TRANSCRIPTIONAL PROFILING OF PANCREATIC PROGENITOR CELLS By Leah Ashley Potter 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 Molecular Physiology and Biophysics December, 2011 Nashville, Tennessee Approved: Professor Maureen Gannon Professor Alyssa Hasty Professor Patricia Labosky Professor David Piston Professor Christopher V.E. Wright MOLECULAR PHYSIOLOGY AND BIOPHYSICS TRANSCRIPTIONAL PROFILING OF PANCREATIC PROGENITOR CELLS LEAH ASHLEY POTTER Dissertation under the direction of Mark A. Magnuson, M.D. Pluripotent stem cells, because of their ability to differentiate into any cell type, have been widely advocated as a means of producing a nearly unlimited source of new insulin- producing β cells for the treatment of diabetic diseases. However, while there has been remarkable progress in learning how to direct the differentiation of human embryonic stem (hES) cells towards pancreatic endocrine cell fates, insulin-expressing cells made in this manner are often polyhormonal and lack a normal response to glucose, thereby suggesting a need for a deeper understanding of the gene regulatory networks that are established in a stepwise manner during pancreas development. My thesis studies explored three main topics, each of which holds potential for the development of improved hES cell directed pancreatic differentiation protocols and the discovery of genes that may specifically affect β cell development. First, we used mice that contained a fluorescent reporter allele and fluorescence-activated cell sorting (FACS) to isolate several discrete pancreatic cell populations which were then analyzed using whole transcriptome sequencing (RNA-Seq). By doing so, we were able to examine the genetic requirement and temporal changes of cells expressing pancreas specific transcription factor 1a (Ptf1a), a marker of the pancreatic multipotent progenitor cells (MPCs) and of acinar- specified cells, during pancreas development. By comparing the transcriptional profiles, we identified five gene clusters, each of which provides insights into the dynamics of gene expression during specific aspects of pancreas development. Second, my studies revealed that Nephrocan, an inhibitor of the TGFβ signaling pathway, was expressed in pancreatic MPCs. Thus, to explore the role of Nepn further, we generated mice containing a single copy insertion of a Nepn-Cherry transgene. Finally, to facilitate the combinatorial sorting of Pdx1- and Ptf1a-expressing cells during early pancreas development, we generated a mouse line expressing a cyan fluorescent protein under control of the endogenous pancreatic and duodenal homeobox 1 (Pdx1) gene. The research that I have performed is part of a larger project focused on generating and characterizing a series of high quality transcriptional profiles representing key stages in the generation of pancreatic endocrine cells that occur naturally in the mouse. We anticipate that further analysis of the datasets I have generated for specific developmental stages, in combination with similarly generated datasets at other developmental stages, will facilitate identification of signaling pathways and gene clusters essential for formation of functional pancreatic β cells in the mouse, thereby stimulating new hypotheses for identifying pro-β cell signals necessary to direct the differentiation of pluripotent stem cells into pancreatic β cells. Approved: Professor Mark A. Magnuson Date: December 2011 ACKNOWLEDGEMENTS Chinese fortunes have always been a fundamental means of communication in the Magnuson laboratory. As I looked through my collection of fortunes on the corner of my desk, the one that stood out the most was, “A solid challenge will bring forth your finest abilities.” By far, graduate school has been my most challenging endeavor, and I would be remiss not to acknowledge the people who have imparted their knowledge and encouraged me along the way. First, I would like to thank the members of the Magnuson laboratory, both past and present. I am forever indebted to Jill Lindner, Susan Hipkens and Pamela Uttz. They have been with me since the very beginning, and I cannot fathom what my time here at Vanderbilt would have been like without them. They encouraged me every step of the way, “taught me the ropes,” and were always there to listen when I needed an ear. I am grateful to Eunyoung Choi, Anna Osipovich, Judsen Schneider, Madhi Saranadasa, Anil Laxman, Jared Burlison, Yanyun Gu, and Weiping Yuan. No lab can function without a scientific ‘sounding board,’ and I am thankful to them for providing an environment that stimulates scientific discussions. I would also like to thank, Rama Gangula, Changqing Zhang, Kathy Shelton, and Jody Peters, whose technical expertise and assistance were invaluable. I would like to thank the members of the Beta Cell Biology Consortium, specifically Chris Stoeckert, Elisabetta Manduchi, Guoqiang Gu, Lori Sussel, and Ray MacDonald, as well as the BCBC coordinating center affiliates, Jean-Philippe Cartailler, Thomas Houfek, Suzannah Green and Kim Cornwell. Within the Vanderbilt Center for Stem Cell Biology, I would like to thank members of the Labosky and Huppert laboratories, specifically Stacey Huppert, Kari Huppert, Erin Sparks, Jennifer Plank and Elise Pfaltzgraff. The work presented iv within this dissertation would not have been possible without the expertise and assistance from numerous shared resource facilities and their personnel, including Kevin Weller, Dave Flaherty, and Brittany Matlock from the Flow Cytometry Core and Shawn Levy, Vicky Amann, Chelsea Baker, Travis Clark, Christian Shaffer, David Sexton, and Robert Woodall from the Genome Sciences Resource. Additionally, a number of individuals at Vanderbilt have been generous in their assistance and in sharing their scientific expertise and reagents, including Anna Means, Al Powers, Michelle Southard-Smith, Jeffery Raum, Yan Hang, Michelle Guney, Chunhua Dai, Ashley Cantrell, Stephanie Byers, Clay Spencer, and Rebecca McWhirter. I am grateful to Roland Stein for providing constructive criticism, for cheering me on from the front row, and for simply making me smile even in the midst of difficult moments. I am especially grateful to the members of my dissertation committee who have guided me through this entire process. To my chair, Maureen Gannon, who has gone above and beyond what is required, and to Alyssa Hasty, Trish Labosky, David Piston and Chris Wright, who have provided much needed encouragement at exactly the right moments, demonstrated what it means to be a successful scientist, provided constructive criticism and critiques, and who have seen me at my worst and still believed I had the potential to succeed. While a great amount of support came from within my scientific community, a significant amount of support came from those outside this realm. First, I want to thank my family, Anthony, Charlotte, Rachel and Joshua. From the very beginning, they instilled in me the importance of hard work and nurtured my inquisitive mind. They have supported me every step of the way, from late night calls to weekend visits; they have been my one constant source of encouragement and for that I am grateful. To my extended family, Papa, Mrs. Doris, Nanny, JD, Dickey, KK, Jeff, Bob, Michelle, and Trey who were there v encouraging me at every step. To my close girlfriends, Amanda, Jessica, Mariruth, Mariam, Michelle, Stephanie, Katie, Heather and Stephanie, who were there to listen to every story and offer advice, an escape, a shoulder to cry on, or a glass of wine. To Eric, who has proved to be a wonderful distraction in the midst of what have been the most trying months of graduate school. I am blessed to call him my own. To Jennifer, who was one of the reasons I stayed, who was the solution to my FACS crisis, who was the one to practice my dissertation talk with, who was the shoulder to cry on, who was the one cheering when I succeeded, and who was the one has encouraged me at every point. For her, I am truly grateful. Last, but certainly not least, I would like to thank Mark. Scientifically, he has afforded me the luxury of working in a laboratory outfitted with the best resources to perform my experiments. The colleagues he has surrounded himself with and the collaborations he has engaged in have furthered my scientific development and permitted my project to move beyond what was capable in my hands alone. He has given me the opportunity to pursue a project, that while difficult, has been intellectually rewarding and forced me to ‘blaze a trail.’ He has provided constructive criticism and encouraged me along the way, which is evident by the numerous fortunes and quotes that are displayed on my desk. While he once told me, “You won’t get a Ph.D. if you listen to me every day,” I must admit that it has been by his example, guidance, criticism, and encouragement that I have accomplished what I have. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ..................................................................................................... iv LIST OF TABLES ................................................................................................................... xi LIST OF FIGURES ................................................................................................................. xii LIST OF ABBREVIATIONS ...............................................................................................