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Zebrafish Ryanodine Receptors Gene Protein Transcript Protein Identity Identity Genome Location Exons Γ Γ Name Name (Bp) (Aa) (1) (2)

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Zebrafish Ryanodine Receptors Gene Protein Transcript Protein Identity Identity Genome Location Exons Γ Γ Name Name (Bp) (Aa) (1) (2) The role of ryanodine receptors in development Wu, Houdini Ho-Tin The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author For additional information about this publication click this link. http://qmro.qmul.ac.uk/jspui/handle/123456789/1329 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] THE ROLE OF RYANODINE RECEPTORS IN DEVELOPMENT by HOUDINI HO-TIN WU A thesis submitted to The University of London for the degree of DOCTOR OF PHILOSOPHY School of Biological and Chemical Sciences Queen Mary, University of London, Mile End Road, London E1 4NS United Kingdom April 2011 1 ABSTRACT Calcium ions (Ca2+) are fundamental to the regulation of many cellular processes; however, the coordination of these signals during embryogenesis is not well understood. Ryanodine receptors (RyR) are a family of important intracellular ion channels that are responsible for the release of Ca2+ and they regulate the cytosolic Ca2+ concentration. Humans have three differentially expressed ryr genes (ryr1, ryr2 and ryr3) and mutations can cause both skeletal and cardiac diseases. Although the primary function of RyR is to mediate excitation-contraction coupling in muscle, they may also regulate Ca2+ signalling during developmental processes. The project has addressed the role of RyR during embryonic development, using the zebrafish as an in vivo vertebrate model. Five zebrafish RyR genes (ryr1a, ryr1b, ryr2a, ryr2b and ryr3) were characterised and a comprehensive overview of their spatial and temporal expression in the embryo was determined. At 24 hours post- fertilisation (hpf), ryr1a, ryr1b and ryr3 are expressed in the skeletal muscle, ryr2a in specific neuronal populations and ryr2b in the cardiac muscle. Semi-quantitative PCR data and wholemount in situ hybridisation revealed strong maternal expression of ryr3 during the cleavage and blastula periods and into adulthood. The early expression of the ryr3 gene suggests that this receptor functions during the initial stages of development; a role that has not been described previously. The functional significance of RyR3 during early embryogenesis was investigated in a loss-of- 2 function model using antisense morpholino oligonucleotides. The ryr3 specific knockdown experiments appeared to affect the establishment of embryonic axis prior to the segmentation periods (before 10 hpf). In addition, by 19 to 20 hpf ryr3 morphants failed to exhibit spontaneous muscle contractions and displayed a defect in neuromuscular development. In conclusion, this study has characterised the ryr genes and provided an overview on their temporal and spatial expression. The work provides evidence that ryr3 expression provides the Ca2+ vital for myofibrils organisation and that is required for the spontaneous movements during zebrafish embryonic development. The knowledge of RyR tissue distribution in zebrafish has provided a strong foundation for loss-of-function studies aimed at addressing their role in development. In the long term, the work will also facilitate more focused studies on disease. 3 DECLARATION This work recorded in this thesis was carried out in the School of Biological and Chemical Sciences at the Queen Mary College, University of London, United Kingdom, during the period of October 2007 to September 2010. The work is original except where acknowledged by reference. No portion of the work is being or has been submitted for any other qualification at any other universities. 4 ♥ For my parents I.I. & Margaret and my wife Oona ♥ The thesis is also dedicated to my family ♥ 5 ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor, Dr. Rachel Ashworth for giving me this opportunity to be her first Ph.D. student to gain gratifying fruitful experience in her laboratory. I am very grateful to her for allowing me to present my work at conferences on many occasions. I would like to extend my thanks to Rachel and my second supervisor, Dr. Caroline Brennan for all the patience, support and invaluable advices they have given me throughout my Ph.D. Their inspiration in science has not only given me the motivations during the project, but also set me an excellent example to pursue a life-long career in scientific research, which has taken me to this point. A BIG THANK YOU TO BOTH OF YOU! Importantly, I would like to thank my parents I.I. and Margaret, my grandmother Lam Wai-Ying, my ‘great grandparents’, my brother Andy and my wife Oona for all their endless love, support and encouragements, enabling me to achieve my ambitions and to maintain my sanity. I would also like to thank my parents-in-law Leo and Katherine, my brothers-in-law Eddy and Gee, my sisters-in-law Jane and Gigi, uncle Allain and auntie Pauline for their caring and support (and their kindness :P). Thank you to both Uncle Two and Auntie Two, who have always treated me like their family members and bought me to golfing (a sport that I could never imagine I would be playing) and this really has broaden my horizons. All you guys mean so much to me and I really cannot imagine how I could do this without you! Particularly, I would like to thank my beloved one, Oona, who always loves me and my family. My life has been fantastic since your presence (28th July, 2006 just to remind myself in case I forgot ) and I know it always is my biggest fortune to have you in my life. Thank you so much for always being so supportive and kind to me. So, please keep it up :P and thank you! 6 Furthermore, I would also like to express my sincere gratitude to my college, SBCS for the pre-doctoral research studentship that funds my tuition fees, living and research costs, as well as my travel expenses, which has given me the opportunity to attend scientific workshop. In addition, I am very grateful to the Central Research Fund and Physiological Society travel grants for financially supporting my project to generate isoform specific RyR antibodies and enabling me to travel around the globe to present my work at conferences. A special thanks to Maggie & Haidee for the administrative help, Emma for the computing support, Ray for printing me poster up to the deadlines :P and my panel members (Prof Ralf Stanewsky, Drs Robin Maytum & Lesley Dobson) for their constructive comments for my project. Additionally, I would like to thank all my friends in Birmingham, Cambridge, Milton Keynes, London and Hong Kong. Thank you, Drs Abayomi Ogunbayo & Lynn Dover (for giving me an early interest into molecular biology), Carly Nichols & Heather Callaway (for the fish and eggs all these years), Chun Ming Chan (for your long-term support and encouragements), Prof David Minnikin (for keeping Oona in Birmingham), Debbie Goode & Dr Paul Piccinelli (for the advices on bioinformatics), Derek, Leo & Timothy (for our precious friendship), Eagle (for bringing me and Oona together), Dr Frank Michelangeli (for providing me an early ambition to do research and your invaluable advices), Dr Heidi Welch (for the great subcloning project to practise my molecular biology techniques and your encouragement), Drs John Puddefoot & Stewart Barker (for lending me your Western blotting equipment), Johnny Lau & Robert Li (for sharing golfing and magic tips), Drs Manuela Lahne & Yaniv Hintis (for sharing your scientific advices and brilliant ideas), Dr Michela Egertova (for teaching me how to do cryostat sectioning), Dr Raju Tatituri (for giving me an early ambition to pursue a Ph.D.), Miral Parmar (for your big support and sharing many funny jokes and video clips with me), Rosina Tsang (for the best pastries in Milton Keynes), Drs Sarah Batt, Usha Veeraraghavan & Natacha Veerapen (for your caring, support and taking good care of Oona for me while I’m in London). I really feel that it is my pleasure to have you all here throughout this journey. Thank you people!! ;) Finally, I have to say it always has been my pleasure to work on this fascinating project in Rachel’s lab and I hope my work will contribute to the community. 7 LIST OF ABBREVIATIONS ANOVA Analysis of variance AP Alkaline phosphatase ATP Adenosine 5’-triphosphate BCIP 5-bromo-4-chloro-3-indolyl phosphate BLAST Basic local alignment and search tool bp Base pairs Ca2+ Calcium ion [Ca2+] Concentration of calcium ions 2+ 2+ [Ca ]i Concentration of intracellular calcium ions (cytosolic Ca ) cAMP 3’, 5’-cyclic adenosine monophosphate cDNA Complementary DNA CICR Ca2+ induced Ca2+ release CNS Central nervous system Da Dalton DAB Diaminobenzidine DEPC Diethylpyrocarbonate DHPR Dihydropyridine receptor DICR Depolarisation induced Ca2+ release DIG Digoxygenin DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid dNTPs Deoxynucleotidyl triphosphates DTT Dithiothreitol ECC Excitation-contraction coupling ECL Enhanced chemiluminescence EDTA Ethylenediaminetetraacetic acid EGTA Ethyleneglycoltetraacetic acid ELISA Enzyme linked immunosorbant assay 8 ER Endoplasmic reticulum EST Expressed cDNA sequence tag gDNA Genomic DNA HEPES N-[2-hydroxyethyl] piperazine-N’-[2-ethane sulphonic acid] hpf Hours post fertilisation HRP Horseradish peroxidase HS Horizontal myoseptum ICC Immunocytochemistry IgG Immunoglobulin G IHC Immunohistochemistry IP3 Inositol-1,4,5-trisphosphate IP3R IP3 receptor kb Kilo base pairs (103) kDa Kilo Dalton (103) MO Morpholino oligonucleotides
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