HOX6 FUNCTION IS NECESSARY FOR ENDOCRINE PANCREAS DEVELOPMENT IN VIVO AND IN VITRO by Brian Matthew Larsen A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cellular and Molecular Biology) in the University of Michigan 2015 Doctoral Committee: Associate Professor Deneen M. Wellik, Chair Assistant Professor Benjamin L. Allen Professor Gregory R. Dressler Assistant Professor Marina Pasca Di Magliano Assistant Professor Jason R. Spence © Brian Matthew Larsen 2015 To my family for all of their support throughout my life and through school and to my fiancé Nicole for all of her encouragement, support, and advice. Thank you. ii TABLE OF CONTENTS DEDICATION ..................................................................................................... ii LIST OF FIGURES .............................................................................................. v LIST OF TABLES ............................................................................................... vii ABSTRACT ...................................................................................................... viii CHAPTER 1: INTRODUCTION .......................................................................... 1 Hox genes ..................................................................................................... 1 Pancreas development in vivo ...................................................................... 5 The role of the mesenchyme in pancreas development .............................. 11 Signaling in pancreas development ............................................................. 12 Differentiation of ESCs to insulin-producing cells in vitro ............................ 16 Unanswered questions in the field ............................................................... 24 CHAPTER 2: MESENCHYMAL HOX6 FUNCTION IS REQUIRED FOR PANCREATIC ENDOCRINE CELL DIFFERENTIATION ................................. 26 Summary ...................................................................................................... 26 Introduction ................................................................................................... 28 Results ......................................................................................................... 30 Discussion .................................................................................................... 38 Materials and Methods ................................................................................. 40 Acknowledgments ........................................................................................ 43 Figures ......................................................................................................... 45 iii CHAPTER 3: HOX6 FUNCTION IN THE IN VITRO DIFFERENTIATION OF MESCS TO INSULIN-PRODUCING CELLS ..................................................... 68 Summary ...................................................................................................... 68 Introduction ................................................................................................... 69 Results ......................................................................................................... 71 Discussion .................................................................................................... 76 Materials and Methods ................................................................................. 77 Acknowledgments ........................................................................................ 79 Figures ......................................................................................................... 80 CHAPTER 4: CONCLUSION ............................................................................ 93 Summary of Findings ................................................................................... 93 Contributions to the field ............................................................................... 96 Future directions ........................................................................................... 97 APPENDIX: PUBLICATIONS AND MANUSCRIPTS ...................................... 105 REFERENCES ................................................................................................ 106 iv LIST OF FIGURES Figure 1.1. Homeotic transformations in Hox6 and Hox10 mutant mice. ....... 3 Figure 1.2. Model of early pancreas development. ......................................... 6 Figure 1.3. Pancreas duct development. ......................................................... 8 Figure 1.4. Strategies for β-cell generation in vitro. ....................................... 20 Figure 2.1. Hox6 mutant embryos are externally indistinguishable from controls ........................................................................................ 45 Figure 2.2. Loss of Hox6 function results in abnormal pancreas morphology, but normal amylase expression ............................. 46 Figure 2.3. Hox6 mutant pancreata have slightly reduced proliferation, but no overall changes in volume ............................................... 47 Figure 2.4. Hox6 mutant pancreata demonstrate a drastic reduction of endocrine hormone expression .................................................. 49 Figure 2.5. Immature endocrine cell genes are decreased in the mutant but there is no defect in endocrine progenitor delamination ...... 50 Figure 2.6. Ngn3 is unchanged at E18.5 ....................................................... 51 Figure 2.7. Hox6 expression is limited to the pancreas mesenchyme .......... 52 Figure 2.8. Hox6 gene expression is restricted to the mesenchyme ............ 54 Figure 2.9. There is no change in vascularization in the Hox6 null pancreas ..................................................................................... 56 Figure 2.10. Expression of Wnt5a and Wnt inhibitors Sfrp3 and Dkk1 is lost in the Hox6 mutant pancreas .............................................. 57 Figure 2.11. Expression of Wnt2b, Wnt4, Wnt7b, and Wnt11 appears similar between control and Hox6 mutant pancreata ................ 58 v Figure 2.12. Expression Wnt inhibitors Sfrp3 and Dkk1 is lost in endocrine progenitors in the Hox6 mutant pancreas ................ 60 Figure 2.13. Wnt inhibitor expression is reduced in the Hox6 mutant pancreas .................................................................................... 62 Figure 2.14. Exogenous Wnt5a treatment rescues endocrine cell differentiation in Hox6 mutant pancreas .................................... 63 Figure 2.15. Hox6 regulates mesenchymal Wnt5a expression to promote endocrine cell differentiation ...................................................... 64 Figure 3.1. Differentiation protocol. .............................................................. 80 Figure 3.2. mESCs differentiate to endoderm. ............................................. 81 Figure 3.3. Both exocrine and endocrine pancreas cells are represented in in vitro differentiation. ............................................................ 83 Figure 3.4. qRT-PCR analyses for genes primarily expressed in other foregut derived organs ............................................................... 85 Figure 3.5. Hox genes are expressed and the Hox code is maintained in vitro ............................................................................................. 86 Figure 3.6. Hox6 mutant ESCs differentiate to endoderm and exocrine cells normally but exhibit lower Ins+ cell populations. ................ 87 Figure 3.7. BMP4 increases the amount of mesoderm early in differentiation but does not increase the amount of Ins expression overall. ..................................................................... 89 vi LIST OF TABLES Table 2.1. List of primary and secondary antibodies ..................................... 65 Table 2.2. List of qRT-PCR primers for embryonic tissue ............................. 66 Table 3.1. List of primary antibodies ............................................................. 91 Table 3.2. List of qRT-PCR primers for cultured cells ................................... 92 vii ABSTRACT Diabetes affects millions of Americans and is caused by a disruption in β- cell abundance or function. β-cells are one of the five types of endocrine cells found in the pancreas. A promising potential cellular therapy for the treatment of diabetes is the differentiation of human embryonic stem cells to insulin-producing β-cells. The study of endocrine pancreas development has been critical for the design of in vitro protocols for the differentiation of insulin-producing cells. The mesoderm is critical for the differentiation of endocrine cells and despite significant advances in our understanding of endocrine cell development, the function of the pancreatic mesodermal niche in this process is poorly understood. Fully elucidating the role of the mesoderm in endocrine cell development can greatly contribute to the design of protocols for the successful generation of functional β-cells in vitro. We have discovered a novel role for mesodermally expressed Hox6 genes in endocrine pancreas development. Inactivation of all three Hox6 paralogs leads to a dramatic loss of endocrine cells, including β-cells, as well as mild delays and disruptions in branching and exocrine cell differentiation. Loss of Hox6 function in the mesenchyme of the pancreas results in reduced expression of Wnt5a in the viii pancreatic mesenchyme.