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Effects of Gap Junction Blockage on Regenerating Body Segments of Lumbriculus Variegatus

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Effects of Gap Junction Blockage on Regenerating Body Segments of Lumbriculus Variegatus EFFECTS OF GAP JUNCTION BLOCKAGE ON REGENERATING BODY SEGMENTS OF LUMBRICULUS VARIEGATUS By Monica K. Richmond A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology Committee Membership Dr. Bruce O’Gara, Committee Chair Dr. Ethan Gahtan, Committee Member Dr. John Reiss, Committee Member Dr. Amy Sprowles, Committee Member Dr. John Steele, Committee Member Dr. Paul Bourdeau, Program Graduate Coordinator May 2020 ABSTRACT EFFECTS OF GAP JUNCTION BLOCKAGE ON REGENERATING BODY SEGMENTS OF LUMBRICULUS VARIEGATUS Monica Richmond Lumbriculus variegatus is a freshwater annelid that is well known for its regenerative capacity. There are many different factors that affect regeneration in animals. Cell-to-cell communication is a key component of regeneration. Gap junctions are made up of proteins that create a channel that connects the cytoplasm of two cells. Many molecules and ions pass through the gap junctions that can affect regeneration. The purpose of this study was to determine if blocking gap junctions would have an impact on regenerating body segments of L. variegatus and what effects it would produce. L. variegatus was exposed to a known gap junction blocker, octanol, for differing time periods following transection, then measured and photographed for six days. Worms treated with octanol experienced regenerative growth that was notably slower and also resulted in abnormal morphology of the regenerated tail. Octanol is known to leave the system immediately after treatment is taken away; however, L. variegatus did not show immediate recovery of growth when placed back into pond water. It is not clear whether there is a critical time period for exposure to octanol that affects regeneration; however, the duration of the exposure corresponded to the severity of abnormal morphology. The observations of the outcome of gap junction blockage on ii regenerative growth will contribute to the on-going research in areas of biology related to regeneration and gap junction communication. iii ACKNOWLEDGEMENTS This thesis work would not have been possible without the support for so many people. First, I would like to thank my thesis advisor, Dr. Bruce O’Gara and my committee members Dr. Ethan Gahtan, and Dr. John Reiss, Dr. Amy Sprowles. and Dr. John Steele. You have all been so helpful and supportive. Without Dr. O’Gara’s support, this project could not have been possible. I thank him for his patience and constant advice and wisdom along the way. I would like to give a special thank you to Dr. Amy Sprowles who allowed me to use her Zeiss microscope. She graciously let me become a pseudo member of her lab and gave me advice whenever I asked. In all the time I spent physically in her lab, I became friends with the intelligent and talented graduate and undergraduate women in her lab. I would like to thank Sharon Otis, Rachel Brewer, Kristine Teague, Linh Pham and Elizabeth Zepeda for the many science conversations and discussions we had about our projects. I would like to thank Susan Wright in the Biological Sciences Stockroom. Susan was instrumental in assisting me with all of my supplies as well as giving me emotional support. I would also like to thank Dave Baston in the CNRS Core Research Facility. The use of the Core Research Facility was invaluable. Lastly I would like to thank all of the people who have supported me in this very long journey. Thank you to all the incredible professors and lecturers who I taught labs for: Dr. Frank Shaughnessy, Leslie VanderMolen, Dr. Elizabeth Whitchurch, Jane iv Monroe, and Dr. Brigitte Blackman. They taught me how to teach and mentor students and how to be a better graduate student. Thank you to the HSU Children’s Center and Elizabeth Rice and Jennifer Black. Without them I would not have been able to be in school. Thank you to my first graduate school friend and co-lab teaching assistant, Aleika Lippman whose friendship and support I value very much. Tidepooling and collecting seaweeds and other botany samples together was a beautiful way to get to know Humboldt County. And, an enormous thank you to my husband Aaron, and my kids Avery and Charlie, and my mom Linda Backer. I wouldn’t have been able to do any of this without your support and encouragement. Thank you to everyone. All of your dedication to helping support me as I pursue this degree has been an incredible gift. v TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGEMENTS ............................................................................................... iv LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix INTRODUCTION .............................................................................................................. 1 METHODS ......................................................................................................................... 8 Animal culture ................................................................................................................ 8 Treatment solution, concentrations and testing .............................................................. 8 Determination of abnormal morphological growth ...................................................... 10 Cell proliferation assay ................................................................................................. 13 Selection of worms for EdU staining ............................................................................ 13 EdU staining protocol for assessment of cellular proliferation .................................... 13 Determination of cell proliferation ............................................................................... 14 Statistical analysis of regeneration ............................................................................... 15 RESULTS ......................................................................................................................... 16 Regeneration of control worms ..................................................................................... 16 Treatment with 0.01% octanol had a significant negative effect on regeneration compared to untreated control worms. ......................................................................... 16 Mean rate of regeneration showed that growth varied each day for all treated and untreated worms ............................................................................................................ 33 Comparison of heads and tails within the same treatment show similar regeneration trends to each other ....................................................................................................... 37 vi EdU cell proliferation assay .......................................................................................... 41 DISCUSSION ................................................................................................................... 46 Comparison of treated and non-treated worm regeneration ......................................... 48 Recovery after octanol exposure ................................................................................... 52 Critical time period for regeneration ............................................................................ 54 Comparison of mean daily overall regeneration and mean rate of regeneration .......... 56 EdU results inconclusive .............................................................................................. 56 CONCLUSIONS............................................................................................................... 58 REFERENCES ................................................................................................................. 59 Appendix ........................................................................................................................... 63 vii LIST OF TABLES Table 1: Treatment protocols for transected fragments of worms. Immediately following transection (day zero), worms were placed in the following solutions. Group Octanol 0 (n = 40) were placed in 0.01% octanol for day zero followed by pond water for days one through six. Group Octanol 0-1(n = 34) were placed in 0.01% octanol for day zero through day one followed by pond water for days two through six. Group Octanol 0-2 (n = 30) were placed in 0.01% octanol for day zero through day two followed by pond water for days three through six. Group Octanol 0-6 (n = 26) were placed in 0.01% octanol solution for days zero through six. Group Hexanol (n = 31) were placed in 0.01% hexanol solution for days zero through six. Group Control (n = 47) were placed in pond water for days zero through six. ........................................................................................ 10 Table 2: Table of significant differences for head regeneration between treatments and days. Empty cells do not have significant differences for head regeneration. ................. 34 Table 3: Table of significant differences for tail regeneration between treatments and days. Empty cells do not have significant differences for tail regeneration. ................... 36 viii LIST OF FIGURES Figure 1: Illustration describing body fragments of worms
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