Characterization of Williams-Beuren Syndrome Mouse Models: Linking Genes with Cognition and Behaviour by Emily Lam A thesis submitted in conformity with the requirements for the degree of Master of Science Institute of Medical Science University of Toronto © Copyright by Emily Lam (2012) Characterization of Williams-Beuren Syndrome Mouse Models: Linking Genes with Cognition and Behaviour Emily Lam Master of Science Institute of Medical Science University of Toronto 2012 Abstract Deletion (Williams-Beuren syndrome (WBS)) and duplication (Dup7q11.23) of a common interval spanning 26 genes on chromosome 7q11.23 cause disorders with a spectrum of clinical, cognitive and behavioural symptoms. Studies of individuals with atypical deletions have implicated two genes, GTF2IRD1 and GTF2I. Here I describe the behavioural characterization of mice hemizygous for Gtf2i, or Gtf2ird1 and Gtf2i together, as well as mice with additional Gtf2i copies. Dosage changes in Gtf2i were associated with working memory impairment and separation anxiety, and possibly with general anxiety and repetitive behaviours. A potential cause of these phenotypes was found in brain tissue, where subcellular localization of the calcium channel TRPC3, which is regulated by GTF2I, was found to be altered. Collectively, these results provide a better understanding of the contributions of GTF2I to the cognitive and behavioural profile of WBS and Dup7q11.23 and identify a potential biological mechanism that may underlie some of the symptoms. ii Acknowledgements I would like to extend my deepest gratitude to my supervisor, Dr. Lucy Osborne, for her continuous guidance and support throughout the duration of my graduate studies. Lucy’s bright laughter in the lab is always an encouraging sign that, despite the bad days, there are always good times to look forward to in grad school! I would also like to thank the members of my program advisory committee, Dr. Howard Mount and Dr. Vincent Tropepe, for their assistance and advice that guided my research toward the right direction. In particular, I am grateful to Howard for providing not only scientific guidance but also for imparting valuable insights about the diverse paths of life. Current and former colleagues in the lab have made my time in the Osborne lab an absolute pleasure. Ingredients to fun and laughter included late-night working, storytelling, commiserating, and celebrating in the lab with Jen O’Leary, Ted Young, Amy Oh, Elaine Tam, Eli Brimble, Emma Strong, and Joana Dida. I owe my gratitude to Joana for her expertise in ultrasonic vocalizations of mouse pups, and to Elaine for maintaining the animal colony and assisting with the genotyping of test animals. I would also like to thank our collaborators at the Toronto Centre for Phenogenomics as well as in Dr. John Roder’s lab (especially Laleh Sinai) for their assistance. My proficiency in behavioural assays would not have been possible without the help and invaluable advice of Keith Ho and Beverly Francis, both of whom have become treasured friends. Outside of the lab, I am grateful to the many friendships that have endured from high school and my undergraduate studies, as well as to the new friendships that I have formed during my graduate studies. The times spent with each and every friend have provided countless memories of fun, relaxation, and social support. Aymun Qayume, in particular, has always been there to share the good and the bad, to empathize about the miseries of adulthood, and to laugh and dream together. The hardships of grad school were made much easier with the unwavering love and support from my parents and grandparents. They encouraged me to always reach higher and persevere, and thus, I am indebted to them for where I am in life today. My sisters, Pamela and Carmen, are both able to elicit a smile from me even on the gloomiest of days, and their company and care ensured that I came home every weekend while I lived downtown. Lastly, the final stretch of my graduate studies was made brighter by Wilson, whose love, support, and comforting reassurance, as well as chocolates and desserts, have conveyed a myriad of possibilities ahead. iii Table of Contents Table of Contents ........................................................................................................................... iii List of Abbreviations ................................................................................................................... viii List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii Chapter 1 Introduction .....................................................................................................................1 1.1 Williams-Beuren syndrome .................................................................................................1 1.1.1 History of Williams-Beuren syndrome ....................................................................1 1.1.2 WBS clinical profile ................................................................................................2 1.1.3 WBS cognitive profile .............................................................................................6 1.1.4 WBS behavioural profile .........................................................................................8 1.1.5 Mutational mechanisms in WBS, duplication and triplication of 7q11.23 ..............9 1.2 Genotype-phenotype correlations in WBS ........................................................................11 1.2.1 Atypical deletions in WBS: implications for GTF2I and GTF2IRD1 ...................12 1.2.2 GTF2I family of transcription factors ....................................................................14 1.3 Duplication and Triplication of 7q11.23 ............................................................................20 1.4 Previously studied mouse models ......................................................................................24 1.4.1 Gtf2ird1-knockout mice .........................................................................................24 1.4.2 Gtf2i-heterozygous mice ........................................................................................26 1.4.3 Mice with a deletion encompassing Limk1 to Gtf2i...............................................27 1.4.4 Other single-gene deletion mouse models of WBS ...............................................28 Chapter 2 Behavioural analyses of Gtf2i and Gtf2i/Gtf2ird1 mouse models ................................35 2.1 Introduction ........................................................................................................................35 2.1.1 Generation of Mouse Models .................................................................................35 iv 2.1.2 Research Aims .......................................................................................................37 2.1.3 Hypothesis..............................................................................................................38 2.2 Materials and Methods .......................................................................................................38 2.2.1 Expression Analysis ...............................................................................................39 2.2.2 Animals ..................................................................................................................40 2.2.3 Statistical Analyses ................................................................................................40 2.2.4 Grip Strength ..........................................................................................................41 2.2.5 Rotarod Performance .............................................................................................42 2.2.6 Contextual and Cued Fear Conditioning ................................................................42 2.2.7 Morris Water Maze ................................................................................................43 2.2.8 Barnes Maze...........................................................................................................44 2.2.9 Resident Intruder ....................................................................................................45 2.2.10 Novel Object Recognition ......................................................................................45 2.2.11 Elevated Zero Maze ...............................................................................................46 2.2.12 Open Field ..............................................................................................................47 2.2.13 Maternal Separation-Induced Ultrasonic Vocalizations ........................................48 2.3 Results ................................................................................................................................48 2.3.1 Gene and protein expression levels ........................................................................48 2.3.2 Gtf2i+/- and Gtf+/del mice exhibit impaired rotarod performance but normal grip strength ...........................................................................................................52 2.3.3 Cued, but not contextual, fear conditioning altered in Gtf2i+/- and Gtf+/del mice ........................................................................................................................55