AXON GUIDANCE IN THE MOUSE OLFACTORY SYSTEM A Dissertation Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Eric Owen Williams February 2010 © 2010 Eric Owen Williams AXON GUIDANCE IN THE MOUSE OLFACTORY SYSTEM Eric Owen Williams, Ph. D. Cornell University 2009 Within the mouse olfactory system, 1200 different types of olfactory sensory neurons use molecular cues to appropriately target stereotyped locations within the olfactory bulb. Recent studies reveal that some of these molecular cues are regulated by olfactory receptor mediated neuronal activity. Within this thesis we develop a hypothesis drive microarray based method to identify novel axon guidance molecules expressed by the target of this process, the olfactory bulb. This method is designed to identify genes that are differentially expressed within the developing olfactory bulb during the developmental stages when olfactory sensory neurons are first reaching and converging within the olfactory bulb. We identify an entire gene family, the δ protocadherins, which fulfill these characteristics and are expressed within subsets of olfactory sensory neurons in the olfactory epithelium. Furthermore, we find that many of the δ protocadherins are regulated by neuronal activity within the olfactory system and hippocampus. Biochemical analysis of the δ protocadherins reveals novel heterophilic interactions between the extracellular domains of some family members, as well as heterophilic interactions between Pcdh10 and some classical cadherins. Furthermore, we identify the ability of the cytoplasmic domains of these proteins to translocate to the nucleus. Ectopic expression through transgenic analysis of one family member, Pcdh10, is sufficient to disrupt proper axon targeting of SR1 olfactory neurons. Based on this data we propose that the δ protocadherins are excellent axon guidance candidate molecules in the mouse olfactory system. BIOGRAPHICAL SKETCH Eric Williams was born in Manchester, New Hampshire on January 21st, 1977. He grew up in Salem, New Hampshire where he attended Salem High School. In 1995, he attended Cornell University where he earned his Bachelor’s degree in genetics and developmental biology in 1999. In the summer of 1999, he worked in Dr. Ruth Collin’s lab, studying secretory pathways using the model system, sacchoromyces cerevisiae. Later that year he went on to work in the transcription division of Cereon Genomics in Boston, Massachusetts. In late 1999, he went to work for Dr. Jing Zhou using mouse models to study polycystic kidney disease. In, 2001 he began work with Dr. Davide Trotti studying glutamate uptake in mouse models of amyotrophic lateral sclerosis. In, 2002, Eric married Cynthia Barrie Carlson Williams in Lambertville, New Jersey. In 2003, he began his PhD, studying axon guidance in the mouse olfactory system. His thesis was completed in February of 2010. Eric plans to move back to Boston, where he will work with Dr. Leonard Guarente, studying the therapeutic effects of Sirtuins on neurodegenerative disorders. iii I dedicate this thesis to my wife Cynthia for all her love and support iv ACKNOWLEDGMENTS Many thanks to Heather Sickles and Christine Kobre for their many technical contributions to the experiments outlined in this thesis. In addition, Leslie Lee cloned and transfected many of the GFP tagged cytoplasmic domains of the δ protocadherins. Kristen Blauvelt performed the initial co-immunoprecipitation experiments with the extracellular domains of protocadherins. Allison Dooley first characterized the differential expression of Pcdh10 in the olfactory system. Patrick Sturgis identified Kelchlike8 interaction with Pcdh10. Special thanks for Ashok Gopinath and Steve Rodriguez for helpful discussions and technical advice. This work was funded through the neurobiology and behavior neuroethology as well as the cell and molecular training grant. Finally, special thanks to David Lin for excellent mentorship, intellectual discussions, experimental design, technical expertise, and contributions to experiment execution. v TABLE OF CONTENTS iii Biographical Sketch iv Dedication v Acknowledgments vi Table of Contents vii List of Figures xii List of Tables xiii List of Abbreviations xvii List of Symbols pg 1 Chapter 1 Introduction: Axon Guidance in the Mouse Olfactory System pg 43 Chapter 2 The role of the olfactory bulb in olfactory sensory neuron axon guidance: Identifying putative axon guidance molecules pg 73 Chapter 3 The δ protocadherins, a family of putative axon guidance molecules in the mouse olfactory system pg 115 Chapter 4 Protocadherin 10 is an axon guidance molecule in the olfactory system that is regulated by PKA and CamKII pg 143 Chapter 5 Future Directions pg 157 Appendix vi LIST OF FIGURES Chapter 1 Figure 1 pg 4 Organization of the mouse olfactory system Figure 2 pg 5 Organization of the mouse olfactory system Figure 3 pg 6 Olfactory sensory neuron convergence Figure 4 pg 8 Lactosamine expression in the olfactory bulb Figure 5 pg 9 Receptor swap experiments Figure 6 pg 11 Contextual model of axon guidance Figure 7 pg 14 Olfactory receptor signaling Figure 8 pg 15 Olfactory receptor signaling is important for axon convergence Figure 9 pg 16 Gs and Golf are expressed at different developmental stages of olfactory neuron maturation Figure 10 pg 18 Posterior positioning of I7 glomeruli is regulated through cAMP Figure 11 pg 19 Model of local olfactory sensory neuron axon guidance Figure 12 pg 20 P2 olfactory sensory neurons lose stereotyped convergence without an olfactory bulb Figure 13 pg 23 Radial glia are required for olfactory sensory neuron innervation Figure 14 pg 24 Presorting in the axon bundles Figure 15 pg 25 Model of olfactory sensory neuron presorting Figure 16 pg 26 IGF signaling in the olfactory sensory neuron axon guidance Figure 17 pg 27 Slit-1 is required for proper convergence of dorsal olfactory neurons Chapter2 Figure 1 pg 45 Developmental time course of OSN convergence vii Figure 2 pg 46 Method to identify differentially expressed genes within external plexiform layer of the olfactory bulb Figure 3 pg 51 MCAM is differentially expressed in the olfactory bulb Figure 4 pg 52 CTGF is differentially expressed in the olfactory bulb Figure 5 pg 53 Nasal Ablation Paradigm Figure 6 pg 55 Zcsl3 is differentially expressed in the olfactory bulb Figure 7 pg 57 Notch receptors are not expressed in the external plexiform layer Figure 8 pg 58 Jagged1 is differentially expressed in the olfactory bulb Figure 9 pg 59 Neat2 is differentially expressed in the olfactory bulb Figure 10 pg 60 Neat2 is coexpressed with SC35 Figure 11 pg 61 Neat2 common sequence contains a microRNA sequence Figure 12 pg 62 Blast results for common sequence Chapter 3 Figure 1 pg 73 Phylogenetic analysis of nonclustered protocadherins using intracellular domains Figure 2 pg 74 Cytoplasmic homology of delta protocadherins Figure 3 pg 74 Phylogenetic analysis of nonclustered protocadherins using extracellular domain Figure 4 pg 76 Pcdh1 expression in the olfactory system Figure 5 pg 77 Pcdh7 expression in the olfactory system Figure 6 pg 79 Pcdh8 expression in the olfactory system Figure 7 pg 80 Pcdh9 expression in the olfactory system Figure 8 pg 82 Pcdh11x expression in the olfactory system Figure 9 pg 83 Pcdh17 expression in the olfactory system Figure 10 pg 84 Pcdh19 expression in the olfactory system viii Figure 11 pg 85 δ Protocadherins are expressed in mature olfactory sensory neurons Figure 12 pg 88 Double fluorescent in situ hybridization of Pcdhs in E17.5 olfactory epithelium Figure 13 pg 89 Double fluorescent in situ Figure 14 pg 90 Coexpression of olfactory receptors and delta protocadherins Figure 15 pg 91 Double fluorescent in situ hybridization in E17.5 olfactory epithelium Figure 16 pg 92 Heterophilic interactions of protocadherins and cadherins Figure 17 pg 92 Pcdh7 and Pcdh10 are expressed in complementary patterns Figure 18 pg 93 Putative protocadherin NLS Figure 19 pg 94 Pcdh cytoplasmic GFP fusions are transported to the nucleus Figure 20 pg 95 Cytoplasmic Pcdh10 is transported to the nucleus in vivo Figure 21 pg 96 Pcdh10 NLS is not necessary or sufficient for nuclear localization Figure 22 pg 97 Nasal Ablation Figure 23 pg 100 Structure of EC1 cadherin domains Figure 24 pg 101 Cytoplasmic Pcdh10 interacts with Kelch like 8 Chapter 4 Figure 1 pg 117 Protocadherin 10 expression in the olfactory system Figure 2 pg 118 Immunohistochemical analysis of Pcdh10 in the olfactory system Figure 3 pg 119 Coexpression of Pcdh10 and olfactory receptors Figure 4 pg 119 Pcdh10 Trangene Figure 5 pg 120 Pcdh10 trangene expression at P3 Figure 6 pg 120 Pcdh10 transgene western blot ix Figure 7 pg 121 Pcdh10 trangene expression in adult Figure 8 pg 121 Olfactory marker in Pcdh10 transgene Figure 9 pg 122 Whole mount GFP in Pcdh10 transgene Figure 10 pg 122 Pcdh10 trangene expression at E17 Figure 11 pg 122 Pcdh10 transgene coexpression with olfactory receptors Figure 12 pg 123 SR1 olfactory sensory neurons are misrouted during early convergence in the Pcdh10 transgenic animals Figure13 pg 123 SR2 olfactory sensory neurons are misrouted at P0 Figure 14 pg 124 Quantification of interglomerular distance in Pcdh10 transgene Figure 15 pg 125 Coexpression of Pcdh10 and activity regulated olfactory axon guidance molecules in E17.5 olfactory epithelium Figure 16 pg 126 Model of olfactory axon guidance molecule regulation by activity Figure 17 pg 127 Nasal