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Eptatretus Stoutii and Myxine Glutinosa) Biogenesis of Hagfish Slime: Timing and Process of Slime Gland Refilling in Hagfishes (Eptatretus stoutii and Myxine glutinosa) by Sarah Schorno A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Integrative Biology Guelph, Ontario, Canada © Sarah Schorno, September 2018 ABSTRACT Biogenesis of Hagfish Slime: Timing and Process of Slime Gland Refilling in Hagfishes (Eptatretus stoutii and Myxine glutinosa) Sarah Schorno Advisors: University of Guelph, 2018 Dr. D. S. Fudge Dr. T. E. Gillis Mucus and slime production is widely represented throughout the animal kingdom. Many animals produce mucus for locomotion, to prevent desiccation, in mating, or to physically or chemically ward off predators and pathogens. However, the crown for volume of defensive slime formation goes to the hagfishes, who release components producing 0.9 litres of a gill-clogging slime within 100 milliseconds of an attack by a predator. Their slime is unique in that it contains protein fibres that have one of the highest tensile strengths of any biomaterial. Hagfishes produce slime components in specialized slime glands, which are found in pairs along the whole length of their body. These slime glands contain two main cell types, gland thread cells and gland mucous cells, which produce, respectively, the thread and mucous components of the defensive slime. Each gland thread cell produces a single, coiled, proteinaceous thread formation known as a thread skein, while each gland mucous cell produces thousands of tiny mucin vesicles. While much is known about the biochemistry of the gland thread cells and gland mucous cells, little was known about where these cells were produced within the slime gland, or how these cells were replenished after a sliming event. The objective of this thesis was to examine in detail the process of slime gland emptying and refilling. Using a number of cell manipulation, imaging and bioinformatics techniques, I reveal: (1) how long it takes for depleted slime glands to refill, (2) how the slime exudate composition changes during emptying and refilling of the gland, (3) the cellular mechanisms of refilling, (4) the site of slime cell proliferation, (5) the possible function of a third, previously undescribed cell type within the slime gland, and finally (6) the differential gene expression of slime glands during early refilling compared to full glands. Together, these findings provide a comprehensive analysis of the process of slime gland refilling in hagfish. Understanding more about how the slime glands refill and produce their cells may provide new insights into how the gland thread cells and gland mucous cells produce their respective components, which are useful biomimetic models for producing renewable, protein-based textiles and hydrogels. ACKNOWLEDGEMENTS I would like to start by thanking my advisor, Dr. Douglas Fudge, for providing me with the opportunity to not only start a MSc in your lab, but to then transfer to the PhD program. Your support, guidance, and patience over the years has been instrumental in helping me complete this thesis, and you have provided me with the opportunity to travel all over the continent for my work. It has not always been easy having a long-distance advisor, but I feel that this experience has made me a stronger, more independent researcher. Dr. Todd Gillis, thank you for accepting me into your lab when Dr. Fudge took another position in California. Your support here at home has been essential for my continued growth. Drs. Andreas Heyland, Fred Laberge, and Cezar Khursigara, thank you for being a part of my advisory committee throughout my graduate career, and for all your helpful advice. Thank you to all the past/present faculty and students in the Comparative Physiology Research group, including: Elizabeth Johnston, Andy Turko, Evan McKenzie, Lauren Gatrell, Calli Freedman, Atsuko Negishi, and Jean-Luc Stiles. Specifically, I would like to thank Shannon Ferraro and Sarah Boggett for all their help in the lab, and for their amazing continued friendship. Our “S&S&S” kitchen adventures are some of my fondest memories. I would also like to thank some people for their technical and administrative support: Matt Cornish, Mike Davies (Hagen Aqualab); Helen Coates, Jodi Morrison (OVC Histoprep Lab); Dr. Michaela Strüder-Kypke, Bob Harris (Molecular and Cellular Imaging Facility); Dr. Dyanne Brewer (Advanced Analysis Centre); as well as Lori Ferguson and Debbie Bailey (Integrative Biology Administrative Services). Lastly, but most of all, I would like to thank my family for their love and support during my graduate career and over my whole life. As a first-generation Canadian and first-generation university student, my parents have provided me with everything I have ever needed to achieve my dreams. Thank you for allowing the little girl who liked bugs and slimy things to explore and be curious about the world around her. I would especially like to thank my amazing husband, Kevin Stinson, for his patience and for lifting me up on my bad days. You are my cheerleader, and always lend me a shoulder to lay my head on at the end of a long day. “Alone we can do so little; together we can do so much” – Helen Keller iv TABLE OF CONTENTS Abstract .............................................................................................................................ii Acknowledgements ............................................................................................................iv Table of Contents .............................................................................................................. v List of Tables ................................................................................................................... vii List of Figures ................................................................................................................. viii List of Supplemental Material ............................................................................................. x 1.0 GENERAL INTRODUCTION ...................................................................................... 1 2.0 EMPTYING AND REFILLING OF SLIME GLANDS IN ATLANTIC (Myxine glutinosa) AND PACIFIC (Eptatretus stoutii) HAGFISHES ............................................................... 33 3.0 CELLULAR MECHANISMS OF SLIME GLAND REFILLING OF PACIFIC HAGFISH (Eptatretus stoutii) ........................................................................................................... 42 4.0 DISCOVERY OF A THIRD ABUNDANT CELL TYPE IN THE HAGFISH SLIME GLAND – GLAND INTERSTITIAL CELLS ..................................................................... 54 4.1 Abstract ............................................................................................................. 55 4.2 Introduction ....................................................................................................... 56 4.3 Materials and Methods ........................................................................................ 59 4.4 Results .............................................................................................................. 64 4.5 Discussion ......................................................................................................... 74 5.0 DIFFERENTIAL GENE EXPRESSION IN REFILLING PACIFIC HAGFISH (Eptatretus stoutii) SLIME GLANDS ................................................................................................. 84 5.1 Abstract ............................................................................................................. 85 5.2 Introduction ....................................................................................................... 86 5.3 Materials and Methods ........................................................................................ 88 5.4 Results .............................................................................................................. 95 v 5.5 Discussion ....................................................................................................... 105 6.0 GENERAL DISCUSSION ........................................................................................ 116 Appendix 1.0 – Chapter 5 Supplemental Material .............................................................. 136 Appendix 2.0 – Permissions ............................................................................................ 141 vi LIST OF TABLES Table 5.1 Summary of Pacific hagfish transcriptome data ...........................................................96 Table 5.2 Gene ontology (GO) analysis of differentially expressed genes in refilling (2 days post sliming) Pacific hagfish slime glands ..........................................................................................100 Table 5.3 Genes potentially involved in rotation of the nucleus of the gland thread cells in Pacific hagfish slime glands.........................................................................................................104 vii LIST OF FIGURES Figure 1.1 External morphology of the New Zealand hagfish (Eptatretus cirrhatus) ....................8 Figure 1.2 Anatomy of slime glands and ejection of slime cells ..................................................11 Figure 1.3 Hagfish slime as a defense against gill-breathing predators........................................12 Figure 1.4 Thread skeins and mucin vesicles of hagfish slime exudate .......................................14
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