Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish Austrofundulus Limnaeus
Total Page：16
File Type：pdf, Size：1020Kb
Portland State University PDXScholar Dissertations and Theses Dissertations and Theses Fall 11-30-2017 Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish Austrofundulus limnaeus Amie L. Romney Portland State University Follow this and additional works at: https://pdxscholar.library.pdx.edu/open_access_etds Part of the Biology Commons, and the Genetics and Genomics Commons Let us know how access to this document benefits ou.y Recommended Citation Romney, Amie L., "Transcriptomic Regulation of Alternative Phenotypic Trajectories in Embryos of the Annual Killifish Austrofundulus limnaeus" (2017). Dissertations and Theses. Paper 4033. https://doi.org/10.15760/etd.5917 This Dissertation is brought to you for free and open access. It has been accepted for inclusion in Dissertations and Theses by an authorized administrator of PDXScholar. Please contact us if we can make this document more accessible: [email protected]. Transcriptomic Regulation of Alternative Phenotypic Trajectories in embryos of the Annual Killifish Austrofundulus limnaeus by Amie Lynn Thomas Romney A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Dissertation Committee Jason Podrabsky, Chair Suzanne Estes Bradley Buckley Todd Rosenstiel Dirk Iwata-Reuyl Portland State University 2017 © 2017 Amie Lynn Thomas Romney ABSTRACT The Annual Killifish, Austrofundulus limnaeus, survives the seasonal drying of their pond habitat in the form of embryos entering diapause midway through development. The diapause trajectory is one of two developmental phenotypes. Alternatively, individuals can “escape” entry into diapause and develop continuously until hatching. The alternative phenotypes of A. limnaeus are a form of developmental plasticity that provides this species with a physiological adaption for surviving stressful environments. The developmental trajectory of an embryo is not distinguishable morphologically upon fertilization and phenotype is believed to be influenced by maternal provisioning within the egg based on observations of offspring phenotype production. However, incubation temperature may override any such maternal pattern suggesting an environmental influence on the regulation of developmental trajectory. We hypothesize that maternally packaged gene products coordinate the cellular events prior to the maternal-to-zygotic transition (MTZ) that determine developmental trajectory in embryos of A. limnaeus . In addition, we propose that environmentally responsive gene expression after the MTZ can sustain or override any such maternal provisioning. Using high-throughput RNA-sequencing, we have generated transcriptomic profiles of protein-coding messenger RNA and noncoding RNA during development in A. limnaeus . Embryos destined for either the diapause or escape phenotypes display unique expression profiles immediately upon fertilization that support hormone synthesis, well before the stage when phenotypes are morphologically distinct. At stages when the i trajectories diverge from one another, differential expression of the vitamin D receptor signaling pathway suggests that vitamin D signaling may be a key regulator of developmental phenotype in this species. These data provide a critical link between maternal and environmental influences on the genetic regulation of phenotypic plasticity. These results will not only impact our understanding of the genetic mechanisms that regulate entrance into diapause, but also provide insight into the epigenetic regulation of gene expression and development. Uncovering genetic mechanisms in a system exhibiting alternative developmental trajectories will elucidate the role of maternal packaging in regulating developmental decisions, and in sustaining metabolic depression during diapause. ii DEDICATION This work is dedicated to my parents, and my parents’ parents. iii ACKNOWLEDGEMENTS My dissertation research in the Podrabsky Lab would not have been possible without the support of many people. I am indebted to Jason Podrabsky, my research advisor, who was supportive and encouraging throughout my project. This body of work is a testament to his contribution and insight on all aspects of my research. I would like to thank my graduate advisory committee: Suzanne Estes, Todd Rosenstiel, Dirk Iwata- Reuyl, Bradley Buckley, and Deborah Lutterschmidt. Their advice throughout the years enriched my scientific development. Further research support came from Rahul Raghavan and his advice on bioinformatics analysis. I would also like to thank the many lab mates I have had throughout my dissertation project: Josiah Wagner, Claire Riggs, Daniel Zajic, S. Cody Woll, Ian Garrett, and Kristin Culpepper. Our collaborations fostered a better approach to my own research and our personal relationships further enhanced my doctoral experience. I am grateful to the faculty and graduate students of the Biology department at Portland State for creating an environment that invoked growth and achievement. iv TABLE OF CONTENTS ABSTRACT ......................................................................................................................... i DEDICATION ................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................... iv LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii CHAPTER 1: The influence of genetics and environment on phenotype .......................... 1 CHAPTER 2: The maternally provisioned transcriptome in embryos of A. limnaeus ..... 10 CHAPTER 3: Small noncoding RNAs along alternative developmental trajectories in A. limnaeus ............................................................................................................................ 59 CHAPTER 4: Vitamin d is (not) for diapause .................................................................. 89 CHAPTER 5: Connecting gene expression associated with alternative developmental trajectories across life-history stages .............................................................................. 118 CHAPTER 6: Ecologically relevant developmental programs ...................................... 135 REFERENCES ............................................................................................................... 142 APPENDIX A: Tissue sampling and RNA-sequencing from diapause- and escape-bound embryos ........................................................................................................................... 162 APPENDIX B: Tissue sampling and small RNA-sequencing from temperature induced diapause- and escape-bound embryos ............................................................................. 172 APPENDIX C: WGCNA Analysis ................................................................................. 176 APPENDIX D: Supplemental files ................................................................................. 208 v LIST OF TABLES Table 2.1 Frequency of poly-A genes at expression bins ..................................................51 Table 2.2: Top 20 most abundant poly-A RNA transcripts expressed in 1-2 cell stage embryos of A. limnaeus ......................................................................................................52 Table 2.3: Most differentially expressed orthologous genes between A. limnaeus and D. rerio ....................................................................................................................................53 Table 2.4: Mitochondrial genes of A. limnaeus and D. rerio and their expression summary in the 1-2 cell stage transcriptome .....................................................................54 Table 2.5: Top twenty most abundantly expressed small RNA transcripts expressed across all libraries in 1-2 cell stage embryos of A. limnaeus ............................................. 55 Table 2.6: Number of genomic alignments for the top 8 most abundant small RNA sequences as a function of the number of bases that must match identically starting from the 5’ end............................................................................................................................56 Table 2.7: Differentially expressed sncRNAs in diapause- and escape-bound embryos of A. limnaeus ......................................................................................................................... 57 Table 2.8: Number of genes designated in the minimal stress proteome (or MSP) identified in the A. limnaeus and D. rerio transcriptomes at the 1-2 cell stage .................58 Table 3.1: Summary of potential miR-430 gene targets ....................................................88 Table 4.1: Summary of modules and hub genes ..............................................................117 Table 5.1: Differentially expressed exons in 1-2 cell stage embryos of A. limnaeus ...... 134 vi LIST OF FIGURES Figure 2.1. Alternative splicing of poly-A RNA in embryos of A. limnaeus that will develop along two alternative developmental trajectories .................................................45 Figure 2.2: Top 10 genes with developmental trajectory-specific splice variants based on statistical