Molecular evolution of zebrafish (Danio rerio) visual pigment function by James Michael Morrow A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Cell & Systems Biology University of Toronto © Copyright by James Michael Morrow 2014 Molecular evolution of zebrafish (Danio rerio) visual pigment function James Michael Morrow Degree of Doctor of Philosophy Department of Cell & Systems Biology University of Toronto 2014 Abstract Photoreception in vertebrates is mediated by opsins, members of the G protein-coupled receptor (GPCR) superfamily of proteins. In the dark, opsins are covalently bound to a light-sensitive chromophore, 11-cis-retinal, which acts as an inverse agonist to suppress dark state activation. When exposed to light, the chromophore isomerizes to its all-trans conformation, activating opsin and initiating a signaling cascade within the photoreceptor cell. Zebrafish (Danio rerio) has a large complement of visual opsins, and serves as a model for the vertebrate visual system on a developmental, physiological, and behavioural level, making it an ideal candidate for the study of opsin evolution and function. Following a general introduction, chapter 2 of my thesis presents the initial characterization of a novel rhodopsin-like gene that we identified in zebrafish, rh1- 2. Chapter 3 further investigates rh1-2, showing rh1-2 expression in photoreceptor cells of the zebrafish retina and using in vitro expression to identify functional characteristics of rh1-2 that are similar to rhodopsin, despite the fact that it is a much less stable pigment. Chapter 4 describes biochemical investigations of zebrafish rhodopsin and, in ii particular, addresses the question of the key molecular mechanisms mediating retinal release rate differences among vertebrate rhodopsins. Finally, chapter 5 investigates duplicated rh2 cone opsin genes in teleost fish and notes that many with multiple rh2 genes have at least one copy with Q122 and one with E122, a discrepancy that is known to help in establishing the contrasting functional identities of rhodopsins and cone opsins. This thesis contributes significantly to the characterization of visual pigment function in zebrafish. These experiments have also increased the understanding of functional variation within rhodopsins and cone opsins, particularly by investigating the molecular mechanisms of retinal release in rhodopsin, and the role of duplicated cone opsin genes, and will serve as an important non-mammalian reference against which future studies of the vertebrate visual system will be compared and contrasted. iii Acknowledgments At this moment in time, I feel very lucky. From the age of 23 to 29, I was granted the opportunity to think for a living. While my time in graduate school did not allow for a frivolous lifestyle, this was an opportunity that very few people are afforded during this period of their lives, and I have done my very best to take advantage of my fortunate situation. Throughout my studies, I have grown both as an academic and an individual. Since it won’t be possible to list the names of everyone that has contributed to this growth and supported me during this time, I want to quickly acknowledge my friends, extended family, fellow lab members, collaborators, CSB Faculty and staff. If you are reading this right now, chances are you are a part of this list, so thank you! I also want to extend this thanks to my enemies, critics, competitors and saboteurs; I am often motivated by the challenges presented to me, and am often able to reach new heights of accomplishment only when told they are beyond my capacities. The most central figure to my academic success has without a question been my supervisor, Belinda Chang. I have been extremely fortunate to have a supervisor that always gave me a chance to both prove and improve my worth. To be able to openly discuss any topic or idea with my supervisor, academic or otherwise, and to be able to trust that her thoughts and actions are always in my best interest is a feeling that I greatly appreciate and have never taken for granted. Both Vince Tropepe and Melanie Woodin were also central in guiding my research goals as members of my advisory committee, iv and I would like to thank them for their helpful insights and patience. I would also like to thank Dr. Shoji Kawamura, my external examiner, for a very thoughtful and thorough thesis appraisal. Mom, dad, and Matt, I hope you all know how important your support has been throughout this process. I know that we have a very hands-off family dynamic, allowing each part to have its much needed independence, but I never forget nor take for granted the security that the three of you have provided and continue to provide for me. I feel that one of the reasons that I’ve been able to avoid falling down in life is because I have the confidence in knowing that the three of you would be there to catch me if I ever do. Last but not least, my puppy dog Cody. You’ve only been at my side for 18 months, but you’re probably the main reason that I have remained in a healthy psychological state over that period of time, especially during the latter stages of this writing process. You’re a very good boy and yes, it is time for a walkie. v Table of Contents Abstract ........................................................................................................................... ii Acknowledgments .......................................................................................................... iv Table of Contents ........................................................................................................... vi List of Figures ................................................................................................................ ix List of Tables ................................................................................................................. xi List of Abbreviations .................................................................................................... xii Publications and author contributions ........................................................................... xv Chapter 1 – General introduction ............................................................................... 1 1.1 Visual pigments ..................................................................................................... 2 1.2 Phototransduction .................................................................................................. 7 1.3 The retinoid cycle .................................................................................................. 9 1.4 Cells of the retina ................................................................................................. 11 1.5 Heterologous opsin expression ............................................................................ 15 1.6 Visual pigment structure and function ................................................................ 18 1.7 Zebrafish .............................................................................................................. 23 1.8 Thesis objectives ................................................................................................. 25 1.9 Thesis overview ................................................................................................... 26 Figures........................................................................................................................... 30 References ..................................................................................................................... 34 Chapter 2 – A novel rhodopsin-like gene expressed in zebrafish retina ................ 51 2.1 Abstract ............................................................................................................... 52 2.2 Introduction ......................................................................................................... 53 2.3 Materials and methods ......................................................................................... 56 2.3.1 Opsin sequences ...................................................................................... 56 2.3.2 Protein expression .................................................................................... 57 2.3.3 Localizing expression of zebrafish rh1-2 ................................................ 58 2.3.4 Phylogenetic analyses .............................................................................. 58 2.4 Results ................................................................................................................. 60 2.4.1 Sequence analysis of rh1-2 ...................................................................... 60 2.4.2 In vitro expression ................................................................................... 62 2.4.3 Zebrafish rh1-2 expression pattern .......................................................... 62 2.4.4 Phylogenetic analyses .............................................................................. 63 2.5 Discussion ........................................................................................................... 64 Figures........................................................................................................................... 71 Tables ............................................................................................................................ 77 References ....................................................................................................................