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COPYRIGHT AND USE OF THIS THESIS This thesis must be used in accordance with the provisions of the Copyright Act 1968. Reproduction of material protected by copyright may be an infringement of copyright and copyright owners may be entitled to take legal action against persons who infringe their copyright. Section 51 (2) of the Copyright Act permits an authorized officer of a university library or archives to provide a copy (by communication or otherwise) of an unpublished thesis kept in the library or archives, to a person who satisfies the authorized officer that he or she requires the reproduction for the purposes of research or study. The Copyright Act grants the creator of a work a number of moral rights, specifically the right of attribution, the right against false attribution and the right of integrity. You may infringe the author’s moral rights if you: - fail to acknowledge the author of this thesis if you quote sections from the work - attribute this thesis to another author - subject this thesis to derogatory treatment which may prejudice the author’s reputation For further information contact the University’s Director of Copyright Services sydney.edu.au/copyright The Morphological and Genetic Characterisation of Pelvic Finless Zebrafish Emily Kate Don The Laboratory of Development and Disease Models A thesis submitted in fulfilment of the requirements for the degree Doctor of Philosophy (Medicine) in the Discipline of Anatomy and Histology, Bosch Institute The University of Sydney 2013 i Declaration I hereby certify that this thesis does not contain, without appropriate acknowledgement, any material previously submitted for a degree or diploma in any university, except where due reference is made. I also certify that this thesis does not contain, to the best of my knowledge, any material previously published or written by another person, except where due reference is made. Emily Kate Don Dr Nicholas Cole ii Acknowledgments I would like to say thank you to all the people from the Anderson Stuart and zebrafish communities who have assisted me during my time as a PhD student. Firstly, I would like to deeply thank my supervisor Dr. Nicholas Cole who has lived and breathed this project alongside me, through all its ups and downs. In particular, thank you for allowing me this opportunity and the invaluable training you have provided. Thank you for your ongoing enthusiasm, input and support (both professionally and personally). It has been an interesting ride from setting up a new laboratory all the way through to changing institutions. I wish you the best of luck in your new endeavour over at ASAM. I would also like to thank the Australian government for awarding me an Australian Postgraduate Award to allow me to undertake my doctoral research. A big thank you needs to go to my mapping team: Dr. Tanya de Jong-Curtain and Associate Professor Joan Heath. Thank you for taking me into your lab and teaching positional cloning to me. Thank you for your ongoing assistance and input. To Dr. Louise Cole, Dr. Donna Lai, Dr. Dane King, Dr. Errin Johnson and Dr. Ian Kaplin, thank you for your technical knowledge that made this project possible. To the lab, and in particular, Claire and Andy, I’m not sure that thank you is a big enough word. You have shared this project with me, significantly contributed, been there for my little victories, listened to my complaints and most of all, put up with me on a daily basis! My time in the Anderson Stuart building would not have been the same without the social club. Thank you to all who have made my Friday evenings over the past few years just that little bit more entertaining. Keep up the tradition, we all know that the best collaborations are made over a nice cold beer! I also owe a huge thank you to my family. Mum and Dad, thank you for instilling in me your love of education and knowledge. Thank you for teaching me, by example, that you can have anything you want in life, if you are prepared to work hard enough for it. Thank you for the proofreading of this thesis. If that doesn’t show unconditional love, I don’t know what does. To Claire and Hannah, thank you for your support during this project and always inspiring me to finish and get a real job My biggest and most heartfelt thanks must go to Kjetil. This would not have been possible without you, and your love and support. You have always been there to celebrate iii the highs with me and provide a hug that fixed everything during the lows. I’m sorry you now know so much more about pelvic fins than you ever wanted to know. I am so excited for our wedding in February and cannot wait to begin of the rest of our lives together. I love you. iv Table of contents Declaration................................................................................................................................ ii Acknowledgments .................................................................................................................. iii Table of contents ...................................................................................................................... v Abstract .................................................................................................................................... ix Research publications and awards ..................................................................................................................... xi List of abbreviations .......................................................................................................................................... xiii Nomenclature ...................................................................................................................................................... xv List of Tables and Figures ................................................................................................................................. xvi List of Tables ...................................................................................................................................................... xix 1. Introduction .......................................................................................................................... 1 1.1. Introduction to development ........................................................................................................................ 1 1.1.1. Introduction to zebrafish as developmental models .................................................................................... 2 1.1.2. Advantages of utilising zebrafish as developmental models ........................................................................ 3 1.1.3. Zebrafish fins as a model for development .................................................................................................. 6 2. Literature Review ................................................................................................................ 8 2.1. Updates to literature review........................................................................................................................ 29 2.1.1. Examination of Euphanerops ...................................................................................................................... 29 2.1.2. Publication of the African coelacanth genome........................................................................................... 29 3. Materials and methods ...................................................................................................... 30 3.1. Production and manipulation of zebrafish ................................................................................................ 30 3.2. Molecular methods ...................................................................................................................................... 34 3.3. Staining ......................................................................................................................................................... 42 3.4. Microscopy ................................................................................................................................................... 46 3.5. Statistical methods ....................................................................................................................................... 46 3.6. Sequence analysis and alignments .............................................................................................................. 47 4. Morphological characterisation of pelvic finless zebrafish ............................................ 48 4.1. Introduction ................................................................................................................................................. 48 4.2. Results ........................................................................................................................................................... 49 4.2.1. Embryonic morphology of pelvic finless zebrafish ..................................................................................... 49 4.2.2. Larval morphology of pelvic finless zebrafish ............................................................................................. 52 4.2.3. Adult morphology of pelvic finless zebrafish .............................................................................................. 54 4.2.4. Pelvic finless zebrafish display abnormal pelvic fin development .............................................................. 57 4.2.5. Pelvic fins of pelvic finless