during the critical time frame was feasible, this study presents fundamental arguments that melanopsin is a promising candidate for light-dependent asymmetry induction in pigeons. The following sections will discuss the implications of these data on the induction of visual asymmetries, possible interaction partners and future studies that should be conducted to finally prove the influence of melanopsin on the induction of lateralization in pigeons.

7.1 Melanopsin is able to influence the ontogeny of visual lateralization in pigeons

Melanopsin represents a “third” photosensitive instance in addition to rods and cones in the retina (Berson, 2003). To elucidate the role of melanopsin in the induction of visual lateralization in pigeons, the four requirements stated in the general introduction will be considered in regard to the data obtained in this thesis. However, this discussion will mainly focus on melanopsin isoform L, since isoform L was demonstrated to be significantly expressed during the critical developmental stage and was proven to form a functional sensory 129

CHAPTER 7: GENERAL DISCUSSION photopigment. Therefore, a closer look at the four main requirements already introduced in the general introduction (cf. 1.1.3 and 1.3) elucidates the promising role of pigeon melanopsin isoform L as an inducer of lateralization (for an graphical overview see Figure 7.1): I. The possible inducer has to be photosensitive. The sequence of pigeon melanopsin (Opn4xS and L) provides the core features of an opsin enabling light detection (cf. Chapter 2). Additionally, calcium imaging as well as patch-clamp experiments in vitro confirmed the activation of pigeon melanopsin upon light incidence (cf. Chapter 5 and 6). II. The possible inducer has to be already functionally present inside the retina during the critical embryonic time frame. As already shown in chicken and mice (Verra et al., 2011; Bertolesi et al., 2014; Diaz et al., 2014), immunohistochemical staining as well as qPCR analyses revealed that pigeon melanopsin isoform L is expressed during the critical time period shortly before (and also after) hatch in pigeons (E16, PH2; cf. Chapter 3 and 4). III. The possible inducer must be activated by wavelengths that are able to pass through the eggshell. In vitro patch-clamp data revealed that pigeon melanopsin forms a blue light-sensitive photopigment, with a maximum absorption peak at 430 nm (cf. Chapter 6) comparable to other species melanopsins (Panda, 2005; Torii et al., 2007). These wavelengths are able to pass through the eggshell (Rogers, 2006; Maurer et al., 2015). IV. The possible inducer must be able to propagate the sensory input to visual areas of the brain to shape the visual pathway into a lateralized system. Previous studies showed that RGCs possess functional connections to the brain before hatch (Theiss and Güntürkün, 2001), and first synapses of RGC axons on tectal neurons were established during embryonic development starting from E15 in pigeons (Manns and Güntürkün, 1997). The tectal tracing conducted in this thesis revealed the expression of melanopsin in RGCs in adult pigeons (cf. Chapter 3). Therefore, a signal propagation of activated RGCs to higher brain areas is conceivable already in pigeon embryos and hatchling. Nevertheless, a direct evidence of melanopsin involvement has to be further investigated (cf. 7.3).

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Figure 7.1: Illustration of the requirements melanopsin has to fullfill as a possible inducer of visual lateralization in pigeons. Melanopsin (isoform L) was characterized in several studies as a potential inducer of the formation of visual asymmetries in pigeons in this thesis. Exemplary data is shown which provides evidence for melanopsin as a potential inducer. Green background marks the components which are proven to be fulfilled by melanopsin, whereas gray background indicates components which have to be further investigated.

Nevertheless, this indicates that pigeon melanopsin is able perceive light already before hatch and can mediate visual information transfer to the brain prior to the functional development of classical photoreceptors. This addresses the question of how melanopsin

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CHAPTER 7: GENERAL DISCUSSION transduces the induction of asymmetries in pigeons. Therefore, the next section will consider possible transfer mechanisms.

7.1.1 Transducing asymmetrical light information – possible transfer mechanism from the retina to the brain

Functional and structural asymmetries which reflect a favorable selective advantage are a widespread phenomenon occurring in all vertebrates and even invertebrates (Vallortigara and Rogers, 2005; Frasnelli, 2013). Nevertheless, little is known about the ontogeny of lateralization. In pigeons an asymmetrical light stimulus during a critical embryonic phase was identified to be responsible for asymmetry formation even though the classical photoreceptors are not functionally developed at this time point (Bagnoli et al., 1985; Rojas et al., 2007). However, this thesis provides the evidence that melanopsin can act as a light-sensitive instance inside the retina already before hatch. This has been already discussed in detail in this thesis, but the precise signal transduction cascade to the brain upon melanopsin activation remains quite speculative. Possible mechanisms will be discussed in the upcoming sections. So far, an asymmetrical light stimulation of the pigeon embryo due to its asymmetrical position inside the egg could lead to a pronounced activation of melanopsin in the RGCs of the right eye of the pigeon embryo. Subsequently, light-dependent melanopsin activation initializes the Gq signaling cascade in RGCs which results in depolarization of the cells due to a Ca2+ influx. This provokes an unilateral retinal activation which might be directly propagated to the TO, since functional connections of RGCs to the TO are already established from E15 onwards in pigeons (Manns and Güntürkün, 1997). Several factors might be able to “convert” the asymmetrical light signal into structural asymmetries in the pigeon brain (e.g. larger somata in the tectal superficial layer 2-12 on the left side (Güntürkün, 1997) and larger rotundal neurons on the same side; Manns and Güntürkün, 1999b). However, structural asymmetries are primarily found on the tectal level and higher visual areas, whereas no asymmetries were located on retinal level (e.g. in retinal soma sizes in normal-incubated pigeons; Güntürkün, 1997). Therefore, these data indicate that there is no morphological change on retinal level due to an asymmetrical light incidence. In addition, this thesis demonstrated that there are no significant expression differences of melanopsin either

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CHAPTER 7: GENERAL DISCUSSION between the left and the right eye in pigeon embryos or between normal- and dark-incubated pigeon embryos (cf. Chapter 4).

Figure 7.2: Possible transfer mechanisms of asymmetrical light stimulation onto higher brain regions during embryonic development in the pigeon. The asymmetrical light stimulus can be transferred onto higher visual areas due to melanopsin-based signaling in the retina. At higher brain areas several factors might interact to manifest functional/behavioral lateralization based on structural asymmetries (blue: activated visual brain areas, dark grey: deprived visual areas, thickness of arrows indicates differences in projection strength, and different cell diameters indicate cell size asymmetries, green: possible melanopsin signaling pathway in the retina, light grey: processes on brain level; abbreviations: Ca2+ = calcium ion, E = Entopallium, R = retina, RT = Nucleus rotundus, TO = optic tectum).

However, several factors might affect the light-triggered induction of asymmetries: under consideration are growth factors, transcription factors, additional genetic influences,

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CHAPTER 7: GENERAL DISCUSSION microRNAs (miRNAs) and possibly also hormones. All factors will be discussed in the subsequent paragraphs (for an overview see Figure 7.2.). In general, growth factors play an important role in cell growth, differentiation and development (McGeachie and Tennant, 1997). These factors might be involved in asymmetry formation on different levels in the central nervous system. The growth factor BDNF (brain-derived neurotrophic factor) was suggested to be involved in asymmetry formation in pigeons. If BDNF plays a role in the asymmetrical retinal signal transduction due to asymmetrical light stimulation, it would be expected that BDNF injections into the retina of dark-incubated pigeons would be able to mimic an asymmetrical light stimulation. However, this assumption was proven to be wrong. Studies in pigeons revealed that retinal injections of BDNF in dark-incubated pigeons shift the superiority to the non-injected side (Manns et al., 2008). This led to the insight that BDNF is unlikely to mediate the effects of asymmetrical light stimulation on retinal level, whereas it is conceivable that retinal input regulates the BDNF release in the TO. The altered soma sizes in the TO might be a secondary consequence of BDNF expression (Manns et al., 2008; Manns and Ströckens, 2014). A similar effect was already demonstrated four years earlier by Prior et al. (2004). Tetrodotoxin (TTX) injections into the retina resulted in an enhancement of the non-injected eye in pigeons instead of a suppression of the injected eye. This indicated an activity-dependence of asymmetry formation (Prior et al., 2004). In addition, another study revealed that the trkB (tyrosine receptor kinase B) signaling cascade is asymmetrically activated in response to embryonic light stimulation in the TO (Manns et al., 2005). Ras activity, which is controlled by BDNF and its corresponding receptor trkB, was downregulated on the stronger stimulated TO in pigeons (Manns et al., 2005). Even though, no retinal immunolabeling of BDNF and NT-3 (neurotrophin-3) was evident (Theiss and Güntürkün, 2001), expression on tectal level was detectable which probably shapes the intra-tectal wiring pattern. However, due to the late expression of BDNF and NT-3 in pigeons, an involvement in asymmetry formation is questionable, and these factors might be more important in post-hatch processes (Theiss and Güntürkün, 2001). Moreover, recent studies demonstrated the influence of melanopsin on growth factors inside the retina in mice. The effect on the outgrowth of retinal vasculature has been investigated in mice exhibiting a mutated melanopsin gene or in mice, which were reared in darkness from a late gestation state on. Both variations resulted in an overgrowth of retinal vasculature. This study indicates that a light-dependent pathway based on melanopsin photoreception can

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CHAPTER 7: GENERAL DISCUSSION control VEGF (vascular endothelial growth factor) expression. Additionally, this study highlights that visceral cavity photon flux is probably sufficient to activate ipRGCs containing melanopsin in the mouse fetus (critical period E16-17). This suggests a pathway in that the mouse fetus is able to respond to light via ipRGCs already in utero (Rao et al., 2013). This represent a very important point, hence converting to birds ambient light reaching the pigeon embryo has first to pass through the eggshell. The eggshell is translucent to a certain degree (Rogers, 2006; Maurer et al., 2015), and the ambient light transmitting the eggshell is be sufficient for appropriate melanopsin activation if already photon flux through the visceral cavity of maternal mice is sufficed. In addition, G-protein-coupled receptor signaling has proven to be essential for axon guidance and targeting during development in mice (Li et al., 2016). The serine/threonine protein kinase mTor (mechanistic target of rapamycin or formerly mammalian target of rapamycin) regulates for example cell growth and proliferation. Li et al. (2016) demonstrated that melanopsin-containing ipRGCs exhibit high levels of mTor after axotomy in mice, whereas melanopsin knock-out mice show constant levels of mTor. Importantly, the upregulation in mTor and axon regeneration depends on light-activated melanopsin signaling via the Gq-coupled signaling pathway and probably on the elevation of intracellular Ca2+ (Li et al., 2016). Both studies indicate that melanopsin is also able to directly influence growth factors and axon outgrowth. In addition, even low illumination intensities (inside the visceral cavity of maternal mice) are sufficient for activation of melanopsin. Taken together, these data presented above indicate that melanopsin might also contribute to the network of light-induced asymmetry formation in pigeons in addition to simply transducing the asymmetrical light incidence to the TO. In addition to growth factors, transcription factors could be considered to play a role in establishing structural visual asymmetries in the pigeons. The transcription factor Brn3 (comprising Brn3a, Brn3b and Brn3c), for example, belongs to the POU family and is required for differentiation of vertebrate RGCs and for axon outgrowth from the retina to the brain (Xiang et al., 1995; Liu et al., 2000; Erkman et al., 2000; Contin et al., 2006). In mice Brn3b is found in 65% of melanopsin-positive cells (ipRGCs) which are involved in image and non-image forming vision (Ecker et al., 2010; Jain et al., 2012). Brn3b ablation leads to impaired pupillary light reflex but does not change in circadian entrainment, suggesting different roles of ipRGCs, since Brn3b-positive cells do not innervate the SCN (Chen et al., 2011). In 2015, García-Ayuso et al. demonstrated in a rat model of Retinitis pigmentosa (rats which exhibit retinal

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CHAPTER 7: GENERAL DISCUSSION degeneration comparable to Retinitis pigmentosa in human), that a small number of melanopsin-containing ipRGCs express Brn3a and importantly, that the number increases with progressive loss of rods and cones. They suggested two possibilities to explain the phenomenon: a preferential surviving of cells expressing Brn3a or increased expression in surviving cells. This led to the speculation that an increase in vision by ipRGCs might compensate the loss of photoreceptors (García-Ayuso et al., 2015). The co-expression of melanopsin and Brn3 might hold true for pigeons as well. Since Brn3 is involved in axon outgrowth it might also have implications on the establishment of lateralization in the visual system. These melanopsin-activated networks assembled of growth factors and transcription factors might lead to further networks establishing visual asymmetries. Of course, genetic influences play an important role in the ontogeny of asymmetries as well. Genetic factors determine the asymmetrical position of the chicken embryo inside the egg (Ramsdell and Yost, 1998; Chiandetti et al., 2013) which initially leads to the asymmetrical light stimulation during embryonic development. The hypothesis of genetically regulated mechanisms which influence asymmetry formation have been studied in detail in zebrafishes. Especially, the Nodal signaling pathway is able to control ventral forebrain development in zebrafish. In zebrafish embryo, developmental processes establish left/right (L/R) asymmetries in the epithalamus which is divided in a left and a right habenular nucleus and the adjacent pineal complex (Roussigné et al., 2012). Nodal, as a member of the transforming growth factor-β family (TGF- β), is involved in the directionality of asymmetries and promotes asymmetrical timing of early habenular neurogenesis (Concha et al., 2012; Matsui and Bessho, 2012; Roussigné et al., 2012). Therefore, Nodal signaling represents a required component in the establishment of L/R asymmetries, and its absence leads to symmetrical neuron formation in the habenulae (Roussigné et al., 2012). In zebrafish Nodal is transiently activated on the left side of the brain (Halpern et al., 2005; Matsui and Bessho, 2012). Nodal and Nodal-target genes (e.g. lefty) participate in stablishing asymmetry and laterality in the human embryo as well and mutations can cause a situs inversus for instance (Vallortigara and Rogers, 2005; Roussigné et al., 2012; Lambert et al., 2016). In 2013, Chiandetti et al. suggested a second route of asymmetry formation in addition to the asymmetrical light stimulation in a critical period shortly before hatch in chickens. By exposing chick embryos to light during the first three days after fertilization, hatched chicks still exhibit the same behavioral lateralization as animals exposed to light during the critical phase shortly before hatch. Importantly, both early

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CHAPTER 7: GENERAL DISCUSSION and late light stimulation were able to affect the lateralized behaviors in the same direction. They proposed a second route that might be genetically regulated and is able to genetically affect expression of photosensitive regions before the development of a functional visual system (Chiandetti et al., 2013). However, additional genetically determined mechanisms in the induction of visual asymmetries in the pigeons cannot be excluded in general, even though no second genetically defined instance has been identified yet in addition to the genetically predisposed position inside the egg. A genetically determined asymmetrical expression of melanopsin for example can be excluded, since no differences between the left and the right eye as well between normal- and dark-incubated pigeons were detectable by qPCR analysis (cf. Chapter 4). A further important regulator of asymmetry formation can be mircoRNAs (miRNA or miR). MiRNAs are small non-coding RNAs which are able to regulate the expression of complementary mRNA (Ambros, 2004). Therefore, they play a crucial role in cell proliferation, developmental timing as well as patterning of the nervous system just to name of few responsibilities (for review see: Ambros, 2004). Potentially, miRNAs represent the missing link between an asymmetrical activation of melanopsin-containing RGCs and structural lateralization on tectal and rotundal level in the tectofugal pathway. Two independent studies published in 2011 demonstrated the involvement of miR-132 in ocular dominance plasticity in mice. Ocular dominance in mammals can be shifted by covering one eye during development. This monocular deprivation results in expansion of columns serving the open eye (Hensch, 2005): miR-132 is involved in dendritic spine plasticity and monocular deprivation reduced its expression in the visual cortex contralateral to the deprived eye (Mellios et al., 2011; Tognini et al., 2011). Tognini et al. (2011) demonstrated that dark-rearing from birth blocked the cortical increase of miR-132 during development and they found a visual experience-dependent expression. Similar data were published simultaneously by Mellios et al. (2011). Using qPCR analysis they were able to demonstrate that miR-132 expression increases after eye opening while dark rearing delayed the elevation of expression levels. In addition, they were able to show that inhibition of miR-132 in vivo prevents ocular dominance plasticity in mice. This indicates that the light-/activity-induced elevation of miR-132 is essential for the initiation of ocular dominance plasticity in mice. This strongly supports the assumption of both research groups that miR-132 plays a critical role in plasticity of visual cortex circuits (Mellios et al., 2011; Tognini et al., 2011). Moreover, a rather similar mechanism

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CHAPTER 7: GENERAL DISCUSSION seems to be feasible in the manifestation of structural asymmetries in the pigeon brain. An asymmetrical/unilateral activation (light-induced) is given by melanopsin-containing RGCs which might induce asymmetrical miRNA expression in the visual system. These miRNAs might in turn influence for example the more extensive growth of neurons in the superficial layers 2-12 of the left TO and left RT. However, further investigation of miRNA occurrence and expression patterns in pigeons are necessary to investigate this assumption. Furthermore, hormones were documented to influence lateralization in addition to gene expression and the environmental factor light. Hormones are able to change the degree/strength of lateralization (Vallortigara and Rogers, 2005). Injections of steroid hormones like estrogen or corticosterone prevent the development of asymmetries in response to light in chickens and additionally effects of stress on lateralization were demonstrated (Rogers and Deng, 2005; Halpern et al., 2005). Altogether, hormones might play a modulating role in the formation of asymmetries in pigeons as well. Moreover, lateralization is characterized by an interplay of the asymmetrical bottom-up system and top-down control. Top-down control is based on the tractus septomesencephalis (TSM) whose neurons of origin are mainly located in the visual wulst. These fibers terminate within the TO (Leresche et al., 1983; Manns and Güntürkün, 2009). These intrahemispheric processes stabilize the asymmetries post-hatch. Top-down control is shown to more intensively modify the left hemisphere than the right hemisphere which is less modulated (Freund et al., 2016). How these asymmetrical top-down influences emerge is uncertain. However, a development during learning, due to ontogenetic experiences and possibly (but not necessarily) pre-wiring are under consideration (Manns and Ströckens, 2014; Freund et al., 2016). Additionally, tecto-tectal interactions might influence lateralization (Güntürkün and Böhringer, 1987). The left and the right TO are connected via commissures (Voneida and Mello, 1975; Hunt and Künzle, 1976) which are able to transfer inhibitory signals (Hardy et al., 1984; Robert and Cuénod, 1969; Keysers et al., 2000). Moreover, the left TO inhibits the right TO to a larger extent than vice versa (Keysers et al., 2000; Manns and Güntürkün, 2009). In conclusion, the cell size asymmetries which comprise larger somata of neurons of the superficial layer 2-12 in the left hemisphere and larger rotundal neurons on the same side (Güntürkün, 1997; Manns and Güntürkün, 1999b) might be explained by increased right tectal activation. This pronounced left hemispheric activation might evoke growth factors, miRNA and different factors predominantly on the left side, resulting in more

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CHAPTER 7: GENERAL DISCUSSION intense cell growth. However, this does not explain the projection asymmetries with more fibers projecting from the right TO to the left RT than from the left TO to the right RT (Güntürkün et al., 1993; Güntürkün et al., 1998). In this case, axon guidance factors emanating from the left RT might enhance axon outgrowth from the right (actually deprived) TO to the left RT based on the stronger activation of the left hemispheric RT. Furthermore, time-delayed maturation/development of the neuronal circuits in the light-deprived visual components might be considered as an important factor as well. Light-activation in the right eye/left brain hemisphere might be melanopsin-depended possibly already before hatch, whereas activation of the left eye/right hemisphere is first possible after hatch due to the asymmetrical position of the embryo inside the egg. In summary, in this thesis I suggest melanopsin as a potential transducer of asymmetrical light stimulation during the crucial developmental phase in the pigeon embryo. The asymmetrical light incidence results in a unilateral depolarization of RGCs due to melanopsin activation. However, subsequently different factors might translate this asymmetrical activation into structural and later behavioral asymmetries (cf. Figure 7.2). These factors might constitute a complex network of miRNA, growth factors and transcription factors and many more.

7.2 Melanopsin and cryptochrome: Possible interaction partners?

In addition to the classical photoreceptors, rods and cones as well as the newly investigated “third” photoreceptor system, melanopsin, several studies have been conducted on further photoreceptive instances in the retina. Miyamoto and Sancar (1998) demonstrated the expression of two additional blue light receptors in the RGCs and INL of the mouse retina: Cryptochrome 1 and 2 (Cry1 and 2). They also described Cry1 expression in the SCN which oscillated in a circadian manner. Furthermore, they proposed cryptochrome as a vitamin

B2-based protein which is involved in the entrainment of the circadian clock (Miyamoto and Sancar, 1998). Cryptochromes belong to the photolyase family of blue-light photoreceptors and are found in organisms ranging from cyanobacteria to humans (Öztürk et al., 2007). Cryptochromes resemble folate- and flavin-based proteins (Öztürk et al., 2007). In plants and mammals cryptochromes are composed of two domains: an N-terminal

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CHAPTER 7: GENERAL DISCUSSION photolyase-related (PHR) domain comprising binding sites for FAD (flavin adenine dinucleotide) and MTFH (methylene tetrahydrofolate) and a C-terminal extension. The C-terminus is responsible for nuclear/cytosolic trafficking and protein/protein interactions (Lin and Shalitin, 2003; Sancar, 2004; Lin and Todo, 2005; Owens et al., 2012). In general, cryptochromes share structural similarities to photolyases (Sancar, 2004; Bouly et al., 2007). Nevertheless, no photolyase activity in human or other placental mammals is exhibited by cryptochrome (Sancar, 2004). Further studies confirmed the existence of cryptochromes in RGCs (Sancar, 2000; Gooley et al., 2001) which are mainly excited by light in the blue portion of the visible spectrum (maximal absorption peak human Cry: 420 nm, Sancar, 2000; plant Cry: 420 nm, Bouly et al., 2007). Studies on cryptochrome knock-out mice (either lacking Cry1 or 2) demonstrated an impaired light-dependent induction of the clock gene mPer1, defining cryptochrome as central component in the regulation of the molecular clock (Sancar, 2000, 2004). In contrast, Cry2 is involved in photoperiodism and regulates diverse developmental processes in plants (Miyamoto and Sancar, 1998; Liu et al., 2016). In 2003, Drivenes et al. suggested that several light detecting molecules might play a role in circadian entrainment including cryptochrome and melanopsin. However, cryptochrome can function pleiotropically in both circadian rhythm and photic entrainment of behavioral responses (van Gelder et al., 2002) such as the regulation of pupil responses (van Gelder et al., 2002; van Gelder, 2003b). Additionally, in addition to circadian rhythm and pupillary light responses, cryptochrome can function as a magneto-sensor in magnetoreception. Foley et al. (2011) demonstrated that human Cry is able to rescue light-dependent magnetoreception in Cry-deficient flies (Drosophilia). In general, Cry is involved in sensing the earth magnetic field in migratory birds and turtles based on light-sensitive chemical reactions (Wiltschko and Wiltschko, 2005; Foley et al., 2011). The fact that Cry (especially Cry1) might subserve magnetoreception also in various mammalian species has been recently proposed (Nießner et al., 2016). In addition, recent studies propose that Cry1 and 2 might regulate the abundance of transcription factors (Liu et al., 2016). Cryptochrome is activated in response to blue light which induces a conformational change allowing propagation of light signals to downstream components (Liu et al., 2016). The redox state of the FAD is important for Cry-based signaling. The inactive form of cryptochrome incorporates an oxidized FAD and subsequent absorption of light leads to a photo-reduction of FAD to a semi-reduced neutral FAD radical (FADH*), representing the signaling state of Cry

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(Bouly et al., 2007; Liu et al., 2016). Three tryptophan residues (Trp-triad) in α-helix might serve as electron donor (Liu et al., 2016). Cryptochrome is phosphorylated under blue light which plays an important role in photo-activation. This might be ascribed to its auto-phosphorylation activity (Öztürk et al., 2007). Similar mechanisms in plant and animal cryptochromes were proposed (Bouly et al., 2007). Besides the activation via blue light, partial inhibitory effects of green light on Cry which are comparable to melanopsin were described (Bouly et al., 2007; Liu et al., 2016; cf. Chapter 6). The Cry-based signal might be transduced via interactions of downstream proteins (Bouly et al., 2007) and the regulation of transcription factors (Liu et al., 2016). Hence, photo-induction of gene expression in the SCN and light-induced pupillary constriction plays a central role in circadian oscillator (Cashmore, 2003). Unfortunately, no further information about the photochemical reaction carried out by cryptochrome are conversant up to now. Importantly, a recent study suggested that Cry1b (an isoform of Cry1) could be involved in the induction of visual lateralization in pigeons. This study demonstrated Cry1b protein expression during the critical embryonic time frame in pigeons, therefore, proving the evidence of another light-sensitive instance aside from rods and cones (Ströckens and Güntürkün, 2016). Unfortunately, no direct activation of these Cry-containing cells could be shown in the critical time frame due to methodical issues. However, this raises the question whether cryptochrome and melanopsin might interact to transduce the asymmetrical embryonic light stimulation in pigeons. The study of Ströckens & Güntürkün (2016) demonstrated a broad cryptochrome expression in the majority of RGCs whereas this thesis revealed a widespread expression of melanopsin isoform L in RGCs of pigeons. Thus, co-localization and possible interactions of these two molecules are conceivable. Several studies suggested that cryptochrome might be involved in melanopsin-based photoreception (Hattar et al., 2002; Drivenes et al., 2003). Drivenes et al. (2003) proposed that Cry1 and 2 might be involved in ipRGC-dependent light detection, and in 2004 Sancar suggested that melanopsin and cryptochrome might work cooperatively to generate robust retino-hypothalamic photosignals. However, studies demonstrated that ipRGCs of melanopsin knock-out mice are not photoresponsive anymore, but that circadian entrainment was not severely impaired. This leads to the conclusion that rods, cones and cryptochrome are sufficient for circadian entrainment and melanopsin adds additional input (van Gelder, 2003a).

Furthermore, if melanopsin knock-out (-/-) mice are crossed with mice lacking functional rods

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CHAPTER 7: GENERAL DISCUSSION and cones, all responses to light are lost (Hattar et al., 2003; Panda et al., 2003; Hankins et al., 2008). Experiments validated that in absence of rods, cones and melanopsin no other photoreceptor is sufficient to provide signaling to the brain. Hence, a melanopsin knock-out in retinal degenerate mice leads to a loss of all photo-responses like significant pupil reflexes, entrainment of light/dark cycles and any masking response is vanished. On the contrary, in mice which have a cryptochrome knock-out and show retinal degeneration some light responses e.g. pupillary light responses can still persist (Hattar et al., 2003). This leads to the conclusion that cryptochrome might either depend on melanopsin or does not signal the light responses investigated in this study (Hattar et al., 2003). These studies suggest that rods, cones and melanopsin-containing ipRGCs can fully account for all light detection within the eye. Hence, these triple knock-out studies argue that cryptochrome might not act as a photopigment. The complete loss of light responses in rods/cone- and melanopsin knock-out animals leaves little room for a Cry-based photopigment (based on action spectra data; Hattar et al., 2003; Peirson et al., 2005). This leads to the conclusion that cryptochromes do not form photopigments in mammals (Peirson et al., 2005; Hankins et al., 2008), resembling a statement of Cashmore (2003), that cryptochrome is not a direct photoreceptor. In addition, these findings are supported by a recent study, which illustrated that loss of cryptochrome in retina-degenerate mice reduces sensitivity of pupillary light response at all wavelength but does not alter the form of the action spectrum (Owens et al., 2012). And the remaining action spectrum matches that of melanopsin. Therefore, they assumed that cryptochrome does not function as a photopigment as well. Melanopsin appears to be both necessary and sufficient for photoreception whereas cryptochrome is an accessory light-sensitive instance to modulate melanopsin-dependent photoreception (Owens et al., 2012). Furthermore, melanopsin expression (both mRNA and protein) is not influenced by a cryptochrome knock-out (van Gelder et al., 2002), indicating that cryptochrome does not propagate its effects via modulation ipRGC development or gene expression (van Gelder, 2003a). Therefore, rods, cones and melanopsin provide a sufficient and necessary system for all photic input of these accessory visual functions (Hattar et al., 2003; Peirson et al., 2005). Moreover, melanopsin is responsible for photoreception aside from rods and cones, whereas cryptochrome is only necessary to achieve the full sensitivity of the system (van Gelder, 2003a).

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Interactions between melanopsin and cryptochrome in the induction of asymmetries cannot be excluded. Van Gelder (2003a) suggested that cryptochrome might amplify melanopsin-dependent signals in a light-dependent manner and that it might confer additional photoreceptive properties on the signal. This might hold true for the induction of asymmetries as well. Melanopsin might be the main inducer, whereas cryptochrome could contribute to signal intensification. Comparing melanopsin and cryptochrome strongly implicates that melanopsin is the functional photopigment of ipRGCs which can transduce the asymmetrical light stimulation. However, it is difficult to speculate about possible mechanisms and how cryptochrome might intensify the melanopsin-based signal, since little is known about the exact signaling pathway of cryptochromes (Hattar et al., 2003; Liu et al., 2016). The regulation of melanopsin expression by cryptochrome has to be excluded, since melanopsin expression was not altered by a cryptochrome knock-out (van Gelder et al., 2002). Another possibility could be represented by a cryptochrome-induced stronger depolarization of the cell upon light activation. Melanopsin signals most likely via a Gq-coupled signaling pathway in the retina which leads to influx of intracellular Ca2+ and consequently to depolarization of the cell. However, studies disproved the direct involvement of cryptochrome in an increase of intracellular Ca2+(Lin and Shalitin, 2003). Therefore, this kind of interaction between melanopsin and cryptochrome has to be excluded as well. Further investigations of the cryptochrome signaling pathway are indispensable to examine the possible interaction mechanism between melanopsin and cryptochrome. However, melanopsin seems to play a major role in the induction of asymmetries, while cryptochrome seems to be important in circadian entrainment as well as magnetoreception. Nevertheless, an interaction and an enhancement of the melanopsin-dependent asymmetrical light signal transduction by cryptochrome in the embryonic development must be seriously considered.

7.3 Conclusion and outlook

The aim of this thesis was to characterize and further analyze the photopigment melanopsin in regard to its photosensitive properties and its expression during the critical embryonic time frame in pigeons when visual asymmetries are established. This thesis

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CHAPTER 7: GENERAL DISCUSSION provides evidence for the involvement of melanopsin in the induction of visual asymmetries in the pigeon. I suggest that the asymmetrical light stimulation inside the pigeon egg induces a unilateral activation of pigeon melanopsin isoform L containing RGCs which results in an increase in intracellular Ca2+ and a depolarization of the cells (cf. Figure 7.2, green background). The increased activation of the right eye might initiate a complex network of mechanisms in the brain “translating” asymmetrical light stimulation into the formation of structural asymmetries (cf. Figure 7.2, light grey background). The current status of asymmetry research in pigeons demonstrates the complexity of the ontogeny of lateralization. Understanding the complex network of mechanisms step by step might also contribute to understand the interplay of genetic, epigenetic and environmental factors in the ontogeny of human asymmetries as well. Moreover, this thesis may provide an important piece in the puzzle of understanding the ontogeny of lateralization. Nonetheless, further investigations are needed to strengthen the hypothesis that melanopsin is the crucial factor in the induction of light-mediated visual asymmetries. An important cornerstone for upcoming experiments is provided by this thesis. Since patch-clamp analyses revealed an activation of melanopsin in the blue portion of light (420 nm to 470 nm) whereas light between 540 nm and 560 nm is able to actively deactivate melanopsin. Therefore, to finally prove the involvement of melanopsin in the ontogeny of visual asymmetries, behavioral and anatomical experiments could clarify the influence of melanopsin. If melanopsin actually mediates the asymmetrical light stimulation during embryonic development, incubation of eggs under monochromatic blue light (e.g. 450 nm) should result in the typical lateralized behavioral responses in discrimination tasks (Güntürkün, 1985; Skiba et al., 2002) and histological studies should reveal the characteristic anatomical asymmetries on the one hand (Güntürkün, 1997; Güntürkün and Hahmann, 1999; Manns and Güntürkün, 1999b). On the other hand incubation of eggs under monochromatic green light (ranging between 540 and 560 nm) should lead to melanopsin deactivation and therefore, to loss of lateralization on behavioral and structural level comparable to dark-incubated pigeons. This study could serve as a final proof of the involvement of melanopsin in the induction of lateralization. To further investigate the up-stream processes that might result from a unilateral melanopsin-dependent RGC activation, miRNA expression patterns in the TO and the RT should be taken into account (as depicted in 7.1.1). Therefore, the investigation of miRNAs in

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CHAPTER 7: GENERAL DISCUSSION the pigeon and subsequent expression profiling of possible left and right hemispheric expression differences might contribute essentially to the understanding of asymmetry formation. In summary, pigeon melanopsin is a highly interesting candidate to transduce the light-induced asymmetry formation in the visual system of pigeons. This thesis delivers a crucial basis for further investigation of the processes of the ontogeny of lateralization in the model system of the pigeon.

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APPENDIX

A. CHEMICALS AND EQUIPMENT

A.1 List of chemicals and reagents

Agar agar Fisher Scientific, Germany Agarose Biozym and Sigma-Aldrich, Germany and Gibco-BRL, USA Ammonium chloride (NH4Cl) Riedel-de Häen, Germany Ammonium hydroxide (NH4OH) J.T. Baker, Netherlands Ammonium nickel sulfate (NiNH4SO4) Alfa Aesar, Germany Ampicillin Invitrogen, Germany ß-D-glucose Sigma, USA Boric acid Sigma-Aldrich, Germany Bromophenol blue Sigma-Aldrich, Germany Calcium chloride (CaCl2) Merck, Germany Carbenicillin Invitrogen, Germany Chloral hydrate Sigma, USA Cholera toxin subunit b (Tracer) Sigma-Aldrich, Germany Cobalt (II)-Chloride (CoCl2) Merck, Germany Concentrated hydrochloric acid (HCl) Sigma-Aldrich, USA DAPI-Fluoromount Southern Biotech, USA Diaminobenzidine (DAB) pellets Sigma, USA Dipotassium phosphate (K2HPO4) J.T. Baker, Netherlands Dithiothreitol (DTT) AppliChem, Germany DPX mounting solution Sigma-Aldrich, Germany Ethylenediaminetetraacetic acid (EDTA) Merck, Germany Ethylene glycol tetraacetic acid (EGTA) Sigma-Aldrich, Germany Ethanol (C2H6O, EtOH) Sigma-Aldrich, Germany Ethidium bromide Sigma, Germany Fetal bovine serum (FBS) Gibco®, Germany G418 (Geneticin) Gibco, Germany Gelatine Merck, Germany Glucose oxidase Sigma-Aldrich, Germany Glycerin J.T. Baker, Netherlands HEPES-NaOH Sigma-Aldrich, Germany Hydrogen peroxide (H2O2) J.T. Baker, Netherlands Isopentane (C5H12) VWR, Germany Isopropanol (C3H8O) VWR Chemicals, Belgium and Sigma-Aldrich, Germany Kanamycin Invitrogen, Germany Magnesium chloride (PCR) Gene Craft, Germany Magnesium chloride (MgCl2) Merck, Germany Magnesium sulfate (MgSO4) J.T. Baker, Netherlands 178

A. CHEMICALS AND EQUIPMENT

MgATP Sigma-Aldrich, Germany NaGTP Sigma-Aldrich, Germany Neutral red Merck, Germany Normal donkey serum Vector laboratories, USA Nuclease-free water Thermo Scientific and Roche, Germany Nucleoside triphosphates (dATP, dCTP, dGTP, dGTP) PeqLab, Germany Paraformaldehyde (PFA) Prolabo, Germany Penicillin (Pen Strep) Invitrogen, Germany Pentobarbital Sigma-Aldrich, USA phys. 0.9% NaCl Braun, Germany Poly-L-lysine Sigma-Aldrich, Germany Potassium aspartate Merck, Germany Potassium acetate (KOAc) Merck, Germany Potassium chloride (KCl) Merck, Germany Propane-1,2-diol (C3H8O2) Merck, Germany RNAlater® Sigma-Aldrich, Germany RNaseAWAY™ Thermo Scientific, Germany RNaseZap® Ambion, USA Roti®-Mount FluorCare Roth, Germany Sodium acetate (CH3COONa, NaAc) J.T. Baker, Netherlands Sodium chloride (NaCl) VWR Chemicals, Belgium and Roth, Germany Sodium dihydrogen phosphate (NaH2PO4) J.T. Baker, Netherlands Sodium dodecyl sulfate Sigma-Aldrich, Germany Sodium hydroxide (NaOH) Merck, Germany SsoFastTM EvaGreen® Supermix Bio-Rad, Germany Sucrose (C12H22O11) AppliChem, Germany Streptomycin (Pen Strep) Invitrogen, Germany Tissue Tek Jung, Germany Tris-acetate Sigma-Aldrich, Germany TRIS-borate-EDTA (TBE) buffer VWR, Germany Tris-HCl Roth, Germany Triton-X100 Sigma-Aldrich, Germany Trypton Invitrogen, Germany X-gal Thermo Scientific, Germany Xylol J.T. Baker, Netherlands Yeast extract Invitrogen, Germany

A.2 Enzymes and corresponding buffers

Buffer B4 (10x) NEB, Germany Buffer BiothermTM (10x) Gene Craft, Germany CIP NEB, Germany NheI NEB, Germany SacII NEB, Germany T4 Buffer (10x) NEB, Germany T4 Ligase NEB, Germany

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A. CHEMICALS AND EQUIPMENT

Taq Polymerase Gene Craft and Qiagen, Germany

A.3 Pharmaceuticals

Heparin Ratiopharm, Germany Ketavet® (Ketamine) Zoetis, Germany Rimadyl Zoetis, Germany Rompun® (Xylazine) Bayer Healthcare, Germany Tyrosur® powder Engelhard Arzneimittel, Germany

A.4 Antibodies

Biotinylated secondary α-goat antibody Vector Laboratories, USA α-CTB from goat Calbiochem, Germany α-goat Alexa 594 secondary antibody Invitrogen, USA α-melanopsin 2 from rabbit Bethyl, USA Sequence: CLSSSTEFLEAVEKFLS α-rabbit Alexa 488 secondary antibody Invitrogen, USA

A.5 Buffer and solutions

Polymerase chain reaction (PCR) 1 kb Marker Fermentas, Germany dNTP-Mix 2.5 mM dATP 2.5 mM dCTP 2.5 mM dGTP 2.5 mM dTTP

Ethidium bromide 1% in TBE or TAE GeneRulerTM 100 bp Fermentas, Germany

Loading buffer 0.25% Bromophenol blue 34.5% glycerin

MgCl2 (PCR) 50 mM

Midori Green Advance Nippon Genetics GmbH, Germany

1xTAE 40 mM tris-acetate 20 mM NaAc 2mM EDTA, pH 8.0 H2O

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A. CHEMICALS AND EQUIPMENT

1xTBE 89 mM tris-HCl 89 mM boric acid 2 mM EDTA H2O

Perfusion and preparation Equitisin 5 g chloral hydrate 1.15 g pentobarbital 2.5 g MgSO4 12 ml 96% EtOH 54 ml propane-1,2-diol 50 ml Aqua dest.

4% PFA solution 40 g PFA 800 ml Aqua dest. 8 drops 40% NaOH 200 ml 0.6 M PBS stock solution

0.9% physiological NaCl 90 g NaCl 10 l Aqua dest.

Postfix 15 g sucrose [30%] 50 ml 4% PFA solution

Sucrose solution 15 g sucrose [30%] 50 ml 0.12 M PBS

Immunohistochemistry Bleaching Solution 5 ml 1.8% NaCl [1.54 mM] 4 ml 30% H2O2 [12%] 1 ml H2O 1 drops 25% NH4OH

Phosphate buffered saline (PBS)

- 0.6 M stock solution PBS 0.5 M K2HPO4 [87.09 g] 0.1 M NaH2PO4 [13.80 g] pH 7.4

- 0.12 M working solution PBS 200 ml 0.6 M PBS stock solution 800 ml 0.9% NaCl

- 0.3% PBS-T 1.5 ml Triton-X-100 [0.3%] 498.5 ml 0.12 M PBS

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A. CHEMICALS AND EQUIPMENT

Sodium acetate buffer - 0.2 M Sodium acetate buffer 16.4 g water-free NaAc 1000 ml Aqua dest. pH 5.5 – 5.8

- 0.1 M Sodium acetate buffer 500 ml 0.2 M sodium acetate buffer 500 ml Aqua dest. pH 6.0

Mounting solution 800 ml Aqua dest. 1 g gelatine dissolve with mild heating (40 °C) 100 ml 0.6 M PBS 100 ml 100% EtOH filtrate

Cell culture and media Agar plates 25 mg LB-Agar agar [1.2%] 1 l dH2O

DMEN (Dulbecco´s modified Eagles medium) Gibco®, Germany

LB broth medium 1% Trypton 0.5% Yeast extract 1% NaCl

LB medium + Kanamycin LB broth medium 50 µg/ml Kanamycin/Ampicillin

LB medium + Carbenicillin LB broth medium 100 µg/ml Carbenicillin

Opti-MEM® Gibco®, Germany

SOC medium Invitrogen, Germany

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A. CHEMICALS AND EQUIPMENT

Plasmid mini preparation Buffer B1 50 mM tris-HCl pH 8.0 10 mM EDTA 100 µg/ml RNase A

Buffer B2 200 mM NaOH 1% SDS

Buffer B3 3 M KOAc pH 5.5

Cell culture and electrophysiological methods HEK 1.2 medium 10% FBS 400 µg/ml G418 100 U/ml Penicillin 100 µg/ml Streptomycin in DMEM

HEK 1.2 bath solution 20 mM NaCl (Extracellular solution) 120 mM KCl 2 mM CaCl2 10 mM HEPES-NaOH pH 7.3

HEK 1.2 pipette solution 100 mM K-aspartate (Intracellular solution) 40 mM KCl 5 mM MgATP 10 mM HEPES-NaOH 2 mM EGTA 2 mM MgCl2 0.01 mM NaGTP pH 7.3

A.6 Kits and manuals

I. Kits α-goat Vectastain Elite-ABC-Kit Vector Laboratories, USA α-rabbit Vectastain Elite-ABC-Kit Vector Laboratories, USA Agilent RNA 6000 Pico Kit Agilent, USA Fluo-4 Direct Calcium Assay Kit Invitrogen, Germany Fugene HD Transfection Reagent Promega, Germany Invisorb® Fragment Cleanup Kit Invitec, Germany iScript™ Select cDNA Synthesis Kit Bio-Rad, Germany Lipofectamine 2000 Invitrogen, Germany NucleoBond® Xtra Midi & Maxi Machery-Nagel, Germany

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A. CHEMICALS AND EQUIPMENT

Nucleo Spin RNA II Kit Machery & Nagel, Germany One Shot® TOP10 Chemically Competent E. coli Invitrogen, Germany Qiagen Plasmid Midi Kit 100 Qiagen, Germany QIAEX II Gel Extraction Kit Qiagen, Germany QIAquick Extraction Kit (250) Qiagen, Germany QuantiTect® Reverse Transcription Kit Qiagen, Germany RNase-free DNase Set Qiagen, Germany RNeasy Mini Kit Qiagen, Germany Sensiscript RT Kit Qiagen, Germany Taq DNA Polymerase Kit Qiagen, Germany TOPO® TA Cloning® Kit Life Technologies, Germany Wizard SV Gel and PCR Clean-Up System Promega, Germany

II. Manuals α-goat/rabbit Vectastain Elite-ABC-Kit www.vectorlabs.com/data/protocols/PK-6100.pdf Agilent RNA 6000 Pico Kit http://www.chem.agilent.com/library/usermanuals/Public/G2938-90049_RNA6000Pico_QSG.pdf Invisorb® Fragment Cleanup Kit http://www.invitek.de/e1531/e1658/e2414/e2443/e2490/ip_manual6620/MSBRapace,Vario,FragmentCleanu pundDNAExtraction.pdf iScript™ Select cDNA Synthesis Kit http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10001023B.pdf Nanodrop ND-1000 Spectrophotometer http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NucleBond® Xtra Midi/Maxi http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/ UM_pDNA_NuBoXtra.pdf Nucleo Spin RNA II Kit http://www.mnnet.com/Portals/8/attachments/Redakteure_Bio/Protocols/RNA%20and%20mRNA/UM_TotalR NA.pdf Qiagen Plasmid Midi Kit (100) https://www.qiagen.com/de/resources/resourcedetail?id=c164c4ce-3d6a-4d18-91c4-f5763b6d4283&lang=en QIAEX II Gel Extraction Kit https://www.qiagen.com/de/resources/resourcedetail?id=13d33145-9f64-426a-a43b-394211d8cf2b&lang=en QIAquick Extraction Kit (250) https://www.qiagen.com/de/resources/resourcedetail?id=3987caa6-ef28-4abd-927e-d5759d986658&lang=en QuantiTect® Reverse Transcription Kit https://www.qiagen.com/de/resources/resourcedetail?id=f0de5533-3dd1-4835-8820-1f5c088dd800&lang=en RNeasy Mini Kit https://www.qiagen.com/de/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en Sensiscript RT Kit https://www.qiagen.com/resources/download.aspx?id=11b31d9c-8eb0-4c12-bc9f-0bfd92cd6027&lang=en. SsoFast™ EvaGreen® Supermix http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10014647A.pdf 184

A. CHEMICALS AND EQUIPMENT

Taq DNA Polymerase https://www.qiagen.com/de/resources/resourcedetail?id=c73208eb-a83e-40c4-a9b6-ea5c4c94b9f4&lang=en

A.7 Technical equipment and consumable supplies

2100 Bioanalyzer Instrument Agilent Technologies, USA AccuBlock™ Digital Dry Baths Labnet International, Inc., USA Beckmann CoulterTM DU®530 Beckmann Coulter, USA BioTek’s Take3™/Gen5™ Biotek, Germany Camera AxioCam MRm Zeiss, Germany Centrifuge Eppendorf 5415 Eppendorf, Germany Centrifuge Eppendorf 581012 Eppendorf, Germany Centrifuge Eppendorf 5417 C Eppendorf, Germany CFX384 Touch Real-Time PCR detection system Bio-Rad, Germany Chemidoc MP Bio-Rad, Germany Cryostat Leica 3050S Leica, Germany Dissecting set FST by Dumont, Switzerland Electrophoresis power supply Bio-Rad, Germany Egg incubator (Brutmaschine Typ 3000) Bruja GmbH, Germany Eppendorf Research® plus Pipettes Eppendorf, Germany epT.I.P.S.®-Reloads PCR clean Eppendorf, Germany Filter tips Sarstedt, Germany Hard-Shell® 96-Well Semi-Skirted PCR plates Bio-Rad, Germany Hard-Shell® Thin-Wall 384-Well Skirted PCR Plates Bio-Rad, Germany Heating cabinet Memmert and Hereaus, Germany Eppendorf Thermomixer ® F Eppendorf, Germany Leica TCS SP5 confocal laser scanning microscope (Prof. Herlitze) Leica, Germany LSM510 Meta confocal laser scanning microscope (Prof. Theiss) Zeiss, Germany Microscope Imager M1 Zeiss Zeiss, Germany Microscoppy slides Menzel-Gläser, Thermo Scientific, Germany Microseal® 'B' Adhesive Seals Bio-Rad, Germany Microtome Leica RM 2135 Leica, Germany MiniSpin® Centrifuge Eppendorf, Germany Mini-Sub® Cell GT Cell/Sub-Cell® GT Cell Bio-Rad, Germany NanodropND-1000 Spectrophotometer Thermo Scientific, Germany Optical Flat 8-Cap Strips Bio-Rad, Germany PCR tubes Sarstedt, Germany Perfusion pump Ismatec, Germany Pipettes Eppendorf, Germany Polytron PT1200 (homogenizer) Kinematica AG, Switzerland Power supply Power-Pac-300 Bio-Rad, Germany Reaction tubes Sarstedt and Eppendorf, Germany 185

A. CHEMICALS AND EQUIPMENT

Shaker device Heidolph, Germany Small animal guillotine World Precision Instruments, USA T100TM Thermal Cycler Bio-Rad, Germany Mastercycler ® Eppendorf, Germany Thermocycler T-Professional Biometra, Germany Tips, TipOne Starlab, Germany Tumbling shaker Heidolph, Germany Vilber Lourmat Fusion SL PeqLab, Germany Vortex-Genie 2 Bender & Hobein AG, Switzerland WPI Nanoliterinjector 2000 World Precision Instruments, USA

A.8 Software

Axio Vision 4.8.10 Zeiss, Germany Basic local alignment search tool (BLAST) NCBI, USA (http://blast.ncbi.nlm.nih.gov/Blast.cgi) CFX Manager™ Software Bio-Rad, Germany Corel Draw X5 Corel, USA Corel Photo Paint X5 Corel, USA DNA Copy Number Calculator ThermoFisher Scientific, Germany (https://www.thermofisher.com/de/de/home/brands/thermo-scientific/molecular-biology/molecular-biology- learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/dna-copy-number- calculator.html) ExPASy SIB Swiss Institute of Bioinformatics, Switzerland (http://web.expasy.org/translate/) G*Power 3.1 G*Power, Germany Igor Pro WaveMetrics, USA ImageJ ImageJ, USA; Schneider et al., 2012 Leica Application Suite Leica, Germany LSM Image Browser Zeiss, Germany Microsoft Excel Microsoft, USA Microsoft Excel 2013 Microsoft, USA Microsoft Word 2013 Microsoft, USA PredictProtein Biosof LLC, USA (https://www.predictprotein.org/) Primer3 (http://primer3.ut.ee/) Whitehead Institute for Biomedical Research, USA Pulse & PulseFit HEKA Elektronik, Germany Reverse Complement Bioinformatics, Canada (http://www.bioinformatics.org/sms/rev_comp.html) Serial cloner SerialBasics, USA SigmaPlot Systat Software Inc, Germany SnapGene Viewer® GSL Biotech LLC, USA SPSS Statistics 20 IBM, USA Test Time Analyzer ImageJ, USA; Schneider et al., 2012 TMpred tool Hofman and Stoffel, 1992, (http://www.ch.embnet.org/software/TMPRED_form.html) Victor 2030 Workstation software Perkin Elmer, USA

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A.9 Accession numbers for BLAST

Nucleotide accession Species Protein accession number number Anolis carolinensis XM_003221173.1 XP_003221221.1 (Carolina anole) Danio rerio NM_001258223.1 NP_001245152.1 (Zebrafish) Gadus morhua AY126448.1 AAM95160.1 (Atlantic cod) Gallus gallus isoform S EU124631.1 ABX10831.1 (Chicken) Gallus gallus isoform L EU124630.1 ABX10830.1 (Chicken) Homo sapiens BC143688.1 AAI43689.1 (Human) Meleagris gallopavo XM_003205485.1 XP_003205533.1 (Turkey) Mus musculus isoform S EU303117.1 ACA01962.1 (Mouse) Mus musculus isoform S EU303118.1 ACA01963.1 (Mouse) Podarcis sicula DQ013043.2 AAY34941.2 (Ruin lizard) Rattus norvegicus NM_138860.1 NP_620215.1 (Brown rat) Taeniopygia guttata XM_002191902.1 XP_002191938.1 (Zebra finch) Xenopus laevis BC169653.1 AAI69653.1 (African clawed frog)

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A.10 Plasmids

Label Description Source Standard vector contains Melanopsin S kanamycin resistance with full Life Technologies, Germany T_Opn4x_mit8c_S length ORF of pigeon melanopsin isoform S (1329 bp) Standard vector contains Melanopsin L kanamycin resistance with full Life Technologies, Germany T_Opn4x_ohne8c_L length ORF of pigeon melanopsin isoform L (1713 bp) pCR2.1 Subcloning Invitrogen, Germany pCR2.1 vector with full length Melanopsin S sequence of pigeon melanopsin in house production pCR2.1-Mel_S isoform S without STOP codon pCR2.1 vector with full length Melanopsin L sequence of pigeon melanopsin in-house production pCR2.1-Mel_L isoform L without STOP codon N1 Vector for C-terminal fusion of pmCherry-N1 Clontech, Germany mCherry Pigeon melanopsin S C-terminally Melanopsin S tagged with mCherry on in-house production pmCherry-N1_Mel_S pmCherry-NI expression vector Pigeon melanopsin L C-terminally Melanopsin L tagged with mCherry on in-house production pmCherry-N1_Mel_L pmCherry-NI expression vector Vector for C-terminal fusion of pEGFP-N1 Addgene, Germany EGFP Pigeon melanopsin isoform L Melanopsin L C-terminally tagged with EGFP on in-house production pEGFP-N1_Mel_L pEGFP-N1 vector

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A.11 Oligonucleotide primers

Primer Primer sequence Target Application direction 5´ 3´ GTAAAACGACGGCCAGTGAATTCTCCAGCCA F Melanopsin CATTCTGTGACCAAG part I CAGGAAACAGCTATGACTCTAGATGG R Sequencing of GTGAATTATTGCATATATGATGG pigeons melanopsin GTAAAACGACGGCCAGTGAATTCTATTGTGT F (Chapter 2) Melanopsin ATGTCTTGTCCTGGT part II CAGGAAACAGCTATGACTCTAGATAA R CTCTGTAGATGAGCTAAGCCC Pigeon F GCTGTTCCCTGCTTAAGGTTCC Analysis of pigeon specific specific melanopsin insert R GCTCTACAGGGTCAAGCTCTACATC insert (Chapter 2)

F GCTAGCCACCATGGGCACCCAGCCCCACG Melanopsin isoform S R CCGCGGTCTTCCCTTTTCTGTGGTTGCTGCG Addition of NheI + SacII restriction sites F GCTAGCCACCATGGGCACCCAGCCCCACG (Chapter 5) Melanopsin isoform L R CCGCGGTGACAGAAATTTCTCAACAGCTTCA

All primers used for qPCR analysis (Chapter 4) can be found in E.2 Supplementary data CHAPTER 4, paragraph 6 qPCR oligonucleotides.

189

B. LIST OF ABBREVIATIONS

7TM seven transmembrane µl microliter µM micro molar A (amino acid) alanine A (developmental stage) adult aa amino acid Actb β-actin ANOVA analysis of variance approx. approximately Aqua dest. distilled water ARP acidic ribosomal protein BDNF brain-derived neurotrophic factor BLAST basic local alignment search tool C (amino acid) cysteine Ca2+ calcium ion CaCl2 calcium chloride cAMP cyclic adenosine monophosphate cDNA complementary DNA cf. conferre (Latin), compare cGMP cyclic guanosine monophosphate Cq quantification cycle Cry Cryptochrome CNI Close-Neighbor-Interchange CTB Cholera Toxin Subunit B CV coefficient of variation Cytb Cytochrome b D (amino acid) aspartic acid DAB 3,3'-Diaminobenzidine DAG diacylglycerol DDBJ DNA Data Bank of Japan dLGN dorsal lateral geniculate nucleus DMEM Dulbecco’s modified Eagle Medium DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate dRGC displaced retinal ganglion cell dT deoxythymidine dz effect size E (amino acid) glutamic acid E (brain region) Entopallium E (developmental stage) embryonic day E.coli Escherichia coli EDTA ethylenediaminetetraacetic acid e.g. exempli gratia (Latin), for example EGFP enhanced green fluorescent protein 190

B. LIST OF ABBREVIATIONS

EGTA ethylene glycol tetraacetic acid EMBL European Molecular Biology Laboratory EPCR primer efficiency etc. et cetera EtOH ethanol E value expect value F (amino acid) phenylalanine FAD flavin adenine dinucleotide G (amino acid) glycine Gapdh glyceraldehyde3-phosphate dehydrogenase GCL ganglion cell layer GIRK G-protein-coupled inwardly-rectifying potassium GLd Nucleus geniculatus lateralis pars dorsalis GPCR G-protein-coupled receptor GΩ gigaohm h hour H (amino acid) histidine HEK human embryonic kidney HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HEPES-NaOH HEPES sodium hydroxide Hprt1 hypoxanthine phosphoribosyltransferase1 HRP horseradish peroxidase I (amino acid) isoleucine INL inner nuclear layer IP3 inositol triphosphate IPL inner plexiform layer ipRGC intrinsically photosensitive retinal ganglion cell IRC inter-run calibration IS inner segments K (amino acid) lysine K+ potassium ion K-Aspartate potassium aspartate kb kilo base KCl potassium chloride L (amino acid) leucine L (isoform) long LB lysogeny broth LGN lateral geniculate nucleus LOD limit of detection LOQ limit of quantification M (qPCR) gene stability measure M (amino acid) methionine MAPK 1 mitogen activated (MAP) kinase 1 MgATP magnesium adenosine triphosphate MgCl2 magnesium chloride min minute miRNA/miR microRNA 191

B. LIST OF ABBREVIATIONS

MIQE minimum information for publication of qPCR experiments mm millimeter mOsm milliosmole mRNA messenger RNA MTFH methylene tetrahydrofolate mTOR mechanistic target of rapamycin mV millivolt MΩ megaohm N (amino acid) asparagine Na2+ sodium ion NaCl sodium chloride Nad4 nicotinamide adenine dinucleotide dehydrogenase subunit 4 NaGTP guanosine 5′-triphosphate sodium salt hydrate nBOR nucleus of the basal optic root NCBI National Center for Biotechnology Information NFL nerve fiber layer nl nanoliter nM nanomolar nm nanometer NT-3 neurotrophin 3 NTC no template control OM Tractus occipitomesencephalicus ONL outer nuclear layer OPL outer plexiform layer Opn4 melanopsin Opn4m mammalian melanopsin Opn4x non-mammalian/invertebrate melanopsin ORF open reading frame OS outer segments P (amino acid) proline PBS phosphate buffered saline PCR polymerase chain reaction PFA paraformaldehyde PH (developmental stage) post-hatch day PHR domain N-terminal photolyase-related domain PIP2 phosphatidylinositol 4,5-bisphosphate PK protein kinase PKC protein kinase C PLC phospholipase C pmol picomolar Ppia peptidylprolyl isomerase A pRGC photosensitive retinal ganglion cell PRL photoreceptor layer PTX pertussis toxin Q (amino acid) glutamine QC query coverage 192

B. LIST OF ABBREVIATIONS q-(RT)PCR quantitative polymerase chain reaction R retina R (amino acid) arginine RGC retinal ganglion cell RHT retinohypothalamic tract RIN RNA integrity number RNA ribonucleic acid ROI region of interest RPE retinal pigmentepithelium rpm revolutions per minute RT (PCR) reverse transcriptase RT (brain region) nucleus rotundus S (amino acid) serine S (isoform) short s seconds SCN suprachiasmatic nucleus SEM standard error of the mean S.O.C. super optimal broth containing glucose T (amino acid) threonine TBE Tris/Borate/EDTA buffer TGF-β transforming growth factor-β Tm melting temperature TM transmembrane TO optic tectum, tectum opticum trkB tyrosine receptor kinase B Trp transient receptor potential channel TSM tractus septomesencephalis TTX teterodotoxin UV ultraviolet V (amino acid) valine VEGF vascular endothelial growth factor W (amino acid) tryptophan Y (amino acid) tyrosine

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C. LIST OF FIGURES

Figure 1.1: Schematic overview of the vertebrate retina...... 5 Figure 1.2: Two pathways in the avian visual system...... 7 Figure 1.3: Ontogeny of lateralization in the tectofugal system...... 11

Figure 1.4: Melanopsin Gq (A)/Gi/o (B) signaling pathway...... 18 Figure 2.1: Position of the selected primers on the putative pigeon melanopsin gene...... 25 Figure 2.2: Position of the selected primers for analysis of the pigeon specific melanopsin insert...... 30 Figure 2.3: Illustration of the entire melanopsin gene sequence (exon 1 to 9) in pigeons without introns...... 32 Figure 2.4: PCR analysis of pigeon specific melanopsin insert in different pigeons...... 34 Figure 2.5: Deduced amino acid sequence of pigeon melanopsin protein...... 35 Figure 2.6: A Minimum-Evolution tree regarding evolutionary relationships of melanopsin...... 40 Figure 2.7: Phylogenetic tree of the Vertebrata subphylum...... 46 Figure 3.1: Immunolabeling of the pigeon retina with an anti-melanopsin antibody...... 56 Figure 3.2: Fluorescent immunolabeling of the pigeon’s retina according to tectal CTB injections...... 57 Figure 4.1: Comparison of the relative melanopsin expression levels of isoform S and L in the retina of adult pigeons...... 77 Figure 4.2: Relative expression levels of melanopsin isoform L in the retina of pigeon embryos and hatchlings...... 79 Figure 4.3: Comparison of melanopsin isoform expression values between normal-incubated and dark-incubated pigeon embryos...... 80 Figure 4.4: Analysis of variance between normal- and dark-incubated pigeon embryos...... 81 Figure 5.1: Schematic overview representing the general cloning strategy of pigeon melanopsin into pm-Cherry-N1 vector...... 92 Figure 5.2: Representative photomicrographs showing the heterologous expression of melanopsin isoform S and L C-terminally tagged with mCherry in HEK cells. .... 102 Figure 5.3: Light-induced changes in intracellular Ca2+ levels by pigeon melanopsin in transfected HEK cells...... 103 194

C. LIST OF FIGURES

Figure 5.4: Direct comparison of light-induced changes in intracellular Ca2+ levels of melanopsin isoform S and L...... 104 Figure 5.5: Representative photomicrographs showing the heterologous expression of either melanopsin isoform S (A) or L (B)...... 105 Figure 5.6: Light-induced changes in intracellular Ca2+ levels of melanopsin isoform S and L...... 106 Figure 6.1: Schematic overview representing the recloning strategy of pigeon melanopsin...... 113 Figure 6.2: Schematic drawing of whole cell patch-clamp measurement configuration...... 116 Figure 6.3: Simple stimulation of pigeon melanopsin isoform L in vitro...... 119 Figure 6.4: Long-term stimulation of pigeon melanopsin isoform L in vitro...... 120 Figure 6.5: Repetitive activation of pigeon melanopsin isoform L in vitro...... 121 Figure 6.6: Dependency of activation pulse duration on the relative amplitude of the GIRK currents...... 122 Figure 6.7: Wavelength-dependency of pigeon melanopsin isoform L...... 123 Figure 7.1: Illustration of the requirements melanopsin has to fullfill as a possible inducer of visual lateralization in pigeons...... 131 Figure 7.2: Possible transfer mechanisms of asymmetrical light stimulation onto higher brain regions during embryonic development in the pigeon...... 133

195

D. LIST OF TABLES

Table 2.1: Melanopsin reverse primers used for the transcription of retinal RNA into cDNA...... 28 Table 2.2: Melanopsin oligonucleotide primers used for amplification of the putative melanopsin gene in pigeons via PCR...... 29 Table 2.3: Melanopsin oligonucleotide primers used for amplification of the pigeon specific melanopsin DNA insert sequence via PCR...... 29 Table 2.4: Exon length of the melanopsin DNA sequence found in pigeons...... 33 Table 2.5: Pigeon melanopsin protein sequence analysis (PredictProtein, PROSITE)...... 36 Table 2.6: Pigeons melanopsin gene ORF sequence compared to other melanopsin genes (BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi)...... 38 Table 2.7: Pigeons melanopsin amino acid sequence compared to other melanopsin genes...... 39 Table 3.1: Antibodies used for fluorescent immunohistochemically staining...... 55 Table 4.1: Overview of potential reference genes for quantitative melanopsin expression analysis in pigeon retina...... 68 Table 4.2: Overview of primer used for amplification of putative reference genes for sequencing...... 69 Table 4.3: Overview of primers used for evaluation of putative reference genes for qPCR...... 72 Table 4.4: Overview of reference genes used for quantification of melanopsin...... 75 Table 4.5: Overview of primers used for quantification of melanopsin...... 76 Table 5.1: Overview of primer used for addition of NheI and SacII restriction sites...... 94 Table 5.2: Application protocol for Fluo-4 calcium assay...... 99 Table 6.1: Application protocols of different patch-clamp experiments in HEK 1.2 cells expressing pigeons melanopsin isoform L...... 117

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E.1 Supplementary data Chapter 2

Table E.1.1: Positions and length of the transmembrane domains found in pigeon melanopsin. Putative transmembrane domains were calculated using TMpred tool (transmembrane prediction tool; Hofman and Stoffel, 1992, http://www.ch.embnet.org/software/TMPRED_form.html). Opsin typical seven transmembrane domains were found (aa = amino acid). Transmembrane Position [aa] Length [in aa] domain 1 16 - 43 27 2 54 - 80 26 3 92 - 114 22 4 132 - 157 25 5 184 - 204 20 6 241 – 260 19 7 279 - 297 18

Figure E.1.1: BLAST comparison of pigeon melanopsin isoform L and mouse melanopsin isoform S. Both melanopsin isoforms show a higher identity in the N-terminal region up to amino acid 357 (of pigeon melanopsin).

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Table E.1.2.a: Nucleotide sequence of pigeon melanopsin gene (isoform S) and corresponding amino acid sequence. Whole ORF of pigeon melanopsin isoform S sequence. In bold red letters the START codon (ATG) and the STOP codon (first: TAA) are marked, bold blue letters indicate the exon/exon boundaries. Red background and white letters mark the pigeon specific insert of 87 bp (exon 8a) and blue background with white letters indicate the additional sequence insert of 86 bp containing a premature STOP codon found in the short isoform (Abbreviations: A = adenine, bp = base pair, C = cytosine, G = guanine, ORF = open reading frame, T = thymine).

Nucleotide sequence of pigeons melanopsin short isoform (isoform S) ATGGGCACCCAGCCCCACGCTCTGACCAAGGCAGAGATACCCGCCCATGTTCTTTACACCGTAG GAACATGCGTGCTCATTATCGGCTCCATTGGCATCATTGGAAACCTCCTAGTTCTCTACGCATTT exon 1 TACAGCAACAAGAAGCTGAGGACACCCCAAAACTACTTTATAATGAATTTGGCCATGAGTGAC

TTCTTGATGTCGGCTTCTCAGGCACCCATGTGCTTTGTGAACAGCTTGCACGGAGAGTGGATAC exon 2 TTGGAGATATAGGCTGTGACATGTACGCTTTCTGTGGGGCACTCTTTGGAATAACCTCAATGAT GACTTTATTGGCTATTTCTGTGGATCGCTACCTTGTGATCACGATGCCCCTGCGATCCATCCAGT GGACGTCCAAGAAGCGCACGGTGCAGATCATCGCTGCCGTCTGGCTGTACTCGCTGGGCTGGA exon 3 GCGTGGCTCCGCTCTTCGGGTGGAGTTCCTATGTGCCTGAGGGCTTGATGATATCCTGTACGTG GGACTACGTAACCTACTCCCCTGCAAACAGAAGTTACACCATGATTTTATGTTGCTGCGTGTTTT TTATTCCTCTTATAATAATATTCCATTGTTATTTATTTATGTTCCTGGCCATAAGACGTACTGGCA exon 4 GAGACGTTCAGAAGCTGGGCTCCTGCAGCCGGAAATCCTACCTCTCTCAGTCCATGAAGAATG

AATGGAAACTGGCAAAAATTGCTTTTGTGGTCATCATTGTGTTTGTCTTGTCTTGGTCTCCATAT GCTTGTGTCACCTTGATTGCCTGGGCAGGTCGAGGCAACACCCTAACACCATATTCCAAATCCG exon 5 TGCCAGCAGTTATTGCCAAAGCTTCTGCAGTCTACAACCCCATCATCTACGCAATAATTCACCCA AGATACAGAAAAACCATCCACAACGCTGTTCCCTGCTTAAGGTTCCTTATACGAATATCGAAGA exon 6 ATGACCTCCTGAGAGGGTCCATAAATGAATCCTCATTTAGGACATCTCTCTCCAGCCATCACTCT CTTGCTGGCAGGACCAAGAGCACTTGTGTTTCATCCATTTCCACCGGAGAAGCTACCACTGATT exon 7

ACACAATGATGCATGAGAGTATCAACTGTGATGATTTCATGAACTCTAAAATATCCAGACCATC exon8a CTTGCAGAATGAGACCTGGAGCGATGTAGAGCTTGACCCTGTAGAGCCAGCTCATAAGAAGCT GCAGCCCCGCCGAAGTCATTCTTTCTCAGCAAGTGTGAGACAGGAGAAGAGGGACCTGCTCCC exon 8b AAAGACCTGCAGCTGCGACGCAGCAACCACAGAAAAGGGAAGATAAGAGAAAGAAACTCCCA TTTCTCCCTAAGTGGGAGAGCTCTGGGAGCTGCCACCCTTCCCTCACTGAAAGTGGAGATGGTC TCATTCTGCTCCAGCTATCTGGAGAAGGTGCTTGGGCGGGCAGTCCTACACAGTCCCCCTGCAA exon 8c

CACTCGTGACCAGCTCCCTAAAAGCAGCTTCTCTACCTGTTGGTTTGAACAGCAGCAGCATCAG

CAGAGGAGGCGACACAGACACTTCAGAGATAGCAAGTCAAGAAAGTCAAGTTAACGGAGTCC TGGGCTCTATCATTAGCAATACTGTCCCTCGCATCATCATCATTCCTACCTCAGAGACCAACCTA TTTCGAGAGGAACTAGAAGAGGAAGAGACTCAGTTGTTCCACTTTCCYGACAAAAAGGGCAAT exon9 CTGCTGGACTTGGAAGGGCTTAGCTCATCTGTAGAGTTTCTTGAAGCTGTTGAGAAATTTCTGT CATGA

Corresponding amino acid sequence of isoform S MGTQPHALTKAEIPAHVLYTVGTCVLIIGSIGIIGNLLVLYAFYSNKKLRTPQNYFIMNLAMSDFLMSASQAPMCFVNSLHG EWILGDIGCDMYAFCGALFGITSMMTLLAISVDRYLVITMPLRSIQWTSKKRTVQIIAAVWLYSLGWSVAPLFGWSSYVPE GLMISCTWDYVTYSPANRSYTMILCCCVFFIPLIIIFHCYLFMFLAIRRTGRDVQKLGSCSRKSYLSQSMKNEWKLAKIAFVVII VFVLSWSPYACVTLIAWAGRGNTLTPYSKSVPAVIAKASAVYNPIIYAIIHPRYRKTIHNAVPCLRFLIRISKNDLLRGSINESSF RTSLSSHHSLAGRTKSTCVSSISTGEATTDYTMMHESINCDDFMNSKISRPSLQNETWSDVELDPVEPAHKKLQPRRSHSFS ASVRQEKRDLLPKTCSCDAATTEKGR

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Table E.1.2.b: Nucleotide sequence of pigeon melanopsin gene (isoform L) and corresponding amino acid sequence. Whole ORF of pigeon melanopsin isoform L sequence. In bold red letters the START codon (ATG) and the STOP codon (TGA) are marked, bold blue letters indicate the exon/exon boundaries. Red background and white letters mark the pigeon specific insert of 87 bp (exon 8a; Abbreviations: A = adenine, bp = base pair, C = cytosine, G = guanine, ORF = open reading frame, T = thymine).

Nucleotide sequence of pigeons melanopsin long isoform (isoform L) ATGGGCACCCAGCCCCACGCTCtGACCAAGgCAGAGATACcCGCcCATGTtCTtTACACcGTAGG exon 1 AACATGCGTGCTCATtATCGGCTCCATTGGCATCATTGGAAACCTCCTAGTTCTCTACGCATTTT ACAGCAACAAGAAGCTGAGGACACCCCAAAACTACTTTATAATGAATTTGGCCATGAGTGACT exon 2 TCTTGATGTCGGCTTCTCAGGCACCCATGTGCTTTGTGAACAGCTTGCACGGAGAGTGGATACT

TGGAGATATAGGCTGTGACATGTACGCTTTCTGTGGGGCACTCTTTGGAATAACCTCAATGATG ACTTTATTGGCTATTTCTGTGGATCGCTACCTTGTGATCACGATGCCCCTGCGATCCATCCAGTG exon 3 GACGTCCAAGAAGCGCACGGTGCAGATCATCGCTGCCGTCTGGCTGTACTCGCTGGGCTGGAG CGTGGCTCCGCTCTTCGGGTGGAGTTCCTATGTGCCTGAGGGCTTGATGATATCCTGTACGTGG GACTACGTAACCTACTCCCCTGCAAACAGAAGTTACACCATGATTTTATGTTGCTGCGTGTTTTT exon 4 TATTCCTCTTATAATAATATTCCATTGTTATTTATTTATGTTCCTGGCCATAAGACGTACTGGCAG

AGACGTTCAGAAGCTGGGCTCCTGCAGCCGGAAATCCTACCTCTCTCAGTCCATGAAGAATGA

ATGGAAACTGGCAAAAATTGCTTTtGTGGTCATCATTGTGTTTGTCTTGTCTTGGTCTCCATATG exon 5 CTTGTGTCACCTTGATTGCCTGGGCAGGTCGAGGCAACACCCTAACACCATATTCCAAATCCGT GCCAGCAGTTATTGCCAAAGCTTCTGCAGTCTACAACCCCATCATCTACGCAATAATTCACCCAA exon 6 GATACAGAAAAACCATCCACAACGCTGTTCCCTGCTTAAGGTTCCTTATACGAATATCGAAGAA TGACCTCCTGAGAGGGTCCATAAATGAATCCTCATTTAGGACATCTCTCTCCAGCCATCACTCTC exon 7 TTGCTGGCAGGACCAAGAGCACTTGTGTTTCATCCATTTCCACCGGAGAAGCTACCACTGATTA exon8a CACAATGATGCATGAGAGTATCAACTGTGATGATTTCATGAACTCTAAAATATCCAGACCATCC

TTGCAGAATGAGACCTGGAGCGATGTAGAGCTTGACCCTGTAGAGCCAGCGAGTCATAAGAA exon 8b GCTGCAGCCCCGCCGAAGTCATTCTTTCTCAGCAAGTGTGAGACAGGAGAAGAGGGACCTGCT CCCAAAGACCTGCAGCTGCGACGCAGCAACCACAGAAAAGGTCTCATTCTGCTCCAGCTATCTG

GAGAAGGTGCTTGGGCGGGCAGTCCTACACAGTCCCCCTGCAACACTCGTGACCAGCTCCCTA

AAAGCAGCTTCTCTACCTGTTGGTTTGAACAGCAGCAGCATCAGCAGAGGAGGCGACACAGAC exon9 ACTTCAGAGATAGCAAGTCAAGAAAGTCAAGTTAACGGAGTCCTGGGCTCTATCATTAGCAAT ACTGTCCCTCGCATCATCATCATTCCTACCTCAGAGACCAACCTATTTCGAGAGGAACTAGAAG AGGAAGAGACTCAGTTGTTCCACTTTCCYGACAAAAAGGGCAATCTGCTGGAcTTGGAAGGGC TTAGCTCATCTGTAGAGTTTCTTGAAGCTGTTGAGAAATTTCTGTCATGA

Corresponding amino acid sequence of isoform L MGTQPHALTKAEIPAHVLYTVGTCVLIIGSIGIIGNLLVLYAFYSNKKLRTPQNYFIMNLAMSDFLMSASQAPMCFVNSLHG EWILGDIGCDMYAFCGALFGITSMMTLLAISVDRYLVITMPLRSIQWTSKKRTVQIIAAVWLYSLGWSVAPLFGWSSYVPE GLMISCTWDYVTYSPANRSYTMILCCCVFFIPLIIIFHCYLFMFLAIRRTGRDVQKLGSCSRKSYLSQSMKNEWKLAKIAFVVII VFVLSWSPYACVTLIAWAGRGNTLTPYSKSVPAVIAKASAVYNPIIYAIIHPRYRKTIHNAVPCLRFLIRISKNDLLRGSINESSF RTSLSSHHSLAGRTKSTCVSSISTGEATTDYTMMHESINCDDFMNSKISRPSLQNETWSDVELDPVEPASHKKLQPRRSHSF SASVRQEKRDLLPKTCSCDAATTEKVSFCSSYLEKVLGRAVLHSPPATLVTSSLKAASLPVGLNSSSISRGGDTDTSEIASQES QVNGVLGSIISNTVPRIIIIPTSETNLFREELEEEETQLFHFXDKKGNLLDLEGLSSSVEFLEAVEKFLS

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Figure E.1.2a: PROSITE data for pigeon melanopsin isoform S. Protein sequence analysis using the online tool PredictProtein (http://ppopen.informatik.tu-muenchen.de/; Yachdav et al., 2014).

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Figure E.1.2b: PROSITE data for pigeon melanopsin isoform L. Protein sequence analysis using the online tool PredictProtein (http://ppopen.informatik.tu-muenchen.de/; Yachdav et al., 2014).

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E.2 Supplementary data CHAPTER 4

The following pages list information required by the MIQE guidelines that are considered essential according to Bustin et al., 2009 Table 1.

1. Experimental design a) Definition of experimental and control Adult pigeons groups Pigeon hatchling (PH1-2) Pigeon embryos (E16) Dark-incubated pigeon embryos (ED15-16) b) Number within each group Adult pigeon retina: n=8 for each left and right Hatchling, embryonic and dark-incubated embryonic pigeon retina: n = 8 for each left and right

2. Sample a) Description Retinal tissue from adult, hatchling, embryonic and dark-incubated embryonic pigeons b) Volume/mass of sample proceeded Up to 20 mg of RNAlater® stabilized tissue c) Microdissection or macrodissection Macrodissection d) Processing procedure Upon decapitation, the retina was prepared and immediately immersed in RNAlater® (Sigma- Aldrich, Germany) e) If frozen, how and how quickly? Stored in RNAlater® at 4 °C for one day, than frozen in -80 °C; preparation time: max. 10 min for adult pigeons and max. 6:30 min for hatchlings, embryos and dark-incubated embryos f) If fixed, with what and how quickly? No fixation g) Sample storage conditions and duration Stored in RNAlater® (Sigma, Germany) at 4 °C for (especially for FFPE samples) one day, then at -80 °C until further processing

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3. Nucleic acid extraction a) Procedure and/or instrumentation Total RNA extraction b) Name of kit and details of any RNeasy® Mini Kit (Qiagen, Germany) modifications Elution step was modified to 2 elution steps with 30 + 20 µl and an incubation time of 2 min per elution step c) Details of DNase or RNase treatment DNase treatment was integrated according to the manufacturer's recommendation using the DNaseI Kit that is compatible with the RNeasy® Mini Kit (Qiagen, Germany) d) Contamination assessment (DNA or DNA digestion only RNA) e) Nucleic acid quantification RNA concentration was determined using spectrophotometric measurement at 260 nm f) Instrument and method BioTek’s Take3™ and Gen5™, Biotek, Germany g) Purity (A260/A280) See Table E.2.4a and b h) Yield See Table E.2.4a and b i) RNA integrity: method/instrument 2100 Bioanalyzer Instrument (Agilent Technologies, USA) j) RIN/RQI or Cq of 3' and 5' transcripts For RIN see section 4.2.3 and Table E.2.4a and b and Figure E.2.1a and b k) Electrophoretic traces See Figure E.2.1a and b l) Inhibition testing (Cq dilutions, spike, or Standard curve of cDNA with 10 fold dilution steps other) over 4 data points

4. Reverse Transcription See manual of QuantiTect Reverse Transcription Kit a) Complete reaction conditions (Qiagen, Germany) Adult retina: 100 ng b) Amount of RNA and reaction volume Hatchling, embryonic, dark-incubated embryonic retina: 1 µg c) Priming oligonucleotide (if using GSP) RT primer mix of oligo-dT and random primers, and concentration according to manufacturer's recommendation Quantiscript Reverse Transcriptase including RNase d) Reverse transcriptase and concentration inhibitor, according to manufacturer's recommendation 15 min at 42 °C and subsequent RTase inactivation e) Temperature and time at 95 °C for 3 min f) Manufacturer of reagents and catalogue Qiagen, Germany numbers Cat No./ID 205311 Dilution adult 1:100 Dilution hatchling, embryonic, dark-incubated g) Storage conditions of cDNA embryonic retina: 1:10 Frozen in 20 µl Aliquots and stored at -80 °C

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5. qPCR target information d) In silico e) Location of each b) Sequence f) What splice a) Gene c) Amplicon specificity primer by exon or accession variants are symbol length screen intron (if number targeted? (BLAST…) applicable) No accession Exon only Actb number, 87 bp R-Primer is exon- N/A sequences spanning have been Exon only ARP identified for 82 bp R-Primer is exon- N/A the first time spanning in pigeons Exon only Cytb (Columba 87 bp consists of only one N/A livia) exon Exon only Gapdh AF 036934.1 91 bp F-Primer is exon- N/A spanning Using Primer3, Exon only Hprt1 86 bp Mispriming F-Primer is exon- N/A Library (repeat spanning library) was set Exon only Mapk1 81 bp to "rodent and R-Primer is exon- N/A No accession simple" spanning number, Exon only Nad4 sequences 90 bp consists of only one N/A have been exon identified for the first time Exon only Ppia in pigeons 89 bp R-Primer is exon- N/A (Columba spanning livia) Exon only Opn4xS 90 bp F-Primer is exon- Isoform S spanning Exon only Opn4xL 91 bp F-Primer is exon- Isoform L spanning

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6. qPCR oligonucleotides c) Location and d) Manufacturer a) Gene e) Purification b) Primer sequences identity of of symbol methods any mod. oligonucleotides TGTACCCTGGCATTGCTGAC Actb CAGGCGGGGCAATGATCTT CGGCAGCATTTACAATCCCG ARP GACGTTACGAACACCCTCCA CAGACACCACCCTAGCCTTT Cytb CGTTTGCATGGAGGTTTCGG CATACACAGAGGACCAGGTTGT Gapdh TCATTCAGTGCAATGCCAGC TCATGGACAGGACGGAGAGG Hprt1 ACCCTTGAGTACACAGAGAGC No Invitrogen, Desalted TGTGCTTCATCGTGACCTCA modifications Germany Mapk1 GCCAGTCCAAAGTCACAAATCTT CAACTCCTGAACTGGCCTCC Nad4 GGTAGTCATAGGTGCAGGCC ATCCTGTCCATGGCCAACG Ppia TGCTTGCCATCCAACCACTC CAGCAACCACAGAAAAGGGAAG Opn4xS CCCAAGCACCTTCTCCAGAT AGAAAAGGTCTCATTCTGCTCCA Opn4xL TGGTCACGAGTGTTGCAGG

7. qPCR protocol a) Complete reaction conditions See section 4.2.8 b) Reaction volume and amount of 5 µl reaction volume cDNA/DNA Adult animals: 100 ng RNA for RT, then diluted 1:100 in H2O Hatchlings, embryos and dark-incubated embryos: 1 µg RNA for RT, then diluted 1:10 in H2O 1 µl was used in each reaction volume c) Primer, (probe), Mg2+ and dNTP Primer concentration: 300 nM of each forward and concentrations reverse primer Rest was performed according to the manufacturer's recommendations d) Additives (SYBR Green I, DMSO and so EvaGreen® forth) e) Manufacturer of plates/ tubes and Bio-Rad, Germany catalog number HSP-3805R Hard-Shell 384-Well Plates Clear shell/white well f) Complete thermocycling parameters See section 4.2.8 g) Manufacturer of qPCR instrument Bio-Rad, Germany Thermocycler CFX384 Touch Real-Time PCR Detection System 205

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8a. qPCR validation b) Specificity d) Calibration e) PCR c) For SYBR f) r2 of a) Gene (gel, melt, curves with efficiency Green I, Cq calibration symbol sequence or slope and y calculated of the NTC curve digest) intercept from slope Slope: -3.272 Melting curve Actb y-intercept: 34.382 102.1% 0.996 Data not shown Data not shown Melting curve Slope: -3.332 ARP see Figure E.2.2a y-intercept: 36.152 99.6% 0.997 B See Figure E.2.2a A Slope: -3.314 Melting curve Cytb y-intercept: 36.158 100.3% 0.997 Data not shown Data not shown Melting curve Slope: -3.391 Gapdh see FigureE.2.2a y-intercept: 36.668 97.2% 0.999 B see Figure E.2.2a A Slope: -3.392 Melting curve Hprt1 No SYBR Green, y-intercept: 35.978 99.7% 0.998 Data not shown SsoFast™ Data not shown EvaGreen® Slope: -3.442 Melting curve instead, Mapk1 y-intercept: 42.938 95.2% 0.979 Data not shown no Cq signal Data not shown Slope: -3.462 Melting curve Nad4 y-intercept: 35.707 96.1% 1.000 Data not shown Data not shown Melting curve Slope: -3.108 Ppia see Figure E.2.2a y-intercept: 35.936 109.8% 0.964 B See Figure E.2.2a A Melting curve Slope: -3.300 Opn4xS see Figure E.2.2b y-intercept: 42.586 100.9% 0.991 B See Figure E.2.2a B Melting curve Slope: -3.587 Opn4xL see Figure E.2.2b y-intercept: 43.520 90.0% 0.990 B See Figure E.2.2a B

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8b. qPCR validation a) Gene b) Linear c) Cq variation d) Evidence e) If multiplex, efficiency and symbol dynamic range at LOD (SD) for LOD LOD of each assay

Actb N/A N/A N/A

See Figure E.2.2a A See section ARP 0.64 and E.2.3 4.2.7

Cytb N/A N/A N/A

See Figure E.2.2a A See section Gapdh 1.26 and E.2.3 4.2.7

Hprt1 N/A N/A N/A No multiplex analysis Mapk1 N/A N/A N/A

Nad4 N/A N/A N/A

See Figure E.2.2a A See section Ppia 0.84 and E.2.3 4.2.7 See Figure See section Opn4xS 0.37 E.2.2b A 4.2.7 See Figure See section Opn4xL 0.44 E.2.2b A 4.2.7

9. Data analysis a) qPCR analysis program (source, version) CFX Manager™ Software 3.1 (Bio-Rad, Germany) b) Method of Cq determination Threshold set by program c) Outlier identification and disposition Based on triplicates d) Results for NTCs No amplification curves observant e) Justification of number and choice of Pairwise variation using the CFX Manager™ reference genes Software 3.1 (Bio-Rad, Germany) f) Description of normalization method ∆∆Cq using CFX Manager™ Software 3.1 (Bio-Rad, Germany) g) Number and stage (reverse transcription Each experiment was repeated three times with or qPCR) of technical replicates triplicates of each sample (nine data points per sample) h) Repeatability (intra-assay variation) Each plate has been repeated three times i) Statistical methods for results Paired and un-paired sample t-Test significance j) Software (source, version) SPSS Statistics 20 (IBM, USA) 207

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Table E.2.1: Partial sequences of reference genes for qPCR analysis. Forward and reverse primer sequences for the qPCR experiment are highlighted in green and yellow (bold letters), respectively. Bold blue letter show the exonal borders. Letters highlighted in red mark polymorphisms (A: adenine; ARP: acidic ribosomal protein; C: cytosine; Gapdh: Glyceraldehyde 3-phosphate (GAP) dehydrogenase; G: guanine; Ppia: peptidylprolyl isomerase A; T: thymine). Partial ARP mRNA sequence (Columba livia) ACTACTTCTTGAAAATCATCCAACTCCTGGATGATTACCCAAAATGTTTCATTGTGGGAGCAGACAATGTGGGATCCAA GCAGATGCAGCAGATCCGTATGTCCCTGCGAGGGAAGGCTGTTGTGCTGATGGGGAAGAACACGATGATGCGCAAAG CTATTCGTGGTCATCTGGAGAATAACCCTGCCTTAGAAAAGCTGCTCCCTCATATCCGTGGGAATGTGGGCTTTGTGTT CACCAAGGAGGATCTGACCGAGATCCGYGACATGCTGCTGGCTAACAAGGTGCCAGCAGCTGCCCGTGCYGGCGCTA TTGCTCCTTGTGATGTGACGGTGCCGGCACAGAACACCGGTCTCGGCCCGGAGAAGACCTCCTTTTTCCAGGCCTTGG GCATCACCACGAAGATTTCCAGAGGGACCATTGAAATTCTGAGCGATGTGCAGCTCATCAAGACTGGAGACAAAGTGG GTGCCAGCGAAGCCACCCTGCTGAACATGCTGAACATCTCCCCCTTCTCTTTCGGGCTGGTGATCCAGCAGGTCTTTGA CAACGGCAGCATTTACAATCCCGAAGTGCTGGACATCACCGAGGAGACCTTGCACAAGCGCTTCCTGGAGGGTGTTC GTAACGTCGCCAGCGTCTGTCTGCAGATCGGGTACCCGACCATCGCTTCTGTGCCCCACTCCATCATCAACGGGTACAA GCGGGTCCTGGCCGTGGCGGTGGAGACGGACTACACCTTCCCGCTGGCTGAGAAGGTGAAGGCCTTCCTGGCAGACC CCTCTGCCTTCGTGGTGGC Partial Gapdh mRNA sequence (Columba livia) GGAATCAACGGATTTGGCCGCATCGGCCGCCTGGTCACCCGGGCTGCTATTCTCTCTGCCAAAGTCCAAGTGGTGGCC ATCAACGATCCCTTCATTGATCTGAACTACATGGTTTACATGTTCAAATATGACTCTACTCATGGTCACTTCCGGGGCAC TGTCAAGGCCGAGAATGGGAAACTTGTGATTAATGGGAATGCTATCACCATCTTCCAGGAGCGTGATCCCAGCAACAT AAAGTGGGCAGATGCTGGTGCTGAATATGTTGTGGAGTCCACTGGTGTCTTTACCACCATGGAGAAGGCTGGGGCTCA CCTGAAGGGTGGTGCTAAGCGTGTTATCATCTCTGCCCCCTCAGCTGATGCTCCCATGTTTGTGATGGGTGTCAACCAT GAAAAGTATGACAAGTCCCTGAAAATTGTCAGCAATGCCTCTTGCACTACTAACTGCCTGGCGCCCTTGGCCAAGGTCA TCCATGACAACTTCGGCATCGTGGAGGGTCTTATGACCACTGTCCATGCTATCACAGCCACACAGAAGACAGTGGATG GGCCCTCTGGGAAGCTGTGGAGrGATGGCAGAGGTGCTGCCCAGAACATTATTCCAGCATCTACTGGAGCTGCTAAGG CTGTGGGAAAAGTCATCCCTGAGCTCAATGGGAAACTTACTGGAATGGCTTTCCGTGTGCCAACCCCCAATGTCTCTGT TGTGGACCTGACCTGCCGTCTGGAAAAACCAGCCAAGTATGATGACATTAAGAGGGTAGTGAAGGCTGCTGCTGATG GGCCCCTGAAGGGCATCCTGGCATACACAGAGGACCAGGTTGTCTCATGTGACTTCAACGGTGACAGCCATTCCTCCA CCTTTGATGCGGGTGCTGGCATTGCACTGAATGACCACTTTGTCAAGCTGGTTTCCTGGTATGATAATGAGTATGGATA CAGCAACCGTGTTGTGGACTTGATGGTCCACATGGCATCCAAGGAGTGAGCCKAGCACACAGCCCCCCCTGCTGCCTA GGGAAGCAGGACTCTCCTTTGTTGGAGCCCCTGCCCTTCTTCACCACCGCTTAGCTCTGCATCCTGCAGTGAGAGGCCA GTTCTGTTCCCTTGTCTCCCCCACTCCTCC Partial Ppia mRNA sequence (Columba livia) AGCTGTTTGCAGACAAGGTCCCCAAGACAGCAGAAAACTTCCGTGCCCTGAGCACCGGTGAGAAGGGATTTGGCTACA AGGGGTCCTGCTTCCACAGAATCATTCCTGGGTTCATGTGCCAGGGTGGCGACTTCACGCGCCACAAYGGCACCGGCG GCAAATCCATCTATGGGGAGAAGTTCCCCGATGAGAACTTCATCCTGAAGCACACAGGCCCTGGCATCCTGTCCATGG CCAACGCTGGCCCCAACACAAATGGTTCCCAGTTCTTCATCTGCACTGCCAAGACCGAGTGGTTGGATGGCAAGCACG TTGTCTTCGGCCGCGTCAAGGAGGGGATGAACGTGGTGGAGGCCATGGAGCGCTGG

Table E.2.2: Partial sequences calibration gene used for qPCR analysis. Forward and reverse primer sequences for the qPCR experiment are highlighted in green and yellow (bold letters), respectively (A: adenine; C: cytosine; G: guanine; Nad4: NADH dehydrogenase subunit 4; T: thymine). Partial Nad4 mRNA sequence (Columba livia) ACCCTCATCAGTTCTCTACCACTACTAGTTGCAATACTATACCTACACATACAAATCGGCACCCTACACCTCACAATACTC AAACTAACCCACCCTCCCCTTAACAACTCCTGAACTGGCCTCCTATCTAGCCTAGCACTACTAATAGCATTCATAGTAAA AGCACCCTTATACGGCCTGCACCTATGACTACCCAAAGCCCACGTCGAAGCACCAATCGCAGGATCAATGTTACTCGCA GCCCTACTTCTTAAGCTAGGCGGCTATGGCATCATACGAGTTACCCTCCTAATAAACCCCACCTCCAACCACCTATACTA CCCATTCATTACCTTAGCCCTATGGGGCGCCCTAATAACTAGCTCAATCTGCTTGCGCCAAACGGACCTAAAGTCCCTCA TTGCCTACTCTTCCGTTAGCCATATAGGCCTAGTCATCGCTGCAAGCATAATCCAAACCCACTGAGC

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Table E.2.3: Melanopsin gene sequence. Forward and reverse primer sequences for the qPCR experiment are highlighted in green and yellow (bold letters), respectively. Bold blue letter show the exonal borders. Letters highlighted in red mark START and STOP codons. Red background and white letters mark the pigeon specific insert of 87 bp (exon 8a) and blue background with white letters indicate the additional sequence insert of 86 bp found only in the short isoform (A: adenine ; C: cytosine; G: guanine, T: thymine).

Melanopsin isoform S (Columba livia) ATGGGCACCCAGCCCCACGCTCtGACCAAGgCAGAGATACCCGCCCATGTTCTTTACACCGTAGGAACATGCGTGCTCA TTATCGGCTCCATTGGCATCATTGGAAACCTCCTAGTTCTCTACGCATTTTACAGCAACAAGAAGCTGAGGACACCCCA AAACTACTTTATAATGAATTTGGCCATGAGTGACTTCTTGATGTCGGCTTCTCAGGCACCCATGTGCTTTGTGAACAGCT TGCACGGAGAGTGGATACTTGGAGATATAGGCTGTGACATGTACGCTTTCTGTGGGGCACTCTTTGGAATAACCTCAA TGATGACTTTATTGGCTATTTCTGTGGATCGCTACCTTGTGATCACGATGCCCCTGCGATCCATCCAGTGGACGTCCAAG AAGCGCACGGTGCAGATCATCGCTGCCGTCTGGCTGTACTCGCTGGGCTGGAGCGTGGCTCCGCTCTTCGGGTGGAGT TCCTATGTGCCTGAGGGCTTGATGATATCCTGTACGTGGGACTACGTAACCTACTCCCCTGCAAACAGAAGTTACACCA TGATTTTATGTTGCTGCGTGTTTTTTATTCCTCTTATAATAATATTCCATTGTTATTTATTTATGTTCCTGGCCATAAGACG TACTGGCAGAGACGTTCAGAAGCTGGGCTCCTGCAGCCGGAAATCCTACCTCTCTCAGTCCATGAAGAATGAATGGAA ACTGGCAAAAATTGCTTTTGTGGTCATCATTGTGTTTGTCTTGTCTTGGTCTCCATATGCTTGTGTCACCTTGATTGCCTG GGCAGGTCGAGGCAACACCCTAACACCATATTCCAAATCCGTGCCAGCAGTTATTGCCAAAGCTTCTGCAGTCTACAAC CCCATCATCTACGCAATAATTCACCCAAGATACAGAAAAACCATCCACAACGCTGTTCCCTGCTTAAGGTTCCTTATACG AATATCGAAGAATGACCTCCTGAGAGGGTCCATAAATGAATCCTCATTTAGGACATCTCTCTCCAGCCATCACTCTCTTG CTGGCAGGACCAAGAGCACTTGTGTTTCATCCATTTCCACCGGAGAAGCTACCACTGATTACACAATGATGCATGAGA GTATCAACTGTGATGATTTCATGAACTCTAAAATATCCAGACCATCCTTGCAGAATGAGACCTGGAGCGATGTAGAGCT TGACCCTGTAGAGCCAGCTCATAAGAAGCTGCAGCCCCGCCGAAGTCATTCTTTCTCAGCAAGTGTGAGACAGGAGAA GAGGGACCTGCTCCCAAAGACCTGCAGCTGCGACGCAGCAACCACAGAAAAGGGAAGATAAGAGAAAGAAACTCCC ATTTCTCCCTAAGTGGGAGAGCTCTGGGAGCTGCCACCCTTCCCTCACTGAAAGTGGAGATGGTCTCATTCTGCTCCA GCTATCTGGAGAAGGTGCTTGGGCGGGCAGTCCTACACAGTCCCCCTGCAACACTCGTGACCAGCTCCCTAAAAGCAG CTTCTCTACCTGTTGGTTTGAACAGCAGCAGCATCAGCAGAGGAGGCGACACAGACACTTCAGAGATAGCAAGTCAAG AAAGTCAAGTTAACGGAGTCCTGGGCTCTATCATTAGCAATACTGTCCCTCGCATCATCATCATTCCTACCTCAGAGACC AACCTATTTCGAGAGGAACTAGAAGAGGAAGAGACTCAGTTGTTCCACTTTCCYGACAAAAAGGGCAATCTGCTGGAC TTGGAAGGGCTTAGCTCATCTGTAGAGTTTCTTGAAGCTGTTGAGAAATTTCTGTCATGA Melanopsin isoform L (Columba livia) ATGGGCACCCAGCCCCACGCTCtGACCAAGgCAGAGATACcCGCcCATGTtCTtTACACcGTAGGAACATGCGTGCTCATt ATCGGCTCCATTGGCATCATTGGAAACCTCCTAGTTCTCTACGCATTTTACAGCAACAAGAAGCTGAGGACACCCCAAA ACTACTTTATAATGAATTTGGCCATGAGTGACTTCTTGATGTCGGCTTCTCAGGCACCCATGTGCTTTGTGAACAGCTTG CACGGAGAGTGGATACTTGGAGATATAGGCTGTGACATGTACGCTTTCTGTGGGGCACTCTTTGGAATAACCTCAATG ATGACTTTATTGGCTATTTCTGTGGATCGCTACCTTGTGATCACGATGCCCCTGCGATCCATCCAGTGGACGTCCAAGAA GCGCACGGTGCAGATCATCGCTGCCGTCTGGCTGTACTCGCTGGGCTGGAGCGTGGCTCCGCTCTTCGGGTGGAGTTC CTATGTGCCTGAGGGCTTGATGATATCCTGTACGTGGGACTACGTAACCTACTCCCCTGCAAACAGAAGTTACACCATG ATTTTATGTTGCTGCGTGTTTTTTATTCCTCTTATAATAATATTCCATTGTTATTTATTTATGTTCCTGGCCATAAGACGTA CTGGCAGAGACGTTCAGAAGCTGGGCTCCTGCAGCCGGAAATCCTACCTCTCTCAGTCCATGAAGAATGAATGGAAAC TGGCAAAAATTGCTTTtGTGGTCATCATTGTGTTTGTCTTGTCTTGGTCTCCATATGCTTGTGTCACCTTGATTGCCTGGG CAGGTCGAGGCAACACCCTAACACCATATTCCAAATCCGTGCCAGCAGTTATTGCCAAAGCTTCTGCAGTCTACAACCC CATCATCTACGCAATAATTCACCCAAGATACAGAAAAACCATCCACAACGCTGTTCCCTGCTTAAGGTTCCTTATACGAA TATCGAAGAATGACCTCCTGAGAGGGTCCATAAATGAATCCTCATTTAGGACATCTCTCTCCAGCCATCACTCTCTTGCT GGCAGGACCAAGAGCACTTGTGTTTCATCCATTTCCACCGGAGAAGCTACCACTGATTACACAATGATGCATGAGAGT ATCAACTGTGATGATTTCATGAACTCTAAAATATCCAGACCATCCTTGCAGAATGAGACCTGGAGCGATGTAGAGCTTG ACCCTGTAGAGCCAGCGAGTCATAAGAAGCTGCAGCCCCGCCGAAGTCATTCTTTCTCAGCAAGTGTGAGACAGGAGA AGAGGGACCTGCTCCCAAAGACCTGCAGCTGCGACGCAGCAACCACAGAAAAGGTCTCATTCTGCTCCAGCTATCTGG AGAAGGTGCTTGGGCGGGCAGTCCTACACAGTCCCCCTGCAACACTCGTGACCAGCTCCCTAAAAGCAGCTTCTCTAC CTGTTGGTTTGAACAGCAGCAGCATCAGCAGAGGAGGCGACACAGACACTTCAGAGATAGCAAGTCAAGAAAGTCAA GTTAACGGAGTCCTGGGCTCTATCATTAGCAATACTGTCCCTCGCATCATCATCATTCCTACCTCAGAGACCAACCTATT TCGAGAGGAACTAGAAGAGGAAGAGACTCAGTTGTTCCACTTTCCYGACAAAAAGGGCAATCTGCTGGAcTTGGAAGG GCTTAGCTCATCTGTAGAGTTTCTTGAAGCTGTTGAGAAATTTCTGTCATGA

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Table E.2.4a: RNA concentration, purity (A260/280) and RNA integrity of samples used in qPCR analysis. The ratio of A260 nm and A280 nm is an indicator for purity of the RNA, with values around 2.0 indicating pure RNA. RNA integrity is determined by the RIN (RNA integrity number) values ranging from 1 (degraded) to 10 (intact). RINs ≥6 are considered sufficient for subsequent qPCR experiments (PH: hatchling, post-hatch; T: Taube = pigeon). Concentration Sample/Animal A260/A280 RIN [ng/µl] R 152.22 2.08 8.10 T317 L 153.57 2.08 8.00 R 152.15 2.09 8.10 T337 L 106.29 2.08 8.30 R 102.27 2.06 7.90 T338 L 141.80 2.07 7.50 R 168.51 2.08 7.50 T345 L 120.53 2.06 7.20 R 142.59 2.07 7.30 T432 L 173.28 2.08 7.50 R 123.80 2.09 8.00 T433 L 165.61 2.07 7.70 R 125.99 2.09 7.80 T438 L 136.07 2.08 7.50 R 356.36 2.10 7.60 Txx1 L 305.74 2.08 8.30 R 203.64 2.09 7.80 PH2 (20) L 361.70 2.04 7.90 R 282.22 2.09 9.40 PH1 (21) L 190.38 2.01 8.40 R 465.00 1.94 6.80 PH2 (22) L 313.90 2.04 7.80 R 247.49 2.05 9.60 PH2 (23) L 262.18 2.07 10.00 R 359.08 2.07 9.90 PH2 (26) L 357.56 2.00 8.90 R 141.83 2.07 9.80 PH1 (27) L 342.28 2.06 9.40 R 429.74 2.10 9.20 PH2 (28) L 369.58 2.08 9.50 R 408.09 1.94 9.70 PH2 (29) L 124.19 2.08 10.00

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Table E.2.4b: RNA concentration, purity (A260/280) and RNA integrity of samples used in qPCR analysis. The ratio of A260 nm and A280 nm is an indicator for purity of the RNA, with values around 2.0 indicating pure RNA. RNA integrity is determined by the RIN (RNA integrity number) values ranging from 1 (degraded) to 10 (intact). RINs ≥6 are considered sufficient for subsequent qPCR experiments (E: embryo; ED: dark-incubated embryo). Concentration Sample/Animal A260/A280 RIN [ng/µl] R 373.06 2.00 7.50 E16 (14) L 384.15 2.00 7.60 R 321.88 2.03 6.80 E16 (15) L 382.68 2.05 7.70 R 457.02 2.07 8.30 E16 (16) L 397.73 2.04 9.20 R 371.02 2.07 9.10 E16 (17) L 426.82 2.09 9.10 R 191.60 2.06 9.30 E16 (22) L 235.66 2.07 9.90 R 298.42 2.04 9.30 E16 (23) L 454.85 1.97 9.30 R 463.90 2.06 9.70 E16 (24) L 106.40 2.15 9.00 R 500.44 2.07 9.00 E16 (25) L 537.81 2.00 9.30 R 316.76 2.06 8.90 ED16 (1) L 289.12 2.07 9.60 R 358.92 2.05 8.40 ED15 (4) L 233.78 2.08 9.20 R 469.11 2.07 9.50 ED 15 (6) L 197.99 2.03 8.80 R 262.16 2.09 9.30 ED16 (7) L 173.86 2.09 9.00 R 222.05 2.08 9.20 ED16 (11) L 294.31 2.00 8.90 R 194.46 2.06 9.60 ED16 (12) L 195.94 2.04 8.20 R 165.16 2.03 9.20 ED16 (13) L 464.23 2.04 8.60 R 66.31 2.06 8.80 ED16 (15) L 93.06 2.06 8.80

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Figure E.2.1a: Electrophoretic traces of RNA integrity measurements. RIN data can be found in Table E.2.4a (PH: hatchling, post-hatch; T: Taube = pigeon).

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Figure E.2.1b: Electrophoretic traces of RNA integrity measurements. RIN data can be found in Table E.2.4b (E: embryo; ED: dark-incubated embryo).

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Figure E.2.2a: Primer specificity and efficiency of reference genes used in qPCR. Amplification of serial dilutions for each primer pair of reference gene. Melting curves demonstrating that all primer pair just 2 2 amplify only a single gene product. ARP: E = 99.6% (R = 0.997), Tm = 82.5 °C; Gapdh: E = 97.2% (R = 2 0.999), Tm = 82 °C; Ppia: E = 109.8% (R = 0.964), Tm = 83.5 °C (ARP: acidic ribosomal protein, Gapdh: Glyceraldehyde 3-phosphate (GAP) dehydrogenase, Ppia: peptidylprolyl isomerase A).

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Figure E.2.2b: Primer specificity and efficiency of melanopsin isoform S and L for qPCR. Amplification of serial dilutions for each primer pair of melanopsin gene. Melting curves demonstrating that all 2 primer pairs just amplify only a single gene product. Opn4xS: E = 100.9% (R = 0.991), Tm = 82 °C; 2 Opn4xL: E = 90.0% (R = 0.990), Tm = 83 °C (Opn4xS: melanopsin isoform S, Opn4xL: melanopsin isoform L).

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A A

B B

C C

Figure E.2.3: Linear dynamic range of the used reference genes, ARP (A), Gapdh (B) and Ppia (C). Linearity is given up to at least 1,000,000 copies.

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Figure E.2.4: Limit of detection (LOD) and Limit of quantification (LOQ). Determination of LOD and LOQ by standard curves with 2-fold dilution steps. Coefficient of determination ARP: R² = 0.9989, Gapdh: R² = 0.9993, Ppia: R² = 0.9958, melanopsin isoform S: R² = 0.9989 and melanopsin isoform L: R² = 0.9989 (ARP: acidic ribosomal protein Gapdh: Glyceraldehyde 3-phosphate (GAP) dehydrogenase, melanopsin isoform L: Opn4xL, melanopsin isoform S: Opn4xL, Ppia: peptidylprolyl isomerase A).

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Table E.2.5: Overview of LOD and LOQ values. LOD sets the last value when all replicates of a sample can still be detected, while LOQ describes the point when values start to deviate than 20% from the nominal value. This table shows the copy numbers and their corresponding Cq values of used reference genes and melanopsin (ARP: acidic ribosomal protein Gapdh: Glyceraldehyde 3-phosphate (GAP) dehydrogenase, melanopsin isoform L: Opn4xL, melanopsin isoform S: Opn4xL, Ppia: peptidylprolyl isomerase A).

LOD LOQ Gene # of copies Cq # of copies Cq ARP < 2.5 34.67 40 32.34 Gapdh 10 37.15 160 32.48 Ppia 10 34.49 40 32.47 Opn4xS 20 37.75 1280 33.98 Opn4xL < 2.5 35.63 10 33.77

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Table E.2.6a: Raw melanopsin Cq values of adult Table shows inter-run calibrated values of three qPCR reads (L = left, Opn4xS/L = melanopsin isoform S/L, R = right, T = Taube = adult pigeon).

Target Sample Expression Expression SEM Mean Cq Cq SEM Opn4xS T317 R 0.60712 0.35009 34.88 0.80546 L 0.37679 0.18027 35.51 0.65071 T337 R 0.14074 0.08824 36.65 0.86114 L 0.32503 0.20649 35.62 0.88659 T338 R 0.32484 0.15506 35.31 0.64750 L 0.28094 0.12953 34.86 0.63720 T345 R 0.15429 0.06421 37.28 0.54452 L 0.25718 0.11550 36.41 0.58666 T432 R 0.44321 0.21595 35.36 0.63275 L 1.06130 0.50900 34.11 0.63609 T433 R 0.15473 0.07850 36.65 0.66797 L 0.30394 0.15323 35.63 0.68168 T438 R 0.41685 0.18061 36.14 0.55585 L 0.17680 0.05626 37.05 0.36408 Txx1 R 0.69451 0.43849 34.47 0.89151 L 1.27347 0.45551 33.03 0.47821 Opn4xL T317 R 1.13898 0.20627 28.71 0.15800 L 1.37228 0.24884 28.39 0.14599 T337 R 0.93869 0.23350 28.66 0.22838 L 1.09192 0.23741 28.62 0.22444 T338 R 1.15201 0.25484 28.23 0.21648 L 1.00000 0.19873 27.78 0.21401 T345 R 1.27007 0.32835 28.98 0.27419 L 1.17807 0.27226 28.96 0.20962 T432 R 0.83204 0.23518 29.20 0.32217 L 0.77835 0.22833 29.30 0.33648 T433 R 1.04969 0.27397 28.63 0.24928 L 0.98390 0.26104 28.68 0.29891 T438 R 1.20369 0.26796 29.36 0.19890 L 1.17930 0.28165 29.06 0.22183 Txx1 R 0.55512 0.08098 29.54 0.09695 L 0.69020 0.13812 28.66 0.22341

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E. SUPPLEMENTARY DATA

Table E.2.6b: Raw melanopsin Cq values of post-hatch pigeons. Table shows inter-run calibrated values of three qPCR reads (L = left, Opn4xL = melanopsin isoform L, PH = post-hatch pigeon, R = right).

Target Sample Expression Expression SEM Mean Cq Cq SEM Opn4xL PH2 (20) R 1.64968 0.07368 29.67 0.03723 L 1.62656 0.08385 29.50 0.05598 PH1 (21) R 1.29692 0.07472 29.44 0.05654 L 0.88132 0.04124 29.88 0.05002 PH2 (22) R 1.31995 0.06115 30.13 0.05287 L 1.33705 0.12453 30.03 0.12351 PH2 (23) R 1.21685 0.05712 29.33 0.04121 L 0.89986 0.05212 29.69 0.07809 PH2 (26) R 1.57740 0.13856 29.12 0.09337 L 0.78084 0.04544 30.23 0.04039 PH1 (27) R 0.29462 0.01698 31.24 0.06952 L 0.51549 0.05229 30.61 0.03481 PH2 (28) R 0.58539 0.02884 30.72 0.06572 L 0.59686 0.04700 29.86 0.11052 PH2 (29) R 0.69007 0.05684 31.44 0.10425 L 0.33426 0.02983 31.79 0.12348

Table E.2.6c: Raw melanopsin Cq values of embryonic pigeons. Table shows inter-run calibrated values of three qPCR reads (E = embryonic pigeon, L = left, Opn4xL = melanopsin isoform L, R = right).

Target Sample Expression Expression SEM Mean Cq Cq SEM Opn4xL E16 (14) R 1.13158 0.25122 31.29 0.22786 L 0.83798 0.15140 31.32 0.15616 E16 (15) R 0.94065 0.16856 30.43 0.16294 L 0.50937 0.08203 31.05 0.07915 E16 (16) R 0.43008 0.07288 32.33 0.10275 L 0.63325 0.12058 30.89 0.15506 E16 (17) R 0.80762 0.13653 30.05 0.14937 L 0.58566 0.09034 30.36 0.08986 E16 (22) R 0.60096 0.11483 30.75 0.18739 L 0.37883 0.05809 31.07 0.11930 E16 (23) R 0.30586 0.04317 31.24 0.06683 L 0.62295 0.11590 30.75 0.12585 E16 (24) R 0.51552 0.07048 30.59 0.10546 L 0.18045 0.02546 32.52 0.08722 E16 (25) R 0.73343 0.10302 29.94 0.12352 L 1.00000 0.13911 29.25 0.13113 220

E. SUPPLEMENTARY DATA

Table E.2.6d: Raw melanopsin Cq values of dark-incubated embryonic pigeons. Table shows inter-run calibrated values of three qPCR reads (ED = dark-incubated embryonic pigeon, L = left, Opn4xL = melanopsin isoform L, R = right).

Target Sample Expression Expression SEM Mean Cq Cq SEM

Opn4xL ED16 (1) R 0.43390 0.10581 31.66 0.33546 L 0.54203 0.09015 31.37 0.22125

ED15 (4) R 0.64989 0.11635 30.78 0.23511 L 0.32763 0.06696 31.60 0.27313

ED 15 (6) R 0.36745 0.07103 31.36 0.25791 L 0.19879 0.02550 31.83 0.16014

ED16(7) R 1.44977 0.27725 29.45 0.25929 L 0.48728 0.08271 30.96 0.22979

ED16 (11) R 0.87646 0.14850 30.17 0.22800 L 1.69340 0.25504 29.58 0.19235

ED16 (12) R 0.16144 0.02769 32.92 0.23807 L 0.22106 0.04819 31.80 0.28844

ED16 (13) R 0.58632 0.06857 30.65 0.14862 L 0.62231 0.10069 30.84 0.17673

ED16 (15) R 0.52718 0.08458 30.36 0.20993 L 0.54594 0.16008 30.87 0.35631

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E. SUPPLEMENTARY DATA

Table E.2.7: Delta and Delta absolute values of normal- and dark-incubated pigeon embryos. Table shows mean expression values (inter-run calibrated) of three qPCR reads for melanopsin isoform L, the Delta and Delta absolute values which are the basis of the variance analysis (E = normal-incubated embryonic pigeons, ED = dark-incubated embryonic pigeons). Mean expression Sample/Animal Delta Delta absolute value R 1.132 E16 (14) 0.29 0.29 L 0.838 R 0.941 E16 (15) 0.43 0.43 L 0.509 R 0.430 E16 (16) -0.20 0.20 L 0.633 R 0.808 E16 (17) 0.22 0.22 L 0.586 R 0.601 E16 (22) 0.22 0.22 L 0.379 R 0.306 E16 (23) -0.32 0.32 L 0.623 R 0.516 E16 (24) 0.34 0.34 L 0.180 R 0.733 E16 (25) -0.27 0.27 L 1.000 R 0.434 ED16 (1) -0.11 0.11 L 0.542 R 0.650 ED15 (4) 0.32 0.32 L 0.328 R 0.367 ED 15 (6) 0.17 0.17 L 0.199 R 1.450 ED16 (7) 0.96 0.96 L 0.487 R 0.876 ED16 (11) -0.82 0.82 L 1.693 R 0.161 ED16 (12) -0.06 0.06 L 0.221 R 0.586 ED16 (13) -0.04 0.04 L 0.622 R 0.527 ED16 (15) -0.02 0.02 L 0.546

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CURRICULUM VITAE

Personal Details Name: Rena Klose

Date and place of birth: 06.09.1987 in Remscheid

Nationality: German

Marital status: single

Academic qualifications since 12/2012 PhD student at the biopsychology lab of Prof. Dr. Drs. h.c. Onur Güntürkün, Department of Psychology, Ruhr-University Bochum

10/2010-08/2012 Master degree: Biochemistry Focal point: Biochemistry of the nervous system, Department of Chemistry and Biochemistry, Department of Psychology, Ruhr-University Bochum

10/2007-08/2010 Bachelor degree: Biochemistry Focal point: Biochemistry of the nervous system, Department of Chemistry and Biochemistry, Ruhr- University Bochum

Education 1998-2007 Secondary school: Städtisches Gymnasium Wermelskirchen (Abitur, equivalent to A-level)

1994-1998 Elementary school: Grundschule Ost Wermelskirchen

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CURRICULUM VITAE

Teaching Experience 2016 Supervision of the neuroanatomical course “The painting and crafting workshop of the human brain” (Bachelor course) 2015/2016 Supervision of the seminar “Evolution of higher cognitive functions in non-human animals” (Bachelor and Master seminar) 2015 Supervision of the neuroanatomical course “The painting and crafting workshop of the human brain” (Bachelor course) 2014/2015 Supervision of the concomitant seminar of the lecture “Left brain – Right brain” (Master seminar) 2014 Supervision of the neuroanatomical course “The painting and crafting workshop of the human brain” (Bachelor course) 2013/2014 Supervision of the concomitant seminar of the lecture “Left brain – Right brain” (Master seminar) 2013 Assistant supervision of the neuroanatomical course “The painting and crafting workshop of the human brain” (Bachelor course)

Student Supervision 2012 – Present Several internships: Psychology, biology and biochemistry students 2015 Stephanie Lor (M.Sc. thesis) Luisa Betke (B.Sc. thesis) 2014 Pascal Röderer (B.Sc. thesis) 2013 Stephanie Lor (B.Sc. thesis)

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PUBLICATIONS

Submitted manuscripts

 Schmitz J., Lor S., Klose R. and Ocklenburg S. (2016). Shedding light on the genetic determinants of functional lateralization: A commentary on Karlebach and Francks, 2015. Cortex (75): 260–261.

Submissions to international conferences

 Klose R., Ströckens F. and Güntürkün G. (2014). Melanopsin – a possible trigger of lateralization in pigeons? Annual Meeting of the Society for Neuroscience, Washington, D.C., USA.

 Klose R., Ströckens F., Spoida K., Herlitze S. and Güntürkün G. (2015). How visual asymmetry starts in pigeons – characterizing melanopsin as a potential trigger. Annual Meeting of the Society for Neuroscience, Chicago, IL, USA.

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ACKNOWLEDGEMENTS/DANKSAGUNG

Als erstes möchte ich Prof. Dr. Onur Güntürkün danken, dass er mir die Möglichkeit gegeben hat dieses Projekt in seinem Labor durchführen zu können. Onur, ich bedanke mich für das mir entgegengebrachte Vertrauen, die persönliche Unterstützung und die konstruktive Kritik während der gesamten letzten Jahre.

Des Weiteren möchte ich Prof. Dr. Jörg T. Epplen für die Übernahme des Korreferats danken. Jörg, vielen Dank für das aufmerksame Verfolgen meiner Arbeit und die kontinuierliche Unterstützung während meiner gesamten Promotionsphase.

Zudem danke ich Dr. Gabriele Dekomien, Manuela Meyer und Dr. Sabrina Schreiber aus dem Labor von Prof. Epplen für ihre Unterstützung bei der Sequenzierung. Mit euch konnte erst der Grundstein für diese Arbeit gelegt werden.

Zusätzliche danke ich Prof. Dr. Robert Kumsta für die Möglichkeit das gesamte Equipment in seinem Labor für die Durchführung meines Projekts verwenden zu können. Ein besonderer Dank gilt dabei auch Dr. Dirk Moser. Dirk, danke für deine unermüdliche Unterstützung bei der Etablierung der qPCR, die zahlreichen Ideen und das Interesse an diesem Projekt.

Weiterhin möchte ich Prof. Dr. Stefan Herlitze, Dr. Katharina Spoida und Dennis Eickelbeck für ihre Unterstützung während des gesamten Projekts danken. Katha und Dennis, ohne eure Hilfe wäre die Durchführung des Calcium Imagings und der Patch-Clamp Experimente so nicht möglich gewesen!

Prof. Dr. Stephan Hahn und vor allem Matthias Becker danke ich für die Bereitstellung des Equipments für die RIN-Messung und für die Hilfe bei der Durchführung. Des Weiteren danke ich Prof. Dr. Carsten Theiss für die Möglichkeit das konfokale Mikroskop an seinem Lehrstuhl nutzen zu können.

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ACKNOWLEDGEMENTS/DANKSAGUNG

Ein großer Dank gilt auch der ganzen Abteilung der Biopsychologie für die wunderbare Atmosphäre, die vielen spannenden Gespräche während der Mittagspause, die unzähligen interessanten Diskussion und die vielen Labortage mit allen Höhen und Tiefen. Ein besonderer Dank gilt dem Büro mit dem besten Dresscode! Annika, Charlotte, Martin, Sara und Steffi, vielen Dank für die vielen Ratschläge, Einschätzungen und unzähligen Gespräche während der gesamten Zeit! Sebastian, vielen Dank für tausende beantwortete Statistik Fragen und die ständige Versorgung mit großen Zuckermengen. Ohne euch alle hätte es nur halb so viel Spaß gemacht!

Ein ganz besonderer Dank gilt natürlich meinen Eltern, die mich bedingungslos unterstützen und immer an mich glauben! Ein riesiger Dank gilt auch Olaf, der unendlich Geduldig mit mir ist, mich in jeglicher Hinsicht unterstützt und immer an meiner Seite ist!

DANKE! THANKS! KIITOS!

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ERKLÄRUNG

Hiermit erkläre ich, dass ich die vorgelegte Dissertation selbständig und ohne unzulässige fremde Hilfe angefertigt und verfasst habe, dass alle Hilfsmittel und sonstigen Hilfen angegeben und dass alle Stellen, die ich wörtlich oder dem Sinne nach aus anderen Veröffentlichungen entnommen habe, kenntlich gemacht worden sind. Weiterhin erkläre ich, dass die Dissertation in der vorgelegten oder einer ähnlichen Fassung noch nicht zu einem früheren Zeitpunkt an der Ruhr-Universität Bochum oder einer anderen in- oder ausländischen Hochschule als Dissertation eingereicht worden ist.

Bochum, den

______Rena Klose

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