MOLECULAR PROFILING OF CELLULAR IDENTITY AND PLASTICITY IN THE NERVOUS SYSTEM OF APLYSIA CALIFORNICA By CALEB JAMES BOSTWICK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2019 © 2019 Caleb James Bostwick To my family ACKNOWLEDGMENTS First, I’d like to acknowledge the people who helped me to bring this dissertation to fruition. My advisor Dr. Leonid Moroz helped me come up with ideas and questions and encouraged me to generate more data and perform more extensive analyses than I would have previously thought possible. Dr. Andrea Kohn helped keep the lab organized and running. Yelena Bobkova provided animal care, dissection, and molecular biology expertise as well as friendship and support. Tanya Moroz assisted in cDNA library construction and the ganglia plasticity experiments in addition to brightening the general lab atmosphere. Dr. Peter Williams aided me in finding ways to better explain and document my computational biology techniques. Dr. Shaun Mukherjee was a friend who made lab meetings more bearable. Dr. Gabrielle Winters assisted me by providing assistance with molecular biology in the lab, asking insightful questions, proofreading my dissertation, and by being a generous and supportive partner during our many years of graduate study. Dr. Emily Dabe was a colleague and friend with whom I discussed bioinformatics methods and techniques and shared laughs. I thank my university and my graduate committee (Dr. Thomas Foster, Dr. David Borchelt, and Dr. Richard Yost), as well as Dr. Jada Lewis for her support and encouragement. I also thank my colleagues who have graduated from the University of Florida IDP program before me for their support and friendship. Finally, I thank my family, whose love and encouragement allowed me to become the person I am today. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES ........................................................................................................ 10 LIST OF OBJECTS ....................................................................................................... 13 LIST OF ABBREVIATIONS ........................................................................................... 14 ABSTRACT ................................................................................................................... 17 CHAPTER 1 INTRODUCTION .................................................................................................... 19 Simple Models and Powerful Technologies for Understanding Neuroscience ........ 19 Aplysia californica as a Model Organism for Learning and Memory Studies .... 20 Next-generation Single Cell RNA Sequencing (scRNA-seq) ............................ 22 Biology of Identified Single Neurons ................................................................. 24 Aplysia Learning and Memory Circuits and cAMP Signaling .................................. 27 Aplysia: Model for a Reductionist Approach to Studying Learning and Memory ......................................................................................................... 27 Molecular Biology of Short- and Long-term Memory ........................................ 28 2 METHODS .............................................................................................................. 32 Molecular Biology for Construction of RNA-sequencing Libraries ........................... 32 Animals and Cell/Tissue Isolation ..................................................................... 32 Treatment of CNS Ganglia with 8-Br cAMP...................................................... 33 RNA Extraction ................................................................................................. 34 cDNA Synthesis and Verification ...................................................................... 34 External RNA Spike-ins .................................................................................... 35 Sequencing Library Preparation ....................................................................... 35 Library Quality Control and Validation .............................................................. 36 RNA-Sequencing Quality Control, Read Mapping, and Exploratory Data Analysis ............................................................................................................... 36 Read Summarization ........................................................................................ 37 Data Visualization ............................................................................................. 38 Principal Component Analysis .......................................................................... 38 Saturation Curves ............................................................................................. 39 Linear Regression Curve Fitting ....................................................................... 39 Differential Expression Analysis ....................................................................... 39 Calculating Relative scRNA-seq Transcript Abundance ................................... 40 5 Absolute scRNA-seq Transcript Abundance .................................................... 40 Localization of Cellular mRNA ................................................................................ 41 Molecular Cloning ............................................................................................. 41 Synthesis of Antisense Probes Labelled with Digoxygenin .............................. 42 3 ABSOLUTE QUANTIFICATION OF MESSENGER RNAS FROM IDENTIFIABLE SINGLE NEURONS IN APLYSIA .................................................. 46 Overview of Single-Cell RNA Sequencing .............................................................. 46 Mammalian Single Cell RNA-seq Studies ........................................................ 47 Characteristics of the Aplysia californica Genome ........................................... 49 Results of Absolute mRNA Quantification in Identifiable Single Neurons ............... 50 Transcriptomic Evaluation of the Characteristics of Identified Neurons ............ 52 Shallow Transcriptomic Profiling of 96 Individual Neurons, Including VC Sensory Neurons .......................................................................................... 55 Insights Regarding Absolute Molecular Quantification of Messenger RNAs .... 56 Functional Biology of Identified Neurons .......................................................... 59 Data transformation used for differential expression analysis .................... 59 R2 vs L7 differentially expressed ion channel-related genes ..................... 60 DE cell cycle and synaptic genes ............................................................... 61 Five RNA modification/editing/methylation genes are DE between L7 and R2 .................................................................................................... 63 Transcription factors and chromatin remodeling genes DE in R2 versus L7 ............................................................................................................ 64 Overall comparison of DE genes between R2 shared with five other neuron classes ........................................................................................ 65 Functional annotation of enriched genes using DAVID .............................. 66 Enriched genes found between R2 and other neurons .............................. 67 Principal component analysis reveals features of cell type-specific clustering ................................................................................................ 69 Discussion of Absolute Quantitation of mRNAs from Identifiable Single Neurons .. 70 Rapidly Advancing Knowledge of Secretory Molecules from the Phylum Mollusca .............................................................................................................. 71 4 DISCOVERY OF NOVEL MEMORY-RELATED GENES IN A CAMP-TREATED MOLLUSCAN NERVOUS SYSTEM ..................................................................... 118 Introduction ........................................................................................................... 118 Results of RNA-seq Reveal Canonical and Novel Gene Expression .................... 123 RNA-seq of Established cAMP-Dependent Genes ......................................... 123 Novel Differentially Expressed Genes in Response to 8-Br cAMP Treatment 129 AG Molecular Function GO ............................................................................ 130 AG Biological Process GO ............................................................................. 132 AG Cellular Component GO ........................................................................... 133 Right Pleural Molecular Function GO ............................................................. 134 Classification of Unique Ganglia-Specific Transcripts ........................................... 136 Time-course Analysis of Transcripts Influenced by 8-Br cAMP Treatment ........... 137 6 Unsupervised Clustering of 8-Br cAMP-Treated Ganglia ............................... 139 Abdominal Ganglion 0.5-hour cAMP Treatment vs Control (FSW) ................. 140 Abdominal