TRANSDIFFERENTATION IN Turritopsis dohrnii (IMMORTAL JELLYFISH): MODEL SYSTEM FOR REGENERATION, CELLULAR PLASTICITY AND AGING A Thesis by YUI MATSUMOTO Submitted to the Office of Graduate and Professional Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Chair of Committee, Maria Pia Miglietta Committee Members, Jaime Alvarado-Bremer Anja Schulze Noushin Ghaffari Intercollegiate Faculty Chair, Anna Armitage December 2017 Major Subject: Marine Biology Copyright 2017 Yui Matsumoto ABSTRACT Turritopsis dohrnii (Cnidaria, Hydrozoa) undergoes life cycle reversal to avoid death caused by physical damage, adverse environmental conditions, or aging. This unique ability has granted the species the name, the “Immortal Jellyfish”. T. dohrnii exhibits an additional developmental stage to the typical hydrozoan life cycle which provides a new paradigm to further understand regeneration, cellular plasticity and aging. Weakened jellyfish will undergo a whole-body transformation into a cluster of uncharacterized tissue (cyst stage) and then metamorphoses back into an earlier life cycle stage, the polyp. The underlying cellular processes that permit its reverse development is called transdifferentiation, a mechanism in which a fully mature and differentiated cell can switch into a new cell type. It was hypothesized that the unique characteristics of the cyst would be mirrored by differential gene expression patterns when compared to the jellyfish and polyp stages. Specifically, it was predicted that the gene categories exhibiting significant differential expression may play a large role in the reverse development and transdifferentiation in T. dohrnii. The polyp, jellyfish and cyst stage of T. dohrnii were sequenced through RNA- sequencing, and the transcriptomes were assembled de novo, and then annotated to create the gene expression profile of each stage. Comparative functional gene enrichment analyses with the cyst as the central stage of comparison reported significant GO categories that were over-expressed, such as telomere maintenance and DNA repair, in the cyst as compared to other stages. The enrichment analyses also reported ii significantly under-expressed categories, such as mitotic cell division and cellular differentiation, in the cyst as compared to the other stages. Additionally, candidate genes, such as the Yamanaka (Oct4, Sox2, Klf4, c-Myc) and Thompson Factors (Lin28, Nanog) that exhibit potential association with the transdifferentiation processes were found among the three stages for downstream differential gene expression analyses. Ultimately, our work produced a foundation to develop an alternative model system to further investigate and comprehend regeneration, cellular plasticity and aging in metazoans. iii ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my M.S. graduate adviser and committee chair, Dr. Maria Pia Miglietta, for consistently providing invaluable guidance throughout the duration of my thesis project. Her patience, enthusiasm and knowledge have been fundamental to my deep understanding of genomics and the advancement of my project. Furthermore, Dr. Miglietta’s investment in the success of my research and M.S. career has facilitated my further development as a scientist, and has inspired my future as a researcher in the academic system. Additionally, I would like to thank the rest of my thesis committee, Dr. Jaime Alvarado-Bremer, Dr. Noushin Ghaffari and Dr. Anja Schulze, and essential collaborator, Dr. Stefano Piraino, for the encouragement and support they provided throughout the progression of my research. Without their provision, my project would have failed to incorporate different perspectives which drastically expanded the scope of my project. Sample collection would not have been possible without the research facilities of the Smithsonian Tropical Research Institute and support from the members of the Systematics and Biology of Hydrozoa Summer Course (2015) who invested long hours helping me collect specimens in Bocas del Toro, Panama. This project would also not have been possible without the troubleshooting and bioinformatic support from the Texas A&M Genomics and Bioinformatics Service team. Funding for this project was iv generously provided by Texas A&M University’s Agrilife Research Center and Texas Sea Grant. In addition, graciously being awarded the Graduate Student Boost Funding scholarship significantly assisted the efficiency and intricacy of my research during my final year as a graduate student as it enabled the complete devotion to the progression of my project. v CONTRIBUTORS AND FUNDING SOURCES Contributors This work was supported by a thesis committee consisting of Professor Maria Pia Miglietta [adviser], Jaime Alvarado-Bremer, and Anja Schulze of the Department of Marine Biology, and Dr. Noushin Ghaffari of the Department of Electrical and Computer Engineering. All work for this thesis was completed by the student, under the advisement of Dr. Maria Pia Miglietta of the Department of Marine Biology. Funding Sources Graduate study was supported by a Graduate Student ‘Boost’ Funding Scholarship (2016-2017) from Texas A&M University, Galveston. This work was made possible in part by Texas Sea Grant College Program’s Grants-In-Aid of Graduate Research Program. Additionally, this work was made possible in part by TAMUG’s Marine Biology Mini Travel Grants, Galveston Graduate Student Association Travel Grant, and Erma Lee and Luke Mooney Graduate Student Travel Grant, TAMU’s Agrilife Research Division and TAMU’s PESCA Research Division. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Texas Sea Grant College Program and TAMUG. vi NOMENCLATURE BUSCOs Benchmarking Universal Single-Copy Orthologs ESc Embryonic Stem cell FDR False Discovery Rate GO Gene Ontology iPSc Induced Pluripotent Stem cell KEGG Kyoto Encyclopedia of Genes and Genomes NCBI National Center for Biotechnology Information PE Paired Ends RNA-seq RNA-sequencing TAMUG Texas A&M University Galveston vii TABLE OF CONTENTS Page ABSTRACT ....................................................................................................................... ii ACKNOWLEDGEMENTS .............................................................................................. iv CONTRIBUTORS AND FUNDING SOURCES ............................................................ vi NOMENCLATURE ........................................................................................................ vii TABLE OF CONTENTS ................................................................................................ viii LIST OF TABLES ............................................................................................................. x LIST OF FIGURES ......................................................................................................... xii 1. INTRODUCTION ......................................................................................................... 1 1.1 Background ........................................................................................................ 1 1.2 Study system: Transdifferentiation in Turritopsis dohrnii ................................ 9 1.3 Research approach: RNA-Sequencing and de novo assembly ........................ 14 1.4 Research goals ................................................................................................. 17 2. METHODOLOGY ...................................................................................................... 19 2.1 Specimen collection/Species identification ..................................................... 19 2.2 RNA extraction/Library construction/RNA-sequencing ................................. 21 2.3 De novo transcriptome assembly ..................................................................... 22 2.4 Completeness of transcriptomes ...................................................................... 23 2.5 Functional annotation of transcriptomes .......................................................... 24 2.6 Functional enrichment analyses ....................................................................... 25 2.7 Screening of candidate genes ........................................................................... 26 3. RESULTS AND DISCUSSION .................................................................................. 27 3.1 Transcriptome sequencing and assembly ......................................................... 27 3.2 Completeness of transcriptomes ...................................................................... 35 3.3 Functional annotation and categorization of transcriptomes ........................... 38 3.4 Functional enrichment analyses ....................................................................... 53 3.5 Candidate genes of interest .............................................................................. 68 viii Page 4. CONCLUSION ............................................................................................................ 83 REFERENCES ................................................................................................................ 85 APPENDIX A ................................................................................................................ 104 APPENDIX B ...............................................................................................................