USING THE MOUSE RETINA TO MODEL THE ROLE OF SOX2 IN NEURAL INDUCTION Whitney E. Heavner A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for a degree of Doctor of Philosophy in the Program in Genetics and Molecular Biology Chapel Hill 2013 Approved by Robert Duronio, Ph.D. Stephen Crews, Ph.D. Norman Sharpless, M.D. Ellen Weiss, Ph.D. Mark Zylka, Ph.D. © 2013 Whitney E. Heavner ALL RIGHTS RESERVED ii ABSTRACT WHITNEY E. HEAVNER: Using the Mouse Retina to Model the Role of SOX2 in Neural Induction (Under the direction of Dr. Larysa Pevny) Neural competence is the ability of a progenitor cell to generate a neuron. The eye is one of the few tissues derived from the neural ectoderm that contains both neurogenic and non-neurogenic cells, all of which arise from a common progenitor pool. Therefore, the eye is a particularly useful model to study the molecular mechanisms that confer neural competence. Moreover, this cell fate dichotomy is highly reminiscent of the earlier process of neural induction, or the decision of an ectoderm precursor cell to become neural plate or epidermis. The HMG-box transcription factor SOX2 is crucial for both of these processes. Little is known about the role of SOX2 in neural induction, and what is known has been worked out primarily in lower vertebrates. Humans and mice with mutations in SOX2 exhibit a range of neural defects; therefore, from the perspective of human health, it is important to understand SOX2’s function in mammalian neuroepithelium. This project takes advantage of the accessibility of the embryonic mouse eye to identify pathways that SOX2 regulates to maintain neural competence. Chapter I gives an overview of neural induction from an historical perspective, with particular attention to the role of SOXB1 factors. A detailed description of iii mammalian eye development and the similarities between optic cup regionalization and neural induction are described. Chapter II investigates one potential mechanism of how SOX2 specifies neural fate: antagonism of Pax6. Using mouse genetics, we show that ablation of Sox2 in the OC leads to increased Pax6 expression and loss of neural competence. This phenotype is partially rescued by lowering Pax6 in Sox2cond/cond; P0CreiresGFP cells. Chapter III investigates the increased WNT signaling activity observed upon SOX2 ablation. Using mouse genetics, we show that removal of -Catenin from Sox2-negative cells rescues some aspects of the Sox2-mutant phenotype. Chapter IV offers some ideas of how these studies potentially relate to the treatment of degenerative eye disease, focusing on identifying candidate genes that could rescue neurogenesis in a Sox2-depleted background. To put these ideas into context, an overview of the current state of gene therapy for the treatment of eye disease is offered. iv This work is dedicated to my beloved advisor, Larysa, who taught me to revere mouse genetics and adore the developing embryo. v ACKNOWLEDGMENTS Scientific inquiry is often a solitary endeavor. Discoveries made in isolation can be thrilling and deeply satisfying. However, science by nature is collaborative, and its aims, ideally, are selfless. The work presented here stands on the shoulders of intellectual giants. Indeed, it often seems that eye development has not gone untouched by any major development biologist in the past few decades. Although I could never do adequate justice to those who came before me, directly contributed to this project or shared in the trials and joys associated with my six years in graduate school, I hope this work does some honor to their efforts and accomplishments. I owe a tremendous amount of gratitude to the members of the Pevny lab. There was never a day that I did not look forward to being in the lab and working with my “Pevny” colleagues and friends. I can’t imagine future lab meetings ever being as enjoyable as our weekly 8th floor conference room science tell-all hour. I am particularly indebted to the lovely and talented Danielle Matsushima. Without her this thesis would have never materialized, at least not in its present form. Her pioneering work on the genetic relationship between Sox2 and Pax6 allowed me to contribute to a “working” project as soon as I joined the lab. This rarity in graduate school gave me the freedom to spend a lot of time “experimenting” (messing up), learning and growing as a scientist without the risk of lost time. vi I am also particularly indebted to Dr. Scott Hutton, who took on much more responsibility than any senior graduate student or post-doc should ever have to. And if he ever resented this fact, he never showed it. From how to deal with lab safety compliance to running qPCR experiments and culturing neurospheres, if Scott didn’t know how to do it, then no one did. But most of all, Scott stepped in as an unofficial advisor when I needed it the most. For that I am extremely grateful. I never cease to be amazed by the amount of support one can receive in a time of need. For the help and encouragement coming from members of the Neuroscience Center and my committee, I am particularly thankful. Dr. Bill Snider, Dr. Eva Anton, Dr. Steve Crews and Dr. Bob Duronio were all instrumental in helping me finish the thesis and find a post-doc. I am also eternally thankful for the support and enthusiasm of Dr. Raluca Dumitru. If I was ever unsure about something, Raluca would give me the confidence and positive energy to go for it. And for the many belays and good advice from Dr. JrGang Cheng. I am also indebted to Dr. Scott Magness for reagents and technical expertise, Dr. Vladimir Ghukasyan for knowing more about confocal microscopy than I could ever hope to learn, Dr. Chandri Yandava for bioinformatics help, and Megumi Aita for all the in situ hybridizations. I’ll never be able to adequately express my gratitude to Josiah Zayner of the Sosnick lab at the University of Chicago for purified proteins, help with circular dichroism experiments and general inspiration for the possibilities inherent in science and computer programming. vii For community, colleagues and training I owe the Fundamental Issues in Vision Research course supported by the NEI and the UNC Curriculum in Genetics and Molecular biology. Lastly and most of all, I am grateful and truly lucky to have been a student of Larysa’s. It’s very rare in life to meet someone who not only meets your expectations but constantly defies them. In her humble demeanor and quiet perseverance, Larysa was full of surprises. She didn’t speak often, but when she spoke, it was captivating and meticulous. Larysa welcomed me into her lab with enthusiasm and warmth. I knew beyond a doubt that I had found my lab home when I hadn’t even finished my third rotation and she had already cleared off a bench for me. Her lab and she were just perfect. In fact, I knew before even meeting her that she is who I wanted to work for. Her name on my list of possible rotations had asterisks and circles around it. I joined her lab with an exclamation point and was not disappointed. The science was fascinating, the lab was fun and the PI was fantastic. She encouraged me, a timid but endlessly curious young graduate student, to speak at international conferences, and she would work with me tirelessly until my talks were perfect. She was genuinely excited to see me do well, like a coach for an athlete, and this was the best encouragement I could receive. Larysa’s passion for science was contagious. She once told me that when she was young, she would collect antique Erlenmeyer flasks and other glassware. It was like the allure of the laboratory, the mysteries and the challenges therein, drew her to the life of a scientist. Indeed, she could see great beauty where others just saw a retina or a neural viii tube or an antique Erlenmeyer flask. This was the type of excitement that Larysa embodied. And she would get frustrated when others didn’t appreciate what she saw. Her science world was rich and full, and often no one appreciated it more than she did. I don’t say that Larysa was caring in passing. I think it is quite rare for someone to be as empathetic as she was, in science or elsewhere. Her passion for her work was so pure, almost dutiful. It seems like during the last months of her life, she felt extra pressure to pursue her ideas in the lab. Like she only had so much time left to paint this beautiful picture for the world. Like she had to get it out there, what she saw under the microscope. And I would get angry with her for working so hard when her body was obviously so weak. But she had so much drive, and nothing would make her happier than to be in the lab. This is the most visible irony of Larysa’s life: that she put so much effort into the health of her cells and her mice and her lab while in the end, no one could do anything to save her. But also how typical of Larysa’s life; she gave everything she could and never asked for anything in return. You couldn’t help but love her. Larysa excelled as a scientist. To watch her at her best was awe-inspiring. She also excelled at making me feel better about the world. She had a firm belief in the beauty of the embryo, the possibilities of science and the sanctity of truth.