The Identification and Characterization of a Novel, Vertebrate Specific Gene, with Potential Roles in Human Cri Du Chat Syndrome
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IDENTIFICATION AND ANALYSIS OF THE NOVEL GUMBY GENE AND ITS VERTEBRATE SPECIFIC ROLES IN THE MOUSE by ELENA RIVKIN A thesis submitted in conformity with requirements for the degree of Doctor of Philosophy. Graduate Department of Molecular Genetics University of Toronto © Copyright by Elena Rivkin, 2010 Identification and analysis of the novel gumby gene and its vertebrate roles in the mouse Elena Rivkin Doctor of Philosophy Department of Molecular Genetics University of Toronto 2010 ABSTRACT Forward genetic screens in the mouse are contributing significantly to our understanding of basic mammalian development and human disease. In one such screen, our laboratory has identified the novel mouse gumby mutant, which affects the development of the neural and vascular systems. Here, I describe the characterization of the gumby mutant phenotype and the identification of its causative mutation in a novel, vertebrate-specific gene, which is one of the genes deleted in patients affected by Cri du Chat Syndrome that exhibit mental retardation and craniofacial deficits. Expression and phenotypic analyses revealed a requirement for the gumby gene in the facial nerve axon guidance and angiogenesis. Lately, it has become evident that many common mechanisms and molecules operate during neural and vascular development. My results suggest that the gumby gene is an attractive candidate for regulating both processes and its analysis in the future may help us understand how the navigational mechanisms for both systems are intertwined. ii My studies show that gumby is a cytoplasmic protein that is present in many embryonic and adult tissues. In yeast-two-hybrid assays gumby interacts with a member of the highly conserved Wnt pathway - Dishevelled 2 (Dvl2). In both Dvl2-/- and gumby homozygotes, the level of the cardiac neural crest cell marker Plexin2A is decreased. The three branches of the Wnt pathway have been shown to regulate a wide range of events during embryogenesis and adult homeostasis, and subsequently have been implicated in multiple human pathologies. Taken together my data suggest that gumby may be required for Wnt signaling in angiogenesis and/or facial nerve guidance. Given that Wnt signaling has been shown to play key roles in axon guidance, gumby and its roles in Wnt signaling may also contribute to the mental retardation seen in patients with Cri du Chat Syndrome. Thus, further analyses of molecular and biologic roles of gumby will provide important avenues for understanding the cell biology of human disease. iii ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Dr. Sabine Cordes for giving me the opportunity to work with her and for being a great mentor throughout this project. Without her guidance and persistent help, this dissertation would not be possible. I am truly grateful for my committee members, Dr. C. C Hui and Dr. Joseph Culotti who have made their support and assistance available in a number of ways, and whose advice has shaped this project tremendously. I would like to acknowledge my fellow lab members with whom I had the pleasure of working over the years. These include Stephanie Almeida, Michael Huynh, Dennis Kim, Teresa MacLean, Ryan Mui, Angela Sing, Kendra Sturgeon, and Joanna Yu. You have created a great research environment and I will remember and cherish you forever. Lastly, I would like to offer my eternal gratitude to my family and friends who offered me support and encouragement during the completion of this project. I am especially grateful to my wonderful husband and our little creation Julia, who mean the world to me, and constantly inspire me to work harder and to aim higher. iv TABLE OF CONTENTS Abstract...............................................................................................................................ii Acknowledgements............................................................................................................iv Table of contents.................................................................................................................v List of tables.......................................................................................................................ix List of figures......................................................................................................................x List of appendices.............................................................................................................xii Abbreviation key..............................................................................................................xiii 1.0 INTRODUCTION....................................................................................................... 1 1.1 Overview of work that has led to this project............................................................... 1 1.1.1 Forward genetic screen to identify cranial nerve mutations in the mouse..........1 1.2 Overview of vertebrate cranial nerves and their early development ............................ 8 1.2.1 The unique development of the facial cranial nerve.........................................12 1.2.1.1 Basic neuroanatomical development of facial cranial nerve .................... 12 1.2.1.2 Migration of facial branchiomotor neurons .............................................. 13 1.2.1.3 Axon guidance of facial cranial nerves..................................................... 15 1.3 Human cranial dysinnervation disorders .................................................................... 19 1.4 Cri du Chat Syndrome ................................................................................................ 21 1.5 Overview of Angiogenesis.......................................................................................... 22 1.5.1 VEGF/VEGFR signaling in vascular development ..........................................23 1.5.2 Notch signaling in angiogenesis .......................................................................25 1.5.3 Wnt signaling in vascular development............................................................27 1.5.3.1 Short summary of Wnt pathways.............................................................. 27 1.6 Common mechanisms of nerve and blood vessel development ................................. 35 1.6.1 Pathways with roles in facial nerve axon guidance and angiogenesis..............37 1.6.2 Pathways with roles in FBM migration and angiogenesis................................40 1.6.2.1 Neuropilins and VEGF ............................................................................. 40 1.6.2.2 Wnt signaling pathway ............................................................................. 41 1.6.3 Other axon guidance pathways with roles in cranial nerve development and...... angiogenesis.....................................................................................................43 1.6.3.1 Eph-Ephrins .............................................................................................. 43 1.6.3.2 Netrins and Unc5b .................................................................................... 45 1.6.3.2 Slits and Robos ......................................................................................... 46 1.7 Summary..................................................................................................................... 47 2.0 MATERIALS AND METHODS ............................................................................. 49 2.1 Mouse Manipulations.................................................................................................. 49 v 2.2 Embryos harvesting .................................................................................................... 49 2.3 Synthesis of first strand cDNA ................................................................................... 49 2.4 Mouse genotyping....................................................................................................... 49 2.4.1 Standard PCR assay ..........................................................................................50 2.5 Genetic mapping of the gumby mutation.................................................................... 50 2.5.1 Initial mapping done by other members of our laboratory ...............................50 2.5.2 Fine mapping of the gumby mutation ...............................................................50 2.5.3 Sequencing the genes within gumby critical interval........................................52 2.5.4 Sequencing Fam105b........................................................................................52 2.5.5 Genotyping the T285A mutation using ARMS-PCR analysis .........................53 2.6 Cloning of Fam105b................................................................................................... 54 2.6.1 Synthesis of first strand cDNA .........................................................................54 2.6.2 Cloning of Fam105b-pBluescript SKII.............................................................54 2.7 Histological Analysis.................................................................................................. 55 2.8 Northern Blot analysis .........................................................................................55 2.9 Apoptosis and Proliferation Assays............................................................................ 56 2.10 Whole-mount in situ hybridization (WISH) ............................................................. 56 2.10.1 Fam105b