The Role of CASK in Neuronal Morphogenesis and Brain Size in Drosophila: A Genetic Model of Human Intellectual Disability with Microcephaly Item Type text; Electronic Dissertation Authors Tello Vega, Judith Arane Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 29/09/2021 20:08:46 Link to Item http://hdl.handle.net/10150/630252 THE ROLE OF CASK IN NEURONAL MORPHOGENESIS AND BRAIN SIZE IN DROSOPHILA: A GENETIC MODEL OF HUMAN INTELLECTUAL DISABILITY WITH MICROCEPHALY by Judith Arane Tello Vega Copyright © Judith Arane Tello Vega 2018 A Dissertation Submitted to the Faculty of the GRADUATE INTERDISCIPLINARY PROGRAM IN NEUROSCIENCE In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2018 1 2 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of the requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that an accurate acknowledgement of the source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: Judith Tello 3 Acknowledgements Firstly, I would like to express my sincere gratitude to my advisor Dr. Linda Restifo for her continuous support and patience. Her skillful guidance helped me enormously during all these years of research and writing of this dissertation. My appreciation also extends to Dr. Wulfila Gronenberg, for sharing expertise and always supporting me in numerous ways. I thank the rest of my dissertation committee: Dr. Julie Miller and Dr. Naomi Rance for insightful comments and especially for their encouragement during this process. My deepest thanks go to Dr. Lee Ryan and Dr. Konrad Zinsmaier who helped me through difficult times. Also, I wish to express my sincere thanks to Kirsten Cloutier Grabo for always taking care of me behind the scenes. I am grateful to Mark Yanagihashi, for his continuous encouragement and understanding. I am grateful to numerous Restifo Lab members who collaborated with me over the years: Monica Chaung, Dailu Chen, Rachel Bear, Lauren Pisani, and Dave Andrew. Especially I thank John Clark for the stimulating discussions. My deep gratitude goes for Sara Lewis and Cecilia Brown, my fellow lab mates and friends, for practical help and continual support, especially through hard times. I am extremely thankful and indebted to my great friends during the PhD: Romina Barria, Lilian Patron, Grace Samtani, Emilia Basantes, Claudia Barahona and Israel Lopez. Their friendship, cheering, laughs and affection were absolutely essential to survive. I take this opportunity to express gratitude to all my Chilean friends in Tucson, particularly Liss and Pablo for simply making me part of their family and keeping me sane. I am forever indebted to my surrogate parents, Mami Lupita and Mr. Heleno, for the unceasing support and love. I am also thankful to Ulises, my partner, who supported me no matter what through this venture. Above all, I would like to thank my wonderful parents, Judith and Julio, for the faith in me and for loving and supporting me in all the ways possible. To my sister Karinna and my brother Julio Cesar for the laughs and unique words of encouragement. Last but not least, I am grateful to God, for giving me the strength necessary to get through this journey. 4 Dedication To my beautiful grandmother, Carmen Muñoz, my “Mami” & In loving memory of my grandfather, Ramón Tello “I remember their love when they can no longer remember” 5 Table of Contents List of tables and figures .................................................................................................................8 List of abbreviations ........................................................................................................................9 Abstract ..........................................................................................................................................11 Chapter I: Introduction I.A. Intellectual Disability (ID) ..................................................................................................13 I.A.1. Definition, epidemiology, and associated features.......................................................13 I.A.2. Etiology and genetics of ID ..........................................................................................15 I.B. Microcephaly .......................................................................................................................16 I.C. CASK mutations cause X-linked developmental brain disorders ........................................17 I.C.1. ID and microcephaly with pontine and cerebellar hypoplasia (MIM# 300749) ..........18 I.C.2. FG syndrome 4 and X-linked ID with or without nystagmus (MIM# 300422) ...........25 I.C.3. Genotype-phenotype relationships ...............................................................................26 I.D. CASK a multi-domain protein, localization and hypothesized function ............................28 I.D.1. Discovery of CASK and its structure ..........................................................................28 I.D.2. The CaMK-like domain of CASK: a pseudo kinase ....................................................29 I.D.3. CASK protein localization: guilt by association ..........................................................30 I.D.4. Other roles for CASK: insights from mouse models ...................................................40 I.E. Drosophila CASK tools and contributions ..........................................................................42 I.F. Problem statement and research questions addressed in this dissertation ...........................51 CHAPTER II: Neurite-arborization abnormalities of CASK-mutant neurons in dissociated cell culture II.A. Rationale ...........................................................................................................................54 II.B. Materials and Methods .......................................................................................................57 II.B.1. Drosophila culture and genetics ..................................................................................57 II.B.2. Primary neuronal cell cultures ....................................................................................60 II.B.3. Immunostaining and morphometric analysis of 2D neuron image .............................61 II.B.4. Linear mixed modeling with nlme ..............................................................................66 II.C. Results. ...............................................................................................................................66 II.C.1. CASKΔ18 homozygous neurons have a “bushy” phenotype ......................................66 6 II.C.2. Linear mixed modeling to combine results from multiple experiments .....................74 II.C.3 CASK “bushy” phenotype is semi-dominant................................................................77 II.C.4. Two chromosomal deficiencies of CASK uncover the bushy phenotype ...................77 II.C.5. Transgenic CASK+ improves the bushy phenotype ....................................................86 II.D. Discussion. .........................................................................................................................90 II.D.1. Key findings and their significance to human CASK phenotypes ..............................90 II.D.2. Potential mechanisms underlying the CASK-mutant neurite arbor phenotype ...........91 II.D.3. Contributions of primary culture for understanding neurite-arbor morphogenesis ....93 CHAPTER III: CNS-size abnormalities in Drosophila CASK loss-of-function mutants III.A. Rationale ..........................................................................................................................96 III.B. Materials and Methods ...................................................................................................101 III.B.1. Sample collection and photography .........................................................................101 III.B.2. Photography and estimation of body length ............................................................101 III.B.3. Histology ..................................................................................................................102 III.B.4. Image acquisition and analysis ................................................................................104 III.B.5. Statistical analysis and graphing ..............................................................................105 III.C. Results. ...........................................................................................................................105 III.C.1. The small-brain phenotype of CASK mutant flies ...................................................105