INVESTIGATING THE MECHANISM OF COURTSHIP ACCEPTANCE IN DROSOPHILA by JOSEPH SCHINAMAN Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biology CASE WESTERN RESERVE UNIVERSITY August, 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Joseph Schinaman candidate for the degree of Doctor of Philosophy*. Committee Chair Michael Bernard Committee Member Rui Sousa-Neves Committee Member Claudia Mieko Mizutani Committee Member Peter Harte Committee Member Mark Willis Date of Defense June 29th, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION To my grandfather Joe, who always insisted on providing his children with the sort of education he could never have, and to my parents, who have provided me with a better education than I could have ever hoped for. TABLE OF CONTENTS Abstract ........................................................................................................................................... 1 Chapter 1: Background and Significance ........................................................................................ 3 1.1- The Genetic Basis of Behavior .............................................................................................. 4 1.2- The Drosophila Courtship Display ......................................................................................... 5 1.3- The Sensory Inputs of Drosophila Courtship ........................................................................ 7 1.3.1- The Role of Olfaction in Courtship Stimulation ............................................................. 9 1.3.2- The Role of Audition in Courtship Stimulation ............................................................ 20 1.4- Regions of the Central Brain Controlling Receptivity ......................................................... 25 1.5- Genes Influencing Courtship and Receptivity ..................................................................... 27 1.6- Hypothesis of Integration of Courtship Signals .................................................................. 34 1.7- Summary ............................................................................................................................. 37 Chapter 2: A Novel Genetic Tool for Clonal Analysis of Fourth Chromosome Mutations ......... 46 2.1- Abstract ............................................................................................................................... 47 2.2- Introduction ........................................................................................................................ 49 2.3- Results ................................................................................................................................. 50 2.3.1- Genotypes and Chromosomes .................................................................................... 50 2.3.2- Clone Induction and Marking ...................................................................................... 51 2.3.3- Frequency of Clone Recovery ...................................................................................... 53 2.4- Discussion ........................................................................................................................... 54 2.5- Materials and Methods....................................................................................................... 57 2.5.1- Fly Culture Conditions and Genotypes Used to Generate Haplo-4 Clones ................. 57 2.5.2- Heat Shock Regimens .................................................................................................. 58 2.5.3- Brain Dissection, Multiplex in situ, and Confocal Microscopy .................................... 58 2.5.4- Fluorescence Signal Normalization.............................................................................. 60 i Chapter 3: The KRÜPPEL-Like Transcription Factor DATILÓGRAFO Is Required in Specific Cholinergic Neurons for Sexual Receptivity in Drosophila Females ............................................ 67 3.1- Abstract ............................................................................................................................... 68 3.2- Introduction ........................................................................................................................ 69 3.3- Materials and Methods....................................................................................................... 71 3.3.1- Mutants, Transgenes, Genotypes, and Stocks............................................................. 71 3.3.2- Recording of Mating Behavior ..................................................................................... 73 3.3.3- Quantification of Discrete Female Behaviors .............................................................. 73 3.3.4- Analysis of Locomotor Behavior .................................................................................. 74 3.3.5- Antibodies and Immunostaining ................................................................................. 74 3.3.6- Automated Image Analysis and Cell Counting ............................................................. 75 3.3.7- Clonal Analyses ............................................................................................................ 76 3.3.8- Olfactory Behavioral Assays ........................................................................................ 76 3.3.9- Mushroom Body and Antennal Lobe Image Analysis .................................................. 77 3.3.10- Statistical Analysis ..................................................................................................... 77 3.4- Results ................................................................................................................................. 78 3.4.1- Identification of dati on the Drosophila Fourth Chromosome .................................... 78 3.4.2- dati Mutant Females Are Courted Normally But Fail to Accept Male Courtship ....... 79 3.4.3- dati is Required In Neurons for Normal Acceptance and Locomotion ........................ 80 3.4.4- The Removal of dati in Cholinergic Neurons Impairs Normal Female Acceptance But Not Locomotion ..................................................................................................................... 81 3.4.5- dati Mutants Exhibit Abnormal γ-Lobes of the Mushroom Bodies, But These Defects Do Not Cause Female Rejection ............................................................................................ 83 3.4.6- dati Is Expressed in a Large Set of Neurons But in a Small Subset of the Cholinergic Neurons.................................................................................................................................. 83 3.4.7- Mapping Brain Regions Where dati Is Required to Generate Acceptance Reveals Discrete Brain Foci ................................................................................................................. 84 3.4.8- Rejection Foci Contain Few dati-Positive, Cholinergic Neurons .................................. 85 3.4.9- dati Is Required to Generate a Subtype of Cholinergic Neurons ................................ 86 3.4.10- Nuclear Bar Coding Reveals That DATI CHA Neurons Mediate Short- and Long-Range Connections ........................................................................................................................... 87 3.5- Discussion ........................................................................................................................... 89 ii 3.5.1- dati Encodes a Conserved ZNF Transcription Factor Related to Rotund/Squeeze and ZNF384 Required for Female Decision Making and Locomotion .......................................... 89 3.5.2- Locomotor Defects Are Separable From the Inability to Make Decisions .................. 91 3.5.3- dati Adds an Additional Layer to the Identity of Cholinergic Neurons That Is Shared by Noncholinergic Neurons ........................................................................................................ 91 3.5.4- The Regions Where dati Is Required Agree with Previous Mapping and Suggest the Existence of a Core Circuit for Female Decision Making ....................................................... 92 3.5.5- dati 's Requirement in Few Excitatory Neurons in Three Discrete Brain Foci Reveals a Simple, Yet Fundamental, Mechanism of Female Decision Making in Drosophila ............... 94 Chapter 4: Conclusions and Future Directions ........................................................................... 114 4.1- dati and the Neural Circuitry of Courtship Acceptance .................................................... 115 4.2- Revisiting the Summation Hypothesis .............................................................................. 117 4.3- Future Directions for Investigating the Neural Circuitry of Receptivity ........................... 119 4.4- Further Analysis of Receptivity ......................................................................................... 122 4.5- The Molecular Mechanism of the dati Gene in Cell Specification .................................... 128 References ................................................................................................................................... 136 iii LIST OF FIGURES Chapter 1 Figure 1.1- The steps of Drosophila courtship .....................................................