Investigating the genetic basis of altered activity profiles in the blind Mexican cavefish, Astyanax mexicanus A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences of the McMicken College of Arts and Sciences by by Brian M. Carlson B.S. Biology, Xavier University, May 2010 Committee Chair: Dr. Joshua B. Gross June 2015 ABSTRACT Organisms that have evolved to exploit extreme ecological niches may alter or abandon survival strategies that no longer provide a benefit, or may even impose a cost, in the environment to which they have adapted. Cave environments are characterized by perpetual darkness, isolation and relatively constant temperature and humidity. Accordingly, cave-adapted species tend to converge on a suite of regressive and constructive morphological, physiological and behavioral alterations, including loss or reduction of eyes and pigmentation, increased locomotor activity and reduction or alteration of behavioral rhythmicity. The cave environment and the associated changes in locomotor behavior make species of cavefish prime natural models in which to examine the complex genetic architecture underlying these behavioral phenotypes. The principal goal of this dissertation was to investigate the genetic basis of altered locomotor activity patterns in the blind Mexican tetra, Astyanax mexicanus. Initially, a custom locomotor assay rig and experimental protocols were developed to assess, characterize and compare activity patterns in surface and Pachón cavefish. The results of these assays clarified differences between the morphotypes, provided evidence that Pachón cavefish retain a weakly-entrainable circadian oscillator with limited capacity to self-sustain entrained rhythms and suggested that patterns in spatial “tank usage” data may be the result of a positive masking effect in response to light stimulus in both morphotypes. In order to facilitate downstream genetic analysis, a high-density genotyping-by-sequencing (GBS) based linkage map was constructed. This map provided much finer mapping ability than any previously published linkage map in this species. Further, the large number of markers enabled the anchoring of ~80% of the total length of the unassembled Astyanax mexicanus draft genome to the newly developed linkage map, which in turn assisted with downstream comparison of linkage groups and quantitative trait loci (QTL) between maps. ! ii! This greatly facilitated the process of screening for potential candidate genes. Employing the new linkage map and the assay protocols developed for this work, a large F2 surface x Pachón mapping pedigree was assayed and the resulting data was subjected to QTL analysis. QTL results suggest that loci on at least six different linkage groups are associated with patterns in either velocity or tank usage; however, the regions of the genome mediating each of these components are distinct from one another. Further, comparison of linkage groups between maps indicated that the QTL for locomotor activity described here are different than those published in previous studies. Finally, critical genomic intervals underlying activity QTL were screened for candidate genes. A total of 36 potential candidates were identified and genomic and transcriptomic resources were leveraged to highlight several prime candidates for further analysis, as well as to demonstrate that it is unlikely that either catastrophic changes to components of the core molecular clockwork or truncation of members of well-characterized photoreceptor families are behind the altered activity profiles observed in the Pachón cave population. ! iii! ! iv! ACKNOWLEDGEMENTS As my doctoral training draws to a close and I ready myself to begin what I hope will be a long, successful and satisfying career as an educator, a scientist and a mentor, I would like to take this opportunity to express my sincerest gratitude and heartfelt appreciation to the following people, all of whom have contributed in some significant way to my success in this endeavor: Drs. Dorothy Engle, George Farnsworth and the rest of the biology faculty at Xavier University, who provided me with my first opportunities to explore what it’s like to be a “real biologist,” ensured that I was extremely well-prepared to excel in a doctoral program, and provided an example of effective teaching and genuinely interested mentoring that continues to inspire me to this day. It remains my sincere hope that I might some day have the opportunity to return to my alma mater and help provide future students with the same excellent learning environment and educational opportunities that were given to me. Dr. Irene Luken and Mrs. Hedy Fye for giving me the knowledge and skills necessary to correctly pronounce words like “zeitgeber” and work my way through old journal articles with titles like, “Untersuchungen über den Lichtsinn und dessen Lokalisation bei dem Höhlenfisch Anoptichtys jordani Hubbs and Innes (Characidae).” Drs. Daniel Buchholz, John Layne, Herman Mays and Kenneth Petren for taking time out of your busy schedules to provide me with the guidance and support necessary to ensure that I was successful in my coursework and research efforts over the past five years. Dr. Joshua Gross for being the most intelligent, kind, supportive and inspiring doctoral advisor that I could have ever asked for. I cannot imagine any better environment in which to ! v! grow as a student, a scientist and a person than working with you against the backdrop of the warm and inviting lab culture that you so carefully cultivate. You’ve somehow managed to find the perfect balance between lab boss and lab dad and I am confident that any students fortunate enough to spend time in your lab will be forever changed for the better because of it; no one could be a stronger advocate for his or her students than you’ve been for us. Drs. Kathryn Rafferty and Kathleen Koenig for providing me the tools, training and opportunities that have allowed me to develop and grow as an educator throughout the course of my graduate career. Mandy Powers for being my confidant, lunch buddy and partner in crime for the last several years. I wish you success and many happy Pandora jam sessions as you work toward a red robe and funny hat of your own. Bethany Stahl for keeping me on my toes. Slacking off was never an option when I knew I’d constantly have to compare my own drive, work ethic and scientific output to the bar you somehow always managed to set just the tiniest bit higher than mine. Keep it up! Tyler Bussian, Allison Furterer, Ian Klinger, Wendy Lu, Brad Meyer and Sam Onusko for dedicated assistance in pushing my projects forward, no matter how tedious the task. I hope that the time and effort that you contributed toward making my projects a success comes back to you three fold! The rest of the Gross Lab family, both past and present. I wish you all nothing but success and happiness in whatever awaits you. ! vi! Dr. Merritt Oleski for letting me take over your conference room while I worked furiously to finish my dissertation and for distracting me with conversations about our shared belief in the value of basic science research when you could tell I needed a break. Please consider the contents of this dissertation as a few humble data points for inclusion in the Library of Alexandria, Version 2.0. Mr. L. for being the person most genuinely excited to talk with me about my research. My friends, family and the Brethren of J.B. Covert Lodge #437 and Alembic Lodge #793 (“Ole 793”) for supporting me, keeping me grounded and providing me with much needed distractions as I’ve worked hard to achieve my goals and make something of myself. Most importantly, however, I would like to dedicate this dissertation and all of the time and labor that went into writing it to the following, without any of whom it is absolutely inconceivable that I would be where I am today: To my wife, Julie Carlson, who has never waivered in her love and support for me and whose quiet strength and encouragement were all that kept me going through the roughest, most stressful portions of this experience. After well over a hundred pages of tedious proofreading and weeks of late nights, cranky moods, neglected chores and general unpleasantness on my part, I am positive that someone is traveling to the Vatican to advance her cause for living sainthood even as we speak. To my parents, Mary and Harry Brownfield and Daniel and Kathy Carlson, who have invested untold amounts of time, energy and resources into making sure that I always had the very ! vii! best education that they could afford, that I never truly wanted for anything and that there was never any doubt in my mind that I was loved and that they are proud of me. As a small expression of my gratitude for everything that you’ve done for me: Dad: Please consider the results of all of my analyses as the solution to the biggest damn “fish problem” I could come up with. Mom: Please consider the figures and tables contained within this tome as a poor substitute for all of the “artist’s renderings” that you’ve missed since I went away to college. Finally, to the memory of my late grandfather, Rudolph Flis, who taught me through word and deed the importance of family, faith and service to others… and that it’s nearly impossible to stay stressed or unhappy when you’re listening to a good polka. Thanks especially for that last one, Grandpa; it’s come in quite handy these past few weeks. ! viii! PREFACE Once we were blobs in the sea, and then fishes, and then lizards and rats, and then monkeys, and hundreds of things in between.