DISTINCT TRANSCRIPTOMES DEFINE ROSTRAL AND CAUDAL 5HT NEURONS by CHRISTI JANE WYLIE Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Dr. Evan S. Deneris Department of Neurosciences CASE WESTERN RESERVE UNIVERSITY May, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS TABLE OF CONTENTS ....................................................................................... iii LIST OF TABLES AND FIGURES ........................................................................ v ABSTRACT ..........................................................................................................vii CHAPTER 1 INTRODUCTION ............................................................................................... 1 I. Serotonin (5-hydroxytryptamine, 5HT) ....................................................... 1 A. Discovery............................................................................................... 1 B. Processing of Serotonin ........................................................................ 1 1. Synthesis of 5HT (see Figure 1) ....................................................... 2 2. Sites of 5HT synthesis ...................................................................... 2 3. Storage and sequestration ................................................................ 3 4. Reuptake .......................................................................................... 4 5. Degradation ...................................................................................... 5 C. 5HT Receptors ...................................................................................... 5 D. 5HT Neuron Development .................................................................. 12 II. Heterogeneity of 5HT Neurons ................................................................ 13 A. Rhombomere of Origin ........................................................................ 14 B. Transcription Factor Requirements ...................................................... 14 C. Axonal Projections ............................................................................ 15 D. Cotransmission ................................................................................... 17 CHAPTER 2 PURIFICATION AND GENE EXPRESSION PROFILING OF GENETICALLY LABELED 5HT NEURONS REVEALS DISTINCT TRANSCRIPTOMES FOR ROSTRAL AND CAUDAL 5HT NEURONS ........................................................ 24 SUMMARY ......................................................................................................... 24 INTRODUCTION ................................................................................................ 25 RESULTS ........................................................................................................... 27 DISCUSSION ..................................................................................................... 41 iii MATERIALS AND METHODS ............................................................................ 47 ACKNOWLEDGEMENTS ................................................................................... 54 CHAPTER 3 CONDITIONAL DELETION OF NEUROPILIN 2 IN 5HT NEURONS ................. 73 SUMMARY ......................................................................................................... 73 INTRODUCTION ................................................................................................ 74 RESULTS ........................................................................................................... 79 MATERIALS AND METHODS ............................................................................ 83 CHAPTER 4 DISCUSSION ................................................................................................ 117 I. Utility of ePet-EYFP transgenic mice ................................................... 117 II. Development of methodology to purify embryonic rostral and caudal 5HT neurons. ....................................................................................... 118 III. Gene array profiling of rostral and caudal 5HT neurons. .................. 118 A. Identification of developing 5HT neuron transcriptomes ............... 130 B. Genes with enriched expression in 5HT neurons .......................... 130 C. Unique transcriptional profiles of rostral and caudal 5HT neurons 131 1. Homeodomain transcription factors enriched in rostral 5HT neurons ......................................................................................... 132 2. Hox genes mark caudal 5HT neurons ........................................... 133 3. Imprinted gene expression in 5HT neurons .................................. 134 Appendix: Supplemental Tables ..................................................................... 133 LITERATURE CITED ....................................................................................... 277 iv TABLE LIST CHAPTER 2 TABLE 1: Top 50 genes with enriched expression in R+C+ 62 5HT neurons (cluster I). TABLE 2: Gene ontology and pathway enrichment in R+C+ 65 5HT neurons. TABLE 3: Top genes with enriched expression in R+ (cluster II) 66 and R+R- (cluster III). TABLE 4: Top genes with enriched expression in C+ (cluster IV) 69 and C+C- (cluster V). TABLE 5: Gene ontology and pathway enrichment in R+ 72 versus C+ 5HT neurons. FIGURE LIST CHAPTER 1 FIGURE 1: 5HT biosynthesis and breakdown. 20 FIGURE 2: Specification and differentiation of 5HT neurons. 22 CHAPTER 2 FIGURE 1: ePet-EYFP expression marks rostral and caudal 5HT 56 neurons in the embryonic ventral hindbrain. FIGURE 2: Purification and expression profiling of rostral and 58 caudal 5HT neurons. FIGURE 3: Unsupervised hierarchical clustering of serotonergic 60 gene expression. v FIGURE 4: Verification of 5HT neuron-enriched gene 63 expression. FIGURE 5: Verification of rostral 5HT neuron-enriched HD gene 67 expression. FIGURE 6: Verification of Hox gene expression in caudal 5HT 70 neurons CHAPTER 3 FIGURE 1: Genomic organization and alternative splice variants 89 of NRP2 in mouse. FIGURE 2: Classes of semaphorins. 91 FIGURE 3: Semaphorins and their receptors. 93 FIGURE 4: Neuropilin, Semaphorin, and Plexin gene expression 95 in E12.5 5HT neurons and surrounding neural tube as detected by Affymetrix gene arrays. FIGURE 5: Confirmation of NRP2 protein enrichment in rostral 97 5HT neurons. FIGURE 6: Transgenics and conditional deletion of NRP2 in 99 5HT neurons. FIGURE 7: Conditional deletion of NRP2 in 5HT neurons 101 (5HTΔNRP2) results in a loss of 5HT immunoreactive axons and cell bodies. FIGURE 8: High magnification of sections in Figure 7 to detail 103 the profound loss of 5HT axonal fibers in the absence of NRP2. vi FIGURE 9: Loss of NRP2 results in a severe disruption of 5HT 105 neurons at the midline. FIGURE 10: High magnification of stereo confocal images in 107 Figure 9 to illustrate dystrophic growth cone endings in the absence of NRP2. FIGURE 11: 5HT and the 5HT metabolite 5HIAA are decreased 109 in the brains of adult 5HTΔNRP2 mice. FIGURE 12: 5HT immunoreactive axons are decreased in the 111 cortex of 5HTΔNRP2 mice. FIGURE 13: 5HT immunoreactive axonal fibers are decreased 113 in the dentate gyrus of adult 5HTΔNRP2 mice. FIGURE 14: Ingenuity Pathway Analysis (IPA) of Neuropilin 115 associated genes expressed in rostral 5HT neurons. CHAPTER 4 FIGURE 1: Dissociated ePet-EYFP neurons grown in culture. 129 FIGURE 2: Verification of enriched imprinted gene expression 131 in 5HT neurons. Supplemental Datasets (available as annotated excel files at www.jneurosci.org as supplemental material) Dataset 1: An annotated master list of expression signals and Present/Absent calls for 12 arrays. Dataset 2: A database of E12.5 5HT neuron expressed genes (called Present on 3 biological replicates at a minimum threshold intensity of the lowest known 5HT gene) containing 12,559 probe sets corresponding to 8,186 genes, 866 RIKEN cDNAs, and 435 ESTs. vii Dataset 3: R+C+ gene ontology and pathway enrichment. Dataset 4: R+ versus C+ gene ontology and pathway enrichment. Supplemental Tables Supplemental Table S1. 133 Microarray detection of Tph2-like genes identified by the Allen Institute for Brain Science. We filtered the Allen data set for genes with a Tph2-like expression pattern in adult brain and identified 248 genes. Expression of 155 of these Tph2- like genes (with 95 duplicate probe sets) was detected in our R+ and C+ arrays. Supplemental Tables S2-S7 – Expressed Genes 5HT neuron gene expression according to the molecular categories of DNA binding proteins, axon pathfinding/cell adhesion, ion channels, solute carriers, G- protein coupled receptors, and kinases. A combination of Gene Ontology (GO) nomenclature, Ingenuity Pathway Analysis (IPA) software, web-based databases, and Microsoft Access was used for filtering Dataset 3. Each table shows