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Thesis Abstract Post-transcriptional Regulatory Mechanisms Controlling Development of Murine Cerebral Cortical Precursors by Gianluca Amadei A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Gianluca Amadei (2016) Post-transcriptional Regulatory Mechanisms Controlling Development of Murine Cerebral Cortical Precursors Gianluca Amadei Doctor of Philosophy Department of Molecular Genetics University of Toronto 2016 Thesis Abstract The complex neural circuitry of the mammalian nervous system arises from a small pool of neural precursors that, during development, sequentially gives rise to neurons, astrocytes and oligodendrocytes. Several molecular mechanisms and environmental stimuli regulate the expansion of the neural precursor pool and the subsequent generation of differentiated progeny. In this thesis, I sought to investigate whether post-transcriptional regulation plays a role in the development of mammalian neural precursors. In the first part of this thesis, I show that the double stranded RNA binding protein Staufen2 is part of a repressive complex, with Pumilio2 and DDX1, that prevents early differentiation of neural precursors into neurons by repressing the translation of neurogenic mRNAs such as prox1. In the second part of the thesis I show that Smaug2, a translational repressor, also prevents early generation of neurons by repressing nanos1 mRNA, which encodes another RNA-binding protein. I also show that nanos1 repression occurs by its inclusion in a P-body like granule with 4E-T, another known repressor of mRNA translation. In the last chapter I identify mRNAs associating with Smaug2 and I suggest that Smaug2 may have additional functions that are independent of nanos1 regulation and that unlike nanos1, which is regulated together by Smaug2 and 4E-T, the majority of Smaug2 mRNAs are regulated independently from 4E-T. Together, these studies suggest that several RNA-binding ii proteins are crucial regulators of cortical development and that they perform this role by forming several, largely independent, repressive RNA-protein complexes. I suggest that these repressive complexes prime neural precursors to generate progeny at the appropriate time by repressing proneurogenic mRNAs until the appropriate developmental cue. iii Acknowledgments Upon completion of this doctoral degree, I realize more than ever that this journey could not have been completed without the help of so many amazing people. First and foremost, I would like to thank my supervisors Freda and David for all their help along this PhD. Your support, mentoring, and guidance meant so much to me and your passion for science has been a constant inspiration. Thank you for taking a chance on me and letting me study in your laboratory. It has been a terrific opportunity and a life changing experience! I would like to thank my committee members Dr. Bret Pearson and Dr. Howard Lipshitz, who have constantly been evaluating my progress over the years. Your invaluable intellectual input and criticism was instrumental in guiding this thesis along and I am very grateful for all your help. Many many thanks are also due to our wonderful collaborators, Dr. Craig Smibert and Dr. Jason Dumelie. When we were not sure where to go next, you were always able to offer advice and technical expertise and I am very grateful to you for that! My gratitude also goes to my wonderful colleagues and friends in the Kaplan/Miller laboratory. I could not have asked for a more talented group of people to work with. Over the years, the incredible display of talent, dedication and passion for science that I have witnessed in each and every one of you is both humbling and inspiring. I feel every one of you, in his/her own way, has taught me something valuable and I hope to put these lessons to good use in my future work. I will always cherish our friendship and the memory of our years together. A special place in this PhD journey belongs to two wonderful people, Peter and Sally Cant. Over the past few years I have been fortunate enough to benefit from the scholarship they established to honour the memory of their son, David S. Cant and I have also had the privilege of getting to know them better on a number of occasions. It is hard to find two people who are more kind, gentle, and generous than Sally and Peter and I will never forget their commitment to supporting brain research and students. It is also thanks to the contribution of people like them that research can move forward. Peter, Sally, I will never forget you and your generosity! Last but not least, many thanks to my family and good friends. I do not know where I would be without your unwavering support. I am so fortunate to have you in my life! To all of you, thank you, thank you, thank you, from the bottom of my heart! iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v List of Abbreviations ................................................................................................................... viii List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................. xiii Chapter 1 Literature Review ........................................................................................................... 1 1.1 Overview of thesis objectives: .................................................................................................. 1 1.2 Overview of murine cortical development: .............................................................................. 2 1.3 Neural diversity results from a heterogeneous pool of cortical precursors: ............................. 5 1.3.1 Neuroepithelial stem cells (NESCs): ..................................................................................... 5 1.3.2 Radial precursor cells (RP): ................................................................................................... 7 1.3.3 Basal or intermediate progenitor cells: .................................................................................. 8 1.3.4 Outer radial glial precursor cells: ........................................................................................... 9 1.3.5 Oligodendrocyte precursor cells: ......................................................................................... 10 1.4 Mechanisms regulating cortical neural precursor self-renewal versus differentiation: .......... 10 1.4.1 Mitotic polarity as a potential regulator of neural precursor cell fate: ................................ 11 1.4.2 RNA-binding proteins as asymmetrically-segregated determinants in stem cell biology: .. 12 1.4.2.1 The RNA-binding protein Staufen2: ................................................................................. 15 1.4.2.2 The post-transcriptional regulator Smaug2: ...................................................................... 18 1.4.2.3 The function of mammalian Smaug: ................................................................................. 22 1.4.3 A potentially conserved mRNA repression pathway: .......................................................... 27 1.4.3.1 Nanos: ............................................................................................................................... 27 1.4.3.2 4E-T: ................................................................................................................................. 28 Chapter 2 Experimental Procedures .............................................................................................. 30 Chapter 3 An asymmetrically localized Staufen2-dependent RNA complex regulates maintenance of mammalian neural stem cells .............................................................................. 41 3.1 SUMMARY ............................................................................................................................ 42 3.2 BRIEF INTRODUCTION AND RATIONALE .................................................................... 42 3.3 RESULTS ............................................................................................................................... 43 3.3.1 Staufen2 is apically localized in embryonic radial glial precursors in the developing murine cortex ............................................................................................................................................. 43 3.3.2 Staufen2 is part of an apical RNA complex in radial glial precursors ................................. 46 3.3.3 Asymmetric localization of Staufen2 and prox1 mRNA in dividing radial glial precursors49 3.3.4 Knockdown of Staufen2 promotes neurogenesis and depletes radial glial precursors ........ 52 3.3.5 Knockdown of either Pumilio2 or DDX1 phenocopies the effects of Staufen2 knockdown ....................................................................................................................................................... 58 3.3.6 Disruption of Staufen2-RNA interactions affects prox1 mRNA localization and expression and causes differentiation of radial glial precursors
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