University of Cincinnati March 2, 2016 Investigating the roles of Neurogenin 3 in human pancreas and intestine development and disease. A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Program in Molecular and Developmental Biology of the College of Medicine by Patrick Sean McGrath Bachelor of Science, Colorado State University, May 2009 Committee Chair: James M. Wells, Ph.D. Brian Gebelein, Ph.D. Michael A. Helmrath, MD, MS Stacey S. Huppert, Ph.D. Aaron M. Zorn, Ph.D. Summary The incidence of diabetes mellitus, a disease defined by the inability to properly regulate glucose, is increasing at an alarming rate and affects an estimated 26 million people in the U.S. creating an economic cost of $245 billion per year (American Diabetes, 2013). Glucose homeostasis is regulated through a complex interplay of hormones produced by both the pancreatic and intestinal endocrine systems. Neurogenin 3 is a basic helix-loop-helix (bHLH) transcription factor which acts as a “master regulator” of endocrine cell specification. Mouse studies have shown Neurog3 to be necessary and sufficient for the formation of all endocrine lineages in both the pancreas and intestine. Human patients harboring NEUROG3 mutations have a complete loss of intestinal endocrine cells, but surprisingly retain at least some endocrine pancreas function, calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly, we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. Directed differentiation of NEUROG3-/- hESC lines efficiently formed pancreatic progenitors, but lacked detectible NEUROG3 protein and did not form any endocrine cells in vitro. Moreover, NEUROG3-/- hESC lines were unable to form mature pancreatic endocrine cells following engraftment of PDX1+/NKX6.1+ pancreatic progenitors into mice confirming that NEUROG3 is required for human pancreatic endocrine development. Similarly, we found that NEUROG3 is essential for endocrine specification in PSC derived human intestinal organoids (HIOs). The NEUROG3-/- hPSC line was then used as a null background in which we could ectopically express NEUROG3 using an rtTA inducible lentiviral vector. Furthermore, NEUROG3 was also mutated to include the patient mutations R107S, L135P, or E28X. NEUROG3R107S, but not NEUROG3L135P, was functional and induced endocrine cell ii formation in pancreatic precursors but at significantly reduced levels compared to wild type NEUROG3. None of the NEUROG3 mutants were able to rescue endocrine formation in HIOs, perfectly mimicking the human phenotype. Furthermore, we showed NEUROG3R107S has a significantly shorter half-life which may contribute to its reduced function. Lastly, we utilized a NEUROG3ERT2 construct to identify NEUROG3 direct and indirect target genes in differentiated human pancreas. In summary, these studies help define the requirement for NEUROG3 function in human pancreatic and intestinal endocrine development. More broadly, the methods utilized here are a robust approach by which we can interrogate human development or disease entirely in vitro. iii iv Table of Contents Chapter 1. Introduction .............................................................................................................. 6 Introduction ....................................................................................................................... 7 In vivo development of pancreas and β cells ........................................................ 7 Directed differentiation of human pluripotent stem cells into pancreas ................. 8 The function of Neurogenin 3 in endocrine specification ....................................... 9 Mouse versus human requirement for Neurog3 .................................................. 11 Gene editing and disease modeling in PSC derived tissues ............................... 13 References ...................................................................................................................... 17 Figure Legends ............................................................................................................... 22 Figures ............................................................................................................................ 23 Chapter 2. The basic helix-loop-helix transcription factor NEUROG3 is required for development of the human endocrine pancreas ................................................................... 25 Summary ......................................................................................................................... 26 Introduction ..................................................................................................................... 27 Methods .......................................................................................................................... 28 Results ............................................................................................................................ 31 Discussion ....................................................................................................................... 37 References ...................................................................................................................... 40 Figure Legends ............................................................................................................... 44 Tables ............................................................................................................................. 51 Figures ............................................................................................................................ 53 Chapter 3. Functional characterization and disease modeling of NEUROGENIN 3 in human pluripotent stem cell-derived pancreas and intestinal organoids ........................... 61 Summary ......................................................................................................................... 62 Introduction ..................................................................................................................... 64 Materials and Methods .................................................................................................... 66 Results ............................................................................................................................ 72 Discussion ....................................................................................................................... 81 References ...................................................................................................................... 86 Figure Legends ............................................................................................................... 89 Tables ............................................................................................................................. 96 Figures .......................................................................................................................... 105 Chapter 4. Discussion ............................................................................................................ 117 Major Findings ............................................................................................................... 118 Using hPSCs to study pancreatic endocrine development and the importance of studying NEUROG3 mutations in the correct context ............................................... 119 Modeling NEUROG3 mutations and some experimental limitations ............................. 121 Differential requirement for NEUROG3 in pancreatic versus intestinal endocrine development and mechanisms leading to tissue specific target genes and endocrine lineages ............................................................................ 126 Modeling human development and disease .................................................................. 133 References .................................................................................................................... 135 Figure Legends ............................................................................................................. 138 Figures .......................................................................................................................... 139 v CHAPTER 1 Introduction. Patrick S. McGrath1 and James M. Wells*1,2. 1Division of Developmental Biology, 2Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039 ***Portions of this chapter have been reproduced from (McGrath et al., 2015). 6 In vivo development of pancreas and β cells Embryonic development of pancreatic endocrine cells can be broadly subdivided into several steps including endoderm formation, organ specification, endocrine specification and maturation (Gittes, 2009; Murtaugh, 2007; Sinner et al., 2006; Zorn and Wells, 2009). The definitive endoderm is derived from a bipotential mesendoderm progenitor marked by Brachyury and Mixl1 (Pearce and Evans, 1999; Wilkinson et al., 1990). Specification into definitive endoderm is then marked by a number of transcription factors including Sox17, Foxa2, and Goosecoid (Blum et al., 1992; Hudson et al., 1997; Sasaki and Hogan, 1993). A series of signaling events then subdivides the definitive