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Staufen 1 Does Not Play a Role in NPC Asymmetric Divisions but Regulates Cellular Positioning During Corticogenesis

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Staufen 1 Does Not Play a Role in NPC Asymmetric Divisions but Regulates Cellular Positioning During Corticogenesis Staufen 1 does not play a role in NPC asymmetric divisions but regulates cellular positioning during corticogenesis by Christopher Kuc A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Molecular and Cellular Biology Guelph, Ontario, Canada © Christopher Kuc, September 2018 ABSTRACT INVESTIGATING THE ROLE OF STAUFEN1 IN ASYMMETRIC NEURAL PRECURSOR CELL DIVISIONS IN THE DEVELOPING CEREBRAL CORTEX Christopher Kuc Advisors: Dr. John Vessey University of Guelph, 2018 Cerebral cortex development relies on asymmetric divisions of neural precursor cells (NPCs) to produce a recurring NPC and a differentiated neuron. Asymmetric divisions are promoted by the differential localization of cell fate determinants between daughter cells. Staufen 1 (Stau1) is an RNA-binding protein known to localize mRNA in mature hippocampal neurons. However, its expression pattern and role in the developing mammalian cortex remains unknown. In this study, Stau1 mRNA and protein were found to be expressed in all cells examined and was temporally and spatially characterized across development. Upon shRNA-mediated knockdown of Stau1 in primary cortical cultures, NPCs retained the ability to self-renew and generate neurons despite the loss of Stau1 expression. This said, in vivo knockdown of Stau1 demonstrated that it may play a role in anchoring NPCs to the ventricular zone during cortical development. ACKNOWLEDGMENTS I would first like to thank my advisor Dr. John Vessey. Throughout these 2 years, you have provided me with an invaluable opportunity and played an instrumental role in shaping me as a scientist. The guidance, support and expertise you have provided me will be always appreciated and never forgotten. I would next like to thank all the members of Vessey lab who I had a pleasure of knowing. Thank you for being everything I needed when I needed it, whether that be a supportive friend, competition or instructor. Thank you to Dendra for all your patience in training me when I was just beginning my adventure in the Vessey lab. Thank you to Hayley for sharing this experience with me side by side. I always felt that we were a team and could rely on you through thick and thin. I really appreciate all these great people I met and I hope we stay in touch. Thank you to all the members of the molecular and cellular biology department. There are a lot of extraordinary people that I had the pleasure of calling my friends. Your laughter and companionship through my journey has helped me more than you could know. Last but not least, I would like to thank my best friend Jennifer. I never met anyone like you who I could share anything and everything with. As we did our degree together we guided each other towards the finish line and supported each other when we needed it most. I will never forget our frustrating, yet enjoyable late night sessions in the lab. Without you, this experience would not be the same and for that I am forever thankful. iii DECLARATION OF WORK PERFORMED I, Christopher Kuc, declare that all the work reported in this thesis was performed by me with the exception of the fluorescent in-situ hyrbidization performed by Julia Brott, the leptomycin B treatment performed by Anastasia Smart and the production of mature cortical culture performed by Hayley Thorpe. iv TABLE OF CONTENTS ABSTRACT ............................................................................................................................. ii ACKNOWLEDGMENTS ....................................................................................................... iii DECLARATION OF WORK PERFORMED ........................................................................iv TABLE OF CONTENTS .......................................................................................................... v LIST OF FIGURES .................................................................................................................vi LIST OF ABBREVIATIONS ................................................................................................ vii Chapter One: Introduction ....................................................................................................... 1 Development of the cerebral cortex .................................................................................................. 1 Asymmetric divisions ........................................................................................................................ 6 The Staufen family of RNA-binding proteins ................................................................................. 10 Staufen: Asymmetric divisions during neurogenesis ...................................................................... 11 Staufen: Synaptic plasticity and memory ....................................................................................... 13 Staufen: Translational control ........................................................................................................ 15 Staufen: mRNA stability ............................................................................................................... 16 Staufen1-mediated decay regulates cortical neurogenesis ............................................................. 17 Rationale .......................................................................................................................................... 19 Hypothesis........................................................................................................................................ 20 Objectives ........................................................................................................................................ 21 Chapter Two: Materials and Methods ................................................................................... 22 Animals ............................................................................................................................................ 22 Cell culture ...................................................................................................................................... 22 Western blotting .............................................................................................................................. 24 RT-PCR ........................................................................................................................................... 24 Transient transfection of cultured cells .......................................................................................... 25 In utero electroporation .................................................................................................................. 26 Immunocytochemistry and immunohistochemistry ....................................................................... 27 Microscopy and quantification ....................................................................................................... 28 Statistics ........................................................................................................................................... 29 Chapter Three: Results ........................................................................................................... 30 Stau1 mRNA and protein is expressed in NPCs, newly born neurons, and intermediate progenitors in the developing murine cortex .................................................................................. 30 Stau1 is expressed by all cells of the developing cortex, is predominantly cytoplasmic, and can shuttle in and out of the nucleus...................................................................................................... 33 Stau1 is dispensable for NPC self-renewal and for neuronal differentiation................................. 41 Chapter Four: Discussion ....................................................................................................... 50 Chapter Five: Summary and Future Directions .................................................................... 58 Future directions ............................................................................................................................. 58 Summary ......................................................................................................................................... 59 References ............................................................................................................................... 61 Appendix: Supplementary Data ............................................................................................. 69 v LIST OF FIGURES Figure 1.1. Cytoarchitecture of the developing cerebral cortex. ............................................ 4 Figure 1.2. Timeline of murine brain development. .............................................................. 5 Figure 1.3. Structural comparison between Stau1 isoforms. ................................................ 12 Figure 3.1. stau1 mRNA and protein is expressed by NPCs and newborn neurons during cortical development. ................................................................................................ 31 Figure 3.2. Stau1 is expressed throughout the developing murine cortex. .......................... 35 Figure 3.3. Stau1 is expressed in NPCs, as well as newborn and mature cortical neurons. 39 Figure 3.4. Stau1 shuttles between the cytoplasm and nucleus of NPCs. .......................... 42 Figure 3.5. Stau1 shRNAs knock-down Stau1 protein
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