INTESTINE OF ZEBRAFISH: REGIONALIZATION, CHARACTERIZATION AND STEM CELLS WANG ZHENGYUAN (M.Sci., NUS) (B.Eng., NPU) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN COMPUTATION AND SYSTEMS BIOLOGY (CSB) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements I want to thank my supervisors, professor Matsudaira Paul and professor Gong Zhiyuan, whose time, knowledge and wise guidance has constituted a key com- ponent ensuring the continual progression of my research work. I want to thank my thesis committee members, professor Lodish Harvey and assistant professor Bhowmic Sourav, for following my progress, evaluating and steering my work. Thanks also go to professor Rajagopal Gunaretnam, who initiated the project and helped much to get the project started several years ago. This study was supported by funding and a graduate fellowship from the Singapore-MIT Alliance. Special thanks to my labmates, Zhan Huiqing, Wu Yilian, Du Jianguo, Tavakoli Sahar, Li Zhen, Zheng Weiling, Tina Sim Huey Fen, Liang Bing, Ung Choon Yong, Lam Siew Hong, Yin Ao, Mintzu, Li Caixia, Grace Ng, Sun Lili, Cecilia, Lana and others. Four years of doctoral study in the laboratory would by no means be joyful and fun-filled without the company of them. Last but not least, thanks go to my family members, including my parents, sisters, wife and son for their full support during my doctoral study. Time spent in the laboratory could not be spent with my family. Thank them for i bearing with me during the years of scientific training. Thank my son, the little lovely creature, for bringing oceans of joy to me. ii Contents 1 Introduction 1 1.1 Introduction to the digestive system . .2 1.2 Tissue architecture and cell types of the intestinal epithelium . .5 1.3 Turnover of the intestinal epithelium . .6 1.4 Significance of the study of the digestive system . .7 1.5 Intestinal stem cells . .8 1.5.1 Location of intestinal stem cells . .9 1.5.2 Intestinal stem cell number . 11 1.5.3 Intestinal stem cell marker . 12 1.6 Intestines of different vertebrate models . 13 1.6.1 Mouse intestine . 13 1.6.2 Chicken intestine . 14 1.6.3 Frog intestine . 16 1.6.4 Zebrafish intestine . 17 1.7 Establishing zebrafish as a vertebrate model for study on intestine 19 1.8 Research goals of the current work . 20 1.8.1 Morphological and histological features of zebrafish in- testine . 21 1.8.2 Characterization of regionalization of zebrafish intestine through genome-wide gene expression analysis . 21 1.8.3 Study of the cell fate decision in zebrafish intestine . 22 1.8.4 Responsive nature of intestine during regeneration . 22 1.8.5 Computational analysis of intestinal stem cells and their adaptive changes . 23 2 Functional organization along the rostrocaudal axis of the in- testine 24 2.1 Background . 25 i 2.2 Materials and Methods . 27 2.2.1 Maintenance of zebrafish and dissection of zebrafish in- testine . 27 2.2.2 Paraffin sectioning of zebrafish intestine . 27 2.2.3 Hematoxylin and eosin and alcian blue staining . 28 2.2.4 Quantitative real-time PCR (qRT-PCR) . 28 2.2.5 Microarray experiments . 29 2.2.6 Identification of differentially expressed genes from the microarray data . 30 2.2.7 Gene ontology (GO) analysis by GO Tree Machine . 30 2.2.8 Pathway analysis using WebGestalt . 31 2.2.9 Gene Set Enrichment Analysis . 32 2.3 Results . 32 2.3.1 Architectural differences along the zebrafish intestinal tract . 32 2.3.2 Distinct molecular signatures along the zebrafish intesti- nal tract . 37 2.3.3 Molecular features of the small and large intestine-like functions . 43 2.3.4 Analysis of gene ontology along the anterior-posterior axis 48 2.3.5 Cross-species Gene Set Enrichment Analysis (GSEA) in- dicates the segments S1-S5 to be multi-functional . 53 2.3.6 Stomach-like functions of the intestine . 56 2.3.7 Pathway analysis for zebrafish intestine . 59 2.3.8 Discussion . 66 2.3.9 Conclusions . 69 3 Regulation of cell fate and composition of the intestinal ep- ithelium 70 3.1 Background . 71 3.2 Materials and Methods . 72 3.2.1 DAPT treatment of zebrafish . 72 3.2.2 Alcian blue and Periodic Acid in Schiff's reagent staining 73 3.2.3 Whole mount in situ hybridization . 74 3.2.4 Cryosection of zebrafish intestine . 74 3.2.5 Immunohistochemistry . 75 3.3 Results . 76 3.3.1 Inhibition of Notch signaling in larval zebrafish intestine 76 ii 3.3.2 Verification of inhibition of Notch signaling in adult ze- brafish intestine . 77 3.3.3 Reduction in the pool of intestinal progenitor cells upon inhibition of Notch . 78 3.3.4 Increase of secretory lineages after inhibition of Notch signaling . 81 3.3.5 Enhanced expression of gata6 upon inhibition of Notch in the intestine . 84 3.3.6 Enhanced activity of BMP signaling due to inhibition of Notch signaling . 85 3.3.7 Suppression of glycogen-rich intestinal subepithelial my- ofibroblasts (ISEMFs) along the villus axis due to inhi- bition of Notch signaling . 89 3.4 Discussion . 93 3.4.1 Notch signaling and binary lineage allocation . 93 3.4.2 Involvement of a distinct cohort of glycogen-rich ISEMFs in cell lineage allocation . 95 3.4.3 Preferable targeting of secretory cells in cancer . 97 3.4.4 Cooperative BMP and gata6 activities in epithelial dif- ferentiation . 98 3.5 Conclusion . 100 4 Regeneration of zebrafish intestine following whole body gamma- radiation 101 4.1 Introduction . 102 4.2 Methods . 105 4.2.1 Experiment setup for radiation . 105 4.2.2 Sampling schedule . 105 4.2.3 RNA extraction and real-time PCR . 106 4.2.4 Paraffin embedding and AB-PAS staining . 106 4.2.5 Alkaline phosphatase staining . 106 4.3 Results . 110 4.3.1 Survival of zebrafish after whole body gamma radiation . 110 4.3.2 Two rounds of elimination of intestinal villi . 110 4.3.3 Two waves of Wnt/beta-catenin signaling: a driver of proliferation . 113 4.3.4 Cell proliferation as measured by pcna staining . 116 4.3.5 Radiation induced cell apoptosis . 118 4.3.6 Changes in the intestinal epithelium renewal . 122 iii 4.3.7 Regeneration of the secretory epithelial cells . 124 4.3.8 Maintenance of basic intestinal functions following radi- ation . 126 4.3.9 Active involvement of intestinal stem cells during tissue restitution . 130 4.3.10 Elevated mesenchymal activities . 133 4.4 Discussion . 135 4.4.1 Concerns regarding the radiation setup . 135 4.4.2 Impressive regenerative capacity of zebrafish intestine . 138 4.4.3 Differential sensitivity of intestine to radiation and can- cer rate . 139 4.4.4 Implications for colorectal cancer therapy . 140 4.4.5 Future directions . 141 5 STORM: A General Model to Investigate Stem Cell Number and Their Adaptive Changes 143 5.1 Background . 144 5.2 Materials and Methods . 146 5.2.1 Development of the STORM model . 146 5.2.2 Maintenance of zebrafish . 156 5.2.3 Tissue sectioning . 156 5.2.4 Immunohistochemistry . 156 5.3 Results . 158 5.3.1 General characteristics of the crypt-villus system . 158 5.3.2 Determination of the number of epithelial stem cells in a 2D section of the inter-villi pocket of zebrafish (Danio rerio) intestine . 159 5.3.3 Determination of the stem cell number in each crypt of mouse small intestine . 163 5.3.4 Determination of the stem cell number in each crypt of human duodenum . 165 5.3.5 Comparison of the intestines of different species . 168 5.3.6 Uncontrolled expansion of the capacity of stemness upon impaired feedback mechanism . 171 5.3.7 Application of the model to help evaluate hyperplasia in human duodenitis and ulcer . 172 5.4 Discussion . 173 iv 5.4.1 Epithelium apoptosis is actively initiated in zebrafish in- testine before mature cells get exfoliated at the tips of villi . 173 5.4.2 Achieving the optimal epithelium renewal rate might be a fundamental principle of the crypt-villus system design by nature . 174 5.4.3 The number of stem cells is largely conserved in the small intestines of teleost, murine and human . 175 5.4.4 A general model for analysis of stem cell number with equal applicability to teleost, murine and human intesti- nal tracts . 176 5.4.5 Homeostasis of intestinal secretory cells takes high pri- ority to ensure the integrity of the feedback mechanism . 176 5.4.6 Growing evidence for validity of the model . 177 5.5 Conclusion . 178 6 Conclusion 179 6.1 Conclusion . 180 6.2 Future research directions . 182 Appendix A 208 v Abstract Unlike the mammalian digestive tract that has been developed into distinct regions for different functions, fish have a relatively simple intestine and many fishes have no recognizable stomach. We used the zebrafish microarray ap- proach to characterize its intestine. By dividing the zebrafish intestine into seven segments along its length, we found that the first five segments resem- ble the mammalian small intestine and the last two segments resemble the mammalian large intestine. We then investigated the role of Notch signaling and found that a specific group of glycogen-rich fibroblasts were involved in the Notch-mediated cell fate decision process. Further, we studied the effects of radiation and found an interesting pattern of regeneration in the intestine. Moreover, the number of intestinal stem cells was investigated through a novel computational model, which was applicable not only to zebrafish, but also to mammalian intestinal tracts.