NEURON-SATELLITE GLIAL CELL INTERACTIONS IN SYMPATHETIC NERVOUS SYSTEM DEVELOPMENT by Erica D. Boehm A dissertation submitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland July 2020 © 2020 Erica Boehm All rights reserved. ABSTRACT Glial cells play crucial roles in maintaining the stability and structure of the nervous system. Satellite glial cells are a loosely defined population of glial cells that ensheathe neuronal cell bodies, dendrites, and synapses of the peripheral nervous system (Elfvin and Forsman 1978; Pannese 1981). Satellite glial cells are closely juxtaposed to peripheral neurons with only 20nm of space between their membranes (Dixon 1969). This close association suggests a tight coupling between the cells to allow for possible exchange of important nutrients, yet very little is known about satellite glial cell function and development. How neurons and glial cells co-develop to create this tightly knit unit remains undefined, as well as the functional consequences of disrupting these contacts. Satellite glial cells are derived from the same population of cells that give rise to peripheral neurons, but do not begin differentiation and proliferation until neurogenesis has been completed (Hall and Landis 1992). A key signaling pathway involved in glial specification is the Delta/Notch signaling pathway (Tsarovina et al. 2008). However, recent studies also implicate Notch signaling in the maturation of glia through non- canonical Notch ligands such as Delta/Notch-like EGF-related Receptor (DNER) (Eiraku et al. 2005). Interestingly, it has been reported that levels of DNER in sympathetic neurons may be dependent on the target-derived growth factor, nerve growth factor (NGF), and this signal is prominent in sympathetic neurons at the time in which satellite glial cells are developing (Deppmann et al. 2008; Hall and Landis 1992). Here, we find that the close association of satellite glial cells with sympathetic neuronal cell bodies is mediated by neuronal expression of DNER. In mice, conditional deletion of DNER from sympathetic neurons disrupts neuron-glia contacts. Loss of ii DNER and/or neuron-glia contacts has profound effects on the neurons, resulting in hyper-innervation of targets, decreased neuron soma size, and an increase in activity. Additionally, DNER expression is regulated by target-derived NGF. This suggests a tripartite system in which the development of neurons and glial cells may be coordinated by the same distant signal, and supports a role for neuron-satellite glia contacts in neuronal architecture and activity. Advisor: Rejji Kuruvilla, Ph.D. Second Reader: Haiqing Zhao, Ph.D. Committee Members: Mark Van Doren, Ph.D. Seth Blackshaw, Ph.D. Hey-Kyoung Lee, Ph.D. Haiqing Zhao, Ph.D. iii PREFACE Nothing I have done in life prior to graduate school could have prepared me for the trials and errors of my time here. It has been an emotional and intellectual rollercoaster with twists, turns, and loops of failures and successes. I believe I have come off this ride a better, more confident person in my work life and in my personal life. I have discovered the aspects of science that I love as well as the aspects of it that I do not like so much, and the growth I have gone through as a person is something that I will carry with me for the rest of my life wherever it leads me. During my time as a graduate student in Dr. Rejji Kuruvilla’s lab, I have learned how to think for myself, how to design experiments, lead a project, and mentor students with patience and kindness. I have gotten to dip my hands into many different techniques, some of which were very helpful while others did not bear fruit as we had hoped. I have learned how to deal with failure and disappointment when experiments didn’t work or when they gave confusing results, and I learned how to think outside the box to find new solutions to problems. My time here has transformed me into a person I never thought I could be; someone who can provide valuable insight during lab meetings, who can think critically about data being presented, and who figured out how to appreciate the small successes as much as the big ones. I have a number of people to thank for my growth throughout graduate school. First, I would like to thank my mentor, Rejji, who constantly kept pushing me to be better, think harder, and dig deeper for answers. I’d also like to thank the Mouse Tri-lab as a community and the individuals within who have really helped me along the way. Without the Mouse Tri-lab, I would not be the thoughtful person I am today. The unique iv environment of the Tri-lab community nurtured my scientific curiosity. I would especially like to thank those who have helped me with my project experimentally and through their critical eyes. Particularly I would like to give thanks to my lab mates, current and former, Dr. Alexis Ceasrine, Dr. Ami Patel, Aurelia Mapps, Blaine Connor, Dr. Chantal Bodkin-Clarke, Dr. Chih-Ming Chen, Dr. Emily Scott-Solomon, Dr. Eugene Lin, Dr. Jessica Houtz, and Nelmari Ruiz-Otero for helping with experiments, lending expert advice, and being shoulders to lean on. Without these people, I would not be the scientist I am. I also want to thank a collaborator, Dr. Bryan Jones, for his contribution to my work. Without his help, this work would not be where it is today. Finally, I would like to thank the professors from the Mouse Tri-lab and my committee for their support and generosity throughout my time here. So, thank you to Dr. Haiqing Zhao, Dr. Hey- Kyoung Lee, Dr. Mark van Doren, Dr. Samer Hatter, and Dr. Seth Blackshaw. I am grateful to everyone, friends, family, and colleagues, who supported me and kept me afloat while obtaining my Ph.D. v TABLE OF CONTENTS ABSTRACT………………………………………………………………………….......ii PREFACE………………………………………………………………………………..iv TABLE OF CONTENTS…………………………………………………………..…...vi LIST OF FIGURES………………………………………………...….…....................viii LIST OF ABBREVIATIONS…………………………………………………...............x CHAPTER ONE: INTRODUCTION…………………………………………..……....1 CHAPTER TWO: REGULATION AND EXPRESSION OF DNER IN THE SYMPATHETIC NERVOUS SYSTEM……………………………………………....16 INTRODUCTION………………………………………………………………...17 RESULTS………………………………………………………………................22 DISCUSSION………………………………………………………………..........31 METHODS………………………………………………………………..............33 CHAPTER THREE: NEURONAL DNER IS REQUIRED FOR SYMPATHETIC- SATELLITE GLIAL CELL CONTACTS……………………………………………40 INTRODUCTION………………………………………………………………...41 RESULTS………………………………………………………………................46 DISCUSSION………………………………………………………………..........58 METHODS………………………………………………………………..............61 CHAPTER FOUR: DISRUPTION OF NEURONAL MORPHOLOGY AND ACTIVITY IN DNER CKO MICE……………………………………………………67 INTRODUCTION………………………………………………………………...68 RESULTS………………………………………………………………................73 vi DISCUSSION………………………………………………………………..........88 METHODS………………………………………………………………..............93 CHAPTER FIVE: GENETIC ABLATION OF SATELLITE GLIAL CELLS DURING SYMPATHETIC NERVOUS SYSTEM DEVELOPMENT……………100 INTRODUCTION…………………………………………………………….…101 RESULTS………………………………………………………………..............105 DISCUSSION………………………………………………………………........115 METHODS………………………………………………………………............117 CHAPTER SIX: MOLECULAR CHANGES IN DNER CKO SYMPATHETIC GANGLIA………………………………………………………..……………………123 INTRODUCTION………………………………………………………….........124 RESULTS………………………………………………………………..............126 DISCUSSION………………………………………………………………........130 METHODS………………………………………………………………............131 CLOSING REMARKS………………………………………………………..…...….133 REFERENCES………………………………………………………..…….................141 CURRICULUM VITAE………………………………………………………………168 vii LIST OF FIGURES Chapter Two: Figure 2.1. DNER is expressed in all three major compartments of sympathetic neurons…………………………………………………………………………………...26 Figure 2.2. DNER has a half-life around 4 hours and partially localizes to the cell surface membrane………………………………………………………………………………...28 Figure 2.3. DNER is regulated by NGF-TrkA signaling………………………………...29 Figure 2.4. Disruption of satellite glial cell sheaths in TrkA knockout sympathetic ganglia…………………………………………………………………………………....30 Chapter Three: Figure 3.1. DNER alters satellite glial cell morphology in vitro………………………...51 Figure 3.2. DNER-Fc is capable of activating Notch signaling in cultured satellite glial cells………………………………………………………………………………………52 Figure 3.3. DNER is knocked down in DNER cKO sympathetic ganglia……………….53 Figure 3.4. Cell proliferation is unaffected in neonatal DNER cKO sympathetic ganglia……………………………………………………………………………………54 Figure 3.5. Satellite glial cell ring structure is lost in sympathetic ganglia of DNER cKO mice………………………………………………………………………………………55 Figure 3.6. Sympathetic ganglia morphology is disrupted in DNER cKO mice………...56 Figure 3.7. Neuron-satellite glial cell contacts are lost in DNER cKO ganglia………….57 Chapter Four: viii Figure 4.1. Loss of adrenergic biosynthetic pathway enzyme expression in DNER cKO ganglia……………………………………………………………………………………78 Figure 4.2. DNER cKO neurons are smaller in size……………………………………..79 Figure 4.3. Neuronal survival is unaffected in DNER cKO animals………………….....80 Figure 4.4. DNER cKO neurons hyper-innervate the heart. …………………………….83 Figure 4.5. Salivary glands are hyper-innervated in DNER cKO mice………………….84 Figure 4.6. Cultured DNER cKO neurons do not exhibit changes in neurite length or branching…………………………………………………………………………………85 Figure 4.7. DNER cKO neurons have a sustained calcium response to depolarization….86 Figure 4.8.