SMALL INTESTINAL TUFT CELL SPECIFICATION AND BEHAVIOR IN HOMEOSTASIS AND INTESTINAL INFLAMMATORY DISEASE By Amrita Banerjee Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY in Cell and Developmental Biology May 10, 2019 Nashville, TN Approved: Guoqiang Gu, Ph.D. Christopher Wright, D.Phil. Robert J. Coffey, M.D. Christopher Williams, M.D., Ph.D. Ken S. Lau, Ph.D. For my family, without whose support this work would not have been possible. ii ACKNOWLEDGEMENTS I am deeply indebted to my mentor Dr. Ken Lau for his support and guidance during my five years in his lab. He has afforded me every opportunity to develop my research, writing, and computational skills. He encourages me to think deeply about my experiments without forgetting about the big picture. My progress in all aspects of my training are due to his patience and tutelage. Thank you for giving me the freedom to explore my intellectual curiosity as well as the support to ensure that I did not go astray. I would like to thank my committee members: my chair Dr. Guoqiang Gu, my co-chair Dr. Chris Wright, Dr. Robert Coffey, and Dr. Christopher Williams for their many years of guidance and encouragement. From my first class with Dr. Gu, he has always expressed a willingness to offer his mentorship and assistance in making me a better scientist. I am grateful to Dr. Wright for acting as the co-chair of my committee and providing his support to ensure that everything remained on track. I am thankful to Dr. Coffey, who in classes, one-on-one meetings, and presentations, has always asked the most perspicacious questions to broaden my thinking. And finally, I am thankful toward Dr. Williams, whose insight into my work I have always valued. I am deeply grateful for all the constructive criticism, insight, and encouragement each has offered towards my science and my career. Science does not happen in a vacuum and my work is no exception. I am incredibly thankful for the supportive and collaborative atmosphere that Dr. Lau has fostered in his lab, which made all the difference during my training. Chuck, thank you for your scientific contributions to my work but also for offering your wit and humor from as near as the next bay over to as far as Western Australia. Joey, I cannot overstate how valuable your help and advice has been to my graduate training. Cherie’, thanks for always thinking the same and keeping your Slack window open at all times. Hyeyon, my undergraduate extraordinaire, I appreciate your persistence with any project that I handed you as your efforts were invaluable in the completion of this dissertation. Thank you all for your friendship and support these past four and a half years. I also want to thank iii all the members of the Lau, Coffey, and Goldenring labs, past and present, for their advice, encouragement, and guidance throughout my graduate training. I am incredibly fortunate to have been a part of the Cell and Developmental Biology Department and the Epithelial Biology Center these past years. I also want to acknowledge the funding sources that have supported my graduate training, including NIH Grant RO1 DK103831 as well as support from the Crohn’s and Colitis Foundation of America and the Vanderbilt Microbiome Institute. Finally, the shared resources from the Vanderbilt DDRC, Flow Cytometry Core, TPSR, and DAC were essential to this work. Outside of the lab, I have been blessed with the most wonderful friends and family. Joan, thank you for your friendship from our first days in Alspaugh on East Campus to graduate school in Nashville for me and medical school in Cleveland for you. I am so excited to see and share in all the amazing things you are sure to accomplish. I want to thank my family, both present and absent, for shaping me into the person I am. To the memory of Lt. Col. Bimal Chandra Banerjee and Smt. Amita Roy, who left behind a wonderful legacy as well as the finest parents a person could ask for. To the late Dr. Minakshi Midya, whose presence and wisdom is still felt after more than a decade. To my grandmother, Smt. Anjali Banerjee, who always keeps me in her thoughts and lets me recite Keats to her. To my grandfather, Dr. Indra Shekhar Roy, whose devotion to the health and welfare of his community has been an inspiration. I am also grateful to my entire family in Kolkata and elsewhere who, despite the distance, have shown me nothing but support and encouragement. Finally, to Ma, Baba, and Dada – I cannot say enough times or with sufficient accuracy what our foursome means to me. Thank you for all the “one last thing(s)” you have to say to me and for celebrating all the highs and cheering me on through the lows. I cherish the adventures we have experienced thus far and am so excited for the numerous that lie ahead. iv TABLE OF CONTENTS Section Page Dedication ................................................................................................................................... ii Acknowledgements .................................................................................................................... iii List of Figures ............................................................................................................................ ix List of Tables ............................................................................................................................. xi Preface ..................................................................................................................................... xii Chapter I. Background – The small intestinal epithelium in health and disease ..................................... 1 Inflammatory bowel disease .................................................................................................... 1 The intestinal stem cell niche ................................................................................................... 3 Paneth cell differentiation and function .................................................................................... 5 The commensal microbiome and microbial-derived metabolites .............................................. 7 Population-level microbiome analysis ...................................................................................... 9 Host metabolites .................................................................................................................... 10 Organoid culture .................................................................................................................... 12 Systems biology and single-cell analysis .............................................................................. 14 Background on DISSECT-CyTOF ......................................................................................... 15 Background on droplet-based single-cell RNA-sequencing (scRNA-seq) .............................. 15 Background on p-Creode ....................................................................................................... 16 Mouse models ....................................................................................................................... 17 TNFΔARE/+ model of Crohn’s-like ileitis .................................................................................... 17 II. Background – Specification and function of small intestinal tuft cells .................................. 19 Small intestinal tuft cells are a rare chemosensory cell type .................................................. 19 Tuft cell morphology .............................................................................................................. 20 Intracellular tuft cell structure ................................................................................................. 22 v Small intestinal tuft cell specification ...................................................................................... 25 Tuft cell gene signature ......................................................................................................... 28 Colonic tuft cells .................................................................................................................... 29 Tuft cells in intestinal epithelial damage response ................................................................. 30 Tuft cells in gastrointestinal helminth infection ....................................................................... 34 III. Single-cell analysis of differential response to inflammatory stimuli ..................................... 40 Introduction ............................................................................................................................ 40 Results .................................................................................................................................. 43 A novel disaggregation procedure for investigating epithelial signaling heterogeneity ........ 43 DISSECT application of CyTOF identifies a differentially signaling enterocyte subpopulation that is sensitized to TNF-α-induced cell death .................................................................... 44 Divergently responding enterocytes are neighbors within the intestinal epithelium ............. 48 Discussion ............................................................................................................................. 50 Methods ...............................................................................................................................