Hyperactive Alpha2-Chimaerin Reveals the Complexity of Axon Guidance Signaling Pathways in Motor Neuron Development
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Hyperactive alpha2-chimaerin reveals the complexity of axon guidance signaling pathways in motor neuron development The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Nugent, Alicia Anne. 2016. Hyperactive alpha2-chimaerin reveals the complexity of axon guidance signaling pathways in motor neuron development. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493414 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Hyperactive alpha2-chimaerin reveals the complexity of axon guidance signaling pathways in motor neuron development A dissertation presented by Alicia Anne Nugent to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Neurobiology Harvard University Cambridge, Massachusetts March 2016 © 2016 Alicia Anne Nugent All rights reserved. Dissertation Advisor: Dr. Elizabeth C. Engle Alicia Anne Nugent Hyperactive alpha2-chimaerin reveals the complexity of axon guidance signaling pathways in motor neuron development Abstract !2-chimaerin has striking importance for proper neural circuit formation. Individuals with gain-of-function mutations in !2-chimaerin have Duane retraction syndrome, a common neurogenic eye movement disorder, and !2-chimaerin knockout mice have a rabbit-like hopping gait, resulting from aberrant motor neuron wiring. While molecular mechanisms underlying altered gait circuitry in the loss-of-function mouse have been characterized, mechanisms underlying the etiology of DRS caused by mutant CHN1 remain unclear. Here, we report the first Chn1 gain-of-function DRS mouse model (Chn1KI), which harbors a knock-in point mutation identified in human patients. Chn1WT/KI and Chn1KI/KI embryonic mice exhibit abducens nerve stalling, aberrant trochlear nerve branching, and first cervical spinal segment (C1) misrouting, which result from axon guidance defects. The Chn1KI mouse recapitulates the human DRS phenotype, thus providing a novel mouse model of DRS. Chn1KO/KO embryos display abducens nerve wandering and defasciculation, establishing that human DRS-causing mutations are indeed gain-of-function. We combine detailed 3D whole embryo imaging with novel in vitro approaches to demonstrate that hyperactivated !2-chimaerin acts downstream of ephrin forward and reverse signaling selectively in abducens neurons to modulate nerve development, as C1 neurons use only ephrin forward signaling, and trochlear neurons do not significantly use ephrin signaling during nerve guidance. In vivo, we find that selectively removing ephrin forward or reverse iii signaling via EphA4 dramatically impacts the development of the abducens nerve, distinct from bidirectionally removing EphA4 signaling. Further experimentation reveals that alpha2-chimaerin and EphA4 can signal through other pathways. We find that hyperactivated alpha2-chimaerin modulates BDNF, GDNF, NGF, and HGF signaling in vitro, thus suggesting its role as a broad regulator of axon guidance pathways. Our studies lend insight into the complexity of axon guidance during development and highlight mechanisms that cause the abducens nerve to be selectively vulnerable to alpha2- chimaerin misregulation in DRS. iv TABLE OF CONTENTS Abstract………………………………………………………………………………………..….iii Table of Contents……………….…………………………………………………………………v Table of Figures……………….………………………………………………………………....vii Acknowledgements……………….……………………………………………………………....ix CHAPTER 1 - Introduction……………….………………………………………………………1 References……………………………………………………………………………………18 CHAPTER 2 - Methods……………….…………………………………………………………25 References…………………………………………………………………………...……….41 CHAPTER 3 - Bidirectional ephrin signaling alters abducens nerve development via !2- chimaerin…………………………….……….…………………………………………………..42 Abstract………………………………………………………………………………………45 Introduction……………………………………………………………………………….….46 Results………………………………………………………………………………………..50 Discussion……………………………………………………………………………………99 References……………………………………………………………………………….….106 CHAPTER 4 - Neurotrophic factors regulate abducens outgrowth through hyperactive !2- chimaerin-mediated pathways……………….…………………………………………...…….110 Abstract………………………………………………………………………………….….112 Introduction…………………………………………………………………………………113 Results……………………………………………………………………………………....116 Discussion……………………………………………………………………………….….133 References…………………………………………………………………….…………….138 v CHAPTER 5 - Discussion……………….……………………………………………………..141 Summary of findings…………………………………….……………………………....…142 References…………………………………………………………………….…………....152 APPENDIX……………….……………………………………………………………………154 Appendix I: Human Disorders of Axon Guidance………………………………...…….…154 Appendix II: Loss of MAFB function in human and mouse causes Duane syndrome, aberrant extraocular muscle innervation, and inner ear defects..........................……………......177 Appendix III: Supplemental Figures: Loss of MAFB function in human and mouse causes Duane syndrome, aberrant extraocular muscle innervation, and inner ear defects.........200 Appendix IV: Movies from Chapter 3: Bidirectional ephrin signaling alters abducens nerve development via !2-chimaerin........................................................................................207 vi TABLE OF FIGURES Figure 1.1……………………………………………………………………………………….....5 Figure 1.2....……………………………………………………………………………………….9 Figure 1.3…..…………………………………………………………………………………….11 Figure 1.4……..………………………………………………………………………………….14 Table 2.1..………………………………………………………………………………………..28 Figure 2.1.……….……………………………………………………………………………….37 Figure 3.1..……………………………………………………………………………………….51 Figure 3.2..………………………….…………………………..……………………………..…52 Figure 3.3..……………………………………………………………………………………….55 Figure 3.4.………………………………………………………………………………………..58 Figure 3.5…………………………………………………...……………………..…………......60 Figure 3.6..……………………………………………………………………………………….62 Figure 3.7..……………………………………………………………………………………….66 Figure 3.8..……………………………………………………………………………………….68 Figure 3.9..……………………………………………………………………………………….70 Figure 3.10..…………..………………………………………………………………………….72 Figure 3.11..…………..………………………………………………………………………….74 Figure 3.12..……………………..……………………………………………………………….77 Figure 3.13..……………………..……………………………………………………………….79 Figure 3.14..……………………..……………………………………………………………….83 Figure 3.15..……………………..……………………………………………………………….84 Figure 3.16..……………………..……………………………………………………………….90 vii Figure 3.17..……………………..……………………………………………………………….92 Figure 3.18..……………………..……………………………………………………………….95 Figure 3.19..……………………..……………………………………………………………….97 Figure 3.20..……………………..……………………………………………………………...102 Figure 4.1..……………………..…………………………………………………………….....117 Figure 4.2..……………………..…………………………………………………………….....119 Figure 4.3..……………………..…………………………………………………………….....122 Figure 4.4..……………………..…………………………………………………………….....126 Figure 4.5..……………………..…………………………………………………………….....128 Figure 4.6..……………………..…………………………………………………………….....131 Figure AI.1..…………………..…………………………………………………………..….....159 Figure AI.2..…………………..…………………………………………………………..….....169 Figure AII.1..…………………..………………………………………………………..…........184 Table AII.1..…………………..………………………………………………………..….........185 Figure AII.2..…………………..………………………………………………………..…........186 Figure AII.3..…………………..………………………………………………………..…........191 Table S1..…………………..…………………………………………….....…………..…........201 Figure S1..…………………..………………………………………….....…………..…...........202 Figure S2..…………………..………………………………………….....…………..…...........203 Table S2..…………………..…………………………………………….....…………..…........204 Figure S3..…………………..………………………………………….....…………..…...........205 Figure S4..…………………..………………………………………….....…………..…...........206 viii ACKNOWLEDGEMENTS There are so many people who have contributed to the success of my graduate work. To my advisor, Elizabeth Engle, you have helped me gain confidence and intelligence as a scientist, strive for perfection, and have always encouraged me to follow my interests. I thank you for your constant support over the past six years. I thank all members of the Engle Lab, past and present. It’s been incredible to be surrounded by such fantastic colleagues and friends. I will miss sharing the joys and frustrations of science with you all. Michelle DeLisle, Yan Wei, and Jong Park - thanks for the countless hours you poured into helping with various projects. Special thanks to Jo Chan, Long Cheng, Max Tischfield, Sherin Shaaban, Alessandro DiGioia, Mary Whitman, Tammy Chu, Yi Su, Lingyun Jia, Adrianne Kolpak, and Maya Peeva for guidance, feedback, training, and technical assistance. To my students - Ariel Garcia, Hannah Marudzinski, and John Masland - you have taught me invaluable lessons, thank you for your hard work. Thank you to my Dissertation Advisory Committee, Tom Schwarz, Jeff Lichtman, and Davie Van Vactor, for the valuable feedback on which experiments to persevere with and which ones to give up on. I will always remember Tom’s advice: If you keep too many experiments in your back pocket, you’ll never be able to sit down. Touché. Thank you also to the people who made the Program in Neuroscience a great place to grow up: Rachel Wilson, Roz Segal, Rick Born, Karen Harmin, and all of my incredibly talented and fun classmates. To my prior mentors - Chris Tsang, Rich Kramer, Asheeta Prasad, Jeroen Pasterkamp,