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Daneshshahab.Pdf (3.352Mb) BMP SIGNALING THROUGH BMPR1A IS REQUIRED FOR ESTABLISHMENT OF PANCREATIC LATERALITY APPROVED BY SUPERVISORY COMMITTEE Ondine Cleaver, Ph.D. Melanie H. Cobb, Ph.D. Raymond J. MacDonald, Ph.D. Eric N. Olson, Ph.D. THIS DISSERTATION IS DEDICATED TO MY FAMILY, PAST, PRESENT AND FUTURE BMP SIGNALING THROUGH BMPR1A IS REQUIRED FOR ESTABLISHMENT OF PANCREATIC LATERALITY by SHAHAB SHAUN MALEKPOUR DANESH DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas May, 2009 Copyright by SHAHAB SHAUN MALEKPOUR DANESH, 2009 All Rights Reserved ACKNOWLEDGEMENTS I would like to thank my thesis advisor, Dr. Ondine Cleaver for her guidance and support. Ondine accepted me into her lab as a fifth year graduate student who had already been in three labs and had no background in developmental biology or mouse work. Her undying guidance, motivation and enthusiasm for science have allowed me to grow as a scientist and complete my thesis in two short years. I would like to thank my committee members Dr. Melanie Cobb, Dr. Eric Olson, and especially my thesis chair, Dr. Raymond MacDonald. They have provided invaluable suggestions and guidance that have facilitated my project that has allowed me to complete a substantial amount of work in a short time. I would like to thank the members of the Cleaver lab for providing a collaborative environment for science. Aly Villasenor was a great resource for exchange of valuable scientific ideas. Ke Xu was very helpful with reagents, techniques, and for important scientific discussion. I‟d also like to thank Diana Chong for her assistance with experiments. I am very thankful to my fiancée, Christa Bennett for her patience, love and support through four labs and six years of graduate school. I‟d also like to thank Mali Malekpour, Shahriar Sarkeshik, Golemreza Malekpour and the rest of my family for their support. I‟d especially like to thank my parents Dan and Nazanin Danesh, my grandparents, Nasser Sarkeshik and Omah. This dissertation is also dedicated in loving memory of my late father Ali Malekpour and my grandmother Forough Sarkeshik, who always wanted me to be a scientist. This wouldn‟t have been made possible without support and motivation from all of you. v vi BMP SIGNALING THROUGH BMPR1A IS REQUIRED FOR ESTABLISHMENT OF PANCREATIC LATERALITY SHAHAB SHAUN MALEKPOUR DANESH, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2009 Supervising Professor: ONDINE CLEAVER, Ph.D. Pancreatic development begins in the mouse, at embryonic day 8.5, as two patches within the gut tube that are located between the stomach and the duodenum. These patches begin to bud, proliferate, and differentiate to become the dorsal and ventral pancreas. During pancreatic bud development several tissues that are adjacent to the gut tube provide important signals for pancreatic development. Various bone morphogenetic proteins (Bmps) and Bmp receptors (Bmprs) are expressed in the pancreas and in the tissues that flank the pancreatic bud during pancreatic development. Bmp7, BmpR1a, and BmpRII are expressed in the pancreatic endoderm during pancreatic bud development. Bmp2, Bmp4, BmpR1a and BmpRII are expressed in the dorsal aorta which transiently flanks the pancreatic bud. Bmp4, Bmp7, BmpR1a, BmpR1b, and BmpRII are expressed in the pre-pancreatic mesenchyme. Interestingly, Bmp4 is expressed asymmetrically along the gut in the mesogastrium, preceeding gut turning. Transgenic knock down of BMP signaling via the secreted BMP antagonist Noggin in the pre-pancreatic milieu results in vii reduced pancreas, spleen, stomach and in failure of pyloric sphincter formation. Interestingly, BMP knockdown also results in defects in lateral growth of the pancreas. Specifically, BMP knockdown prevents formation of the splanchnic mesodermal plate (SMP), the asymmetrically forming mesothelial structure that accompanies leftward growth of the pancreas. Conversely, over expression of Bmp2 in the pre-pancreatic milieu does not result in defects in SMP formation or lateral growth of the pancreas. However, overexpression of Bmp2 in the pancreas epithelium resulted in failure to differentiate into endocrine and exocrine cell lineages. Global knockout of BmpR1a, but not BmprII, prior to pancreatic bud development results in developmental defects in SMP formation and pancreatic laterality. Bmpr1a knockout results in reduced SMP expression of Bapx1, a gene required for SMP formation and lateral growth of the pancreas. Therefore, defects in lateral growth of the pancreas in Bmpr1a mutants are likely mediated by Bapx1 and, accordingly, Bmpr1a mutant pancreata exhibit misregulation of genes that require Bapx1, including Fgf9 and Fgf10. Additionally, deletion of Bmpr1a also leads to misregulation of Barx1, another gene with SMP restricted expression. Deletion of BmpR1a specifically from the pre-pancreatic endoderm or endothelial (aorta) cells did not show defects with respect to pancreatic growth, development, or differentiation. Finally, work herein shows that BMP-BMPR1A signaling to the endothelium specifically is required for vascularization of the pancreatic bud and SMP formation. This study presents the first evidence of a cell-cell signaling molecule playing a role in left-right patterning during organogenesis. viii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................. V ABSTRACT ................................................................................................................... VII TABLE OF CONTENTS ................................................................................................ IX PRIOR PUBLICATIONS ............................................................................................. XV LIST OF FIGURES ..................................................................................................... XVI LIST OF TABLES ........................................................................................................ XX LIST OF ABBREVIATIONS ......................................................................................... 22 CHAPTER 1 INTRODUCTION ................................................................................ 24 OVERVIEW ............................................................................................................. 25 CHAPTER 2 LITERATURE REVIEW ..................................................................... 29 STRUCTURE AND FUNCTION OF THE PANCREAS ....................................... 30 EXOCRINE PANCREAS ................................................................................. 30 ENDOCRINE PANCREAS ............................................................................... 31 PANCREATIC DYSFUNCTION ............................................................................ 33 DIABETES ........................................................................................................ 33 PANCREATIC CANCER ................................................................................. 34 DEVELOPMENT OF THE PANCREAS ................................................................. 35 PANCREATOGENESIS OVERVIEW .............................................................. 36 EXTRINSIC AND INTRINSIC FACTORS DURING PANCREATOGENESIS ................................................................................................ 43 ix BACKGROUND: BONE MORPHOGENETIC PROTEINS ................................... 44 SIGNALING ...................................................................................................... 44 BMP CROSS-TALK ......................................................................................... 47 ROLE OF BMPS IN DEVELOPMENT ............................................................ 49 BMP LIGANDS ................................................................................................ 49 BMP RECEPTORS ............................................................................................ 53 LEFT-RIGHT ASYMMETRY DURING EMBRYONIC AND PANCREATIC DEVELOPMENT ........................................................................................................... 56 EARLY EMBRYONIC LEFT-RIGHT (LR) PATTERNING ........................... 56 SITUS-SPECIFIC ORGANOGENESIS ............................................................ 60 THE ROLE OF BMPS IN THE PANCREAS ......................................................... 63 THESIS RATIONALE AND SIGNIFICANCE ...................................................... 65 CHAPTER 3 METHODOLOGY ............................................................................... 67 TRANSGENIC CONSTRUCTS .............................................................................. 68 IMMUNOHISTOCHEMISTRY ............................................................................... 70 ANIMAL HUSBANDRY ........................................................................................ 70 TISSUE PREPARATION ........................................................................................ 72 WHOLE MOUNT IN SITU HYBRIDIZATION .................................................... 73 IN SITU HYBRIDIZAITON ON SECTIONS ......................................................... 75 HISTOLOGY ...........................................................................................................
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