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Molecular Cues in Pathfinding of Axial Motoneurons in the Developing Zebrafish

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Molecular Cues in Pathfinding of Axial Motoneurons in the Developing Zebrafish Molecular cues in pathfinding of axial motoneurons in the developing zebrafish DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Jona Dela Cruz Hilario Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2010 Dissertation Committee: Christine Beattie, Advisor Susan Cole James Jontes Harald Vaessin Copyright by Jona Hilario 2010 Abstract Establishing neuromuscular specificity is an important step during development to ensure proper motor function. Motor axons may travel over relatively long distances from their positions in the CNS to muscle targets in the periphery. The zebrafish model has been useful for investigating the process of motor axon pathfinding. The embryonic motor system of the zebrafish is relatively simple and has been well studied. Both forward and reverse genetics have been used to study axon pathfinding of zebrafish trunk motor axons. Our study of two motor axon mutants, stumpy and topped, has led to the identification of two molecules that guide trunk motor axons to their muscle targets. stumpy mutants exhibit a phenotype wherein motor axons stall for prolonged periods at intermediate targets prior to them reaching their final target. Positional cloning mapped the mutation to the zebrafish homolog of the collagenXIXa1 (colXIX). colXIX is expressed at known intermediate targets during the time of axon outgrowth. Knocking down ColXIX using morpholinos (MO) phenocopies stumpy. Also stumpy mutants were rescued by knocking down ColXIX and adding back mouse colXIX RNA. This suggests that ColXIX functions to enable growth cones to navigate intermediate target during development. ii Semaphorin 5A (Sema5A) was initially identified as a candidate gene for the topped mutant but this does not appear to be a case. However, Sema5A appears to play a role in motor axon pathfinding. Sema5A is expressed in the myotome during the time of axon outgrowth. Knocking down Sema5A using MOs specifically affects the Caudal Primary (CaP) motor axon, inducing a delay in its extension to its muscle target as well as axon branching. This MO phenotype can be rescued by adding back rat sema5A RNA. Sema5A has been previously shown to act as a bifunctional cue in the rat habenula. In zebrafish, we saw that adding back RNA encoding the sema domain alone rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. These data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The addition of ColXIX and Sema5A to the list of molecules that are involved in this seemingly simple pathfinding process demonstrates that numerous factors and pathways may be involved in establishing precise neuromuscular connections. Both these molecules have been shown to function in other contexts in the nervous system thus, understanding the roles these molecules play in axonal pathfinding can reveal novel mechanisms involved in wiring the nervous system. iii To Paul, who not only helped me with stats and feeding, but also believed I could do this when I couldn’t see that for myself. You have my love and gratitude. To my Family, for their love and support that could be felt over oceans. To our Li’l One, can’t wait to meet you! iv Acknowledgments I am very grateful to my adviser Dr. Christine Beattie for being a mentor in the truest sense of the word throughout my graduate career. Her enthusiasm for science helped me see past the routine disappointments bench science often throws your way and look for the bigger more important picture. I would also like to thank my committee Dr. Susan Cole, Dr. Jamie Jontes and Dr. Harald Vaessin for your encouragement, input and help when my project ran up against a wall. I would also like to thank Louise Rodino-Klapac and Chunping Wang who helped and trained me early on and whose projects I eventually took over. I would not have had anything to work on if not for both your hard work! I would also like to thank all Beattie lab members past and present for their friendship and camaraderie as well as for their generosity in sharing their knowledge, reagents and their lives with me. Thanks too to the rest of the zebrafish group at CMN (PDH and JDJ labs) for making this a fun environment to work in. Also thanks to the fishroom staff who allow all of us to do our work well. I thank Dr. Catherina Becker, Dr. Sarah Childs and Dr. Michael Granato for graciously providing reagents. This work has been supported by the National Science Foundation. v Vita March 1998 ....................................................Philippine Science High School-Diliman April 2002 ......................................................B.S. Molecular Biology and Biotechnology, University of the Philippines-Diliman 2004 to present ..............................................Graduate Research Associate, Department of Neuroscience, The Ohio State University Publications Hilario, J.D., Rodino-Klapac, L.R., Wang, C., Beattie, C.E., 2009. Semaphorin 5A is a Bifunctional Axon Guidance Cue for Axial Motoneurons in Vivo. Dev. Biol. 1, 190-200. Fields of Study Major Field: Molecular, Cellular and Developmental Biology vi Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii Publications ....................................................................................................................... vii Fields of Study .................................................................................................................. vii Table of Contents ............................................................................................................. viii List of Tables ................................................................................................................... xiv List of Figures ................................................................................................................... xv Abbreviated terms .......................................................................................................... xviii Chapter 1. Introduction to Motor Axon guidance .............................................................. 1 Overview ......................................................................................................................... 1 Growth cones and axon pathfinding ................................................................................ 2 Axon guidance molecules ............................................................................................... 5 Netrins and its receptors .............................................................................................. 6 Eph receptors and Ephrins ......................................................................................... 10 vii Slits and Robo ............................................................................................................ 11 Semaphorins and its receptors ................................................................................... 12 The role of the extracellular matrix in axon pathfinding .............................................. 16 Proteoglycans............................................................................................................. 18 Fibrillar ECM Molecules ........................................................................................... 20 Laminins .................................................................................................................... 20 Tenascins ................................................................................................................... 21 Collagens ................................................................................................................... 22 The zebrafish as a model for studying axon guidance .................................................. 25 Forward genetics reveals molecules responsible guiding primary motoneurons ...... 28 diwanka mutants ........................................................................................................ 29 unplugged mutants ..................................................................................................... 30 stumpy mutants .......................................................................................................... 32 topped mutants ........................................................................................................... 33 Reverse Genetics confirms roles of known molecular motor axon guidance cues in zebrafish..................................................................................................................... 33 Chapter 2: Collagen XIXa1 is critical for intermediate target navigation by primary motor axons .............................................................................................................................. 43 Abstract ........................................................................................................................
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