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Cell Cycle, Fate, Stem Cells, and the Planarian Schmidtea Mediterranea

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Cell Cycle, Fate, Stem Cells, and the Planarian Schmidtea Mediterranea CELL CYCLE, FATE, STEM CELLS, AND THE PLANARIAN SCHMIDTEA MEDITERRANEA by Hara Kang A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Neurobiology and Anatomy The University of Utah May 2009 Copyright © Hara Kang 2009 All Rights Reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submittedby HaraKang This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. - Z / 7 [VjL Cljmi£ Alej andro Sanchez Alvarado �I-Z/() t} I I Monica Vetter Richard Dorsky U §t24 H Shannon Odelberg Charles Murtangh THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate COlmcil of the University of Utah: have read the dissertation of Kang in jts f m aj famI and have found that (1) its fonnat, citations,Ham and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the manuscript is satisfactory� to the supervisory committee and is � submissionfmal to The Graduate School. re y foci Date Ak>f£tndroJ"efrcz'A lvarado /\Oiair: Supervisory Committee Approved for the Major Department MonicaChairlDean Vetter Approved for the Graduate Council David S. 0JapmaA Dean of The Graduate School ABSTRACT The replacement of differentiated cells is a major challenge for all multicellular organisms throughout their life spans. Humans, for example, must replace an estimated 10 billion cells every day, while some animals replace body parts as a result of continuous tissue homeostasis. Such turnover and body part replacement can be regulated through the maintenance, proliferation, and differentiation of somatic stem cells. Although it is clear that the fate decision of somatic stem cells to proliferate or differentiate is tightly controlled by regulating the cell cycle machinery, little is known about the cell cycle of somatic stem cells due to difficult experimental accessibility. We used an excellent in vivo model system, the planarian Schmidtea mediterranea to study the cell cycle of somatic stem cells. S. mediterranea has a relatively large population of stem cells distributed through the entire body except the area in front of photoreceptors and the pharynx. First of all, we developed and optimized methods to characterize the cell cycle of planarian stem cells using flow cytometry and fluorescent- activated cell sorting. Using these methods, we described the cell cycle of planarian total cells or specific populations. Second, for an in-depth understanding of the planarian cell cycle, we identified and characterized planarian homologs of the regulatory molecules controlling the cell cycle in other organisms. Among these molecules, we are highly interested in Smed-cdc73 due to the strong phenotype caused by RNAi and lack of functional studies of Cdc73 in stem cells. Finally, we therefore characterized the function of Smed-cdc73 in the decision for proliferation or differentiation of planarian stem cells using RNAi and molecular markers for stem cells and differentiated progeny of stem cells. We observed that stem cell proliferation was prevented, while differentiation of stem cells was accelerated in the absence of Smed-cdc73, suggesting that Smed-cdc73 is required for self-renewal of planarian stem cells. Taken together, our studies demonstrate the ability to analyze cell cycle of planarian stem cells in vivo and to identify cell cycle regulators required for stem cell functions. Studies on the cell cycle of planarian stem cells will allow valuable basic understanding of stem cell biology. TABLE OF CONTENTS ABSTRACT iv ACKNOWLEDGMENTS viii Chapter 1. INTRODUCTION 1 Abstract 1 The Planarian Schmidtea mediterranea 1 Neoblasts 2 Neoblasts and the Cell Cycle 6 Research Summary 8 References 9 2. DEVELOPMENT OF METHODOLOGIES FOR THE ANALYSIS OF STEM CELL PROLIFERATION IN PLANARIANS 12 Abstract 12 Introduction 13 Materials and Methods 15 Results 17 Discussion 34 Acknowledgments 36 References 37 3. IDENTIFICATION AND CHARACTERIZATION OF GENES THAT AFFECT THE CELL CYCLE 39 Abstract 39 Introduction 39 Materials and Methods 45 Results 52 Discussion 71 Acknowledgments 74 References 74 4. cdc73 IS REQUIRED FOR SELF-RENEWAL OF PLANARIAN STEM CELLS 78 Abstract 78 Introduction 78 Materials and Methods 81 Results 85 Discussion 115 Acknowledgments 125 References 125 5. DISCUSSION 132 What Was Previously Known About the Cell Cycle in Planarians?.. 132 What We Learned from This Study 133 Future Studies 135 References 137 APPENDIX: CHARACTERIZATION OF ADDITIONAL MOLECULES THAT AFFECT MITOTIC ACTIVITIES OF NEOBLASTS 138 vii ACKNOWLEDGMENTS I have often heard that people gain three chances of lifetime. It was my second golden chance to meet with Dr. Alejandro Sanchez Alvarado and to study in his laboratory. During my Ph.D. studies with him, I came to have my dream of becoming a good scientist after the example of his enthusiasm for science. I sincerely appreciate the chance to be in his laboratory and his care of me. I would also like to thank all members of the laboratory, especially Dr. Carrie Adler. I could not have overcome all my various difficulties without her valuable and thoughtful advice. Also, it was my pleasure to be with Dr. George Eisenhoffer and Sofia Robb who are very delightful persons. I really enjoyed spending time with them in the laboratory. I would like to thank my committee members, Drs. Monica Vetter, Richard Dorsky, Shannon Odelberg, and Charles Murtaugh, for helping me to complete the long course. I also appreciate the encouragement from all my friends who are in Korea and those I met in Salt Lake City. I especially thank Eonjoo Park and Sanghee Yun for their simple friendship. Finally, I would like to thank my family who are always at my side for their devoted love with my whole heart. I cannot thank my husband, Sunghwan Kim who gave me my first lucky chance, enough. CHAPTER 1 INTRODUCTION Abstract All multicellular organisms depend on stem cells for survival and perpetuation. The central role of stem cells in reproductive, embryonic, and postembryonic processes, combined with their wide phylogenetic distribution in both the plant and animal kingdoms intimates that the emergence of stem cells may have been a prerequisite in the evolution of multicellular organisms. Somatic stem cells may be ancestral, with germ line stem cells being derived later in the evolution of multicellular organisms. Current studies of stem cell biology are likely to benefit from studying the somatic stem cells of simple metazoans such as the planarian Schmidtea mediterranea. This chapter will provide an overview of the merits of known neoblast functions, and the merits of studying these experimentally accessible cells to dissect the fundamental in vivo biology of stem cells. The Planarian Schmidtea mediterranea Planarians are bilaterally symmetric, triploblastic animals that possess abundant somatic stem cells known as neoblasts |1], Planarians belong to the phylum Platyhelminthes, and are phylogenetically placed in the Lophotrochozoa, a sister group to both the Ecdysozoa (which include D. melanogaster and C. elegans) and the Deuterostomes (to which vertebrates belong) [2], The Lophotrochozoa comprise animals 2 displaying the largest collection of body plans on the planet (squids, mollusks, annelids, ribbonworms, etc), yet have remained underrepresented in current molecular and cellular investigations. There are thousands of different known species [3], but only several dozen have been characterized in any detail. Of these, the free-living, freshwater hermaphrodite Schmidtea mediterranea emerged as a good candidate for in depth analyses due to their robust regenerative properties and their stable diploid state (2n=8) and relatively small 8 1 genome size of -4.8x10 bp (nearly half that of other common planarians) [2]. The ease of maintenance and manipulation of S. mediterranea has allowed us [4] to develop the requisite molecular tools to dissect the remarkable biology of these animals. We have established loss-of-function assays [5, 6], large collections of cDNAs (71, have recently completed a large-scale RNAi-based screen |8|, a sequenced genome, and a genome browser, SmedGD [9, 10]. These advances have permitted us to commence systematic cellular and molecular genetic studies on animal regeneration, tissue homeostasis and the attendant stem cells driving these phenomena. Neoblasts Neoblasts are the adult somatic stem cells of planarians and are distributed throughout the planarian body (Figure 1.1) [11]. Morphologically, neoblasts share many attributes with the stem cells of other organisms, such as large nuclei with extensively decondensed DNA, and largely undifferentiated, highly basophilic cytosols. Neoblasts are the only mitotically active somatic cells in planarians [11], and their division progeny generate the -40 different cell types found in the adult organism [2], In intact planarians, neoblasts replace cells lost to normal physiological turnover [1 1], while in amputated animals, they give rise to the blastema, the structure in which missing tissues are 3 FIGURE 1.1. Planarian stem cells. (A) Immunostaining of S. mediterranea using a phosphorylated histone H3 (H3P) antibody that detects mitotic cells. (B) Immunostaining of S. mediterranea using a BrdlJ (Bromodeoxyuridine) antibody. Animals were stained 48 hours after BrdU-injection.
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