TOOTH DEVELOPMENT: LEARN FROM “THE NORMAL” AND “THE ABNORMAL” by Shih-Kai Wang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Oral Health Sciences) in the University of Michigan 2014 Doctoral Committee: Professor James P. Simmer, Chair Professor Deborah L. Gumucio Professor Paul H. Krebsbach Associate Professor Yuji Mishina Shih-Kai Wang © ——————————— 2014 All Rights Reserved DEDICATION To my parents Su-Ching Wu and Wen-Da Wang ii ACKNOWLEDGEMENTS I would like to thank all individuals who encouraged and supported me throughout the doctoral training: Dr. James Simmer and Dr. Jan Hu, as my research advisors, have been providing me with exceptional scientific resources and environments. Their personal commitment to excellence in science has greatly inspired me and is very much appreciated. Also, Dr. Jan Hu has given me personal guidance and support for life in the United States, which I am deeply grateful for. I would like to express my appreciation to the members of my dissertation committee: Dr. Deborah Gumucio, for her contributions as a thesis committee member and for being a wonderful teacher who inspired me in developmental biology. Dr. Paul Krebsbach, for support of my research from the Biologic and Materials Sciences Department and for being a role model of leadership for me. Dr. Yuji Mishina, for his continuous support and mentoring since my second lab rotation in 2009. His work ethics and attitude in science are always valued. I have received tremendous and diverse support and training for experiments and scientific discussions and want to thank the following individuals for their generosity of sharing knowledge and experimental materials. iii Present and past members of Simmer and Hu laboratory: Dr. Charles Smith, for inspiring me with his personal commitment and enthusiasm to excellence in dental enamel research and for being a role model for me. Dr. Yuanyuan Hu, for support in molecular biology, mouse genetics, and histology. Dr. Yasuo Yamakoshi, for providing fundamental teaching in protein biochemistry. Dr. Hui-Chen Chan, for providing fundamental teaching in human mutational analyses. Amelia Richard, for support in molecular biology, mouse genetics, and histology and for her wonderful friendship and partnership. Andrew Samann, for support in protein biochemistry and for his “fruddy” friendship. Bryan Reid, for support in mutational analyses, molecular biology, and mouse genetics. Soumya Pal, for support in mutational analyses and mouse genetics. Dr. Stephanie Núñez, for the peer support and friendship in Oral Health Sciences Ph.D. program. Dr. Enamul Kabir, Rachel Milkovich, Rangsiyakorn Lertlam, Dr. Yuhe Lu, Dr. Yonghee Chun, Fumiko Yamakoshi, Dr. Hsun-Liang Chan, Dr. Shuhei Tsuchiya, Dr. Jie Yang, for support in experiments and for creating a vibrant work environment. Dr. Yoshihiro Komatsu, from Mishina laboratory, for inspiring me in science and for being a role model for me. Dr. Nobuhiro Kamiya, from Mishina laboratory, for providing fundamental teaching in bone biology. Dr. Hongxiang Liu, from Mistretta laboratory, for discussion in histology. iv Dr. Peter Polverini, the former dean of School of Dentistry, University of Michigan, for creating a unique scientific environment in the school and for providing financial support in the first two years of my doctoral training. Dr. Robert Lyons and Dr. Brendan Tarrier, at the DNA Sequencing Core, University of Michigan, for whole exome sequencing. Dr. James Cavalcoli, Dr. Yongsheng Bai, and Dr. Manjusha Pande, at the Bioinformatics Core, University of Michigan, for analyses of exome sequencing data. The Yale Center for Genome Analysis, Yale University, for whole exome sequencing. Dr. Murim Choi, at Seoul National University, for analyses of exome sequencing data. Dr. Kathrin Wilczak, at the Keck Biotechnology Resource Laboratory, Yale University, for conducting mass-spectrometry experiments and analyses. I am very grateful to the staff of the Oral Health Sciences Research Office, Patricia Schultz, Landon Manette, Charlene Erickson, Kimberly Smith, and Misty Gravelin, for their administrative support and kindness. All current and past Oral Health Sciences Ph.D. students are appreciated for their comradeship, particularly Christopher Donnelly, for his peer support and inspiring friendship. I would like to thank faculty and staff of the Biologic and Materials Sciences Department for their support. I am very grateful to the faculty of my alma mater, National Taiwan University School of Dentistry, for their commitment to dental education and for well preparing me with knowledge and skills for pursuing doctoral training. v Dr. Ming-Kuang Guo, for inspiring me to higher education and academic dentistry. Dr. Bing-Yu Wang, for his inspiring friendship and for encouraging me to explore myself. Finally, I want to express my deepest appreciation to my parents, family, and friends for their unconditional love and support. vi PREFACE This thesis includes significant work of Drs. Jan Hu, James Simmer, Hui-Chen Chan, Murim Choi, James Cavalcoli, Manjusha Pande, Yongsheng Bai, Rachel Milkovich, Yuanyuan Hu, Amelia Richardson, Bryan Reid, Soumya Pal, Yasuo Yamakoshi, M. Enamul Kabir, and Andrew Samann. Description of my contribution to the work presented in this thesis: I wrote all chapters of this thesis. Some work of Chapter 2 and most of Chapter 3 have been published as 6 manuscripts which work contribution can be referred to. I have updated the references and content to the discussion. Drs. James Simmer and Jan Hu contributed to the experimental design and thesis writing. I wrote Chapter 1 “Introduction,” summarizing the general hypotheses and specific aims, and providing background information about topics discussed in following chapters. Chapter 2 and 3 describe studies in mutational analysis of human inherited dental defects, including familial tooth agenesis (FTA) and amelogenesis imperfecta (AI). The work presented in these two chapters is part of the ongoing human genetic projects in the lab. All IRB protocols and consents were accomplished by Dr. Jan Hu, who also established collaborations, recruited subjects, acquired patient information, oral photographs, radiographs, and DNA samples. Genomic DNA isolation from blood/saliva was performed by several lab members, including Dr. vii Hui-Chen Chan, Rachel Milkovich, Bryan Reid, Soumya Pal, and myself. Target gene analyses of FTA described in Chapter 2 were mainly conducted by myself and partly by Rachel Milkovich, Soumya Pal, and Bryan Reid. Exome sequencing and analyses were conducted at 3 different institutes. Sequencing data generated at the University of Michigan DNA Sequencing Core was analyzed by Drs. James Cavalcoli, Manjusha Pande, and Yongsheng Bai at the University of Michigan Bioinformatics Core. Commercial services from Edge BioSystems (Gaithersburg, MD, U.S.A.) were also used. Dr. Murim Choi at Seoul National University analyzed all of the exome sequencing data generated from Yale Center for Genome Analysis. He called the sequence variants, filtered the data, and narrowed the list of reasonable candidate variants, occasionally even suggesting the specific variant that caused the disease. I routinely further analyzed the filtered data. The results of my analyses are provided as tables in the Chapter 2. Bryan Reid generally performed the PCR amplifications for segregation analyses of the final candidate genes. In Chapter 4, the purified recombinant FAM83H protein used for kinase reaction was provided by Drs. Yasuo Yamakoshi and M. Enamul Kabir. The in vitro kinase assays of FAM83H were conducted mainly by Drs. Jan Hu and Yasuo Yamakoshi and partly by myself. All mass spectrometry experiments and data analyses were conducted by Keck Biotechnology Resource Laboratory at Yale University. Many expression constructs were generated by Custom DNA Constructs, LLC (Cleveland, OH, U.S.A.). All other work described in Chapter 4 was designed and performed by myself with advice and assistance from Drs. James Simmer, Jan Hu, Yasuo Yamakoshi, and Andrew Samann. The work from Chapter 4 is not yet published. In Chapter 5 “Conclusion,” I summarized the work accomplishment of this thesis and discussed about prospects and potential future directions of the work. viii TABLE OF CONTENTS DEDICATION............................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................ iii PREFACE .................................................................................................................................... vii LIST OF FIGURES ..................................................................................................................... xi LIST OF TABLES ..................................................................................................................... xiii ABSTRACT ................................................................................................................................ xiv CHAPTER 1 ...................................................................................................................................1 INTRODUCTION Problem Statement .......................................................................................1 General Hypothesis ......................................................................................3 Specific