Telomere Elongation in Regenerating Tissues of the Grey Starfish
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TELOMERE ELONGATION IN REGENERATING TISSUES OF THE GREY STARFISH, LUIDIA CLATHRATA by Rebecca Varney B.S., University of California Davis, 2011 A thesis submitted to the Department of Biology Hal Marcus College of Science and Engineering The University of West Florida In partial fulfillment of the requirements for the degree of Master of Science 2016 © 2016 Rebecca Michelle Varney The thesis of Rebecca Michelle Varney is approved: ____________________________________________ _________________ Alexis M. Janosik, Ph.D., Committee Member Date ____________________________________________ _________________ Hui-Min Chung, Ph.D., Committee Member Date ____________________________________________ _________________ Christopher M. Pomory, Ph.D., Committee Chair Date Accepted for the Department/Division: ____________________________________________ _________________ Christopher M. Pomory, Ph.D., Interim Department Chair Date Accepted for the University: ____________________________________________ _________________ Jay Clune, Ph.D., Interim AVP for Academic Programs Date ACKNOWLEDGMENTS This project was made possible by a grant from the University of West Florida Scholarly and Creative Activities Committee. I thank my committee members for guidance in and out of the lab, and for being equally generous with patience and time. I thank Reena Torrance and Nick Honeycutt of the Pomory Lab who were welcoming from my first day there, the Chung lab (especially Kendra Buer) for support and equipment training, and the Janosik Lab for inclusion and friendship. I offer my gratitude to Colton Seals for gifting me starfish, animal care assistance, and humor. For help collecting and maintaining additional animals I thank Katie Vaccaro, Kathy McCarthy, Mary Cvetan, and especially Stacy Cecil (who braved cold water to discover our first baby Luidia). Thank you to Jim Hammond, who helped keep my starfish alive and the greenhouse running, and to Karen Gibbs, who keeps the world turning in our department and offered essential motivational sass. Thanks to innumerable other members of the UWF Biology Department for support, including Mariah Pfleger, Bethany McAcy, Hillary Skowronski, and Dr. Kari Clifton. Also thanks to Melissa Nehmens for being an inspiring scientist and friend. Thank you to my students across all my semesters, in research laboratories and classrooms alike, who provided constant inspiration and reminded me daily why I was here. Lastly, to all those who said yes when they could have more easily said no, my sincere and infinite thanks. iv TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................................ iv LIST OF TABLES ....................................................................................................................... vi LIST OF FIGURES ..................................................................................................................... vii ABSTRACT ............................................................................................................................... viii CHAPTER I. INTRODUCTION ............................................................................................1 A. Reproduction in Asteroids ...........................................................................2 B. Autotomy in Asteroids .................................................................................2 C. Regeneration in Asteroids ............................................................................3 D. Telomeres and Telomerase ..........................................................................5 E. The Grey Starfish, Luidia clathrata ..........................................................13 F. Purpose and Hypotheses .............................................................................15 CHAPTER II. MATERIALS AND METHODS .....................................................................16 A. Collection, Housing, and Injury ..................................................................16 B. Experimental Manipulation ........................................................................16 C. Genetic Analysis of Telomere Length ...................................................... 19 D. Genetic Analysis of Telomerase Expression ............................................ 20 E. Statistical Analysis .................................................................................... 20 CHAPTER III. RESULTS ....................................................................................................... 22 A. Experiment 1: Telomere Length in Regenerating Arm Tissues ............... 22 B. Experiment 2: Telomere Length after a Simulated Fission ....................... 23 C. Experiment 3: Telomere Length in Twice-Regenerated Arms ................. 26 D. Experiment 4: Telomere Length in Juvenile Luidia clathrata .................. 26 E. Experiment 5: Telomerase Expression in Regenerating Tissues ............. 27 CHAPTER II. DISCUSSION ................................................................................................. 29 A. Simulated Fission and Regeneration ......................................................... 29 B. Presence of Telomere Elongation ............................................................. 30 C. Reasons for Elongation ............................................................................. 31 D. Limitations to Telomere Elongation ......................................................... 33 E. Telomerase Expression .............................................................................. 34 F. Summary and Future Studies ...................................................................... 35 REFERENCES ............................................................................................................................ 38 v LIST OF TABLES 1. Experiment 1, Luidia clathrata, individual telomere DNA Ct (cycle threshold) values for experimental starfish regenerating two arms and non-regenerating controls for two time points………………………………………………..……………………………….……… 23 2. Experiment 2, Luidia clathrata, individual telomere DNA Ct (cycle threshold) values for experimental three-armed half starfish regenerating two arms and non-regenerating controls for two time points ………………………………………………..……………………...… 24 3. Experiment 3, Luidia clathrata, individual telomere DNA Ct (cycle threshold) values for experimental starfish regenerating two arms with one of the two removed twice.…………...…………………………………………………………………………… 26 4. Experiment 4, Luidia clathrata, individual telomere DNA Ct (cycle threshold) values for non-regenerating juveniles and adults ……………………………………………………... 27 5. Experiment 5, Luidia clathrata, individual telomerase mRNA Ct (cycle threshold) values for experimental starfish regenerating one arm at three time points ………………………...… 28 . vi LIST OF FIGURES 1. Diagram of DNA forming a T-loop and subsequently formed D-loop ……………………... 6 2. Replication fork at the end of a chromosome, demonstrating the portion of DNA lost to replication of DNA ………………………………………………..……………………...… 7 3. Phylogenetic relationships of telomere sequences across Metazoans ………….…………… 9 4. Differing views of echinoderm evolutionary relationships ……………..…………………. 12 5. Phylogenetic tree of asteroid orders and families, illustrating groups where fissiparous reproduction occurs and the location of Luidia clathrata……………………...…………… 14 6. Experiments 1, 2, 3, and 5 wounding patterns of Luidia clathrata for experimental regeneration ………………………………………………………………………………… 18 7. Adult and juvenile Luidia clathrata side by side to illustrate size difference …………...… 19 8. Regeneration of one individual of Luidia clathrata during experiment 2, illustrating protruding stomach and uneven initial arm regeneration ………………………………….. 25 vii ABSTRACT TELOMERE ELONGATION IN REGENERATING TISSUES OF THE GRAYSTARFISH, LUIDIA CLATHRATA Rebecca Michelle Varney Regeneration of body tissues in starfish remains poorly understood despite centuries of study. In 2015 elongation of telomere sequences was documented in the asexually reproducing starfish Coscinasterias tenuispina, the first time such a phenomenon had ever been observed in somatic tissues. Here, telomere sequences were investigated in Luidia clathrata, a sexually reproducing species. Telomere elongation was confirmed in Luidia clathrata, after both arm injury and a simulated asexual split. Telomeres of juvenile starfish were consistently longer than those of adults. Telomerase expression was detected prior to injury as well as during regeneration, suggesting constitutive expression. As Luidia clathrata are not immediately related to Coscinasterias tenuspina, the presence of telomere elongation suggests that this ability may be common to all starfish. viii CHAPTER I INTRODUCTION The regenerative ability of echinoderms has long fascinated scientists (Cuvier 1797; Lamarck 1818). Though other animals are certainly proficient at regenerating extremities, few are capable of regenerating internal organs (Maginnis 2006). Additionally, the regenerated appendages of other groups frequently lack the complexity of the original (Jamison 1964). Echinoderms by contrast are capable of regenerating both limbs and their respective internal organs, without a loss of function in either (Hyman 1955). Regeneration is fundamentally a method by which echinoderms can maintain or increase their fitness within, or in spite of, their environment. In asteroids (starfish), regeneration of