University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2016-03-18 Molecular Mechanisms of Assembly and Long-term Maintenance of Neuronal Architecture: A Dissertation Cassandra R. Blanchette University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Developmental Neuroscience Commons, and the Molecular and Cellular Neuroscience Commons Repository Citation Blanchette CR. (2016). Molecular Mechanisms of Assembly and Long-term Maintenance of Neuronal Architecture: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/M2FS37. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/829 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. MOLECULAR MECHANISMS OF ASSEMBLY AND LONG-TERM MAINTENANCE OF NEURONAL ARCHITECTURE A Dissertation Presented By CASSANDRA R. BLANCHETTE Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MARCH 18TH, 2016 NEUROBIOLOGY MOLECULAR MECHANISMS OF ASSEMBLY AND LONG-TERM MAINTENANCE OF NEURONAL ARCHITECTURE A Dissertation Presented By CASSANDRA R. BLANCHETTE This work was undertaken in the Graduate School of Biomedical Sciences Program in Neuroscience The signature of the Thesis Advisor signifies validation of the Dissertation content Claire Bénard, Ph.D., Thesis Advisor The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation Vivian Budnik, Ph.D., Member of the Committee Patrick Emery, Ph.D., Member of the Committee Hong-Sheng Li, Ph.D., Member of the Committee Maxwell Heiman, Ph.D., External Member of the Committee The Signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee Michael Francis, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Science signifies that the student has met all graduation requirements of the School. Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences March 18th, 2016 iii ACKNOWLEDGEMENTS First I would like to thank my mentor, Claire, who has molded me into the scientist that I am today. Not only has she transformed me scientifically, but she has also helped me to become a more confident and assertive person through her positive and compassionate mentoring. She has provided me with unconditional support during all of the ups and downs of graduate school, and has always made me feel valued, appreciated, and understood. Claire has invested much of her time and energy to my training and provided me with many opportunities for scientific growth. She is genuine and puts her heart and soul into the people in her life, and I have been so fortunate to be a part of that. I especially would like to thank Andrea, for everything she does to make our lab run smoothly, for her scientific rigor and support with my projects, and for being an admirable and compassionate friend. Paola, thank you for being a strong role model for me in lab and in life, for making me laugh, and for your help with my research. Thank you to my good friend Gillian not only for her assistance with my projects, but also for always being so thoughtful and encouraging. Devyn, it has been a joy getting to work with you in the lab, thank you for your positive energy and enthusiasm for science. I would also like to thank all other past and present members of the Bénard Lab, in particular Anagha Khandekar who went through all of graduate school with me side by side, Janine Recio who trained me during my rotation, Brother James Ritch, Avery Fisher, and Catherine Harwood. I am thankful to all of the staff and faculty of the Neurobiology Department for making our department a one-of-a-kind place. Thank you to my past and present committee members, for all of their insights into my project and advice for my future, especially to my Dissertation Exam Committee members Vivian Budnik, Patrick Emery, Hong-Sheng Li, external examiner Maxwell Heiman, and committee chair Michael Francis. I thank my parents and my brother who have always unconditionally supported me in any endeavor that I’ve undertaken. Thank you for always believing in me. I am so grateful for my best friend and future husband Joe, who has been by my side every step of the way. I would also like to thank the rest of my family and my friends for their encouragement and for being the most supportive and loving group of people anyone could ever wish for. iv ABSTRACT Nervous system function is closely tied to its structure, which ensures proper connectivity and neural activity. Neuronal architecture is assembled by a series of morphogenetic events, including the coordinated migrations of neurons and axons during development. Subsequently, the neuronal architecture established earlier must persist in the face of further growth, maturation of the nervous system, and the mechanical stress of body movements. In this work, we have shed light on the molecular mechanisms governing both the initial assembly of the nervous system and the long-term maintenance of neural circuits. In particular, we identified heparan sulfate proteoglycans (HSPGs) as regulators of neuronal migrations. Our discovery and analysis of viable mutations in the two subunits of the heparan sulfate co-polymerase reveals the importance of the coordinated and dynamic action of HSPGs in neuronal and axon guidance during development. Furthermore, we uncovered that the HSPG LON-2/glypican functions as a modulator of UNC-6/netrin signaling through interactions with the UNC-40/DCC receptor. During larval and adult life, molecules such as the protein SAX-7, homologous to mammalian L1CAM, function to protect the integrity of nervous system architecture. Indeed, loss of sax-7 leads to progressive disorganization of neuronal architecture. Through a forward genetic screen, we identified LON-1 as a novel maintenance molecule that functions post- embryonically with SAX-7 to maintain the architecture of the nervous system. Together, our work highlights the importance of extracellular interactions to v modulate signaling events during the initial development of the nervous system, and to subsequently maintain neuronal architecture for the long-term. vi TABLE OF CONTENTS Title Page.............................................................................................................. i Signature Page .................................................................................................... ii Acknowledgements ............................................................................................ iii Abstract ............................................................................................................... iv Table of Contents ............................................................................................... v List of Tables ...................................................................................................... ix List of Figures ..................................................................................................... x List of Third Party Copyrighted Materials Produced by the Author ............. xiii List of Symbols, Abbreviations, or Nomenclature ......................................... xiv Preface ............................................................................................................... xv Chapter I: General Introduction ........................................................................... 1 Nervous system development .................................................................... 2 Axon guidance ........................................................................................... 3 C. elegans as a model to study nervous system development .................. 7 Postnatal challenges to the nervous system ............................................ 10 C. elegans as a model to study long-term maintenance of nervous system architecture .................................................................................. 13 Chapter II: Heparan sulfate proteoglycans are required for the guidance of migrations during development........................................................................... 17 Abstract .................................................................................................... 18 Introduction .............................................................................................. 19 vii Results ..................................................................................................... 22 Discussion ............................................................................................... 54 Materials and Methods............................................................................. 66 Acknowledgements .................................................................................. 76 Tables ...................................................................................................... 77 Chapter III: Glypican is a modulator of netrin-mediated axon guidance ............ 83 Abstract ...................................................................................................