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Hyperosmotic Stress Enzyme Signaling Modulates Oct4, Nanog, and Rex1 Expression and Induces Prioritized Differentiation of Murine Embryonic Stem Cells Jill A

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Hyperosmotic Stress Enzyme Signaling Modulates Oct4, Nanog, and Rex1 Expression and Induces Prioritized Differentiation of Murine Embryonic Stem Cells Jill A Wayne State University Wayne State University Dissertations 1-1-2012 Hyperosmotic Stress Enzyme Signaling Modulates Oct4, Nanog, And Rex1 Expression And Induces Prioritized Differentiation Of Murine Embryonic Stem Cells Jill A. Slater Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Cell Biology Commons, and the Developmental Biology Commons Recommended Citation Slater, Jill A., "Hyperosmotic Stress Enzyme Signaling Modulates Oct4, Nanog, And Rex1 Expression And Induces Prioritized Differentiation Of Murine Embryonic Stem Cells" (2012). Wayne State University Dissertations. Paper 700. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. HYPEROSMOTIC STRESS ENZYME SIGNALING MODULATES OCT4, NANOG, AND REX1 EXPRESSION AND INDUCES PRIORITIZED DIFFERENTIATION OF MURINE EMBRYONIC STEM CELLS by JILL SLATER DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2013 MAJOR: PHYSIOLOGY Approved by: ______________________________ Advisor Date ______________________________ ______________________________ ______________________________ ______________________________ © COPYRIGHT BY JILL SLATER 2013 All Rights Reserved DEDICATION This dissertation is dedicated to my husband, Bruce, and my adult children, Christy, Andrew, and Colin: I thank you for allowing me the freedom to pursue this goal, even though it meant fending for yourselves for meals, wading through papers piled high in every room, interrupting family times for trips to the lab, and living with a distracted wife/mother. I love you all very much, and so look forward to re-joining your lives. I also dedicate this work of my hands and mind to God. It has been a privilege to watch His work appear before my eyes. May He indeed confirm the work of my hands. “Let Your work appear to Your servants And Your majesty to their children. Let the favor of the Lord our God be upon us; And confirm for us the work of our hands; Yes, confirm the work of our hands.” Psalm 90:16-17 ii ACKNOWLEDGEMENTS My mentor and advisor: Dr. Dan Rappolee, Thank you for giving me a research home and for teaching me how to think about science. My long-suffering thesis committee: Dr. Jose Cibelli, Dr. Don DeGracia, Dr. Joe Dunbar, Dr. Todd Leff, Dr. Jerry Sanders, Dr. Assia Shisheva: Your counsel, insight, and friendship was invaluable to me and will long be warmly remembered. My colleagues and friends from the Rappolee Lab: Dr. Yufen Xie, Dr. Sichang Zhou, Dr. Awoniyi Awonuga, Dr. George Proteasa, Simona Proteasa: It was a privilege to work with each one of you! My colleagues and friends from the C.S. Mott Center, who graciously shared their time, expertise, and equipment: Dr. Husam Abu-Soud, Dr. Ghassan Saed, Dr. Steve Krawetz, Dr. Randy Armand, Susan Clark, Leona Culpepper, Nicole Kelly, Dr. Dhiman Maitra: Many thanks! My statistician colleagues: Michael Kruger, C.S. Mott Center, Dave Childers, University of Michigan So glad your brains work that way! My colleagues, mentors, and friends from the Physiology Department: Dr. Jeff Ram – a true mentor and friend, Drs. Doug Yingst, Patrick Mueller, Robert Lasley, Steve DiCarlo, Heidi Lujan, Debra Skafar: Your support helped me through some difficult days. Thank you. Christine Cupps: would that everyone had an advocate like you. Without your gracious assistance, listening ear, and the occasional kick-in-the-pants, this would never have become a reality. “Thank you” is inadequate recognition for all you do on behalf of graduate students. Wayne State University School of Medicine: Your financial support was greatly iii appreciated. My fellow graduate students, many of whom beat me to the finish line: Dr. Sijana Dzinic, Dr. Tim McFarland, Dr. Monique Lewis, Jannifer Tyrrell, Ruth Watts: Your support is remembered with genuine fondness. My extended family and friends, who have been neglected far too long! iv TABLE OF CONTENTS Dedication ii Acknowledgments iii List of Tables viii List of Figures ix List of Abbreviations xi Chapter 1 Background and Introduction 1 Early mouse development 2 Preimplantation 2 Peri-implantation 3 Gastrulation 9 In vitro models of early murine development 12 Mouse trophoblast and embryonic stem cells 12 Embryoid bodies 13 Osmoregulatory mechanisms of the murine embryo 15 Hyperosmolarity as a stressor 16 Hyperosmotic stress effects on stem cells of early embryos 18 Hyperosmotic stress activates MAPK and PI3K signaling pathways 20 MAPK and PI3K signaling during early murine development 20 JNK (MAPK8) 21 JNK knockout 22 Double knockout of JNK family members 23 JNK during murine preimplantation 23 JNK in mESC 23 Pharmacological inhibition of JNK 24 p38 MAPK (MAPK11-14) 25 v p38 knockout 26 p38 during murine preimplantation 26 p38 in mESC 26 Pharmacological inhibition of p38 27 Extracellular signal regulated kinase (ERK1/2) MAPK 27 ERK/MEK knockout 28 ERK during murine preimplantation 28 ERK in mESC 29 Pharmacological inhibition of ERK 30 Introduction - phosphatidylinositol 3-kinase (PI3K) 31 PI3K knockout 31 PI3K during murine preimplantation 32 PI3K in mESC 32 Pharmacological inhibition of PI3K 33 MAPK and PI3K signaling in extraembryonic lineages of gastrulation 34 MAPK and PI3K signaling leading to development of the three germ layers 36 Summary of stress enzymes and development 39 Conclusion 39 Chapter 2 Stress Enzyme Activation Primes Murine Embryonic Stem Cells to Differentiate toward Extraembryonic Lineages 41 Abstract 41 Introduction 42 Materials and Methods 44 Results 49 Discussion 66 Chapter 3 Hyperosmotic Stress Induces the Early-Developing Primitive Endoderm and Suppresses the Later-Developing Mesoderm in Murine Embryoid Bodies 71 vi Abstract 71 Introduction 71 Materials and Methods 73 Results 76 Discussion 88 Chapter 4 Conclusions and Future Directions 94 Appendix A 99 References ` 105 Abstract 133 Autobiographical Statement 136 vii LIST OF TABLES Table 1. Protein markers of early embryonic lineages 4 Table 2. Total cell numbers in the embryonic germ layers 8 Table 3. Mean duration of cell cycle 9 Table 4. MAPK and PI3K signaling during murine preimplantation development 24 Table 5. MAPK, PI3K involvement in normal development of extraembryonic lineages 35 Table 6. MAPK, PI3K involvement in normal development of germ layer lineages 37 Table 7. Inhibitor only effects on OCT4, Nanog, and REX1 expression 46 Table 8. RT-PCR primer sequences 48 Table 9. Doubling rate of mESC in monolayer culture 51 Table 10. Transcription factor expression levels during 4h of hyperosmotic stress +/- enzyme inhibition in mESC monolayer culture 58 Table 11. Transcription factor expression levels during 24h of hyperosmotic stress +/- enzyme inhibition in mESC monolayer culture 61 Table 12. Summary of enzyme effects on OCT4, NANOG, and REX1 following hyperosmotic stress in mESC monolayer culture 66 Table 13. Primer sequences for qPCR of second study 76 Table 14. Diameter of embryoid bodies grown for 7 days under varying culture conditions 79 Table 15. In vivo induction of various lineage markers 80 viii LIST OF FIGURES Figure 1. Segregation of the first three unique lineages of the mouse blastocyst 2 Figure 2. Tissue types in the embryo at five developmental stages 6 Figure 3. Schematic of the differentiation steps from the morula stage through gastrulation 11 Figure 4. Embryoid bodies resemble peri-implantation embryos 13 Figure 5. Schematic of the hanging drop culturing technique utilized to develop embryoid bodies 14 Figure 6. Adaptive responses of somatic cells to hyperosmotic stress 17 Figure 7. Summary of MAPK signaling during early murine development 38 Figure 8. Hyperosmotic stress effects on cell proliferation in mESC 50 Figure 9. Hyperosmotic stress effects on apoptosis in mESC 51 Figure 10. Activation of p38, JNK, MEK1/2, PI3K by hyperosmolarity 52 Figure 11. Efficacy of enzyme inhibitors 53 Figure 12. Hyperosmotic stress in mESC mediates loss of OCT4, SOX2, NANOG, and REX1 protein 54 Figure 13. Hyperosmotic stress in mESC mediates loss of Oct4, Nanog, and Rex1 mRNA transcripts 55 Figure 14. MG132 effects on OCT4, Nanog, REX1 during sorbitol stimulation of mESC 56 Figure 15. Lactacystin effects on OCT4, Nanog, REX1 during sorbitol stimulation of mESC 57 Figure 16. Enzymes activated during 4h of hyperosmotic stress initiate the differentiation program in mESC 59 Figure 17. Nanog expression in mESC maintained following 72h of stimulation with 200mM sorbitol 60 Figure 18. p38 rescues pluripotency of mESC during 24h of hyperosmotic stress 62 Figure 19. PI3K, JNK and MEK1/2 target continue to modulate transcription factor expression during 24h hyperosmotic stress 63 ix Figure 20. REX1 reversal following withdrawal of sorbitol stimulation 64 Figure 21. Hyperosmotic stress primes mESC toward primitive endoderm 65 Figure 22. 7d culture of embryoid bodies in various culture conditions 77 Figure 23. JNK and p38 rescue aggregation of EBs during hyperosmotic stress 78 Figure 24. JNK/SAPK activation in mESC during 24h of exposure to sorbitol (10mM) 79 Figure 25. Fold change in lineage-specific expression due to hyperosmotic stress in EBs 81 Figure 26. Visceral endoderm markers during hyperosmotic stress in EBs 86 Figure 27. EBs grown in the presence of MAPK inhibitors 87 Figure 28. MEK1
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