UNIVERSITY of CALIFORNIA, IRVINE Netrin-Mediated Signaling

UNIVERSITY OF CALIFORNIA, IRVINE Netrin-mediated signaling directs central neuron dendrite growth and connectivity in the developing vertebrate visual system DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biological Sciences by Anastasia Nicole Nagel Dissertation Committee: Professor Susana Cohen-Cory, Chair Associate Professor Karina S. Cramer Professor Georg F. Striedter 2015 © 2015 Anastasia Nicole Nagel DEDICATION To those who have supported me in my pursuit of understanding the natural world: my husband, family, friends and those who we have lost along the way but who remain present in spirit. “There are naive questions, tedious questions, ill-phrased questions, questions put after inadequate self-criticism. But every question is a cry to understand the world. There is no such thing as a dumb question.” Carl Sagan, The Demon-Haunted World: Science as a Candle in the Dark ii TABLE OF CONTENTS Page <ul style="display: flex;"><li style="flex:1">iv </li><li style="flex:1">LIST OF FIGURES </li></ul>ACKNOWLEDGMENTS CURRICULUM VITAE vi vii <ul style="display: flex;"><li style="flex:1">x</li><li style="flex:1">ABSTRACT OF THE DISSERTATION </li></ul><ul style="display: flex;"><li style="flex:1">INTRODUCTION </li><li style="flex:1">1</li></ul>CHAPTER 1: Molecular signals regulate visual system development Netrin regulates neurodevelopment through diverse receptor binding The role of netrin-1 in retinotectal circuit formation Summary and objectives 49 19 27 <ul style="display: flex;"><li style="flex:1">CHAPTER 2: Netrin directs dendrite growth and connectivity in vivo </li><li style="flex:1">29 </li></ul>29 31 36 64 Introduction Materials and Methods Results Discussion <ul style="display: flex;"><li style="flex:1">CHAPTER 3: DCC and UNC5 Netrin receptor signaling guides dendritogenesis </li><li style="flex:1">70 </li></ul>70 72 Introduction Materials and Methods <ul style="display: flex;"><li style="flex:1">Results </li><li style="flex:1">77 </li></ul><ul style="display: flex;"><li style="flex:1">Discussion </li><li style="flex:1">102 </li></ul><ul style="display: flex;"><li style="flex:1">CHAPTER 4: Decreases in Netrin signaling impact visual system function </li><li style="flex:1">113 </li></ul>113 114 116 121 Introduction Materials and Methods Results Discussion CHAPTER 5: Summary and Conclusions REFERENCES 122 129 iii LIST OF FIGURES Page Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 1.6 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 Figure 2.9 Neuron during general developmental processes 58Signaling molecules that direct visual system formation Schematic representation of a netrin-1 gradient in the spinal cord Diagram of the X. laevis retinotectal system 13 21 24 26 38 39 41 44 45 47 51 54 55 57 59 60 63 79 80 DCC-mediated netrin-1 signaling and RGC axon differentiation Netrin-1 has differential effects on axons due to stage Expression of Netrin-1 and of its receptors DCC and UNC5 DCC and UNC-5 expression in the CNS Protein diffusion after treatment Rapid remodeling of dendritic arbors upon acute manipulations Altering netrin levels decreases branch number and total length Blocking DCC signaling induces changes in dendritic arbor shape Manipulations in netrin levels induce rapid dendrite remodeling Altered netrin-1 and DCC signaling impact postsynaptic remodeling Postsynaptic cluster addition and stabilization quantification Figure 2.10 Overlays of sample neurons illustrate altered arbor morphology Figure 2.11 Perturbations in netrin signaling alter dendritic arbor directionality Figure 2.12 Individual branches change orientation in response to treatment Figure 2.13 Neuron outcome not affected by altered netrin signaling Figure 3.1 Figure 3.2 DCC and UNC5 morphant neurons 24h after transfection DCC and UNC5 morphant neurons 48h after transfection iv Figure 3.3 Figure3.4 Figure3.5 Figure3.6 Figure3.7 Figure3.8 Figure3.9 <ul style="display: flex;"><li style="flex:1">DCC and UNC5 morphant neurons 72h after transfection </li><li style="flex:1">81 </li></ul>Receptor down-regulation affects dendrite growth and directionality 85 Overlays of down-regulated neurons reveal abnormal morphology Receptor knock down affects filopodia 86 90 Down-regulation of DCC and UNC5 induces ectopic basal projections Altered tectal neuron morphology with receptor down-regulation Receptor signaling prevents cell division and promotes survival 91 93 97 Figure3.10 Netrin receptors influence growth and maturation Figure 3.11 Self-avoidance is not regulated by netrin signaling 98 101 118 120 Figure 4.1 Figure 4.2 Sequestration of endogenous netrin-1 affects vision Total swim time is not affected by altered netrin signaling vACKNOWLEDGMENTS I would like to express the deepest appreciation to my committee chair, Professor Susana Cohen-Cory, who is the embodiment and provider of patience during my studies at UC Irvine. Her mentorship guided me through difficult times and she always offered genuine support for me during hardships, whether they were difficulties in science or troubles from real life. I have learned so much in the time that we have worked together, and I am grateful for the opportunity to develop myself not only as a scientist but as a well-rounded individual. I thank my committee members, Associate Professor Karina Cramer and Professor Georg Striedter, whose work helped me improve my investigations and direct my research into a cohesive project. Thank you for your advocacy, consideration, and for helping me balance my research and my other duties during my time teaching with you both. In addition I thank the Chair of Neurobiology and Behavior, Professor Marcelo Wood, the man who conducted a challenging albeit invigorating interview with me many years ago. I feel that this interview helped my admission into INP and affirmed my aspirations to be a scientist. Over the years your continued support helping students with teaching and offering encouragement was greatly appreciated by all of us. Thank you for your advocacy. I thank my collaborators: Dr. Sonia Marshak, Dr. Miki Igarashi, Rommel A. Santos, and Sussie Gill; those who have helped me with my research as technical support: Lilly Zuniga and Sarah D. Mortero; and my 199 or volunteer students: Rommel A. Santos, Marc Piercy, Anthony Gallegos, Solomon Tang, Nathan Cruz, Gail Suchoknand, Arsany Bechay , Leslie Ocampo, Bryan Carbaugh, Myrna Leal, Tydus Thai, Mamdouh Hanna, Jimmy Villa, Rozhin Eskandary, Sabrina Lebron, Ruthy Mamo. Lastly, I thank Professor Oswald Steward, Dr. David Kinker, and Professor Clark Lindgren, all of whom helped me get to graduate school. I would also like to thank my family. I give a special thanks to my uncle, Dr. Robert Kunkle, for telling me his dissertation story so that I would be inspired during difficult times. I thank John Nagel for his proofreading and emotional support, and I especially thank Dr. Brian Marion, my husband who has helped me with my research and with generally being whole and sane during this process. Financial support was provided by the National Eye Institute grant EY-011912 to Susana Cohen-Cory and a Department of Education GAANN award P200A120165 to Professor Karina Cramer. vi CURRICULUM VITAE Anastasia Nicole Nagel 2005 2006 B.A. with Honors in Biology, Grinnell College, Grinnell, IA Research Technician, Department of Genetics, Duke University, Durham, NC 2007-2009 Research Assistant, Department of Anatomy and Neurobiology, University of California Irvine, Irvine, CA 2009-2015 Graduate Student Researcher, Department of Neurobiology and Behavior, University of California Irvine, Irvine, CA 2011-2014 Teaching Assistant, Ayala School of Biological Sciences, University of California, Irvine, Irvine, CA 2014 2015 Teaching Assistant for Graduate Student Cellular Neuroscience Lab Course, Ayala School of Biological Sciences, University of California, Irvine, Irvine, CA Ph.D. in Neurobiology and Behavior, Ayala School of Biological Sciences, University of California, Irvine FIELD OF STUDY Neurobiology of central nervous system wiring during development PUBLICATIONS Marion B*; Mclean K*; and Nagel A*. 2005. Blocking of muscarinic receptors does not affect post tetanic response in frog neuromuscular junction. Pioneering Neuroscience, 6, 5-8. *indicates shared first co-authorship Nagel AN*, Marshak S*, Manitt C, Santos RA, Piercy MA, Mortero, SD, Shirkey-Son, NJ, and Cohen-Cory S. “Netrin-1 directs dendritic growth and connectivity of vertebrate central neurons in vivo.” Neural Development (in press) 2015. *indicates shared first coauthorship CONFERENCE PRESENTATIONS <ul style="display: flex;"><li style="flex:1">2011 </li><li style="flex:1">Marshak S*, Nagel A*, Manitt C, and Cohen-Cory S. Netrin influences the </li></ul>directionality of dendritic arbor growth and postsynaptic connectivity in the vii developing Xenopus visual system. 2011 Abstract. Washington, DC: Society for Neuroscience, 2011. Online. <ul style="display: flex;"><li style="flex:1">2013 </li><li style="flex:1">Nagel A*, Marshak S*, Santos RA, Hung V, and Cohen-Cory S. DCC-mediated </li></ul>netrin-1 signaling influences dendritic arbor orientation and growth dynamics in the developing Xenopus visual system. 2013 Abstract and Poster. San Diego, CA: Society for Neuroscience, 2013. PROFESSIONAL MEMBERSHIPS, SCHOLARSHIPS, AND AWARDS 2012 2012 HHMI Graduate Fellow Award for Teaching Belegarth Scholarship Award (Academic Performance & Community Service in BMCS) <ul style="display: flex;"><li style="flex:1">2012-2014 </li><li style="flex:1">GAANN Fellowship </li></ul>2010- 2015 Society for Neuroscience Member TEACHING AND MENTORSHIP Instructor Neurobiology Lab Teaching Assistant Neurobiology and Behavior Teaching Assistant/ Substitute Neural Development Instructor Neurobiology Lab 2011 Spring 2011 Spring 2011 Fall 2012 Winter 2012 Spring 2012 Summer 2012 Summer 2012 Fall Teaching Assistant Functional Neuroanatomy Session I Introductory Biology <ul style="display: flex;"><li style="flex:1">Session II Introductory Biology </li><li style="flex:1">(Online Course) </li></ul>Introductory Biology 2013 Winter 2014 Winter Teaching Assistant Functional Neuroanatomy Teaching Assistant Cellular Neuroscience Graduate Level Lab <ul style="display: flex;"><li style="flex:1">2010 – Present </li><li style="flex:1">Mentored 11 undergraduate students in Undergraduate Research </li></ul>Opportunities Program (UROP), Excellence in Research, and BRIDGES. All of the students I mentored in Excellence in Research received this award and published in the Journal of Undergraduate Research in the Biological Sciences. Two students in Excellence in Research won additional awards. 2011-2012 2014 Mentored undergraduates in the Minorities in Science Program (MSP) Mentored 2 students in the UC Irvine Summer Internship Program viii SKILLS &TRAINING Teaching, undergraduate student mentorship and project coordination, in vivo imaging, confocal fluorescent microscopy, 2-photon confocal microscopy, micro-dissection, transfection, single cell and multi-cell electroporation, histology, immunohistochemistry, spectrophotometry, in situ hybridization, sterile technique, electrophoresis, veterinary technician, lab management, event coordination ix ABSTRACT OF THE DISSERTATION Netrin-mediated signaling directs central neuron dendrite growth and connectivity in the developing vertebrate visual system By Anastasia Nicole Nagel Doctor of Philosophy in Biological Sciences University of California, Irvine, 2015 Professor Susana Cohen-Cory, Chair Netrins are a family of extracellular proteins that function as chemotropic guidance cues for migrating cells and axons during neural development. In visual system development, Netrin-1 has been shown to play a key role in retinal ganglion cell (RGC) axon growth and synaptogenesis where presynaptic RGC axons form partnerships with the dendrites of tectal neurons. However, the signals that guide the dendrites of tectal neurons and facilitate connections between these postsynaptic cells and RGC axons are yet unknown. This work in collaboration with other lab members has explored the dynamic cellular mechanisms by which netrin-1 influences visual circuit formation, particularly those that impact postsynaptic neuronal morphology, connectivity, and function during retinotectal wiring. Immunohistochemistry reveals that netrin-1 and its receptors DCC and UNC5 are expressed in the optic tectum at the developmental stage when circuit formation occurs. Time-lapse in vivo imaging of individual Xenopus laevis optic tectal neurons co-expressing tdTomato and PSD95-GFP revealed rapid remodeling and reorganization of dendritic arbors following acute manipulations in netrin-1 levels. Within four hours of treatment, tectal injection of recombinant netrin-1 or sequestration of endogenous netrin with an UNC-5 receptor ectodomain induced significant changes in the xdirectionality and orientation of dendrite growth and in the maintenance of already established dendrites away from the tectal neuropil, demonstrating that relative levels of netrin are important for these functions. In contrast, altering DCC-mediated netrin signaling with function blocking antibodies induced postsynaptic specialization remodeling and changed growth directionality by turning stable dendrites. Reducing netrin signaling also decreased avoidance behavior in a visually guided task, suggesting that netrin is essential for emergent visual system function. In vivo 2 Photon imaging examining the cell autonomous effects of translation blocking morpholino oligonucleotides to netrin receptors UNC5 and DCC in single cell electroporated neurons revealed altered neuronal morphologies, decreases in branch length and number, and changes to the directionality of treated dendritic arbors. Together the findings in the following chapters suggest netrin mediated DCC and UNC5 signaling play a critical role in the normal development of vertebrate central neuron dendritic arbors during the establishment of the functional visual system. xi INTRODUCTION The nervous system is a highly complex network, where simple molecular changes can have profound effects on development; a fundamental question both in basic and clinical neuroscience is how molecular signals influence neuronal growth and maturation to create such a network. The signals involved in establishing neural networks appear highly conserved in form and function across different species and between invertebrates and invertebrates. During the establishment of the nervous system, these molecules play many roles and are reused in different locations and developmental stages. Much like an actor with multiple roles in a single play, the molecule netrin-1 has exemplified the ability to regulate neuron development through diverse mechanisms and at various times during circuit formation. Netrin-1 has been shown to instruct: the axons and dendrites of neurons during guidance to their target [1-8], whether projections become axons or dendrites [9], the branching of axonal and dendritic arbors [4, 10-12], establishing the patterning of dendrites [13-15] and the formation of synaptic connections with their partners [16-18]. Yet the study of how this signaling molecule plays a particular role or coordinates with other signals in different aspects of development remains in its infancy. Many of the developmental roles for these signals were first elucidated in simpler models such as Drosophila melanogaster and Caenorhabditis elegans. Current investigations focus on enhancing our understanding of how signals such as netrin-1 converge to direct growth and coordinate development in ways that produce an integrated, complex nervous system. This gap between our understanding of the function of these molecules in invertebrates and more complex 1organisms highlights the necessity of investigations that examine the roles of these signals in vertebrate models of central nervous system development. Previous work investigating netrin-1 signaling has used invertebrates or cell culture systems from mouse models that often involves the isolation of neurons from their in vivo environments. These preparations cannot reflect the mechanisms of compensation seen in whole organisms. These models also involve the creation of mutations or the use of manipulations that affect large populations of neurons, making it more difficult to parse out differences between causal and compensatory effects. Both of these models may not fully represent the conditions in a more complex and complete system. Fortunately the Xenopus laevis visual system can be used as a model to specifically examine the effects of netrin-1 in vertebrate neuron development and circuit formation while avoiding these issues. The transparency of embryos allows investigators to examine morphological maturation in individual neurons in the intact brain to understand cellautonomous molecular mechanisms of development. Additionally the functionality of visual system circuitry can be assessed by examining the behavioral responses of developing X. laevis tadpoles in a visually guided avoidance task [19], allowing for researchers to correlate the effects of manipulations on neuron morphology and connectivity observed in vivo with observations of how the same manipulations affect behavior. All of the above make this model system ideal for investigations of how netrin-1 influences neural circuit formation. Although we have learned much about netrin-1 since it was first discovered in C. elegans [20], new evidence suggests that netrin-1 may play additional roles. Netrin-1 has already been extensively studied in vertebrate models of spinal cord and retinal development and has been 2specifically shown to play a role mediating axon guidance in the spinal cord and retina [1, 3, 21- 24]. Even though the coordination of netrin-1 with its receptors deleted-in-colorectal cancer (DCC) and uncoordinated 5 (UNC-5) during axon guidance is often described as a canonical process in development [7], the role of netrin-1 in the development of dendrites is not well understood. Recent work in C. elegans suggests that netrin-1 participates in important aspects of dendritic development, including dendrite branching [25] and the self-avoidance of dendrites during the establishment of dendritic patterns [26]. In vertebrates, the evidence for netrin-1 participating in dendritic development is particularly sparse as only one paper examining zebrafish has demonstrated that netrin plays any role in dendritogenesis [6]. As netrin can be located in regions where both presynaptic axons and postsynaptic dendritic arbors form connections [11], it is important to ask whether netrin-1 can simultaneously play dual roles influencing both axon development and dendritogenesis at the time when they form synaptic connections. The Xenopus laevis visual system is well suited to investigate this potential new role of netrin-1 in dendritogenesis for several reasons. Most importantly netrin-1 in known to be expressed in the optic tectum of the central nervous system in regions where both presynaptic axons and postsynaptic neurons reside [11], and its role in retinal ganglion cell axon development has been characterized extensively [3, 11, 21, 22]. Yet, no studies to date have investigated whether it also influences the dendrites of postsynaptic neurons. This body of work is among the first to use an in vivo, vertebrate model to identify the potential roles for netrin-1 in influencing dendrite growth and connectivity as well as the receptors that mediate this signaling. In the following chapters, I will demonstrate that netrin-1 influences dendrite growth and connectivity and that the developmental changes in dendritic architecture are primarily due to signaling mediated by two netrin-1 receptors: DCC and UNC5. 3CHAPTER 1 MOLECULAR SIGNALS REGULATE VISUAL SYSTEM DEVELOPMENT Signaling molecules play multiple roles in neuron development and have been shown to instruct both the axons and dendrites of neurons during the establishment of neural circuits. This requires the convergence of many molecular signals to establish patterns of wiring with specificity. This review will briefly discuss some of the processes and signals that direct the formation of neural circuits that ultimately comprise complex nervous systems and will specifically focus on visual system development. The simplified steps of forming a neural circuit include: 1) the migration of neurons to their ultimate location in specific areas; 2) establishing the identity of neuronal projections as axons or dendrites; 3) guiding the directionality of those projections to their targets; 4) growing and branching projections so they occupy their territories in specific patterns; 5) forming connections between projections by creating synapses between partnering neurons so that functional connections are made (Figure 1.1). The first aspects of establishing a circuit is for neuronal precursors to reach their target area through migration and to have their neurites, early neuronal projections, determine whether they will become future axons or dendrites. Signaling molecules have been demonstrated to participate in both of these first two roles in both invertebrates and vertebrates [9, 27, 28]. 4Figure 1.1: Schematic of neuron during undergoing the general developmental processes that establish functional neural circuitry. (A) A neuron is born from radial glial progenitor cell and then it must migrate to its correct location in the brain. (B) Early in development it must polarize to determine which projections will be dendrites and which will be the axon. (C) The growth of dendritic branches must then be directed so they project to their target area. (D) As the neuron matures and grows, the dendritic arbor elaborates, dynamically adding and eliminating branches so that the arbor becomes larger. Dendrites branch into specific patterns and the arbor grows into a specified territory. (E) Dendrites ultimately connect with their correct partnering cells, forming synapses with arriving axons to establish a functional circuit. 5The emphasis of this proposal is concentrated on the later developmental steps mentioned above: 3) the guidance and directionality of dendrites, 4) their branching and growth, and 5) synaptogenesis and connectivity between axons and dendrites to build functional circuitry. After neural projections have an identity (axons or dendrites), they need to find their targets, the partnering neurons with whom they will form connections. This guidance is often accomplished through diffusible chemical attractants or repellants [1, 20, 29, 30]. These signals have been shown to direct the growth cones of neural projections in both in vivo and in vitro experiments [2, 3, 23, 27]. Netrin-1 is an example of one of these signals that has been shown to guide axons in the spinal cord, out of the retina, and in other neurons of the central nervous system [1, 7, 21, 31]. An example model developed from in vivo and in vitro studies illustrates how signals can guide projections throughout development (Figure 1.2). In this example, a retinal ganglion cell (RGC) axon is shown navigating from the eye to the optic tectum using many different molecular signals. Initially, axons are repulsed from the periphery of the retina by chondroitin sulfate and are attracted to the optic disc, where they exit the eye by short-range, attractive netrin-1 mediated DCC signaling. Axons then travel along a path to the optic chiasm due to a distribution of semaphorins and inhibitory Slit/Robo interactions. At the optic chiasm, Slits repulse axons from inhibitory zones and ephrin-B2 repulses some axons so that projections are guided either to their ipsilateral or contralateral targets (Figure 1.2A). After the optic chiasm, semaphorins and Slit guide the axon through the brain too the dorsal optic tectum. Here RGC axons make connections with the tectal neurons of the tectum and signaling through Ephrins and Eph ligands establishes the topographic maps of projections in the visual system (Figure 1.2B). Although much is known about the cues for axon guidance from vertebrate models in this

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