CONTINUOUSLY ACTIVE TRANSCRIPTIONAL PROGRAMS ARE REQUIRED TO BUILD EXPANSIVE SEROTONERGIC AXON ARCHITECTURES By LAUREN JANINE DONOVAN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Evan S. Deneris Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY January 2020 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Lauren Janine Donovan candidate for the degree of Doctor of Philosophy*. Committee Chair Jerry Silver, Ph.D. Committee Member Evan Deneris, Ph.D. Committee Member Heather Broihier, Ph.D. Committee Member Ron Conlon, Ph.D. Committee Member Pola Philippidou, Ph.D. Date of Defense August 29th, 2019 *We also certify that written approval has been obtained for any proprietary material contained therein. ii TABLE OF CONTENTS List of Figures……………………………………………………………………….….vii Abstract………………………………………….………………………………..….…1 CHAPTER 1. INTRODUCTION………………………………………………...……..3 GENERAL INTRODUCTION TO SEROTONIN………………………………….….4 Serotonin: Discovery and function………………………….……………...4 Serotonin Biosynthesis…………………………..…………………………..6 Manipulation of the serotonin system in humans……………………….6 Human mutations in 5-HT related genes………………………………….9 SEROTONIN NEURON NEUROGENESIS……………..………………………….11 5-HT neuron specification……………..………………………………...…11 Development of 5-HT neurons……………..………………………………13 NEUROANATOMY……………..……………………………………………………..13 Cytoarchitecture ……………..………………………………………………13 Adult Ascending 5-HT axon projection system ………………………..14 5-HT axon innervation patterns in the forebrain……………………14 Serotonergic axon tracing studies……………..…………………….16 Varicosities in ascending 5-HT axons……………………………….18 Adult Descending 5-HT axon projection system……………………….19 DEVELOPMENT OF SEROTONERGIC AXON PROJECTION SYSTEM……...20 Development of ascending projection system ………………………...20 Primary axon pathway formation…………………………………….21 Selective routing ………………………………………………………22 Terminal target arborization…………………………………………..24 iii Known molecules affecting 5-HT neuron ascending axon development…………………………………………………………………..26 Wnt planar cell polarity pathway……………………………………..26 Slit-Robo signaling…………………………………………………….28 EphrinA5………………………………………………………………..28 Gap43…………………………………………………………….….....29 STOP (Map6) ………………………………………………………….30 Pcdhac2………………………………………………………………...31 5-HT……………………………………………………………………..34 Development of descending projection system ……………………….37 PET-1…………………………………………………………………………………...38 Gene/protein expression……………………………………………………38 Protein structure………………………………………………………….…..39 Function………………………………………………………………………..39 LMX1B………………………………………………………………………………….40 Gene/protein structure………………………………………………………40 Lmx1b in peripheral tissues………………………………………………..41 Lmx1b in the CNS…………………………………………………………….42 Lmx1b in midbrain-hindbrain patterning…………………………….42 Lmx1b in midbrain Dopaminergic neurons………………………….43 Lmx1b in Noradrenergic neurons…………………………………….44 Lmx1b in Serotonergic neurons……………………………………...44 iv CHAPTER 2. PET-1 CONTROLS TETRAHYDROBIOPTERIN PATHWAY AND SLC22A3 TRANSPORTER GENES IN SEROTONIN NEURONS. Summary……………………………………………………………………………....60 Introduction…………………………………………………………………..……….61 Methods………………………………………………………………………………..65 Results and Discussion………………………………………………………….....68 CHAPTER 3. LMX1B IS REQUIRED AT MULTIPLE STAGES TO BUILD EXPANSIVE SEROTONERGIC AXON ARCHITECTURES Summary……………………………………………………………………………….90 Introduction……………………………………………………………………………91 Materials and Methods………………………………………………………………94 Results………………………………………………………………………………..104 Lmx1b controls formation of ascending 5-HT projection pathways…………….104 Lmx1b controls formation of descending 5-HT projection pathways…………...107 Delayed primary pathway formation and aborted selective pathway routing in Lmx1bcKO mice………………………………………………………………….......108 Lmx1b acts temporally to control 5-HT axon selective pathways…………..…..110 Lmx1b switches function to control terminal arborization...…………………......112 Targeting of 5-HT synthesis does not impair formation of forebrain and spinal cord 5-HT arbors………………………………………………………………..…....113 Lmx1b controlled axon-related transcriptomes…………………………………...114 An ascending-specific axonal Lmx1Pet1 regulatory cascade………………...117 Lmx1b acts through Pet1 to temporally control postnatal stage-specific gene expression and forebrain arborization……………………………………………...120 Discussion…………………………………………………………………………...123 v CHAPTER 4. DISCUSSION AND FUTURE DIRECTIONS Serotonergic Heterogeneity………………………………………………………196 Lmx1b is continuously required for the formation of 5-HT axon architectures………………………………………………………………………...198 Lmx1b in intrinsic 5-HT axon growth or guidance?.....................................200 Short-range versus long-range 5-HT axon growth……………………….200 Lmx1b regulates axon growth and guidance genes …………………….202 Potential mechanisms of Lmx1bcKO axon failure to innervate…………206 From identity to axon development………………………………………….…..211 Transcription factor regulation of ascending vs descending 5-HT axon architectures…………………………………………………………………………212 Lmx1bPet1 regulatory cascade functions in 5-HT arborization…………214 Possible role for Lmx1b in regeneration?.....................................................217 Investigation of human mutations in the 5-HT GRN………………………….219 Conclusion…………………………………………………………………………...220 Bibliography………………………………………………………………………….228 vi LIST OF FIGURES CHAPTER 1 Figure 1. Enzymatic steps of 5-HT synthesis……………………………....47 Figure 2. Transcription factor regulatory network that defines serotonergic identity……………………………………………….…………..49 Figure 3. Cytoarchitecture of 5-HT neurons in the mouse brain………….51 Figure 4. 5-HT axon innervation patterns in the forebrain………………...53 Figure 5. Single labeled 5-HT neurons reveal a great diversity of 5-HT axon innervation……………………………………………………………….55 Figure 6. Embryonic time series of 5-HT axon selective routing in the mouse…………………………………………………………………………..57 CHAPTER 2 Figure 1. Schematic of BH4 de novo synthesis, salvage, and regeneration pathways and its role in 5-HT synthesis………………………………….…77 Figure 2. Isolation of ePet-EYFP and Pet-1-/-;ePet-EYFP 5-HT neurons.……79 Figure 3. Microarray analyses………………………………………………..81 Figure 4. Slc22a3 expression………………………………………………...83 Figure 5. Slc22a3 and Htr1a expression in the adult dorsal raphe……....85 Figure 6. Pet-1 control of the 5-HT neuron-type gene battery…………….87 CHAPTER 3 Figure 1. Lmx1b is required for the formation of ascending 5-HT axon projection pathways…………………………………………………………..130 Figure 1—figure supplement 1. Surrogate marking of 5-HT cell bodies and axons and Lmx1b conditional targeting……………………………….132 Figure 1—figure supplement 2. Lmx1b deficiency disrupts 5-HT axon patterns in the forebrain……………………………………………………...135 Figure 2. Lmx1b is required for the formation of descending 5-HT axon projection pathways…………………………………………………...……...137 Figure 2—figure supplement 1. Conditional targeting of Lmx1b in the descending 5-HT projection pathway……………………………………….139 vii Figure 2—figure supplement 2. Progressive deficits of 5-HT axon fibers in Lmx1b deficient spinal cord white and gray matter…………………....141 Figure 3. Initial axon outgrowth is delayed and selective pathway routing fails in Lmx1b deficient 5-HT neurons………………………….....143 Figure 4. Lmx1b is temporally required for 5-HT projection pathway formation……………………………………………………………………....145 Figure 4—figure supplement 1. Efficiency of postnatal tamoxifen inducible targeting of Lmx1b………………………………………………...147 Figure 5. Lmx1b temporally controls postnatal 5-HT terminal arborization…………………………………………………………………....149 Figure 5—figure supplement 1. P3 targeted Lmx1bicKO mice display normal 5-HT axon routing but decreased 5-HT terminal arbors………...151 Figure 6. Specific targeting of 5-HT synthesis does not alter 5-HT arborization patterns…………………………………………………….…....153 Figure 7. Ascending and descending Lmx1b regulated transcriptomes…………………………………………………………......….155 Figure 7--supplement table 1. Lmx1b-regulated axon-related genes in rostral and caudal 5-HT neurons at E17.5……………………………..….169 Figure 8. Distinct transcription factor requirements in the formation of ascending and descending 5-HT projection pathways………………..….158 Figure 8—figure supplement 1. Pet1cKO and Lmx1bcKO mice exhibit distinct axon defects in thalamus…………………………………………...160 Figure 8—figure supplement 2. DKO and Pet1-/- analyses………………162 Figure 9. An ascending specific Lmx1bPet1 cascade controls stage specific 5-HT gene expression and postnatal terminal arborization….…164 Figure 9—figure supplement 1. Lmx1bPet1 cascade acts postnatally to control 5-HT terminal arborization…………………...…………………..167 CHAPTER 4 Figure 1. Lmx1b regulates both growth and guidance genes in 5-HT neurons………………………………………………………………………...222 Figure 2. The glial sling is a transient barrier in the embryonic mouse brain........................................................................................................224 viii Figure 3. Gap43 is significantly upregulated in maturing 5-HT neurons into postnatal stages………………………………………………………………226 ix Continuously Active Transcriptional Programs are Required to Build Expansive Serotonergic Axon Architectures Abstract by LAUREN JANINE DONOVAN Neurons express unique neurotransmitters, extend axons either short or long distances, and distribute axon processes in specific patterns in order to functionally integrate into the complex network that is the brain. Neuronal diversity is ultimately shaped by gene expression diversity. Serotonin (5-HT)