University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 8-23-2013 Conservation and Diversification of Appendage Identity Specification Mechanisms Along the Anteroposterior and Proximodistal Axes in Panarthropoda Frank W. Smith III University of Connecticut, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Smith, Frank W. III, "Conservation and Diversification of Appendage Identity Specification Mechanisms Along the Anteroposterior and Proximodistal Axes in Panarthropoda" (2013). Doctoral Dissertations. 161. https://opencommons.uconn.edu/dissertations/161 Conservation and Diversification of Appendage Identity Specification Mechanisms Along the Anteroposterior and Proximodistal Axes in Panarthropoda Frank Wesley Smith III, PhD University of Connecticut, 2013 A key characteristic of arthropods is their diverse serially homologous segmented appendages. This dissertation explores diversification of these appendages, and the developmental mechanisms producing them. In Chapter 1, the roles of genes that specify antennal identity in Drosophila melanogaster were investigated in the flour beetle Tribolium castaneum. Antenna-to-leg transformations occurred in response to RNA interference (RNAi) against homothorax, extradenticle, spineless and Distal-less. However, for homothorax/extradenticle RNAi, the extent of transformation along the proximodistal axis differed between embryogenesis and metamorphosis. This suggests that distinct mechanisms specify antennal identity during flour beetle embryogenesis and metamorphosis and leads to a model for the evolution of the Drosophila antennal identity mechanism. Homothorax/Extradenticle acquire many of their identity specification roles by acting as Hox cofactors. In chapter 2, the metamorphic roles of the Hox genes, extradenticle, and homothorax were compared in T. castaneum. homothorax/extradenticle RNAi and Hox RNAi produced similar body wall phenotypes but different appendage phenotypes. These results suggest that Hox genes require extradenticle and homothorax to specify sclerite identities in the thorax and abdomen during metamorphosis. On the other hand, the Hox genes act independently of extradenticle or homothorax to specify appendage identities along the body axis, while Frank Wesley Smith III – University of Connecticut, 2013 extradenticle and homothorax do not require Hox genes to impart proximal identity to appendage podomeres. Tardigrades are closely related to arthropods, but their ventral appendages retain the lobopodal leg-like morphology that is ancestral for arthropods. In Chapter 3, the body plan of the tardigrade Hypsibius dujardini was characterized using anti-!-tubulin immunostaining and phalloidin staining. These methods revealed differences in the nervous system and musculature that make each segment unique. These results raise the possibility that the arthropod/tardigrade ancestor already exhibited morphologically differentiated segments. In Chapter 4, the embryonic role of the gene Distal-less was investigated in H. dujardini. It is expressed in the pharyngeal stylets and across the entire proximodistal appendage axis. The uniform expression of Distal- less in developing appendages is consistent with the lesser degree of morphological regionalization exhibited in tardigrade appendages relative to arthropod appendages. The appendix provides an in situ hybridization protocol for H. dujardini. Conservation and Diversification of Appendage Identity Specification Mechanisms Along the Anteroposterior and Proximodistal Axes in Panarthropoda Frank Wesley Smith III A.S., Community College of Rhode Island, 2004 B.S., University of Rhode Island, 2007 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2013 Copyright by Frank Wesley Smith III 2013 APPROVAL PAGE Doctor of Philosophy Dissertation Conservation and Diversification of Appendage Identity Specification Mechanisms Along the Anteroposterior and Proximodistal Axes in Panarthropoda Presented by Frank Wesley Smith III, B.S. Major Advisor__________________________________________________________________ Elizabeth L. Jockusch Associate Advisor_______________________________________________________________ Steven Q. Irvine Associate Advisor_______________________________________________________________ Carl D. Schlichting Associate Advisor_______________________________________________________________ David L. Wagner University of Connecticut 2013 ii Dedicated to my parents, Ann and Ken Hoover iii Acknowledgments Chapters 3 and 4 of this dissertation were supported by the following sources: The Betty Decoursey Endowment and the James A. Slater Endowment Fund to the EEB Dept. and Connecticut State Museum of Natural History. Sigma Xi Grants-in-Aid of Research Award Society for the Study of Evolution (SSE) Rosemary Grant Graduate Research Award EDEN Research Exchange Fund (NSF/EDEN grant IOS# 0955517) I am deeply indebted to my advisor, Elizabeth L. Jockusch, for the wonderful opportunities and the invaluable advice she has provided for me during my journey through graduate school and growth as a scientist. Elizabeth was always happy to discuss my interests with me, and push me to take on challenges that I would have been reluctant to accept without her encouragement. Elizabeth taught me a wealth of knowledge about evolution and development and thanks to her, a window to an incredibly exciting world has been opened to me. She has revealed mysteries that I will spend the rest of my career working to solve. As a scientist, I cannot imagine a greater gift. I would like to thank my other committee members for all that they have taught me. Carl Schlichting has been an incredible influence on the way I perceive organismal evolution. Dave Wagner has instilled in me an immense appreciation for the diversity of insects. Steve Irvine provided me with my first evo-devo research experience, which gave me the confidence to pursue a PhD. I want to thank all my committee for their excellent comments and advice concerning my dissertation. I owe them all for the excitement I feel about this tremendous accomplishment. iv I would like to thank Dave Angelini for his excellence mentorship when I first started my PhD. He really got me excited about appendage development and evolution. I did not know that I would become an appendage biologist when I arrived at UCONN! I would like to acknowledge Bob Goldstein for encouraging me to study tardigrades. Bob’s encouragement extended all the way to hosting me in his lab during the summer of 2011. During my visit, I developed skills that were incredibly useful for my dissertation research. I cannot wait to join his lab for my postdoc. There is not another lab in the world that I would rather join! I feel incredibly lucky for the opportunity to work along side so many great colleagues during my time in the Jockusch lab. I would like to thank my fellow graduate students in the Jockusch lab: Roberta Engel, Cera Fisher, Beth Timpe, and Tobias Landberg. They have provided me with incredible support. I would like to thank Iñigo Martinez-Solano, who was a postdoc in the Jockusch lab, for sharing his phylogenetic expertise with me and for being a great friend. Finally, I would like to thank all the undergraduates I have been lucky enough to mentor: Derek Cornetta, Matthew Gaudio, Daniel Madden, Devin O’Brien, and Chelsea Willet. It was incredibly exciting to watch them grow as scientists. I would like to thank my professors in the EEB Department: Kurt Schwenk, Janine Caira, Charlie Henry, Chris Simon, Louise Lewis, Paul Lewis, and Andy Bush. The knowledge I accrued in their classes and seminars was critical for the development of my dissertation. I would like to thank Marie Cantino, Bruce Goodwin, and Stephen Daniels of the University of Connecticut Electron Microscopy Laboratory. I would also like to thank Carol Norris of the University of Connecticut Confocal Microscopy Facility. Members of both v imaging facilities were kind enough to share their expertise, which enabled me to collect invaluable data for my dissertation research. Special thanks to all of the graduate students in the EEB Department. It has been a treat! I would like to thank my parents, Ann and Ken, my brother, Nick, sister in law Amy, and nieces, Halle and Hannah for their love and support. Finally, I would like to thank Jackie Meier for her love and support for the last four years. I love you so much Jackie!! I hope you find some great artifacts!! vi Table of Contents Chapter 1: Distinct mechanisms specify antennal identity during embryogenesis and metamorphosis of the flour beetle Tribolium castaneum...............................................pg. 1 Chapter 2: Hox genes require homothorax and extradenticle for sternite and tergite identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum………………………………………………………………………………pg. 63 Chapter 3: All trunk segments exhibit distinct ventral morphologies in the tardigrade Hypsibius dujardini………………………………………………………………………………..pg. 118 Chapter 4: Distal-less specifies legs and stylets in the tardigrade Hypsibius dujardini ………………………………………………………………………………………….pg. 161 Appendix: Embryonic in-situ hybridization for the tardigrade Hypsibius dujardini ………………………………………………………………………………………….pg. 192 vii Chapter 1. Distinct mechanisms specify antennal identity