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Regulating Distinct Cell Lineages in the Pancreatic Islet Joshua a Levine

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Regulating Distinct Cell Lineages in the Pancreatic Islet Joshua a Levine Regulating Distinct Cell Lineages in the Pancreatic Islet Joshua A Levine Submitted in partial fulfillment of the Requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 © 2012 Joshua A Levine All Rights Reserved ABSTRACT Regulating Distinct Cell Lineages in the Pancreatic Islet Joshua A Levine Type I and type II diabetes mellitus are associated with a loss of functioning insulin- producing β cells in the pancreas. Understanding the mechanism of normal islet and β cell development will be an important step in developing possible treatments for the disease. Nkx2.2 is essential for proper β cell differentiation. Nkx2.2-/- mice show a complete absence of insulin-producing β cells, a 90% reduction of glucagon-producing α cells, and an increase in ghrelin-producing cells. Nkx2.2 contains three conserved domains: the tinman domain (TN), homeodomain (HD), and NK2-specific domain (SD). The SD domain is highly conserved among Nk2 family members and across species. However, its function remains largely unknown. In order to further understand the molecular interactions involving Nkx2.2 in the developing mouse pancreas, we have generated a mouse line containing mutations in the NK2-SD domain. We show that SD mutant mice have a decrease in β cell numbers as well as a decrease in the β cell markers, NeuroD, Nkx6.1, Ins1 and Ins2. However, there is no change in α cell numbers or the α cell markers, Glucagon and Irx2. Unlike the persistent upregulation of ghrelin in the Nkx2.2-/- mice, Nkx2.2SD/SD mice display a transient increase in ghrelin expression, which normalizes by birth. Additionally, polyhormonal cells are seen as early as e12.5 and persist postnatally. Postnatally, the mice show morphological changes in islet size and the proximity of their islets to the ducts. Moreover, they show a continuing loss of β cells and the persistence of polyhormonal cells resulting in severe hyperglycemia. Mechanistically, Nkx2.2 has been shown to interact in a protein complex involving several methylation factors. We show that the SD domain is necessary for the interaction of Nkx2.2 and Dnmt1, the maintenance methyltransferase. We further show that there is a loss of methylation in the a cell gene Arx in sorted β cells of the Nkx2.2SD/SD mice as well as global hypomethylation in the Nkx2.2SD/SD mice. These data suggest that Nkx2.2 is responsible for proper methylation patters of islet specific genes in the developing pancreas, which is important for β cell development and the formation of normal islet cell identities. Table of Contents 1. Introduction 1 The Pancreas 1 Diabetes Mellitus 1 Current Treatments 3 Islet Transplantation 4 Pancreatic Endocrine Cell Differentiation 7 Islet Specific Transcription Factors 8 Ngn3 11 Nkx2.2 12 Nkx2.2 Domains 14 Epigenetic Regulation 17 DNA Methyltransferase 18 DNA Methyltransferases in Islet Development 21 Methylation and Gene Regulation 22 2. Islet Cell Identity: The role of the Nkx2.2 NK2-SD Domain in Differentiating Endocrine Cells 25 Introduction 25 Materials and Methods 29 Generation of Nkx2.2SD mice 29 Animal Maintenance 30 Immunohistochemistry 30 RNA Analysis 31 i ChIP Analysis 32 In Vitro Pulldown, Coimmunoprecipitation and Western Blot 33 FACS 34 Methylation Sequencing Analysis 35 Results 35 Generation of Nkx2.2SD/SD mice 35 Nkx2.2SD/SD mice display a defect in β cell development 36 Nkx2.2SD/SD mice fail to form distinct mono-hormonal islet cells 42 SD domain interacts with Dnmt1 46 Mutation of the SD domain leads to gene specific hypomethylation 48 Nkx2.2SD/SD mice have global hypomethylation 50 Discussion 52 3. Conclusions and Perspectives 60 Nkx2.2 SD domain Function 61 In Vitro Differentiation of β Cells 65 Conclusions 67 References 68 A. Transcriptome Analysis of Nkx2.2SD/SD Mice 79 β-cell Specific Genes 79 Non-Annotated Genes 80 Gon-4-Like Pseudogene 80 Conclusions and Future Directions 81 ii B. Proteomic Analysis of Nkx2.2 SD Domain Binding Partners 83 Histone Variants 83 Other SD Interacting Proteins 84 Gain of Function Proteins 84 Conclusions and Future Directions 85 iii List of Figures and Tables 1. Introduction 1 Figure 1-1: Pancreatic Endocrine Development 9 Figure 1-2: Pancreatic Tip and Trunk Domains 10 Figure 1-3: Nkx2.2 Domains 16 Figure 1-4: DNA Methyltransferases 19 2. Islet Cell Identity: The role of the Nkx2.2 NK2-SD Domain in Differentiating Endocrine Cells 25 Table 2-1: List of AOD and Primer/Probe Sets for qRT-PCR analysis 32 Figure 2-1: SD domain mutations 36 Figure 2-2: Insulin is decreased in Nkx2.2SD/SD mice 38 Figure 2-3: Ghrelin is transiently increased in Nkx2.2SD/SD mice 39 Figure 2-4: Insulin is decreased in Nkx2.2SD/SD mice at E18.5 40 Figure 2-5: Islet size and cell numbers are decreased in Nkx2.2SD/SD mice at E18.5 41 Figure 2-6: A small population of ε cells differentiate into δ cells of Nkx2.2SD/SD mice 43 Figure 2-7: Proliferation and apoptosis of ghrelin cells are relatively unchanged at E16.5 44 Figure 2-8: Nkx2.2SD/SD islets contain polyhormonal cells 46 Figure 2-9: Nkx2.2SD/SD islets contain polyhormonal cells postnatally 47 Figure 2-10: SD domain is required for the interaction between Nkx2.2 and Dnmt1 49 Figure 2-11: SD domain is required for proper methylation and gene expression in β cells 51 Figure 2-12: SD domain is required for proper methylation patterns 53 iv A: Transcriptome Analysis of Nkx2.2SD/SD mice 79 Figure A-1: Somatostatin is unchanged in Nkx2.2SD/SD mice at E16.5 82 B: Proteomic Analysis of Nkx2.2 SD Domain Binding Partners 83 Table B-1: List of proteins identified by mass spectrometry 87 v Acknowledgements I would like to thank all of those who have helped me throughout my graduate years. First, I would like to thank my mentor, Lori Sussel, for her continual guidance and support. She has always maintained an open door policy allowing me to ask her any question at any point in the day. She has fostered my interest in development and has continued to push me to improve my scientific thought and writing, crucial steps towards become a successful scientist. Working with Lori has also given me ample opportunity to realize my true passion for the medical field of endocrinology and am fortunate that I will be able to bridge the gap between the basic science and clinical medicine that is needed to improve patient care. Our relationship will continue as our careers continue to evolve. I am also indebted to the amazing colleagues in the Sussel lab. I look forward to the daily interactions that occur when I step foot into the lab everyday. You continue to ask interesting questions that have steered my research towards new and interesting avenues. I particularly want to thank Jonathon Hill who welcomed me into the lab during my rotation and who answered all of my questions no matter how trivial. I was also fortunate to be able to mentor a rotation student, Matt Borok, and an undergraduate Diana Garofalo. One learns the most from their students and I appreciate all that they have taught me over the years. Finally I want to thank by family for all of their support during these last few years. My wife Rebecca has given me unconditional support throughout my research years. She has given me the greatest gift of all this past year with the birth of our son Jamie. I also want to thank my parents and in-laws for their support over the past few years. You have all encouraged me to strive for success as a physician scientist. vi Dedication I dedicate this work to my wife Rebecca and my son Jamie. vii 1 Chapter 1 Introduction The Pancreas The mouse pancreas is a dual organ that lies between the stomach, spleen, and small intestine. 95% of the pancreas consists of exocrine tissue, which is responsible for the secretion of enzymes that are necessary for digestion. The other 5% of the pancreas consists of the endocrine compartment, which is responsible for glucose homeostasis and consists of clusters of cells called the Islets of Langerhans. The adult islet is composed of 4 cell types. 80% of the islet consists of a core of insulin-producing β cells. The remainder of the islet cells surround this core and consist of the glucagon-producing α cell, the somatostatin-producing δ cell, and the pancreatic polypeptide-producing PP cell. A fifth cell type, the ghrelin-producing ε cell, is found only in the embryonic pancreas. A portion of these ghrelin expressing cells co-express glucagon during development, but after birth ghrelin is turned off while glucagon expression remains. The remaining ghrelin-expressing cells differentiate into PP cells (Arnes et al., 2012, in press). Diabetes Mellitus 25 million people in the United States are currently afflicted with Diabetes mellitus. Moreover, 79 million others are considered to be prediabetic with a high likelihood of progressing to a full-fledged diabetic state. Diabetes is the seventh leading cause of death in the United States and is the leading cause of kidney failure, nontraumatic lower limb amputation, and blindness. Diabetes is also a major cause of heart disease and stroke (CDC 2011). It is of great interest to understand the disease and the possible modes of treatment. 2 Diabetes mellitus consists of two main subgroups, Type 1 and Type II diabetes. Type I diabetes (T1DM) is caused by the autoimmune destruction of the insulin producing β cells in the pancreatic islet (Bottazzo et al., 1974; MacCuish et al., 1974).
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