INVESTIGATING MAMMALIAN DEVELOPMENT USING MOUSE FORWARD GENETICS A Dissertation Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Joel Michael Brown May 2018 © 2018 Joel Michael Brown INVESTIGATING MAMMALIAN DEVELOPMENT USING MOUSE FORWARD GENETICS Joel Michael Brown, Ph.D. Cornell University 2018 Forward genetics allows the identification of novel genes involved in a particular biological process. We performed an ENU forward mutagenesis screen in mice aimed at identifying genes which regulate early organ morphogenesis during development. Fifteen mouse mutants were identified in the screen which possess cardiovascular, craniofacial, extraembryonic, or general body morphology defects. After performing preliminary characterization of all 15 lines, we identified a likely causative mutation in 14 of the 15 mutants. 1D and 13B mutants both exhibit craniofacial defects and were selected for in-depth characterization to identify the molecular mechanisms whereby they regulate embryogenesis. 1D mutants are characterized by an open neural tube in the hindbrain region. This phenotype is similar to human embryos with exencephaly, a congenital birth malformation of high incidence in the human population. Positional cloning of 1D identified a mutation in SPCA1, a Golgi-localized pump that controls calcium homeostasis. Results from the molecular characterization of mouse 1D mutants, as well as from time lapse microscopy of chicken embryos, revealed that calcium is tightly regulated during neural tube closure and that calcium homeostasis is required to promote apical constriction of neuroepithelial cells. These results show that SPCA1 activity is required to regulate the actomyosin dynamics that propel apical constriction, and that the actin severing protein, Cofilin 1, is a key mediator of SPCA1 function. Together, my findings provide the first genetic evidence that calcium homeostasis is needed for neural tube closure, opening a new window into understanding the etiology of human neural tube defects. 13B mutants have neural tube defects accompanied by a suite of other malformations including randomized L-R patterning and maxillary overgrowth. Molecular characterization revealed that all 13B phenotypes result from the absence of cilia, an organelle important for neural tube patterning and for the establishment of L-R asymmetry. Exome sequencing of 13B embryos identified a nonsense mutation in Pibf1. I show that PIBF1 is required for ciliogenesis during early embryonic development and identify a novel role for PIBF1 in regulating centrosome duplication. These findings highlight the importance of PIBF1 in regulating multiple aspects of centrosome biology, and provide a model for understanding how defects in these processes contribute to human ciliopathies. BIOGRAPHICAL SKETCH Joel Brown, the son of Glen and Dorothy Brown, was born in rural Iowa in 1988. Along with his seven siblings, Joel was educated at home by his mother from elementary school through high school. From an early age, Joel expressed a broad range of interests, exploring art, music, and science. Joel pursued these interests by attending Pensacola Christian College in Pensacola, FL, where he earned a B.S. degree in Biology Education (2009), with minors in fine art and piano. After graduating, Joel joined the lab of Dr. Hui-Min Chung at the University of West Florida where he studied gamma-secretase function using fruit flies and completed an M.S. degree in Biology (2012). It was during this time that he took a developmental biology coarse taught by Dr. Charles D’Asaro which inspired him to pursue a career in developmental biology. In 2012, Joel was accepted into the graduate program in Genetics, Genomics, and Development at Cornell University. Joel joined the lab of Dr. Maria Garcia-Garcia where he completed his dissertation research. iii ACKNOWLEDGMENTS I would like to recognize the many individuals who contributed to this work and to my training as a doctoral student at Cornell University. I wouldn’t be where I am today without the patient guidance of my mentor, Dr. Maria Garcia-Garcia. I cannot begin to the express how grateful I am for the amount of time that Maria has invested in my technical, scientific, and academic training. I am only beginning to understand how much she has influenced my growth as a scientist. I also want to thank my committee members, Dr. Natasza Kurpios and Dr. John Schimenti, for their advice and support over the course of the project. The ENU mutagenesis screen described in this work was accomplished by a team of individuals in the Garcia-Garcia Lab including Dr. Iván Durán, Dr. Bärbel Ulmer, Dr. Kate Alexander, and Yitong Li. I especially want to thank my lab mate, fellow graduate student, and friend, Dr. Kate Alexander, who was an inspiration during my time at Cornell. Her enthusiasm about science was and is contagious. In the latter years of my graduate work, the Marcos Simões- Costa lab also became an important source of technical and intellectual (and social) support. Katherine Strednak was an invaluable member of the mouse room staff who kept our mice happy and healthy. I was funded in part by the Cornell GGD Training grant (Cornell University) as well as the Cornell Center for Vertebrate Genomics. This work made use of the Cornell Stem Cell and Transgenic Core Facility (supported by NYSDOH Contract #C029155), the Cornell Center for Materials Research Facilities (supported by the National Science Foundation under Award Number DMR-1120296), and the Cornell Biotechnology Resource Center Imaging Facility (supported by NIH S10RR025502 and NIH S10OD012287). iv TABLE OF CONTENTS CHAPTER 1 MOUSE—A MODEL FOR MAMMALIAN DEVELOPMENT .................. 1 1.1 Congenital Birth Defects ................................................................................................. 2 1.2 A Model for Mammalian Development ......................................................................... 3 1.3 Mammalian Development .............................................................................................. 4 1.4 Summary of Thesis Research .......................................................................................... 7 CHAPTER 2 A FORWARD GENETIC SCREEN IN MOUSE TO IDENTIFY GENES THAT REGULATE EARLY ORGAN MORPHOGENESIS .................................................. 8 2.1 Introduction ........................................................................................................... 9 2.1.1 Forward Genetics .................................................................................................... 9 2.1.2 The Ins and Outs of Mouse Forward Genetic Screens .......................................... 10 2.1.3 Scope of Present Study ......................................................................................... 16 2.2 Materials and Methods ........................................................................................ 17 2.2.1 ENU Injections & Screening .................................................................................. 17 2.2.2 Mutation Identification ......................................................................................... 17 2.2.3 Mouse Embryo Analysis ........................................................................................ 18 2.3 Results and Discussion ......................................................................................... 20 2.3.1 Fifteen mutants affecting a wide range of developmental processes were identified in a forward mutagenesis screen in mice............................................. 20 2.3.2 Mutants with craniofacial defects (1D, 2F, 13B, 27B) .......................................... 26 2.3.3 Mutants with heart defects (21A, 31C, 13E)......................................................... 29 2.3.4 Mutants with general body morphology defects (27E, 9D, 20F, 22A) ................. 35 2.3.5 Mutants with extraembryonic defects (12C, 19C, 2G, 34A) ................................. 38 2.4 Summary and Conclusions .................................................................................... 42 CHAPTER 3 THE SECRETORY PATHWAY CA2+ ATPase SPCA1 PROMOTES NEURAL TUBE CLOSURE BY REGULATING CYTOSKELETAL DYNAMICS ......................... 45 3.1 Introduction ......................................................................................................... 46 3.1.1 Neural Tube Defects ............................................................................................. 46 3.1.2 Mechanisms of Neural Tube Closure .................................................................... 49 3.1.3 The Role of Calcium in Neural Tube Closure ........................................................ 52 3.2 Materials and Methods ........................................................................................ 53 3.2.1 Mice ...................................................................................................................... 53 3.2.2 Linkage Analysis and Positional Cloning ............................................................... 54 3.2.3 Mouse Embryo Analysis ........................................................................................ 54 3.2.4 Cell Culture ............................................................................................................ 56 3.2.5 Chick Electroporation and Live Imaging ............................................................... 58 3.2.6