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Characterization of SPECC1L function in cell adhesion and migration in craniofacial morphogenesis By © 2017 Nathan Wilson B.Sc., Rockhurst University, 2010 Submitted to the graduate degree program in Anatomy and Cell Biology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Dr. Irfan Saadi, Ph.D. (Chair) Dr. András Czirók, Ph.D. Dr. William Kinsey, Ph.D. Dr. Paul Trainor, Ph.D. Dr. Pamela Tran, Ph.D. Dr. Jinxi Wang, M.D., Ph.D. Date Defended: 19 April 2017 The dissertation committee for Nathan Wilson certifies that this is the approved version of the following dissertation: Characterization of SPECC1L function in cell adhesion and migration in craniofacial morphogenesis Dr. Irfan Saadi, Ph.D. (Chair) Date approved: 12 May 2017 ii Abstract Orofacial clefts are frequent congenital malformations, which can result from reduced contribution of cranial neural crest cells (CNCCs) to the developing cranium. Upon delamination from embryonic neural folds, CNCCs migrate to pharyngeal arches, which give rise to mid-facial structures. Previously, mutations in the cytoskeletal gene SPECC1L were implicated in rare, severe atypical facial clefting. Overexpression of a patient isolated SPECC1L mutation showed disrupted association with acetylated microtubules. We show a similar disruption upon overexpression of SPECC1L variants isolated from patients with less severe syndromic cleft lip and palate. Severe Specc1l deficiency in homozygous mouse mutants is embryonic lethal, showing a reduction in pan-AKT levels and arrested CNCC delamination from the neural folds. Staining of adherens junction (AJ) proteins is increased in mutant neural folds, consistent with impaired CNCC delamination, a process requiring AJ dissolution. In vitro, AJ changes induced by SPECC1L-kd are rescued by activating PI3K-AKT signaling. We also looked at cell migration properties of SPECC1L-deficient cells in vitro. Collective cell migration is a cooperative process fundamental to embryonic development. Wound-repair assays using primary mouse embryonic palatal mesenchyme (MEPM), derived from CNCCs, with moderate Specc1l deficiency show impaired collective migration with reduced correlation lengths. These data indicate SPECC1L as a novel modulator of AJs and PI3K-AKT signaling in CNCC delamination and migration of CNCC- derived cells in craniofacial development. iii Acknowledgements So many people have contributed to both the completion of this project, as well as my progress in completing this degree, that I struggle to adequately list their names, let alone describe their contributions – there are many. First and foremost are my parents, who created every possible educational opportunity, for all of us. I also wish to prominently acknowledge and thank the members of my committee – Dr. András Czirók, Dr. William Kinsey, Dr. Irfan Saadi, Dr. Paul Trainor, Dr. Pamela Tran and Dr. Jinxi Wang, who have offered me invaluable guidance and feedback. I deeply appreciate the time each of them has dedicated to my progress. Each has contributed, in a specific way, to help me move forward and complete this project, as well as to each of their individual contributions towards my scientific training. Importantly, I also need to acknowledge our collaborators, which includes two members of my committee. I wish to acknowledge and thank deeply Dr. Paul Trainor and Dr. Kristin Watt, for collaborating with us, as well as the group of Drs. András Czirók, Brenda Rongish and Charles Little, who have each offered me guidance and shared their expertise, facilities and other resources with me, throughout this project. Also, I enjoy acknowledging here the profound efforts of Edina Kósa, Dona-Gréta Isai and Mike Fila throughout that collaboration. Their hard work made many difficult things possible, and for that I will always be thankful. There are collaborators I wish to acknowledge, whom I have not yet met, such as Dr. Paul Kruszka and his colleagues, at Children’s Hospital of Philadelphia, as well as Dr. Bryan Bjork and his, at Midwestern University. The staff of the Anatomy and Cell Biology department deserve my heartiest acknowledgments, as my progress as a student would not be possible without their efforts. I wish to thank and acknowledge the efforts of our previous graduate education director, Dr. Peggy Petroff, and especially the current director, Dr. Julie Christianson, for guiding me throughout this iv process. Also, I wish to thank and acknowledge both our departmental chair, Dr. Dale Abrahamson, and our Dean of Graduate studies, Dr. Mike Werle, for their invaluable guidance throughout this stage in my education, as that guidance was ultimately both invaluable and warmly offered. This project was made possible through the tireless contributions of the many fine core facilities we all rely upon. I wish to acknowledge the guidance and support offered by Pat St. John and Barbara Fegley, in the Electron microscopy core, as well as Richard Hastings, in the Flow cytometry core and Jing Huang, in the histology core. Also, I wish to acknowledge Stan Fernald and Phil Shafer, of the Integrative imaging core, for their assistance with all things aesthetic. Many friends have also helped me along this path, including Dr. Pratik Home, who has always loaned me his ear and given his me wise advice, along with a lot of laughs. Also, my friend John Schupp, who often loaned me his dining room table, mainly for the purposes of writing, yet also for dining and stimulating conversation. I couldn’t have done this without with either of them. I have also had the honor of making many good friends, throughout this journey. I should begin by acknowledging Adam Olm-Shipman and Dr. Syed Rafi, whom I met shortly after the founding of Dr. Saadi’s lab. Later, I also had the honor of working with Diana Acevedo, an effective and amazing lab manager. I also acknowledge Dr. Kanagaraj Palaniyandi, a friend and colleague, with whom working together was a great pleasure. We have also enjoyed the company of hard-working, visiting summer students I wish to acknowledge – Kelly Stumpff, Guerin Smith, Abby Stork, Lauren Hipp and Shahnawaz Yousuf. They all contributed much and made the journey quite a bit more fun and warm, from my perspective. v Lastly, I acknowledge my mentor and our current lab members. I offer my heartfelt thanks and best wishes to Everett Hall, my friend and lab mate. His enthusiasm, hard work, humor and friendship have made the latter part of this journey most enjoyable for me, for which I am very thankful all around. I predict and hope for all the best things in his future. Luke Wenger and Jeremy Goering are much more recent lab members and friends, but no less deserving of acknowledgment, as their efforts are moving the project I soon leave in exciting new directions. To my most valued and esteemed mentor, Dr. Irfan Saadi, no words can adequately express the gratitude in my heart, for not just the effort he has taken in educating me and helping me to become a scientist, but also his great friendship. Further, I need to acknowledge and thank him for the grand sum of time he has spent helping me investigate, shape and complete the project described in this document. He will always have my endless gratitude, as he has been both the mentor I needed and wanted. Last, and most importantly, I lovingly acknowledge my wife Lina Yu, who has always supported me throughout the good and also the challenging times, and who contributed the hierarchical edge bundling analysis included in this appendix of this document. She will defend her doctoral dissertation a few weeks after me, and thus I am very proud of her and excited for our future together. She is the light of my life, and I am the most fortunate man alive to share these challenges, opportunities and joys with her. vi Dedication I dedicate this work to four generations. The first and eldest are my beautiful grandparents. Together, they taught me many things but specifically the vital elements of persistence, scholarship and how to best appreciate the fruits which blossom from them, in time. Next and foremost, my parents, who created every opportunity I had in my childhood. They made everything possible for all of us and made it look easy. As l look backward and forward, I know that it was not easy, but they made it seem so – loving and always nurturing. I equally dedicate this work to the love of my life, my wife Lina. She always makes my heart and eyes shine bright, and lifts me when I need love, support and every other wonderful thing that she brings. Last, but always first in my mind, I dedicate this work to the next generation – my three nieces, Lilly, Olivia and Sage, our daughter Toni and the child we will welcome soon, to accompany her throughout her life and ours. vii Table of Contents List of figures ................................................................................................................................ xii List of tables ................................................................................................................................. xiv List of abbreviations ..................................................................................................................... xv List of protein symbols and names ............................................................................................. xvii Chapter One: General introduction ................................................................................................

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