University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2009 PHYSIOLOGICAL GENOMICS OF SPINAL CORD AND LIMB REGENERATION IN A SALAMANDER, THE MEXICAN AXOLOTL James Robert Monaghan University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Monaghan, James Robert, "PHYSIOLOGICAL GENOMICS OF SPINAL CORD AND LIMB REGENERATION IN A SALAMANDER, THE MEXICAN AXOLOTL" (2009). University of Kentucky Doctoral Dissertations. 703. https://uknowledge.uky.edu/gradschool_diss/703 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION James Robert Monaghan The Graduate School University of Kentucky 2009 PHYSIOLOGICAL GENOMICS OF SPINAL CORD AND LIMB REGENERATION IN A SALAMANDER, THE MEXICAN AXOLOTL ________________________________ ABSTRACT OF DISSERTATION ________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By James Robert Monaghan Lexington, KY Director: Dr. S. Randal Voss, Associate Professor of Biology Lexington, KY 2009 Copyright © James Robert Monaghan 2009 ABSTRACT OF DISSERTATION PHYSIOLOGICAL GENOMICS OF SPINAL CORD AND LIMB REGENERATION IN A SALAMANDER, THE MEXICAN AXOLOTL Salamanders have a remarkable ability to regenerate complex body parts including the limb, tail, and central nervous system. Although salamander regeneration has been studied for several hundred years, molecular-level studies have been limited to a relatively few important transcription factors and signaling molecules that are highly conserved among animals. Physiological genomic approaches were used here to investigate spinal cord and limb regeneration. Chapter 2 reports that hundreds of gene expression changes were identified during spinal cord regeneration, showing that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. Chapter 3 presents results that identify the salamander ortholog of mammalian Nogo-A, a gene known to inhibit mammalian nerve axon regeneration. Nogo-A gene expression was characterized during salamander development and adulthood in order to address the roles of Nogo-A in the nervous system. Chapters 4 and 5 use physiological genomic approaches to examine limb regeneration and why this process is dependent upon an intact nerve supply. Results presented in Chapter 4 showed that many processes regulated during early limb regeneration do not depend upon nerve-derived factors, but striking differences arise between innervated and denervated limbs by 14 days after amputation. Chapter 5 identified genes associated with peripheral nerve axon regeneration and identified gene candidates that may be secreted by nerves to support limb regeneration. Lastly, chapter 6 characterizes the expression of a developmentally important family of genes, matrix metalloproteinases, during tail regeneration. These results suggest that matrix metalloproteinases play multiple roles throughout the regeneration process. Primarily, this dissertation presents data from the first genomic studies of salamander regeneration. The results suggest genes such as matrix metalloproteinases, and molecular pathways such as the Wnt and FGF signaling pathways that can be exploited to enhance regenerative ability in humans. KEYWORDS: Axolotl, regeneration, microarray, spinal cord, limb James Robert Monaghan April 1, 2009 PHYSIOLOGICAL GENOMICS OF SPINAL CORD AND LIMB REGENERATION IN A SALAMANDER, THE MEXICAN AXOLOTL By James Robert Monaghan S. Randal Voss, Ph.D. Director of Dissertation Brian C. Rymond, Ph.D. Director of Graduate Studies RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ DISSERTATION James Robert Monaghan The Graduate School University of Kentucky 2009 PHYSIOLOGICAL GENOMICS OF SPINAL CORD AND LIMB REGENERATION IN A SALAMANDER, THE MEXICAN AXOLOTL ________________________________ DISSERTATION ________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By James Robert Monaghan Lexington, KY Director: Dr. S. Randal Voss, Associate Professor of Biology Lexington, KY 2009 Copyright © James Robert Monaghan 2009 ACKNOWLEDGMENTS I would like to thank the many people that have supported my dissertation experience during my time at the University of Kentucky. I would first like to thank my advisor, Randal Voss, for providing me with a stimulating scientific environment, more than ample financial support, and exceptional mentoring over the past five years. I feel lucky to have happened upon such a productive lab and such an interesting project. I would also like to thank my committee members Elizabeth Debski, Doug Harrison, and Tim McClintock for their advice and support over the years. I would also like to thank past and present Voss lab members including John Walker, Amy Smith, Jeremiah Smith, Robert Page, Meredith Boley, Sri Putta, Kevin Kump, and Laura and Chris Muzinic. Each lab member has been more than willing to give a helping hand on my projects, regardless of reciprocation. Each member has furthered my career in their own way and I thank them for the help. I thank my wife, Stephanie, for listening to me talk about my projects and all of her support and patience with my frustrations and successes. I also thank my family for teaching me the value of education, hard work, patience, and ambition. My journey through graduate school has led to a very satisfying and enjoyable scientific experience and I hope my upcoming career is as rewarding as my graduate school experience. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii LIST OF FILES ................................................................................................................. ix CHAPTER 1: Introduction ..................................................................................................1 Spinal cord regeneration..........................................................................................1 Limb regeneration....................................................................................................3 Wound healing and the wound epidermis................................................................4 Nerve-dependency of limb regeneration..................................................................5 CHAPTER 2: Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum ........................................................................9 Introduction............................................................................................................10 Materials and Methods...........................................................................................11 Results....................................................................................................................16 Discussion..............................................................................................................21 CHAPTER 3: Identification and characterization of nogo-A in a naturally regenerating salamander (Ambystoma mexicanum) ..............................................................................41