University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2016 Multiple Roles of Ret Signaling in Mechanosensory Neuron Development Michael Scott Fleming University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular Biology Commons, and the Neuroscience and Neurobiology Commons Recommended Citation Fleming, Michael Scott, "Multiple Roles of Ret Signaling in Mechanosensory Neuron Development" (2016). Publicly Accessible Penn Dissertations. 1718. https://repository.upenn.edu/edissertations/1718 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1718 For more information, please contact [email protected]. Multiple Roles of Ret Signaling in Mechanosensory Neuron Development Abstract Somatosensation is critical for interaction with the surrounding environment. Somatosensory stimuli are detected by primary somatosensory neurons of the dorsal root ganglia and trigeminal ganglia, which detect distinct classes of stimuli, such as temperature, pain, and pressure. In Chapters 2 and 3 of this thesis, we focus on rapidly adapting low-threshold mechanoreceptors (RALTMRs), which mediate the detection of light touch. RALTMRs are molecularly defined yb the early embryonic expression of the receptor tyrosine kinase Ret. Ret is required for the development of central axonal projections of RALTMRs into the dorsal spinal cord. RET responds to the glial cell line-derived family of neurotrophic factors, which activate RET in combination with GPI-linked GFRα co-receptors. In vitro, RET can be activated by co-receptor expressed in the same cell (cis signaling) or by co-receptor expressed by neighboring cells (trans signaling), but previous studies suggest that trans RET signaling may not play a physiologically relevant role in vivo. Here, we show that RET in mouse RALTMRs can be activated by both GFRα2 expressed in the same cell (cis signaling) and GFRα1 expressed by neighboring cells (trans signaling), and that trans RET signaling is sufficient for the vde elopment of RALTMR central projections in vivo. Peripherally, Ret is required for the development of vibration sensitive Pacinian corpuscle RALTMR end organs. We show that Ret mediates the neuronal expression of the ETS transcription factor Er81, which is also required for Pacinian corpuscle development, and that deficient axon/Schwann cell communication is the primary deficit in Pacinian corpuscle development in Er81 mutant mice. Furthermore, we show that Neuregulin-1, an important mediator of axon/Schwann cell interactions, is required for Pacinian corpuscle development, and that Er81 regulates the expression of specific Neuregulin-1 isoforms. In total, we demonstrate that RET signaling drives the development of distinct developmental processes in both the central and peripheral axonal branches of RALTMRs. In Chapter 4, we describe the expression of itch-related neuropeptides GRP and NMB and their receptors in somatosensory neurons and the dorsal spinal cord. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Neuroscience First Advisor Wenqin Luo Keywords Development, Molecular Biology, Neuroscience, Neurotrophic Factors Subject Categories Molecular Biology | Neuroscience and Neurobiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1718 MULTIPLE ROLES OF RET SIGNALING IN MECHANOSENSORY NEURON DEVELOPMENT Michael S. Fleming A DISSERTATION in Neuroscience Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation ______________ Wenqin Luo, MD, PhD Assistant Professor of Neuroscience Graduate Group Chairperson _________________ Joshua I. Gold, PhD Professor of Neuroscience Dissertation Committee: Gregory J. Bashaw, PhD, Professor of Neuroscience Douglas J. Epstein, PhD, Professor of Genetics Robert O. Heuckeroth, MD, PhD, Professor of Pediatrics Steven S. Scherer, MD, PhD, Professor of Neurology MULTIPLE ROLES OF RET SIGNALING IN MECHANOSENSORY NEURON DEVELOPMENT COPYRIGHT 2016 Michael S. Fleming ACKNOWLEDGEMENT I would like to thank my mentor, Dr. Wenqin Luo. Wenqin has been an amazingly supportive and inspiring mentor over the past five years. Her insights and encouragement have been crucial in performing the work in this thesis. Wenqin is a brilliant scientist and our countless conversations about my project and science in general have been invaluable in teaching me how to think scientifically. I would also like to thank members of the Luo lab for insightful comments, suggestions, and friendship. Thank you to Dr. Jingwen Niu, Dr. Lian Cui, Dr. Ishmail Abdus-Saboor, Dr. Nathan Fried, Will Olson, Peter Dong, Kim Kridsada, and Anna Vysochan for making the Luo lab such a great environment. Thank you to Dr. Minghong Ma and the members of her lab for helpful comments during joint lab meetings. Many thanks to Drs. Greg Bashaw, Doug Epstein, Steve Scherer, and Robert Heuckeroth for serving on my thesis committee. Their expertise has been a valuable resource in designing and interpreting experiments, and their insights in and out of committee meetings have strengthened my thesis work in many ways. Thank you to my family and friends for all of your support. My parents have always supported my interest in science and I want to thank them for encouraging me to pursue my goals. Most importantly, thank you to my wife, Melissa, for constant love, support, and understanding. iii ABSTRACT MULTIPLE ROLES OF RET SIGNALING IN THE DEVELOPMENT OF MECHANOSENSORY NEURONS Michael S. Fleming Wenqin Luo Somatosensation is critical for interaction with the surrounding environment. Somatosensory stimuli are detected by primary somatosensory neurons of the dorsal root ganglia and trigeminal ganglia, which detect distinct classes of stimuli, such as temperature, pain, and pressure. In Chapters 2 and 3 of this thesis, we focus on rapidly adapting low-threshold mechanoreceptors (RALTMRs), which mediate the detection of light touch. RALTMRs are molecularly defined by the early embryonic expression of the receptor tyrosine kinase Ret. Ret is required for the development of central axonal projections of RALTMRs into the dorsal spinal cord. RET responds to the glial cell line- derived family of neurotrophic factors, which activate RET in combination with GPI- linked GFRα co-receptors. In vitro, RET can be activated by co-receptor expressed in the same cell (cis signaling) or by co-receptor expressed by neighboring cells (trans signaling), but previous studies suggest that trans RET signaling may not play a physiologically relevant role in vivo. Here, we show that RET in mouse RALTMRs can be activated by both GFRα2 expressed in the same cell (cis signaling) and GFRα1 expressed by neighboring cells (trans signaling), and that trans RET signaling is sufficient for the development of RALTMR central projections in vivo. Peripherally, Ret iv is required for the development of vibration sensitive Pacinian corpuscle RALTMR end organs. We show that Ret mediates the neuronal expression of the ETS transcription factor Er81, which is also required for Pacinian corpuscle development, and that deficient axon/Schwann cell communication is the primary deficit in Pacinian corpuscle development in Er81 mutant mice. Furthermore, we show that Neuregulin-1, an important mediator of axon/Schwann cell interactions, is required for Pacinian corpuscle development, and that Er81 regulates the expression of specific Neuregulin-1 isoforms. In total, we demonstrate that RET signaling drives the development of distinct developmental processes in both the central and peripheral axonal branches of RALTMRs. In Chapter 4, we describe the expression of itch-related neuropeptides GRP and NMB and their receptors in somatosensory neurons and the dorsal spinal cord. v TABLE OF CONTENTS ACKNOWLEDGEMENT………………………………………………………………..iii ABSTRACT……………………………………………………………………………...iv LIST OF TABLES……………………………………………………………………...viii LIST OF ILLUSTRATIONS…………………………………………………………......ix CHAPTER 1: INTRODUCTION………………………………………………………....1 MAMALIAN SOMATOSENSATION………………………………………………………………………2 MEISSNER’S CORPUSCLES……………………………………………………………………………….4 PACINIAN CORPUSCLES………………………………………………………………………………….9 LANCEOLATE ENDINGS………………………………………………………………………………...14 MERKEL CELLS…………………………………………………………………………….…………......20 RUFFINI CORPUSCLES………………………………………………………………………………......29 RET STRUCTURE AND FUNCTION……………………………………………………………………..32 RET SIGNALING…………………………………………………………………………………………..35 ER81…………………………………………………………………………………………………….…..38 SCHWANN CELLS AND NEUREGULIN-1……………………………………………………………...41 BOMBESIN-RELATED PEPTIDES AND ITCH………………………………………………………….43 CHAPTER 2: CIS AND TRANS RET SIGNALING CONTROL THE SURVIVAL AND CENTRAL PROJECTION GROWTH OF RAPIDLY ADAPTING MECHANORECEPTORS……………………………………………………………….47 ABSTRACT………………………………………………………………………………………….……..48 INTRODUCTION……………………………………………………………………………………..……49 RESULTS……………………………………………………………………………………………….…..52 DISCUSSION…………………………………………………………………………………………….…70 vi MATERIALS AND METHODS…………………………………………………………………………...78 CHAPTER 3: A RET-ER81-NEUREGULIN1 SIGNLAING PATHWAY DRIVES THE DEVELOPMENT OF PACINIAN CORPUSCLES…………………………………...117 ABSTRACT…………………………………………………………………………………………….…118 INTRODUCTION………………………………………………………………………………………....119 RESULTS………………………………………………………………………………………………….121 DISCUSSION……………………………………………………………………………………………...135