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The Proliferation of Satellite Cells in the Developing Sturgeon

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The Proliferation of Satellite Cells in the Developing Sturgeon Lake Sturgeon (Acipenser fulvescens) Myotome Development: The Proliferation of Satellite Cells in the Developing Sturgeon by Amber Hiebert A thesis submitted to the Faculty of Graduate Studies in partial fulfillment of the requirements to the degree of MASTER OF SCIENCE Department of Biological Sciences University of Manitoba Winnipeg, Manitoba Copyright © 2018 by Amber Hiebert 1 Contents Abstract ............................................................................................................................... 4 Acknowledgements ............................................................................................................. 5 List of Tables ...................................................................................................................... 6 List of Figures ..................................................................................................................... 7 Introduction ......................................................................................................................... 9 Chapter 1: Literature Review ............................................................................................ 10 1.1 Skeletal Muscle Development ................................................................................ 10 1.1.1 Mammalian Development ................................................................................ 11 1.1.2 Teleost Development ....................................................................................... 15 1.2 Myofiber Characteristics ......................................................................................... 16 1.2.1 Types of Myofibers .......................................................................................... 16 1.2.2 Anatomy and Physiology ................................................................................. 18 1.2.3 Innervation ....................................................................................................... 21 1.3 Satellite Cell Characteristics ................................................................................... 22 1.3.1 Satellite Cell Heterogeneity ............................................................................. 25 1.3.2 Satellite Cell Activation and Proliferation ....................................................... 29 1.4 Lake Sturgeon Development................................................................................... 36 1.4.1 Sturgeon Life History and Development ......................................................... 36 1.4.2 Sturgeon Muscle Development ........................................................................ 37 1.5 Isolated fibers in culture as a model system ........................................................... 38 1.6 Research Question/Rationale .................................................................................. 39 Chapter 2: Methodology ................................................................................................... 41 2.1 Dissection ................................................................................................................ 41 2.2 Fiber Isolation ......................................................................................................... 43 2.3 Myofiber Plating ..................................................................................................... 44 2.4 Cryosectioning ........................................................................................................ 47 2.5 Staining ................................................................................................................... 48 2.5.1 IHC Staining for Isolated Myofibers (HRP-DAB) .......................................... 48 2.5.2 H&E Staining for Sections............................................................................... 50 2.6 Counting .................................................................................................................. 50 2 2.7 Statistical Analysis .................................................................................................. 52 Chapter 3: Results ............................................................................................................. 53 3.1 Body length and weight .......................................................................................... 53 3.2 Fiber length, width, volume, and configuration ...................................................... 55 3.3 Muscle histology in sections ................................................................................... 56 3.4 Immunohistochemistry ........................................................................................... 57 3.4.1 Satellite cell cycling – BrdU+ satellite cells .................................................... 57 3.4.2 Myogenic precursor cells – Pax7+ satellite cells ............................................. 60 3.4.3 Fiber myonuclei and myonuclear domains ...................................................... 61 3.5 Ratio of satellite cells to total nuclei ....................................................................... 62 3.6 Other relationships .................................................................................................. 63 Chapter 4: Discussion ....................................................................................................... 95 4.1 SC Cell-Cycle Variations ........................................................................................ 97 4.2 Myofiber changes during development ................................................................ 100 4.3 SC Population Variations ...................................................................................... 103 4.4 Regressions ........................................................................................................... 104 4.5 Limitations and Future Directions ........................................................................ 105 4.6 Significance........................................................................................................... 107 References ....................................................................................................................... 110 Appendix ......................................................................................................................... 129 Antibodies ................................................................................................................... 129 Solutions ..................................................................................................................... 129 Disposal....................................................................................................................... 130 3 Abstract The Lake Sturgeon (Acipenser fulvescens) continues to grow throughout life, though the process by which muscle stem cells, called satellite cells (SCs) contribute to formation of fibers in the myotome is largely unknown in this endangered fish. Since muscle function and growth are critical to survival, it is important to understand the functional basis of fiber growth, and how SCs provide daughter cells that fuse into fibers in myotome development and regeneration. The hypotheses are that during aging: the cell cycle of SCs lengthens, the ratio of SCs to myonuclei decreases, and myonuclear domain increases. This experiment used the single fiber model and a pulse-chase design in which exposure to bromodeoxyuridine (BrdU) labeled S-phase for the first 2 hr in culture. Myofibers were isolated from 20-40 fish per age (1-6 months post-hatch). After fiber culture for 24 hours, fibers were fixed and stained for BrdU or Pax7 (expressed by myogenic stem cells) with colour detection. The number of Pax7- or BrdU-positive nuclei and the total number nuclei associated were counted per fiber in 8-20 fibers per dish. Results showed a significant change in the ratio of Pax7+ SCs to myonuclei as the fish age (p < 0.05) with an apparent decrease in cell cycle duration with increasing age (p < 0.05). This investigation adds to our understanding of SC contributions to myofiber growth in the developing Lake Sturgeon and results will be a new foundation for future research on the role of environmental influences on muscle in Lake Sturgeon. 4 Acknowledgements I want to thank several people for helping me through the past 2 years of graduate school, and for continual support, guidance and encouragement. My research and this thesis would not have been possible without the people in my life. I must start by thanking my advisor, Judy Anderson. I cannot thank you enough for your patience, guidance and support though my research and writing, but also in my personal life. Your expertise and understanding made it possible to work on and complete research on this topic that was of great interest to me. It was such a pleasure to work with you. I am grateful to the members of my committee, Dr. Peeler and Dr. G. Anderson. Your guidance and help contributed greatly to this project. Thank you to the members of the lab, Ziba and Nasibeh. Your assistance, support and company were appreciated daily. Thank you to the funding provided by the NSERC Discovery grant program and the Graduate Enhancement of Tri-Council Stipends program at the University of Manitoba (both to Dr. Judy Anderson), as well as collaborative support of research funding to Dr. Gary Anderson (NSERC-Manitoba Hydro Industrial Research Chair; NSERC Discovery grant program).
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