Axon guidance in the zebrafish visual system analysis of esrom mutant

AXON GUIDANCE IN THE ZEBRAFISH VISUAL SYSTEM: ANALYSIS OF THE esrom MUTANT JASMINE JOYCE D’SOUZA (M.Sc. University of Mumbai) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2005 i ACKNOWLEGEMENTS I would like to thank my supervisor, Dr. Suresh Jesuthasan, for his excellent guidance and encouragement. His constant support made this thesis possible. My heartfelt thanks also go to my Ph.D committee members, Dr Karuna Sampath, Dr. Peng Jinrong and Dr. Xiaohang Yang for their excellent advice. I would also like to thank all Principal Investigators at Temasek Life Sciences Laboratory for their scientific feedback during annual work presentations. Special thanks to the administrative staff for their help as well as Temasek Life Sciences Laboratory for their Funding. I would like to thank all members of the Developmental Neurobiology group. Special thanks to Michael Hendricks and Sylvie Le Guyader. My heartfelt thanks also go to Sivan Subburaju, Song Choon and He Fang for their help in research work. I would like to Dr. Alan Coulson form the Wellcome Trust Sanger Institute for his help is sequencing zebrafish genome. Special thanks to my family, especially my mother for her patience in waiting for my Ph.D. degree. Thanks to all my friends. ii DEDICATION This thesis is dedicated to my father and mother. iii Table of Contents Chapter I: General introduction 1.1 Vertebrate nervous system 1.2 Neurogenesis 1.3 Steps involved in the formation of neural network 1.4 Mechanism of axon guidance 1.5 Axon guidance molecules 1.5.1 Netrin 1.5.2 Ephrins 11 1.5.3 Slits 16 1.5.4 Semaphorins 19 1.5.5 Other guidance molecules 23 1.6 Cell adhesion molecules 24 1.6.1.1 NCAM 24 1.6.1.2 L1/ NILE/ NgCAM/ G4/ 8D9 25 1.6.1.3 Neuroglian 26 1.6.1.4 Nr-CAM 26 1.6.1.5 TAG-1/axonin 27 1.6.1.6 Contactin/F3/F11 27 1.6.1.7 DM-GRASP/BEN/IrreC 28 1.6.1.8 Dscam 28 1.6.2 Cadherins 29 1.6.3 SAM 30 iv 1.7 Growth Cone Machinery 33 1.8 Integrating guidance signals 35 Chapter II: Materials and Method 40 2.1 Fish stock maintenance 40 2.2 DNA extraction from single embryos 40 2.3.1 Bulked segregant analysis (BSA) 41 2.3.2 Mapping 41 2.4 Polymerase Chain Reaction 41 2.5 Genomic Library Screening 42 2.6 Pulse field gel electrophoresis and DNA fingerprinting 43 2.7 Genome walk 43 2.8 Construction of an eye cDNA library 44 2.9 cDNA library array 46 2.10 cDNA library screening 47 2.11 Sequencing 47 2.12.1 RT-PCR 48 2.12.2 Single-Cell RT PCR 49 2.13 RNA In-situ hybridization 50 2.14 DNA injections in zebrafish embryo 51 2.15 Gene knockdown using morpholino 52 2.16 DiI labeling of the eye 53 2.17 Motor neuron labeling 53 2.18 3′ and 5′ RACE 53 v 2.19 Cloning of large genes 54 2.20 Purification of GST fusion protein 55 2.21 Western Blot 56 2.22 E3 ligase Assay 57 2.23 Retinal culture 57 2.24 Immuno-Histochemistry 58 2.25 Preadsorbtion and antibody specificity 58 2.26 Ligand Binding Assay 59 2.27 Generation of Dominant-Negative esrom construct 59 2.28 Assay with LPA 60 Chapter III: Characterization of the mutant 63 3.1 Introduction 63 3.2 Isolation of the mutant 66 3.3 Results 68 3.4 Esrom gene function is required in the eye 75 3.5 Positional identity in the retina 78 3.6 Discussion 78 Chapter IV: Positional cloning of esrom 82 4.1 Introduction 82 4.2 Genetic mapping and isolation of closely linked markers 83 vi 4.3 Genomic walk 92 4.4 Defining a critical interval 92 4.5 Gene discovery cloning of the gene 99 4.6 Cloning of esrom 102 4.7 Discussion 106 Chapter V: Localization and molecular characterization of Esrom 110 5.1 Introduction 110 5.2 Expression and localization of esrom 110 5.3 Esrom acts as an E3 ligase 115 5.4 Esrom is involved in signal transduction 117 5.5 Generating a Dominant-negative fragment 119 Chapter VI: General Discussion 122 Chapter VII: References 131 vii Summary During development of the visual system, precise connections are formed between the eye and the brain. Retinal ganglion cells (RGCs) are the only neural cells present in the retina that send connections to a specific region in the brain known as the optic tectum. A spatial correlation is maintained between the position of the RGCs within the retina and the termination zone on the optic tectum. This highly ordered connection gives rise to a retino-tectal map. Little is known about the molecules that orchestrate the formation of a retino-tectal map. In an effort to understand this process better, a large-scale mutant screen was carried in zebrafish. This has led to the isolation of a mutant called esrom. This study involves the identification and characterization of the esrom gene. Analysis of the zebrafish mutant esrom shows that it is required for axon fasciculation, targeting, branching and skin pigmentation. In the visual system esrom is necessary for mapping along the A-P and D-V axes, correct innervation of the pretectal targets and axon bundling in the optic tract. The gene function is required in the eye and not in the brain for retinal ganglion cell (RGC) targeting. The mapping of esrom revealed that there is a strong synteny between the esrom locus and human chromosome 13 q22.3. Using positional cloning, esrom was established to encode a very large protein homologous to human PAM (Protein Associated with Myc)/ Drosophila Highwire/ C. elegans Regulator of Presynaptic Morphology 1. The cranofacial motoneurons in esrom mutants have expanded synapses at the neuromuscular junction like in Drosophila hiw mutants, and branch ectopically. However RGC axon branching appears normal in size, unlike in hiw, viii indicating that esrom has a different function in the vertebrate visual system. This large multidomain protein has an E3 ligase activity. Esrom is ubiquitously expressed in the embryo unlike its invertebrate homologues HIW/RPM-1. In the RGCs it is required for proper activation of the p38 MAPK signal transduction pathway in response to lysophosphatydic acid (LPA). Esrom is also known to regulate phosphorylated Tuberin, a tumor suppressor within the growth cones. These data suggest that esrom might act as a molecular switch that integrates signal transduction events in axons. ix List of Tables Table1: Genetic linkage of esrom 91 Table 2: Clones obtained from eye cDNA library screening 94 Table 3: RNA injection results 97 Table 4: Morpholino1 (RCC1 morpholino) results 99 Table 5: Morpholino2 (B-box morpholino) results 101 Table 6: Standard morpholino control results 101 x LPA induced activation of the p38 MAPK signaling pathway in esrom RGC axons is abnormal. The activation of the MAPK cascade leads to the phosphorylation of appropriate target substrates in reponse to extracelluar signaling (Shi and Gaestel, 2002). In neutrophils the p38 signaling cascade is required for chemotaxis (Cara et al., 2001). In leucocytes activation of p38 MAPK by selectin and its ligand have an impact on downstream event of leucocyte rolling, including adhesion and directed migration (Smolen et al., 2000; Cara et al., 2001). Esrom may regulate axon fasciculation by controlling the activation of p38 phosphorylation. In retinal ganglion cells it is well established that axon guidance molecules activate the MAPK cascade which in turn regulates protein synthesis and protein degradation (Campbell and Holt, 2003). As LPA induced growth cone response requires only protein degradation, it is tempting to speculate that the substrate which Esrom ubiquitinates is required for p38 activation. We currently lack data to demonstrate that E3 ligase activity of esrom is absolutely essential for establishment of retino-tectal phenotype although the B-Box morpholino data supports the hypothesis. MAPK is not only required for axon steering but also for axon resensitization which helps the growth cone to readjust sensitivity over a concentration gradient of a guidance factor. Interestingly the C terminus end of PAM binds to Tuberin, a tumor suppressor (Murthy et al., 2004). In esrom mutants there is a marked elevation in the levels of ser939phosphorylated Tuberin (D'Souza et al., 2005). This could be either due to misregulated intracellular signaling or accumulation of Tuberin in the axons since Tuberin is also degraded by ubiquitin mediated proteolysis (Plas and Thompson, 2003). Both 127 phosphorylatated Tuberin and phosphorylated p38 MAPK activate signal transduction pathways that converge at the phosphorylation of eIF4E a key component of the translation initiation machinery (Knauf et al., 2001; Tee et al., 2002; Campbell and Holt, 2003; Tee et al., 2003; Tee et al., 2003; Duncan et al., 2005). Finally in mammalian cells, the membrane localization of PAM has been shown to be regulated by signaling downstream of G protein coupled receptors that recognize the phospholipid sphingosine 1-phosphate (Ehnert et al., 2004). This is essential for longterm inhibition of adenylyl cyclase by PAM. In summary, Esrom can regulate several different signal transduction pathways that are known to be involved in axon guidance. By influencing phosphorylation of p38 MAPK. Esrom may modulate the signaling through netrin-1, BMPs, Dscam and ephrin B1 (Iwasaki et al., 1999; Campbell and Holt, 2001; Xu et al., 2003; Li and Guan, 2004; Zuzarte-Luis et al., 2004). By regulating phosphorylated Tuberin it may alter signaling through mTOR and influence protein synthesis (D'Souza et al., 2005). Eph-A2, a receptor tyrosine kinase involved in topographic mapping has been shown to be under translational regulation in axons (Brittis et al., 2002). TOR pathway also regulates Rho activation thereby altering cell adhesion and migration that could potentially affect axon fasciculation. Activation of G protein coupled receptors may recruit the rapid localization of PAM/Esrom to the plasma membrane resulting in the inhibition of adenylyl cyclase activity, causing a change in the cytosolic cAMP concentration (Pierre et al., 2004). cAMP being a secondary messenger may influence the growth cone steering (Song et al., 1997). Alternatively Esrom being an E3 ligase may directly cause rapid ubiquitination of 128 129 its substrate leading to its modification or degradation in the growth cones. Rapid, localized protein turnover in the axons modulates chemotropic response (Campbell and Holt, 2001). While navigating, growth cones continually assess the guidance molecules present in the environment. 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Dev Biol 272:39-52. 142 [...]... expressed by the axons that navigate the midline (Kidd et al., 1998) Longitudinal axons which express Robo never cross the midline but in robo mutants these axons freely cross the midline suggesting that there is a midline repellent cue sensed by Robo Similarly commisural axons which cross the midline once do not recross the midline again but in robo mutants they recross the midline Two lines of evidence... and several axon guidance molecules The ECM include heparin binding growth associated molecule (HB-GAM), glycoproteins like laminin, tenascin, fibronectin, vitronectin, thrombospondin and glycoaminoglycans like heparin sulfate and hyaluronate The CAMs include N-Cadherin, L1 family of molecules, NCAM, axonin/TAG-1 and contactin The neurotrophic factors include nerve growth factor (NGF), brain derived... tyrosine kinases with sequence identities of approximately 60-90% in the kinase domain and approximately 30-70% in the extracellular domain Their ligands, the ephrins are also membrane bound All ligands share a conserved core sequence of about 125 amino acids, including four invariant cysteine residues probably corresponding to receptor binding domain This is followed by the anchorage domain The ephrin A... establishing a retino-tectal topographic map Although there is no direct evidence of c-Myc in axon guidance, it might indirectly regulate molecules involved in axon guidance The role of Eph related receptors is diverse in other neuronal types including motor neurons which express the receptors only in a subset of neurons (O'Leary and Wilkinson, 1999) They are also required for targeting the vomeronasal axons... develops to form the brain, spinal cord and eye; components of the central nervous system (Sporle and Schughart, 1997) The neural plate on the dorsal side of the embryo buckles in at the midline to give rise to the neural tube (Fig 1.1) The dorsal region of the neural tube forms the roof plate by closure of the dorsal tips of the neuroectoderm whereas the ventral region forms the floor plate The floor plate... identified including two Drosophila homologues netrin A and netrinB (Harris et al., 1996) and zebrafish homologues netrin1, netrin-2, and netrin-4 (Chisholm and Tessier-Lavigne, 1999) The role of Netrin has been evolutionarily conserved in guiding axons towards the midline The N terminus contains a signal peptide followed by domains of type V and VI B1 laminin chain, three predicted EGF repeats and a C terminal... done in the visual system (Flanagan and Vanderhaeghen, 1998; O'Leary and Wilkinson, 1999) The retino-tectal projections from the RGCs terminate orderly in the tectum in a manner that maintains their neighborhood relationship In chick retina, the Eph-A receptors and ephrin-A ligands were found to have graded distribution along the anterior posterior axis within the retino-tectal system, confirming Sperry’s... Steps involved in the formation of neural network One of the earliest steps in the development of the central and peripheral nervous system is the initiation of axon outgrowth from the newly born neurons Once differentiated, neurons send out long processes called axons and several short processes called dendrites Axons travel long distances in order to find their right target to synapse The growing axons... the accessory olfactory bulb (Knoll et al., 2001) Loss- of -function studies of Eph and ephrins have been limited to focused analysis of mice Defects in the formation of forebrain commissures have been observed in mice lacking Eph-B2, Eph-B3 and Eph-A8 Patterning of the forebrain and hindbrain structures were disrupted in the presence of dominant –negative Eph-A4 (O'Leary and Wilkinson, 1999) Nonetheless... family proteins and repulsion by Unc-5 family proteins (Fig 1.3) Within the developing nervous system Netrin and its receptors are expressed in diverse group of structures One particular example is the visual system The DCC receptor is expressed in the retinal ganglion cell and netrin is expressed in the optic nerve head (Gad et al., 2000) By analyzing both the DCC and netrin mutants netrin was shown . i AXON GUIDANCE IN THE ZEBRAFISH VISUAL SYSTEM: ANALYSIS OF THE esrom MUTANT JASMINE JOYCE D’SOUZA ( M.Sc. University of Mumbai) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK. carried in zebrafish. This has led to the isolation of a mutant called esrom. This study involves the identification and characterization of the esrom gene. Analysis of the zebrafish mutant esrom. targets and axon bundling in the optic tract. The gene function is required in the eye and not in the brain for retinal ganglion cell (RGC) targeting. The mapping of esrom revealed that there is