Current Topics in Developmental Biology
Volume 60 Stem Cells in Development and Disease
Series Editor Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, PA 15213
Editorial Board Peter Gru¨ss Max-Planck-Institute of Biophysical Chemistry Go¨ttingen, Germany
Phillip Ingham University of Sheffield, United Kingdom
Mary Lou King University of Miami, Florida
Story C. Landis National Institutes of Health National Institute of Neurological Disorders and Stroke Bethesda, Maryland
David R. McClay Duke University, Durham, North Carolina
Yoshitaka Nagahama National Institute for Basic Biology, Okazaki, Japan
Susan Strome Indiana University, Bloomington, Indiana
Virginia Walbot Stanford University, Palo Alto, California
Founding Editors A. A. Moscona Alberto Monroy
Current Topics in Developmental Biology Volume 60 Stem Cells in Development and Disease Edited by
Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn-Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, PA 15213
Cover Photo Credit: LBs example of a-Syn Inclusions. Photo courtesy of Mark S. Forman, MD, PhD, from the Center of Neurodegenerative Disease Research at the University of Pennsylvania, Philadelphia. For more details see Chapter 2, Table 1. Elsevier Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK
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All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2004 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0070-2153/2004 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail:
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ISBN: 0-12-153160-0 PRINTED IN THE UNITED STATES OF AMERICA 04 05 06 07 08 9 8 7 6 5 4 3 2 1
Contents
Contributors Preface xi
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1 Therapeutic Cloning and Tissue Engineering Chester J. Koh and Anthony Atala I. II. III. IV. V.
Introduction 1 Principles of Tissue Engineering 2 Stem Cells 5 Therapeutic Cloning 6 Applications of Tissue Engineering and Therapeutic Cloning References 12
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2 -Synuclein: Normal Function and Role in Neurodegenerative Diseases Erin H. Norris, Benoit I. Giasson, and Virginia M.-Y. Lee I. II. III. IV. V. VI. VII. VIII. IX. X.
Introduction to the Synuclein Protein Family 18 Synuclein in Synaptic Development and Maintenance 20 Synuclein Protein Interactions 22 Synucleinopathies 23 Structural Properties of -Synuclein Protein 30 Implications of Oxidative and Nitrative Injury in Synucleinopathies Effects of Oxidation and Nitration on -Synuclein Fibril Formation Possible Role of Pesticides and Other Toxic Chemicals in PD 36 Transgenic and Nontransgenic Animal Models of Synucleinopathies Concluding Remarks 39 Acknowledgments 40 References 40
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3 Structure and Function of Eukaryotic DNA Methyltransferases Taiping Chen and En Li I. II. III. IV.
DNA Methylation: A Heritable Epigenetic Mark Eukaryotic DNA Methyltransferases 58 Function of DNA Methylation 72 Concluding Remarks 79 Acknowledgments 80 References 80
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4 Mechanical Signals as Regulators of Stem Cell Fate Bradley T. Estes, Jeffrey M. Gimble, and Farshid Guilak I. II. III. IV. V. VI.
Introduction 92 Mechanical Regulation of Limb Development 94 Theoretical Models of Mechanical Stress in Development In Vivo Experimentation 97 In Vitro Experimentation 103 Conclusions 121 Acknowledgment 121 References 121
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5 Origins of Mammalian Hematopoiesis: In Vivo Paradigms and In Vitro Models M. William Lensch and George Q. Daley I. II. III. IV. V. VI.
Introduction: The Hematopoietic System 128 Hematopoiesis: De Muribus et Maribus 130 Points of Divergence Between Murine and Human Blood Formation Hematopoiesis from Embryonic Stem Cells 161 What Remains To Be Learned? 171 Summary 174 Acknowledgments 174 References 175
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6 Regulation of Gene Activity and Repression: A Consideration of Unifying Themes Anne C. Ferguson-Smith, Shau-Ping Lin, and Neil Youngson I. Introduction 197 II. Regulation of Imprinted Gene Activity and Repression III. A Consideration of Recurrent Themes 207 References 210
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7 Molecular Basis for the Chloride Channel Activity of Cystic Fibrosis Transmembrane Conductance Regulator and the Consequences of Disease-Causing Mutations Jackie F. Kidd, Ilana Kogan, and Christine E. Bear I. Introduction 216 II. Role of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function in Tissue Physiology and Pathophysiology 218 III. Cell Biology of CFTR and the Major CF Mutant (F508) 220 IV. Quaternary Structure of CFTR 222 V. The Permeation Pore of CFTR 224 VI. Molecular Basis for Regulation by Phosphorylation 228 VII. Molecular Basis for Regulation by Nucleotides 230 VIII. Future Directions 239 Acknowledgments 241 References 241 Index 251 Contents of Previous Volumes
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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.
Anthony Atala (1), Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157 Christine E. Bear (215), Programme in Structural Biology and Biochemistry Research Institute, Hospital for Sick Children, Departments of Physiology and Biochemistry, University of Toronto, Toronto, Canada M5G 1X8 Taiping Chen (55), Cutaneous Biology Research Center and Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129 George Q. Daley (127), Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115; Division of Pediatric Hematology/Oncology, Children’s Hospital Boston, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 Bradley T. Estes (91), Department of Surgery, Division of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina 27710 Anne C. Ferguson-Smith (197), Department of Anatomy, University of Cambridge, Cambridge CB4 1AW, England Benoit I. Giasson (17), Center for Neurodegenerative Disease Research and, the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Jeffrey M. Gimble (91), Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808 Farshid Guilak (91), Department of Surgery, Division of Orthopaedic Surgery, Duke University Medical Center, Durham, North Carolina 27710 Jackie F. Kidd (215), Programme in Structural Biology and Biochemistry Research Institute, Hospital for Sick Children, Departments of Physiology and Biochemistry, University of Toronto, Toronto, Canada M5G 1X8 ix
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Ilana Kogan (215), Programme in Structural Biology and Biochemistry Research Institute, Hospital for Sick Children, Departments of Physiology and Biochemistry, University of Toronto, Toronto, Canada M5G 1X8 Chester J. Koh (1), Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston Salem, North Carolina 27157 Virginia M.-Y. Lee (17), Center for Neurodegenerative Disease Research and, the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 M. William Lensch (127), Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142; Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 En Li (55), Cutaneous Biology Research Center and Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129; Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139 Shau-Ping Lin (197), Department of Anatomy, University of Cambridge, Cambridge CB4 1AW, England Erin H. Norris (17), Center for Neurodegenerative Disease Research and, the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Neil Youngson (197), Department of Anatomy, University of Cambridge, Cambridge CB4 1AW, England
Preface This volume of Current Topics in Developmental Biology, entitled ‘‘Stem Cells in Development and Disease,’’ showcases important Wndings across regenerative and molecular medicine related to stem cells. This exceptional edition covers therapeutic cloning and tissue engineering, stem cell biomedicine, a protein implicated in a host of neurodegenerative diseases – which may well be the Wrst class of diseases to be cured using stem cells, the generation of hematopoeisis from embryonic stem cells, the inXuence of stem cell fate using mechanical stress, the function and regulation of the cystic Wbrosis gene, and provides two comprehensive examinations of imprinting and methylation, events in early embryonic development that are important to the success of cloning and transgenics. Therapeutic Cloning and Tissue Engineering by Chester Koh and Anthony Atala of Children’s Hospital Boston and Harvard emphasizes the shortage of suitable donor organs and the great need for continued advancement toward the creation of functional replacement organs and tissues. Assessing the current state of the art, the authors go on to elaborate on their own extraordinary, landmark work using both nuclear transfer and seeded scaVolds to produce genetically compatible muscle and renal tissue in Bos taurus. -Synuclein: Normal Function and Role in Neurodegenerative Diseases by Erin Norris, Benoit Giasson, and Virginia Lee of the University of Pennsylvania is a comprehensive primer on the newly-named class of diseases called ‘‘synucleinopathies.’’ A protein of the central nervous system, -synuclein seems to be a factor in Parkinson’s and related neurodegenerative diseases with similar Lewy body inclusions. While the exact role of synuclein in these diseases is not yet known, the authors’ summation of the Weld raises intriguing and important questions about the interplay of protein, genes, and environment in neurodegenerative disease etiology. In Structure and Function of Eukaryotic DNA Methyltransferases by Taiping Chen of Harvard and En Li of Harvard and Novartis, the authors review a host of model organisms, few of which exhibit the same methylation function, suggesting that DNA methylation is an evolutionary ‘‘device’’ that has achieved diVerent ends in diVerent beings. In mammals, methylation plays an important role in gene expression during development, including X-inactivation and maintenance of genomic integrity, perhaps most crucially in the short time span before embryo implantation. Mechanical Signals as Regulators of Stem Cell Fate by Bradley Estes of Duke and JeV Gimble and Farshid Guilak of Louisiana State University considers the role of physical stress on the diVerentiation of progenitor cells. xi
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This is important for understanding embryonic development, and because replication of these forces in vitro will contribute to the production of superior, engineered tissues for clinical applications. Mammalian Hematopoiesis: In Vivo Paradigms and In Vitro Models by William Lensch of the Whitehead Institute and the Massachusetts Institute of Technology and George Q. Daley of Harvard, is a comprehensive overview of human and murine hematopoietic development, of attempts to generate hematopoietic tissue from embryonic stem cells, and of questions remaining to be answered in this Weld of crucial, translatable research. In Regulation of Imprinted Gene Activity and Expression: A Consideration of Unifying Themes by Anne Ferguson-Smith, Shau-Ping Lin, and Neil Youngson of the University of Cambridge, the authors seek commonalities among diVerentially imprinted parental germlines, thereby shedding light on the evolution and function of this intriguing process of early development. Finally, Molecular Basis for the Chloride Channel Activity of Cystic Fibrosis Transmembrane Conductance Regulator and the Consequences of Disease-Causing Mutations by Jackie Kidd, Ilana Kogan, and Christine Bear of the University of Toronto examines the function and regulation of the CFTR gene, and the challenges inherent in studying it. This is vital work that will lead to molecular and perhaps stem cell therapies to treat and even cure this devastating disease. This volume has beneWted from the ongoing cooperation of a team of participants who are jointly responsible for the content and quality of its material. The authors deserve the full credit for their success in covering their subjects in depth yet with clarity, and for challenging the reader to think about these topics in new ways. The members of the Editorial Board are thanked for their suggestions of topics and authors. I also thank Laura Hewitson and Leah KauVman for their fabulous scientiWc insights and Rhonda Genes for her exemplary administrative support. Finally, we are grateful to everyone at the Pittsburgh Development Center of MageeWomens Research Institute here at the University of Pittsburgh School of Medicine for providing intellectual and infrastructural support for Current Topics in Developmental Biology Jerry Schatten Pittsburgh Development Center, Pennsylvania
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Therapeutic Cloning and Tissue Engineering Chester J. Koh and Anthony Atala Wake Forest Institute for Regenerative Medicine Wake Forest University School of Medicine Winston Salem, North Carolina 27157 I. II. III. IV. V.
Introduction Principles of Tissue Engineering Stem Cells Therapeutic Cloning Applications of Tissue Engineering and Therapeutic Cloning A. Muscle B. Kidney C. Mitochondrial DNA Analysis References
A severe shortage of donor organs available for transplantation in the United States leaves patients suVering from diseased and injured organs with few treatment options. Scientists in the field of tissue engineering apply the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Therapeutic cloning, where the nucleus from a donor cell is transferred into an enucleated oocyte in order to extract pluripotent embryonic stem cells, oVers a potentially limitless source of cells for tissue engineering applications. The present chapter reviews recent advances that have occurred in therapeutic cloning and tissue engineering and describes applications of these new technologies that may oVer novel therapies for patients with end-stage organ failure. ß 2004, Elsevier Inc.
I. Introduction The fields of regenerative medicine and tissue engineering aim to restore the form and function of damaged tissue and organs that have suVered from disease and injury. Many disorders, such as congenital anomalies, cancer, trauma, infection, inflammation, iatrogenic injuries, and other conditions, can lead to organ damage or loss and to the eventual need for reconstruction. It has been estimated that in the United States, one person in five reaching 65 years of age will receive temporary or permanent organ replacement therapy during his or her remaining life span (Lysaght and O’Loughlin, 2000). Furthermore, using the kidney as an example, over 2 million patients are projected to suVer from Current Topics in Developmental Biology, Vol. 60 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00
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end-stage renal disease by 2010 and aggregate health care costs for treating these patients has been estimated to be over $1 trillion (Lysaght, 2002). Most current reconstructive techniques rely on donor tissue for replacement. However, a shortage of donor tissue may limit these types of reconstructions, and usually some degree of morbidity is associated with the harvest procedure. Furthermore, these approaches rarely reproduce the entire functional aspects of the organ that it is replacing and may even lead to complications because of the inherently diVerent functional parameters. While caution and disease prevention may never completely eradicate the incidence of disease and injury, physicians and scientists in regenerative medicine hope to relieve each patient’s suVering from these unfortunate entities and, if possible, to repair or restore the damaged tissue with the hope of regenerating essentially normal body parts. Organ transplantation was one of the first methods to restore form and function in modern medicine. In 1955, Murray performed the first successful organ transplant (kidney), and in the early 1960s, he performed the first allogeneic kidney transplantation from a genetically dissimilar donor into an unrelated recipient (Murray et al., 1955). This transplant was the first of many such operations to overcome the immunologic barrier, which marked the modern era in which transplantation could be used as means of therapy for diseased and injured organs. Since then, there have been tremendous advances in immunosuppressive medications, in the matching of similar donors to recipients, and in the treatment of rejections, all of which have resulted in thousands of patients each year undergoing successful transplantation of donor organs. In 2001, over 23,000 patients received a transplanted organ in the United States (UNOS, 2002) However, despite the advances in transplantation since the early 1950s, shortage of donor organs limits the eVectiveness of this treatment, such that in 2001, nearly 80,000 patients were awaiting an available donor organ, and over 6000 patients were reported to have died while awaiting an organ transplant (UNOS, 2002). This has spawned the search for alternate therapies, such as tissue engineering and therapeutic cloning, that have the potential for replacement therapy. This chapter reviews the general principles of tissue engineering and therapeutic cloning and highlights some applications of these two technologies in organ replacement therapy.
II. Principles of Tissue Engineering In general, an individual’s native tissues are usually preferable for reconstruction, as they do not undergo rejection. Skin, for example, is available in suYcient quantities such that a graft may be taken from an alternate
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donor site and placed over the reconstruction area. For select tissues, a diVerent autologous tissue type may be used as the donor tissue. In the example of an injured bladder that requires augmentation or replacement, a native gastrointestinal segment may be used if insuYcient bladder tissue is available. However, for many tissue types, the use of native tissue may be limited by a shortage of donor tissue, and the morbidity of donor tissue harvest may limit its availability. When native tissue is unavailable in suYcient amounts for reconstruction, other potential sources of tissue include homologous tissues from cadavers, heterologous tissues from animal sources (bovine), and artificial materials (silicone, polyurethane, Teflon). Over the past century, synthetic materials have been developed that could serve to replace or rebuild diseased human tissues or body parts. Advances in the fields of chemistry and natural science have led to the production of several types of new man-made materials, such as tetrafluoroethylene (Teflon) and silicone, which could be manufactured into a wide variety of devices that could be applied for human use. These devices were noted to be biocompatible and could provide structural replacement; however, the functional component of the original tissue was usually not recovered. Because of the shortcomings in replacement therapy just listed, the field of tissue engineering has emerged since the 1960s. Scientists in this relatively new field aim to combine the principles of cell transplantation, material science, and engineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Over the past two decades, scientists have attempted to engineer tissue replacements for virtually every tissue and part of the human body. Tissue engineering strategies generally fall into two categories: acellular matrices, where matrices are used alone and which depend on the body’s natural ability to regenerate for proper orientation and direction of new tissue growth, and matrices with cells. Acellular tissue matrices are usually prepared by removing cellular components from tissues via mechanical and chemical manipulation to produce collagen-rich matrices (Chen et al., 1999; Dahms et al., 1998; Piechota et al., 1998; Yoo et al., 1998b). These matrices tend to slowly degrade on implantation and are generally replaced by the extracellular matrix (ECM) proteins that are secreted by the ingrowing cells. When cells are used for tissue engineering, a small piece of donor tissue is dissociated into individual cells. These cells are either implanted directly into the host or are expanded in culture, attached to a support matrix, and then reimplanted into the host after expansion. The source of donor tissue can be heterologous (such as bovine), allogeneic (same species, diVerent individual), or autologous. Ideally, both structural and functional tissue replacement will occur with minimal complications. The most
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preferred cells to use are autologous cells, where a biopsy of tissue is obtained from the host, the cells are dissociated and expanded in culture, and the expanded cells are implanted into the same host (Amiel and Atala, 1999; Atala, 1999; Atala and Lanza, 2001; Atala and Mooney, 1997; Atala et al., 1993, 1994, 1999; Cilento et al., 1994; Fauza et al., 1998a,b; Godbey and Atala, 2002; Kershen and Atala, 1999; Machluf and Atala, 1998; Oberpenning et al., 1999; Park et al., 1999; Yoo and Atala, 1997; Yoo et al., 1998a,b). The use of autologous cells avoids rejection, and thus the deleterious side eVects of immunosuppressive medications can be avoided. One of the initial factors that inhibited progress in cell-based tissue engineering in the past was the diYculty of growing the cells in culture in quantities suYcient for transplantation. While cells could be grown in the laboratory setting for in vitro studies, there was the inherent diYculty in expanding them into large enough quantities in the laboratory, such that tissue formation could occur in vivo after transplantation. This was thought to be due to a natural senescence that was diYcult to overcome. However, over the past two decades, protocols have been developed in many laboratories that allow for suYcient expansion in vitro that produced adequate numbers of cells for tissue formation. As an example, urothelial cells are now able to be harvested and expanded without the use of any enzymes or serum (Cilento et al., 1994; Liebert et al., 1997; Puthenveettil et al., 1999; Scriven et al., 1997). With urothelial cells, a single specimen of cells that initially covers a surface area of 1 cm2 can be expanded into enough cells that cover a surface area of 4202 m2 (the equivalent area of one football field) within 8 weeks (Cilento et al., 1994). This serves as an example that a small sample of autologous cells can be collected from a human patient, can be expanded in culture, and then can be returned to the same patient in suYcient quantities for reconstructive purposes. For transplantation, the expanded cells are seeded onto a scaVold synthesized with the appropriate biomaterial. In tissue engineering, biomaterials replicate the biologic and mechanical function of native ECM found in tissues in the body by serving as an artificial ECM. As a result, biomaterials provide a three-dimensional space for the cells to form into new tissues with appropriate structure and function, and also can allow for the delivery of cells and appropriate bioactive factors (e.g., cell adhesion peptides, growth factors) to desired sites in the body (Kim and Mooney, 1998). As the majority of mammalian cell types are anchorage dependent and will die if no cell-adhesion substrate is available, biomaterials provide a celladhesion substrate that can deliver cells to specific sites in the body with high loading eYciency. Biomaterials can also provide mechanical support against in vivo forces such that the predefined three-dimensional structure is maintained during tissue development. In addition, bioactive signals, such as
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cell-adhesion peptides and growth factors, can be loaded along with cells to help regulate cellular function. The ideal biomaterial should be biocompatible in that it is biodegradable and bioresorbable to support the replacement of normal tissue without inflammation. Incompatible materials are destined for an inflammatory or foreign body response that eventually leads to rejection and/or necrosis. In addition, the degradation products, if produced, should be removed from the body via metabolic pathways at an adequate rate that keeps the concentration of these degradation products in the tissues at a tolerable level (Bergsma et al., 1995). Furthermore, the biomaterial should provide an environment in which an appropriate regulation of cell behavior (e.g., adhesion, proliferation, migration, diVerentiation) can occur such that functional tissue can form. Cell behavior in the newly formed tissue has been shown to be regulated by multiple interactions of the cells with their microenvironment, including interactions with cell-adhesion ligands (Hynes, 1992) and with soluble growth factors (Deuel, 1997). Furthermore, biomaterials provide temporary mechanical support that allows the tissue to grow in three dimensions while the cells undergo spatial tissue reorganization. The properly chosen biomaterial should allow the engineered tissue to maintain suYcient mechanical integrity to support itself in early development, and while in late development, the properly chosen biomaterial should have begun degradation such that it does not hinder further tissue growth (Kim and Mooney, 1998).
III. Stem Cells Most current strategies for tissue engineering depend on a sample of autologous cells from the diseased organ of the host. However, for many patients with extensive end-stage organ failure, a tissue biopsy may not yield enough normal cells for expansion and transplantation. In these situations, pluripotent human embryonic stem cells are envisioned as a viable source of cells, as they can serve as an alternative source of cells from which the desired tissue can be derived. Combining the techniques learned in tissue engineering over the past few decades with this potentially endless source of versatile cells could lead to novel sources of replacement organs. Embryonic stem cells exhibit two remarkable properties: the ability to proliferate in an undiVerentiated, but pluripotent state (self-renew) and the ability to diVerentiate into many specialized cell types (Brivanlou et al., 2003). They can be isolated by immunosurgery from the inner cell mass of the embryo during the blastocyst stage (5 days postfertilization) and are usually grown on feeder layers consisting of mouse embryonic fibroblasts or human feeder cells (Richards et al., 2002). More recent reports have
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shown that these cells can be grown without the use of a feeder layer (Amit et al., 2003) and thus avoid the exposure of these human cells to mouse viruses and proteins. These cells have demonstrated longevity in culture by maintaining their undiVerentiated state for at least 80 passages when grown using current published protocols (ReubinoV et al., 2000; Thomson et al., 1998). Human embryonic stem cells have been shown to diVerentiate into cells from all three embryonic germ layers in vitro. Skin and neurons have been formed, indicating ectodermal diVerentiation (ReubinoV et al., 2001; Schuldiner et al., 2000, 2001; Zhang et al., 2001). Blood, cardiac cells, cartilage, endothelial cells, and muscle have been formed, indicating mesodermal diVerentiation (Kaufman et al., 2001; Kehat et al., 2001; Levenberg et al., 2002), and pancreatic cells have been formed, indicating endodermal diVerentiation (Assady et al., 2001). In addition, as further evidence of their pluripotency, embryonic stem cells can form embryoid bodies, which are cell aggregations that contain all three embryonic germ layers, while in culture, and can form teratomas in vivo (Itskovitz-Eldor et al., 2000). However, the harvesting of human embryonic stem cells requires the destruction of human embryos, which has raised significant ethical and political concerns in the United States. On August 9, 2001, in a compromise between the stem cell research advocates and critics, the federal government ordered that only previously generated human embryonic stem cell lines could be approved for federal research funding, and over 70 diVerent cell lines met this criterion at that time. However, as stated in National Institutes of Health (NIH) testimony before Congress in April 2003, only 11 stem cell lines are currently available, which has had deleterious eVects on the progress of stem cell research in the United States (Kennedy, 2003). In addition, most of the approved cell lines were grown in the presence of mouse cells (feeder cells), which can supply many needed growth factors, but which also expose the human cells to potential contamination from mouse viruses or proteins. This may render the current cell lines unsuitable for human therapeutic purposes.
IV. Therapeutic Cloning Nuclear cloning, which has also been called nuclear transplantation and nuclear transfer, involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate a cloned embryo. A better understanding of the diVerences between the two types of nuclear cloning, reproductive cloning and therapeutic cloning, may help alleviate some of the controversy that surrounds these revolutionary technologies (Colman and Kind, 2000; Vogelstein et al., 2002). Banned in most countries, reproductive
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cloning is used to generate an embryo that has the identical genetic material as its cell source. This embryo can then be implanted into the uterus of a female to give rise to an infant that is a clone of the donor cell. However, therapeutic cloning is used to generate cloned embryos that are explanted in culture and then can give rise to embryonic stem cell lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue engineering and tissue replacement applications (Hochedlinger and Jaenisch, 2003). Some useful applications would be in the treatment of diseases, such as end-stage kidney disease, neurodegenerative diseases, and diabetes, for which there is limited availability of immunocompatible tissue transplants. As a result, therapeutic cloning provides an alternative source of cells that theoretically may be limitless. Figure 1 shows the strategy of combining therapeutic cloning with tissue engineering to develop tissues and organs. According to data from the Centers for Disease Control, it has been estimated that approximately 3000 Americans die every day of diseases that could have been treated with embryonic stem cell-derived tissues (Lanza et al., 2001). Because of immunologic incompatibility, rejection is a frequent complication of allogeneic tissue transplantation, with immunosuppressive drugs usually being given to treat the host-versus-graft disease (Hochedlinger and Jaenisch, 2003). Somatic cell nuclear transfer may lead
Figure 1
Strategy of therapeutic cloning with tissue engineering. (See Color Insert.)
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to the prevention of immune responses that typically are associated with transplantation of all nonautologous tissues. With this technology, the variety of serious and potentially life-threatening complications associated with immunosuppressive drugs can be avoided (Lanza et al., 1999).
V. Applications of Tissue Engineering and Therapeutic Cloning We applied the principles of both tissue engineering and therapeutic cloning in an eVort to produce genetically identical renal, cardiac, and skeletal muscle tissue in a large animal model, the cow (Bos taurus) (Lanza et al., 2002). Unfertilized bovine donor oocytes were retrieved from abbatoir-derived ovaries, and the nuclear material from these eggs was removed by mechanical enucleation 18–22 h after maturation. Complete enucleation of the metaphase plate was confirmed with bis-benzimide dye under fluorescence microscopy. Bovine skin fibroblasts from adult Holstein steers were obtained by ear notch, and single donor cells were isolated and microinjected into the perivitelline space of the enucleated oocytes (nuclear transfer). Neoembryogenesis with the fusion of cell–oocyte complexes was initiated by applying a single electrical pulse of 2.4 kV/cm for 15 ms. The resulting blastocysts were implanted into progestrin-synchronized recipients to allow for further in vivo growth. After 12 weeks, cloned renal and muscle cells were harvested, expanded in vitro, and then seeded onto biodegradable scaVolds. The constructs, which consist of the cells and the scaVolds, were then implanted into the subcutaneous space of the same steer from which the cells were cloned to allow for tissue growth. A. Muscle Cloned bovine cardiac and skeletal muscle cells seeded on polyglycolic acid scaVolds were retrieved 6 weeks after transplantation. Histologic examination of the cloned muscle constructs revealed well-organized cellular organization with spindle-shaped nuclei (Fig. 2D and E), and immunohistochemical analysis identified muscle fibers within the implanted constructs. In addition, Figure 2 Applications of therapeutic cloning and tissue engineering. (A) Illustration of the tissueengineered renal unit. (B) Renal unit seeded with cloned cells, 3 months after implantation, showing the accumulation of urine-like fluid. (C) A clear unidirectional continuity exists among mature glomeruli, their tubules, and the polycarbonate membrane. (D) Retrieved cloned cardiac muscle tissue shows a well-organized cellular orientation 6 weeks after implantation. (E) Retrieved cloned skeletal muscle tissue shows spatially oriented muscle fibers 12 weeks after implantation. (F) Elispot analyses of the frequencies of T cells that secrete IFN- after primary and secondary stimulation with allogeneic renal cells, cloned renal cells, or nuclear donor fibroblasts. (See Color Insert.)
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semiquantitative reverse transcriptase-polymerase chain reaction and Western blot analysis confirmed the expression of muscle-specific mRNA and proteins. Furthermore, histologic examination revealed extensive vascularization within the implants, which is essential for the survival and future growth of the cloned tissue. Control implants seeded with allogeneic cells were noted to have a lack of muscle bundle formation, and histology revealed an abundance of inflammatory cells, fibrosis, and necrotic debris consistent with acute rejection. Using immunocytochemical analyses and CD4- and CD8-specific antibodies, control implants were noted to have an approximately twofold increase in CD4þ and CD8þ T cells as compared to cloned implants. Furthermore, Western blot analyses indicated a significant increase in the expression intensities of both CD4 and CD8 in control implants when compared with cloned implants.
B. Kidney The kidney is a complex organ with multiple cell types and a complex functional anatomy that renders it one of the most diYcult to reconstruct (Amiel and Atala, 1999; Auchincloss and Bonventre, 2002). Previous eVorts in tissue engineering of the kidney have been directed toward the development of extracorporeal renal support systems made of biological and synthetic components (Aebischer et al., 1987; Amiel et al., 2000; Humes et al., 1999; Ip et al., 1988; Joki et al., 2001; Lanza et al., 1996; MacKay et al., 1998), and ex vivo renal replacement devices are known to be life-sustaining. However, there would be obvious benefits for patients with end-stage kidney disease if these devices could be implanted long term without the need for an extracorporeal perfusion circuit or immunosuppressive drugs. For the production of cloned kidney tissue, renal cells were isolated and expanded in vitro. The presence of renal-specific proteins, such as synaptopodin (produced by podocytes), aquaporin-1 (produced by proximal tubules and the descending limb of the loop of Henle), aquaporin-2 (produced by the ascending loop of Henle), Tamm-Horsfall protein (produced by the ascending loop of Henle), and factor VII (produced by endothelial cells), was confirmed by immunohistochemistry on in vitro samples. In addition, renal cells were noted to produce two important kidney proteins: erythropoietin and 1,25-dihydroxyvitamin D3. After the renal cells were characterized and expanded, the cloned cells were seeded on scaVolds consisting of three collagen-coated cylindrical polycarbonate membranes (Fig. 2A). The ends of the three membranes of each scaVold were connected to catheters that terminated into a collecting reservoir. This created a renal neo-organ with a mechanism for collecting the
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excreted urinary fluid. These scaVolds with the collecting devices were transplanted subcutaneously into the same steer from which the genetic material originated and were retrieved 12 weeks after implantation. Upon retrieval, a straw-yellow fluid was seen in the reservoirs of the cloned cells (Fig. 2B). Chemical analysis noted the presence of urea nitrogen and creatinine in the fluid at levels that approached physiologically normal levels. In addition, after analysis of the electrolyte levels, specific gravity, and glucose concentrations of the collected fluid, the implanted renal cells were noted to possess filtration, reabsorption, and secretory capabilities. Histological examination of the retrieved implants revealed extensive vascularization and self-organization of the cells into glomeruli- and tubule-like structures. The glomeruli structures were noted to have a variety of cell types with abundant red blood cells, whereas the tubule-like structures were lined with cuboidal epithelial cells with large, spherical, and pale-stained nuclei. A clear continuity among the glomeruli, the tubules, and the polycarbonate membrane was noted that allowed the passage of urine into the collecting reservoir (Fig. 2C). Immunohistochemical analysis with renal-specific antibodies revealed the presence of renal proteins corresponding to tubular (AQP1), collecting tubule (AQP2), and glomerular (synaptopdin) segments within the cloned constructs. RT-PCR analysis confirmed transcription of the corresponding renal-specific RNA in the cloned specimens, and Western blot analysis confirmed the presence of elevated renal-specific protein levels.
C. Mitochondrial DNA Analysis Since previous studies have shown that bovine clones harbor oocyte mitochondrial (mt) DNA (Evans et al., 1999; Hiendleder et al., 1999; Steinborn et al., 2000), mitochondrial DNA of the donor egg was thought to be a potential source of immunologic incompatibility. DiVerences in mtDNAencoded proteins expressed by cloned cells could stimulate a T-cell response specific for mtDNA-encoded minor histocompatibility antigens when the cloned cells are implanted back into the original nuclear donor (Fischer Lindahl et al., 1991). We used nucleotide sequencing of the mtDNA genomes of the clone and fibroblast nuclear donor to identify potential antigens in the muscle constructs. Only two amino acid substitutions were noted to distinguish the clone and the nuclear donor and, as a result, a maximum of two minor histocompatibility antigens could be defined. Given the lack of knowledge regarding peptide-binding motifs for bovine MHC class I molecules, there is no reliable method to predict the impact of these amino acid substitutions on bovine histocompatibility. Oocyte-derived mtDNA was also thought to be a potential source of immunologic incompatibility in cloned renal cells. Maternally transmitted
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minor histocompatibility antigens in mice have been shown to stimulate both skin allograft rejection in vivo and cytotoxic T lymphocytes expansion in vitro (Fischer Lindahl et al., 1991) that could prevent the use of these cloned constructs in patients with a chronic rejection of major histocompatibility-matched human renal transplants (Hadley et al., 1992; Yard et al., 1993). We tested for a possible T-cell response to the cloned renal devices using delayed-type hypersensitivity testing in vivo and Elispot analysis of interferon- secreting T cells in vitro. Both analyses revealed that the cloned renal cells showed no evidence of a T-cell response, suggesting that rejection will not necessarily occur in the presence of oocyte-derived mitochondrial DNA (Fig. 2F). This finding may represent a step forward in overcoming the histocompatibility problem of stem cell therapy (Auchincloss and Bonventre, 2002). These studies demonstrated that cells derived from nuclear transfer can be successfully harvested, expanded in culture, and transplanted in vivo with the use of biodegradable scaVolds on which the single suspended cells can organize into tissue structures that are genetically identical to that of the host. These studies were the first demonstration of the use of therapeutic cloning for regeneration of tissues in vivo. The eventual goal would be to take a single skin cell from a patient and then generate any type of tissue that could be replaced or transplanted. The advantage of this system, as opposed to current transplantation techniques, is that the transplanted tissue would be genetically identical to that of the recipient and would be fully immunocompatible. Conceivably, each patient could have a ready-made supply of their own tissues available for their own use, should the need arise in the future.
References Aebischer, P., Ip, T. K., Panol, G., and Galletti, P. M. (1987). The bioartificial kidney: Progress towards an ultrafiltration device with renal epithelial cells processing. Life Support Syst. 5, 159–168. Amiel, G. E., and Atala, A. (1999). Current and future modalities for functional renal replacement. Uro. Clin. North Am. 26, 235–246, xi. Amiel, G. E., Yoo, J. J., and Atala, A. (2000). Renal therapy using tissue-engineered constructs and gene delivery. World Urol. 18, 71–79. Amit, M., Margulets, V., Segev, H., Shariki, K., Laevsky, I., Coleman, R., and ItskovitzEldor, J. (2003). Human feeder layers for human embryonic stem cells. Bio. Reprod. 68, 2150–2156. Assady, S., Maor, G., Amit, M., Itskovitz-Eldor, J., Skorecki, K. L., and Tzukerman, M. (2001). Insulin production by human embryonic stem cells. Diabetes 50, 1691–1697. Atala, A. (1999). Future perspectives in reconstructive surgery using tissue engineering. Uro. Clin. North Am. 26, 157–165, ix–x.
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Atala, A., Freeman, M. R., Vacanti, J. P., Shepard, J., and Retik, A. B. (1993). Implantation in vivo and retrieval of artificial structures consisting of rabbit and human urothelium and human bladder muscle. J. Urol. 150, 608–612. Atala, A., Guzman, L., and Retik, A. B. (1999). A novel inert collagen matrix for hypospadias repair. J. Urol. 162, 1148–1151. Atala, A., Kim, W., Paige, K. T., Vacanti, C. A., and Retik, A. B. (1994). Endoscopic treatment of vesicoureteral reflux with a chondrocyte-alginate suspension. J. Urol. 152, 641–643, discussion 644. Atala, A., and Lanza, R. P. (2001). Preface. In ‘‘Methods of Tissue Engineering’’ (A. Atala and R. P. Lanza, Eds.), p. xi. Academic Press, San Diego. Atala, A., and Mooney, D. (1997). Preface. In ‘‘Tissue Engineering’’ (A. Atala, Ed.), p. xi. Birkhauser Press, Boston, MA. Auchincloss, H., and Bonventre, J. V. (2002). Transplanting cloned cells into therapeutic promise. Nature Biotechnol. 20, 665–666. Bergsma, J. E., Rozema, F. R., Bos, R. R., Boering, G., de Bruijn, W. C., and Pennings, A. J. (1995). In vivo degradation and biocompatibility study of in vitro pre-degraded as-polymerized polyactide particles. Biomaterials 16, 267–274. Brivanlou, A. H., Gage, F. H., Jaenisch, R., Jessell, T., Melton, D., and Rossant, J. (2003). Stem cells: Setting standards for human embryonic stem cells. Science 300, 913–916. Chen, F., Yoo, J. J., and Atala, A. (1999). Acellular collagen matrix as a possible ‘‘off the shelf’’ biomaterial for urethral repair. Urology 54, 407– 410. Cilento, B. G., Freeman, M. R., Schneck, F. X., Retik, A. B., and Atala, A. (1994). Phenotypic and cytogenetic characterization of human bladder urothelia expanded in vitro. J. Urol. 152, 665–670. Colman, A., and Kind, A. (2000). Therapeutic cloning: Concepts and practicalities. Trends Biotechnol. 18, 192–196. Dahms, S. E., Piechota, H. J., Dahiya, R., Lue, T. F., and Tanagho, E. A. (1998). Composition and biomechanical properties of the bladder acellular matrix graft: Comparative analysis in rat, pig and human. Br. J. Urol. 82, 411–419. Deuel, T. F. (1997). Growth factors. In ‘‘Principles of Tissue Engineering’’ (R. P. Lanza, R. Langer, and W. L. Chick, Eds.), pp. 133–149. Academic Press, New York. Evans, M. J., Gurer, C., Loike, J. D., Wilmut, I., Schnieke, A. E., and Schon, E. A. (1999). Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep. Nature Genet. 23, 90–93. Fauza, D. O., Fishman, S. J., Mehegan, K., and Atala, A. (1998a). Videofetoscopically assisted fetal tissue engineering: Bladder augmentation. J. Pediatr. Surg. 33, 7–12. Fauza, D. O., Fishman, S. J., Mehegan, K., and Atala, A. (1998b). Videofetoscopically assisted fetal tissue engineering: Skin replacement. J. Pediatr. Surg. 33, 357–361. Fischer Lindahl, K., Hermel, E., Loveland, B. E., and Wang, C. R. (1991). Maternally transmitted antigen of mice: A model transplantation antigen. Annu. Rev. Immunol. 9, 351–372. Godbey, W. T., and Atala, A. (2002). In vitro systems for tissue engineering. Ann. N. Y. Acad. Sci. 961, 10–26. Hadley, G. A., Linders, B., and Mohanakumar, T. (1992). Immunogenicity of MHC class I alloantigens expressed on parenchymal cells in the human kidney. Transplantation 54, 537–542. Hiendleder, S., Schmutz, S. M., Erhardt, G., Green, R. D., and Plante, Y. (1999). Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle. Mol. Reprod. Dev. 54, 24–31. Hochedlinger, K., and Jaenisch, R. (2003). Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N. Engl. J. Med. 349, 275–286.
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Humes, H. D., Buffington, D. A., MacKay, S. M., Funke, A. J., and Weitzel, W. F. (1999). Replacement of renal function in uremic animals with a tissue-engineered kidney. Nature Biotechnol. 17, 451–455. Hynes, R. O. (1992). Integrins: Versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25. Ip, T. K., Aebischer, P., and Galletti, P. M. (1988). Cellular control of membrane permeability: Implications for a bioartificial renal tubule. ASAIO Trans. 34, 351–355. Itskovitz-Eldor, J., Schuldiner, M., Karsenti, D., Eden, A., Yanuka, O., Amit, M., Soreq, H., and Benvenisty, N. (2000). Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol. Med. 6, 88–95. Joki, T., Machluf, M., Atala, A., Zhu, J., Seyfried, N. T., Dunn, I. F., Abe, T., Carroll, R. S., and Black, P. M. (2001). Continuous release of endostatin from microencapsulated engineered cells for tumor therapy. Nature Biotechnol. 19, 35–39. Kaufman, D. S., Hanson, E. T., Lewis, R. L., Auerbach, R., and Thomson, J. A. (2001). Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 98, 10716–10721. Kehat, I., Kenyagin-Karsenti, D., Snir, M., Segev, H., Amit, M., Gepstein, A., Livne, E., Binah, O., Itskovitz-Eldor, J., and Gepstein, L. (2001). Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 108, 407–414. Kennedy, D. (2003). Stem cells: Still here, still waiting. Science 300, 865. Kershen, R. T., and Atala, A. (1999). New advances in injectable therapies for the treatment of incontinence and vesicoureteral reflux. Urol. Clin. North Am. 26, 81–94, viii. Kim, B. S., and Mooney, D. J. (1998). Development of biocompatible synthetic extracellular matrices for tissue engineering. Trends Biotechnol. 16, 224–230. Lanza, R. P., Chung, H. Y., Yoo, J. J., Wettstein, P. J., Blackwell, C., Borson, N., Hofmeister, E., Schuch, G., Soker, S., Moraes, C. T., West, M. D., and Atala, A. (2002). Generation of histocompatible tissues using nuclear transplantation. Nature Biotechnol. 20, 689–696. Lanza, R. P., Cibelli, J. B., and West, M. D. (1999). Prospects for the use of nuclear transfer in human transplantation. Nature Biotechnol. 17, 1171–1174. Lanza, R. P., Cibelli, J. B., West, M. D., Dorff, E., Tauer, C., and Green, R. M. (2001). The ethical reasons for stem cell research. Science 292, 1299. Lanza, R. P., Hayes, J. L., and Chick, W. L. (1996). Encapsulated cell technology. Nature Biotechnol. 14, 1107–1111. Levenberg, S., Golub, J. S., Amit, M., Itskovitz-Eldor, J., and Langer, R. (2002). Endothelial cells derived from human embryonic stem cells. Proc. Nat. Acad. Sci. USA 99, 4391–4396. Liebert, M., Hubbel, A., Chung, M., Wedemeyer, G., Lomax, M. I., Hegeman, A., Yuan, T. Y., Brozovich, M., Wheelock, M. J., and Grossman, H. B. (1997). Expression of mal is associated with urothelial differentiation in vitro: Identification by differential display reverse-transcriptase polymerase chain reaction. Differentiation 61, 177–185. Lysaght, M. J. (2002). Maintenance dialysis population dynamics: Current trends and longterm implications. J. Am. Soci. Nephrol. 13, S37–S40. Lysaght, M. J., and O’Loughlin, J. A. (2000). Demographic scope and economic magnitude of contemporary organ replacement therapies. ASAIO J. 46, 515–521. Machluf, M., and Atala, A. (1998). Emerging concepts for tissue and organ transplantation. Graft 1, 31. MacKay, S. M., Funke, A. J., Buffington, D. A., and Humes, H. D. (1998). Tissue engineering of a bioartificial renal tubule. ASAIO J. 44, 179–183. Murray, J. E., Merrill, J. P., and Harrison, J. H. (1955). Renal homotransplantation in identical twins. Surg. Forum 6, 432–436.
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Oberpenning, F., Meng, J., Yoo, J. J., and Atala, A. (1999). De novo reconstitution of a functional mammalian urinary bladder by tissue engineering. Nature Biotechnol. 17, 149–155. Park, H. J., Yoo, J. J., Kershen, R. T., Moreland, R., and Atala, A. (1999). Reconstitution of human corporal smooth muscle and endothelial cells in vivo. J. Urol. 162, 1106–1109. Piechota, H. J., Dahms, S. E., Nunes, L. S., Dahiya, R., Lue, T. F., and Tanagho, E. A. (1998). In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft. J. Urol. 159, 1717–1724. Puthenveettil, J. A., Burger, M. S., and Reznikoff, C. A. (1999). Replicative senescence in human uroepithelial cells. Adv. Exp. Med. Biol. 462, 83–91. Reubinoff, B. E., Itsykson, P., Turetsky, T., Pera, M. F., Reinhartz, E., Itzik, A., and Ben-Hur, T. (2001). Neural progenitors from human embryonic stem cells. Nature Biotechnol. 19, 1134–1140. Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A., and Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. [erratum appears in Nat. Biotechnol. 18(5), 559 (2000)]. Nature Biotechnol. 18, 399–404. Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., and Bongso, A. (2002). Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nature Biotechnol. 20, 933–936. Schuldiner, M., Eiges, R., Eden, A., Yanuka, O., Itskovitz-Eldor, J., Goldstein, R. S., and Benvenisty, N. (2001). Induced neuronal differentiation of human embryonic stem cells. Brain Res. 913, 201–205. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D. A., and Benvenisty, N. (2000). Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Nat. Acad. Sci. USA 97, 11307–11312. Scriven, S. D., Booth, C., Thomas, D. F., Trejdosiewicz, L. K., and Southgate, J. (1997). Reconstitution of human urothelium from monolayer cultures. J. Urol. 158, 1147–1152. Steinborn, R., Schinogl, P., Zakhartchenko, V., Achmann, R., Schernthaner, W., Stojkovic, M., Wolf, E., Muller, M., and Brem, G. (2000). Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nature Genet. 25, 255–257. Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M. (1998). Embryonic stem cell lines derived from human blastocysts. [erratum appears in Science 282(5395), 1827 (1988)]. Science 282, 1145–1147. UNOS (2002). 2002 OPTN/SRTR annual report.www.optn.org. Vogelstein, B., Alberts, B., and Shine, K. (2002). Genetics: Please don’t call it cloning! Science 295, 1237. Yard, B. A., Kooymans-Couthino, M., Reterink, T., van den Elsen, P., Paape, M. E., Bruyn, J. A., van Es, L. A., Daha, M. R., and van der Woude, F. J. (1993). Analysis of T cell lines from rejecting renal allografts. Kidney Int. Suppl. 39, S133–S138. Yoo, J. J., and Atala, A. (1997). A novel gene delivery system using urothelial tissue engineered neo-organs. J. Urol. 158, 1066–1070. Yoo, J. J., Lee, I., and Atala, A. (1998a). Cartilage rods as a potential material for penile reconstruction. J. Urol. 160, 1164–1168; discussion 1178. Yoo, J. J., Meng, J., Oberpenning, F., and Atala, A. (1998b). Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 51, 221–225. Zhang, S. C., Werning, M., Duncan, I. D., Brustle, O., and Thomson, J. A. (2001). In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nature Biotechnol. 19, 1129–1133.
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-Synuclein: Normal Function and Role in Neurodegenerative Diseases Erin H. Norris, Benoit I. Giasson, and Virginia M.-Y. Lee Center for Neurodegenerative Disease Research and the Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104
I. II. III. IV.
V. VI. VII. VIII. IX. X.
Introduction to the Synuclein Protein Family Synuclein in Synaptic Development and Maintenance Synuclein Protein Interactions Synucleinopathies A. Parkinson’s Disease (PD) B. Dementia with Lewy Bodies (DLB) C. Multiple System Atrophy (MSA) D. Other Synucleinopathies Structural Properties of -Synuclein Protein Implications of Oxidative and Nitrative Injury in Synucleinopathies EVects of Oxidation and Nitration on -Synuclein Fibril Formation Possible Role of Pesticides and Other Toxic Chemicals in PD Transgenic and Nontransgenic Animal Models of Synucleinopathies Concluding Remarks Acknowledgments References
Synucleins are a family of small, highly charged proteins expressed predominantly in neurons. Since their discovery and characterization during the last decade, much has been learned about their structure, potential functions, interactions with other proteins, and roles in disease. One of these proteins, -synuclein (-syn), is the major building block of pathological inclusions that characterize many neurodegenerative disorders, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and neurodegeneration with brain iron accumulation type 1 (NBIA-1), which collectively are termed synucleinopathies. Furthermore, genetic and biological studies support a role for -syn in the pathophysiology of these diseases. Therefore, research must be continued in order to better understand the functions of the synuclein proteins under normal physiological conditions as well as their role in diseases. ß 2004, Elsevier Inc. Current Topics in Developmental Biology, Vol. 60 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00
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I. Introduction to the Synuclein Protein Family The first synuclein (syn) protein was identified in 1988 by Maroteaux and colleagues upon its purification from the electric organ of the Pacific ray, Torpedo californica, as well as isolation from rat brain (Maroteaux et al., 1988). Approximately 4 years later, the second member of the syn protein family was identified in bovine brain and initially named phosphoneuroprotein 14 (Tobe et al., 1992). Upon realization of the homology between these proteins, they were correspondingly termed -synuclein (-syn) and -synuclein (-syn) (Jakes et al., 1994). The - and -syn genes were mapped to chromosomes 4q21.3-q22 and 5q35, respectively (Spillantini et al., 1995). A third member of the syn family, initially termed breast cancer gene-specific product 1, was found in metastatic breast cancer tissue in 1997 (Ji et al., 1997), and this same gene was also cloned and characterized the following year by Ninkina et al. (1998), where it was called persyn. It then became known as -synuclein (-syn) due to its significant homology to - and syn proteins (Lavedan et al., 1998), and its gene was mapped to chromosome 10q23.2-q23.3 (Lavedan et al., 1998; Ninkina et al., 1998). -Syn is a heat-stable, cytoplasmic protein of 140 amino acids with incompletely defined function that is found most abundantly in neurons of the central nervous system (CNS) (Davidson et al., 1998; George et al., 1995; Iwai et al., 1995a; Jakes et al., 1994). The amino-terminal region, which contains 60 amino acid residues, is predicted to form amphipathic helices in the presence of synthetic lipids (Clayton and George, 1999). The region contains four imperfect KTKEGV repeats (Fig. 1) and has homology to a lipid-binding domain of apolipoproteins (Clayton and George, 1999; George et al., 1995; Weinreb et al., 1996). It has been suggested that -syn be classified as a protein with a class A2 lipid-binding helix, as it has a cluster of basic residues at the polar–apolar surface that contains a high proportion of lysine residues versus arginine residues and also has a high number of glutamate residues in the polar region of the protein (Perrin et al., 2000; Segrest et al., 1990, 1992). It was demonstrated directly that -syn can bind loosely to lipid vesicles, and the presence of vesicles might induce -syn to acquire an -helical structure (see Section V). Residues 61–95 of -syn form the hydrophobic and amyloidogenic central region, referred to as the nonamyloid component (NAC) of the amyloid precursor protein, which includes two additional KTKEGV imperfect repeat motifs (Han et al., 1995). A peptide corresponding to the NAC region of -syn was isolated from amyloid-rich senile plaques in Alzheimer’s disease (AD) patient brains (Ueda et al., 1993). However, further research failed to confirm that the NAC peptide is an integral component of senile plaques (Bayer et al., 1999), and it is likely that the presence of the NAC peptide in biochemical fractions of senile
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Figure 1 Amino acid sequence alignment of human -, -, and -synuclein proteins. Imperfect repeats (KTKEGV) are designated by blue boxes, and the hydrophobic NAC region (residues 61–95) of -syn is delineated by the red bar. Pathological mutations of -syn, A30P and A53T, are circled. Sequences for - and -syn were obtained from Jakes et al. (1994), and the sequence for -syn was obtained from Ji et al. (1997). (See Color Insert.)
plaques was due to Lewy bodies (LBs) and Lewy neurites (LNs) contaminating these fractions (see Section IV). While -syn protein is expressed more highly in neurons of brain regions such as the frontal cortex, hippocampus, striatum, and olfactory bulb (Iwai et al., 1995a), it is also found in other brain regions (Iwai et al., 1995a; Maroteaux and Scheller, 1991), in olfactory receptor neurons and basal cells of the olfactory epithelium (Duda et al., 1999), and at low levels in platelets (Hashimoto et al., 1997), hematopoietic cells (Shin et al., 2000), cells of the neuromuscular junction and skeletal muscle (Askanas et al., 2000; Lavedan, 1998), cardiac tissue (Iwanaga et al., 1999), and cultured Schwann cells (Mori et al., 2002) and oligodendrocytes (Richter-Landsberg et al., 2000). In contrast, -syn protein is found more uniformly throughout the brain (Abeliovich et al., 2000; Nakajo et al., 1994; Shibayama-Imazu et al., 1993) in addition to being expressed in native and cultured astrocytes (Tanji et al., 2001) and Sertoli cells of the testis (Nakajo et al., 1996; ShibayamaImazu et al., 1998). - and -syn proteins share a high level of homology; however, they diVer mainly in the NAC region as -syn lacks many of these amino acid residues (Fig. 1). In neurons, - and -syn are localized primarily in presynaptic terminals. Through the use of cellular fractionation and microscopy techniques, it was
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shown that - and -syn are enriched in synaptosomal preparations (but are not found in highly purified synaptic vesicle fractions) (George et al., 1995; Irizarry et al., 1998; Kahle et al., 2000; Shibayama-Imazu et al., 1993) and are localized in close proximity to synaptic vesicles (but are clearly not bound to them) (Iwai et al., 1995a; Nakajo et al., 1994). In accordance with these findings, electron microscopy (EM) immunogold labeling was used to show that - and -syn form a moderately dense, amorphous ‘‘matrix’’ just behind or beneath the region of abundant synaptic vesicles (Clayton and George, 1999). -Syn is also expressed in brain and spinal cord, but is expressed most abundantly in cells of the peripheral nervous system (Buchman et al., 1998b; Lavedan et al., 1998) as well as the epidermis (Ninkina et al., 1999) and retina (George, 2002). In addition, much higher levels of -syn are expressed in advanced-stage breast cancer tissue compared to normal breast tissue or benign breast lesions (Bruening et al., 2000; Ji et al., 1997; Ninkina et al., 1998), implicating its involvement in the progression of cancer. The recent investigation of over 80 brain tumors showed the presence of -, -, and -syn proteins in glial tumors and/or medulloblastomas, which further associates syn proteins in cancer progression and possibly cancer cell survival (Fung et al., 2003). Unlike - and -syn, -syn protein is distributed diVusely throughout the cytosol of neurons, where it may be involved in cytoskeletal maintenance (Buchman et al., 1998a). It also has been proposed that -syn acts as a centrosome protein; it was localized by double-labeling immunocytochemistry to centrosomes of cells in interphase and to the spindle poles of cells in mitosis (Surguchov et al., 2001). -Syn protein is smaller than - and -syn proteins due to a shorter C-terminal region, yet it contains much of the NAC region that is missing in -syn (Fig. 1).
II. Synuclein in Synaptic Development and Maintenance Much research has been carried out in humans, mice, and chickens to study the role of syn proteins in development of the nervous system and synaptic plasticity. Although -syn is localized predominantly to axon terminals of mature neurons, it is initially present in the cell body. Studies of hippocampal and nigral cultures showed translocation of -syn from perikarya to terminals between 15 and 18 weeks of gestation (Galvin et al., 2001; Murphy et al., 2000). Therefore, -syn does not appear to be involved in synapse formation, as it is expressed later in development (Murphy et al., 2000; Withers et al., 1997). Moreover, -syn knockout (KO) mice are vital and fertile, show no morphological deficits (Cabin et al., 2002), and produce normal dopaminergic cell bodies, fibers, and synapses (Abeliovich et al., 2000), providing further evidence that -syn is not necessary for synaptic development.
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-Syn is also initially localized to the perikarya and has an even greater delay in translocation to nerve terminals, which takes place at approximately 17 to 20 weeks of gestation (Galvin et al., 2001; Murphy et al., 2000). In addition, it was shown that syn expression occurs much later than synaptophysin and synapsin I, two other synaptic proteins, in primary cultures (Murphy et al., 2000; Withers et al., 1997). Because these syn proteins appear to be expressed and potentially functional after synaptic development, it is presumed that they are not necessary for the development of synapses but could play a role in synapse modulation or maturation (Murphy et al., 2000). In order to investigate the role of -syn as a synaptic modulator or maintainer, a series of experiments were carried out that involved knockdown of -syn expression in cultured neurons, transection of cultured neurons, and generation of -syn (KO) mice. EM data show that downregulation of syn expression by antisense oligonucleotides in mouse hippocampal neurons resulted in a reduction in the distal pool of vesicles, although the size of the docked vesicular pool was unaltered. In addition, synaptic proteins, synapsin I and synaptophysin, were also downregulated (Murphy et al., 2000). Furthermore, two studies support the possible involvement of syn proteins in neuronal regeneration. -Syn expression was demonstrated in continuously regenerating olfactory receptor neurons and basal cells of the olfactory epithelium (Duda et al., 1999). Also, a study by Quilty et al. (2003) demonstrated that syn accumulates rapidly in axons after neural transection. Additionally, Cabin et al. (2002) reported that -syn is necessary for synaptic maintenance as -syn KO mice displayed faulty synaptic transmission in which docked and distal vesicles are depleted upon prolonged stimulation, whereas control mice only show depletion of their docked vesicular pools. Moreover, nigral neurons from -syn KO mice respond to paired electrical stimuli with an increased release of dopamine (DA) compared to wild-type (WT) animals. This altered release pattern leads to abnormal reduction in striatal DA levels in addition to a decreased amphetamine-induced locomotor response (Abeliovich et al., 2000). Therefore, a great deal of evidence supports the involvement of -syn in synaptic maintenance, most likely by regulating mobilization of the distal/reserve pool of vesicles in the presynaptic terminal of neurons. To further support the role of -syn in the synapse, studies have shown that it inhibits phospholipase D2 (PLD2) (Jenco et al., 1998). PLD2 catalyzes the production of phosphatidic acid (PA) (Exton, 1997), an essential molecule for proper vesicle budding (Chen et al., 1997). Because -syn binds vesicles containing PA, it has been suggested that high levels of intracellular PA may influence its interaction with and inhibition of PLD2 in vivo, therefore halting the further production of PA (Clayton and George, 1999). In addition, it has been proposed that mutation or an abnormal conformation of -syn could prevent or alter PLD2 inhibition and hence wrongly interfere with synaptic membrane biogenesis and turnover, potentially causing
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deleterious events in cells that contain mutant or damaged -syn (Clayton and George, 1999).
III. Synuclein Protein Interactions The direct binding of the -syn carboxy terminus to the microtubule-binding region of tau has been shown by Jensen et al. (1999). Tau is a neuronal microtubule-associated protein and also forms aberrant brain inclusions in patients with a group of neurodegenerative diseases collectively termed tauopathies, including Alzheimer’s disease (Buee et al., 2000; Duda et al., 2000c; Goedert et al., 1998; Lee et al., 2001b). This interaction was identified using human brain cytosol in an -syn aYnity chromatography column. Other studies led to the proposition that -syn modulates soluble axonal tau by causing phosphorylation at serine residues, therefore aVecting the stability of microtubules in vivo (Jensen et al., 1999). More recently, studies provided further support for this tau–syn association. The coexistence of tau and syn pathological inclusions (see following sections) has been demonstrated as common in both familial and sporadic neurodegenerative diseases (Duda et al., 2002b; Kosaka and Iseki, 1996; Trembath et al., 2003). Furthermore, expression of -syn can stimulate the formation of tau inclusions in transgenic mice (Giasson et al., 2003). Giasson et al. (2003) also demonstrated that -syn can act as a seed to induce tau fibrillization in vitro, and this coincubation is synergistic in promoting the fibril formation of both proteins. Therefore, not only could -syn interfere with the normal function of tau in microtubule assembly by causing phosphorylation of the protein, but it could also initiate and promote rapid fibril formation, thus leading to the pathological inclusions found in neurodegenerative diseases. Ostrerova et al. (1999) demonstrated an association between -syn and 14-3-3 proteins (chaperone proteins that are expressed ubiquitously in the brain) as well as protein kinase c (PKC) and BAD, a pro-apoptotic protein. These investigators found that the -syn sequence has some homology (40%) to regions of 14-3-3 in that amino acid residues 8–61 of -syn correspond well with amino acid residues 45–102 of 14-3-3. Immunoprecipitates of either 14-3-3 proteins or -syn from rat brain homogenate were immunoreactive for both proteins, indicating that these proteins at least bind indirectly to each other (Ostrerova et al., 1999). This protocol was also used to show the binding capacity of -syn for PKC, BAD, and extracellular signal-related kinase, ERK (Ostrerova et al., 1999). Moreover, due to the interaction between -syn and 14-3-3, Souza et al. (2000b) investigated the role of -syn as a molecular chaperone using common chaperone activity assays involving insulin and alcohol dehydrogenase. These authors concluded that the entire syn protein family acts as chaperones, but showed that
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the A30P mutation in -syn prevented this activity. Moreover, this activity may be dependent on the carboxy terminus of syn protein, as C-terminal truncation resulted in a loss of chaperone activity (Souza et al., 2000b). Interactions between -syn and the dopamine transporter (DAT) have also been identified. Lee et al. (2001) reported that the NAC region of syn binds directly to the C terminus of DAT in neurons and in -syn/ DAT-transfected cells and that this complex leads to the accumulation of DAT at the cellular membrane. This upregulation of transporters at the cell surface hence leads to increased DA uptake and cell death, and they also proposed that this increase in intracellular DA is toxic to cells and causes degeneration in patients with disease (Lee et al., 2001a). However, Wersinger and Sidhu (2003) published conflicting data, demonstrating more direct evidence of an -syn–DAT complex by coimmunoprecipitation. Their work demonstrated the inhibition of DAT activity by -syn in several cell lines, which instead causes a decrease in DA uptake (without a change in DAT aYnity for DA) without resulting in cell death (Wersinger and Sidhu, 2003). Although the diVerence in results regarding the interaction of DAT and -syn has not been resolved, it can be concluded that DAT function can be modulated in some way by -syn protein. -Syn has also been reported to interact with kinases of G protein coupled receptors (Pronin et al., 2000), Src (Ellis et al., 2001), casein (Ingelsson and Hyman, 2002), synphilin (Engelender et al., 1999), and tubulin (Payton et al., 2001).
IV. Synucleinopathies Synucleinopathies are a collection of neurodegenerative diseases grouped together by the presence of pathological brain inclusions that are composed mainly of filamentous, insoluble -syn protein. The syn proteins had been isolated for nearly a decade before their direct involvement in neurodegenerative disease pathology was appreciated. Significant findings were made in 1997 and 1998 that linked -syn protein to Parkinson’s disease (PD), and soon thereafter it was implicated in numerous other neurodegenerative disorders. Genetic analysis of Mediterranean and Greek families with early-onset PD uncovered a missense mutation in -syn, namely A53T, and thereafter a second mutation in -syn (A30P) was identified in a German family (Kruger et al., 1998; Polymeropoulos et al., 1997). Not long after the recognition of the A53T mutation, it was determined that LBs and LNs, the proteinaceous brain inclusions found in PD and other neurodegenerative disorders, were mostly composed of -syn protein (Mezey et al., 1998; Spillantini et al., 1997). The -syn protein found in pathological inclusions has a filamentous form as shown by transmission and immunogold EM (Baba et al., 1998; Crowther et al., 1998, 2000; Gai et al., 1999; Serpell et al., 2000; Spillantini et al., 1998a; Tu et al., 1998).
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Table I Major Diseases with -Synuclein Pathology (See Color Insert.) Synucleinopathy Disease
Parkinson’s disease
Major Brain Region Affected
Pathological Inclusion
Brain stem
Lewy bodies Lewy neurites
Dementia with Lewy bodies
Neocortex and brain stem
Lewy bodies Lewy neurites
Lewy body variant of Alzheimer’s disease
Neocortex and brain stem
Lewy bodies NF tangles amyloid plaques
White matter
Glial cytoplasmic inclusions
Multiple system atrophy
Examples of -Syn Inclusions
-Syn-positive inclusions can be present diVusely in brains such as in cases of Dementia with Lewy bodies (DLB), Lewy body variant of AD (LBVAD), and multiple system atrophy (MSA) (Irizarry et al., 1998; Spillantini et al., 1997, 1998a,b; Takeda et al., 1998; Tu et al., 1998). In other diseases, such as familial Alzheimer’s disease, NBIA-1, and Down’s syndrome, the presence of -syn appears to be more limited to certain regions (Table I). A. Parkinson’s Disease (PD) Parkinson’s disease, the most common movement disorder and second most common neurodegenerative disorder, was first defined by James Parkinson in 1817 as the ‘‘shaking palsy.’’ It aVects over one million people in North America (Lang and Lozano, 1998) and over four million worldwide and influences all races and both sexes. The prevalence of PD increases with age; 0.5 to 1% of the population from 65 to 69 years of age is diagnosed with PD, and this percentage increases to 4% at the age of 85 (Tanner and Goldman, 1996). However, 5 to 10% of PD patients are diagnosed before the age of 40, and this type is termed early-onset PD. More AfricanAmericans experience PD than African blacks, and there are higher numbers of PD patients from rural, farming communities than from city-dwelling or suburban areas, implicating environmental exposure and surroundings as a causative factor (Barbeau et al., 1986; Gorell et al., 1998; Hertzman et al., 1990; Liou et al., 1997; Semchuk et al., 1993; Tanner et al., 1989).
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PD is defined clinically by four characteristic motor symptoms: uncontrollable resting tremor, slowness in movement (bradykinesia), stooped posture and instability, and muscle rigidity (Hoehn and Yahr, 1967). Dementia is not a factor in all PD patients, yet it is about six times more likely in a PD patient than in a normal elderly person (Aarland et al., 1996; Brown and Marsden, 1984; Marttila and Rinne, 1976; Mayeau et al., 1990; Rajput et al., 1987; Riggs, 1993). Other secondary symptoms of PD include sleep disturbances and autonomic instability (Arai et al., 2000; Boeve et al., 1998; Kaufmann et al., 2001; Olson et al., 2000), and 25% of PD patients experience depression (Tandberg et al., 1996). Due to the wide variation in secondary symptoms, it is believed that some patients have a combination of neurodegenerative diseases, e.g., AD and PD, which suggests that these diVerent neurodegenerative disorders could share a common mechanism. In some instances, functional imaging allows clinicians to distinguish PD from other forms of parkinsonism (reviewed in Lang and Lozano, 1998), as it can detect some of the neurodegenerative features specific to PD. First, there is a progressive and extensive loss of dopaminergic neurons in the substantia nigra pars compacta, which results in greatly decreased DA levels in the striatum. This striatal DA reduction is believed to be the cause of the motor discoordination found in PD patients (Damier et al., 1999; Lang and Lozano, 1998; Pakkenberg et al., 1991). Cell loss is also found in other brain regions, including the locus coeruleus, basal nucleus of Meynert, and raphe nuclei (Mayeau et al., 1984; Whitehouse et al., 1983). Second, intracellular proteinaceous inclusions (LBs and LNs) are present in regions of the brain, and these inclusions are mostly composed of polymers of fibrillized -syn protein along with lesser amounts of other cellular proteins (Baba et al., 1998; Gai et al., 2000; Irizarry et al., 1998; Kuzuhara et al., 1988; Pollanen et al., 1993; Spillantini et al., 1997, 1998b). Frederick Lewy first described these inclusions in 1912 and therefore are referred to as LBs and LNs, depending on their structure and location within cells. LBs are found in the perikarya of the remaining dopaminergic cells of the substantia nigra (Forno, 1996; Pollanen et al., 1993), but are also present in other catecholaminergic and serotonergic cells of the brain stem, cholinergic cells of the basal nucleus of Meynert, and cells of the dorsal nucleus of vagus, ventral tegmental region, peduncolo-pontine nucleus, thalamus and hypothalamus, cingulate gyrus, entorhinal cortex, olfactory bulb, locus coeruleus, and raphe nucleus (Forno, 1996; Kosaka and Iseki, 1996; Louis and Fahn, 1996). LBs in the brain stem and basal forebrain are usually over 15 m in diameter and have a spherical, dense core with a clear halo (Lang and Lozano, 1998; Okazaki et al., 1961), and filaments of -syn contained in LBs are 200 to 600 nm long and 5 to 10 nm wide (Crowther et al., 2000). It has been proposed that these intracellular proteinaceous clumps disrupt organelle function and protein transport, as evidenced by distortion of the Golgi
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apparatus, mitochondria, nucleus, and endoplasmic reticulum in aVected cells (DuVy and Tennyson, 1965; Hill et al., 1991; Schmidt et al., 1991), and these abnormalities could potentially lead to cell death. In addition to these proteinaceous aggregates, brains of PD patients show signs of extracellular melanin released from dying neurons as well as reactive gliosis. Pathology in brain regions other than the striatum may cause the secondary symptoms of PD listed earlier (discussed in Lang and Lozano, 1998), and depression most likely arises from the massive degeneration of serotonergic and noradrenergic neurons (Mayeau et al., 1984).
B. Dementia with Lewy Bodies (DLB) In 1961, Okazaki and colleagues described a severely demented patient with quadriparesis and cortical LBs. This disease, which generally seems to combine the pathology of PD and clinical symptoms of AD, became known as DLB and is now considered to be the second most common form of neurodegenerative dementia (after AD) (McKeith et al., 1996). The more clearly defined clinical signs of DLB are progressive dementia with visual hallucinations and fluctuating cognition, and many patients also present with muscular rigidity that is more prominent than bradykinesia and a pronounced sensitivity to neuroleptic drugs (Crystal et al., 1990; Galasko et al., 1994; Gibb et al., 1987; Hansen et al., 1990; Louis et al., 1997; McKeith et al., 1996; Mega et al., 1996; Perry et al., 1990). l-Dopa, the most common drug treatment for PD patients, is also eYcacious in alleviating motor complications in DLB patients, but it usually induces psychiatric side eVects. Pathologically, diseased brains contain a large number of -syn-positive LBs and LNs in the cerebral cortex, which is less common in PD, and in the substantia nigra (Kosaka, 1978; McKeith et al., 1996; Spillantini et al., 1997). However, if a patient has clinical symptoms similar to DLB but has numerous cortical and subcortical LBs, tangles, and senile plaques upon postmortem examination, the patient is diagnosed as having had LBVAD (Hansen et al., 1990; Katzman et al., 1995; Samuel et al., 1996). The number of LBs correlates with the severity of the patients’ dementia in both DLB and LBVAD (Haroutunian et al., 2000; Hurtig et al., 2000; Samuel et al., 1996). Cortical LBs are smaller than the LBs found in the brain stem of PD patients and typically do not contain a core (Lang and Lozano, 1998; Okazaki et al., 1961). They are often distributed unevenly throughout the brain, but are most abundant in the cingulate gyrus, entorhinal cortex, amygdala, temporal cortex, and insular cortex (Kosaka, 1990; Perry et al., 2000; Rezaie et al., 1996). They are found less prominently in the hippocampus, frontal lobes, and parietal lobes. Because the presence and location of LBs cannot be determined until a patient’s brain has been analyzed postmortem, it is
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significant that 25% of clinically defined demented PD patients actually suVered instead from DLB (Hughes et al., 1993). In addition to LB pathology, LNs are fairly abundant in the striatum of DLB brains, and these inclusions may contribute to the parkinsonian symptoms in DLB (Duda et al., 2002a). Because LBs and LNs in DLB and LBVAD are so similar to those in PD and because they are mostly composed of filamentous -syn protein (Baba et al., 1998; Crowther et al., 2000; Spillantini et al., 1997, 1998b), it has been suggested that PD and DLB share a common disease mechanism. In addition, it has been shown that LBs and LNs of LBVAD and DLB contain nitrated -syn protein (Duda et al., 2000a; Giasson et al., 2000a), and therefore it is speculated that oxidative and nitrative stress may contribute to the formation of LBs and LNs as well as to cell death (see following sections).
C. Multiple System Atrophy (MSA) In 1900, Dejerine and Thomas described two patients with a sporadic neurological disorder that consisted of cerebellar features, parkinsonism, and dysautonomia. Upon postmortem examination of these brains, neurodegeneration was found in the inferior olives, pons, and cerebellum, and the disease was termed olivopontocerebellar atrophy (OPCA) (Dejerine and Thomas, 1900). Decades later, two patients presented with the features of OPCA, but degeneration was also found in the substantia nigra, striatum, and intermediolateral cell columns. However, autonomic dysfunction in these patients was prominent, and this syndrome became known as Shy– Drager syndrome (SDS) (Shy and Drager, 1960). Following this report, more patients were evaluated with a disease that had similarities to OPCA and SDS, but included predominant parkinsonism features, which was termed striatonigral degeneration (SND) by Adams, van Bogaert, and van der Eecken (Adams et al., 1964). In 1969, Graham and Oppenheimer suggested that OPCA, SDS, and SND were the same syndrome and called it multiple system atrophy (MSA). Since then, the discovery of the specific pathological lesions unique to MSA, known as glial cytoplasmic inclusions (GCIs), has confirmed this notion (see later) (Papp et al., 1989). Most MSA patients display typical symptoms of PD, including rigidity, bradykinesia, and unstable balance and posture, although they often do not show signs of resting tremor (Wenning et al., 1994, 1997). Nearly all patients develop autonomic symptoms, ranging from impotence to urinary incontinence to orthostatic hypotension (drop in blood pressure when patient stands upright). The majority of patients display cerebellar signs, such as gait ataxia, and upper motor neuron disturbances. Mild dementia is not uncommon, but severe dementia is atypical in MSA (Wenning et al., 1997). The median age of onset is 55 years, but ranges from 33 to 76 years of age and
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aVects more men than women (Wenning et al., 1995). The symptoms of MSA progress faster than those of PD, and average patient survival is 6 to 7 years (Wenning et al., 1994). Postmortem examination of MSA brains showed that neurodegeneration and gliosis are found in the substantia nigra, striatum, external globus pallidus, locus coeruleus, inferior olives, pontine nuclei, and cerebellum in addition to the intermediolateral cell columns and Onuf’s nucleus of the spinal cord (Wenning et al., 1997). Wenning et al. (1995) correlated the orthostatic hypotension with degeneration of the intermediolateral cell columns and ataxia with atrophy of the olives, pontine nuclei, and cerebellum. Demyelination also occurs in MSA, giving some areas of the white matter in MSA brains a yellowish tint (Matsuo et al., 1998). The defining pathological lesions of MSA are known as GCIs. These proteinaceous aggregates are found mainly in oligodendrocytes and were discovered in 1989 by Papp and colleagues. As with LBs and LNs, GCIs are composed mostly of insoluble filamentous polymers of -syn protein (Dickson et al., 1999; Duda et al., 2000b, 2002a; Gai et al., 1998, 2003; Tu et al., 1998), some of which are nitrated (Giasson et al., 2000a). These -syn-positive GCIs are found predominantly in the neocortex, hippocampus, basal ganglia, substantia nigra, spinal cord, medulla, and cerebellum (Arima et al., 1992; Duda et al., 2000b; Lantos, 1997). Similar to LBs and LNs, it has been proposed that GCIs disrupt normal protein and organelle traYcking within aVected cells, thereby leading to cellular dysfunction and death (Arima et al., 1998; Tu et al., 1998); however, it is enigmatic how GCIs in oligodendrocytes lead to the degeneration of neurons. Although it generally appears that the proteinaceous inclusions of PD and DLB have similarities to those of MSA, there are some notable diVerences. Most obviously, -syn aggregates are found mainly in neurons of PD and DLB brains, whereas they are located primarily in oligodendrocytes of MSA brains. Second, -syn filaments in GCIs are wider in diameter than those of LBs and LNs and are sometimes found in a twisted formation (Crowther et al., 2000; Spillantini et al., 1998a). In addition, antibodies raised against diVerent epitopes of -syn protein show that most regions of -syn protein are equally exposed in LBs of PD and DLB diseased brains (Duda et al., 2000b; Giasson et al., 2000b); however, some of the epitopes in -syn are more diYcult to recognize in GCIs (Duda et al., 2000b). Another dissimilarity between LBs and GCIs is the accumulation of diVerent associated protein components in these inclusions. LBs of PD and DLB mainly contain -syn along with some ubiquitin and neurofilament subunits; GCIs of MSA mainly contain -syn in addition to B-crystallin, ubiquitin, and - and -tubulin, but lack neurofilament subunits (Gai et al., 1999). It is probable that diVerences in cell types wherein filamentous -syn inclusions form cause the variation in protein components that accumulate in LBs versus
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GCIs and, depending on which proteins interact with -syn within the inclusions, conformational and structural changes of -syn may result.
D. Other Synucleinopathies Neurodegeneration with brain iron accumulation (NBIA-1) known previously as Hallervorden–Spatz disease, is a rare familial and sporadic neurode generative disorder (Dooling et al., 1974; Halliday, 1995; Jellinger, 1973). Disease onset is typically in late adolescence or early adulthood and the disease is unremitting in its progression (Dooling et al., 1974; Jankovic et al., 1985; Jellinger, 1973; Taylor et al., 1996). The clinical symptoms of NBIA-1 include motor disabilities and rigidity, language diYculties, and dementia (Dooling et al., 1974; Jankovic et al., 1985; Jellinger, 1973; Sugiyama et al., 1993; Swaiman, 1991; Taylor et al., 1996). The pathology in NBIA-1 consists of cerebral atrophy and lesioning in the globus pallidus; however, an intense accumulation of iron in the substantia nigra and globus pallidus (which appears as an ‘‘eye of the tiger’’ on magnetic resonance images) is the signature feature of NBIA-1 (Dooling et al., 1974; Halliday, 1995; Jankovic et al., 1985; Jellinger, 1973; Nishiyama et al., 1997; Tripathi et al., 1992). Abnormalities have also been found in the liver and pituitary of patients, and bone marrow biopsies have shown histocytes and osmophilic inclusions (Swaiman, 1991; Zupanc et al., 1990). Mutations in the pantothenate kinase 2 (PANK2) gene, located on chromosome 20p12.3-p13 (Taylor et al., 1996), have been linked to familial forms of NBIA-1 (Zhou et al., 2001). PANK2 is one of the four putative PANKS in humans, and these enzymes are necessary for the synthesis of coenzyme A and are therefore essential in fatty acid metabolism. Many types of mutations of the PANK2 gene, including missense mutations, nonsense mutations, insertions, deletions, and frameshifts, have been found in familial NBIA-1 patients. So far, all mutations in PANK2 are believed to be recessive, suggesting a loss-of-function mutation. Axonal spheroids, LB-like lesions, and glial inclusions composed of insoluble, fibrillar -syn are present in brains of sporadic NBIA-1 patients (Galvin et al., 2000). These inclusions are found mostly throughout the cortex, subcortical regions, and brain stem (Arawaka et al., 1998; Saito et al., 2000; Tu et al., 1998; Wakabayashi et al., 1999). Interestingly, Galvin et al. (2000) also discovered the existence of - and -syn accumulations in some spheroids of NBIA-1 patients. However, it is unclear whether -syn pathology is found in aVected individuals with PANK2 mutations. Many other diseases have joined the list of synucleinopathies, and it is likely that more will be recognized in the future. In addition to those discussed in this chapter, -syn pathology is present in familial AD (Lippa et al.,
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1998), autosomal-recessive juvenile parkinsonism (Kitada et al., 1998), Down’s syndrome (Lippa et al., 1999), the parkinsonism–dementia complex of Guam (Forman et al., 2002; Yamazaki et al., 2000), rapid eye movement sleep behavior disorder (Boeve et al., 1998; Olson et al., 2000), and progressive autonomic failure (Arai et al., 2000; Kaufmann et al., 2001). However, -syn inclusions are distributed less widely in these diseases and/or are not prominent pathological features.
V. Structural Properties of a-Synuclein Protein -Syn is normally found in a ‘‘natively unfolded’’ soluble form in aqueous solutions and is predicted to be in such a form in the cytosol of cells (Eliezer et al., 2001; Murray et al., 2003; Serpell et al., 2000; Weinreb et al., 1996). What makes soluble -syn protein transform into insoluble, filamentous inclusions found in neurodegenerative diseases (Conway et al., 2000a; Crowther et al., 2000; Serpell et al., 2000) has been the focus of studies by many research groups. Recombinant syn proteins are expressed easily in and purified from Escherichia coli, which facilitated the study of -syn polymerization into fibrils. Recombinant WT -syn protein, in addition to recombinant -syn with pathogenic mutations and various C-terminal truncations, readily forms fibrils in vitro (Conway et al., 1998; Crowther et al., 1998; El Agnaf et al., 1998a,b; Giasson et al., 1999; Han et al., 1995; Iwai et al., 1995b; Narhi et al., 1999; Yoshimoto et al., 1995). These fibrils are easily visualized by negative-staining EM and atomic force microscopy (AFM) (Conway et al., 2000a; Giasson et al., 1999; Uversky et al., 2001a). The formation of -syn fibrils has been shown to be temperature, concentration, buVer, and pH dependent (Clejan and Cederbaum, 1989; Dickson et al., 1999; Giasson et al., 1999, 2001; Narayanan and Scarlata, 2001; Paik et al., 1999, 2000; Uversky et al., 2001a,b, 2002). EM analysis shows great similarity between fibrils assembled in vitro to those extracted from pathological inclusions of diseased brains (Conway et al., 1998, 2000a; Crowther et al., 2000; Giasson et al., 1999; Hashimoto et al., 1998; Narhi et al., 1999). The kinetics of in vitro -syn fibril formation have been studied intensely, and this process is nucleation dependent in that fibril formation increases rapidly over time after an initial lag phase (Li et al., 2001; Narhi et al., 1999; Uversky et al., 2001a; Wood et al., 1999). The entire process may consist of a slow initial phase in the conversion of monomeric -syn into a small oligomeric intermediate/protofibril and finally into polymeric mature fibrils (Norris et al., 2003; Uversky et al., 2001a). Adding ‘‘seeds’’ to monomeric -syn protein reduces the lag phase and allows for faster filament formation (Han et al., 1995; Murray et al., 2003). Moreover, recombinant forms of the A53T and A30P
2. -Synuclein
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-syn proteins form oligomeric protofibrils faster than the WT -syn (Conway et al., 2000b,c). However, A53T mutant -syn forms polymeric fibrils easily and does so even faster than WT -syn (Conway et al., 1998; Giasson et al., 1999; Serpell et al., 2000). The C-terminal region of -syn can modulate the assembly of -syn into fibrils. The negatively charged residues within the carboxy terminus counteract fibril formation; therefore, truncation or mutation of this region allows for the rapid production of filaments in vitro compared to WT -syn protein (Crowther et al., 1998; Murray et al., 2003; Serpell et al., 2000). For example, truncated -syn proteins 1–89, 1–102, and 1–110 demonstrate faster rates of filament formation compared to full-length -syn, and these artificial truncated species of -syn act as seeds to induce rapid WT -syn assembly (Murray et al., 2003). Substitution of negatively charged glutamate residues at positions 104, 105, and 114 and aspartate residue 115 to alanine residues promotes fast fibrillization of -syn as well (Murray et al., 2003). It has also been proposed that proline residues of the C terminus suppress the ability of the protein to form fibrils in vitro (Uversky and Fink, 2002). In addition, it has been suggested that the C terminus of -syn does not bind lipids, making it available for protein–protein interactions (Eliezer et al., 2001). The assembly of -syn into fibrils is associated with a dramatic change in the secondary structure. Circular dichroism data show that recombinant -syn protein in a filamentous form predominantly assumes a -pleated sheet structure (Giasson et al., 2001; Murray et al., 2003; Serpell et al., 2000; Uversky et al., 2001a). This conformation was also demonstrated using amyloid-binding dyes, such as thioflavin T and K114 [(trans,trans)-1bromo-2,5-bis(4-hydroxy)styrylbenzene], to bind fibrils in solution (Conway et al., 2000a; Crystal et al., 2003; Murray et al., 2003; Uversky et al., 2001a) and in situ as pathological inclusions in tissue sections. Furthermore, A53T and A30P mutant -syn proteins form -pleated sheet structures under fibril assembly conditions as shown by circular dichroism and EM (Conway et al., 2000a; Giasson et al., 1999; Lashuel et al., 2002; Serpell et al., 2000). Because -syn fibrillizes readily in vitro, the finding that recombinant -syn protein did not fibrillize or form a sheet was puzzling (Biere et al., 2000; Giasson et al., 2001; Serpell et al., 2000). However, these data correlate well with in vivo data in which -syn protein is not found in fibrillar inclusions in neurodegenerative disease brains. As stated earlier, - and -syn amino acid sequences are very similar except that -syn lacks part of the hydrophobic middle (or so-called NAC) region of -syn. Using recombinant -syn protein with this region deleted, it was shown that part of the NAC region, specifically residues 71–82, is required for -syn fibril formation in vitro (Giasson et al., 2001). Investigation of the -syn peptide 71–82 showed that this peptide will fibrillize by itself, form amyloidogenic -pleated sheets, and seed the rapid fibril formation of WT -syn in vitro
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(Giasson et al., 2001). Since this discovery, antibodies directed against various epitopes of -syn protein have shown that this central hydrophobic region is buried in the core of pathological inclusions and often requires formic acid pretreatment for epitope exposure (Duda et al., 2000b; Giasson et al., 2000b; Murray et al., 2003). Therefore, it is thought that this central hydrophobic NAC region is highly responsible for the ability of -syn to form filaments in vivo. Protofibrils of -syn are functionally diVerent from those of -syn, which may help explain how -syn protofibrils may inhibit the formation of -syn protofibrils in vitro (Park and Lansbury, 2003). Importantly, it has been suggested that small oligomers of -syn stabilized in this form may be a toxic, pathogenic species, and -syn could potentially act as a therapeutic strategy for disease states (Conway et al., 2001; Park and Lansbury, 2003). Ample evidence also supports the notion that the polymerization of -syn is directly responsible for cellular dysfunction and demise (Giasson and Lee, 2003; Goldberg and Lansbury, 2000). In the presence of acidic phospholipids, -syn can change shape and stabilize in an -helical conformation (Davidson et al., 1998; Eliezer et al., 2001), which had been expected due to the presence of the proposed lipidbinding domain in -syn (Clayton and George, 1999; George et al., 1995). Narayanan and Scarlata (2001) used real-time equilibrium methods to determine the types of lipids to which syn proteins bind, and their studies indicate that -syn binds strongly to both negatively charged and neutral lipids, but that this lipid binding did not aVect the secondary structure. However, Jo et al. (2000) and Davidson et al. (1998) reported that -syn binds only acidic lipid vesicles and not neutral lipids in vitro, and upon binding, -syn transforms from random coil conformation to -helix. In addition, Jensen and colleagues (1998) showed that WT -syn binds to vesicles at its N-terminal repeat domain, whereas the A30P mutant -syn disrupts this binding capability. It was shown that the A30P -syn protein is less likely to transform from its unordered, random coil conformation to -helix, possibly due to the insertion of the proline residue, which is known to be a helix breaker (Jo et al., 2002). This lack in conformational change may be responsible for its reduced aYnity to bind lipid vesicles (Jo et al., 2002), and Jensen et al. (1998) proposed that this lack of lipid binding of A30P -syn may eventually lead to the formation of -syn inclusions. McLean et al. (2000) used fluorescence resonance energy transfer (FRET) to determine interactions between -syn protein and lipid membranes in -syn transiently transfected primary cortical neurons and to determine the conformation of -syn in these neurons. In disagreement with other studies, McLean and colleagues (2000) reported a tight association of N and C termini of WT -syn to membranes of these neurons, and A53T and A30P mutant forms of -syn share similar membrane interactions. A separate study by Jo et al. (2000) showed that
2. -Synuclein
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WT -syn disrupts planar lipid bilayers and forms amorphous aggregates and fibrils, whereas A53T -syn did so at a slower rate. These lipid-binding observations may help determine if WT -syn is involved in membrane and/ or synapse maintenance and how this interaction is disrupted in the presence of mutant -syn proteins.
VI. Implications of Oxidative and Nitrative Injury in Synucleinopathies Many hypotheses have been proposed for what leads to the pathology and clinical symptoms of PD and other synucleinopathies. Oxidative injury, which arises from a ‘‘state of imbalance between the production of reactive oxygen species and their clearance by cellular antioxidant systems’’ (Lotharius and Brundin, 2002), has been implicated in the pathophysiology of neurodegenerative disorders. For example, lipid, protein, and DNA oxidation is increased and mitochondrial complex I activity is decreased in the substantia nigra of PD patients’ brains (reviewed in Lotharius and Brundin, 2002). The exposure of nucleic acids to reactive oxygen species can result in strand breakage, cross-linking, and mutations (Breen and Murphy, 1995), whereas amino acids, especially methionine, cysteine, and histidine, are sensitive to oxidative stress in that this can lead to irreversible structural changes in proteins (Davies et al., 1987). In addition, levels of two naturally occurring free radical scavengers, superoxide dismutase and glutathione, are altered in the nigra of PD brains (reviewed in Lotharius and Brundin, 2002; Mann et al., 1992). The intrinsic metabolic properties of dopaminergic neurons are believed to render them more vulnerable to oxidative stress. First, the enzymatic metabolism of DA generates H2O2, which may then generate OH in the iron-mediated Fenton reaction, and second, DA autooxidation forms semiquinones, which allows for the generation of reactive oxygen species. Moreover, nitrative injury was found to exist in synucleinopathy brains when antibodies specific for 3-nitrotyrosine (3-NT) were used to label LBs and LNs in PD and DLB (Duda et al., 2000a). It was later found that much of this labeling was due to nitrated, aggregated -syn protein (Giasson et al., 2000a). Protein nitration (i.e., the formation of 3-NT) is believed to be largely due to the formation of peroxynitrite (ONOO ) (Fig. 2) (Ischiropoulos and al Mehdi, 1995; van der Vliet et al., 1994). ONOO is formed naturally in the reaction between superoxide and nitric oxide (Beckman and Koppenol, 1996; Huie and Padmaja, 1993; Ischiropoulos and Beckman, 2003). Further, because ONOO is a strong oxidant, it can also cause DNA oxidation (Inoue and Kawanishi, 1995), nucleic acid strand breakage (Salgo et al., 1995), lipid peroxidation (Radi et al., 1991a), and oxidation of cysteine, methionine, tryptophan, phenylalanine, and tyrosine residues of proteins (Ischiropoulos, 1998; Radi et al., 1991b; van der Vliet et al., 1994).
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Figure 2 Formation of peroxynitrite from superoxide and nitric oxide and its impact on tyrosine residues of proteins. Peroxynitrite (ONOO ) forms naturally in cells by the reaction of nitric oxide (NO) and superoxide (O2 ). ONOO interacts with and damages proteins by nitration and oxidation. 3-Nitrotyrosine is formed when ONOO nitrates tyrosine residues, and dityrosine cross-linking also results from the ONOO interaction with protein tyrosine residues.
It is still unclear if oxidative and nitrative damage leads to other types of cellular dysfunction, which later involves -syn aggregate formation, or if -syn is first aVected by the oxidative/nitrative insult, which then leads to cellular dysfunction. Furthermore, familial PD patients with mutations in the -syn gene (i.e., A53T, A30P) present with the disease at a much earlier age than sporadic PD patients. It is possible that these mutations render syn more susceptible to oxidative and/or nitrative damage, allowing for disease onset to be much faster. Some cell model systems have shown faster -syn aggregate formation and/or higher levels of cell death in oxidatively stressed cells that express mutant -syn, particularly A53T, than in those cells expressing WT -syn protein or no -syn protein at all (Alves da Costa et al., 2000; Junn and Mouradian, 2002; Ko et al., 2000; Norris et al., 2003; Ostrerova-Golts et al., 2000; Paxinou et al., 2001; Tabrizi et al., 2000). These findings greatly support the idea that oxidative and nitrative mechanisms play a role in the pathophysiology of synucleinopathies.
VII. Effects of Oxidation and Nitration on a-Synuclein Fibril Formation In order to more fully investigate the oxidative/nitrative injury hypothesis, recombinant -syn proteins were exposed to common oxidative and nitrative reagents, and structural and functional changes were analyzed. It was found by Paik et al. and others that -syn recombinant proteins oligomerize when incubated with metals and either cross-linking agents or H2O2 (Paik et al., 1999, 2000; Uversky et al., 2001b). Oxidation can influence the ability of -syn to form fibrils in vitro, but the levels and types of oxidation appear to aVect this process in a variable manner. Uversky et al. (2002) showed that full oxidation at all four methionine (Met) residues prevented fibrillization of
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the protein, yet much lower levels of H2O2, which presumably do not oxidize all four Met residues, do not prevent filament formation (Norris et al., 2003). Yamin et al. (2003a) suggested that extensive Met oxidation stabilizes -syn in its monomeric form, but that the addition of certain metals, namely Ti3+, Zn2+, Al3+, and Pb2+, changes the protein folding and allows for fibril formation. In addition, if fibrils of -syn are already formed, oxidation by metals and H2O2 stabilizes the protein in this fibrillar form (Norris et al., 2003). Another oxidative process that can influence -syn fibrillization is its interaction with DA. Conway et al. (2001) showed that exposure to DA prevents the fibril formation of -syn proteins, and it appears that the -syn is stabilized at a protofibril state by its ‘‘dopaminization.’’ These authors speculate that these data provide further evidence for the ‘‘protofibril toxicity’’ model discussed earlier. Oxidation of tyrosine (Tyr) residues may have an important influence in the formation of stable -syn polymers. These amino acids are easily nitrated and oxidized, and the C terminus of -syn contains a highly conserved region with three Tyr residues (i.e., Tyr 125, Tyr 133, and Tyr 136). When ONOO is used to nitrate -syn in vitro, this process causes oligomerization of the protein through o,o’-dityrosine cross-linking and formation of 3-NT (Krishnan et al., 2003; Norris et al., 2003; Souza et al., 2000a). Also, all four Tyr residues of -syn can be nitrated through this process as shown by the use of highly specific antibodies against nitrated -syn protein (Giasson et al., 2000a; Norris et al., 2003). Furthermore, nitration of recombinant -syn protein by ONOO prevents fibril formation (Norris et al., 2003; Yamin et al., 2003b). As is the case with oxidation, preformed filaments of -syn are protected against chaotropic treatment if stabilized by nitration (Norris et al., 2003). Cell culture has also been used to investigate the involvement of oxidative and nitrative mechanisms, and it was found that the intracellular formation of ONOO (using superoxide and nitric oxide donors) induces the formation of filamentous WT -syn aggregates (Norris et al., 2003; Paxinou et al., 2001). Interestingly, nitrative events are dependent on the presence of Tyr residues in the protein, but these residues are irrelevant in oxidative processes (Norris et al., 2003). Figure 3 depicts a possible model for these oxidative/nitrative events and their eVects on the structure of -syn protein. Monomeric -syn is necessary for membrane/synapse interactions, but this monomeric form can naturally convert to nonfunctional small oligomers/protofibrils or polymeric filaments in vivo. Nitration or extensive oxidation of monomeric -syn promotes the conversion to and stabilizes the protein in protofibril form, preventing its further conversion to mature fibrils. However, oxidation or nitration stabilizes already formed fibrils. Because these fibrils are found in pathogenic brain inclusions, it is more likely that the toxic species of -syn are the mature fibrils that are stabilized by further oxidation and/or nitration,
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Figure 3 Putative model for pathological -syn inclusion formation. (A) Under normal conditions, -syn protein is in a soluble, monomeric form. (B) This structural form of -syn converts to an intermediate, protofibrillar form naturally or is influenced more strongly by oxidation and/or nitration conditions. (C) Unmodified protofibrils of -syn transform to larger filaments of -syn protein. Oxidized -syn protofibrils may form polymeric filaments, depending on the degree of oxidation of the protein. Nitrated -syn protofibrils, however, will not form mature -syn fibrils. The ‘‘protofibril toxicity’’ model proposes that these nitrated and oxidized protofibrils, incapable of forming mature filaments, may be deleterious to the cells. (D) Mature fibrils can be stabilized by oxidative and nitrative modifications, including cross-linking of the polymers. (E) These inclusions of -syn fibrils trap other cellular proteins and can presumably shut down cellular processes and lead to cell death. However, it is still a possibility that -syn inclusions are just a by-product of another cellular mechanism and are not a cause of cell death in neurodegenerative diseases. (See Color Insert.)
although some evidence supports the idea that stable, oxidized/nitrated protofibrils are the noxious species.
VIII. Possible Role of Pesticides and Other Toxic Chemicals in PD As environmental factors have been associated with neurodegenerative diseases, chemical agents, such as pesticides and pollutants, found unnaturally in water and soil are scrutinized as being putative causative elements. Studies have shown that an increased risk of PD exists in people who live on farms or in rural areas, drink well water, or are exposed to agricultural chemicals (Gorell et al., 1998; Hertzman et al., 1990; Liou et al., 1997; Semchuk et al.,
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1993). Also, case-controlled studies in China, Spain, and Canada showed that exposure to pesticides or industrial chemicals increased the risk of PD greatly (Barbeau et al., 1986, 1987; Hertzman et al., 1990; Jimenez-Jimenez et al., 1992; Tanner et al., 1989). Many of these chemicals are believed to act, at least in part, through oxidative mechanisms, as they are capable of generating reactive species, alkylating reduced thiols, and inhibiting mitochondrial complex I (reviewed in Ischiropoulos and Beckman, 2003). For example, a metabolite of MPTP, the contaminant in synthetic heroine, can cause severe parkinsonism in humans (Langston et al., 1983) by inhibiting complex I of the mitochondrial electron transport chain and hence increasing free radical formation (see Section IX) (Nicklas and Heikkila, 1985; Tipton and Singer, 1993). After the discovery that paraquat, a commonly used pesticide, bears structural similarity to the MPTP metabolite, this pesticide, as well as others, came under investigation as a potential instigator of neurodegeneration. In order to further examine the eVects of paraquat exposure, mice and rats have been administered this substance in various ways. Oral administration of paraquat to mice caused behavioral deficits and a decrease in DA and its metabolites in brain regions (Fredriksson et al., 1993). Also, direct intracerebral injection of paraquat into rats caused a dose-dependent depletion of DA in the striatum, marked degeneration in the nigra, and behavioral deficits (Liou et al., 1996). To study the involvement of -syn in these paradigms, intraperitoneal injections of paraquat into mice resulted in an upregulation of -syn expression, formation of -syn-positive inclusions with a -sheet structure (Manning-Bog et al., 2002), and selective dopaminergic nigral degeneration (McCormack et al., 2002). It was also shown that recombinant -syn protein assembled into mature fibrils more quickly in the presence of pesticides, including paraquat (Manning-Bog et al., 2002; Uversky et al., 2001c). Furthermore, control and transgenic (TG) mice expressing human WT or A53T -syn under the control of the tyrosine hydroxylase promoter were exposed to paraquat. Non-TG, control mice that were administered paraquat developed -syn-positive inclusions and nigrostriatal degeneration. However, exposure of paraquat to -syn TG mice resulted in -syn-positive aggregates, but they did not show brain degeneration. These results are puzzling, but they may imply that -syn plays a protective role in pesticide exposure in mice (Manning-Bog et al., 2003). Another highly studied pesticide regarding neurodegeneration is rotenone, a naturally occurring substance that is extracted from plant roots. It is used commonly in vegetable gardens as a pesticide and in lakes and ponds to control fish populations. Studies show that rotenone is also a high-aYnity inhibitor of mitochondrial complex I and crosses lipid membranes easily (Betarbet et al., 2000). Chronic exposure of human neuroblastoma cells to rotenone leads to an increase in soluble -syn expression, which ultimately leads to the accumulation of insoluble -syn and ubiquitin. The cells
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eventually undergo apoptosis, which appears to be due to an increased susceptibility to oxidative stress, as the cells show decreased glutathione levels and increased DNA and protein oxidation (Sherer et al., 2002). Betarbet et al. (2000) showed that a continuous administration of rotenone to rats resulted in highly specific degeneration of the nigrostriatal pathway, motor deficits (including some behaviors reminiscent of resting tremor and hunched posture found in human PD patients), and filamentous intracellular inclusions containing ubiquitin and -syn. Similar studies involving a subcutaneous injection of rotenone to mice resulted in dopaminergic cell death and -syn-positive neuronal inclusions (Sherer et al., 2003).
IX. Transgenic and Nontransgenic Animal Models of Synucleinopathies Intense research has been conducted to generate animal models that closely resemble PD. Before the discovery of the involvement of -syn in neurodegenerative diseases, parkinsonism was examined in rats and mice by chemical administration. The most popular models involved MPTP administration in mice and 6-OHDA infusion in rats (reviewed in Lotharius and Brundin, 2002). Both of these models are believed to induce parkinson-like pathology and/or phenotype through oxidative mechanisms. The active metabolite of MPTP, 1-methyl-4-phenypyridium or MPP+, acts as a selective dopaminergic neurotoxin as it is taken up into dopaminergic cells through DAT. From there, MPP+ is believed to inhibit complex I of the mitochondrial electron transport chain (Nicklas and Heikkila, 1985; Tipton and Singer, 1993). Mice treated with MPTP show selective degeneration of dopaminergic neurons of the nigra and often present with behavioral phenotypes characteristic of human PD. Although MPTP is a widely used method for studying parkinsonism in animals, the characteristic -syn inclusions are not present in mouse brains or in human individuals exposed to MPTP (Langston et al., 1999). As with 6-OHDA administration in rats, this reagent is highly selective in eliminating dopaminergic neurons in the brain but does not lead to inclusion formation. Therefore, to study synucleinopathies in animals more thoroughly and realistically, various WT and mutant -syn TG and -syn KO mice were created. In 2000, Abeliovich et al. reported the generation of the first -syn KO mouse line. These mice show no overt morphological or behavioral abnormalities, especially regarding dopaminergic cell bodies, processes, and terminals. However, these authors noticed an increase in DA release upon repeated electrical stimulation of nerve terminals, which then results in decreased levels of stored DA in the striatum. The authors hypothesized that -syn acts as a negative regulator of DA release and that inhibition of -syn
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function increases synaptic DA release and therefore could aid in the slowing of PD progression (Abeliovich et al., 2000). Another research group created an -syn KO mouse line, which proved to be resistant to the toxicity of MPTP (Dauer et al., 2002). Dopaminergic neurons are spared in these MPTP-treated mice, striatal DA levels are unaltered, and the DAT functions normally. Interestingly, rotenone, which is believed to confer its toxicity through complex I inhibition, is still toxic to these -syn KO mice (Dauer et al., 2002). Masliah et al. (2000) reported the generation of a TG mouse line that expresses human WT -syn in neurons using the platelet-derived growth factor- (PDGF-) promoter. These mice exhibited some behavioral dysfunctions, and histological studies showed the loss of dopaminergic terminals, but not processes, in the striatum. -Syn- and ubiquitin-positive aggregates were present in neurons of the neocortex, hippocampus, and substantia nigra. However, EM examination revealed that these aggregates were dense but not filamentous as they are in human disease brain (Masliah et al., 2000). Nevertheless, coexpression of -syn with -syn in bigenic mice attenuated the eVects of -syn in these TG mice (Hashimoto et al., 2001). Furthermore, attempts to more closely recapitulate features of PD in a TG mouse model were made using the TH promoter to drive the expression of -syn in dopaminergic cells. Although high levels of -syn were detected in dopaminergic cell bodies, -syn aggregates did not develop and cell counts remained unaVected (Matsuoka et al., 2001). Two research groups were more successful in creating a synucleinopathy mouse model. Giasson et al. (2002) and Lee et al. (2002) generated WT and mutant -syn transgenic mice using the prion protein (PrP) promoter. WT and A30P -syn mice showed high expression levels of -syn protein in CNS neurons, but exhibited no parkinsonian-like phenotype or pathology at 14–24 months of age. However, A53T -syn mice developed a severe movement disorder that progressed to paralysis and death (Giasson et al., 2002; Lee et al., 2002). Histological studies showed the presence of filamentous -syn-positive intraneuronal inclusions that are characteristic of LBs in human disease brain (Giasson et al., 2002). Thus, this A53T -syn TG mouse model recapitulates key features of human synucleinopathies, making it an attractive mouse model for pathological, physiological, and pharmacological studies of -syn inclusions and neurodegeneration similar to authentic human diseases.
X. Concluding Remarks The recent recognition of synucleinopathies, the group of neurodegenerative diseases grouped together due to the common presence of -syn
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proteinaceous inclusions, has led to intense investigation of the role of -syn in neurodegeneration. Despite the rapid progress in research on the pathogenesis of -syn inclusions in disease in recent years, the exact role of -syn and other proteins is not completely understood. The changes in the cellular milieu that precipitate the conversion of a monomeric soluble protein to highly insoluble forms, leading to brain amyloid deposits, are still unclear. Hence, many studies now focus on whether -syn inclusions are detrimental to the cell or if another form of -syn (i.e., protofibril) is truly the toxic cellular species. Further investigations using WT and mutant recombinant proteins, various cell models, and TG mice will provide more insight into the pathobiology of these disorders and will hopefully lead to the development of better treatments for patients with a synucleinopathy disease.
Acknowledgments Inclusion micrographs in Table I were generously donated by Mark S. Forman, M.D., Ph.D., and Benoit I. Giasson, Ph.D., from the Center for Neurodegenerative Disease Research at the University of Pennsylvania, Philadelphia.
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Structure and Function of Eukaryotic DNA Methyltransferases Taiping Chen* and En Li *,{ *Cutaneous Biology Research Center and Cardiovascular Research Center Massachusetts General Hospital Harvard Medical School Charlestown, Massachusetts 02129 { Novartis Institutes for Biomedical Research Cambridge, Massachusetts 02139
I. DNA Methylation: A Heritable Epigenetic Mark II. Eukaryotic DNA Methyltransferases A. The Dnmt1 Class B. The Dnmt2 Class C. The Dnmt3 Class D. The Chromomethylase Class E. The Masc1 Class III. Function of DNA Methylation A. DNA Methylation and Genome Stability B. DNA Methylation and Gene Expression C. DNA Methylation and Development IV. Concluding Remarks Acknowledgments References
DNA methylation is a common epigenetic modification found in eukaryotic organisms ranging from fungi to mammals. Over the past 15 years, a number of eukaryotic DNA methyltransferases have been identified from various model organisms. These enzymes exhibit distinct biochemical properties and biological functions, partly due to their structural diVerences. The highly variable N-terminal extensions of these enzymes harbor various evolutionarily conserved domains and motifs, some of which have been shown to be involved in functional specializations. DNA methylation has divergent functions in diVerent organisms, consistent with the notion that it is a dynamically evolving mechanism that can be adapted to fulfill various functions. Genetic studies using model organisms have provided evidence suggesting the progressive integration of DNA methylation into eukaryotic developmental programs during evolution. ß 2004, Elsevier Inc. Current Topics in Developmental Biology, Vol. 60 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00
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Table I DNA Methylation in Model Organisms
S. cerevisiae S. pombe Neurospora Ascobolus Arabidopsis C. elegans Drosophila Xenopus Mouse
CpG methylation
CpNpG methylation
— — Commona Common Common — Rare Predominant Predominant
— — Rare Rare Common — Rare Rare Rare
Asymmetric methylation
Common Common Common Predominant Rare Rare
a Relative frequencies of methylated cytosines in diVerent sequence contexts in each organism.
Methylation of the C5 position of cytosine is a common covalent modification found in the genomic DNA of many eukaryotic organisms, including fungi, plants, and vertebrate and invertebrate animals. However, DNA methylation is not universal among eukaryotes, as some organisms, such as the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans, lack detectable methylation and do not have conventional DNA methyltransferase genes in their genomes (Colot and Rossignol, 1999) (Table I). While the biological significance and function of DNA methylation vary greatly among species, genetic studies have demonstrated the involvement of DNA methylation in various aspects of development in several model organisms. In mammals, DNA methylation is essential for embryonic development and plays important roles in gene expression, genomic imprinting, X chromosome inactivation, and maintenance of genome integrity. Aberrant changes of genomic methylation patterns or abnormal interpretation of DNA methylation signals are associated with several human disorders, most notably the immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, Rett syndrome, and cancer. This chapter focuses on the structure and function of eukaryotic DNA methyltransferases. Other aspects of DNA methylation have been the subjects of several other reviews (Bird, 2002; Jaenisch and Bird, 2003; Li, 2002).
I. DNA Methylation: A Heritable Epigenetic Mark In 1975, Holliday and Pugh and Riggs independently proposed a very influential theory, which suggested that DNA methylation represents a heritable epigenetic modification for cellular memory (Holliday and Pugh, 1975;
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Riggs, 1975). This theory was based on the observation that methylated cytosines reside predominantly in CpG dinucleotides in mammalian cells (and in both CpG and CpNpG in plants). Because CpG and CpNpG sites are diagonally symmetric, these sites provide a molecular basis for semiconservative inheritance of DNA methylation patterns after DNA replication (Fig. 1). An important prediction made by the theory was the existence of at least two distinct DNA methyltransferase activities. De novo methyltransferase methylates unmodified DNA and establishes DNA methylation patterns. Maintenance methyltransferase recognizes hemimethylated DNA and copies the preexisting methylation patterns onto the nascent strands after each round of DNA replication (Fig. 1). Consistent with this prediction, many species have multiple DNA methyltransferases and, as discussed later, they exhibit distinct biochemical properties, enzymatic activities, and biological functions.
Figure 1 Inheritance of symmetric (CpG and CpNpG) and asymmetric (CpNpN) methylation. Methylation of cytosines in all contexts is initially established by de novo methyltransferases, which methylate unmodified DNA. After each round of DNA replication, symmetric methylation is maintained by maintenance methyltransferases, which recognize hemimethylated DNA and copy the parental-strand methylation pattern onto the daughter strand, whereas asymmetric methylation is maintained by persistent de novo methylation (black dots represent methyl groups).
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Besides CpG and CpNpG sites, cytosine methylation can occur in other contexts, resulting in asymmetric methylation. Asymmetric methylation is believed to be maintained by persistent de novo methylation (Fig. 1). In keeping with this notion, non-CpG methylation is more prevalent in mouse embryonic stem (ES) cells, which have high de novo methyltransferase activity, than in somatic tissues (Dodge et al., 2002; Ramsahoye et al., 2000). Non-CpG methylation is also detected in other organisms and almost always associates with active de novo methylation (Table I). In plants, a unique phenomenon called RNA-directed DNA methylation leads to de novo methylation of cytosine residues in all sequence contexts (CpG, CpNpG, and nonCpG) within the region of sequence identity between the triggering RNA and the target DNA (Matzke et al., 2001; Pelissier et al., 1999). In fungi, asymmetric methylation is associated with the repeat-induced point mutation (RIP) phenomenon in Neurospora crassa (Selker, 1997) and the methylation-induced premeiotically (MIP) phenomenon in Ascobolus immersus (Rossignol and Faugeron, 1994). The fruit fly Drosophila melanogaster, long thought to be devoid of DNA methylation, has a DNA methyltransferase gene in its genome (Hung et al., 1999; Tweedie et al., 1999) and contains very low levels of methylated cytosines, mostly in the context of non-CpG dinucleotides (Gowher et al., 2000; Lyko et al., 2000a). The biological function of asymmetric methylation remains unclear. Studies in plants suggest that non-CpG methylation may contribute to gene silencing (Dieguez et al., 1997, 1998) and transposon control (Kato et al., 2003).
II. Eukaryotic DNA Methyltransferases DNA (cytosine-5) methyltransferases (C5-MTases) catalyze the transfer of a methyl group (-CH3) from S-adenosyl-l-methionine (AdoMet) to the C5 position of cytosine residues in DNA. Most of our knowledge about the mechanism of this enzymatic reaction came from studies of bacterial C5-MTases (Cheng and Roberts, 2001). Eukaryotic C5-MTases are larger proteins, but the sequence homology they share with bacterial enzymes and the available experimental data suggest that they act using the same catalytic mechanism (Dong et al., 2001). C5-MTases contain 10 characteristic sequence motifs, 6 of which are strongly conserved (Posfai et al., 1989; Wilson, 1992). Based on structural analysis using X-ray crystallography (Cheng et al., 1993; Dong et al., 2001; Klimasauskas et al., 1994; Reinisch et al., 1995; Roberts and Cheng, 1998), C5-MTases are organized into a two-domain structure. The large domain encompasses most of the conserved motifs and contains the catalytic center in motif IV (PC motif) and the cofactor AdoMet-binding pocket, formed by motifs I and X, as well as a number of residues from motifs II–V. The small domain comprises the variable
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Figure 2 Schematic diagram of representative members of the five classes of eukaryotic DNA methyltransferases. The catalytic domains of these enzymes are conserved (the most conserved signature motifs, I, IV, VI, IX, and X, are shown), but there is little similarity among their N-terminal regulatory domains. PCNA, PCNA-interacting domain; NLS, nuclear localization signal; RFT, replication foci-targeting domain; CXXC, a cysteine-rich Zn2þ-binding domain; BAH, bromo-adjacent homology domain; GK, glycine–lysine repeat hinge region; PWWP, a domain containing a highly conserved ‘‘proline–tryptophan–tryptophan–proline’’ motif; ATRX, an ATRX-related cysteine-rich region containing a C2–C2 zinc finger and an atypical PHD domain; Chromo, chromodomain. (See Color Insert.)
region between motifs VIII and IX and is responsible for DNA target recognition. The two domains form a cleft where the DNA substrate fits with the major groove facing the small domain and the minor groove facing the large domain. The target cytosine flips out of the DNA helix and projects into the concave catalytic pocket. A number of eukaryotic C5-MTases have been identified. A prominent feature of these enzymes (with the exception of Dnmt2 family members) is the presence of N-terminal extensions in addition to the catalytic domains that contain the highly conserved C5-MTase motifs (Fig. 2). The highly variable nature of these extensions is believed to be the structural basis for the diVerences in biochemical properties and biological functions displayed by various enzymes. Based on structural and functional similarities, the known eukaryotic C5-MTases can be grouped into several classes, represented by the mammalian Dnmt1, Dnmt2, Dnmt3a/3b, the plant chromomethylases (CMTs), and the Ascobolus Masc1, respectively (Table II).
A. The Dnmt1 Class The first eukaryotic DNA methyltransferase gene, Dnmt1, was cloned from murine cells through protein purification and subsequent peptide sequencing (Bestor et al., 1988). The Dnmt1 locus has several transcription start sites
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Chen and Li Eukaryotic DNA Methyltransferases and Their Functions
Genes Dnmt1 class Dnmt1
Species
Major activity
Mouse
Maintenance
xDnmt1
Xenopus
Maintenance
MET1
Arabidopsis Maintenance
Masc2 Dim-2
Ascobolus ? Neurospora Maintenance de novo?
Dnmt2 class Dnmt2 dDnmt2
Pmt1 Dnmt3 class Dnmt3a
Mouse Drosophila
No activity? De novo (non-CpG)
S. pombe
No activity
Major phenotypes of mutants
Global demethylation, embryonic lethality at E9.5, abnormal expression of imprinted genes, ectopic X chromosome inactivation, transcription of IAP Premature zygotic gene expression, embryonic defects, apoptosis of embryonic cells Global loss of CpG methylation, activation of transposons, abnormal gene expression, late flowering, floral homeotic transformations No obvious phenotypes Global demethylation, activation of transposons, no developmental phenotypes No obvious phenotypes Depletion by RNAi results in global demethylation, but no obvious developmental phenotypes No obvious phenotypes in mutant haploid cells
De novo, repair Postnatal lethal around 4 weeks, male sterility, failure to establish methylation imprints Dnmt3b Mouse De novo, repair Demethylation of minor satellite DNA, neural tube defects, embryonic lethality around E14.5, Dnmt3a Dnmt3b double mutants fail to initiate de novo methylation after implantation and embryos die around E9.5, double mutant ES cells show progressive global demethylation Dnmt3L Mouse No activity Failure to establish maternal methylation imprints, male sterility DRM1 and 2 Arabidopsis De novo Failure to initiate de novo methylation, loss of asymmetric methylation, drm1 drm2 cmt3 triple mutants show developmental retardation, reduced plant size, and partial sterility
CMT class CMT1 CMT2 CMT3 Masc1 class Masc1 Rid
Mouse
Arabidopsis No activity Arabidopsis ? Arabidopsis Maintenance (CpNpG)
? ? Global loss of CpNpG methylation, activation of transposons, abnormal gene expression
Ascobolus De novo? Neurospora De novo?
Defects in MIP Defects in RIP
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(Mertineit et al., 1998; Rouleau et al., 1992). A somatic cell-specific promoter is activated shortly after implantation, and it drives the expression of a transcript that includes a somatic cell-specific exon (exon 1s). Translation initiation within exon 1s results in the full-length Dnmt1 protein, which consists of 1620 amino acids (Tucker et al., 1996; Yoder et al., 1996). An upstream oocyte-specific promoter drives the expression of a transcript that contains an oocyte-specific exon (exon 1o), which causes translation to initiate within exon 4. The resulting protein, Dnmt1o, lacks the N-terminal 118 amino acids of the somatic form (Mertineit et al., 1998). Both Dnmt1 and Dnmt1o are enzymatically active (Ding and Chaillet, 2002; Gaudet et al., 1998). The human DNMT1 protein, which consists of 1616 amino acids, is 78% identical to mouse Dnmt1 at the amino acid level (Tucker et al., 1996; Yen et al., 1992; Yoder et al., 1996). Dnmt1 is expressed constitutively in proliferating cells and ubiquitously in somatic tissues throughout mammalian development. The enzymatic properties of Dnmt1 have been studied extensively using in vitro assays and in vivo genetic approaches. Although Dnmt1 can methylate both unmethylated and hemimethylated CpG dinucleotides in vitro, its activity toward hemimethylated substrates is 5- to 50-fold higher, depending on the specific study (Hitt et al., 1988; Pradhan et al., 1999; Ruchirawat et al., 1987; Yoder et al., 1997a). Dnmt1 is a nuclear protein, but its localization changes dynamically during the cell cycle. Dnmt1 localizes diVusely in the nucleoplasm during G1 and G2 phases but associates with replication foci during the S phase (Leonhardt et al., 1992), suggesting that Dnmt1-mediated methylation is coupled to DNA replication. Importantly, inactivation of Dnmt1 in ES cells and mice leads to global demethylation of genomic DNA, but has little eVect on de novo methylation of newly integrated retrovirus DNA (Lei et al., 1996; Li et al., 1992). Overexpression of Dnmt1 in the absence of the de novo methyltransferases Dnmt3a and Dnmt3b (see later) fails to induce de novo methylation in mouse ES cells (Chen et al., 2003) or in Drosophila (Lyko et al., 1999). Furthermore, genetic studies have shown that Dnmt1o functions as a maintenance methyltransferase in oocytes and preimplantation embryos and is essential for maintaining, but not establishing, allele-specific methylation at imprinted loci (Howell et al., 2001), in contrast to a previous prediction that Dnmt1o may carry out de novo methylation during oogenesis (Mertineit et al., 1998). Taken together, these findings demonstrate that Dnmt1 represents the major mammalian enzyme responsible for the maintenance of CpG methylation and that it alone has little or no de novo methyltransferase activity. Because most bacterial C5-MTases, which do not display maintenance activity, lack an N-terminal domain, the unique maintenance methyltransferase activity of Dnmt1 is thought to be determined by its large N-terminal region. However, the domain responsible for such intrinsic maintenance
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methylation activity has yet to be identified. Several functional domains have been defined in the N-terminal region of Dnmt1 (Fig. 2). These include a nuclear localization signal (NLS) and several domains involved in targeting the enzyme to replication foci during the S phase. Initially, a single region (amino acids 325–573) was thought to be responsible for targeting to replication foci and, therefore, designated the replication foci-targeting (RFT) domain (Leonhardt et al., 1992). Later studies indicated the involvement of other regions as well, including a domain that mediates the interaction with proliferating cell nuclear antigen (PCNA, an auxiliary factor for DNA replication and repair) (Chuang et al., 1997) and a bromo-adjacent homology (BAH) domain that is implicated in mediating protein–protein interactions (Callebaut et al., 1999; Liu et al., 1998). Between the replication foci-targeting domain and the BAH domain lies a cysteine-rich CXXC domain, a conserved Zn2þ-binding sequence that is also present in a number of other proteins, including trithorax (HRX) (Ma et al., 1993), CpG-binding protein (CGBP) (Voo et al., 2000), and methyl-CpG binding domain protein 1 (MBD1) (Cross et al., 1997; Hendrich and Bird, 1998). Studies have shown that the CXXC domains of these proteins are able to bind DNA sequences containing unmethylated CpG dinucleotides (Birke et al., 2002; Fujita et al., 1999; Lee et al., 2001a; Voo et al., 2000). The N-terminal region of Dnmt1 is linked to the C-terminal catalytic domain via a stretch of alternating glycyl and lysyl residues. The functional role of these GK repeats is unclear. Homologs of Dnmt1 have been identified in many other eukaryotes. Arabidopsis thaliana has at least three classes of DNA methyltransferase genes (Finnegan and Kovac, 2000; Martienssen and Colot, 2001). The MET1 class is most similar to Dnmt1 in both sequence and function. The MET1 cDNA was initially isolated in a cDNA library screening with a genomic DNA probe generated by polymerase chain reaction (PCR) using degenerate oligonucleotides, based on homology between prokaryotic C5-MTases and mouse Dnmt1. It encodes a protein of 1534 amino acids, with the catalytic domain and the N-terminal region showing 50 and 24% homology, respectively, to the corresponding parts of Dnmt1 (Finnegan and Dennis, 1993). The catalytic domain, which has 8 of the 10 signature motifs, is separated from the N-terminal region by a short glycine/lycine-rich sequence that resembles the GK repeats in Dnmt1. While the functional domains of MET1 have not been well defined, the N-terminal region harbors a putative NLS and a BAH domain, but lacks the Zn2þ-binding CXXC domain (Finnegan and Dennis, 1993). Loss-of-function MET1 mutants (also known as ddm2 mutants) and antisense MET1 transgenic plants show a severe loss of CpG methylation and a slight reduction of CpNpG and asymmetric methylation (Bartee and Bender, 2001; Cao and Jacobsen, 2002a; Finnegan et al., 1996; Kankel et al., 2003; Kishimoto et al., 2001; Ronemus et al., 1996). Thus, the major function of MET1 is to maintain
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CpG methylation. The reduction of non-CpG methylation observed in MET1 mutants might be a secondary eVect caused by the primary loss of CpG methylation (Cao and Jacobsen, 2002a). In Ascobolus immersus, two putative methyltransferase genes, masc1 and masc2, have been identified (Martienssen and Colot, 2001). The masc2 gene encodes a protein similar to Dnmt1 in structural organization: a large N-terminal region and a C-terminal catalytic domain containing all 10 conserved motifs arranged in canonical order (Chernov et al., 1997; Goyon, 1998). Purified Masc2 protein expressed in baculovirus-infected insect cells displays C5-MTase activity in vitro (Chernov et al., 1997). However, disruption of the masc2 gene alone or both the masc1 and masc2 genes had no obvious eVect on maintenance of the preexisting methylation of single gene copies previously subjected to MIP, natural retroelement-like repeats, and tandemly repeated rDNA. The masc2 mutation did not alter MIP or the de novo methylation process that operates in vegetative cells, nor did it impair de novo methylation during the meiotic process (Malagnac et al., 1999). These results suggest the existence of at least a third C5-MTase gene that is responsible for maintenance methylation in A. immersus. The Neurospora crassa genome contains two putative C5-MTase genes, dim-2 (defective in methylation) and rid (RIP defective). Dim-2 encodes a 1454 amino acid protein, including a C-terminal catalytic domain that is homologous to those of Dnmt1, MET1, and Masc2 and a large N-terminal region that is less conserved. Notable sequence features include a putative NLS, a BAH domain, and an ATP/GTP-binding motif in the N-terminal region and a 220 amino acid segment rich in acidic residues downstream of the conserved C5-MTase motifs in the C-terminal region (Kouzminova and Selker, 2001). Disruption of the dim-2 gene had no eVect on RIP but eliminated DNA methylation completely (Foss et al., 1995; Kouzminova and Selker, 2001). As discussed later, rid is essential for RIP, but not required for methylation in vegetative tissues. These findings led to the conclusion that DIM-2 is responsible for all DNA methylation in vegetative tissues (Kouzminova and Selker, 2001). While it is possible that DIM-2 has both de novo and maintenance methylation activities, expression of DIM-2 in a heterologous system such as Drosophila would help confirm whether DIM-2 is able to carry out de novo methylation in the absence of RID.
B. The Dnmt2 Class By searching EST databases, several groups independently identified a second putative mammalian C5-MTase gene, named Dnmt2, which encodes a protein of 391 amino acids in human or 415 amino acids in mouse (Okano et al., 1998b; Van den Wyngaert et al., 1998; Yoder and Bestor,
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1998). Interestingly, Dnmt2 appears to be the most conserved eukaryotic C5-MTase gene, as its homologs have been identified in many species, including some organisms that have little or no methylated cytosine, such as the fission yeast Schizosaccharomyces pombe and the fruit fly Drosophila (Hung et al., 1999; Lyko et al., 2000b; Tweedie et al., 1999; Wilkinson et al., 1995). In fact, the Dnmt2-like genes pmt1 and dDnmt2 each represent the only putative C5-MTase gene present in the fully sequenced genomes of S. pombe and Drosophila. The protein products of pmt1 and dDnmt2 are 330 and 345 amino acids, respectively, and they are 42–50% identical to Dnmt2 at the amino acid level (Hung et al., 1999; Okano et al., 1998b; Wilkinson et al., 1995). Structurally, Dnmt2 family members are more closely related to prokaryotic C5-MTases than to other eukaryotic counterparts in that they all lack an N-terminal regulatory domain (Fig. 2). Human DNMT2 has been shown to form a two-domain structure that closely resembles M.HhaI, a prokaryotic C5-MTase (Dong et al., 2001). Despite the presence of all 10 conserved C5-MTase motifs and the ability to bind DNA (Dong et al., 2001), Dnmt2 and its family members do not have detectable catalytic activity in standard in vitro assays (Okano et al., 1998b; Tweedie et al., 1999; Van den Wyngaert et al., 1998; Wilkinson et al., 1995). pmt1 is unlikely to encode a functional C5-MTase because its protein product contains the sequence Pro-Ser-Cys instead of Pro-Pro-Cys at the active site in motif IV (Wilkinson et al., 1995). Indeed, deletion of the Ser residue has been reported to create an active enzyme that methylates the internal cytosine within the sequence CCWGG (W ¼ A or T), the same sequence that is modified by the product of the Escherichia coli dcm gene (Pinarbasi et al., 1996). While the reason other Dnmt2 family members are inactive in vitro is unknown, it is possible that their activity requires other factors or that the DNA substrates used in in vitro assays do not contain their preferred target sites. Studies have shown that at least some Dnmt2-like proteins are functional C5-MTases in vivo, and their primary target appears to be non-CpG sites (Kunert et al., 2003; Liu et al., 2003; Tang et al., 2003). It remains to be determined whether mammalian Dnmt2 is involved in non-CpG methylation as well. Genetic evidence, however, has demonstrated that Dnmt2 does not play a major role in global de novo or maintenance methylation of CpG sites in mammals. Targeted disruption of the Dnmt2 gene in mouse ES cells has no eVect on preexisting genomic methylation patterns or on the ability to methylate newly integrated retrovirus DNA de novo (Okano et al., 1998b). Dnmt2 is also not essential for mammalian development, as mice homozygous for a Dnmt2 mutation are viable and fertile with no obvious defects (M. Okano and E. Li, unpublished results). Similarly, it has been shown that depletion of dDnmt2 by RNA interference (RNAi) has no detectable eVect on the embryonic development of Drosophila (Kunert et al., 2003).
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C. The Dnmt3 Class Following the identification of Dnmt1, it was debated for a long time as to whether de novo methylation and maintenance methylation in mammals are carried out by Dnmt1 alone or by two or more distinct enzymes. The finding that mouse ES cells homozygous for a null Dnmt1 mutation contained residual levels of methyl cytosine and retained the ability to methylate provirus DNA de novo provided the first genetic evidence for the existence of independently encoded de novo methyltransferases (Lei et al., 1996). The identification of Dnmt3a and Dnmt3b genes in human and mouse (Okano et al., 1998a) led to the eventual settlement of the debate. Several lines of evidence strongly suggest that Dnmt3a and Dnmt3b function primarily as de novo methyltransferases. First, the expression of these genes correlates with de novo methylation during mouse development. Dnmt3a and Dnmt3b are highly expressed in ES cells, early embryos, and developing germ cells, where de novo methylation is known to take place, but are downregulated in somatic tissues of postnatal animals (Okano et al., 1998a). Second, recombinant Dnmt3a and Dnmt3b proteins methylate CpG dinucleotides in various native and synthetic DNA substrates in vitro and show no preference for hemimethylated DNA (Aoki et al., 2001; Okano et al., 1998a). Third, Dnmt3a and Dnmt3b cause de novo methylation when expressed ectopically in mammalian cells or transgenic flies (Chen et al., 2003; Hsieh, 1999; Lyko et al., 1999). Fourth, inactivation of both Dnmt3a and Dnmt3b by gene targeting blocks de novo methylation in ES cells and early embryos, as well as for de novo methylation of imprinted genes in the germ cells (Hata et al., 2002; Okano et al., 1999). Consistent with genetic data obtained from the murine system, mutations in human DNMT3B have been shown to cause ICF syndrome (Hansen et al., 1999; Okano et al., 1999; Xu et al., 1999), a rare autosomal-recessive disorder characterized by hypomethylation of classical satellite DNA (Jeanpierre et al., 1993). The majority of ICF mutations identified so far are missense mutations that occur in the catalytic domain (Wijmenga et al., 2000). It should be noted that the de novo methyltransferase activity of Dnmt3a and Dnmt3b is not only responsible for the establishment of DNA methylation patterns, but is also essential for the faithful inheritance of these patterns during mitosis. Studies have demonstrated that both Dnmt1 and Dnmt3 families of methyltransferases are required for the stable maintenance of global methylation patterns in mouse ES cells, and these two types of enzymes have distinct and nonredundant functions but act cooperatively in this respect (Chen et al., 2003; Liang et al., 2002; Okano et al., 1999). It has been proposed that Dnmt1 is the major maintenance methyltransferase, which, in association with the DNA replication machinery, methylates hemimethylated CpG sites with high eYciency but not absolute accuracy,
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whereas Dnmt3a and Dnmt3b, via their de novo methylation activity, function as methylation repair enzymes by filling the gaps of hemimethylated CpG sites missed by Dnmt1 (Chen et al., 2003). In human cancer cell lines, however, the roles of DNMT1 and DNMT3 families of methyltransferases in maintaining methylation patterns are still being debated. Rhee et al., (2000, 2002) showed that targeted disruption of DNMT1 or DNMT3B in HCT116 colorectal carcinoma cells caused only a minor reduction in CpG methylation, whereas disruption of both DNMT1 and DNMT3B resulted in a severe loss of methylation, suggesting that the two enzymes have redundant functions in maintaining CpG methylation. In contrast, Robert et al. (2003) showed that selective depletion of DNMT1 alone using antisense or siRNA in HCT116 and other human cancer cell lines resulted in global and gene-specific demethylation, consistent with the current view that DNMT1 is the major maintenance enzyme. More studies are necessary to determine whether normal and cancer cells use diVerent mechanisms to maintain DNA methylation patterns. The murine Dnmt3a gene encodes at least two protein products, Dnmt3a and Dnmt3a2, which consist of 908 and 689 amino acids, respectively. These isoforms derive from transcripts initiated from two diVerent promoters and, as a result, Dnmt3a2 lacks the N-terminal region of Dnmt3a (Chen et al., 2002; Okano et al., 1998a; Xie et al., 1999). The human DNMT3A and DNMT3A2 proteins have 912 and 689 amino acids, respectively, and show high sequence identity to their murine counterparts (98%). Dnmt3a and Dnmt3a2 are both enzymatically active in vitro and in vivo, but they show diVerent localization patterns (Chen et al., 2002, 2003). Dnmt3a is concentrated in heterochromatic foci, whereas Dnmt3a2 localizes diVusely in the nucleus (Bachman et al., 2001; Chen et al., 2002). The Dnmt3b gene encodes multiple isoforms through alternative splicing of exons 10, 21, and/or 22. At least five human isoforms (DNMT3B1–DNMT3B5) and four murine isoforms (Dnmt3b1, Dnmt3b2, Dnmt3b3, and Dnmt3b6) have been identified, and the longest forms from human (DNMT3B1) and mouse (Dnmt3b1) consist of 853 and 859 amino acids, respectively, and show 94% sequence identity (Chen et al., 2002; Hansen et al., 1999; Okano et al., 1998a; Robertson et al., 1999; Xie et al., 1999). Dnmt3b1 and Dnmt3b2 are active enzymes, as shown by in vitro and in vivo methyltransferase assays, whereas the other Dnmt3b isoforms are inactive because they lack either part of motif IX or both motifs IX and X in the catalytic domain (Aoki et al., 2001; Chen et al., 2003; Hsieh, 1999; Okano et al., 1998a). Like Dnmt3a, Dnmt3b1 has been shown to localize to heterochromatin (Bachman et al., 2001). Dnmt3a/3b isoforms show diVerent expression patterns during development. Dnmt3a2 and Dnmt3b1 are highly expressed in ES cells, germ cells, and early embryos, but are almost undetectable in most somatic tissues, whereas Dnmt3a and Dnmt3b3 are expressed at low levels in almost
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all somatic tissues and cell lines examined (Chen et al., 2002). Human DNMT3B4 and DNMT3B5 are expressed predominantly in the testis (Robertson et al., 1999), and mouse Dnmt3b6 is detected only in ES cells (Chen et al., 2002). Dnmt3a2 and Dnmt3b1 are probably the major enzymes that carry out de novo methylation in early postimplantation embryos to establish the initial methylation pattern, and Dnmt3a, in cooperation with Dnmt1, may be involved in maintaining tissue-specific methylation patterns (Chen et al., 2003). Dnmt3b3 and the other ‘‘inactive’’ Dnmt3b isoforms may function as negative regulators of DNA methylation (Chen et al., 2003; Saito et al., 2002). The Dnmt3a and Dnmt3b proteins are very similar in structural organizations (Fig. 2). Sequence comparison of the catalytic domain with prokaryotic and other eukaryotic C5-MTases indicates that the Dnmt3 family of methyltransferases is related more closely to type II bacterial C5-MTases than to other eukaryotic enzymes, including Dnmt1 and Dnmt2 family members, suggesting that the Dnmt3 family probably originates from a distinct ancestral prokaryotic C5-MTase in evolution. The N-terminal extensions of Dnmt3a and Dnmt3b contain a variable region (280 amino acids in Dnmt3a, 60 amino acids in Dnmt3a2, and 220 amino acids in Dnmt3b proteins) followed by two conserved regions, i.e., the PWWP domain and a cysteine-rich region (Okano et al., 1998a; Xie et al., 1999). The cysteine-rich region is unrelated to the CXXC domain of Dnmt1, but shares homology with a similar region found in ATRX, a member of the SNF2/SWI family (Picketts et al., 1996). This region is often referred to as a plant homeodomain (PHD) in the literature. In fact, it consists of a C2–C2 zinc finger and a PHD-like sequence that lacks the characteristic histidine residue present in typical PHD domains (Aasland et al., 1995). Dnmt3a and Dnmt3b has been shown to interact with HDAC1 and RP58 (a putative transcriptional repressor) via the ATRX-related region (Bachman et al., 2001; Fuks et al., 2001). The role of the PWWP domain in the function of the Dnmt3 family of enzymes is still unknown. The PWWP domain is a protein module characterized by a region of sequence similarity of 100–150 amino acids containing a highly conserved ‘‘proline–tryptophan–tryptophan–proline’’ motif (Stec et al., 2000). The Dnmt3a and Dnmt3b PWWP domains span 143 and 135 amino acids, respectively, with a conserved SWWP motif (Qiu et al., 2002). The crystal structure of the Dnmt3b PWWP domain reveals that the N-terminal half of the domain resembles a barrel-like five-stranded structure and that the C-terminal half contains a five-helix bundle, and the two halves are packed against each other to form a single structural module (Qiu et al., 2002). The presence of a basic (positively charged) area on the surface of the structure and the ability of the Dnmt3b PWWP domain alone to bind DNA in vitro led to the proposal that this domain functions as a DNA-binding module
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(Qiu et al., 2002). However, most of the positively charged residues believed to be involved in DNA binding are not conserved (Slater et al., 2003), suggesting that DNA binding may not be a universal property of the PWWP domain. In fact, the barrel of the PWWP domain is remarkably similar to those of the Tudor domain and the chromodomain, which have both been shown to mediate protein–protein interactions (Ball et al., 1997; Jacobs and Khorasanizadeh, 2002; Maurer-Stroh et al., 2003; Nielsen et al., 2002; Selenko et al., 2001). Therefore, it is likely that the PWWP domain also has a protein-binding function. The PWWP domain has been found in several dozens of eukaryotic proteins, many of which are known to associate with chromatin (Qiu et al., 2002; Stec et al., 2000). Indeed, we have found that the PWWP domain of Dnmt3a and Dnmt3b is required for these enzymes to localize to heterochromatin (Chen and Li, unpublished results). It is likely that the PWWP domain interacts with a chromatin component and thus plays a role in targeting the Dnmt3 family of enzymes to certain genomic regions. The importance of the PWWP domain for the function of the Dnmt3 family of methyltransferases is underscored by the identification of a missense mutation (S270P) in the DNMT3B PWWP domain from two patients with ICF syndrome (Shirohzu et al., 2002). The highly conserved serine 270 is part of strand 4 in the -barrel structure (Qiu et al., 2002; Slater et al., 2003), and the mutation may have a profound eVect on the overall folding of the PWWP domain. A third member of the Dnmt3 family, DNMT3L (DNMT3-like), was originally isolated by database analysis of the human genome sequence (Aapola et al., 2000). Its murine homolog, Dnmt3L, was subsequently identified (Aapola et al., 2001; Hata et al., 2002). The human and mouse Dnmt3L proteins consist of 387 and 421 amino acids, respectively, and share 60% sequence identity. Dnmt3L contains an ATRX-related domain that is closely related to that of Dnmt3a and Dnmt3b, but lacks a PWWP domain. Its C-terminal region is related to the catalytic domain of Dnmt3a and Dnmt3b, but lacks some residues known to be critical for enzymatic activity, including the PC dipeptide at the active site in motif IV and the ENV sequence in motif VI (Aapola et al., 2000, 2001; Hata et al., 2002). Recombinant Dnmt3L protein has no methyltransferase activity in vitro (Hata et al., 2002). Therefore, it is highly unlikely that Dnmt3L would encode a functional C5-MTase. However, genetic studies have demonstrated that Dnmt3L is essential for the establishment of maternal methylation imprints and the appropriate expression of maternally imprinted genes, suggesting that Dnmt3L may function as a regulator of DNA methylation (Bourc’his et al., 2001; Hata et al., 2002). The expression pattern of Dnmt3L is strikingly similar to that of Dnmt3a and Dnmt3b during mouse development (Hata et al., 2002). The Dnmt3L protein has been shown to interact with Dnmt3a and Dnmt3b in the nuclei of mammalian cells (Hata et al., 2002). When
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overexpressed in human cells, DNMT3L stimulates de novo methylation by Dnmt3a (Chedin et al., 2002). Moreover, Dnmt3L has been shown to associate with HDAC1 via its ATRX-related domain and function as a transcriptional repressor in reporter systems (Aapola et al., 2002; Deplus et al., 2002). Taken together, Dnmt3L may regulate genomic imprinting by targeting Dnmt3a and Dnmt3b to imprinted loci, by altering the accessibility of these enzymes to imprinted loci, or by enhancing the activity of these enzymes. The function of Dnmt3 class of enzymes is likely conserved, as homologous enzymes have been found in other vertebrates and plant. A putative zebrafish homolog has been identified in the EST database (GenBank accession No. AF135438). The C-terminal portion of the deduced zebrafish protein shares significant similarity to the corresponding portion of the mammalian Dnmt3 family, whereas the N-terminal portion is not homologous to any known protein (Xie et al., 1999). It remains to be determined whether this protein is an active methyltransferase and what its function is. Several C5-MTase genes that show sequence similarity to mammalian Dnmt3 have been identified in plants, including ZMET3 from maize and DRM1 and DRM2 from Arabidopsis (Cao et al., 2000). The catalytic domains of these gene products show an average of 28% amino acid identity to those of Dnmt3a and Dnmt3b, but there is no significant similarity between the N-terminal regions of the plant and animal proteins. These plant methyltransferases exhibit a unique arrangement of the conserved catalytic motifs. Unlike all other known eukaryotic C5-MTase, which show a consecutive order of motifs I–X from the N terminus to the C terminus, the DRM/Zmet3 proteins display a rearranged order so that motifs VI–X precede motifs I–V (DRM stands for domains rearranged methyltransferase) (Cao et al., 2000). Rearrangement of methyltransferase motifs also occurs in bacterial C5-MTases (Xu et al., 1997), suggesting that the usual arrangement is not absolutely required for function. It is possible that the juxtaposition of motifs I and X in the primary sequence does not have a significant eVect on the overall folding of the catalytic domain because these motifs are adjacent to each other in the tertiary structure (Cheng et al., 1993; Dong et al., 2001; Klimasauskas et al., 1994; Reinisch et al., 1995). DRM/Zmet3 proteins lack the PWWP and the ATRX-related domains present in Dnmt3a and Dnmt3b, but contain a series of ubiquitin-associated (UBA) domains in their N termini (Cao et al., 2000). The UBA domain, which is often found in proteins involved in the ubiquitin pathway, has been implicated in mediating protein–protein interactions (Hofmann and Bucher, 1996). Genetic evidence suggests that the DRM genes encode the major de novo methyltransferases in Arabidopsis. Inactivation of both DRM1 and DRM2 blocks the de novo methylation of cytosines in all known sequence contexts (CpG, CpNpG, and asymmetric), but show no eVect on the maintenance of CpG methylation and gene silencing (Cao and Jacobsen, 2002a; Cao and
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Jacobsen, 2002b). However, DRM proteins are required for maintaining the methylation of asymmetric sites (Cao and Jacobsen, 2002a), consistent with the notion that asymmetric methylation is maintained by the persistent activity of de novo methyltransferases.
D. The Chromomethylase Class Chromomethylases were first identified in a database search for genes containing chromodomains (HenikoV and Comai, 1998; Rose et al., 1998). This class of DNA methyltransferases is unique to the plant kingdom, and there are three related CMT genes in Arabidopsis: CMT1, CMT2, and CMT3. CMT1 has been deemed nonessential because several Arabidopsis ecotypes contain genes with a retroelement insertion that disrupts the coding region or point mutations that result in frameshift or truncated proteins (HenikoV and Comai, 1998). CMT2 and CMT3 are predicted to encode functional proteins (McCallum et al., 2000; Rose et al., 1998). Several studies have provided genetic evidence that CMT3 functions primarily as a maintenance enzyme for CpNpG methylation. CMT3 loss-of-function mutants show a genome-wide loss of CpNpG methylation and a reduction of asymmetric methylation at some loci, but no significant changes in global CpG methylation (Bartee et al., 2001; Cao and Jacobsen, 2002a; Lindroth et al., 2001; McCallum et al., 2000). A maize CMT-like gene, Zmet2, has also been shown to be essential for maintaining CpNpG methylation (Papa et al., 2001). These findings provide an explanation for the fact that plants contain high levels of CpNpG methylation in their genomes relative to animals. CMT3 has been shown to preferentially methylate transposon-related sequences and to contribute to the silencing of transposons in Arabidopsis (Kato et al., 2003; Papa et al., 2001). Furthermore, CpNpG methylation mediated by CMT3 appears to play a critical role in epigenetic silencing at some loci, such as the SUPERMAN (SUP) and the phosphoribosylanthranilate isomerase (PAI) genes (Bartee and Bender, 2001; Lindroth et al., 2001). The function of CMT2 remains to be determined. A distinguishing feature of CMT proteins is the presence of a chromodomain embedded between catalytic motifs I and IV (Fig. 2). Chromodomains are believed to mediate interactions between chromatin components. However, the molecular mechanisms whereby diVerent chromodomains interact with chromatin appear to diVer. For example, the chromodomain of HP1 proteins has been shown to bind methylated lysine 9 of histone H3 (Bannister et al., 2001; Lachner et al., 2001), whereas the chromodomain of MOF1 has been shown to bind RNA (Akhtar et al., 2000). While the function of the chromodomain in CMTs remains to be determined, an intriguing possibility is that CMT enzymes interact with small RNAs through the
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chromodomain and mediate RNA-directed DNA methylation. It is also conceivable that the chromodomain of CMTs may target these enzymes to specific genomic regions by interacting with certain forms of chromatin modifications. The N-terminal regions of CMTs are smaller than those found in the Dnmt1 class of maintenance methyltransferases, but they contain a BAH domain (like Dnmt1/MET1), which might couple the activity of CMTs to DNA replication during the S phase.
E. The Masc1 Class The masc1 gene from A. immersus was isolated by cloning a genomic DNA fragment that hybridized with a probe generated by PCR using degenerate primers corresponding to part of the conserved C5-MTase motifs (Malagnac et al., 1997). The predicted Masc1 protein consists of 537 amino acids. Its C-terminal catalytic domain contains all 10 conserved motifs arranged in the standard order, and its N-terminal region contains a putative NLS and a region poorly homologous to the BAH motif (Freitag et al., 2002; Malagnac et al., 1997) (Fig. 2). No enzymatic activity is detected in vitro for the purified Masc1 protein expressed in either E. coli or baculovirus-infected insect cells. However, the masc1 gene is absolutely required for the de novo methylation associated with MIP, a process that scans the genome for DNA duplications and subsequently methylates the cytosines in these repeats (Malagnac et al., 1997). This indicates that Masc1 is most likely the de novo methyltransferase responsible for MIP, although the possibility that Masc1 lacks intrinsic methyltransferase activity but functions as a regulator of MIP cannot be excluded. Masc1 activity is dispensable for viability but is essential for fertility, as crosses between parents harboring a masc1 mutation are arrested at an early stage of sexual reproduction (Malagnac et al., 1997). As discussed earlier, masc1 is not required for maintenance methylation and gene silencing (Malagnac et al., 1997, 1999). In N. crassa, most DNA methylation is found in relics of RIP (Selker et al., 2003), a process that is somewhat similar to MIP in A. immersus (Selker, 2002). RIP detects repeated DNA in a pairwise manner and riddles them with G:C to A:T transition mutations (Selker, 2002). While the mechanism of RIP is not fully understood, the favored model involves unrepaired deamination of C or mC to U or T, respectively, perhaps catalyzed by a DNA methyltransferase (Selker, 1990). In support of this model, the putative C5-MTase gene rid has been shown to play an essential role in this process (Freitag et al., 2002). Rid was identified by searching Neurospora sequence databases. The predicted RID protein, which consists of 845 amino acids, is most similar to Masc1 in both the catalytic domain and the N-terminal region. Unlike Masc1, however, RID is a tripartite protein that contains, in addition to a catalytic domain and an N-terminal region, a long (270
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amino acids) C-terminal extension that lacks any known conserved motif. Similar to Masc1, RID shows no enzymatic activity in vitro, despite the presence of all conserved catalytic motifs in the center domain. Disruption of rid has no eVect on DNA methylation in vegetative tissues (Freitag et al., 2002), but results in recessive defects in RIP, as duplications of a number of genes fail to be inactivated in crosses homozygous for the mutation. Rid function is not essential for fertility, growth, or development (Freitag et al., 2002), in contrast to the situation with masc1 mutants in A. immersus, which are sterile in homozygous crosses (Malagnac et al., 1997).
III. Function of DNA Methylation Some model organisms, such as Neurospora, Arabidopsis, and all vertebrates (zebrafish, Xenopus, chick, and mouse), have DNA methylation in their genomes, whereas others, such as S. cerevisiae and C. elegance, contain no methyl cytosine. The genome of Drosophila contains low levels of methyl cytosine, but its function, if any, remains unknown (Gowher et al., 2000; Lyko et al., 2000a). The lack of DNA methylation in some organisms suggests that the DNA methylation system might be present in all ancestral eukaryotic organisms but was lost in some organisms during evolution. For instance, a DNA methyltransferase-like gene (pmt1) is present in the genome of S. pombe, but it does not appear to encode a functional enzyme (Wilkinson et al., 1995). Evidently, DNA methylation is not a conserved mechanism for the regulation of complex morphogenetic processes as worms (and probably flies) can do without. Why then is DNA methylation preserved? What developmental or cellular events are regulated by DNA methylation? There is no simple answer to this question, as the function of DNA methylation appears to be quite divergent in diVerent organisms (Table II). In filamentous fungi, DNA methylation has been shown to be involved in the repression of repeats (Selker, 2002). In plants, DNA methylation is required for silencing repeated genes and retrotransposons (Martienssen and Colot, 2001). In mammals, DNA methylation is also required for silencing retrotransposons such as IAP (Walsh et al., 1998), but it acquires additional functions such as regulating the allelic expression of imprinted genes and silencing of one of the two X chromosomes in females. Antisense inactivation of Dnmt1 in Xenopus activates zygotic genes prematurely and disrupts development (Stancheva and Meehan, 2000). These findings support a theory that DNA methylation is an evolutionary device that provides a unique means for setting up a variety of functions (Colot and Rossignol, 1999). It is likely that many cellular and developmental functions controlled by DNA methylation are still unknown.
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A. DNA Methylation and Genome Stability The ancestral function of DNA methylation appears to protect the organism from infection by foreign pathogens such as DNA and RNA viruses. Such a defense mechanism is relatively conserved across species. In fungi, transgene duplications are methylated eYciently by RIP in N. crassa and MIP in A. immersus (Selker, 2002). Silence of the viral genome and transgenes by DNA methylation has also been observed in plants and mammals (Martienssen and Colot, 2001; Verma, 2003). In plants, this might be a vital mechanism in preventing viral pathogens from spreading. However, DNA methylation does not appear to be an eYcient ‘‘immune’’ mechanism against infectious viruses. A related function for DNA methylation might be to silence endogenous transposable elements (or parasitic DNA). DNA methylation is associated primarily with transposons in N. crassa (Selker et al., 2003). Loss of methylation by treatment with the methylation inhibitor 5-azacytidine (5-azaC) or by inactivation of dim-2 has been shown to reactivate the expression and transposition of Neurospora transposons (Zhou et al., 2001). In plants, a large part of the genome is occupied by transposons, most of which are heavily methylated and inactivated (Martienssen and Colot, 2001; Tompa et al., 2002). Mutations in ddm1, which encodes a SNF2-like chromatin-remodeling protein, result in a loss of methylation and reactivation and mobilization of transposons in Arabidopsis (Jeddeloh et al., 1999; Miura et al., 2001; Singer et al., 2001). Mammalian genomes contain far more transposons than exonic DNA. For example, over 45% of the human genome is occupied by transposons, whereas less than 2% represents exonic DNA (Lander et al., 2001). Like much of the mammalian genome, transposable elements are highly methylated and silenced in somatic cells. Mouse embryos deficient in Dnmt1 show a massive transcription of IAP retroelements, suggesting that DNA methylation is required for their suppression (Walsh et al., 1998). However, definitive evidence that increased transcription of these elements leads to transposition is still lacking. It is therefore possible that the methylation of transposable elements and various repeats is necessary to reduce transcriptional background noise in organisms with a large genome size (Bird, 1995). Another function for DNA methylation relevant to genome stability might be to suppress homologous recombination. DNA methylation has been shown to reduce the frequency of meiotic crossing over in Ascobolus (Maloisel and Rossignol, 1998). Similarly, a significant increase in somatic recombination has been observed in Dnmt1-deficient ES cells (Chen et al., 1998) or in cells from patients with ICF syndrome (Miniou et al., 1997). This is of potential importance for tumorigenesis as both genome-wide DNA hypomethylation and increased mitotic recombination are seen at early stages of cancer (Laird and Jaenisch, 1994). Indeed, studies have shown that DNA hypomethylation
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plays a causal role in tumor formation in mice, possibly by promoting chromosomal instability (Eden et al., 2003; Gaudet et al., 2003).
B. DNA Methylation and Gene Expression It is well documented that DNA methylation is involved in the regulation of gene expression. Usually, hypermethylation correlates with gene silencing and demethylation correlates with gene activation. DNA methylation appears to regulate gene expression at three diVerent levels. DNA methylation has been shown to be involved in silencing of a whole chromosome as seen in the case of X inactivation. DNA methylation can also aVect gene expression by acting on a large chromosome domain containing multiple genes, such as some imprinted loci. Furthermore, DNA methylation can act directly on transcription either by blocking transcription factors binding to the promoter sequences or by interfering with transcription elongation. In all cases, DNA methylation is believed to act via several CpG-binding proteins to regulate chromatin structure and promoter activity. X chromosome inactivation is a process unique to mammals, and several reviews have covered this topic in detail (Avner and Heard, 2001; BrockdorV, 2002; Lee, 2003; Li, 2002). The following sections discuss the roles of DNA methylation in genomic imprinting and tissue- and developmental stage-specific gene expression in various organisms. 1. Genomic Imprinting Genomic imprinting is well known as a regulatory property of some specific chromosomal regions and leads to diVerential behavior of maternally and paternally inherited alleles. This phenomenon is observed in fission yeast, insects, flowering plants, and mammals. Although DNA methylation most likely is not the epigenetic mark for imprinting in yeast and insects, it plays a critical role in imprinting in mammals and flowering plants. In mammals, over 70 genes have been identified as imprinted genes. Almost all imprinted loci show allele-specific DNA methylation within specific domains. Most imprinted genes acquire their methylation imprints in female germ cells, whereas a few imprinted genes, such as H19 and Rasgrf1, acquire their methylation imprints in male germ cells (Reik and Walter, 2001). Although the mechanisms whereby gametic imprints are established are poorly understood, studies of Dnmt3L have provided genetic evidence that DNA methylation is an essential epigenetic mark for the establishment of genomic imprinting (Bourc’his et al., 2001; Hata et al., 2002). As discussed earlier, Dnmt3L encodes a protein that shares homology with Dnmt3a and Dnmt3b, but lacks methyltransferase activity. Although the zygotic function
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of Dnmt3L is not essential for embryonic development and global DNA methylation, disruption of Dnmt3L shows a maternal eVect on embryonic development. Embryos derived from Dnmt3L / oocytes display neural tube and placental defects and die around 10.5 days (E10.5) of gestation (Bourc’his et al., 2001; Hata et al., 2002). Analysis of DNA methylation patterns shows that Dnmt3L / females fail to establish maternal methylation imprints in oocytes, which leads to a loss of monoallelic expression of maternally (but not paternally) imprinted genes in the oVspring (Bourc’his et al., 2001; Hata et al., 2002). Dnmt3L may cooperate with the de novo methyltransferases Dnmt3a and Dnmt3b to establish DNA methylation patterns in germ cells. In fact, Dnmt3a / , Dnmt3bþ/ females also fail to establish maternal methylation imprints, but Dnmt3aþ/ , Dnmt3bþ/ female mice are fertile (Hata et al., 2002). These observations suggest that Dnmt3a probably contributes more, if not entirely, to the de novo methylation of imprinted genes. Taken together, these findings suggest that the Dnmt3 family of de novo methyltransferases is responsible for the establishment of maternal genomic imprinting. Interestingly, a recessive mutation in a case of human familial complete hydatidiform mole (CHM) has been shown to disrupt the establishment of maternal imprints (Judson et al., 2002), similar to the phenotype of Dnmt3L mutant mice. This mutation is not located in the known DNMT genes (including DNMT3L) (Hayward et al., 2003), indicating that it aVects another trans-acting factor that may play an important role in de novo methylation during oogenesis. A unique feature of imprinting is that diVerential methylation patterns are maintained during preimplantation development when most methyl groups in the genome are erased. While the mechanism for this maintenance is still unclear, maternally derived Dnmt1o appears to play a role in this process (Howell et al., 2001). However, given that Dnmt1o is localized predominantly in the cytoplasm of the preimplantation embryo, except a transient nuclear localization at the eight cell stage (Ratnam et al., 2002), other DNA methyltransferases, derived maternally or zygotically, may also be involved in maintaining the methylation of imprinted loci during preimplantation development. Indeed, it has been shown that Dnmt3a and Dnmt3b are required for the stable maintenance of methylation of imprinted genes in ES cells (Chen et al., 2003). After implantation of the blastocyst, the methylation of imprinted loci is probably maintained mainly by the somatic form of Dnmt1 (Li et al., 1993). Genetic studies have demonstrated that maintaining the diVerential methylation patterns in somatic cells is crucial for the monoallelic expression of imprinted genes. In Dnmt1-deficient embryos, alleles of both Igf2 and Igf2r, which are normally expressed from the paternal and maternal alleles, respectively, are silenced, whereas the H19 gene, which is normally maternally expressed, is transcribed biallelically (Li et al., 1993). The mechanisms by which DNA methylation regulates imprinted gene expression have been studied extensively. Methylation of the diVerentially
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methylated regions (DMRs) in imprinted loci can silence some imprinted genes directly (e.g., H19) and activate others indirectly by secondary mechanisms, such as blocking the binding of CTCF (CCCTC-binding factor) to insulators (e.g., Igf2) and repressing the production of antisense transcripts (e.g., Igf2r) (Bell and Felsenfeld, 2000; Hark et al., 2000). In flowering plants, imprinting aVects the extraembryonic endosperm, but not the embryo (Martienssen, 1998). The embryo and the endosperm are separate fertilization products, and the endosperm plays an analogous role to the mammalian placenta in transferring maternal resources to the embryo. So far, about half a dozen imprinted genes have been identified in plants. In maize endosperm, imprinted zein genes are only expressed when inherited from the seed parent, and these loci are methylated at fewer sites on the maternal alleles as compared to the paternal alleles (Lund et al., 1995). DiVerential methylation also corresponds with parent-specific expression of the R locus (Kermicle and Alleman, 1990). In the Arabidopsis endosperm, the MEDEA (MEA) gene, which encodes a SET-domain Polycomb protein, has been shown to be imprinted (Kinoshita et al., 1999; Vielle-Calzada et al., 1999). Normally, only the maternal MEA allele is expressed throughout endosperm development while the paternal allele is silenced. Thus, inheritance of a mutant maternal mea allele results in seed abortion, even when the paternal MEA allele is wild type. However, mutations at the ddm1 locus are able to rescue mea seeds probably by reactivating paternally inherited MEA alleles during seed development (Vielle-Calzada et al., 1999). Loss of imprinting is also observed in crosses using plants with DNA methylation reduced by a MET1 antisense transgene (Adams et al., 2000). It has been shown that the DNA glycosylase gene DEMETER is required for the maternal allele expression of MEA in the endosperm and its precursor, the central cell (Choi et al., 2002). While the mechanism by which DEMETER regulates MEA expression is unknown, it is speculated that MEMETER might modify the chromatin structure by excising 5-methylcytosines (Choi et al., 2002). 2. Tissue-Specific and Developmental Stage-Specific Gene Expression While the primary function of DNA methylation remains controversial (Bird, 2002; Yoder et al., 1997b), accumulating evidence suggests that it is involved in the regulation of gene expression during development. Some developmental phenotypes caused by hypomethylation, as described later, are probably due to alterations in gene expression. In Arabidopsis, the floral homeotic transformations observed in MET1 antisense plants are associated with ectopic expression of the floral homeotic genes AGAMOUS and APETALA3 (Finnegan et al., 1996), and the late flowering phenotype observed in met1 mutants may result from hypomethylation at the FWA
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locus and creation of an fwa epiallele (Kankel et al., 2003). Arabidopsis ddm1 mutations lead to an immediate loss of methylation in the repetitive fraction of the genome but only to a delayed and gradual hypomethylation of single copy genes (Kakutani et al., 1996; Vongs et al., 1993). This may partly explain the observation that some phenotypes of ddm1 mutants become progressively more severe as the mutants are inbred (Kakutani et al., 1996). In Xenopus, gene expression is suppressed during the first 12 cleavages of the zygote, and transcription normally initiates at the midblastula transition. In embryos depleted of xDnmt1, however, many developmentally regulated genes are activated two cell cycles earlier due to the loss of methylation at promoter regions, suggesting a role for DNA methylation in regulating the timing of gene expression in Xenopus development (Stancheva et al., 2002; Stancheva and Meehan, 2000). In mice, DNA methylation is not essential for the survival of embryonic cells, and the eVect of disrupting methylation patterns only becomes apparent during or after gastrulation when the pluripotent embryonic cells begin to diVerentiate (Lei et al., 1996; Li et al., 1992; Okano et al., 1999). In agreement with these results, mouse ES cells are fully viable even with their DNA almost completely demethylated, but they undergo rapid cell death upon diVerentiation (Chen et al., 2003; Lei et al., 1996; Tucker et al., 1996). Inappropriate expression of imprinted genes and ectopic X chromosome inactivation probably contribute to the lethality of mutant ES cells and embryos (Li et al., 1993; Panning and Jaenisch, 1996). Abnormal expression of other developmentally regulated genes may also play an important role in causing the phenotypes. Deletion of Dnmt1 in cultured fibroblasts results in widespread gene activation, with 10% of all genes being activated aberrantly (Jackson-Grusby et al., 2001). These results raise the possibility that DNA methylation plays an important role in the regulation of tissue-specific or developmental stage-specific gene expression during mammalian development. Indeed, several examples of tissue-specific genes regulated by promoter methylation have been reported. One study has shown that expression of the gene encoding the glial fibrillary acidic protein (GFAP) is regulated by methylation of a CpG dinucleotide within the STAT3-binding element in the GFAP promoter region during astrocyte diVerentiation (Takizawa et al., 2001). The CpG site is hypermethylated in neuronal epithelial cells prior to diVerentiation and becomes demethylated upon astrocyte diVerentiation and accessible by STAT3, which activates transcription of the GFAP gene. Another study has shown that demethylation of the interleukin (IL)-4 promoter region during T-cell diVerentiation leads to a high-level expression of IL-4 (Lee et al., 2001b). Promoter demethylation is clearly one of the mechanisms for the activation of tissue-specific genes during development. It remains to be seen how prevalent such a mechanism is used.
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C. DNA Methylation and Development Both the levels and the patterns of DNA methylation vary during development in almost all organisms that display this modification, but to diVerent degrees. In mammals, complex changes in DNA methylation patterns occur during embryonic development and cell diVerentiation. During preimplantation development, both the paternal and the maternal genomes undergo a wave of demethylation, which erases the methylation marks (except those at imprinted genes) inherited from the gametes. Shortly after implantation, a wave of global de novo methylation reestablishes the overall methylation pattern, which is then maintained throughout life in the somatic cells of the animal. During diVerentiation of somatic tissues, a subset of tissue-specific genes become demethylated (Li, 2002). In other vertebrates, changes in methylation during development appear to be much less dramatic. For example, in Xenopus and zebrafish, there is no evidence of global demethylation after fertilization, although changes of methylation in individual genes may occur (Macleod et al., 1999; Martin et al., 1999; Stancheva and Meehan, 2000 Stancheva et al., 2002). In plants, measuring methylation levels during early development is hampered by the inaccessibility of very early embryos, which are encased in maternal tissue. However, postembryonic tissues of increasing age show a gradual increase of methylation levels (Richards, 1997). In several fungi, changes in overall methylation levels are readily detectable during the dimorphic transition from yeast-like to mycelial growth (Colot and Rossignol, 1999). Variations in methylation levels, patterns, and dynamics observed in diVerent organisms could reflect diVerences in the function of methylation in the development of these organisms. Most of our knowledge concerning the significance of DNA methylation in development comes from the genetic manipulation of DNA methyltransferase genes or genes that regulate DNA methylation in various model organisms. From available experimental data, it appears that DNA methylation becomes increasingly integrated into eukaryotic developmental programs during evolution. In Neurospora, disruption of either dim-2 or rid shows no obvious phenotype, although these mutations result in genome-wide demethylation and defects in RIP, respectively (Freitag et al., 2002; Kouzminova and Selker, 2001). In Ascobolus, disruption of masc1 results in sterility but no defects in vegetative growth (Malagnac et al., 1997). DNA methylation also does not appear to play an important role in Drosophila development, as depletion of dDnmt2 by RNAi shows no obvious defects, despite significant demethylation (Kunert et al., 2003). In contrast, the eVects of methylation perturbations are more conspicuous in plants and animals. In Arabidopsis, mutants displaying demethylation of CpG sites (as seen in antisense MET1 or met1 mutant plants), non-CpG sites
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(as seen in drm1 drm2 cmt3 triple mutant plants), or both (as seen in ddm1 mutant plants) are viable, but exhibit a number of developmental abnormalities, including late flowering, decreased fertility, reduced plant size, and altered leaf and floral morphology (Cao and Jacobsen, 2002a; Finnegan et al., 1996; Kakutani et al., 1996; Kankel et al., 2003; Ronemus et al., 1996). In several vertebrate species, DNA methylation appears to be essential for normal development. Zebrafish embryos treated with the DNA methylation inhibitor 5-azaC show gastrulation defects and abnormal somite patterning, although 5-azaC is known to have nonspecific cytotoxicity (Martin et al., 1999). Depletion of Xenopus Dnmt1 by antisense RNA results in severe developmental defects and apoptosis of embryo cells (Stancheva et al., 2001; Stancheva and Meehan, 2000). The role of DNA methylation in mouse development has been studied extensively. A hypomorphic Dnmt1 mutant allele shows embryonic lethality at E10.5 (Li et al., 1992). Complete elimination of the Dnmt1 function by disrupting the catalytic center results in more severe phenotypic changes. Development of the mutant embryo is arrested between presomite stage and 8 somite stage around E9.5 (Lei et al., 1996). Disruption of Dnmt3b also results in embryonic lethality with multiple developmental defects and growth impairment after E9.5 and lethality after E12.5, whereas Dnmt3a mutant mice develop to term but die at about 4 weeks of age. Inactivation of both Dnmt3a and Dnmt3b results in a similar phenotype to Dnmt1-null mutation, and homozygous embryos die around E9.5 (Okano et al., 1999). These results demonstrate that DNA methylation in mammals is not only essential for early embryogenesis but is also required for later development and even for the normal physiological functions in adults. This conclusion is further supported by findings from the analysis of conditional mutant mice that exhibit developmental stage- and cell lineage-specific demethylation. For example, studies using conditional Dnmt1 mutants have shown that DNA methylation plays a critical role in the function and survival of embryonic fibroblasts, neuronal cells, and T lymphocytes (Fan et al., 2001; JacksonGrusby et al., 2001; Lee et al., 2001b). Conceivably, the complexity of DNA methylation as a regulatory mechanism may have reached its evolutionary peak in mammals.
IV. Concluding Remarks The ever-expanding sequence databases have greatly facilitated the discovery of C5-MTase genes over the past several years. Genetic manipulation of these genes in various model organisms has provided compelling evidence for the involvement of DNA methylation in a variety of biological processes. Much less is known, however, about the mechanisms by which
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DNA methylation patterns are generated during development. Studies have established that DNA methylation can be regulated by other epigenetic mechanisms (Li, 2002). Lysine 9 methylation of histone H3 has been shown to control DNA methylation in fungi, plants, and mammals (Jackson et al., 2002; Lehnertz et al., 2003; Tamaru and Selker, 2001). Chromatin remodeling proteins such as DDM1, ATRX, and Lsh (the mammalian homolog of DDM1) have also been shown to modulate DNA methylation in plants and mammals (Dennis et al., 2001; Gibbons et al., 2000; Jeddeloh et al., 1999). The challenge we are facing now is to determine the mechanistic links between the DNA methylation system and the chromosome modification and remodeling systems. Further characterization of the functional domains of C5-MTases and identification of their interacting partners will undoubtedly accelerate our understanding of the nature of the interplay between various epigenetic regulators. Such knowledge will help elucidate the molecular events in many diseases and design eVective therapeutic strategies.
Acknowledgments Research in our laboratory is supported by Grants CA82389 and GM52106 from the National Institutes of Health (to E.L.). T.C. is a recipient of a long-term fellowship from the Human Frontier Science Program.
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Mechanical Signals as Regulators of Stem Cell Fate Bradley T. Estes,* Jeffrey M. Gimble,{ and Farshid Guilak* *Department of Surgery Division of Orthopaedic Surgery, Duke University Medical Center Durham, North Carolina 27710 { Pennington Biomedical Research Center, Louisiana State University Baton Rouge, Louisiana 70808
I. II. III. IV.
Introduction Mechanical Regulation of Limb Development Theoretical Models of Mechanical Stress in Development In Vivo Experimentation A. Continuous Passive Motion B. Hydrostatic Pressure C. Tensile Stress D. Compressive Stress
V. In Vitro Experimentation A. Compressive Stress B. Cyclic Stretch (Biaxial Tensile Loading) C. Multiaxial Loading D. Hydrostatic Pressure E. Shear Stress F. Shock waves VI. Conclusions Acknowledgment References
In recent years, stem cells have shown significant promise for their potential to provide a source of undiVerentiated progenitor cells for therapeutic applications in tissue or organ repair. Significant questions still remain, however, as to the genetic and epigenetic signals that regulate the fate of stem cells. It is now well accepted that the micro-environment of the stem cell can have a significant influence on its diVerentiation and phenotypic expression. Although emphasis has been placed in previous work on the role of soluble mediators such as growth factors and cytokines on stem cell diVerentiation, there is now significant evidence, both direct and indirect, that mechanical signals may also regulate stem cell fate. We review a number of in vivo and in vitro studies that have provided evidence that mechanical factors have the ability to influence the diVerentiation of a Current Topics in Developmental Biology, Vol. 60 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00
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number of cells that have been classified as either precursor, progenitor, or stem cells. Taken together, these studies show that specific mechanical signals may promote cell diVerentiation into a particular phenotype, potentially having an eVect on embryonic development. The use of such mechanical signals in vitro in specially designed ‘‘bioreactors’’ may provide important adjuncts to standard biochemical signaling pathways for promoting engineered tissue growth. A further understanding of the biomechanical and biochemical pathways involved in mechanical signal transduction by stem cells will hopefully provide new insight for the improvement of stem-cell based therapies. ß 2004, Elsevier Inc.
I. Introduction Over the past several years, there has been increasing interest in understanding stem cell biology, particularly as it relates to the control of stem cell fate. It is widely held that stem cells hold significant promise for therapeutic applications in tissue or organ repair in the treatment of diseases such as heart disease, arthritis, and neural disorders, such as Parkinson’s disease and paralysis, as well as other pathologies related to trauma or degeneration. A critical step in developing successful treatment regimens for these diseases will require a more thorough understanding of the diVerentiation pathways that guide the uncommitted cell toward a specific tissue phenotype and promote the repair or regeneration of damaged or diseased tissue. However, it is noted that this is a complicated endeavor, as the diVerentiated phenotype is governed by a myriad of factors, including the micro- and macroenvironment of the cell, which include both the biochemical and the biomechanical environment of the cell. Particularly in the area of mesenchymal-derived tissue development as well as neuronal development, many studies have been undertaken to investigate the ability of the biochemical environment to modulate the phenotype of the uncommitted cell (Ashjian et al., 2003; Bianco and Cossu, 1999; Bruder et al., 1994; Dorheim et al., 1993; Erickson et al., 2001, 2002; Gimble et al., 1994, 1996; Halvorsen et al., 2000, 2001a,b; Jaiswal et al., 1997; Johnstone et al., 1998; Kuznetsov et al., 1997; Mackay et al., 1998; Mizuno et al., 2002; Pittenger et al., 1999, 2000; SaVord et al., 2002; Saladin et al., 1999; Sen et al., 2001; Wickham et al., 2003; Yoo et al., 1998; Zuk et al., 2001, 2002). In many of these studies, human mesenchymal stem cells (hMSC), human adipose-derived adult stem cells (hADAS), or humanprocessed lipoaspirate (PLA) cells were induced along several mesodermal lineages including fat, muscle, bone, and cartilage. The use of adiposederived stem cells (hADAS and PLA) has gained significant interest in recent
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years, as these cells can be harvested in abundance through a minimally invasive liposuction procedure, thereby avoiding painful and potentially iatrogenic sequelae from committed cell isolation or from autologous bone marrow harvest (Erickson et al., 2001, 2002; Halvorsen et al., 2000, 2001a,b; Mizuno et al., 2002; Zuk et al., 2001). While the aforementioned studies have provided great insight into the biochemical milieu necessary to aVect diVerentiation, very few studies have directly investigated the influence of mechanical stress on the phenotype of these and other types of progenitor cells. The influence of such ‘‘biophysical’’ signals are thought to be critical in regulating the phenotype of the stem cell and may have additive or synergistic eVects in combination with soluble mediators. This realization, coupled with the complexity of the issue, emphasizes the importance of a multidisciplinary approach to the study of stem cells involving the coalescence of many disciplines, including cell and molecular biology, biochemistry, materials science, and biomechanical engineering. To aid in this eVort, a set of principles have been proposed to facilitate the study of the role of mechanical factors in tissue regeneration, giving rise to the field of ‘‘functional tissue engineering’’ (FTE) (Butler et al., 2000; Guilak, 2002; Guilak et al., 2001). The tenets of FTE can be summarized as (1) understanding the native mechanical environment of the tissue that is being replaced; (2) understanding the relevant mechanical properties of the native undamaged tissue; (3) using a subset of these mechanical properties as design parameters; and (4) studying the influence of mechanical factors on cells in vivo as well as in vitro in bioartificial matrices to aVect an improved therapeutic outcome (Butler et al., 2000; Guilak, 2002; Guilak et al., 2001). It follows from these tenets that in using stem or uncommitted cells for FTE, knowledge of the biochemical signals that arise from mechanical stress (mechanotransduction pathways) may be a crucial step in promoting the appropriate diVerentiation pathway for stem cells. To this end, a number of studies over the past century provide convincing evidence, both direct and indirect, that mechanical stress may control cell fate in stem or stem-like cells undergoing diVerentiation. More recently, investigators have attempted to elucidate these mechanotransduction pathways through more controlled biomechanical experiments. This chapter reviews these recent published works; because these studies are few in number, the articles reviewed cover a variety of tissue types and include both in vitro and in vivo experimentation. In addition, because the nomenclature for stem or stem-like cells varies greatly, no eVort has been made to categorize the studies by the potential of the cell. If the studies used cells in an undiVerentiated state, whether or not the cells were labeled as precursor or progenitor cells or multipotent, pluripotent, totipotent, or mesenchymal stem cells, and so on, we included the work.
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II. Mechanical Regulation of Limb Development In 1922, Carey observed that ossification of the femur in a developing pig model formed on the convex side of the femoral shaft, where the tensile stresses were largest, causing him to hypothesize that these stresses acted as a mechanical morphogen in skeletogenesis. Later in the 1920s and 1930s, experiments were conducted to determine if the removal of chick or rabbit fetal limb bones from the embryo would result in loss of development. These membranes were excised and cultured in nonfunctional host sites such as chorioallantoic membranes (Murray and Huxley, 1925; Murray and Selby, 1930), brains (Willis, 1936), and petri dishes (Fell and Robison, 1929) to determine various environmental eVects on the diVerentiation/development of the tissue. Some of these membranes continued to form recognizable limbs; however, the progress of epiphyseal, periosteal, and osseous development was slowed significantly. In some cultures, these limbs tended to become more slender and shorter over time with abnormal articular cartilage and bone marrow cavity formation. It was postulated that the limbs were formed by an intrinsic program in the anlagen and that the external features of the limbs were formed by an extrinsic mechanical factor (Murray and Selby, 1930). At that time, it would have been diYcult, if not impossible, to fully understand the molecular signals directing development and whether continued growth in the nonfunctional environment may have been a result of signals sent prior to removal from the embryo. Nonetheless, the observation that a lack of mechanical load had a profound eVect on development was duly noted. In a series of experiments performed in the late 1930s and early 1940s, Glu¨cksmann (1939) cultured chick rudiments under static compression after displacement of the periosteum and perichondrium and showed a characteristic cartilage phenotype under this loading model. He later observed that the application of tensile stresses on these rudiments promoted bone formation (Glu¨cksmann, 1942). Many other more recent studies have also been conducted on membranous bone with similar results. For example, when membranous bones were articulated from chick embryos, secondary cartilage was formed, in contrast to none formed when the same culture was held under constant pressure or no mechanical stress (Hall, 1968). Paralysis of chicken embryos is known to inhibit cartilage formation (Fang and Hall, 1995; Hall, 1979; Hall and Herring, 1990; Murray and Drachman, 1969). Further, at late developmental stages, paralysis also resulted in the absence of menisci of the tibiofemoral joint and plantar tarsal sesamoid of the tibiotarsal joint, both of which serve mechanical purposes in joint motion (Mikic et al., 2000). The immobilization of chick embryo muscles was found to alter bone development significantly, as measured by collagen expression and mechanical properties
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(Wong et al., 1993). Conversely, increasing the mobility of the joint during development resulted in an increase in joint cavity size during formation compared to a decrease under paralysis (Ruano-Gil et al., 1985). In addition, calcification of fetal mouse cartilaginous rudiment was enhanced significantly with intermittent dynamic mechanical compression (Klein-Nulend et al., 1986, 1987a), as was sulfate content and the size of chondroitin sulfate chains (Klein-Nulend et al., 1987b).
III. Theoretical Models of Mechanical Stress in Development Regardless of the type of mechanical stress (or lack of mechanical stress) applied to the tissues in the aforementioned studies, significant evidence suggests that mechanical factors contribute to the control of embryonic development, but the mechanism(s) of diVerentiation remains unanswered. Since the late 1800s, many authors have proposed theoretical or mathematical models in an attempt to explain limb bone development patterns. As early as 1895, by experimentally manipulating the environment of developing eggs and embryos, Roux hypothesized that mechanical stress was responsible for the diVerentiation of connective tissues. More specifically, he proposed that tensile stresses resulted in fibrous tissue, compressive stresses resulted in bone formation, and the combination resulted in cartilage formation (Roux, 1895, 1912a,b). In 1911, Gephardt introduced a photoelastic celluloid model with point loading on the entire cartilaginous end of the developing long bone. This analysis resulted in a model showing principal stress trajectories that crossed in the center of the epiphysis, consistent with the location of the secondary ossific nucleus (Fig. 1). This finding resulted in his conclusion that this secondary bone formation is greatest at the area of greatest accumulated stress. Subsequently, others postulated alternative hypotheses. BenninghoV (1924) hypothesized that cartilage formation was a result of shear stress, whereas Krompecher (1937, 1955, 1956, 1958) hypothesized that compressive stresses resulted in hyaline cartilage formation. However, it was not until 1965 (later translated into English from German in 1980) that Pauwels investigated mathematical modeling in an attempt to understand the stress states in developing limbs (Pauwels, 1965, 1980). In this work, Pauwels revised Gephardt’s photoelastic model by changing the boundary conditions to distributed loads, using a loading block, along the periphery of the developing end of the long bone (Pauwels, 1965, 1980). With this change in boundary conditions, Pauwels’ results showed the central epiphyseal region to be under pure hydrostatic pressure, thus resulting in his conclusion that
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Figure 1 Gephardt’s (1911) point-loading model of chondroepiphysis.
this secondary ossification nucleus is due to the principal compressive and tensile stresses being of equal magnitude or, in other words, pure hydrostatic pressure. However, in 1988, Carter and Wong postulated that the boundary conditions of Pauwels’ model were also incorrect and modified them to rigid fixation at the ossified ends of the bone (i.e., at the wedges; see Fig. 1) and uniform joint contact pressures (5 MPa) at the end of the developing bone. Using a two-dimensional plane stress, finite element model with these boundary conditions, the authors showed results significantly diVerent from those obtained by Pauwels. Finite element analysis resulted in significant compressive hydrostatic stress near the joint surfaces and areas of high shear stress in the central region of the chondroepiphysis. From these findings, the authors concluded that secondary bone formation was due to high shear stresses, whereas cartilage formation was due to compressive hydrostatic stress (Carter and Wong, 1988). The authors further conjectured that high compressive hydrostatic pressures would also inhibit ossification of the cartilage at the ends of the developing long bones (Carter and Wong, 1988). These results are consistent with other finite element work of the authors in which the development of bone formation and cartilaginous formation from the femoral anlage correlated well with areas of shear stresses and compressive hydrostatic stresses, respectively (Carter et al., 1987). In summary, these mathematical models provided potential insight into the nature of the stress–strain environment of the diVerentiating limb and its local influence on cell fate. Regardless, such hypotheses on the specific influence of stress on cell diVerentiation must be verified and modified rigorously, if need be, with appropriate experimental design and implementation. The following sections review in vivo and in vitro research since the early
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1990s that elucidates the role of specific types of mechanical stress on progenitor cell diVerentiation.
IV. In Vivo Experimentation A. Continuous Passive Motion Other evidence apart from developing limb studies of the influence of mechanical factors on stem cell diVerentiation has been demonstrated through in vivo experimentation. Periosteal tissue with the cambium layer intact has been used to study the eVects of continuous passive motion on chondrogenesis following the creation of the defect. The use of periosteum tissue was based on the knowledge that this cambium layer contains a source of osteochondro-undiVerentiated cells (O’Driscoll and Salter, 1984; Ohlsen, 1978) and the hypothesis that under the correct stimuli, these cells could be induced along the chondrogenic lineage. Early experiments in fracture healing showed cartilaginous tissue callus formation after the fractured membrane or bone was subjected to motion (Hall, 1968; Hall and Jacobson, 1975; Kernek and Wray, 1973; Tonna and Pentel, 1972). In one of these studies (Hall and Jacobson, 1975), convincing evidence was provided as to the cell source for chondrogenesis in that removal of the periosteum, which included the cambium layer at the time of fracture, resulted in significantly less cartilage than if the cambium layer were left intact. Using free periosteal grafts in cartilage defects in rabbits, Rubak and colleagues were able to demonstrate qualitatively some cartilage formation when animals were allowed to ambulate intermittently compared to much less cartilage production when the joint was completely immobilized (Rubak, 1982; Rubak et al., 1982a,b). Further evidence of mechanical-induced diVerentiation was provided in a series of studies by O’Driscoll and colleagues, who investigated the eVects of periosteal tissue for articular cartilage generation in New Zealand white rabbits (O’Driscoll and Salter, 1984, 1986; O’Driscoll, et al., 1986). The authors studied the eVects of both placing loose (free graft) periosteum in the joint (O’Driscoll and Salter, 1984) and the eVects of creating full thickness cartilage defects (i.e., the authors removed a section of cartilage penetrating to the underlying subchondral bone) and filling (via a press fit or suture) these defects with autogenous periosteal grafts with the cambium layer intact and facing outward (O’Driscoll and Salter, 1986; O’Driscoll et al., 1986). Under continuous passive motion in this series, the authors were clearly able to show an increase in chondrogenesis compared to the immobilized joint (O’Driscoll and Salter, 1984, 1986). Although the mechanical environment at the cell or tissue level is not strictly quantifiable in this setting, the motion imparts stress to the joint and to the underlying tissues
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and implies that mechanical stress has the ability to aVect the phenotype of the undiVerentiated cell population.
B. Hydrostatic Pressure In a tibial metaphases study, Tagil and Aspenberg (1999) tested the hypothesis of Carter and colleagues (1988; Carter and Wong, 1988), who stated that cartilage would form from undiVerentiated mesenchymal tissue with increasing hydrostatic stress and perhaps decreasing oxygen tension. By modifying their bone conduction chamber (Aspenberg and Wang, 1993) to impart a controlled load on the anteromedial aspect of the proximal tibia metaphases, the authors could test this hypothesis directly. In this loading experiment, four out of the seven loaded chambers resulted in the formation of an apparent cartilage layer next to the loading piston compared to zero of the seven controls (P ¼ 0.03). Seven weeks of loading clearly detailing a zone consisting of large rounded cells in lacunae separated in a soft tissue matrix (Fig. 2). Indeed, the large rounded cells in this cartilage-like region stained positively in their territorial matrix for aggrecan, suggesting cartilage formation; however, the authors were quick to note a limitation on the conclusions drawn
Figure 2 H&E staining after 7 weeks of loading. (Left) A marrow cavity is seen at the bottom in contrast to the top, which shows a zone of chondrocytes separated by trabecular bone (20). (Right) A 100 magnified chondrocyte region (Tagil and Aspenberg, 1999). Reprinted from Journal of Orthopaedic Research (1999) with permission from Orthopaedic Research Society.
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from this study, as they did not stain for collagen type II. Although it is unclear from this study what the source of chondroprogenitor cells were, one could conjecture that the undiVerentiated cell type is derived from the bone marrow, as the screw was tapped into the underlying bone marrow. In addition, the fact that the periosteum was incised and raised before the chamber was placed into the proximal tibia further suggests that the source of undiVerentiated cells may have been within the bone marrow. Regardless of the exact source of progenitor cell, the authors concluded that a controlled mechanical hydrostatic stress of 2 MPa at the frequency used in this bone chamber study induced cartilage formation, again consistent with the hypotheses of Carter and Wong (1988), Carter et al. (1988).
C. Tensile Stress Several studies have been undertaken to describe the eVects of mechanical stress on midpalatal suture cartilage (Saitoh et al., 2000; Takahashi et al., 1996, 2003). This tissue lies between the maxillary bones and behind the incisal foramen at the level of the first molars. This midpalatal suture tissue is characterized by two tissue types: a precartilaginous tissue layer containing osteochondral progenitor cells and a secondary layer with mature cartilage (Mizoguchi et al., 1990) (Fig. 3). Histologically, it has been shown that this tissue contains a population of mesenchymal-like stem cells and osteochondral progenitor cells (Hall, 1979; Silbermann et al., 1987; Strauss et al., 1990). In two of these studies (Takahashi et al., 1996, 2003), the role of tensile forces on the midpalatal suture in rats was examined to understand the mechanical force eVects on cell diVerentiation. Briefly, orthodontic wires were placed between the first and the second molars, and a tensile force of 10 to 20 g was applied to the wires, resulting in tensile forces on the midpalatal suture cartilage in addition to bending stresses and some compressive stresses (i.e., combined stress state), all of which are unquantifiable. Further, the expansive force applied throughout the 14 days was likely to decrease; nonetheless, the authors noted that some force was applied throughout the experimental time frame. Strong staining for type I collagen was observed in the precartilaginous tissue layer as well as in the maxillary bone; however, strong type II collagen was noted in the mature cartilage layer concurring with a previously published description of these two regions of tissue (Mizoguchi et al., 1990). The experimental group showed a progressive phenotypic change in the precartilaginous tissue, consisting of fibroblastic cell realignment in the direction of the tensile forces, coupled with a narrowing of the cartilaginous tissue layer in comparison with the controls. These findings suggested that the mature cartilage was being resorbed by application of these tensile
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Figure 3 Microphotographs of normal midpalatal suture cartilage from 4-week-old Sprague– Dawley rats. P: precartilaginous cell layer; C: cartilaginous cell layer; B: bone (Takahashi et al., 2003). Reprinted with permission from European Journal of Cell Biology (2003).
forces. This thinning of the cartilaginous tissue layer progressed throughout the experimental time frame and, concurrent with this time frame, blood vessels invaded the precartilaginous tissue layer. By day 14 in the experimental group, cartilaginous and precartilaginous tissues were divided into two distinct tissue bodies separated by a newly formed intramembranous bone formation, which showed partial staining to osteocalcin. Osteochondral progenitor cells formed a blastema on the surface of the cartilage surrounded by a fibrous matrix, which eventually, in later times in the study (i.e., by day 14), was surrounded by a deposition of calcified matrix. Immunohistochemical data on the presence of types I and II collagen, as well as osteocalcin, suggested that the midpalatal suture osteochondral progenitor cells or
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mesenchymal stem-like cells diVerentiated along the osteoblast lineage in response to the mechanical tensile forces.
D. Compressive Stress In an attempt to further elucidate how the secondary cartilage of the midpalatal suture cartilage responds to mechanical loads, Saitoh and colleagues (2000) investigated the eVects of mechanical compression on this same tissue using an orthodontic wire apparatus for imparting compressive forces on the midpalatal suture cartilage. As in the previous study, the application of compressive forces on the orthodontic appliance resulted in a state of combined stress in the midpalatal suture cartilage; however, it is again important to note that these stresses were not quantified. For the 14-day study, gross morphologic observations of the unloaded controls revealed cartilaginous masses separated from one another by a layer of polygonal cells along the midline of the suture, which matched that of the previous tensile study from the authors (Takahashi et al., 1996). The unstressed, control tissue was also studied via immunohistochemistry for collagen types I and II, as well as type X, which confirmed the observed tissue type. In this control tissue, type I collagen staining was noted in the precartilaginous tissue layer and the maxillary bone, with strong type II collagen staining in the cartilaginous tissue. In contrast to the control group, the 20-g experimental group showed a decrease in the thickness of the midline precartilaginous tissue and an increase in the lateral cartilaginous layers up to day 7. By day 14, the precartilaginous layer was not detectable, as it appeared to be replaced by well-diVerentiated cartilage tissue composed of hypertrophic chondrocytes. The gross morphologic changes in response to an increase in mechanical compressive force resulted in the same trend, albeit in an accelerated fashion (Fig. 4). In addition to the gross morphological findings, the authors noted strong type II collagen immunoreactivity in the thickened cartilage layer in the experimental group coinciding with a decrease in collagen type I staining over the time course of the study. Figure 5 shows immunohistochemistry results for collagen type II for a control animal, 20 g of loading, 30 g of loading, and 40 g of loading (all loading groups are for 7 days). In addition to investigating the distribution of collagens throughout the midpalatal tissue, the authors investigated the accumulation of other markers for chondrogenic diVerentiation. Indeed, increasing immunoreactivity in the experimental groups for common glycosaminoglycans (GAGs) found in cartilage, namely chondroitin-4-sulfate, chondroitin-6-sulfate, and keratin sulfate antibodies, was observed over the 14 days of the study. Figure 6, in contrast to the controls, which showed no increase in GAG
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Figure 4 Photomicrographs of midpalatal suture cartilage in frontal sections of 5-week-old control animals. (a) The 20-g compressive force experimental group at day 14. (b) The 40-g compressive force experimental group. (c) Two carrot-shaped cartilaginous masses that included mature chondrocytes were separated from each other between right and left maxilla. P: precartilaginous cell layer; C: cartilaginous cell layer; B: maxillary bone. Original magnification, x50. Toluidine blue staining (Saitoh et al., 2000). Reprinted from The Anatomical Record (2000), with permission by Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.
accumulation, shows an increasing intensity of immunoreactivity in experimental conditions for all GAGs over the time course of the study. In this study, the authors were clearly able to demonstrate a phenotypic change under mechanical compression in midpalatal cartilage suture tissue, strongly suggesting chondrogenesis. This was noted not only by an appearance of collagen type II, but also the progressive accumulation of GAG within this midline tissue. This finding was in contrast to their earlier work (Takahashi et al., 1996) in which tensile forces resulted in osteoinduction in this secondary cartilage layer.
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Figure 5 Collagen type II immunohistochemistry; left side (A) Control; (a) 7 day, 20 g; (b) 7 day, 30 g; and (c) 7 day, 40 g (Saitoh et al., 2000). Note the increasing collagen type II reactivity in the midpalatal suture cartilage as a function of compressive load. Reprinted from The Anatomical Record (2000), with permission by Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.
V. In Vitro Experimentation A. Compressive Stress Following their in vivo work in midpalatal suture cartilage, Takahashi and colleagues (1998) isolated limb bud cells from E10 stage embryos from timed-pregnant Swiss Webster mice at 10 days of gestation. Cells were resuspended in a collagen I gel, and static compressive forces were used to apply stresses of 1, 1.5, and 2 kPa to the constructs. Type II and X collagens were shown to increase qualitatively compared to unloaded controls. In addition, using semiquantitative reverse transcriptase polymerase chain reaction (RTPCR), the authors found a direct increase in collagen type II, a marker for chondrogenic diVerentiation, in direct proportion to the magnitude of stress applied. When the 2.0-kPa stress group was compared to unloaded controls, an 8.3-fold increase in collagen II production was noted. In addition, increasing the magnitude of loading from 1.0 to 1.5 and 2.0 kPa resulted in 1.5 and 3.8-fold increases, respectively, in the expression of collagen type II mRNA (Fig. 7). Aggrecan was also shown to be significantly upregulated under load over the time course of the study (Fig. 7).
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Figure 6 Photomicrographs of sections for immunohistochemistry of GAGs in the 40-g experimental group. Experimental groups at day 4 (a–c), day 7 (d–f), and day 14 (g–i). Left column: chondroitin-4-sulfate, a, d, and g. Center column: chondroitin-6-sulfate, b, e, and h. Right column: keratan sulfate, c, f, and i. Original magnification, 31.25 (Saitoh et al., 2000). Reprinted from The Anatomical Record (2000), with permission by Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.
The authors also investigated the expression via RT-PCR of a positive regulator of chondrogenesis, Sox9, along with the expression of the catabolic cytokine, interleukin 1 (IL-1), and its receptor, IL-1RI, via RT-PCR and ELISA. Although Sox9 was detectable in both compressed and control constructs, statistically significant greater amounts of Sox9 transcripts in the compressed groups were noted at days 7 and 10 in culture. IL-1, was also downregulated to statistically significant lower levels compared to uncompressed controls. In this in vitro static compression study, Takahashi et al. (1998) were able to show that static mechanical compression of E10 mouse limb bud cells in general resulted in an upregulation of Sox9, which coincided with an increase in collagen type II and X and aggrecan, all of which are positive markers of chondrogenesis. Placing the constructs under mechanical compression also resulted in a downregulation of the catabolic cytokine IL-1, which has been shown to regulate chondrogenesis negatively by inhibiting GAG production and reducing collagen II production. Using a similar protocol, Elder et al. (2000, 2001) isolated stage 23/24 chick limb bud cells from Hamburger–Hamilton White Leghorn chick
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Figure 7 Compressive forces increased type II collagen and aggrecan expression assayed by competitive RT-PCR. Time course of type II collagen mRNA expression level (A) and aggrecan mRNA expression level (B) and force magnitude dependency of type II collagen mRNA expression (C) are shown. Open and solid bars represent control and compressed cultures, respectively. Standard deviation bars are placed on top of each respective data bar. Statistically significant diVerences are indicated by *P < 0.05, **P < 0.01, n ¼ 4 – 5 (A, B), n ¼ 3 (C) (Takahashi et al., 1998). Reprinted from Journal of Cell Science (1998), with permission from The Company of Biologists Ltd.
embryos, as these cells are known to possess the ability to diVerentiate down multiple mesenchymal lineages, including the chondrocytic lineage (Ahrens et al., 1977; O’Driscoll et al., 1986). After isolating the cells and cell aggregates, the authors cast the cells in agarose, obtaining a final concentration of 3% (w/v) with a cell concentration 3 106 cells/ml. Using a custom-built instrument, the 11 2-mm-diameter constructs were loaded cyclically (n ¼ 14) with a sinusoid wave form with peak-to-peak stresses of 0.25 to 9.0 kPa at a frequency of 0.33 Hz. This wave form was applied for 2 h in three diVerent loading sessions: within 24 h of cell isolation and then subsequently at 24 and 48 h following the first loading session. Results showed an approximate doubling of both cartilage nodule density and [35S]sulfate incorporation compared to the static loading group and the two control groups, which were statistically significant ( ¼ 0.05). No diVerences were noted between the static-loaded group and the two control groups.
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The authors then investigated the cyclic frequency eVects and the duration of cyclic loading on chondrogenic diVerentiation (Elder et al., 2001). The frequencies of loading were 0 (control), 0.03, 0.15, and 0.33 Hz (n ¼ 7/group) with a loading duration of 2 h. The duration of loading was also assessed with durations of 0 min (control), 12 min, 54 min, and 2 h with a frequency of 0.33 Hz. In addition to assessing cartilage nodule density and [35S]sulfate incorporation, collagen type II production was also assessed via immunohistochemistry. An approximately linear increase in chondrogenesis was noted in response to an increase in cyclic frequency as assayed by sulfate incorporation and cartilage nodule density (Fig. 8). The authors also showed an increase in GAG synthesis and cartilage nodule density correlating with duration of loading (12 to 120 min) at 0.33 Hz although the chondrogenic response was not linearly related to duration as it was with increasing frequency. A significant increase in chondrogenesis of approximately 50% was also noted when loading duration increased from 12 to 54 min or 2 h. No diVerences were noted between 0 and 12 min of loading. An example of the presence of collagen type II is shown in Fig. 9. These two studies by Elder and colleagues (2000, 2001) point to the ability of dynamic mechanical stress to induce chondrogenic diVerentiation of state 23/24 chick limb bud cells embedded in an agarose gel. This was shown over various frequencies with relatively short durations of loading time through
Figure 8 Chondrogenesis as assayed by [35S]sulfate incorporation (GAG synthesis) and cartilage nodule density (Elder et al., 2001). Reprinted from Annals of Biomedical Engineering (2001), with permission from Biomedical Engineering Society.
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Figure 9 (Left) Presumptive cartilage nodule viewed under transmitted light. (Right) confocal laser microscopy image of the same microscopic field (Elder et al., 2001). Reprinted from Annals of Biomedical Engineering (2001), with permission from Biomedical Engineering Society.
GAG synthesis rates, cartilage nodule density, and the presence of type II collagen. B. Cyclic Stretch (Biaxial Tensile Loading) Biaxial tensile loading has also been applied to progenitor cell populations to understand the role of tensile strains on development and diVerentiation events. Based on earlier work, which demonstrated that the unloading of bones in vivo resulted in a significant reduction in the number of osteogenic precursor cells in the stromal cell population and decreased expression of bone growth factors and markers (Keila et al., 1994; Zhang et al., 1995), Thomas and El Haj (1996) hypothesized that bone marrow stromal cells in vitro would be responsive to mechanical strain. Following isolation of bone marrow cells from male Wistar rats, the authors plated the cells on flexible dishes and subjected the membranes to cyclic biaxial stretch for 30 min at 1 Hz at three strain magnitudes: 35, 230, and 2500 mstrain. Following 10 days of culture, one loading regimen, and a recovery time of 2 additional days, the overall alkaline phosphatase production and the number of alkaline phosphatase positive cells were measured via flow cytometry using a specific antibody to alkaline phosphatase and a FITC-conjugated secondary antibody. All strain regimens resulted in an increase in alkaline phosphatase production, although only the 230 mstrain regimen resulted in a significant increase in the number of alkaline-positive cells compared to the unstrained controls. The authors further investigated the eVects of loading at diVerent time points, again with 30 min of loading duration for each time point, and the ensuing eVects on alkaline phosphatase activity. Results for this portion of the experiment varied depending on the day of loading and the day of
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analysis. Interestingly, the authors showed no increases in alkaline phosphatase-positive cells and overall production when loading was carried out on days 5 and 8 and stopped on days 7 and 10, respectively. However, if flow cytometry analysis was performed on day 12 instead of day 7 or 10, increases in both alkaline phosphatase-positive cells and overall alkaline phosphatase production were noted, although to nonsignificant levels. In addition, if the cells were loaded at 10 days and analyzed 2 days later, significant increases in the percentage of alkaline phosphatase-positive cells and alkaline phosphatase production were observed. In contrast, if loading occurred at 12 or 14 days and flow cytometry was performed 2 days later (for each respective group), a significant increase was noted in the percentage of alkaline phosphatase-positive cells, but no significant diVerences in alkaline phosphatase production were noted. The authors attribute these findings to the possibility that the significant increases observed on day 12 could be a result of increases in alkaline phosphatase production from mature osteoblasts in addition to the diVerentiation of osteoprogenitor cells. The lack of alkaline phosphatase production after this time point was attributed to the possibility that the cells are at an advanced diVerentiation stage where mineralization could occur. Nonetheless, this study established a preliminary link between osteoprogenitor diVerentiation and biaxial strain, although the mechanisms for diVerentiation remained unknown. To this end, the authors (Thomas and el Haj, 1996) also hypothesized that the mechanical strain induction of osteogenesis is mediated by prostaglandins, as they have been shown to be upregulated in response to load in previous studies (Binderman et al., 1988; Brighton et al., 1991; Murray and Rushton, 1990; Reich and Frangos, 1991). Using indomethacin, a general inhibiter of prostaglandins, the authors were able to inhibit the straininduced response, thereby implicating prostaglandins as a regulator in the strain-induced mechanotransduction pathway. In a series of studies by Rubin and colleagues (1999, 2000, 2002, 2003), the authors sought to understand the factors that aVect osteoclast diVerentiation for a bone marrow precursor cell population. Using 0.2–5% biaxial dynamic strain on collagen I-coated silicon membranes (Flexcell International Inc., Hillsborough, NC), the authors inhibited the osteoclast stimulation eVect of 1,25-dihydroxyvitamin D [1,25(OH)2D3] in a straindependent fashion compared to unstimulated control cultures (Rubin et al., 1999). This eVect was particularly noted through 5 days of culture but was lost from days 5 to 7 of culture (Rubin et al., 1999). In a follow-up study, these authors used 1.8–2.0% dynamic biaxial strain on a culture of murine primary stromal cell cultures to discern whether the osteoclast diVerentiation factor or transforming necrotic factor (TNF)-related activation-induced cytokine (ODF or TRANCE) was modulated by biaxial strain (Rubin et al., 2000).
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Using Northern analysis and RT-PCR for ODF/TRANCE, the authors demonstrated a significant decrease in ODF/TRANCE expression in the biaxial strain group after stimulation with 1,25(OH)2D3, a known stimulator of osteoclast diVerentiation. With the addition of exogenous soluble ODF, the authors were able to rescue the strain-mediated inhibition (Rubin et al., 2000). In order to discern possible mechanotransduction pathways, the authors investigated the interaction with 1.8% dynamic biaxial strain, extracellular signal-related kinase, ERK-1/2, and receptor activator of nuclear B ligand (RANKL), a marker for osteoclasts and N-terminal c-Jun kinase (JNK) (Rubin et al., 2002). In a previous study, downregulation in RANKL expression resulted in a reduction in osteoclast recruitment coupled with lower bone resorption (Fan et al., 2001). The results of this study showed a rapid but transient upregulation of ERK-1/2 (peak expression obtained by 5 min returning to baseline by 60 min), as measured by Western blotting, in response to dynamic strain coupled with an approximate 50% downregulation of RANKL expression as measured with RT-PCR, although this straininduced inhibition was transient and disappeared following an additional 24 h of unstrained culture. Inhibiting ERK-1/2 with PD98059 (Calbiochem) resulted in complete rescue of the strain-mediated inhibition of RANKL, indicating that ERK-1/2 is necessary to inhibit RANKL expression following strain. However, the authors noted that this repression of RANKL expression via mechanical strain by ERK-1/2, which is upregulated rapidly, is alone not suYcient, as approximately 6 h of loading are required for this biaxial strain-mediated repressive eVect. In contrast to the transient activation of ERK-1/2, dynamic mechanical strain maintained an upregulated JNK expression as measured by a JNK assay and RT-PCR. This finding suggests that the persistence of JNK expression may modulate the cascade of signaling events in response to dynamic biaxial strain to inhibit osteoclastogenesis. In a similar study, Rubin and colleagues (2003) demonstrated that in addition to the upregulation or ERK-1/2 and downregulation of RANKL, as shown in their previous study (Rubin et al., 2002), a concomitant upregulation of endothelial nitric oxide synthase (eNOS) was induced when bone marrow osteoprogenitor cells were loaded with dynamic biaxial strain. eNOS expression results in the production of nitric oxide (NO), which has been shown to promote anabolism in bone and inhibit osteoclast activity (Collin-Osdoby et al., 2000; Kasten et al., 1994; Koyama et al., 2000; Ralston, 1997). In addition to reconfirming downregulation of RANKL under dynamic mechanical strain, the authors, using a specific primer for eNOS, performed RT-PCR and showed an approximate doubling of eNOS expression under this loading regimen. Further, the authors demonstrated an expression eVect for eNOS that was correlated with increasing strain magnitude and an inverse correlation for RANKL expression (0.25–2% strain). An increase in NO was observed after 24 h of continuous mechanical
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stimulation to approximately two times that of control cultures. Blocking ERK-1/2 activity with PD98059 resulted in no significant increases of NO production compared to unloaded controls, implicating ERK-1/2 activity as an upstream signal of eNOS. In addition, to ensure that eNOS was the producer of NO, a-competitive inhibitor of eNOS, N-nitro-l-arginine methyl ester (l-NAME) was used under the dynamic biaxial strain regimen, which resulted in a successful blockade of NO production. The authors further demonstrated that blocking NO production with l-NAME had no eVect on strain-induced ERK-1/2 activation, resulting in their conclusion that NO does not play a major role in the strain-induced upregulation of ERK-1/2. To confirm these strain-induced results, bone marrow stromal cells were transfected with an adenovirus to constitutively deliver activated MEK, which is upstream to and activates ERK-1/2, to the cells. This constitutively active MEK expression, without loading, resulted in significant increases in eNOS expression and a significant downregulation of RANKL expression as measured by RT-PCR. These results were consistent with the earlier conclusions that upregulation of ERK-1/2 was an upstream regulator of eNOS and RANKL. However, applying dynamic strain while inhibiting JNK, which was shown in their previous study to be constitutively active when subjected to dynamic biaxial strain (Rubin et al., 2002), the expressions of eNOS and RANKL are unaVected, thus indicating that JNK is aVected downstream of these important regulators of bone homeostasis. In summary, the authors concluded that these important regulators of bone homeostasis are, in part, governed by activation of the ERK-1/2 signaling cascade, but that it is probable that many other coregulators govern the expression of these two genes by mechanisms not yet elucidated (Rubin et al., 2003). Takahashi and colleagues (2003) sought to expand their study on the eVects of tensile loading using both in vivo and in vitro systems in order to elucidate the role of extracellular matrix adhesion on the inhibition of chondrogenesis. Based on their earlier work, Takahashi and colleagues showed an inhibition of chondrogenesis and an induction of osteogenesis in midpalatal suture cartilage under tensile forces (Takahashi et al., 1996). Four-week-old Sprague–Dawley rats were used for the in vivo experiments with immunohistochemical analysis of the cytoskeletal proteins, paxillin and vinculin, in addition to 5 and 1 integrins and ERK-1/2 and phosphorylated extracellular signal-related kinase (pERK-1/2) at time points of 6 h, 12 hr, 24 h, and 2 days. Furthermore, the authors investigated fibronectin and filamentous actin via immunohistochemistry and immunocytochemistry at 0, 24, and 48 h. The in vivo tensile loading apparatus was as described previously (Takahashi et al., 1996). The in vitro tensile loading system consisted of cells on a stretchable silicone membrane controlled by the activation of a screw (Fig. 10). For the in vitro study, E12 stage embryos were harvested from Sprague– Dawley rats and were processed as described previously (Takahashi et al.,
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Figure 10 In vitro loading schematic (Takahashi et al., 2003). Reprinted with permission from European Journal of Cell Biology (2003).
1998). Approximately 80,000 cells were plated on the center of the flexible membrane and subjected to tensile loading (Fig. 10). In addition to confirming the results for the in vivo experimentation of their earlier study (Takahashi et al., 1996), the authors were also able to investigate the response of several cytoskeletal proteins to this mechanical stress. The authors noted the accumulation of fibronectin around the precartilaginous cells as they become spindle shaped in response to the mechanical forces. After 6 h of loading, confocal microscopy revealed an accumulation of fibronectin and actin in the cells, both of which were immunolocalized to focal adhesion complexes. An upregulation of paxillin and vinculin was also observed at 3 h in the mechanically loaded group compared to unstressed controls (Fig. 11). In the control tissues, both cartilaginous and precartilaginous cells stained positive for 5 and 1 integrins. In contrast, the experimental group demonstrated a statistically significant upregulation (P < 0.01) of 1 expression over the 2 days of the study (Fig. 12). Statistically significant diVerences were noted for 1 integrin and FAK compared to controls as expression was upregulated 40 and 36%, respectively, compared to controls. The expression level of 1 integrin peaked at 3 h and then decreased to approximately 125% of that of controls at 12 h. The expression of FAK decreased rapidly after the 1-h peak to values of approximately 10% greater than control. Conversely, the expression of 5 integrin was not statistically diVerent from control animals over the time course of 12 h. In the in vitro system, stretching resulted in a smaller cell size and an inhibition of collagen type II expression as determined by Alcian blue staining. Conversely, the unloaded control cultures demonstrated appearances of chondrogenesis. Interestingly, addition of the glycine–arginine–glycine–aspartic acid–serine (GRGDS) peptide to stretched cells at
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Figure 11 Immunolocalization of paxillin (A, C) and vinculin (B, D). Arrowheads indicate the focal adhesion contacts observed in stretched cells. (A and B) Zero-hour control group. (C and D) Three-hour group. Original magnification, 126 (Takahashi et al., 2003). Reprinted with permission from European Journal of Cell Biology (2003).
Figure 12 Immunolocalization of 5 (A, C) and 1 integrins (B, D). Arrowheads indicate immunoreaction against 5 and 1 integrin-specific antibodies. (A and B) Zero-day control group. (C and D) Two-day group. Original magnification, 40 (Takahashi et al., 2003). Reprinted with permission from European Journal of Cell Biology (2003). (See Color Insert.)
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concentrations of 1 and 100 mM completely rescued the chondrogenic phenotype from tension-mediated inhibition, suggesting that certain cell–ECM contacts can regulate chondrogenesis negatively. In this regard, the authors stated that downregulation of the 1 integrin may be critical in regulating chondrogenesis as has been observed in a previous study by Imoto et al. (2002). This in vitro finding is consistent with the in vivo results, which showed a trend toward osteoinduction, concomitant with an increase in 5 and no change in 1 integrin expression. In addition, based on earlier work by Oh and colleagues (2000), who showed a decrease in ERK-1/2 activity during chondrogenesis, sustained ERK-1/2 activity throughout the in vivo experiment would be expected. Indeed, ERK activity peaked at 3 h and was maintained throughout the time course of the experiment. In this study, the authors clearly demonstrated that osteochondroprogenitor cells from midpalatal suture cartilage changed phenotype in response to mechanical tensile stress by observing significant changes in protein, integrin, and GAG expression. Based on this work and some of their previous work (Saitoh et al., 2000; Takahashi et al., 1996, 1998), the authors conclude by stating that tensile forces result in the maintenance of cell–extracellular matrix interactions by the upregulation of integrins and FAK, thus prohibiting chondrogenesis and the ensuing upregulation of collagen type II. Simmons et al. (2003) also employed biaxial stretch (modified Flexercell FX-2000 system; Flexcell International Inc., Hillsborough, NC) to investigate the eVects of mechanical stress on a purified human mesenchymal stem cell (hMSC) population (Cambrex Bio Science) extracted from adult bone marrow aspirates. In this study, a continuously applied 3% strain magnitude at 0.25 Hz for 16 days was used. Briefly, the authors plated hMSCs on a collagen type I-coated (1 mg/cm2) silicone well at a low seeding density of 1000 cells/cm2 (seeding area 5 cm2). The authors used a low density to ensure that the cells would not be contact inhibited and that diVerentiation would not occur. Using osteogenic media, the authors first investigated the proliferative capabilities of these hMSCs followed by an investigation of two mitogen-activated protein kinase (MAPK) pathways, ERK-1/2 and p38 MAPK. To inhibit these two pathways, 10 mM of U0126 (Cell Signaling) and 15 mM of SB203580 (Calbiochem) were used to block the ERK-1/2 (via inhibition of MEK1/2) and p38 MAPK pathways, respectively. Although the proliferative capabilities of hMSCs were maintained in both strained and control cultures, a statistically significant greater number of cells were observed in the control cultures coupled with a significant increase in calcium matrix deposition over the 16-day time period (Fig. 13) (Simmons et al., 2003). These data alone suggested that cyclic biaxial strain induces osteogenic diVerentiation of hMSCs, although the path by which this occurs was not
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Figure 13 Von Kossa stain indicating greater mineralization in strain-induced cultures (A) Unstrained (B) Strained. Original magnification, 100. (Simmons et al., 2003) Reprinted from Journal of Biomechanics (2003), with permission from Elsevier.
elucidated. To examine the potential mechano-transduction pathways, ERK1/2 was blocked with U0126, leading to decreases in both alkaline phosphatase (ALP) expression ( 55 21%, P < 0.001) and mineral production ( 31 18%, NS) at 16 days compared to uninhibited control cells; however, no statistically significant diVerences were noted between strained and control cells in their respective groups (control or inhibited). U0126 also resulted in a statistically significant decrease in strain-induced mineral deposition (55 27%, P < 0.05) compared to uninhibited cultures, suggesting that ERK1/2 was necessary for strain-induced mineral deposition. Blocking p38 MAPK with SB203580 resulted in a significant increase (P < 0.05) in ALP expression at 16 days in control cultures. Conversely, SB203580 resulted in a statistically significant decrease in ALP activity in strained cultures relative to uninhibited strained cells. This was coupled with an approximate five-fold increase in strain-induced calcium mineralization relative to the p38 MAPKinhibited control culture (P < 0.02). These data suggest that p38 MAPK plays an inhibitory role in osteogenesis, as blocking its action resulted in mineralization around the cell. In contrast, ERK-1/2 appeared to be actively involved in strain-induced osteogenesis, as blocking its activity resulted in a significant downregulation of osteoinduction. This latter observation is consistent with the findings of Takahashi et al. (2003), who observed using immunohistochemistry that ERK-1/2 was upregulated following mechanical loading, which resulted in osteogenesis in a zone of midpalatal suture cartilage. This ERK-1/2 activity was maintained at elevated levels over controls throughout their experimental time frame during osteoinduction. Simmons et al. speculated that the target for ERK-1/2 expression may be Cbfa1, a transcription factor implicated in osteoblast diVerentiation (Ducy et al., 1997). However, the authors also noted that soluble signals are also mediated by ERK-1/2 during osteogenesis (Gallea et al., 2001; Jaiswal et al., 2000; Lai et al., 2001; Takeuchi et al., 1997; Xiao et al., 2002), as are cell–matrix interactions (Lai et al., 2001; Takeuchi et al., 1997; Xiao et al., 2000, 2002), possibly implicating ERK-1/2 as a common mediator of diVerent signal types
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in osteoblast diVerentiation. Regardless, it appears that the strain-induced diVerentiation of hMSCs is regulated by the MAPK signaling pathway.
C. Multiaxial Loading Altman and colleagues (2002) isolated mesenchymal progenitor cells from both human and bovine sources and seeded the cells on collagen type I matrices with the goal of inducing ligament diVerentiation with the application of complex multiaxial loading, somewhat mimicking the combinations of strains experienced in vivo. The loading groups were subjected to 2% axial tensile strain and 90 rotational strain at a frequency of 0.0167 Hz. Using 35-mm photography and transmission electron microscopy (TEM), the authors noted a more organized collagen fibril organization in the loaded group compared to the static control, in addition to elongated cells consistent with ‘‘ligament-like’’ cell morphology. The authors were also able to quantify, via RT-PCR, diVerences in diVerentiation by measuring a significant upregulation in mRNA production of typical markers of ligament cells, collagen types I and III, and tenascin-C. From these analyses, the authors concluded that biaxial mechanical strain significantly enhances the ability of the progenitor cells to diVerentiate along the ligament lineage, although the mechanical signaling mechanism remains unknown.
D. Hydrostatic Pressure To understand the eVects of hydrostatic pressure on osteoclastogenesis, Rubin and colleagues (1997) obtained tartrate-resistant, multinucleated [through exposure to 1,25(OH)2D3] murine bone marrow cells and stimulated the cells with 1, 1.37, or 2.0 atm of pressure over varying periods of time up to 7 days. The hypothesis posed was that osteoclastogenesis was, in part, regulated by hydrostatic pressure and, by applying hydrostatic pressure at early times in culture, the authors could inhibit the biochemical induction of osteoclast diVerentiation as mediated by 1,25(OH)2D3. This hypothesis was partially driven by the observation that low pressures exerted in joints while walking have an eVect on osteoblast function (Ozawa et al., 1990) and that the use of hydrostatic pressure could alter the osteoclast/ osteoblast balance. The cells were exposed to hydrostatic pressure from days 2 to 7, 2 to 4, or 5 to 7 and then fixed on day 8. On other days in which hydrostatic pressure was not applied, the cells were maintained at 1 atm. By staining for tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase after exposure to 1.37 atm of hydrostatic pressure for 6 days,
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the authors noted a significant decrease of osteoclast-like cell number to 67% of control cells (1 atm). While there were no diVerences in cells exposed to either 2 to 4 or 2 to 7 days, the reduction in the number of osteoclast-like cells was similar to that found in the 6-day loading period. In contrast, loading from days 5 to 7 had no eVect on the reduction of the number of osteoclast-like cells. An increase in magnitude of hydrostatic pressure from 1.37 to 2.0 atm also had no eVect on the reduction of osteoclast-like cells. The macrophage colony-stimulating factor (MCSF) was also shown to be downregulated in the membrane-bound form under hydrostatic pressure, which suggested that this downregulation may inhibit osteoclastogenesis further by not providing proper osteoclast diVerentiation signals to monocytic precursors. Many studies since the early 1990s on the eVects of hydrostatic pressure on articular chondrocytes have been conducted with clear physiological eVects on the expression of cartilaginous matrix (Ikenoue et al., 2003; Lammi et al., 1994; Lee et al., 2003a,b; Parkkinen et al., 1993a,b, 1995; Smith et al., 1996, 2000; Suh et al., 1999; Tagil and Aspenberg, 1999). With this knowledge, Angele et al. (2003) applied hydrostatic pressure to hMSC aggregates in an attempt to promote chondrogenic cellular diVerentiation in vitro. hMSCs were isolated from iliac crest bone marrow of patients undergoing spine fusion and were plated until 80% confluent. After removal of the cells from the plate, a cell aggregate of 2 105 cells was placed in a polypropylene tube with 1 ml of a defined chondrogenic medium (Johnstone et al., 1998; Yoo et al., 1998) added to fill the tube completely. The tubes were then placed in a water bath with a computer-controlled hydraulic actuator. Using this instrument, the authors were able to control the hydrostatic pressure within each of the tubes with a minimum physiologic pressure of 0.55 MPa and a maximum physiologic pressure of 5.03 MPa (Mow et al., 1992) for 4 h per day at 1 Hz. The three experimental loading groups were (a) single day loading on day 1, (b) single day loading on day 3, and (c) loading on each day (days 1–7). Two sets of controls were used for the study: one with identical treatment as the aggregates subjected to hydrostatic stress and the second with standard culture conditions (n ¼ 3 for all groups.). Also, the groups were further subdivided into two analysis time points: day 14 or day 28. Results of the study (Angele et al., 2003) showed no significant diVerences between either of the control groups at any time during the experiment. DNA content, as measured by the Hoechst 33258 dye assay, also remained the same throughout the experiment regardless of treatment group or control. No significant diVerences were noted in proteoglycan or collagen concentration in either group a or group b. This applied to both 14-day and 28-day harvest time points. In contrast, group c revealed significant diVerences in the loaded group compared to the control in both proteoglycan
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and collagen concentrations, indicating significantly more matrix accumulation as a result of 7 days of applied hydrostatic pressure. However, immunohistochemical analysis revealed no qualitative diVerences in collagen distribution between loaded and unloaded controls of group c (7 days of loading). Interestingly, though, the authors showed a significant increase in the size of the aggregate with more matrix accumulation and greater distance between cells for the 7-day loading group. This was shown at either the 14-day or the 28-day time point. The authors noted that since the chondrogenic eVect was equivalent to that of multiloading days, hydrostatic loading is cumulative, consistent with previous work on hydrostatic loading and chondrocytes (Ellison et al., 1994). In other words, if the mechanism for triggering chondrogenesis was a short-term hydrostatic event, for example, one would expect little to no diVerence in groups a, b, or c at either day 14 or day 28 in culture. In contrast, results seem to suggest the need for hydrostatic pressure applied over a longer time period to induce chondrogenic diVerentiation. Finally, the influence of hydrostatic pressure on the diVerentiation capabilities of hMSC cell aggregates is suggested, but further studies will be needed to delineate the mechanisms of chondrogenesis and the interplay between the mechanical and the biochemical induction of chondrogenesis.
E. Shear Stress Shear stress has also been used to stimulate stem cell diVerentiation. In one study, purified basal cell cultures from adult olfactory epithelium were stimulated with mechanical shear stress or biochemical stress to promote neurogenesis (Feron et al., 1999). The authors isolated cells from the nasal septum olfactory epithelium of adult, pathogen-free, Wistar rats and from a transgenic mouse strain with the LacZ gene driven by the olfactory marker protein (OMP) promoter, which is found exclusively in fully diVerentiated olfactory sensory neurons (Keller and Margolis, 1975). At day 6 after plating the basal cells, the cultures were treated with successive baths of EDTA (200 ml/cm2) and 50 ml/cm2 of trypsin. Cells released from the plates after these sequential treatments were replated and placed in the biochemical stressed group (i.e., those cells removed from the plate by washes with EDTA and trypsin). Both the mechanical stress group and the control group consisted of plated basal cells, which were not released following EDTA and trypsin treatment. The mechanical group was stressed by applying a coverslip over the cells and gently agitating the dish for days 7 and 8 of culture, presumably imparting unquantifiable mechanical shear stresses on the basal cell culture. All three groups were maintained in serum-free and epidermal growth factor (EGF)-free medium for the remainder of the study in an attempt to isolate the mechanical or biochemical treatment. Using immunocytochemistry, the
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authors stained for the neuronal markers, anti--tubulin III, anti-MAP5, and anti-OMP, which proved to be present in approximately 25% of the mechanically stressed group, mostly along the periphery of the coverslip. Likewise, the authors stated that the biochemically stressed groups also resulted in olfactory neuronal diVerentiation, although quantitatively, it is diYcult to compare these two stressed groups. In contrast, the control cultures did not stain for these neuronal markers, but instead had markers not found in the mechanically induced basal cell culture, including antiSUS1 (an immature neuronal maker) and anti-keratin, a horizontal basal cell marker. The authors confirmed their results from the transgenic mouse by driving the LacZ gene with the OMP promoter after stressed induction. The authors concluded that mature olfactory neurons can be induced by single mechanical or biochemical events on basal cells without supporting cells such as astrocytes, growth factors, or conditioned medium. In a separate study, fluid shear stress was used to stimulate nitric oxide and prostaglandin production in preosteoclast-like cells derived from bone marrow (McAllister et al., 2000). In earlier work by these same authors, a significant increase of nitric oxide was noted when osteoblasts were stimulated with fluid shear stress (Johnson et al., 1996). Because bulk flow is directed toward the periosteum in a radially oriented direction, the authors postulated that the endosteal surface, which contains a precursor cell population, may not be exposed to known osseous paracrine signaling molecules [e.g., prostaglandins E2 and I2 (PGI2), cyclic adenosine monophosphate (cAMP), intracellular-free calcium (Ca2þ), inositol trisphosphate (IP3), and transforming growth factor (TGF-)] (Hung et al., 1995; Johnson et al., 1996; Klein-Nulend et al., 1995, 1996; Reich and Frangos, 1991; Reich et al., 1997). Because paracrine signaling via diVusion was deemed unlikely by the authors, they hypothesized that fluid shear stress may drive this precursor cell population to osteoclast diVerentiation (McAllister et al., 2000). To test this hypothesis, a mixed population of bone marrow-derived cells was isolated from the femurs and tibiae of Sprague–Dawley rat neonates, which included osteoclast precursors, osteoblasts, fibroblasts, and macrophages. The specific isolation protocol, which utilizes a vitamin D3 treatment to induce osteoclastogenesis, yielded a cell population that was approximately 90% TRAP positive. The remaining cell population stained weakly for nonspecific esterase (NSE) activity, a marker for macrophages, and the cells were negative for alkaline phosphatase activity, which is an early marker for osteoblasts. All of these markers point to the preosteoclast nature of this population of cells. Within 24 h of plating the cells on tissue culture slides, the cells were exposed to a 16-dynes/cm2 fluid shear stress in a parallel plate fluid flow system (Reich and Frangos, 1991). Results of the fluid flow for a 6-h period showed an initial stimulation of nitric oxide (NO) production of 19.5 nmol/mg protein/h, which was reduced to a steady production
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rate of 7.5 nmol/mg protein/h, for the last 5 h of fluid flow. This production of NO was significantly greater than that of static controls (P < 0.001). The authors also added NG-amino-l-arginine (L-NAA) in a separate fluid shear stress treatment group to ensure that NO was induced endogenously from the fluid shear stress. Results indicated that the addition of L-NAA completely inhibited the endogenous production of NO. In addition to an upregulation in NO production, both PGE2 and PGI2 production were stimulated significantly compared to unstressed controls with sustained levels throughout the 6-h experiment. Even though the authors experimented on a mixed population of cells, results suggest that marrow-derived cells, which include osteoblasts and macrophages, as well as osteoclast precursors, are sensitive to fluid shear stress. Further, the authors noted that because the osteoblast fraction of the mixture is less than 10% (alkaline phosphatase positive), it is unlikely that the upregulation of NO and prostaglandins is from this population, especially considering that similar levels of NO were obtained in an enriched osteoblast culture in a previous fluid shear stress study (Johnson et al., 1996).
F. Shock Waves Based on their clinical success in the treatment of bone fractures and tendonopathies with the use of shock waves (Wang et al., 2001a,b,c), Wang and colleagues (2001d, 2002a,b) performed several studies under the driving hypothesis that the in vitro use of shock waves on bone marrow-derived stem cells would be beneficial in promoting osteogenic diVerentiation. The shock wave is produced by creating a high-voltage spark discharge underwater, which in turn results in an explosion that causes a rapid evaporation of water, which produces high-energy acoustic waves. The wave is then reflected directly to the cells or the tissue being stimulated. Using either human bone marrow-derived stromal cells (Wang et al., 2001d) or Sprague–Dawley rat bone marrow-derived stromal cells (Wang et al., 2002b), the authors were able to show an increase in osteogenesis in this cell population (Wang et al., 2001d, 2002b). Briefly, the authors subjected the stromal cells to a shock wave at 0.16 mJ/mm2 with varying pulses ranging from 0 to 3000 impulses. In both studies, 500 impulses of the shock wave resulted in a plateau of osteoprogenitor colony-forming units (CFU-O), which were defined by colonies showing more than 32 segregate cells and staining positive for alkaline phosphatase activity. Greater than 500 impulses resulted in a loss of CFU-O, most likely due to damage inflicted on the cells. Similarly, 500 impulses also resulted in the maximum upregulation (as compared to other impulse durations) of TGF- (via ELISA), CBFA1, osteocalcin, bone nodule formation (measured by Von Kossa stain, see Fig. 14), and Ras; the latter has been
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Figure 14 Von Kossa staining in an untreated group (left) and in a shock wave group (right) (Wang et al., 2002b). Reprinted with permission from Journal of Bone and Joint Surgery (Br) (2002).
shown in to be induced by membrane potential changes (Zwick et al., 1997). By transfecting the cells with a dominant-negative form of Ras, the authors tested the hypothesis that through hyperpolarization of the membrane following the shock wave, Ras was upregulated, ultimately leading to activation of its downstream target, CBFA1, an osteogenic transcription factor. Indeed, this transfection completely blocked CBFA1 upregulation and also abrogated the shock wave-induced osteocalcin and bone nodule upregulation, thereby confirming the hypothesis (Wang et al., 2001d). In an eVort to discern the mechanotransduction pathway, the authors investigated various oxygen radicals and signaling molecules that could be the primary signal induced by the shock wave (Wang et al., 2002a). This reasoning was based on evidence that shock waves increase the permeability of the cell membrane and therefore allow the rapid infiltration of biological substances (Huber et al., 1999; Zhong et al., 1999). By selectively blocking potential signaling molecules, O2 , Ca2þ , H2 O2 , or PGE2 just prior to subjecting the cells to a shock wave and measuring the downstream eVect on [3H]thymidine uptake as a measure of bone marrow stromal cell growth, the authors were able to determine that only superoxide dimutase (SOD), a scavenger of O2 , significantly reduced [3H]thymidine uptake, alluding to the role of superoxide in initiating the signaling event. SOD also suppressed the ERK signal following the shock wave and the expression of CBFA1,
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thus inhibiting the ability of the shock wave to induce osteogenic diVerentiation. To further elucidate the potential signaling cascade following superoxide induction, protein kinase C, protein kinase A, and tyrosine kinase were selectively blocked, and results demonstrated that only one of these kinases, tyrosine kinase, when blocked, aVected ERK activation, suggesting that ERK activation is mediated through a tyrosine kinase pathway (Wang et al., 2002a). Through these studies investigating the eVects on osteogenic diVerentiation in bone marrow stromal cell populations, the authors have demonstrated compelling evidence of the involvement of superoxide, Ras, tyrosine kinase, MEK, and ERK-1/2 during shock wave-induced osteogenesis (Wang et al., 2001d, 2002a,b).
VI. Conclusions In summary, there is significant evidence that mechanical signals play a critical role in regulating stem cell fate. These influences are apparent during the normal diVerentiation process of embryonic stem cells during development, but may also manifest in situations that require the recruitment of undiVerentiated progenitor cells, such as wound healing or tissue repair. Importantly, it is clear that specific mechanical signals may promote cell diVerentiation into a particular phenotype. The use of such mechanical signals in vitro in specially designed ‘‘bioreactors’’ may provide important adjuncts to standard biochemical signaling pathways for promoting engineered tissue growth. It is hoped that further understanding of the biomechanical and biochemical pathways involved in mechanical signal transduction by stem cells will provide new insight in the improvement of stem cell-based therapies.
Acknowledgment This study was supported in part by NIH Grants GM08555, AR49294, AG15768, AR48182, and AR50245.
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Origins of Mammalian Hematopoiesis: In Vivo Paradigms and In Vitro Models M. William Lensch*,{ and George Q. Daley *,{,{,§ *Whitehead Institute for Biomedical Research Cambridge, Massachusetts 02142 { Biotechnology Process Engineering Center Massachusetts Institute of Technology Cambridge, Massachusetts 02139 { Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School Boston, Massachusetts 02115 § Division of Pediatric Hematology/Oncology Children’s Hospital Boston Dana-Farber Cancer Institute Boston, Massachusetts 02115
I. Introduction: The Hematopoietic System II. Hematopoiesis: De Muribus et Maribus A. In Vivo Blood Development in Mus musculus B. In Vivo Blood Development in Homo sapiens III. Points of Divergence Between Murine and Human Blood Formation IV. Hematopoiesis from Embryonic Stem Cells A. The Beginnings of Pluripotent Cell Research B. Endothelial Cells and Blast Colony-Forming Cells C. Hematopoietic Cells D. In Vivo Hematopoietic Reconstitution with Murine Embryonic Stem Cell Derivatives E. Hematopoiesis and Human Embryonic Stem Cells V. What Remains To Be Learned? VI. Summary Acknowledgments References
Though a topic of medical interest for centuries, our understanding of vertebrate hematopoietic or ‘‘blood-forming’’ tissue development has improved greatly only in recent years and given a series of scientific and technical milestones. Key among these observations was the description of procedures that allowed the transplantation of blood-forming activity. Beyond this, other advances include the creation of a variety of knock-out animals (mice and more recently zebrafish), microdissection of embryonic and fetal blood-forming tissues, hematopoietic stem (HSC) and progenitor Current Topics in Developmental Biology, Vol. 60 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00
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cell (HPC) colony-forming assays, the discovery of cytokines with defined hematopoietic activities, gene transfer technologies, and the description of lineage-specific surface antigens for the identification and purification of pluripotent and diVerentiated blood cells. The availability of both murine and human embryonic stem cells (ESC) and the delineation of in vitro systems to direct their diVerentiation have now been added to this analytical arsenal. Such tools have allowed researchers to interrogate the complex developmental processes behind both primitive (yolk sac or extraembryonic) and definitive (intraembryonic) hematopoietic tissue formation. Using ES cells, we hope to not only gain additional basic insights into hematopoietic development but also to develop platforms for therapeutic use in patients suVering from hematological disease. In this review, we will focus on points of convergence and divergence between murine and human hematopoiesis in vivo and in vitro, and use these observations to evaluate the literature regarding attempts to create hematopoietic tissue from embryonic stem cells, the pitfalls encountered therein, and what challenges remain. ß 2004, Elsevier Inc.
I. Introduction: The Hematopoietic System Hematopoiesis is the term applied to the myriad processes resulting in blood cell production. The description of hematopoietic stem cell activity stands as a tremendous scientific milestone of the last century and was a culmination of many studies (e.g., Becker et al., 1963; Bradley and Metcalf, 1966; DanchakoV, 1916b; Dexter and Lajtha, 1974; Dick et al., 1985; Eglitis et al., 1985; Hodgson and Bradley, 1979; Jacobson et al., 1949; Jordan and Lemischka, 1990; Joyner et al., 1983; Keller et al., 1985; Lemischka et al., 1986; Lorenz et al., 1951; Spangrude et al., 1988; Till and McCulloch, 1961; Till et al., 1964; Williams et al., 1984). Moreover, the detailed analysis of hematopoiesis also directly facilitated the development of a profoundly beneficial medical intervention, namely bone marrow transplantation (Thomas et al., 1957) (reviewed in Thomas, 1999), a stunning example of the key role that basic research plays in improving human health. Information regarding the biology of hematopoiesis remains largely unknown in all but a handful of organisms (Tavassoli, 1991). In mice and humans the establishment of the hematopoietic system occurs in two waves in utero: the first being ‘‘primitive,’’ transitory, extraembryonic, and (at least at its earlier stages) incapable of reconstituting blood formation long term in transplant recipients (Huang et al., 1994; Medvinsky et al., 1993; Moore and Metcalf, 1970; Muller et al., 1994). The earliest, primitive blood elements are mainly erythroid in nature and consist of large, nucleated red cells. Whether or not the progenitors of these early erythroid cells contribute to the establishment of definitive, lifelong blood formation or are truly ephemeral
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remains a topic of much deliberation. A second round of hematopoietic cell development occurs after somite initiation, apparently arising within the embryo proper. This later-occurring, ‘‘definitive’’ form of intraembryonic hematopoiesis is durable and transitions temporally through visceral sites of blood formation via chemotaxis and the upregulation of adhesion molecules that facilitate stem cell retention in the liver, thymus, and spleen. These definitive hematopoietic cells will eventually seed the bone marrow and produce blood tissue throughout the remaining life span of the organism. Here we see a defined hematopoietic ontogeny originating with self-renewing, pluripotent hematopoietic stem cells (PHSC) capable of producing all elements of the myeloid and lymphoid lineages. The PHSC is defined by its capacity to rescue all aspects of long-term hematopoiesis when transplanted into a lethally myeloablated recipient through a combination of self-renewal, expansion of derivative progenitor cell pools, and ultimate diVerentiation to mature progeny. Several investigators have successfully correlated the activity of the PHSC with a defined surface phenotype and have thus facilitated eYcient enrichment from mixed populations (e.g., Benveniste et al., 2003; Christensen and Weissman, 2001; Osawa et al., 1996; Spangrude et al., 1988). Just distal to the PHSC in the hematopoietic ontogeny is a functional bifurcation that restricts the lymphoid compartment from a myeloid lineageproducing, multipotent hematopoietic progenitor cell. A common lymphoid progenitor (CLP) has been characterized at the molecular level and shown to be capable of producing both B and T cells from a single starting cell (Kondo et al., 1997). The CLP has restricted developmental potential and produces components of only the lymphoid lineage, including B lymphocytes, T lymphocytes, and other entities such as natural killer (NK) and plasma cells, although NK cells have not been derived from a single starting cell that also produced B- and T-cell progenitors and thus their exact origins relative to the CLP remain somewhat unclarified (Akashi et al., 2000a). Additionally, it has been shown that certain T-cell progenitors found within the thymus derive from a cell that also possesses limited myeloid developmental potential, is unresponsive to interleukin (IL)-7 stimulation, and persists in the absence of the Ikaros transcription factor, all clear distinctions from the CLP, suggesting that the ontogeny of lymphopoiesis requires further study (Allman et al., 2003) (reviewed in Montecino-Rodriguez and Dorshkind, 2003). The common myeloid progenitor (CMP) ultimately gives rise to seven myeloid lineages in response to environmental cues (Dexter, 1984; Metcalf, 1985): erythrocytes (anucleate, oxygen-fixing red cells), monocytes (macrophage progenitors), megakaryocytes (which produce clotting factor-containing thrombocytes or platelets), and four types of secretory granule-producing cells—eosinophils, basophils, granulocytes [also known as neutrophils, polymorphonucleocytes (PMNs)], and finally the mast cell (a bane of seasonal
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allergy suVerers). A molecular signature has also been suggested for the CMP (Akashi et al., 2000b). Functioning normally, the hematopoietic system provides for eYcient tissue oxygenation, assists in the repair of compromised vascular tissue, scavenges debris as well as pathogens, establishes and maintains immunity, and through the secretion of proinflammatory and other factors mediates a host of cellular responses throughout the body. These many functions are carried out while the system continuously reconstitutes itself, beginning proximally with stem cell self-renewal through the distal elaboration of functional progeny, at a rate of literally billions of cellular components per day. The complexity of the regulatory networks that govern this process is staggering, although the advent of whole genome-scale expression analysis partnered with computational biology and bioinformatics in addition to a wealth of studies using standard molecular biology have revealed a great deal (Ivanova et al., 2002; Quackenbush, 2001, 2002). However, there is still much to learn regarding the onset and maintenance of blood cell production. Having provided this brief overview of hematopoiesis, we now turn to a more detailed review of what is known of the origins of vertebrate, mammalian hematopoiesis using the mouse as a model. This is followed by a description of this same process in humans and a comparison of these two systems relative to one another. Among the key points that will hopefully emerge is the observation that even in the mouse, where a vast amount of experimentally derived data is available concerning all aspects of blood cell formation and development, the precise definition of the origins of long-term repopulating hematopoietic stem cells remains incomplete. Embryonic stem cells (ESC) have demonstrated a capacity to form hematopoietic tissue in vitro that functions in vivo, which presents us with powerful tools to inform our analysis of not only the very beginnings of normal blood development, but also disease processes and potential therapeutics.
II. Hematopoiesis: De Muribus et Maribus Although not the first organism studied in any detail, arguably the best understood vertebrate hematopoietic system is that of the laboratory mouse (Mus musculus), although recent work places the zebrafish (Danio rerio) a very close (and gaining) second (reviewed in Bahary and Zon, 1998; Hsu et al., 2001). By facilitating the analysis of strains with defective blood formation, such as the W or white-spotting mouse [mapping to (Chabot et al., 1988) and mutant at the c-kit locus (Geissler et al., 1988) and causing severe, often fatal, neonatal anemia], murine models quickly established themselves as robust systems for the study of hematopoiesis (e.g., De Aberle, 1924; Detlefsen, 1923). That both mice and zebrafish also permit the design of
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specific transgenic animals has given them a provocative advantage over other vertebrates as experimental platforms, although landmark studies have been conducted using both amphibian and avian systems. An excellent review of hematopoiesis in four model organisms and humans has been published by Galloway and Zon (2003).
A. In Vivo Blood Development in Mus musculus 1. Primitive Hematopoiesis In the mouse, early or ‘‘primitive’’ blood formation initiates in the yolk sac (YS) mesenchyme during gastrulation at postcoitum embryonic days 7.0–7.5 (d7.0–7.5, presomite; for somite staging, see Downs and Davies, 1993) (reviewed in Auerbach et al., 1996) (Fig. 1). Primitive hematopoiesis occurs in the extraembryonic splanchnopleura and is manifested as the formation of blood islands in the ectodermally derived mesoderm (i.e., from the epiblast) (Haar and Ackerman, 1971; Moore and Metcalf, 1970; Silver and Palis, 1997). The presentation of blood islands immediately follows the development of vascular progenitors (angioblasts) in the YS at d6.5 (Drake and Fleming, 2000). Structurally, blood islands consist of clusters of hemogenic cells surrounded by a flattened endothelium, thus making yolk sac hematopoiesis intravascular in nature. DiVerentiation of primitive hematopoietic elements in the yolk sac is of a more expeditious nature than what is seen in adult bone marrow. Here, the terminal cells of the erythroid lineage are large [10 mm in diameter (Mucenski et al., 1991)] and remain nucleated (Attfield, 1951) [see DanchakoV (1918) and references therein for early studies]. Sparse macrophages can be found, despite no visible monocyte precursors (Naito et al., 1996). The rare megakaryocytes present from d8.5 to 13.5 are of lower ploidy than their later marrow counterparts (Ferkowicz et al., 2003; Xu et al., 2001b); as bone marrow-derived megakaryocytes mature, their nuclei replicate, although the cells do not divide, thus making them progressively hyperploid with up to 128N or 128 haploid equivalents per cell (Long and HoVman, 1995). Taken together, these findings support a shortened maturation interval for primitive, extraembryonic hematopoietic cells. The abundant, yolk sac erythroid elements, termed ‘‘megaloblasts’’ (Attfield, 1951), express two embryonic forms of -globin-like hemoglobin ( and H1) (Barker, 1968; Brotherton et al., 1979; Craig and Russell, 1964; Fantoni et al., 1967; Keller et al., 1993; Whitelaw et al., 1990; Wong et al., 1986), as well as both embryonic and adult forms of -globin-like hemoglobin ( and , respectively) (Leder et al., 1992; Whitelaw et al., 1990; Wong et al., 1986). These four globins combine to make three tetrameric
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Figure 1 A composite depicting key events in hematopoiesis in vivo for the mouse (on top and scaled in days) and human (on bottom and scaled in weeks). Both murine and human figures are interpreted using the same key as follows: vertical red dashed line at left, onset of circulation; vertical green solid line at right, birth; EryP, primitive, nucleated erythroblasts in peripheral blood (PB); EryD, definitive, anucleate erythrocytes in peripheral blood (PB); P-Sp, onset of hematopoiesis in paraaortic splanchnopleura (down-slanting hash marks); AGM, onset of hematopoiesis in aorta-gonad-mesonephros (up-slanting hash marks); blue horizontal bars, -like globin gene expression; black horizontal bars, -like globin gene expression; yellow area, yolk sac hematopoiesis; green area, transition from yolk sac to liver; blue area, liver hematopoiesis; purple area, transition from liver to bone marrow; red area, bone marrow hematopoiesis. See text for the exact timing and duration of events summarized here. (See Color Insert.)
complexes known as EI ( 22), EII (22), and EIII (2H12) (Fantoni et al., 1967). Cells bearing them are termed primitive erythroblasts (EryP) and their embryonic forms of hemoglobin are uniquely adapted to the diVusionlimited, oxygen-poor microenvironment of the precirculation embryo (Bauer et al., 1975; Leder et al., 1980; Petschow et al., 1978). This early erythroid tissue requires growth factor stimulation via soluble burst-promoting activity (BPA) that is intrinsic to the yolk sac and is surveyed in vitro via the burst-forming unit erythroid (BFU-E) assay in methylcellulose (Labastie et al., 1984). Erythropoietin (epo) is not required for this early red cell activity (Lin et al., 1996; Wu et al., 1995). At d6.5 the yolk sac tissue is strongly positive for expression of the hematopoietic transcription factor Gata3 (Keller et al., 1993). The angioblasts of the blood islands are positive for the transcription factor Tal1 (T-cell acute lymphocytic leukemia, also known as Scl or stem cell leukemia hematopoietic
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transcription factor) (Robb et al., 1995; Shivdasani et al., 1995), as well as the receptor tyrosine kinase Flk-1 [for fms-like tyrosine kinase, also known as KDR in human cells and the receptor for vascular endothelial growth factor (VEGF)] (Matthews et al., 1991). The hemogenic cells are positive for these same markers (Palis et al., 1999) in addition to the transcription factors Gata-1 (GATA-binding protein 1) (Palis et al., 1999; Weiss and Orkin, 1995) and Brachyury (Palis et al., 1999). The creation of knockout mice has aided in the identification of transcriptional and signaling machinery important for the establishment of hematopoiesis (see Car and Eng, 2001). Among the genes whose mutation results in absent or significantly impaired yolk sac blood formation (and the resulting demise of the midgestation conceptus) is the aforementioned transcription factor Scl (Shivdasani et al., 1995), as well as the transcription factors Lmo2 (LIM domain only 2, also known as rhombotin 2 or rbtn2) (Warren et al., 1994), Gata1 (Fujiwara et al., 1996), Gata2 (Silver and Palis, 1997; Tsai et al., 1994), and the receptor tyrosine kinase Flk-1 (Shalaby et al., 1995, 1997). Double knockout of both Gata1 and Gata2 creates a more severe phenotype than either alone and leads to a complete loss of YS hematopoiesis similar to what is seen in the Scl / mouse (Fujiwara et al., 2004). Knockout of the soluble factor Bmp-4 (bone morphogenetic protein four) is embryonic lethal and has been shown to result in loss of Brachyury expression and the near complete absence of mesodermal structures (including blood islands) (Winnier et al., 1995). Expression of these genes is present in the yolk sac prior to the emergence of primitive erythroblasts (Quinn et al., 1993; Silver and Palis, 1997; Yamaguchi et al., 1993). The intersection of shared markers between endothelial and hematopoietic progenitors in the yolk sac lends evidence to the notion that they derive from a common progenitor. Observations made more than a century ago in the chicken [(His, 1900; see also Murray, 1932; Sabin, 1920) and a superb review of Sabin’s work by Downs (2003)], wherein the primordial hemogenic tissue (erythroid) was noted to develop in conjunction with the nascent yolk sac vascular endothelium [also described in more recent studies by Drake and Fleming (2000)], suggested a common progenitor. This hypothetical bipotential progenitor was called the hemangioblast (Murray, 1932). These results were controversial when first suggested (e.g., DanchakoV, 1916a; Stockard, 1915), although the concept is much less so today, thanks in part to work using ESC to define this population (see Section IV). The hemangioblast has not been isolated as a single cell per se although the yolk sac population that contains this activity has been characterized extensively (reviewed in Forrai and Robb, 2003; Lacaud et al., 2001). The hemangioblast is likely positive for the surface glycoprotein CD34 (Young et al., 1995), a marker of both endothelial (Fina et al., 1990) and hematopoietic cells (Krause et al., 1994). The analysis of knockout mice that demonstrate a
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phenotype in both yolk sac vascular endothelium and primitive hematopoiesis such as Flk-1 (Shalaby et al., 1995, 1997), the Creb-binding protein CBP (Oike et al., 1999), and the receptor tyrosine kinase Tie-2 (Takakura et al., 1998) provides additional evidence for the existence of the hemangioblast. Furthermore, in vitro studies using endothelial cells sorted from microdissected embryos (Nishikawa et al., 1998b) or derivatives of embryonic stem cells also point to the existence of a progenitor with both endothelial and hematopoietic activity, although not all embryonic endothelial cells are hemogenic (even those interrogated as early as the 1sp stage), as shown by the lack of blood-forming capacity in the developing allantois (Downs et al., 1998; Palis et al., 1999). In addition, surface phenotypes of the endothelial and hematopoietic derivatives of the hemangioblast soon diverge. Embryonic endothelial cells are positive for the calcium-dependent adhesion protein VE-cadherin and negative for CD45 (also known as the leukocyte common antigen), whereas hematopoietic progenitor cells are just the opposite (i.e., VE-cad , CD45þ) (Nishikawa et al., 1998b). Also, VE-cadherinþ CD45 cells are capable of reconstituting long-term lymphomyeloid hematopoiesis (Fraser et al., 2002). Finally, mounting evidence shows that the bipotential nature of hemangioblast-like cells exists into adulthood, as purified adult bone marrow stem cells are also capable of giving rise to cells of both endothelial and hematopoietic lineages (Bailey et al., 2004; reviewed in Hirschi and Goodell, 2001). Yolk sac hematopoietic cells are negative for major histocompatibility complex (MHC) class I and II antigens, although they slowly transition to immunoreactivity by d11.0–12.0. They are likewise negative at d7.0 and positive at d8.0 for the antigen recognized by the AA4.1 antibody (Auerbach et al., 1996). The AA4.1þ fraction is enriched for hematopoietic activity (Godin et al., 1999; Harrison et al., 1997; Huang and Auerbach, 1993; Jordan et al., 1990, 1995; Lu et al., 1996), particularly as stem cell antigen (Sca-1, also known as Ly-6A/E) immunoreactivity increases in the fetal liver stage of hematopoiesis (Jordan et al., 1995). However, adult bone marrowderived HSCs are AA4.1 negative (Auerbach et al., 1996; Rebel et al., 1996). Auerbach et al. (1996) also noted that yolk sac hematopoietic tissue is not immunoreactive for the CD34 surface antigen until d9.0 [although it is polymerase chain reaction (PCR) positive at d7.0–8.0], whereas Young et al. (1995) have shown that a fraction of d7.5 YS hematopoietic cells are CD34 positive. The majority (approximately 94%) of hematopoietic progenitors in the d9.5 YS are within the fraction positive for the surface antigen CD41 (also known as platelet glycoprotein IIb) (Mitjavila-Garcia et al., 2002), whereas in another experiment, the entirety of EryP colony-forming activity (as well as the primitive megakaryocyte and macrophage-forming activity) could be isolated from the d7.0 yolk sac by purification using CD41 (Ferkowicz et al., 2003). However, endothelial progenitors are noted
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to be CD41 negative (Mikkola et al., 2003a), suggesting that this integrin is perhaps one of the first diVerentially expressed genes distal to the hemangioblast. CD41 expression persists through the transition to definitive hematopoiesis, although it identifies a fractionally smaller percentage of cells as the fetal liver and bone marrow become the dominant hematopoietic organs (Ferkowicz et al., 2003; Mikkola et al., 2003a; Mitjavila-Garcia et al., 2002). The appearance of lymphoid cells is not a prominent feature of precirculation, yolk sac hematopoiesis (Ogawa et al., 1988; Owen et al., 1976) and serves as a demacration in the transition to definitive blood formation. Detectable lymphoid precursors have been obtained from explanted yolk sac tissue taken from postcirculation embryos and cocultured on stromal cell monolayers (Cumano et al., 1993; Godin et al., 1995; Palacios and Imhof, 1993). YS-derived tissue from d8.0 to 9.0 mice has also been shown to generate T-cell precursors in ex vivo, thymic organ culture (Liu and Auerbach, 1991). Furthermore, when conditioned by stromal coculture, YS tissue from d8.0 mice generates cells positive for Thy1 [a marker of T lymphocytes (RaV, 1971)] and B220 [a marker of B-cell precursors and part of the CD45 family of receptor-linked protein tyrosine phosphatases along with Ly5 (Charbonneau et al., 1988)] and fosters long-term reconstituting activity in myeloablated recipients (Matsuoka et al., 2001). Although in vitro coculture of explanted embryonic cells is a useful experimental model, allowing one to interrogate the developmental capacity of isolated tissues, it is a synthetic environment and thus some experiments may reflect the plasticity of early hematopoietic tissue more accurately than the true nature of hematopoiesis in vivo as lymphocyte progenitors generally do not appear in the yolk sac until d8.0–9.0 (Cumano et al., 1993, 1996; Huang et al., 1994; Ogawa et al., 1988; Yoder et al., 1997a) in what is perhaps the postcirculation embryo (see later), hitting their maximum circulating levels at d12.0 (Delassus and Cumano, 1996). Studying explanted tissue from firmly precirculation embryos (d7.5), Cumano and colleagues (1996) were incapable of observing lymphoid progeny from yolk sac derivatives, even given extended stromal cell coculture and the addition of exogenous cytokines with lymphoid activity. Additionally, lymphoid development in the d8.5 (10sp) yolk sac parallels that occurring within the embryo proper in the paraaortic splanchnopleura (P-Sp; see later) (Godin et al., 1995). It seems unlikely that identical cellular activities would arise at two sites independently within an embryo possessing active circulation, which suggests that the emerging lymphoid progenitors arise in one tissue but then comingle in the postcirculation embryo or, alternatively, that they arise in two distinct locations but are coordinated by soluble growth factors produced in one locale that circulate in the embryo.
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2. The Onset of Circulation Recognizing that the temporal onset of circulation relative to the location of cells under study is of particular importance when considering the origins of hematopoietic cells in d8.0 to 9.0 extraembryonic mouse tissues, a careful review of the literature indicates that the assignment of the beginning of murine development or ‘‘day zero’’ by researchers is somewhat arbitrary and does not mark the moment of fertilization accurately, varying by as much as 12 h between authors [e.g., Huang and colleagues (1994) assign ‘‘day zero’’ as the morning a vaginal plug is discovered, whereas Godin and colleagues (1999) denote plug discovery as day 0.5]. Even such a short interval as one halfday may be very important when assigning a time line to the fine scale of events in early murine development in utero. Thus, the precise temporal staging of the onset of circulation has a profound impact on the interpretation of the literature describing the origins of definitive hematopoiesis, something that should be kept in mind by the reader when evaluating events occurring close to the onset of blood circulation. Studies making use of somite staging for the timing of early events are perhaps more accurate than those relying on ‘‘gestational days,’’ although strain-to-strain diVerences do exist (Downs and Davies, 1993). Timing aside, the onset of circulation in the embryo marks a major developmental transition in hematopoiesis and is dependent on the union of the yolk sac to the embryonic vasculature as well as the initiation of cardiac function. Primitive megaloblasts (or any other component of the blood for that matter) previously confined to the yolk sac vitilline plexus are now capable of egress from the extraembryonic structures into the embryo proper and vice versa for cells (or factors) formerly sequestered within the embryo. Cardiac contractions begin at 5sp (d8.25) (Fig. 1) with some degree of vascular flow by 7sp (d8.25–8.5) (Ji et al., 2003), although steady-state circulation remains unestablished until d10.5 (35sp) (McGrath et al., 2003). EryP enter the embryonic bloodstream at the onset of circulation (Ji et al., 2003) and persist beyond the establishment of liver hematopoiesis (d10.0, 28sp) (Houssaint, 1981; Johnson and Moore, 1975), although their progenitors are largely extinguished by d9.0 (20sp) (Palis et al., 1999). EryP are the only erythroid cells found in the embryo at d10.0. Thereafter, until their near absence at d15.0, EryP coexist peripherally with smaller, anucleate, definitive liver erythrocytes (EryD) (Mucenski et al., 1991). The hallmark feature of nuclear expulsion by red cells indicates the transition from intravascular erythropoiesis (as occurs in the yolk sac) to extravascular hematopoiesis (such as that found in the fetal liver and bone marrow) (reviewed in Tavassoli, 1991). The onset of circulation also marks the development of a site of long-term repopulating hematopoietic stem cell generation within the embryo proper in the region of the paraaortic splanchnopleura (P-Sp) at
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d8.5–10.0 (Godin et al., 1993). This area is later termed the aorta-gonadmesonephros (AGM) and will manifest blood cell-forming activity in the proximal portion of the umbilical arteries, vitilline arteries, and aorta after additional maturation from d10.0 to 12.0 (de Bruijn et al., 2000; Medvinsky et al., 1993; Muller et al., 1994; Wood et al., 1997). 3. The P-Sp/AGM The origin(s) of definitive hematopoiesis in the d10.0 liver (Houssaint, 1981; Johnson and Moore, 1975) remains at the heart of an intense scientific debate. There are three main theories regarding this issue, one termed ‘‘monophyletic’’ or ‘‘monoclonal’’ and two referred to as ‘‘diphyletic’’ or ‘‘biclonal.’’ In the first, it is held that blood cell progenitors within the yolk sac transition from primitive hematopoietic potential to mature, long-term reconstituting stem cells that then seed the fetal liver (Moore and Metcalf, 1970). The second model is a collaborative one and suggests that yolk sac HSCs migrate to the fetal liver and, in concert with those from an independent, intraembryonic source (the AGM), constitute the definitive stem cell pool. Finally, it has also been proposed that primitive yolk sac hematopoiesis is truly transient and contributes nothing durable to definitive hematopoiesis, with all long-term repopulating activity being solely derivative of the AGM. In this independent biclonal model, the primitive hematopoietic activity of the yolk sac functions there alone, and any reconstituting activity noted in the YS is coincidental and entirely due to circulating HSCs originating from within the embryo. The most convincing support for this last model comes from studies in birds where chicken yolk sacs were eVectively grafted onto quail embryos, yet the derivative quails did not bear hematopoietic tissue of chick origin (Dieterlen-Lievre, 1975). The intraembryonic region believed to be this second site of hematopoiesis in mammals is the aforementioned AGM that develops from the paraaortic splanchnopleura. The postulate that hematopoiesis arises from an intraembryonic hemogenic site is not new. It was over 100 years ago that van der Stricht (1899) first published the occurrence of intraaortic, epivascular ‘‘leukocytes’’ in developing mammals (the bat) (cited in Jordan, 1918). Eight years later, DanchakoV described clusters of blood cell progenitors (hemoblasts) in the 3- to 4-day chicken embryo that appeared to elaborate directly from the ventral endothelium of the dorsal aorta (DanchakoV, 1907). These findings were in agreement with the bipotential ‘‘angioblast’’ theory of yolk sac vasculogenesis/hematopoiesis proposed by His (1900). DanchakoV’s findings were followed by similar reports in the rabbit (Emmel, 1916; Maximow, 1909), mouse (Emmel, 1916), pig (Emmel, 1916; Jordan, 1916), and human (Jordan, 1918; Minot, 1912) and have since been substantiated in several other vertebrate species (for a review of early work see Smith and Glomski,
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1982) as disparate as the Jamaican mongoose and the loggerhead turtle (Jordan, 1917). The first precise description of the intraembryonic, aortic ‘‘hemogenic endothelium’’ using electron microscopy was reported by Smith and Glomski (1982) in the Mongolian gerbil. Their study also included serial sections of a 9.5-mm human embryo (ca. week 6) and the d10.0 mouse again, in agreement with the aforementioned earlier reports that hemoblasts appeared to arise directly from (or were at the least very intimate to) aortic endothelial cells (Smith and Glomski, 1982). A series of more recent papers have refined the appearance and activity of the intraembryonic, lymphomyeloid hematopoietic tissue of the d10.0–12.0 AGM (Garcia-Porrero et al., 1995; Godin et al., 1999; Medvinsky and Dzierzak, 1996; Medvinsky et al., 1993, 1996; Muller et al., 1994; Sanchez et al., 1996) (Fig. 1). Cells isolated from the d10.0–11.5 AGM bear long-term reconstituting hematopoietic stem cell activity in a fraction of recipients following 4 days of organ culture ex vivo (Cumano et al., 2001). The scale of cells generated in the murine AGM at this stage has been estimated to be on the order of 500 (Godin et al., 1999), although the number of long-term repopulating units contained within this population is much lower (Kumaravelu et al., 2002), perhaps reflecting the need for additional maturation in order to acquire such activity. In the murine AGM, the bipotential, endothelial/hematopoietic progenitor (hemangioblast) likely resembles a cell of the endothelial lineage and expresses the transcription factor Runx1 (also known as AML1) (North et al., 2002), Flk-1 (Shalaby et al., 1997), c-kit [also known as CD117, the receptor for stem cell factor (SCF)], CD31 [also known as platelet-endothelial cell adhesion molecular (PECAM)], and CD34 (de Bruijn et al., 2002; Wood et al., 1997; Young et al., 1995). However, in contrast to the developing yolk sac, endothelial cell growth in the nascent aorta precedes hematopoietic cell formation (i.e., the aorta is fully formed when the intravascular clusters of hemogenic cells arise), thus suggesting a distinct hemangioblastic activity within the embryo proper, with asynchronous vessel and blood cell-forming capacity (de Bruijn et al., 2002). It should be noted that the CD34 low to negative fraction [identified as either CD34lo/ c-kitþ Sca-1þ lineage (Osawa et al., 1996) or CD34lo/ Hoechstlo Sca-1þ (Goodell et al., 1997)] contains long-term reconstituting activity. This latter observation presents some diYculty in terms of interpretaion, as CD34 would have a biphasic expression profile: on in the bipotential endothelial/ hematopoietic progenitor, oV in the HSC (CD34lo/ ), back on again in the restricted hematopoietic progenitor cell, and then finally declining in expression for good as the cells diVerentiate into the myeloid lineages. Such complicated kinetics are certainly not without precedent in development. Freshly isolated progenitor cells from the P-Sp/AGM demonstrate a reduced capacity to engraft recipients long term. Additional maturation is likely required in order to become fully functional hematopoietic stem cells
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and suggests pre-HSCs. Intraembryonic cells with lymphomyeloid hematopoietic potential have been characterized in murine d9.5 embryonal body fragments by stromal cell coculture and shown to express B lymphocyte markers, whereas yolk sac tissues from the same developmental stage were negative (Ogawa et al., 1988; see also Marcos et al., 1991). Godin and colleagues (1993) observed B lymphoid potential in the d8.5–9.0 mouse (10–18sp) paraaortic splanchnopleura by engraftment experiments in immunodeficient hosts. Day 11.5 AGM-derived hematopoietic cells acquire greater long-term repopulating (LTR) activity after coculture on fetal liver stroma in vitro (Takeuchi et al., 2002). Repopulating cells are to be found in the yolk sac at these same time points, although the necessity for maturation of pre-HSCs is required here as well. Day 9.5 cells obtained from yolk sac and enriched for c-kitþ CD34þ CD41þ are capable of long-term reconstitution of neonates (Ferkowicz et al., 2003). Day 9.0 P-Sp cells are also capable of reconstituting primary neonatal recipients (Yoder et al., 1997a). Adult mice can then be reconstituted as secondary recipients provided one uses bone marrow from the previously transplanted neonates as input material (Yoder et al., 1997a). A similar requirement for transplanted hematopoietic tissue to transition through a neonatal mouse before acquiring the capacity to engraft an adult animal had also been noted with cells from the d10.0 yolk sac (Yoder and Hiatt, 1997), although a previous experiment by Toles et al. (1989) demonstrated that d9.0 yolk sac directly injected transplacentally to W mice not only contributed to definitive hematopoiesis in a fraction of neonates but a small number of the mothers as well. While the exact route that the injected YS cells would take to facilitate engraftment of the pregnant mothers is unclear, it has been suggested that additional maturation may have been achieved in the neonatal intermediates and was required for proper homing to hematopoietic tissues in the adult, secondary transplant recipients (Yoder and Hiatt, 1997; Yoder et al., 1997a,b). Additional maturation likely occurred in the primary recipients and is supported by the observation that precirculation YS hematopoietic tissue cocultured on stroma in vitro prior to transplantation allows direct repopulation of primary adult recipients (Matsuoka et al., 2001). While these studies present provocative evidence in support of a model wherein the YS contributes to definitive hematopoiesis, they nevertheless fall a bit short of defining what actually occurs in utero. Although experimentally approachable in oviparous organisms such as the chicken (Dieterlen-Lievre, 1975), identification of the origins of hematopoietic cells within the YS and embryo proper in postcirculation mammals presents a profound and currently insurmountable technical obstacle. Ideally, one would diVerentially mark cells in both the precirculation yolk sac and the P-Sp/AGM in order to identify the origins of definitive hematopoietic
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stem cells later in development. Without such a system in the mouse, one is left to infer the origin of HSCs based on their location at the time of analysis. Short of this, identifying whether YS cells possess at least the capacity to transition to definitive products would lend support (albeit indirect) to the theory that YS cells contribute true HSC activity. The observations of Matsuoka and colleagues (2001) mentioned earlier support the notion that YS progenitors are capable of maturing to HSCs given the proper environment. An additional set of experiments by Kyba et al. (2002) further indicates that this is possible. Here, the authors showed that enforced expression of the HoxB4 transcription factor combined with stromal cell coculture confers long-term lymphomyeloid repopulating activity to d8.25 (2-4sp) YS cells (Kyba et al., 2002). This activity was further capable of being transferred to secondary recipients, although lymphoid activity became less robust over time (Kyba et al., 2002). Both of these studies indicate that precirculation YS hematopoietic tissue is capable of definitive HSC activity given proper induction. What is the nature of the eVect produced by stromal cell coculture or HoxB4 expression? It remains to be clarified if these conditions impose a new fate on otherwise nonrepopulating YS tissue or rather encourage proper homing to and/or retention in areas of long-term hematopoiesis. However, a more basic question is: does such an inductive environment exist in the yolk sac in vivo and how might it function in promoting the transition to definitive hematopoiesis? The answers will likely remain unknown pending the capacity to diVerentially identify distinct mammalian cells in utero. However, these experiments, as well as those mentioned earlier, all suggest that there is likely an embryonic pre-HSC that must mature to the state of long-term reconstituting activity, regardless of its origin. While the outcome of this maturation likely relates, at least in part, to improved homing and/or retention (see later), it might also be due to an acquired capacity to avoid immune surveillance by upregulating MHC class I antigens. Cumano and colleagues (2001) demonstrated that freshly purified d7.5–8.5 yolk sac cells conferred no reconstitution in Rag2 mice, immunodeficient animals that lack functional B and T cells (Shinkai et al., 1992). However, some animals showed myeloid engraftment short term with YS, provided the recipients were Rag2c mice bearing no NK cells in addition to their B and T lymphocyte deficiencies (Colucci et al., 1999; Cumano et al., 2001). Importantly, the use of Rag2c recipients allowed long-term repopulation to be achieved in both myeloid and lymphoid lineages with precirculation d7.5–8.0 P-Sp cells (Cumano et al., 2001). These results suggest that low levels of class I MHC expression could be one explanation for direct adult engraftment failure, as NK cells selectively target MHC class I low cells; class I cells are not expressed embryonically until approximately d10.5 (Ozato et al., 1985). Additionally, the immune system is less robust in neonates compared to adults, which may explain how transplanted cells
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can be spared by aVording them a greater opportunity to develop class I expression prior to recognition by NK cells in the young mice. One key question is that if the P-Sp/AGM is the only site of definitive HSC generation, then why is such potent repopulating activity found in the yolk sac relative to the embryo proper at early time points? Yoder et al. (1997a) showed that the d9.0 YS contains 37 times more CD34þ c-kitþ LTR cells than the P-Sp at this same time point, from which they draw the conclusion that LTR cells are created in the yolk sac. While the CD34þ c-kitþ LTR cells were functionally equivalent between the YS and the P-Sp, the basic observation was that many more were present in the YS. An obvious answer is simply that long-term repopulating activity initiates in the YS (or, at the very least, does so in conjunction with the AGM), eventually seeding more distal sites of HSC activity, such as the liver. While there is much convincing data in support of this theory, it remains possible that the YS contributes no HSCs to definitive hematopoiesis. By d9.0–10.0, a time point when several investigators would agree that abundant hematopoietic activity is present in the extraembryonic tissues, the yolk sac has been a site of blood cell production for 2–3 days, primitive such as it is. Even given that the P-Sp has begun to make hematopoietic cells de novo at this same time point, it is at best a naive blood-forming tissue in comparison to the yolk sac, which is enriched for hematopoiesis-promoting factors relative to the embryo proper. The observation that YS-derived hematopoietic tissue is more highly proliferative than that found in subsequent sites of blood formation was made some time ago (Moore and Metcalf, 1970). It has been shown that certain endothelial stromal cell lines derived from the yolk sac produce several key hematopoietic growth factors, including VEGF, stem cell factor (SCF), Flt-3 ligand (fms-like tyrosine kinase), and IL-6 (Fennie et al., 1995), whereas others are capable of stimulating greater hematopoietic colony-forming activity than bone marrow stromal cells (Yoder et al., 1994). Of course, not all stromal cell lines are equal in terms of their capacity to facilitate the expansion and diVerentiation of hematopoietic stem cells (Wineman et al., 1996). It should be noted that some YS–stromal cell lines are capable of supporting only hematopoiesis of a more primitive nature (Fennie et al., 1995; Yoder et al., 1994). Whereas others support definitive hematopoiesis, including lymphoid progenitors (Lu et al., 1996). AGM-derived stromal lines also support hematopoiesis (Oostendorp et al., 2002). That being said, could an enriched growth-promoting environment in the YS account for the heightened activity of AGM-derived hematopoietic cells located there at d9.0–10.0? It seems plausible that given a crude circulation is present at d9.0, any pre-LTR cell that managed to enter the YS from the embryo proper might encounter a heightened capacity to proliferate compared to one in the distal vasculature where pre-LTR cells would be obliged to rely on locally produced growth factors or those that had circulated from the
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distal YS. Such a premise would suggest a dual role for the yolk sac as an initial, transient source of primitive blood cells and then as a stromal resource once circulation has been established, facilitating the expansion of newly minted HSCs from the P-Sp/AGM prior to liver colonization, thus supporting a biclonal model. In support of this, it has been shown that an initial wave of long-term repopulating activity is present at d11.0 in the AGM that is followed by a second wave in the YS at d12.0, although with the caveat that the authors could not discern if the YS–HSCs were generated in situ or rather only expanded there after being produced elsewhere (Kumaravelu et al., 2002). With regard to the ontogeny of the hematopoietic tissues in utero, a rather provocative, observation-based hypothesis has been proposed by Godin and colleagues (1999) that speaks to the relationship between sites of hematopoietic stem cell production and the locations where these cells expand in number and diVerentiate. In short, this theory states that early sites of hematopoietic stem cell production are by their very nature ‘‘incompatible’’ with the expansion and diVerentiation of stem cells, with these functions being carried out in other locations where stem cells are not generated (Godin et al., 1999). Sites of apparent HSC production, such as the AGM, contain neither lineage-restricted erythromyeloid colonies (at levels above control tissues such as limb bud) nor bear much colony-forming activity in vitro unless conditioned a priori with stromal coculture (Godin et al., 1999). At the other end of the spectrum, while lymphomyeloid progenitors may be found in the d15.0 omentum (derivative of the P-Sp and fated to give rise to the splenic rudiment) they do not contribute long-term repopulating activity, indicating that the omentum, while containing hematopoietic tissue, is not a site where progenitors arise (Godin et al., 1999). Based on gene expression studies, Palis et al. (1999) could not verify the existence of clonogenic progenitors in the P-Sp/AGM region of the d9.5–10.5 embryo. However, the authors also indicated that the sensitivity of their assay was perhaps insuYcient to detect small numbers of cells or that hematopoietic progenitors do not diVerentiate in the AGM, with the latter notion being supported by the theory of Godin and colleagues (1999). In all, such a system would unify the apparently contradictory observations of LTR cells being generated within the embryo proper, yet being more abundant (and proliferative) in the extraembryonic yolk sac. Finally, a more provocative question might be if the biclonal model holds and developing mice (and humans as we shall see) do in fact maintain two distinct sites of hematopoietic tissue formation, then why? What development advantage is there to transient extraembryonic hematopoiesis that does not transition to permanent sites of blood formation? The observation that such yolk sac hematopoietic cells would be by definition nonrepopulating (and their early stroma incapable of maintaining a bona fide HSC) perhaps
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hints at an explanation. Continued experimentation will no doubt shed more light on this question. However, despite uncertainties regarding the source of HSCs deposited within the fetal liver, the blood-forming activity derivative of fetal liver and its eventual transition to other sites within the mouse including the bone marrow has been well described. 4. Definitive Hematopoiesis Definitive hematopoiesis in utero initially matures in a manner opposite of that found in the adult. That is, cells with no capacity to self-renew, bearing only colony-forming activity (CFU-C), are temporally the first to arise at d9.0, prior to progenitors with limited proliferative potential [colonyforming unit-spleen (CFU-S), d10.0], which are then followed by true hematopoietic stem cells at d11.0 (Muller et al., 1994). This inverted ontogeny suggests that a progressive enrichment of microenvironmental cues, perhaps corresponding to increased stromal cell activity in the various sites of blood formation, leads to greater long-term repopulating activity as in utero development ensues (Muller et al., 1994). The transition of blood-forming activity between developing sites of intraembryonic hematopoiesis is facilitated by the circulating blood, which at d11.0 is a veritable elixir of enriched hematopoietic stem cells (Delassus and Cumano, 1996). Thereafter, the frequency of HSCs relative to all cells in the hematopoietic system begins to decline. After d11.0 the fractional percentage of HSCs in the peripheral blood will shrink rapidly as the cells take up residence in the fetal liver (Delassus and Cumano, 1996). Hematopoietic stem cell migration via the peripheral blood was shown to occur in adult animals (Goodman and Hodgson, 1962) and has been experimentally estimated to be on the order of about 100 HSCs in the adult mouse peripheral blood at any given time (Wright et al., 2001). Based on a value of approximately 74,000 HSCs in the adult mouse bone marrow (a frequency of 0.017%) (Morrison et al., 1997), the fraction of the stem cell pool in transit through the peripheral circulation is quite small. The fractional HSC content of hematopoietic organs likely declines over time as fetal liver has been shown to have approximately five times greater long-term repopulating activity than adult bone marrow (Harrison et al., 1997). First described as a blood-forming organ in 1824 by Pre´vost and Dumas (cited in Minot, 1912), the fetal liver is seeded at d10.0 (Houssaint, 1981; Johnson and Moore, 1975) and contains a hematopoietic hierarchy similar to that found in the adult marrow (Traver et al., 2001), consisting of HSCs as well as committed, lineage-restricted progenitors [such as the colonyforming unit granulocyte and macrophage (CFU-GM)] (Fig. 1). Following liver colonization, which persists as a blood-forming organ until the neonatal period, the splenic rudiment is seeded at d12.5, becoming fully hematopoietic at d14.5 (Godin et al., 1999). This activity persists throughout the life
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span of the animal, perhaps as compensation for the small intramedullary volume of the mouse skeleton. The thymus is colonized at d10.5 (Fontaine-Perus et al., 1981; Jotereau et al., 1987; Owen and Ritter, 1969) and is fundamental to the development of mature B and T lymphocytes, although athymic animals are capable of continued NK cell generation. Bone marrow is colonized at d16.0 (Ogawa et al., 1988) and, following the cessation of hepatic hematopoiesis in the neonatal period, serves as the primary site of blood formation in the juvenile and adult mouse. Definitive erythroblasts (EryD or ‘‘macrocytes’’) are found in the liver as early as d9.0 (Leder et al., 1992) and are slightly smaller [8 mm (Mucenski et al., 1991)] than their YS counterparts. They are distinguished molecularly by a transition in -like and -like globin gene expression from embryonic to adult forms (Whitelaw et al., 1990). Here we see the expression of three main types of globin, -globin and two forms of -globin ( major and minor), that combine to make two forms of the adult hemoglobin heterodimeric tetramer: 2 2major and 22minor (Wong et al., 1983). This transition to adult type -globin production is preceded by an increase in the expression of the Gata-1 transcription factor (Whitelaw et al., 1990). Also, whereas primitive erythroid tissue requires burst-promoting activity (BPA) alone, a combination of BPA antecedent to erythropoietin from intraembryonic sources is required to transition to adult erythroid colony-forming units (CFU-E) (Cudennec et al., 1981; Johnson and Barker, 1985; Labastie et al., 1984). CFU-E are first detectable in the d9.5 (26–29sp) yolk sac with higher colony-forming activity detectable in the peripheral blood and liver by day 10.5 (40–43sp) (Palis et al., 1999). Lymphoid progenitors are first detected in the d8.5–9.5 YS and embryo body (Cumano et al., 1993; Godin et al., 1993; Ogawa et al., 1988). While capable of both proliferation and diVerentiation (the latter being demonstrated by IgM production and immunoglobulin rearrangement), extrahepatic lymphoid progenitors are more immature than those found later in the liver (Cumano et al., 1993). While they are both AA4.1 and B220 positive, early circulating progenitors remain Sca-1/Ly6A negative, suggesting that additional maturation is achieved in the liver (Cumano et al., 1993). This notion is supported by the observation that d9.0 B220þ cells require longer stromal coculture in vitro in order to respond to Salmonella lipopolysaccharide (LPS) than d12.0 B220þ liver cells (Cumano et al., 1993). Circulating lymphoid progenitors can be found as early as d10.0 and are virtually undetectable by d14.0, as they are retained in the liver (Delassus and Cumano, 1996). Several genes have been described that are key to definitive hematopoiesis and include the DNA-binding proteins jumonji (Kitajima et al., 1999) and AML-1 [also known as core-binding factor (CBF) or runx1] (Okuda et al., 1996), the oncogene c-myb (Mucenski et al., 1991; Palis et al., 1999),
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and the receptor tyrosine kinase Tie2 (Takakura et al., 1998). Mutants in these genes have partially or fully spared yolk sac hematopoiesis. Of interest, mutations in at least two of these genes (Tie2 and AML-1) have also been correlated with reduced or absent P-Sp/AGM hematopoietic activity (North et al., 1999; Takakura et al., 1998). The observation that loss of genes such as c-myb does not impact both primitive and definitive hematopoiesis has been used as evidence in support of a biclonal hematopoietic model (Mucenski et al., 1991). However, it is also plausible that some definitive hematopoiesis-specific genes merely reflect adaptive changes in YS hematopoietic activity as it evolves toward the capacity to engraft the fetal liver (and away from aYnity for the yolk sac) in a monoclonal model. Additionally, the role of a gene may be diYcult to ascertain if it also has important functions during primitive hematopoiesis and results in early embryonic lethality. Are ‘‘complete’’ hematopoietic mutants (i.e., those lacking both primitive and definitive blood formation) incapable of generating definitive hematopoietic stem cells or are they rather demonstrative of ineYcient HSC homing, retention, and/or expansion during a YS-to-fetal liver transition? In order to address this diYcult issue experimentally, some studies have attempted to create chimeric animals by introducing mutant embryonic stem (ES) cells into normal blastocysts, with the thought being that chimeric animals would survive embryonically and perhaps foster a blood tissue derivative of mutant ES cells later during definitive hematopoiesis. Two notable works in this regard attempted to define the hematopoietic contributions of Scl / (Porcher et al., 1996) and Flk-1 / (Shalaby et al., 1997) ES cells. While none of the resulting animals demonstrated hematopoiesis (or endothelial cell production for that matter) derivative of the mutant cells in either case, Flk-1 / ES cells were capable of forming some blood elements after diVerentiation in vitro (Shalaby et al., 1997) (see Section IV), whereas the Scl / ES cells had no such capability (Porcher et al., 1996). In addition, through the use of conditional Scl knockouts it has been demonstrated that while Scl activity is vital to HSC generation, the gene is dispensable for the maintenance of lymphomyeloid hematopoietic activity, including self-renewal, engraftment, and homing (Hall et al., 2003; Mikkola et al., 2003b). Although a challenge experimentally, continued investigation using assays such as these will no doubt shed further light on the genetic determinants of both primitive and definitive hematopoiesis in the mouse. 5. Homing and Retention Regarding the deposition of circulating HSCs into the fetal liver and any other future site of blood production, two key questions present themselves: (1) how do cells locate the hematopoietic spaces and (2) what mechanisms
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regulate HSC retention? It has long been thought that chemotactic signals were involved, emanating from the target organ and perhaps directly leading to the elaboration of cell surface adhesion molecules that would facilitate stromal interactions. In relation to both of these questions, mice carrying targeted deletions of the stromal cell-derived factor 1 (SDF-1) chemokine have a profound hematological phenotype. Fifty percent of SDF-1 mutants die prior to birth, with the remainder expiring in the first neonatal hour with cardiac defects, reduced fetal liver lymphoid progenitors, and the near complete absence of all lymphomyeloid bone marrow hematopoiesis (Nagasawa et al., 1996). That myeloid progenitors were present in the fetal livers of d18.5 SDF-1 knockout embryos suggests at least two distinct activities for this chemokine, one leading to the proliferation of lymphoid precursors and another contributing to the homing of all hematopoietic activity to the bone marrow (but not the fetal liver) (Nagasawa et al., 1996). SDF-1 was shown to be expressed in the wild-type bone marrow stroma (Nagasawa et al., 1996), and CD34þ progenitors migrate chemotactically up an SDF-1 gradient (Aiuti et al., 1997). Mice bearing knockouts of the gene for the SDF-1 receptor (CXCR4) have a nearly identical phenotype as the SDF-1 knockouts, which further defines the importance of this pathway in the homing of fetal liver HSCs to the bone marrow in utero (Zou et al., 1998). By using in vitro clonal assays, Zou and colleagues (1998) further showed that the lack of lymphoid progenitors in the fetal liver was due to impaired proliferation and/or lineage commitment and not to a defect of homing to the intrahepatic space, thus lending additional support to earlier observations that the SDF-1/CXCR4 pathway is likely not a component in the establishment of liver hematopoiesis, although key in the transition to the bone marrow. That being said, it has been shown that SDF-1 may play a later role in the homing of hematopoietic cells to the liver in response to stress (SDF-1 is expressed in liver bile duct epithelium) (Kollet et al., 2003). Finally, treatment of adult mice with granulocyte colony-stimulating factor (G-CSF) or the chemotherapeutic agent cyclophosphamide (used clinically to ‘‘mobilize’’ bone marrow HSCs to the peripheral blood for harvest and use in transplantation therapy) has been shown to result in N-terminal cleavage of CXCR4 on hematopoietic cells, as well as the proteolytic reduction of SDF-1 [via the activity of neutrophil elastase (Petit et al., 2002)], with both events leading to the loss of chemotaxis (Levesque et al., 2003; Petit et al., 2002) followed by increased, likely compensatory, gene expression for both SDF-1 and CXCR4 (Petit et al., 2002). As these observations were made in adults with active marrow hematopoiesis, SDF-1/CXCR4 was further implicated to be important for the retention of stem cell activity in the marrow in addition to its initial function in homing.
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B. In Vivo Blood Development in Homo sapiens While the literature contains many analyses regarding the onset and ontogeny of human hematopoiesis [the most authoritative being that of Kelemen, Calvo, and Fliedner (Kelemen et al., 1979)], the primary limitation of specimen availability, especially at the earliest stages of development in utero, is as much the case today as at the time of the first reports. Rare and often poorly preserved human concepti have been obtained as the result of spontaneous and therapeutic pregnancy termination and used to delineate key events in the development of the human blood system (Rowley et al., 1978). In marked contrast, the ease by which one might biopsy hematopoietic tissue from adult volunteers (bone marrow or mobilized peripheral blood stem cells) has facilitated a multitude of studies that describe the biology of hematopoietic stem and progenitor cells in vivo, in vitro, and in detail. It remains an unfortunate informational paradox that the best understood of all human stem cells, the adult blood stem cell, bears such a descriptive dearth regarding its origins. However, work from several groups has imparted a considerable degree of detail to our understanding of embryonic and fetal hematopoiesis in situ. Combined with in vitro molecular studies of gene expression and protein function, a great deal has become known of blood development in humans, although compared to the information obtained from lower species, it remains but a small fraction. 1. Primitive Hematopoiesis Formation of the human yolk sac diVers somewhat from the mouse. In humans, the primary yolk sac disaggregates at D12.0–13.0 (presomite) to form what are known as exocoelomic cysts, as a second outgrowth of the hypoblast forms the definitive or secondary yolk sac; referred to hereafter as simply ‘‘yolk sac’’ or YS (Larsen, 1993). Studying a cohort of wellpreserved, embedded human embryos ranging in gestational age from the prehematopoietic period at embryonic day 13 (D13.0) through early secondary yolk sac hematopoiesis (D18.5, coincident to early head formation) to the terminus of YS blood production in approximately the early ninth week (W9.0), Bloom and Bartelmez (1940) presented the first staged, high-resolution analysis of the onset of in vivo human hematopoiesis (Fig. 1). This rather elegant study pays particular attention to the form and variety of hematopoietic cells in the secondary yolk sac, chiefly ‘‘hemocytoblasts,’’ primitive hematopoietic progenitors, and occasional macrophages (Bloom and Bartelmez, 1940). These cells were present in multiple extravascular foci until at least D19.0 (presomite) when isolated blasts could be found within the precirculation vitilline vessels (Bloom and Bartelmez, 1940).
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More contemporary analyses confirm earlier observations that yolk sac hematopoiesis begins in the second week with the formation of blood islands in the extraembryonic mesoderm of the secondary yolk sac (Luckett, 1978). Colony-forming activity has been assayed at D25.0–30.0 (20–35SP) and was rather modest with approximately 100 of both large erythroid (megaloblasts) and nonerythroid (granulocyte and macrophage) colonies per YS studied, with the yolk sacs containing roughly 100,000 total nucleated cells (Huyhn et al., 1995). Additionally, although the total number of erythroid colonies per yolk sac increased (one to threefold) up to D40.0 (with nonerythroid colonies actually declining somewhat), their relative quantity decreased (and was dwarfed by the roughly 40,000 to 90,000 colonies also present per fetal liver at D40.0) (Huyhn et al., 1995). Human erythropoietin (Jacobs et al., 1985; Miyake et al., 1977) modulates the expansion of erythroid progenitors in response to stresses such as reduced oxygen tension or irradiation (Gurney and Fried, 1965). The progenitors of megaloblasts form BFU-E in methylcellulose colony-forming assays in an Epo-independent manner, requiring only erythroid-potentiating activity (EPA). EPA was characterized as a purified protein (Westbrook et al., 1984), cloned (Gasson et al., 1985), and found to be identical to the tissue inhibitor of matrix metalloproteinases TIMP1 (Docherty et al., 1985). YS erythroid progenitors have been shown to be responsive to hematopoietic growth factors in methylcellulose colony-forming assays that included combinations of Epo, IL-3, and SCF, although this eVect was roughly one order of magnitude less robust than concurrent experiments using fetal liver (Huyhn et al., 1995). As in the mouse, the architecture of mesenchymal blood islands demonstrates an outer, endothelial layer surrounding foci of large, nucleated erythroid cells within the developing mesoderm. However, the origin of human yolk sac mesoderm appears to diVer from what is known of the mouse in that it derives from the primitive endoderm of the hypoblast rather than the ectodermal epiblast (Bianchi et al., 1993; Takashina, 1987; reviewed in Palis and Yoder, 2001) and is thus considered extraembryonic or placental [although not to be confused with trophectodermal (Luckett, 1978)]. While detailed studies such as these mentioned here have provided exquisite data regarding the origins of the human extraembryonic mesoderm, spontaneously occurring abnormal events in nature provide additional levels of insight (as is often the case). Here, a certain type of nonviable human conceptus that entirely lacks an embryo and contains only placental tissue may shed additional light on the origins of the yolk sac mesoderm in our species. This is known as a complete hydatidiform mole (CHM) (Jacobs et al., 1980; Ohama et al., 1981) and derives from fertilization of an oocyte without a pronucleus and is thus genetically entirely paternal, although diploid following the duplication of the haploid sperm genome (termed an androgenote).
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CHMs occasionally contain primitive nucleated erythroblasts, further supporting the notion that the earliest hematopoiesis in humans may not have its origin in tissues derivative of the embryo proper (Paradinas et al., 1997; Zaragoza et al., 1997). While one must take care to ensure that contaminating embryonic material is not present in a suspected CHM that contains blood products [such as in cases of twinning (Fisher et al., 1997), ruled out easily by microsatellite PCR], it is rather intriguing to consider that diVerential imprinting may be an additional distinction between YS and intraembryonic hematopoiesis, as the CHM contains primitive hematopoietic tissue and yet is entirely paternal. This idea has been broached previously in describing a case involving a hemidysmorphic 14-month-old child that was chimeric for parthenogenetic (i.e., purely maternal) tissue (Strain et al., 1995). In this individual, all cells of the peripheral blood were shown to be homozygous in a genome-wide scan, containing alleles of only maternal origin, whereas fibroblasts demonstrated genetic contribution from both parents (Strain et al., 1995). This presents the interesting notion that distinct genetic imprints may exist in the primitive versus definitive hematopoietic compartments, with the paternal genome lending itself to extraembryonic, primitive hematopoiesis and the maternal genome contributing to intraembryonic, definitive hematopoiesis. Mouse chimeras actively exclude parthenogenetic cells from many (but not all) embryonic and adult tissues, although several investigators have noted variable contributions to the murine primitive ectoderm, extraembryonic mesoderm, yolk sac, and/or adult blood (Clarke et al., 1988; Fundele et al., 1989; Kaufman et al., 1977; Nagy et al., 1987, 1989; Paldi et al., 1989; Stevens et al., 1977; Surani et al., 1977, 1987, 1988; Thomson and Solter, 1988). Again, as both murine extra- and intraembryonic hematopoiesis ultimately derive from the epiblast, these data are not in conflict with observations made in the human cases stated here. Human globins have been studied in exhaustive detail (for review, see Karlsson and Nienhuis, 1985) due in large part to the occurrence of severe, often fatal, genetic diseases caused by globin mutation, including sickle cell anemia and / thalassemia (Weatherall and Clegg, 2001). The primitive erythroid megaloblasts of the yolk sac express three forms of embryonic hemoglobin (Hb): the major form Hb Gower 1 ( 22) (Gale et al., 1979; Hecht et al., 1966; Kazazian, 1974), which is also the first expressed (Peschle et al., 1985); the lesser abundant Hb Gower 2 (22) (Gale et al., 1979; Hecht et al., 1966); and the minor Hb Portland ( 2 2) (Capp et al., 1967). Primitive erythroid megaloblasts expressing these embryonic globins will eventually circulate into the embryo proper (beginning at W3.0, see later), but will vanish by W10.0–12.0 (Gale et al., 1979; Peschle et al., 1985), circulating with definitive erythrocytes expressing fetal and adult globins from W8.0 to 12.0 (Peschle et al., 1985). As in the mouse, the expression patterns of
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-like and -like globins are regulated asynchronously, and the embryonic -like -globin will decline in abundance at W6.0 prior to the decrease in the -like -globin at W8.0 (Peschle et al., 1984b). Finally, the constituents and gene expression patterns of globins are shared between humans and Old World monkeys and are much more comparable than those of humans to New World monkeys, giving one a sense of globin phylogeny (Johnson et al., 2000). To say there is a minute body of literature describing the gene expression and molecular surface phenotype of human yolk sac hematopoietic cells would be generous. This is almost certainly due to the diYculty in obtaining early fetal tissues. Nevertheless, it has been shown that CD15þ (committed myeloid) cells are present from D19.0 onward (Tavian et al., 2001). CD34þ endothelial and hematopoietic cells are noted at D25.0–30.0 in the early postcirculation yolk sac (Huyhn et al., 1995; Labastie et al., 1998; Tavian et al., 1999, 2001), as well as cells positive for glycophorin-A (also known as CD235a, a marker of red cells), although the latter were CD34 (Tavian et al., 1999). With the curious exception of CD56þ CD94þ NK cells, lymphoid progenitors do not arise during yolk sac hematopoiesis as noted in studies using D19.0 YS explants and stromal cell coculture (Oberlin et al., 2002; Tavian et al., 2001). Tavian et al. (1999) have noted CD45þ cells in the D24.0 YS (15SP), although they add the caveat that these likely circulated to the YS from within the embryo. Expression of the KG-1 kinase (a serine/threonine kinase isolated from the KG-1 myeloid leukemia cell line) is detected via in situ hybridization as early as 5SP (D22.0–23.0) in the yolk sac and mirrors the expression of CD34 (Labastie et al., 1998). Low-level expression of the SCF gene has been noted in unfractionated D25.0–30.0 (25–30SP) yolk sac via RT-PCR (Huyhn et al., 1995; Teyssier-Le Discorde et al., 1999), as well as that of its receptor, c-kit (Teyssier-Le Discorde et al., 1999). These were the only specimens available for analysis and as expression was noted at the earliest time points, it was perhaps present prior to that time. Oberlin et al. (2002) suggested that all endothelial cells in the W3.0–6.0 embryo are positive for CD31 and CD34 but negative for CD45 and VEcadherin [as stated earlier for the mouse (Nishikawa et al., 1998b)]. In further agreement with findings in the mouse, the authors went on to demonstrate that endothelial cells sorted by CD31þ34þ45 and cocultured on stromal cell monolayers generated hematopoietic cells (Oberlin et al., 2002). This activity was present in endothelial cells obtained from the D26.0–40.0 YS, D28.0–44.0 P-Sp/AGM (see later), D27.0–56.0 fetal liver, and W11.0–24.0 bone marrow (Oberlin et al., 2002), although it was lacking in other sites such as D23.0–27.0 P-Sp/AGM, D66.0 aorta, umbilical cord, spleen, and newborn vascular endothelial cells (HUVEC) (Oberlin et al., 2002). That
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hemogenic activity was also present in the endothelium of liver and bone marrow is of added interest and deserving of future studies in order to further define what factors promote this apparent endothelial to hematopoietic lineage transition. Considering the aforementioned controversy regarding whether HSCs originate in the murine YS, one is left to wonder if human yolk sac tissue bears the capacity to engraft transplant recipients long term? In a rather unorthodox transplantation procedure conducted in the early 1970s, combined infusion of unprocessed YS and liver from a woman’s own 7-week (22-mm) embryo resulted in apparent recovery from her transfusion-dependent, aplastic anemia (diagnosed over 1 year before the onset of her pregnancy) (Kelemen, 1973, 1996). However, this outcome remains ambiguous as the author stated that donor cell engraftment was not confirmed. Immunodeficient murine hosts [such as the severe combined immunodeficient (SCID) mouse] have proven capable of being engrafted by human fetal liver, thymus, peripheral blood, and bone marrow (Gallacher et al., 2000; Kollmann et al., 1994; McCune et al., 1988; Plum et al., 1994) and might also be used to determine if and when long-term repopulating activity arises in the human yolk sac. Blood circulation initiates at the beginning of W3.0 (around D21.0–22.0) when the human embryo is roughly 2.5 mm in length (Larsen, 1993) [see the spectacular illustrations by Bro¨del in Cullen (1916) regarding the architectural features of the major embryonic vasculature relative to the yolk sac, one of which is reproduced in modified form in Fig. 2]. As confirmation of an active circulation, glycophorin-Aþ CD45þ CD34 cells have been noted inside the cardiac cavity at D21.0 (3SP) (Tavian et al., 1999). Following the establishment of circulation, the human liver becomes hematopoietic at W5.0–6.0 (Migliaccio et al., 1986; Peschle et al., 1984b) and the number of hematopoietic progenitors in the YS will decline rapidly (Migliaccio et al., 1986), demarcating the transition toward definitive hematopoiesis. 2. The P-Sp/AGM As in the mouse, clusters of ‘‘hemoblasts’’ have been noted in the ventral endothelium of the dorsal aorta of H. sapiens (Jordan, 1918; Labastie et al., 1998; Marshall et al., 1999; Minot, 1912; Smith and Glomski, 1982; Tavian et al., 1996, 1999). While Minot was the first to publish observations of these cells in human embryos over 90 years ago, it was his conclusion that they did not evolve from the underlying endothelium but rather aggregated there from the circulating blood (Minot, 1912). This theory was due in part to his observation that few mitoses were present within the associated endothelium, although all things being equal, such a notion would agree with the
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Figure 2 The 2.5-mm human embryo (embryonic day, 21–23; coincident to the onset of circulation). The yolk sac extends to the left, and the amniotic cavity containing the embryo is to the right. Superimposed on the embryo proper (solid white area within the amniotic cavity) is the major embryonic vasculature, including the aorta and umbilical arteries/vessels. The region of the hemogenic aorta-gonad-mesonephros (AGM) has been indicated by a bracket (and green oval) and includes the vental portion of the dorsal aorta, as well as the proximal preumbilical and vitilline arteries. Modified and taken from Cullen (1916). Image courtesy of the Department of Art as Applied to Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland, where the original art (without color) is in the Max Bro¨del archive. (See Color Insert.)
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predictions made by the model of Godin and colleagues (1999) regarding sites of hematopoietic stem cell generation being largely nonproliferative. As the scale of CD34þ hemogenic cells produced in the AGM is likely on the order of less than 1000 over a physical distance of two somite pairs at D35.0 (Tavian et al., 1996), the rare mitoses present in a static section (such as those analyzed by Minot where rare mitotic figures were in fact noted) may be suYcient to produce this low number of cells in a period of roughly 2 weeks. This time frame was first suggested by Jordan in the year 1918 when he noted many large clusters of aortic hemoblasts in a study of the 7-mm embryo [ca. D32.0–33.0 (Jordan, 1918; Larsen, 1993)]. Few if any were to be found in either the 5- or the 13-mm human embryo (Jordan, 1918), establishing a temporal window for their generation from about D28.0 to D41.0 or W4.0 to W6.0 [using Larsen’s (1993)] scale (Fig. 1). Such a developmental interval is in perfect concordance with current observations (Labastie et al., 1998; Tavian et al., 1996, 1999). Some studies have noted that the subaortic endothelium just beneath the clusters of hematopoietic cells is either disrupted or absent and were interpreted to suggest that endothelial cells may have diVerentiated directly into hematopoietic cells (Oberlin et al., 2002; Tavian et al., 1999). As in the mouse (Garcia-Porrero et al., 1995), the sites of intraembryonic hematopoietic precursor generation also extend into the proximal portions of the preumbilical and vitilline arteries (Tavian et al., 1999) (Fig. 2). Tissues obtained from the D35.0 AGM demonstrate a greater capacity to generate hematopoietic progenitors than comparable tissues obtained from age-matched fetal livers, both after culture on stroma (Tavian et al., 1996). As other body tissues (such as heart and limb) had negligible activity, it was felt that the progenitor activity in these assays could not be accounted for by circulating cells (Tavian et al., 1996). Using explanted tissues and stromal coculture, Tavian et al. (2001) demonstrated that lymphomyeloid activity was present in the P-Sp/AGM as early as D21.0 (1–2SP), although activity was also present at D19.0 using cultured whole embryo. Endothelial cells of the human AGM are VE-cadherinþ CD34þ CD45 , whereas hematopoietic cells are VE-cadherinþ CD34þ CD45þ (Marshall et al., 1999; Oberlin et al., 2002), further expressing CD43 (sialophorin), CD44 (H-CAM), and CD164 (sialomucin) (Watt et al., 2000). Cortes and colleagues (1999) suggested that the earliest hematopoietic (and endothelial) progenitor arising in the predecessor of the AGM (the P-Sp) is CD34 , with the latter point (at least as far as the hematopoietic stem cell is concerned) being in agreement with some (e.g., Goodell et al., 1997; Wang et al., 2003) but not all (e.g., Aversa et al., 1998; Ziegler et al., 1999) findings in adult human bone marrow. Of course, while data derivative of adult bone marrow are certainly instructive, they may not accurately reflect the conditions present within the early embryo in the area of the P-Sp/AGM. The refinement of
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methods to prospectively identify the hematopoietic stem cell via surface phenotype (both adult and embryonic) is certain to continue. Gene expression in the human P-Sp/AGM is similar to that seen in the murine system. Hematopoietic cell clusters in the ventral margin of the aorta express SCL, GATA-2/3, and c-myb (Labastie et al., 1998) in addition to VEGF and its receptor KDR (the human homolog of Flk-1) plus FLT-3L (FLT-3 ligand) and its receptor FLT-3, indicating an environment of likely autocrine/paracrine stimulation and signaling (Marshall et al., 1999). Labastie et al. (1998) have further characterized the expression of KDR relative to the diVerentiation of hematopoietic cells on the aortic endothelium in that expression declines as they move away from the endothelium and into the aortic lumen, similar to what was noted in the mouse as cells matured in the liver and bone marrow (Kabrun et al., 1997). Endothelial cells lining the AGM express a variety of stromal cell factors and receptor/ligand pairs as before, including KDR (Cortes et al., 1999; Marshall et al., 1999) and VEGF, as well as FLT-3L and FLT-3, and the transcription factor SCL (Marshall et al., 1999). The endothelium just beneath the hematopoietic clusters in the AGM is positive for FLK-1/VEGF but negative for FLT-3L/FLT-3 and SCL (Marshall et al., 1999). Furthermore, molecules specific to the mesenchyme just below the intraaortic clusters of blood cells have been identified, suggesting a direct role in generating/maintaining blood-forming activity, and include BMP-4 (Marshall et al., 2000) and tenascin-C (Marshall et al., 1999). Tenascin-C is an extracellular matrix glycoprotein found in bone marrow stroma (Klein et al., 1993). While the tenascin-C knockout mouse is viable and shows no apparent hematological defects in vivo (Forsberg et al., 1996; Saga et al., 1992), the marrow demonstrates a mild-to-moderate impairment of colony-forming activity in vitro (Ohta et al., 1998). Shared gene expression between human embryonic/fetal endothelial and hematopoietic progenitors supports a common bipotential progenitor. Some findings suggest that this hemangioblast activity continues into adulthood. The BCR /ABL fusion oncogene is the causative factor in the human hematological disorder chronic myeloid leukemia (CML) (Daley et al., 1990; de Klein et al., 1982; GroVen et al., 1984). BCR/ABL may also be found in myocardial endothelial cells from CML patients or in endothelial cells obtained from patients and cultured in vitro (Gunsilius et al., 2000). One mystery regarding these data relates to an uncertainty as to whether it was the hemangioblast that becomes transformed in these rare cases or rather that transformation forced a more primitive phenotype to emerge in the leukemic stem cell. Findings also suggest that human cord blood contributed endothelial tissue to stably engrafted immunodeficient murine hosts (Cogle et al., 2004). After engraftment, retinal ischemia was induced and human endothelial cells were subsequently noted in the neovascularized areas (Cogle et al., 2004).
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3. Definitive Hematopoiesis The transition from YS to liver is gradual, occurring over a 2-week period beginning at D27.0–30.0 (Huyhn et al., 1995; Oberlin et al., 2002; Tavian et al., 1999) as hematopoietic cells enter the extravascular trabeculae of the hepatic parenchyma (Migliaccio et al., 1986; Minot, 1912; Thomas and YoVey, 1964) (Fig. 1). Enucleate macrocytes do not enter the hepatic sinusoids and peripheral blood until W8.0 (Peschle et al., 1985), a point at which liver hematopoiesis is quite robust (Migliaccio et al., 1986). By D42.0 the liver has completely replaced the yolk sac as the major hematopoietic organ (Tavian et al., 1999). In the period from W5.0 to 10.0, the liver will expand greatly in size. At W5.0, the yolk sac and liver contain roughly the same number of total nucleated cells (approximately three to five million), although in the liver this will increase by roughly two orders of magnitude by W10.0 with a concurrent increase in the absolute numbers of colonyforming progenitors (BFU-E, CFU-E, and CFU-GM) (Migliaccio et al., 1986). At W10.0–16.0 the liver is a powerhouse of hematopoietic activity and CD34þ cells obtained from the fetal liver have a two to three orders of magnitude increased proliferative capacity compared to cord blood and adult bone marrow CD34þ cells, respectively (Lansdorp et al., 1993). Fetal liver CD34þ cells have also been shown to express CD164 and the stem cell marker AC133, although their levels declined as the liver cells became more CD38 positive (CD38 generally varies inversely with CD34 on hematopoietic progenitors, rising in expression as the cells become lineage committed) (Watt et al., 2000). The transition from embryonic to fetal and adult types of hemoglobin begins in the liver at W6.0 (Gale et al., 1979; Peschle et al., 1984a). Humans have a transitional form, known as fetal hemoglobin (HbF), that is a complex of -globin and the -like gamma-globin (2 2) (reviewed in Karlsson and Nienhuis, 1985). -globin is uniquely suited to the fetal oxygenation requirements of animals with a long gestational interval such as cattle (Efremov and Braend, 1965) and humans, as it has higher aYnity for oxygen than HbA (reviewed in Perutz, 1979). In the precirculation human, embryonic globins are adapted to the diVusion-limited oxygen environment. With the onset of circulation, the fetal blood benefits from the left-shifted (i.e., higher aYnity) oxygenation profile of -globin (reviewed in Bunn, 1981) relative to the adult globin of the mother, resulting in oxygen being taken up by HbF in the placenta (Purves et al., 2001). HbF persists throughout the remainder of fetal life and into the neonatal period, although it begins to decline at W34.0 as it is gradually replaced by the adult hemoglobin (HbA) molecule (22) (Kazazian, 1974; Migliaccio et al., 1986; Peschle et al., 1984a, 1985). Unlike embryonic BFU-E, fetal BFU-E containing HbF require erythropoietin (Linch et al., 1982; Peschle et al., 1981) and are highly Epo sensitive (Papayannopoulou et al., 1977), demonstrating
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peak responsiveness at one-sixth the dose of that required for the maximal stimulation of HbA-containing BFU-E (Linch et al., 1982). Adult hemoglobin (HbA), the heterodimeric tetramer of adult -globin and -globin (22), is not present during primitive hematopoiesis (Peschle et al., 1985). Humans have but a single adult -globin that is expressed at very low levels as early as W6.0–8.0 [less than 2% at W7.0 and approximately 10% of all non--globin at W10.0 (Peschle et al., 1985)] and is upregulated late in gestation at approximately W34.0 (Migliaccio et al., 1986; Peschle et al., 1984a, 1985). Near birth, the -globin fraction experiences its steepest increase (Bard et al., 1970), although at birth, it is still a minor component compared to fetal -globin (less than 20% of all non--globin), transitioning to dominance by postnatal W6.0, and the vast majority of non--globin by W12.0 of the infant’s life (reviewed in Karlsson and Nienhuis, 1985). Additionally, humans have rare forms of both -like and -like adult globins, known as - and -globin, respectively. Identified originally in nonhuman primates (Marks et al., 1986a,b), the human -globin-like, -globin gene has been shown to be transcribed (Hsu et al., 1988; Leung et al., 1987) and is expressed beginning at W5.0–8.0 (Albitar et al., 1992). Although -globin mRNA can be translated using a cell-free system in vitro (Leung et al., 1989), it is unknown if a protein is present in vivo. Rabbits have a -globin pseudogene (Cheng et al., 1986), although no pseudogene (or gene) has yet been described in the mouse. The -globin-like, -globin is first expressed at W12.0 (similar to -globin) and forms 2 2, also known as HbA2, although as this hemoglobin is only 2–3% of that in the adult, the
-globin protein is much less abundant (Gilbert, 2000). The mouse genome contains a pseudogene (H3) related to the human -globin sequence (Hutchison et al., 1984). The importance of these rare globins in development is unclear. Circulating hematopoietic progenitor cells are found in the fetal blood (Linch et al., 1982). CD34þ38 cells are present at W12.0–19.0 at a higher frequency than in term cord blood, suggesting a greater fractional percentage of blood stem cell activity at earlier stages of gestation (Campagnoli et al., 2000; Gallacher et al., 2000). These early circulating progenitors were also noted to be diVerentially responsive to hematopoietic cytokines, needing only exogenous SCF and Flt3-ligand in vitro to maintain NOD/ SCID repopulating activity, whereas term cord blood progenitors also required IL-3, IL-6, and G-CSF (Murdoch et al., 2002). In this same developmental window of circulation, one may identify various primitive and lineage-committed blood cells expressing CD33 (also known as myeloid diVerentiation antigen), CD15, the B-cell markers CD19 and CD20, and very low numbers of CD4þ8þ T-cell progenitors (Gallacher et al., 2000). These cells presumably originate from the liver and are key to the development of the bone marrow and other sites of hematopoiesis such as the spleen,
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which will contain mixed lineage hematopoietic cells beginning at W18.0– 19.0 (gone prior to birth), and the thymus, containing low-level activity also from W19.0 (Hann et al., 1983). The liver remains hematopoietic into early infancy, although its activity begins to decline in midgestation (Linch et al., 1982). Although scattered granulocytes may be found at time points when the liver is the primary hematopoietic organ, significant granulopoiesis does not occur until the onset of bone marrow blood production (Charbord et al., 1996; Thomas and YoVey, 1962, 1964). The observation that the medulla of the developing bone was a site of hematopoiesis was made by Neumann and Bizzozero, working independently, in the year 1868 (cited in Minot, 1912). Bone marrow is initially seeded at W10.5 and is in its final form by W16.0 (Charbord et al., 1996). The marrow of the vertebrae becomes hematopoietic slightly later at W14.0 and aVords a more approachable platform for the study of hematopoiesis in situ compared to bone, as vertebral marrow is chiefly encased in cartilage until later stages of fetal life (Chen and Weiss, 1975). In the bone marrow of the vertebrae we see a sparse scaVold of extracellular matrix and reticular cells that contain the proliferating hematopoietic tissue and fill the space between cartilage or bone in the periphery and the endothelium of the central vascular sinus (through which blood cells must pass to enter the circulation) (Chen and Weiss, 1975). The architecture of the long bone marrow is a bit diVerent as it contains ‘‘logettes,’’ which number less than 10 per long bone at W11.0– 12.0 and extend parallel to the axis of the medullary space (Charbord et al., 1996). Logettes have a central arteriole with CD34þ endothelial cells at the interior and an -smooth muscle actin (SM actin)-positive cellular media (Charbord et al., 1996). Clusters of hematopoietic cells are just beyond the SM actin media and are bordered at the outer margin by a second CD34þ endothelium (Charbord et al., 1996). Hematopoietic cells within the logettes are a mixture of progenitors and more diVerentiated progeny and may express CD34, CD45, CD68 (monocytes), CD15, or glycophorin A (erythrocytes) (Charbord et al., 1996). CD34þThy-1þ multipotential fetal bone marrow progenitors (approximately 0.05% of fetal, mononuclear, bone marrow cells) are capable of initiating long-term cultures in vitro as well as engrafting SCID mice (Baum et al., 1992). By W16.0 the expansion of hematopoietic cells in the bone marrow is such that the logettes are indefinable (Charbord et al., 1996). At this point, the final architecture of the bone marrow is largely established, although the amount of hematopoietic tissue within the expanse of the intramedullary space will decrease slowly in adult life, being replaced by fat in keeping with the hypothesis that the fractional percentage of lymphomyeloid progenitors is greatest in early human development (e.g., Campagnoli et al., 2000; Gallacher et al., 2000; Lansdorp et al., 1993). As but one proof of this, fetal bone marrow has been shown to contain a greater proportion of HSCs than either juvenile or adult
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marrow (Brashem et al., 1982). Finally, the expression of genes in the hematopoietic tissue of the bone marrow is quite comparable to that seen in the AGM and fetal liver and includes SCL, c-myb, GATA-2/3, c-kit, and KDR (Labastie et al., 1998), likely indicating that a large part of hematopoietic identity is established early on in gestation. 4. Homing and Retention Homing of hematopoietic activity to and retention in blood-forming organs are as vital to H. sapiens as they are to mice. In a fascinating experiment, Zanjani and colleagues (1993) demonstrated that the stromal environment of the target tissue bears a significant role in the homing of blood-forming activity. Here, preimmune fetal sheep were used as transplant recipients for human W12.0–14.0 fetal liver HSCs. Given the introduction of identical human cells, it was noted that the ultimate location to which the injected cells would migrate was dependent on the age of the recipient in that a prebone marrow animal was populated in the liver and spleen. However, if an older recipient was used, one with active fetal liver hematopoiesis as well as bone marrow blood formation, the injected cells homed exclusively to the marrow, indicating that an engraftment ‘‘hierarchy’’ was present. This preferential targeting was less likely due to the phenotype of the injected cells (as they were identical between recipients) than that of the engrafted tissues, suggesting that the shift from one site of hematopoiesis to another can be influenced by the maturation of distinct stromal environments and not only the evolution of the hematopoietic cells themselves. As described earlier in the mouse, the homing of hematopoietic activity to the human marrow is dependent on the activity of the chemokine SDF-1 and its receptor CXCR4. In humans, CXCR4 has been shown to be expressed on CD34þ lymphomyeloid progenitors that migrate chemotactically toward an SDF-1 source (Aiuti et al., 1997, 1999). Using blocking antibodies, SDF-1 and CXCR4 were shown to be vital to the engraftment of human hematopoietic progenitors in the bone marrow of immunodeficient mice (Peled et al., 1999). CXCR4 is induced on CD34þ cells by exposure to the hematopoietic growth factors SCF and IL-6 (Peled et al., 1999). Furthermore, SDF1 has been shown to activate the integrins VLA-4, VLA-5, and LFA-1 (Peled et al., 2000). VLA-4 has been shown to be of importance to CFU-S homing to bone marrow (Vermeulen et al., 1998). LFA-1 is important in the transendothelial migration of human hematopoietic progenitors (Mohle et al., 1997; Yong et al., 1998), which is significant as definitive hematopoiesis occurs extravascularly (Tavassoli, 1991). Mobilized peripheral blood stem cells are less responsive to SDF-1 (Aiuti et al., 1997). As before in mice, treatment of human patients with G-CSF or cyclophosphamide has been shown to result in N-terminal cleavage of CXCR4 on hematopoietic cells and the
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proteolysis of SDF-1 in the bone marrow (Levesque et al., 2003; Petit et al., 2002), which is followed by increased expression of both SDF-1 and CXCR4 (Petit et al., 2002). Now that we have outlined the broad strokes of hematopoietic ontogeny in both mouse and human, it behooves us to make a few comparisons regarding the manner by which each generates blood tissue.
III. Points of Divergence Between Murine and Human Blood Formation Although the preceding paragraphs indicate that the origin of human hematopoiesis is very similar to that found in the mouse, the roughly 75 million years of evolutionary divergence from a common ancestor (Waterston et al., 2002) has resulted in some understandable dissimilarity in the manner by which each makes blood. Notable among these are the diVerent origins of the murine and human mesoderm, the former arising from the epiblast (primary ectoderm) following gastrulation and the latter from the hypoblast (primary endoderm) prior to formation of the primitive streak and gastrulation. Given that blood islands of the yolk sac arise from diVerent sources in each organism (and thus from distinct inductive environments), it would be reasonable to expect that we might see divergence in the initial events of EryP formation. However, such is not the case, as murine and human primitive hematopoiesis parallel one another fairly well. Thus we must conclude that a certain ‘‘leveling of the field’’ occurs once the mesoderm has formed, regardless of the source from whence it derives. How might we explain the diVerences? One minor distinction is the brevity of human yolk sac hematopoiesis relative to the mouse (Fig. 1). Although humans have a much longer gestational interval than mice (40 weeks as opposed to 20 days), fractionally speaking, EryP are present in roughly 40% of mouse gestation compared to approximately 25% in humans. Also, the onset of circulation, serving as a milestone in the transition between extraembryonic and intraembryonic hematopoiesis, diVers significantly between the two species. Circulation in mice occurs at d8.25, after roughly 42% of gestation has transpired (Ji et al., 2003). In comparison, circulation in the human embryo begins relatively early in development at D21.0, when less than 8% of the gestational interval has passed (Larsen, 1993). How the murine embryo can progress through nearly half of its gestational period, making use of only what oxygen is present via diVusion (although some mixing must occur as the mother ambulates), is almost certainly due to the small size of the developing animal and the kinetics of its hemoglobin saturation profile. Additionally, these
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two factors likely have bearing upon another apparent distinction between the two species, namely the onset and duration of intrauterine bone marrow hematopoiesis. In mice, the liver is seeded at d10.0 (Houssaint, 1981; Johnson and Moore, 1975) and maintains hematopoietic potential throughout the remaining 50% of fetal life to the perinatal period. Murine marrow is not seeded until d16.0 (Ogawa et al., 1988), and active marrow hematopoiesis does not occur until the following day when only 15% of gestation remains. In marked contrast, blood-forming activity begins in the human liver at roughly weeks 5–6 (Migliaccio et al., 1986; Peschle et al., 1984b) and begins to wane before the third trimester (W26) (Linch et al., 1982). Human bone marrow hematopoiesis has its beginnings in long bone diaphyses at W10.5, is in its final form by W16.0 (Charbord et al., 1996), and contributes hematopoietic tissue for the remainder of gestation, or roughly 24 weeks (and beyond of course). Overall, the marrow in our species is a site of blood cell production for roughly 30 weeks or 75% of our gestational period. Why such a diVerence relative to the mouse? As discussed earlier, when we considered the diVerential origins of the extraembryonic mesoderm, the distinction is perhaps not as great as it initially appears. Here, if we evaluate the timing of the onset of bone marrow hematopoiesis relative to the size of murine and human embryos, the distinction vanishes. At the onset of circulation (again, D21.0 in humans versus d8.25 for the mouse), the human embryo is roughly three times larger than the mouse (2.0 mm as opposed to 0.7 mm, respectively) (Larsen, 1993; Nagy et al., 2003). The size of the growing human fetus will ultimately, of course, experience a vast increase over its murine counterpart. However, at the point where each species seeds the bone marrow with hematopoietic activity, they are roughly the same size. The mouse at the onset of marrow hematopoiesis (d16.0) is 14–17 mm in length (average 15.5 mm) (Nagy et al., 2003) and the human at this same hematopoietic stage (D40.0–45.0) is roughly 11–14 mm (average 12.5 mm) (Larsen, 1993). If we look to other physiological indicators of hematopoiesis, such as globin gene expression, we do see some discordance (Fig. 1). Mice have a form of -like globin (-H1 globin), not present in humans, that is expressed strongly by d7.5 and concurrently (although of shorter duration) to the early, -like, -globin (Keller et al., 1993; Whitelaw et al., 1990). Fetal -globin is not found in mice (Leder et al., 1980) and is adapted uniquely to the oxygenation requirements of the human fetus. Additionally, mice express two forms of adult -globin, major and minor (both being expressed in liver onward from d11.5) (Whitelaw et al., 1990), whereas humans have a single adult -globin that, although expressed from early on, is upregulated quite late in gestation in the liver and bone marrow at around W34.0 (Kazazian, 1974; Migliaccio et al., 1986; Peschle et al., 1984a,
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1985). Finally, humans have rare forms of both -like and -like adult globins, namely - and -globin, respectively. The importance of these rare globins in development is unclear. While there are additional diVerences between murine and human blood tissues, including the nuclear morphology of some leukocytes, the manner by which each makes (and uses) blood is generally quite similar. This notion has been supported innumerable times, as basic observations made in the mouse have been shown to have cognates in the human system. However, although investigations conducted at the level of the whole organism are imperative for understanding the ‘‘big picture’’ of hematopoiesis as it relates to development, studies carried out on a finer scale using explanted organs or single cell suspensions (either isolated directly from blood-forming tissues or as immortalized cell culture lines) have aVorded researchers the opportunity to address the molecular details of blood development. In 1981, the field of hematology research gained a powerful new tool (as did many other disciplines for that matter) when two groups published the first derivation of embryonic stem cell lines from the preimplantation murine blastocyst (Evans and Kaufman, 1981; Martin, 1981). This was followed many years later by the isolation of comparable human lines (Thomson et al., 1998) that, together with those from the mouse, brought a new perspective to hematological investigation. This is especially true in the human system where the types of observations that are common in murine models are simply not possible. For the first time, researchers have the capability to manipulate primordial human tissues in order to define the earliest molecular determinants of blood cell formation. Data obtained using human embryonic stem cells are certain to not only broaden our understanding of hematopoiesis in our own species, but also present an incredibly promising avenue of investigation leading to the improvement of therapies for the treatment of hematological disease.
IV. Hematopoiesis from Embryonic Stem Cells A. The Beginnings of Pluripotent Cell Research Early work investigating the biology of pluripotential mammalian cells came from Stevens and Little (1954) regarding their studies of spontaneous testicular teratomas in the 129 inbred mouse strain. This was followed by several manuscripts detailing the analysis of both murine and human embryonal carcinoma (EC) cell lines (ECs are the proliferative component or ‘‘stem cells’’ of the teratocarcinoma) (for review, see Andrews, 2002). Not only were many murine EC cell lines capable of producing teratomas in serial transplantation experiments [even clonal, multidiVerentiated teratomas produced from a
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single input cell (Kleinsmith and Pierce, 1964)], but the introduction of murine EC cells into preimplantation blastocysts could establish chimerism in the resulting adult mice (Brinster, 1974). Mouse EC lines were later shown to have the capacity for not only clonal growth in vitro, but also the formation of multicellular, cystic structures known as embryoid bodies (EB) that resembled the early blastocyst and also contained derivatives of all three embryonic germ layers (ectoderm, mesoderm, and endoderm) (Martin and Evans, 1975). Murine embryonic stem cell (mESC) lines derived from the inner cell mass of d2.5 blastocysts were first developed in the early 1980s by two teams, that of Evans and Kaufman (1981) in the United Kingdom and Martin (1981) in the United States. It was subsequently discovered that the cytokine leukemia inhibitory factor [LIF, also known as human interleukin in DA cells (HILDA)] could maintain the undiVerentiated state of mESCs in the absence of murine embryonic fibroblast (MEF) feeder layers (Gearing et al., 1987; Moreau et al., 1988; Smith et al., 1988; Williams et al., 1988). Seventeen years later, studies detailing similarly derived pluripotential, human embryonic stem cell (hESC) lines from preimplantation blastocysts (approximately D6.0) were published by Thomson and colleagues (1998) (for review, see Smith, 2001) and thereby ignited a veritable powder keg of both scientific inquiry as well as heated ethical debate. Pluripotent cell lines have also been derived from murine (Matsui et al., 1991; Resnick et al., 1992) and human (Shamblott et al., 1998) primordial germ cells. Human ESCs are most often fostered on MEF feeder layers supplemented with basic fibroblast growth factor (bFGF, also known as FGF2), although conditions supporting their growth on human fibroblast feeders have been described (Amit et al., 2003; Hovatta et al., 2003; Richards et al., 2002, 2003). Techniques have also been reported that allow feeder-free growth (Xu et al., 2001a), a condition that is likely required for any body of work aimed at eventual therapeutics. Both murine and human ES cells have high telomerase activity, are capable of being passaged for extended periods of time in vitro, maintain a diploid karyotype, and, upon withdrawal of LIF from murine cultures and bFGF/MEFs from human ES cells, lead to the formation of EBs in vitro [each mouse EB containing roughly 20,000 to 40,000 cells maximally (Dang et al., 2002)]. Human ESCs form teratomas in vivo that demonstrate all three embryonic germ layers (ReubinoV et al., 2000; Thomson et al., 1998; reviewed in Odorico et al., 2001; Smith, 2001). Additionally, human ESCs give rise to trophectoderm (Xu et al., 2002), a capacity that mESCs do not appear to have in vitro (Beddington and Robertson, 1989) unless the transcription factor Oct-4 is downregulated (Niwa et al., 2000). Human ES cells are capable of being grown clonally from a single starting hESC, although the process is ineYcient (Amit et al., 2000).
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Murine and human ESCs are positive for alkaline phosphatase (ReubinoV et al., 2000; Thomson et al., 1998) and markers indicative of an undiVerentiated, pluripotent state, including TRA-1–60 (Andrews et al., 1984), OCT-3/4 (ReubinoV et al., 2000; Scholer et al., 1990), Cripto (Ciardiello et al., 1991), Rex-1 (Hosler et al., 1989), and Nanog (Chambers et al., 2003). UndiVerentiated hESCs also are positive for the stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4) and negative for SSEA-1 (Thomson et al., 1998), which is the opposite of murine ES cells (Henderson et al., 2002; Kannagi et al., 1983; Solter and Knowles, 1978). While the developmental capacity of mESCs is defined by their ability to give rise to a viable animal [nonchimeric animals derived entirely from cultured mESCs implanted into tetraploid blastocysts have become common (Forrester et al., 1991; Nagy et al., 1990, 1993)], no such platform exists for their human counterparts, for obvious reasons. However, a standard has been suggested that takes into consideration the aforementioned cohort of gene and marker expression, capacity for extended passage in vitro, diploid karyotype, and teratoma formation (Brivanlou et al., 2003). A handful of hESCs meet this suggested standard and are currently available for study using public funding (see NIH, 2003), although other lines meeting the standard exist as well. The capacity of ES cells to form blood tissue (Fig. 3) has been well documented, although it appears that ectodermal (neural) derivatives are the ‘‘preferred’’ developmental pathway in spontaneously diVerentiating murine ES cells (Tropepe et al., 1999; for review, see Munoz-Sanjuan and Brivanlou, 2002). First reported by Doetschman and colleagues in 1985, mouse EBs are capable of forming blood islands containing primitive nucleated megaloblasts that express embryonic globins similar to those seen in the murine yolk sac. Blood island formation has subsequently been used as a developmental standard for determining the pluripotency of mESCs cultured in vitro (Gossler et al., 1986). Murine EB-derived blood islands recapitulate their yolk sac cognates fairly well, as indicated by the formation of CD34þ vascular channels (Young et al., 1995) containing luminal hemogenic progenitors (Doetschman et al., 1985; Risau et al., 1988; Wang et al., 1992). One key question that comes to mind when observing the development of endothelial as well as hematopoietic tissue in cystic EBs is whether a cell like the hemangioblast is present in vitro. Were the answer in the aYrmative, the experimental implications would be obvious and would include an ability to manipulate genes and/or environmental factors on a fine scale within the tissue culture flask, thus facilitating a robust functional definition of the bipotential cell. A number of well-done experiments indicate that a bipotential endothelial/hematopoietic progenitor is in fact present, a cell referred to as the blast colony-forming cell (BL-CFC) (Choi et al., 1998; Kennedy et al., 1997).
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Figure 3 The process of hematopoietic cell formation from ES cells as well as the scale of the intermediates generated therein. (A) A mouse (line CCE) embryoid body at day 14 of culture. Several prominent blood islands may be noted. EBs are then disrupted (most often by day 7) and replated in coculture with stromal cell underlayers. Following coculture, hematopoietic precursors are replated into methylcellulose that contains hematopoietic growth factors in order to determine (retrospectively) the progenitor content of the input material. (B) a typical day 10 colony-forming unit granulocyte, erythrocyte, macrophage, and megakaryocyte (CFUGEMM). (C) A composite of representative myeloid cell types found in CFU-GEMM (following staining with Wright’s/Geimsa). EB and CFU-GEMM images are courtesy of Dr. Michael Kyba, University of Texas Southwestern Medical Center at Dallas. The stained cytospin image is courtesy of Dr. Yuan Wang, Whitehead Institute. (See Color Insert.)
B. Endothelial Cells and Blast Colony-Forming Cells The endothelial component of mES cells has been shown to progressively mature as EBs form in methylcellulose containing VEGF, Epo, bFGF, and IL-6 (Vittet et al., 1996). Both CD31 and Tie-2 were present at d0.0 (although curiously absent until d4.0 when both reappeared), whereas other
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markers of endothelial cells came up slowly over time, including Flk-1 (d3.0), Tie-1 (d5.0), and VE-cadherin (d5.0) (Vittet et al., 1996). Of note, targeted deletion of VE-cadherin in mES cells results in the absence of the EB vascular network, although other markers of endothelial cells were present (and blood cell formation was unimpaired), suggesting a role in the assembly or stability of higher order vascular structures (Vittet et al., 1997). The in vitro equivalent of the hemangioblast is the BL-CFC (for review, see Lacaud et al., 2001). The BL-CFC was first demonstrated as a cell derived from the limited window of d2.5–3.5 in mouse EB development, just proximal to the time point when EryP appear. The BL-CFC was capable of generating both endothelial and hematopoietic progenitors in vitro (Choi et al., 1998; Kennedy et al., 1997). The BL-CFC is generated by disaggregating d3.25 EBs and replating them in a cocktail of VEGF and SCF (Choi et al., 1998; Kennedy et al., 1997), although thrombopoietin (Tpo) alone was shown to give equivalent activity in isolation and enhanced activity when used in combination with VEGF and SCF (Perlingeiro et al., 2003). The BL-CFC is positive for CD34 (Choi et al., 1998; Chung et al., 2002; Kennedy et al., 1997), VE-cadherin (Nishikawa et al., 1998a), and Flk-1 (Choi et al., 1998; Faloon et al., 2000; Kennedy et al., 1997), in agreement with observations made in mouse and human hemangioblastic tissues. The endothelial derivatives of the BL-CFC show immunoreactivity for CD31, Flt-1, and Tie-2 and uptake of acetylated low-density lipoprotein [LDL, an indicator of endothelial cell function (Voyta et al., 1984)] (Choi et al., 1998). The blood cell derivatives express hematopoiesis-specific genes (see later) and form hematopoietic colonies in methylcellulose (Choi et al., 1998; Kennedy et al., 1997).
C. Hematopoietic Cells Mouse embryonic stem cells are capable of spontaneously forming all hematopoietic colony types found in the murine yolk sac (i.e., in the absence of exogenously added cytokines but containing 3% fetal calf serum), although not all mouse ES cells surveyed were equally eYcient: D3 (Doetschman et al., 1985) and CCE (Evans and Kaufman, 1981), both on the strain 129/Sv background, are among the most robust (Burkert et al., 1991). The observation that added cytokines are not required for the development of EryP has been verified in independent experiments, although the authors suggested that fetal calf serum, used at 15%, may have contained suYcient growth factor activity to stimulate primitive colonies (Keller et al., 1993). ES cell-derived blood progenitors likely condition their own environment to a fair degree, as developing EBs express growth factor genes, including both ligands and receptors for Epo, macrophage colony-stimulating factor [M-CSF, also
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known as colony-stimulating factor-1 (CSF-1)], and IL-4 (Schmitt et al., 1991). Although Epo does not alter the temporal appearance of erythropoiesis (Wiles and Keller, 1991), it has been shown to enhance erythroid colony formation (Burkert et al., 1991). Also, while mixed myeloid colonies were present alongside erythroid colonies, these progenitors were unable to produce CFU-S in vivo (Burkert et al., 1991). This result is similar to what has been seen in early (prior to d10.0) murine yolk sac (Muller et al., 1994). Multiple hematopoietic colony types are formed following EryP in the discrete temporal order of macrophages prior to EryD prior to mast cells (Keller et al., 1993). This progression begins at day 4.0 of culture in vitro, suggesting (along with gene expression studies) that early EB blood development follows closely that of the yolk sac (Keller et al., 1993). Conditions have been published that facilitate enrichment for neutrophils using stromal cell coculture and exogenous cytokines (Lieber et al., 2004). Low-level BP-1 aminopeptidase [a marker of murine B-cell diVerentiation (Wu et al., 1991)] and CD3 (T-cell receptor) immunoreactivity indicate that lymphoid cells may be present in murine EBs (although no CD4 or CD8 positive cells were noted) (Chen et al., 1992). Furthermore, if these EB-derived cells were first immortalized with Abelson murine leukemia virus (A-MuLV), they were capable of generating limited chimerism (spleen) in murine recipients (as well as forming tumors in many tissues) (Chen et al., 1992). At day 6.0 of EB formation, several hematopoietic cell surface markers are present, including CD34 (on both endothelial and blood cells) (Kabrun et al., 1997), CD41 (Mikkola et al., 2003a), c-kit, CD31, Flk-1, and Ter-119 (also known as erythroid diVerentiation-related factor) (Mitjavila-Garcia et al., 2002). Lindenbaum and Grosveld (1990) defined an in vitro model for murine globin gene expression in EBs that demonstrates the upregulation of embryonic forms (H1) at d6.0 that correctly transition to adult forms (H1 and from the -cluster, and from the -cluster) by d9.0. This was accompanied by expression of the NF-E1 erythroid/megakaryocytic transcription factor (Lindenbaum and Grosveld, 1990), providing an independent molecular correlate for the proper temporal activity of the globin genes. Keller et al. (1993) showed that the majority of hematopoiesis-associated genes are initially expressed just prior to or beginning at d4.0 of diVerentiation in vitro and include Brachyury, H1 globin, -major globin, c-myb, Gata1, and SCF with others such as c-kit and the Epo receptor coming up at d7.0. The ephrin receptor B4 [EphB4, ephrins being receptor tyrosine kinases that mediate migration and adhesion during patterning and somite formation (Durbin et al., 1998)] has been shown to impact early mesoderm formation in murine ES cells as EphB4 / EBs have reduced mesodermal structures, including impaired blood cell formation (Wang et al., 2003b). Additionally, negative regulators of EB hematopoiesis that function in the pre-BL-CFC have been proposed and include members of the Id
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helix–loop–helix (HLH) proteins as their expression level declines during BL-CFC formation around d3.0 (Nogueira et al., 2000). The hematopoietic lineage specifying transcription factor Scl is expressed from d3.5 (Robertson et al., 2000) following the upregulation of Flk-1 (Chung et al., 2002). Of interest, Scl / mES cells are incapable of forming blood cells in vivo or in vitro (Porcher et al., 1996). A more detailed examination reveals a functional hierarchy for this transcription factor in that the expression of hematopoiesis-specific genes, such as Gata1, globins, and the transcription factors PU.1 and Eklf (Kruppel-like factor 1), was abrogated in Scl / EBs, whereas genes specifying general mesodermal induction and/or endothelial cells (including sequences believed to be key to hemangioblast function) were spared, including Brachyury, Bmp4, Flk-1, Gata2, CD34, and c-kit (Elefanty et al., 1997). The observation that Flk-1 is expressed proximally to Scl has been firmly established in an interesting study wherein the human CD4 gene was knocked in at the murine Scl locus, allowing cells to be sorted based on the surface expression of Flk-1 and human CD4 (Chung et al., 2002). This study demonstrated that fractions expressing Scl were enriched for hematopoietic derivatives of the BL-CFC, whereas endothelial cells were largely dependent on Flk-1 alone (Chung et al., 2002). Another type of EB-derived colony known as the transitional colony was subsequently identified that lacks expression of Brachyury but that did express genes involved in endothelial and hematopoietic specification, including Scl and -major globin (Fehling et al., 2003; Robertson et al., 2000). This indicates that the transitional colony is committed to mesoderm but not yet specified in its ultimate fate. The authors went on to show that transitional colonies also lacked significant EryP formation as well as cellular components indicative of definitive hematopoiesis (including EryD), although they maintained robust colony formation for other definitive lineages (Robertson et al., 2000). This placed the transitional colony proximal to the d2.5–3.5 BL-CFC and distal to the d0.0 EB-forming cell in development in vitro (Robertson et al., 2000). As stated earlier, Scl / EBs do not generate hematopoietic cells in vitro, but show no deficiencies in transitional colony formation. This implicates Scl as a key gene regulating formation of the BL-CFC but not the more proximal transitional colony (Robertson et al., 2000). Runx1 is a transcription factor implicated in the determination of definitive hematopoietic cell fate (North et al., 1999, 2002). Runx1 is expressed at low levels in the ES cell, but its expression increases beginning at d4.0, similar to Flk-1 (Lacaud et al., 2002). Runx1 / EBs demonstrate defective BL-CFC formation with 10- to 20-fold fewer colonies than wild-type EBs, although they maintain primitive erythroid formation, suggesting that there may be two populations of BL-CFC in vitro, one corresponding to the hemangioblast of the YS (and producing EryP) that is not reliant on Runx1 function
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and another requiring Runx1 and capable of generating EryD, similar to the putative hemangioblast of the P-Sp/AGM. The capacity of Runx1 gene function to distinguish EryP and EryD suggests a biclonal model of hematopoiesis within the EB (Lacaud et al., 2002). This last point is supported by independent experiments noting diVerential growth factor requirements for EryP and EryD cells emerging in vitro as cultured on OP9 stromal cell monolayers (Nakano et al., 1994, 1996). Finally, it has been shown that haploinsuYciency for Runx1 in EBs (i.e., Runx1 heterozygotes) actually promotes hemangioblast development, suggesting diVerential activity for this transcription factor in early (stimulating the formation of mesoderm) versus late (hemangioblast specification) stages of development (Lacaud et al., 2004).
D. In Vivo Hematopoietic Reconstitution with Murine Embryonic Stem Cell Derivatives Studies such as those outlined earlier demonstrate the power of in vitro analysis using ES cells to define pathways involved in hematopoietic cell development. Another avenue of experimentation involves intentionally inducing hematopoietic candidate genes and then evaluating blood cell formation by gene expression, colony assay, or that gold standard of HSC research, in vivo long-term multilineage reconstitution. After identifying the initiation of spontaneous hematopoietic colonyforming activity in the d4.0 EB, Hole et al. (1996) inoculated lethally irradiated adult recipients with d4.0 EB cells and noted variable repopulation, with significant survival compared to control animals receiving a bolus of splenic cells alone. These data suggested that ES-cell derived hematopoietic tissue was capable of at least short-term reconstitution. Building upon the observation that transformation of the murine HSC with the BCR/ABL oncogene conferred a disease similar to the human disorder chronic myelogenous leukemia (CML) in which myeloid blood elements expand in number (leukocytosis) yet retain their capacity to diVerentiate into function progeny (Daley et al., 1990; Gishizky et al., 1993; Li et al., 1999), the BCR/ABL gene was employed to attempt expansion of the HSC activity derivative of murine ES cells. Using a conditional BCR/ABL transgene, Era and Witte (2000) were able to demonstrate that the induction of expression led to an expansion of myeloid progenitor activity in mESCs. Peters and colleagues (2001) isolated two subclones of BCR/ABL-containing murine ES cells that demonstrated a limited number of hematopoietic cell surface markers (including c-kit, Sca-1, and CD44 but negative for AA4.1 and CD34) that were then injected into sublethally irradiated murine hosts. Although this resulted in acute leukemia with granulocytosis in the recipients, it also demonstrated
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that the ESC derivatives were retained in hematopoietic organs, in this case the spleen (Peters et al., 2001). Additional studies went on to show that while the BCR/ABL-transformed EB cells formed only primitive erythroid tissue in vitro (expressing -H1 globin), the leukemic derivatives obtained from transplanted animals expressed markers, indicating lymphoid maturation (B220, CD4, and CD8) as well as transition to the -major globin, both verifying that maturation of the hematopoietic activity (and thus, development something like that occurring in vivo) was possible (Perlingeiro et al., 2001). In an attempt to recapitulate engraftment in recipients while sparing them from the leukemia due to transformation by BCR/ABL, downstream signaling components of the oncoprotein were investigated for their capacity to promote hematopoiesis in ES cells. The transcription factor STAT5 (signal transducer and activator of transcription 5) is a downstream transducer of BCR/ABL signaling (Carlesso et al., 1996; Frank and Varticovski, 1996; Ilaria and Van Etten, 1996; Shuai et al., 1996). STAT5 significantly enhances the colony-forming potential of d4.0–6.0 EBs, although they engraft recipients only transiently (Kyba et al., 2003). Expression of another factor, the homeobox gene HoxB4, has been shown to enhance the primitive bloodforming capacity of adult marrow (Sauvageau et al., 1995), as well as to augment hematopoietic cell formation from an ESC starting point (Helgason et al., 1996). Not only was expression of a conditional HoxB4 gene capable of generating engraftable lymphomyeloid hematopoietic cells in vivo (following OP9 coculture with exogenous SCF, VEGF, and Tpo in vitro) (Kyba et al., 2002), but in combination with a complex protocol including somatic cell nuclear transfer (SCNT) and gene therapy was also able to serve as the first proof of principle that embryonic stem cell-based therapy could be used to treat genetic disease [in this case, the immunodeficient (Rag2 / ) mouse] (Kyba et al., 2002; Rideout et al., 2002; reviewed in Daley, 2003; Hochedlinger and Jaenisch, 2003).
E. Hematopoiesis and Human Embryonic Stem Cells To date, there is not a wealth of papers describing the hematopoietic products of human ES cells cultured in vitro and none that speak to reconstitution of the blood-forming compartments in vivo. It is after all a new field and, coupled with the added challenges of a restrictive research environment, is just beginning to generate interesting data. What has been shown is that hESCs are capable of generating mesodermal structures (muscle, cartilage, and kidney) in teratoma formation experiments (ReubinoV et al., 2000; Thomson et al., 1998). Also, and as stated earlier, hESCs form EBs in vitro
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and markers of hematopoietic cell formation can be noted therein by RT-PCR and include expression of - and -globin in response to growth factors (nerve growth factor, hepatocyte growth factor, epidermal growth factor, and BMP4) (Schuldiner et al., 2000), -globin without stimulation (Itskovitz-Eldor et al., 2000), and - and -globin (unpublished results). Chadwick et al. (2003) have shown that exogenous cytokines (SCF, Flt3L, IL-3, IL-6, and G-CSF) in combination with BMP4 increase the proliferation (but not the diVerentiation) of hESC-derived CD45þ hematopoietic tissue, first appearing at D10.0 of culture. Fractions of these hEB blood derivatives were also positive for CD34 and AC133 with low levels of c-kit and, in colony-forming assays, morphologically appeared similar to macrophages, granulocytes, and erythrocytes (expressing CD33 and CD13 while glycophorin-AþCD45 cells were also noted) (Chadwick et al., 2003). This same study also showed that genes for the transcription factors GATA-1 and PU.1 were upregulated on D3.0 and D15.0, respectively, following cytokine treatment, although RUNX1 was expressed continually from D0.0 onward (Chadwick et al., 2003). BMP4 has also been shown to be a potent inducer of hematopoietic activity in rhesus monkey ESCs and in combination with stromal (S17) coculture, SCF, IL-3, IL-6, G-CSF, VEGF, Flt-3L, and Epo led to the upregulation of Scl, the IL-6 receptor and VE-cadherin (Li et al., 2001). These hematopoietic derivatives were also CD34 positive (Li et al., 2001) and it is reasonable to expect a similar impact on hESCs, especially considering that cognate activities between rhesus and H. sapiens exist for bone marrow-derived HSCs, including those pertaining to homing, the cell cycle, and overall hematopoietic diVerentiation (Lu et al., 2002). Finally, Kaufman et al. (2001) noted that by stromal cell coculture using the S17 line (from mouse bone marrow) and 20% fetal bovine serum without exogenously added growth factors, cobblestone areas of hematopoietic cell formation can be noted by D5.0 in vitro. After 17 days in culture, CD34þ31þ38 45 cells were observed (Kaufman et al., 2001). If CD34 positive cells were sorted, this fraction was enriched for colony-forming activity above the CD34 negative subset and yielded CFU-M (macrophages), CFUGM (granulocytes and macrophages), BFU-E, CFU-E, and rare CFUGEMM (granulocytes, erythrocytes, macrophages, and megakaryocytes) (Kaufman et al., 2001), with the latter being identical in appearance to the murine CFU-GEMM colony shown in Fig. 3. These cells also expressed hematopoiesis-specific genes such as SCL and GATA-2 at D7.0, although LMO-2 and FLK-1 were expressed in undiVerentiated hESCs (Kaufman et al., 2001). Comparison of these data to the kinetics of marker expression in the mouse indicates a more latent time frame for the development of hematopoietic cells from hESCs [e.g., in vitro SCL expression in the mouse ramps up at d3.5 (Robertson et al., 2000) and D7.0 in the human (Kaufman et al., 2001)].
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V. What Remains To Be Learned? As is the case with any body of inquiry, the results beg more questions than they answer. Such is certainly true in the field of hematology research. One specific matter stemming from these pages speaks to the relationship of the NK cell to the development of B and T lymphocytes (reviewed in Colucci et al., 2003). It is intriguing that Tavian and colleagues (2001) reported the detection of NK cells positive for CD56 (a marker of multiple stages of NK cell maturity) in explants from the D19.0 human P-Sp or YS days before B- and T-cell progenitors appear in the region of the P-Sp. That neither Bnor T-cell progenitors were noted at any time point in explanted YS in these experiments (even taken as late as D32.0) is of added interest. Similar results were obtained in independent studies using a slightly diVerent methodology (Oberlin et al., 2002). Does the human NK cell derive from the same progenitor as the B and T cell? Furthermore, although the answer may be in the aYrmative, it remains to be defined what activities regulate the preferential elaboration of NK progeny in the human P-Sp at early stages and restricts altogether the emergence of B and T cells in the yolk sac. Regarding the relationship between cells of the hemogenic endothelium (be they in the YS or AGM), the basic question remains nearly the same today as when it was posed by Vera DanchakoV some 90 years ago, ‘‘What are the conditions and the causes for the development of certain groups of morphologically identical mesenchyme cells into morphologically diVerent products of diVerentiation? ’’ (DanchakoV, 1916a). One by one, the factors that define the hemangioblast are being described. In the end, we shall see if the hemangioblast ‘‘exists’’ as a single cell or represents an activity of a more complex nature, such as that occurring along a morphogen gradient (reviewed in Gurdon and Bourillot, 2001) and thus, more akin to a population than a discrete, cellular entity. However, that hemangioblastic (i.e., BLCFC) activity has been shown repeatedly in chaotic, cystic embryoid bodies where such gradients are likely hard pressed to form suggests either that hemangioblast isolation is possible or that we have a lot to learn about ‘‘polarity’’ in EBs (the latter being likely and a topic deserving investigation). The development of additional systems for regulated transgene expression (including RNA interference), as well as reporters of ‘‘naturally’’ occurring expression [such as those utilized by Fehling et al., (2003)], will continue to prove extremely valuable in defining the hemangioblast. There are other unanswered questions that are more diYcult to address at present. Despite many decades of work attempting to define the origins of definitive hematopoiesis in mammals, the jury is still out as to whether all adult hematopoietic stem cells originate in the yolk sac (monoclonal), in both the yolk sac and P-Sp/AGM (collaborative biclonal), or in the P-Sp/AGM
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alone (independent biclonal with two ‘‘eras’’ of blood formation in the YS and P-Sp/AGM, with the former being purely transient and primitive and the latter being durable and definitive). The mammalian embryo is currently unapproachable for the sorts of diVerential marking experiments that have made studies in the chick so instructive in defining the origins of long-term hematopoietic activity. While improved methodologies will likely allow us to leap this hurdle in the years to come, we can currently only infer the origins of mammalian HSCs based on finely staged studies in the embryonic mouse. Added to this are findings made in vitro using stromal cell coculture, exogenously added hematopoietic growth factors, and/or enforced gene expression to interrogate the capacity of YS tissue to be ‘‘made definitive.’’ What we refer to as ‘‘stroma’’ is itself a complex tissue in vivo (reviewed in Dennis and Charbord, 2002) and although key elements of how stromal cells instruct such maturation events are unknown (in vivo or in vitro), several studies suggest that precirculation YS cells are capable of transitioning to longterm lymphomyeloid engraftment given the proper inductive environment or genetic modification. However, this is not the same as proving what occurs in vivo in the undisturbed embryo. One might expect that early blood tissue bears some degree of plasticity that allows it to become definitively hematopoietic. After all, both primitive and definitive blood cells within an individual contain the same genome. However, we are far from a firm understanding of how key genes are regulated diVerentially between the YS and the P-Sp/AGM (and in most other tissues and systems for that matter). A small cohort of studies would suggest that diVerential imprinting is one defining feature. Should it be shown that human primitive blood-forming tissue bears a diVerent epigenetic state than definitive tissue, it would lead us to conclude that the pre-HSC nucleus is reprogrammed to a certain degree (in a monoclonal model) during the transition to definitive hematopoiesis and would further suggest experiments designed to assess and/or influence patterns of DNA methylation in the YS. However, it is important to observe that as the human extraembryonic mesoderm (and thus primitive hematopoiesis) does not derive from the embryo proper, definitive hematopoiesis would reflect a permanent contribution of placental tissue to the developing fetus and adult in a monoclonal model. Such a circumstance appears to be without precedent and rather supports an independent biclonal model. In the murine system, as both extra- and intraembryonic hematopoiesis derive from the epiblast, we are unable to make predictions regarding the origins of definitive hematopoiesis in this context. It would be premature to state with any certainty, based on the few reports of blood cell formation in parthenogenotes and androgenotes cited here, that diVerential imprinting is a phenomenon aVecting the switch between primitive and definitive hematopoiesis. It remains, however, an intriguing hypothesis. The impact of epigenetic
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modifications in development is a fascinating area of inquiry and one of significant importance in the field of embryonic stem cell research (reviewed in Gurdon et al., 2003). Although few studies to date have addressed the hematopoietic potential of human embryonic stem cells and none speak to the capacity of hESC derivatives to engraft recipients, there is every reason to expect that they will have comparable hematopoietic activity to stem cells derived from mice and nonhuman primates. Studies employing human ES cells may facilitate an understanding of how epigenetic programming impacts embryonic hematopoiesis. This is in addition to the many experiments that have already been carried out in the mouse now requiring evaluation in the human system. Beyond this, we look to the use of embryonic stem cells as not only tools to study hematopoietic ontogeny, but also as direct therapeutic material for patients suVering from diseases of the blood. These potential therapies would be possible pending the successful description of methods by which enriched populations of hematopoietic tissue could be fostered from human ES cells. It has been proposed that a ‘‘bank’’ of embryonic stem cells might be established containing the most common histocompatibility groups in order to have suYcient resources to transplant the majority of candidates for ES cell-based therapies (Drukker et al., 2002). The success of stem cell-based therapy making use of somatic cell nuclear transfer to create haploidentical hematopoietic material in the mouse (Kyba et al., 2002; Rideout et al., 2002) suggests that this might one day be possible for humans as well. The use of nuclear transfer to create ‘‘custom’’ ES cells would not only benefit individuals that do not have an HLA match in the proposed haplotype bank, but could also be used to create disease-specific cell lines to aid in understanding human illness. Much work needs to be completed before we can truly begin to look to therapeutic uses of hES cells, especially those created using SCNT, as the epigenetic stability of ES cells is quite low (Humpherys et al., 2001), resulting in abnormal gene expression profiles in cloned animals (Humpherys et al., 2002). However, studies attempting to create only defined cell types (such as blood stem cells) likely do not bear a complexity that is comparable to that of an entire cloned organism. Furthermore, systems of in vitro blood cell development aVord a greater opportunity to screen for cells bearing a normal phenotype (reviewed in Hochedlinger and Jaenisch, 2003), as well as facilitating the capacity to prospectively engineer systems that allow one to ablate abnormal, potentially dangerous cellular outgrowths prior to transplantation and thereafter (i.e., by incorporating conditional ‘‘suicide genes’’) (Schuldiner et al., 2003). That being said, the current literature contains many more publications reviewing the potential for the therapeutic use of ESCs (e.g., see Bhatia, 2003; Carpenter et al., 2003; Daley, 2003; Hochedlinger and Jaenisch,
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2003; Kaufman and Thomson, 2002; Odorico et al., 2001) than actual research manuscripts describing experimentally derived data. The coming days are certain to bring more reports detailing conditions and methods that promote the generation of engraftable, long-term lymphomyeloid repopulating cells in both murine and human ES cell systems. Without a doubt, these basic observations will prove to be as important to the future development of the field of regenerative medicine as experiments in the last century were to the current field of hematopoietic stem cell transplantation.
VI. Summary By reviewing murine and human blood formation, we have sought to create a standard with which to evaluate the current literature on the use of embryonic stem cells in the study of hematopoiesis. While mice and humans diVer greatly on several levels, we have argued that they are generally quite similar as far as the ontogeny of hematopoiesis is concerned. As such, the many published works using the mouse as a model hematopoietic system have imparted much valuable knowledge to our overall understanding of this complex and fascinating biological process in our own species. However, significant diVerences do exist and suggest that the best conducted studies in the mouse will simply leave gaps in what is known of the establishment and maintenance of the human blood system. The fact that both murine and human embryonic stem cells are now available for study has instigated a new era of inquiry into the foundations of normal blood cell development. This is especially true in the human system where detailed, prospective studies would be otherwise untenable. By using ES cells to model human blood development, investigators have already begun to not only solve many biological mysteries (such as putting a finger on the elusive hemangioblast), but also to address those diseases of the blood that, despite the best current therapies, continue to go without eVective treatment. In this regard, basic research has a rich history of paving the way for tomorrow’s cures. The authors of this review firmly believe that because human embryonic stem cells are now part of this eVort, they will have a tremendous positive impact on the understanding of human disease and the improvement of medical practice.
Acknowledgments We extend our gratitude to the members of the Daley laboratory for being exceptionally generous with both collegiality and intelligent commentary and to Drs. Michael Kyba and Yuan Wang in particular for the use of photographic images. We acknowledge Drs. Michael
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Kyba, Paige Larrabee, Chet Glomski, and Mark L. Damen for insightful comments regarding the preparation of the manuscript, Ms. Melissa Withers for the kind contribution of equipment, and Mr. Dave Richardson, Ms. Mary Sears, and Drs. Marshall Lichtman and Tian-Jian Hsu for assistance in obtaining early published works. The authors further acknowledge Drs. Grover Hutchins and Ranice W. Crosby for kind assistance in obtaining images from the Bro¨del Archive at the Johns Hopkins University, School of Medicine. MWL specifically acknowledges Dr. Grover C. Bagby for planting the seed of what will undoubtedly continue to be a lifelong interest in blood. MWL is a Career Development Fellow of the Leukemia and Lymphoma Society. GQD is a Birnbaum Scholar of the Leukemia and Lymphoma Society and a Burroughs Wellcome Fund Career Awardee in the Biomedical Sciences. This work was funded in part by the NIH (CA76418, CA86991, DK59279, and HL71265) and by the Biotechnology Process Engineering Center (BPEC) at the Massachusetts Institute of Technology (MIT).
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Yoder, M. C., Hiatt, K., Dutt, P., Mukherjee, P., Bodine, D. M., and Orlic, D. (1997a). Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity 7, 335–344. Yoder, M. C., Hiatt, K., and Mukherjee, P. (1997b). In vivo repopulating hematopoietic stem cells are present in the murine yolk sac at day 9.0 postcoitus. Proc. Natl. Acad. Sci. USA 94, 6776–6780. Yoder, M. C., Papaioannou, V. E., Breitfeld, P. P., and Williams, D. A. (1994). Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells. Blood 83, 2436–2443. Yong, K. L., Watts, M., Shaun Thomas, N., Sullivan, A., Ings, S., and Linch, D. C. (1998). Transmigration of CD34þ cells across specialized and nonspecialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31). Blood 91, 1196–1205. Young, P. E., Baumhueter, S., and Lasky, L. A. (1995). The sialomucin CD34 is expressed on hematopoietic cells and blood vessels during murine development. Blood 85, 96–105. Zanjani, E. D., Ascensao, J. L., and Tavassoli, M. (1993). Liver-derived fetal hematopoietic stem cells selectively and preferentially home to the fetal bone marrow. Blood 81, 399–404. Zaragoza, M. V., Keep, D., Genest, D. R., Hassold, T., and Redline, R. W. (1997). Early complete hydatidiform moles contain inner cell mass derivatives. Am. J. Med. Genet. 70, 273–277. Ziegler, B. L., Valtieri, M., Porada, G. A., De Maria, R., Muller, R., Masella, B., Gabbianelli, M., Casella, I., Pelosi, E., Bock, T., Zanjani, E. D., and Peschle, C. (1999). KDR receptor: A key market defining hematopoietic stem cells. Science 285, 1553–1558. Zou, Y. R., Kottmann, A. H., Kuroda, M., Taniuchi, I., and Littman, D. R. (1998). Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393, 595–599.
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Regulation of Gene Activity and Repression: A Consideration of Unifying Themes Anne C. Ferguson-Smith, Shau-Ping Lin, and Neil Youngson Department of Anatomy University of Cambridge Cambridge CB4 1AW, England
I. Introduction II. Regulation of Imprinted Gene Activity and Repression A. Imprinting Control at the Igf2r Domain on Mouse Chromosome 17 B. Control at Two Imprinted Subdomains Associated with the Overgrowth Disorder, Beckwith–Wiedemann Syndrome C. Imprinting at the Prader–Willi/Angelmann Syndrome D. The Dlk1-Gtl2-Imprinted Domain on Distal Mouse Chromosome 12 III. A Consideration of Recurrent Themes References
I. Introduction Genomic imprinting is a normal process that causes a subset of mammalian genes to be expressed or repressed depending on their parental origin. Hence, the chromosomes that an individual inherits from the mother contribute diVerent functions compared to those inherited from the father, which results in a requirement of both parental genomes in normal mammalian development (Ferguson-Smith and Surani, 2001). In humans, several genetic diseases have been described that are associated with disorders exhibiting parental origin eVects in their patterns of inheritance. These diseases fall into three categories: neurological disorders, disorders of prenatal growth and development, and syndromes in which there are defects in both prenatal development and postnatal behavior (Hitchins and Moore, 2002; Paulsen and Ferguson-Smith, 2001). To date, about 70 imprinted genes have been identified in the mammalian genome and these map to about 15 genomic locations (Beechey, 2003). Many, although not all, imprinted genes are located in clusters, suggesting longrange cis-acting control elements that regulate multiple genes within the Current Topics in Developmental Biology, Vol. 60 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00
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cluster. Furthermore, genes expressed from the maternal chromosome can reside alongside genes that are silent on the maternal chromosome; hence, imprinted domains can contain closely linked but reciprocally imprinted genes. Nonimprinted genes are also found within these domains. So how does the transcriptional machinery of the cell tell the diVerence between the two parental alleles of a gene at an imprinted domain? Imprinted chromosomal regions on the two parental homologues are marked diVerentially by heritable epigenetic modifications to the DNA and chromatin. These include DNA methylation at CpG dinucleotides and modifications such as acetylation and methylation to the lysine residues on core histones (Li, 2002). These diVerential modifications are likely to aVect the overall conformation of the chromatin influencing interactions between the regulatory machinery and the genomic template. Data examining the relationship between DNA methylation and histone modifications are still limited; however, it is clear that regions that have diVerential histone modification on the two parental chromosomes do not necessarily have diVerential DNA methylation (Grandjean et al., 2001; Perk et al., 2002). In all examples examined to date, areas of diVerential DNA methylation also harbor diVerential histone modifications. In several in vitro situations, enzymes involved in histone modification have been found to associate with DNA methyltransferases and with proteins that specifically bind methylated DNA (Fuks et al., 2003a,b). These interactions can often be correlated with transcriptional activity or repression. Epigenetic diVerences important for genomic imprinting on the two parental genomes are established in the germlines during spermatogenesis and oogenesis (primary imprints). After fertilization, additional diVerential marks often accumulate at other regions, notably at the CpG-rich promoters of some, although not all, imprinted genes, perhaps in response to the postfertilization activity or repression of that gene (secondary imprints). The dynamic changes associated with the establishment, maintenance, and germline reversibility of DNA methylation imprints are illustrated in Fig. 1. Immediately after fertilization, most of the genome loses methylation and then reacquires it around the time of implantation—a process termed nuclear reprogramming (Monk et al., 1987; Reik et al., 2001). It is believed that the primary imprints established in the germline are resistant to genomewide demethylation events during preimplantation development; however, this has not been assessed comprehensively. While understanding the mechanism, function, and regulatory consequences of primary imprints is an exciting prospect, secondary imprints are also significant and are useful diagnostic tools for human-imprinted disorders. How and why do the two parental genomes establish diVerential epigenetic marks in their respective germlines? For example, are there particular genomic sequences or features that the male and female germlines recognize and epigenetically ‘‘treat’’ diVerently? One thought-provoking idea pertaining
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Figure 1 Establishment, heritability, and erasure of genomic methylation imprints. A small population of diploid somatic cells gives rise to germ cell progenitors at early postimplantation stages of mouse development. These cells erase their methylation imprints as they migrate toward the genital ridge and acquire new imprints depending on the sex of the gonad. During this reprogramming phase, female germ cells will acquire maternal methylation imprints (red) and sperm will acquire paternal methylation imprints (blue). After fertilization, and before implantation, the two parental germlines lose most of their DNA methylation in a second round of nuclear reprogramming. Some methylation imprints are resistant to this reprogramming and retain their methylation imprints throughout preimplantation development. At the blastocyst stage around implantation, the cells progressively acquire secondary methylation de novo. (See Color Insert.)
to the evolution of the imprinting mechanism suggests that intragenomic parasitic elements, including retrotransposons and other related elements, are heritably and variably silenced by DNA methylation in order that they are rendered transcriptionally inactive, ‘‘hidden’’ by heterochromatinization and/or immobilized (Bird, 1997; Martienssen, 1998; Whitelaw and Martin, 2001; Yoder et al., 1997). Extending this to imprinted domains, one could postulate that particular genomic elements may be recognized and silenced in only one of the parental germlines and/or the two germlines may use diVerent epigenetic-modifying mechanisms to suppress these elements. A consequence of this germline-specific silencing may be the parental origin-specific gene activity and repression within these regions in somatic tissues after fertilization. Regardless, in order to address the role of germline-modified genomic features in imprinting control, several teams
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working on diVerent imprinted loci have identified and characterized germline-derived diVerentially methylated regions (DMRs) and have assessed their contributions to both short- and long-range imprinting control. Although no single mechanism has emerged, there are several recurrent themes. The following section summarizes the roles of germline-derived DMRs in the control of imprinted gene activity and repression in five diVerent imprinted gene clusters.
II. Regulation of Imprinted Gene Activity and Repression To date, five imprinted domains have been characterized in which germlinederived DMRs have been identified and shown to act as long-range cis-acting imprinting control elements for multiple imprinted genes. Two of the five DMRs acquire their methylation in the sperm and are unmethylated in the egg, and three become methylated in the egg and are unmethylated in sperm. These are depicted in Fig. 2 illustrating schematically the organization of each domain and the location and properties of the germline-controlling element(s) within them. Confirmation of the function of the DMRs as long-range imprinting control elements has come primarily from targeted deletion experiments in mice or from the study of patients with imprinted disorders. In these analyses, consequences of the mutation on imprinting are considered and compared after transmission through male and female germlines. Three models to explain the epigenetic control of imprinted activity and repression involving DMRs have been described. The three models are (1) the antisense transcript model, (2) the ‘‘insulator’’ model, and (3) the bipartite imprinting center model. No one of these models is mutually exclusive or comprehensive. Indeed, each imprinted domain might involve features of more than one model.
A. Imprinting Control at the Igf2r Domain on Mouse Chromosome 17 1. An Antisense Transcript Mechanism The Igf2r domain contains a cluster of three imprinted genes: the insulin-like growth factor 2/mannose-6-phosphate receptor and two tissue-specific cation membrane-spanning cation transporter proteins, Slc22a2 and Slc22a3, which are imprinted in the placenta. A third cation transporter in the domain, Slc22a1, is expressed in adults only and is not imprinted (Zwart et al., 2001). A germline DMR has been identified in the second intron of Igf2r. DMR methylation is established in oocytes and the region is unmethylated in sperm (Stoger et al., 1993). The DMR is maintained in a diVerentially
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methylated state throughout pre- and postimplantation development. In contrast, the promoter of Igf2r is also a DMR but acquires methylation on the repressed paternal allele late in gestation. CpG islands in the vicinity of Slc22a2 and Slc22a3 promoters are not methylated diVerentially, suggesting imprinting control by other elements (Zwart et al., 2001). The intronic germline DMR is a promoter for an imprinted antisense transcript (Air) that is repressed on the maternal chromosome and active on the paternal chromosome (Fig. 2a). Large transgenes with deletion of the methylated CpG island on the paternal chromosome have activation of the three paternally repressed genes and loss of Air transcription (Wutz et al., 1997; Zwart et al., 2001). A maternally inherited deletion of the methylated intronic Air promoter has no eVect on imprinting—the three genes remain active on the maternal chromosome (Wutz et al., 1997). In situations where the methylated Air promoter on the maternal chromosome fails to maintain methylation, Igf2r is downregulated (Li et al., 1993). Importantly, truncation of the Air transcript on the paternal chromosome results in loss of repression of the three imprinted genes, indicating that the noncoding RNA itself is required for imprinting in cis (Sleutels et al., 2002). Because imprinting is lost at genes transcribed from promoters both upstream and downstream of the imprinting control element, the RNA is believed to have a bidirectional function. The precise mechanism of Air action remains to be determined.
B. Control at Two Imprinted Subdomains Associated with the Overgrowth Disorder, Beckwith–Wiedemann Syndrome Beckwith–Wiedemann syndrome (BWS) is one of the best studied imprinted growth disorders. Clinical features include somatic overgrowth and an increased incidence of embryonal tumors (Engel et al., 2000). BWS is caused by genetic and epigenetic changes in a 1-Mb region containing at least 15 genes, the majority of which are imprinted. The organization and imprinting of the orthologous region in the mouse are shown in Figs. 2b and 2c. In BWS, 25–50% of patients have biallelic IGF2 expression and 50% have biallelic expression of the LITI/KCNQ1 noncoding RNA (Fig. 2b). About 5% of patients have mutations in CDKN1C (Li M et al., 2001). The organization of the region is conserved in mouse (Figs. 2b and 2c) and can be divided into two diVerent imprinted subdomains consistent with the identification of two imprinting control elements—one located in the Kcnq1 gene and the second located within the Igf2–H19 domain. The noncoding transcript LIT1/KCNQ1OT1 is expressed from the paternally inherited chromosome from an intronic CpG island promoter within and antisense to KCNQ1. The intronic CpG island promoter is a germline DMR that is hypomethylated on the active paternal allele and methylated
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Figure 2 For all schematics, maternally expressed transcripts are represented in red and paternally expressed transcripts are in blue. Yellow bars represent nonimprinted genes, and dark gray bars represent the repressed alleles of imprinted genes. DMRs are represented by black-filled circles on the methylated allele, and unfilled clear circles represent the unmethylated allele. Arrows represent the direction of transcription. (a) The Igf2r locus on mouse chromosome 17. (b) The Kcnq1 locus on distal mouse chromosome 7. (c) The Igf2-H19 locus on distal mouse chromosome 7. The methylation-sensitive zinc finger DNA-binding insulator
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on the maternally inherited inactive allele (Fig. 2b). Paternal inheritance of a deletion of this CpG island results in no Lit1/Kcnq1ot1 transcription and activation of all genes usually repressed on the paternal chromosome (Fitzpatrick et al., 2002; Horike et al., 2000). This activation is bidirectional; both Mash2 and Tssc3 are expressed from the paternal allele. Deletion of the methylated promoter on the maternal chromosome has no eVect on the imprinting of genes in the cluster. However, maternally inherited translocations within the KCNQ1 gene are seen frequently in BWS. The translocations result in hypomethylation at the maternal LIT1 CpG island promoter and this is associated with its inappropriate expression on the maternal chromosome and repression of the normally expressed alleles on that chromosome independent of any eVect on IGF2 (Lee et al., 1999). Taken together, these data indicate that the unmethylated CpG island and/or Lit1 transcription is required for imprinting on the paternal chromosome. This is reminiscent of imprinted regulation at the Igf2r domain and suggests that, like for Igf2r, this BWS region might be regulated by an antisense transcript mechanism. 1. An Insulator Mechanism Over two dozen targeted deletion and transgenic experiments have been conducted to elucidate the mechanism regulating imprinting at the Igf2–H19 domain (Arney, 2003). Igf2 and H19 are imprinted, reciprocally, are located approximately 90 kb apart, and are coexpressed in multiple tissues during development. Key elements involved in their regulation are illustrated schematically in Fig. 2c and are reviewed by Verona and colleagues (2003). The two genes compete for shared enhancers for their mesodermal and endodermal expression and these are located downstream of H19. A germline DMR, located 2–4 kb upstream of H19, acquires methylation in sperm, is unmethylated in eggs, retains this state at preimplantation stages, and hence is resistant to the global genome-wide demethylation event that occurs during preimplantation development (Warnecke et al., 1998). This element has two diVerent functions. On the unmethylated maternally inherited chromosome, the germline DMR is an insulator element that contains multiple binding sites for the methylation-sensitive zinc finger DNA-binding protein CTCF. Normally, CTCF binds to the unmethylated element on the maternal chromosome and inhibits the interaction between Igf2 and protein is indicated in purple, and green bars represent downstream endodermal and mesodermal enhancers. Black arrows represent enhancer/promoter interactions. (d) The mouse orthologue of the PWS/AS domain. (e) The mouse Dlk-Gtl2-imprinted domain on distal chromosome 12. Hairpin structures represent preprocessed micro-RNAs that are expressed from the maternally inherited chromosome in an antisense orientation to the paternally expressed Rtl1 mRNA. Because of this orientation relationship, micro-RNAs are 100% complementary to the Rtl1 mRNA and hence might interact with this potential target. (See Color Insert.)
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the downstream enhancers. CTCF does not bind to the methylated paternal chromosome, thus allowing interaction between Igf2 and the enhancers. Methylation on the paternal chromosome is required for H19 repression and Igf2 activity and for the prevention of insulator function. Deletion of the entire DMR results in a symmetrical loss of imprinting; on the maternal chromosome, Igf2 is activated and H19 expression is reduced proportionally, whereas the opposite happens on the paternal chromosome: H19 is activated and Igf2 expression is reduced proportionally. Other distinct elements also contribute to tissue-specific repression of H19 on the paternal allele (Drewell et al., 2000) and Igf2 on the maternal allele (Constancia et al., 2000). To date, there is no well-established second example of imprinting on the two parental chromosomes regulated by a germline-derived DMR that is also a methylation-sensitive insulator.
C. Imprinting at the Prader–Willi/Angelmann Syndrome 1. A Bipartite Imprinting Center Mechanism Prader–Willi (PWS) and Angelman (AS) syndromes are two distinct neurobehavioral disorders associated with the same imprinted domain on human chromosome 15. The domain contains a cluster of paternally expressed genes and at least two maternally expressed imprinted genes in human and one in mouse (Cavaille et al., 2000; Ferguson-Smith and Surani, 2001; Horsthemke et al., 1997; Jiang et al., 1998) (Fig. 2d). PWS is a result of molecular defects resulting in repression of the paternally expressed imprinted genes, and AS is associated with a loss of expression of maternally expressed imprinted genes. In contrast to the regulation of imprinting at the three aforementioned domains, two elements, one essential for imprinting on the maternal chromosome (the AS imprinting center, AS-IC) and one on the paternal chromosome (the PWS imprinting center, PWS-IC), control imprinting at this domain. Only the unmethylated PWS-IC, encompassing the SNRPN promoter and its first exon on the paternal chromosome, is required for activity and repression of all the genes with the domain; hence it has a bidirectional function. When it is deleted on the paternal chromosome, there is no SNRPN transcription and the region becomes hypermethylated. When it is deleted on the maternal chromosome, there is no eVect on the maternally repressed alleles (Buiting et al., 2001; Dittrich et al., 1996; Ferguson-Smith, 1996). The AS-IC is located 35 kb upstream of the PWS-IC. Although no DMR has been identified in the AS-IC, diVerential chromatin conformation and allele-specific histone modifications have been described in a region that maps to the appropriate locus as defined from genetic studies. On the maternal chromosome, the region is hyperacetylated at histone H4, methylated at
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lysine 4 of histone H3, and hypersensitive to DNase I in the absence of clear diVerential methylation (Perk et al., 2002). Deletions that include the AS element on the maternal chromosome aVect the PWS-IC (SNRPN promoter) and cause an epigenotype switch, hypomethylation, and activation of usually repressed alleles and repression of the two maternally active alleles. The bipartite imprinting center model at the PWS/AS domain might also include a role for antisense transcription. Normally, Snrpn transcription from the paternal chromosome, regulated by the PWS-IC, overlaps with the paternally repressed Ube3a gene and is therefore transcribed in an antisense orientation to Ube3a (Fig. 2d). In the absence of Snrpn transcription on the paternal allele, the Ube3a gene becomes activated (Chamberlain and Brannan, 2001). Hence, the ‘‘antisense’’ transcription of Snrpn might be required for the imprinting of Ube3a in cis. Mutations in UBE3A have been identified in Angelman syndrome (Fang et al., 1999).
D. The Dlk1-Gtl2-Imprinted Domain on Distal Mouse Chromosome 12 One of the more recently studied imprinted domains is the Dlk-Gtl2 domain on mouse distal chromosome 12. Orthologous imprinted domains have been identified on ovine chromosome 18 and on human chromosome 14. The domain spans at least 1 Mb and contains at least three paternally expressed imprinted genes: Dlk1 (Schmidt et al., 2000; Takada et al., 2000), Rtl1 (Seitz et al., 2003), and Dio3 (Tsai et al., 2002). Dlk1 encodes the delta-like 1 protein, a member of the Notch-Delta family of developmental signaling molecules; however, its function in development is not well defined (Laborda, 2000). Dio3 encodes a protein that functions to inactivate the thyroid hormones T3 and T4 and is therefore a negative regulator of thyroid hormone metabolism. Rtl1 is a retrotransposon-like gene lacking LTRs (Seitz et al., 2003; Youngson et al., manuscript in preparation). It is conserved in at least three mammalian species and encodes an open reading frame. The maternal chromosome expresses a cluster of noncoding RNAs (Cavaille et al., 2002; Schmidt et al., 2000; Seitz et al., 2003; Takada et al., 2000). The maternally expressed transcripts are all expressed in the same orientation and have features suggesting functional RNAs. These include clusters of C/D sno-RNAs and, of particular interest, a pair of micro-RNA genes (mir-127 and mir-136) expressed from the maternal chromosome in an antisense orientation to the Rtl1 gene (Fig. 2e). The micro-RNAs are therefore 100% complementary to an endogenous mRNA and are the first animal micro-RNAs with this property. A germline DMR located 13 kb upstream from the Gtl2 promoter is required for imprinting on the maternal chromosome (Lin et al., 2003). Deletion of the unmethylated element causes
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silencing of all the maternally expressed noncoding RNAs and activation of the repressed Dlk1, Rtl1, and Dio3 genes. In contrast, embryos in which the deletion has been inherited from the father have no change in imprinting status; paternally expressed genes are expressed and the repressed noncoding RNAs remain inactive. While loss of imprinting of Dlk1 and Dio3 in the maternal germline DMR deletion results in a double dose of their expression, levels of Rtl1 transcription are considerably higher than a double dose. It has been proposed that the Rtl1 transcripts may have become stabilized in the absence of the micro-RNAs that are now repressed on the maternal chromosome (Lin et al., 2003). Identification of a long-range cis-acting imprinting regulator at this domain allows the comparison of regulation at this locus with the three models outlined earlier. Although an insulator function for the Dlk-Gtl2 DMR cannot yet be ruled out, it is unlikely to regulate the genes in the same way as the Igf2–H19 domain. This is because at Igf2–H19 a reciprocal eVect is observed after its DMR deletion from each of the parental germlines. Three more likely models can be considered. First, the element might be a transcriptional regulator of the noncoding transcript Gtl2 and associated transcripts. Gtl2 noncoding RNAs might then function as bidirectional cis-acting negative regulators of the maternally repressed imprinted genes Dlk1, Rtl1, and Dio3 in a manner reminiscent of the function of Air at the Igf2r locus. In a second model, the unmethylated DMR might act directly as a long-range regulator of the whole domain conferring a particular epigenetic state and chromatin conformation associated with activity and repression over the 1-Mb domain on the maternal chromosome. The absence of an eVect after deletion of the element on the paternal chromosome indicates that repression of the noncoding RNAs and activity of the protein-coding genes on that chromosome might be regulated by a diVerent element. This idea suggests a model of imprinting control similar to that described at the PWS-AS locus in which two diVerent regions regulate imprinting on the two parental chromosomes in a bipartite model of imprinting control. The search for additional imprinting control elements at that locus is in progress. Other imprinted domains with germline DMRs have been identified and their characterization is underway. For example, a pair of paternally expressed imprinted genes, Rasgrf1 and A19, have been identified on mouse chromosome 9 (de la Puente et al, 2002; Plass et al., 1996). Both genes are expressed from the paternally inherited chromosome, and an intergenic germine DMR has been identified that is unmethylated in eggs and acquires its methylation imprint in sperm. A repetitive sequence immediately adjacent to the DMR is required for the establishment of its methylation in the male germline but has no role to play in the prevention of methylation on the maternally inherited locus (Yoon et al., 2002). Loss of methylation of the DMR on the paternal chromosome results in the repression of Rasgrf1 (A19 was
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not assessed in this assay). It has been suggested that the DMR can act as an insulator in enhancer-blocking assays (Yoon et al., 2002).
III. A Consideration of Recurrent Themes In considering common features of imprinting control, it is apparent that the five imprinting control elements identified to date do have some features in common. All are germline-derived diVerentially methylated regions, although some acquire methylation during oogenesis and others during spermatogenesis. Most exert a similar eVect on the imprinted genes around them—deletion of the unmethylated element causes the activation of usually repressed adjacent genes, whereas deletion of the methylated copy usually has no eVect. In the case of Snrpn, Igf2r, and Kvlqt1 loci, this is probably because the methylated element is an inactive promoter for a transcript involved in imprinting control, and deleting that promoter is also consistent with transcriptional repression. This indicates that methylation of the DMR renders it inactive or nonfunctional and suggests that mechanisms may exist to protect unmethylated imprinted domains from de novo methylation. However, the Igf2–H19 domain represents an exception to this rule— deletion of the methylated element on the paternal chromosome does have an eVect on imprinting. This means that, in contrast to other imprinted regions, the methylated element per se is required for imprinting on the paternally inherited chromosome. Most imprinted domains contain noncoding RNAs with functional potential. Examples of noncoding RNAs expressed from the maternally inherited chromosome and others expressed from the paternally inherited chromosome have been found. Furthermore, a hypothesis suggesting that unmethylated DMR elements are associated with active noncoding RNAs and repressed mRNAs and, conversely, that methylated DMR elements are associated with active mRNAs and repressed noncoding RNAs continues to hold true, in general (with the exception of 50 mRNAs in the PWS cluster). Noncoding RNAs fall into the following three categories. 1. Small RNAs that are most likely functional. These include the paternally expressed C/D sno-RNAs found at the PWS/AS region and the maternally expressed ones at the Dlk1-Gtl2 domain. C/D sno-RNAs are 70 nucleotides long, have a characteristic structure, and function in posttranscriptional RNA modification. Micro-RNAs, another class of small RNA, were found in the Dlk1-Gtl2-imprinted domain. Micro-RNAs are 20–25 nucleotides long, single-stranded RNAs that, in diverse organisms, have been implicated in translational repression, in RNA interference, and in mediating heterochromatinization. Sno-RNAs and micro-RNAs are processed from larger imprinted primary transcripts. The specific functions of
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the small RNAs located in the imprinted domains have not been established; however, sno-RNAs in the PWS domain have been implicated in the etiology of the disorder, and imprinted micro-RNAs have been implicated in target mRNA transcript stability at the Dlk1-Gtl2-imprinted domain. 2. Noncoding RNAs producing defined transcripts visible on Northern blots that do not overlap with protein coding genes. These include the abundant developmentally regulated H19 gene and the Gtl2 gene. Evidence suggests that H19 RNA plays no role in repressing other imprinted genes in the domain (Schmidt et al., 1999), although two reports have indicated a developmental role for H19 (Hao et al., 1993; Kono et al., 2002). The function of Gtl2 remains to be determined. 3. Antisense transcripts that are expressed over many kilobases of genomic DNA and overlap with protein coding genes. These include Lit1, Air, and Snrpn/Ube3a-AS. Interestingly, these are all expressed from the paternally inherited chromosome; however, it is unknown whether this apparent common feature has any significance. Noncoding RNAs are not exclusive to imprinted domains and can be found in many genome databases as expressed sequence tags (ESTs). In this postgenomic era, there is considerable emerging interest in assessing their role, if any, in the regulation of genome function. It is also useful to consider whether some noncoding RNAs may be passive bystanders with their activity reflecting the ability of the DNA/chromatin in which they reside to be transcribed. Alternatively, their activity might contribute to an active chromatin state that influences the expression of other genes in the region. In the case of sno-RNAs, it has been proposed that the tandem arrangement of sno-RNA clusters may provide important structural features that are recognized and modified diVerently in the two germlines. Interestingly, sno-RNAs within the PWS are expressed from the paternally inherited chromosome and, conversely, those in the Dlk1-Gtl2 domain are expressed from the maternally inherited chromosome. These two diVerent clusters of sno-RNAs are not related at the sequence level and those at the PWS region are GC-rich flanked by AT-rich sequences, whereas those at the Dlk1-Gtl2 domain are AT-rich flanked by GC-rich sequences (Cavaille et al., 2002). These interesting sequence isochores may contribute to the diVerent parental origin-specific behavior in the two unlinked sno-RNA domains. Repetitive elements have been associated with parental origin-specific diVerential modifications. Within imprinted domains, SINE elements are underrepresented compared to the published average values for mouse and human nonimprinted sequences with similar G þ C contents (Greally, 2002; Paulsen et al., 2001). It has been suggested that long interspersed nuclear element (LINE)-1 transposon sequences may be mediators of the
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spread of X inactivation (Lyon, 2000). No enrichment for LINE 1 elements has been observed at imprinted domains (Greally, 2002; Paulsen et al., 2001). However, conserved short tandem repeat sequences are frequently observed in the vicinity of germline DMRs. Indeed, at the Rasgrf1 locus on mouse chromosome 9, a series of 40 short tandem repeats are required to confer paternal methylation in the germline, and in more divergent rodent species that do not imprint Rasgrf1, these repeats are not present (Yoon et al., 2002). At the Dlk-Gtl2 locus, the intergenic germline DMR contains a series of tandem repeats conserved in their location within the DMR (Paulsen et al., 2001). Interestingly, the imprinted mouse Impact gene possesses a CpG island that is characterized by a series of tandem repeats, whereas in the nonimprinted human IMPACT gene, such repeats are absent (Okamura et al., 2000). It is therefore likely that short tandem repeats may be a recurrent theme at imprinted domains. Eukaryotic genomes are vulnerable to proliferative parasitic elements such as transposons, retrotransposons, and retroviruses. These intragenomic sequences are CG rich and can become repressed by methylation, which may occur in a parental origin-specific manner depending on the element. For example, the IAP class of retroviral elements is methylated diVerentially, acquiring methylation in the paternal germline; maternally inherited copies are unmethylated. Alu elements apparently show the opposite eVect (Yoder et al., 1997). It is therefore interesting that an imprinted retrotransposon-like gene Rtl1, lacking LTRs and with a conserved open reading frame, has been found to be imprinted in the Dlk-Gtl2-imprinted domain (Seitz et al., 2003). This gene is a member of the Sushi-ichi family of retrotransposons and there are only a handful of copies within the mammalian genome (Youngson et al., manuscript in preparation). Most of these are located on the mouse X chromosome, but four map to autosomes. Of particular interest is the finding that another member of this family, Peg 10, has been shown to be imprinted (Ono et al., 2001); being expressed from the paternally inherited chromosome, like Rtl1. These findings suggest that this family of retrotransposon-like genes might attract parental origin-specific epigenetic modification, resulting in allele-specific gene expression. To test this, a third conserved autosomal member of this family was assayed for imprinting and was found to be expressed from both parental chromosomes. Consistent with this lack of imprinting is the finding that two CpG islands within Rtl1 are methylated on both parental chromosomes. Hence, despite a ‘‘host parasite response’’ that appears to be attempting to silence this retrotransposon, selective pressures have kept it active, and indeed it may have acquired imprinted regulation because of the genomic environment in which it has found itself (Youngson et al., manuscript in preparation). In conclusion, understanding how epigenetic modifications control gene activity and repression within imprinted domains has the potential to
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contribute much to our understanding of the epigenetic control of genome function. For example, genomic imprinting is a good model mammalian system for contributing to our understanding of the function of large and small noncoding RNAs. The relationships between chromatin modifications and DNA methylation can be studied in relation to gene expression, because for an imprinted gene, comparison between active and repressed DNA, RNA, and chromatin allows diVerences to be correlated with function. However, the sequence itself probably holds the key. Are there genomic features that confer imprinting control and what germline mechanisms act upon them to contribute to the epigenetic diVerences between the two parental chromosomes? Are there biological functions in each germline for which the establishment of epigenetic modification is essential or is it the postfertilization developmental requirement for diVerent biological functions between maternal and paternal sets of chromosomes that is the driving force behind the evolution of this remarkable process?
References Arney, K. (2003). H19 and Igf2-enhancing the confusion? Trends Genet. 19(1), 17–23. Beechey, C. (2003). Mouse imprinted genes regions and phenotypes. http://www.mgu.har. mrc.ac.uk/imprinting/all impmaps.html. Bird, A. (1997). Does DNA methylation control transposition of selfish elements in the germline? Trends Genet. 13(12), 469–470. Buiting, K., Barnicoat, A., Lich, C., Pembrey, M., Malcolm, S., and Horsthemke, B. (2001). Disruption of the bipartite imprinting center in a family with Angelman syndrome. Am. J. Hum. Genet. 68(5), 1290–1294. Cavaille, J., Buiting, K., Kiefmann, M., Lalande, M., Brannan, C. I., Horsthemke, B., Bachellerie, J. P., Brosius, J., and Huttenhofer, A. (2000). Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization. Proc. Natl. Acad. Sci. USA 97(26), 14311–14316. Cavaille, J., Seitz, H., Paulsen, M., Ferguson-Smith, A., and Bachellerie, J.-P. (2002). Identification of tandemly-repeated C/D snoRNA genes at the imprinted human 14q32 domain reminiscent of those at the Prader-Willi locus. Hum. Mol. Genet. 11(13), 1–12. Chamberlain, S., and Brannan, C. (2001). The Prader-Willi syndrome imprinting centre activates the paternally expressed murine Ube3a antisense transcript but represses paternal Ube3a. Genomics 73(3), 316–322. Constancia, M., Dean, W., Lopes, S., Moore, T., Kelsey, G., and Reik, W. (2000). Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19. Nat. Genet. 26(2), 203–206. de la Puente, A., Hall, J., Wu, Y. Z., Leone, G., Peters, J., Yoon, B. J., Soloway, P., and Plass, C. (2002). Structural characterization of Rasgrf1 and a novel linked imprinted locus. Gene 291(1–2), 287–297. Dittrich, B., Buiting, K., Korn, B., Rickard, S., Buxton, J., Saitih, S., Nicholls, R., Poustka, A., Winterpacht, A., Zabel, B., and Horsthemke, B. (1996). Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene. Nat. Genet. 14(2), 163–170.
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Drewell, R., Brenton, J., Ainscough, J., Barton, S., Hilton, K., Arney, K., Dandolo, L., and Surani, M. A. (2000). Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic switch. Development 127(16), 3419–3428. Engel, J., Smallwood, A., Harper, A., Higgins, M., Oshimura, M., Reik, W., Schofield, P., and Maher, E. (2000). Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome. J. Med. Genet. 37(12), 921–926. Fang, P., Lev-Lehman, E., Tsai, T., Matsuura, T., Benton, C., Sutcliffe, J., Christian, S., Kubota, T., Halley, D., Meijers-Heijboer, H., Langlois, S., Graham, J., Beuton, J., Willems, P., Ledbetter, D., and Beaudet, A. (1999). The spectrum of mutations in UBE3A causing Angelman syndrome. Hum. Mol. Genet. 8(1), 129–135. Ferguson-Smith, A. (1996). Imprinting moves to the centre. Nat. Genet. 14, 119–122. Ferguson-Smith, A., and Surani, M. A. (2001). Imprinting and the epigenetic asymmetry between parental genomes. Science 293, 1086–1089. Fitzpatrick, G., Soloway, P., and Higgins, M. (2002). Regional loss of imprinting and growth deficiency in mice with a targeted deletion of KvDMR1. Nat. Genet. 32(3), 426–431. Fuks, F., Hurd, P., Deplus, R., and Kouzarides, T. (2003a). The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res. 31(9), 2305–2312. Fuks, F., Hurd, P. J., Wolf, D., Nan, X., Bird, A. P., and Kouzarides, T. (2003b). The methylCpG-binding protein MeCP2 links DNA methylation to histone methylation. J. Biol. Chem. 278, 4035–4040. Grandjean, V., O’Neill, L., Sado, T., Turner, B., and Ferguson-Smith, A. (2001). Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Ig2-H19 domain. FEBS Lett. 488(3), 165–169. Greally, J. (2002). Short interspersed transposable elements (SINEs) are excluded from imprinted regions in the human genome. Proc. Natl. Acad. Sci. USA 99(1), 327–332. Hao, Y., Crenshaw, T., Moulton, T., Newcombe, E., and Tycko, B. (1993). Tumour-supressor activity of H19 RNA. Nature 365(6448), 764–767. Hitchins, M. P., and Moore, G. E. (2002). Genomic imprinting in fetal growth and development. Exp. Rev. Mol. Med. http://www.expertreviews.org/0200457Xh.htm. Horike, S., Mitsuya, K., Meguro, M., Kotobuki, N., Kashiwagi, A., Notsu, T., Schulz, T. C., Shirayoshi, Y., and Oshimura, M. (2000). Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in BeckwithWiedemann syndrome. Hum. Mol. Genet. 9(14), 2075–2083. Horsthemke, B., Dittrich, B., and Buitung, K. (1997). Imprinting mutations on human chromosome 15. Hum. Mutat. 10(5), 329–337. Jiang, Y., Tsai, T., Bressler, J., and Beaudet, A. (1998). Imprinting in Angelman and PraderWilli syndromes. Curr. Opin. Genet. Dev. 8(3), 334–342. Kono, T., Sotomaru, Y., Katsuzawa, Y., and Dandolo, L. (2002). Mouse parthenogenetic embryos with monoallelic H19 expression can develop to day 17.5 of gestation. Dev. Biol. 243(2), 294–300. Laborda, J. (2000). The role of the epidermal growth factor-like protein, Dlk1, in cell differentiation. Histol. Histopathol. 15(1), 119–129. Lee, M. P., DeBaun, M. R., Mitsuya, K., Galonek, H. L., Brandenburg, S., Oshimura, M., and Feinberg, A. P. (1999). Loss of imprinting of a paternally expressed transcript, with antisense orientation to KVLQT1, occurs frequently in Beckwith-Wiedemann syndrome and is independent of insulin-like growth factor II imprinting. Proc. Natl. Acad. Sci. USA 96(9), 5203–5208. Li, E. (2002). Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3(9), 662–673.
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Li, E., Beard, C., and Jaenisch, R. (1993). Role for DNA methylation in genomic imprinting. Nature 366(6453), 362–365. Li, M., Squire, J., Shuman, C., Fei, Y., Atkin, J., Pauli, R., Smith, A., Nishikawa, J., Chitayat, D., and Weksberg, R (2001). Imprinting status of 11p15 genes in Beckwith-Wiedemann syndrome patients with CDKN1C mutations. Genomics 74(3), 370–376. Lin, S.-P., Youngson, N., Takada, S., Seitz, H., Reik, W., Paulsen, M., Cavaille, J., and Ferguson-Smith, A. (2003). Asymmetric regulation of imprinting on the maternal and paternal chromosomes at the Dlk1-Gtl2 imprinted cluster on mouse chromosome 12. Nat. Genet. 35(1), 97–102. Lyon, M. (2000). LINE-1 elements and X chromosome inactivation: A function for ‘‘junk’’ DNA? Proc. Natl. Acad. Sci. USA 97(12), 6248–6249. Martienssen, R. (1998). Transposons, DNA methylation and gene control. Trends Genet. 14(7), 263–264. Monk, M., Boubelik, M., and Lehnert, S. (1987). Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development 99(3), 371–382. Okamura, K., Hagiwara-Takeuchi, Y., Li, T., Vu, T. H., Hirai, M., Hattori, M., Sakaki, Y., Hoffman, A. R., and Ito, T. (2000). Comparative genome analysis of the mouse imprinted gene impact and its nonimprinted human homolog IMPACT: Toward the structural basis for species-specific imprinting. Genome Res. 10(12), 1878–1889. Ono, R., Kobayashi, S., Wagatsuma, H., Aisaka, K., Kohda, T., Kaneko-Ishino, T., and Ishino, F. (2001). A retrotransposon-derived gene, PEG10, is a novel imprinted gene located on human chromosome 7q21. Genomics 73(2), 232–237. Paulsen, M., and Ferguson-Smith, A. (2001). DNA methylation in genomic imprinting, development and disease. J. Pathol. 195(1), 97–110. Paulsen, M., Takada, S., Youngson, N., Benchaib, M., Charlier, C., Segers, K., Georges, M., and Ferguson-Smith, A (2001). Detailed sequence analysis of the imprinted Dlk1-Gtl2 locus in three mammalian species reveals highly conserved genomic elements and refines a comparative map with the Igf2-H19 region. Genome Res. 11(12), 2085–2094. Perk, J., Makedonski, K., Lande, L., Cedar, H., Razin, A., and Shemer, R. (2002). The imprinting mechanism of the Prader-Willi/Angelman regional control center. EMBO J. 21(21), 5807–5814. Plass, C., Shibata, H., Kalcheva, I., Mullins, L., Kotelevtseva, N., Mullins, J., Kato, R., Sasaki, H., Hirotsune, S., Okazaki, Y., Held, W., Hayashizaki, Y., and Chapman, V. M. (1996). Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M. Nat. Genet. 14(1), 106–109. Reik, W., Dean, W., and Walter, J. (2001). Epigenetic reprogramming in mammalian development. Science 293(5532), 1089–1093. Schmidt, J., Levorse, J., and Tilghman, S. M. (1999). Enhancer competition between H19 and Igf2 does not mediate their imprinting. Proc. Natl. Acad. Sci. USA 96(17), 9733–9738. Schmidt, J., Matteson, P., Jones, B., Guan, X., and Tilghman, S. M. (2000). The Dlk1 and Gtl2 genes are linked and reciprocally imprinted. Genes Dev. 14(16), 1997–2002. Seitz, H., Youngson, N., Lin, S.-P., Dalbert, S., Paulsen, M., Bachellerie, J.-P., FergusonSmith, A. C., and Cavaille, J. (2003). Imprinted microRNAs transcribed antisense to a reciprocally imprinted retrotransposon-like gene. Nat. Genet. 34(3), 261–262. Sleutels, F., Zwart, R., and Barlow, D. (2002). The non-coding Air RNA is required for silencing autosomal imprinted genes. Nature 415(6873), 810–813. Stoger, R., Kubicka, P., Liu, C. G., Kafri, T., Razin, A., Cedar, H., and Barlow, D. (1993). Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell 73(1), 61–71.
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Takada, S., Tevendale, M., Baker, J., Georgiades, P., Campbell, E., Freeman, T., Johnson, M. H., Paulsen, M., and Ferguson-Smith, A. (2000). Delta-like and Gtl2 are reciprocally expressed, differentially methylated, linked imprinted genes on mouse chromosome 12. Curr. Biol. 10(18), 1135–1138. Tsai, C.-E., Lin, S.-P., Ito, M., Takagi, N., Takada, S., and Ferguson-Smith, A. (2002). Genomic imprinting contributes to thyroid hormone metabolism in the mouse embryo. Curr. Biol. 12, 1221–1226. Verona, R., Mann, M., and Bartolomei, M. (2003). Genomic imprinting: Intricacies of epigenetic regulation in clusters. Annu. Rev. Cell Dev. Biol. 19, 237–259. Warnecke, P., Mann, J., Frommer, M., and Clark, S. (1998). Bisulfite sequencing in preimplantation embryos: DNA methylation profile of the upstream region of the mouse imprinted H19 gene. Genomics 51(2), 182–190. Whitelaw, E., and Martin, D. (2001). Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nat. Genet. 27(4), 361–365. Wutz, A., Smrzka, O., Schweifer, S., Schellander, K., Wagner, E., and Barlow, D. (1997). Imprinted expression of the lgf2r gene depends on an intronic CpG island. Nature 389(6652), 745–749. Yoder, J., Walsh, C., and Bestor, T. (1997). Cytosine methylation and the ecology of intragenomic parasites. Trends Genet. 13(8), 335–340. Yoon, B. J., Herman, H., Sikora, A., Smith, L. T., Plass, C., and Soloway, P. D. (2002). Regulation of DNA methylation of Rasgrf1. Nat. Genet. 30(1), 92–96. Zwart, R., Sleutels, F., Wutz, A., Schinkel, A. H., and Barlow, D. P. (2001). Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes. Genes Dev. 15(18), 2361–2366.
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Molecular Basis for the Chloride Channel Activity of Cystic Fibrosis Transmembrane Conductance Regulator and the Consequences of Disease-Causing Mutations Jackie F. Kidd, Ilana Kogan, and Christine E. Bear Programme in Structural Biology and Biochemistry Research Institute, Hospital for Sick Children Departments of Physiology and Biochemistry University of Toronto, Toronto Canada M5G 1X8
I. Introduction II. Role of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function in Tissue Physiology and Pathophysiology A. Respiratory Tract B. Pancreatic Duct C. Gastrointestinal Tract D. Sweat Duct III. Cell Biology of CFTR and the Major CF Mutant (F508) IV. Quaternary Structure of CFTR V. The Permeation Pore of CFTR A. Chloride Ion Permeation B. Interaction of Other Physiologically Significant Anions with the Pore VI. Molecular Basis for Regulation by Phosphorylation VII. Molecular Basis for Regulation by Nucleotides A. Biochemical Studies of the Relative Roles of NBD1 and NBD2 of CFTR in Nucleotide Binding and Hydrolysis B. Structural Studies of Prokaryotic ABC Transporters and Their Relationship to CFTR C. Electrophysiological Studies of the Relative Roles of NBD1 and NBD2 in Channel Gating D. Insight Regarding Structural Interaction between Nucleotide-Binding Domains and the Pore-Forming Domain of CFTR Provided by Structural Studies of Intact Prokaryotic ABC Proteins VIII. Future Directions Acknowledgments References
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I. Introduction Cystic fibrosis (CF) is a lethal disease caused by mutations in the gene cystic fibrosis transmembrane conductance regulator (CFTR) gene (Kerem et al., 1990). CF aVects multiple organs, including the respiratory tract, sweat ducts, pancreatic ductules, the gastrointestinal tract, and reproductive organs (Frizzell, 1999; Welsh and Fick, 1987). Typically, the airways are aVected the most severely, becoming obstructed with mucus, with patients succumbing eventually to recurrent bacterial infection (Pilewski and Frizzell, 1999). The secretion of pancreatic and salivary enzymes, as well as intestinal transit through the gastrointestinal tract, is impaired due to mucus plugging (Durie, 2000). These hallmark clinical features of cystic fibrosis are thought to develop as a result of fundamental changes in epithelial electrolyte and water transport. We now know that the protein product of the CF gene, CFTR, is an anion channel localized to the apical surface of epithelia and that anion flux through CFTR is essential for maintaining proper hydration and viscosity of the fluid lining the airways, gastrointestinal tract, ducts of the pancreas, sweat glands, and the male reproductive system. CFTR is a multidomain membrane protein possessing an internal duplication similar to other related members of the ATP-binding cassette (ABC) superfamily of transport proteins (Higgins, 1995, 2001; Higgins and Linton, 2001; Riordan et al., 1989). The CFTR protein is composed of two halves, each possessing a membrane-spanning domain (MSD) composed of several helical membrane segments (probably six) with intervening extracellular and intracellular loops followed by a nucleotide-binding domain (NBD) (Fig. 1). In the first half of the molecule, the putative nucleotide-binding fold (NBD1) is followed by a large cytoplasmic domain called the ‘‘R’’ domain, which contains multiple serine residues that are phosphorylated by protein kinase A and protein kinase C to regulate CFTR channel function (Chang et al., 1993; Chappe et al., 2003; Mathews et al., 1998a; Seibert et al., 1995, 1999). The ‘‘R’’ domain connects the first and the second half of the molecule, with the second half of the molecule composed of the second membrane-spanning domain (MSD2) and the second NBD (NBD2). Since cloning of the gene in 1989 (Riordan et al., 1989), researchers studying CFTR have been engaged in active debate regarding its function and the regulation of this function. For the most part, other family members of the ABC superfamily of membrane proteins function as ATP-dependent transporters that mediate the unidirectional translocation of diverse substrates. Therefore, acceptance of the idea that CFTR functions as a regulated chloride ion channel, which mediates the electrodiVusion of anions, required several years of rigorous experimentation, including its expression
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Figure 1 The multidomain structure of CFTR, the two membrane-spanning domains (MSD1 and MSD2), the two nucleotide-binding domains (NBD1 and NBD2), and the relatively flexible ‘‘R’’ domain are shown. (See Color Insert.)
in multiple, distinct expression systems and eventually its purification and functional reconstitution (Anderson et al., 1991; Bear et al., 1992; Kartner et al., 1991; Rommens et al., 1991). CFTR remains the only known member of the ABC superfamily of membrane proteins that functions as an anion channel. Definition of the molecular mechanisms underlying channel activity by CFTR has been and continues to be challenging. There is no significant homology between the putative membrane-spanning domains of CFTR and those of other chloride channels. Therefore, we lack guideposts with which to model the molecular basis for anion conduction through CFTR. Furthermore, regulation of the channel function of CFTR exhibits several intriguing and novel properties. For example, the channel activity by CFTR exhibits obligatory regulation by phosphorylation of its large cytosolic regulatory ‘‘R’’ domain, a structure that is unique to CFTR in the ABC family (Seibert et al., 1997). The ‘‘R’’ domain is phosphorylated at multiple sites (Chang et al., 1993) and is thought to mediate complex interactions with the rest of the molecule (Ostedgaard et al., 2000; Winter and Welsh, 1997). Therefore, defining the molecular detail underlying its role in channel function is challenging. Further, CFTR channel activity is also regulated by nucleotide binding and hydrolysis (Anderson et al., 1991; Vergani et al., 2003). While this property is consistent with its membership in the ABC superfamily, intrinsic ATP hydrolysis has not been linked to the gating mechanism for any other channel to date. Therefore, investigations of the structure–function relationships of CFTR are exciting because they lead researchers into uncharted areas. Finally, such studies are also important for our understanding of the molecular basis for CF disease caused by mutations throughout the gene.
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The following sections summarize current understanding of the physiological role of CFTR as an anion channel and the pathogenesis of disease in certain CF-aVected epithelia. We then discuss current findings relating to the mechanisms underlying the anion channel function of this distinctive and highly provocative molecule as well as the basis for disease in mutant CFTR proteins.
II. Role of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function in Tissue Physiology and Pathophysiology As a regulated chloride channel, CFTR plays a major, tissue-dependent role in fluid transport. It is now well recognized that improper regulation of CFTR function in CF patients severely aVects the function of many organs, resulting in pulmonary abnormalities, pancreatic insuYciency, and gastrointestinal abnormalities, as well as elevated sweat electrolytes. Due to the phenotypic variability and large number of cellular defects observed in CF patients, it has been proposed that disease-causing mutations may not only aVect chloride conductance through CFTR. For example, studies have shown that CFTR mediates the conductance of other anions of physiological significance, including bicarbonate ion (implicated in pH regulation of pancreatic ducts) (Ahn et al., 2001; Choi et al., 2001) and glutathione (implicated in the regulation of oxidative stress in airways) (Gao et al., 1999; Hallows et al., 2000; Hudson, 2001; Kogan et al., 2003; Linsdell and Hanrahan, 1998; Velsor et al., 2001). In addition to its function as an anion channel, several studies have suggested that CFTR also contributes to epithelial transport function by regulating the function of neighboring membrane transporters and channels. For example, compelling evidence supports a role for CFTR in regulation of the amiloride-sensitive sodium channel (ENaC) in both airway epithelial cells and sweat duct cells (Reddy et al., 1999; Stutts et al., 1997). Furthermore, a functional interaction between CFTR and the chloride/bicarbonate exchanger on the apical surface of pancreatic ductular epithelia has been described and is thought to be important for proper regulation of the pH of ductular secretions (Ahn et al., 2001; Choi et al., 2001; Ko et al., 2002). A. Respiratory Tract CF-related airway disease is the major cause of mortality, characterized by mucus obstruction in the large airways, persistent pulmonary infections, intense neutrophil-dominated airway inflammation, and bronchiecstasis. Chronic infection of CF airways by gram-negative Pseudomonas aeruginosa is the major cause of morbidity and mortality among CF patients (Gibson
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et al., 2003; Pier, 2002; Rajan and Saiman, 2002). Canadian and U.S. CF data registries have shown that Burkholderia cepacia is also an independent predictor of mortality in CF (Gibson et al., 2003). An exaggerated inflammatory response to infection by these pathogens plays a major role in the progression of disease (Berger, 2002; Chmiel et al., 2002; Puchelle, 2002). These hallmark clinical features of cystic fibrosis are thought to develop as a result of fundamental changes in the transport and barrier functions of airway epithelial tissues. A number of hypotheses have been proposed to link the basic defect in CFTR protein to the deregulated host defense observed in the airways of CF patients. These hypotheses evoke the role of CFTR in ion transport and modification of mucus fluidity (Boucher, 2002; Donaldson and Boucher, 2003), the regulation of inflammatory cytokine production (DiMango et al., 1998), and its potential role in the modification of cell surface receptors for bacterial binding (Zar et al., 1995).
B. Pancreatic Duct The pancreatic function of CF patients seems to be consistently aVected. Among the various clinical symptoms associated with CF, pancreatic function is the only one that can be relatively well predicted from the CFTR genotype (Kristidis et al., 1992). CF patients can be classified as ‘‘pancreatic suYcient’’ (PS) or ‘‘pancreatic insuYcient’’ (PI), depending on the ability of their pancreas to produce and secrete digestive enzymes. There is general agreement that PI is the major cause of growth failure and nutritional deficiencies that occur in CF children. In general, patients who are PI are homozygous or compound heterozygous for severe CFTR mutations. However, patients with a PS phenotype have one or two mild CFTR mutations (Kristidis et al., 1992). Although the progressive loss of pancreatic function in CF patients is highly variable, its course is gradual. The absence of functional CFTR on the luminal membrane of the duct cells interferes with the Cl , HCO3 , and water secretion of the cells, resulting in the limited flow of protein-rich pancreatic juice (Kopelman et al., 1985). Obstruction of the small pancreatic ducts by viscous calcium-rich secretions and cellular debris is an early feature of pancreatic malfunction, which is later followed by dilation of the secretory duct lumen, causing intra- and interlobular fibrosis. With time, acinar secretions are blocked and damaged acinar cells are replaced with small cysts, fibrous tissue, and fat. Late in the course of CF, due to tissue scarring, the function of the islets of Langerhans also becomes diminished. Consequently, the impaired production of insulin and glucagon leads to the development of insulin-dependent diabetes (Kopito and Shwachman, 1976; Lebenthal, 1992; Quinton, 1999).
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C. Gastrointestinal Tract Although symptoms related to the gastrointestinal (GI) tract are very common among CF patients, they are usually not life-threatening if treated properly (Welsh and Smith, 1995). The intestinal tract of patients usually displays little change. However, Brunner’s glands of the duodenum are hypertrophied, displaying dilated ducts and acinar lumens that are filled with mucus. Ten to 20% of CF infants exhibit symptoms of meconium ileus. In these children, the small intestine is blocked with a thick, dehydrated, rubbery, and tenacious mucoid plug. The precise cause of the increased viscosity of this meconium is currently unknown. However, a failure in pancreatic enzyme secretion in utero, as well as decreased water content, is thought to give rise to the accumulation of undigested proteins in the intestine. Mixing of these proteins with intestinal mucus is believed to contribute to the production of an impervious, hyperviscid meconium substance (Quinton, 1999). Small bowel obstruction may also occur in CF patients in later stages during development, usually above the age of 15 (Andersen et al., 1990). GI involvement in CF, together with pancreatic insuYciency, results in nutrient malabsorption and predisposes patients to malnutrition (Lebenthal, 1992).
D. Sweat Duct Sweat secretion has proven over the years to be the most useful criterion for diVerentiating CF patients from non-CF individuals. Analysis of the electrolyte composition in sweat, particularly the concentration of Cl , has become an internationally accepted protocol for CF diagnosis. Abnormally high Cl levels in sweat, i.e., greater than 60 mEq/liter, are often observed in CF patients (Gibson and Cooke, 1959; LeGrys, 1996). Further, Naþ and Kþ concentrations are also increased in CF patients’ sweat. It is currently known that a defect in Cl transport, down its electrochemical gradient across both apical and basolateral duct membranes, causes a defect in Cl reabsorption along the sweat gland duct. As a result, following extensive sweating, children with CF often ‘‘taste salty’’ or may display salty crystals on their skin (Welsh and Smith, 1995).
III. Cell Biology of CFTR and the Major CF Mutant (F508) The cell biology of CFTR has garnered significant attention among CF researchers, as the maturation and traYcking of CFTR through the endocytic pathway are aVected in the major CF-causing mutant: CFTRF508. Several laboratories have shown that misfolding due to the F508 mutation causes its relative retention in the ER and subsequent delivery to the
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proteosomal degradation pathway (Cheng et al., 1990; Denning et al., 1992; Kartner et al., 1992; Meacham et al., 2001; Ward et al., 1995; Zhang et al., 1998). Therefore, the misfolding of CFTRF508 results in its negligible expression at the cell surface where its function as a regulated channel is required. Various experimental manipulations (lower temperature, phenylbutyrate, and chemical chaperones) can partially correct this defect in maturation of the mutant, leading to its surface expression and the appearance of regulated channel function, albeit with somewhat impaired phosphorylation-dependent gating properties (Brown et al., 1997; Cheng et al., 1995; Hwang et al., 1997). Therefore, rescue of this processing defect in CFTRF508 has the potential to eVectively treat disease in patients bearing this mutation. Importantly, many other disease-causing mutations scattered throughout the CFTR gene result in temperature-dependent misfolding, supporting the idea that these additional disease-causing mutants can also be rescued through similar strategies. Presently, it is unclear why CFTRF508 and so many other CF-related mutations result in misfolding of CFTR. However, clues are evident in the analysis of related proteins. As mentioned previously, CFTR is a member of the ABC family of membrane proteins. Like other mammalian members of the ABC superfamily, CFTR is a multidomain membrane protein bearing membrane domains and complex cytoplasmic domains. Mutagenesis studies conducted in the KATP channel, composed of the ABC protein SUR1 and the potassium channel Kir6.2, revealed that correct assembly of this multidomain, bimolecular complex was required for exit from the endoplasmic reticulum (Zerangue et al., 1999). Similarly, appropriate interaction among domains within CFTR and its assembly as a functional complex may precede its exit from the endoplasmic reticulum. It has been shown that CFTRF508 disrupts dimerization between isolated nucleotide-binding domains of CFTR, supporting the idea that correct intramolecular interaction among the domains of CFTR precedes its exit from the endoplasmic reticulum (DeCarvalho et al., 2002; deCarvalho et al., 2002). Rees and colleagues hypothesized, on the basis of their alignment of the sequence of CFTR with the crystal structure solved for the ABC transporter protein BtuCD, that F508 would reside at the interface between the nucleotide-binding domain of CFTR and the membrane domain (Locher et al., 2002). Furthermore, residues in one of the two putative membrane domains forming an interface with the nucleotide-binding domains constitute a hot spot for diseasecausing mutations. Therefore, structural data combined with genetic analyses in yeast suggest that the susceptibility of CFTR to misfolding may be associated with the complexity of intramolecular, domain–domain interactions occurring during its biosynthesis and folding. Compounding this defect in the delivery of CFTRF508 to the cell surface, the stability of CFTRF508 at the cell surface is also impaired.
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Even after rescue of CFTRF508 by means of one or all of the experimental maneuvers described earlier, it exhibits a shorter residence time at the cell surface (Lukacs et al., 1993). This specific defect in cell surface stability is also exhibited by certain disease-causing mutations, resulting in truncation of the carboxy terminus (Benharouga et al., 2001; Ostedgaard et al., 2003). Current experimentation is directed toward understanding the mechanisms underlying this decreased stability of CFTRF508 (and possibly other processing mutants) and carboxy terminus truncation mutants. It is not yet clear whether an increase in the rate of mutant CFTR internalization and/or a decrease in its rate of redelivery, via recycling endosomes to the cell surface, underlies this cellular phenotype. Clearly, correction of this defect will necessarily complement eVorts to overcome the biosynthetic defect inherent in certain CFTR mutants. Although the mechanism underlying the decreased cell surface stability of carboxy terminus truncation mutants of CFTR is not yet understood, it may relate to the disruption of interactions mediated by the PDZ-binding motif on the extreme terminus. The PDZ-binding motif on CFTR has been shown to mediate its interaction with multiple proteins, including CAL, NHERF, or ezrin-radixin-moesin-binding phosphoprotein (EBP50) (Ahn et al., 2001; Moyer et al., 2000; Raghuram et al., 2001; Sun et al., 2000; Wang et al., 2000). Interaction with NHERF has been shown to provide a scaVold for a macromolecular complex (Chang et al., 2002; Naren and Kirk, 2000; Naren et al., 2003). Through formation of this complex, CFTR can be regulated more tightly by protein kinase A, which phosphorylates CFTR, thereby regulating its channel activity. In addition to its role in CFTR channel function, phosphorylation has also been shown to enhance the cell surface stability of CFTR (Lukacs et al., 1997). Therefore, future studies may be directed toward determining whether the decreased cell expression of C terminus mutants can be corrected by pharmacological agents that enhance their phosphorylation status, i.e., phosphatase inhibitors.
IV. Quaternary Structure of CFTR Biochemical studies suggest that the regulated channel activity of CFTR can be mediated by the monomeric form of the protein (Chen et al., 2002; Ramjeesingh et al., 2003). Although purified CFTR exists in monomeric and dimeric structures following reconstitution into phospholipid liposomes, stable monomers could be purified following fractionation and were found to be capable of mediating chloride electrodiVusion. Furthermore, detailed single channel analysis revealed that purified, monomeric CFTR can mediate regulated chloride channel and ATPase activity (a function discussed in depth in subsequent paragraphs) (Ramjeesingh et al., 2003). Riordan and
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colleagues (1989) confirmed these findings using an alternative approach wherein concatamers composed of wild-type and pore-defective mutants were linked covalently, expressed, and then studied by patch-clamp electrophysiology. As expected for a functional monomer, each protein in the concatamer conferred a unique conductance pore, with normal or altered single channel conductance, respectively, rather than a hybrid conductance (Chen et al., 2002). The suggestion that CFTR is fully functional as a monomer is consistent with results from structural and biochemical studies of related members of the ABC transporter superfamily. As discussed in detail in a later section of this text, X-ray crystal structures of prokaryotic ABC proteins, including the lipid A transporter MsbA (Chang, 2003; Chang and Roth, 2001) and the vitamin B12 transporter BtuCD (Locher et al., 2002), reveal multisubunit proteins composed of two membrane-spanning domains and two nucleotidebinding domains. These essential components of the functional proteins are contained within a single CFTR polypeptide. Similarly, low-resolution structures generated of the mammalian eZux pump P-glycoprotein by electron crystallography (Rosenberg et al., 2001) or by electron microscopy and image analysis of detergent-solubilized protein revealed that it is a monomer (Lee et al., 2002). Furthermore, the monomeric structure undergoes conformational changes upon addition of the activating nucleotide, supporting the idea that this is the minimal functional form of the protein (Lee et al., 2002; Rosenberg et al., 2001). Interestingly, studies of purified CFTR, reconstituted into phospholipid liposomes and CFTR expressed in mammalian cells, suggest that this protein self-associates (Ramjeesingh et al., 2001). Purified CFTR associates to form functional dimers, with each protein apparently functioning independently. However, biochemical studies of the quaternary structure of CFTR in biological membranes have yielded mixed results, possibly reflecting the diVerential structure of CFTR in discrete subcellular compartments (Chen et al., 2002; Marshall et al., 1994; Ramjeesingh et al., 2003). DiVerentially tagged versions of CFTR fail to show coimmunoprecipitation from whole cell homogenates in a variety of detergents, arguing that in studies representative of the total cellular pool of CFTR, there is no detectable association between CFTR molecules (Chen et al., 2002; Marshall et al., 1994). Gel filtration studies of detergent-solubilized CFTR derived from whole cell homogenates showed that CFTR migrated primarily as a monomer, consistent with previous coimmunoprecipitation studies (Ramjeesingh et al., 2003). However, analysis of CFTR solubilized from samples enriched in plasma membranes revealed that CFTR-containing complexes migrated as dimers. Furthermore, CFTR-containing complexes, the size of dimers, can be crosslinked at the cell surface using cross-linkers with short spacer arms of ˚ , consistent with the close contact of these proteins. approximately 11 A Together, these data suggest that CFTR expressed at the cell surface may
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exist as a dimeric complex. A similar scenario is envisioned for many membrane proteins and has received considerable support for rhodopsin-like G-protein-coupled receptors (George et al., 2002; Lee et al., 2003). Further studies are required to determine the stage in its biosynthesis in which CFTR forms a dimeric complex and the structural basis for dimer formation. It is not known whether CFTR molecules interact via their cytosolic domains and/or their membrane domains. Interestingly, the glycophorin-like dimerization motifs (GXXXG) (Russ and Engelman, 2000) exist in CFTR, in the predicted membrane-spanning helices 8 and 11 and in the second nucleotide binding domain. Furthermore, the consequences of dimer formation remain unresolved. Previous single channel studies of purified, reconstituted dimers suggest that both proteins may function as independently gated channels (Ramjeesingh et al., 2001). However, the functional significance of the dimeric structure in the context of the plasma membrane in epithelial cells remains to be determined. Hypothetically, a distinct structural motif relevant for its activation by certain signaling molecules may be presented only in the context of the dimeric complex. In fact, cooperative channel gating by multiple CFTR molecules has been observed consistently in patch-clamp studies on epithelial membranes (Hanrahan et al., 1995; Krouse and Wine, 2001). In addition, CFTR dimerization may have an impact on its traYcking to and residence at the cell surface. Currently, there is considerable interest in assessing whether the misfolding of mutant CFTR proteins, such as CFTRF508, could be rescued in the endoplasmic reticulum through association with the wild-type protein. Thus, studies by Chen and colleagues (2002) suggest that if heterodimers can form between CFTR and CFTRF508, there appears to be no impact on maturation and processing of either protein. However, the impact of such an association on the cell surface stability of either protein remains to be determined. Finally, as innovative and sensitive methods for studying membrane protein oligomerization in living cells (i.e., bioluminescence resonance energy transfer, fluorescence resonance energy transfer, and yeast-based genetic screens for membrane protein assembly) continue to be developed, we will acquire greater insight and resolution of the molecular basis for and the potential relevance of dimeric CFTR structures.
V. The Permeation Pore of CFTR A. Chloride Ion Permeation The chloride-selective permeation path through the membrane-spanning domains of CFTR has been examined extensively in electrophysiological studies (Akabas, 2002; Cheung and Akabas, 1997; Dawson et al., 1999;
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Linsdell et al., 2000; Tabcharani et al., 1997). Whole cell and single channel patch-clamp studies reveal that the phosphorylation and nucleotideregulated CFTR conductance path is nonrectifying in the presence of symmetrical chloride concentrations and exhibits the following relative anion conductances: Cl > NO3 >Br >F >>I >>SCN (Linsdell et al., 2000). The single channel conductance for the chloride ion at 150 mM symmetrical chloride ion concentrations is fairly low, approximately 8–10 pS. There are several excellent reviews of the pharmacological agents eVective in modifying CFTR function (Hwang and Sheppard, 1999; Sheppard and Welsh, 1999). Chloride conduction through CFTR is blocked by three classes of compounds, including disulfonic stilbenes, arylaminobenzoates, and sulfonylurea compounds, such as glybenclamide (Hwang et al., 1999; Linsdell, 2001). Blockage by 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid only occurs when the drug is applied to the cytoplasmic side of the membrane (Linsdell and Hanrahan, 1996). However, it has been acknowledged in the field that the specificity of these compounds for CFTR is relatively poor and that non-CFTR channels can also be inhibited by certain compounds, including the sulfonylurea compound glybenclamide (Gyomorey et al., 2000, 2001). Furthermore, the molecular basis of action of these pharmacological agents remains to be determined. Interestingly, certain compounds, i.e., genistein, can exert both activating and inhibitory eVects, depending on the dose applied, implicating the role of multiple sites of action on the CFTR molecule (Lansdell et al., 2000). The inhibitory binding site for genistein appears to be abolished in the disease-causing mutation CFTRG551D, leaving its activating binding site intact (Bulteau-Pignoux et al., 2002; Derand et al., 2002; Zegarra-Moran et al., 2002). Therefore, there is a clear clinical rationale for assessing the functional consequences of structural modifications of these lead compounds with the goal of optimizing their therapeutic eYcacy and specificity. High throughput methods have been employed eVectively in the functional screening of such derivatives and in the identification of other potential novel therapeutics (Ma et al., 2002). The structural basis for chloride conduction through CFTR remains incompletely understood. One would predict, on the basis of the structures of procaryotic ABC proteins (Chang, 2003; Chang and Roth, 2001; Locher et al., 2002), that the translocation path would require both MSDs (MSD1 and MSD2) of CFTR (each composed of six putative helical segments). There may, however, be significant redundancy in the chloride ion-binding sites within the membrane domains. For example, a construct coding for the first half of CFTR (i.e., MSD1-NBD1) alone was found to dimerize to confer an anion-selective path (Sheppard et al., 1994). Therefore, dimerization of MSD1 appeared to reconstitute the pore normally formed by the interaction between MSD1 and MSD2. The sixth putative transmembrane (TM6) helix within MSD1 has been studied most extensively for its role in
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chloride conduction, either through mutagenesis and/or through monitoring the accessibility of substituted cysteine residues along its length using sulfhydryl reagents (Akabas, 2002; Cheung and Akabas, 1996; Dawson et al., 1999; Gupta et al., 2001; Linsdell, 2000, 2001a,b; Linsdell et al., 2000; Smith et al., 1999; Tabcharani et al., 1997). Multiple residues appear to contribute to anion binding in TM6, including residues [335–344] (Linsdell et al., 2000). Studies by Gong and Linsdell (2003) and by Smith and colleagues (2001) highlight the potential importance of the positively charged residues— arginine at position 334 and lysine at position 335—in forming an external vestibule that attracts anions toward the selectivity filter. The selectivity filter appears to be confined to a narrower segment [comprising residues F337 to S341 (Gong and Linsdell, 2003)]. To date, it is not clear which other putative transmembrane helices in MSD1 contribute to formation of the chloride conductance path. The role for specific helices or residues in the second membrane-spanning domain (MSD2) in anion conduction is somewhat less clear. Ramjeesingh et al. (2003) examined the biophysical properties of MSD2 (residues 857–1158) purified and reconstituted into phospholipid bilayers. As for the experiments performed by Sheppard et al. (1994) with the first half of CFTR, it was found that MSD2 can dimerize to reconstitute a chloride-selective pore. However, dimerization of MSD2 may be less eVective than MSD1 as its expression led to the appearance of chloride channel activity in some, but not all, expression systems (Chan et al., 2000; Devidas et al., 1998; Ramjeesingh et al., 2003; Sheppard et al., 1994). Certain site-directed mutagenesis studies also support a role for MSD2 in channel pore formation. Gupta et al. (2001) reported that mutations in TM12 (T1134, M1137, S1141) altered anion binding within the pore, although they had relatively minor eVects on the anion selectivity and unitary conductance of the channel. McCarty (2000) also suggested that T1134 (in TM12) plays an indirect role in determining selectivity. Finally, as for MSD1, mutagenesis studies of MSD2 have been limited to one or two of six potential helical segments. Therefore, future studies must employ alternative approaches to assess the contribution of other regions of both membrane-spanning domains in channel pore formation.
B. Interaction of Other Physiologically Significant Anions with the Pore Studies have highlighted the potential significance of the capacity of CFTR to mediate the flux of other physiologically significant anions in addition to chloride ion, including bicarbonate ion and glutathione. Impaired bicarbonate secretion into the lumen of certain epithelial tissues has been linked to
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pathogenesis in CF as bicarbonate ion is known to play a key role in maintaining the solubility of macromolecules, including pancreatic enzymes, and in aVecting mucus rheology (Durie, 1989; Joo et al., 2001; Kopelman et al., 1989; Quinton, 2001; Wine, 2003). Therefore, understanding the molecular basis for bicarbonate ion transport in aVected epithelia is vital for our understanding of disease pathogenesis. As revealed in patch-clamp studies, CFTR can mediate the conduction of bicarbonate ion across the apical membrane of CFTR-transfected cells, although the relative permeability of bicarbonate ion is significantly less than that of chloride ion (PHCO3 /PCl 0.1– 0.3) (Gray et al., 2001; Illek et al., 1997; Linsdell et al., 1997; Tabcharani et al., 1997). There has been debate about whether the relative permeability of CFTR for bicarbonate may be too low to account for the net bicarbonate secretion across the apical surface of epithelia. Data have been presented arguing in favor of a CFTR-coupled mechanism wherein CFTR functions to activate the electrogenic Cl :HCO3 exchanger (SLC26) and this coupled mechanism mediates net bicarbonate flux across the apical membrane of pancreatic ductular epithelia (Ko et al., 2002). However, in other studies, the apical secretion of bicarbonate ion across pancreatic, sweat duct, and airway epithelia has been shown to be mediated predominantly by CFTR (Bridges, 2002; Illek et al., 1997; Ishiguro et al., 2002; Reddy and Quinton, 2003). Therefore, it remains to be resolved in which tissues CFTR mediates a direct and/or an indirect role in bicarbonate ion secretion. Reddy and Quinton (2003) reported that the chloride and bicarbonateconducting functions of CFTR can be regulated diVerentially by intracellular glutamate and ATP. Further, these authors suggested that due to their distinct regulatory properties, disease-causing mutations may exert distinguishable eVects on bicarbonate and chloride flux. For example, the CF mutation CFTRR117H retains near-normal bicarbonate conductance, despite a substantive reduction of chloride conductance. These authors argued that the relative ‘‘sparing’’ of the bicarbonate conductance by CFTR may account for the milder pancreatic phenotype associated with this mutation in patients. Similarly, conductance of another physiologically important anion, glutathione (GSH), is regulated diVerently than chloride conductance. Linsdell and Hanrahan (1998) were the first to show that glutathione could be conducted through membranes expressing CFTR, although its permeability relative to that of chloride ion is very low; PGSH/PCl 0.08. Furthermore, these authors reported that glutathione conductance exhibited a distinctive dependence on nucleotide interaction (to be discussed in more detail in a subsequent section of the text). Kogan et al. (2003) also determined that the nucleotide requirements for glutathione conductance through purified
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CFTR were diVerent than those required for chloride conductance. These findings complement those discussed earlier for bicarbonate ion and suggest that the structure of the conductance pore through CFTR may be malleable and dependent on the nucleotide occupancy of the cytosolic domains (nucleotide-binding domains). Like bicarbonate flux, glutathione flux is an important consideration in understanding normal tissue physiology. Glutathione is a major extracellular and intracellular antioxidant. Lack of an eZux path in the apical membrane of respiratory epithelia may be expected to alter the concentration of glutathione in the airway surface fluid and impact negatively on the antioxidant load in this tissue (Gao et al., 1999; Hudson, 2001; Kelly, 1999). Future experiments will address the molecular basis for the diVerential permeation properties of CFTR and the consequences of specific disease-causing mutations.
VI. Molecular Basis for Regulation by Phosphorylation It is well known that phosphorylation of the ‘‘R’’ domain of CFTR by PKA is absolutely required for the nucleotide-dependent channel activity of CFTR (Chang et al., 1993; Chappe et al., 2003; Mathews et al., 1998; Seibert et al., 1995, 1999; Tabcharani et al., 1991). There are 10 dibasic consensus sites for PKA phosphorylation on the ‘‘R’’ domain, and it has been proposed that upon phosphorylation, they act in a coordinated manner, as no single phosphoserine residue appears to be critical for function (Chang et al., 1993). However, the mechanisms underlying phosphorylation-dependent regulation remain unclear. Studies suggest that the functional ‘‘R’’ domain likely extends from a residue located somewhere between position 634–709 and residue 835 of CFTR (Chan et al., 2000; Winter and Welsh, 1997). Analyses by circular dichroism and nuclear magnetic resonance showed that this same peptide is largely unstructured, regardless of its phosphorylation status, prompting speculation regarding the mechanism by which the domain activates CFTR (Ostedgaard et al., 2000). These authors suggested that the ‘‘loose’’ structure of the ‘‘R’’ domain likely permits its interaction via its multiple phosphoserine residues with multiple regions of the protein. Deletion mutants of the ‘‘R’’ domain and the functional analysis of isolated peptides derived from the ‘‘R’’ domain reveal that there may be structures responsible for inhibition or stimulation within the ‘‘R’’ domain. Deletion of residues 760–783 led to constitutive, phosphorylation-independent activity of CFTR, suggesting that this region may have an inhibitory eVect on CFTR channel gating (Xie et al., 2002). However, studies by Winter and Welsh (1997) showed that addition of a PKA-phosphorylated ‘‘R’’ domain peptide, corresponding to residues 708–831, caused activation of
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the CFTR channel (missing this region), suggesting that this large peptide includes regions that can engage in stimulatory interactions after phosphorylation. As this large ‘‘R’’ domain peptide enhanced nucleotide-dependent gating to the channel open state, primarily by enhancing the nucleotide aYnity of the activity, it was suggested that it interacts with and modifies the NBDs. Indeed, in a study performed on recombinant NBD1-R and NBD2 of CFTR, it was suggested that phosphorylation controls the interaction between them (Howell et al., 2003). Currently, however, it remains unclear whether the ‘‘R’’ domain may also interact with other regions of the protein, i.e., membrane-spanning domains. As mentioned previously, the major mutant CFTRF508 is largely absent from the plasma membrane because it is misfolded during biosynthesis and degraded from the endoplasmic reticulum. CFTRF508 is capable of channel activity if this maturation defect is overcome by manipulations such as low temperature tissue culture conditions and the addition of phenylbutyrate (Brown et al., 1997; Denning et al., 1992; Zeitlin, 2000). However, the channel activity of the rescued mutant protein exhibits a specific defect in phosphorylation-dependent gating. The rate of activation following the addition of purified PKA plus MgATP (possibly reflecting incremental phosphorylation at multiple phosphorylation sites) was significantly slower for the mutant channels than observed for the wild-type protein (Wang et al., 2000). However, following maximal phosphorylationdependent activation, the channel open probability of both the wild type and the mutant channel was essentially identical, as were the kinetics of nucleotide-dependent gating. Interestingly, similar open probabilities of wild-type and mutant CFTR channels were determined following their expression in the insect cell line, a finding that may correlate with the relative phosphorylation status of CFTR molecules in this expression system (Li et al., 1993). As the rate of phosphorylation may be impaired in CFTRF508, it is tempting to speculate that the conformation of the ‘‘R’’ domain or the conformation of other regions of the protein (i.e., intracellular loops connecting the intramembrane helices and/or NBDs) with which the ‘‘R’’ domain interacts is altered in this mutant. As discussed later, a model of CFTR based on a comparison with BtuCD (a prokaryotic ABC superfamily member) places F508 on the surface of the nucleotide-binding domain where it interacts with a loop region extending from the membrane-spanning domain. Therefore, assuming that functional interactions between NBDs and MSDs are modified by phosphorylation to cause channel gating, CFTRF508 may exhibit altered gating because such phosphorylation-dependent modifications are impaired. The fidelity of this model requires verification with analysis of the impact of the F508 mutation on domain–domain interaction with the intact CFTR protein.
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VII. Molecular Basis for Regulation by Nucleotides As discussed previously, the chloride channel activity of phosphorylated CFTR is regulated by nucleotide interaction with its two nucleotide-binding domains, NBD1 and NBD2, and by ATP hydrolysis (Anderson et al., 1991; Bear et al., 1997; Gadsby et al., 1994; Hanrahan et al., 1996; Hunt, 2002; Ramjeesingh et al., 1999; Seibert et al., 1997; Sheppard and Welsh, 1999; Welsh and Anderson, 1993). To date, CFTR is the only ion channel known to possess measurable ATPase activity, and this property has galvanized ion channel physiologists to study the potential link between this enzymatic activity and CFTR channel gating. The following paragraphs discuss the biochemical and electrophysiological studies that contributed to current models describing the nucleotide-dependent channel function of CFTR. A. Biochemical Studies of the Relative Roles of NBD1 and NBD2 of CFTR in Nucleotide Binding and Hydrolysis ATP binding and hydrolysis by CFTR in membrane preparations have been studied using photoactivatable nucleotides or directly in the preparation of a purified protein reconstituted in phospholipid liposomes. In photolabeling studies, it was shown that CFTR can be labeled stably (for minutes) by [-32P]8-N3ATP, and this label was associated with the first half of the molecule, encompassing NBD1 (isolated following proteolysis and immunoprecipitation) (Szabo et al., 1999; Aleksandrov et al., 2001) or expressed as a half molecule (Basso et al., 2003). These findings suggest that ATP interacts stably with NBD1. ATPase activity by CFTR in transfected membranes has been assessed using [-32P]8-N3ATP in conjunction with the inorganic phosphate analogue orthovanadate (Vi). In other ATPases, Vi has been shown to bind tightly in the catalytic site in place of released phosphate to form a stable complex with the other hydrolysis product, e.g., ADP (Lindquist et al., 1973) or [-32P]8-N3ADP. In studies of CFTR, treatment with Vi induced trapping of [-32P]8-N3ADP in NBD2, not NBD1. Together, these findings support the idea that nucleotides bind to both NBDs, but NBD1 is the site of stable ATP binding and NBD2 is the primary site for ATP hydrolysis (Aleksandrov et al., 2001). Direct measurements of the ATPase activity of CFTR require its purification and reconstitution in phospholipid liposomes. While challenging experimentally, such direct assessments are essential for quantitation of the kinetic properties of ATPase activity by the intact protein. Li et al. (1996) found that CFTR is a slow ATPase, exhibiting a low catalytic rate of 0.2 ATP molecules hydrolyzed per second. This finding was reproduced by Aleksandrov et al. (2002). However, calculation of the actual turnover number requires an accurate assessment of the quantity of functionally reconstituted protein. On
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the basis of planar lipid bilayers, Bear et al. (1992) estimated that 10–20% of CFTR molecules were functionally reconstituted as a chloride channel. Therefore, assuming that only 10% of the CFTR protein is functionally reconstituted, the turnover rate of the hydrolytic activity of CFTR may be as high as one to two ATP molecules per second, a rate still considerably slower than other ATPases, including the related ABC protein P-glycoprotein (Romsicki and Sharom, 1998). Li et al. (1996) also found that, as in the case of the channel-gating function of CFTR, PKA phosphorylation caused an increase in the apparent aYnity for MgATP of the ATPase activity. These findings support the idea that the function of NBDs is modified by the phosphorylated ‘‘R’’ domain. However, in experiments using [-32P]8-N3ATP, investigators observed only a slight diVerence in ATP binding to CFTR under phosphorylating or nonphosphorylating conditions (Basso et al., 2003), possibly reflecting a frequently observed diVerence between binding aYnity and apparent aYnities determined from the action of an enzyme. In order to further assess the relative contribution of the two nucleotide-binding domains of CFTR to its ATPase activity, we compared the consequences of mutating the conserved lysine residues in the canonical Walker A, ATP-binding motif of either NBD1 or NBD2. Studies done on Walker A mutants suggested that the two NBDs of CFTR may function as a complex. For example, mutation of the conserved lysine residue in either Walker A (i.e., K464A in NBD1 or K1250A in NBD2) caused a decrease in ATPase activity to significantly less than 50% of that exhibited by the intact protein (Ramjeesingh et al., 1999). Therefore, neither the Walker A motif in NBD1 nor the Walker A motif in NBD2 can function independently in catalysis. Further, the eVects of mutating the Walker A lysines in the two NBDs were nonequivalent. For example, the ATPase activity exhibited by CFTRK464A was decreased by 70–80%, whereas the ATPase activity of CFTRK1250A was approximately 1% of wild-type activity. Hence, there may be one primary catalytic site formed by the NBDs of CFTR (involving the Walker A lysine in NBD2 K1250) and one modulatory site (involving the Walker lysine in NBD1 K464), a model initially proposed by Kopito and Gunderson in 1995. These findings are also consistent with the vanadatetrapping experiments described in the preceding paragraph (Aleksandrov et al., 2001; Basso et al., 2003; Szabo et al., 1999).
B. Structural Studies of Prokaryotic ABC Transporters and Their Relationship to CFTR Structural studies of prokaryotic ABC transporters have provided some insight into the potential physical basis for functional interaction between the two nucleotide-binding domains of mammalian ABC protein, possibly
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including CFTR. Crystallographic data from both intact prokaryotic ABC transporters and isolated NBDs have demonstrated that there is a conserved structural interaction between the two NBDs (Chang, 2003; Fetsch and Davidson, 2002; Hopfner and Tainer, 2003; Hopfner et al., 2000; Locher et al., 2002). The key residues involved in binding ATP exist in the interface between the NBDs and include residues in the Walker A, B, and C motifs, as well as the switch and Q-loop. These are heavily conserved between diVerent members of the ABC transporter superfamily, including both NBDs of CFTR (Locher et al., 2002). In the structures of MsbA, BtuCD, Rad50, and MJ0796, two ATP-binding sites formed by apposition of the NBDs coming together in a head-to-tail orientation, wherein each site is formed by the Walker A motif of one NBD and the Walker C of the other NBD (Chang, 2003; Hopfner et al., 2000, 2001; Locher et al., 2002; Smith et al., 2002) (Fig. 2). The crystallographic evidence for this arrangement was further supported by biochemical studies showing nucleotide-dependent dimerization between NBDs of Rad50 and MJ0796 (Hopfner et al., 2000; Smith et al., 2002). In addition, these studies showed that mutations in key residues in the Walker A or C motifs prevented dimer formation (Hopfner et al., 2000; Smith et al., 2002). Biochemical cross-linking and biophysical studies of the maltose-binding protein (Fetsch and Davidson, 2002) and P-glycoprotein (Loo et al., 2002)
Figure 2 Interaction of the two nucleotide-binding domains of BtuCD in a ‘‘head–tail’’ orientation. Each nucleotide-binding domain is colored diVerently (blue or green). The cyan arrow indicates the Walker A motif and vanadate ion (purple). The orange arrow indicates the Walker C motif. Asterisks show the putative location of disease-causing mutants of CFTR. The single asterix represents Q129 R in the conserved Q-loop. The double asterix indicates the location of L96 in BtuCD, corresponding to the F508 residue. (Kraulis, 1991; Merritt and Bacon, 1997) Figure rendered by D. G. Smith. (See Color Insert.)
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suggest that dimerization in this ‘‘head-to-tail’’ orientation may occur throughout the ABC family of transporters. Indeed, biochemical studies performed on recombinant NBD1 and NBD2 CFTR proteins have shown that they interact physically (Howell et al., 2003; Lu and Pedersen, 2000). Furthermore, ATPase experiments on Walker A mutants from NBD1 or NBD2 demonstrated that the two NBDs function cooperatively to bind and hydrolyze ATP (Ramjeesingh et al., 1999). However, the molecular basis for their interaction has yet to be determined. Despite a high degree of similarity, the primary sequences of NBD1 and NBD2 of CFTR are not identical to NBDs of the procarytic ABC proteins across all of the canonical motifs described earlier, i.e., Walker A, B, and C motifs as well as the switch and Q-loop (Fig. 3). Furthermore, the overall degree of sequence identity between NBD1 and NBD2 of CFTR is moderate (approximately 30%) and it is therefore possible that the two ATP-binding sites formed between the two NBDs (possibly in a head-to-tail configuration) are nonidentical. Interestingly, mutations in either the Walker A sequence of NBD2 (K1250A) or the Walker C sequence of NBD1 (G551D; a CF disease-causing mutation) completely abrogate ATPase activity (Li et al., 1996; Ramjeesingh et al., 1999), suggesting that these motifs contributed by NBD2 and NBD1, respectively, form a single catalytically active site as predicted by the aforementioned crystal structures. However, disruption of the Walker A sequence of NBD1 (K464A) reduces but does not completely eliminate ATPase activity (Ramjeesingh et al., 1999). Unfortunately, the eVect of disrupting Walker C in NBD2 (G1349D) has not yet been studied with respect to its eVect on ATPase activity. Therefore, reiterating our previous observations and those of others (Aleksandrov et al., 2001; Gunderson and Kopito, 1995; Ramjeesingh et al., 1999; Vergani et al., 2003), CFTR may not have two symmetrical sites for ATPase activity and we may find that the structure–function relationships of the NBDs of CFTR diverge somewhat from those of the prokaryotic ABC proteins that have been crystallized.
Figure 3 Alignment of conserved ATP-binding motifs in BtuD, Rad 50, NBD1, and NBD2 of CFTR (Locher et al., 2002). (See Color Insert.)
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C. Electrophysiological Studies of the Relative Roles of NBD1 and NBD2 in Channel Gating As described previously, CFTR channel opening and closing, i.e., gating, have been linked to nucleotide binding and ATPase activity by one or both of its NBDs (Aleksandrov et al., 2000; Ikuma and Welsh, 2000; Ramjeesingh et al., 1999; Sugita, et al., 1998; Vergani et al., 2003). Numerous studies have been conducted to examine whether nucleotide binding and/or hydrolysis is required for channel gating by CFTR. For the most part, this question has been addressed through comparative single channel studies of the functional consequences of the addition of various nucleotides (hydrolyzable and poorly hydrolyzable nucleotides) and variations in the divalent cations employed. Initial studies of the nucleotide dependence of CFTR channel open probability revealed that the addition of MgATP was required for optimum channel open probability (Po values ranging from 0.3 to 0.6 for fully phosphorylated CFTR), and dose–response analyses revealed that concentrations of 0.04 to 0.1 mM were required for half-maximal eVects (Anderson and Welsh, 1992; Baukrowitz et al., 1994; Gunderson and Kopito, 1994; Schultz et al., 1996). Magnesium ion was the preferred cation for this activity in some, but not all, studies (Ikuma and Welsh, 2000; Schultz et al., 1996). Subsequent studies have revealed that relatively high concentrations (5–10 mM) of magnesium salts of nonhydrolyzable analogues (i.e., MgAMP-PNP and MgATPS) are also capable of initiating channel gating, albeit with a low maximal open probability of (Po ¼ 0.01) (Aleksandrov et al., 2000). For the most part, however, substitutions of ATP with various nucleotides wherein the pentose ring, nitrogenous heterocyclic base, or phosphate chain is modified would be expected to have altered binding aYnity (Aleksandrov et al., 2002). Hence, it is somewhat problematic to use these various ATP analogues to try to separate the relative role of binding and hydrolysis on channel opening. That being said, however, the inability of the poorly hydrolyzable analogues (AMP-PNP and ATPS) to mediate Po greater than 0.01, even at high concentrations of 10 mM, argues that chloride channel open probability may be regulated by events secondary to ATP binding, namely hydrolysis, product dissociation, and/or conformational changes in protein structure. Coupling between Channel-Gating Activity and ATPase Activity by Nucleotide-Binding Domains of CFTR While there is consensus that nucleotide binding and hydrolysis are required for normal gating of the CFTR channel, the mechanisms underlying this regulation remain controversial. Some of this uncertainty reflects the
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variability in the kinetics of nucleotide-dependent gating of wild-type and mutant CFTR channels reported by diVerent laboratories. This variability primarily reflects diVerences in cell type and experimental conditions employed and possibly the level of channel phosphorylation, a modification known to aVect nucleotide interaction with CFTR (Mathews et al., 1998a,b). Furthermore, it is diYcult to compare the kinetics of single channel gating to the kinetics of ATP hydrolysis by CFTR. Detailed studies of the kinetics of the ATPase activity of CFTR require its purification and functional reconstitution and are intrinsically diYcult because of the low level of this activity (Bear et al., 1992; Ramjeesingh et al., 1999). Furthermore, whereas channel gating studies reflect the activity of a single functional molecule, the ATPase activity measured reflects a population of purified molecules of which only a certain percentage has been functionally reconstituted. Ideally, an assay of the ATPase activity of a single molecule of CFTR needs to be developed so that these kinetics could be compared directly to the kinetics of single molecule gating. In fact, fluorescence assays have been developed successfully for the characterization of the activity of single molecular motors such as myosin ATPase and have provided tremendous insight into the mechanisms underlying coupling between catalytic activity and molecular movement (Ishijima et al., 1998; Shih et al., 2000). There have been many studies of nucleotide-dependent gating of CFTR, and papers by Gadsby and colleagues provide an excellent review of many of these findings (Basso et al., 2003; Vergani et al., 2003). The following paragraphs discuss our view of certain general concepts that emerge from such studies. First, opening of the CFTR channel is likely regulated by events secondary to ATP binding, namely conformational changes in protein structure and/or hydrolysis. As mentioned previously, the inability of magnesium salts of nonhydrolyzable analogues to stimulate normal rates of channel opening, even at concentrations two- to threefold greater than the Kd for MgATP (Aleksandrov et al., 2000), suggests that ATP binding alone may be insuYcient to gate the channel eVectively. Vergani et al. (2003) noted that the saturable dependence of opening on [MgATP] suggests that some (relatively slow) step unrelated to nucleotide binding sets the maximal rate of channel opening at saturating [MgATP]. At present, the results of studies of Walker A lysine mutants of NBD1 (K464A) and NBD2 (K1250A) cannot unequivocally support or refute the idea that an event secondary to binding is required for channel opening. Photolabeling studies suggest that mutations in either motif will aVect ATP binding to the mutated domain (Aleksandrov et al., 2001; Basso et al., 2003). Hence, the diVerential eVect of these mutations on the channel opening rate observed—K464A (channel opening rate) >K1250A (channel opening rate) (Powe et al., 2002; Ramjeesingh et al., 1999) — may reflect events secondary to binding. It is not yet clear whether this diVerential opening rate is directly
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related to the relative impact of these mutations on the ATPase activity of CFTR, although there is the same trend, i.e., the ATPase activity of K464A is greater than that of K1250A (Ramjeesingh et al., 1999). Channel closure may also be linked (possibly directly or indirectly) to ATPase activity at one or more sites in CFTR (Vergani et al., 2003). Disruption of the Walker A lysine residue in NBD2 dramatically decreases ATPase activity by the whole molecule and decreases the rate of channel closure (Carson et al., 1995; Gunderson and Kopito, 1995; Powe et al., 2002; Ramjeesingh et al., 1999; Sugita et al., 1998). Furthermore, disruption of the Walker A lysine in NBD1 increased the closing rate (in some studies) at saturating ATP concentrations, suggesting possibly a modifying role for NBD1 in nucleotide-dependent closing (Powe et al., 2002). It appears that a nonhydrolytic interaction of ATP with NBD1 plays a role in channel closure. This is suggested by a study showing that channel gating occurred on the order of seconds, whereas at the same temperature, nucleoside triphosphate remained associated with NBD1 for many minutes (Basso et al., 2003). Finally, in our view, while there is a clear relationship between the ATPase activity and the channel gating of CFTR, it is unlikely that this represents a tight, stoichiometric coupling between these two activities. In other words, there may be a loose coupling ratio with several cycles of channel opening and closing occurring for every catalytic cycle (see Fig. 4). In support of this concept, we found that mutation of the Walker A lysine in NBD1 caused a significant reduction in ATPase activity by the whole protein (reducing it by 70–80%), yet changes in channel gating are very subtle (Powe, et al., 2002; Ramjeesingh et al., 1999). Furthermore, although the Walker A mutation in NBD2 abolished ATPase activity completely, the CFTR channel could still gate, albeit with significantly altered kinetics (Ramjeesingh et al., 1999). In another set of experiments, Aleksandrov and colleagues (2001) compared the ability of CFTR to hydrolyze 30 -dATP versus ATP and found
Figure 4 Diagram illustrating the two possible models describing coupling (tight or loose) between catalytic and channel gating activities of CFTR. In both diagrams the yellow wheel represents the ATPase cycle and the green wheel represents the opening and closing of the channel pore. (See Color Insert.)
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that the Vmax for hydrolysis of these two nucleotides was approximately 6-fold diVerent [Vmax(ATP) ¼ 47 nmol/mg/min >>Vmax(dATP) ¼ 8.5 nmol/ mg/min]. However, there was only a 2.5-fold change in rates of channel opening and closing (Aleksandrov et al., 2002). Together, these studies support the concept that these two functions may be coupled loosely rather than tightly as shown in the model in Fig. 4.
D. Insight Regarding Structural Interaction between Nucleotide-Binding Domains and the Pore-Forming Domain of CFTR Provided by Structural Studies of Intact Prokaryotic ABC Proteins As for other members of the ABC superfamily of transporters, conformational changes occurring in the NBDs of CFTR are thought to transmit long-range conformational changes in the MSDs via connecting structures or interfacial surfaces. Clearly, information regarding the nucleotide occupancy of the NBDs must be transmitted to the MSDs in order to open and close the channel gate of CFTR (Vergani et al., 2003). Conversely, as shown in previous work (Kogan et al., 2001), the ATPase activity by the NBDs of CFTR is modified by pore-blocking inhibitors and by mutations in the membrane-spanning domain, supporting a role for long-range conformational changes extending from the membrane to the NBDs. The recent crystal structures of intact ABC transporters have provided CFTR researchers with plausible models with which to envision possible molecular mechanisms underlying the transduction of information between the nucleotide-binding domains and the membrane domains. The channel function of CFTR requires transmission of information regarding the nucleotide occupancy of the nucleotide-binding domains to the membrane domains via conformational change. Such intramolecular signal transduction likely occurs through the interface of the membranespanning domains and the NBDs. For Rad50 and possibly BtuCD, ATPase activity and the formation of the high-energy transition state induce a tighter interaction between the two NBDs (Hopfner et al., 2000, 2001; Locher et al., 2002), a conformational change predicted to cause secondary, long-range conformational changes in the membrane-spanning domains that comprise the permeation path. A plausible interface between the membrane-spanning domains and the nucleotide-binding domains was identified by Locher et al. (2002) through alignment of the primary sequence of CFTR with the interfacial regions determined in the crystal structure of the prokaryotic ABC transporter, BtuCD. On the basis of the crystal structure of BtuCD, Locher et al. (2002) suggested that helical segments (L1–L2) extending from the membrane domain intercalate with and provide a docking site for the nucleotide-binding domains (see Fig. 5).
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Figure 5 Structure of the intact BtuCD transporter (Locher et al., 2002). L1–L2 regions of the membrane domains (MSDs) that interact with the NBDs. The Putative location of diseasecausing mutations Q1291R and F508 is indicated. Backbones of membrane-spanning helices (BtuC) are shown as purple and red. Ribbon diagrams of nucleotide-binding domains (BtuD) are shown as blue and green. Figure rendered by D. G. Smith. (See Color Insert.)
As mentioned earlier, Locher et al. (2002) defined an L1–L2 region on the membrane-spanning domains that interacts with the NBDs (Fig. 5). The two helical segments L1–L2 are bisected by a glycine residue and this glycine confers a bend, critical in creating the interface. This loop region in BtuD was aligned with the region in MSD1 (residues 248–261) and MSD2 (1056– 1069) of CFTR by Locher et al. (2002). The conserved glycine, found in 13 of 13 NBDs from diVerent ABC proteins, is also conserved in CF-MSD1 and CF-NBD2. Of the four conserved residues in the L1–L2 region (including the glycine residue GEIxxR), CF-MSD1 has three (GKIxxR) and CFMSD2 has two (GLWxxR) conserved residues. However, there is little conservation of the interfacial residues defined on the nucleotide-binding domain of the BtuCD structure. Hence, the specificity of the interaction between the membrane and the nucleotide-binding domains likely resides in the L1–L2 loops. Disease-causing mutations exist in the L1–L2 region identified in MSD1 (CFTRR258G) and MSD2 of CFTR (i.e., CFTRG1061R, CFTRL1065F/ P/R, CFTRR1066C/S/H/L, and A1067T/D), supporting the idea that this
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region plays an important role in channel structure and function (CF mutations database). The L1–L2 region in MSD1 is contained within the intracellular loop CL2 (as defined in the secondary structure predictions initially published) (Riordan et al., 1989). Seibert and colleagues (1997) studied the eVect of the disease-causing mutation R258G in CL2 and found that it caused protein misfolding, with the protein failing to exhibit complex glycosylation and cell surface expression. The putative L1–L2 region in MSD2 is contained within the intracellular loop CL4 (1037–1095), as defined in the original topology of CFTR (Riordan et al., 1989). This region is a hot spot for disease-causing mutations, many of which have been shown to lead to protein misprocessing (i.e., CFTRL1065P, CFTRR1066C/H/L, and CFTRA1067T). Although all of the mutants exhibited poor cell surface expression following transient expression in HEK293 cells, there was suYcient expression of CFTRR1066L and CFTRA1067A to perform single channel studies using the patch-clamp technique. Both of these mutants exhibited altered channel gating with relatively low open probability due to decreased open times for R1066L or increased long closed times between openings. Together, these findings suggest that mutations in the putative connection region extending from the membrane domains to the NBDs of CFTR lead to protein misfolding and to altered channel gating in some cases (Cotten et al., 1996; Seibert et al., 1996). Hence, this putative interface appears to be important in mediating correct folding during biosynthesis and in transmitting the information required for gating of the chloride channel pore of CFTR. Significantly, the major mutant of CFTR, CFTRF508, aligns with a residue in BtuD (the NBD domain of the functional complex, BtuCD) located at the L1,L2–NBD interface (Locher et al., 2002). It is well known that CFTRF508 is misprocessed (Cheng et al., 1990) and exhibits defects in phosphorylation-dependent gating (Hwang et al., 1997), supporting an important role for this putative interfacial region for both processes (Fig. 5).
VIII. Future Directions As evident from the preceding discussion, there is still much to be learned about CFTR with respect to the molecular basis for its function as a chloride channel and the consequences of disease-causing mutations on its expression and function at the cell surface. This section summarizes a subset of important questions in the field. In future experiments, we plan to address the functional significance of CFTR dimerization. As discussed previously, functional analyses of purified and reconstituted monomeric CFTR support the idea that this structure is fully functional as a chloride channel and as an ATPase (Ramjeesingh
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et al., 2001). However, biochemical analyses of the quaternary structure of CFTR in the plasma membrane suggest that dimeric CFTR can exist at the cell surface. Our challenge for the future is to develop fluorescence and/or bioluminescence based methods for examining the quaternary structure of CFTR in living cells, in intracellular membranes, and at the cell surface. This approach has been employed eVectively in the study of the oligomeric assembly of various transporters and receptor molecules in living cells (Ayoub et al., 2002; Iino et al., 2001; Sorkina et al., 2003) and will be invaluable for our assessment of the in vivo relevance of CFTR dimers. Further, such methods will permit our definition of the subcellular compartment in which dimerization occurs, thereby providing clues regarding its role in protein maturation and/or cell surface stability. Application of these methods for the study of mutant versions of CFTR will also elucidate the relevance of dimerization as a molecular target for certain disease-causing mutants. Our understanding of the molecular basis for anion permeation through CFTR remains limited to studies of the role of the sixth putative transmembane helix of MSD1. Although CFTR is not related to the C1C family of chloride channels structurally, we have learned from the crystal structure of prokaryotic C1C channels (Dutzler et al., 2002, 2003) that residues conferring the chloride selective pore are contributed from multiple regions (four), spread throughout the primary sequence. Therefore, novel search strategies need to be developed to encompass all of the putative membrane helices in CFTR. Studies of the chloride channel function of purified MSD2 (Ramjeesingh et al., 2003) highlight the feasibility of reconstituting the intact chloride channel pore by the assembly of purified membrane protein fragments, fragments that may eventually be more amenable to structural studies than the full-length protein. Novel approaches for the detection of molecular dynamics will also need to be developed to advance our understanding of how the CFTR channel is regulated and how mutations may disrupt this function. Future progress in this area requires that we specifically identify those regions of the intact CFTR protein that undergo dynamic conformational change upon phosphorylation, nucleotide binding, and hydrolysis. For example, fluorescence labels, targeted toward specific amino acids, i.e., substituted cysteine residues, have been very eVective in studies of real-time dynamics in membrane proteins such as lac permease (Kaback and Wu, 1997) and cyclic nucleotidegated cation channels (Zheng and Zagotta, 2000, 2003). Assuredly, several research laboratories are currently engaged in optimizing the research tools for similar studies of CFTR. Finally, the field of CF research has witnessed many technological achievements and breakthroughs, not the least of which was the identification of the gene itself in the late 1980s (Rommens et al., 1989). The next
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decade of CF research will introduce the development of methods that provide insight into the molecular dynamics underlying CFTR function, the structural defects caused by disease-associated mutations, and the molecular targets for therapeutics.
Acknowledgments We acknowledge continuous support from the Canadian Cystic Fibrosis Foundation in terms of personnel awards to J.F.K and I.K and operating grants to C.E.B. The authors are also grateful to Dr. D. G. Smith (Hospital for Sick Children, Toronto) for rendering structural models. We would also like to thank Dr. Fiona Stratford for careful proofreading of the text.
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Index A ABC. See ATP-binding cassette AD. See Alzheimer’s disease African Americans/African blacks PD and, 24 Aggrecan, upregulation of, 105f AGM. See Aorta-gonad-mesonephros Air (antisense transcript) DMR and, 201 truncation of, 201 Airway disease, CF related, 218 Alkaline phosphatase expression (ALP) U0126, decreases in, 113 ALP. See Alkaline phosphatase expression Alzheimer’s disease (AD), 18 Dementia with Lewy Bodies and, 26 -syn pathology in, 29 A-MuLV (Abelson murine leukemia virus), 166 Antisense transcript mechanism, 200–1 Aorta-gonad-mesonephros (AGM) CD34+ hemogenic cells produced in, 153 human endothelial cells, 153 stromal lines derived from, 141 Arabidopsis CMT genes in, 70 gene imprinting in, 76 Articular chondrocytes hydrostatic pressure’s effects on, 116 AS (Angelman) syndrome IC, 204 Ascobolus immersus, 63 Asymmetric methylation, 58 ATPase activity channel-gating activity coupling with, 234–37 ATP-binding cassette (ABC) CFTR and, 217, 221, 230 family of transport proteins, 216 ATP-dependent transporters, ABC proteins and, 216
Autologous cells expansion of, 4 tissue engineering and, 5
B BCR/ABL fusion oncogene CML caused by, 154 1 downregulation of, 112 upregulation of, 112f BFGF (basic fibroblast growth factor), 162 Biaxial tensile loading flow cytometry variances and, 107–8 Bicarbonate ion transport, 227 Biomaterial characteristics of ideal, 5 Bipotential endothelial/hematopoietic progenitor, 163 Blast colony-forming cell (BL-CFC), 163, 164–65 pre, 166–67 two populations in vitro of, 167–68 Blood island formation, 163 Blood-forming tissue, human primitive, 172 Bone conduction chamber, modification of, 98 Bone fractures shock wave treatment of, 119 Bone marrow fetal, 157 hematopoietic cell expansion in, 157 homing hematopoietic activity to, 158 stromal cell populations, 120 undifferentiated cells within, 99 vertebral, 157 BtuCD CFTR compared with, 229 nucleotide-binding domain interaction of, 232f, 237 BWS (Beckwith-Wiedemann syndrome) clinical features of, 201
251
252 C C5-MTase genes Dnmt3 class similarities to, 69 Calbiochem (15 M of SB203580), 113 Cancer cell lines Dnmt1, Dnmt3 and, 66 Cartilage formation hyaline, 95 shear stress hypothesis of, 95 CD4+, CD8+ T cells, 10 CD4 , CD8-specific antibodies, 10 CD34+ hemogenic cells, 153 CXCR4 induction on, 158 Cell differentiation shear stress and, 117 stress’s effect on, 96 Cell Signaling (10 M of U0126), 113 Cell-adhesion peptides, 5 Cellular function regulation of, 4–5 Center for Disease Control, 7 CF. See Cystic fibrosis CFTR F508, 220–22 CFTR and, 224 channel activity capability of, 229 phosphorylation, impaired, in, 229 rescue of, 222 stability, impaired, of, 221, 222 CFTR. See Cystic fibrosis transmembrane conductance regulator CFU-C (colony forming activity), 143 Channel-gating activity ATPase activity coupling with, 234–37 CFTR’s, 236f Chaperone proteins, 22 Chick embryos experiments involving, 94 Hamburger-Hamilton White Leghorn, 104 Chloride ion permeation, 224–26 Chloride-selective permeation path, CFTR and, 224–25 CHM (complete hydatidiform mole), 148, 149 Chondroepiphysis, point-loading model of, 96f Chondrogenesis assay of, 106 ERK-1/2 activity during, 113 inhibition of, 110 mechanical stress induction of, 106 Chondrogenic differentiation, 103 cyclic loading on, 105 mechanical stress induction of, 106
Index Chromomethylases (CMT proteins) chromodomain’s function in, 70–71 distinguishing feature of, 70 N-terminal regions of, 71 Chronic myeloid leukemia (CML) BCR/ABL fusion oncogene and, 154, 168 murine HSC and, 168 Cloned muscle histologic examination of, 9–10 seeding of, 9 Cloning nuclear, 6 reproductive, 6–7 therapeutic, 6–9 CLP (common lymphoid progenitor), 129 CML. See Chronic myeloid leukemia CMT proteins. See Chromomethylases Collagen(s) fibril organization, 115 type II immunohistochemistry, 103f type II mRNA, 103 Type II, X, 103 Collagen type II immunohistochemistry, 103f presence of, 107f Compressive stress, 101–2 Continuous passive motion, 97–98 increased chondrogenesis and, 97 periosteal tissue, cambium layer, and, 97 CpG island promoter, 201–2 CpG methylation Dnmt1 and, 61 Dnmt2 and, 64 DRM1, DRM2 inactivation and, 69 MET1 and, 62–63 CSF (colony-stimulating factor). See Macrophage colony-stimulating factor (MCSF) CTCF Igf2 inhibition of, 203–4 methylation-sensitive zinc finger DNA-binding protein, 203 CXCR4 N-terminal cleavage of, 146, 158 Cyclic Stretch (Biaxial Tensile Loading), 107–14 progenitor cell population and, 107 Cystic fibrosis (CF) cause of, 216 CFTR and, 216 CFTR, improper regulation in, 218
Index clinical features of, 216 GI tract and, 220 pancreatic duct and, 219 Pseudomonas aeruginosa, 218 respiratory tract and, 218–19 sweat duct and, 220 Cystic fibrosis (CF) airways, chronic infection of, 218–19 Cystic fibrosis transmembrane conductance regulator (CFTR), 216 ATP binding by, 230 BtuCD compared with, 229 cell biology of, 220–22 CFTRF508 and, 224 channel activity regulation of, 217, 218–20, 222, 230 chloride, bicarbonating functions of, 227 chloride conduction through, 225 chloride-selective permeation path and, 224–25 composition of, 216 dimerization, 224 epithelial transport function of, 218 future directions regarding, 239–41 GXXXG motifs exist in, 224 hydrolysis by, 230 mediation by, 226 mutant F508, 220–22 NBDs structure-function relationship with, 233 nucleotide binding domains of, 231 nucleotide-dependent gating of, 235 PDZ-binding motif on, 222 permeation pore of, 224–28 pore forming domain of, 237–39 prokaryotic ABC transporter’s relationship to, 231–33 purified, monomeric, 222, 223 quaternary structure of, 222–24 ‘‘R’’ domain of, 217, 228–29 Cytosine methylation, 58 D DAT. See Dopamine transporter Definitive erythroblasts (EryD/‘‘macrocytes’’), 144 Definitive hematopoiesis, Homo sapiens, 155–58 Definitive hematopoiesis, Musmusculus genes, 144–45
253 theories regarding, 137 in utero v. adult, 143 Degradation products removal of, 5 Dementia, 25 Dementia with Lewy bodies (DLB), 17 PD, AD, and, 26 -synuclein and, 17 Differential imprinting, 172 Differentially methylated regions (DMRs), 199–200 Air and, 201 enhancer-blocking assays and, 207 germline-derived, 200, 202f intronic germline, 201 methylation of, 75–76 three models involving, 200 Distal mouse chromosome Dlk1-Gtl2-imprinted domain on, 205–7 DLB. See Dementia with Lewy bodies Dlk-Gtl2 domain on distal mouse chromosome, 205 DMR insulator function of, 206 small RNAs found at, 207 DMRs. See Differentially methylated regions DNA, epigenetic modifications to, 198 DNA hypomethylation, 73–74 DNA methylation. See also Asymmetric methylation; Cytosine methylation; Eukaryotic DNA methyltransferases; Gene expression; Genomic imprinting ancestral function of, 73 chromatin modification and, 210 CpG-binding proteins and, 74 development and, 78 differential, 198 Dnmt3L and, 68 establishment, maintenance, germline reversibility of, 199f as evolutionary device, 72 filamentous fungi and, 72 function of, 72–80 gene expression and, 74, 75 genome stability and, 73–74 heritable epigenetic modification theory of, 56–57 histone modifications and, 198 homologous recombination suppression by, 73 inheritance patterns of, 57f
254
Index
DNA methylation (cont.) lack of, 72 mammalian, 56, 78 mouse development and, 79 N-terminal extensions of, 55 plants and, 72 RNA-directed, 58 DNA methyltransferases Arabidopsis thaliana and, 62 chromomethylase class of, 70–71 eukaryotic, 58–72 multiple, 57 Dnmt1 class disruption of, 79 Dnmt3 class cooperation DNMT3, 65 homologs of, 62 human, 61 MET1 class, similar to, 62 N-terminal region of, 61 transcription start sites of, 59, 61 Dnmt1o, 61 Dnmt2, 63–64 CpG and, 64 prokaryotic C5-MTase and, 64 targeted disruption of, 64 Dnmt3 class, 65–70 C5-MTase genes similarities to, 69 de novo methyltransferase activity in, 65 Dnmt1 class cooperation with, 65 genomic imprinting and, 75 ICF syndrome and, 65 isoforms of, 66 PWWP domain and, 67, 68 Dnmt3a/Dnmt3b genes, 66 disruption/inactivation of, 79 Dnmt3L interaction with, 68–69 gene imprinting and, 75 similarities of, 67 Dnmt3L (Dnmt3-like), 68 DNA methylation regulation by, 68 Donor tissue, 3 Dopamine transporter (DAT) -syn interaction with, 23 Dopaminic neurons metabolic properties of, 33 DRM/Zmet3 proteins, 69
EC (embryonal carcinoma) cell lines murine, human, 161, 162, 163 ECM. See Extracellular matrix proteins Embryo, human, 152f circulation’s beginning in, 159 Embryo, mammalian, experimental unapproachability of, 172 Embryonic stem cells (ESC). See also Human embryonic stem cells blood disease studies using, 173 blood tissue formation and, 163, 163f, 174 hematopoiesis from, 161–70 hematopoietic forming capacity of, 130 human, 6, 162 potential therapeutic uses of, 173–74 properties of, 5 in vitro analysis using, 168 Endothelial cells, blast-colony forming and, 164–65 eNOS (endothelial nitric oxide synthase), 109 EPA (erythroid-potentiating activity), 148 Epigenetic modifications, understanding, 209–10 Epo, 165–66 erythropoiesis and, 166 ERK. See Extracellular signal-related kinase Erythropoietin, human, 148 ESC. See Embryonic stem cells Eukaryotic DNA methyltransferases, 58–72. See also Dnmt1 class C5, 58–59 Dnmt1 class of, 59, 61–63 five classes of, 59f, 60f Eukaryotic genomes, vulnerability of, 209 Evolutionary divergence, 159 Extracellular matrix proteins, 3, 4 replication of, 4 Extracellular signal-related kinase (ERK), 109, 110, 113 10 M of U0126 blocking of, 113 15 M of SB203580 blocking of, 113 Extraembryonic mesoderm, 148 Extraembryonic/intraembryonic hematopoieses transition between, 159
E
F
E10 mouse limb bud cells static mechanical compression of, 104
Fetal limb bones, experiments on, 94 Fluorescence resonance energy transfer (FRET), 32
Index [fourteen]14-3-3 proteins -syn association wtih, 22 Fracture healing, early experiments in, 97 FTE (functional tissue engineering), tenets of, 93 G GAG. See Glycosaminoglycans Gastrointestinal tract (GI), CF and, 220 GCIs (Glial cytoplasmic inclusions), 27, 28 -syn filaments in, 28 Gene expression hypomethylation and, 76 maternal chromosome, 198 tissue-specific, developmental stage-specific, 76–77 Xenopus development and, 77 Genomic imprinting, 74–76 DNA methylation and, 74 Dnmt3 methyltransferases and, 75 heterochromatinization and, 199 loss of, 76 process of, 197 regulatory properties of, 74 Germline-specific silencing, consequence of, 199 GI. See Gastrointestinal tract Glial cytoplasmic inclusions. See GCIs Globins adaptations of, 155 human, 149 human’s rare forms of, 161 shared gene expression patterns of, 150 Glutathione conduction, 227–28 Glycophorin-like dimerization motifs (GXXXG), 224 Glycosaminoglycans (GAG) increased immunoreactivity for, 101 photomicrographs of, 104f GRGDS (glycine-arginine-glycine-aspartic acid-serine) peptide added to stretched cells, 111 GXXXG. See Glycophorin-like dimerization motifs H HADAS (human adipose-derived adult stem cells), 92 Hallervorden-Spatz disease. See NBIA-1
255 HbA (adult hemoglobin), 156 HbF (fetal hemoglobin), 155 Hemangioblast-like cells bipotential nature of, 134 Hemangioblasts, 133 factors defining, 171 Hematopoiesis, human high-resolution analysis of, 147 Hematopoiesis, mammalian active marrow, 146 AGM-derived stromal lines support, 141 definitive, 137, 143–45 intraembryonic, 143 introduction to, 128–30 key events in, 132f knockout mice, creation of, and, 133 physiological indicators of, 160 precirculation, yolk sac, 135 primitive, 131–35 questions remaining about, 171–74 transient extraembryonic, 142 in utero, 128, 143 yolk sac differentiation in, 131 Hematopoietic cells circulation’s onset in, 136–37 murine development/‘‘day zero’’ and, 136 progenitor, 134 yolk sac, 134 Hematopoietic colony-forming activity, spontaneous, 168 Hematopoietic ontogeny CLP and, 129 PHSC and, 129 Hematopoietic system functions of, 130 Hematopoietic tissues ontogeny in utero of, 142 Hemoblasts theories regarding, 151–52 ventral endothelium and, 151 Hemogenic endothelium intraembryonic, aortic, 136 Hemoglobin, adult, 156 embryonic, three forms of, 149 transitions of, 155 HESCs. See Human embryonic stem cells HILDA (human interleukin in DA cells), 162 HoxB4 expression, 140
256 HSC (human cell stem) production origination controversy regarding, 151 sites of, 142 Human chromosome 15, 204 Human embryonic stem cells (HESCs) differentiation of, 6 harvesting of, 6 hematopoiesis and, 169–70, 173 mesodermal structures generated by, 169 NIH and, 6 Human-processed lipoaspirate (PLA) cells, 92 Hyaline cartilage formation compressive stress hypothesis of, 95 Hydrostatic pressure, 98–99 articular chondrocytes and, 116 MCSF and, 115 osteoclastogenesis and, 115 Hypomethylation, gene expression and, 76
Index In Vivo blood development, Homo sapiens definitive hematopoiesis and, 155–58 hematopoiesis, human embryonic stem cells and, 169–70 honing, retention and, 158–59 primitive hematopoiesis and, 147–51 P-Sp/AGM and, 151, 153–54 In Vivo blood development, Musmusculus circulation onset and, 136–37 definitive hematopoieses and, 143–45 honing, retention and, 145–46 primitive hematopoiesis and, 131–35 P-Sp/AGM and, 137–43 Indomethacin stain-induced response and, 108 Intracellular proteinaceous inclusions (LBs, LNs) PD and, 25 Islets of Langerhans, CF and, 219
I IC imprinting center, 204 ICF syndrome, Dnmt3B mutations and, 65 Igf2-HI9 domain, 203 Dlk-Gtl2 and, 206 reciprocal effect observed at, 206 regulation elements of, 203 Igf2r domain imprinting control at, 200–1 three imprinted gene cluster at, 200 Igf2r domain on mouse chromosome imprinting control at, 200–1 Immunodeficient murine hosts, 151 Immunologic incompatibility, 7 Imprinted genes Dlk-Gtl2, 205 Igf2r domain, 200 location of, 197–98 noncoding RNAs and, 208 number of, 197 SINE elements and, 208 small RNAs located in, 208 Imprinting control, common features of, 207 In Vitro experimentation compressive stress and, 103–6 cyclic stretch (biaxial tensile loading) and, 107–14 hydrostatic pressure and, 115–17 multiaxial loading and, 114–15 shear stress and, 117–19 shock waves and, 119–21 In Vitro loading schematic, 111f
J JNK. See N-terminal c-Jun kinase K KG-1 kinase, expression of, 150 Kidney histologic examination of implants for, 11 RT-PCR analysis of, 11 tissue engineering efforts of, 10 Knockout mice, creation of, 133, 134 L L1-L2 region disease causing mutations in, 238–39 MSD2 and, 239 NBD1/NBD2 and, 238 LacZ gene OMP driven, 117, 118 LBs (intracellular proteinaceous inclusions) location of, 25, 26, 27 L-Dopa PD and, 26 Limb development. See also Compressive stress; Continuous passive motion; Hydrostatic pressure; Tensile stress mathematical models of, 96 mechanical regulation of, 94–95 Pauwels, F., model of, 95, 96 in vitro experimentation and, 103–6 in vivo experimentation and, 97–102
257
Index LIT1/KCNQ1T1, noncoding transcript, 201 L-Name (N-nitro-L-arginine methyl ester), 110 NO production blocked by, 110 Loading experiment, stained cells from, 98f Lymphoid progenitors, 144 M Macrophage colony-stimulating factor (MCSF) hydrostatic pressure and, 115 Malnutrition, CF and, 220 Maltose-binding protein biochemical cross-linking/biophysical studies of, 232–32 Mammalian cell types anchorage dependent, 4 MAPK (mitogen-activated protein kinase) pathways, 38, 113 Marrow, stem cell activity in, 146 Masc1 class, 71–72 fertility and, 71 MIP requiring of, 71 RIP protein similarities to, 71 MCSF. See Macrophage colony-stimulating factor Mechanical compressive force morphological changes caused by, 101 phenotypic changes under, 102 Mechanical stress, theoretical models of, 95–97 Mechanotransduction pathways, 93 discerning, 109 Mediterranean/Greek families PD and, 23 MEF (murine embryonic fibroblast) feeder layers human ESCs fostered on, 162 Membranous bones, studies on, 94 MESC. See Murine embryonic stem cell lines Mesenchymal progenitor cells, isolation of, 114–15 MET1, 62 CpG methylation and, 62–63 Micro-RNAs, 207 Midpalatial suture cartilage collagen distribution throughout, 101 immunohistochemical data relating to, 100–1 mechanical stress’ effect on, 99
micrographs of, 100f, 102f osteochondral progenitor cells on, 100 secondary cartilage response of, 101 thinning of, 100 two tissue types in, 99 Mitochondrial DNA analysis, 11–12 MSA. See Multiple system atrophy MSD2. See Second membrane-spanning domain mtDNA-encoded proteins differences in, 11 oocyte-derived, 11–12 Multiaxial loading, 114–15 Multiple system atrophy (MSA), 27–29 pathological lesions of, 28 PD and, 27 Murine development/‘‘day zero,’’ 136 Murine embryonic stem cell lines (MESC), 162 myelin progenitor activity expansion in, 168 Murine/human blood formation divergence between, 159–61 Muscle, cloned, 9–10 spindle-shaped nuclei of, 8f Musmusculus hematopoietic studies of, 130 in vivo blood development in, 131–46 N NBD1. See Nucleotide-binding domain NBD2. See Second nucleotide-binding domain NBIA-1, 29 Neurospora crassa, 63 Neutrophils, enrichment for, 166 NIH (National Institute of Health) stem cell research and, 6 NK cell B, T lymphocyte’s relationship to, 171 NO (nitric oxide) eNOS production results in, 109, 110 Noncoding RNAs, 207–10 imprinted domains and, 208 Northern blots and, 208 Nonimprinted genes, 198 NSE (nonspecific esterase) activity, 118 N-terminal c-Jun kinase (JNK), 109 persistence of, 109 N-terminal region Dnmt1’s, 62 functions of, 62
258 Nuclear programming, 198 Nucleotide-binding domain (NBD1), 216, 217f. See also Second nucleotide-binding domain (NBD2) ATP interaction with, 230, 231, 236 CFTR structure-function relationship with, 233 channel gating and, 234–37 L1-L2 region and, 238f NBD2 similarities with, 233 nucleotide occupancy of, 237 Walker A mutants from, 233 Nucleotide-dependent gating CFTR and, 235
O Olivopontocerebellar atrophy. See OPCA OLP (olfactory marker protein) promoter, 117 OPCA (Olivopontocerebellar atrophy), 27–29 Organ damage disorders leading to, 1 Organ transplantation, 2 Osteoclast differentiation factors affecting, 108 Osteoclastogenesis hydrostatic pressure’s effects on, 115 MCSF downregulation inhibition of, 115–16 vitamin D3 induced, 118 Osteogenesis induction of, 110 mechanical strain induction of, 108 shock wave induced, 120 Oxidative injury causes of, 33 Oxidative/nitrative injury hypothesis, investigation of, 34 P Pancreatic insufficient (PI), CF patients and, 219 Pancreatic sufficient (PS), CF patients and, 219 PANK2 (pantothenate kinase 2), 29 Parental origin, disorders exhibiting, 197 Parkinson, James, 24
Index Parkinson’s Disease (PD) clinical definition of, 25 increased risk of contracting, 36–37 intracellular proteinaceous inclusions and, 25 L-Dopa and, 25 Mediterranean, Greek families and, 23 pesticide’s role in, 36–38 prevalence of, 24 secondary symptoms of, 25 -synuclein and, 17, 23 PD. See Parkinson’s Disease PDZ-binding motif, CFTR and, 222 Peroxynitrite (ONOO ), formation of, 34f Pesticides, exposure to, 37–38 P-glycoprotein ABC protein, 231 biochemical cross-linking/biophysical studies of, 232–33 Phosphatidic acid (PA) -syn and, 21 Phospholipase D2 (PLD2) -syn inhibition of, 21 Photoelastoid cellular model, 95 PHSC (pluripotent hematopoietic stem cells), 129 PI. See Pancreatic insufficient PKA phosphorylation, 231 Pluripotential cell research, 161–63 Prader-Willi/Angelmann Syndrome human chromosome 15 and, 204 imprinting at, 204–5 Progenitor cells ES cell-derived blood, 165 hematopoietic, 134, 156 lymphoid, 144 osteochondral, 100 P-Sp/AGM, 138 Promoter demethylation, 77 PS. See Pancreatic sufficient Pseudomonas aeruginosa, CF and, 218 P-Sp/AGM, 137–43 HSC generation and, 141 human gene expression of, 154 progenitor cells from, 138 reduced hematopoietic activity of, 145 PWS (Prader-Willi) syndrome, 204 IC (imprinting center), 204, 205
Index PWS/AS region C/D sno-RNAs found at, 207 PWWP domain barrell of, 68 characteristics of, 67–68 DNA binding nature of, 67–68 protein binding function of, 68 R ‘‘R’’ domain deletion mutants of, 228 phosphorylation of, 217, 228–29 Rag2c recipients, use of, 140 RANKL (Receptor activator of nuclear B ligand) downregulation of, 109 Reconstruction tissue used for, 2, 3 Recurrent themes, consideration of, 207–10 Red cell nuclear expulsion, hallmark feature of, 136 Regenerative medicine aim of, 1 Rejection, 7 Replacement therapy shortcomings in, 3 Replication foci-targeting domain, 62 Retrotransposon-like genes, 209 Retroviral elements, IAP class of, 209 RID protein, 71–72 RNAs, noncoding imprinted domains and, 207 three categories of, 207–10 Runx1 capacity of, 168 transcription factor, 167 S SDF 1. See Stromal cell-derived factor 1 SDS (Shy-Drager syndrome), 27 Second membrane-spanning domain (MSD2), 216 dimerization of, 226 helices, residues roles in, 226 Second nucleotide-binding domain (NBD2), 216. See also Nucleotide-binding domain (NBD1) ATP-binding motif of, 231
259 CFTR structure-function relationship with, 233 channel gating and, 234–37 L1-L2 region and, 238f NBD1 similarities with, 233 nucleotide occupancy of, 237 Shear stress cell differentiation stimulation by, 117 nitric oxide stimulation by, 117 prostaglandin production stimulated by, 117 Shock waves treatment with, 119 Von Koss staining and, 120f Shy-Drager syndrome. See SDS Sickle cell anemia, 149 Sno-RNAs, 207 cluster’s tangent arrangement, 208 Snrpn transcription PWS-IC regulation of, 205 SOD (super oxide dimutase) induction, 120 Stem cells, 5–6 bone-marrow derived, 119, 120 embryonic, 5 hematopoietic migration of, 143 increased interest in, 92 micro-environment of, 91 promise of, 91, 92 Stem cells, hematopoietic methods identifying, 154 Strain-induced response indomethacin inhibition of, 108 Stromal cell-derived factor 1 (SDF 1) CXCR4, receptor of, 146 integrins activated by, 158 mutants, 146 Sweat secretion, CF and, 220 Synaptic development syn proteins and, 20–21 -synuclein (-syn) binding properties of, 32 brain, locations of, 19 characteristics of, 18 C-terminal region of, 31 DLB and, 17 dopamine transporter interaction with, 23 14-3-3 protein association with, 22 location of, 20 mouse lines expressing, 39 oxidation’s effects on, 34, 35, 35f pathologies of, 24f
260 -synuclein (-syn) (cont.) PD and, 17, 23 pesticides and, 37 structural properties of, 30–33 synaptic maintenance and, 21 -synuclein (-syn) fibril formation oxidation, nitration and, 34–36 -synuclein (-sy), 18 neuronal locations of, 19 -synuclein (-syn), 18 differences with -syn, -syn, 20 locations of, 20 Synuclein protein(s) amino acid sequence alignment of, 19f defined, 17 interactions of, 22–23 recombinant, 30–31 roles of, 20 tau protein association with, 22 types of, 18–20 Synucleinopathies, 17, 23–30 insoluble -syn protein, 23 mouse models exhibiting, 39 oxidative, nitrative injury in, 33–34 transgenic, nontransgenic animal models of, 38–39
Index categories of, 3 current strategies for, 5 factors limiting progress in, 4 kidneys and, 10–11 preferred cells for, 4 principles of, 2–5 therapeutic cloning combined with, 7f Tissue, newly formed cell behavior in, 5 Transgene expression, regulating, 171 Tyrosine (Tyr) residues, oxidation of, 35 U UBA domains (ubiquitin associated domains) protein-protein interactions mediated by, 69 Ube3a gene, activation of, 205 Urothelial cells, 4 V Ventral endothelium, hemoblasts in, 151 Viral genome, DNA methylation silencing of, 73 Vitamin D3 osteoclastogenesis and, 118
T Tau protein synuclein association with, 22 Tendonopathies shock wave treatment of, 119 Tensile stress, 99–101 / thalassemia, 149 Therapeutic cloning, 6–9 applications of, 8f, 9–12 cloned embryos generated by, 7 tissue engineering combined with, 7f Tibial metaphases study, 98 TIMP1 (tissue inhibitor or matrix metalloproteinases), 148 Tissue development mesenchymal-derived, 92 Tissue engineering aim of, 1 applications of, 8f, 9–12 autologous cells and, 5
W Walker A mutants, 233 NBD1/NBD2 and, 235 Walker motifs, 233f X Xenopus, gene expression suppression in, 77 Y YS (yolk sac) blood islands of, 159 DNA methylation in, 172 dual role of, 142 hematopoiesis of, 148 hematopoietic cells and, 134, 142, 171 human, 147 secondary, 147 transition to liver, 155
Contents of Previous Volumes Volume 47 1 Early Events of Somitogenesis in Higher Vertebrates: Allocation of Precursor Cells during Gastrulation and the Organization of a Moristic Pattern in the Paraxial Mesoderm Patrick P. L. Tam, Devorah Goldman, Anne Camus, and Gary C. Shoenwolf
2 Retrospective Tracing of the Developmental Lineage of the Mouse Myotome Sophie Eloy-Trinquet, Luc Mathis, and Jean-Franc¸ois Nicolas
3 Segmentation of the Paraxial Mesoderm and Vertebrate Somitogenesis Olivier Pourqule´
4 Segmentation: A View from the Border Claudio D. Stern and Daniel Vasiliauskas
5 Genetic Regulation of Somite Formation Alan Rawls, Jeanne Wilson-Rawls, and Eric N. Olsen
6 Hox Genes and the Global Patterning of the Somitic Mesoderm Ann Campbell Burke
7 The Origin and Morphogenesis of Amphibian Somites Ray Keller
8 Somitogenesis in Zebrafish Scott A. Halley and Christiana Nu¨sslain-Volhard
9 Rostrocaudal Differences within the Somites Confer Segmental Pattern to Trunk Neural Crest Migration Marianne Bronner-Fraser
261
262
Contents of Previous Volumes
Volume 48 1 Evolution and Development of Distinct Cell Lineages Derived from Somites Beate Brand-Saberi and Bodo Christ
2 Duality of Molecular Signaling Involved in Vertebral Chondrogenesis Anne-He´le`ne Monsoro-Burq and Nicole Le Douarin
3 Sclerotome Induction and Differentiation Jennifer L. Docker
4 Genetics of Muscle Determination and Development Hans-Henning Arnold and Thomas Braun
5 Multiple Tissue Interactions and Signal Transduction Pathways Control Somite Myogenesis Anne-Gae¨lle Borycki and Charles P. Emerson, Jr.
6 The Birth of Muscle Progenitor Cells in the Mouse: Spatiotemporal Considerations Shahragim Tajbakhsh and Margaret Buckingham
7 Mouse–Chick Chimera: An Experimental System for Study of Somite Development Josiane Fontaine-Pe´rus
8 Transcriptional Regulation during Somitogenesis Dennis Summerbell and Peter W. J. Rigby
9 Determination and Morphogenesis in Myogenic Progenitor Cells: An Experimental Embryological Approach Charles P. Ordahl, Brian A. Williams, and Wilfred Denetclaw
Volume 49 1 The Centrosome and Parthenogenesis Thomas Ku¨ntziger and Michel Bornens
2 g-Tubulin Berl R. Oakley
Contents of Previous Volumes
263
3 g-Tubulin Complexes and Their Role in Microtubule Nucleation Ruwanthi N. Gunawardane, Sofia B. Lizarraga, Christiane Wiese, Andrew Wilde, and Yixian Zheng
4 g-Tubulin of Budding Yeast Jackie Vogel and Michael Snyder
5 The Spindle Pole Body of Saccharomyces cerevisiae: Architecture and Assembly of the Core Components Susan E. Francis and Trisha N. Davis
6 The Microtubule Organizing Centers of Schizosaccharomyces pombe Iain M. Hagan and Janni Petersen
7 Comparative Structural, Molecular, and Functional Aspects of the Dictyostelium discoideum Centrosome Ralph Gra¨f, Nicole Brusis, Christine Daunderer, Ursula Euteneuer, Andrea Hestermann, Manfred Schliwa, and Masahiro Ueda
8 Are There Nucleic Acids in the Centrosome? Wallace F. Marshall and Joel L. Rosenbaum
9 Basal Bodies and Centrioles: Their Function and Structure Andrea M. Preble, Thomas M. Giddings, Jr., and Susan K. Dutcher
10 Centriole Duplication and Maturation in Animal Cells B. M. H. Lange, A. J. Faragher, P. March, and K. Gull
11 Centrosome Replication in Somatic Cells: The Significance of the G1 Phase Ron Balczon
12 The Coordination of Centrosome Reproduction with Nuclear Events during the Cell Cycle Greenfield Sluder and Edward H. Hinchcliffe
13 Regulating Centrosomes by Protein Phosphorylation Andrew M. Fry, Thibault Mayor, and Erich A. Nigg
14 The Role of the Centrosome in the Development of Malignant Tumors Wilma L. Lingle and Jeffrey L. Salisbury
15 The Centrosome-Associated Aurora/IpI-like Kinase Family T. M. Goepfert and B. R. Brinkley
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Contents of Previous Volumes
16 Centrosome Reduction during Mammalian Spermiogenesis G. Manandhar, C. Simerly, and G. Schatten
17 The Centrosome of the Early C. elegans Embryo: Inheritance, Assembly, Replication, and Developmental Roles Kevin F. O’Connell
18 The Centrosome in Drosophila Oocyte Development Timothy L. Megraw and Thomas C. Kaufman
19 The Centrosome in Early Drosophila Embryogenesis W. F. Rothwell and W. Sullivan
20 Centrosome Maturation Robert E. Palazzo, Jacalyn M. Vogel, Bradley J. Schnackenberg, Dawn R. Hull, and Xingyong Wu
Volume 50 1 Patterning the Early Sea Urchin Embryo Charles A. Ettensohn and Hyla C. Sweet
2 Turning Mesoderm into Blood: The Formation of Hematopoietic Stem Cells during Embryogenesis Alan J. Davidson and Leonard I. Zon
3 Mechanisms of Plant Embryo Development Shunong Bai, Lingjing Chen, Mary Alice Yund, and Zinmay Rence Sung
4 Sperm-Mediated Gene Transfer Anthony W. S. Chan, C. Marc Luetjens, and Gerald P. Schatten
5 Gonocyte–Sertoli Cell Interactions during Development of the Neonatal Rodent Testis Joanne M. Orth, William F. Jester, Ling-Hong Li, and Andrew L. Laslett
6 Attributes and Dynamics of the Endoplasmic Reticulum in Mammalian Eggs Douglas Kline
7 Germ Plasm and Molecular Determinants of Germ Cell Fate Douglas W. Houston and Mary Lou King
Contents of Previous Volumes
265
Volume 51 1 Patterning and Lineage Specification in the Amphibian Embryo Agnes P. Chan and Laurence D. Etkin
2 Transcriptional Programs Regulating Vascular Smooth Muscle Cell Development and Differentiation Michael S. Parmacek
3 Myofibroblasts: Molecular Crossdressers Gennyne A. Walker, Ivan A. Guerrero, and Leslie A. Leinwand
4 Checkpoint and DNA-Repair Proteins Are Associated with the Cores of Mammalian Meiotic Chromosomes Madalena Tarsounas and Peter B. Moens
5 Cytoskeletal and Ca2+ Regulation of Hyphal Tip Growth and Initiation Sara Torralba and I. Brent Heath
6 Pattern Formation during C. elegans Vulval Induction Minqin Wang and Paul W. Sternberg
7 A Molecular Clock Involved in Somite Segmentation Miguel Maroto and Olivier Pourquie´
Volume 52 1 Mechanism and Control of Meiotic Recombination Initiation Scott Keeney
2 Osmoregulation and Cell Volume Regulation in the Preimplantation Embryo Jay M. Baltz
3 Cell–Cell Interactions in Vascular Development Diane C. Darland and Patricia A. D’Amore
4 Genetic Regulation of Preimplantation Embryo Survival Carol M. Warner and Carol A. Brenner
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Contents of Previous Volumes
Volume 53 1 Developmental Roles and Clinical Significance of Hedgehog Signaling Andrew P. McMahon, Philip W. Ingham, and Clifford J. Tabin
2 Genomic Imprinting: Could the Chromatin Structure Be the Driving Force? Andras Paldi
3 Ontogeny of Hematopoiesis: Examining the Emergence of Hematopoietic Cells in the Vertebrate Embryo Jenna L. Galloway and Leonard I. Zon
4 Patterning the Sea Urchin Embryo: Gene Regulatory Networks, Signaling Pathways, and Cellular Interactions Lynne M. Angerer and Robert C. Angerer
Volume 54 1 Membrane Type-Matrix Metalloproteinases (MT-MMP) Stanley Zucker, Duanqing Pei, Jian Cao, and Carlos Lopez-Otin
2 Surface Association of Secreted Matrix Metalloproteinases Rafael Fridman
3 Biochemical Properties and Functions of Membrane-Anchored Metalloprotease-Disintegrin Proteins (ADAMs) J. David Becherer and Carl P. Blobel
4 Shedding of Plasma Membrane Proteins Joaquı´n Arribas and Anna Merlos-Sua´rez
5 Expression of Meprins in Health and Disease Lourdes P. Norman, Gail L. Matters, Jacqueline M. Crisman, and Judith S. Bond
6 Type II Transmembrane Serine Proteases Qingyu Wu
7 DPPIV, Seprase, and Related Serine Peptidases in Multiple Cellular Functions Wen-Tien Chen, Thomas Kelly, and Giulio Ghersi
Contents of Previous Volumes
267
8 The Secretases of Alzheimer’s Disease Michael S. Wolfe
9 Plasminogen Activation at the Cell Surface Vincent Ellis
10 Cell-Surface Cathepsin B: Understanding Its Functional Significance Dora Cavallo-Medved and Bonnie F. Sloane
11 Protease-Activated Receptors Wadie F. Bahou
12 Emmprin (CD147), a Cell Surface Regulator of Matrix Metalloproteinase Production and Function Bryan P. Toole
13 The Evolving Roles of Cell Surface Proteases in Health and Disease: Implications for Developmental, Adaptive, Inflammatory, and Neoplastic Processes Joseph A. Madri
14 Shed Membrane Vesicles and Clustering of Membrane-Bound Proteolytic Enzymes M. Letizia Vittorelli
Volume 55 1 The Dynamics of Chromosome Replication in Yeast Isabelle A. Lucas and M. K. Raghuraman
2 Micromechanical Studies of Mitotic Chromosomes M. G. Poirier and John F. Marko
3 Patterning of the Zebrafish Embryo by Nodal Signals Jennifer O. Liang and Amy L. Rubinstein
4 Folding Chromosomes in Bacteria: Examining the Role of Csp Proteins and Other Small Nucleic Acid-Binding Proteins Nancy Trun and Danielle Johnston
268
Contents of Previous Volumes
Volume 56 1 Selfishness in Moderation: Evolutionary Success of the Yeast Plasmid Soundarapandian Velmurugan, Shwetal Mehta, and Makkuni Jayaram
2 Nongenomic Actions of Androgen in Sertoli Cells William H. Walker
3 Regulation of Chromatin Structure and Gene Activity by Poly(ADP-Ribose) Polymerases Alexei Tulin, Yurli Chinenov, and Allan Spradling
4 Centrosomes and Kinetochores, Who needs ‘Em? The Role of Noncentromeric Chromatin in Spindle Assembly Priya Prakash Budde and Rebecca Heald
5 Modeling Cardiogenesis: The Challenges and Promises of 3D Reconstruction Jeffrey O. Penetcost, Claudio Silva, Maurice Pesticelli, Jr., and Kent L. Thornburg
6 Plasmid and Chromosome Traffic Control: How ParA and ParB Drive Partition Jennifer A. Surtees and Barbara E. Funnell
Volume 57 1 Molecular Conservation and Novelties in Vertebrate Ear Development B. Fritzsch and K. W. Beisel
2 Use of Mouse Genetics for Studying Inner Ear Development Elizabeth Quint and Karen P. Steel
3 Formation of the Outer and Middle Ear, Molecular Mechanisms Moise´s Mallo
4 Molecular Basis of Inner Ear Induction Stephen T. Brown, Kareen Martin, and Andrew K. Groves
5 Molecular Basis of Otic Commitment and Morphogenesis: A Role for Homeodomain-Containing Transcription Factors and Signaling Molecules Eva Bober, Silke Rinkwitz, and Heike Herbrand
Contents of Previous Volumes
269
6 Growth Factors and Early Development of Otic Neurons: Interactions between Intrinsic and Extrinsic Signals Berta Alsina, Fernando Giraldez, and Isabel Varela-Nieto
7 Neurotrophic Factors during Inner Ear Development Ulla Pirvola and Jukka Ylikoski
8 FGF Signaling in Ear Development and Innervation Tracy J. Wright and Suzanne L. Mansour
9 The Roles of Retinoic Acid during Inner Ear Development Raymond Romand
10 Hair Cell Development in Higher Vertebrates Wei-Qiang Gao
11 Cell Adhesion Molecules during Inner Ear and Hair Cell Development, Including Notch and Its Ligands Matthew W. Kelley
12 Genes Controlling the Development of the Zebrafish Inner Ear and Hair Cells Bruce B. Riley
13 Functional Development of Hair Cells Ruth Anne Eatock and Karen M. Hurley
14 The Cell Cycle and the Development and Regeneration of Hair Cells Allen F. Ryan
Volume 58 1 A Role for Endogenous Electric Fields in Wound Healing Richard Nuccitelli
2 The Role of Mitotic Checkpoint in Maintaining Genomic Stability Song-Tao Liu, Jan M. van Deursen, and Tim J. Yen
3 The Regulation of Oocyte Maturation Ekaterina Voronina and Gary M. Wessel
4 Stem Cells: A Promising Source of Pancreatic Islets for Transplantation in Type 1 Diabetes Cale N. Street, Ray V. Rajotte, and Gregory S. Korbutt
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Contents of Previous Volumes
5 Differentiation Potential of Adipose Derived Adult Stem (ADAS) Cells Jeffrey M. Gimble and Farshid Guilak
Volume 59 1 The Balbiani Body and Germ Cell Determinants: 150 Years Later Malgorzata Kloc, Szczepan Bilinski, and Laurence D. Etkin
2 Fetal–Maternal Interactions: Prenatal Psychobiological Precursors to Adaptive Infant Development Matthew F. S. X. Novak
3 Paradoxical Role of Methyl-CpG-Binding Protein 2 in Rett Syndrome Janine M. LaSalle
4 Genetic Approaches to Analyzing Mitochondrial Outer Membrane Permeability Brett H. Graham and William J. Craigen
5 Mitochondrial Dynamics in Mammals Hsiuchen Chen and David C. Chan
6 Histone Modification in Corepressor Functions Judith K. Davie and Sharon Y. R. Dent
7 Death by Abl: A Matter of Location Jiangyu Zhu and Jean Y. J. Wang
