THE ROLE OF SCHWANN CELLS IN PERIPHERAL NERVE DEVELOPMENT IN ZEBRAFISH A DISSERTATION SUBMITTED TO THE DEPARTMENT OF DEVELOPMENTAL BIOLOGY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Alya Raphael January 2011 © 2011 by Alya Rachel Raphael. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/dm455gb2152 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. William Talbot, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Ben Barres I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Thomas Clandinin I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. David Kingsley Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii ABSTRACT Myelin is a specialized sheath that insulates axons and allows for the rapid conduction of action potentials. In the peripheral nervous system, myelin is made by glial cells called Schwann cells. Whereas Schwann cells have received attention mainly because of their role in generating myelin, they play many other important roles. Using the posterior lateral line nerve in zebrafish, I have investigated two aspects of Schwann cell development. First, I demonstrate a new role for Schwann cells in repositioning peripheral nerves. Second, I find that ErbB signaling and Schwann cell proliferation are required during radial sorting, a process during which Schwann cells change their interactions with axons and reorganize the structure of the nerve itself. The posterior lateral line nerve is a prominent peripheral nerve in zebrafish that innervates sensory organs called neuromasts. I find that the posterior lateral line nerve initially grows out within the epidermis but is then rapidly transitioned across the basement membrane into the subepidermal space. Schwann cells are required for this process; in three different mutants that lack Schwann cells, the nerve is consistently mislocalized to the epidermis. This mislocalization results in significant disorganization of the nerve that worsens over time. When wildtype Schwann cells are transplanted into mutants lacking Schwann cells the position of the nerve is rescued. These results provide evidence that moving the posterior lateral line nerve out of the epidermis, across the basement membrane, protects the nerve from the migration of its targets. iv Before Schwann cells can make myelin around an axon, they must go through several developmental steps. Immediately after Schwann cells and axons have completed their migration, each Schwann cell associates with many axons. In order for myelination to proceed, however, a Schwann cell must associate with only a single axon. This transition, termed radial sorting, results in significant reorganization of the nerve. Neuregulin signaling from axons through ErbB receptors on Schwann cells controls many aspects of Schwann cell development and in mammals both Neuregulin/ErbB signaling and Schwann cell proliferation have been implicated in radial sorting. This prompted me to investigate whether ErbB signaling was required to regulate Schwann cell proliferation during radial sorting. To test this I took advantage of the zebrafish model system and used small molecule inhibitors to inhibit either ErbB signaling or proliferation at the time when radial sorting is beginning. I find that ErbB signaling and Schwann cell proliferation are critical for radial sorting. ErbB signaling was also required for Schwann cells to extend processes into the axon bundle, while proliferation was not. Therefore, I propose that ErbB signaling is directly required during radial sorting to regulate Schwann cell process extension, in addition to the previously established role of ErbB signaling in stimulating Schwann cell proliferation. v ACKNOWLEDGEMENTS This thesis could never have been completed without the help and support of many, many people. And the internet. Listing all of them would make for a document longer than the thesis itself, so, thank you everyone! None of this would have been possible without the amazing support staff for Developmental Biology. Thanks especially to Vanessa Bravo, Sue Elliott, and Yong Chong. And very especially to Todd Galitz, who fixes everything, finds everything, and does it all with a smile on his face. I will miss stalking him through the halls of Beckman by following the sounds of his laughter. I spent at least half of my time at Stanford in the TEM facility, so I owe a major debt of gratitude to John Perrino and Lydia-Marie Jorbert for always making it such a welcoming, cheerful, and pleasant environment. And for answering all of my questions! And for being wonderful people. I want to thank Will Talbot, for letting me join his lab and for giving me the freedom, time, and guidance to find my way to two projects that I really love. For all of his guidance and support and knowledge over the years, and especially in the final year as everything was coming together and finishing. I want to thank him especially for his amazing attention to detail and for going through every line of everything that I wrote, making the writing better and tighter and more scientific. vi Thanks to my committee: David Kingsley, Ben Barres, Tom Clandinin and my chair, Kang Shen. Thanks to David Kingsley for always being in my head, making me a better scientist, asking me why I’m doing an experiment. Ben Barres for being the fastest email responder ever, and always remembering my science. Tom Clandinin for spending an entire afternoon teaching me how to use our microscope better – it has revolutionized the way we take images in our lab. Kang Shen for always being interested in what I’m doing, for his support and encouragement. Thanks to Lucy Shapiro for really embracing me and welcoming me into the McShap lab family. And for all her advice and enthusiasm and warmth. Matt Scott for convincing me to come to Stanford and for all the stories and pictures. Sue McConnell for making me a better teacher and speaker. All the members of the Talbot lab throughout my time here have been fantastic – they are an incredibly fun and wise group of people. Thanks to everybody for all your help over the years and for dealing with my total insanity. And for generally putting things back in the right spot. Mrwah. Dave Lyons for his infinite wisdom, good cheer, guidance, support, and for suggesting the experiment that led to chapter 3. Sara Mercurio for her endless cheerleading and for being the best landlady ever. Kelly Monk for all the songs, sage science and shopping advice, tasty meals, and telling me to go home. Tom Glenn for the evening vii conversations about science and everything else. Marguax Bennett for being the most fun and enthusiastic person around. Harwin Sidik for bringing such joy and fun back into the lab – you’re going to do great! Tuky Reyes for always being unbelievably helpful and kind and taking amazing care of our fish. Chenelle Hill for making every day fun – my life is going to be so boring without you in it! Nitzan Sternheim for being an amazing role model. Matt Voas for being as an insane as I am, writing the best protocols imaginable, and for choreographing our show stopping rendition of the Time Warp. Thanks to everyone else in the Talbot lab throughout the years: Ian, Heather, Naomi, Isaac, Ali, Claudia, Stephen, Ryan, Jeanette, and Celia. I think it is safe to say that Julie Perlin is just the best all around human being I have ever met. Her constant enthusiasm, support, kindness, and lunch company made coming to lab everyday a pleasure. She is the best friend anyone could ask for. Thanks to the McShap lab poker gang, especially Franklin, Lisandra, Ayla, Leticia, Grant, Anya, Katya, Antonio, Nikki, Paola, Monica, and Jen. Thanks to my parents for always telling me that I could do anything I wanted to and for supporting me on my quest. Thanks for trying so hard (and mostly succeeding!) to understand what it is that I do, and why we care about Schwann cells. Thank you for always being there with guidance, support, and love. viii I have the awesomest siblings on the planet – Naomi, Jake, and Goldie. It’s been so much fun to watch them grow into themselves and I’m so proud of who they are becoming. They always make me laugh…I’ll put it in a box, and put that box in another box, and then I’ll mail it to myself, and then I’ll smash it with a HAMMER! If there’s one person that this really couldn’t have been done without, it would be Esteban Toro. He’s been there for me since the beginning, and has been unbelievable generous with his encouragement, support, and advice. And love. Thanks for talking to me in the silly voice when I need cheering up and introducing me to all sorts of new and exciting foods and places and experiences.