Nfatc1 in Cardiac Valve Development and EPDC Invasion
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University of Cincinnati Date: 11/30/2010 I, Michelle D Combs , hereby submit this original work as part of the requirements for the degree of Doctor of Philosophy in Developmental Biology. It is entitled: NFATc1 in cardiac valve development and EPDC invasion Student's name: Michelle D Combs This work and its defense approved by: Committee chair: Katherine Yutzey Committee member: Walter Keith Jones Committee member: Jeff Molkentin Committee member: James Wells Committee member: Yi Zheng 1317 Last Printed:1/13/2011 Document Of Defense Form NFATc1 in cardiac valve development and EPDC invasion A dissertation submitted to the Division of Graduate Studies and Research 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 2010 by Michelle D. Combs Bachelor of Science, Quincy University, 1996 Committee Chair: Katherine E. Yutzey, Ph.D. W. Keith Jones, Ph.D. Jeffery D. Molkentin, Ph.D. James M. Wells, Ph.D. Yi Zheng, Ph.D. Abstract Congenital malformations are the most common cause of death in infancy in the United States. Of these birth defects, most are malformations of cardiac valvuloseptal structures and a significant number are coronary vessel malformations. Therefore, identifying molecular mechanisms that regulate cardiac valve and coronary artery development is of great clinical importance. Cardiac valve morphogenesis begins with growth of endocardial cushions in the atrioventricular canal and outflow tract regions of the looping heart. After growth, endocardial cushions are remodeled into thin leaflets, characteristic of mature heart valves. Nuclear Factor of Activated T-cells cytoplasmic 1 (NFATc1) is a transcription factor necessary for heart valve development. The studies detailed here demonstrate that Vascular Endothelial Growth Factor A (VEGF)/ NFATc1 pathway function promotes endocardial cushion growth, while Receptor Activator of NFκB (RANKL)/ NFATc1 pathway function is associated with valve remodeling. These studies further demonstrate that NFATc1 serves as a nodal point in the transition from endocardial cushion growth to valve remodeling via ERK1/2 or JNK1/2 copathway activation. In the course of these studies NFATc1 expression by PE, epicardium, and EPDCs was discovered. During heart looping, PE cells migrate onto the myocardium and proliferate to form the epicardium. A subset of epicardial cells undergo epithelial-to- mesenchymal transformation (EMT) and invade the subepicardium and myocardium as epicardium-derived cells (EPDCs). EPDCs differentiate into coronary endothelial and ii smooth muscle cells, as well as adventitial fibroblasts that produce the fibrous matrix. Studies detailed in this dissertation demonstrate that conditional loss of NFATc1 expression in EPDCs in mice causes embryonic death by E18.5 with reduced coronary vessel and fibrous matrix penetration into myocardium. These studies further demonstrate that RANKL/NFATc1 pathway components are expressed in EPDCs and loss of NFATc1 in EPDCs causes loss of CtsK expression in the myocardial interstitium in vivo. Likewise, RANKL treatment induces CtsK expression in PE-derived cell cultures via a calcineurin-dependent mechanism. In chicken embryo hearts, RANKL treatment increases the distance of EPDC invasion into myocardium, and this response is calcineurin-dependent. Together, these data demonstrate a critical role for the RANKL/ NFATc1 signaling pathway in promoting invasion of EPDCs into myocardium by induction of extracellular matrix-degrading enzyme gene expression. iii iv Table of Contents ABSTRACT ii TABLE OF CONTENTS 1 LIST OF FIGURES AND TABLES 6 CHAPTER 1. Introduction Overview 12 Overview of Heart Development 12 Valve Development 14 Coronary Vessel and Fibrous Matrix Development 19 NFATs 25 Calcineurin and NFATs in Heart Development 27 VEGF/ NFATc1 29 RANKL/ NFATc1 30 Experimental Rationale 33 References 36 Figure Legends 56 Figures 60 CHAPTER 2. VEGF and RANKL regulation of NFATc1 in heart valve development Abstract 68 Non-Standard Abbreviations and Acronyms 68 1 Introduction 70 Materials and Methods 73 Results 74 Discussion 85 Acknowledgements 88 Sources of Funding 88 Disclosures 88 References 89 Figure Legends 95 Figures 99 Supplementary Methods 107 References 114 Supplemental Figure Legends 116 Supplemental Figures 119 CHAPTER 3. NFATc1 promotes epicardium-derived cell (EPDC) invasion into myocardium Summary 125 Introduction 126 Materials and Methods 128 Results 135 Discussion 146 2 Acknowledgements 151 References 152 Figure Legends 160 Figures 165 Supplemental Figure Legends 172 Supplemental Figures 175 CHAPTER 4. Summary and Discussion Major Findings 182 NFATc1 in Cardiac Valve Maturation 182 NFATc1 in EPDC Invasion 184 Experimental Limitations and Alternative Approaches 186 Calcineurin Inhibitors 191 A Broad View of NFATc1-regulated Mechanisms 192 References 194 Figure Legend 201 Figures 202 APPENDIX I. Heart valve development: regulatory networks in development and disease Abstract 205 Introduction 206 3 Overview of Valve Development 206 Endocardial Cushion Formation and EMT 211 Growth of Endocardial Cushions and Valve Primordia 216 Diversification of Valve Cell Types 223 Heart Valve ECM Maturation and Organization 227 Heart Valve Development and Disease 230 Conclusions and Perspectives 232 Acknowledgements 233 Sources of Funding 234 Disclosures 234 References 235 Figure Legends 264 Tables and Figures 266 APPENDIX II. Transcriptional regulation of heart valve progenitor cells Abstract 273 Introduction 274 Overview of Endocardial Cushion Formation and Valve Remodeling 274 Transcriptional Regulation of Valve Development 277 Twist1 278 Tbx20 279 Msx1 and Msx2 281 4 NFATc1 282 Sox9 284 Scleraxis 286 Overall Conclusions and Future Perspectives 287 References 289 Tables 303 5 LIST OF FIGURES AND TABLES CHAPTER 1. Figure 1. Graphical overview of heart development 60 Figure 2. Stratified ECM compartments are evident in mature semilunar and atrioventricular valves 61 Figure 3. Schematic representation of embryonic AV valve development 62 Figure 4. Schematic overview of coronary vessel and fibrous matrix development 63 Figure 5. Illustration depicting the structure of human NFATc1, NFATc2, NFATc3, and NFATc4 mRNA transcripts 64 Figure 6. Model of VEGF/NFATc1 pathway signaling in human pulmonary valve endothelial cells 65 Figure 7. Model of RANKL/NFATc1 pathway signaling in osteoclasts 66 CHAPTER 2. Figure 1. E10.5 NFATc1-/- mouse ECC endothelial and mesenchymal cells exhibit decreased proliferation 99 Figure 2. VEGF treatment of ECC cells induces NFATc1 nuclear localization 100 Figure 3. VEGF-induced proliferation of ECC cells is dependent upon calcineurin signaling 101 Figure 4. NFATc1, RANKL, and CtsK mRNA expression in developing chick AVC 102 6 Figure 5. RANKL treatment of ECC cells induces NFATc1 nuclear localization and increased CtsK expression via a Cn-dependent mechanism 103 Figure 6. Ligand-specific effects on ECC cell proliferation and CtsK expression. RANKL inhibits VEGF-induced ECC cell proliferation 104 Figure 7. JNK1/2 activation is not seen in E11.5 mouse ECCs, but is detected in E12.5 mitral and tricuspid valve endothelial cells in vivo 105 Figure 8. VEGF-induced ECC cell proliferation is MEK1-ERK1/2-dependent. RANKL-induced CtsK expression and RANKL inhibition of VEGF-induced cell proliferation is JNK1/2 dependent 106 Supplemental Figure 1. NFATc1-positive cells co-express endothelial markers, and VEGF induces proliferation of endothelial cells in cultured ECCs. 119 Supplemental Figure 2. Expression of endothelial and mesenchymal markers by cultured ECC cells over time mimics gene expression observed in maturing ECCs/ mitral valves in vivo 120 Supplemental Figure 3. OPG does not inhibit VEGF-induced cell proliferation and sFlt1 does not inhibit RANKL-induced CtsK expression 121 Supplemental Figure 4. Percent diphosphorylated ERK1/2 positive cells is significantly reduced in cultures treated with U0126 compared to controls 122 Supplemental Figure 5. Model of NFATc1 function in the transition from ECC growth to valve remodeling 123 7 CHAPTER 3. Figure 1. NFATc1 is expressed by the PE and coronary vessels and colocalizes with WT1 in the epicardium and EPDCs 165 Figure 2. WT1-Cre(+);NFATc1(fl/fl) embryos have reduced NFATc1 positive epicardial cells and EPDCs in addition to increased myocardial compaction 166 Figure 3. WT1-Cre(+);NFATc1(fl/fl) embryos lack interstitial fibrous matrix and have reduced penetration of Collagen1a1-expressing cells 167 Figure 4. WT1-Cre(+);NFATc1(fl/fl) embryos have reduced investment of activated fibroblasts, and reduced intramyocardial vessel penetration 168 Figure 5. RANKL/NFATc1 pathway components are expressed in mouse and chick embryos during EPDC invasion 169 Figure 6. WT1-Cre(+);NFATc1(fl/fl) embryos have reduced CtsK expression in the myocardial interstitium 170 Figure 7. RANKL increases EPDC migration distance and CtsK expression via a calcineurin/ NFAT-dependent mechanism 171 Figure S1. NFATc1 is expressed by PE, epicardium, EPDCs, and coronary vessels in chick demonstrating a conserved expression pattern in vertebrates 175 Figure S2. NFATc1 mRNA is expressed in epicardial cells and EPDCs of chick and mouse 176 Figure S3. An intact epicardium is apparent with systemic loss of NFATc1 177 Figure S4. Control and WT1-Cre(+);NFATc1(fl/fl) embryos are grossly indistinguishable until E17.5 when WT1-Cre(+);NFATc1(fl/fl) hearts show 8 signs of