Dissecting mechanisms controlling neural network formation in Drosophila melanogaster Hong Long Department of Neurology and Neurosurgery McGill University Montreal, Quebec, Canada August 2009 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the Ph.D. degree in Neuroscience © Hong Long, 2009 1 Library and Archives Bibliothèque et Canada Archives Canada Published Heritage Direction du Branch Patrimoine de l’édition 395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada Your file Votre référence ISBN: 978-0-494-66458-2 Our file Notre référence ISBN: 978-0-494-66458-2 NOTICE: AVIS: The author has granted a non- L’auteur a accordé une licence non exclusive exclusive license allowing Library and permettant à la Bibliothèque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par télécommunication ou par l’Internet, prêter, telecommunication or on the Internet, distribuer et vendre des thèses partout dans le loan, distribute and sell theses monde, à des fins commerciales ou autres, sur worldwide, for commercial or non- support microforme, papier, électronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats. 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Table of contents List of figures and tables 8 List of abbreviations used 10 Abstract 21 Résumé 23 Acknowledgements 25 Contribution of co-authors 26 Original contributions to knowledge 27 Chapter 1. Introduction 28 1.1 Axonal guidance, targeting, and tiling 28 1.1.1 Growth cone 28 1.1.2 Guidepost cell, Pioneer axon, and the labelled pathway 29 1.1.3 Layer-specific targeting 31 1.1.4 Topographic Mapping 32 1.1.5 Axon tiling and self-avoidance 33 1.1.6 Drosophila visual system 35 1.1.7 Review of molecules and signalling pathways involved in R-cell axon guidance, targeting, and tiling. 39 1.1.7.1 Nuclear proteins 39 1.1.7.1.1 Brakeless 39 2 1.1.7.1.2 Runt 41 1.1.7.1.3 Nuclear factors YC 42 1.1.7.1.4 Sequoia 44 1.1.7.2 Cytoplasmic signalling proteins 46 1.1.7.2.1 Dreadlocks 46 1.1.7.2.2 Misshapen 47 1.1.7.2.3 Bifocal 49 1.1.7.2.4 p21-activated kinase 51 1.1.7.2.5 Trio 53 1.1.7.2.6 Rho-GTPase Rac 57 1.1.7.3 Cell surface proteins and secreted proteins 58 1.1.7.3.1 Insulin Receptor 58 1.1.7.3.2 N-Cadherin 60 1.1.7.3.3 Flamingo 68 1.1.7.3.4 Leukocyte common antigen-related RPTP 71 1.1.7.3.5 PTP69D 74 1.1.7.3.6 Capricious 76 1.1.7.3.7 Golden Goal 78 1.1.7.3.8 Anaplastic lymphoma kinase and Jelly Belly 80 1.1.7.3.9 Off-track and Semaphorin-1a 84 1.1.7.3.10 Dfrizzled2 and DWnt4 87 1.1.7.3.11 Transforming Growth Factor β /Activin 89 1.1.8 Review of genes involved in glia and target field development 91 3 1.1.8.1 Genes involved in glia development, migration, and function 91 1.1.8.1.1 Decapentaplegic, Hedgehog, and Gilgamesh 91 1.1.8.1.2 Wingless, Dpp, and Glial cell missing 93 1.1.8.1.3 Non-stop, SAGA complex and Histone H2B 94 1.1.8.1.4 JAB1/CSN5 95 1.1.8.2 Genes involved in the lamina-lobula boundary formation 96 1.1.8.2.1 Robo and Slit 96 1.1.8.2.2 Egghead 98 1.1.8.3 Genes involved in target neuron development 99 1.2 Dendrite patterning and function 101 1.2.1 Dendrite development, arborisation, and patterning 101 1.2.1.1 Dendrite outgrowth 102 1.2.1.2 Dendrite guidance and targeting 104 1.2.1.3 Dendrite branching and spine formation 106 1.2.1.4 Dendrite heteroneuronal tiling 108 1.2.1.5 Dendrite isoneuronal self-avoidance 110 1.2.1.6 Dendrite maintenance 112 1.2.1.7 Dendrite remodelling 115 1.2.2 Drosophila PNS dendritic arborisation (da) neurons 117 1.3 An overview of my studies and their contributions to our knowledge of the 121 related fields 1.3.1 My work in axon guidance and tiling in the fly visual system 121 1.3.2 My analyses in dendritic patterning in the fly da neurons 124 4 Chapter 2. De Novo GMP Synthesis Is Required for Axon Guidance in Drosophila 126 Summary 128 Introduction 129 Materials and methods 132 Genetics 132 Molecular Biology 132 Histology and Immunohistochemistry 133 Results 134 Figures 144 Table 160 Discussion 161 Acknowledgements 165 References 166 Chapter 3. Dendrite branching and self-avoidance are controlled by Turtle, a conserved IgSF protein in Drosophila 171 Summary 173 Introduction 174 Materials and methods 177 Fly stocks and genetics 177 Imaging and quantification 178 Immunohistochemistry 178 5 Results 180 Figures 189 Table 213 Discussion 214 Acknowledgements 221 References 222 Chapter 4. Characterizing gene Dnrk in Drosophila 227 Summary 229 Introduction 231 Materials and methods 243 Genetics 243 Molecular biology 243 Histology and Immunohistochemistry 244 Results 246 Figures 259 Table 278 Discussion 279 Acknowledgements 282 References 283 Chapter 5. General discussion 296 5.1 The involvement of de novo GMP synthesis in R-cell axon guidance 298 6 5.1.1 de novo GMP synthesis versus the salvage synthesis pathway 298 5.1.2 How GMP level is controlled within the cell? 299 5.1.3 Signaling events of GMP 301 5.1.4 bur also functions in motor axon guidance 302 5.1.5 Defasciculation, a requirement for layer-specific R1-R6 targeting? 303 5.1.6 Is the de novo GMP synthesis required for neural network formation in mammals? 304 5.2 The function of Turtle in dendrite branching and patterning 304 5.2.1 Domain requirement and the possible homotypic function of Tutl 304 5.2.2 The dendrite patterning phenotype in class I neuron 306 5.2.3 Functional relationship between tutl and other known genes in dendrite development 307 5.2.4 Potential role of tutl in the VNC? 307 5.2.5 Tutl functions in R7 axon tiling in the fly visual system 308 5.2.6 The function of Tutl in fly behavior 308 5.2.7 The function of the mammalian homolog of Tutl 309 5.3 The function of Dnrk in fly 310 5.3.1 The potential redundancy between Dnrk and Dror 310 5.3.2 The potential function of Dnrk in the VNC 311 5.3.3 Genetic interaction between Dnrk and DWnt5 312 References 314 7 List of figures and tables Chapter 2, figure 1: Molecular characterization of the bur gene 144 Chapter 2, figure 2: Mutations in the bur gene disrupted R-cell axon pathfinding 146 Chapter 2, figure 3: bur is not required for lamina-specific termination of R2-R5… 148 Chapter 2, figure 4: R-cell differentiation and patterning remained normal in bur… 150 Chapter 2, figure 5: Rescue of the R-cell pathfinding phenotype by expressing… 152 Chapter 2, figure 6: Mutations in the ras gene caused a bur-like phenotype in R… 154 Chapter 2, figure 7: Depleting guanine from the fly food did not affect R-cell axon 156 Chapter 2, figure 8: Reducing the dosage of bur enhanced the Rac phenotype 158 Chapter 2, table 1: Transgenic rescue of R-cell axonal hyperfasciculation… 160 Chapter 3, figure 1: Tutl protein structure, tutl alleles, and Tutl expression in da… 189 Chapter 3, figure 2: tutl is required to restrain dendrite branching in class I da… 191 Chapter 3, figure 3: Class II and class III DA neurons are unaffected in tutl23… 193 Chapter 3, figure 4: tutl is required for dendrite self-avoidance in class IV da… 195 Chapter 3, figure 5: tutl mutants exhibit normal dendritic tiling among class IV… 197 Chapter 3, figure 6: Overexpression of Tutl inhibits dendrite branching in class… 199 Chapter 3, figure 7: The cytoplasmic tail of Tutl is dispensable for function in… 201 Chapter 3, figure S1: Additional examples of dendrite branching and self-… 203 Chapter 3, figure S2: Quantification of the effects of tutl mutations on additional… 205 Chapter 3, figure S3: Overexpression of Tutl is not sufficient to induce dendrite… 207 Chapter 3, figure S4: Tutl expression in mutants of ab, ss, kn, and cut 209 8 Chapter 3, figure S5: Expression of Tutl and TutlΔcyto in class I da neurons of tutl… 211 Chapter 3, table S1: Genetic Interaction Experiments for tutl and trc 213 Chapter 4, figure 1: Predicted domain structure of Ror family proteins 259 Chapter 4, figure 2: Molecular characterization of the Dnrk gene 261 Chapter 4, figure 3: Mutation in the Dnrk gene disrupted R-cell axon guidance… 263 Chapter 4, figure 4: The adult R-cell axon projection pattern in Dnrk mutant was… 266 Chapter 4, figure 5: Dnrk mutant did not display axon guidance defect in the VNC… 268 Chapter 4, figure 6: Dendrite development and tiling pattern in wild type and… 270 Chapter 4, figure 7: Potential functional redundancy between Dnrk and Dror in… 272 Chapter 4, figure 8: Overexpression of UASDnrk in the eye and in the da… 274 Chapter 4, figure 9: The overexpression of UASDnrk in the wing could change… 276 Chapter 4, table 1: Modifier screen on the loss-of-cross-vein phenotype