ROLE OF FZR1 IN EMBRYOGENESIS SEAH KAY YI MICHELLE BSC. (HONS I) PH.D THESIS Statement of Originality This thesis contains no material which has been accepted for the award for any other Degree or Diploma in any University or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University library, being made available for loan and photocopying subject to the provisions of the Copyright Act 1968. Seah Kay Yi Michelle 17th December 2012 Page | ii Acknowledgements I would like to sincerely express my appreciation and gratitude to my supervisors, Keith and Janet for giving me this opportunity and to share their wisdom and guidance throughout my PhD. I would also like to extend my thanks to all the lab members including Evan, Jess, Julie, Kyra, Nicole, Phoebe, Simon, Sophia, Suzanne and Yan. Thank you for your company and help throughout my PhD, it has made this an enjoyable experience in the lab. To my family especially Popo, Mummy and Daddy, thank you for the endless love, support and understanding that you have showered upon me. For that, I will be eternally grateful. Thank you for moulding me into the person that I am today, for without all of you, I will not be where I am today. I love you and will always be your little girl. To Clara, my BFF, thank you for your encouragements and to always be there for me. I will treasure our intellectual and bimbo moments. The fun and laughter have been missed. To Huili, thank you for the visits, support and the crazy times we shared. To David, for introducing me into this wonderful world of research, your guidance was invaluable. To all my friends and relatives back home, thank you for all the love and support. To Brown Bear and Zee Zee, for all your endless love and company, you will be missed dearly. To Timbre, your bountiful happiness and affection has made the trying times easier and the journey more interesting. Last but not least, I would like to thank Wai Shan. Thank you for being there for me through the good times and bad, the happy and sad, the crazy and sane, you have been a great source of strength and inspiration. Thank you for always believing in me and sharing this journey. Thank you for all the wonderful memories, they will be cherished. I await for what lies ahead in the future. Page | iii Abstract The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that targets many cell cycle associated proteins for degradation by the 26S proteasome. APC activity is in part controlled by binding to one of its co-activators, and here the focus of the study was on the co-activator Fizzy- Related 1 (Fzr1). When this thesis was started, antisense knockdown studies had shown that Fzr1 is involved in the maintenance of Germinal Vesicle (GV) stage arrest; the fidelity of chromosome segregation during the first meiotic division; and measurable APCFzr1 activity had been observed following fertilization. Although these antisense experiments had used controls, it was still appropriate to question whether any of these observations were due to off-target effects. Moreover, it was important to ask how relevant Fzr1 was to the process of meiosis in vivo, given thus far only in vitro studies had been performed. During the course of my studies, a transgenic knockout of Fzr1 was independently created, and using embryos from these mice, it was found that this APC activator has a crucial role in endoreduplication, and was needed for trophoblast giant cell formation and placentation. However, due to the presence of maternal Fzr1 contribution, its role in early preimplantation embryogenesis or meiosis could not be addressed. Here in this thesis, I created an oocyte-specific Fzr1 knockout from breeding mice that had loxP elements inserted into the Fzr1 gene with those carrying the Zp3-Cre recombinase promoter. Breeding results in progeny that have an oocyte-specific deletion in their Fzr1 gene. I was able to determine that knockout females produce viable metaphase II eggs capable of generating live pups when fertilized. Therefore, even though measurable activity of APCFzr1 has been detected at various stages of meiosis, this APC activator was not an essential meiotic gene. I investigated an optimal culture media suitable to sustain preimplantation development of B6CBF1 hybrid and C57Bl6 fertilized embryos and parthenotes to the blastocyst stage. Using parthenotes from knockout females, I was able to show that Fzr1 is involved in the process of pronuclear fusion at syngamy in zygotes. Its loss also resulted in delayed and asynchronous cleavage divisions of blastomeres; which led either to arrest of embryos at the 2-cell stage or premature initiation of compaction in 4-cell stage embryos that then failed to develop to form blastocysts. Additionally, I developed a miRNA construct that achieved Fzr1-/- knockdown in embryos and also showed a 2-cell arrest phenotype. Examination of 1- and 2-cell Fzr1-/- parthenotes also showed an increased incidence of chromosomal instability, demonstrated by the formation of micronuclei and fragmented DNA. In summary, work of this thesis found that Fzr1 is not an essential meiotic gene, as knockdown studies may have suggested, but instead was remarkably essential for early embryonic development. Page | iv Table of Contents Statement of Originality ................................................................................................................ ii Acknowledgements ...................................................................................................................... iii Abstract ........................................................................................................................................ iv List of Tables ............................................................................................................................. xiv List of Figures ............................................................................................................................. xv List of Abbreviations ................................................................................................................. xix 1. Introduction ....................................................................................................................... - 1 - 1.1. Female gametogenesis .............................................................................................. - 1 - 1.1.1. Overview of female meiosis ............................................................................. - 2 - 1.1.2. Fertilization and egg activation ......................................................................... - 4 - 1.1.3. Syngamy ........................................................................................................... - 6 - 1.2. Overview of Embryogenesis ................................................................................... - 10 - 1.2.1. Timing of cell cycle in early embryogenesis .................................................. - 11 - 1.2.2. Embryonic genome activation ........................................................................ - 12 - 1.2.3. 2-cell block ...................................................................................................... - 14 - 1.2.4. Compaction ..................................................................................................... - 17 - 1.2.6. Blastocyst formation ....................................................................................... - 22 - 1.3. The Anaphase Promoting Complex ........................................................................ - 23 - 1.3.1. Co-activators of the anaphase-promoting complex ......................................... - 24 - 1.4. Role of APC in mitosis ........................................................................................... - 25 - 1.4.1. Role of APC in mitotic entry .......................................................................... - 25 - 1.4.2. Involvement of APC at anaphase .................................................................... - 25 - Page | v 1.4.3. APC in regulating mitotic exit ........................................................................ - 27 - 1.4.4. G1/S maintenance by APC activity................................................................. - 29 - 1.5. Roles of APCFzr1 outside of the cell cycle ............................................................... - 29 - 1.5.1. Cell cycle exit and G0 maintenance ................................................................ - 30 - 1.5.2. Role of Fzr1 as a tumour suppressor and in genomic integrity maintenance .. - 30 - 1.5.3. Role in endoreduplication ............................................................................... - 32 - 1.5.4. Role of Fzr1 in cellular differentiation ............................................................ - 32 - 1.6. Role of APC in meiosis ........................................................................................... - 34 - 1.6.1. APC mediating homolog and sister chromatid separation .............................. - 34 - 1.6.2. Importance of APC in maintaining GV arrest ................................................. - 35 - 1.6.3. Importance of APC in Prometaphase I progression ........................................ - 37 - 1.6.4. APC activity during meiosis II .......................................................................