University of the Pacific Scholarly Commons University of the Pacific Theses and Dissertations Graduate School 2017 Studies on development in Euphilomedes ostracods: Embryology, nervous system development, and the genetics of sexually dimorphic eye development Kristina Koyama University of the Pacific, [email protected] Follow this and additional works at: https://scholarlycommons.pacific.edu/uop_etds Part of the Biology Commons Recommended Citation Koyama, Kristina. (2017). Studies on development in Euphilomedes ostracods: Embryology, nervous system development, and the genetics of sexually dimorphic eye development. University of the Pacific, Thesis. https://scholarlycommons.pacific.edu/uop_etds/2978 This Thesis is brought to you for free and open access by the Graduate School at Scholarly Commons. It has been accepted for inclusion in University of the Pacific Theses and Dissertations by an authorized administrator of Scholarly Commons. For more information, please contact [email protected]. 1 STUDIES ON DEVELOPMENT IN EUPHILOMEDES OSTRACODS: EMBRYOLOGY, NERVOUS SYSTEM DEVELOPMENT, AND THE GENETICS OF SEXUALLY DIMORPHIC EYE DEVELOPMENT by Kristina H. Koyama A Thesis Submitted to the Office of Research and Graduate Studies In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE College of the Pacific Biological Sciences University of the Pacific Stockton, California 2017 2 STUDIES ON DEVELOPMENT IN EUPHILOMEDES OSTRACODS: EMBRYOLOGY, NERVOUS SYSTEM DEVELOPMENT, AND THE GENETICS OF SEXUALLY DIMORPHIC EYE DEVELOPMENT by Kristina H. Koyama Approved by Thesis Advisor: Ajna Rivera, Ph.D. Committee Member: Douglas Weiser, Ph.D. Committee Member: Tara Thiemann, Ph.D. Department Chair: Craig Vierra, Ph.D. Dean of Graduate School: Thomas H. Naehr, Ph.D. 3 DEDICATION For everyone who gave me their support, encouragement, and patience during this crazy journey. It wouldn’t have been possible without you (no contribution too small)! 4 ACKNOWLEDGEMENTS I would like to thank Dr. Ajna Rivera for her mentorship and support during the writing of this thesis, and for giving the opportunity to explore and learn. I would also like to thank my other two committee members, Dr. Thiemann and Dr. Weiser, for their support and guidance. I also would like to thank my lab mates, past and present, for your lab help, comradery, and friendship. It’s been a pleasure working with you all. Finally, I would like to thank the staff, faculty, and graduate students of the Biology department, for your support. You are all wonderful people to work with, and truly inspiring. Thank you all for believing in me. 5 Studies on development in Euphilomedes ostracods: Embryology, nervous system development, and the genetics of sexually dimorphic eye development Abstract By Kristina H. Koyama University of the Pacific 2015 Model organism studies have been fundamental in understanding evolutionary and developmental biology. However, non-model organisms present opportunities to study unique characteristics and as comparisons to model organisms, leading us toward broader and more relevant perspectives on diversity. The Euphilomedes genus of ostracods is an example of a non-model group with potential for evolutionary and developmental studies. Ostracoda is an ancient, basally branching lineage of Crustaceans with a complete and prodigious fossil record. Despite the group’s promise for evolutionary studies, much remains unknown about the basic biology of this clade. There are a limited number of embryogenesis studies in Ostracoda; here, I study development in Euphilomedes. In Part 1, I study the main events in Euphilomedes’ embryology, focusing on cleavage and cell migration. I describe the general embryology of Euphilomedes, and devise a visual staging scheme for their development. Using fluorescent nuclear staining and microscopy, I visualize nuclei in cleavage throughout development of nuclear 6 divisions and migrations during development. The meroblastic cleavage observed in Euphilomedes resembles that of another Myodocopid ostracod, Vargula hilgendorfii. Finally, immunostaining for acetylated-alpha tubulin and phalloidin staining are used to visualize the general anatomy of the embryonic brain. This provides new protocols for visualizing the nervous system, enabling more detailed nervous system studies in the future. In Part 2, I explore differential gene expression patterns in the developing eyes of juvenile Euphilomedes. Euphilomedes have sexually dimorphic eye types – males have lateral compound eyes, while females instead have eye rudiments. Previous studies in E. carcharodonta show that genes in the retinal determination and phototransduction gene networks have differential expression in males and females during eye development. In this thesis, we attempt to compare these patterns to expression in a sister species, E. morini. 7 TABLE OF CONTENTS LIST OF TABLES ...........................................................................................................10 LIST OF FIGURES .........................................................................................................11 LIST OF ABBREVIATIONS ..........................................................................................12 CHAPTER 1. General introduction ......................................................................................14 2. Introduction to Part 1: Embryogenesis and Embryonic Nervous System in Euphilomedes carcharodonta ........................................................................18 Euphilomedes ostracods as representatives of Myodocopid ostracods ....18 Embryogenesis in Myodocopid ostracods ...............................................20 Euphilomedes as a model for the superfamilly Sarsielloidea ..................21 Euphilomedes ostracods as representatives in Crustacea .........................21 Neurophylogeny in Crustacea and Arthropoda........................................23 The nervous system in Ostracoda ............................................................24 Practical techniques for embryology studies in Euphilomedes ...............25 3. Methods..........................................................................................................27 Collection of E. carcharodonta ...............................................................27 Immunohistochemistry, phalloidin, and DAPI staining ..........................27 Fluorescent microscopy and microphotography ......................................29 Color staging and photography ................................................................30 Estimation of brooding duration ..............................................................30 Improvement of artificial rearing .............................................................31 4. Results ............................................................................................................32 8 General embryology.................................................................................32 Early nuclear divisions and cell migrations .............................................34 Limb bud formation and late development ..............................................37 Improvement of artificial rearing technique ............................................38 Nervous system visualization ..................................................................39 Brain structure in Euphilomedes embryos ...............................................41 5. Discussion ......................................................................................................43 6. Introduction to Part 2: Differential gene expression patterns in the sexually dimorphic eyes of Euphilomedes ostracods ...................................................46 Why study sexual dimorphism? ...............................................................47 Euphilomedes ostracods as models for arthropod eye evolution .............49 Ommatidial development .........................................................................52 Phototransduction ....................................................................................54 Morphological differences from similar genetic backgrounds ................55 Can the same morphology be determined by different gene expression patterns? ...................................................................................................56 Studying differential gene expression in Euphilomedes ..........................57 7. Methods..........................................................................................................60 Collection of Euphilomedes morini .........................................................60 Dissection .................................................................................................60 RNA isolation and cDNA synthesis ........................................................61 Generation of qPCR standard curves .......................................................61 8. Results ............................................................................................................64 9. Discussion ......................................................................................................68 9 REFERENCES ................................................................................................................69 10 LIST OF TABLES Table Page 1. Expression data for TOY ...............................................................................64 2. Expression data for ELAV .............................................................................65