University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2019-01-22 Semaphorin3f as a Spatial Regulator of Embryogenesis Halabi, Rami Halabi, R. (2019). Semaphorin3f as a Spatial Regulator of Embryogenesis (Unpublished doctoral thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/109507 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Semaphorin3f as a Spatial Regulator of Embryogenesis by Rami Halabi A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN NEUROSCIENCE CALGARY, ALBERTA JANUARY, 2019 © Rami Halabi 2019 Abstract During embryogenesis, cells integrate both spatial and temporal information from their surroundings to influence proliferation, migration, differentiation and physiological functions. Understanding the molecular mechanisms which confer spatial identity is essential to our understanding of tissue development and human disease. In this thesis I explore multiple roles for the secreted chemotactic ligand Semaphorin3f (Sema3f) in different biological contexts. Using zebrafish (Danio rerio) as a model I take advantage of the duplicated genome to study loss of function of both orthologs, Sema3fa and Sema3fb, in discrete contexts due to their differential expression. First, I show that in the eye Sema3fa produced by progenitors is necessary for the generation of amacrine cells within the temporal retina and the spatially organized transcriptome of stem cells in the ciliary marginal zone (CMZ). Second, I define an endogenous role of Sema3fa to maintain the avascularity of the neural retina and refine the branch pattern of intraocular vessels. Loss of Sema3fa results in the pathologic angiogenesis of leaky blood vessels into the neural retina. Last, I unveil a role for Sema3fb produced by cardiomyocyte progenitors in the differentiation of the ventricle of the developing heart. Overall, my work provides the first evidence of a Sema3 involved in retinal progenitor cell and cardiomyocyte differentiation, and elucidates the endogenous role of Sema3fa as a negative regulator of retinal blood vessels in the embryo and adult. My data exemplifies the necessity of spatial information conferred by a single chemotactic molecule, Sema3f, to impact differentiation and cellular biology. 1 Acknowledgements I thank Sarah McFarlane for all her help and mentorship over the last five years. You are a great supervisor and I appreciate your support in developing my own skills, both inside and outside of the lab. Thank you to my committee members, Sarah Childs and Peng Huang, for your input during my PhD. Thank you to both my external examiners Jeff Biernaskie and Vince Tropepe for your readiness to be part of the examination committee. I thank all the members of the McFarlane lab for their support, guidance and contributions over the years. Thank you to Carrie Hehr for all your assistance in running experiments and maintaining the lab so we can all work at full capacity. Thank you Karen Atkinson-Leadbeater for your career guidance, and Gabriel Bertolesi for all your help in consolidating experimental ideas. I thank Paula Cechmanek for all the great conversations, both scientific and life related, and for the excellent collaboration we had by the end of our respective PhDs. Thank you Jonathan Yang and Zachary Nurcombe - our chats in the “cove” will be missed! Amira Kalifa and Risa Mori- Kreiner, I appreciate all the help you have given me this last little while and the passion you have shown for your work is brightening. I also extend my thanks to all those that have made this PhD memorable in many ways: Debbie Kurrasch, Cairine Logan, Lian Willets, Dierdre Lobb, Cam Teskey, Andy Bulloch, Kelly Cook and Carol Schuurmans. Thank you for the conversations, the advice, and the opportunities you have afforded me over the years. To my entire family – Samar, Riad, Rabih, Marwan, Amal, Stella, Alexander, Anabella, Firas, Rosa and Sarah – thank you for supporting me and asking what experiments I am in the middle of despite not understanding a thing of what I said. Thank you to 2 the administrative and education personnel – Tianni Song, Lesley Towill and Jason Ng - whom I have worked closely with on side projects. Thank you to all my friends, both old and new, you have made my time in Calgary wonderful. Thank you to Charlene Watterston, the honorary McFarlane Lab Member - you are an amazing friend and collaborator. Thank you to Rami Abu Zeinab – you are an excellent friend and have been great help in preparing me for my next steps post PhD. Thank you to Raquel Cruz for everything you have done and continue to do for me. And to Matt Lemieux, Becky Klein, Cody Sahlin, Yamile Jasaui, Tooka Collette, Amlish Munir, Julie Dang, Alexandra Sull, Judith Sull and Graham Ring for always being there when I needed a break from writing. And thank you to all others I have not directly mentioned here - the last 5 years would not have been the same without you. Lastly, I thank the Department of Neuroscience, ACHRI, Hotchkiss Brain Institute, REALISE, AIHS, BFF and NSERC. Without your support and without the opportunities you have afforded me I would not be where I am now. Thank you. All writing, experiments, quantifications, images and figures were completed by myself, with the following exceptions: Figures 3.20 and 5.20 – explants of tissues were done by Carrie Hehr. Plastic sectioning and in situ hybridization of several genes were also carried out by Carrie Hehr. Figures 5.23 and 5.24 – figures made by Dr. Paula Cechmanek Figures 5.11, 5.16, and 5.19 – confocal images taken by Dr. Paula Cechmanek. Figures 4.3, 4.4, 4.5, 4.6, 4.7 – confocal images taken by Charlene Watterston. 3 Dedication To my Mother, for her continued encouragement. 4 Table of Contents Abstract ................................................................................................................................1 Acknowledgements ..............................................................................................................2 Dedication ............................................................................................................................4 Table of Contents .................................................................................................................5 List of Tables .......................................................................................................................9 List of Figures and Illustrations .........................................................................................10 List of Symbols, Abbreviations and Nomenclature ...........................................................14 CHAPTER ONE: INTRODUCTION ................................................................................17 1.1 Spatial Cues in Development ...................................................................................17 1.1.1 Neurovascular Guidance .................................................................................18 1.1.2 Chemotactic Molecules involved in Cellular Guidance ..................................19 1.1.3 Sema3 Signalling During Development ..........................................................23 1.1.4 Spatially Regulated Tissues in Development ..................................................25 1.2 Development of the Zebrafish Neural Retina ..........................................................28 1.2.1 Organization of the neural retina .....................................................................28 1.2.2 Development of the zebrafish retina and lens .................................................30 1.2.3 Neurogenesis of the zebrafish retina ...............................................................31 1.2.4 Neuronal differentiation in the zebrafish retina ...............................................32 1.2.5 Development of the RGC axon projection ......................................................34 1.2.6 Class 3 Semas in Retinal Development and Injury .........................................35 1.3 Development of the Vasculature of the Zebrafish Retina ........................................35 1.3.1 Hyaloid and Retinal Vasculature Development ..............................................36 1.3.2 Choroidal Vasculature Development ..............................................................38 1.3.3 Blood-Retina-Barrier .......................................................................................40 1.3.4 Age Related Macular Degeneration ................................................................40 1.3.5 Class 3 Semas in Orbital Vessel Dynamics .....................................................42 1.4 Development of the Zebrafish Cardiovascular System ...........................................43 1.4.1 Specification and Differentiation of the Zebrafish Heart ................................43 1.4.2 Cardiac Chamber Morphogenesis ...................................................................46 1.4.3 Cardiac Neural Crest and Second Heart Field .................................................47