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Bioinformatic Analysis of Epithelial:Mesenchymal Crosstalk During Mouse Gut Development and Patterning

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Bioinformatic Analysis of Epithelial:Mesenchymal Crosstalk During Mouse Gut Development and Patterning BIOINFORMATIC ANALYSIS OF EPITHELIAL:MESENCHYMAL CROSSTALK DURING MOUSE GUT DEVELOPMENT AND PATTERNING by Xing Li A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Bioinformatics) in The University of Michigan 2009 Doctoral Committee: Professor Deborah L. Gumucio, Chair Professor Andrzej A. Dlugosz Professor David J. States Associate Professor Kerby A. Shedden Assistant Professor Zhaohui S. Qin © Xing Li 2009 DEDICATION To my wife and my son ii ACKNOWLEDGEMENTS I would like to extend my sincere thanks to my advisor, Deborah Gumucio, for her full support, excellent training, amazing insights, incredible patience and inspiration, and great passion for science. Her enduring zeal and persistent diligence set a wonderful example for me to follow as a scientist and person. Without her complete support and encouragement, this thesis work was impossible. I have learned a great deal from her. Dr. Gumucio has always been very supportive and considerate in many aspects of my family, especially for my son’s birth. I cannot give enough thanks to her. It has been a great pleasure having her as my mentor and I am grateful for this invaluable opportunity. I am also grateful to other members of my thesis committee: Drs. Andrzej A. Dlugosz, Zhaohui Qin, Kerby Shedden and David States. I really appreciated all of your incredible support, advice and guidance. Dr. States has been my co-advisor and offered excellent insights and direction both in bioinformatics and biology fields； I also sincerely thank Dr. States for his invaluable support and motivation, especially in the first couple years when I arrived at The University of Michigan. Dr. Shedden was the mentor of my first rotation. He gave me excellent advice and direction on microarray data analysis, statistics and computing programming with great patience. Dr. Qin has been a very supportive and talent advisor on microarray data mining, statistical analysis, and many other fields. I have learned a lot from him, especially for microarray data analysis, transcription factor binding site analysis, clustering, and iii programming in R and BioConductor. Dr. Dlugosz has been offering me outstanding advice and inspiration Hedgehog patterning and its roles in controlling gut development. His helpful comments in my Gut group presentations were greatly appreciated. It is truly an honor to have such an excellent committee. I also offer my gratitude to the past and present members in the Gumuco lab. I could not imagine being able to work with more enthusiastic, dedicated, smart and cooperative people. Specifically, in the epithelium and mesenchyme project, I would like to thank Will Zacharias for teaching me how to make cDNA and perform RT-PCR; Åsa Kolterud for in situ hybridization. In the pyloric border microarray project, I thank Chunbo Hu for in situ hybridization, Tracy Qiao for tissue collection and Sox2 staining, Neil Richards for PCR analysis and probe production. A special thank you to Aaron Udager, who is a co-first author on the border microarray paper. Aaron performed tissue collection, PCR and in situ hybridization. He also helped with data assembly and table formatting. He will continue to study this interesting dataset for his thesis work. I am also grateful to Jierong Long for taking care of the mice and genotyping. Thanks to Kate Walton for the help with formatting problems; you really save me. Thanks to Mike Czrewinski and other previous lab managers for helping me with ordering and other issues. I also extend my gratitude to the rest of the Gumucio lab, Ann Grosse, Andrea Waite. Thank you all for your inspiring discussions, kind help, and for creating such a productive, enjoyable and friendly atmosphere. I have so many great memories of fun times that we shared in the lab, potlucks, Christmas parties, North Michigan party, etc. I really could not have asked for a more wonderful group of labmates. Many thanks are extended to Becky Pinter in Center for the Organogenesis for her iv kind help with many things. My thanks also to Yili Chen, Ji Chen and other members in the States Lab for the great discussions and fun interactions. I thank the Gut Group for inspiring discussions. I also thank the UM Cancer Center Microarray Core personnel for performing an microarray hybridizations. My gratitude is also given to the staff in the Bioinformatics Program, CCMB and CDB for their complete support. In particular, I am extremely grateful for my beautiful wife, Qingling, and my lovely son, Ruihao, for their love, happiness and complete support during my Ph.D study. I also greatly appreciate my parents, parents-in-law, brothers, sisters-in-law and brother-in-law for their love, constant support and encouragement. Thank you all. v Table of Contents DEDICATION..............................................................................................................ii ACKNOWLEDGEMENTS ...................................................................................... iii List of Figures..............................................................................................................ix List of Tables................................................................................................................xi ABSTRACT................................................................................................................xii CHAPTER I. INTRODUCTION ...............................................................................1 The biology of gut development............................................................................1 Brief introduction to gut development...........................................................1 Epithelial-mesenchymal crosstalk .................................................................4 Specific pathways important in intestinal development ................................6 Pyloric border formation in gut development..............................................12 Microarray technology and development ............................................................13 Brief introduction to microarray ..................................................................14 General procedure for microarray experiments ...........................................18 Identification of significantly changed genes ..............................................25 Correction for multiple hypothesis tests ......................................................28 Microarray data mining and Bioinformatics................................................30 Verification and follow-up experiments for microarray results...................46 Summary..............................................................................................................47 Bibliography ........................................................................................................53 CHAPTER II. DECONVOLUTING THE INTESTINE: MOLECULAR EVIDENCE FOR A MAJOR ROLE OF THE MESENCHYME IN THE MODULATION OF SIGNALING CROSSTALK..................................................68 ABSTRACT.........................................................................................................68 INTRODUCTION ...............................................................................................70 MATERIALS AND METHODS .........................................................................72 Separation of epithelium and mesenchyme .................................................72 Microarray Analysis.....................................................................................74 Search for Hnf4 binding sites ......................................................................75 vi RT-PCR verification.....................................................................................75 In situ hybridization .....................................................................................76 RESULTS.............................................................................................................77 Validation of clean tissue separation............................................................77 Functional attributes of genes expressed in mesenchyme and epithelium ..80 A tissue-specific signature held by the epithelium ......................................81 Compartmentalized transcription factors in mouse intestine.......................81 Enrichment of transcription factor sub-types...............................................84 Hnf4 binding sites in epithelial genes..........................................................85 The mesenchyme as a modulator of signal transduction .............................87 The Bmp pathway: modulation by numerous mesenchymal factors...........92 DISCUSSION......................................................................................................94 ACKNOWLEDGEMENTS.................................................................................98 BIBLIOGRAPHY..............................................................................................139 CHAPTER III. PATTERNING OF THE EPITHELIAL PYLORIC BORDER IS ACCOMPANIED BY A DRAMATIC INDUCTION OF GENE EXPRESSION IN THE INTESTINAL EPITHELIUM .................................................................144 Abstract..............................................................................................................144 Introduction........................................................................................................145 Materials and Methods.......................................................................................147
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