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Regulation of Muscle Stem Cell Function by the Transcription Factor Pax7

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Regulation of Muscle Stem Cell Function by the Transcription Factor Pax7 Regulation of Muscle Stem Cell Function by the Transcription Factor Pax7 Alessandra Pasut Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate in Philosophy degree in Cellular and Molecular Medicine Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Alessandra Pasut, Ottawa, Canada, 2015 ...above all don’t fear difficult moments, the best comes out from them. [Rita Levi Montalcini-Nobel laureate, 1909-2012] ii Abstract Pax7 is a paired box transcription factor expressed by all satellite cells which are critically required for muscle regeneration and growth. The absolute requirements of Pax7 in the maintenance of the satellite cell pool are widely acknowledged. However the mechanisms by which Pax7 executes muscle regeneration or contributes to satellite cell homeostasis remain elusive. We performed cell and molecular analysis of Pax7 null satellite cells to investigate muscle stem cell function. Through genome wide studies, we found that genes involved in cell- cell interactions, regulation of migration, control of lipid metabolism and inhibition of myogenic differentiation were significantly perturbed in Pax7 null satellite cells. Analysis of satellite cells in vitro showed that Pax7 null satellite cells undergo precocious myogenic differentiation and have perturbed expression of genes involved in the Notch signaling pathway. We showed that Notch 1 is a novel Pax7 target gene and by using a genetic approach we demonstrate that ectopic expression of the constitutively active intracellular domain of Notch1 (NICD1) in Pax7 null satellite cells is sufficient to maintain the satellite cell pool as well as to restore their proliferation. Instead of differentiating into myogenic cells and in the absence of a myogenic cue, NICD1 Pax7 null satellite cells become a source of ectopic brown fat within muscles and give rise to brown adipocytes both in vivo and in vitro. In conclusion we showed that Notch 1 partially rescues Pax7 deficient satellite cells loss and proliferation. Additionally we provide the first evidence that Notch signalling contributes to satellite cell fate by inhibiting terminal myogenic differentiation and inducing brown adipogenesis. iii Aknowledgements My first BIG thank you goes to my supervisor. Michael, you inspire me to be creative and fearless. Your leadership showed me what it takes to be at the forefront of innovation and research and I hope the skills I’ve learned in your lab and under your guidance will be the foundation for reaching even bigger achievements. Ad majora semper. A SINCERE thank you goes to my lab mates, past and present. Too many to mention here. You know who you are. A SPECIAL thank you goes to Jennifer Ritchie, Hong Ming and Fan Xiao. Your help has been invaluable throughout these years. During my PhD I had the pleasure to work for and collaborate with excellent scientists both within OHRI and outside. I’d like to thank my PhD advisory committee members Dr Valerie Wallace, Dr Alan Mears, Dr Lynn Megeney and Dr Jeff Dilworth for their guidance as well as Dr Vittorio Sartorelli for being a long distant but precious advisor. A WARM and affectionate thank you goes to my dearest Canadian friends, Sarah, Mari, Alex , the 977 Shefford girls Tammy and Laura, Erika, Andrea, Paul and Sandy. A SUPER thank you goes to those people that have known me forever and still haven’t gotten tired of me: Vane and Le Sacilesi, grazie per essermi state vicino anche se lontane; Elena e Piera abbiamo iniziato e finito i rispettivi dottorati in tre paesi diversi e ne abbiamo condiviso gioie e dolori! My final and MOST IMPORTANT thank you goes to my family. Thank you for giving me this incredible opportunity. I hope I made you proud. Grazie per avermi supportato e sopportato in questi anni, spero di avervi resi orgogliosi. A special thank you goes to my grandma, your b-day cards and Christmas messages sent over the ocean and across thousands miles made me feel closer to home. La tua forza e la tua dolcezza sono un esempio di vita che rimarra’ con me per sempre. iv Contributions of co-authors All experimental work was performed by the thesis author unless otherwise specified. The writing and preparation of the manuscript in Chapter 2 were carried out by the author, with help from Dr Michael Rudnicki and Paul Oleynik for FACS analysis and interpretation of results. The writing and preparation of the manuscript in Chapter 3 were carried out by the author with help from Michael Rudnicki. RNA-seq data presented in Figure 1C and Supplementary Figure 2 of Chapter 3 were carried out by Chris Porter. Data presented in Figure 4A and C of Chapter 3were provided by Hong Ming. Dr Vahab Soleimani helped with the analysis of ChIP-seq data and provided all cell lines used to perform experiments in Figure 4A. Dr Robin Parks provided adenovirus constructs used in Figure 7 and Supplementary Figure 7. Dr Melanie Lacaria performed cardiotoxin injury and provided technical help with experiments shown in Figure 8. Jennifer Ritchie contributed to the maintenance of all mouse lines used throughout this thesis. v Contents Abstract…………………………………………………………………………………… iii Acknowledgments……………………………………………………………………….... iv Contributions of co-authors……………………………………………………………….. v Contents………………………………………………………………………………….... vi List of Figures……………………………………………………………………………… viii List of Tables………………………………………………………………………………. ix List of Abbreviations………………………………………………………………………. x Chapter 1-General Introduction................................................................................................................................... 1 1.1 The anatomy and mechanics of the adult skeletal muscle tissue…………………………. 2 1.2 The embryogenesis of the skeletal muscle tissue………………………………………… 3 1.3 Molecular determinants of muscle differentiation………………………………………... 8 1.4 Satellite cells regulate muscle regeneration and post natal growth………………………. 10 1.5. Heterogeneity of satellite cells…………………………………………………………… 14 1.6. Regulation of satellite cells quiescence…………………………………………………... 15 1.7 Pax7 is an absolute requirement of satellite cells in adult myogenesis…………………... 17 1.8. Pax7 transcriptional network…………………………………………………………….. 19 1.9. Regulation of satellite cells by the muscle stem cell niche………………………………. 21 1.10. Role of Notch signalling in adult myogenesis………………………………………….. 22 1.11. Alternative sources of muscle regeneration…………………………………………….. 26 1.12. Relevance of studying muscle regeneration…………………………………………….. 26 1.13. Hypothesis and rationale………………………………………………………………... 27 Chapter 2- Isolation of Muscle Stem Cells by Fluorescence Activated Cell Sorting Citometry……………………………………………………………………………………… 28 Summary……………………………………………………………………………………… 29 Introduction…………………………………………………………………………………… 30 Materials………………………………………………………………………………………. 31 Methods……………………………………………………………………………………….. 32 Conclusions…………………………………………………………………………………… 38 References…………………………………………………………………………………….. 41 Chapter 3- Notch1 Rescues Pax7-deficient Satellite Cell Loss and Induces Brown Adipogenesis.................................................................................................................................42 vi Summary……………………………………………………………………………………… 44 Introduction…………………………………………………………………………………… 45 Results………………………………………………………………………………………… 47 Discussion…………………………………………………………………………………….. 56 Material and methods…………………………………………………………………………. 59 Acknowledgments…………………………………………………………………………….. 65 Contributions of authors………………………………………………………………………. 65 References…………………………………………………………………………………….. 65 Figures………………………………………………………………………………………… 69 Supplementary information…………………………………………………………………… 94 Chapter 4- General Discussion.................................................................................................. 99 4.1. General overview………………………………………………………………………. 100 4.2. FACS based approach for gene expression studies of stem cells………………………. 100 4.3. Gene expression studies of Pax7 null satellite cells……………………………………. 101 4.4. Regulation of Notch signaling by Pax7…………………………………………………. 103 4.5. Notch signaling partially fulfills Pax7 requirements in adult myogenesis……………… 104 4.6. Role of Pax7 and Notch in lineage progression………………………………………… 106 4.7. Future directions………………………………………………………………………… 107 4.8. Relevance……………………………………………………………………………….. 109 4.9. Conclusive remarks……………………………………………………………………... 109 References...................................................................................................................................111 Appendix A-Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle................................................................................................................. 127 Appendix B-MicroRNA-133 Controls Brown Adipose Determination in Skeletal Muscle Satellite Cells by Targeting Prdm16............................................................................................135 Appendix C-Polycomb EZH2 Controls Self -renewal and Safeguard the Transcriptional Identity of Skeletal Muscle stem Cells..................................................................................................... 151 vii List of Figures Chapter 1- General Introduction Figure 1.2 Embyonic development of the skeletal muscle tissue…………………………… 7 Figure 1.4. Satellite cells in adult myogenesis………………………………………………. 13 Figure 1.10. The Notch signaling pathway………………………………………………….
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