University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2019 The Functional and Structural Analysis of Drosophila robo2 in Axon Guidance LaFreda Janae Howard University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/etd Part of the Cell Biology Commons, Computational Biology Commons, Genetics Commons, and the Molecular and Cellular Neuroscience Commons Recommended Citation Howard, LaFreda Janae, "The Functional and Structural Analysis of Drosophila robo2 in Axon Guidance" (2019). Theses and Dissertations. 3379. https://scholarworks.uark.edu/etd/3379 This Dissertation is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected]. The Functional and Structural Analysis of Drosophila robo2 in Axon Guidance A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Molecular Biology by LaFreda Janae Howard Fort Valley State University Bachelor of Science in Biology, 2014 August 2019 University of Arkansas This dissertation is approved for recommendation to the Graduate Council. Timothy A. Evans, Ph.D. Dissertation Advisor Jeffrey Lewis, Ph.D. Ines Pinto, Ph.D. Committee Member Committee Member Michael Lehmann, Ph.D. Paul Adams, Ph.D. Committee Member Committee Member ABSTRACT In animals with complex nervous systems such as mammals and insects, signaling pathways are responsible for guiding axons to their appropriate synaptic targets. Importantly, when this process is not successful during the development of an organism, outcomes include catastrophes such as human neurological diseases and disorders. It is vital to determine the underlying causes of such diseases by understanding the development of the nervous system. There are many pathways that have been identified to play a role in this, however, we lack an understanding of how these pathways can promote such diverse outcomes in different populations of neurons. These pathways include conserved ligand and receptor complexes that can either synergistically or antagonistically determine the fate of axons. Among these complexes include Slit and Robo, the first ligand and receptor complex to be identified in Drosophila. Previous studies show that disrupting this complex causes ectopic midline crossing of axons in a wide range of animals. Here, to analyze the structural foundation of the diverse activities of Robo2, I examine the relative contributions of its Ig domains by generating transgenic animals expressing variant proteins. I show that Ig1 and Ig3 domains are vital for protein stabilization and localization in vivo. I also use a cell overlay assay to examine the structural and functional importance of all domains of Drosophila Robo2. Deleting the Ig1, Ig5, and Fn2 domains of Robo2 reduce Slit binding in cultured Drosophila cells. The other domains of Robo2 are individually dispensable in Robo2’s ability to bind to Slit in vitro. ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Timothy Evans for believing in me even when I could not see the light at the end of the tunnel. Thank you for being a listening ear and pushing me to be the best scientist that I can be. I would also like to say a huge thank you to my parents, Frederick and Wilma Howard. You are the epitome of great parents and I am forever grateful. To my twin, LaFrada Howard, you are my best friend and I would not have chosen anyone else to be by my side throughout this doctoral process. I love you all. I would like to dedicate this dissertation to my late grandmother, Mildred Anderson Howard. May she continue to rest in peace. TABLE OF CONTENTS Chapter One. Introduction: Development of Drosophila melanogaster and an introduction to Roundabout 2............................................................................................................................. ....... 1 Significance ...............................................................................................................................2 Part One: Development of the central nervous system .............................................................3 Part Two: Conservation of axon guidance ................................................................................9 Part Three: Ig superfamily: neural cell adhesion molecules of the immunoglobulin superfamily..........................................................................................................................................13 Conclusions................................................................................................................................16 References..................................................................................................................................18 Chapter Two. The effects of expressing modified Robo2 proteins on cultured Drosophila cells……………………………………………………………………………………………..21 Abstract ....................................................................................................................................22 Background ............................................................................................................................. .22 Materials and Methods .............................................................................................................28 Results ............................................................................................................................. .........30 Discussion ................................................................................................................................34 Conclusions ..............................................................................................................................35 References ................................................................................................................................37 Chapter Three. Analyzing CRISPR-Cas9 modified Robo2 protein expression of Drosophila melanogaster....................................................................................................................................39 Abstract ....................................................................................................................................40 Background ..............................................................................................................................40 Materials and Methods .....................................................................................................................49 Results.................................................................................................................................................55 Discussion .......................................................................................................................................60 Conclusions ........................................................................................................................................61 References ........................................................................................................................ .................63 Chapter Four. Analyzing the function of CRISPR-Cas9 modified Robo2 expressing embryos………………………………………………………………………………………………..65 Abstract .............................................................................................................................................66 Background .......................................................................................................................................67 Materials and Methods ........................................................................................................................72 Results ............................................................................................................................. ..................74 Discussion .........................................................................................................................................81 Conclusions ........................................................................................................................................83 References .........................................................................................................................................84 Conclusions and Future Directions................................................................................................87 Appendix 1. Institutional Biosafety Committee Approval Letter................................................91 Appendix 2. List of Generated Stocks ............................................................................................92 Appendix 3. Plasmid Construction Maps ......................................................................................93 Appendix 4. Ectopic Crossing Scoring and Lateral Positioning Data…………………………...113 Appendix 5. CRISPR Data..............................................................................................................127 Appendix 6. Reannotation of Robo2 Fn deletions .........................................................................130 DEFINITIONS 1. Fruit fly – Drosophila melanogaster 2. Axon guidance – developmental process by which neurons send axons out to reach their synaptic targets. 3. DCC – deleted in colorectal cancer 4. CNS – central nervous system