ANALYSIS OF INTESTINAL SPLANCHNOPLEURE DEVELOPMENT By Nichelle I. Winters Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Cell and Developmental Biology August, 2012 Nashville, Tennessee Approved: Professor Chin Chiang Associate Professor Ambra Pozzi Professor Agnes B. Fogo Professor Robert J. Coffey Professor David M. Bader ACKNOWLEDGEMENTS This work would not have been possible without the support of many. I gratefully acknowledge the financial support of the Vanderbilt Medical Scientist Training Program, the American Heart Association, the Vanderbilt Digestive Disease Research Center, and the National Institutes of Health. I have been fortunate to work with many individuals who have generously offered training, intellectual input, and provided essential reagents. In particular, Dr. Michael Stark (BYU) and Dr. Jeanette Hyer (UCSF) hosted me in their laboratories and provided invaluable training in electroporation and lineage tracing techniques. I would like to thank my committee members, Dr. Chin Chiang, Dr. Agnes Fogo, Dr. Ambra Pozzi and Dr. Robert Coffey for their insight and thought provoking conversation. I have greatly valued their career guidance and instruction on how to become a better scientist. The Vanderbilt MSTP has provided an essential network of friends and training. I have benefited greatly from a supportive laboratory environment led by Dr. David Bader who has been a mentor and a friend. I have learned a lot about how to do good science, how to do effective science and how to have fun while working hard from David. I am very grateful to my colleagues and friends in the Bader Lab who have kept things interesting the past few years—Rebecca Thomason, Emily Cross, Hillary Carter, Kt Gray, Michelle Robertson, Elise Pfaltzgraff, Paul Miller, Sam Reddy, Cheryl Seneff, Pierre Hunt, Ryan Roberts, and Adam Greer. Finally, I would like to thank my family—my parents, Cody, Ben, Kate, Desmond and Conner—who I can always count on. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................ ii LIST OF TABLES ............................................................................................................ v LIST OF FIGURES ......................................................................................................... vi Chapter I. INTRODUCTION ......................................................................................................1 Adult mesothelium: structure and function ........................................................... 1 Coelomic organogenesis ...................................................................................... 2 Coelomic vasculogenesis ..................................................................................... 5 Endocardium ............................................................................................6 Other coelomic organs ............................................................................. 7 Cardiac mesothelial development ...................................................................... 10 Discovery of the proepicardium .............................................................. 10 Origin of coronary endothelium ............................................................... 12 Origin of the proepicardium .................................................................... 16 Induction of the proepicardium ............................................................... 18 Signaling functions of the epicardium ..................................................... 19 Hepatic mesothelium development .................................................................... 21 Pulmonary mesothelium development ............................................................... 23 Pancreatic mesothelium ..................................................................................... 24 Intestinal mesothelium development .................................................................. 24 Summary ........................................................................................................... 25 Dissertation hypothesis and summary of aims ................................................... 26 References ........................................................................................................ 28 II. A COMPREHENSIVE TIMELINE OF MESODERMAL DEVELOPMENT IN THE QUAIL SMALL INTESTINE..................................................................................... 37 Abstract ............................................................................................................ 37 Introduction ...................................................................................................... 38 Materials and methods ..................................................................................... 42 Results ............................................................................................................. 44 Establishment and maturation of the major intestinal components.......... 44 Development of the outer epithelium ...................................................... 49 Expansion of the mesenchymal component ........................................... 50 Development of the muscularis layers and myofibroblasts ..................... 54 The organization of the endothelial plexus .............................................. 55 Generation of muscularized surface blood vessels ................................. 62 Discussion ......................................................................................................... 63 iii Appearance of the intestinal anlage ........................................................ 68 Development of the mesenchymal compartment: E1.9-E5 ..................... 68 Completion of intestinal tube formation: E5-E6 ....................................... 69 Maturation of visceral smooth muscle and vascular components: E6-16 ................................................................................ 71 References ........................................................................................................ 73 III. IDENTIFICATION OF A NOVEL DEVELOPMENTAL MECHANISM IN THE GENERATION OF MESOTHELIA ......................................................................... 78 Abstract ............................................................................................................. 78 Introduction ........................................................................................................ 78 Materials and methods ....................................................................................... 81 Results ............................................................................................................... 85 Trilaminar organization of the intestine is established prior to tube formation ....................................................................................... 85 Mesothelial progenitors are resident to the splanchnic Mesoderm .............................................................................................. 87 Intestinal mesothelial progenitors are localized broadly throughout the splanchnic mesoderm ..................................................... 97 Discussion ....................................................................................................... 100 References ...................................................................................................... 106 IV. CHICK-TRANSGENIC QUAIL CHIMERAS IN STUDIES OF EMBRYONIC DEVELOPMENT ................................................................................................ 110 Abstract ........................................................................................................... 110 Introduction ...................................................................................................... 110 Materials and methods ..................................................................................... 112 Results ............................................................................................................. 113 Generation of chick-transgenic quail splanchnopleure chimeras .......... 113 Generation of chick-transgenic quail somatopleure chimeras ............... 117 Discussion ............................................................................................ 119 References ...................................................................................................... 122 V. CONCLUSIONS AND FUTURE DIRECTIONS ................................................... 125 References ...................................................................................................... 131 iv LIST OF TABLES Table Page 2.1. Stages at which key developmental events occur throughout the development of quail intestinal mesoderm .......................................................... 67 v LIST OF FIGURES Figure Page 1.1. Avian gastrulation and organogenesis .................................................................. 3 1.2. Development of the proepicardium ..................................................................... 11 2.1 Schematic depicting intestinal primordium, primitive intestinal tube, and adult intestine .................................................................................................... 39 2.2 Early basement membrane