AXON GUIDANCE GENES ARE ESSENTIAL IN THE ADULT NERVOUS SYSTEM by Aarya Vaikakkara Chithran Bachelor of Technology (B. Tech) in Biotechnology, VIT University, 2013 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2016 © Aarya Vaikakkara Chithran, 2016 ii Abstract Axon guidance cues are extracellular signals that direct the growth and steering of neuronal growth cones. Both attractive and repulsive cues are required to guide developing axons to their targets. Nonetheless, after axons have reached their targets and established functional circuits, many neurons continue to express these developmental cues. The expression of these genes in the adult indicates that there are likely additional roles for guidance cues beyond the initial phase of neuronal process outgrowth, growth cone navigation, and target innervation. The central goal of my work is to determine the functions of these cues in the mature nervous system. I hypothesize that axon guidance genes expressed in the adult nervous system have functional roles in the maintenance of neural circuits. Work in the past few decades has led to the discovery of numerous axon guidance genes and identified their functions during development. This study is the first to perform an RNA-mediated interference (RNAi) screen for axon guidance genes that have functional roles in the mature nervous system. Axon guidance genes expressed in the adult Drosophila melanogaster nervous system were identified using bio-informatics tools. In Drosophila, more than 96% of embryonic cues continue to be expressed in the adult. The axon guidance genes were knocked down in adult neurons using RNAi via spatial and temporal control of GAL4-UAS system. I have identified 15 axon guidance genes that are essential for survival and normal behavior (climbing, mobility, activity/rest cycle) in adult Drosophila. The results suggests that axon guidance genes are functional in the adult nervous system and may be involved in the maintenance of neural circuits underlying these phenotypes. Further studies on circuit morphology are required to understand better how axon guidance genes contribute to the maintenance of neuronal structure in adult brains. iii Preface The experiments presented in this thesis were designed together by my supervisor Dr. Tim O’ Connor, my co-supervisor Dr. Douglas W. Allan, and myself. I conducted all aspects of the experiment, such as fly genetics, survival and behavioural assays, expression analyses and performed all data analyses, under the guidance of Drs. O’ Connor and Allan. This work was conducted at the Life Sciences Institute at the University of British Columbia, Vancouver. Some experimental findings presented in this thesis were submitted in abstract form, oral/poster presentation at 2014 VanFly Meeting in Vancouver (British Columbia), 2015 UBC Graduate Program in Cell and Developmental Biology Retreat at Loon Lake (British Columbia), and 2016 Canadian Developmental Biology Meeting at Banff (Alberta). I was the lead author of the abstracts and posters and I presented the work at the mentioned research meetings. Studies presented in this thesis have not been submitted for publication at the time of thesis submission. iv Table of Contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ............................................................................................................................... viii List of Figures ............................................................................................................................... ix List of Abbreviations .................................................................................................................... xii Acknowledgements ..................................................................................................................... xix Chapter 1: Introduction .................................................................................................................. 1 Axon Guidance .................................................................................................................. 1 Role of axon guidance cues during the development of nervous system .......................... 3 Netrins .................................................................................................................... 4 Slits ......................................................................................................................... 5 Semaphorins ........................................................................................................... 5 Ephrins .................................................................................................................... 6 Morphogens ............................................................................................................ 7 v Cell adhesion molecules ......................................................................................... 8 Cytoskeletal-associated proteins ........................................................................... 10 Others ................................................................................................................... 11 Role of axon guidance cues beyond development ............................................................ 12 CNS regeneration ................................................................................................. 12 Regulation of adult synaptic plasticity .................................................................. 12 Drosophila melanogaster as model organism ................................................................... 13 GAL4-UAS System............................................................................................... 14 RNA-mediated Interference ................................................................................. 15 Hypothesis ............................................................................................................ 17 Chapter 2: Materials and Methods ............................................................................................... 18 Identifying axon guidance genes ..................................................................................... 18 Fly genetics ...................................................................................................................... 18 Fly stocks ......................................................................................................................... 19 GAL4 driver lines ................................................................................................ 19 UAS-RNAi lines .................................................................................................. 20 Protein trap lines .................................................................................................. 24 Control lines ......................................................................................................... 24 vi Survival assay .................................................................................................................. 24 Climbing assay ................................................................................................................. 25 Drosophila Activity Monitor System ................................................................................ 26 Video-assisted movement tracking ................................................................................... 26 Immunohistochemistry ..................................................................................................... 27 Chapter 3: Results ........................................................................................................................ 29 Axon guidance related genes are expressed in the adult Drosophila melanogaster nervous system ………………………………………………………………………………..… 29 Axon guidance genes are essential for survival in adults ………………………...……. 32 Axon guidance genes control adult climbing behavior in Drosophila ………………… 44 Axon guidance genes are required for normal adult activity ……………………………. 46 Video-assisted movement tracking ………….………………………………………….. 63 Expression patterns of axon guidance proteins in the adult CNS ……………………….. 66 Chapter 4: Discussion .................................................................................................................. 69 Expression of developmental axon guidance genes in adulthood has functional relevance ........................................................................................................................................... 69 Axon guidance genes are required for survival …………………………………………. 70 Loss of axon guidance genes causes aberrant mobility ……….………………………… 71 vii Axon guidance genes may be acting on motor neurons ………………………………… 73 Future work and directions ……………………………………………………………… 73 Conclusion ……………………………………………………………………………… 73 Bibliography …………………………………………………………………………….……… 75 Appendix ………………………………………………………………………………..……… 93 viii List of Tables Table 2.1 List of UAS-RNAi lines used in the study …………………………………………… 20 Table 2.2 List of protein trap lines used in the study ……………………………………………. 24 Table 3.1 Expression profile categories of axon guidance related genes in the adult Drosophila melanogaster nervous system ………………………………………………………………….. 29 Table 3.2 Categories of axon guidance genes based on Molecular function GO terms ……….. 31 Table 3.3 Summary of survival analysis of Gene Switch screen …………………………….... 33 Table 3.4 Summary of survival analysis of TARGET screen ……….………………………… 33 ix List of Figures Figure 1.1 Diverse mechanisms involved in axon guidance ………………………………...…… 2 Figure 1.2 The four classical families of axon guidance cues and their receptors ………………. 4 Figure 1.3 GAL4-UAS, TARGET and Gene Switch systems …………………………………... 15 Figure 1.4 Basic in-vivo F1 RNAi screen ………………………………………………………. 16 Figure 2.1 Timeline for survival analysis experiment …………………………………………... 25 Figure 2.2 Experimental setup for video-assisted movement tracking ………………………….. 27 Figure 3.1 Comparison study of axon guidance related genes expressed in Drosophila melanogaster in the embryo and adult nervous system …………………………………………. 30 Figure 3.2 Survival analysis curves from the Gene Switch screen ……………………………… 34 Figure 3.2 Survival analysis curves from the TARGET screen ………………………………… 37 Figure 3.4 Effect of knockdown of axon guidance genes (using elavGS) on adult climbing behavior …………………………………………………………………………………………………... 45 Figure 3.5 Effect of knockdown of axon guidance genes (using OK371-GAL4) on adult climbing behavior ………………………………………………………………………………………… 46 Figure 3.6 Activity plots for day 9 old flies with injection stock control BDSC #36303 ………. 48 Figure 3.7 Inactive time plots for day 9 old flies with injection stock control BDSC #36303 …. 49 Figure 3.8 Activity indices for day 9 old flies with injection stock control BDSC #36303 …….. 51 x Figure 3.9 Activity plots for day 14 old flies with injection stock control BDSC #36303 ……... 52 Figure 3.10 Inactive time plots for day 14 old flies with injection stock control BDSC #36303.. 53 Figure 3.11 Activity indices for day 14 old flies with injection stock control BDSC #36303 ….. 55 Figure 3.12 Activity plots for day 14 old flies with injection stock control VDRC #60000 …… 56 Figure 3.13 Inactive time plots for day 14 old flies with injection stock control VDRC #60000.. 58 Figure 3.14 Activity indices for day 14 old flies with injection stock control VDRC #60000 …. 59 Figure 3.15 Activity plots for day 14 old flies with injection stock control BDSC #36304 ……. 60 Figure 3.16 Inactive time plots for day 14 old flies with injection stock control BDSC #36304.. 61 Figure 3.17 Activity indices for day 14 old flies with injection stock control BDSC #36304 ….. 63 Figure 3.18 Effect of knockdown on the average velocity in a video-assisted movement tracking experiment ……………………………………………………………………………………… 64 Figure 3.19 Effect of knockdown on the distance moved in a video-assisted movement tracking experiment ……………………………………………………………………………………… 64 Figure 3.20 Heat maps of the positions of day 9 old flies from a video-assisted movement tracking experiment ……………………………………………………………………………………… 65 Figure 3.21 Heat maps of the positions of day 14 old flies from a video-assisted movement tracking experiment ……………………………………………………………………………………… 66 Figure 3.22 Sema-1a expression pattern in the adult CNS ……………………………………… 67 Figure 3.23 Fas3 expression pattern in the adult CNS ………………………………………….. 68 Figure 3.24 Comparison of Fas3 expression to other GAL4 driver line expressions in the adult CNS …………………………………………………………………………………………….. 68 xi Figure A.1 Survival analysis curves for male flies from the Gene Switch screen ………...……. 93 Figure A.2 Survival analysis curves for female flies from the Gene Switch screen ……………. 96 Figure A.3 Survival analysis curves for male flies from the TARGET screen …………………. 99 Figure A.4 Survival analysis curves for female flies from the TARGET screen ………………. 102 xii List of Abbreviations +TIPs Plus-End-Tracking Proteins °C Degree Celsius Abl Abelson tyrosine kinase ACF7 Actin crosslinking family 7 Act-β Activin β Alk Anaplastic lymphoma kinase ANOVA Analysis Of Variance APC Adenomatous polyposis coli Babo Baboon BDSC Bloomington Drosophila Stock Center Beat Beaten path Bif Bifocal BMPs Bone morphogenetic proteins C. elegans Caenorhabditis elegans CadN N-cadherin CAMs Cell adhesion molecules xiii Caps Capricious Chic Chickadee CKAP5 Cytoskeleton associated protein 5 cm centimeter CNS Central nervous system DAM Drosophila Activity Monitor Daw Dawdle DCC Deleted in colorectal cancer Dg Dystroglycan Dhc64C Dynein heavy chain 64C Dlp Dally-like Drl Derailed dsRNA double stranded RNA Dys Dystrophin ECM Extracellular matrix elavGS elav-Gene Switch Ena Enabled Eph Ephrin EVL Enabled/Vasodilator-stimulated phosphoprotein- like xiv F-actin Filamentous-actin Fas I (Fas 1) Fasciclin 1 Fas II (Fas 2) Fasciclin 2 Fas III (Fas 3) Fasciclin 3 Fmi Flamingo Fra Frazzled GAL80ts GAL80 temperature sensitive GAPs GTPase activating proteins GDP Guanosine diphosphate GEFs Guanine nucleotide exchange factors GFP Green Fluorescent Protein GO Gene Ontology GPI Glycosylphophatidylinositol Gr Gustatory receptor GS Gene Switch GTP Guanosine triphoshpate GTPases Guanosine triphosphatases Hh Hedgehog Hip1 Huntingtin interacting protein 1 xv hnRNP Heterogeneous nuclear ribonucleoprotein HSPGs Heparan sulphate proteoglycans Ig Immunoglobulin ISN Inter segmental nerve Jeb Jelly belly Lar Leucocyte antigen related like Lis1 Lissencephaly 1 MAPs Microtubule-associated proteins MATLAB Matrix laboratory Mena Mouse enabled homolog Mew Multiple edematous wings Mical Molecule interacting with CasL mm millimeter modENCODE Model organism Encyclopedia Of DNA Elements mRNA Messenger RNA Msps Mini spindles MT Microtubules Mys Myospheroid NMJ Neuromuscular junction xvi Nrg Neuroglian Nrt Neurotactin PBS Phosphate buffered saline PBT PBS + 0.01% Triton-X PBTN PBT + 5% Normal donkey serum PFA Paraformaldehyde Plex Plexin PNS Peripheral nervous system ppk Pickpocket Put Punt RNA Ribonucleic acid RNAi RNA-mediated interference RNA-seq RNA sequencing Robo Roundabout RT Room temperature RU486 Mifepristone Sdc Syndecan sec Second Sema Semaphorin xvii Shh Sonic hedgehog Shot Shortstop shRNA Small hairpin RNA Sli Slit Sm Smooth Smo Smoothened SN Segmental nerve Stan Starry night TARGET Temporal And Regional Gene Expression Targeting TGFβ Transforming Growth Factor β Tlr Tolloid-related Tok Tolkin TRiP Transgenic RNAi Project Tutl Turtle UAS Upstream Activating Sequence Uzip Unzipped VASP Vasodilator-stimulated phosphoprotein VDRC Vienna Drosophila Resource Center Wit Wishful thinking xviii WNK With No lysine Kinases βSpec β-Spectrin xix Acknowledgements I would like to express my sincerest gratitude to many people who have provided me with the support without which the completion of this thesis would not have been possible. First of all, I would like to thank my supervisor, Dr. Tim O’Connor, for his unwavering patience, encouragement and guidance. Your mentorship has truly enabled me to realize my own abilities and passion for science. I would like to thank my co-supervisor, Dr. Douglas Allan, for his ongoing support and encouragement. I am grateful for your prompt replies and flexibility in accommodating unpredictable schedules intricately linked to co-supervision. Thank you to my committee members, Dr. Micheal Gordon and Dr. Shernaz Bamji, for their thoughtful insight and constructive criticism in helping to shape my project and review this thesis and also for putting up with my tight schedule. I would like to show my deepest gratitude to all past and present members of the O’Connor lab and Allan lab for their help throughout my time here. Without their help, I surely would not have been equipped with skills and knowledge to complete my research. I must specially thank the Gordon lab, Auld lab and Tanentzapf lab for the various fly strains they provided. Many thanks goes to Dr. Micheal Gordon and Dr. Eric Accili for letting me use their Drosophila Activity Monitor systems. I would also like to specially thank Jeff Stafford (former graduate student from the Gordon lab) for his guidance in using MATLAB and R software for data analyses. I am grateful to MITACS for awarding me the Globalink Graduate Fellowship during 2013-2015. And most importantly, I would like to show my deepest appreciation to all my family and friends, without whose support I would not have made it this far. 1 Chapter 1 Introduction The most extraordinary physical characteristic of the nervous system is the complexity and accuracy of its wiring. There are multiple mechanisms involved in shaping the pattern of these connections. However, one of the most important mechanism is guiding the axons to their specific targets. During the development of the nervous system, billions of neurons navigate to their targets, in response to various guidance cues in the extracellular environment (Tessier-Lavigne and Goodman, 1996). It has been widely accepted that this process of navigation and pathfinding is accomplished by a highly dynamic, fan-shaped, actin rich structure located at the tip of the axon, called the growth cone. It was first described by Santiago Ramón y Cajal over a century ago, and has been extensively studied for its role in axon guidance since then. The growth cone actively steers the growing axon in the correct direction in response to guidance cues, undergoing classic behaviours such as turning, fasciculation, collapse, retraction and stalling. The growth cone, thus, acts as a sensor, a signal transducer and a motility device (Suter and Forscher, 2000). Axon guidance Guidance cues can be either attractive or repulsive. Attractive cues attract axonal growths toward the source of the cue whereas, repulsive cues function as inhibitory barriers or repel axonal growth (Tessier-Lavigne and Goodman, 1996). These cues can act over short or long distances. Short range cues are often membrane associated molecules anchored to stationary cells or tethered to a particular substrate and mediate contact attraction or repulsion. Meanwhile, long-range cues are often secreted molecules that establish concentration gradients and function in chemoattraction or chemorepulsion from a distance (Figure 1.1) (Kolodkin and Tessier-Lavigne, 2011). Depending on the type of receptors present on the growth cone and the downstream effector pathways, the 2 expression of one guidance molecule can mediate different effects on a growth cone (Grunwald and Klein, 2002; Huber et al., 2003; Meyer and Feldman, 2002). Furthermore, it is common for a growth cone to respond sequentially to different guidance cues along the way to its target, thus the growth cone must have the ability to integrate incoming signals, terminate its responsiveness to certain cues and begin responding to others in order to make the correct guidance decisions (Kolodkin and Tessier-Lavigne, 2011). Figure 1.1 Diverse mechanisms involved in axon guidance (Kolodkin and Tessier-Lavigne, 2011) The ability to transduce extracellular signals into changes in neuronal morphology is a critical part of axon guidance. This change in morphology can result in growth cone extension, steering or retraction (Dent and Gertler, 2003; Lowery and Van Vactor, 2009). It is the motility and changes in the dynamics of cytoskeletal components that allow for a growth cone to respond to guidance cues by advancing or withdrawing its filopodia and the leading edge of the growth cone. Filamentous actin (F-actin) is found within the periphery of the growth cone and bundled microtubules (MT) inhabit the axon shaft and the growth cone central domain. Actin polymerization occurs at the leading edge of the advancing growth cone and actin depolymerisation concurrently occurs in the central domain. There is a myosin driven retrograde flow of F-actin from the leading edge towards the central domain (Suter and Forscher, 2000). Guidance cues influence the growth cone trajectories by altering the assembly, disassembly and dynamics of one or more of these cytoskeletal components (Kolodkin and Tessier-Lavigne, 2011). The downstream signalling events of guidance cues are known to involve regulation of the Rho family of guanosine triphosphatases (GTPases). Guidance cue receptor activation modulates 3 Rho GTPases by switching them between an inactive GDP-bound state and an active GTP-bound state through action of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively (Kolodkin and Tessier-Lavigne, 2011). Attractive cues, via activation of Rho GTPases, result in stabilization and extension of actin filaments. Conversely, repulsive cues inactivate Rho GTPases leading to destabilization and collapse of the growth cone (Meyer and Feldman, 2002; Zhang et al., 2003). In addition to the Rho family of GTPases, guidance cue receptor activation can also function through other signaling cascades such as kinase phosphorylation, which in turn recruits adaptor proteins and/ or additional kinases that are known to regulate cytoskeletal dynamics (Bashaw and Klein, 2010; Kolodkin and Tessier-Lavigne, 2011). Role of axon guidance cues during the development of nervous system Work in the past several decades has advanced our understanding of the function of numerous guidance cues and their receptors, their underlying signaling cascades and the mechanism they use to integrate information from multiple guidance cues (Dickson, 2002; Timothy and Bargmann, 2001; Huber et al., 2003). Through various biochemical, genetic and tissue culture approaches in both invertebrates and vertebrates, several ‘classical’ families of guidance cues and their receptors have been characterized (Figure 1.2). These families of guidance molecules and receptors, namely, Netrins, Slits, Semaphorins, Ephrins were initially identified as central mediators of axon guidance and were considered to be ‘classical’ guidance cues. More recently, additional classes of guidance cues have also been discovered (Yaron and Zheng, 2006). Netrins Netrins are a small family of evolutionarily conserved guidance cues, initially discovered through genetic screens in Caenorhabditis elegans (C. elegans) and biochemical studies in vertebrates. Studies in C. elegans showed that the unc-6 gene, which encodes Netrin, is required for guidance along the dorso-ventral axis (Hedgecock et al., 1990; Ishii et al., 1992). In vertebrates, Netrin-1 mediates neuronal outgrowth and chemoattractant activity for commissural axons to the ventral midline (Kennedy et al., 1994; Serafini et al., 1994). Netrin receptors also were identified in C.elegans, encoded by the unc-40 and unc-5 genes (Leung-Hagesteijn et al., 1992). Later, 4 vertebrate homologues of unc-40 (Deleted in Colorectal Cancer, DCC) and unc-5 were identified as well (Keino-Masu et al., 1996; Leonardo et al., 1997). Netrins act bifunctionally, mediating attraction through DCC and unc-40, whereas it acts through unc-5 to mediate repulsion (Hong et al., 1999; Keleman and Dickson, 2001). In Drosophila, Netrins are known to attract axons to the nervous system midline, and function through the receptor Frazzled (Fra) (Harris et al., 1996; Mitchell et al., 1996; Kolodziej et al., 1996). There is one isoform of Netrin in C. elegans (UNC-6), two isoforms in Drosophila (Netrin-A and Netrin–B), one in chicks (Netrin-2) and two in mammals (Netrin-1 and Netrin-3). Netrins can act both at short and long ranges (Kennedy et al., 1994; Deiner et al., 1997; Brankatschk and Dickson, 2006). Figure 1.2 The four classical families of axon guidance cues and their receptors (Yaron and Zheng, 2006). 5 Slits Slits are secreted proteins, involved in axonal repulsion along with its receptor Roundabout (Robo). Studies on guidance of commissural axons at the Drosophila midline showed that Slit- Robo 1 signalling prevents the axons from re-crossing the midline (Seeger et al., 1993; Kidd et al., 1998). In Drosophila, there are three Robo isoforms (Robo 1, Robo 2, Robo 3) and one Slit isoform (Sli). In C. elegans, the Robo and Slit homologues are named Sax-3 and Slt, respectively. In vertebrates, there are four isoforms of Robo (Robo 1, Robo 2, Robo 3, Robo 4) and three isoforms of Slit (Slit 1, Slit 2, Slit 3). In Drosophila, combinatorial expression of the three members of the Robo family on commissural axons determines the lateral positions of these axons after midline crossing (Rajagopalan et al., 2000; Simpson et al., 2000). A spliced isoform of Robo 3 (Robo 3.1) in vertebrates and Robo 2 in Drosophila have been shown to inhibit the respulsive actions of other Robo receptors (Sabatier et al., 2004; Chen et al., 2008; Spitzweck 2010). In addition to the axonal repulsion at the midline, Slit also acts as a repellent cue on retinal and olfactory bulb axons (Li et al., 1999; Wu et al., 1999; Plump et al., 2002). Semaphorins The Semaphorins are a large, evolutionarily conserved protein family of secreted and membrane-tethered guidance cues, implicated mainly as axonal repellents. Semaphorin 1a (Sema-1a), originally named ‘Fasciclin IV’, was the first Semaphorin to be identified and was shown essential for correct pathfinding of pioneer sensory axons in a developing grasshopper limb (Kolodkin et al., 1992). Semaphorin 3A (Sema 3A), originally named ‘Collapsin 1’, was the first vertebrate Semaphorin to be discovered (Luo et al. 1993). The Semaphorin family can be categorized into eight classes: classes 1 and 2 are found in invertebrates, classes 3-7 in vertebrates and last, there is a class of viral Semaphorins. A class 5 Semaphorin, Semaphorin-5c (Sema-5c) was identified in invertebrates as well (Bahri et al., 2001). The first set of binding receptors identified for Semaphorins were Neuropilins (He and Tessier-Lavigne, 1997; Kitsukawa et al., 1997; Kolodkin et al., 1997). Vertebrates have two Neuropilins whereas invertebrates have none. The fact that Neuropilins do not have signaling ability led to the discovery of Plexins, a conserved family of receptors for Semaphorins (Comeau et al., 1998; Takahashi et al., 1999; Tamagnone et al., 1999). Plexins are a transmembrane protein family divided into four classes, namely A, B, C, 6 D. In Drosophila, Plexin-A (Plex A) serves as receptor for Sema-1a and Semaphorin 1b (Sema-1b) that controls motor and central nervous system (CNS) axon guidance (Winberg et al., 1998). Whereas, Plexin-B (Plex B) is a receptor for Semaphorin 2a (Sema-2a) and Sema-2b, and is also involved in motor neuron axon guidance (Cho et al., 2012). In vertebrates, different secreted Semaphorins require specific combinations of the co-receptor Neuropilins and the receptor Plexins for guidance responses (Tran et al., 2007). Plexin receptor activation is known to initiate intracellular signaling events that result in the local disassembly of growth cone cytoskeletal components and substrate attachments (Zhou et al., 2008). Although Semaphorins serve as key repulsive cues during nervous system development in vertebrates and invertebrates, many Semaphorins are known to be bifunctional in certain circumstances (Wong et al., 1997; Kitsukawa et al., 1997; Kolodkin and Tessier-Lavigne, 2010). Semaphorins themselves serve as receptors sometimes and participate in regulating dendritic targeting events in the Drosophila olfactory system and photoreceptor guidance in the Drosophila visual system (Sweeney et al., 2011; Cafferty et al., 2006). Ephrins Ephrins are cell-surface signaling molecules that play an important role in axon guidance during development (Klein, 2004). Ephrins and their receptors Eph were discovered during a search for graded guidance cues that guide the retinal axons to their correct topographic locations in the optic tectum (Drescher et al., 1995). Ephrins and Eph receptors can be divided into two types: Ephrin As which are membrane-anchored via a glycosylphophatidylinositol (GPI) linkage and bind to Eph A receptors; and Ephrin Bs that have a transmembrane domain and bind to Eph B receptors (Wilkinson, 2001; Dickson, 2002). There are 13 Eph receptors and 8 Ephrins in vertebrates, whereas C. elegans and Drosophila have just one Eph receptor that binds to four and one Ephrin ligands, respectively (Dickson, 2002). Ephrins act as short range attractive and repellent cues. Ephrin A ligands and their receptors Eph A serve as repellent cues in the visual system for mapping the retinal axons along the anterior-posterior axis (Wilkinson, 2001). Ephrin B ligands and Eph B receptors mediate attractive signaling along the dorso-ventral axis (Hindges et al., 2002; Mann et al., 2002). Recent findings also indicate that Ephrins and their receptors play an important role in axonal pruning, regulation of dendrite morphology, synatopgenesis and synaptic plasticity (Shen and Cowan, 2010). 7 Morphogens Morphogens were primarily described as developmental cues that specify cell fate by modulating the cellular response to their gradient concentration (Teleman et al., 2001). Recent studies imply that these graded morphogens guide axons to their specific targets. Members of three different morphogen families, Hedgehog (Hh), Transforming Growth Factor β (TGFβ), and Wnts, are known to act as guidance cues in both vertebrates and invertebrates. Similar to the ‘classical’ guidance cues, morphogens are multifunctional cues (Yaron and Zheng, 2006). Sonic hedgehog (Shh) was identified as an additional attractant for commissural axons to the midline, along with Netrin-1 (Charron et al., 2003). Shh was also shown to act as a repellent cue after the axons have crossed the midline (Bourikas et al., 2005). This bifunctional activity is achieved through two receptors, Smoothened (Smo) for attraction and Hip1 for repulsion (Charron et al., 2003; Bourikas et al., 2005). Shh are also known to be repulsive cues for a subset of retinal axons (Trousse et al., 2001). TGFβ family of receptors bind primarily to the ligands, bone morphogenetic proteins (BMPs) and activins. BMPs have been shown to act as repellent cues to direct the commissural axons towards the midline (Yaron and Zheng, 2006). Activin-β (Act-β), also known as dActivin, was the first invertebrate activin gene to be described (Kutty et al., 1998). It is a ligand for type I receptor Baboon (Babo), and type II receptors Punt (Put) and Wishful thinking (Wit) and is implicated in axonal and dendritic remodelling (Grueber and Jan, 2004). Dawdle (Daw) is another TGF-β ligand that binds to Babo and Put, and functions in motor neuron axon guidance. Daw mutants show mistargeting of inter segmental nerve b (ISNb) and segmental nerve a (SNa) axons, similar to the phenotypes observed in Babo mutants and Put mutants (Parker et al, 2006). These mutant phenotypes were also observed in loss of function experiments of Tolkin (Tok), also known as Tolloid-related (Tlr), a member belonging to a family of proteases (Serpe and O’Connor, 2006; Meyer and Aberle, 2006). In Drosophila, Wnt5 acts via its receptor Derailed (Drl) to repel commissural axons (Yoshikawa et al., 2003). In vertebrates, Wnt4 acts via its receptor Frizzled to attract commissural axons along the rostral-caudal axis to the brain after they have crossed the midline (Lyuksyutova et al., 2003). On the other hand, Wnt1 and Wnt5a function as repellents for corticofugal axons as 8 they descend in the opposite direction from the brain to spinal cord (Liu et al., 2005). Wnts have also been involved in directing synapse formation and topographic mapping in the vertebrate visual system (Salinas and Zou, 2008). Heparin sulphate proteoglycans (HSPGs) like Dally-like (Dlp) are required for Wnt signalling (Baeg et al., 2001). Dlp is known to known to have distinct roles in axon guidance and visual system assembly in Drosophila. Mutations in Dlp displayed phenotypes similar to that of Syndecan (Sdc) mutants, another HSPG (Rawson et al., 2005). Sdc is also implicated in mediating Slit signaling (Chanana et al., 2009). Sdc and Dlp serve as extracellular ligands for Leucocyte antigen related (Lar), a receptor protein tyrosine phosphatase, to promote synaptic growth and maturation at Drosophila neuromuscular junction (NMJ). In addition, the intracellular domain of Lar is known to interact with N-cadherin (CadN) to promote R7 photoreceptor targeting in the Drosophila visual system (Hofmeyer and Treisman, 2009; Prakash et al., 2009) and is also implicated in the normal targeting and pathfinding of segmental nerve b (SNb) and segmental nerve d (SNd) motor axons (Krueger et al, 1996). Drosophila Wnk (With No lysine Kinases) are also known positive regulators of the Wnt pathway (Serysheva t al., 2013). Wnk has been shown to regulate neuronal wiring in the Drosophila visual system (Berger et al., 2008). Cell Adhesion Molecules (CAMs) The two major classes of CAMS are Cadherins and the Immunoglobin (Ig) superfamily. Of these, the Ig superfamily CAM Fasciclin II (Fas II or Fas 2) is known to regulate axonal fasciculation and motor neuron guidance (Harrelson and Goodman, 1988; Lin et al., 1994; Lin and Goodman, 1994). Fasciclin I (Fas I or Fas 1) and Fasciclin III (Fas III or Fas 3) are novel Drosophila neural CAMs. Fas 1 is a membrane associated glycoprotein expressed on the surface of all peripheral nervous system (PNS) axons and a subset of fasciculating CNS axons during development (Bastiani et al., 1987; Zinn et al., 1988). Although Fas 1 mutants show no gross neuronal morphology, it has been shown to have a strong interaction with Abelson tyrosine kinase (Abl). Double mutants of Fas 1 and Abl exhibit commissural axon pathfinding defects, particularly in the RP1 neurons (Elkins et al., 1990). Fas 3 is expressed in and serves as a synaptic target recognition molecule for RP3 motor neurons and its synaptic partners muscles 6 and 7 (Chiba et al., 1995). Capricious (Caps), a member of the ‘leucine-rich repeat’ family of CAMs, selectively mediates the formations of connections between RP5 motor neurons and muscle 12 (Shishido et 9 al., 1998; Taniguchi et al., 2000; Abrell and Jäckel, 2001). Caps is also implicated in the layer specific targeting of photoreceptor neurons in the medulla (Shinza-Kameda et al., 2006). In Drosophila, the Ig superfamily member Beaten Path (Beat) serves as a receptor in motor neurons for Sidestep, another Ig superfamily member expressed by intermediate neurons, thus acting as an attractant cue for these axons (Siebert et al;., 2009). Turtle (Tutl) are phylogenetically conserved Ig proteins found in both vertebrates and invertebrates. In Drosophila, Tutl serves as axonal attractants that promotes midline crossing via a Netrin and Slit independent mechanism. Loss of function experiments display stalled or missing axonal projections, whereas gain of function experiments display ectopic axonal branching (Al-Anzi and Wyman, 2009). In Drosophila, Neuroglian (Nrg), a member of the Ig superfamily, is the homolog of the vertebrate CAM L1 (Bieber et al., 1989). Nrg is shown to regulate motor neuron target recognition, and morphology and patterning of sensory neurons (Hall and Bieber, 1996). Another CAM, Neurotactin (Nrt), is widely expressed during nervous system development. Nrt mutants display defasciculation of ocellar pioneer axons and stalling of CNS axons. Nrt/Nrg double mutants display phenotypic synergy showing enhanced thinning of longitudinal connectives and fusion of commissures (Spiecher et al., 1998). Dystroglycan (Dg) is a transmembrane protein that interacts with Dystrophin (Dys) to link extracellular matrix (ECM) and the cytoskeletal actin. This interaction is highly conserved from invertebrates to vertebrates. In Drosophila, mutations in Dg and Dys are shown to result in muscular dystrophy phenotypes (decline in mobility and age-dependent muscle degeneration) and defective axonal pathfinding in photoreceptor neurons (Shcherbata et al., 2007). Integrins, highly expressed in growth cones, serve as a link between ECM and the growth cone cytoskeleton (Takagi et al., 1998; Takagi et al., 2000; Stevens and Jacobs, 2002). Axon defasciculation and axonal tract displacement are seen at the midline in Drosophila mutants for the intergrin gene α subunit, multiple edematous wings (mew), and β subunit, myospheroid (mys) (Stevens and Jacobs, 2002). Starry night (Stan), also known as Flamingo (Fmi), belongs to the Cadherin family of proteins. Stan is implicated in photoreceptors R8 axon-axon and axon-target interactions (Senti et al., 2003). Stan mutants display phenotypes similar to that observed in Jelly belly (Jeb) mutants. Jeb is the ligand for Anaplastic lymphoma kinase (Alk). Jeb/Alk signalling is implicated in 10 mediating neuronal circuit assembly in the Drosophila visual system (Bazigou et al., 2007). Drosophila CadN is known to regulate axonal targeting in the lamina (Nern et al., 2008). Drosophila Unzipped (Uzip), is a CAM expressed in longitudinal glia and longitudinal axonal tracts. Uzip is shown to genetically interact with CadN and Wnt5 to mediate axon guidance and patterning of Sema-2b expressing neurons and pioneer neurons, respectively (Ding et al., 2011). Cytoskeletal-associated proteins Although many cytoskeletal-associated proteins are expressed in the nervous system, relatively few are implicated in axon guidance (Dent et al., 2011). Cytoskeletal-associated proteins include actin-associated proteins, microtubule (MT) associated proteins (MAPs) and actin-MT crosslinking proteins. Among actin-associated proteins, the Ena/VASP proteins were some of the first to be implicated in axon guidance (Drees and Gertler, 2008). There are three Ena/VASP proteins in vertebrates (Mena, VASP, EVL), and one each in Drosophila (Enabled ‘Ena’) and C. elegans (UNC-34). In the developing nervous system, Ena/VASP proteins are known be highly expressed in filopodia and lamellipodia (Lanier et al., 1999). In Drosophila, Ena is required for axon guidance of the ISNb neurons to their appropriate targets (Wills et al., 1999) and mutations in Ena/VASP proteins in both vertebrates and invertebrates are known to result in midline crossing defects (Lanier et al., 1999; Menzies et al., 2004; Wills et al., 2002; Timothy et al., 2002; Gitai et al., 2003). Ena/VASP proteins are also suggested to have roles in Netrin and Slit signalling (Bashaw et al., 2000; Timothy et al., 2002; Moore et al., 2007). The Abelson tyrosine kinase (Abl) is an actin-binding non-receptor tyrosine kinase that is implicated in several signaling cascades (Dent et al., 2011). The role of Abl in axon guidance was identified in Drosophila. Abl was shown to mediate cell-cell interactions via CAMs like Fas I and Nrt during nervous system development (Elkins et al., 1990; Liebl et al., 2003). Abl also regulates axon guidance of ISNb by antagonizing the effect of Ena (Wills et al., 1999; Dent et al., 2011). Abl is also known to regulate midline crossing by regulating Netrin and Slit pathways via the receptors Fra and Robo, respectively (Forsthoefel et al., 2005; Bashaw et al., 2000). During guidance responses, Abl coordinates actin and MT dynamics through interactions with Mini spindles (Msps) (Lowery et al., 2010). 11 β-Spectrin (β-Spec), a member of the Spectrin family of F-actin binding proteins, is implicated in midline axon guidance in Drosophila (Hulsmeier et al., 2007). Genetic interactions studies also suggest a role for β-Spec in Slit/Robo mediated midline repulsion (Garbe et al., 2007). Molecule interacting with CasL (Mical), an F-actin depolymerizing protein has been shown to mediate motor neuron axon guidance via Sema-1a/Plex A repulsive signaling in Drosophila (Terman et al., 2002; Hung et al., 2010). Profilin, an actin-monomer binding protein, is abundantly present within cells and serves multiple functions (Kaiser et al., 1999; Dent et al., 2011). Mutations in the Drosophila homolog of Profilin, Chickadee (Chic), was shown to affect axonal outgrowth and navigation of ISNb, a phenotype similar to Abl mutants (Van-Vactor et al., 1993; Wills et al., 1999). Spectraplakins, a family of actin-MT crosslinking proteins, are another class of cytoskeletal-associated proteins that are implicated in axon guidance. The vertebrate Spectraplakin, ACF7, and its Drosophila homolog, Shortstop (Shot), play important roles during nervous system development by coordinating the cytoskeletal networks during axonal growth (Sanchez-Soriano et al., 2009). The Bifocal (Bif) protein, an actin-binding as well as MT-binding protein, is required for normal photoreceptor axon targeting in Drosophila (Sisson et al., 2000; Ruan et al., 2002). Adenomatous polyposis coli (APC) protein, a class of MT-associated plus-end-tracking protein (+TIPs), have two isoforms in Drosophila- APC1 (or ‘Apc’) and APC2, both of which are required for normal optic lobe development (Hayden et al., 2007). Drosophila Lissencephaly-1 (Lis1) is a regulator of the MT cytoskeleton and is required for dendrite growth, branching and maturation. Lis1 mutants exhibit phenotype similar to the cytoplasmic dynein heavy chain (Dhc64C) mutants (Liu et al., 2000). Drosophila Minispindles (Msps), a class of MT stabilizing proteins, is an ortholog of the human cytoskeleton associated protein 5 (CKAP5). Msps mutants show defects in axonal morphology and ectopic midline crossing in Drosophila embryos, and is implicated to interact strongly with Abl (Lowery et al., 2010). Others Smooth (Sm), a heterogeneous nuclear ribonucleoprotein (hnRNP), is widely expressed in neuronal cells during development. Sm mutants display axonal defects in chemosensory neurons and also show defective feeding behavior (Layalle et al., 2005). 12 Role of axon guidance cues beyond development Following nervous system development, the expression of numerous axon guidance molecules changes, whereas others retain embryonic expression levels. Many mature neurons continue to express receptors for guidance cues even after they reach their targets, which implies additional roles for these developmental cues beyond the initial phase of neuronal process outgrowth, growth cone navigation, and target innervation (Giger et al., 2010). The role of axon guidance cues in a mature nervous system is less understood. CNS regeneration The expression of a number of axon guidance cues are regulated following CNS injury. Since many neurons continue to express guidance receptors, it is implied that these neurons remain responsive to guidance cues throughout life. It has been speculated that the up-regulation of the expression of many guidance cues with known inhibitory activity, including members of the Semaphorin family, is significant for inhibiting nervous system regeneration. The presence of class 3 Semaphorins (Sema3s) in the mature nervous system have been proposed to contribute to the growth inhibitory nature of injured CNS tissue (Pasterkamp and Verhaagen, 2006). In an in vivo adult rabbit cornea injury model, Sema 3 had shown to inhibit collateral nerve sprouting, proving these neurons retain their ability to respond to Sema 3 in a fully developed adult nervous system (Tanelian et al., 1997). In a rat model of olfactory nerve axotomy, it was shown that blocking Sema 3 function enhances axon regeneration (Kikuchi et al., 2003). Similar to Semaphorins, blocking the function of Ephrins had also shown to promote axonal sprouting in spinal cord injury rat models (Fabes et al., 2007). The Wnt family is another set of developmental guidance cues implicated to have a role in the inhibition of nervous system regeneration (Liu et al., 2008). Collectively these studies attribute the inability of severed axons to undergo spontaneous repair in the adult CNS, to the presence of the very same guidance molecules that established them in the first place. Regulation of adult synaptic plasticity Guidance molecules are known to have specific roles during various phases of synaptogenesis including, synaptic pre-patterning, filopodial motility, contact stabilization and synaptic maturation (Shen and Cowan, 2010). Once fully developed, neuronal circuits in the 13 mature nervous system become more stable, however, the strength and number of mature synapses are altered in response to experience, injury, and aging (Shen and Cowan, 2010; Giger et al., 2010). In vertebrates, Netrin-1 and its receptor DCC are known to regulate synaptogenesis during development but their contribution to synaptic plasticity in the mature CNS was unknown until recently. Conditional deletion of DCC in adult mice forebrain neurons using the cre/loxp gene targeting strategy was shown to result in shorter dendritic spines, loss of long term potentiation, and impaired spatial and recognition memory (Horn et al., 2013). Drosophila melanogaster as model organism Important work contributing to our understanding of axon guidance and guidance cues has been carried out in both vertebrates and invertebrates. In many instances, this has demonstrated the conservation of principle guidance molecules and mechanisms across the animal kingdom. The use of genetically tractable invertebrate model organisms like the fruit fly Drosophila melanogaster has improved our insight into axon guidance. The strength of this organism lies in its amenability to genetic manipulation and the fact that its nervous system is composed of relatively lower number of cellular elements. The principal structures of vertebrate and invertebrate neurons have been proposed to be homologous despite there being certain organisational differences. Drosophila screens have been employed extensively during developmental stages to identify new proteins or signalling pathways that are critical for key conserved developmental events. However, screens in adult Drosophila have not been as extensively employed. This is possibly because many mutations cause embryonic or larval lethality, making the utility of adult flies for genetic screens limited (Sánchez-Soriano, N et al., 2007). However, the wide availability of RNA-mediated interference (RNAi) lines and the possibility of spatial and temporal regulation of genetic tools such as ‘Temporal And Regional Gene Expression Targeting’ (TARGET) and ‘Gene Switch’ (GS) systems have overcome this problem. These tools allow one to knockdown any target gene, in any targeted cell at any time. 14 GAL4-UAS System GAL4- Upstream Activating Sequence (UAS) system is a standard technique, adapted from yeast, for targeted gene expression studies in Drosophila. This system utilizes a cell-type specific cloned promoter or endogenous enhancer to direct the expression of the yeast transcriptional activator GAL4 in a spatially restricted fashion. The GAL4 then drives the expression of any gene of interest that has been cloned downstream of a UAS binding site. The advantage of this system is that the transcriptional activator GAL4 (‘GAL4 driver line’) and the UAS-based transgene (‘UAS target line’) are carried in different parental lines, thus ensuring their viability and enabling a combinatorial approach with different driver and target lines to the biological question of interest. Once generated, a line expressing GAL4 in a given spatial pattern can be crossed with any UAS target line, allowing the GAL4 line to be used as a general resource. Similarly, when a given UAS target line is generated, the target gene can be transcribed anywhere in the fly by crossing it to the appropriate GAL4 line (McGuire et al., 2004). The GAL4 driver line used primarily in this study is Elav-GAL4. ‘Elav’ is a neuronal cell specific enhancer that spatially controls the GAL4 activity to all post mitotic neurons (Robinow and White, 1991). We also used OK371-GAL4 in certain experiments to limit the GAL4 activity to motor neurons (Mahr and Aberle, 2006). Temporal control of GAL4 was achieved by two methods called the TARGET and Gene Switch systems. In the TARGET system, the conventional GAL4-UAS system is conditionally regulated by a temperature-sensitive allele of GAL80 (GAL80ts). At 18°C, transcription of UAS-transgene is repressed, whereas this repression is relieved by a temperature shift to 29°C, leading to high levels of expression of UAS-transgene in a specific tissue. The second system, termed Gene Switch, is based on a GAL4-progesterone receptor chimera that is hormone-inducible. In this system, the DNA binding domain of the GAL4 protein is fused to the p65 activation domain and a mutant progesterone receptor ligand binding domain to generate ligand-stimulated chimeric activators. In the absence of hormone, the Gene Switch is in the inactive state. In the presence of hormone, the Gene Switch molecule changes to an active conformation in which it can bind to a UAS sequence and activate transcription of a transgene (McGuire et al., 2004). Figure 1.3 illustrates how spatial and temporal control of GAL4 has been employed in this study to activate UAS-RNAi in specific cell populations at a given time. 15 Figure 1.3 GAL4-UAS, TARGET and Gene Switch systems. In a conventional GAL4-UAS system, the yeast transcriptional activator GAL4 is driven in a specific spatial pattern by either a defined promoter or an endogenous enhancer. The GAL4 protein, in turn, binds to its cognate UAS binding site and constitutively activates the transcription of RNAi cloned downstream of the UAS. The TARGET and Gene Switch systems allow simultaneous spatial and temporal control of this GAL4 activity. RNA-mediated Interference RNA-mediated interference (RNAi) utilizes gene specific reagents that are introduced into the cell, triggering gene knockdown via sequence-specific degradation and translational interference of mRNA transcripts. RNAi screening provides a powerful reverse genetic approach for large scale functional analysis in cultured cells and in an increasing number of in vivo systems 16 (Mohr and Perrimon, 2011). In Drosophila, RNAi is cell autonomous and can be triggered by the expression of double-stranded hairpin RNA from a transgene containing a gene fragment cloned as an inverted repeat (Figure 1.4). These double-stranded RNAs are processed by Dicer into shRNAs (short hairpin RNAs) which direct sequence-specific degradation of the target mRNA. Using the binary GAL4-UAS expression system, such RNAi transgenes can be used to target gene knockdown in any desired cell type at any stage of the Drosophila lifespan. This, combined with the availability of Drosophila genome-wide library of transgenic RNAi strains, has made it possible to conduct systematic RNAi screens targeted to specific cell types in the intact fly (Dietzl et al., 2007). Figure 1.4 Basic in-vivo F1 RNAi screen (http://igtrcn.org/rnai-screening/). This combined with temporal control (using either TARGET or Gene Switch system) has been employed in this study. 17 Hypothesis Axon guidance cues are extracellular signals that direct the growth and steering of neuronal growth cones. Both attractive and repulsive cues are required to guide developing axons to their targets. Work in the past few decades has led to the discovery of numerous axon guidance genes and identified their functions during development. Nonetheless, after axons have reached their targets and established functional circuits, many neurons continue to express these developmental cues. The expression of these genes in the adult indicates that there are likely additional roles for guidance cues beyond the initial phase of neuronal process outgrowth, growth cone navigation, and target innervation (Giger et al., 2010; French and Pavlidis, 2011). The central goal of my work is to determine the functions of these cues in the mature nervous system. I hypothesize that axon guidance genes expressed in the adult nervous system have functional roles in the maintenance of neural circuits. This study is the first to perform an RNAi screen of axon guidance genes expressed in the mature nervous system and to determine their impact on the structure and function of adult neurons. The study consists of the following aims: 1) Identify the various axon guidance genes expressed in the adult Drosophila melanogaster nervous system using bio-informatics tools 2) Generate adult knockdown flies using RNAi and GAL4-UAS, TARGET, Gene Switch systems 3) Analyze the effect of knockdown on adult survival 4) Analyze the resulting behaviour (climbing, mobility) of knockdown adult flies 5) Identify strategies to examine the impact of knockdown on circuit morphology 18 Chapter 2 Materials and Methods Identifying axon guidance genes The RNA-Seq Expression Profile search tool from FlyBase (www.flybase.org) version FB2013_05 (released September, 2013) was used to search for genes expressed in Drosophila melanogaster in the embryo (stage-wise expression, 10-24 hours) and in the adult (tissue-wise expression in head, 1- 20 days post eclosion). This search tool allowed us to query FlyBase records using the modENCODE high-throughput RNA-sequence (RNA-seq) data published in Graveley et al., (2010). An ‘expression on’ filter was used to search for genes expressed above a certain expression profile threshold, namely very low, low, moderate, moderately high, high, very high and extremely high (Gelbart and Emmert, 2013). The resulting dataset was further refined using Biological process gene ontology (GO) terms ‘axon guidance’ and ‘dendrite morphogenesis’. Transcription factors were excluded using Cellular component GO term ‘nucleus’. Gene list venn diagram (http://genevenn.sourceforge.net/) was used to categorize the genes into discrete expression levels, ranging from very low to extremely high, and also to compare the genes expressed in the embryo and adult Drosophila. This data combined with their previously known roles in receptor activity and signalling during neuronal pathfinding were used to prioritize genes. Fly genetics Reducing axon guidance gene expression by RNAi in the adult nervous system was carried out using two well-characterized systems: 1) Gene Switch and 2) TARGET. GAL4 driver line virgin females were crossed with UAS-RNAi males and injection strain males (negative control). In case of Gene Switch screen, crosses were set up at 25°C. The progeny (F1) were fed with food containing 6.5µg/mL mifepristone (RU486) from day 1 adult onwards to activate the GAL4-UAS 19 system. In case of TARGET screen, crosses were set up at 18°C and the progeny (F1) were switched to 29°C immediately after eclosion to repress GAL80ts and activate the GAL4-UAS system. Fly stocks Drosophila melanogaster stocks used in this study were maintained on standard cornmeal food and at 18, 25, or 29°C in environment rooms set at 70% humidity. The stocks used in this study are as follows: GAL4 driver lines The following GAL4 driver lines were built to drive RNAi expression in all neurons in the adult. All of these lines were used for the survival assay and showed similar results, thus the elav-gene switch driver line (elavGS) was used in all other experiments. 1) Elav-GAL4, UAS.Dicer2 ; tubGAL80ts, UAS nGFP ; +/+ 2) Elav-GAL4, UAS.Dicer2 ; +/+ ; tubGAL80ts, UAS nGFP 3) UAS.Dicer2 ; +/+ ; elavGS, UAS nGFP The following line was built to restrict the RNAi expression to glutamatergic neurons in the adult. 1) UAS.Dicer2 ; OK371-GAL4 ; tubGAL80ts, UAS nGFP The following driver- reporter lines (a gift from Dr. Micheal Gordon) were used to study the expression of Fas3 in adult Drosophila CNS. 1) w ; Gr64f-GAL4, UAS GCaMP6f/ Cyo ; +/+ 2) w ; LexAop-CD2::GFP/ Cyo ; Gr5a-LexA::VP16(1-3)/ TM6 3) w ; ppk28-LexA::VP16/ Cyo ; LexAop-CD2::GFP 20 UAS-RNAi lines The UAS-RNAi lines used to knock down various axon guidance genes are listed in the Table 2.1. Table 2.1 List of UAS-RNAi lines used in the study Gene Library Stock# Genotype Abl BDSC 28325 y1 v1; P{TRiP.JF02960}attP2 Actβ BDSC 42493 y1 v1; P{TRiP.HMJ02057}attP40 BDSC 29597 y1 v1; P{TRiP.JF03276}attP2 VDRC 108663 P{KK101617}VIE-260B Apc BDSC 28582 y1 v1; P{TRiP.HM05070}attP2 BDSC 34869 y1 sc* v1; P{TRiP.HMS00188}attP2 babo BDSC 40866 y1 v1; P{TRiP.HMS02033}attP40 BDSC 25933 y1 v1; P{TRiP.JF01953}attP2 VDRC 106092 P{KK108186}VIE-260B beat-Ic VDRC 105066 P{KK113293}VIE-260B bif BDSC 28372 y1 v1; P{TRiP.JF03009}attP2 VDRC 109722 P{KK105557}VIE-260B cac BDSC 27244 y1 v1; P{TRiP.JF02572}attP2 caps BDSC 28020 y1 v1; P{TRiP.JF02854}attP2 chic BDSC 34523 y1 sc* v1; P{TRiP.HMS00550}attP2 VDRC 102759 P{KK112358}VIE-260B dlp BDSC 34089 y1 sc* v1; P{TRiP.HMS00875}attP2 BDSC 34091 y1 sc* v1; P{TRiP.HMS00903}attP2 daw BDSC 50911 y1 v1; P{TRiP.HMJ03135}attP40 BDSC 34974 y1 sc* v1; P{TRiP.HMS01110}attP2 VDRC 105309 P{KK110248}VIE-260B Dhc64C BDSC 36698 y1 sc* v1; P{TRiP.HMS01587}attP2 Dg BDSC 34895 y1 sc* v1; P{TRiP.HMS01240}attP2 VDRC 107029 P{KK100828}VIE-260B 21 ena BDSC 39034 y1 sc* v1; P{TRiP.HMS01953}attP2 BDSC 31582 y1 v1; P{TRiP.JF01155}attP2 Fas1 BDSC 42887 y1 sc* v1; P{TRiP.HMS02580}attP40 VDRC 23014 w1118; P{GD12817}v23014 VDRC 23015 w1118; P{GD12817}v23015 Fas3 VDRC 939 w1118; P{GD80}v939 VDRC 940 w1118; P{GD80}v940 VDRC 3091 w1118; P{GD2576}v3091 VDRC 26850 w1118; P{GD13161}v26850 fra BDSC 31469 y1 v1; P{TRiP.JF01231}attP2 BDSC 31664 y1 v1; P{TRiP.JF01457}attP2 BDSC 40826 y1 sc* v1; P{TRiP.HMS01147}attP2 VDRC 29910 w1118; P{GD14401}v29910 VDRC 29909 w1118; P{GD14401}v29909/TM3 jeb VDRC 103047 P{KK111857}VIE-260B Lar BDSC 40938 y1 sc* v1; P{TRiP.HMS00822}attP2 BDSC 34965 y1 sc* v1; P{TRiP.HMS00822}attP2 VDRC 107996 P{KK100581}VIE-260B Lis-1 BDSC 35043 y1 sc* v1; P{TRiP.HMS01457}attP2 BDSC 28663 y1 v1; P{TRiP.JF03078}attP2 msps BDSC 38990 y1 sc* v1; P{TRiP.HMS01906}attP40/CyO BDSC 31138 y1 v1; P{TRiP.JF01613}attP2 Mical BDSC 31148 y1 v1; P{TRiP.JF01625}attP2 VDRC 105837 P{KK102751}VIE-260B mew BDSC 27543 y1 v1; P{TRiP.JF02694}attP2 BDSC 44553 y1 sc* v1; P{TRiP.HMS02849}attP2 VDRC 44890 w1118; P{GD1230}v44890 mys BDSC 33642 y1 v1; P{TRiP.HMS00043}attP2 VDRC 29620 w1118; P{GD15002}v29620/CyO; MKRS/ TM6B, Tb Atpα VDRC 100619 P{KK108782}VIE-260B 22 VDRC 12330 w1118; P{GD3093}v12330 Nrg BDSC 37496 y1 sc* v1; P{TRiP.HMS01638}attP40 BDSC 28724 y1 v1; P{TRiP.JF03151}attP2 VDRC 107991 P{KK100482}VIE-260B VDRC 6688 Nrg RNAi Nrt BDSC 28742 y1 v1; P{TRiP.JF03170}attP2 VDRC 106080 P{KK106657}VIE-260B plexA BDSC 30483 y1 sc* v1; P{TRiP.HM05221}attP2 VDRC 107004 P{KK101499}VIE-260B plexB BDSC 28911 y1 v1; P{TRiP.HM05122}attP2 VDRC 8382 w1118; P{GD2500}v8382 VDRC 8383 w1118; P{GD2500}v8383 VDRC 12165 w1118; P{GD3150}v12165 VDRC 12167 w1118; P{GD3150}v12167 VDRC 27219 w1118; P{GD14473}v27219 VDRC 27220 w1118; P{GD14473}v27220 VDRC 46687 w1118; P{GD16420}v46687 VDRC 6873 w1118; P{GD3148}v6873/TM3 put BDSC 27514 y1 v1; P{TRiP.JF02664}attP2 BDSC 39025 y1 sc* v1; P{TRiP.HMS01944}attP40 VDRC 107071 P{KK102676}VIE-260B robo BDSC 31287 y1 v1; P{TRiP.JF01230}attP2 BDSC 35768 y1 sc* v1; P{TRiP.HMS01517}attP2 BDSC 31663 y1 v1; P{TRiP.JF01456}attP2 BDSC 39027 y1 sc* v1; P{TRiP.HMS01946}attP40 VDRC 100624 P{KK108817}VIE-260B Sema-1a BDSC 29554 y1 v1; P{TRiP.JF03231}attP2 BDSC 34320 y1 sc* v1; P{TRiP.HMS01307}attP2 VDRC 104505 P{KK109430}VIE-260B Sema-1b BDSC 28588 y1 v1; P{TRiP.HM05076}attP2 23 VDRC 107233 P{KK104666}VIE-260B Sema-2a BDSC 29519 y1 v1; P{TRiP.HM05196}attP2 VDRC 15810 w1118; P{GD5476}v15810/TM3 VDRC 15811 w1118; P{GD5476}v15811/CyO Sema-5c BDSC 29436 y1 v1; P{TRiP.JF03372}attP2 VDRC 1052 w1118 P{GD5}v1052 shot BDSC 28336 y1 v1; P{TRiP.JF02971}attP2 sli BDSC 31467 y1 v1; P{TRiP.JF01228}attP2 BDSC 31468 y1 v1; P{TRiP.JF01229}attP2 sm VDRC 108351 P{KK108588}VIE-260B stan BDSC 35050 y1 sc* v1; P{TRiP.HMS01464}attP2 BDSC 26022 y1 v1; P{TRiP.JF02047}attP2 VDRC 107993 P{KK100512}VIE-260B Syb BDSC 39067 y1 sc* v1; P{TRiP.HMS01987}attP40/CyO Syt12 BDSC 38978 y1 v1; P{TRiP.HMS01894}attP40/CyO Syt14 BDSC 28365 y1 v1; P{TRiP.JF03001}attP2/TM3, Sb1 Sdc BDSC 51723 y1 sc* v1; P{TRiP.HMC03265}attP2 VDRC 13322 w1118; P{GD4545}v13322 tok VDRC 2656 w1118; P{GD245}v2656 tutl BDSC 54850 y1 v1; P{TRiP.HMJ21587}attP40 VDRC 108746 P{KK108880}VIE-260B unc-5 BDSC 33756 y1 sc* v1; P{TRiP.HMS01099}attP2 VDRC 110155 P{KK102074}VIE-260B uzip BDSC 29558 y1 v1; P{TRiP.JF03237}attP2 wit BDSC 41906 y1 sc* v1; P{TRiP.HMS02298}attP2 BDSC 25949 y1 v1; P{TRiP.JF01969}attP2 VDRC 103808 P{KK100911}VIE-260B Wnk BDSC 42521 y1 v1; P{TRiP.HMJ02087}attP40 VDRC 106928 P{KK102654}VIE-260B β-Spec BDSC 38533 y1 sc* v1; P{TRiP.HMS01746}attP40 24 Protein trap lines The protein trap lines used to determine the expression patterns of various Green Fluorescent protein (GFP) tagged axon guidance proteins are listed in the Table 2.2. Table 2.2 List of protein trap lines used in the study BDSC# Gene Genotype 59761 Abl y1 w*; Mi{PT-GFSTF.1}AblMI00347-GFSTF.1/TM6C, Sb1 Tb1 50841 Fas3 w*; P{PTT-GA}Fas3G00258 59809 Fas3 y1 w67c23; Mi{PT-GFSTF.1}Fas3MI03674-GFSTF.1/SM6a 59835 Fra y1 w67c23; Mi{PT-GFSTF.1}fraMI06684-GFSTF.1 50816 Sema-1a y1 w*; P{PTT-GA}Sema-1aCA07125 60140 Sema-1a y1 w67c23; Mi{PT-GFSTF.2}Sema-1aMI00031-GFSTF.2/SM6a 60548 Sema-2a y1 w67c23; Mi{PT-GFSTF.0}Sema-2aMI05602-GFSTF.0 Control lines The following were used as negative controls in all the experiments. 1) y1 v1; P{CaryP}attP2 (attP2 insertion TRiP line injection strain, BDSC #36303) 2) y1 v1; P{CaryP}attP40 (attP40 insertion TRiP line injection strain, BDSC #36304) 3) y,w1118; P{attP,y+,w[3`]} (KK line injection strain, VDRC #60100) 4) w1118 (GD line injection strain, VDRC #60000) 5) w1118; P{UAS-GFP.dsRNA.R}143 (UAS GFP RNAi on 2nd chromosome, BDSC #9331) 6) w1118; P{UAS-GFP.dsRNA.R}142 (UAS GFP RNAi on 3rd chromosome, BDSC #9330) Survival assay F1 progeny males and females were collected and separated immediately after eclosion. They were maintained in vials of 10 and their survival was recorded for the next 20 days. A timeline of the experiment is illustrated in Figure 2.1. Fresh food was supplied every week. 25 Survival analysis for the Gene Switch screen was carried out at 25°C and that for the TARGET screen was carried out at 29°C. In both cases, multiple RNAi lines were tested for each axon guidance gene. For each RNAi line, 3 vials containing 10 flies were tested per sex. The results were analysed using two-way Analysis of Variance (ANOVA) and Tukey’s multiple comparison tests using Graphpadprism software. Figure 2.1 Timeline for survival analysis experiment Climbing assay To identify adult climbing defects, assays were carried out as described in Perkins et al., (2014) with minor modifications. Male flies were collected immediately after eclosion, maintained in groups of 10 flies per vial and assayed at a desired age. During the climbing assay, flies were transferred to a clean, empty vial with a line drawn 7.5 cm from the bottom. Flies were allowed to acclimatize for 1 minute and then tapped to the bottom to induce an innate climbing response. The number of flies that successfully reached the 7.5 cm line in 8 seconds were recorded. For each genotype, 3 vials were assayed and 3 replicates per vial were performed to ensure an accurate reading for each vial. The results were analysed using one-way ANOVA and Tukey’s multiple comparison tests using Graphpadprism software. The assay was repeated using OK371-GAL4 (along with GAL80ts) to test if knockdown of these genes in motor neurons in the adult could cause any climbing defects. The flies were grown at 18°C until eclosion and switched to 29°C from day 26 1 adult to repress GAL80ts and activate the GAL4-UAS system. The remaining aspects of the climbing assay were kept same as before. Drosophila Activity Monitor System The Drosophila Activity Monitor (DAM) system from TriKinetics was used to monitor the activities of individual flies for 3 days on a 12 hours light/ 12 hours dark cycle. Knockdown of genes were carried out using Gene Switch. Male flies were collected immediately after eclosion, maintained in groups of 10 flies per vial and assayed at a desired age at 25°C. They were fed with food containing 6.5µg/mL mifepristone (RU486) starting at adult day 1. Flies were placed inside the activity monitor tubes for at least 24 hours before each experiment to acclimatize to the experimental set-up. 32 flies were placed in 5 mm diameter tubes, with one end containing 1 cm worth of food. Drosophila activity was recorded every 5 minutes. Activity phenotypes were assessed using the actmon R package publicly available online at: https://github.com/kazi11/actmon (kindly provided by Jeff Stafford, UBC). Data from flies that died during the course of an experiment were discarded. Video-assisted movement tracking To characterize the locomotor behavior of adult flies, a video tracking system was developed to record the trajectory of a single fly walking in a circular field arena. Knockdown of genes were carried out using Gene Switch. Male flies were collected immediately after eclosion, maintained in groups of 10 flies per vial and assayed at a desired age at 25°C. They were fed with food containing 6.5µg/mL mifepristone (RU486) from day 1 adult onwards. The experimental setup was as shown in Figure 2.2. Fly tracking arenas were 9 cm diameter Petri dishes (Corning) placed in a box open at two sides (left and right) to allow uniform diffusion of light into it. A hole was cut out from the top of the box through which a camera was used to record the movement of a single fly for 90 seconds. Flies were cooled on ice for 3–4 minutes to anaesthetize them before placing in the arena. Flies were left to acclimatize for 30 minutes before being recorded. 10 flies 27 were video-tracked for each genotype. ‘fly_tracker’- a collection of MATLAB algorithms (kindly provided by Jeff Stafford, UBC) was used to track and quantify the behavior of individual flies from the videos. A region of interest in the captured video was analysed to extract multiple parameters including position traces, heat maps, average velocities and distance travelled by the fly. The statistical significance of the resulting data were determined using one-way ANOVA and Tukey’s multiple comparison tests using Graphpadprism software. Figure 2.2 Experimental setup for video-assisted movement tracking Immunohistochemistry Adult male Drosophila CNS were dissected in ice cold 1x PBS, over a period of 30 minutes. The CNS were fixed, on poly-Lysine coated slides, in 4% PFA at room temperature (RT) for 30 minutes followed by 3 x 5 minutes washes with 0.1% PBT. The samples were blocked in 5% PBTN for 2 hours at RT, and then incubated in primary antibodies overnight at 4oC. The next day, samples were washed 3 x 30 minutes in 0.1% PBT. They were then blocked in 5% PBTN for 1 hour at RT and incubated in secondary antibody for 3 hours at RT in the dark. The samples were washed 3 x 30 minutes in 0.1% PBT. They were mounted in vactashield mounting media (Vector Laboratories Inc.), coversliped and sealed with nail-polish. Primary antibodies used in this study were as follows: chicken-anti-GFP (Abcam, 1:1000), mouse-anti-Fasciclin III 7G10 (DSHB, 1:400), mouse-anti-Sema I 6F8 (DSHB, 1:1), mouse-anti-Sema II 19C2 (DSHB, 1:1), mouse-anti-chickadee 1J (DSHB, 1:1) and mouse-anti-Robo 13C9 (DSHB, 1:40). Secondary antibodies used in this study were as follows: donkey-anti-chicken cy3 (Jackson, 1:500), donkey-anti-chicken Alexa 647 (Jackson, 1:500), donkey-anti-mouse cy3 (Jackson, 1:250) and donkey-anti-mouse 28 Alexa 647 (Jackson, 1:250). All images were acquired on an Olympus FV1000 confocal microscope as TIFF files and Z-stacks. 29 Chapter 3 Results Axon guidance related genes are expressed in the adult Drosophila melanogaster nervous system Query results from a RNA-Seq Expression Profile search tool indicated that around 161 genes involved in ‘axon guidance’ (including 48 transcription factors) and 122 genes involved in ‘dendrite morphogenesis’ (including 66 transcription factors) are expressed in the adult Drosophila melanogaster nervous system. After excluding the transcription factors, these axon guidance related genes were categorised using Gene Venn (http://genevenn.sourceforge.net/) diagram into discrete expression profiles, namely very low, low, moderate, moderately high, and high. The results are summarised in Table 3.1. Table 3.1 Expression profile categories of axon guidance related genes in the adult Drosophila melanogaster nervous system Expression level ‘Axon guidance’ ‘Dendrite morphogenesis’ Very low 16 4 Low 37 52 Moderate 31 0 Moderately high 29 0 High 0 0 30 Gene Venn diagram was also used to compare the axon guidance related genes expressed in the embryo and those expressed in the adults. The results from this comparison showed that more than 96% of embryonic guidance cues continue to be expressed in the adult (summarised in Figure 3.1). Figure 3.1 Comparison study of axon guidance related genes expressed in Drosophila melanogaster in the embryo and adult nervous system. Considering those genes common to both of Biological process GO term categories, there were 151 genes expressed in the adult Drosophila melanogaster nervous system that are involved in either ‘axon guidance’ or ‘dendrite morphogenesis’. Out of this 151 genes, 44 were prioritized on the basis of their higher expression profiles and previously known roles in receptor activity and signalling during neuronal pathfinding. Throughout the rest of the study, these 44 genes will be mentioned as ‘axon guidance genes’. Based on the Molecular function GO terms provided on FlyBase, these 44 axon guidance genes were categorised as ‘receptor activity’, ‘microtubule/ actin binding’, ‘guidance ligand/ protein binding’ and ‘cell adhesion’ (Table 3.2). These 44 genes were further analysed for their roles in the survival and behavior of adult flies. 31 Table 3.2 Categories of axon guidance genes based on Molecular function GO terms Molecular function GO terms Axon guidance genes Receptor activity punt (put) roundabout (robo) frazzled (fra) baboon (babo) Syndecan (Sdc) starry night (stan) unc-5 (unc-5) plexin A (plexA) plexin B (plexB) wishful thinking (wit) Leukocyte-antigen-related like (Lar) Dystroglycan (Dg) Microtubule binding/ Actin binding APC-like (Apc) β-Spectrin (β-Spec) enabled (ena) short stop (shot) Molecule interacting with CasL (Mical) mini spindles (msps) bifocal (bif) chikadee (chic) Guidance ligands/ Protein binding Activin-β (Actβ) Sema-1a Sema-1b Sema-2a Sema-5c slit (sli) Lissencephaly (Lis-1) 32 Abl tyrosine kinase (Abl) WNK homolog (Wnk) dally-like (dlp) dawdle (daw) jelly belly (jeb) tolkin (tok) smooth (sm) Cell adhesion capricious (caps) beaten path Ic (beat-lc) myospheroid (mys) Neuroglian (Nrg) turtle (tutl) Neurotactin (Nrt) Fasciclin 1 (Fas 1) Fasciclin 3 (Fas 3) multiple edematous wings (mew) unzipped (uzip) Axon guidance genes are essential for survival in adults In the Gene Switch RNAi screen, knockdown of 15 out of the 44 genes showed significant reduction in the survival of males and 7 genes showed significant reduction in the survival of females. Of these positive hits, 5 genes caused significant reduction in the survival of both males and females. These results are summarised in Table 3.3. In the TARGET RNAi screen, knockdown of 32 out of the 44 genes showed significant reduction in the survival of males and 12 showed significant reduction in the survival of females. Of these positive hits, 11 genes were common to both sexes. These results are summarised in the Table 3.4. In both screens, multiple RNAi lines were used for each gene. The survival analysis curves from the RNAi knockdown that gave 33 statistically significant data are represented in Figures 3.2 and 3.3. Survival assay curves for the remaining RNAi lines can be found in the Appendix section of this thesis. Table 3.3 Summary of survival analysis of Gene Switch screen Abl Daw Msps Robo Sdc Actβ Dg Mical Sema-1a Tok Apc Ena Mew Sema-1b Tutl Babo Fas1 Mys Sema-2a Unc-5 Beat-Ic Fas3 Nrg Sema-5c Uzip Bif Fra Nrt Shot Wit Caps Jeb PlexA Sli Wnk Chic Lar PlexB Sm Β-Spec Dlp Lis-1 Put Stan No significant lethality in adults Significant lethality in adult males Significant lethality in females Significant lethality in both sexes Table 3.4 Summary of survival analysis of TARGET screen (see the above legend) Abl Daw Msps Robo Sdc Actβ Dg Mical Sema-1a Tok Apc Ena Mew Sema-1b Tutl Babo Fas1 Mys Sema-2a Unc-5 Beat-Ic Fas3 Nrg Sema-5c Uzip Bif Fra Nrt Shot Wit Caps Jeb PlexA Sli Wnk Chic Lar PlexB Sm Β-Spec Dlp Lis-1 Put Stan 34 a b c 35 d e f 36 g h i 37 Figure 3.2 Survival analysis curves from the Gene Switch screen. Data shown are mean + SEM. Each data point is the average of three replicates. ‘*’ denotes statistical significance from Control I and II (p < 0.05, two-way ANOVA and Tukey’s multiple comparison tests). Figures a, b, c, d, e, f, g show survival analysis curves of males. Figures h, i, j show survival analysis curves of females. Control I (Injection stock) varies with respect to the RNAi line. Injection stock for figures a, b, c, d, e, h, i is BDSC stock #36303. Injection stock for figure ‘f’ is BDSC stock #36304. Injection stock for figures g, j is VDRC stock #60000. Control II (GFP RNAi) used for figure ‘f’ is BDSC stock #9331 (on the 2nd chromosome) and for the remaining figures is BDSC stock #9330 (on the 3rd chromosome). j a 38 b c d 39 e f g 40 h i j 41 k l m 42 n o p 43 Figure 3.2 Survival analysis curves from the TARGET screen. Data shown are mean + SEM. Each data point is the average of three replicates. ‘*’ denotes statistical significance from Control I and II (p < 0.05, two-way ANOVA and Tukey’s multiple comparison tests). Figures a, b, c, d, e, f, g, h, i, j, k show survival analysis curves of males. Figures l, m, n, o, p show survival analysis curves of females. Control I (Injection stock) varies with respect to the RNAi line. Injection stock for figures g, h, i, j, k, o, p is BDSC stock #36303. Injection stock for figures d, e, f, n is BDSC stock #36304. Injection stock for figures a, b, l is VDRC stock #60000. Injection stock for figures c, m is VDRC stock #60100. Control II (GFP RNAi) used for figures a, b, c, d, e, f, l, m, n is BDSC stock #9331 (on the 2nd chromosome) and for the remaining figures is BDSC stock #9330 (on the 3rd chromosome). Almost all of the axon guidance genes that were knocked down and resulted in a significant reduction in the survival of males in the Gene Switch screen also reduced survival in the TARGET screen. The exception to this was Lis-1 which only showed reduced survival in the Gene Switch screen. The greater number of genes that showed reduced survival in the TARGET screen could be due to the heat-induced stress at 29°C. At the end of the survival assays on the 22nd day, there were 96.7% survival in all the negative controls, whereas the percentage survival in the knockdowns were significantly less. The lowest survival percentage was found in Chic knockdowns, which was 26.7%. In Sema-1a knockdowns, only 30% survival was observed. 33.3% of Fra and Sema-1b knockdowns survived until the end of the assays. The survival percentage in the other knockdowns were 36.7% in Sema-5c, 40% in PlexB, 53.3% in Abl, Dg, Msps, 56.7% in Fas3, 60% in Apc, 66.7% in Lis-1, PlexA, Robo, and 76.7% in Sema-2a. For our additional analyses, I decided to concentrate on those 15 genes from the Gene Switch screen that showed the greatest overlap with the TARGET screen. These 15 genes include Abl, Apc, Chic, Dg, Fas3, Fra, Lis-1, Msps, PlexA, PlexB, Robo, Sema-1a, Sema-1b, Sema-2a and Sema-5c. These 15 genes were roughly categorised into two groups based on the two-way ANOVA results from their survival analysis graphs: 1) Genes that showed significant reduction from approximately day 9 onwards (Sema-1a, Sema-1b, Sema-2a, Sema-5c, PlexA, PlexB, and Chic), and 2) Genes that showed significant reduction from approximately day 14 onwards (Abl, 44 Apc, Dg, Fas3, Fra, Lis-1, Msps, and Robo). Using these two time points as rough estimates of gene knockdown, I conducted all of our behavioral assays in two groups with one group starting at day 9 and the second group starting at day 15 after the initiation of RNAi. Axon guidance genes control adult climbing behavior in Drosophila The 15 axon guidance genes that were shown to be essential for adult survival in males were further analysed for their role in adult climbing behavior. Of these 15 genes, Sema-1a, Sema-1b, Sema-2a, Sema-5c, PlexA, PlexB, and Chic were assayed at day 9 after eclosion and initiation of knockdown. Whereas, Abl, Apc, Dg, Fas3, Fra, Lis-1, Msps, and Robo were assayed at day 14 after eclosion. During the climbing assays, number of flies that crossed the 7.5cm line in 8 seconds were counted. All of the 15 axon guidance genes tested showed a significant climbing defect (Figure 3.4). In day 9 adults, Chic knockdowns had the most severe climbing difficulty. Only 10% of the total number of flies could climb all the way. Flies that failed to climb to the endpoint line were either very slow or did not start climbing even after tapping the vials. In the control flies, success of climbing ranged from 87% to 94%. The success rates in other knockdown flies were 37.8% in PlexA, 36.7% in PlexB, 72.2% in Sema-1a, 37.7% in Sema-1b, 63.3% in Sema-2a, and 58.8% in Sema-5c. In case of day 14 adults, Dg knockdowns were the most severely affected. Only 16.7% of the Dg knockdown flies were marked a success. The climbing success in the other knockdowns were 32.2% in Abl, 31.1% in Apc, 44.4% in Fas3, 21.2% in Fra, 21.1% in Lis-1, 27.8% in Msps, and 33.3% in Robo. 45 Figure 3.4 Effect of knockdown of axon guidance genes (using elavGS) on adult climbing behavior. Data shown are mean + SEM. *Significant from the respective controls (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests) Since a significant decline in the adult climbing behavior was observed after knockdown using a pan-neuronal driver (Elav-Gene Switch), I tested whether knocking down these genes in motor neurons would induce a similar climbing behavior phenotype. The climbing assay was repeated after knocking down these axon guidance genes using a glutamatergic neuron specific GAL4 driver line (UAS.Dicer; OK371-GAL4; tubGAL80ts, UAS nGFP). The flies were grown at 18°C until eclosion and switched to 29°C from day 1 adulthood. The climbing assays were performed at 29°C at day 9 or day 14, depending on the gene being knocked down. All 15 genes again showed defects in adult climbing behavior (Figure 3.5). When comparing the severity of climbing defects caused by the knockdown of each gene using the ElavGS as well as OK371-Gal4, only Chic and Apc showed lesser severity in their climbing defects in the OK371-GAL4 experiments than the ElavGS experiments. For the remaining 13 genes, there was no significant difference in the severity of climbing phenotype between the two GAL4 drivers. 46 Figure 3.5 Effect of knockdown of axon guidance genes (using OK371-GAL4) on adult climbing behavior. Data shown are mean + SEM. *Significant from the respective controls (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests) Axon guidance genes are required for normal adult activity Since a decline in the adult climbing behavior was observed after the knockdown of axon guidance genes, I examined the mobility of adult flies for a longer period of time. For this, a DAM system was used to monitor flies in individual channels for 3 days. Knockdown was carried out using Gene Switch and the DAM system experiments were conducted at 25°C on a 12 hours dark/ 12 hours light cycle. Similar to the climbing assay, Sema-1a, Sema-1b, Sema-2a, Sema-5c, PlexA, PlexB, and Chic were assayed from day 9 after eclosion. Whereas, Abl, Apc, Dg, Fas3, Fra, Lis-1, Msps, and Robo were assayed from day 14 after eclosion. The raw data from the DAM system were processed using the actmon R package to assess the following: 1) Activity (counts): an absolute measure of the locomotor activity of the flies. The activity count equals the number of beam crosses per 12 hour time period. 47 2) Inactive time duration (minutes): the cumulative amount of inactive time in a 12 hour period measured in minutes. An episode of continuous immobility lasting for at least five consecutive minutes is considered as an inactive episode or bout. 3) Activity index (AI) (counts/minutes): a relative measure of locomotor health. AI is calculated by dividing the total activity counts by the total time spent active in a 24 hour period. This is a way to normalize activity among flies with different total sleep durations. (Gilestro, G. F., 2012) Activity and inactive time duration were calculated separately for light and dark cycles (per 12 hours). AI was calculated for a 24 hour period. For the presentation of the results, I categorized flies into 4 groups based on the age of analysis (day 9 and day 14) and the injection stock controls. Different injection stocks were used as controls due to the different RNAi libraries. Activity plots for the day 9 old flies with injection stock control BDSC #36303 are shown in Figure 3.6. Chic knockdown flies showed significantly lower activity counts than the control throughout the day (both light and dark cycles). Sema-1a knockdown flies showed significantly lower activity counts only during the dark cycle. Interestingly, Sema2a knockdown, PlexA knockdown and PlexB knockdown flies exhibited higher activity counts than the control during the dark cycle (Figure 3.6). 48 Figure 3.6 Activity plots for day 9 old flies with injection stock control BDSC #36303. This group 49 includes Sema-1a, Sema-1b, Sema-2a, PlexA, PlexB and Chic. In the first figure, each activity plot of a genotype is the average activity of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the activity counts. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). Inactive time plots for the day 9 old flies with injection stock control BDSC #36303 are shown in Figure 3.7. Sema-1a knockdown flies remained more inactive than the control throughout the day (both light and dark cycles). Sema-1b knockdown and Chic knockdown flies remained more inactive only during the light cycle. The reduced activity of Chic flies was also apparent when examining the activity index (Figure 3.8). In contrast, the activity indices of Sema-2a knockdown, PlexA knockdown and PlexB knockdown flies were significantly higher than control (Figure 3.8). 50 Figure 3.7 Inactive time plots for day 9 old flies with injection stock control BDSC #36303. This group includes Sema-1a, Sema-1b, Sema-2a, PlexA, PlexB and Chic. In the first figure, each time plot of a genotype is the average of the percentage of inactive time duration of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the total inactive time durations. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). 51 Figure 3.8 Activity indices for day 9 old flies with injection stock control BDSC #36303. This group includes Sema-1a, Sema-1b, Sema-2a, PlexA, PlexB and Chic. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). Activity plots for the day 14 old flies with injection stock control BDSC #36303 are shown in Figure 3.9. Robo knockdown flies showed significantly higher activity counts than the control throughout the day (both light and dark cycles). Abl knockdown flies showed significantly higher activity counts only during the dark cycle. Interestingly, no knockdown flies had significantly lower activity counts than the control. 52 Figure 3.9 Activity plots for day 14 old flies with injection stock control BDSC #36303. This group 53 includes Abl, Apc, Dg, Fra, Lis-1 and Robo. In the first figure, each activity plot of a genotype is the average activity of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the activity counts. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). Inactive time for the day 14 old flies with injection stock control BDSC #36303 are presented in Figure 3.10. Robo knockdown flies remained significantly less inactive than the control throughout the day (both light and dark cycles). Abl knockdown flies exhibited less inactive time only during the dark cycle. Again, no knockdown flies had significantly higher inactive time duration than the control. Activity indices are plotted in Figure 3.11. As expected from the activity and inactive time pots, Abl knockdown and Robo knockdown flies exhibited higher activity indices than the control. 54 Figure 3.10 Inactive time plots for day 14 old flies with injection stock control BDSC #36303. This group includes Abl, Apc, Dg, Fra, Lis-1 and Robo. In the first figure, each time plot of a genotype is the average of the percentage of inactive time duration of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the total inactive time durations. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). 55 Figure 3.11 Activity indices for day 14 old flies with injection stock control BDSC #36303. This group includes Abl, Apc, Dg, Fra, Lis-1 and Robo. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). Fas3 RNAi was the only RNAi used for this experiment from the GD library of VDRC. The control used in this study was the injection stock VDRC #60000. The activity plots for Fas3 knockdown flies are given in Figure 3.12. Fas3 knockdown flies exhibited significantly lower number of activity counts than the control throughout the day (both light and dark cycles). However, the Fas3 knockdown flies spent significantly more time inactive only during the light cycle (Figure 3.13). There was no significant difference in the activity indices (Figure 3.14). 56 Figure 3.12 Activity plots for day 14 old flies with injection stock control VDRC #60000. This 57 group includes Fas3. In the first figure, each activity plot of a genotype is the average activity of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the activity counts. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). 58 Figure 3.13 Inactive time plots for day 14 old flies with injection stock control VDRC #60000. This group includes Fas3. In the first figure, each time plot of a genotype is the average of the percentage of inactive time duration of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the total inactive time durations. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). 59 Figure 3.14 Activity indices for day 14 old flies with injection stock control VDRC #60000. This group includes Fas3. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). For experiments with Msps RNAi, the control used was the injection stock BDSC #36304. The injection stock was different from the rest of the BDSC RNAi lines due to the difference in the chromosomal insertion sites. The activity plots for Msps knockdown flies are given in Figure 3.15. Msps knockdown flies exhibited significantly lower number of activity counts than the control during the light cycle. However, there was no significant difference in the duration of time spent inactive (Figure 3.16). Msps knockdown flies also had lower activity indices than the control flies (Figure 3.17). 60 Figure 3.15 Activity plots for day 14 old flies with injection stock control BDSC #36304. This 61 group includes Msps. In the first figure, each activity plot of a genotype is the average activity of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the activity counts. 62 Figure 3.16 Inactive time plots for day 14 old flies with injection stock control BDSC #36304. This group includes Fas3. In the first figure, each time plot of a genotype is the average of the percentage of inactive time duration of multiple flies from 3 days. The grey portion (0-12 hours) represents dark cycle and the white portion (12- 24 hours) represents the light cycle. The second figure is a bar graph showing the differences in the total inactive time durations. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). 63 Figure 3.17 Activity indices for day 14 old flies with injection stock control BDSC #36304. This group includes Msps. Data shown are mean + SEM. *Significant from the control (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests). Video-assisted movement tracking In order to more closely assess mobility of individual flies after gene knockdown, I used a movement tracking system to examine velocity, distance and exploration behaviors of individual flies. Knockdown was carried out using Gene Switch and the video tracking experiments were conducted at 25°C at either 9 days after eclosion or 14 days after eclosion (as described above). The raw data from the videos were processed using fly_tracker’- a collection of MATLAB algorithms. A region of interest in the captured video was analysed to extract multiple parameters including position traces, heat maps, average velocities and distance travelled by the fly. The average velocities and the distance travelled by the knockdown flies were compared and studied. Only Chic knockdown flies showed a significantly lower average velocity (Figure 3.18) and total distance travelled (Figure 3.19). The average velocity of the Chic knockdown flies were 2.74 mm/sec whereas that of the controls were 4.35 mm/ sec. The mean distance travelled by the Chic knockdowns in 90 seconds were 97.42mm whereas that of the controls were 464.82 mm. 64 Figure 3.18 Effect of knockdown on the average velocity in a video-assisted movement tracking experiment. Data shown are mean + SEM. *Significant from the respective controls (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests) Figure 3.19 Effect of knockdown on the distance moved in a video-assisted movement tracking experiment. Data shown are mean + SEM. *Significant from the respective controls (p < 0.05, one-way ANOVA and Tukey’s multiple comparison tests) 65 The heat maps of positions of the controls and the knockdown flies were extracted to study the positional preference of the flies and examine any specific walking pattern. The heat maps are shown in Figure 3.20 and Figure 3.21. In day 9 old flies, a significant difference was observed in the heat maps of Chic knockdown flies. There was a spot in the top center of the tracking arena where the flies spent most of their time. The heat map shows significantly less area covered by the Chic knockdowns compared to the controls. There is also some noticeable difference in Sema-2a and Sema-5c knockdowns. Figure 3.20 Heat maps of the positions of day 9 old flies from a video-assisted movement tracking experiment. Control I is the injection stock BDSC #36303. In day 14 old flies, noticeable differences can be observed in the heat maps of Abl, Apc, Fra, Msps and Fas3 knockdown flies, when compared to their respective controls (Figure 3.21). 66 is VDRC #60000. Figure 3.21 Heat maps of the positions of day 14 old flies from a video-assisted movement tracking experiment. Control I is injection stock BDSC #36303, Control II is BDSC #36304 (control for Msps RNAi) and Control III is injection stock VDRC #60000 (control for Fas3 RNAi). Expression patterns of axon guidance proteins in the adult CNS In order to unravel the changes in the neural circuitry that may underlie the behavioral changes observed when I knocked down guidance genes in the adult nervous system, antibodies and protein trap lines were used to determine the expression patterns of different axon guidance 67 proteins in the adult CNS. Antibody staining for Sema-1a, Chic, Robo appeared non-specific and widespread whereas protein trap lines for Abl, Fra, Sema-2a were inconclusive (images not shown). Protein trap line for Sema-1a showed expression in brain and ventral nerve cord (VNC) regions that looks like cell bodies (Figure 3.22). Protein trap lines and antibody staining of Fas3 expression was reproducible and showed similar expression patterns in the antennal lobes, optic lobes, sub-oesophageal ganglion (SOG) and three thoracic segments of the VNC (Figure 3.23). Fas3 expression was compared to various other GAL4 driver line expressions in order to identify the specific neuron types. Fas3 expression was found similar, but not identical, to Gustatory receptors 5a (Gr5a) and 64f (Gr64f) (Figure 3.24) and pickpocket 28 (ppk28) (image not shown) expression patterns in the adult CNS, indicating that additional GAL4 lines will need to be explored to knockdown Fas3 in the precise neurons where it is expressed. Figure 3.22 Sema-1a expression pattern in the adult CNS. (a), (b), (c) are immunostaining images of Sema-1a protein trap line (BDSC #60140). (a) GFP tagged Sema-1a, no primary antibody (b) GFP tagged Sema-1a + ck α GFP primary antibody (Abcam, 1:1000) (c) Merged image. All three images are of the same tissue (adult male CNS). Scale bar represents 50µm. 68 Figure 3.23 Fas3 expression pattern in the adult CNS. (a) Fas3 protein trap line (BDSC #59809) adult male CNS expressing GFP tagged Fas3 (green) and nc82 (purple. (b) m α Fas3 (DSHB, 1:400) expression in adult male CNS. Scale bar represents 50µm. Figure 3.24 Comparison of Fas3 expression to other GAL4 driver line expressions in the adult CNS. (a) Gr5a expression pattern (Gr5a-LexA-VP16 driving LexA-op::CD2 GFP) compared to Fas3 expression (m α Fas3). (b) Gr64f expression pattern (Gr64f-GAL4 driving UAS-GCaMP6f) compared to Fas3 expression (m α Fas3). (c) SOG region from ‘a’ enlarged (dotted line). (d) SOG region from ‘b’ enlarged (dotted line). Scale bar represents 50µm. 69 Chapter 4 Discussion Axon guidance cues are extracellular signals that direct the growth and steering of neuronal growth cones. Work in the past few decades has led to the discovery of numerous axon guidance genes and identified their functions during development. Current research indicates that many of these proteins and their receptors are expressed in the mature nervous system as well. The expression of these genes in the adult indicates that there are likely additional roles for guidance cues beyond the initial phase of neuronal process outgrowth, growth cone navigation, and target innervation. This study has begun to elucidate the functions of axon guidance genes in adult Drosophila, and the impact that they have on the survival and mobility of adult flies. This study is the first to perform an RNAi screen of axon guidance genes expressed in the mature nervous system to determine their role in the maintenance of neural circuits. The major findings from this study and their implications are discussed below. Expression of developmental axon guidance genes in adulthood has functional relevance Using various bio-informatics tools, I have compared the axon guidance gene expressions of Drosophila in the embryonic (10-24 hours) and adult (day 1- day 20) stages. The results showed that more than 96% of embryonic guidance cues continue to be expressed in the adults as well. The expression levels of some genes have changed (that is, decreased or increased) whereas some of them retain their embryonic expression levels. Gene expression studies in adult mouse brain suggest that although, the expression patterns of many genes change dramatically during development, the brain retains a degree of the embryological transcriptional imprint that is important for the maintenance of established units in the adult brain (Zapala et al., 2005). Gene expression studies in monkeys have identified sets of genes that are important equally for the 70 formation as well as the maintenance and plasticity of connections in the adult thalamus (Murray et al., 2007). A recent study has also shown that the genes involved in neuronal development and axon guidance have the highest correlation with neuro-anatomical connectivity in the adult brain (French and Pavlidis, 2011). All these imply that the expression of axon guidance genes in the adulthood are functionally relevant. It is tempting to speculate that the roles of these axon guidance genes in the adult are similar to their developmental roles and that they maintain the established neural circuits. Axon guidance genes are required for survival Bio-informatics search revealed expression of 151 axon guidance genes in the adult Drosophila nervous system. Of these, 44 genes were prioritized based on their higher expression profiles and their previously known roles in signalling during neuronal pathfinding. The main goal of this study was to screen for axon guidance genes that are essential and have functional roles in the adult as well. After prioritizing the 44 axon guidance genes, I was interested to see how many among these would be essential for adult survival. This screen was carried out using two systems, Gene Switch and TARGET, to assess the necessity of a particular gene for survival. In the Gene Switch screen, knockdown of 15 axon guidance genes caused significant reduction in the survival of males, and 7 in females, of which 5 were common to both sexes. There was 96.7% survival in the controls, whereas survival in the male knockdown flies ranged from 76.7% to 26.7%. This clearly implies that that these 15 axon guidance are required for adult survival. Adult survival was more affected in the males than females. This could be because Drosophila males have a super-active X chromosome due to increased acetylation at K16 residue of histone H4 (Krebs et al., 2009). This causes transcriptional activation and upregulation of the UAS.Dicer2 on the X chromosome, which in turn enhances RNAi potency (Dietzl et al., 2007). The two genes that caused significant reduction only in females (Mys and Shot) could be either false positives or that these two genes act differently in males and females. There were far more positive hits in the TARGET screen (32 in males, 12 in females including 11 in both sexes). This could be due to the heat induced stress at 29°C. It is important to note that all 15 axon guidance genes that caused significant reduction in the survival of adult males in the Gene Switch screen, also caused the same 71 in the TARGET screen (with an exception of Lis-1). Mutations in these 15 axon guidance genes are previously known to cause lethality during development. This implies that axon guidance genes are essential throughout the lifespan of Drosophila, from development through adulthood. Loss of axon guidance genes causes aberrant mobility The reduction in adult survival could result from the dysfunction of any number of neuron types in the adult nervous system. It is presumed that the functions of the affected neurons is compromised before the flies die. To narrow down the range of neuron types that could underlie premature death, I used a more subtle assay to examine whether their mobility was affected. The resulting behaviour of knockdown in male flies were studied using a climbing assay, a Drosophila Activity Monitor (DAM) system, and a video-assisted movement tracking assay. The climbing and mobility of knockdown flies were significantly affected. Since the RNAi-mediated knockdown was restricted to adult flies, these locomotor phenotypes are not developmental defects, but are more likely due to defects in the maintenance of neuronal circuits. Recent studies have started to uncover the mechanisms underlying maintenance of neuronal integrity. It has been shown that cell autonomous neuronal activity along with Wnt signalling and downstream Glycogen synthase kinase-3β (Gsk-3β) function is required for the maintenance of adult neuron morphology and survival in the Drosophila olfactory system (Chiang et al., 2009). Gsk-3β is a known regulator of the microtubule cytoskeleton and neuron morphology (Salinas, 2007). Similarly, I propose that axon guidance gene knockdown may affect one of many different signalling pathways that regulate the microtubule and actin cytoskeleton and thus modulating neuron morphology. Another possible explanation for the locomotor phenotypes is that the knockdown could be affecting muscle function, possibly by having a potential role at NMJs (Perkins and Tanentzapf, 2014). The activity/rest cycles of the adult flies were studied using the DAM system after gene knockdown. Chic, Sema-1a, Robo and Fas3 knockdown flies showed significant difference in their activity/rest cycles throughout the day. Whereas the activity/rest cycles of Abl, Sema-2a, Plex A and Plex B knockdowns were affected only during the dark cycle and that of Sema-1b and Msps 72 only during the light cycle. These differences in the activity/rest cycles may be attributed as a locomotor defect or as a defect in circadian rhythm. For example, Abl knockdowns showed increased activity and also decreased rest duration during the dark cycle. This implies that their sleep rhythm may be affected. This could also be true for Robo knockdowns that had higher baselines of activity than the control flies throughout the day. Robo mutants have been previously shown to alter the pace of their circadian clock (Berni et al., 2008). Sema-2a, Plex A and Plex B also showed higher activity levels than their controls. Such abnormally high levels of activity and decreased consolidated rest have been reported in previous studies. The transitions between the physiological states of sleep and wakefulness or rest and activity are shown to be mediated through various signalling pathways (Hendricks et al., 2003; Kume et al., 2005; Gilestro et al. 2009; Ishimoto and Kitamoto, 2010). Knockdowns of Sema-1a, Sema-1b, Chic, Fas3 and Msps have lower activity levels and increased rest durations than control flies. Sema-1a, Sema-1b and Fas3 knockdown flies remained hypoactive because they have shorter periods of activity. Lower activity indices of Msps and Chic knockdown flies imply that these flies were less active in any given activity period, meaning that their mobility was affected. Periods of rest in Drosophila are considered behaviorally similar to mammalian sleep. One of the proposed functions for rest (sleep) is to remove undesirable by-products that accumulate during the active (awake) state (Hartmann, 1973). Hence, it can be implied that rest may be essential for fly survival. Mutations in the genes that have been implicated in the regulation of rest, have also caused shortening of lifespan in adult flies (Shaw et al., 2002; Hendricks et al., 2003). The shortened lifespan of adult flies observed following the knockdown of axon guidance genes may be attributed to this. Another interesting function of sleep is that it contributes to the modulation of synapses and neural plasticity (Benington and Frank, 2003). This supports the recent finding that the expression levels of several synaptic marker proteins in the Drosophila brain are altered as a function of activity/ rest cycles (Gilestro et al. 2009). It may be implied that axon guidance genes maintain the structure and function of the adult neurons by modulating their synapses and that any dysfunction in this may be affecting the lifespan of the fly. 73 Axon guidance genes may be acting on motor neurons A decline in climbing ability was observed in adult flies following the knockdown of axon guidance genes using a pan-neuronal GAL4 driver. This phenotype was reproduced using a glutamatergic neuron specific GAL4 driver (OK371-GAL4) as well. All motor neurons in Drosophila are glutamatergic neurons. This could imply any of the two things: axon guidance proteins act cell autonomously on motor neurons or that motor neurons express receptors that respond to a secreted guidance cue released from other neurons. The expression of Sema-1a observed in protein trap lines suggest that Sema-1a may be expressed in motor neurons. Previous studies have shown that the transmembrane Sema I is required for motor axon guidance (Yu et al., 1998). Abl, Chic and Fas3 are the other axon guidance proteins implicated in axonal pathfinding and synaptic recognition of motor neurons during development (Elkins et al., 1990; Van-Vactor et al., 1993; Wills et al., 1999; Chiba et al., 1995). Future work and directions Future work includes identifying a subset of neurons that express axon guidance genes and studying their morphology on an RNAi background. Specific GAL4 driver lines can be used to drive RNAi of the gene of interest and a neuronal membrane marker that will help us study the effect of knockdown on circuit morphology. Analysing the dendrite and axonal morphologies, NMJs and synaptic stability will reveal the perturbations in the maintenance of neural circuits when guidance genes are knocked down in adulthood. Conclusion The main goal of this study was to screen for axon guidance genes that have functional roles in adulthood. I believe that this goal has been accomplished. I have identified 15 axon guidance genes that are essential for survival and normal behavior (climbing, locomotion, rest/activity cycle) in adult Drosophila. These axon guidance genes are implicated in the 74 maintenance of neural circuits underlying these phenotypes. Further studies on circuit morphology are required to understand better how axon guidance genes contribute to the maintenance of neuronal structure in adult brains. 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Survival curves from the Gene Switch screen for male and female flies are presented in Figures A.1 and A.2, respectively. Survival curves for male and female flies from the TARGET screen are presented in Figures A.3 and A.4, respectively. 94 95 96 Figure A.1 Survival analysis curves for male flies from the Gene Switch screen. The control for each group is the injection stock and is shown as attP2, attP40, GD or KK. Data presented are mean + SEM. 97 98 99 Figure A.2 Survival analysis curves for female flies from the Gene Switch screen. The control for each group is the injection stock and is shown as attP2, attP40, GD or KK. Data presented are mean + SEM. 100 101 102 Figure A.3 Survival analysis curves for male flies from the TARGET screen. The control for each group is the injection stock and is shown as attP2, attP40, GD or KK. Data presented are mean + SEM. 103 104 105 Figure A.4 Survival analysis curves for female flies from the TARGET screen. The control for each group is the injection stock and is shown as attP2, attP40, GD or KK. Data presented are mean + SEM.