THE ROLE OF HOXD10 IN THE DEVELOPMENT OF MOTONEURONS IN THE POSTERIOR SPINAL CORD by Veeral Shailesh Shah B.S., University of Pittsburgh, 2000 Submitted to the Graduate Faculty of University of Pittsburgh School of Medicine Department of Neurobiology in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2006 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE DEPARTMENT OF NEUROBIOLOGY This thesis was presented by Veeral Shailesh Shah It was defended on January 17, 2006 and approved by Paula Monaghan-Nichols, Ph. D, CNUP, Neurobiology Debbie Chapman, Ph. D, Biological Sciences Willi Halfter, Ph. D, CNUP, Neurobiology Carl Lagenaur, Ph. D, CNUP, Neurobiology Cynthia Forehand, Ph. D, University of Vermont, Anatomy and Neurobiology Dissertation Advisor: Cynthia Lance-Jones, Ph. D, CNUP, Neurobiology ii Copyright © by Veeral Shailesh Shah 2007 iii THE ROLE OF HOXD10 IN THE DEVELOPMENT OF MOTONEURONS IN THE POSTERIOR SPINAL CORD Veeral Shah University of Pittsburgh, 2007 Hox genes encode anterior-posterior identity during central nervous system development. Few studies have examined Hox gene function at lumbosacral (LS) levels of the spinal cord, where there is extensive information on normal development. Hoxd10 is expressed at high levels in the embryonic LS spinal cord, but not the thoracic (T) spinal cord. To test the hypothesis that restricted expression of Hoxd10 contributes to the attainment of an LS identity, and specifically an LS motoneuron identity, Hoxd10 was ectopically expressed in T segments in chick embryos via in ovo electroporation. Electroporations were carried out at early neural tube stages (stages 13-15) and at the onset of motoneuron differentiation (stages 17-18). Regional motoneuron identity was assessed after the normal period of motor column formation (stages 28-29). Subsets of motoneurons in transfected T segments developed a molecular profile normally shown by anterior LS LMCl motoneurons, including Lim 1 and RALDH2 expression. In addition, motoneurons in posterior T segments showed novel axon projections to two muscles in the anterodorsal limb, the sartorius and anterior iliotibialis muscles. These changes are accompanied by a significant reduction in the number of T motoneurons at stage 29. Analyses of Hoxd10 electroporated embryos at the onset of motor column formation (stage 18) suggest that early and high levels of Hoxd10 expression led to the death of some early differentiating motoneuron. Despite these adverse effects, our data indicate that Hoxd10 expression is sufficient to induce LS motoneuron identity and axon trajectories characteristic of motoneurons in the LS anterior spinal cord. Equivalent changes in motoneuron identity were not found with the ectopic expression of Hoxd9, a gene normally expressed in T as well as LS segments. In an additional series of experiments, Hoxd10 was overexpressed in LS segments via in ovo electroporation at early neural tube stages. Analyses at stage 29 indicated proportionate increases in LMCl (Lim 1+, RALDH2+) motoneurons, and proportionate decreases in LMCm and MMC motoneurons (Isl 1+) motoneurons. These findings suggest that Hoxd10 specifically promotes the development and/or survival of LMCl motoneurons. iv TABLE OF CONTENTS PREFACE.................................................................................................................................XIII 1.0 GENERAL INTRODUCTION................................................................................... 1 1.1 SIGNIFICANCE.................................................................................................. 3 1.2 BACKGROUND.................................................................................................. 3 1.2.1 Early regulation of spinal neurogenesis and the specification of generic motoneuron identity..................................................................................................... 4 1.2.2 Motoneuron organization and early morphological development. .......... 6 1.2.3 Evidence of early specification of morphological features and projections..................................................................................................................... 8 1.2.4 Hox gene structure and expression. ............................................................ 9 1.2.5 Analyses of Hox gene function in the hindbrain ........................................ 9 1.2.6 Analyses of Hox gene function in spinal cord........................................... 12 1.2.7 LIM transcription factors and the development of motoneuron subtypes 13 1.2.8 Establishing links between Hox genes and the establishment of motoneuron subtypes................................................................................................. 16 1.2.9 Secreted signaling molecules that set-up Hox and LIM genes................ 16 1.2.10 The growth cone and general mechanisms of axon pathfinding ............ 19 1.2.11 Evidence of specific and general guidance cues within the limb. ........... 20 1.2.12 Factors affecting exit from the spinal cord and spinal nerve formation. 21 1.2.13 Factors affecting axon growth into the limb............................................. 23 1.2.14 Factors affecting axon pathfinding at choice points. ............................... 23 1.2.15 Establishing links between Hox genes and axon guidance...................... 25 v 1.2.16 Hoxd10 in the Posterior Spinal Cord........................................................ 26 2.0 METHODS AND MATERIALS .............................................................................. 27 2.1 EMBRYOS ......................................................................................................... 27 2.2 HOXD9/EGFP, HOXD10/EGFP, AND EGFP EXPRESSION CONSTRUCTS................................................................................................................... 27 2.3 IN OVO ELECTROPORATION AND EMBRYO SACRIFICE.................. 28 2.4 IMMUNOHISTOCHEMISTRY...................................................................... 29 2.5 IN SITU HYBRIDIZATION ............................................................................ 30 2.6 WHOLEMOUNT NEUROFILAMENT STAINING: ................................... 32 2.7 RETROGRADE LABELING: ......................................................................... 33 2.8 CONFOCAL IMAGING: ................................................................................. 34 2.9 MOTONEURON CELL COUNTS: ................................................................ 34 2.10 STATISTICAL ANALYSIS:............................................................................ 35 3.0 ECTOPIC EXPRESSION OF HOXD10 IN THORACIC SEGMENTS AT EARLY NEURAL TUBE STAGES INDUCES LS MOTONEURON SUBTYPES ............ 36 3.1 INTRODUCTION ............................................................................................. 36 3.2 RESULTS ........................................................................................................... 38 3.2.1 Electroporation of Hoxd10 into the thoracic spinal cord........................ 38 3.2.2 Hoxd10 transfected thoracic motoneurons develop lumbosacral-like LIM-profiles. .............................................................................................................. 39 3.2.3 Electroporations with Hoxd10/EGFP leads to a decrease in motoneuron numbers at stage 28-29. ............................................................................................. 41 3.2.4 Hoxd10 transfection has an early influence on cell survival................... 44 3.2.5 Hoxd10/EGFP transfected thoracic motoneurons express RALDH2.... 46 3.2.6 Hoxd10/EGFP transfected T segments have normal Hoxc8 and Hoxd9 expression patterns .................................................................................................... 49 3.3 DISCUSSION..................................................................................................... 50 3.3.1 Hoxd10 imparts an anterior LMCl phenotype ........................................ 50 3.3.2 Hoxd10 gene activity and the sequential induction of LMCl molecular profile 52 3.3.3 The timing of Hoxd10 expression on the specification of motoneurons 53 vi 3.3.4 Hox function and motor columnar identity.............................................. 55 3.3.5 The effect of Hoxd10 on motoneuron survival and early development .56 4.0 ECTOPIC EXPRESSION OF HOXD10 IN THORACIC SEGMENTS ALTERS NERVE PATTERNS TO LS TARGETS ................................................................................. 59 4.1 INTRODUCTION ............................................................................................. 59 4.2 RESULTS ........................................................................................................... 61 4.2.1 Hoxd10 transfected thoracic motoneurons project to dorsal limb muscles 61 4.2.2 Hoxd10 transfected thoracic motoneurons are likely to reach limb muscles by proximal connectives.............................................................................. 62 4.2.3 The ectopic expression of Hoxd10 in T segments leads to the induction of the c-met receptor and Pea3 transcription factor ................................................... 62 4.3 DISCUSSION..................................................................................................... 65 4.3.1 Misexpression of Hoxd10 in T segments results in altered AP nerve patterns 65 4.3.2 Evidence that Hoxd10 expressing T motoneurons respond to specific guidance