Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2012 Promoting Thymopoiesis with Age: Potential Role of the Transcription Factor Foxn1 Erin Christine Zook Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Immunology and Infectious Disease Commons Recommended Citation Zook, Erin Christine, "Promoting Thymopoiesis with Age: Potential Role of the Transcription Factor Foxn1" (2012). Dissertations. 317. https://ecommons.luc.edu/luc_diss/317 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2012 Erin Christine Zook LOYOLA UNIVERSITY CHICAGO PROMOTING THYMOPOIESIS WITH AGE: POTENTIAL ROLE OF THE TRANSCRIPTION FACTOR FOXN1 A DISERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN CELL BIOLOGY, NEUROBIOLOGY, AND ANATOMY BY ERIN C. ZOOK CHICAGO, IL MAY 2012 Copyright by Erin C. Zook, 2012 All rights reserved ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Phong Le for his mentorship and guidance. His encouragement influenced my decision to apply to graduate school, and his support and patience helped me completed the program. I would also like to thank my committee members, Drs Avinash Bhandoola, Makio Iwashima, Toni Pak, John McNulty, and Pamela Witte for their support, time, and conversations. Also, thank you to Patricia Simms for her friendship and countless hours of cell sorting. Thank you to the past and present members of the Le and Witte laboratories. I am grateful for their friendships and humorous conversations. They made days in the laboratory fun and unpredictable. Finally, I would like to thank my family and friends. Without their support and understanding I would not have been able to achieve this goal. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………… iii LIST OF FIGURES……………………………………………………………….. vii LIST OF TABLES………………………………………………………………..... xii LIST OF ABBREVIATIONS……………………………………………………… xiii CHAPTER I: STATEMENT OF PROBLEM…………………………….………. 1 CHAPTER II: REVIEW OF THE LITERATURE…………..………………….… 4 Introduction to the aged immune system…………………………………... 4 The thymus…………………………………………………………………. 5 The discovery of the thymus as an organ that produces T cells.......… 5 Cellular and structural composition of the thymus………………...… 7 Thymopoiesis……..……………………………………….…………. 13 Thymic organogenesis………….……………………….…………… 22 Age associated thymic involution……...…………………………………... 25 Thymic structure and aging…………………...………….……....….. 26 Aging and thymopoiesis…………...…………….……...………..….. 28 Peripheral effects of thymic involution………………………...……. 29 HSC and hematopoiesis…………………………….……………………… 31 Embryonic origins of HSC……………….……………….……..…... 32 BM HSC niche……………………………………………….………. 35 Development of lymphoid and myeloid progenitors from HSC…..… 43 Development of T cell progenitors in the BM……………………….. 45 Cytokine and Transcriptional Regulation of the Development of Myeloid and Lymphoid Progenitors from HSC…………………... 50 Hematopoiesis and aging……………...…………………………………… 55 HSC and aging…………..….…………………………....................... 55 Aging and the HSC niche….........……………...……..……………... 60 Foxn1 and nude mice………………………………………………………. 60 CHAPTER III: OVER EXPRESSION OF FOXN1 ATTUNATES THYMIC INVOLUTION………………………………………………………..…………. 64 Introduction………………………………………..……………….……… 64 Results…..……………………………………………..………….……….. 65 iv Summary…..………………………………………..……………..………. 99 CHAPTER IV: OVER EXPRESION OF FOXN1 PROMOTES THE GENERATION OF T CELL PROGENITORS IN THE BM……..…………….. 102 Introduction…….…………………………………………...……….…….. 102 Results…..………………………………………………………………….. 103 Summary.…………………………………………………………………... 146 CHAPTER V: B CELL AND MYELOID PROGENITORS IN FOXN1TG BM..........…………………………………………........…………… 147 Introduction….…………………..………………………….……………… 147 Results….…………………………………...………….……………...…… 148 Summary….………………………………….…………………....……..… 161 CHAPTER VI: FOXN1 EXPRESSION IN THE BM…………………….………. 166 Introduction…………………………………………………..…….……… 166 Results…..…………………………………………..……….……..……… 167 Summary…..…………………………………………….……………..….. 183 CHAPTER VII: DISSCUSSION…………...……………………………….…..… 187 Foxn1 and the thymus…………………………………………………...… 188 Regulation of Foxn1 in the thymus……..…………………....……. 188 A role for Foxn1 in thymopoiesis with age……………....………... 190 A role for Foxn1 in maintaining thymic architecture with age…..... 194 Foxn1 and the bone marrow……………………………..…………....…… 197 The effect of Foxn1 on HSC………..………………..…………..… 197 A role for Foxn1 in hematopoiesis…………….…………………... 207 A role for Foxn1 in the development of CTP and CIP…...……….. 207 Expression and regulation of Foxn1 in the bone marrow…….….... 210 Concluding remarks……………………………………….......................... 215 Putting it all together…………...…………………….…….…….... 215 Significance………………………………………........................... 216 CHAPTER VIII: MATERIALS AND METHODS………...……………….……. 220 Wt mice, H2-VEX mice, Foxn1cre-LacZ mice……………………….…... 220 Foxn1 transgenic mice (Foxn1Tg)………………………………………... 220 Isolation of thymocytes from thymic stroma…………..………………..… 221 Isolation of BM cells……………..…………………………………..……. 221 Digestion of thymus and isolation of thymic epithelial cells…….....……... 224 Separation of BM cells using percoll……………………..……...… …...… 225 Quantification of Foxn1 mRNA levels in thymic stroma and BM………… 225 Quantitative RT-PCR analysis of Foxn1 in different BM populations…..... 226 Quantification of HSC, MPP, CLP, ETP, CTP and CIP frequency and total cell number ………………………………………..……………….. 228 Electronically sorting of ETP………………………….…………………... 228 v Electronic Isolation of HSC, MPP, CTP and CIP………..………...………. 230 Cell cycle analysis…………………………………………….…… ……... 230 Flow cytometers and antibodies……………..……………...………………232 OP9 cell cultures……………………….………………..…………………. 232 Colony forming assay methylcellulose cultures……..…………………….. 232 Non-irradiated adaptive transfers…………………………………………... 233 Irradiated adaptive transfers……………………………………................... 233 Immunofluorescence and hematoxylin and eosin (H&E) staining of thymi…………………………………………………………………....... 234 Immunohistochemistry of sternums for Foxn1……………...……... ….….. 234 Identification of Foxn1pos Cells within the Sorted Linneg/low EpCAMpos BM cells ………………………………………………............................ 235 Statistical analysis………………………………………………....…….… 236 REFERENCES………………………………..……………….…………………... 237 VITA………………………………………………..……………… …....... ………271 vi LIST OF FIGURES Figure 1. Thymus structure and localization of thymocyte populations.…..… 10 Figure 2. Development of TEC and TEC subsets.…………………… ……... 12 Figure 3. Cell surface phenotype of developing thymocytes…...……. ……... 14 Figure 4. TCR Vα chain rearrangement and generation of sjTRECs………… 18 Figure 5. Check points of important stages of T cell development………...… 19 Figure 6. Endosteal and vascular HSC niches………………………............... 36 Figure 7. Hematopoiesis in mouse BM.……………………………… ……... 44 Figure 8. Simplified diagram of hematopoiesis with CTP and CIP….. ……... 47 Figure 9. Cytokine and transcriptional regulation of hematopoiesis.... ……... 56 Figure 10. Changes in the BM and thymic populations with age………...….... 58 Figure 11. Design and specificity of primer to the Foxn1 transgene….. ……... 67 Figure 12. Endogenous and transgenic Foxn1 expression in thymic stroma….. 69 Figure 13. Foxn1 expression in Wt and Foxn1Tg thymi……………… …….. 70 Figure 14. Distribution of thymocyte populations with age in Wt and Foxn1Tg………………………………………………………….. 72 Figure 15. Thymocyte number in Wt and Foxn1Tg with age…………. …….. 74 Figure 16. Gross morphology of aged Wt and Foxn1Tg thymi……….. …….. 76 Figure 17. Flow cytometry identification of ETP……………………....……... 77 Figure 18. ETP frequency and number in young and aged Wt and Foxn1Tg.… 78 Figure 19. Flow cytometry analysis of ETP cultures on OP9-DL1(GFP)…..… 81 vii Figure 20. ETP commitment to T lineage and differentiation………… …….. 82 Figure 21. Recombination of the VEX construct in H2-SVEX cells…. …….. 84 Figure 22. Identification of donor CD45.1pos VEXpos ETP in aged Wt and Foxn1Tg hosts…………………………………………………… 86 Figure 23. Frequency of donor ETP and percent of donor ETP that express VEX in aged Wt and Foxn1Tg hosts………………………….… 87 Figure 24. Gross thymic morphology and histology of old Wt and Foxn1Tg mice…………… ………………………………………………… 88 Figure 25. Keratin 8 and keratin 5 staining of Wt and Foxn1Tg thymi . ……... 91 Figure 26. Identification of TEC subsets by flow cytometry………………….. 93 Figure 27. Number of cTEC in young and aged Wt and Foxn1Tg……. ……... 94 Figure 28. Number of MHCIIpos mTEC in young and aged Wt and Foxn1Tg... 95 Figure 29. Number of MHCIIhi mTEC in young and aged Wt and Foxn1Tg..... 96 Figure 30. Frequency of Ki67pos MHCIIhi mTEC in young and aged Wt and Foxn1Tg ………………………………………………………… 97 Figure 31. Identification of HSC and MPP….…………………………….….. 105 Figure 32. Frequency and number of MPP in young and aged Wt and Foxn1Tg………………………………………………………….. 106 Figure 33. Frequency and number of HSC in young and aged Wt and Foxn1Tg. ……………………………………………………….... 108 Figure 34. Frequency of LSK cells within the Linneg population of Wt and Foxn1Tg mice with age…………….……………………. ……... 111 Figure 35. Frequency and number of LSK in young and