Current Topics in Developmental Biology, Volume 67 (Current Topics in Developmental Biology)

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, Pennsylvania 15213

Editorial Board Peter Gru¨ss Max-Planck-Institute of Biophysical Chemistry Go¨ttingen, Germany

Philip 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

Contents

Contributors

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1 Deer Antlers as a Model of Mammalian Regeneration Joanna Price, Corrine Faucheux, and Steve Allen I. Introduction 2 II. Regulation of the Antler Development and Regeneration Acknowledgments 37 References 38

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2 The Molecular and Genetic Control of Leaf Senescence and Longevity in Arabidopsis Pyung Ok Lim and Hong Gil Nam I. II. III. IV. V. VI. VII. VIII.

Introduction 50 Arabidopsis as a Model Plant for Studying Leaf Senescence Senescence Symptoms 52 Identification of Senescence-Associated Genes and Their Functional Analysis 55 Regulatory Mode of Senescence-Associated Genes 62 Regulatory Factors: Molecular Genetic Regulation of Leaf Senescence 62 Biotechnological Application of Senescence 73 Conclusions and Future Challenges 73 Acknowledgments 77 References 77

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3 Cripto-1: An Oncofetal Gene with Many Faces Caterina Bianco, Luigi Strizzi, Nicola Normanno, Nadia Khan, and David S. Salomon I. Introduction 86 II. Structure and Genomic Organization of the EGF-CFC Gene Family III. Function and Expression of EGF-CFC Genes During Embryonic Development 92 IV. EGF-CFC Proteins in Mammary Gland Development 97 V. EGF-CFC Proteins in Transformation and Tumorigenesis 99 VI. Intracellular Signaling Pathways Activated by Cripto-1 103 VII. Expression of Cripto-1 in Human Carcinomas and Premalignant Lesions 109 VIII. Cripto-1 as Target for Therapy in Human Cancer 115 IX. Conclusions and Perspectives 118 Acknowledgments 120 References 121

4 Programmed Cell Death in Plant Embryogenesis Peter V. Bozhkov, Lada H. Filonova, and Maria F. Suarez I. II. III. IV. V.

Introduction 136 Model Embryonic Systems 139 Mechanics of Cell Death 154 Molecular Executioners 162 Concluding Remarks 170 Acknowledgments 171 References 171

5 Physiological Roles of Aquaporins in the Choroid Plexus Daniela Boassa and Andrea J. Yool I. II. III. IV.

Aquaporin Water Channels 182 Development of the Choroid Plexus 183 Ion Channels in the Choroid Plexus 185 Function of AQP1 as a Gated Cation Channel

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Contents V. VI. VII. VIII. IX. X. XI.

Physiological Relevance of AQP1 Ion Channels in Choroid Plexus Regulation of Cerebrospinal Fluid Production 192 Choroid Plexus ‘‘Dark Cells’’ 193 Barrier Function of the Choroid Plexus 194 Neuroendocrine Function 197 Pathophysiology of the Choroid Plexus 198 Conclusions 199 References 199

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6 Control of Food Intake Through Regulation of cAMP Allan Z. Zhao I. II. III. IV. V. VI.

Introduction 207 Regulation of Feeding at the Hypothalamus 208 Hypothalamic EVects of cAMP on Food Intake 210 cAMP as an Orexigenic Second Messenger 210 The Anorectic EVects of cAMP in the PVN 212 Regulation of Food Intake by Leptin Requires a PI3K-PDE3B-cAMP Signaling Pathway 213 VII. What Elevates the Intracellular cAMP Levels in the NPY-Neurons in a Negative Energy Balance State? 215 VIII. A Working Model for Hypothalamic Control of Food Intake Involving Regulation of cAMP—A Perspective from the NPY/AgrP Neurons 217 IX. Dysregulation of cAMP in the Hypothalamus—Implication in Obesity 218 References 218

7 Factors Affecting Male Song Evolution in Drosophila montana Anneli Hoikkala, Kirsten Klappert, and Dominique Mazzi I. Background 226 II. Male Song Variation in D. montana and Other Species of the virilis Group 231 III. Female Preferences for Male Song Characters 236 IV. Song as a Species-Recognition Signal 243

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Contents V. Summary 243 Acknowledgments References 245

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8 Prostanoids and Phosphodiesterase Inhibitors in Experimental Pulmonary Hypertension Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, and Norbert Weissmann I. II. III. IV. V. VI. VII. VIII.

Introduction 252 Animal and Organ Models of Pulmonary Hypertension 257 Prostanoids and PDE Inhibitors in Acute and Chronic Hypoxia Prostanoids and PDE Inhibitors in Monocrotaline-Induced Pulmonary Hypertension 263 Prostanoids and PDE Inhibitors in U46619-Induced Acute Pulmonary Hypertension 267 Less Frequently Used Models of Experimental Pulmonary Hypertension 270 Combination of PDE Inhibitors with Vasodilators 271 Summary and Concluding Remarks 273 Acknowledgments 273 References 274

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9 14-3-3 Protein Signaling in Development and Growth Factor Responses Daniel Thomas, Mark Guthridge, Jo Woodcock, and Angel Lopez I. II. III. IV. V. VI. VII.

Introduction 286 14-3-3: A Dimer with Phosphoserine-Binding Activity 287 14-3-3 Pathways in Drosophila Development 292 Interaction with the Ras-Raf Signaling Pathway 294 14-3-3 and Growth Factor Signaling 295 Phosphorylation of 14-3-3 by Sphingosine-Dependent Kinase Conclusions 298 References 298

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10 Skeletal Stem Cells in Regenerative Medicine Wataru Sonoyama, Carolyn Coppe, Stan Gronthos, and Songtao Shi I. II. III. IV. V. VI. VII.

Introduction 306 Isolation and Characterization of MSCs from Bone Marrow Niche Microenvironment of BMSSCs 309 Therapeutic Uses of BMSSCs 310 Delivery of BMSSCs 314 Alternative Sources of MSCs 315 Future Direction 316 References 316

Index 325 Contents of Previous Volumes

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Steve Allen (1), Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU, United Kingdom Caterina Bianco (85), Tumor Growth Factor Section, Mammary Biology & Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Daniela Boassa (181), Department of Physiology, University of Arizona College of Medicine, Tucson, Arizona 85724 Peter V. Bozhkov (135), Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden Carolyn Coppe (305), Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892 Corrina Faucheux (1), INSERM U-44 1, Pessac, France Lada H. Filonova (135), Department of Wood Science, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden Hossein Ardeschir Ghofrani (251), Department of Internal Medicine II, Justus-Liebig University Giessen, 35392 Giessen, Germany Stan Gronthos (305), Mesenchymal Stem Cell Group, Division of Haematology, Institute of Medical and Veterinary Science, Frome Road, Adelaide SA 5000, Australia Mark Guthridge (285), Cytokine Receptor Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide SA 5000, Australia Anneli Hoikkala (225), Department of Biological and Environmental Science, FIN-40014 University of Jyva¨skyla¨, Finland Nadia Khan (85), Tumor Growth Factor Section, Mammary Biology & Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute. National Institutes of Health, Bethesda, Maryland 20892

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Kirsten Klappert (225), Department of Evolutionary Biology, Dyer’s Brae House, University of St. Andrews, Fife, KY16 9th, Scotland, United Kingdom Pyung Ok Lim (49), National Research Laboratory of Plant Molecular Genetics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Korea* Angel Lopez (285), Cytokine Receptor Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide SA 5000, Australia Dominique Mazzi (225), Department of Biological and Environmental Science, FIN-40014 University of Jyva¨ skyla¨ , Finland Hong Gil Nam (49), National Research Laboratory of Plant Molecular Genetics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea Nicola Normanno (85), Division of Haematological Oncology and Department of Experimental Oncology, ITN-Fondazione Pascale, Naples 80131, Italy Joanna Price (1), Department of Veterinary Basic Sciences, The Royal Veterinary College, London NW1 OTU, United Kingdom David S. Salomon (85), Tumor Growth Factor Section, Mammary Biology & Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Ralph Theo Schermuly (251), Department of Internal Medicine II, Justus-Liebig University Giessen, 35392 Giessen, Germany Songtao Shi (305), Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892 Wataru Sonoyama (305), Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892 Luigi Strizzi (85), Tumor Growth Factor Section, Mammary Biology & Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

*Current address: Department of Science Education, Cheju National University, Jeju-si, Jeju 690-756, Korea.

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Maria F. Suarez (135), Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, SE - 750 07 Uppsala, Sweden and Departamento de Biologia Molecular y Bioquimica, Facultad de Ciencias, Universidad de Malaga, Campus de Teatinos, E-29071 Malaga, Spain Daniel Thomas (285), Cytokine Receptor Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide SA 5000, Australia Norbert Weissmann (251), Department of Internal Medicine II, JustusLiebig University Giessen, 35392 Giessen, Germany Jo Woodcock (285), Cytokine Receptor Laboratory, Division of Human Immunology, Hanson Institute, Institute of Medical and Veterinary Science, Adelaide SA 5000, Australia Andrea J. Yool (181), Department of Physiology and Department of Pharmacology, University of Arizona College of Medicine, Tucson, Arizona 85724 Allan Z. Zhao (207), Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

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Deer Antlers as a Model of Mammalian Regeneration Joanna Price,* Corrine Faucheux,{ and Steve Allen* *Department of Veterinary Basic Sciences, The Royal Veterinary College London NW1 OTU, United Kingdom { INSERM U-44 1, Pessac, France

I. Introduction A. Development of the First Set of Antlers B. Antler Regeneration C. The Early Stages D. Ontogony E. Longitudinal Growth F. Chondrogenesis G. Regenerating Antlers: Ossification and Remodeling II. Regulation of the Antler Development and Regeneration A. External and Systemic Factors B. Local Mechanisms Acknowledgments References

Deer antlers are cranial appendages that develop after birth as extensions of a permanent protuberance (pedicle) on the frontal bone. Pedicles and antlers originate from a specialized region of the frontal bone; the ‘antlerogeneic periosteum’ and the systemic cue which triggers their development in the fawn is an increase in circulating androgen. These primary antlers are then shed and regenerated the following year in a larger, more complex form. Antler growth is extremely rapid—an adult red deer can produce a pair of antlers weighing
30kg in three months, and involves both endochondral and intramembranous ossification. Since antlers are sexual secondary characteristics, their annual cycles of growth have evolved to be closely coordinated to the reproductive cycle which, in temperate species, is linked to the photoperiod. Cessation of antler growth and death of the overlying skin (velvet) coincides with a rise in circulating testosterone as the autumn breeding season approaches. The ‘dead’ antlers remain attached to the pedicle until they are shed (cast) the following spring when circulating testosterone levels fall. In red deer, the species that we study, casting of the old set of antlers is followed immediately by growth of the new set. Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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0070-2153/05 $35.00 DOI: 10.1016/S0070-2153(04)67001-3

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Although the anatomy of antler growth and the endocrine changes associated with it have been well documented, the molecular mechanisms involved remain poorly understood. The case for continuing to decipher them remains compelling, despite the obvious limitations of using deer as an experimental model, because this research will help provide insight into why humans and other mammals have lost the ability to regenerate organs. From the information so far available, it would appear that the signaling pathways that control the development of skeletal elements are recapitulated in regenerating antlers. This apparent lack of any specific ‘antlerogenic molecular machinery’ suggests that the secret of deers’ ability to regenerate antlers lies in the particular cues to which multipotential progenitor/ stem cells in an antler’s ‘regeneration territory’ are exposed. This in turn suggests that with appropriate manipulation of the environment, pluripotential cells in other adult mammalian tissues could be stimulated to increase the healing capacity of organs, even if not to regenerate them completely. The need for replacement organs in humans is substantial. The benefits of increasing individuals’ own capacity for regeneration and repair are self evident. C 2005, Elsevier Inc.

I. Introduction Since ancient times deer antlers have held a fascination for humans as beautiful and spectacular works of nature and as symbols of male superiority and strength. Human alpha males hang antlers on walls as a demonstration of their own wealth and power and because they represent male strength and virility. Extracts of deer antler have also been used for centuries as components of oriental medicine. In many parts of the world, deer are now farmed for the production of antler velvet and this industry has undoubtedly helped rejuvenate antler research in recent years. Antlers have also long been a focus of scientific interest because, while the study of antlers is relevant to many areas of biology—bone biology, developmental biology, zoology, evolutionary biology, and endocrinology—it is their ability to regenerate that makes antlers so important. Mammals have a very limited regenerative ability, whereas most other phyla which include some species which can regenerate large sections of their body plan after injury or amputation. The study of antlers can help shed light on why this may be the case. However, the limitation of antlers for investigating the molecular processes of regeneration must not be overlooked. Few deer gene sequences are known, there is no ‘‘deer genome project’’ on the horizon, and transgenic deer are unlikely to exist outside the realm of science fiction. Although genomic and proteomic approaches are currently being used to identify molecules expressed in antlers, establishing function will always be a

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challenge. As well as being genetically intractable, deer are large wild animals that require specialized management. Notwithstanding, despite not being a mainstream laboratory animal, deer are the only mammals that can regenerate complete appendages and so deserve to retain a place in regeneration research. Regenerative medicine is an expanding field, and, as discussed by Brockes and Martin (2004) at a recent Royal Society discussion meeting on tissue repair and regeneration, the continued study of a variety of natural examples of regeneration can only increase the prospects for the restoration of functional tissues and organs in humans. A. Development of the First Set of Antlers Except in the genus Rangifer (reindeer), antlers develop only in male deer and, in most species, this occurs in the spring of the animal’s second year of life. Thus, while parallels are frequently drawn between antlers and other developing appendages such as limbs, antlers are unique since they develop after birth. Antlers therefore provide a unique model for studying the mechanisms that control the development of a complete bony appendage from tissues that have presumably completed a developmental program. Antlers grow from pedicles, secondary sexual characteristics that are outgrowths of the frontal bone (Fig. 1), and it is from the pedicles that antlers are shed and regenerate each year. However, the presence of a pedicle bone is not an absolute requirement for antler formation because pedicle amputation in a number of species has been shown not to prevent subsequent antler

Figure 1 Two-year-old red deer stag with a set of regenerated antlers at 75 days of growth. The pedicle, the permanent extension of the frontal bone, is marked with the arrow. At this stage of the growth cycle, antlers continue to elongate at the distal tip, but growth in more proximal branches (tines) has stopped.

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development, although the pedicles themselves did not always regenerate (Bubenik and Pavalansky, 1965; Goss, 1961; Jaczewski, 1954, 1955). Li et al. (2001b) have described a potentially interesting relationship between pedicle height and antler phylogeny; more highly evolved deer species, which have larger and more complex antlers, have shorter pedicles, although the functional significance of this is not known. One of the first important issues that antler biologists sought to address was the identity of the tissue(s) that are responsible for initiating pedicle and antler development. A series of transplantation experiments, many carried out several decades ago, demonstrated that periosteum is the tissue involved. Excision of skin and subcutaneous tissues from the frontal bone in young fawns was found to have no eVect on antler development whereas resection of periosteum and surrounding bone prevented development (Goss and Powel, 1985; Goss et al., 1964; Hartwig, 1967; Hartwig and Schrudde, 1974). Transplantation of periosteum to another site on the frontal bone led to antler development at this new location, but not always at the original site (Hartwig, 1968). Hartwig and Schrudde (1974) also showed that transplantation of periosteum to the leg resulted in the development of small antlers which display an annual cycle of growth. Kierdorf and Kierdorf (2000) repeated this experiment and found no ectopic growth until nine years after transplantation and they concluded that the pedicle had to attain a minimum size before an antler could form. This tissue was first described as ‘‘antlerogenic periosteum’’ (AP) (Goss, 1983) and it has been extensively studied and reviewed by Kierdorf and Kierdorf (2001) and by Li and Suttie (2001). Li et al. (2001a) showed that structures resembling antlers, or pedicle-antlers, could be generated if AP is transplanted over the calvarial bones of a nude mouse. As will be discussed in more detail in a later section, an adult derivative of this antlerogeneic periosteum is likely to be the source of progenitor cells from which some, if not all, regenerating antler tissues are derived. Even during fetal life, the sites where future antlers are destined to grow are apparent as small bony elevations on the lateral crests of the frontal bone of the skull (Lincoln, 1973). These anlage of pedicle enlarge between 55 and 100 days of gestation but regress in later stages (Li and Suttie, 2001). However, after birth, the periosteum at this site remains thicker than in other locations on the skull. Initially, the bone beneath this periosteum is made up of flattened plates, characteristic of cranial bones. However, as androgen levels increase at the time of puberty, new trabeculae form beneath the periosteum and a visible pedicle forms (Sempere and Boisson, 1983; Suttie et al., 1984, 1991). Histological studies in red deer by Li and Suttie (1994) have shown that pedicle formation is made up of four ossification stages: (1) intramembranous ossification (direct formation of bone by osteoblasts insignaling cellular periosteum), (2) transitional ossification (formation of osseocartilaginous tissue), (3) pedicle endochondral ossification (when only

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chondrogenesis takes place in the pedicle), and (4) antler endochondral ossification (continued chondrogenesis and the appearance of antler velvet). In vitro studies have shown that insulin-like growth factor I (IGF-I) may be an important systemic regulator of pedicle formation as it stimulates proliferation of antlerogeneic cells from all four ossification stages. Interestingly, testosterone alone has no mitogenic eVect on these cells, even though there are specific binding sites for testosterone in this periosteum; however, in combination with IGF-I, it stimulates proliferation during stages 1 and 2 of pedicle ossification but reduces proliferation of cells from the fourth stage (Li et al., 1999). Initially, pedicles are covered by skin that is identical to that covering the rest of the skull. However, the transition between pedicle growth and antler development (stage 4) is marked by the appearance of characteristic antler skin described as ‘‘velvet’’ (Fig. 1). Velvet contains fewer hair follicles than normal skin does, but each of these has a sebaceous gland associated with it which gives it a ‘‘shiny’’ appearance and it contains no erector pili muscles. These first antlers then continue to elongate, generally as single unbranched spikes, by a modified endochondral process. However, a rise in circulating testosterone levels in autumn leads to cessation of growth, mineralization of the antler, and the consequent shedding of the velvet skin. This leaves a single unbranched antler (Fig. 2) attached to the pedicle until it is shed (cast) the following spring.

Figure 2 The first set of unbranched antlers grown by a red deer stag. These have completed their development and the velvet skin covering them has been shed.

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While the development of the pedicle and first antler has been described at a histological and ultrastructural level (Li and Suttie, 1994, 1998, 2000), the molecular mechanisms that regulate these processes have been little studied, not least because such studies present practical and ethical challenges. For example, a molecular characterization of antlerogeneic periosteum at diVerent developmental stages (before and after birth) needs to be undertaken to help understand the biology of this fascinating tissue. Comparing the molecular pathways which control development of the first antler and regeneration of subsequent antlers may also shed light on the extent to which developmental and regenerative pathways diverge. Furthermore, regenerating antlers in most deer species have branches whereas the first antlers to develop are single spikes and therefore such comparative studies could help to identify molecules that specifically control antler patterning, as distinct from those that control increases in antler size.

B. Antler Regeneration At this stage, it is worth considering why antlers regenerate. A number of explanations have been put forward and the interested reader should refer to Richard Goss’s review of the subject (Goss, 1983). One theory, originally proposed by John Hunter in the eighteenth century, was that stags shed their antlers before calves are born and are thus less likely to cause them harm. Perhaps a more rational explanation may lie in the adage that ‘‘size matters’’; antlers are secondary sexual characteristics and their function is to enable stags to achieve and maintain dominance over a harem of females. Regeneration enables antlers to increase in size each year as stags become more mature; it has been calculated that in red deer, antler size increases one and a half times as much as body size during the course of maturation (Huxley, 1926, 1931). An ability to regrow antlers damaged during fighting is also likely to have conferred a selective advantage. Interestingly, a study in red deer has shown that antler size is heritable and that stags with bigger antlers are the most successful breeders (Kruuk et al., 2002). However, over the 30-year study period, this selection did not generate an evolutionary response in antler size. This led the authors to conclude that environmental factors, in particular, nutrition, also have an important influence on antler size and success in combat (Kruuk et al., 2002). The annual cycle of antler loss and regrowth may also have evolved when deer moved to inhabit more temperate zones. If antlers were to retain their blood supply and continue to grow throughout the year, freezing weather conditions in winter would inevitably lead to tissue damage and necrosis. This could be one explanation for why antlers mineralize and become ‘‘dead’’ during the winter months. Another physiological challenge facing

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deer living in temperate habitats is the significant loss of body mass that takes place during winter. For deer stags (and other short-day breeders such as rams), this presents a particular problem as they have already lost a significant amount of body fat during the few frenzied weeks of sexual activity in autumn. Perhaps this explains why nature has developed a system that releases deer stags from the ‘‘tyranny of testosterone’’ for a few months during the summer, thus enabling them to increase their body mass and, at the same time, grow another set of antlers in time for the next mating season.

C. The Early Stages As described previously, a stag’s first antlers are normally shed in the spring of his second year of life and the process of antler casting has been shrouded in mystery for centuries because lost antlers are rarely found in the wild. In the Middle Ages, folklore had it that stags would deliberately hide their antlers in dark, concealed places in a forest whereas a more likely explanation is that they are tempting food for wild carnivores. Casting is a spontaneous event that appears not to be anticipated by the animal and involves osteoclasts resorbing bone at the interface between the solid antler and the pedicle. In most species, casting is coincident with regeneration; in fact, the velvet skin of the new antler is visible as a swollen ring around the base of the old antler (Fig. 3A). The local mechanisms controlling the process are far from clear although the process is regulated by a decline in circulating testosterone; a number of experimental studies have shown that deer maintained on artificially high levels of testosterone or estrogen do not cast their antlers and castration will result in casting at any time of year (Fletcher, 1978; Goss, 1968; Waldo and Wislocki, 1951). Thus, the trigger for casting appears to be increased osteoclast activity as a consequence of sex hormone withdrawal. As will be discussed in a later section, there is increasing evidence that the testosterone’s eVects on bone cells may be indirect following conversion by aromatase to estrogen (Meinhardt and Mullis, 2002) and we have some evidence that this conversion may also take place in antler tissues. It is known from studies in man and other animals that a decline in estrogen will increase bone resorption and that this is mediated by various cytokines that regulate osteoclast formation and activity (Riggs et al., 2002). Studies in man have shown that estrogen withdrawal is associated with increased synthesis of receptor activator of NF-kB ligand (RANKL), a potent activator of osteoclast diVerentiation (Eghbali-Fatourechi et al., 2003). We have found that RANKL is localized in cells in the blastema (Price and Allen, unpublished observations), although how its synthesis is systemically regulated needs investigation. The surface of the shed antler is concave and has no apparent blood supply (Fig. 3B), whereas the exposed surface of the pedicle bleeds

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Figure 3 Early stages of antler regeneration. (A) Pedicle (arrowhead) with the first antler still attached. The first signs of regeneration are evident as a ‘‘ring’’ of swollen tissue (arrow) at the base of the old antler. (B) Ventral convex surface of a cast antler that has been resorbed by osteoclasts. (C) The regenerating antler a few hours after casting showing the velvet skin (arrows) migrating over the surface of the exposed pedicle. (D) Antler bud 14 days after casting. The epidermis now covers most of the antler surface. The position of the future branches are visible as raised areas (arrows). (E) Section through the central region of a 4-day ‘‘blastema.’’ The wound epithelium (WE) can be seen under the scab migrating over a mass of ‘‘granulationlike’’ tissue and undiVerentiated mesenchymal cells. (F) Higher-power view of boxed region in (E), showing this cellular tissue which contains little extracellular matrix. (G) Section through the outer region of a 4-day blastema showing the transition from wound epithelium (WE) to mature epidermis containing hair follicles (H) and sebaceous glands (S). Scale bars: E,G, 200
m; F, 50
m.

and, within hours, a large scab forms and covers it (Fig. 3C). As in other situations where appendages regenerate, healing of an epidermal wound is required for the process to be initiated. A migratory epithelial layer rapidly covers the exposed pedicle (Fig. 3E) and within 7 to 9 days, epithelialization is complete and after approximately 10 to 14 days, future branches become visible as swollen raised areas at the periphery of this antler ‘‘bud’’ (Fig. 3D). Identifying the source of cells below the zone of amputation that develop into the early antler has been a subject of debate for many years. The tissues that make up the pedicle are skin, bone, periosteum, blood vessels, and nerves. Unlike the situation in the regenerating urodele limb, there is no muscle. Early experimental work involved amputation of the pedicle and/or surrounding regions of the skull (Goss, 1961; Jaczewski, 1955) and this showed that the ‘‘regeneration territory’’ is extensive; however, these surgical procedures involved significant trauma, which makes their interpretation diYcult. There have been no reports that transplantation of pedicle periosteum from a regenerating antler can induce antler growth at another site. However, what became clear from these transplantation studies was that skin of the pedicle cannot give rise to antler tissue; velvet skin transplanted to other sites will survive for several years but does not give rise to antler

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outgrowths (Goss, 1972). In one rather ingenious experiment, ear skin was transplanted onto the distal pedicle bone, which prevented pedicle skin from contributing to formation of the new antler (Goss, 1964). A normal antler formed and it was covered with antler velvet skin, which showed that epidermal diVerentiation is induced by underlying mesenchymal tissues. Notwithstanding, despite the lack of direct evidence, many antler biologists are of the view that the pedicle periosteum is likely to be a source of cells that form the regenerating antler (Kierdorf and Kierdorf, 1992, 2001; Li et al., 2004a,b). What is the evidence? Kierdorf et al. (1994) have shown that double head antlers (these form when the old antler fails to be cast) develop from the pedicle. More recently, Kierdorf et al. (2003) described thickening of the periosteum of the distal pedicle soon after antler casting, which would also suggest expansion of cell populations in this tissue. Our finding that parathyroid hormone-related peptide (PTHrP) is immunolocalized in both periosteum and in mesenchymal cells in the blastema is further evidence that the latter may be derived from the former (Faucheux et al., 1994). Li et al. (2004, 2005) have recently presented convincing morphological evidence that the pedicle bone appears not to contribute to the formation of the early antler and that ‘‘stem’’ cells from the pedicle periosteum proliferate and migrate onto the exposed pedicle surface following antler casting. This has led them to challenge the validity of using the term ‘‘blastema’’ in the context of antlers because they have concluded that cells which form the regenerating antler are stem cell-derived and do not appear to arise by a process of dediVerentiation (Li et al., 2004b, 2005). In contrast, a series of elegant experiments have shown that in newts, cells in the blastema arise by a process of dediVerentiation of mature cell types, including neural ependymal cells and multinucleated muscle fibers (Echeverri and Tanaka, 2002; Echeverri et al., 2001; Kumar et al., 2000; Lo et al., 1993). Thus, if a blastema is defined as a structure that arises exclusively by a process of dediVerentiation, then Li and colleagues are correct and the term should not be used to describe the early stage of antler regeneration. However, Richard Goss, the eminent regeneration biologist, in his excellent book on antler biology (1983), described antlers as ‘‘histologically complex structures derived from a mass of undiVerentiated cells that fits the definition of a blastema’’ and we have also recently described the early antler (<14 days) as a blastema. Goss’s definition of a blastema is ‘‘a rounded mass of cells endowed with the capacity to develop into structure replacing that which was lost’’ (Goss, 1983) and the Collins English Dictionary defines a blastema as ‘‘a mass of undiVerentiated animal cells that will develop into an organ or tissue: present at the site of regeneration of a lost part.’’ Thus, in our opinion, it remains valid to describe specific regions of the early antler as a blastema, or blastema-like, since they contain undiVerentiated cells; the fact that these cells may not arise by a process of dediVerentiation is not relevant. It is worth noting that there is no evidence

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Figure 4 Histology of a 14-day blastema and PCNA staining to show proliferating cells. (A) Section through a blastema at one of the future branches, showing that tissues at diVerent stages of diVerentiation can be distinguished: perichondrium (arrow), undiVerentiated mesenchymal cells (m), chondroprogenitors (cp), cartilage (ca), and bone (bo). Histology (B, D, F, H) and PCNA staining (dark brown nuclei in C, E, G, I) in the diVerent regions of the blastema. (B, C) Mesenchyme: In this very cellular region, a large number of undiVerentiated mesenchymal cells are proliferating; this is the equivalent of the ‘‘growth zone’’ in the growing tip of larger antlers. (D, E) Chondroprogenitors (cp): These cells are no longer dividing and become aligned in columns between which are vascular channels (v); however, there is cell

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for dediVerentiation and metaplasia in the regenerating tail of xenopus, where regeneration resembles normal tissue renewal, and yet it is described as containing a blastema of undiVerentiated tissue (Slack et al., 2004). However, we agree that it is incorrect to describe the entire early antler as a blastema; the term ‘‘bud’’ is more appropriate. It has proved diYcult to establish which cells in the pedicle give rise to regenerating antler tissues because studies of cell lineage, such as those applied in other models of regeneration, are practically diYcult to perform in antlers in vivo. For example, transgenic technology was used to establish cell lineage determination in the xenopus tail (Gargioli and Slack, 2004). In a review paper, Li and Suttie (2001) briefly describe an experiment in which they labeled antlerogeneic periosteum (AP) cells in vivo with LacZ and concluded from this that all cell types in the pedicle and primary antler appear to be the progeny of AP cells. What is not known, because the appropriate studies have not yet been done, is whether cells in the regeneration are derived from true stem cells, as suggested by Li et al. (2004), a strategy used by planaria (Agata et al., 2003; Sanchez Alvarado, 2003, 2004), from multipotent mesenchymal cells in the periosteum (Nakase et al., 1993), or from dediVerentiation of periosteal fibroblasts, osteoblasts, dermal fibroblasts, or nerve cells in the pedicle. Further transplantation studies could help to resolve this issue and these could involve not only whole tissue explants but populations of purified cell types isolated from the pedicle. Genomic and proteomic analyses of cells from antlerogenic petriosteum and its adult derivative would also help to identify the properties of these tissues. The anatomy and histology of the early antler ‘bud’ has recently been described by Li et al. (2004b, 2005) and clearly this is not a morphologically uniform structure like the blastema of regenerating newt limb (Brockes, 1997). For the first few days after casting, the center surface of the pedicle is composed of a granulation-like tissue and interspersed mesenchymal cells below a scab and regenerating wound epithelium (Fig. 3E, F). In contrast, at the periphery of the ‘‘doughnut’’, the pedicle skin is full thickness and contains appendages including hair follicles and sweat glands (Fig. 3G). There is little cell division at this stage and many cells express PTHrP. However, between days 3 and 4 and days 10–14 after casting, there is a significant increase in the number of mesenchymal cells, epithelialization is completed, and diVerent tissue regions are visible in tissue sections (Fig. 4A). There are distinct, relatively avascular ‘‘growth zones’’ of mesenchymal cells beneath the epidermis at sites where the main and subsidiary branches will division associated with perivascular tissue. (F, G) Cartilage: The mature chondrocytes (ca) are surrounded by extracellular matrix and do not divide. (H, I) Bone: In the proximal region where bone (b) is being formed, a proportion of osteoblasts (arrow) are proliferating. Scale bars: A, 1 cm; B, 12.5
m; C, E–I, 25
m; D, 50
m.

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Figure 5 Immunolocalization of parathyroid hormone-related peptide (PTHrP) in the early regenerating antler. Section through a blastema at day 14. (A) Skin: PTHrP is localized (brown stain) in epidermis (e), dermis (d), and around hair follicles (h). (B) Mesenchyme (m)/ chondroprogenitors (cp): The majority of cells stain positive. (C) Cartilage (ca): PTHrP is not present in mature chondrocytes but is localized in perivascular cells. (D) Cellular periosteum: As in the mesenchymal zone, a significant proportion of cells stain positive. Scale bars: A, 100
m; B, 50
m; C, D, 25
m.

develop (Fig. 4B). These cells are proliferating (Fig. 4C) and are not highly diVerentiated as they do not express alkaline phosphatase (ALP), a marker of diVerentiated chondrocytes and osteoblasts. They also express PTHrP (Fig. 5A), which we consider to be a phenotypic marker for antler progenitor cells (Faucheux et al., 2004). Proximally, these mesenchymal cells diVerentiate into chondroblasts that align themselves into columns (Fig. 4D) interspersed with vascular channels. PCNA staining shows that these chondroblasts are not proliferating, although a population of PCNA-positive cells are seen lining vascular spaces. These may be proliferating endothelial cells associated with de novo angiogenesis (Clark et al., 2004) or could be derived from perichondrium/periosteum since RAR mRNA is expressed in the perivascular region of the blastema (Price and Allen, 2004) and in perichondrium (Allen et al., 2002). Lower down the antler bud, these chondroblasts diVerentiate into chondrocytes synthesizing cartilage matrix and this is associated with an increase in ALP activity and decreased synthesis of PTHrP (Fig. 5C). Coincident with the onset of chondrogenesis, increased remodeling activity by osteoclasts and osteoblasts leads to restoration of the integrity of resorbed bony trabeculae on the pedicle surface (Kierdorf et al., 2003). If blastema formation is a defining feature of epimorphic regeneration, so is the presence of a healing wound (Goss, 1983) and antlers will not regenerate if skin is grafted over the pedicle (Goss, 1972). This distinguishes epimorphic regeneration from the process of physiological regeneration

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(e.g., the replacement of red blood cells or skin cells) or tissue repair. For example, fracture repair involves regeneration of bone that is structurally and functionally normal but does not involve wound healing, and thus cannot be defined as epimorphic regeneration. Identification of the local factors that trigger regeneration in response to antler casting (the equivalent of amputation or injury in a newt) is clearly a priority. Pointers can be taken from work in other models of regeneration on the link between regeneration and tissue injury (Brockes, 1997; Brockes et al., 2001). For example, Jeremy Brockes’s group have identified activated thrombin, a critical regulator of the injury response, as being able to regulate cell cycle re-entry in newt myotubes (Tanaka et al., 1999) and in pigment epithelial cells (Simon and Brockes, 2002).

D. Ontogony Antlers elongate with a typical S-shape growth curve (Fig. 6). In the first month to 6 weeks, the branches of a regenerating antler form (Fig. 7) and, at this stage, the growth rate is relatively slow; however, during the summer, growth accelerates rapidly, then slows as autumn approaches. In larger species of deer, this rate of growth is spectacular; for example, in a moose,

Figure 6 The relationship between circulating testosterone (solid line) and antler growth (dotted line). Antlers are cast in the spring when circulating testosterone levels are low. In red deer, the early stages of antler growth do not appear to be dependent on sex steroids. However, as testosterone levels rise in late summer, longitudinal growth of the antlers slows, the bone becomes completely mineralized, and the velvet skin is shed. Polished hard antlers are then used for fighting during the autumn mating season.

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Figure 7 Two-year-old red deer stag with a set of regenerated antlers at approximately 30 days of growth. The major branches (tines) are now visible. The main branch (mt) is most caudal and the brow tine (arrow) most cranial.

antlers can grow up to 1.25 meters long and have a span that exceeds 2 meters. The shape and size is species dependent; small brocket deer which inhabit forests in South America, have simple unbranched antlers, species like red deer and wopiti, have complex branched ‘‘racks’’ (Fig. 1), whereas fallow deer and moose have palmate antlers. The main branch (beam) continues to elongate until the antler’s final size is achieved, whereas proximal branches (tines) of an antler will complete their development and become fully mineralized before the distal branches. Like the developing limb, antlers have a proximodistal axis, an anterioposterior axis, and a dorsoventral axis (Li and Suttie, 2001). Richard Goss demonstrated (1991) that cells in the antlerogenic periosteum control formation of the anteriorposterior axis, as he was able to show that rotation of the periosteum through 180 degrees resulted in antlers forming in a reversed orientation. The signaling molecules that control antler patterning are not known. One possible candidate is retinoic acid (RA), which controls formation of the zone of polarizing activity in the developing limb (Lu et al., 1997) and in the regenerating newt limb induces digit duplication (Scadding and Maden, 1986; Sessions et al., 1999) and conveys positional information (Maden et al., 1982; Pecorino et al., 1996). We have identified retinoic acids and retinoic acid receptors in the early antler bud (Price and Allen, 2004) and application of RA to the pedicle changes antler shape (Kierdorf and Kierdorf, 1998). PTHrP is another candidate, as it is expressed by a large proportion of mesenchymal cells in the antler blastema (Faucheux et al., 2004) and in the mammary gland regulates branching morphogenesis (Wysolmerski et al., 1995). Sonic hedgehog (Shh) is a key regulator of anterioposterior patterning in the limb, however, although we have detected

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the expression of indion hedgehog at sites of chondrogenesis in the antler (Faucheux et al., 2004), we have been unable to detect the expression Shh by RT-PCR. However, we suspect this may be a technical issue; antlers are large structures and if the site of Shh expression is very restricted and/or if its levels of expression are low, it would be easy to miss the critical tissue in any analysis. Alternatively, the lack of expression of Shh may reflect the diVerent embryological origins of the antler from the limb; as frontal bone derivatives antlers are likely to be neural crest-derived (although this has yet to be demonstrated) and Shh’s role in regulating patterning of head structures that are neural crest-derived is limited (Ahlgren and Bronner-Fraser, 1999; Hu and Helms, 1999). As far as the dorsoventral axis is concerned, Wnt-7a is an important regulator in the developing limb and although its expression has yet to be identified in the antler, we have evidence that other components of the Wnt signaling pathway are expressed in antlers (James Mount, unpublished observations).

E. Longitudinal Growth Longitudinal growth of antlers takes place at the distal tip of each branch (Fig. 8, Fig. 9), where mesenchymal cells proliferate and subsequently diVerentiate into chondrocytes. The cartilage zone is very extensive and consists of chondrocytes arranged in trabeculae which proximally become mineralized and then form a scaVold for new bone formation. At the same time, new bone is laid down circumferentially along the antler shaft by intramembranous ossification. Thus, the whole temporal and spatial sequence of cellular diVerentiation that takes place during endochondral and intramembranous bone growth is represented in one antler branch. Inevitably, the anatomy of the growing antler tip has been compared to that of the epiphyseal growth plate but there are a number of important diVerences. What is immediately apparent, even with the naked eye, is that the antler tip is a highly vascularized structure (Fig. 8), whereas epiphyseal cartilage is not. The main artery supplying the antler is a branch of the temporal artery whose branches and anastomoses remain superficial within the dermis and direct blood to the distal tip. Blood is then channeled vertically downward into cartilage and bone. Blood in the large vascular spaces in the center of the antler then drains into the pedicle or into large veins in the velvet skin. Another important diVerence between the antler and a growth plate is that the separation of cells into defined zones is far less distinct. In the antler tip, there is a region where cells proliferate (Faucheux et al., 2004; Matich et al., 2003); however, these cells are not diVerentiated whereas cells in the proliferating zone of the growth plate are chondrocytes (Farnum and Wilsman, 1993; Loveridge and Farquharson, 1993). This ‘‘growth zone’’ is

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Figure 8 Histology of the growing tip of a regenerating antler. (A) Longitudinal section through the distal end of the main branch of an antler at approximately 35 days of growth. vs: velvet skin, m: mesenchyme, cp: chondroprogenitors, ca: cartilage, min ca: mineralized cartilage, ps: primary spongiosa, # sites of intramembranous bone formation below the periosteum. (B) Section through skin: epidermis (e), dermis (d), hair follicles (h), and sebaceous glands (s). (C) The junction between fibrous perichondrium (fpc) and mesenchyme (m). (D) Junction between fibrous periosteum (fpo) and cellular periosteum (cpo). (E) Shows intramembranous ossification at the periphery of the antler shaft; new bone (b) is formed by osteoblasts (arrows). (F) Region of chondroprogenitors (cp), vascular channel (v). (G) cartilage (ca). (H) Junction between mineralized cartilage and primary spongiosa. Cartilage trabeculae are resorbed by osteoclasts (shown by arrows at higher power in inset box) and new bone is being formed by osteoblasts (asterisk). (I) Von Kossa-stained section (brown/black) to show mineralization of cartilage trabeculae. (J) Spongy bone (b) in the midshaft of the antler. Scale bars: A, 2 cm; B, 400
m; C-J, 50
m; insets H, J, 10
m.

Figure 9 Characteristics of mesenchymal cells in a rapidly growing regenerating antler. (A) Cryostat section through an antler tip showing low levels of staining for alkaline phosphatase (ALP (red stain)) in skin (s), perichondrium (p), and mesenchyme (m). ALP activity increases in cartilage (c). (B) Cells cultured from this region show low levels of staining for ALP. (C) In the presence of betaglycerol phosphate (BGP) and ascorbic acid, long-term cultures of mesenchymal cells will form mineralized nodules as detected by Von Kossa staining (black, indicated by asterisk). Scale bars: A, 1 cm; B, 10
m; C, 50
m.

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a relatively avascular region composed of tightly packed small cells (Fig. 8C) below a fibrous perichondrium which is continuous with the fibrous periosteum covering the lower antler shaft. Interestingly, whereas type I collagen mRNA (a phenotypic marker of cells of the osteoblast and fibroblast lineages) is highly expressed in the fibrous perichondrium, the hybridization signal is much weaker in these mesenchymal cells, consistent with there being little matrix synthesis (no metachromatic staining). The nomenclature used to describe this region can be somewhat confusing. It has been variously described as ‘‘hyperplastic perichondrium’’ and ‘‘reserve mesenchyme’’ (Banks and Newbrey, 1983), and we also describe this as the mesenchymal zone. However, we have also, perhaps rather confusingly, described cells cultured from the site as being ‘‘perichondrium-derived’’ because they are dissected from below the fibrous perichondrium. PCNA staining has shown that in both the regenerating and developing antlers these mesenchymal cells are dividing rapidly (Faucheux et al., 1994; Matich et al., 2003) and, in culture, these cells (Fig. 9) have a doubling time of 24 to 48 hours and will even continue to proliferate in low concentrations of serum (Price and Faucheux, 2001). We have also shown that these cells have an extended life span in culture (they can be cultured for over 80 passages), a characteristic which may underpin the ability of antlers to grow at such phenomenal rates (Price and Faucheux, 2001). In vivo and in vitro (Fig. 9A), these cells express only low levels of alkaline phosphatase (Price and Faucheux, 2001; Price et al., 1994) and do not express type II collagen (Price et al., 1996) or aggrecan protein, which reflects their lack of diVerentiation. In fact, like stromal cells derived from bone marrow (Huttmann et al., 2003), these cells appear to have the capacity to diVerentiate along more than one lineage. For example, dexamethasone, a factor that is known to induce osteoblast diVerentiation in marrow stromal cells (Beresford et al., 1994), will induce alkaline phosphatase in antler mesenchymal cells (Price and Faucheux, 2001). In contrast, when cultured in the presence of rabbit serum, adipocyte-like cells will diVerentiate. When cultured for longer periods in the presence of betaglycerol phosphate and ascorbate, these cells will also form mineralized collagenous nodules (Fig. 9C). Factors that regulate the growth of these cells will be discussed in a later section.

F. Chondrogenesis For many years, there was controversy among antler biologists as to the type of ossification taking place in growing antlers. For example, only 24 years ago, Beresford (1980) classified unmineralized tissues in antler as chondroid bone and concluded that antler cartilage was neither hyaline, elastic, nor fibrocartilage. In contrast, a series of histological and ultrastructural studies

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concluded that antler development involved a modified form of endochondral ossification and intramembranous ossification (Banks, 1974; Frasier and Banks, 1973; Frasier et al., 1975). The problem was that, until 1996, only types I and III collagen had been reported to be present in antler cartilage (Newbrey et al., 1983; Speer, 1983), and these collagens are not characteristic of normal hyaline cartilage. However, we were able to demonstrate by in situ hybridization and immunocytochemistry that antler chondrocytes express type II collagen (Fig. 10E), a marker of the chondrocyte phenotype (Price et al., 1996) and type II collagen has also been identified

Figure 10 Characteristics of the cartilage region of a rapidly growing regenerating antler. C: cartilage. V: vascular spaces. (A) In situ hybridization for collagen I (black silver grains) shows that expression is maintained in the ‘‘transition zone’’ where cells start to diVerentiate into chondroprogenitors; note that cells start to become aligned in columns. (B) In situ hybridization for collagen I (black silver grains) shows that expression is now restricted to cells in perivascular tissue that are likely to be of the osteoblast lineage. (C) Alcian blue staining to show proteoglycans in cartilage matrix. (D) Staining for alkaline phosphatase in cryostat sections (red) shows many positive cells associated with the vascular channels. (E, F, G, H) Immunolocalization of collagen types II (E), VI (F), X (G), and aggrecan in cartilage (red stain).

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biochemically, albeit at low levels (Rucklidge et al., 1997). Histologically the boundary between the zone of dividing mesenchymal cells and the onset of chondrogenetic diVerentiation is not distinct; there is a gradual increase in cell size, decreased proliferation, increased synthesis of extracellular matrix, and cells begin to assume a columnar arrangement (Figs. 8 and 10A). We define the onset of chondrogenic diVerentiation as where cells start to express type IIA collagen mRNA, which transiently precedes the onset of type IIB collagen expression (Price et al., 1996). Type IIA collagen is a splice form of type II procollagen synthesized primarily by chondroprogenitor cells in developing cartilage but is not expressed by mature growth plate chondrocytes (Sandell et al., 1994, 2001). However, chondroprogenitors in this region continue to express type I collagen mRNA (Fig. 10A) and thus this is a ‘‘transition zone’’ with poorly defined boundaries. Interestingly, the synthesis of type I and type II collagen has been demonstrated in the cartilage blastema of the developing chick limb between stages 24 and 26 and the predominant form of type II procollagen is the type IIA isoform (Nah et al., 1992). However, whereas type IIB collagen mRNA expression is maintained in chondrocytes throughout antler cartilage, the expression of type IIA is transient; it only persists into the upper regions of longitudinal trabeculae (Price et al., 1996). Intensity of type II collagen immunoreactivity also increases as chondrocytes hypertrophy in more proximal trabeculae. Antler cartilage is a very abundant source of cells for in vitro studies; in a rapidly growing antler, up to 100 million cells can be harvested from nonmineralized cartilage. Cells cultured from antler cartilage have a slower rate of growth than cells from the growth zone (doubling time of 48–96 hours) and undergo replicative senescence after approximately 20 passages. They also express higher levels of alkaline phosphatase than cells cultured from the growth zone, reflecting their more diVerentiated state (Price et al., 1994) (Fig. 11A). However, antler chondrocytes cultured as monolayers synthesize high levels of type I collagen and lose ALP expression with serial passaging, reflecting their tendency to dediVerentiate rapidly. They maintain their phenotype far better when cultured as high-density micromasses (Fig. 11E, F) but continue to synthesize type I as well as type II collagen. Interestingly, antler chondrocytes synthesize very little type X collagen in vitro (unpublished observations), despite the abundance of this collagen in cartilage in vivo (Gibson et al., 1996). Type X collagen is present throughout nonmineralized and mineralized cartilage (Fig. 10F) and thus is distributed far more widely in antler cartilage than in growth plate (Faucheux et al., 2004; Price et al., 1996). The problem with the antler tip, unlike the growth plate, is that it is very diYcult to define the onset of chondrocyte hypertrophy. If it is taken to be the site where type X collagen is first expressed, then essentially most chondrocytes in antler can be considered to be hypertrophic, since chondrocytes which express type IIB collagen also express type X

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Figure 11 In vitro characteristics of cells cultured from antler cartilage. (A) Primary cultures of cells cultured from antler cartilage have high levels of alkaline phosphatase activity but lose this with serial passaging. (B) High-density monolayer of chondrocytes showing proteoglycan synthesis (alcian blue stain). (C) Cells cultured from cartilage synthesize aggrecan (red stain). (D) Mineralized nodules (*) form in cartilage-derived cells cultured with BGP and ascorbate. (E, F) Antler cartilage-derived cells cultured as micromasses showing high levels of alkaline phosphase activity (red stain) and proteoglycan synthesis (alcian blue stain). Scale bars: A, B, 50
m; C, D, 50
m; E, F, 1 mm.

collagen. In contrast, in mammalian growth plate, type X collagen is only expressed in the lower hypertrophic zone (Reichenberger et al., 1991; Sandell et al., 1994). In fact, the pattern of type X synthesis in antler is similar to that in the developing avian limb, where endochondral ossification occurs in an irregular pattern along the entire diaphysis and the zone of hypertrophic cartilage is far more extensive than in mammalian cartilage (Kwan et al., 1989; Oshima et al., 1989). Because type X collagen is first expressed in antler cartilage at least 1 cm distal to where mineralization of cartilage takes place, this suggests that type X does not play a role in initiating the process. Since type X collagen mRNA is first expressed where there is coincident enlargement of vascular channels, this suggests that it may play some role in promoting formation of the extensive network of these channels in the antler tip. Another usual feature of antler cartilage is that it contains type VI collagen (unpublished observations) (Fig. 10E); however, the significance of this is not known. Another very important matrix component of antler cartilage are glycosaminoglycans (GAGs) (Sunwoo and Sim, 2001) that can be detected by alcian blue staining, which increases in intensity as chondrocytes diVerentiate

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(Fig. 10C). The major GAG in antlers is chondroitin sulfate (Sunwoo et al., 1998a,b) which is an important component of the large aggregating proteoglycan aggrecan, a marker of the chondrocyte phenotype (Heinegard et al., 1985). We have found the spatial expression of aggrecan core protein mRNA expression to be similar to that of type IIB collagen mRNA, although the level of expression is lower (unpublished observations). Aggrecan immunoreactivity is also strong in diVerentiated chondrocytes (Fig. 10H), whereas only faint immunoreactivity is detected in chondroprogenitors in the transition zone. Cultured chondrocytes also synthesize aggrecan (Fig. 11C), whereas undiVerentiated cells from the mesenchyme do not. Another important diVerence between antler and growth plate is that there is no type I expression in growth plate (Fukunaga et al., 2003; Sandell et al., 1994), whereas type I collagen mRNA is expressed throughout antler cartilage (Price et al., 1996) in a population of small cells in perivascular tissue adjacent to the columns of chondrocytes (Fig. 10B). This evidence, and the high level of ALP activity in this region, has led us to conclude that these cells are of the osteoblast lineage, the progenitors of diVerentiated osteoblasts found in primary spongiosa. These perivascular cells also express the retinoic acid receptor RAR and the retinoic acid synthesizing enzyme RALDH2 (Allen et al., 2002), which suggests that retinoic acid regulates the diVerentiation of osteoblasts in antlers. The perivascular tissue is also a site of osteoclast diVerentiation and this will be discussed in more detail in a later section.

G. Regenerating Antlers: Ossification and Remodeling As has been mentioned, the zone of nonmineralized cartilage in antlers is extensive; in red deer, it can extend for than more 70 cell layers proximodistally. Calcification of cartilage, as detected by alizarin red staining or von Kossa staining (Molello et al., 1963), occurs initially as discrete foci in the middle of trabeculae, at a depth of
1.5 cm from the top of the cartilage trabeculae. These foci of mineralization then coalesce to completely surround chondrocytes and eventually the entire cartilage spicule contains mineral (Fig. 8I). Matrix vesicles, another important component of epiphyseal growth plate cartilage that contain high levels of the enzyme ALP, are present in antler cartilage and act as an initial focus for mineralization (Newbrey and Banks, 1975). ALP activity can be detected in sections of antler cartilage (Fig. 9A) and bone (Kuhlman et al., 1963; Molello et al., 1963; Price et al., 1994) and cells cultured from cartilage have significant amounts of ALP activity and will form mineralized nodules in vitro (Fig. 11A, D). Interestingly, the ALP activity of cells from antler cartilage is high (up to 3.6 mmol/mgprotein/minute) compared to levels recorded for other skeletal cell types, including rabbit growth plate chondrocytes

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(0.065 mmol/mg protein/min) (Boyan et al., 1988) and is likely to reflect a high rate of cellular diVerentiation in antlers. In more proximal regions calcified cartilage is resorbed by osteoclasts and this leads to trabeculae losing their typical columnar arrangement to become irregularly shaped. Banks and Newbrey (1983) describe this region as the ‘‘primary spongiosa.’’ Eventually, cartilage trabeculae are replaced by woven bone that is remodeled into lamellar bone. At the same time, woven and lamellar bone are being formed around the antler shaft by a process of intramembranous ossification. Compact lamellar bone is found only in the periphery of the antler shaft whereas cancellous lamellar bone occupies the midshaft (Fig. 8J). Osteoblasts at sites of both endochondral and intramembranous ossification synthesize osteocalcin, a marker of diVerentiated osteoblast phenotype (Allen et al., 2002). Antler bone contains both all-trans and 9-cis-retinoic acid and the RA synthesizing enzyme RALDH2, which suggests that RA may control the activity of mature osteoblasts as well as the diVerentiation of osteoblast progenitors. Antlers are similar to other developing bones in being dependent on osteoclasts to remodel mineralized matrix and facilitate the penetration of large vascular channels. However, the unparalleled rate of endochondral ossification in antlers requires very large numbers of osteoclasts to diVerentiate locally. Perivascular tissue in nonmineralized cartilage is the site where osteoclastic diVerentiation is initiated since it contains cells that express tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors (VNRs) (Fig. 12), phenotypic markers of osteoclasts (Faucheux et al., 2001; Price et al., 1994, 1996). As expected, the number of TRAP-positive cells increases as cartilage mineralizes and is replaced by bone (Szuwart et al., 2002). Interestingly, TRAP-positive cells are also present in mesenchymal tissue of the early antler bud. This regulation of osteoclast formation by chondrocytes/mesenchymal cells has also been shown to occur in fetal bones (Thesingh and Burger, 1983; Van De Wijngaert et al., 1989). Molecules responsible for regulating osteoclast diVerentiation are PTHrP and RANKL; both are expressed in perivascular tissues and in antler cartilage (Faucheux et al., 2001, 2002). Macrophage colony stimulating factor (M-CSF) (Faucheux et al., 2001) and transforming growth factor beta (TGF-) (Faucheux et al., 2004) are also present in antler cartilage in vivo and are also likely to support osteoclastogenesis. When chondrocytes from nonmineralized cartilage are cultured at high density in micromasses (200,000 cells per 10
l), they support the formation of multinucleated osteoclast-like cells that have the phenotypic characteristics of mammalian bone-derived osteoclasts (Fig. 12). They express TRAP, VNRs, calcitonin receptors (the ‘‘gold standard’’ of osteoclast phenotypic markers) and, when cultured on a mineralized substrate, form F-actin rings and large resorption pits (Faucheux et al., 2001). We have found this

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Figure 12 Micromass cultures of cartilage-derived cells support the diVerentiation of osteoclasts. (A) Cryostat section of antler cartilage stained for TRAP showing numerous positive cells (red stain) adjacent to the vascular channels (V). (B) TRAP-stained osteoclasts in micromass culture after
14 days. (C) Confocal image of a diVerentiated osteoclast cultured on a dentine substrate. The vitronectin receptor, a specific osteoclast marker, and an actin ring is present, indicating that the osteoclast is actively resorbing. (D) Resorption pits formed by antler osteoclasts in a micromass culture. Scale bars: A, 100
m; B, 25
m; C, 10
m; D, 1 mm.

model to be very reproducible and now use it routinely for investigating the molecular mechanisms that regulate osteoclast diVerentiation. What is particularly useful about this system is that chondrocytes cryopreserved immediately after extraction from an antler can support osteoclastogenesis. However, the numbers of cells that form ‘‘spontaneously’’ is reduced if cells have been previously frozen, and then it is normally necessary to ‘‘drive’’ osteoclastogenesis with exogenous factors such as PTHrP or RANKL. In contrast, in ‘‘fresh’’ micromass cultures, osteoclasts will form spontaneously in the absence of factors normally required to stimulate osteoclasts in vitro (1,25(OH)2D3, PTH, M-CSF, RANKL, etc.). This may reflect the high concentration of PTHrP synthesized into conditioned medium (Faucheux and Price, 1999), a very potent stimulator of antler osteoclast formation, and/or the expression of RANKL and M-CSF in these micromass cultures (Faucheux et al., 2001, 2002). PTHrP’s eVect on osteoclastogenesis is partially mediated via RANKL as OPG, the soluble decoy receptor for RANKL (Simonet et al., 1997; Tsuda et al., 1997), and can partially inhibit PTHrP-induced osteoclast diVerentiation (Faucheux et al., 2002). However, unlike most mammalian osteoclasts, antler osteoclasts express PPRs, evidence that there may be direct eVects of PTHrP on antler osteoclasts (Faucheux et al., 2002). Retinoic acid is another factor that can regulate osteoclast formation in these micromass cultures (unpublished observations) and, as will be described in more detail in a later section, retinoic acids are present and are synthesized in perivascular tissues of antler cartilage.

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II. Regulation of the Antler Development and Regeneration A. External and Systemic Factors Since antlers are secondary sexual characteristics, their annual cycle of regeneration has evolved to be closely coordinated to the reproductive cycle (Lincoln, 1971) and there is a fixed relationship between the stage of the antler cycle, plasma testosterone, testis diameter, and body weight (Fig. 6). The regulation of the annual cycle of antler growth was studied extensively in the 1970s and 1980s, with a number of groups worldwide contributing to advancement of the field. In temperate species such as red deer, which have defined mating seasons, the environmental cue which triggers changes in reproductive activity is the photoperiod (Lincoln and Short, 1980). In these species, decreasing day length triggers pituitary gonadotrophin activity for 2 months before the autumn mating season (Lincoln and Kay, 1979). There have also been studies to indicate an activation of reproductive hormones in the spring (Bubenik et al., 1982; West and Nordan, 1976). However, during the spring increasing day length does not activate reproduction; instead, it is responsible for initiating antler growth. In fact, it may be the change in photoperiod that is the critical trigger because, under laboratory conditions, the antler cycle can be initiated by either increasing or decreasing day length (Goss, 1976). Notwithstanding, in seasonally breeding deer, the photoperiod would appear to modify rather than act as a ‘‘main driver’’ of antler growth cycle since there is an endogenous rhythm to antler growth. For example, animals maintained on less than 24 hours of continuous lighting show cycles of antler growth and reproductive activity (West and Nordan, 1976). In a study which maintained stags under 8 hours of light and 12 hours of dark, antlers were cast 5 months before those in a control group kept under natural lighting conditions. This occurred because a second luteinizing hormone (LH) peak in the spring did not take place and the consequence of this was a fall in plasma testosterone and premature antler casting (Suttie et al., 1984). The mechanism whereby the photoperiod is linked to antler growth and reproductive activity is not clear but may involve changes in melatonin secretion from the pineal gland, which then acts on the hypothalamopituitary axis (Bubenik et al., 1986). In young animals, the pineal gland may play a particularly important role as pinealectomy leads to changes in the antler growth cycle (Plotka et al., 1984) as does the hormone melatonin (Lincoln et al., 1984). In red deer, exogenous melatonin has similar eVects to that of a short photoperiod, namely, advanced growth of the testis and premature cessation of growth (Webster et al., 1991). In contrast, this photoperiodic-pineal link to the reproductive system is absent, or of only

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minor significance, in species of deer from subtropical or tropical zones (Loudon and Curlewis, 1988). 1. Sex Steroids As discussed earlier, testosterone is required for pedicle and first antler development but, in the first year of life, hormone secretion is not regulated by photoperiodic cycles. Sex hormones are also generally considered to be the most important internal regulators of the timing of the annual cycles of regeneration, although they clearly have to function in a complex endocrine environment and in the context of other internal influences such as genotype, which plays a major role in shaping antler size and shape. As illustrated in Fig. 6, the antlers are cast in the spring, when testosterone levels are low and day length is increasing (Lincoln and Kay, 1979). The growth of the new antlers starts slowly in the spring but accelerates exponentially in the summer when testosterone levels remain low; in fact, studies in red deer have shown that this phase of antler growth appears not to require sex hormones (Fennessy et al., 1988; Suttie et al., 1989). However, this is an area where some controversy exists, as Bubenik and colleagues have argued that testosterone, possibly from nongonadal sources, may be required for initiating regeneration and for regulating longitudinal growth (Bartos et al., 2000). What is not in dispute is that as testis size increases and testosterone levels rise above 1 ng/ml, antler growth slows and mineralization of antler bone increases (Muir et al., 1988). The velvet skin is then lost (Fig. 13) and antlers are ‘‘polished’’ in preparation for the mating (rutting) season, with testosterone levels peaking in October and November in most species of deer (Lincoln, 1971). The long-held assumption has been that antlers lose their blood supply after they have completed velvet shedding and are thus ‘‘dead.’’ However, it has been shown that a functioning vascular system and living cells remain in the core of the hard antlers of fallow deer (Rolf and Enderle, 1999). After the mating period (rut) is over, testosterone levels decline and, in species such as reindeer, moose, and caribou, this is associated with antler casting in early winter; however, in most species, this does not occur until the following spring when testosterone levels fall below 1 ng/ml (Muir et al., 1988). Deer maintained on artificially high levels of sex steroids do not cast their antlers (Fletcher, 1978; Wislocki et al, 1947), whereas orchidectomy (castration) when a stag has hard antlers leads to their premature casting (Jaczewski et al., 1976), as will administration of cyproterone acetate, a specific blocker of androgen receptors (Bubenik et al., 1984). The eVects of castrating a stag when his antlers are growing provide further evidence that sex steroids are key regulators of the start and end of

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Figure 13 Shedding of velvet skin in late summer in a 2-year-old red deer stag.

the cycle of antler growth. However, the eVects of androgen withdrawal are species specific; in reindeer, which are considered to be a phylogenetically primitive genus, castration has little eVect (Bubenik, 1983), whereas in phylogenetically intermediate red deer, the first set of antlers grown by a castrated stag often appear normal but they do not complete their mineralization nor shed their velvet. This means that temperature changes in winter can lead to tissue damage which can have an important eVect on antler structure; castrate antlers have been reported to develop multiple branches and/or get bigger. In contrast, in species such as roe deer which have the longest phylogenetic development, castrate antlers can develop into large benign tumors and thus greatly incapacitate the animal bearing them (Bubenik, 1983; Goss, 1983). Why androgen withdrawal should have such species-specific eVects is not known, but it raises intriguing questions relating to the phylogeny of the function of sex steroids. The castrate antlers of roe deer also raise questions about the relationship between regeneration and cancer and, as such, merit further study. Despite the obvious importance of testosterone as an ‘‘internal driver’’ of antler regeneration, its mechanism(s) of action are not well understood. Androgen receptors have been immunolocalized in antlers (J. Price, unpublished observations); however, Li et al. (1999) failed to demonstrate any eVect of testosterone on the proliferation of antler cells, nor does testosterone sensitize antler cells to the proliferative eVects of IGF-I (Sadighi et al., 2001). However, studies that we have undertaken (unpublished) have shown that testosterone will induce the proliferation of cells cultured from antlers and 17- estradiol has a similar eVect, although the proliferative response

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appears to depend on the stage of antler growth and the stage of cell diVerentiation. Estrogen receptors have been localized in antler tissues (Barrell et al., 1999; Lewis and Barrell, 1994) and in vivo studies undertaken many years ago showed that when administered to castrated stags, animals exhibit rutting behavior and fail to cast their antlers (Fletcher and Short, 1974). Richard Goss demonstrated over 30 years ago that estrogen would induce premature mineralization of antlers in male Sikka deer and was actually more eVective than testosterone (Goss, 1968). Bubenik and Bubenik (1978) reported that administration of an estrogen receptor antagonist to white tail deer stags during growth leads to decreased thickness of compact bone of the antler shaft. These diVerent lines of evidence suggest that testosterone may have indirect eVects on antler cells in vivo, via its conversion to estrogen by aromatase. We have undertaken an experiment similar to that undertaken by Richard Goss in red deer stags and administered 17- estradiol on a single occasion during the phase of rapid growth; longitudinal growth stopped and within 2 weeks the antler was solid bone (J. Price, unpublished observations). These observations suggest that estrogen’s role is to prevent continued antler growth from the distal antler tip; this would be consistent with ERs being present at this site. Thus estrogen’s role in the antler would be similar to its role in the growing male human skeleton; a man lacking ER was found to be profoundly osteopenic and did not stop growing in height after the end of puberty. A similar phenotype has been described in patients lacking a functional aromatase gene (Carani et al., 1997; Morishima et al., 1995). That the function of ER should be conserved is perhaps not surprising as that the ER has been shown to be the most ancient steroid receptor (Thornton, 2001). 2. Insulin-like Growth Factor If sex steroids have a minor role to play early in the cycle when antlers are growing rapidly, this begs the question as to the identity of the ‘‘antler growth stimulus’’ (AGS) that was proposed by Wislocki (1943). Over the years, growth hormone, thyroxine, and prolactin and testosterone have been proposed as likely candidates. However, important work by the group of Suttie et al. (1985) led to the conclusion that IGF-I is likely to be AGS. Several studies have now shown a positive correlation between antler growth and circulating IGF-I (Suttie et al., 1985). IGFs I and II have also been detected at the mRNA level, by PCR, in antler tissues (Francis and Suttie, 1998). Furthermore, ligand binding studies have shown that receptors for both IGF I and IGF II are present in the antler tip (Elliott et al., 1992, 1993) and both IGF I and IGF II promote proliferation of cultured cells of the progenitor and cartilage regions of antler in vitro (Fig. 14; Price et al., 1994; Sadighi et al., 1994). There is evidence for an association between

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Figure 14 The eVect of FGF-2 and IGF-I on antler cell proliferation. Cells from mesenchyme and cartilage were cultured as monolayers in 48 well plates. When subconfluent, cells were serum deprived for 12 hours, then treated for 48 hours with FGF-2 (A) or IGF-I (B) in BGJb medium containing 2% fetal bovine serum. Cultures were pulsed for the final 12 hours with 3H thymidine and incorporated into the cells assayed as described previously (Price et al., 1994). IGF-I stimulates proliferation in both cell types whereas FGF-2 only stimulates growth of the less diVerentiated progenitor cells. *p < 0.05, **p < 0.02, ***p < 0.001, ****p < 0.0001.

IGF-I concentrations and serum testosterone (DitchkoV et al., 2001; Li et al., 2003), although whether previously elevated plasma testosterone levels are directly responsible for the subsequent IGF-I peak remains unclear. There is also evidence that IGF-I concentrations can be influenced by the photoperiod (Suttie et al., 1991). The source of IGF-I that regulates antler growth is likely to be the liver, where its secretion is regulated by growth hormone (GH); and in red deer stags pulsatile increases in GH precede by one month increases in circulating IGF-I, weight gain, and antler growth (Suttie et al., 1989). There have been no reports describing the localization of IGFs or IGF-binding proteins in vivo or in vitro, and stags surgically prevented from growing antlers have elevated IGF-I levels, an observation that lead Suttie et al. (1988) to propose that, although the antler is a major target for IGF-I, antler cells are not a

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major source of the growth factor. However, the importance of locally produced, compared with circulating, IGFs remains unclear and deserves further investigation. In fact, the role of IGFs in antler should be revisited in light of recent advances in the biology of IGFs and their receptors, particularly in the context of regeneration. For example, muscle-specific expression of insulin-like growth factor I has been shown to preserve regenerative capacity in senescent skeletal muscle (Musaro et al., 2001) and to preserve muscle function in mice lacking dystrophin (Barton et al., 2002). B. Local Mechanisms One of the keys to understanding antler development and regeneration is to identify the pathways that control the expansion of progenitor cell populations, cell diVerentiation and survival, and epithelial–mesenchymal interactions. Antlers are also unique among regenerating structures in that the interaction between systemic factors and the local signaling pathways has also to be considered. Evidence has accumulated that developmental programs can be recapitulated in the adult under specific circumstances, and studies in other models of regeneration have shown that many developmental signaling molecules are expressed in regenerating tissues (Brockes, 1997; Holstein et al., 2003; Poss et al., 2003; Slack et al., 2004). It is also necessary to understand how injury (in the case of antlers, the process of antler casting) will ‘‘trigger’’ these pathways. Unfortunately, not a great deal is currently known about the mechanisms that control antler development and regeneration. However, this situation is changing since recent technological advances are applied in antler research. For example, the group in New Zealand has generated a database of genes expressed in regenerating red deer antler tissues (Lord et al., 2001, 2004). They have identified 4516 expressed sequence tags (ESTs) that include 930 gene sequences. The first proteomic analysis of growing red deer antler has recently been completed (Park et al. 2004) and this identified
130 proteins including structural proteins, matrix proteins, metabolic enzymes, and signaling and cell growthrelated proteins. The main driver of this type of research is industry; the successful marketing of velvet antler as a nutraceutical/therapeutic product depends on the components of antler being identified and on studies being undertaken to establish eYcacy. The caveat is that although these ‘‘fishing trip’’ approaches will identify the molecules involved, dissecting out their function is extremely labor intensive and not straightforward in an animal that has limited genetics. Unfortunately, there are probably not enough research scientists working in the field to take full advantage of the huge amount of information that these genomic and proteomic studies will generate. A second caveat is that industry is interested in identifying factors that

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regulate the growth of fully formed, rapidly growing antlers, because these provide the starting product for antler extract production. Therefore, practically nothing is known about the pathways that regulate the very early stages of antler development/regeneration. This area will remain neglected unless more support for basic biological research is forthcoming. Unfortunately, many funding bodies are wary of supporting research that utilizes animal models that are not mainstream and which are ‘‘genetically intractable.’’ In addition to components of extracellular matrix, a number of growth factors, cytokines, hormone receptors, and other signaling molecules have been identified in antlers (the most important are listed in Table I), although there is very little experimental evidence regarding their function. It is also Table I Growth Factors, Hormone Receptors, and other Signaling Molecules Shown to be Present in Antler Tissues Molecule EGF EGF, EGF receptor 1,25(OH)2D3 Estrogen receptor

BMP-2, BMP-4 BMP-2, BMP-4, BMP-14, BMPRI, ACTRIII Neurotrophin-3 c-myc, c-fos TGF-1, TGF-II TGF-1 IGF-I, IGF-II IGF I & IGFII receptors RANKL & M-CSF Dermatopontin, glutathione peroxidase all-trans, 9-cis RA RARs& RXRs RALDH2 PTHrP, PTHrP/PTHR, Indian hedgehog (IHH) FGF-2, FGFR1, FGFR2, FGFR3 VEGF, KDR

Deer species

Protein/mRNA

Red deer antler

protein

Cultured roe deer antler cells Red deer antler

protein protein

White tail deer Red deer antler

mRNA protein

Red deer antler Red deer antler Red deer antler

mRNA mRNA mRNA protein mRNA protein

Red deer antler

Reference Barling et al. (2004); Ko et al. (1986) Sempere et al. (1989) Barrell et al. (1999); Lewis et al. (1994); Park et al. (2004) Barling et al. (2004); Feng et al. (1995, 1997) Garcia et al. (1997) Francis et al. (1998) Faucheux et al. (2004); Francis et al. (1998) Elliott et al. (1992, 1993); Francis et al. (1998) Faucheux et al. (2000) Lord et al. (2001)

Red deer antler Red deer antler

mRNA mRNA

Red deer antler

Red deer antler

protein mRNA protein protein

Faucheux et al. (2004)

Red deer antler

protein

Barling et al. (2004)

Red deer antler

protein

Clark et al. (2004)

Allen et al. (2002)

Note: this does not include the 130 proteins identified in the recent proteome analysis of red deer antler (Park et al. 2004).

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diYcult to draw a consensus from the literature when various species of deer and diVerent stages of antler development/regeneration have been used for such studies. The approach that we have taken has been to focus on molecules that are known to play an important role in development and/or regeneration in lower vertebrates and to use a variety of techniques (cell culture, in situ hybridization, immunolocalization, etc.) to try and explore their role in red deer antlers at diVerent stages of regeneration. A class of molecules that are important regulators of skeletal development and regeneration in urodeles are the retinoic acids (RAs) (Cash et al., 1997; Koyama et al., 1999; Lohnes et al., 1994; Pecorino et al., 1994, 1996; Scadding and Maden, 1994; Schilthuis et al., 1993; Tassava, 1992; Yamaguchi et al., 1998). There is also in vivo evidence that application of RA can influence the development of the antlers; injection of RA into the pedicle anlagen (Kierdorf and Kierdorf, 1998) has been shown to lead to an alteration of pedicle and antler shape. Exogenous RA also increased growth rate of the first antler and this was suggested to occur via an increase in the proliferation of periosteal cells (Kierdorf and Bartos, 1999). We have subsequently undertaken several studies which provide evidence to confirm that RA plays a potentially vital role in the regulation of cellular diVerentiation in regenerating antlers (Allen et al., 2002). HPLC analysis showed that significant amounts of all-trans-RA are present in all antler tissues, with the exception of chondroprogenitors, at levels similar to those found in regenerating amphibian limbs (Scadding and Maden, 1994). 9-cis-RA was also found in perichondrium, mineralized cartilage, and in bone, which suggests that it may have a particular function in regenerating bone since 9-cis-RA has not been detected in the developing limb. The RA synthesizing enzyme RALDH2 is present in skin, perichondrium, mesenchyme, perivascular tissue, and in osteoblasts. In contrast, the only site where RALDH2 is expressed in the chick limb is the perichondrium (Berggren et al., 2001). RAs act through nuclear receptors and retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RAR, RAR, and RXR are all highly expressed in velvet antler skin, although we have not addressed what their role may be at this site. The expression pattern of RAR suggests that it also inhibits the diVerentiation of chondroprogenitors and maintains the perichondrium in a ‘‘prechondrogenic state,’’ consistent with its role in the developing limb (Weston et al., 2000). RAR is also expressed in perivascular tissue and its pattern of expression is so similar to that of type I collagen that it suggests a role for this receptor in regulating osteoblast diVerentiation. RAR is expressed in perivascular cells of the osteoblast (or osteoclast) lineage. RAR is not highly expressed in antler tissues, whereas in the developing limb its expression increases with chondrocyte diVerentiation (Dolle et al., 1989; Koyama et al., 1999; Mendelsohn et al., 1992). Only a

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small number of perivascular cells express RXR and RXR, whereas RXR has a widespread expression in hypertrophic chondrocytes. RA will induce dediVerentiation in micromass cultures of antler chondrocytes, detected by a decrease in GAG synthesis (Allen et al., 2002) and this eVect is dependent on RAR signaling. This is entirely consistent with RA’s eVect on chondrocytes in other species (Ballock et al., 1994; Takigawa et al., 1980). We have also studied PTHrP in some detail in the regenerating antler because, acting through the type I PTH/PTHrP receptor (PPR), it is a ‘‘master’’ regulator of chondrocyte diVerentiation in the developing limb (Kronenberg, 2003). This work has demonstrated how a single factor can have multiple roles in regenerating antler. PTHrP can be localized in epidermis, dermis, hair follicles, and sebaceous glands (Fig. 5A); this suggests that PTHrP may play an important role in controlling the growth of skin in regenerating appendages. These results also suggest that it may be important in mediating epithelial mesenchymal interactions in antlers, as it does in developing structures (Foley et al., 2001; Karperien et al., 1994). PTHrP also has an important role in endochondral and intramembranous ossification; it is present in perichondrium, mesenchyme, and chondroprogenitors. However, unlike the situation in the developing limb, PTHrP is not expressed in diVerentiated chondrocytes (Farquharson et al., 2001; Faucheux et al., 2004; Kartsogiannis et al., 1997) (Fig. 5). In vitro PTHRP inhibits antler chondrocyte diVerentiation and increases proliferation (Faucheux and Price, 1999), as it does in the developing limb. The localization of PTHrP and the PPR in antler osteoblasts indicates that it may also have a role regulating osteoblast diVerentiation. PTHrP has previously been shown to promote (Carpio et al., 2001; Motomura et al., 1996) osteoblast diVerentiation in vitro and adult mice heterozygous for PTHrP are osteopenic (Amizuka et al., 2000). The group of Peter Barling (Barling et al., 2004a) have recently cloned and sequenced PTHrP and the PPR from red deer and have studied their distribution in the developing antler by in situ hybridization and immunocytochemistry. They report the localization of PTHrP and PPR in epidermis, in dermis, and in skin appendages as well as in developing cartilage and bone. The expression of PPR mRNA in velvet antler skin is of particular interest since it has only recently been shown that the receptor is expressed in the skin of newborn rats (Errazahi et al., 2003) and changes in PPR mRNA expression have been shown to take place in mice during the hair cycle (Wang et al., 2002). The similar distribution of PTHrP and PPR in developing and regenerating antlers is further evidence that regeneration recapitulates development. Another molecule that may interact with PTHrP to control chondrogenesis is Indian hedgehog (IHH) that is immunolocalized in early hypertrophic

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chondrocytes in regenerating antlers (Faucheux et al., 2004). In the developing limb, IHH is expressed in prehypertrophic and hypertrophic chondrocytes and acts in a feedback loop with PTHrP to control chondrocyte diVerentiation (Chung et al., 2001; Kobayashi et al., 2002; Lanske et al., 1996; Vortkamp et al., 1996). In IHH null mice endochondral ossification is also impaired (St-Jacques et al., 1999) and IHH may have a similar role in antlers since it can be immunolocalized in osteoblasts in spongy bone in the midshaft of the antler, but is not found in osteoblasts at sites of intramembranous ossification. Lord et al. (2004) have also localized IHH mRNA in developing antler cartilage/bone by in situ hybridization. In a screen of antler extracts for factors that could potentially stimulate bone formation, a number of molecules were identified, including IGF-I and IGF II and fibroblast growth factors (Mundy, 2001). This study identified BMP-4 (Feng et al., 1995) and BMP-2 (Feng et al., 1997), two members of the transforming growth factor  superfamily that are known to be involved in embryonic skeletal development (HoVman and Gross, 2001). Barling et al. (2004b) have recently reported that BMP-2, BMP-4, BMP-14, and the BMP receptors BMPRI and ACTRII can be localized in the skin, cartilage, and bone of the developing red deer antlers. Experimental data from our laboratory (unpublished) suggests that BMP-2 regulates the diVerentiation of mesenchymal progenitor cells cultured from developing antlers; BMP-2 decreases proliferation and increased alkaline phosphatase activity (Fig. 15). BMP-2 also stimulates osteoblast diVerentiation of rat pluripotent progenitor cells (Hiraki et al., 1991). That BMPs should regulate antler regeneration is consistent with their role in regulating endochondral ossification and bone repair; both BMP-2 and BMP-4 will induce ectopic bone formation and BMP-2 will heal cortical bone defects by an endochondral process (Toriumi et al., 1991; Wang et al., 1990; Yasko et al., 1992). TGF1 and TGF2 have also been found at the mRNA level (Francis and Suttie, 1998) and TGF1 at the protein level (Faucheux et al., 2004) in antlers during their rapid growth phase. The role(s) of TGFs is currently unclear; however, the widespread distribution of TGF1 in mesenchymal cells, perivascular cells, and in osteoblasts suggests that it may have multiple functions, depending on its target cell type and its local concentration. For example, treatment of cells derived from mesenchyme with TGF1 will increase cellular diVerentiation (ALP activity) and this is associated with a decrease in cellular proliferation, but only at lower treatment doses (Fig. 16). TGF1 is also able to stimulate the production of PTHrP by antler cells in vitro (Faucheux et al., 2004), suggesting it may have a role similar to TGF2 in embryonic limb, where it is produced by the perichondrium and regulates chondrogenesis by inducing PTHrP (Alvarez et al., 2002). A molecule that may interact with TGF in antlers is dermatopontin that it was identified by Lord et al. (2001) in their cDNA library screen of regenerating

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Figure 15 The eVect of BMP-2 on proliferation and alkaline phosphatase activity of progenitor cells cultured from mesenchyme. (A) For proliferation assays, cells were cultured in 48 well plates and treated for 48 hours with BMP-2 in BGJb medium containing 2% fetal bovine serum. Cultures were pulsed for the final 12 hours with 3H thymidine and incorporated into the cells assayed. (B) For alkaline phosphatase assays, confluent monolayers of cells were treated for 72 hours with BMP-2 in BGJb medium containing 2% fetal bovine serum. Cell cultures were then lysed and the supernatants assayed for alkaline phosphatase using a colorimetric assay (Price et al., 1994). Results were corrected for protein content of the cultures and expressed as
mol product/
g protein/minute. BMP-2 inhibits growth but at higher doses promotes diVerentiation. *p < 0.05 compared with control, **p < 0.001 compared with control.

red deer antlers. Dermatopontin has been shown to interact with TGF s and enhance their activity (Okamoto et al., 1999) and its expression is decreased in tissues where fibrosis is occurring (Catherino et al., 2004; Kuroda et al., 1999), suggesting a role for this molecule in inhibiting fibrosis/scar formation in antlers. Another family of growth factors that may be important local regulators are Fibroblast Growth Factors (FGFs). Peter Barling (Barling et al., 2004b) presented data to show the localization of FGF2 and the FGF receptors FGFR1, FGFR2, and FGFR3 in skin, cartilage, and bone of developing red deer antlers. FGF-8 has also been detected in antler skin (Ashery, 1999). We have evidence that FGF-2 may be an important growth factor in antlers; addition of FGF-2 to cell cultures derived from both mesenchyme and, to a lesser extent, cartilage regions increased thymidine incorporation (unpublished observations) (Fig. 14). Consistent with the findings of Barling et al. (2004b), we have also detected FGFR3 receptor mRNA transcripts in cartilage of the regenerating antler tip, further evidence that FGFs may control chondrocyte growth and/or diVerentiation, as they do in the developing limb (Ornitz and Marie, 2002). An epidermal growth factor-like molecule has also been found in antler velvet tissue (Ko et al., 1986) and Barling et al. (2004b) have immunolocalized EGF and the EGF receptor in the developing antlers, although staining for the EGF receptor was far less

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Figure 16 The eVect of TGF-1 on proliferation (A) and alkaline phosphatase activity (B) of progenitor cells cultured from mesenchyme. The protocol was as described for Figs. 14 and 15. Like BMP-2, TGF-1 inhibits cell proliferation and inhibits diVerentiation. p < 0.05 compared with control, **p < 0.001 compared with control.

intense than that of its ligand. In addition to the growth of skin and bone in growing antlers, there is rapid growth of nerves since antlers are highly innervated structures. Garcia et al. (1997) reported widespread expression of Neurotrophin 3, a factor that controls nerve growth, particularly in the epidermis and dermis. Antler growth also depends on angiogenesis; these structures have to be highly vascularized because rapidly growing tissues have high metabolic demands. The expression of vascular endothelial growth factor (VEGF) and the VEGF receptor KDR have been mapped in the rapidly growing red deer antlers and extracts of antler will induce migration and growth of endothelial cells in culture (Clark et al., 2004). The same study showed that the angiogenic and chondrogenic

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factor pleiotrophin is also highly expressed in antler blood vessels and in chondrocytes (Clark et al., 2004). The studies reviewed here involved either developing antlers or regenerating antlers during their exponential growth phase. Surprisingly little is known about the pathways that control the very early stages of antler regeneration. Questions that need to be answered include: What is the trigger that induces cells in the pedicle to enter the cell cycle and to form a ‘‘blastema’’ or antler bud? What molecules prevent scar formation? What regulates branching? How do hormonal factors interact to regulate these pathways? How is infection prevented before epithelialization is complete? A technique that may prove valuable for establishing the function of signaling pathways in the early antler is biolistic particle delivery. This method has been used to transfect cells in the regenerating newt blastema (Pecorino et al., 1994, 1996) and we have used it to successfully transfect cells on the pedicle surface shortly after the previous set of antlers had been cast (Price and Allen, 2004). To date, molecules reported to be expressed in the early regenerating antler (<14 days of growth) include PTHrP, the PPR, TGF, RA, retinoic acid receptors, RALDH2, and TRAP (Faucheux et al., 2001, 2004; Price and Allen 2004). PTHrP and PPRs are both expressed in the regenerating wound epithelium, in ‘‘normal’’ epidermis, and dermis at the periphery of the antler bud, as well as in a large number of mesenchymal cells (Fig. 5). Since PTHrP is also expressed in periosteum, we have taken this to be evidence, albeit indirect, that these mesenchymal cells may be derived from pedicle periosteum. As mesenchymal cells in the blastema diVerentiate into chondrocytes, PTHrP synthesis is downregulated, which suggests that its role is to maintain mesenchymal cells in an undiVerentiated state. TGF1 is also expressed in the early antler and induces PTHrP synthesis in cultured cells from the blastema (Faucheux et al., 2004). TGF1 may also be involved in controlling fibrosis. As discussed in a previous section, we also have evidence that RA may regulate the early stages of antler regeneration since HPLC analysis has detected several RAs in antlers at fewer than 14 days of development, as well as RALDH2, RAR, RAR, and RXR (unpublished observations). To further explore our hypothesis that embryonic signaling pathways are recapitulated in regenerating antlers, we have also studied the localization of FGF-4 in the early antler (day 4) and found it to be immunolocalized in the regenerating epidermis and cells of the dermis (Fig. 17) (unpublished observations), which suggests that it may control epithelial–mesenchymal interactions. FGF-4 is also associated with vascular channels in the early antler (4-day) bud, although the significance of this observation is not clear. At a slightly later stage of growth (Day 14), FGF-4 is expressed in chondroprogenitors but not in fully diVerentiated chondrocytes, which suggests that it may also play a role in chondrogenesis.

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Figure 17 Immunolocalization of FGF 4 in the early regenerating antler. (A & B) Sections through a 4-day blastema. FGF-4 (brown stain) is localized in the (A) wound epithelium (we), cells of the dermis (d), and (B) in cells lining vascular channels (v). (C & D) Sections through a 4-day blastema. There is positive staining in chondroprogenitors but there is no FGF-4 synthesis by mature chondrocytes (c). Scale bars: 50
m.

Clearly, our understanding of the complex signaling pathways that regulate antler development and regeneration remains fairly rudimentary. However, from the pieces of the jigsaw so far available, it would appear that the pathways that control the development of embryonic skeletal elements and the first set of antlers are conserved, even though antlers are appendages that develop after birth. There is also evidence that developmental signaling pathways are recapitulated during the repeated rounds of antler regeneration. Furthermore, it would appear that the molecules involved can have multiple functions, for example, retinoic acid controls the diVerentiation of progenitor cells, chondrocytes, osteoblasts, and osteoclasts. Identifying the components of these signaling pathways, and determining their function and their local and systemic interactions, will not only contribute to our understanding of basic regenerative processes but could, in the longer term, have applications in regenerative medicine, for example, for enhancing the repair of functionally competent cartilage, bone, skin, and nerves.

Acknowledgments This work has been supported by grants from The Wellcome Trust, The Medical Research Council, and the BBSRC. The authors thank Mr. Brian Nichols, Mr. James Mount, Mr. Thnaian AlThnaian, and Dr. Mariusz Muzylak for their contribution to this work and

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their collaborators Dr. Jeanine Danks, Professor Malcolm Maden, Professor Jeremy Brockes, Professor Mike Horton, and Dr. Monica Colitti.

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Li, C., Suttie, J. M., and Clark, D. E. (2005). Histological examination of antler regeneration in red deer (Cervus elaphus). Anat. Rec. A. Discov. Mol. Cell Evol. Biol. 282A, 163–174. Lincoln, G. A. (1971). Puberty in a seasonally breeding male, the red deer stag (Cervus elaphus L.). J. Reprod. Fertil. 25, 41–54. Lincoln, G. A. (1973). Appearance of antler pedicles in early foetal life in red deer. J. Embryol. Exp. Morphol. 29, 431–437. Lincoln, G. A., and Kay, R. N. (1979). EVects of season on the secretion of LH and testosterone in intact and castrated red deer stags (Cervus elaphus). J. Reprod. Fertil. 55, 75–80. Lincoln, G. A., and Short, R. V. (1980). Seasonal breeding: Nature’s contraceptive. Recent Prog. Horm. Res. 36, 1–52. Lincoln, G. A., Fraser, H. M., and Fletcher, T. J. (1984). Induction of early rutting in male red deer (Cervus elaphus) by melatonin and its dependence on LHRH. J. Reprod. Fertil. 72, 339–343. Lo, D. C., Allen, F., and Brockes, J. P. (1993). Reversal of muscle diVerentiation during urodele limb regeneration. Proc. Natl. Acad. Sci. USA 90, 7230–7234. Lohnes, D., Mark, M., Mendelsohn, C., Dolle, P., Dierich, A., Gorry, P., Gansmuller, A., and Chambon, P. (1994). Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development 120, 2723–2748. Lord, E. A., Stanton, J.-A. L., Martin, S. K., Li, C., Clark, D. E., and Suttie, J. M. (2001). Characterisation of genes expressed in the growing velvet antler tip of red deer (Cervus elaphus). In ‘‘Antler Science and Product Technology’’ (J. S. Sim, H. H. Sunwoo, R. J. Hudson, and B. T. Jeon, Eds.), pp. 189–199. Antler Science and Production Technology Research Centre, Canada. Lord, E. A., Clark, D. E., Martin, S. K., Pedersen, G. M., Gray, J. P., Li, C., and Suttie, J. M. (2004). Profiling genes expressed in the regenerating tip of red deer (Cervus elaphus) antler. In ‘‘Antler Science and Product Technology’’ (J. M. Suttie, Ed.), Vol. 2. Loudon, A. S., and Curlewis, J. D. (1988). Cycles of antler and testicular growth in an aseasonal tropical deer (Axis axis). J. Reprod. Fertil. 83, 729–738. Lu, H. C., Revelli, J. P., Goering, L., Thaller, C., and Eichele, G. (1997). Retinoid signaling is required for the establishment of a ZPA and for the expression of Hoxb-8, a mediator of ZPA formation. Development 124, 1643–1651. Loveridge, N., and Farquharson, C. (1993). Studies on growth plate chondrocytes in situ: Cell proliferation and diVerentiation. Acta Paediatr. Suppl. 82(Suppl. 391), 42–48. Maden, M. (1982). Vitamin A and pattern formation in the regenerating limb. Nature 295, 672–675. Matich, J., Basford Nicholson, L. F., and Barling, P. M. (2003). Mitotic activity in the growing red deer antler. Cell Biol. Int. 27, 625–632. Meinhardt, U., and Mullis, P. E. (2002). The essential role of the aromatase/p450arom. Semin. Reprod. Med. 20, 277–284. Mendelsohn, C., Ruberte, E., and Chambon, P. (1992). Retinoid receptors in vertebrate limb development. Dev. Biol. 152, 50–61. Molello, J. A., Epling, G. P., and Davis, R. W. (1963). Histochemistry of the deer antler. Am. J. Vet. Res. 24, 573–579. Morishima, A., Grumbach, M. M., Simpson, E. R., Fisher, C., and Qin, K. (1995). Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J. Clin. Endocrinol Metab. 80, 3689–3698. Motomura, K., Ohtsuru, A., Enomoto, H., Tsukazaki, T., Namba, H., Tsuji, Y., and Yamashita, S. (1996). Osteogenic action of parathyroid hormone-related peptide (1-141) in rat ROS cells. Endocr. J. 43, 527–535.

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The Molecular and Genetic Control of Leaf Senescence and Longevity in Arabidopsis Pyung Ok Lim* and Hong Gil Nam National Research Laboratory of Plant Molecular Genetics, Division of Molecular and Life Sciences, Pohang University of Science and Technology Pohang, Kyungbuk, 790-784, Korea

I. II. III. IV.

Introduction Arabidopsis as a Model Plant for Studying Leaf Senescence Senescence Symptoms Identification of Senescence-Associated Genes and Their Functional Analysis A. Macromolecule Breakdown and Recycling B. Pathogenesis and Defense-Related Genes C. Regulatory Genes

V. Regulatory Mode of Senescence-Associated Genes VI. Regulatory Factors: Molecular Genetic Regulation of Leaf Senescence A. Developmental Aging Factor B. The Role of Sugar Signaling in Leaf Senescence C. The Roles of Phytohormones in Senescence D. Protein Degradation E. Potential Senescence-Regulatory Genes F. Cis-acting Regulatory Elements of Senescence-Induced Genes VII. Biotechnological Application of Senescence VIII. Conclusions and Future Challenges Acknowledgments References

The life of a leaf initiated from a leaf primordium ends with senescence, the final step of leaf development. Leaf senescence is a developmentally programmed degeneration process that is controlled by multiple developmental and environmental signals. It is a highly regulated and complex process that involves orderly, sequential changes in cellular physiology, biochemistry, and gene expression. Elucidating molecular mechanisms underlying such a complex, yet delicate process of leaf senescence is a challenging and important biological task. For the past decade, impressive progress has been achieved on the molecular processes of leaf senescence through *Current address: Department of Science Education, Cheju National University, Jeju-si, Jeju 690-756, Korea Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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0070-2153/05 $35.00 DOI: 10.1016/S0070-2153(04)67002-5

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identification of genes that show enhanced expression during senescence. In addition, Arabidopsis has been established as a model plant for genetic analysis of leaf senescence. The progress on the characterization of genetic mutants of leaf senescence in Arabidopsis has firmly shown that leaf senescence is a genetically controlled developmental phenomenon involving numerous regulatory elements. Especially, employment of global expression analysis as well as genomic resources in Arabidopsis has been very fruitful in revealing the molecular genetic nature and mechanisms underlying leaf senescence. This progress, including molecular characterization of some of the genetic regulatory elements, are revealing that senescence is composed of a complex regulatory network. In this review, we will present current understanding of the molecular genetic mechanisms by which leaf senescence is regulated and processed, focusing mostly on the regulatory factors of senescence in Arabidopsis. We also present a potential biotechnological implication of leaf senescence studies on the improvement of important agronomic traits such as crop yield and post-harvest shelf life. We further provide future research prospects to better understand the complex regulatory network of senescence. C 2005, Elsevier Inc.

I. Introduction Leaf senescence is most typically observed in the leaves at autumn and during the death process of monocarpic plants, such as rice and crysanthemum. Like other senescence events, leaf senescence is the final phase of development, during which cells undergo distinct metabolic and structural changes leading to cell death (Noode´ n, 1988). Leaf senescence and its following death is perhaps one of the most dramatic developmental phenomena encountered in nature. Thus, leaf senescence has been a favorite topic for poets and artists from ancient times and has also evoked curiosity about its underlying mechanisms. Besides artistic instinct and scientific curiosity, leaf senescence also stimulates strong interest in its practical application in improving plant productivity and storage characteristics, which should become fairly feasible by modifying the senescence process. Senescence in higher plants, including leaf senescence, is a type of programmed cell death (PCD) that occurs by a genetic program as a part of a developmental process (Nam, 1997). PCD in higher plants occurs throughout development of most organs and can be triggered by developmental as well as environmental factors, such as pathogen infection and physical injury. While other PCDs such as hypersensitive response involve rather localized cell death, cell death during senescence is mostly observed in a broad area of plant bodies, for example, in organs such as leaves, petals, fruits, or plant bodies as a whole. Cell death during senescence also

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occurs more slowly than in other PCDs, where cell death occurs more acutely. The slower cell death during senescence is, in part, associated with eYcient remobilization of nutrients that are degraded during senescence. For the clarity of this chapter, we distinguish the term ‘‘senescence’’ and ‘‘developmental aging’’ in plants. The term ‘‘senescence’’ is applicable to a process that leads to death of a cell, an organ, or a whole plant and occurs at the final stage of development. By contrast, developmental aging occurs throughout development, from initiation of a leaf primordium throughout senescence and death; conceptually, developmental aging would determine when senescence starts but it is not itself senescence. Plants, like other organisms, show two types of senescence: mitotic senescence and postmitotic senescence (Gan, 2003). Meristematic cells can undergo a given number of mitotic divisions to produce organs such as leaves and flowers. Loss of capacity for further cell divisions in the meristematic cells is called mitotic senescence or replicative senescence. This type of senescence is also observed in yeast and mammalian cells. In contrast, postmitotic senescence occurs in mature organs such as leaves and petals. Cells in these organs rarely undergo cell division, but these cells undergo cell growth, maturation, senescence, and ultimately, death. In this chapter, we focus our attention mostly on leaf senescence, a type of postmitotic senescence. A leaf is initially formed as a leaf primordium that is derived from the shoot apical meristem. After growth and maturation into a photosynthetic organ through a series of cell division and diVerentiation steps, the leaf organ undergoes the final stage of development, namely, senescence. During senescence, leaf cells undergo dramatic changes in cellular metabolism and the sequential degeneration of cellular structures (Nam, 1997; Noode´ n, 1988). The cellular degeneration process occurs in an orderly manner, beginning with the chloroplast. The mitochondria and the nucleus remain intact until the final stages of leaf senescence. The metabolic changes include loss of photosynthetic activities and hydrolysis of macromolecules that have been accumulated during the growth phase. These degenerative activities occur concomitantly with a massive remobilization of the hydrolyzed molecules to the growing parts of plants, such as young leaves, developing seeds, and fruits. Leaf senescence is therefore an important phase in the plant life cycle that critically contributes to the fitness of plants, ensuring better survival of plants and optimal production of their oVspring (Nam, 1997; Noode´ n, 1988). Since 1995, some extensive research has been conducted to understand the molecular genetic mechanisms underlying the leaf senescence process by identifying mutants that show altered senescence processes and genes that show altered expression during senescence, using the model plant Arabidopsis. Analyses of these mutants and genes are beginning to expand our understanding of the nature and regulation of leaf senescence. In this chapter, we present current understanding of the molecular genetic

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mechanisms by which senescence is regulated and processed, using results obtained mostly from the model plant Arabidopsis. We further provide a few points for future research trends.

II. Arabidopsis as a Model Plant for Studying Leaf Senescence Extensive genomic resources are available for Arabidopsis, which makes it useful for the rapid identification and functional analysis of senescenceregulated genes. In addition to the general advantage of Arabidopsis as a molecular genetic model plant, the Arabidopsis leaf has a short life cycle, readily distinguishable developmental stages, and a well-defined and reproducible senescence program (Bleecker and Patterson, 1997). In many monocarpic plants, such as the pea and soybean, leaf senescence is coupled to development of the reproductive organ (Pic et al., 2002). However, mutations in Arabidopsis that caused male sterility, delayed flowering time, or early termination of the inflorescence have little eVect on the timing of senescence in one particular leaf. Instead, the developmental age of the individual leaf is a major factor that governs leaf senescence (Hensel et al., 1993). The lack of correlative controls on leaf senescence in Arabidopsis leads to a more precise examination of the intrinsic process within a leaf that contributes to the observed age-related senescence. However, the reader is reminded that the findings in Arabidopsis may not reveal some of the molecular mechanisms underlying leaf senescence in other plants. Senescence is a complex process that integrates many other aspects of plant physiology, including leaf development and metabolism. A rich resource of well-characterized mutations in Arabidopsis is also beneficial, since some of these mutations can be utilized to understand the relationship between senescence and other plant physiology. Recognized for these points as a favorite model plant for senescence study, Arabidopsis has been productively utilized for identifying senescence regulators and for elucidating the regulatory mechanisms of leaf senescence through molecular and genetic approaches.

III. Senescence Symptoms Obvious visual symptoms of leaf senescence in Arabidopsis are loss of chlorophyll (degreening), desiccation, and eventual death. The events contributing to these visual symptoms involve complex cellular and molecular changes that exhibit a number of distinguishing features. Ultrastructural

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studies in several species provide a consistent picture of the progressive changes that occur during leaf senescence (Noode´ n, 1988; Thomson and Plat-Aloia, 1987). The initial stages of the senescence symptoms involve a breakdown of membrane structure within chloroplast, where >50% of the protein and >70% of the lipid of leaf are present (Dean and Leach, 1982; Forde and Steer, 1976). This chloroplast degeneration is accompanied by the degreening of the leaves and the progressive loss of proteins in the chloroplast, such as ribulose biphosphate carboxylase and chlorophyll a/b binding proteins (Bate et al., 1990). The cytoplasm of cells undergoing senescence is also aVected, exhibiting a decrease in cytoplasmic volume. The number of detectable cytoplasmic ribosomes is also reduced, reflecting a decrease in overall protein synthesis, although there are some proteins that are produced at a higher level during senescence (Hensel et al., 1993; Lohman et al., 1994). In Arabidopsis, the reduced number of cytoplasmic ribosomes in senescing leaves is reflected as detectable decreases in rRNA and protein. On the other hand, the nucleus and the mitochondria, which are essential for gene expression and for energy production, respectively, remain intact until the last stages of senescence (Smart, 1994). This reflects that the senescing cells need to be functional for progression of senescence until a late stage of senescence, possibly for eYcient reutilization of the cellular materials. Visible disintegration of the plasma and vacuolar membranes seems to be a late event. The loss of integrity of the plasma membrane would lead to the end of homeostasis, resulting in death. The biochemical and physiological changes during leaf senescence are most easily understandable from the standpoint of nutrient salvage, hydrolysis of macromolecules, and subsequent remobilization, which requires operation of a complex array of metabolic pathways. These changes also well reflect the ultrastructural changes observed in senescing leaves. During leaf senescence, a decline in the structural and functional integrity of cellular membranes is clearly noticed at an ultrastructural level, which is the result of the hydrolysis and metabolism of membrane lipids. Lipid-degrading enzymes such as phospholipase D, phosphatidic acid phosphatase, lytic acyl hydrolase, and lipoxygenase appear to be involved in this process (Thompson et al., 1998). For example, chloroplast thylakoid lipids are degraded initially by galactolipase and lipolytic acyl hydrolase (Woolhouse, 1984) and provide an abundant carbon that can be mobilized and used as an energy source during senescence (Ryu and Wang, 1995). Although some of the fatty acids released from membrane lipid hydrolysis in senescing leaves may play other roles, such as providing the substrate for jasmonic acid biosynthesis as in the case of linolenic acid, the majority is either oxidized to provide energy for the senescence process or converted to a-ketoglutarate via the glyoxylate cycle. The a-ketoglutarate can be

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converted into phloem-mobile sugars through gluconeogenesis or used for the mobilization of amino acids released during leaf protein degradation (Buchanan-Wollaston, 1997; Smart, 1994). A massive decrease in nucleic acids occurs during leaf senescence. Total RNA levels are rapidly reduced with the progression of senescence. The initial decrease in the RNA levels is distinctively observed with the chloroplast rRNAs and cytoplasmic rRNAs. The amounts of rRNA species are likely to be regulated coordinately, although this aspect has not been analyzed. The decrease in rRNA is followed by the cytoplasmic mRNA and tRNA. The decrease in RNA levels is accompanied by increased activity of several RNases. However, how exactly each RNase functions during senescence has not been revealed. Chloroplast DNA is likely the first DNA to be degraded along with chloroplast degeneration. The nuclear and mitochondrial DNAs are degraded at the later stage of senescence. Concomitantly, there is an increase in several DNAase activity. It is interesting that there is some similarity between the meiotic senescence of animal cells and mitotic senescence of plant cells in terms of metabolism of nuclear DNA. There appears to be involvement of telomerases and chromosome fragmentation in mitotic senescence of plant cells, although these observations need to be established with more experiments. Protein degradation is a major biochemical event in leaf senescence. Thus, leaf senescence is accompanied by increased activity of various proteases, including the cysteine proteases that are specifically expressed during senescence. A senescence-associated RD21 cystein protease is accumulated in the vacuole as an inactive aggregate and slowly matures to produce a soluble active enzyme at later stages of senescence (Yamada et al., 2001), implying that RD21 has a role in protein degrading during the late senescence stage. Involvement of vacuolar enzymes in a progressive amino terminal degradation of the large subunit of Rubisco in French bean leaves has been proposed earlier (Yoshida and Minamikawa, 1996). As senescence is an orderly process with one of its major functions being nutrient remobilization, protein degradation should also occur in a highly regulated manner. However, detailed knowledge on the protein degradation process, such as which proteins are degraded in which order and which specific proteinases are involved in a specific group of proteins, is still lacking. Hydrolysis and subsequent remobilization of proteins present in the chloroplast constitute one of the major metabolic events occurring early in leaf senescence. Degradation of the predominant stromal proteins, such as Rubisco and glutamine synthetase, has been a favorite focus for studying protein metabolism during senescence, due to their major role in remobilization of proteins. Degradation of these proteins can be initiated nonenzymatically by reactive oxygen species, although it is not clear whether increased ROS could initiate the early step of the degradation of the proteins

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during senescence (Ishida et al., 1999, 2002; Roulin and Feller, 1998). A complete hydrolysis of proteins to free amino acids depends on the action of several endo- and exopeptidases (Brouquisse et al., 2001). Several types of endopeptidases, such as aminopeptidase and metalloendopeptidase, have been detected in the chloroplasts and their involvement in stroma protein degradation has been presented by several groups (Bushnell et al., 1993; Roulin and Feller, 1998). Chloroplast localization of members of the Clp protease family was also reported (Roulin and Feller, 1998). These enzymes may have a role in protein turnover during leaf senescence (Majeran et al., 2000; Shikanai et al., 2001). Breakdown of the thylakoid-bound proteins such as LHCPII during senescence is interconnected with degradation of chlorophyll (Hidema et al., 1992; Ho¨ rtensteiner and Feller, 2002). The LHCP proteins exist as a pigment–protein complex with chlorophyll. Their degradation requires the simultaneous catabolism of chlorophyll, since the disassembly of the pigment–protein complex causes release of hazardous chlorophyll which induces photooxidative damage. Once the chlorophyll is degraded, the thylakoid proteins are degraded by proteases present in chloroplast. Interestingly, nitrogen present in chlorophyll is not exported from senescing leaves, but remains in the form of linear tetrapyrrolic catabolites that accumulate in the vacuole. Therefore, the energy-expensive chlorophyll degradation steps do not appear to be carried out in order to mobilize the nutrients, but rather to remove the potentially toxic-free chlorophyll. The role of the vacuole-accumulated tetrapyrrolic catabolites remains to be investigated, for example, as a defense mechanism. Chloroplast constituents become available to vacuolar and other extraplastidal enzymes after rupture of the chloroplast membrane, while by early senescence stage these catabolic enzymes are separated from stroma and thylakoid components by membranes. An example is possible involvement of vacuolar enzymes in a progressive amino terminal degradation of the large subunit of Rubisco in French bean leaves. Massive remobilization of cellular materials during leaf senescence not only involves protein and membrane constituents but also minerals. Accordingly, levels of various leaf constituents including N, P, K, Mo, Cr, S, Fe, Cu, and Zn are significantly reduced in senescent leaves when compared to green leaves (Himelblau and Amasino, 2001).

IV. Identification of Senescence-Associated Genes and Their Functional Analysis Leaf senescence is a genetically programmed process, and thus requires de novo gene expression and protein synthesis that should be controlled in a highly coordinated manner. Since 1995, there have been extensive eVorts to reveal the

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underlying molecular mechanism of plant senescence by identifying and analyzing so-called senescence-associated genes (SAGs) (Buchanan-Wollaston, 1997; Nam, 1997; Noode´ n et al., 1997). We will here summarize the molecular nature of leaf senescence implicated from the analysis of the SAGs. Investigation of SAGs has been greatly aided by technological advances such as DNA microarray that provided information at a genome-wide level. Initially, diVerential screening of genes up-regulated during leaf senescence was utilized to reveal some dozens of SAGs (Park et al., 1998). These mostly included highly up-regulated genes during senescence. Subtractive hybridization circumvented some of the shortcomings of the conventional diVerential screening approaches. For example, a large-scale identification of SAGs via suppression subtractive hybridization has added 70 new members to the current SAG collection in Arabidopsis (Gepstein et al., 2003). We also found several potential regulatory genes of senescence by a similar approach. EST (expressed sequence tag) analysis has been another straightforward approach to identify SAGs. For example, genes preferentially expressed in autumn leaves of field-grown aspen (Populus tremula) were investigated by generating ESTs (Bhalerao et al., 2003). For Arabidopsis leaves, transcriptom associated with leaf senescence was examined by a large-scale EST analysis (Gepstein, 2004; Guo et al., 2004). Microarray analysis generated a genome-wide molecular view of leaf senescence. For example, a DNA microarray with 13,490 aspen ESTs was used to analyze the leaf transcriptom of aspen leaves during autumn senescence (Andersson et al., 2004). For Arabidopsis, a microarray with 8000 genes was utilized to compare gene expression in mature, green, early, and midsequencing leaves (Buchanan-Wollaston et al., 2003). A more specific case is analysis of the mRNA expression profiles of 402 potential transcription factors at diVerent developmental stages and under various biotic and abiotic stresses (Chen et al., 2002). Furthermore, diVerential gene expression associated with darkinduced Arabidopsis leaf senescence was also monitored using DNA microarray (Lin and Wu, 2004). We also analyzed the eVect of some of the delayed senescence mutants, using either the gene chip representing 25,000 genes (collaboration with Buchanan-Wollaston’s group) or AVymetrix Arabidopsis gene chips representing approximately 24,000 Arabidopsis genes. Another approach that allows identification of SAGs and their in planta function simultaneously is the enhancer trap (He et al., 2001) or promoter trap approach. In this approach, senescence-induced genes are identified by examining expression of a reporter gene trapped by the vector. We were able to identify a delayed senescence mutant out of 7 promoter trap lines that showed a senescence-induced expression pattern (Lim et al., unpublished). The SAGs identified by these studies include genes for potential regulatory factors as well as genes executing the senescence program. The spectrum of the SAGs is mostly consistent with known biochemical and physiological

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symptoms but it also provides many new insights into the molecular events and their regulation during leaf senescence. However, it is obvious that we do not know the function of many of these SAGs and that functional characterization of them will be one of the main elements in understanding leaf senescence and utilizing the knowledge for practical applications. In the following, we describe some of the SAGs by grouping them into a few functional categories, based mostly on their predicted functions.

A. Macromolecule Breakdown and Recycling Consistent with the biochemical data, many of the genes involved in protein turnover are induced during leaf senescence. These included the cDNAs encoding cystein proteases, cathepsin B-like cystein protease, aspartic proteases, and vacuolar-processing enzymes (Kinoshita et al., 1999). An increased expression of the genes encoding proteins associated with the ubiquitination cascade, such as polyubiquitin, ubiquitin carrier protein, 26S proteasome ATPase subunit, and SKP-interacting partner, reflects the involvement of ubiquitin/proteasome-mediated proteolysis during leaf senescence (Gepstein et al., 2003). The importance of ubiquitin/proteasome pathways was also revealed during the dark-induced leaf senescence process (Lin and Wu, 2004). Vegetative storage proteins (VSPs) were suggested to serve as a storage buVer for nitrogen source. The steady-state levels of the VSP1 and VSP2 transcripts are increased during senescence, supporting the postulated function. Elevated expression of genes for cytosolic glutamine synthase and aspartate amino transferase during senescence is consistent with the fact that amino acids are modified into organic nitrogen compounds before being loaded into the vascular system (Nam, 1997). As leaf senescence involves degradation of membrane lipid, some of the genes involved in lipid metabolism are up-regulated during leaf senescence. These included genes for lipase (Gepstein et al., 2003), phospholipase Da (Fan et al., 1997), and acyl hydrolase (He and Gan, 2002). A gene for a key glyoxysomal protein, 3-ketoacyl-CoA thiolase, which may function in the remobilization of fatty acid, is induced during leaf senescence along with other glyoxysomal protein genes (Graham et al., 1992). At least a few of the genes involved in lipid metabolism have a clear role in leaf senescence. For example, reduced expression of the Arabidopsis acyl hydrolase gene by antisense RNA interference in transgenic plants delays the onset of leaf senescence, while chemically induced overexpression of the gene caused precocious senescence (He and Gan, 2002). Transgenic plants with reduced expression of a senescence-induced lipase also show delayed leaf senescence (Thompson et al., 2000). It is likely that the delayed senescence in these

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transgenic lines with reduced lipase expression is due to prolonged maintenance of membrane integrity, indicating the importance of membrane integrity during senescence. Alternatively, the lipase may produce a regulatory molecule(s) that hastens the senescence process. A role of phospholipase D (PLD) in plant senescence was also examined using Arabidopsis with reduced PLD activity. The suppression of PLD activity induced a delay in abscisic acid- and ethylene-promoted senescence in detached leaves (Fan et al., 1997), but did not aVect the natural senescence. Involvement of autophagy pathway during leaf senescence is indicated by an increase in the expression of the autophagy genes, such as APG7 and APG8 (Doelling et al., 2002). Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components and is known to be required for nutrient recycling. As observed in yeast, autophagy may contribute to maintaining cell viability during senescence/starvation. Mutants carrying a T-DNA insertion within the Arabidopsis autophagy genes, AtAPG7 and AtAPG9, exhibited earlier leaf senescence phenotype. In these mutants, nutrients may be less eYciently utilized for execution of senescence (Doelling et al., 2002; Hanaoka et al., 2002) or some of the components needed for progression of senescence may not be eYciently provided and could cause the early senescence phenotype. Alternatively, the incompletely degraded cytoplasmic constituents may interfere with cellular processes during leaf senescence.

B. Pathogenesis and Defense-Related Genes A large proportion of SAGs belong to genes encoding proteins related to pathogenesis and defense (Gepstein et al., 2003; Quirino et al., 1999, 2000). Some of these encode products that are similar to the pathogenesis-related proteins (PR proteins). PR proteins are associated with the hypersensitive response and with the systemic acquired resistance (SAR) defense program. Upregulation of PR proteins during senescence implies that there is an overlap between the two pathways, senescence and pathogen response. The overlapping functions may include cellular defense and cell death process. Many stress-inducible genes are up-regulated during senescence, implying that, during senescence, the cells are under a stress condition (Blein et al., 2002; Eriksson et al., 2002; Gepstein et al., 2003). These genes include those for lipid transfer protein and myrosinase-binding protein, which has a role in plant resistance to biotic and abiotic stresses. Other stress-related genes include genes for metallothionein and ferritin, which might be involved in the chelation of metal ions released during cellular degradation and/or functioning as metal-binding proteins for storage or transport into developing organs (Hsieh et al., 1995). The genes involved in the oxidative stress response are also induced, including the genes for Fe2+-ascorbate oxidase

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(Callard et al., 1996), anionic peroxidase (Tournaire et al., 1996), glutathione S-transferase (Smart et al., 1995), and a blue copper-binding protein. These stress-related genes may participate in protecting the cellular integrity required for progression and completion of senescence, although there is no report on the functional eVect of these genes in leaf senescence.

C. Regulatory Genes Identification and functional characterization of regulatory genes associated with leaf senescence have been two of the main interests in the recent study of leaf senescence (Buchanan-Wollaston et al., 2003; Gepstein et al., 2003; Lin and Wu, 2004). Potential regulatory genes for senescence would include genes for various transcription factors and for signal perception and transduction (Table I). Senescence up-regulated transcription factor genes belong to various transcription factor families including WRKY, EREBP, NAC, bZIP, C2H2, and MYB families. Among the senescence up-regulated transcription factor genes, the genes for WRKY53, a MYB protein, and zinc finger protein show transiently increased expression at a very early stage of leaf senescence but decreased expression again at a late stage (Buchanan-Wollaston et al., 2003; Hinderhofer and Zentgraf, 2001). These genes may play an important role in the early event of senescence, as we found one such gene belonging to the RAV family shows such an expression pattern and has a regulatory function in leaf senescence (Lim et al., unpublished data). Other potential regulatory genes include genes for a LIM domain protein (At2g39900), since LIM domain proteins are known to participate in gene transcription, possibly by assembling and stabilizing transcription complexes (Mundel et al., 2000). Several genes for potential signaling components are also upregulated during senescence. For example, a senescence-associated receptor kinase (SARK) in bean was shown to be expressed early in leaf senescence (Hajouj et al., 2000). In Arabidopsis, senescence-induced receptor kinases (At5g48380, At2g19190) were reported (Gepstein et al., 2003; Robatzek and Somssich, 2002). Thesereceptor kinases may function as regulatory factors perceiving or transducing signals that influence leaf senescence. We found that a senescence up-regulated gene belonging to the LRR receptor kinase gene family can hasten leaf senescence when overexpressed in transgenic plants (Koo et al., unpublished data). Possible importance of Ca++ in the regulation of senescence is reflected by elevation of genes for a calcium-binding protein (At1g18210) and a Ca++dependent protein kinase (At5g54250) (Guterman et al., 2003). A gene encoding a small GTP-binding protein (At5g47201) was induced during senescence (Guterman et al., 2003).

Table I

Senescence Regulatory Genes

Gene or Accession

Molecular Nature

Genes that alter senescence ORE4 Plastid ribosomal protein subunit 17 ORE9 F-box protein DLS1 Arginyl t-RNA transferase GIN2 Hexokinase HYS1

Unknown protein

EIN2 (¼ ORE3) ETR1 OLD1

Putative metal ion transporter Ethylene receptor Not identified

DET2 KN1 Sho

Steroid 5a-reductase Homeobox protein Protein with homology to isopentenyl transferases Eukaryotic translation initiation factor 5a-1 Autophagy gene Autophagy gene Acyl hydrolase

EIF5a-1 AtAPG7 AtAPG9 SAG101

Senescence-enhanced potential regulatory genes ATWRKY6 WRKY transcription factor

EVects of Mutations on Senescence Phenotype or Characteristics

ore4 delays leaf senescence only in age-dependent manner and shows a reduced leaf growth rate ore9 delays leaf senescence dls1 delays leaf senescence gin2 delays leaf senescence. Overexpression of HXK induces early senescence hys1 accelerates leaf senescence; allelic to cpr5; constitutive expression of pathogenesis-related genes; enhanced response to sugar ein2 shows ethylene-insensitive phenotype; ein2 delays leaf senescence etr1 shows ethylene-insensitive phenotype; etr1 delays leaf senescence old1 accelerates leaf senescence in age-dependent manner as well as ethylene-induced condition det2 delays chlorophyll loss Delays leaf senescence when overexpressed in senescence stage Overexpression of Sho induces enhanced shooting, reduced apical dominance and delayed senescence Antisense suppression of EIF5a-1 delays leaf senescence

References

1 2 3 4, 5, 6 7 8 9 10 11 12 13 14

Atapg7 knockout line shows early leaf senescence Atapg9 knockout line shows early leaf senescence Antisense suppression of SAG101 delays leaf senescence

15 16 17

Senescence-induced

18, 19

WRKY53 SARK SIRKa (At2g19190) SIRKb (At5g48380) At3g528000 At5g10650 At1g05340 At1g18210 At1g78080 At5g47201 SENU5

WRKY transcription factor Receptor-like kinase (bean) Receptor-like kinase Receptor-like kinase Zinc finger-like protein Zinc finger-like protein TonB-dependent receptor Calcium-binding protein RAP2.4–AP2 domain TF Ras-related small GTP-binding protein NAC domain family

Senescence-induced

20

Senescence-induced, cytokinin or light inhibits SARK gene expression Senescence-induced Senescence-induced Senescence-induced Senescence-induced Senescence-induced Senescence-induced Senescence-induced Senescence-induced

21 19 22 22 22 22 22 22 23

Senescence-induced

24

This table illustrates possible senescence-regulatory genes. Genes that alter leaf senescence phenotype or potential regulatory genes, such as transcription factors or receptor-like kinase, that are induced during leaf senescence were included. This is only a representation of published results and many genes are not included. References: 1. Woo et al., 2002; 2. Woo et al., 2001; 3. Yoshida et al., 2002a; 4. Jang et al., 1997; 5. Dai et al., 1999; 6. Moore et al., 2002; 7. Yoshida et al., 2002b; 8. Oh et al., 1997; 9. Grbic and Bleecker, 1995; 10. Jing et al., 2002; 11. Chory et al., 1991; 12. Ori et al., 1999; 13. Zubko et al., 2002; 14. Thompson et al., 2004; 15. Doelling et al., 2002; 16. Hanaoka et al., 2002; 17. He and Gan, 2002; 18. Robatzek and Somssich, 2001; 19. Robatzek and Somssich, 2002; 20. Hinderhofer and Zentgraf, 2001; 21. Hajouj et al., 2000; 22. Gepstein et al., 2003; 23. Guterman et al., 2003; 24. John et al., 1997.

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V. Regulatory Mode of Senescence-Associated Genes Leaf senescence occurs by age-dependent internal factors and is also influenced by a range of other internal and environmental factors, such as phytochrome, darkness, drought, pathogen attack, and oxidative stress (Hensel et al., 1993; Quirino et al., 2000). Although these senescenceinfluencing factors induce apparently similar symptoms, the underlying molecular programs are not identical. Some SAGs are induced upon senescence caused by various senescence-influencing factors. However, the regulatory modes of other SAGs show that leaf senescence caused by diVerent senescence factors involves the diVerential induction of SAGs (Nam, 1997; Park et al., 1998), indicating that the molecular states of leaf senescence caused by various senescence factors are diVerent. This may imply that the process of leaf senescence involves fine-tuning the expression of SAGs to incorporate complex senescence-inducing signals into the senescence program. Thus, leaf senescence could be considered a complex process in which various external and internal influences are superimposed on the age-dependent developmental program.

VI. Regulatory Factors: Molecular Genetic Regulation of Leaf Senescence As has been described, the expression patterns of SAGs in response to diVerent senescence-inducing treatment have indicated the existence of a complex regulatory network in leaf senescence processes (He et al., 2001; Lim et al., 2003; Quirino et al., 2000) (Fig.1). We will summarize in this part the regulatory factors that govern the complex network of senescence (Table I). An emphasis will be given on the regulatory factors identified by genetic screening of senescence mutants. Genetic screening of mutants exhibiting early or delayed leaf senescence phenotype has become easy in Arabidopsis. Characterization of the mutants and molecular cloning of the corresponding genes in Arabidopsis has been highly valuable in dissecting the pathways and identifying key regulatory factors involved in leaf senescence, revealing some new insight into the regulatory mechanisms in leaf senescence. A. Developmental Aging Factor Senescence is certainly associated with the developmental aging process in Arabidopsis and thus occurs after a certain developmental stage. Therefore, there should be a cellular mechanism(s) that measures the age of a cell, tissue, organ, or whole body for initiation and/or progression of senescence.

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Figure 1 A Hypothetical model for genetic pathways of leaf senescence. Leaf senescence occurs in an age-dependent manner in many species, but the initiation and progression of senescence can be modulated by a variety of environmental factors such as nutrient deficiency, drought conditions, or pathogen infection. It is also known that internal factors such as plant growth regulators, reproduction, and cellular dierentiation also influence senescence. In this model, leaf senescence is viewed as a complex process in which the eVects of various internal and environmental factors are superimposed on the developmental age-dependent senescence pathways. Multiple pathways that respond to various factors are possibly interconnected to form a regulatory network. However, the molecular the nature and the function of the regulatory factors that incorporate developmental signals and other internal and external signals of leaf senescence are largely unknown. The WRKY transcription factors (WRKY6, WRKY53) or senescence-induced receptor kinase (SIRK) are potential candidates for genes that recognize and transducer age-information into senescence-related physiology or the regulation of SAGs. For the senescence program to proceed, there are likely to be genes that execute the degeneration process. Such genes could be involved in many aspects of the degradation process, including chlorophyll breakdown, and nitrogen and lipid remobilization. Studies on the expression of SAGs in response to dierent senescence-inducing treatments have shown that there is extensive overlap between agedependent leaf senescence and senescence induced by other factors.

The evidence for the presence of genes that alter senescence by controlling developmental aging is accumulating. One of the known senescence-associated genes (SAG12) of Arabidopsis is up-regulated in an age-specific manner and minimally regulated by environmental factors (Gan and Amasino, 1997). There has been no formal report on genes that alter senescence by controlling developmental aging, and the nature of genes that control aging in plants is still not known. It is possible that genes that determine the metabolic rate may regulate developmental aging in plants, as has been suggested for

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Caenorhabditis elegans. The daf-2 and clk-1 mutations of C. elegans confer marked increases in longevity. The DAF-2 gene encodes an insulin/insulin-like growth factor receptor homolog and the CLK-1 gene encodes a protein similar to the yeast metabolic regulator Cat5p (Ewbank et al., 1997; Kimura et al. 1997). These results led to the suggestion that metabolic rate is a mechanism that regulates the aging process (Guarente, 1997). Studies of the ore4-1 mutant in Arabidopsis have shown that the mutation causes a delay in leaf senescence during natural age-dependent senescence, but not in hormone or dark-induced senescence (Woo et al., 2002). The ore4-1 mutant has a partial lesion in chloroplast functions, including photosynthesis, which resulted from reduced expression of the plastid ribosomal protein small subunit 17 (PRPS17) gene. It was suggested that the delayed leaf senescence phenotype observed in the ore4-1 mutant is likely due to reduced metabolic rate, because the chloroplasts, the major energy source for plant growth via photosynthesis, are only partially functional in the mutant. Reduced metabolic rate could lead to less oxidative stress, leading to delayed aging (Munne-Bosch and Alegre, 2002). Similar phenotype was observed in transgenic tobacco plants where expression of Rubisco gene was down-regulated. The leaves of these plants have a longer life span, a reduced fresh weight, and a lower photosynthetic activity than those of wild-type tobacco plants (Miller et al., 2000). As the leaves of the transgenic plants mature at the same rate as wild-type leaves, their delayed leaf senescence phenotype is likely due to reduced metabolic rate. This observation is consistent with the findings in animals that suggest metabolic rate is one of the key mechanisms involved in aging (Ewbank et al., 1997; Kimura et al., 1997), Since leaf senescence is the final stage of leaf development, leaf senescence should be intimately related to the previous developmental stages of the leaf, such as leaf initiation, growth, and maturation. Thus, it is possible that genes controlling these processes, including genes that control mitotic senescence, could influence age-dependent senescence. In that respect, we observed that the leaves of the bop1-1 mutant that show enhanced meristematic activity in leaves show a prolonged life span (Ha et al., unpublished data). B. The Role of Sugar Signaling in Leaf Senescence Sugars are known to act as signaling molecules during various stages of plant development and for a diverse physiology of plants. The hexokinase is a glucose sensor and has a central role in modulating multiple signaling pathways (Rolland et al., 2002). Physiological and genetic analyses indicate that sugar signaling might also be involved in controlling leaf senescence. Source-sink balance, which can aVect the partitioning of sugar in plants, might be important in regulating senescence (Ono et al., 2001). Young leaves

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serve as sink organs, whereas old leaves are source organs that provide sugars. When young leaves mature, they develop photosynthetic machinery, which leads to the elevated level of sugars. It is well known that sugar can repress the expression of photosynthesis-associated genes, presumably via an end-product negative feedback system. Thus, it was proposed that accumulation of sugar in mature leaves will lead to a decline of photosynthetic activity and the reduction of photosynthetic activity at a certain threshold level might act as a senescence signal (Bleecker and Patterson, 1997; Hensel et al., 1993). In agreement with this argument, transgenic plants that overexpress hexokinase exhibit reduced photosynthetic activity, resulting in a decline in sugar production. A notable phenotype found in the transgenic plants is accelerated leaf senescence, supporting the theory that reduced photosynthetic activity may be related to premature leaf senescence (Dai et al., 1999; Jang et al., 1997). Moreover, glucose-insensitive Arabidopsis mutant (gin2) with a lesion in one of the hexokinases shows delayed senescence, suggesting that the sugar level sensed by hexokinase might aVect leaf senescence (Moore et al., 2003). Thus, it appears that sugar signaling is involved in modulating leaf senescence. However, the senescence phenotype observed in this case was not thoroughly examined and needs to be analyzed in more detail to support the idea. The hys1 (hypersenescence1) mutant has an increased sensitivity to exogenously applied sugars as well as accelerated leaf senescence phenotype. This observation led to a suggestion that an enhanced sugar signal in the mutant causes reduced photosynthetic activity and induces premature senescence, likely via hexokinase (Yoshida et al., 2002b). It should be noted that control of senescence by sugar signaling is most likely aVected by other factors, such as nitrogen status and the developmental stage (Ono et al., 2001; Paul and Foyer, 2001; Yoshida, 2003). An integration of these factors into the senescence program might be important in properly regulating the timing of onset and progression of leaf senescence.

C. The Roles of Phytohormones in Senescence The eVect of plant growth regulators, including cytokinins, ethylene, abscisic acid (ABA), methyl jasmonate (MJ), brassinosteroid, salicylic acid (SA), and auxin on leaf senescence has been extensively studied during the past several decades for agronomic purposes. Although ethylene and cytokinins are known to have a major eVect on leaf senescence, other plant hormones also aVect senescence. Cytokinins are the most eVective senescence-retarding growth regulator (Gan and Amasino, 1995; McCabe et al., 2001; McKenzie et al., 1998; Ori et al., 1999). Exogenous application of cytokinins delays leaf senescence in Arabidopsis and other plants. A striking example of the

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senescence-suppressing eVect of cytokinins was observed in transgenic tobacco and lettuce plants expressing the IPT gene, an Agrobacterium-originated cytokinin biosynthesis gene, under the control of the senescence-specific SAG12 promoter (Gan and Amasino, 1995; McCabe et al., 2001). These transgenic plants showed markedly delayed leaf senescence without noticeable pleiotropic phenotypes. Delayed leaf senescence was also observed in an activation tagging lines of petunia that over-express Sho, a gene with a similarity to the Arabidopsis IPT genes (Zubko et al., 2002). It is also noted that transgenic tobacco plants that express the maize homeobox gene knotted1 under the control of the SAG12 promoter exhibit delayed leaf senescence (Ori et al., 1999). The delayed leaf senescence in the transgenic lines was accompanied by increased levels of cytokinin in the older leaves. Thus, the eVect of KN1 on senescence was suggested to be mediated through changes in the cytokinin level. Alternatively, expression of KN1 in the transgenic lines may change the developmental status of leaf cells into younger cells, since KN1 controls the meristematic activity. This may lead to delayed leaf senescence and increased cytokinin level might be an indirect eVect of KN1 gene expression. Although the exact mechanism by which KN1 controls leaf senescence needs to be investigated, this explanation is consistent with what we observed in the bop1-1 mutant (Ha et al., 2003). Despite the importance of cytokinins in controlling leaf senescence, the underlying molecular basis for the antisenescing eVect of cytokinins is still not well understood. Recently, an important and interesting link between the anti-senescence eVect of cytokinins and the function of extracellular invertase was suggested, based on the finding that cytokinin-mediated delay of senescence is correlated with extracellular invertase activity (Lara et al., 2004). The transgenic plants with senescence-induced or chemical-induced expression of extracellular invertase showed delayed senescence. Moreover, when extracellular invertase activity is inhibited, senescence was no longer delayed by cytokinins. These findings suggest that extracellular invertase might be an important mediator of cytokine action in delaying leaf senescence. Our observation that a mutation in a cytokinin receptor of Arabidopsis causes delayed leaf senescence through a constitutive cytokinin response adds an exciting insight into the molecular mechanism of cytokinin action in controlling senescence (Kim et al., unpublished result). Ethylene has long been known as a senescence-accelerating phytohormone with its strong eVect on fruit ripening and flower and leaf senescence (Bleecker and Patterson, 1997). The importance of endogenous ethylene in senescence was clearly demonstrated in transgenic plants with altered ethylene synthesis. For example, transgenic tomato plants expressing 1-aminocyclopropane-1carboxylic acid (ACC) deaminase exhibited significant delays in fruit ripening and deterioration (Klee et al., 1991). Identification of genetic mutants with a lesion in ethylene response has permitted a more precise analysis of

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the role of ethylene in senescence. Two ethylene-insensitive mutants, etr1 (ethylene-resistant1) and er (ethylene-resistant), were originally isolated from Arabidopsis thaliana based on their insensitivity to exogenously added ethylene during seedling development. These mutants were later shown to be allelic to ore3, which exhibited a measurable delayed in the initiation of leaf senescence, but has little eVect on progression of senescence (Grbic and Bleeker, 1995; Oh et al., 1997). This observation thus suggested that ethylene plays a role in coordinating the timely transition of the leaf to senescence state but it is not essential to the process itself. Several ethylene-related mutations in tomato are also known to show delayed leaf senescence and fruit ripening. For example, the tomato Nr (Never-ripe) mutant with ethylene insensitivity shows delayed fruit ripening and leaf senescence (Lanahan et al., 1994). This phenotype is consistent with the fact that the Nr gene encodes a protein homologous to ETR1. Several lines of evidence reveal that ethylene-mediated pathways leading to leaf senescence in Arabidopsis depend on age-dependent factors; thus, ethylene can induce senescence only after leaves reach a certain developmental stage (Grbic and Bleeker, 1995; Weaver et al., 1998). Similarly, tomato fruit ripening was induced by exogenously applied ethylene in mature green fruit, but not in immature fruit (Yang, 1987). A potential regulator that may be involved in integrating ethylene signaling into age-dependent pathways has been reported. The onset of leaf death1 (old1-1) mutant displayed a phenotype with earlier onset of senescence in an age-dependent manner. The early senescence phenotype was further accelerated by ethylene exposure, leading to a suggestion that OLD1 might function as a repressor for integrating ethylene action into leaf senescence (Jing et al., 2002). The NahG overexpressor transgenic plants that do not accumulate SA showed reduced expression of senescence-associated genes, indicating that SA induces senescence symptoms. In addition, SA level increases in senescing leaves, which could account for the senescence-enhanced expression of some of the SAG genes (Morris et al., 2000). Moreover, the NahG transgenic plants exhibit delayed leaf senescence phenotype during natural senescence, which is consistent with the molecular data (unpublished data). Thus, there should be a role of the SA pathway in leaf senescence, possibly in the final death phase of senescence. Exogenous treatment of MJ (methyl jasmonate) induced leaf yellowing by activating a subset of SAGs. Furthermore, jasmonic acid (JA) level increases during leaf senescence; JA levels in senescing leaves are 4-fold higher than in nonsenescing ones (He et al., 2002). These results suggest that MJ is a senescence-promoting hormone. This notion is consistent with the observation that expression of SAGs was reduced in jasmonic acid-insensitive mutant coi1, although the coi1 mutant did not show any visible phenotype, presumably due to functional redundancy. cos1 (coi1 suppressor) that restores the

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coi1-related phenotypes, including defect in senescence, was identified. The COS1 encodes lumazine synthease, a key component in the riboflavin pathways. More studies are required to understand the role of the riboflavin pathways in controlling JA-mediated leaf senescence (Xiao et al., 2004). The eVect of brassinosteroids on plant senescence has not been extensively examined. Nonetheless, there is genetic evidence that brassinosteroids may also be involved in leaf senescence. The det2 (de-etiolated2) mutation has a defect in an early step of brassinosteroid biosynthesis and was reported to confer delayed leaf senescence symptoms, although the symptom was observed only by delayed leaf yellowing (Chory et al., 1991). It was also shown that bri1-EMS-suppressor1 (bes1), which exhibits constitutive BR response phenotypes, showed accelerated senescence phenotype. These reports support a role of brassinosteroids in promoting leaf senescence (Yin et al., 2002). However, it will be necessary to examine the senescence phenotype in these mutants in more detail, including expression of SAGs, to establish that the genes related to synthesis or perception of brassinosteroids alter senescence. The role of auxin on leaf senescence has not been clear, although its inhibitory role in abscission was reported (Bleecker and Patterson, 1997). A delayed senescence mutant we have identified exhibited altered auxin response phenotype (unpublished result). This mutation may provide a clue as to the involvement of auxin in controlling leaf senescence. It is notable that plant hormones are involved in correlative control of senescence (Noode´ n, 1988), although it does not happen in Arabidopsis. It is also notable that plant hormones interact or crosstalk with one another, constituting a complex network of regulation. This is likely the case in controlling plant senescence.

D. Protein Degradation Specific control of protein degradation and stability has emerged as a pivotal mechanism that regulates the growth and development of eukaryotic organisms. Genetic studies in Arabidopsis indicate that protein degradation is also involved in controlling leaf senescence. The ore9 mutant of Arabidopsis exhibited a delay in a wide variety of senescence symptoms (Oh et al., 1997). ORE9 was identified as a protein containing an F-box motif, which is a component of the ubiquitin E3 ligase complex (Woo et al., 2001). The SCF complexes are known to ubiquitinate specific target substrates, which lead to subsequent proteolysis (Patton et al., 1998). Thus, ORE9 might function, via ubiquitin-dependent proteolysis, to limit leaf longevity by removing target proteins that are required to delay the leaf senescence program in Arabidopsis. Potential targets might include key negative regulatory molecules of

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senescence, such as transcriptional repressors of SAGs. Alternatively, ORE9 may function as a receptor for the selective degradation of self-maintenance proteins. Identification of the target proteins of ORE9 is in progress and should improve our understanding on the control of leaf senescence. Proteolysis by the N-end rule pathway, one of the ubiquitin pathways, also appears to be a mechanism involved in regulating leaf senescence in Arabidopsis. The delayed-leaf-senescence 1 (dls1) mutant, which is defective in arginyl tRNA:protein transferase (R-transferase), showed delayed development of leaf senescence symptoms (Yoshida et al., 2002a). R-transferase is a component of the N-end rule proteolytic pathway, which transfers arginine to the amino-terminus of proteins with amino terminal glutamyl or aspartyl residues and thereby targets the proteins for ubiquitin-dependent proteolysis (Varshavsky, 1997). Thus, DLS1 might play a role in degradation of proteins that negatively regulate leaf senescence. Nonetheless, the ore9 and dls1 mutations might have diVerent roles in leaf senescence. Unlike R-transferase, the SCF complex is not involved in the N-end rule-dependent pathway. Furthermore, the dls1 mutation delays not only initiation of leaf senescence but also its progression, whereas the ore9 mutation mainly aVects the initiation of leaf senescence. While specific protein degradation mechanisms appear to have regulatory roles in leaf senescence, much more needs to be revealed to understand their exact roles, including identification of the specific substrates.

E. Potential Senescence-Regulatory Genes 1. Transcription Factors Given that senescence is an active process involving up-regulation as well as down-regulation of a large number of genes, it is well expected that various transcription factors are involved in the senescence process. The DNA microarray examination of the mRNA levels of 402 transcription factor genes at diVerent developmental stages and under various stress conditions showed that 43 among the 402 transcription factor genes were up-regulated during senescence. Interestingly, 28 of them were also induced by stress treatment. Senescence is an integrated response of plants to endogenous developmental signals and external environmental responses. Thus, it was suggested that some regulatory genes that are involved in environmental responses would be predicted to regulate leaf senescence. The microarray data is consistent with this notion; the data imply that there is an extensive overlap in the responses to leaf senescence and stress (Chen et al., 2002). Some of the WRKY family of transcription factors are likely important regulatory factors in leaf senescence. The WRKY53 gene is up-regulated at a

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very early stage of leaf senescence but is down-regulated at a late stage (Hinderhofer and Zentgraf, 2001), indicating that the gene may play a regulatory role in the early events of leaf senescence. Expression of another WRKY gene, AtWRKY6, is influenced by several external and internal signals that trigger senescence (Robatzek and Somssich, 2001). AtWRKY6 may also be a regulator of senescence. cDNA-AFLP-based diVerential display analysis of the wrky6 mutant revealed that a senescence-induced receptor kinase gene, SIRK, may be a potential WRKY6 target gene (Robatzek and Somssich, 2002). Other possible downstream candidates of WRKY6 includes SAGs such as the senescence-associated protein 1 (SEN1), the JA regulatory protein NAC2, and a glutathione transferase. Although it is clear that the AtWRKY6 knockout mutation altered expression of senescence-associated target genes, no obvious phenotype was observed in the mutant. This is likely due to redundant genes or functional redundancy derived from other genes. Two members of the leucin zipper family of transcription factors gene of tobacco show senescence-enhanced expression (Yang et al., 2001). One of them, TBZF, is expressed in senescing leaves and flowers, while the other, TBZ17, is only accumulated in senescing leaves. The proteins of these genes are accumulated in the guard cells and vascular tissues of senescing leaves. These cells need to remain functional until the very last stages of senescence, since the guard cells function in response to environmental stimuli for transpiration and air exchange and since the vascular tissues function for mobilization of materials from the senescing leaf. These proteins, thus, may play a role in retarding senescence in these specific cell types. Although this notion needs to be proved, this could be an important case of diVerential regulation of cell-type specific senescence. The tomato SENU5 is a senescence up-regulated gene (John et al., 1997) and encodes a protein that belongs to the NAC domain family. The NAC domain family of proteins include Petunia No Apical Meristem (NAM), Arabidopsis NAP (a target of the homeotic AP3/PI proteins), and GRAB (a protein interacting with Gemini virus RepA protein). Thus, this protein family appears to have regulatory roles in plant growth and diVerentiation (Xie et al., 1999). It is conceivable that the SENU5 gene belonging to this family may have a regulatory role in senescence. Another recent DNA microarray analysis has revealed ~30 diVerent senescence-enhanced regulatory genes encoding proteins, such as MYB, zinc finger, MADS box, and leucine zipper (Buchanan-Wollaston et al., 2003). We and others also found that a few AP2/EREBP transcription factor family genes are up-regulated during senescence. It is thus clear that senescence is a developmental phenomenon with a complex regulation mode involving various families of transcription factors.

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2. Receptor-like Kinases Similar to other known developmental programs in plants, the senescence program also involves the components of signal transduction pathways. The plant cells should perceive and process senescence signals such as age and other internal and external factors. The microarray and other experiments identified that senescence is certainly associated with induction of many genes that are possibly involved in signal perception and transduction. Here, we introduce one class of these genes, receptor kinases. Receptor kinases can potentially function as a key component in the perception of senescence signals and in the subsequent phosphorylation cascades involved in the plant senescence program. The senescence-associated receptor-like kinase (SARK) gene of bean is specifically expressed during senescence, especially prior to the loss of chlorophyll (Hajouj et al., 2000). Treatment with light and cytokinin and with darkness and ethylene, delayed and hastened, respectively, the induction timing of SARK. This expression implies the protein has a role at an early step of senescence initiation, incorporating age information as well as the internal and external factors. We also found a few senescence-induced receptor-like kinase genes in Arabidopsis (Koo et al., unpublished result). One of them shows increased expression at an early leaf senescence stage and reduced expression at later stages. Furthermore, expression of the gene is strongly induced by SA. This expression implies this protein has a role at an early step of senescence initiation, in this case, coordinating an age-dependent senescence program with SA signals possibly regulating pathogen-aVected senescence. Functional analysis of the genes in the signal transduction pathway would be important to reveal the complex regulatory network that includes signal pathways of various senescence-aVecting factors.

3. Other Regulatory Genes Eukaryotic translation factor 5A (eIF-5A) plays an important role in regulation of plant senescence. eIF-5A is synthesized as an inactive form and post-translationally activated through the sequential actions of two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DHH). Earlier studies in yeast and mammalian cells demonstrated that the eIF-5A might function as a nucleocytoplasmic shuttle protein, translocating specific mRNAs from the nucleus to the cytoplasm (Caraglia et al., 2003; Park et al., 1997). The finding that Arabidopsis DHS and eIF-5A1 genes were specifically expressed in senescing tissues raised the possibility that hypusinated eIF-5A in Arabidopsis might facilitate translation of the subset of mRNAs required for senescence (Thompson et al., 2004). This

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possibility is supported by finding that antisense-driven down-regulation of DHS inhibits the onset of leaf senescence. The relationship between the changes in the telomeric structure and senescence has been intensively investigated in various organisms (Greider, 1998; Nimmo et al., 1994). However, there are few analyses conducted on developmental dynamics of plant telomeres. A recent discovery on the formation of a complex between ATBP1/ATBP2 (Arabidopsis telomeric DNA-binding protein) and telomeric DNA at the onset of leaf senescence is intriguing (Zentgraf et al., 2000). This discovery opens up the possibility that telomeric structure could be involved in post-mitotic senescence in plants. It will certainly be interesting to investigate whether changes of the telomere structure are a part of the genetic mechanism regulating the onset of mitotic or post-mitotic senescence in plants.

F. Cis-acting Regulatory Elements of Senescence-Induced Genes Little progress has been made in identification of cis-acting regulatory elements that are responsible for senescence-induced transcription. While it should be important to reveal such elements to understand regulation of senescence-induced genes and the senescence mechanism, it appears that involvements of multiple signaling pathways leading to senescence complicate the search for this sequence. Yet, the promoter sequence of the age-specific upregulated SAG12 of Arabidopsis turned out to contain a highly conserved region that is responsible for senescencespecific expression (Noh et al., 1999a,b). This promoter region does not contain consensus sequences for any known DNA-binding proteins, suggesting that the regulation of developmental senescence involves a new or a diverged class of transcription factors. The promoter for the OPR1 gene encoding the enzyme 12-oxo-phytodienoic acid-10,11-reductase has been identified from an enhancer trap line. In the trap line, the reporter gene GUS driven by the promoter sequence of the OPR1 gene is up-regulated by both senescence and JA (He and Gan, 2001). Promoter deletion analysis identified two regulatory cis elements, JASE1 and JASE2. However, there are no recognizable conserved sequences among JASE1, JASE2, and the regulatory sequence of the SAG12 gene. This suggests that diVerent molecular mechanisms may be employed to regulate SAG12 and OPR during senescence. Furthermore, no conserved regulatory elements were so far found in the promoter sequences of SAGs. Identification of senescence-specific cis acting elements would probably require understanding of multiple and novel regulatory pathways.

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VII. Biotechnological Application of Senescence From a practical point of view, expanding our knowledge of the process of plant senescence has immense potential for economic benefit. The timing and eYciency has an important role in determining the yield and preharvest quality of many cereal and horticultural crops. Also, postharvest senescence and consequent loss of nutrient quality are important agronomical problems. As has been described, the autoregulated production of cytokinin using a senescence-specific SAG12 promoter and the bacterial IPT gene is an excellent example of the successful manipulation of senescence. The transgenic tobacco plants expressing the construct exhibited a clear improvement of several traits important in agronomy, including a 50% increase in both seed yield and total biomass (Gan and Amasino, 1995). This construct was also used to manipulate postharvest senescence in crops such as broccoli and lettuce. Delayed senescence has been shown in transgenic lettuce, both before and after harvest (McCabe et al., 2001). Moreover, in transgenic broccoli, postharvest degreening is also delayed (Chen et al., 2001). Another excellent example was demonstrated by reduced expression of the DHS gene in Arabidopsis. Transgenic Arabidopsis plants expressing a lower level of eIF-5A1, due to introduction of an antisense DHS transgene, exhibited delayed leaf senescence, increased leaf and root biomass, and enhanced seed yield (Thompson et al., 2004). Reduced eIF-5A1 also resulted in delayed premature leaf senescence induced by drought stress, resulting in enhanced survival. In addition, detached leaves of the transgenic plants exhibited delayed postharvest senescence. This strategy has been successfully utilized in delaying petal senescence of carnation and ripening of fruits such as tomato and banana.

VIII. Conclusions and Future Challenges As we have mentioned, leaf senescence is a highly regulated and complex process during which plants try to maximize their fitness by remobilizing the nutrients from the senescing leaf. However, onset and progression of leaf senescence should be finely controlled. If leaf senescence starts too early, leaves can not accumulate enough photosynthetic material. If senescence occurs too late, the plants may not be able to maximally utilize the nutrient for seed setting or for new organs. On the other hand, if the senescence process of cells is not properly regulated, the cells may not remobilize nutrients eYciently. Elucidating molecular mechanisms underlying such a complex, yet delicate, process of leaf senescence is a challenging and important biological task. For the last decade, genetic approaches as well as genomic approaches have yielded exponential advances in this area.

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However, a picture of the entire senescence process is just beginning to emerge and we are now at the stage where we may be able to answer many of the questions which remain. These questions include (1) what is the nature of age signal that leads to senescence? (2) how are the various signals integrated to coordinate senescence? (3) what are the molecular features of senescence caused by various senescence factors? (4) how is the expression of SAGs controlled? (5) how is leaf senescence coordinated with reproduction? (6) what is the molecular process along leaf senescence? (7) how are senescence signals perceived and processed? (8) how is leaf senescence linked to other developmental programs? (9) how does senescence aVect the fitness of plants, and how are nutrients remobilized? (10) are the senescence of leaf cells autonomous or is there a certain degree of coordination? Genomic and other approaches have led to discoveries of many potential candidates for regulatory components. The task is now to identify functions of these components in senescence. Utilization of the large collection of T-DNA insertional lines or the tilling approach in Arabidopsis is a straightforward way to conduct functional analysis of the genes. However, the senescence phenotype of these loss-of-function mutant lines may often be subtle and may not reveal the related functions due to gene redundancy or functional redundancy caused by various pathways leading to senescence. Thus, senescence phenotype needs to be closely examined in a well-controlled growth condition, especially considering that the senescence process is sensitive to environmental conditions. Senescence is controlled by various factors. Thus, an experiment that examines senescence aVected by multiple factors needs to be designed. This could be easily performed after examining the expression pattern of the genes during senescence that have been aVected by diVerent senescence factors. Even if the visible senescence phenotypes are subtle, molecular phenotypes may reveal a distinct feature of the function of the genes, as seen in the case of the wrky6 mutant. Examination of molecular phenotypes may include DNA microarray, proteomic, or metabolic approaches. To see senescence phenotypes in the loss-of-function mutants, it may be necessary to utilize double, triple, and even higher-order combinations of mutants in redundant genes or in redundant functional pathways. Transgenic approaches using overexpression or suppression of redundant genes by RNAi could also complement the limits of the loss-of-function mutants in seeing the senescence phenotype. Many of the genes that alter senescence are also involved in other cellular processes and are not devoted solely to senescence and thus expression patterns of genes during senescence show considerable overlap with other processes (Chen et al., 2002; He et al., 2001; Robatzek and Somssich, 2001) (Fig. 1). Many of the SAGs are also expressed at earlier stages (Buchanan-Wollaston, 1997). Thus, although senescence involves modified expression of thousands of genes, the number of genes specifically involved in

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senescence may be limited. Furthermore, constitutive overexpression of a regulatory gene often causes a pleiotropic eVect. Since senescence is the final stage of leaf development, any eVect of an overexpressed gene in any of the previous developmental stages or metabolic states may hamper assaying the eVect of the gene on senescence. Thus, functional analysis of a senescence regulatory gene by overexpression or RNAi approach may need to be conducted under the control of senescence-specific promoters. This is also applied in practical application in generating transgenic crops with altered senescence behavior (Gan and Amasino, 1999). An alternative approach is to use inducible promoters, such as dexametazone-inducible promoters. This approach may also be useful in identifying downstream target genes in the case of regulatory transcription factors, since the target genes are likely regulated at early timepoints when the regulatory genes are induced, for example, by the chemical. Furthermore, a T-DNA pool in which a senescence-specific cis-element is inserted into the genome may be quite useful in overcoming the limits (He and Gan, 2001). The genetic screening of senescence mutant proved to be a powerful approach in identifying the regulatory components of senescence and their functional analysis in Arabidopsis. We have identified over 10 delayed senescence mutants that are not allelic. It is certainly expected that there will be many more mutants that can be identified with well-designed and defined screening processes, considering the nature of senescence and the functional and gene redundancy in the pathways, as has been mentioned. We also emphasize that it is important that mutants displaying subtle eVects on senescence should not be ignored in the screening. Besides utilizing the T-DNA insertional knockout or activation lines, we argue strongly that chemically mutagenized pools should be utilized. The chemical mutagenesis will provide novel alleles that are not obtainable by the T-DNA mutagenesis and that will be important in characterizing functions of novel senescence regulatory elements. An example would be a cytokinin receptor in which a novel missense mutation revealed its involvement in regulation of senescence. The senescence process, as a tightly regulated process, should be composed of positive elements that induce senescence and negative elements that suppress senescence. Most of the genetic screening was focused on identifying delayed senescence mutants, which allows identification of positive elements of senescence. However, the regulatory elements of senescence should also be important for controlling initiation and progression of senescence in order to prevent senescence from occurring prematurely. Early senescence mutants would allow identification of negative factors involved in the leaf senescence process. However, mutations that show apparent early senescence symptoms may not be directly associated with control of senescence; mutations in many homeostatic or housekeeping genes could also give apparent early senescence symptoms. Screening of the gain-of-function

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mutations utilizing, for example, the T-DNA activation tagging pool of Arabidopsis may be a way to identify a negative regulator of senescence (Lim and Nam, unpublished results). Generation of a T-DNA pool in which the inducible promoters are inserted into the genome would be quite a useful resource for screening of the negative elements by inducing a gene at a maturation or senescence stage, and then by observing the phenotypic eVect. Alternatively, transgenic plants that express a reporter gene under a senescence-specific promoter may be utilized. In this case, the eVect of a mutation or a gene on the expression of the reporter gene may be assayed as a senescence-specific phenotype. This kind of screening may be applied not only to a transgenic whole plant but also to a single cell level, such as a protoplast. It should be also feasible to isolate regulatory transcription factors of senescence by utilizing the promoter sequence of senescence-associated genes such as SAG12 through various approaches, such as the yeast onehybrid system. Senescence may be controlled not only by diVerential expression of a gene but also by regulating activities, stability, and localization of regulatory proteins. A proteomic approach that identifies these properties of regulatory proteins during senescence may identify a novel mechanism of senescence regulation. A further analysis may include use of a DNA chip combined with proteomic approach that would allow examination of other cellular regulation, such as diVerential translation and localization of protein. Likewise, identification of in vivo interacting proteins using approaches such as coimmunoprecipitation and the yeast two-hybrid system should reveal which genes are participating in the senescence signaling pathways through protein–protein interaction. Related technologies are being rapidly developed for these types of analyses. Adopting these technologies would reveal the mechanisms that we are currently unable to see. Leaf senescence is a developmental strategy tightly associated with reproduction and survival and, in certain species, with the life span of whole plants. It is thus expected that diVerent plant species will have diVerent senescence physiology, reflecting the diVerent environments through which a species evolved. For example, in bean plants the development of reproductive structure aVects leaf senescence, whereas, in Arabidopsis, this correlative control of senescence does not occur. Therefore, it should be noted that a knowledge obtained from a plant species may not be directly transferable to other plant species. Comparative study of the functions of homologous senescence regulatory genes among plant species with diVerent senescence physiology that have evolved from diVerent ecological situations may also reveal important aspects of senescence mechanisms. Senescence involves change in expression of some thousands of genes. It is conceivable that there is some coordination mechanism to control groups of these genes. It will be interesting to examine whether there is a change in

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chromatin structure that coordinately regulates a group of genes during senescence. Like any other biological systems, senescence should be understood at a systems biology level. Many of the components of senescence are likely to show complex interactions, whether direct or indirect. These complex interactions are likely to yield so-called emergent properties that cannot be explained via simple extrapolation from the function of each component. These emergent properties may occur at the gene expression level, at the metabolite level, or at the cellular level. Likewise, these interactions are not static but should be highly dynamic. During age-dependent senescence and/or senescence aVected by other endogenous and exogenous senescence signals, the pattern of these interactions will continuously change to reflect the progressive stage of senescence and/or to reflect the senescence state aVected by various senescence signals plants are processing. For better understanding of the senescence process, we perhaps need to be able to see senescence at a systems level and with a dynamic view. We certainly expect that in the coming decade, there will be an exponential increase of knowledge on the regulation and processes of senescence, revealing the secrets of one of the wonders of nature. At the same time, we certainly expect that the knowledge we learn will lead to the generation of several transgenic foods, horticultural, and vegetable crop plants with improved yield, better postharvest quality, and increased shelf life.

Acknowledgments The work by H.G.N. was supported by grants from the National Research Laboratory program from the Ministry of Science and Education of Korea (M1-9911-00-0024) and the Crop Functional Genomics Center (CG1311) of Korea. The work by P.O.L. was partially supported by the Korea Research Foundation (2001-050-D00031).

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Fan, L., Zheng, S., and Wang, X. (1997). Antisense suppression of phospholipase D alpha retards abscisic acid- and ethylene-promoted senescence of postharvest Arabidopsis leaves. Plant Cell 9, 2183–2196. Forde, J., and Steer, M. W. (1976). The use of quantitative electron microscopy in the study of lipid composition of membranes. J. Exp. Bot. 27, 1137–1141. Gan, S. (2003). Mitotic and postmitotic senescence in plants. Sci. Aging Knowl. Environ. 38, 7. Gan, S., and Amasino, R. M. (1995). Inhibition of leaf senescence by autoregulated production of cytokinin. Science 270, 1986–1988. Gan, S., and Amasino, R. M. (1997). Making sense of senescence: Molecular genetic regulation and manipulation of leaf senescence. Plant Physiol. 113, 313–319. Gan, S., and Amasino, R. M. (1999). Developmental targeting of gene expression by the use of a senescence-specific promoter. In ‘‘Inducible Gene Expression in Plants’’ (Reynolds, Ed.), pp. 169–186. CAB International, New York. Gepstein, S. (2004). Leaf senescence—not just a ‘‘wear and tear’’ phenomenon. Genome Biol. 5, 212. Gepstein, S., Sabehi, G., Carp, M. J., Hajouj, T., Nesher, M. F., Yariv, I., Dor, C., and Bassani, M. (2003). Large-scale identification of leaf senescence-associated genes. Plant J. 36, 629–642. Graham, I. A., Leaver, C. J., and Smith, S. M. (1992). Induction of male synthase gene expression in senescent and detached organs of cucumber. Plant Cell 4, 349–357. Grbic, V., and Bleecker, A. B. (1995). Ethylene regulates the timing of leaf senescence in Arabidopsis. Plant J. 8, 595–602. Greider, C. W. (1998). Telomeres and senescence: The history, the experiment, the future. Curr. Biol. 8, 178–181. Guarente, L. (1997). Aging. What makes us tick? Science. 275, 943–944. Guo, Y., Cai, Z., and Gan, S. (2004). Transcriptome of Arabidopsis leaf senescence. Plant Cell Environ. 27, 521–549. Guterman, A., Hajouj, T., and Gepstein, S. (2003). Senescence-associated mRNAs that may participate in signal transduction and protein traYcking. Physiol. Plant. 118, 439–446. Ha, C. M., Kim, G. T., Kim, B. C., Jun, J. H., Soh, M. S., Ueno, Y., Machida, Y., Tsukaya, H., and Nam, H. G. (2003). The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis. Development 130, 161–172. Hajouj, T., Michelis, R., and Gepstein, S. (2000). Cloning and characterization of a receptorlike protein kinase gene associated with senescence. Plant Physiol. 124, 1305–1314. Hanaoka, H., Noda, T., Shirano, Y., Kato, T., Hayashi, H., Shibata, D., Tabata, S., and Ohsumi, Y. (2002). Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol. 129, 1181–1193. He, Y., and Gan, S. (2001). Identical promoter elements are involved in regulation of the Opr1 gene by senescence and jasmonic acid in Arabidopsis. Plant Mol. Biol. 47, 595–605. He, Y., and Gan, S. (2002). A gene encoding an acyl hydrolase is involved in leaf senescence in Arabidopsis. Plant Cell 14, 805–815. He, Y., Fukushige, H., Hildebrand, D. F., and Gan, S. (2002). Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence. Plant Physiol. 128, 876–884. He, Y., Tang, W., Swain, J. D., Green, A. L., Jack, T. P., and Gan, S. (2001). Networking senescence-regulating pathways by using Arabidopsis enhancer trap lines. Plant Physiol. 126, 707–716. Hensel, L. L., Grbic, V., Baumgarten, D. A., and Bleecker, A. B. (1993). Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis. Plant Cell 5, 553–564. Hidema, J., Makino, A., Kurita, Y., Mae, and Tojima, K. (1992). Changes in the levels of chlorophyll and light-harvesting chlorophyll a/b proteins of PSII in rice leaves aged under

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Cripto-1: An Oncofetal Gene with Many Faces Caterina Bianco,* Luigi Strizzi,* Nicola Normanno,y Nadia Khan* and David S. Salomon* *Tumor Growth Factor Section, Mammary Biology & Tumorigenesis Laboratory Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 y Division of Haematological Oncology and Department of Experimental Oncology ITN-Fondazione Pascale, Naples 80131, Italy

I. Introduction II. Structure and Genomic Organization of the EGF-CFC Gene Family A. Physiochemical Properties B. Genomic Organization III. Function and Expression of EGF-CFC Genes During Embryonic Development A. Gastrulation and Germ Layer Formation B. Left–Right Axis Formation and Cardiac Development IV. EGF-CFC Proteins in Mammary Gland Development V. EGF-CFC Proteins in Transformation and Tumorigenesis VI. Intracellular Signaling Pathways Activated by Cripto-1 A. Nodal=ALK4=ALK7=Smad-2 Signaling Pathway B. Glypican-1=c-Src=MAPK=AKT Signaling Pathway VII. Expression of Cripto-1 in Human Carcinomas and Premalignant Lesions A. Gastric Cancer B. Pancreatic Cancer C. Colorectal Cancer D. Gall Bladder Carcinoma E. Breast Cancer F. Endometrial Cancer G. Cervical Cancer H. Ovarian Cancer I. Expression of Cripto-1 in Other Cancers VIII. Cripto-1 as Target for Therapy in Human Cancer IX. Conclusions and Perspectives Acknowledgments References

Human Cripto-1 (CR-1), a member of the epidermal growth factor (EGF)-CFC family, has been implicated in embryogenesis and in carcinogenesis. During early vertebrate development, CR-1 functions as a co-receptor for Nodal, a transforming growth factor
(TGF
) family member and is essential for mesoderm and endoderm formation and anterior–posterior and left–right axis establishment. In adult tissues, CR-1 is Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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expressed at a low level in all stages of mammary gland development and expression increases during pregnancy and lactation. Overexpression of CR-1 in mouse mammary epithelial cells leads to their transformation in vitro and, when injected into mammary glands, produces ductal hyperplasias. CR-1 can also enhance migration, invasion, branching morphogenesis and epithelial to mesenchymal transition (EMT) of several mouse mammary epithelial cell lines. Furthermore, transgenic mouse studies have shown that overexpression of a human CR-1 transgene in the mammary gland under the transcriptional control of the mouse mammary tumor virus (MMTV) promoter results in mammary hyperplasias and papillary adenocarcinomas. Finally, CR-1 is expressed at high levels in approximately 50 to 80% of diVerent types of human carcinomas, including breast, cervix, colon, stomach, pancreas, lung, ovary, and testis. In conclusion, EGF-CFC proteins play dual roles as embryonic pattern formation genes and as oncogenes. While during embryogenesis EGF-CFC proteins perform specific and regulatory functions related to cell and tissue patterning, inappropriate expression of these molecules in adult tissues can lead to cellular proliferation and transformation and therefore may be important in the etiology and=or progression of cancer. C 2005, Elsevier Inc.

I. Introduction Only with respect to our ability to more completely delineate and identify the genetic and molecular pathways that go awry in tumor cells has the convergence of developmental biology and oncology come to be appreciated. In fact, this realization stemmed, in part, from two seminal observations. First, oncogenes were originally thought to be viral genes that were introduced into the host cell genome following viral infection and which contributed to the development of cancer. We now know that a large fraction of these oncogenes, particularly those which have been incorporated into retroviral genomes, are actually cellular host genes (Weinberg, 1989). These host protooncogenes were selected because they normally perform specific and crucial regulatory functions related to cell proliferation, survival, and diVerentiation that were pirated by retroviruses for the purpose of maintaining their own evolutionary survival. In addition, retroviruses can cause cancer by insertional mutagenesis into regions of the host genome that are either adjacent to or within regulatory genes (Callahan, 1996). Second, a variety of mechanisms including viral infection or carcinogens can lead to gene activation by amplification, point mutations, deletions, chromosomal translocations, or overexpression of genes that are also known to perform critical roles during early embryogenesis. In other words, oncogenesis, in many cases, is the recapitulation of embryogenesis in an inappropriate temporal

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and spatial manner. Genes that control cell specification, cell and tissue patterning, epithelial-mesenchymal interactions, competency, apoptosis, and the cell cycle fall within this realm and are prone to lead to the etiology and=or progression of cancer if aberrantly activated. G. Barry Pierce was one of the first researchers to recognize that tumors are basically caricatures of the process of tissue renewal and embryonic development and that this observation could have a significant impact on our ability to design novel therapies to treat cancer (Pierce and Speers, 1988; Sell and Pierce, 1994). Reciprocally, he also speculated that embryonic fields that are controlled by morphogens could be capable of converting malignant stem cells into normal, diVerentiated lineages that would now respond to homeostatic control pathways (Tabata and Takei, 2004). In other words, malignant stem cells are derived from normal tissue stem cells that, in turn, resemble embryonic stem cells in their pluripotentiality and, when situated in an appropriate cellular and tissue-specific niche, can redirect and nullify their malignant potential (Kopper and Hajdu, 2004; Sell, 2004). The pioneering work of Beatrice Mintz experimentally had validated a number of these predictions (Mintz and Illmensee, 1975). Injection of genetically marked malignant embryonal carcinoma cells into blastocysts of mice were able to contribute to normal tissue development in these chimeric adult animals without any evidence for the appearance of tumors. Conceptually, it is essential to appreciate the function and role of morphogens and morphostats in the process of embryogenesis (Meinhardt, 2001; Potter, 2001; Tabata and Takei, 2004). Morphogens are peptides that can be growth factors and that arise from a restricted region in the organizing centers in the embryo, which probably represent primitive embryonic stem cell niches, stem cell niches in adult epithelial tissues or, in the case of morphostats, in mesenchymal tissue niches (Gilboa and Lehmann, 2004; Nelson and Nusse, 2004; Potter, 2001; Schier and Talbot, 2001; Sell, 2004). Morphogens generally function to control tissue microarchitecture, cell specification, pattern formation, and regional cell motility. They are, therefore, important in providing positional information among cells of the same embryonic lineage or communication with other types of cells that are derived from other germ layers. These molecules intrinsically induce cellular responses in a concentration- or threshold-dependent manner and include members of the Hedgehog (Hh), Wnt, or transforming growth factor
(TGF
) families such as Decapentaplegic (Dpp), Activin, or Nodal (Kodjabachian, 2001; Tabata and Takei, 2004). The shaping of morphogenic gradients can be controlled by the localized and graded expression of the morphogen, but it can also be generated by the local expression and=or diVusion of diVerent concentrations of specific morphogenic inhibitors (e.g., Caronte, Cerberus, Chordin, Dickkopf, DAN, Lefty, Follistatin). Finally, the graded expression of receptors for the morphogens or of

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heparan sulphate-containing proteoglycans (HSPGs) might also contribute to the formation of these gradients.

II. Structure and Genomic Organization of the EGF-CFC Gene Family Human Cripto-1 (CR-1=CFC2 also known as teratocarcinoma-derived growth factor-1 [TDGF-1]) is a member of the Epidermal Growth Factor (EGF)-CFC protein family and structurally contains an EGF-like domain and a cysteine-rich region called the Cripto-1=FRL-1=Cryptic (CFC) domain (Adamson et al., 2002; Persico et al., 2001; Salomon et al., 1999, 2000; Shen and Schier, 2000). The EGF-CFC family includes monkey Cripto-1 (Stevens and Berman, personal communication); mouse Cripto-1 (Cr-1=cfc2) (Dono et al., 1993), chicken Cripto-1 (Colas and Schoenwolf, 2000; Schlange et al., 2001), zebrafish one-eyed pinhead (oep) (Zhang et al., 1998), Xenopus FRL-1 (Kinoshita et al., 1995; Shen and Schier, 2000), and mouse cryptic (Cfc1) and human Cryptic (CFC1) (Bamford et al., 2000; de la Cruz et al., 2002; Shen and Schier, 2000; Shen et al., 1997). To date, EGF-CFC genes have been identified in vertebrates but not in invertebrates. The vertebrate EGF-CFC proteins are highly conserved in the structural organization of their individual modular units or domains that, in turn, are coextensive to highly conserved exons between diVerent vertebrate species. This conservation suggests that these genes are true orthologs that are biologically interchangeable among species in their function(s) and that they probably arose from a common ancestor gene (Colas and Schoenwolf, 2000; Minchiotti et al., 2002). There is one caveat to the exclusivity of these genes to only vertebrates, as has been previously reported. Nodal and Antivin have been cloned from sea urchins, which are nonchordate deuterostomes that are invertebrates. Sea urchin Nodal is involved in regulating the formation of the oral– aboral axis (Duboc et al., 2004). Therefore, if as in other Nodal genes, there is an obligatory requirement for EGF-CFC genes for these Nodal genes to biologically function in anterior–posterior (A=P) axis formation (see following text), then the activity of sea urchin Nodal probably relies on the presence of an EGF-CFC orthologous gene in this species. Therefore, a more rigorous assessment for the presence of other Nodal and EGF-CFC genes in other lower organisms should be seriously reassessed. A. Physiochemical Properties Structurally, the EGF-CFC family consists of extracellular soluble or cell membrane-associated proteins that contain an NH2-terminal signal peptide, a modified EGF-like region, a conserved cysteine-rich domain (CFC motif),

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and a short hydrophobic COOH-terminus which, with the exception of FRL-1, contains additional sequences for glycosylphosphatidylinositol (GPI) cleavage and attachment (Ciccodicola et al., 1989; Dono et al., 1993; Gritsman et al., 1999; Kinoshita et al., 1995; Minchiotti et al., 2000; Shen et al., 1997; Zhang et al., 1998). An overall sequence identity of approximately 30% exists between the EGF-CFC members across diVerent species. Within the EGF-like domain, there is nearly a 60 to 70% sequence similarity while, in the CFC region, the similarity ranges from 35 to 48%. The variant EGF-like motif is a region of approximately 40 amino acids containing six cysteine residues that can form two intramolecular disulfide bonds. Unlike the canonical EGF motif that contains three disulfide loops (A, B, and C), the variant EGF-like motif in the EGF-CFC proteins lacks the A loop, possesses a truncated B loop, and has a complete C loop. This modified EGF-like motif diVerentiates the EGF-CFC proteins from other members in the EGF family of peptides such as transforming growth factor  (TG, amphiregulin (AR), heparin-binding growth factor (HB-EGF), or heregulin (HRG) that can bind to and activate diVerent members of the erbB type I receptor family of tyrosine kinases which includes the EGF receptor (EGFR), erbB-2, erbB-3, and erbB-4. Conserved amino acids within the A loop of these peptides are essential for erbB receptor binding. Since the EGF-CFC peptides lack the A loop, these proteins do not directly bind to any of the known erbB-related tyrosine kinase receptors as either homodimers or heterodimers (Bianco et al., 1999; Kannan et al., 1997). Molecular modeling of this modified EGF-like motif has shown a closely packed threedisulfide stacked arrangement with a disulfide
-cross motif for the first and second disulfides (Lohmeyer et al., 1997). In general, the EGF-like motif in the EGF-CFC proteins exhibits less homology to the EGFR ligands and appears to be somewhat more related in structure to the EGF motif in the HRGs that bind to erbB-3 and=or erbB-4. The CFC domain of human CR-1 contains three disulfide bonds in a pattern which structurally resembles the von Willebrand factor C (VWFC)like domains found within the COOH-terminal extracellular portions of the Notch ligands, Jagged1 and Jagged2 (Foley et al., 2003; van Vlijmen et al., 2004). This is particularly intriguing since, like several of the Notch receptor proteins, all of the EGF-CFC proteins also contain a consensus O-linked fucosylation site within the EGF-like motif that is necessary for their ability to function as co-receptors for the TGF
-related protein, Nodal (Haltiwanger and Stanley, 2002; SchiVer et al., 2001; Yan et al., 2002) (see following text). Biochemical characterization of human CR-1 identified Asn-79 as being an N-linked glycosylation site with <90% occupancy, and Ser-40 and Ser-161 as being O-linked glycosylation sites with 80 and 40% occupancy, respectively. Whether mutation of these other glycosylation sites can aVect the biological activity of CR-1 is presently unknown. Finally,

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a relaxed modeling of the combined EGF and CFC domain structure of Cripto-1 showed a significant similarity between mouse and human Cripto-1 and proteins containing a
-trefoil fold structure such as fibroblast growth factor 2 (FGF-2) (Minchiotti et al., 2001). This may be functionally significant since Xenopus FRL-1 was originally isolated by its ability to indirectly activate the FGF receptor–1 (FGFR-1) (Kinoshita et al., 1995). With the exception of zebrafish oep, all other EGF-CFC proteins are glycoproteins that range from 171 to 202 amino acids. The unmodified core proteins within this family range from 18 to 21 kDa. However, the native mouse and human Cripto-1 proteins are 24, 28, and 36 kDa in size and additional proteins ranging from 14 to 60 kDa have also been identified in mouse and human normal tissues and carcinomas (Brandt et al., 1994; Kenney et al., 1996; Minchiotti et al., 2000; Niemeyer et al., 1998; Seno et al., 1998). The variation in size of these protein species may be due to the removal of the hydrophobic signal peptide and=or to additional post-translational modifications of the core protein, such as glycosylation, myristylation, or phosphorylation (Brandt et al., 1994; Minchiotti et al., 2000; Salomon et al., 2000). In addition, in human CR-1, there are furin (Spc1) and stromolysin cleavage sites within the NH2 terminal region between amino acid residues 37 and 45 which might be utilized and which might also account for the presence of some of the smaller species. When mouse Cr-1 or human CR-1 is expressed as recombinant, full-length proteins in mammalian cells, these proteins are retained on and=or in the cells. Studies on zebrafish oep indicated that the COOH-terminal region may contain a putative GPI-anchorage site (Gritsman et al., 1999; Zhang et al., 1998). Removal of the COOH-terminal stretch containing the GPI site generated a soluble form of oep that was biologically active but less so than the full-length, GPI-linked, cell-associated protein, suggesting that oep could function both in a cell autonomous (autocrine or juxtacrine) and nonautonomous (paracrine) fashion (Gritsman et al., 1999). The characterization of mouse Cr-1 and human CR-1 has confirmed the presence of a GPImodification site within the COOH-terminal region of these proteins (Foley et al., 2003; Minchiotti et al., 2000, 2001; SchiVer et al., 2001). Mouse and human cryptic, but not FRL-1, also contain a potential GPI site for cleavage and attachment that is present in the COOH-terminus. In addition, the mouse Cr-1 protein can also be released by treatment of cells with phosphatidylinositol-specific phospholipase C (PI-PLC) (Minchiotti et al., 2000). Removing the COOH-terminal stretch of residues generates mouse and human Cripto-1 forms that are soluble. These soluble forms of Cripto-1 are biologically active and can function as co-ligands for Nodal in a number of diVerent assays in vivo in zebrafish, Xenopus, and mice in vitro in reporter assays containing promoter elements that possess Smad-2, Smad-3, and FAST2 (FoxH1) binding elements, which are engaged in a Nodal-dependent

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signaling pathway (Minchiotti et al., 2001; Xu et al., 1999; Yan et al., 2002). Full activity requires the presence of a peptide containing only an intact EGF domain and CFC domain (Minchiotti et al., 2001; Yan et al., 2002). However, whether there are significant qualitative and=or quantitative diVerences in the biological activities that are regulated by Cripto-1 between the soluble versus cell-associated forms is not yet known. Likewise, there is no information relating to the processing, subcellular localization, and intracellular degradation which might provide information as to the mechanism by which these proteins function.

B. Genomic Organization CR-1 maps centromerically to chromosome 3p21.3 near a region that is frequently deleted or exhibits loss of heterozygosity (LOH) in a subpopulation of head and neck, renal, gastric, bladder, breast, and lung carcinomas (Dono et al., 1991; Saccone et al., 1995; Sekido et al., 1998; Todd et al., 1996). The mouse and human Cripto-1 genes consist of six exons and five introns and possess inverted Alu and B1 sequence elements, respectively. In addition, AUUU(A)-type Kamen-like sequences in a large 30 untranslated region are present in the mouse and human Cripto-1 mRNAs, suggesting that they encode relatively short-lived mRNA species (Dono et al., 1991). The human CR-1 gene contains six exons and is 4.8 kb in length. There is an excellent conservation of the exon-intron structure in the region of exon 4 which contains the EGF-like motif between the mouse and human Cripto-1 genes (Dono et al., 1991). The 50 upstream genomic sequences of mouse Cr-1 and human CR-1 genes between -610 and -1 from the most distal translation start sites are dissimilar (Baldassarre et al., 2001). Several TATA and CAAT boxes are present in the mouse Cr-1 promoter region while, in the human CR-1 promoter region, these sequences are missing. At least five other human CR-1 related-pseudogenes and two mouse Cr-1 pseudogenes have been identified (Dono et al., 1991; Liguori et al., 1996, 1997; Saccone et al., 1995; Scognamiglio et al., 1999). The CR-2, CR-4, and CR-5 pseudogenes are intronless, truncated at the 50 -end and have accumulated point mutations, deletions, and insertions (Scognamiglio et al., 1999). These genes map to chromosomes 2q37, 6p25, and 3q22, respectively, while CR-6 maps to 19q13.1. The CR-3 pseudogene maps to the Xq21-q22 region. The two mouse Cr-1 pseudogenes (Cr1-ps1=Tdgf1-ps1 and Cr1-ps2=Tdgf1-ps2) are intronless genes that have multiple base mutations which represent both silent and replacement amino acid substitutions and that have many characteristics of a retroposon. CR-3 and Cr1-ps1 have the potential to code for functional proteins that diVer from the proteins encoded by either CR-1 or Cr-1 by only five amino acids (Persico et al., 2001).

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CR-1 and Cr-1 encode major mRNA species of approximately 2 kb. In some cases, less abundant transcripts of about 1.7, 3.0, 3.2, and 3.5 kb have been detected in midgestation mouse embryos and in primary and metastatic human colon and hepatic carcinomas, suggesting that they may arise by the use of diVerent polyadenylation sequences, by alternative splicing, or by the use of an alternative initiation of transcription (Baldassarre et al., 2001; Ciccodicola et al., 1989; Dono et al., 1993; Johnson et al., 1994). In this regard, a truncated CR-1 protein of 145 amino acids may be expressed from the 1.7 kb mRNA transcript in metastatic human colorectal carcinomas and in hepatic colon metastases due to the use of a second CUG initiation codon (at leucine 44 in the coding sequence) 129 nucleotides downstream of the start AUG codon, thereby eliminating the use of the first two exons and deleting 43 amino acids from the N-terminus of the native protein. In the mouse, low levels of Cr-1 mRNA expression can be detected by RNAse protection assays in the adult spleen, heart, lung, and in distinct regions of the brain (Dono et al., 1993; Johnson et al., 1994). Adult tissues in the human that express low levels of mRNA transcripts for the 188 amino acid isoform of CR-1 as detected by RT-PCR include lung, kidney, brain, testis, ovary, and spleen (Baldassarre et al., 2001). Tissues expressing only the truncated 145 amino acid form of CR-1 include pancreas, heart, stomach, small intestine, mammary gland, skeletal muscle, and liver. Likewise, mouse cryptic contains two in-frame potential translation initiation AUG start sites in which the second AUG start site would code for a protein that is truncated at the NH2-terminus by 13 amino acids (Shen et al., 1997).

III. Function and Expression of EGF-CFC Genes During Embryonic Development Establishment of the body axes and germ layers results from an orchestrated set of morphogenic movements and diVerentiation that are regulated by the integrated action of genes to generate specific sets of cell lineages (Beddington and Robertson, 1999; Fraser and Harland, 2000). EGF-CFC proteins function as co-receptors for the TGF
ligands, Nodal and Vg1=growth and diVerentiation factor-1 (GDF-1) (Bianco et al., 2002a; Cheng et al., 2003; Yeo and Whitman, 2001). Genetic studies in zebrafish and mice have defined an essential role for Nodal that functions through oep=Cripto-1 in the formation of the primitive streak, patterning of the A=P axis, specification of mesoderm and endoderm (mesoendoderm), and establishment of left–right (L=R) asymmetry (Ding et al., 1998; Hamada et al., 2002; Schier, 2003; Schier and Shen, 2000; Yan et al., 1999). These genetic studies were also the first to implicate Cripto-1 and cryptic as co-receptors for Nodal. Subsequent biochemical work has demonstrated that EGF-CFC proteins bind

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directly to both Nodal and the Activin type I receptor ALK4 (ActRIB), to recruit the Activin type II receptor to facilitate signaling through the phosphorylation of Smad-2=Smad-3 and dimerization with Smad-4 in the presence of the co-transcriptional activator FoxH1 (Adkins et al., 2003; Bianco et al., 2002a; Yeo and Whitman, 2001). Extracellular inhibitors that can block Nodalor Vg1-dependent signaling and that contribute to the formation of a concentration-dependent gradient of Nodal activity include Lefty1, Lefty2, and Antivin. These proteins are monomeric, unlike Nodal and Vg1=GDF-1, which are dimeric. Lefty and Antivin are promiscuous since they can bind to Nodal, Cripto-1, oep, FRL-1, or cryptic (Chen and Schier, 2002; Chen and Shen, 2004; Ciardiello et al., 1990; Sakuma et al., 2002). Similarly, Tomoregulin-1, which is an EGF-like peptide that can weakly activate the erbB-4 receptor, can also antagonize Nodal signaling by directly binding to the CFC domain in Cripto-1 in a cell-autonomous manner (Harms and Chang, 2003). Interestingly, mouse Nodal, Xenopus nodal3 (Xnr3), Xnr5, and tomoregulin-1 can also directly bind to BMP7, BMP4, BMP5, and BMP2, respectively, in the absence of Cripto-1 and can inhibit signaling through a BMP=Smad-1 pathway (Haramoto et al., 2004; Yeo and Whitman, 2001). In fact, Nodal and BMPs can function as mutual antagonists through a Cripto-1-independent pathway which may be important for the specification of anterior neural tissue (Yeo and Whitman, 2001). Brennan et al. (2001) also showed that Nodal could act independently of Cripto-1 and of Smad-2 to promote posterior cell fates but required Cripto-1 for migration of the anterior visceral endoderm (Brennan et al., 2001). Finally, Lefty can function as an inhibitor of wnt signaling during gastrulation in Xenopus (Branford and Yost, 2002). This may be significant since Xnr3 and Cripto-1 are direct target genes in a wnt=
-catenin=Lef-1 signaling pathway (McKendry et al., 1997; Morkel et al., 2003). During early mouse embryogenesis, Cr-1 expression can first be detected by RT-PCR and, in some cases, by immunohistochemistry in extraembryonic trophoblast cells and in the embryo in the inner cell mass (ICM) of the blastocyst at day 4 of development. This expression seems to correlate with the original isolation and identification of CR-1 and Cr-1 genes from cDNA libraries obtained from undiVerentiated human NTERA2=D1 and mouse F9 embryonal carcinoma cells, respectively (Ciccodicola et al., 1989; Ding et al., 1998; Dono et al., 1993; Johnson et al., 1994; Xu et al., 1999). Expression of both genes is lost in NTERA2=D1 and F9 cells following retinoic acid-induced diVerentiation (Ciccodicola et al., 1989; Dono et al., 1993; Minchiotti et al., 2000). The expression pattern of Cr-1 is clearly distinct from that of cryptic, which is only found in diVerentiated mesoderm cells following retinoic acid treatment and not in embryonal carcinoma or ICM stem cells (Shen et al., 1997). Cr-1 mRNA expression in the embryo is found in the embryonic ectoderm following implantation of the blastocyst. Expression increases on day 6.5 of gestation and is found in the ingressing

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epiblast cells in the nascent primitive streak and increases dramatically in the developing mesoderm cells as the epiblast cells undergo epithelial mesenchymal transition (EMT) (Ding et al., 1998; Dono et al., 1993; Johnson et al., 1994; Xu et al., 1999). Expression then decreases by day 7 but later fetal stages show expression of Cr-1 in the myocardium of the truncus arterious of the developing heart. In the developing heart, Cr-1 mRNA can be detected in the epimyocardium but not in the endocardium. With the exception of the developing heart, little if any expression of Cr-1 mRNA can be detected in the remainder of the embryo after day 8. Expression of Cr-1 mRNA in adult tissues is generally several-fold less than in undiVerentiated F-9 mouse embryonal carcinoma cells (Dono et al., 1993; Minchiotti et al., 2000). A. Gastrulation and Germ Layer Formation Gastrulation establishes the body plan of the embryo through the formation of the ectoderm, mesoderm, and endoderm germ layers (Beddington and Robertson, 1999; Fraser and Harland, 2000). Gastrulation physically occurs by morphogenetic cellular migration consisting of the internal movement of ectodermal epiblast cells through the primitive streak and the subsequent epiboly and convergent and extension movement of these cells. Spatial positioning of the germ layers results in the establishment of polarity along the A=P axis of the embryo and also requires the position and orientation of cells by migration. After the A=P axis and dorsal=ventral (D=V) axis are established, L=R axis is formed and this eventually leads to the asymmetric development of diVerent organs. The first inductive event is the formation of the mesoderm from adjacent posterior ectoderm cells as they migrate along the primitive streak and through the underlying ectoderm. As the epiblast=ectodermal cells migrate through and away from the primitive streak, they undergo EMT, resulting in their conversion to mesodermal cells with mesenchymal characteristics (Savagner, 2001). EMT is controlled by the combined and highly coordinated action of multiple signaling molecules that regulate cell–cell and cell–extracellular matrix (ECM) adhesion, cell shape and loss of cell polarization, and cell motility and guidance. Disruption of any of these signaling pathways can impair gastrulation and is therefore embryonically lethal (Sirard et al., 1998; Wacker et al., 1998; Waldrip et al., 1998). Cripto-1 null mice (Cr-1
=
) die at d7.5 due to their inability to gastrulate and form appropriate germ layers (Ding et al., 1998). Fibroblasts that were derived from Cr-1
=
embryos were found in vitro to exhibit negligible migration in a wound healing assay and were impaired in their ability to migrate chemotactically toward either fibronectin or type 1 collagen as compared to embryonic fibroblasts from wild-type embryos (Xu et al., 1999). In zebrafish oep, in conjunction with the two Nodal-related genes

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squint (sqt) and Cyclops (cyc), is necessary for initiating mesoderm, endoderm, and A=P axis formation (Gritsman et al., 1999; Schier and Shen, 2000; Zhang et al., 1998). Mutations in oep result in cyclopia, absence of head and trunk mesoderm, loss of prechordal plate and ventral neuroectoderm, impairment of gastrulation movements, loss of A=P axis patterning and positioning, and L=R laterality defects (Gritsman et al., 1999; Zhang et al., 1998). Rescue of the oep mutant phenotype can be achieved by expression of either full-length or secreted COOH-terminal truncated forms of the oep protein, suggesting that oep can function under certain conditions as a paracrine eVector. Ectopic expression of Xenopus FRL-1 or mouse Cr-1 or overexpression of Activin or activation of downstream components in an Activin-like signaling pathway such as the ALK4 receptor (TARAM-A) or Smad-2 can also rescue oep inactivating mutations (Gritsman et al., 1999). However, rescue of the mutant phenotype cannot be achieved with BMP2 or -4, activated ras, mitogen-activated protein kinase (MAPK), or with diVerent members in the wnt=
-catenin signaling pathway (Gritsman et al., 1999; Schier and Shen, 2000). Oep, like mouse Cr-1, is absolutely required for the migration of mesendoderm cells through the primitive streak (Warga and Kane, 2003). This seminal paper showed that mutations of oep could eVectively block the ability of epiblast cells to migrate and to induce a more cohesive interaction between these cells in a cell-autonomous fashion. This eVect was independent of the presence of either an active cyc or sqt gene, suggesting that these phenotypes were unique to oep and not dependent upon a Nodal (cyc=sqt) signaling pathway. Oep mutant cells were more epithelial in their morphology and less cohesive and could not be rescued by overexpression of Activin and were generally mesenchymal in their appearance. These data also suggest that oep=Cripto-1 regulates the cell adhesion and motility properties of cells, possibly by their capacity to modulate the expression of transcription factors such as snail, slug, or Twist, which could, in turn, regulate expression of diVerent cell adhesion molecules including the cadherins, occuldins, claudins, integrins, or netrins. Concordant evidence for this possibility also was observed for wild-type human Cryptic (CFC1) when overexpressed in zebrafish embryos, where abnormal movements of epiblast cells were observed during the epiboly phase of gastrulation, which were independent of Nodal signaling through a dominant neomorphic phenotype (Bamford et al., 2000). B. Left–Right Axis Formation and Cardiac Development The Nodal=cryptic signaling pathway is also involved in the establishment of the left–right (L=R) embryonic axis, demonstrating that the same signaling molecules are utilized in multiple developmental pathways (Schier and Shen,

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2000). However, at this developmental stage, with the exception of oep, cryptic replaces the function of Cripto-1 as the co-receptor for Nodal. Nodal signaling in zebrafish during L=R asymmetry development also requires a functional oep gene (Gaio et al., 1999; Saijoh et al., 2000; Schier and Talbot, 2001; Yan et al., 1999). Mutation and partial rescue of oep in the zebrafish or targeted disruption of cryptic in mice (Cfc1) and humans (CFC1) results in laterality defects including randomization of cardiac looping and atrial– ventricular septal defects; pulmonary right isomerization; inverted situs of the spleen, pancreas, and stomach; hyposplenia; heterotaxia; and holoprosencephaly (de la Cruz et al., 2002; Schier and Talbot, 2001). Germline deletion of cryptic eventually leads to postnatal death at approximately 2 weeks because of severe cardiac malfunction (Gaio et al., 1999; Yan et al., 1999). In addition, in Xenopus Vg1 can also regulate L=R asymmetry with respect to the leftward migration of ectodermal cells during gastrulation and functions upstream of any of the Xenopus-related Nodal proteins (Chen et al., 2004). In this context, the HSPG syndecan-2 binds to and is an obligatory co-receptor for Vg1 in the same manner as cryptic can function as a co-receptor for Nodal during the establishment of L=R asymmetry. The expression pattern for cryptic is generally distinct from the expression pattern for Cr-1 since it is not expressed in adult mouse tissue and has a partially overlapping yet distinct expression pattern to Cr-1 in the midgestation mouse embryo (Shen et al., 1997; Yan et al., 1999). Cryptic expression can first be detected in the axial mesoderm and then becomes more restricted to the anterior end of the primitive streak and the head process. Expression is absent in the prechordal plate but is present in the lateral plate mesoderm, node, and floor plate of the neural tube. After day 10.5, little expression of cryptic mRNA can be detected in the embryo. EGF-CFC proteins have an important role in diVerent stages of cardiogenesis (Parisi et al., 2003). Mouse Cr-1 performs an essential role during the early stages of cardiac lineage specification and diVerentiation. Mouse Cr-1 is expressed in the precardiac mesoderm (Dono et al., 1993). In this context, homologous recombination that leads to homozygous knockout of the Cr-1 gene (Cr-1
=
) in pluripotential embryonic stem cells (ES cells) impairs their ability to spontaneously diVerentiate in vitro into cardiomyocytes, a process that normally occurs through a Nodal- and Smad-2-dependent pathway without aVecting the ability of ES cells to diVerentiate into other cell types (Parisi et al., 2003; Xu et al., 1998). Interestingly, a similar embryonically lethal phenotype due to myocardial and endocardial defects, is found in day 10 embryos from erbB-4, erbB-2, and HRG-1 knockout mice (Klapper et al., 2000; Xu et al., 1999). This may have functional significance since the human CR-1 protein can indirectly increase the tyrosine transphosphorylation of erbB-4 and since tomoregulin-1 can bind to both CR-1 and erbB-4 (Bianco

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et al., 1999; Harms and Chang, 2003; Uchida et al., 1999). Disruption of Cr-1 in Cr-1
=
embryos results in the formation of embryos that possess a head without a trunk, demonstrating that there is a severe deficiency in embryonic mesoderm and endoderm without a loss of anterior neuroectoderm formation (Xu et al., 1999). Chimeric embryos that were generated between wild-type and Cr-1
=
ES cells were normal, indicating that Cr-1 produced by the wild-type host cells could rescue the mutant phenotype in a paracrine manner (Xu et al., 1998). Cr
1
=
cells also show extensive neural diVerentiation. This may be of some clinical interest since macaque monkeys that have been infected with a Simian AIDS-like virus (SAIDS), and that develop neuro-AIDS-like symptoms resulting from neural degeneration in the brain, exhibit a 20- to 50-fold induction of monkey cripto-1 expression in their brains following SAIDS infection (Stevens and Berman, personal communication). Finally, in zebrafish, the T-box gene Spadetail (spt), which is required for the movement of posterior somite progenitors from caudal regions in the tail by regulating the expression of the cell adhesion molecules paraxial protocadherin (PAPC) and mesogenin, and the T-box ortholog Notail (ntl ), interact genetically with oep to induce cardiac and somatic mesoderm and trunk somatic mesoderm formation and motility, respectively, to and in regulating the formation of the presomitic mesoderm (PSM) (GriYn and Kimelman, 2002).

IV. EGF-CFC Proteins in Mammary Gland Development The mouse mammary gland remains relatively quiescent with respect to morphogenesis and diVerentiation until subjected to hormonal stimulation at sexual maturation and during pregnancy. During the prenatal phase, hormone-independent molecular signaling; sequential gene activation; and cell– cell interactions among epithelium, mesenchyme, and ECM take place and prepare the rudimentary mammary gland for the further development and diVerentiation that will occur during puberty and pregnancy (Haslam and Woodward, 2001; Howlett and Bissell, 1993; Robinson, 2004). Selfrenewing stem and progenitor cells are localized throughout the mammary gland and generate luminal epithelial cells and myoepithelial cells in specialized structures known as terminal end buds, which form the functional unit of the mammary gland (Lochter, 1998). During puberty, ovarian hormones stimulate the end buds, which begin a highly regulated process of proliferation and apoptosis, resulting in extension of the epithelial structures that form a ductal network extending from the nipple region, through the mammary gland adipose tissue, until the epithelial structures meet other developing ducts or reach the periphery of the fat pad. Hormones released

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during pregnancy further stimulate the development of the lobular–alveolar structures responsible for milk production (Daniel and Silberstein, 1987). Ovarian hormones are known to regulate the expression of numerous growth factors, such as TGF
, TGF, FGFs, EGF, and insulin growth factor-1 (IGF-1). All these play a crucial role in the regulation of the development and maturation of the mammary gland (Barcellos-HoV and Ewan, 2000; Borellini and Oka, 1989; Daniel et al., 2001; Kleinberg, 1998; Spencer-Dene et al., 2001). In the mouse, Cr-1 has been detected during diVerent stages of postnatal mammary gland development. In fact, Cr-1 protein was detected in 4- to 12-week-old virgin, midpregnant, and lactating FVB=N mouse mammary glands (Kenney et al., 1995, 2004). In those studies, the levels of Cr-1 increased two- to three-fold during pregnancy, remained elevated throughout lactation, and decreased during involution. In addition, the expression of Cr-1 mRNA and protein in mammary gland epithelium of Balb=c mice was found to be elevated in older breeder mice, which have a high incidence of developing mammary tumors (Herrington et al., 1997). A 2001 study also detected biologically active CR-1 in human milk, demonstrating that CR-1 is a secretory component of the mammary gland and suggests that this secreted form of CR-1 may play a role in the regulation of proliferation and diVerentiation of milk-producing cells (Bianco et al., 2001). In fact, the EGF-like domain of CR-1 was found to specifically bind to the mouse mammary epithelial cells, NMuMG and HC-11, and to mouse mammary gland tissue sections, suggesting the presence of a putative CR-1 receptor that may be involved in autocrine regulation of mammary gland function during pregnancy and lactation (Bianco et al., 2002b). Further support for CR-1 regulation of mammary epithelial function comes from data which shows increased ability of mouse mammary epithelial cells to respond to the lactogenic hormones, dexamethasone, insulin, and prolactin (DIP) when transiently pretreated with CR-1 and inhibition of
-casein expression, via p21ras- and phosphatidylinositol 30 -kinase (PI3K)-dependent pathways, when these cells were simultaneously treated with both CR-1 and DIP (De Santis et al., 1997). Whether the presence of Cripto-1 in milk is also important for normal development of the enteric mucosa needs to be further investigated. However, the role of CR-1 in growth and proliferation in the gastrointestinal epithelium has been suggested. Studies show that the expression of CR-1 mRNA is lower in intestinal epithelium aVected with inflammatory bowel disease (IBD) and in gastric ulcer lesions, where high rates of necrosis and apoptosis take place, but higher in the intestinal epithelium of IBD patients with concomitant colon tumors and in the regenerative gastric epithelium located distant from the gastric ulcer margin (Abe et al., 1997; Alexander et al., 1995).

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V. EGF-CFC Proteins in Transformation and Tumorigenesis Initial clues suggesting a potential role for Cr-1 in the promotion or progression of mammary tumors in vivo in mice were obtained in a study where Cr-1 expression was investigated in diVerent mammary gland tumors from transgenic mice (Kenney et al., 1996). Transgenic mice overexpressing either neu (erbB-2), TGF, int-3, polyoma middle T (PyMT), or simian virus 40 large T antigens were studied. The expressions of these transgenes have previously been shown to lead to the spontaneous development of mammary gland tumors (Guy et al., 1992; Jhappan et al., 1990, 1992; Maroulakou et al., 1994; Muller et al., 1998). In their study, Kenney et al. found that all these tumors did, in fact, express significant levels of Cr-1 as determined by reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry. Expression of Cr-1 was also consistently expressed in hyperplastic mammary glands of PyMT, neu, and TGF mouse transgenic models, suggesting a role for Cr-1 in progression and deregulation of mammary epithelial proliferation (Niemeyer et al., 1999). A large body of evidence has been collected from in vitro studies to give us a clearer understanding of the biological eVects of Cripto-1 at the cellular level. C-Ha-ras or c-Ki-ras have been shown to up-regulate Cr-1 expression in rat CREF embryo fibroblasts or rat FRLT-5 thyroid epithelial cells (Mincione et al., 1998; Su et al., 1993). Interestingly, reversion of the transformed phenotype of the Ha-ras-transformed cells by overexpression of a Krev-1 ras suppressor gene resulted in a loss of expression of Cr-1, suggesting that Cr-1 might be an eVector of ras-induced cell transformation. In another study, co-infection of primary keratinocytes with v-ras (Ha) and Smad-7 retroviruses enhanced keratinocyte proliferation and induced squamous cell carcinoma in vivo (Liu et al., 2003). Notably, v-ras=Smad-7 tumors overexpressed Cr-1 and TGF, suggesting that Smad-7 induced tumor formation through inhibition of TGF
signaling and up-regulation of Cr-1 and other EGF-related peptides. Experiments involving overexpression of CR-1 cDNA in normal mouse fibroblasts induced these cells to grow in soft agar and increased growth rates in diVerent human breast cancer cells (Brandt et al., 1994; Ciccodicola et al., 1989). Exogenous recombinant CR-1 protein also stimulated cell proliferation of diVerentiated and undiVerentiated embryonal carcinoma cells (Baldassarre et al., 1997). CID 9 mouse mammary epithelial cells were used to further characterize the eVects of Cr-1 in vitro (Niemeyer et al., 1998). CID 9 cells are derived from mammary glands of 14.5-day pregnant mice and normally express Cr-1. The levels of Cr-1 increase when these cells are cultured on extracellular matrix substrates, in the presence of lactogenic hormones such as prolactin, where they diVerentiate forming polarized epithelial structures called mammospheres and

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express the milk protein
-casein. This suggests that hormones released during pregnancy may be involved in the regulation of Cr-1 expression. However, CID 9 cells overexpressing Cr-1 do not form mammospheres and show reduced expression of
-casein, indicating that the overexpression of Cr-1 may repress cell diVerentiation. CID 9 cells overexpressing Cr-1 showed significantly higher proliferation rates, decreased apoptosis, and increased colony formation in soft agar. When the Cr-1 overexpressing cells were subsequently transplanted in vivo, increased growth and survival of these cells were observed but they were unable to form tumors. Insertion of Elvax pellets containing the EGF-like motif of CR-1 protein into the mammary gland of ovariectomized virgin mice produced dramatic increases in DNA synthesis in mammary epithelial cells immediately adjacent to the pellets (Kenney, 1997). Also from that study, when FVB=N or C57B1=6 mouse mammary epithelial cells, that were transduced by the introduction of a retroviral expression vector containing Cr-1 cDNA, were transplanted into cleared mammary fat pad of syngenic ovariectomized virgin mice, increased ductal branching and hyperplasias were observed. These hyperplasias could be serially transplanted, suggesting the presence of an immortalized population of mammary epithelial cells. However, mammary carcinomas did not develop from these transplants. Human estrogen receptor-positive MCF-7 breast cancer cells that were overexpressing CR-1 failed to grow in the absence of estrogens. Nevertheless, CR-1-MCF-7 cells showed higher proliferation rates in serum-free medium; increased colony formation in soft agar; increased resistance to apoptosis induced by disruptions in cell–matrix interactions, known as anoikis; and increased invasiveness as demonstrated in a Boyden chamber invasion assay (Normanno et al., 2004a). Again, overexpression of Cr-1 in the mouse mammary epithelial cell line EpH4 caused increased cell proliferation and anchorage-independent growth in soft agar, induced the formation of ductlike structures when grown in collagen type I matrix, and increased chemotaxsis and migration when these cells were cultured on plastic or on porous filters coated with ECM (Wechselberger et al., 2001). The eVect of CR-1 on stimulating cell migration is in concert with previous data, demonstrating that oep regulates the migration of epiblastic cells independently of Nodal in the primitive streak that leads to mesoderm formation (Warga and Kane, 2003). However, the eVects of Cripto-1 observed in vitro may also depend on the type of cells used and their responsiveness to variations in growth conditions, reflecting the fact that the mammary gland epithelium cyclically goes through phases of proliferation and involution, depending on the variable levels of growth factors and hormones present in the environment in which they grow. In fact, exogenous CR-1 was capable of promoting apoptosis in normal confluent HC-11 mouse mammary epithelial cells, maintained in low serum containing medium

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depleted of hormones and growth factors, by activating caspase-3 protease and downregulating the expression of the anti-apoptotic protein Bcl-xl (De Santis et al., 2000). The eVects of Cr-1 in EpH4 cells was the first study which suggested that Cr-1 may play a role in inducing EMT of mammary epithelial cells. EMT is a process which normally takes place during gastrulation, neural crest cell migration, and tissue diVerentiation and repair (Perez-Pomares and MunozChapuli, 2002). During EMT, intercellular contacts between epithelial cells are lost due to decreased E-cadherin expression and disruption of the adherens junction complex. As a consequence, cells become less adhesive and potentially more motile. Also during EMT, alteration in architecture and molecular composition of the cytoskeleton causes epithelial cells to become spindle-shaped, resembling mesenchyme cells (Boyer et al., 2000; Thiery, 2002). Expression of vimentin, an important component of mesenchyme cell cytoskeleton, is characteristically increased in epithelial cells and tumors during EMT (Ackland et al., 2003; Casaroli-Marano et al., 1999; Dandachi et al., 2001; Grille et al., 2003; Steinert and Roop, 1988). In this respect, overexpression of CR-1 in human cervical carcinoma cells increased the expression of vimentin in these cells as they exhibited increased migration and invasion through matrix-coated membranes, suggesting that CR-1-induced vimentin expression may contribute to a more aggressive phenotype of cervical carcinoma (Ebert et al., 2000). In addition to degradation or loss of expression of the intercellular adhesion molecule, E-cadherin and changes in the composition of the cytoskeleton, altered expression pattern of extracellular adhesion molecules, such as the integrins and increased expression of neural (N)-cadherin, can also take place during EMT (Burridge et al., 1992; Islam et al., 1996). Integrins consist of transmembrane - and
-subunits that interact with growth factors, cytokines and other extracellular matrix molecules and have been shown to aVect cell adhesion, proliferation, and migration of tumor cells, especially -v,
-1,
-3, and
-4 integrins (Abdel-Ghany et al., 2001; Beauvais and Rapraeger, 2003; Danen and Sonnenberg, 2003; Maemura et al., 1995), whereas, N-cadherin has been shown to induce a more aggressive phenotype in malignant tumors including breast cancer (Hazan et al., 2000; Nieman et al., 1999). Further evidence of the role that Cr-1 might play in the induction of EMT comes from a recent study where the expression of markers that characteristically define EMT was investigated in mammary gland hyperplasias and tumors from mice expressing the human CR-1 transgene under the control of the MMTV promoter and in the mouse mammary epithelial cell line, HC-11, overexpressing Cr-1 (HC-11=Cr-1) (Strizzi et al., 2004). In that study, E-cadherin expression was significantly decreased in tissue extracts from the mammary tumor lesions that express the human CR-1 transgene and in extracts from HC-11=Cr-1 cells. Those

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extracts also showed significant increases in the expression of N-cadherin, vimentin, and integrins -3, -v,
-1,
-3, and
-4 as well as increases in phosphorylated forms of signaling molecules, such as c-Src, focal adhesion kinase (FAK), and AKT, which are also known to be activated during EMT and which probably play a role in increasing tumor cell invasion (Thiery and Chopin, 1999). Also, in the CR-1 transgenic mammary tumors and HC-11=Cr-1 cells, the zinc-finger repressor transcription factor snail, which is known to down-regulate expression of E-cadherin, was detected, by RT-PCR and Western blot analysis, at significantly higher levels as compared to control nontumorigenic mammary tissue. Interestingly, both the CR-1 transgenic mammary tumors and the HC-11=Cr-1 cells were found to express the phosphorylated or inactive form of glycogen synthease kinase 3
(GSK-3
) as well as the nonphosphorylated or active form of
-catenin.
-catenin, along with E-cadherin, is an important component of the adherens junctions (Savagner, 2001). Continuous turnover of the intercellular adhesion components may lead to cytoplasmic accumulation of
-catenin but this is normally prevented by GSK-3
-dependent phosphorylation of
-catenin, leading to proteosome ubiquitination and degradation of the phosphorylated
-catenin (Henderson and Fagotto, 2002). During wnt signaling, nonphosphorylated
-catenin translocates to the nucleus in a complex with Tcf=Lef-1 and functions as a transcription factor, activating genes such as c-myc, cyclin-D1, and slug, which is related to snail, that have been shown to be involved in increased cell survival, proliferation, and migration (Polakis, 2000). Cr-1 has been shown to be a target gene during canonical wnt=
-catenin signaling (Morkel et al., 2003) and the fact that increased expression of inactive GSK-3
, active
-catenin, and snail in CR-1 transgenic mammary tumors and in HC-11=Cr-1 cells strongly suggests a possible link between CR-1 and a canonical wnt signaling pathway. In addition, increased expression of certain integrins in the MMTV CR-1 transgenic mammary tumors may indicate that integrin-linked kinase (ILK) may also be involved in CR-1 signaling, since ILK has been shown to regulate wnt=
-catenin signaling pathway (Novak et al., 1998). This further supports the possibility of cross-talk between CR-1 and wnt signaling pathways. A 2004 study has shown that wnt=
-catenin signaling, as previously demonstrated for CR-1, plays a fundamental role during cardiogenesis. In fact, wnt=
-catenin signaling has been shown to play an important role in inducing EMT during cardiac cushion formation from which the atrioventricular valves and mesenchymal portion of the cardiac septum arise. Inhibition of the
-catenin gene expression resulted in embryonic lethality due to defects in cardiac development (Liebner et al., 2004). This is further evidence to support the possibility of cross-talk between wnt=
-catenin and CR-1 signaling pathways.

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VI. Intracellular Signaling Pathways Activated by Cripto-1 Cripto-1 was originally classified among the family of EGF-related peptides due to the presence of a modified EGF-like domain (Salomon et al., 2000). However, CR-1 fails to bind directly to any of the four erbB type I receptor tyrosine kinases, as either homodimers or heterodimers. For several years, it had been considered an orphan ligand, but several studies have implicated Cripto-1 in the activation of multiple intracellular signaling pathways, revealing a complex interplay of signaling molecules activated by Cripto-1 during embryonic development and cellular transformation. The major signaling pathways activated by CR-1 are a Nodal=ALK4=ALK7=Smad-2 signaling pathway and a Glypican-1=c-Src=MAPK=AKT signaling pathway (Fig. 1). A. Nodal=ALK4=ALK7=Smad-2 Signaling Pathway EGF-CFC proteins function as cell-surface co-receptors for Nodal signaling through an heteromeric complex composed of Activin type II and type I (ALK4) serine threonine kinase receptors in the plasma membrane (Fig. 1A) (Schier, 2003). Biochemical studies indicate that CR-1 binds to ALK4 and that this interaction is necessary for Nodal binding to the ActRIIB=ALK4 receptor complex (Bianco et al., 2002a; Yeo and Whitman, 2001). Upon ligand binding, the type I and type II receptor complex triggers phosphorylation of Smad-2 and Smad-3 that, in turn, bind to Smad-4, forming a transcriptional complex that translocates to the nucleus to enhance transcription of specific target genes (Shen and Schier, 2000). EGF-CFC proteins can also mediate Nodal signaling via the type I ALK7 receptor (Fig. 1A) (Reissmann et al., 2001). However, the mechanism by which Nodal activates the ALK4 and ALK7 type I receptors is diVerent. In fact, Nodal signaling through ALK4 is fully dependent upon EGF-CFC proteins. In contrast, Nodal can directly bind ALK7 and can signal in the absence of CR-1, although CR-1 significantly enhances its signaling activity (Reissmann et al., 2001). Unlike Nodal, Activin also utilizes the same receptors (ALK4 and ActRIIB), but does not require EGF-CFC co-receptors for binding to the type I ALK4 receptor. Therefore, a critical function of EGF-CFC proteins during development is to render ALK4 competent for activation by Nodal and to enhance the ability of ALK7 to respond to Nodal. Site-directed mutagenesis experiments have demonstrated that CR-1 interacts with Nodal through its EGF-like domain while the CFC domain is essential for ALK4 binding (Yeo and Whitman, 2001). Interestingly, an O-linked fucose modification has been identified at a conserved threonine residue (T88) within the EGF-like motif of human CR-1 (SchiVer et al., 2001). Mutation of the fucosylation

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Figure 1 Intracellular signaling pathways activated by Cripto-1. Agonistic signaling pathways: (A) Nodal=Cripto-1=ActRIB (ALK4=ALK7) signaling pathway. Cripto-1 binds to ALK4 or ALK7 and Nodal, allowing the formation and activation of an ActRIIB-ActRIB-Cripto-1Nodal complex that triggers phosphorylation of Smads; (B) Cripto-1, independently of Nodal and ALK4, binds to Glypican-1 and induces activation of the cytoplasmic tyrosine kinase c-Src, thus leading to the activation of the MAPK and AKT signaling pathways. Antagonistic signaling pathways: (C) Cripto-1 blocks Activin signaling by binding and sequestring Activin from the type II receptor and=or forming a nonproductive complex with ALK4; (D) Lefty antagonizes Nodal signaling by binding to Cripto-1 or Nodal, thus preventing the formation of an active type I and type II Activin receptor complex; (E) Tomoregulin-1 antagonizes Nodal signaling by binding to Cripto-1 and preventing its interaction with ALK4.

consensus sequence (T88A) results in a nonfucosylated CR-1 protein that is unable to interact with Nodal and to induce activation of a Nodal=ALK4 signaling pathway, suggesting that post-translational modifications might modulate EGF-CFC activity (SchiVer et al., 2001). In addition to functioning as a necessary binding partner for Nodal signaling, studies have shown that EGF-CFC co-receptors modulate the use of these Activin receptors by other TGF
-related ligands. For example,

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Xenopus Vg1 and its ortholog in mouse GDF1 are TGF
ligands that share only 35 to 55% identity with Nodal or Activin in the mature domain, but exhibit similar biological activities acting as mesoderm inducers (Cheng et al., 2003). Similar to Nodal, Vg1 and GDF1 can eYciently signal through ActRIIB and ALK4 receptor complex only in the presence of EGF-CFC co-receptors, suggesting a more widespread requirement for co-receptors in TGF
signaling. In addition to facilitating the formation of functional signaling complexes (for example, in the case of Nodal or Vg1=GDF1), Cripto-1 can also act as an inhibitor of Activin signaling in mammalian cells (Fig. 1C). In this respect, two studies demonstrating Cripto-1 antagonism of Activin signaling disagree on several important points (Adkins et al., 2003; Gray et al., 2003). Adkins et al. (2003) demonstrated that CR-1 directly binds to Activin B, but not to Activin A, by immunoprecipitation, ELISA, and Biacore analysis. In contrast, Gray and colleagues (2003) have shown that CR-1 can inhibit both Activin A and Activin B signaling in cell culture assays, but they were unable to detect a direct interaction between Activin A or Activin B and CR-1. Moreover, they demonstrated that Activin A could bind CR-1 only in the presence of ActRII, suggesting the formation of an inactive Activin=ActRII=CR-1 complex. Another discrepancy is the molecular mechanism by which CR-1 inhibits Activin function. Adkins and colleagues (2003) demonstrated that CR-1 interacts with Activin B through its CFCdomain based upon two distinct findings. First, a CFC-specific anti-CR-1 monoclonal antibody (mAb) prevents Activin B from binding to CR-1, suggesting that CFC-domain residues are critical for CR-1=Activin B interaction. Furthermore, the CR-1 EGF-domain fucosylation mutant protein, which does not bind to Nodal, retains Activin B binding activity. In contrast, Gray et al. (2003) showed that an EGF-domain deletion mutant does not interact with Activin A, suggesting that Nodal and Activin compete for binding to similar sites on CR-1. Several studies have clearly demonstrated that Activin is a potent inhibitor of cell growth in diVerent cell lines and disruption of Activin signaling has been associated with tumorigenesis (Adkins et al., 2003). Therefore, antagonism of Activin signaling by Cripto-1 might represent an additional mechanism by which Cripto-1 regulates cell growth and proliferation and may be an important step in promoting transformation. In fact, the ability of a blocking anti-CR-1 mAb to inhibit tumor cell proliferation in two diVerent xenograft models might be due to its ability to inhibit Activin signaling in vivo. In addition to being dependent on co-receptors of the EGF-CFC family for activity, Nodal signaling can be modulated by several antagonists that function at diVerent levels to precisely regulate tissue patterning during early embryonic development. Interestingly, some of these inhibitors, such as Tomoregulin-1, Lefty, or Antivin, can antagonize Nodal signaling

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through EGF-CFC co-receptors (Harms and Chang, 2003). Tomoregulin-1 is a transmembrane protein containing two follistatin domains and an EGF-motif in the extracellular domain and a short cytoplasmic tail (Uchida et al., 1999). Harms and colleagues have demonstrated that Tomoregulin-1 inhibits Nodal signaling in early Xenopus embryos through direct binding to the CFC domain of CR-1 (Fig. 1E) (Harms and Chang, 2003). Since Tomoregulin-1 and ALK4 both interact with CR-1 through its CFCdomain, it is possible that both proteins compete for binding to CR-1 and the interaction of Tomoregulin-1 with CR-1 might exclude binding of CR-1 to ALK4, leading to inhibition of Nodal signaling. Tomoregulin-1, in addition to functioning as an antagonist of Nodal signaling in Xenopus, can specifically bind with low aYnity, through its EGF-like domain, to the type I tyrosine kinase receptor erbB-4 (Uchida et al., 1999). We have previously demonstrated that CR-1 indirectly enhances the tyrosine phosphorylation of erbB-4 in mammary epithelial cells and in breast cancer cell lines (Bianco et al., 1999). Thus, interaction between CR-1 and Tomoregulin-1 might explain the phosphorylation of erbB-4 tyrosine kinase by CR-1 in mammalian cells. The Lefty=Antivin subfamily of TGF
proteins is another example of extracellular antagonists of Nodal signaling (Schier, 2003). Genetic and biochemical studies have shown that Lefty functions as an antagonist of the EGF-CFC co-receptors, interacting directly with CR-1 and competing with Nodal for binding to CR-1 (Fig. 1D) (Cheng et al., 2004). The competitive binding of Lefty to EGF-CFC co-receptors enables Lefty to antagonize Nodal signaling, preventing its interaction with type I and type II Activin receptors. Furthermore, Lefty can also directly interact with Nodal in solution, preventing Nodal from binding its receptor complex (Fig. 1D) (Chen and Shen, 2004). In addition to blocking Nodal signaling, Lefty can also inhibit Vg1=GDF1 signaling by a similar mechanism. In contrast, Lefty is unable to inhibit signaling by Activin A or TGF
1, which are both EGF-CFC-independent (Branford and Yost, 2002; Chen and Shen, 2004; Cheng et al., 2004). Similarly, a Lefty-related protein in Xenopus, Xantivin, antagonizes Nodal signaling via the EGF-CFC proteins. Coimmunoprecipitation assays demonstrated that Xantivin interacts directly with Xenopus FRL-1 and CR-1, thereby inhibiting Nodal signaling (Tanegashima et al., 2004). Collectively, these studies establish an essential role for EGF-CFC proteins in Nodal signaling. However, CR-1 and Nodal may have distinct functional roles independent of one another. For example, Nodal can inhibit BMP-signaling by binding directly to BMP through a Cripto-1-independent pathway (Yeo and Whitman, 2001). Likewise, the zebrafish EGF-CFC protein oep regulates cell motility independently of the Zebrafish Nodal-related signaling molecules Cyc and Sqt (Warga and Kane, 2003).

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B. Glypican-1=c-Src=MAPK=AKT Signaling Pathway In addition to functioning as a co-receptor for Nodal, we have demonstrated that CR-1 can signal in mammalian cells independently from Nodal, leading to activation of intracellular signaling pathways that regulate cell proliferation, cell motility, and survival, through both ras=raf=MAPK and PI3K=AKT=GSK-3
signaling pathways (Fig. 1B) (De Santis et al., 1997; Ebert et al., 1999). Activation of these signaling pathways can be achieved by a soluble GPI-truncated CR-1 recombinant protein or by a refolded peptide containing only the EGF-like domain, suggesting that CR-1 can function as a secreted growth factor in mammalian cells (Kannan et al., 1997). In this respect, it has been reported that cleavage of the GPIlinkage from the Cr-1 protein with PI-PLC generates a functional soluble protein (Minchiotti et al., 2000). Furthermore, CR-1 can also be detected in the conditioned medium of several human carcinoma cell lines overexpressing CR-1, suggesting that a soluble isoform of CR-1 is naturally cleaved from the cell membrane (Brandt et al., 1994; Normanno et al., 2004a). We have previously shown that a soluble CR-1 recombinant protein can enhance the tyrosine phosphorylation of the SH2-adapter protein Shc, triggering the downstream activation of the ras=raf=MAPK and PI3K=AKT=GSK-3
signaling pathways in several diVerent mouse and human cell lines (Ebert et al., 1999; Kannan et al., 1997). Surprisingly, activation of these two intracellular signaling pathways is independent of Nodal and ALK4. In fact, CR-1 can enhance the phosphorylation of MAPK and AKT in EpH-4 mouse mammary epithelial cells and MC3T3-E1 osteoblast cells that lack Nodal and ALK4 expression, respectively (Bianco et al., 2002a). Activation of these two signaling pathways is mediated by direct binding of CR-1 to the GPI-linked HSPG Glypican-1, which can then activate the cytoplasmic tyrosine kinase c-Src triggering activation of MAPK and AKT (Bianco et al., 2003). Moreover, Glypican-1 and c-Src are required by CR-1 to stimulate MAPK and AKT phosphorylation in mammary epithelial cells. Reciprocally, CR-1 can enhance Smad-2 phosphorylation in mammary epithelial cells independently of Glypican-1 and c-Src, suggesting that these are two distinct pathways which are activated by CR-1 in mammalian cells. Finally, an intact c-Src kinase is required by CR-1 to induce in vitro transformation and to enhance migration in mammary epithelial cells, suggesting that inappropriate activation of c-Src by CR-1 in a Nodal- and ALK4-independent manner may play a key role in promoting cellular transformation. Cripto-1 has also been involved in the activation of the FGFR-1 signaling pathway. In this regard, FRL-1, in part, functions during development through a FGFR-1 signaling pathway since this gene was initially identified using a functional screen in yeast to detect novel ligands of the FGFR-1

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(Kinoshita et al., 1995). In Xenopus oocytes and in yeast (S. cerevisiae), simultaneous ectopic overexpression of FRL-1 and the Xenopus FGFR-1 kinase leads to enhanced Ca2+ influx and to an increase in the indirect transphosphorylation of FGFR-1. However, no direct binding of FRL-1 to the Xenopus FGFR-1 could be detected. It has been demonstrated that N-CAM or N-cadherin can activate the FGFR-1 as surrogate ligands in an FGF-independent fashion by binding to the FGFR-1 through the immunoglobulin-like repeats in the extracellular domain of the FGFR-1 (Cavallaro and Christofori, 2004). This may explain the ability of FRL-1 in Xenopus to indirectly activate the FGFR-1, since Xenopus Nodal (xnr3), functioning through FRL1, can upregulate N-CAM expression (Yokota et al., 2003). Although studies strongly support a role for CR-1 in the activation of MAPK and AKT signaling pathways in mammalian cells, very little is known about the activation of these pathways by CR-1 during embryonic development. In this regard, a potential role of EGF-CFC proteins in the activation of the MAPK pathway during early embryonic development is supported by studies in Xenopus. In this respect, Yabe and colleagues demonstrated that in Xenopus FRL-1 regulates neural induction by activating the MAPK pathway through the FGFR-1 (Yabe et al., 2003). In addition, xnr3, a member of the Nodal sub-family of TGF
proteins in Xenopus, has been shown to activate FGFR-1 and the MAPK pathway to regulate cell movements during gastrulation and neurulation (Yokota et al., 2003). Although xnr3 and FRL-1 strongly synergize in an animal cap assay, a direct interaction between xnr3 and FRL-1 has not been demonstrated and it is unclear whether FRL-1 mediates the ability of xnr3 to activate FGFR-1 and MAPK in Xenopus. Although CR-1 can independently activate the Nodal=ALK4=Smad-2 or Glypican-1=c-Src=MAPK=AKT signaling pathways, these two pathways might not be mutually exclusive. For instance, several studies have demonstrated a cross-talk between TGF
family members and the MAPK pathway. In this regard, ALK7, in addition to its ability to phosphorylate components of the Smad pathway, such as Smad-2 and Smad-3, has also been found to activate c-Jun NH2-terminal kinase (JNK) and p38 MAPK in pheocromocitoma, hepatoma, and throphoblast cells, leading to apoptosis (Jornvall et al., 2001; Kim et al., 2004; Munir et al., 2004). However, whether or not CR-1 is involved in the activation of JNK and p38 in these cell lines is unknown. Finally, several studies have shown a potential interaction between the canonical wnt=
-catenin=Lef-1 and Nodal=CR-1 signaling pathways. In this regard, using microarray technology, Cr-1 has been identified as a primary target gene in the wnt=
-catenin signaling pathway during embryonic development and in colon carcinoma cells (Morkel et al., 2003). Likewise, xnr3 is also a direct target of wnt=
-catenin in Xenopus (Yokota et al., 2003). This cross-talk between
-catenin and CR-1 signaling pathways might be

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functionally significant since activation of
-catenin signaling in cells might lead to CR-1 overexpression, thereby promoting cell proliferation and transformation.

VII. Expression of Cripto-1 in Human Carcinomas and Premalignant Lesions Growth factors and growth factor receptors are involved in the pathogenesis of human cancer (Goustin et al., 1986). In fact, tumor cells generally exhibit a reduced requirement for exogenous growth factors when compared with normal cells (Aaronson, 1991; Sporn and Roberts, 1992). This phenomenon may be due to an increase in the synthesis by transformed cells of various growth factors and=or growth factor receptors. These proteins regulate cellular functions that are essential for tumor development and progression, such as tumor growth, neoangiogenesis, and formation of metastasis. Therefore, overexpression of growth factors and their cognate receptors might confer a growth advantage to tumor cells. In this respect, several studies have demonstrated that CR-1 is overexpressed in a number of human breast, colon, gastric, and pancreatic cell lines, suggesting that CR-1 may function as an autocrine growth factor in these tumor cells (Ciardiello et al., 1991a; Kuniyasu et al., 1991; Normanno et al., 1993). More recently, a wide, comparative screening for CR-1 expression in cell lines from diVerent carcinoma types has been completed by using a quantitative real time PCR assay (Normanno et al., 2004b). This study demonstrated that CR-1 transcripts are also expressed in cell lines derived from non-small-cell lung cancer (NSCLC), and from ovarian, testicular, and renal carcinomas. Expression of immunoreactive CR-1 was also demonstrated in these cancer cell lines by using immunocytochemical techniques. More importantly, this study formally demonstrated, by using antisense oligonucleotides, that CR-1 functions as autocrine growth factor in tumor cell lines derived from diVerent carcinoma types. These findings strongly support a role for CR-1 in the pathogenesis of diVerent types of human carcinoma. This hypothesis is confirmed by several studies that have analyzed the frequency and relative expression of CR-1 mRNA and=or immunoreactive protein in diVerent primary human carcinomas (Table I).

A. Gastric Cancer CR-1 has been detected in normal gastric mucosa (Abe et al., 1997). Relatively intense immunoreactivity of CR-1 has been also found in the mature

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Frequency of Immunoreactive CR-1 Expression in Human Tumorsa Frequency of CR-1 expression

Type of cancer Gastric Pancreatic Colorectal Gall bladder Breast Endometrial Cervical Ovarian Bladder Lung (non-small-cell) Testis non-seminomas seminomas

Average%

Range%

42 55 73 68 78 69 39 50 60 91

33–47 45–81 67–84 75–82 65–71 26–54 47–53

100 33

a Number represents the average percentage (%) of positive tumors. Where available, the range of positive tumors reported in the diVerent studies is indicated.

regenerative epithelium located distant from gastric ulcer margin, suggesting that it might be involved in ulcer healing, probably through a paracrine mechanism (Abe et al., 1997). An increase in CR-1 expression as compared with normal gastric mucosa has been observed in premalignant lesions of the stomach, such as intestinal metaplasia of the gastric mucosa, in gastric adenomas, and in 46% of early and advanced gastric carcinomas (Kuniyasu et al., 1991; Saeki, 1994a). In agreement with these data, overexpression of CR-1 mRNA was found to occur in 55% of gastric adenocarcinomas, by using a fluorescent multiplex reverse transcriptase-PCR technique (Rajevic et al., 2001). CR-1 immunoreactivity was also found to correlate with the degree of dysplasia in the intestinal metaplasia and with tumor stage and patient prognosis in gastric cancer (Kuniyasu et al., 1994). In fact, CR-1 overexpression was reported in 62% of late-stage, locally invasive tumors and in 25% of early-stage, noninvasive cancers (Kuniyasu et al., 1994). CR-1 expression in both groups was associated with a poorer patient prognosis. Finally, CR-1 expression has been described in patients with long-term Helicobacter pylori infection and with atrophic gastritis, a risk factor for gastric carcinogenesis (Haruma et al., 2000). Collectively, these studies suggest that CR-1 overexpression might be involved in the development and progression of gastric cancer.

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B. Pancreatic Cancer CR-1 mRNA and protein have been detected by Northern blot analysis and immunohistochemistry in normal pancreas (Friess et al., 1994). However, the levels of expression of CR-1 were significantly increased in pancreatic ductal adenocarcinomas and, to a lesser extent, in chronic pancreatitis, as compared with normal pancreas (Friess et al., 1994; Tsutsumi et al., 1994). CR-1 staining was also found in pancreatic papillary adenomas (Tsutsumi et al., 1994). The expression of CR-1 in pancreatic cancer correlated with advanced tumor stage, but not with the tumor grade or with the postoperative survival of the cancer patients.

C. Colorectal Cancer Expression of high levels of CR-1 mRNA and protein has been demonstrated in 73% of primary and metastatic colorectal cancers. In particular, CR-1 mRNA expression by Northern blot analysis was detected in 68% of primary or metastatic human colorectal cancers but only in 3% of noninvolved adjacent colon mucosa (Ciardiello et al., 1991b). Expression of the CR-1 protein was demonstrated by using immunohistochemistry in 79% of colon tumors, in 57% of tubulovillous or tubular adenomas, and in 12% of the noninvolved normal colonic mucosa adjacent to tumors or adenomas, but not usually in normal colon mucosa specimens (Saeki et al., 1992). The high frequency of expression of CR-1 in human carcinomas has been confirmed in additional studies in which immunoreactive CR-1 was detected in 67 to 84% of colorectal carcinomas and in 42 to 55% of colon adenomas (De Angelis et al., 1999; Saeki et al., 1995). In the study by Saeki et al. (1995), expression of CR-1 in normal colonic mucosa was not observed, whereas 22% of hyperplastic polyps were found to express significant levels of CR-1 protein. Furthermore, the frequency of CR-1 expression in the adenomas was found to correlate directly with the degree of dysplasia, and with the size and the histological subtype of colon adenomas (Saeki, 1994b). In fact, nonpolypoid flat adenomas had a higher frequency of CR-1 expression than did polypoid lesions. The gradual increase in CR-1 expression that is observed in the multistage process that evolves from adenoma to carcinoma, and the finding that CR-1 might be expressed in the mucosa adjacent to the tumors whereas it is not usually expressed in the normal colonic mucosa, suggest that CR-1 may be a tumor marker for human colorectal cancer and may be involved in the early events of colon carcinogenesis (Saeki et al., 1995). In agreement with this hypothesis, CR-1 expression was detected in 62% of normal colon mucosa specimens from individuals belonging to families with a high incidence of colorectal

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carcinomas, whereas only 20% of colon mucosa from low-risk patients’ specimens were positive for CR-1 (De Angelis et al., 1999). In an additional study, CR-1 immunoreactivity has been detected in 71% of primary rectal carcinomas and in 37% of normal rectal mucosa adjacent to carcinoma (Gagliardi, 1994). Interestingly, tumors with CR-1 immunoreactivity extending to the adjacent normal mucosa showed an increase in the incidence of bowel wall penetration, lymph node involvement, and a recurrence rate of 100%, suggesting a correlation between CR-1 immunoreactivity in the non-involved adjacent mucosa and prognosis and survival of rectal tumors.

D. Gall Bladder Carcinoma CR-1 expression has been detected in 6=9 (67%) hyperplasias, 4=7 (58%) adenomas, and 89=132 (68%) adenocarcinomas of the gall bladder (Fujii et al., 1996). The degree of CR-1 expression was not correlated with depth of tumor invasion, tumor stage, or patient prognosis. However, CR-1 expression was significantly higher in papillary and well-diVerentiated adenocarcinomas as compared with moderately and poorly diVerentiated adenocarcinomas.

E. Breast Cancer CR-1 mRNA and protein can be detected in several human breast cancer cell lines and in approximately 80% of primary human infiltrating breast carcinomas, in 47% of ductal carcinoma in situ (DCIS), in 13% of non-involved adjacent breast tissue samples, and in approximately 6% of normal breast specimens (Adkins et al., 2003; Normanno et al., 1993, 1995; Panico et al., 1996; Qi et al., 1994). In the nontumor specimens, a limited number of epithelial cells stain for CR-1 (<25%) (Panico et al., 1996). No significant correlations have been observed between CR-1 mRNA expression or immunoreactivity and various clinicopathological parameters such as tumor stage, estrogen receptor status, lymph node involvement, histologic grade, proliferative index as assessed by Ki-67 staining or flow cytometry, LOH on chromosome 17p, or overall patient survival (Dublin, 1995; Normanno et al., 1995; Panico et al., 1996; Qi et al., 1994). However, the progressive increase in the frequency and in the levels of expression of CR-1 in in situ carcinomas and in the invasive carcinomas as compared with the normal breast mucosa suggests that CR-1 is involved in the early stages of breast carcinogenesis as well as in the progression of this disease. Furthermore, in human primary breast carcinomas, CR-1 is frequently coexpressed with

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other EGF-related peptides, such as TGF, AR, and HRG, implying that diVerent growth factors might cooperate in supporting the autonomous proliferation of breast cancer cells. A positive correlation between nuclear erbB-4 expression and CR-1 expression in primary human breast carcinomas has also been described (Srinivasan et al., 2000). This finding is intriguing since it has been shown that CR-1 can indirectly enhance the tyrosine phosphorylation of erbB-4 (Bianco et al., 1999). F. Endometrial Cancer Expression of CR-1 mRNA has been demonstrated in both normal human endometrium and in endometrial cancer samples by using a semiquantitative reverse-transcription=polymerase chain technique (PfeiVer et al., 1997). CR-1 expression was higher in the endometrial carcinomas as compared with normal endometrium. However, CR-1 levels did not correlate with tumor stage and tumor diVerentiation. Immunoreactive CR-1 was found in 67% of normal endometrium samples, in 30% of hyperplastic endometrium, and in 65% of tumor samples (Niikura et al., 1996). In this study, CR-1 immunoreactivity in the tumor samples was correlated with surgical stage and positive cytological findings in the peritoneal washing. In an additional study, CR-1 immunoreactivity was found in 68=96 (71%) endometrial carcinomas and in most cases of endometrial hyperplasia with atypia, whereas hyperplasias without atypia were weakly stained (Ayhan et al., 1998). G. Cervical Cancer Expression of CR-1 protein has been demonstrated by using Western blot analysis in primary human keratinocytes, in HPV-16 immortalized keratinocytes, and in cervical carcinoma cell lines (A. D. Ebert, J. Stieler, M. Nees, C. Wechselberger, S. Steinberg, C. Woodworth, C. Bianco, W. Gullick, G. Schaller, and D. S. Salomon, unpublished data). More importantly, CR-1 was found to be overexpressed in 54% of human cervical cancer samples as compared with samples of normal cervix. Preliminary analyses did not find statistically significant correlations between CR-1 expression and FIGO (International Federation of Gynecology and Obstetrics) stage, grading, lymph node involvement, or patient age. Expression of CR-1 mRNA has been also found by RT-PCR in both normal cervix and in cervical cancer (PfeiVer et al., 1997). However, in the normal cervix, CR-1 transcripts were detected only in stroma and not in squamous epithelium. Finally, Ertoy et al. (2000) found that CR-1 protein was overexpressed in a

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subpopulation of 26% of early clinical stage cervical carcinomas when compared with non-neoplastic cervical epithelium (Ertoy et al., 2000). More importantly, CR-1 overexpression was correlated with tumor size, lymphovascular space involvement, endometrial, and parametrial involvement. In addition, the levels of CR-1 expression were increased in metastatic lymph nodes compared with their primary tumors. H. Ovarian Cancer CR-1 was initially detected by immunohistochemistry in 53% of 30 ovarian adenocarcinomas, in 100% of 10 borderline tumors, and in 28.6% of 40 cystoadenomas (benign cysts) (Stromberg et al., 1994). These findings were confirmed by Niikura et al. (1997) in a small series of 17 cystoadenomas, 6 mucinous tumors of low malignant potential, and 25 adenocarcinomas (Niikura et al., 1997). In particular, these authors found that immunoreactive CR-1 was expressed in 52% of ovarian adenocarcinomas (90% mucinous of high malignant potential and 50% serous), in 33% of mucinous tumors of low malignant potential, and in 35% of cystoadenomas (Niikura et al., 1997). In addition, a significant correlation was observed with advanced tumor stage when CR-1 was coexpressed with TGF and AR or with the EGFR, suggesting that coexpression of these EGF-related growth factors and the EGFR in a single tumor represents a growth advantage for the cancer cells through potential autocrine or paracrine mechanisms. In another study, a larger number of human ovarian carcinomas (n. 59) of diVerent histologic types have been analyzed by immunohistochemistry for the expression of CR-1, TGF, and AR (D’Antonio et al., 2002). CR-1 was detected in 47% of ovarian carcinomas of diVerent histological types. The highest frequency of expression was found in mucinous tumors (80%), whereas no expression of CR-1 was detected in endometrioid tumors. CR1 expression did not correlate with lymph node involvement, presence of calcification or necrosis in the tumor, or number of mitoses. Interestingly, a statistically significant higher expression of CR-1 was found in extra-ovarian specimens as compared with ovarian tumors, in a subset of patients for which both types of tissues were available. These data strongly support the hypothesis that overexpression of CR-1 might be related to disease progression in ovarian carcinoma patients. I. Expression of Cripto-1 in Other Cancers Expression of CR-1 protein has been investigated in 45 bladder carcinomas and in 6 benign controls (Byrne et al., 1998). All 45 tumors showed positive cytoplasmic staining for CR-1, including areas of carcinoma in situ, whereas

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none of the six benign controls showed any evidence of positive CR-1 staining. Areas of papillary tumor that showed strong positive staining for CR-1 were found in 23 bladder tumors. CR-1 staining was also detected in 29% of the sections of histologically normal urothelium adjacent to tumor. No association between CR-1 staining and tumor grade, stage, or clinical outcome was found. Although Rusch et al. (1997) did not find expression of CR-1 mRNA in lung cancer specimens by using Northern blot technique, in a large group of 195 stage I-IIIA non-small-cell lung carcinomas, immunoreactive CR-1 was found to be expressed at high levels in 91% of the lung carcinomas (Fontanini et al., 1998; Rusch et al., 1997). However, expression of CR-1 was not correlated with prognosis in this group of patients. In testicular carcinomas, CR-1 expression has been detected by Northern blot analysis and by immunostaining in all of the non-seminomatous tumors such as embryonal carcinomas and malignant undiVerentiated teratocarcinomas that have been surveyed, and in only 33% of the seminomas (Baldassarre et al., 1997). The preferential association of CR-1 expression with non-seminomatous tumors, which are more clinically aggressive, suggests a correlation between CR-1 and a more malignant phenotype in germ cell tumors. Finally, by using diVerential hybridization of cDNA arrays, overexpression of CR-1 has been demonstrated in basal cell carcinoma (Welss et al., 2003). The overexpression of CR-1 in these tumors was confirmed by using a semi-quantitative reverse transcriptase PCR technique in 8=10 basal cell carcinomas. Interestingly, no signal for CR-1 expression was detected in 8 of 10 normal skin specimens and in 2 squamous cell carcinomas.

VIII. Cripto-1 as Target for Therapy in Human Cancer The high frequency of expression of CR-1 in several types of human carcinomas, the ability of this growth factor to activate several diVerent signal transduction pathways, and the lack or low levels of expression in normal tissues make CR-1 a potential target for therapeutic intervention. DiVerent approaches can be used to inhibit the activity of a growth factor, such as to block its synthesis, neutralize its activity, or block the activation of its cognate receptor. In this respect, the signaling of CR-1 has not been completely elucidated. In fact, it is evident that CR-1 can activate diVerent receptor signaling pathways and that it can directly interact with other growth factors, such as Activin A and B and Tomoregulin-1. Therefore, therapeutic approaches aimed at blocking CR-1 activity have been based mainly upon the use of antisense oligonucleotides to reduce its expression or neutralizing antibodies to block the activity of the CR-1 protein.

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Sequence-specific antisense (AS) oligonucleotides or AS expression vectors can block the expression of specific proteins by binding to the corresponding mRNA and preventing translation. In this respect, therapeutic antisense approaches have been successfully used to inhibit the growth of several human cancer cell lines in vitro and their growth as xenografts in vivo in nude mice (Neckers et al., 1992). More importantly, AS oligonucleotides directed against diVerent targets are currently in clinical development (Dean and Bennett, 2003). This approach has been successfully utilized to impair CR-1 expression in several diVerent types of human carcinoma cells. Inhibition of CR-1 expression in human GEO and CBS colon cancer cells by using either an amphotropic recombinant CR-1 AS mRNA retroviral expression vector or CR-1 AS phosphorothioate oligonucleotides resulted in a significant growth inhibition in vitro (Ciardiello et al., 1994). GEO cells that were infected with the CR-1 AS retroviral vector exhibited a reduced tumorigenicity in nude mice in which tumors were smaller and appeared after a longer latency period as compared with noninfected GEO cells. Similar results were obtained in NTERA2 human embryonal carcinoma cells. Treatment of NTERA2 cells with 3 diVerent CR-1 AS oligonucleotides or transfection with a CR-1 AS mRNA expression vector resulted in an inhibition of monolayer and soft agar growth in vitro (Baldassarre et al., 1997). Interestingly, the inhibition in CR-1 expression that was achieved in NTERA2 cells resulted in a significant reduction in the expression of a diVerentiation marker on these tumor cells. In another study, a superadditive eVect was observed on reducing the growth of GEO cells in vitro when a CR-1 AS oligonucleotide was combined with a TGF AS or AR AS oligonucleotide, suggesting that diVerent growth factors contribute to regulate the proliferation of colon cancer cells (Normanno et al., 1996). Similarly, an additive growth inhibitory eVect was observed in MDA-MB-468 human breast cancer cells and in several diVerent human ovarian carcinoma cell lines that had been treated with a combination of a CR-1 AS oligonucleotide and either a TGF AS or AR AS oligonucleotide (Casamassimi et al., 2000; De Luca et al., 1999). Provocative data have also been obtained using combinations of CR-1 AS oligonucleotides with conventional chemotherapeutic drugs in colon cancer cells (De Luca et al., 1997). In a clonogenic assay, pretreatment of GEO cells with diVerent concentrations of 5-fluorouracil, adriamycin, mitomycin C, or cis-platinum induced an additive growth inhibitory eVect when the cells were subsequently treated with a CR-1 AS oligonucleotide. Anti-CR-1 second-generation antisense oligonucleotides have been recently developed. These oligonucleotides contain phosphorothioate backbone and a segment of 20 -O-methylribonucleosides modified at both the 50 and 30 ends of the oligonucleotide (MBOs). The advantages of second-generation oligonucleotides over phosphorothioate oligonucleotides are increased

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aYnity to target mRNA, increased biological activity, reduced polyanionicrelated side eVects, and increased in vivo stability (Agrawal and Zhao, 1998). Furthermore, it has been shown that these compounds are active following oral administration (Wang et al., 1999). CR-1 AS MBOs were able to block the in vitro growth of carcinoma cell lines derived from diVerent carcinoma types (De Luca et al., 2000; Normanno et al., 2004b). Treatment of carcinoma cells with CR-1 AS oligonucleotides resulted in a significant reduction in the levels of expression of CR-1 mRNA and protein. Furthermore, treatment with CR-1 AS MBOs produced in colon carcinoma cells a significant reduction in the levels of activation of AKT, but not of p42=p44 MAPK. Administration of these agents by either the intraperitoneal or the oral route resulted in a significant reduction in the growth of colon cancer xenografts in immunocompromised mice (De Luca et al., 2000; Normanno et al., 2004b). Interestingly, treatment with the combination of CR-1, AR, and TGF MBOs produced a more significant reduction in the growth of colon cancer xenografts as compared with treatment with a single oligonucleotide. This combination also produced a significant reduction in the levels of microvessels in the treated tumors as compared with untreated tumors or tumors from animals treated with a single AS oligonucleotide. Finally, CR-1 AS oligonucleotides have been also used in combination with agents that block diVerent intracellular signal transduction pathways (Normanno et al., 1999). In particular, a second generation CR-1 AS oligonucleotide has been used in combination with a humanized antihuman epidermal growth factor receptor antibody mAb C225 and with 8-Cl-cAMP, a specific analog that inhibits type I protein kinase A. Low doses of each agent produced only a 15 to 35% growth inhibition in GEO cells in vitro but when the CR AS oligonucleotide was combined with either the MAb C225 or with 8-Cl-cAMP, a synergistic antiproliferative eVect occurred. Moreover, when the three agents were added together, a nearly complete suppression in the ability of GEO cells to grow in soft agar occurred. Interestingly, treatment with all three compounds induced apoptosis in GEO cells whereas a single treatment or a combination of only two agents failed to induce any apoptosis. Since 2003, monoclonal blocking antibodies directed against CR-1 have been developed. In particular, Adkins and colleaugues have generated mAbs directed against diVerent regions of the CR-1 protein (Adkins et al., 2003). In particular, they found that anti-CFC domain mAbs disrupted CR-1-Nodal signaling by blocking the association of CR-1 with ALK4. Furthermore, these antibodies were able to prevent the binding of CR-1 to Activin B, and, therefore, to reverse the CR-1 blockade of Activin B-induced growth suppression in human breast carcinoma cells. In agreement with these findings, the anti-CR-1 antibodies were able to inhibit tumor cell growth by up to 70% in two xenograft models of testicular and colon cancer. Interestingly, both Nodal and Activin B were found to be expressed in the tumor xenografts,

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suggesting that the antitumor activity of the anti-CR-1 antibodies might be related to its ability to block the interactions of CR-1 with both signaling molecules. Finally, rat monoclonal antibodies directed against the EGF-like domain of the CR-1 peptide have been generated (Xing et al., 2004). Treatment with these IgM antibodies produced a significant inhibition of the in vitro growth of diVerent carcinoma cell lines. The anti-CR-1 mAbs also prevented tumor development in vivo and inhibited the growth of established tumors of LS174T colon xenografts in immuno-compromised mice. Interestingly, treatment with the anti-EGF-like domain antibodies produced a significant reduction in the levels of activation of AKT, suggesting that their mechanism of action is diVerent as compared with the anti-CFC antibodies described by Adkins et al. (2003). In this respect, the anti-EGF-domain antibodies were also able to activate c-Jun-NH2-terminal kinase and p38 kinase signaling pathways and, ultimately, to induce apoptosis in cancer cells. In agreement with previous findings by De Luca et al. (1997), treatment of cancer cells with a combination of CR-1 AS oligonucleotides and conventional cytotoxic drugs, such as 5-fluorouracil, epirubicin, or cis-platinum, resulted in a more significant inhibition of cancer cell growth as compared with treatment with a single agent (De Luca et al., 1997).

IX. Conclusions and Perspectives EGF-CFC proteins are multifunctional glycoproteins that can function either as cell-associated, GPI-linked co-receptors or as soluble ligands for several structurally distinct proteins through either cell-autonomous or cellnonautonomous pathways. Little information is available about the mechanism(s) by which these soluble versus cell-associated forms of EGF-CFC proteins are diVerentially generated, either during embryonic development or in adult tissues and cancer cells. Likewise, the precise subcellular localization of the GPI-linked forms of EGF-CFC proteins is unknown. It is quite likely that like other GPI-linked proteins, the cell-associated forms of the EGF-CFC proteins might be sequestered in lipid rafts on the apical surface of polarized epithelial cells. Information concerning the identity of growth factors or systemic hormones that might regulate the expression, processing, subcellular localization, or activity of EGF-CFC proteins is relatively rudimentary. The mechanism by which the regulation of glycosylation of EGFCFC proteins such as fucosylation might aVect the activity and function of these proteins in vivo in normal and cancer cells is also unknown. This later modification may be analogous to the capacity of diVerent Notch ligands such as Delta and Serrate to selectively activate Notch receptors, depending upon the diVerential fucosylation of Notch by fucosyltransferases of the Fringe family (Haltiwanger and Lowe, 2004; Okajima and Irvine, 2002).

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Finally, since Nodal and GDF1, but not Activin, require a functional EGFCFC co-receptor protein to signal, and since both Nodal and Activin presumably activate the same set of Activin type II and type IB (ALK4) receptors and the same downstream Smads (i.e., 2, 3, and 4) in the presence of FoxH1, it is not entirely clear as to the mechanism by which the biological readout of these ligands is diVerentially translated via this same set of intracellular eVectors. It seems quite possible that this discrimination might be achieved in a context-specific manner by the ability of the EGF-CFC proteins to co-activate, in some cases, an additional and parallel intracellular signaling pathway unrelated to a canonical Smad pathway which might depend upon the downstream eVectors, ras=raf=MAPK and=or PI3K=AKT. This could occur through the upstream binding of Cripto-1 to and activation of Glypican-1=Src and=or to the generation of a complex involving Tomoregulin-1, Glypican-1, erbB-4, and Cripto-1. For example, Cripto-1 and=or cryptic can bind to either Nodal or GDF1 during gastrulation and in L=R asymmetry orientation. These interactions can activate a canonical Smad2=-3 and Smad-4 pathway involving FoxH1. Alternatively, Cripto-1 can also bind to Tomoregulin-1, a peptide that specifically binds to the type 1 erbB-4 tyrosine kinase receptor with low aYnity (Uchida et al., 1999). Tomoregulin1 also functions as a Nodal inhibitor by sequestering Cripto-1. Since human CR-1 has previously been demonstrated to indirectly activate in trans erbB-4, this may occur, in part, through its ability to directly bind Tomoregulin-1 and=or indirectly through Glypican-1 by activation of c-Src in lipid rafts. Therefore, the pattern and stoichiometry of expression of these diVerent proteins (i.e., Nodal, GDF1, Cripto-1, Tomoregulin-1, and Glypican-1) may be important with respect to which signaling pathway is activated in a particular cellular context. These examples serve merely to illustrate the potential complexity by which the EGF-CFC proteins can function as either co-receptors or ligands for one or several distinct groups of structurally unrelated proteins and receptor signaling pathways. Irrespective of the molecular mechanism by which EGF-CFC proteins might function, the diversity of biological eVects that can be induced by these proteins is large and similar to the pleiotropic spectrum of responses induced by other growth factor families. One eVect that is induced by EGF-CFC proteins and that is particularly significant is the ability of these proteins to induce cell motility through a Nodal-independent pathway. This occurs in embryonic epiblast cells of the primitive streak, in mouse mammary epithelial cells, and in human carcinoma cells (Ebert et al., 2000; Strizzi et al., 2004; Warga and Kane, 2003; Wechselberger et al., 2001; Xu et al., 1999). In all cases, this is preceded by an EMT since cells assume a more mesenchymal phenotype. N-cadherin and vimentin expression increase during this conversion (Ebert et al., 2000; Strizzi et al., 2004; Wechselberger et al., 2001). In contrast, there is a loss or reduction in E-cadherin expression. This may be

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particularly relevant to facilitating changes in the distribution of
-catenin that normally is bound to cell membrane-associated E-cadherin in the focal adhesion junctions. Redistribution of soluble
-catenin to the cytoplasm could then potentially facilitate interaction with Lef-1=tcf-4 transcription factors and lead to the activation of a canonical wnt=
-catenin=Lef-1 signaling pathway. Wnt target genes, such as the protease matrilysin, are necessary for morphogenic patterning which occurs during branching morphogenesis of mammary epithelial cells in vitro and in vivo and for the increased migratory and invasive behavior of tumor cells that are overexpressing Cripto-1 (Crawford et al., 1999). Likewise, Cripto-1 and Nodal (xnr3) are wnt target genes, suggesting an autoregulatory loop. It is more than likely that other growth factors such as Nodal, Activin, TGF
1, BMPs, Wnts, and=or FGFs may accentuate or inhibit the diVerent biological responses to diVerent members of the EGF-CFC family. Identification of factors activating transcription factors that can bind to the promoter regions of the EGF-CFC genes will therefore be important in determining the mechanism by which the expression of these genes is upregulated in tumor cells. Reciprocally, identification of downstream genes that might be regulated by EGF-CFC proteins will be useful in ascertaining the mechanism by which EGF-CFC proteins function. In summary, we hope that this somewhat extensive treatise reviewing the biology of the EGF-CFC family members and their role in embryonic and mammary gland development, their biological eVects on mammalian cells, their interactions with other genes and growth factors, and their overexpression in a number of diVerent types of human tumors has convinced the reader of their enormous functional plasticity and their potential to serve as a therapeutic target in cancer. More importantly, it was our goal to use the EGF-CFC family as an example of a family of embryonic genes that can re-express themselves, possibly in stem cells in adult tissues in an inappropriate fashion, and thereby contribute to the pathogenesis of cancer. In this regard, mouse and human cripto-1 and Nodal are expressed in undiVerentiated mouse and human embryonic stem (ES) cells (Calhoun et al., 2004; Ramalho-Santos et al., 2002; Sato et al., 2003; Zeng et al., 2004). Cripto-1 and Nodal are two genes that maintain the pluripotential capacity of ES cells and are therefore considered as stem cell markers (Vallier, 2004). Clearly, other examples, such as members of the Notch, Wnt, and Hedgehog families, substantiate this connection between stem cells and cancer (NickoloV et al., 2003; Polakis, 2000; Thayer et al., 2003).

Acknowledgments This work was supported in part by a grant from the Associazione Italiana per la Ricerca sul Cancro to Nicola Normanno.

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Programmed Cell Death in Plant Embryogenesis Peter V. Bozhkov,* Lada H. Filonova,y and Maria F. Suarez *,z


Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden y Department of Wood Science, Swedish University of Agricultural Sciences SE-750 07 Uppsala, Sweden z Departamento de Biologia Molecular y Bioquimica, Facultad de Ciencias Universidad de Malaga, Campus de Teatinos, E-29071 Malaga, Spain

I. Introduction II. Model Embryonic Systems A. Zygotic Embryogenesis of Arabidopsis thaliana B. Somatic Embryogenesis of Picea abies C. Monozygotic Polyembryony of Pinus sylvestris III. Mechanics of Cell Death A. Role of Autophagy B. Cytoskeletal Changes C. Nuclear Changes D. The Whole Pathway of Cell Dismantling IV. Molecular Executioners A. Plants and Core Cell-Death Machinery B. Emerging Roles of Caspase-Like Proteins in Plants C. Death Function of Plant Orthologues to Yeast Autophagy Proteins V. Concluding Remarks Acknowledgments References

Successful embryonic development in plants, as in animals, requires a strict coordination of cell proliferation, cell diVerentiation, and cell-death programs. The role of cell death is especially critical for the establishment of polarity at early stages of plant embryogenesis, when the diVerentiation of the temporary structure, the suspensor, is followed by its programmed elimination. Here, we review the emerging knowledge of this and other functions of programmed cell death during plant embryogenesis, as revealed by developmental analyses of Arabidopsis embryo-specific mutants and gymnosperm (spruce and pine) model embryonic systems. Cell biological studies in these model systems have helped to identify and order the cellular processes occurring during self-destruction of the embryonic cells. While Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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metazoan embryos can recruit both apoptotic and autophagic cell deaths, the ultimate choice depending on the developmental task and conditions, plant embryos use autophagic cell disassembly as a single universal cell-death pathway. Dysregulation of this pathway leads to aberrant or arrested embryo development. We address the role of distinct cellular components in the execution of the autophagic cell death, and outline an overall mechanistic view of how cells are eliminated during plant embryonic pattern formation. Finally, we discuss the possible roles of some of the candidate plant cell-death proteins in the regulation of developmental cell death. C 2005, Elsevier Inc. Death of cells is the usual accompaniment of embryonic growth and diVerentiation. John W. Saunders Jr., ‘‘Death in Embryonic Systems’’ (1966)

I. Introduction The idea that cell death, like cell proliferation and diVerentiation, as an integral and necessary part of animal development occurred to anatomists in the mid-nineteenth century (for review, see Clarke and Clarke, 1996). It required a hundred years more for similar ideas to take root in plant biology (discussed by Jones, 2001). As a consequence of such relatively late acceptance, the processes of physiological or programmed cell death (PCD) are less well understood in plants than in animals. In the life cycles of both animals and plants, the earliest manifestations of PCD are seen already during embryogenesis, when some cells or even entire tissues and organs have to be sacrificed for the sake of correct embryonic pattern formation. Before discussing the emerging knowledge about PCD in plant embryogenesis, we should like to dwell on the seminal role of studies on embryonic PCD in animals for discovering the conservative core cell-death machinery of apoptosis (one of the two major types of PCD in animals, together with autophagic cell death; Baehrecke, 2002). Gl€ ucksmann (1951) classified embryonic cell death into three categories, based on their presumed function in development. Morphogenetic cell deaths are those participating in the generation of form, for example, formation of interdigital spaces in limbs. Histogenetic deaths play a role in the diVerentiation of organs and tissues, for example, during remodeling of neural system and bone. Finally, phylogenetic deaths function to eliminate transient or vestigial structures, such as the tail in human embryos. ‘‘The death clock is ticking.’’ This is how embryologist John Saunders, Jr., expressed his inspired guesswork of a tight intrinsic regulation of embryonic

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cell death (Saunders, 1966). He speculated that cell death is regulated at genetic, hormonal, and tissue-environmental levels. A few years later, in 1972, John Kerr and colleagues coined the term ‘‘apoptosis’’ (Kerr et al., 1972). Of note, they admitted that ‘‘the implications of the process in tissue kinetics have not hitherto been appreciated, except by embryologists,’’ apparently ‘‘because of its frequently massive dimensions in the embryo’’ (Kerr et al., 1972). A breakthrough came with the application of modern molecular genetic techniques to the question of how developmental cell death is actually controlled. In 1976, John Sulston and H. Robert Horvitz established an ideal model system for studying genetic regulation of cell death. They demonstrated that 13% of embryonic cells in the nematode Caenorhabditis elegans die predictably, shortly after appearing (Sulston and Horvitz, 1977). Genetic screens for C. elegans mutants defective in developmental cell death led to the isolation of genes ced-3, ced-4, and ced-9 (Ellis and Horvitz, 1986), whose products have since been shown to be homologues to the mammalian caspase family, Apaf-1, and Bcl-2 family, respectively, and collectively constitute what is known to date as a ‘‘core cell death machinery’’ (Horvitz, 2003). Developing embryos require normal functioning of this machinery for correct tissue and organ patterning. Transgenic and gene-ablated mice with defects in PCD either die early in development or display a range of physiological and=or anatomical aberrations. The lethal phenotypes have been obtained upon knockout of caspase-3 (brain overgrowth), caspase-8 (heart failure), caspase-9 (brain overgrowth), and Apaf-1 (delayed limb shaping and brain overgrowth) (Ranger et al., 2001; Zheng and Flavell, 2000). Embryogenesis programs of animals and plants are quite diVerent. In viviparous animals such as humans and other mammals, the embryos mimic the basic architecture of the adult organism, as they undergo active morphogenesis to establish all the essential organs in their final shape. In other animals, including amphibia and most invertebrates, embryogenesis results in a first phenotype, the larva, followed by a dramatic remodeling (metamorphosis) to the adult phenotype. Plant embryogenesis pursues a simpler developmental task, as compared to animal embryogenesis. This is related to the unique ability of plant cells for flexible and reversible diVerentiation as well as for totipotency, which together determine the unique capacity for continuous growth of plants (Walbot and Evans, 2003). Accordingly, a mature plant embryo shows relatively simple developmental organization, represented by three concentrically arranged tissue types – protoderm, ground tissue, and provascular tissue – along the radial axis and major organs – shoot apical meristem, cotyledons, hypocotyl, root meristem, and embryonic root – along the

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apical–basal axis (for review, see Goldberg et al., 1994; Laux et al., 2004; Mordhorst et al., 1997). Most morphogenetic processes in higher plants occur during post-embryonic development when the entire sporophyte is continuously produced by the two apical meristems that are formed initially during embryogenesis (Goldberg et al., 1994). As a consequence of such a simplified embryonic pattern formation in plants, PCD does not seem to serve as many functions in plant embryogenesis as it does during animal embryogenesis. However, there are at least three roles of PCD in plant embryogenesis which have fundamental significance for normal embryo development and plant propagation. First is elimination of the embryo suspensor—the earliest terminally diVerentiated structure formed in a plant’s life. The suspensor functions at early stages of embryogenesis by holding the growing embryo in a fixed position within the seed, providing a route for nutrient transport to the embryo (reviewed by Yeung and Meinke, 1993) and participating in the auxin gradient-mediated establishment of the apical–basal axis (Friml et al., 2003). The suspensor is not required during later stages of embryogenesis and is therefore eliminated by PCD (Filonova et al., 2000a,b; Schwartz et al., 1997). The second role of PCD is removal of entire embryos from seed, which happens, for instance, during monozygotic polyembryony. This type of embryogenesis occurs in many plant species and implicates growth competition among multiple embryos developed from the same zygote, usually resulting in survival of one dominant embryo and death of the remaining subordinate embryos in a seed (Filonova et al., 2002). The third role of PCD applies to the establishment of the vascular system at late stages of embryo development. Although the processes of vascular diVerentiation in plant embryos are poorly understood (Fukuda, 1996), anatomical studies reveal the presence of tracheary elements of metaxylem in mature embryos (Esau, 1965; Taylor and Vasil, 1995), suggesting that cell death-associated xylogenesis is an early process in plant development. Although tracheary element cell death is apparently the best characterized form of developmental PCD in plants, it is not an embryogenesis-specific process, and we do not discuss it here but rather refer the reader to detailed reviews on this subject (Fukuda, 1996, 2000, 2004). This chapter is the first attempt to summarize what is known today about PCD in plant embryogenesis. We first describe the classic (Arabidopsis embryogenesis) and novel (gymnosperm embryogenesis) model systems for studying embryonic PCD. Then we explain how dismantling of cellular contents proceeds during this type of PCD. And, finally, we address the question of how this PCD can be regulated. Given a nascent field of molecular regulation of plant embryonic PCD, we refer to better understood cell-death systems and speculate a little when appropriate.

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II. Model Embryonic Systems A. Zygotic Embryogenesis of Arabidopsis thaliana Embryo development in Arabidopsis follows the Onagrad embryonic type (Mansfield and Briarty, 1991; Yakovlev and Alimova, 1976). The zygote divides asymmetrically into a large basal cell and a small apical cell, which subsequently establish the two cell lineages with fundamentally diVerent growth patterns and developmental fates. The apical cell is an ancestor of almost the entire embryo proper (except for the hypophysis cell and its descendants), which develops through a stereotyped sequence of stages to the mature embryo (Fig. 1). The developmental fate of the basal cell is strikingly diVerent, as it gives rise to a single file of seven to nine cells through a series of transversal divisions. The uppermost cell of the basal lineage, the hypophysis, is incorporated into the embryo proper, while the remaining six to eight cells form the suspensor. DiVerentiation of the suspensor is complete at the globular stage (Fig. 1). It begins to be eliminated at the heart stage and therefore is absent from the mature seed (Mansfield and Briarty, 1991). Being composed of a single file of just a few cells, the Arabidopsis suspensor represents an attractive system for studying cell biological and molecular mechanisms of developmental PCD in plants. However, very little is known to date even about how the PCD of Arabidopsis suspensor proceeds at the cytological level. This PCD is believed to first aVect the basal cell of the suspensor and to then progress acropetally toward the cells of the apical lineage (Marsden and Meinke, 1985); however, the cell dismantling pathway per se remains to be characterized. The remarkable progress in the isolation of Arabidopsis embryo-defective (emb) mutants (Tzafrir et al., 2003, 2004; http:==www.seedgenes.org) provides a solid background for studying molecular regulation of cell diVerentiation and PCD in the suspensor using forward and reversed genetic approaches. The 237 tagged emb mutants with sequenced T-DNA insertions at single chromosomal locations identified to date represent disruption of 175 individual EMB genes (http:==www.seedgenes.org). There are postulated to be 500 to 750 genes encoding nonredundant functions during embryogenesis (McElver et al., 2001; Tzafrir et al., 2003, 2004). Experimental mutagenesis projects yielded at least 15 emb mutants with abnormal development of the suspensor, which can be a direct or indirect consequence of arrested or attenuated cell death in this temporary structure (Table I). The common feature of all these mutants is that deregulation of diVerentiation and=or PCD in the suspensor precedes or coincides with the globular stage of embryo proper development (Fig. 1). Another

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Figure 1 Developmental pathways of embryogenesis in wild-type and in suspensor mutants of Arabidopsis. The presence of two nuclei in one cell at the 4-cell proembryo stage indicates that one cell lay above and one beneath the drawing plane. a, apical cell; b, basal cell; ep, embryo proper; s, suspensor. Not drawn to scale.

commonality among suspensor mutants is that the visible changes in the suspensor pattern are usually preceded by morphological aberrations in the embryo proper (Schwartz et al., 1997). For 10 out of 15 suspensor mutants, the genes whose expression was aVected by T-DNA are known (Table I). The earliest and most severe developmental defects of suspensor development have been observed in yda embryos carrying loss-of-function mutation in YODA, a gene-encoding mitogen-activated protein kinase kinase kinase

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(MAPKK kinase; Lukowitz et al., 2004; Table I; Fig. 1). This mutation suppresses elongation of the zygote and alters the pattern of zygotic division in such a way that the two derivative daughter cells have approximately equal size. The loss of geometrical asymmetry at the first zygotic division further leads to the loss of basal cell identity and to the disruption of the apical–basal pattern formation, as the descendants of the basal cell are incorporated into the embryo proper instead of the diVerentiating suspensor (Lukowitz et al., 2004; Fig. 1). At later stages of embryogenesis, yda mutants exhibit a wide spectrum of developmental aberrations, including impaired root development. The plants regenerated from yda embryos are dwarfed, with sterile flowers. The discovery and characterization of the yda mutant is probably the best demonstration of the vital role of the suspensor in embryonic and post-embryonic plant development. The next fundamental question that can be addressed using the yda mutant is, ‘‘By which mechanism does the disruption of the YODA-dependent MAP kinase cascade prevent asymmetric division of the zygote and alter specification of the basal cellular lineage that is normally destined for terminal diVerentiation and PCD?’’ There is a growing body of evidence suggesting involvement of the MAP kinase cascade in the induction of the hypersensitive response-related cell death caused by pathogens (del Pozo et al., 2004; Petersen et al., 2000; Yang et al., 2001), but nothing is known about the role and place of MAP kinases in the cell-death program operating during plant development. The presence of growing vacuoles is ubiquitous in the suspensor cells (De, 2000). Vacuolation of the suspensor cells is thought to be required for the eYcient nutrient transport through the suspensor (Schwartz et al., 1997), and was also shown to be a part of the suspensor death program (Filonova et al., 2000a; Nagl, 1976, 1977). Interestingly, an emb mutant of Arabidopsis lacking vacuoles in the suspensor has been isolated. It was named accordingly to the phenotype as vacuoleless1 (vcl1; Rojo et al., 2001; Table I; Fig. 1). The mutation was caused by the inactivation of the gene encoding an orthologue of the yeast vacuolar protein sorting 16p protein (Vps16p) involved in the regulation of fusion of vacuoles and docking of vesicles at the tonoplast. The earliest penetrance signs of the mutation in VCL1 were visible as early as immediately after the first division of the apical cell, and included the lack of vacuoles in the suspensor, transversal instead of longitudinal divisions of the apical cell, and suppressed elongation of the suspensor cells (Rojo et al., 2001; Fig. 1). These defects at early stages of embryogenesis led to severe perturbations at later stages, including altered planes of cell divisions in both the suspensor and embryo proper. vcl1 embryos died early in development through autophagocytosis in the embryo proper cells (Rojo et al., 2001). This mutant shows that vacuolation of the cytoplasm in embryo suspensor cells is critical for their normal functioning in the transport of nutrients and growth regulators to and=or from the

Table I

Overview of Arabidopsis emb Mutants with Abnormal Development of Suspensor Embryonic phenotype Embryo propera

Mutant

Mutagen

50B

EMSb

Preglobular; degeneration

sus1

T-DNA, X-Ray

sus2

Suspensor

Plant regeneration

Predicted function of gene product

Reference

Embryo-lethal

Unknown

Marsden and Meinke, 1985

Globular; aberrant; degeneration

Increased cell proliferation (15–150 cells) Increased cell proliferation

Embryo-lethal

DICER-LIKE1

T-DNA, EMSb

Globular; aberrant; degeneration

Increased cell proliferation

Abnormal plantlets in vitro

PRP8-like splicing factor

sus3

T-DNA T-DNA

Increased cell proliferation Forms viable secondary embryos

Abnormal plantlets in vitro Twin plants, slow growth

Unknown

twn1

Unknown

Vernon and Meinke, 1994; Vernon et al., 2001

twn2

T-DNA

twn3

T-DNA

Globular; aberrant; degeneration Cotyledonary; defects in cotyledon pattern and morphology Preglobular; degeneration NDc

Golden et al., 2002; Schwartz et al., 1994; http:==www.seedgenes.org Meinke, 1995; Schwartz et al., 1994; http:==www.seedgenes.org Schwartz et al., 1994

Forms viable secondary embryos Forms secondary embryos

Twin plants, slow growth NDc

Valyl-tRNA synthetase Ribosomal protein S9

Zhang and Somerville, 1997; http:==www.seedgenes.org http:==www.seedgenes.org

raspberry1 T-DNA raspberry2 T-DNA raspberry3 T-DNA

Globular; aberrant; degeneration Globular; aberrant; degeneration Globular; aberrant; degeneration

Increased cell proliferation Increased cell proliferation Increased cell proliferation

Embryo-lethal

Unknown

Yadegari et al., 1994

Embryo-lethal

Unknown

Yadegari et al., 1994

Embryo-lethal

Apuya et al., 2002; http:==www.seedgenes.org

Irregular cell division; lack of vacuoles Absent

Embryo-lethal

Novel protein important for chloroplast diVerentiation VCL1 (vacuole biogenesis and protein traYcking) MAPKK kinase

vcl1

T-DNA

Preglobular; degeneration

yda

T-DNA

dbr1

T-DNA

emb1789

T-DNA

Cotyledonary; aberrant morphogenesis Globular to cotyledonary; aberrant morphogenesis Globular

emb1401

T-DNA

Cotyledonary

a

Lukowitz et al., 2004; http:==www.seedgenes.org

Increased cell proliferation

Embryo-lethal

Lariat debranching enzyme

Wang et al., 2004; http:==www.seedgenes.org

Increased cell proliferation Embryogenic transformation

NDc

Zinc finger family protein Translation initiation factor

http:==www.seedgenes.org

Terminal phenotype of a primary embryo proper at seed maturity. Ethyl methanesylfonate. c Not determined. b

Dwarfed plants

Rojo et al., 2001; http:==www.seedgenes.org

NDc

http:==www.seedgenes.org

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embryo proper. Also, a lack of the vacuoles in the vcl1 suspensors may indicate that the suspensor cells have not acquired the diVerentiation state that is required for normal development of the embryo proper. All other suspensor mutants of Arabidopsis exhibit varied degrees of embryogenic ‘‘transformation’’ of suspensor when the suspensor cells acquire the potential to proliferate and give rise to one or more viable secondary embryos or developmentally arrested embryo-like structures. Partial transformation of suspensor without forming viable secondary embryos is exemplified by 50B, sus, raspberry, dbr1, and emb1789 mutants (Table I; Fig. 1; http:==www.seedgenes.org). There are both morphological and anatomical aberrations in the primary embryo proper of these mutants observed at preglobular to globular stages. These aberrations usually precede visible changes in the organization of the suspensor and account for the further degeneration of the primary embryo proper. As a result, all the emb mutants with partial transformation of suspensor are lethal (Table I; Fig. 1). Tween (twn) mutants are unique in the sense that they are capable of complete transformation of suspensor into relatively normal secondary embryos (Table I; Fig. 1; http:==www.seedgenes.org). In the twn1 mutant, secondary embryos start to develop within the suspensor when the primary embryo proper has attained globular stage (Vernon and Meinke, 1994). Even though some aberrations in the pattern formation of the primary twn1 embryos have been observed, they were much less severe than those of the mutants with partial transformation of suspensor, thus allowing complete development of the primary embryo (Vernon and Meinke, 1994; Fig. 1). Approximately 9% of twn1 seeds gave rise to multiple seedlings, the frequency being 400 to 500 times that of spontaneous polyembryony in wild-type Arabidopsis (Akhundova et al., 1979; Vernon and Meinke, 1994). The only, but significant, developmental diVerence between twn1 and twn2 mutants is that the primary embryos in twn2 seeds degrade at the preglobular stage, so that twin plantlets develop exclusively from the secondary, suspensor-derived embryos (Zhang and Somerville, 1997; Fig. 1). Both twn1 and twn2 plants exhibit slow growth when compared to wild-type plants. Molecular mechanisms underlying the embryogenic transformation of the suspensor in the previously described mutants are still unknown. In all the mutants where the target genes have been identified, the encoded proteins serve basic cellular functions, mainly transcriptional and translational controls (Table I). From the developmental viewpoint, the ability of suspensor cells to rediVerentiate into embryogenic cells at early stages of embryogenesis, up to the globular stage but not later, suggests that it is at the globular stage when suspensor cells become terminally diVerentiated and committed to death. Which of the two cell lineages of the embryo yields the signal triggering PCD in the suspensor? Based on the developmental analysis of the suspensor transformation mutants, two scenarios are equally plausible.

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The first assumes that the cell-death signal is produced by the normally developing embryo proper and moves basipetally toward the cells of the basal lineage. Only diVerentiated suspensor cells can perceive this signal but not the cells in the apical lineage. Accordingly, defects in the embryo proper development can either abolish or weaken the production of this signal or impair its transduction pathway, leading to the altered fate of the suspensor cells. In the alternative scenario, cell death is triggered by the depletion of a specific anti-death factor in the basal cell lineage, which moves acropetally and is required for normal development of the embryo proper. The developmental pathway of suspensor transformation mutants agrees with this second scenario as well; that is, disruption of the acropetal transport of this hypothetical anti-death factor and its accumulation in the basal cell lineage would suppress PCD in the suspensor and disturb the patterning in the apical cell lineage. Further analysis of suspensor mutants of Arabidopsis can provide important clues for understanding the cell biological and molecular regulation of the normal developmental PCD in the suspensor as well as of the pathological cell death that is activated in the apical cell lineages of embryo-lethal mutants.

B. Somatic Embryogenesis of Picea abies Picea abies (Norway spruce) is a member of the family Pinaceae, which, in turn, belongs to Coniferales, the largest order of modern gymnosperms. Many conifer genera, including Picea, are of great economic importance as sources of lumber, paper pulp, and chemical compounds. Picea abies can be clonally propagated through somatic embryogenesis—the process of asexual embryo development from somatic cells cultured in vitro (reviewed in von Arnold et al., 2002). Somatic embryogenesis in P. abies has been thoroughly studied at physiological and developmental levels, so that the whole process can now be synchronized and eVectively controlled by a series of specific treatments, with each treatment designed to stimulate transition through the particular developmental stage (Bozhkov et al., 2002). These advances, together with the considerable resemblance of somatic embryo development to zygotic embryogenesis (Filonova et al., 2000b) have made somatic embryogenesis of P. abies a versatile model system for studying the cell and molecular biology of conifer embryo development. In particular, this biological system was used for the first systematic studies of the embryonic PCD in plants (Bozhkov et al., 2002, 2004; Filonova et al., 2000a; Smertenko et al., 2003; Suarez et al., 2004). We shall first explain the process of Picea zygotic embryogensis, which has a number of fundamental diVerences compared to that in Arabidopsis, and then present how this process can be modeled and regulated in vitro using somatic embryos.

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Figure 2 Embryonic pattern formation in Picea abies including 16-cell proembryo, early embryo, and embryo at the end of late embryogeny (from left to right). There is a gradient of autophagic PCD through successive layers of suspensor cells, along the apical–basal axis of an early embryo. The absence of nuclei in the basal layer of suspensor cells (Es1) illustrates complete removal of the protoplasts by the PCD. The presence of two nuclei in one cell at the 16-cell proembryo stage indicates that one cell lay above and one beneath the drawing plane. E, embryonal group; dS, dysfunctional suspensor; U, upper tier; EM, embryonal mass; et, embryonal tubes; Es1–Es4, four layers of embryonal suspensor (according to Singh, 1978). Not drawn to scale.

Zygotic embryo development in Picea, just as in all gymnosperms, can be divided into three major phases: proembryogeny, early embryogeny, and late embryogeny (Singh, 1978). Proembryogeny includes all the stages before the elongation of the suspensor. A distinctive feature of gymnosperm proembryo development is a free-nuclear stage, that is, the absence of cell walls between the nuclei throughout two or more initial divisions. In Picea, the number of free nuclei at wall formation is eight. A fully formed proembryo of Picea has four tiers of four cells each (Fig. 2). The first two cell tiers situated toward the chalasal pole constitute the embryonal group of the proembryo. In Pinaceae, the four cells of the lowest tier within the

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embryonal group are actually the ancestors of all the cells in the developing embryo (Fig. 2). The third tier is dysfunctional suspensor, which can, in some coniferous species (e.g., Pinus), undergo a series of irregular cell divisions, giving rise to the unorganized cell masses called ‘‘rosette embryos.’’ The cells of the fourth, upper tier have no cell walls toward the micropylar end (Fig. 2). In Picea, neither dysfunctional suspensor nor upper-tier cells divide (Runions and Owens, 1999), suggesting that they are the first cells committed to death during embryogenesis. Early embryogeny begins with the elongation of cells from the second tier of the embryonal group to form the first crop of suspensor (Singh, 1978). The other three crops of suspensor cells are then added sequentially via three rounds of synchronized asymmetric transversal divisions of embryonal group cells. These asymmetric cell divisions contribute to the growth of the embryonal group too, which results in the formation of the embryonal mass—a functional homologue of the embryo proper of angiosperms (cf. Figs. 1 and 2). The proximal (i.e., adjacent to suspensor) layer of embryonal mass cells continues to divide asymmetrically, with one half of the daughter cells elongating and acquiring suspensor cell identity and the other half retaining embryonal mass cell identity (Fig. 2). To emphasize the origin from the embryonal mass of new crops of suspensor cells formed during early embryogeny, these cells were named ‘‘embryonal tube cells’’ (Singh, 1978). The tube cells do not normally divide but rather enter the PCD pathway as soon as they are formed. Therefore, tube cells represent the earliest stage of terminal diVerentiation and PCD in the suspensor. A typical Picea embryo in the beginning of early embryogeny possesses a strict apical– basal pattern and is composed of a compact embryonal mass at the apical pole, four layers of highly elongate suspensor cells extended toward the basal pole, and the intermediately situated layer of elongate tube cells (Fig. 2). Using somatic embryos at a similar developmental stage, we were able to demonstrate that there is a gradient of successive stages of autophagic PCD (discussed in Section III) along the apical-basal axis, from the embryonal tube cells committed to death to the cell corpses at the basal end of the suspensor (Smertenko et al., 2003). This gradient of PCD in the P. abies suspensor is maintained throughout the whole period of early embryogeny, as long as new layers of tube cells are added to the suspensor. A failure to diVerentiate new tube cells heralds the end of early embryogeny and the onset of late embryogeny, where the entire suspensor starts to be gradually eliminated, beginning with the oldest, most basally situated cells. The period of late embryogeny is hallmarked by active histogenesis and diVerentiation of the primary shoot and root meristems. At this time, the embryo can be arbitrarily divided into a micropylar part, where the root cap forms, and a chalasal part bearing the primary shoot meristem and involved in embryo elongation (Fig. 2; Singh, 1978). A mature zygotic embryo of

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Picea has several cotyledons, a primary root and shoot meristems, and distinct procambium, cortex, pith, and root cap (Fig. 2; Singh, 1978). A comparison of early embryo development in Picea versus Arabidopsis points to three fundamental distinctions between the two species in the biogenesis and PCD of suspensor: (1) In Arabidopsis, all suspensor cells are formed through the cell lineage originating from a single basal cell of the two-cell proembryo (Fig. 1), whereas in Picea they are continuously derived from recurring asymmetric divisions in the embryonal mass (Fig. 2). Consequently, the cell lineages establishing the embryonal mass and suspensor in Picea are not separated, as in the case of Arabidopsis. (2) Suspensor cells in the proembryos and preglobular embryos of wild-type Arabidopsis undergo a few rounds of transversal mitotic divisions to complete suspensor development (Fig. 1), whereas in Picea the suspensor cells (including tube cells) do not divide. (3) Therefore, Arabidopsis suspensor cells are committed to PCD only after suspensor growth is complete, as opposed to suspensor cells in Picea embryos, which initiate PCD as soon as they are formed via asymmetric cell divisions in the embryonal mass. Embryogenesis in planta takes place under multiple layers of tissues so that its direct observation is obscured. Furthermore, even within the same plant, diVerent ovules develop asynchronously, which hampers collecting embryos at a common developmental stage for subsequent analyses. To obviate these obstacles to studying cell and molecular mechanisms of conifer embryo development, we have developed a model system of somatic embryogenesis for P. abies. This system provides a unique opportunity to control the entire pathway of embryo development in laboratory conditions and to isolate large numbers of embryos at common developmental stages (reviewed by von Arnold et al., 2002). Somatic embryogenesis of P. abies includes a stereotyped sequence of developmental stages and corresponding regulatory treatments (Fig. 3). Somatic embryos develop not directly but from proembryogenic masses (PEMs), which pass through a series of three characteristic stages distinguished by cellular organization and cell number (PEM I, PEM II, and PEM III). Proembryogenic mass will not diVerentiate to somatic embryos unless a critical mass of cells (i.e., stage PEM III) has been formed. Plant growth regulators (PGR), auxin and cytokinin, stimulate proliferation of PEMs and, in contrast, suppress correct embryo pattern formation. Consistently, withdrawal of PGR triggers the PEM-to-embryo transition, providing a synchronous start of early embryogeny. Early embryo development is accomplished within approximately seven days in PGR-free medium. Subsequently, early embryos require exogenous abscisic acid to undergo late embryogeny and maturation (Fig. 3). The morphology and anatomy of somatic embryos are similar to those of zygotic embryos (Figs. 2 and 3), even though embryo origin is diVerent (i.e., somatic cells in PEMs versus zygote; Filonova et al., 2000b).

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Figure 3 Developmental pathway of somatic embryogenesis in Picea abies (adapted from Filonova et al., 2000a,b). Proliferation of PEMs is stimulated by PGR—auxin and cytokinin. An individual PEM should pass through a series of three characteristic stages (I, II, and III) to become competent for PEM-to-somatic embryo (SE) transition. Early embryogeny is induced by withdrawal of PGR, whereas late embryogeny and embryo maturation require abscisic acid. Shown in red are the living cells, which stain in situ red with acetocarmine. Blue color denotes dying or dead cells, which stain in situ blue with Evan’s blue (Filonova et al., 2000a). Autophagic PCD first kills PEM cells at the PEM-to-embryo transition and then aVects terminally diVerentiated suspensor cells during early embryogeny. The suspensors begin to be eliminated in the beginning of late embryogeny (shown by dashed blue lines). Not drawn to scale.

Autophagic PCD was shown to be critically involved in both the formation of somatic embryos from PEMs and the early embryo patterning (Filonova et al., 2000a; Smertenko et al., 2003). The first wave of PCD kills PEM cells at the PEM-to-embryo transition induced by withdrawal of PGR (Fig. 3). Consistently, artificial inhibition of the cell death in PEMs suppresses embryo formation, indicating that PCD in PEMs and normal embryo development are closely interlinked processes (Bozhkov et al., 2002). This idea has been strengthened by the experiments with the cell lines composed of PCD-deficient PEMs, which are unable to form embryos regardless of treatment (Smertenko et al., 2003; van Zyl et al., 2003). The second wave of PCD in this model system aVects terminally diVerentiated suspensors of early embryos. This PCD occurs in a basipetal gradient of increased degree of cell disassembly toward the basal end of the suspensor where the oldest cells (i.e., those formed first) are located (Smertenko et al., 2003). In animal systems, the gradients of PCD are often observed when morphogenesis of the organ or structure is followed by its programmed removal (reviewed by Sanders and Wride, 1995). Embryonic motoneurons are another example, where the cells are generated and then doomed following the same spatio-temporal order (Gould et al., 1999).

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Table II Pharmacological Manipulation of Autophagic Cell Death in Embryogenic Cell Cultures of Picea abiesa

Target process Ca2+ influx Mitochondrial transition pore opening Polymerization of F-actin Autophagocytosis Activation of VEIDase caspase-like activity

Agent (final conc. in
M)

EVect of agent

TUNEL positive nuclei, % of controlb

A23187 (100) LaCl3 (150) Betulinic acid (10) Cyclosporin A (10) Cytochalasin D (5) Latrunculin B (0.1) 3-Methyladenine (100) zVEID-fmkc (2)

Stimulation Inhibition Stimulation Inhibition Disruption Disruption Inhibition Inhibition

191 53 130 61 192 168 72 49

a The data are from Bozhkov et al., 2004; Smertenko et al., 2003; L. H. Filonova and P. V. Bozhkov, unpublished results. b More than 103 nuclei were observed in 40 -6-diamino-2-phenylindole=TUNEL stained samples for each treatment. Individual agents were added to the fresh PGR-free medium just prior to inoculation of cell aggregates, with the exception for the actin-depolymerizing drugs, which were added 24 h after inoculation. The samples were taken for analysis 96 h after the start of the treatments (120 h for the actin-depolymerizing drugs). The controls were untreated or mock-treated (0.1%, v=v DMSO) cultures. p < 0.01 versus control. c zVEID-fluoromethyl ketone.

The addition of each new layer of cells to the suspensor during early embryogeny takes approximately 24 h, as follows from the time-course analysis of zygotic embryogenesis (Ha˚ kansson, 1956) and time-lapse tracking of somatic embryo development (Filonova et al., 2000b; our unpublished data). Therefore, and given that there are always 5 cell layers in the suspensor, it should take several (at least 5) days for a suspensor cell to autodestruct its protoplast and to leave the hollow-walled cell corpse at the basal end of the suspensor (see Section III.D for the cell dismantling pathway). The slowness of cell death in the suspensor appears to be important for eVective transport of nutrients and growth factors to the developing embryo. As in zygotic embryos, the addition of new tube cells to the suspensors of somatic embryos ceases at the beginning of late embryogeny, followed by a gradual elimination of the whole suspensor (Fig. 3). One of the significant advantages of the P. abies somatic embryogenesis as a model system to study developmental PCD is that it enables the investigator to manipulate cell death by specific agents that either stimulate or inhibit distinct processes of the cell-death pathway (Table II). The eVects of such treatments on PCD and embryogenesis can be investigated using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) of nuclei with fragmented DNA (a robust

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marker of this PCD; Table II) or a diVerent biochemical or cytochemical marker of the downstream cell death process, and by direct microscopic observation of the defects in embryo patterning in response to dysregulated PCD (Bozhkov et al., 2002, 2004; Smertenko et al., 2003; Suarez et al., 2004). C. Monozygotic Polyembryony of Pinus sylvestris During evolution of multicellular eukaryotes, certain approaches to clonal propagation have been developed. Some organisms have embryogenesis programs involving physical splitting of cells, after the first few mitotic divisions, into separate embryos. In the metamorphosing animals, selfcloning can also occur at the larval stage. In both cases, oVspring are derived from the same zygote and therefore are genetically identical to each other, but distinct from their parent(s). This reproductive phenomenon is called ‘‘monozygotic polyembryony.’’ In animals, obligate monozygotic polyembryony has been documented on at least 18 occasions within bryozoans, hydrozoans, parasitoid wasps, flatworms, and armadillos (for review, see Craig et al., 1997). Accidental or ‘‘sporadic’’ polyembryony in the form of monozygotic twinning occurs at a very low frequency in humans and in some other mammals under natural conditions, but the process can be augmented in vitro by embryo splitting technique (Chan et al., 2000). In the latter case, the success of embryo rescue is largely dependent on overcoming high levels of cell death in split embryos (Chan et al., 2000). Genetic control of monozygotic polyembryony is unknown. Development of more than one embryo in a seed is a normal reproductive strategy of many plant species. Supernumerary embryos in plants can emerge from a variety of seed tissues, but can also originate from a single zygotic proembryo or early embryo (for review, see Lakshmanan and Ambegaokar, 1984; Tisserat et al., 1979). Monozygotic polyembryony is especially common among gymnosperms (Singh, 1978). A paradigm of monozygotic polyembryony is that only one embryo in a polyembryonic seed usually develops to maturity and gives rise to a viable plant, while all the other embryos are eliminated at early developmental stages (Dogra, 1967; Singh, 1978). The major mechanism responsible for eliminating all but one monozygotic embryo in a seed has been unraveled using natural polyembryony of Pinus sylvestris (Scots pine) as a model system (Fig. 4; Filonova et al., 2002; Zhivotovsky, 2002). In this system, multiple embryos arise from the same zygote, coexist in the same environment within the corrosion cavity surrounded by the female gametophyte (haploid maternal tissue, which has similar functions as the endosperm in angiosperm seeds), and develop

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Figure 4 The pathway of monozygotic polyembryony in a Pinus sylvestris seed leading to elimination of subordinate embryos. The entire period of polyembryonic development can be divided into three phases, in terms of embryo competition. During the first, brief phase, multiple embryos formed from a single zygote-derived early embryo (1 week after fertilization, waf ) have similar growth rates and thus have an equal opportunity for dominance (2 waf ). The onset of the second, longer phase, coincides with one embryo’s ‘‘gaining a victory’’ in the competition and becoming dominant (3 waf ). The growth of remaining, subordinate embryos is progressively reduced (4 waf ), until it stops completely when the dominant embryo has reached the cotyledonary stage (6 waf ). During the third phase, subordinate embryos are progressively eliminated, while the mature dominant embryo enters the dormancy period (from 6 waf onward). Scale bars: 1 waf, 50
m; 2-11 waf, 500
m. Reproduced with permission from Filonova et al. (2002), Nature Publishing Group.

in close proximity to each other until one embryo becomes dominant and able to complete development, while the remaining, subordinate embryos begin to be eliminated (Fig. 4). We have shown that subordinate embryos are eliminated by the wave of autophagic PCD (see Section III), with the first dying cells detected by TUNEL in the basal parts (adjacent to suspensor) of the embryonal masses at 6 weeks after fertilization. Interestingly, the spatial progression of PCD occurred in a strict basal-to-apical gradient, and it required approximately 4 weeks for the PCD to reach apical cells of the embryonal masses (Filonova et al., 2002). Embryonal masses of the subordinate embryos appeared to cease growth just before the onset of the PCD at their basal poles (Filonova et al., 2002), indicating that apical cells did not proliferate after that time. Why, in this case, were the cells lying closer to the apical region of the embryonal mass always scheduled to die later than the cells situated at the greater distance from the apical region? We are not ready to give a precise answer to this intriguing question yet, although one can assume that the basal-to-apical gradient of PCD in the subordinate embryos is controlled by the balance between a cell death-inducing signal (transmitted acropetally) and a survival signal (pre-accumulated in the apical region and then gradually depleted), specifically adjusted along the apical–basal axis of the embryos (see also Jacobson et al., 1997).

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Figure 5 Programmed cell death in the development of polyembryonic seed of Pinus sylvestris. Not drawn to scale. Reproduced with permission from Filonova et al. (2002), Nature Publishing Group.

These signals can be either produced locally or transported to the subordinate embryos from the female gametophyte, which undergoes massive PCD just before the earliest signs of cellular demise in the subordinate embryos (Filonova et al., 2002). Death of the female gametophyte may halt the supply of an unknown survival factor (e.g., abscisic acid, which is required for embryo development and also plays a death-protective role in plants; Young and Gallie, 2000) to the embryos, thus triggering cell death in the subordinate embryos. Unlike autophagically cleaned protoplasts in the subordinate embryos, the dying cells of the female gametophyte are not subjected to rapid destruction but rather persist as depositories of carbon (in lipid bodies) and nitrogen (in protein storage vacuoles) that will be used by the embryo upon germination (Filonova et al., 2002). A similar type of PCD operates in the endosperm of cereal seeds (Young and Gallie, 2000). The manifold functions of PCD in plant development are made possible through a high degree of plasticity of cell dismantling pathways, resulting, depending on the developmental task, in diVerent magnitudes of cell corpse autoprocessing (Beers and McDowell, 2001; Jones, 2000, 2001). The cooperative action of the two phenotypically distinct cell-death programs initiated in tandem in two diVerent structures in the developing P. sylvestris seed is schematically shown in Fig. 5. During early development, a zygotic pine embryo (stage I) cleaves to form multiple embryos of the same genotype, starting to compete for limited amounts of growth factors (stage II). Each embryo is composed of living embryonal mass and terminally diVerentiated suspensor undergoing PCD (stages II–IV). Embryo competition requires a larger room to accommodate the rapidly growing dominant embryo, thus triggering cell death in the region of the female gametophyte lying on the path of its growth (stage III). This locally induced cell death rapidly progresses throughout the whole female gametophyte (stage IV). Dead cells of the female gametophyte are not removed but rather persist as depositories of nutrients that will be required for embryo germination. Once the female gametophyte is dead, another cell-death program is

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activated in a seed that eliminates subordinate embryos through complete destruction of cell protoplasts. First aVected are the most basally situated cells of embryonal masses (stage V). The autophagic cell death then spreads toward the apical regions until all the cells of subordinate embryos are autodestructed (stages VI and VII). Orchestrated action of these two celldeath programs creates a viable seed containing a single living embryo and nutrient reserves packed within preserved cell corpses in the female gametophyte (stage VIII). Polyembryonic seeds of P. sylvestris represent an attractive model system in which to address the plethora of questions related to the regulation of PCD during normal plant development.

III. Mechanics of Cell Death A. Role of Autophagy The process of eukaryotic PCD can be divided into three general stages: induction, execution, and degradation of cellular content. Execution and degradation can be pursued either by the same dying cell or by two diVerent cells, when a phagocytic cell degrades the dying cell. The latter scenario is characteristic for apoptosis, which is activated in isolated cells and involves caspase-mediated breakdown of the cytoskeleton, membrane blebbing, strong condensation of the chromatin, ordered DNA fragmentation, and formation of membrane-enclosed vesicles (apoptotic bodies), which are engulfed by phagocytes and degraded by phygocytic lysosomes (Baehrecke, 2002; Kerr et al., 1972). Apoptotic death occurs exclusively in metazoan animals, including mammals, and only in a case when isolated cells or small groups of cells are to be eliminated. If in the developing animal the removal of a large group of cells or an entire tissue or organ is needed, then the alternative PCD type, known as autophagic cell death, is activated (Baehrecke, 2002; Clarke, 1990; Golstein et al., 2003). The growing body of experimental evidence suggests that autophagic PCD also is a bona fide type of cell death operating during development of phylogenetically older organisms including plants, Dictyostelium, and fungi (Beers and McDowell, 2001; Golstein et al., 2003; Jones, 2000, 2001; Zhivotovsky, 2002). Apoptosis does not occur in these species because of the presence of cell walls and lack of specialized phagocytic cells and is therefore replaced by autophagic cell death, whereupon the execution and degradation phases are united within the same dying cell. Degradation of cellular content during autophagic cell death is just one example of the multifunctional phenomenon of autophagy, which is also involved in biosynthesis (the Cvt pathway), prevention of disease and aging (e.g., selective removal of damaged organelles), and regulation of the

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metabolism through the elimination of specific enzymes (for reviews, see Klionsky, 2004). Unlike the thoroughly studied apoptotic cell-death machinery, the molecular regulation of autophagic cell death is poorly understood. In particular, the central question of which molecular switch regulates the cellular choice between ‘‘metabolic or selective autophagy’’ and ‘‘bulk autophagy’’ degrading the entire cell content (except for the cell walls) remains to be answered. Cytological events during autophagic cell death resemble those occurring during macroautophagy in yeasts and mammals, the only diVerence being the magnitude of cellular degradation. In both processes, the portions of cytoplasm are sequestered within double-membrane vesicles known as autophagic provacuoles, vacuoles, or autophagosomes. For brevity, we use the term ‘‘autophagosomes.’’ The latter dock and fuse with the lysosome (in animals) or lytic vacuole (in fungi and plants) where the cargo breakdown is complete (Klionsky and Emr, 2000). Very little is known about the biogenesis of lytic vacuoles in plant cells. In the diVerentiated cells that function as nutrient reservoirs (e.g., in the endosperm=female gametophyte in angiosperms=gymnosperms or in the cotyledons), the conversion of protein storage vacuoles into lytic vacuoles may take place when a rapid mobilization of nutrients is required. Ultrastructural analysis of Vigna mungo cotyledon cells during seed germination has shown that protein storage vacuoles are converted into lytic vacuoles through hydrolysis of storage proteins by a papain-type proteinase transported to protein storage vacuoles by endoplasmic reticulum-derived vesicles. Newly formed lytic vacuoles then degrade starch granules through typical autophagic sequestration (Toyooka et al., 2000). In addition to the lytic vacuole-mediated autophagy, autophagosome-mediated degradation of cellular components was detected, indicating at least two distinct autophagic processes in the dying cells of V. mungo cotyledons (Toyooka et al., 2001). At early stages of plant embryogenesis, meristematic cells of the embryo proper=embryonal mass contain neither protein storage vacuoles nor lytic vacuoles. Ultrastructural analysis of the P. abies embryo suspensor revealed that lytic vacuoles are formed de novo via the fusion and growth of numerous autophagosomes (Filonova et al., 2000a). By the time the central lytic vacuole has formed, most cellular components are already autophagocytosed. Therefore, formation of a central lytic vacuole is a late PCD event in this developmental system, followed only by a rupture of the tonoplast, which completes protoplast degradation and leaves a hollow-walled cell corpse at the basal end of the suspensor (Filonova et al., 2000a; Smertenko et al., 2003). This diVers markedly from the kinetics of tracheary element cell death, where tonoplast rupture is a major force of cellular disassembly (Obara et al., 2001).

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Figure 6 Three alternative ways of autophagosome formation in the embryo suspensor: from (1) Golgi (G), (2) proplastids (PP), or (3) endoplasmic reticulum (ER). Degradation of the cellular content inside autophagosomes contributes to the growth of lytic vacuoles (LV). The scheme was drawn based on studies of Nagl (1977), Ga¨ rtner and Nagl (1980), Filonova et al. (2000a), and Wredle (2004).

The origin of autophagosomes per se during suspensor PCD can occur via at least three alternative pathways (Fig. 6). There are two pathways simultaneously activated in embryonal tube cells and cooperating during autophagic cell disassembly in P. abies embryo suspensor. The first pathway begins with the multiplication of large (diameter 90–130 nm) vesicles budding oV the exterior regions of the Golgi cisternae (Fig. 6; Filonova et al., 2000a). The second pathway is more plant-specific, when autophagosomes are formed through the elongation and the following closure of the filamentous proplastids that sequester and digest the portions of the cytoplasm into the lumen (Fig. 6; Filonova et al., 2000a). The same pathway has been earlier described by W. Nagl (1977) using the Phaseolus suspensor as a model system. A third pathway found in Phaseolus (Ga¨ rtner and Nagl, 1980) and

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Vicia faba (Wredle, 2004) involves the membranes of endoplasmic reticulum as precursors of autophagosomes (Fig. 6). At least some of these autophagic pathways are sensitive to 3-methyladenine (the classical inhibitor of autophagy; Seglen and Gordon, 1982), as follows from the inhibitory eVect of this drug on embryonic cell death in the P. abies embryonic system (Table II). B. Cytoskeletal Changes Cytoskeleton-associated proteins can act as both positive and negative regulators of PCD in animals (Cohen et al., 1997; Inbal et al., 2002; Li et al., 1998; Mollinedo and Gajate, 2003; Puthalakath et al., 1999). During cell dismantling, the morphological organization of the cytoskeleton per se undergoes stereotyped changes, which are diVerent for apoptotic and autophagic cell deaths (Bursch et al., 2000; Coleman and Olson, 2002; Jochova et al., 1997; Martin and Baehrecke, 2004). These changes are critically important for the proper functioning of the cell degradation machinery. The roles of the cytoskeleton in plant developmental PCD have been addressed by Smertenko et al. (2003), using P. abies somatic embryogenesis as a model system. This work emphasizes that reorganization of microtubule network and actin filaments through successive stages of autophagic PCD occurs along the apical–basal axis of early embryos. This cell death involved gradual disassembly of the microtubule network, with the first signs of microtubule disorganization detected in the embryonal tube cells. Highly vacuoled cells at the basal end of the suspensor contained only microtubule fragments and tubulin aggregates. Intriguingly, a microtubule-associated protein with molecular weight around 65 kDa (MAP-65), which is involved in the microtubule bundling and formation of the cytokinetic phragmoplast (Hussey et al., 2002; M€ uller et al., 2004; Smertenko et al., 2000), was not bound to the fragments of cortical microtubules in the embryonal tube cells with a partially disorganized microtubule network, suggesting that the dissociation of MAP-65 from microtubules occurs very early in this PCD pathway, concomitantly with the initiation of microtubule disorganization (Smertenko et al., 2003). Further studies are required to determine whether the dissociation of MAP-65 from the microtubules in the terminally diVerentiated cells is a critical regulatory checkpoint of the autophagic cell-death pathway or a passive outcome of microtubule proteolysis. The organization of F-actin successively changed from a fine network in the embryonal masses to thick cables in the suspensor cells where the microtubule network was completely degraded (Smertenko et al., 2003). The F-actin depolymerization drugs, cytochalasin D and latrunculin B, impaired normal embryonic pattern formation and associated PCD in the suspensor, which, in turn, triggered cell death in the embryonal masses (Table II;

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Smertenko et al., 2003), indicating that the actin network is vital in suspensor diVerentiation and PCD. The actin network may provide the scaVold to the components that control autophagosome formation and also direct the movements and engulfment of the cytoplasm by the autophagosomes, as has been previously shown for autophagic PCD in animals (Alpin et al., 1992; Cohen et al., 1997; Inbal et al., 2002; Klionsky and Emr, 2000). C. Nuclear Changes The nucleus is a primary target of the apoptotic cell degradation machinery. The nuclear disintegration process encompasses chromatin events and nuclear envelope events, which occur simultaneously in the same cell (for review, see Buendia et al., 2001; Earnshaw, 1995). A lack of DNA fragmentation in some studies of non-apoptotic cell deaths (Schwartz et al., 1993; Zakeri et al., 1995, and references therein) led to belief that DNA fragmentation is unique for apoptosis. However, subsequent studies showed that autophagic midgut and salivary gland cells show DNA fragmentation in Drosophila (Jiang et al., 1997; Jochova et al., 1997). Likewise, plant cells, which do not have major molecular components of the apoptotic machinery, often display DNA fragmentation at developmentally regulated or stress-induced demise. DNA fragmentation is a part of autophagic PCD in the plant embryo suspensor (Filonova et al., 2000a; Giuliani et al., 2002; Wredle et al., 2001) and it also occurs in the embryonal masses of both subordinate zygotic embryos (Filonova et al., 2002) and somatic embryos with suppressed PCD in the suspensor (Smertenko et al., 2003). These data have been obtained using the TUNEL technique. Electrophoretic analysis of the integrity of the nuclear DNA has been carried out in P. abies using DNA extracted from intact PEMs and somatic embryos, as well as from the dying and living embryonic and PEM cells separated through protoplast isolation (Filonova et al., 2000a). This study has shown accumulation of long (50 kbp), chromatin loop-sized fragments and internucleosomal (180 bp and multiples thereof) fragments in the dying cells (Fig. 7; Filonova et al., 2000a). Strong condensation of chromatin—a major chromatin event during apoptosis—was not found to accompany DNA fragmentation in P. abies embryos (Filonova et al., 2000a). While chromatin events are well studied in diVerent types of cell death in both animals and plants, the nuclear envelope events in non-apoptotic cell deaths are much less understood. In particular, it will be interesting to investigate whether nuclear envelope events are needed for DNA fragmentation in autophagic PCD. In PEM and suspensor cells of P. abies, the earliest morphological change of nuclear envelope is the lobing of the nuclear surface (Fig. 7). The nuclear lobes can then detach to release nuclear

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Figure 7 Chromatin and nuclear envelope events during autophagic embryonic PCD in plants. Left and right halves of the schematic nucleus illustrate intact nucleus and the nuclear changes occurring during PCD, respectively. Chromatin loops detach to give rise to 50-kbp fragments. The DNA is also cleaved into internucleosomal (180 bp) fragments and multiples thereof. At the same time, the nuclear envelope forms lobes; the nuclear pore complex (NPC) disassembles leading to nuclear segmentation. The scheme was drawn based on studies of Filonova et al. (2000a, 2002). h, histones; sp, scaVold protein.

segments, the process associated with disassembly of the nuclear pore complex within the areas of segmentation and concomitant leakage of chromatin into the cytoplasm (Fig. 7; Filonova et al., 2000a). These changes resemble nuclear envelope events during apoptosis (Buendia et al., 2001; Earnshaw, 1995; Willingham, 1999). Being the late process in plant embryonic PCD, the nuclear disassembly does not, however, show a strict timely interdependence with the formation of a large central lytic vacuole; the latter can form either before nuclear breakdown or afterwards, when much of the nucleus is already digested within the smaller lytic vacuoles (Filonova et al., 2000a). In contrast, cell death during tracheary element diVerentiation implies a strict coordination of nuclear and vacuolar events, with the collapse of the large lytic vacuole leading to release of ZEN1 nuclease that attacks nuclear DNA (Ito and Fukuda, 2002). D. The Whole Pathway of Cell Dismantling In the model system of P. abies somatic embryogenesis, the early embryos establish a gradient of autophagic PCD along their apical–basal axis, thus displaying all the successive stages of cell death, from commitment to PCD in

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Figure 8 Cytological characteristics of successive stages of autophagic PCD occurring in apical-to-basal gradient in early embryos of Picea abies. Stage 0 applies to meristematic cells of the embryonal mass (EM), which divides asymmetrically to produce two daughter cells with contrasting developmental fates. One daughter cell retains meristematic identity and remains within EM, whereas its sister cell undergoes terminal diVerentiation to becomes an embryonal tube (et) cell, which is committed to death (stage I). This cell is added to the suspensor, composed of several layers of dying cells with increased age reflected by increased degree of autodestruction (stages II to V) toward the basal end of the suspensor. The scheme was drawn based on studies of Filonova et al. (2000a,b) and Smertenko et al. (2003).

the embryonal tube cells to cell corpses with cleaned protoplasts at the basal end of the embryo suspensor (see Section II.B; Filonova et al., 2000a; Smertenko et al., 2003). Shown in Fig. 8 is a schematic representation of the whole cytological process of cell dismantling occurring in an apical-to-basal gradient in early embryos of P. abies. We do not include mitochondrial changes, because mitochondria look morphologically intact till very late in this PCD (Filonova et al., 2000a). Even though mitochondrial permeability transition appears to be involved (Table II; our unpublished data), more extensive studies are required to provide compelling evidence for the role of mitochondria in the activation of autophagic cell death (see also Lemasters et al., 1998). Stage 0 applies to living meristematic cells of the embryonal mass. These cells are small and have isodiametric shape. They contain dense cytoplasm

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and a rounded nucleus, which occupies a substantial proportion of the protoplast. F-actin and microtubules in these cells form fine networks; MAP-65 is bound to a subset of microtubules (Fig. 8). The cells located in the basal part of embryonal mass divide asymmetrically in the plane perpendicular to the apical–basal axis of the embryo and give rise to two daughter cells with fundamentally diVerent developmental fates. One daughter cell retains meristematic identity and remains in the embryonal mass (stage 0), whereas its sister cell elongates and becomes a terminally diVerentiated tube cell, which is added to the growing suspensor (see Section II.B; Fig. 8). The earliest cytological changes in the tube cells are formation of autophagosomes from the Golgi and proplastids, the major hallmark of the commitment phase of the PCD (stage I). The execution phase of the PCD includes stages II to V, corresponding to the successive layers of cells in the suspensor starting already from the tube cells (Fig. 8). During stage II, MAP-65 dissociates from the microtubules and the microtubule network is disrupted, while F-actin forms thick longitudinal bundles. The cells at stage II are much larger than the cells at the previous stages and they expand further as they progress toward stage III. At the same time, autophagosomes increase in size and number, which sometimes leads to the formation of small lytic vacuoles. It is during stage III when the earliest nuclear envelope events, nuclear lobing, and dismantling of nuclear pore complex, occur. By this stage, several large lytic vacuoles occupy most of the cell volume. No microtubules are left by this stage and only microtubule fragments can be seen in the cytoplasm. In contrast to microtubules, the actin is still preserved in the suspensor cells and forms thick longitudinal cables. By stage IV, the nuclear DNA is fragmented and the nuclei are sometimes segmented. The cells at this stage contain a thin layer of the cytoplasm confined between the plasma membrane and the tonoplast of a large lytic vacuole. Once the vacuole collapses, the remaining cytoplasm is degraded, leaving a hollow-walled cell corpse (stage V; Fig. 8). Autophagic PCD in the suspensor is a slow process; it takes approximately 5 days for complete autodestruction of an individual suspensor cell in P. abies. The transition from one stage of the PCD to the next stage takes approximately 24 hours (i.e., the frequency of the addition of the new layers of cells to the suspensor; Filonova et al., 2000b), with the only exception being the transition from the penultimate to the last stage (i.e., rupture of the tonoplast and the clearance of the remaining cytoplasm), which is apparently a very rapid process (see also Obara et al., 2001). The PCD pathway resembling the one shown in Fig. 8 for suspensor also operates in the other embryonic cell deaths, including death of the PEM cells (Filonova et al., 2000a) and abortion of the whole subordinate embryos in polyembryonic seeds (Filonova et al., 2002).

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IV. Molecular Executioners A. Plants and Core Cell-Death Machinery In metazoan animals, three core molecular components (CED-3=caspases, CED-4=Apaf-1, and CED-9=Bcl-2 family proteins) suYce to regulate several options to die under control in organisms as phylogenetically distant as humans and worms (Aravind et al., 1999, 2001; Koonin and Aravind, 2002). For example, although distinct at the morphological levels, both apoptotic and autophagic cell deaths can require caspase activation (Martin and Baehrecke, 2004) and the mitochondrial permeability transition (Lemasters et al., 1998). A wealth of data accumulated in the past few years suggest that PCD evolved long before the origin of metazoans, since both single-celled eukaryotes and plants exhibit cell autodestruction at various times during their life cycles (Ameisen, 2002; Koonin and Aravind, 2002). Based on the functional classification of the ‘‘domains of death’’ and their phylogenetic distribution, a common ancestor cell-death machinery has been proposed for animals and plants (Aravind et al., 1999). Although studies of plant PCD have suggested some candidate genes and proteins responsible for cell death, we still lack a basic understanding of how PCD operates in plants. A lack of obvious orthologues of core molecular components of metazoan cell death in plant genomes can be interpreted as that plants may have evolved a unique plantspecific cell-death machinery distinct from the one regulating apoptotic cell death. At the same time, a growing body of bioinformatics and experimental data favors the opposite, that is, that plants might actually possess some, if not all, molecular components functionally equivalent to those acting during animal PCD. One case of such a controversy is the existence of ‘‘plant caspases’’ (for review, see Watanabe and Lam, 2004; Woltering et al., 2002). Plants do not have direct homologues of animal caspases, but both synthetic (Bozhkov et al., 2004; del Pozo and Lam, 1998) and natural (del Pozo and Lam, 2003; Hansen, 2000; Lincoln et al., 2002) caspase inhibitors have been shown to exert anti-cell death eVect in plants. These data, together with the identification of the first plant proteins with the substrate specificity characteristic for canonical caspases (CoVeen and Wolpert, 2004; Hatsugai et al., 2004), provide strong evidence for the existence in plants of cell-death pathways regulated by caspase-like proteases (see the following section). Bcl-2 family members are proteins located at diVerent cellular compartments and participating in a number of functions in animal cells. One of the functions is a control of mitochondrial permeability, which is a key factor that determines cell survival (Antonsson et al., 2000; Korsmeyer et al., 2000).

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By transgenic approaches, it has been shown that diVerent animal Bcl-2 family proteins can regulate PCD in plants and yeast. Tobacco plants expressing a pro-apoptotic protein Bax exhibit a cell-death phenotype resembling the hypersensitive response induced by Tobacco mosaic virus (TMV) (Lacomme and Santa Cruz, 1999). In transgenic tobacco plants, overexpression of anti-apoptotic Bcl-xL or CED-9 confers resistance to cell death induced by TMV, UV-irradiation, or chemical stress (Mitsuhara et al., 1999). Although no Bax-related genes have been identified in plant genomes, plants have homologues of the human Bax-inhibitor 1 (BI-1). BI-1 homologues exist in yeast, plants, and animals, providing cytoprotection against a number of stress stimuli. Thus, it is believed that BI-1 regulates evolutionarily conserved mechanisms of stress resistance (Chae et al., 2003). Direct genetic approaches have demonstrated that plant BI-1 is biologically active in suppressing the mammalian Bax action in yeast (Kawai et al., 1999; Kawai-Yamada et al., 2004; Sanchez et al., 2000), plant (Kawai-Yamada et al., 2001, 2004), and animal cells (Bolduc et al., 2003). Based on expression analysis, plant BI-1 is suggested to play a role in response to abiotic and biotic stresses (reviewed in Lam, 2004; Watanabe and Lam, 2004) as well as during senescence (Coupe et al., 2004). Although direct evidence is absent, together these findings present plant BI-1 as a good candidate for molecular regulator of the plant cell-death machinery (Watanabe and Lam, 2004). In agreement with this hypothesis, down-regulation of BI-1 correlates with increased autophagy in tobacco cells subjected to carbon starvation and hypo-osmotic shock (Bolduc and Brisson, 2002). The Arabidopsis genome also contains two other BI-1 homologues, BI-2 and BI-3, which have 5 to 7 predicted transmembrane alpha-helices characteristic for all known BI-1 related proteins. Database searches have revealed 13 BI-2 related genes in Arabidopsis genome (Lam et al., 2001). This gene family seems to have evolved specifically in plants and has been speculated to represent the functional equivalent of the mammalian Bcl-2 family (Lam et al., 2001). Conservation of both ‘‘cell-death domains’’ and their functional properties has occurred during evolution associated with a great level of diversification (Ameisen, 2002). Organisms with increased complexity in development and body plan organization show increased complexity and redundancy in PCD machinery, as well as in the regulation of death-inducing signaling pathways. Biochemical, physiological, and genetic approaches suggest that protein domains playing critical roles in animal PCD are conserved in plants and fungi. Proteins performing a central function in PCD of varied organisms may share functionally relevant motifs, which enables them to occupy appropriate places in the varied cell-death pathways derived from the common ancestral molecular machinery. The sequence of these conserved motifs themselves may not be enough to allow reliable

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sequence-based prediction in genome-wide searches, necessitating the use of sophisticated computational bioinformatics procedures based on domain architecture analysis.

B. Emerging Roles of Caspase-Like Proteins in Plants The caspases are a family of cysteinyl aspartate-specific proteases regulating and executing apoptotic cell death in metazoans (Cohen, 1997; Hengartner, 2000; Thornberry and Lazebnik, 1998). Members of the caspase family are constitutively expressed in almost all cell types as inactive enzymes (zymogens) that require processing to become active proteases. Activation of caspases, more than any other event, defines a cellular response to the apoptotic signal. At the execution of apoptosis, active caspases cleave key structural components of cytoskeleton and nucleus, numerous proteins involved in signal transduction, gene expression, and cell cycle control, as well as several pro- and anti-apoptotic regulatory proteins (reviewed in Earnshaw et al., 1999). Collectively, these proteolytic events disrupt survival pathways and underlie those stereotypic morphological changes that characterize apoptotic cell death. Since ‘‘life requires death’’ and caspases regulate and execute apoptosis, these proteases are indispensable for normal embryonic and post-embryonic development. Examples are caspase 3-, 7-, 8-, or 9-deficient mice, which die prenatally or perinatally, owing to profound abnormalities in embryo pattern formation (for review, see Zheng and Flavell, 2000; Baehrecke, 2002). Studies on developmental cell death during Drosophila metamorphosis have shown that caspases are not only the major executioners of apoptosis, but also participate in the regulation of autophagic PCD. This death is induced by the hormone ecdysone through up-regulation of the caspaseencoding gene dronc (Cakouros et al., 2002; Martin and Baehrecke, 2004) and proceeds with increased caspase activity responsible for the cleavage of a number of structural proteins in the cells (Martin and Baehrecke, 2004). In addition to caspases per se, Drosophila homologues of diverse mammalian regulators of caspases are also involved in autophagic cell-death pathway, as evidenced from the serial analysis of gene expression (Gorski et al., 2003) and microarray analysis (Lee et al., 2003) during Drosophila metamorphosis. Despite a lack of direct homologues of canonical caspases in plants, two groups of plant proteases involved in cell death have been shown to possess caspase-like substrate specificity. The first group is represented by the subtilisin-like serine proteases of Avena sativa, which are constitutively present in an active form and are relocalized to the extracellular fluid after induction of cell death by either the fungal toxin victorin or by heat shock (CoVeen and Wolpert, 2004). These proteases were named ‘‘saspases’’ based on their

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enzymatic activity to cleave a wide range of caspase peptidic substrates containing Asp residue in the P1 position (i.e., serine proteases with aspartate specificity). Like caspases, plant caspases also displayed diVerential preference toward particular amino acid residues in the P2, P3, and P4 positions. They are processive enzymes released to the extracellular fluid before any signs of cell death, which suggests their involvement in a signaling pathway associated with the PCD response rather than in the control of cell autodestruction itself (CoVeen and Wolpert, 2004). Interestingly, mammalian cells also possess a serine protease with caspase-like activity that, once released by cytotoxic lymphocytes, induces apoptosis in virus-infected and tumor cells. This enzyme, called Granzyme B, is able to enter target cells and trigger cell death in diverse ways, depending on the cellular context, such as by direct caspase activation, inducing DNA fragmentation through derepressing caspase-activated DNase, or cleaving key structural components in the cytoskeleton and nuclear membrane (reviewed by Trapani and Sutton, 2003). Vacuolar processing enzymes (VPEs) are another group of plant proteases with caspase-like enzymatic activity. They were shown to account for YVADase (caspase 1-like) activity during TMV-induced hypersensitive response in tobacco (Hatsugai et al., 2004). Both VPE inhibitor and caspase1 inhibitor reduced this activity and, at the same time, protected leaves from the lesion formation in response to TMV infection. Vegetative members of the VPE family are localized in lytic vacuoles, where they are thought to mediate the maturation of vacuolar proteins during senescence and in response to stress-induced cell death (Kinoshita et al., 1999; Kuroyanagi et al., 2002). Plant embryo development involves activation of the unknown protease(s) with the preference to cleave VEID-containing sequences, which are optimal substrates for mammalian caspase-6 (Bozhkov et al., 2004). The VEIDase caspase-like activity is critically important at the early stages of embryogenesis when terminal diVerentiation and autophagic PCD of the suspensor are an integral part of normal embryo patterning. In the early somatic embryos of P. abies, the active VEIDase is localized in the suspensor, including the layer of embryonal tube cells, which are at the commitment stage of the PCD (Fig. 8). Pharmacological inhibition of the VEIDase in vivo suppressed embryonic cell death and impaired embryo development by means of blocking the diVerentiation of the suspensor (Fig. 9A; Bozhkov et al., 2004). Saspases and VPEs are the only plant cell-death proteins with caspase-like substrate specificity identified to date (CoVeen and Wolpert, 2004; Hatsugai et al., 2004). Yet these proteases do not actually have any phylogenetic relationship with caspases based on the sequence. Metacaspases are another group of proteins that have been discovered in protozoa, plants, and fungi and can theoretically perform as functional homologues of animal caspases

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Figure 9 Developmental eVect of the pharmacological inhibition of the VEIDase activity (A) and silencing of the mcII-Pa gene (B) on early embryo development in Picea abies. Note a lack of suspensor as a major developmental abnormality in both cases. The embryos were stained with DAPI (panel A) and acetocarmine=Evan’s blue (panel B). EM, embryonal mass; ES, embryonal suspensor; ET, embryonal tube cells. Reproduced with permission from Bozhkov et al. (2004), Nature Publishing Group (for panel A), and from Suarez et al. (2004), Elsevier (for panel B).

in plant cell-death machinery (Uren et al., 2000). Together with their close relatives from Dictyostelium and metazoans (paracaspases), metacaspases are thought to represent ancestors of canonical caspases (Fig. 10; Koonin and Aravind, 2002). The homology of meta- and paracaspases to caspases is not restricted to the primary sequence, including the catalytic core of histidine and cysteine, but extends to the secondary structure as well (Aravind and Koonin, 2002). The predicted tertiary structure of meta- and paracaspases has also revealed a strong homology to caspases (the so-called caspase-hemoglobinase fold; Aravind and Koonin, 2002). At the same time, recombinant forms of two out of nine Arabidopsis metacaspases, Atmc4 and Atmc9, have been shown to possess a strong preference for cleaving oligopeptides with arginine or lysine and not aspartic acid at the P1 position (Vercammen et al., 2004). Therefore, the enzymatic activity of metacaspases in vivo may likewise be diVerent from that of canonical caspases. Because of their structural and evolutionary relationship to caspases, the possibility that meta- and paracaspases may be involved in an ancient

Figure 10 Domain structure and substrate specificity of the subset of caspase-hemoglobinase fold-containing proteins, including canonical caspases 1 and 6 (csp1 and csp6), paracaspases MALT-1 and PC-Dd, yeast metacaspase YCA1 and plant type-I (Atmc3), and type-II (Atmc5 and mcII-Pa) metacaspases. The position of catalytic dyad of histidine and cysteine is shown by arrowheads and dashed line, respectively. CARD, caspase recruitment domain; CHFD, caspase-hemoglobinase fold domain; DD, death domain; IgD, immunoglobulin domain; PRD, proline-rich domain; ZFD, zinc finger domain. Question marks and ND indicate not studied and not determined substrate specificity, respectively.

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process of genetically driven cell death has commanded the attention of celldeath researchers. To date, the function has been reported for three caspaserelated proteins: the human paracaspase MALT-1, the yeast metacaspase YCA1, and the plant metacaspase mcII-Pa. The cell-death function of paracaspases is doubtful, at best. Human paracaspase MALT-1 seems not to perform as a classical caspase, since its overexpression did not induce apoptosis in mammalian cells (Uren et al., 2000). Furthermore, when expressed in Escherichia coli, MALT-1 does not appear to undergo typical caspase-like autoprocessing and no activity on a number of caspase substrates has been detected. More recently, this protein was demonstrated to be an essential regulator of adaptive immune responses in vivo. MALT-1 acts downstream of Bcl10 to induce nuclear factor kappaB (NF-kappaB) activation, which is required for both the development and function of B cells (Ruefli-Brasse et al., 2003). Although a definite role of a single paracaspase of Dictyostelium remains to be investigated, it does not seem to be involved in the regulation of autophagic PCD of this model organism, as neither the level of cell death nor the developmental pathway of the slime mold was aVected in a paracaspase-null strain (Roisin-BouVay et al., 2004). In contrast to paracaspases, metacaspases are important regulators of PCD. A single yeast metacaspase YCA1 is required for the execution of oxidative stress-induced and senescence-associated cell deaths (Madeo et al., 2002). Unlike human paracaspase MALT-1, YCA1 can undergo caspase-like processing, which appears to be necessary for its activation in vivo. Interestingly, strong amplification of the cell death induced by hydrogen peroxide in the yeast overexpressing YCA-1 gene correlates with the increased level of the VEIDase activity (Madeo et al., 2002), which is also a principal caspase-like activity involved in embryonic cell death in plants (Bozhkov et al., 2004). It remains to be investigated whether this activity is directly attributed to metacaspases. Cell-death function of metacaspases in plants has been demonstrated using P. abies somatic embryogenesis as a model system (Suarez et al., 2004). We found that the expression of the mcII-Pa gene encoding type II metacaspase of P. abies (Fig. 10) during embryo development is restricted to those tissues and structures that are committed to death. Transcriptional regulation of caspases occurs mainly at the hormone-dependent autophagic cell death (e.g., during insect metamorphosis, as discussed previously in this section). Embryonic PCD in plants is likewise a hormonally controlled and autophagy-based process (Filonova et al., 2000a, 2002; Smertenko et al., 2003; Zhivotovsky, 2002), where diVerential expression of metacaspase could constitute a master control mechanism maintaining the proper balance between cell proliferation and death required for embryonic pattern formation.

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Silencing of mcII-Pa led to the suppression of cell death and failure of normal embryonic pattern formation (Suarez et al., 2004). Silenced clones had decreased levels of both the VEIDase caspase-like activity and nuclear DNA fragmentation. These clones were phenotypically similar to the cell lines treated with the VEIDase inhibitors (Bozhkov et al., 2004), with the major developmental defect in both cases being the blocked suspensor diVerentiation (cf. Fig. 9A and B). These data suggest that a single, nonredundant molecular pathway, including the type II metacaspase and protease(s) with the VEIDase activity as principal components, regulates PCD during plant embryogenesis. Purification of the elusive VEIDase(s) and identification of substrate specificity of the type II metacaspase in vivo are necessary for studying molecular interaction among these components in the developmental cell-death pathway.

C. Death Function of Plant Orthologues to Yeast Autophagy Proteins As discussed in the previous section (III.A), autophagocytosis is a major process of cellular degradation and removal during yeast and plant cell death, as well as during autophagic cell death in metazoans. Molecular regulation of this cell-death process is poorly understood, whereas the molecular machinery of macroautophagy required for normal cellular physiology in response to stress has been well characterized in yeast. Genetic screens in Saccharomyces cerevisiae autophagy-deficient mutants have identified 14 genes essential for autophagy (autophagy or apg genes), most of them directly involved in autophagosome formation (Tsukada and Ohsumi, 1993). Orthologues of yeast apg genes or ATG (autophagy-related) genes, according to the new nomenclature (Klionsky et al., 2003), have been found in many organisms including animals and plants, suggesting that in multicellular organisms, autophagy represents an evolutionarily conserved mechanism of a degradative process present in yeast. As the functional domains and the amino acid residues required for yeast autophagy are conserved in the corresponding Atg proteins in animals (Liang et al., 1999) and plants (Hanaoka et al., 2002), the Atg system may function in a similar manner in all eukaryotes and can also be recruited at autophagic PCD. In mammals, certain of the Atg proteins have been proved to be essential for autophagy (Liang et al., 1999; Mizushima et al., 1998; Tanida et al., 1999). Beclin-1 (a functional homologue of yeast ATG6 ) is the first identified gene with a role in mediating mammalian autophagy (Liang et al., 1999). Curiously, this gene is essential for early embryonic development: Beclin -=- mutant cells display clear abnormalities in the induction of autophagy, which contribute to embryonic lethality (Yue et al., 2003). Beclin-1 is able to interact with the apoptotic regulator Bcl-2 (Liang et al.,

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1999), indicating a possible relationship between autophagic and apototic cell deaths. The suppression of autophagic death by caspase-8 through regulation of ATG7-beclin 1 pathway (Yu et al., 2004) also indicates that apoptosis and autophagy may be related processes sharing some of the regulatory proteins and biochemical pathways. Downregulation of mammalian ATG7 or beclin-1 by RNAi leads to inhibition in autophagosome formation and concomitant reduction in cell death (Yu et al., 2004). In Arabidopsis, senescence- and starvation-induced responses are accelerated by the disruption of either AtAPG7 or AtAPG9 autophagic genes (Doelling et al., 2002; Hanaoka et al., 2002). It is tempting to speculate that ATG genes in plant cells, like their orthologues in mammalian cells, may be involved in the regulation of autophagic cell death. However, AtAPG7 or AtAPG9 disruption may also accelerate cell death indirectly, owing to impaired relocation of nutrients in ATG-deficient plants. A similar eVect seems to be encountered in the vcl1 Arabidopsis embryonic mutant (Fig. 1; Table I; Rojo et al., 2001) obtained through inactivation of the plant homologue of yeast vacuolar protein sorting gene Vps16, which is required for autophagy and the cytoplasm-to-vacuole targeting (cvt) pathway (Harding et al., 1995). Yeast Vps16, in a complex with other Vps proteins, facilitates the docking and fusion of transport vesicles with acceptor compartments (Sato et al., 2000). The absence of vesicle traYcking in vcl1 embryos results in defective diVerentiation of the suspensor (Fig. 1), leading to the embryo proper abortion through autophagocytosis at the preglobular stage (Rojo et al., 2001). More experimental data are needed for elucidating the molecular function of Atg, Vps, and Cvt proteins in the normal developmental autophagic PCD (e.g., in the embryo suspensor) and in the pathological cell death induced by deregulation of autophagic machinery (e.g., in the embryo proper of the vcl1 mutant). Unscheduled autophagy in the latter case may actually be regulated in a way similar to the normal physiological cell-death process.

V. Concluding Remarks Despite the ever increasing number of examples of developmental cell death in plants, its mechanisms are still poorly understood. In this chapter, we have attempted to draw attention to the developmental, cell biological, and molecular aspects of PCD at the earliest and perhaps most critical period of plant life—embryogenesis. Most of this information appears to be also relevant to the other developmental cell deaths occurring during post-embryonic plant growth, as most, if not all, of them recruit a common autophagic pathway.

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The major challenge for future research into the nascent field of plant developmental PCD will be to tackle the following problems: Which molecular checkpoint controls the switch from ‘‘metabolic or selective autophagy’’ required for cell survival to ‘‘destructive autophagy’’ involved in the degradation of cellular contents during PCD? How and in which cellular compartments are metacaspases and other plant caspase-like proteases activated in vivo? And which cellular substrates are the targets for these proteases at PCD? What is a molecular mechanism triggering terminal diVerentiation and PCD in one daughter cell at asymmetric cell divisions during early embryonic patterning? The latter problem has been closely approached in a discovery of the critical role of MAPKK kinase in the cellular fate specification at asymmetric division of the Arabidopsis zygote (Lukowitz et al., 2004). The completed genome sequencing and the large collection of T-DNA insertional mutants of A. thaliana, coupled with a well-studied and eYciently controlled pathway of embryonic PCD in the P. abies model system, represent a solid background for answering these fundamental questions. The knowledge gained should also be invaluable for a better view of the evolution of PCD machinery and, ultimately, for the elucidation of whether varied forms of eukaryotic PCD are due to redundancy or diversification of celldeath domains. In addition, practical application of this knowledge will bring many economic benefits, such as those related to plant protection against stress and pathogens, delayed senescence, wood production, and vegetative propagation.

Acknowledgments We thank D. Clapham, A. Smertenko, S. von Arnold, and B. Zhivotovsky for their dependable and stimulating exchanges of ideas on the subject and their helpful comments. Work on PCD in our group has been supported by the Carl Tryggers Foundation, the Spanish Ministry of Education, the Swedish Foundation for International Cooperation in Research and Higher Education, the Thematic Research Project (a joint project of the Swedish University of Agricultural Sciences=the Forestry Research Institute of Sweden) and the Troedssons Fund.

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Physiological Roles of Aquaporins in the Choroid Plexus Daniela Boassa* and Andrea J. Yool*,,y *Department of Physiology and y

Department of Pharmacology, University of Arizona College of Medicine, Tucson Arizona 85724

I. II. III. IV. V. VI. VII. VIII. IX. X. XI.

Aquaporin Water Channels Development of the Choroid Plexus Ion Channels in the Choroid Plexus Function of AQP1 as a Gated Cation Channel Physiological Relevance of AQP1 Ion Channels in Choroid Plexus Regulation of Cerebrospinal Fluid Production Choroid Plexus ‘‘Dark Cells’’ Barrier Function of the Choroid Plexus Neuroendocrine Function Pathophysiology of the Choroid Plexus Conclusions References

The choroid plexus is a specialized tissue that lines subdomains within the four ventricles of the brain where most of the cerebrospinal fluid is produced. Maintenance of an equilibrium in volume and composition of the cerebrospinal fluid (CSF) is vital for a normal brain function, ensuring an optimal environment for the neurons. The necessarily high water permeability of the choroid plexus barrier is made possible by the abundant expression of a water channel, Aquaporin-1 (AQP1), on the apical side of the membrane from early stages of development through adulthood. Data from studies of AQP1 suggest that it also can contribute as a gated ion channel, and suggest that the AQP1-mediated ionic conductance has physiological significance for the regulation of cerebrospinal fluid secretion. The regulation of AQP1 ion channels could be one of several transport mechanisms that contribute to the decreased CSF secretion in response to endogenous signaling molecules such as atrial natriuretic peptide. Numerous classes of ion channels and transporters are targeted specifically to each side of the cellular membrane, and they all work in concert to secrete CSF. Several signaling cascades have a direct eVect on transporters and ion channels present in the choroid plexus epithelium, altering their transport activity and therefore modulating the net transcellular movement Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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of solutes and water. Several neurotransmitters, neuropeptides, and growth factors can influence CSF secretion by direct eVect on transport mechanisms of the epithelium. The mammalian choroid plexus receives innervation from noradrenergic sympathetic fibers, cholinergic and peptidergic fibers that modulate CSF secretion. Water imbalance in the brain can have life-threatening consequences resulting from altered excitability and neurodegeneration, disruption of the supply of nutrients, loss of signaling molecules, and the accumulation of unwanted toxins and metabolites. Understanding the mechanisms involved in the modulation of CSF secretion is of fundamental importance. An appreciation of AQP1 as an ion channel in addition to its role as a water channel should oVer new targets for therapeutic strategies in diseases involving water imbalance in the brain. C 2005, Elsevier Inc.

In the central nervous system (CNS), there are two main types of extracellular fluids: the cerebrospinal fluid (CSF), contained in the ventricles and subarachnoid space, and the interstitial fluid (ISF) that bathes the neurons and glial cells in the brain parenchyma. The CSF is a clear, colorless fluid that is actively secreted from the blood into the ventricles of the brain by the choroid plexus, and circulates in the spaces within and surrounding the spinal cord and brain to protect and maintain homeostasis in the CNS. The choroid plexus is a specialized tissue that lines domains within the four ventricles of the brain, where 80 to 90% of the cerebrospinal fluid is produced. Maintenance of an equilibrium in volume and composition of the CSF is vital for normal brain function, to ensure an optimal environment for the neurons. The necessarily high water permeability of the choroid plexus membrane is made possible by the abundant expression of a specialized type of water channel, Aquaporin-1 (AQP1), which is found in a subset of mammalian tissues that require high transmembrane water movement, including kidney, eye, brain (choroid plexus), lung, and others.

I. Aquaporin Water Channels Aquaporins (AQPs) are members of the Major Intrinsic Protein (MIP) family. To date, eleven members, designated AQP0 through 10, have been characterized in mammals, and more than 150 related members of the MIP family are found in plants, bacteria, invertebrates, and nonmammalian vertebrates (reviewed by Amiry-Moghaddam and Ottersen, 2003). In choroid plexus, the contribution of aquaporin water channels is essential, considering that this tissue serves as the dominant route for water entry

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into the CNS coupled with the net transport of salt into the lumen of the ventricles. AQP1 is abundantly and specifically localized at the apical membrane of choroid plexus cells (Nielsen et al., 1993; Speake et al., 2003). To date, no aquaporins have been identified at the basolateral membrane, leaving open the question of how equivalent amounts of water move across this membrane (Nielsen et al., 1997; Speake et al., 2003). It is possible that other aquaporins, not yet identified, might contribute to water movement at the blood–CSF barrier, but this will require further investigation. For example, AQP4, found in the cytoplasm of rat choroid plexus (but not in either the apical or basolateral membranes), suggested a possible expression in the membranes of intracellular organelles (Speake et al., 2003); however, other studies did not confirm AQP4 expression (Nielsen et al., 1997).

II. Development of the Choroid Plexus Choroid plexus develops on the dorsal side of the neural tube following its closure. It originates from neuroepithelium, and appears between the sixth and eighth weeks of gestation in humans (Netsky and Shuangshoti, 1975a,b), initially in the roof of the fourth ventricle, then in the lateral ventricles, and finally in the third ventricle. By 9 weeks gestation, the fetal choroid plexus is proportionately larger than in the adult (relative to total brain size), and fills
75% of the lateral ventricles. Subsequently, growth of the fetal choroid plexus relative to that of the ventricles slows, until the choroid plexus has assumed its adult appearance by the 20th week of gestation. In adults, the choroid plexus mass is very small compared to the mass of brain tissue. The choroid plexus–CSF system is critical to the development of the neurons that make up the cerebral hemispheres, because it provides the primary source of nourishment to neural tissue early in development. At birth, the anatomical organization of the ventricular system is similar to that in adulthood. In the rat, the choroid plexus is present very early during development, by 14 to 16 days of gestation (Keep and Jones, 1990; Keep et al., 1986), and its capacity for CSF secretion increases with age. From immunocytochemical studies, AQP1 protein is strongly expressed in the developing rat choroid plexus by 14 days of gestation (Fig. 1a), suggesting that AQP1 expression does not appear to represent the limiting factor for CSF secretion during early stages of development. The localized expression of AQP1 protein at the apical (lumen) side of the membrane is also retained in rat primary choroid plexus cell culture (Fig. 1b,c), which makes it a good in vitro model for investigating the mechanisms of modulation of CSF secretion.

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Figure 1 Apical localization of AQP1 in choroid plexus in vivo and in vitro. (A) Localization of AQP1 to choroid plexus epithelial cells of fetal rat (embryonic day E14) in the lateral ventricle by fluorescence microscopy. Frozen rat brain tissue is double-stained with AQP1 antibody (red, AF555-conjugated secondary antibody) and Naþ-Kþ=ATPase antibody (green, FITC-conjugated secondary antibody). Scale bar ¼ 50
m. (B) Localization of AQP1 (red, AF555conjugated secondary antibody) in cultured neonatal rat choroid plexus epithelial cells by confocal microscopy: top view of the cell layer. Cells were transfected with an eGFP expression vector (blue)

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III. Ion Channels in the Choroid Plexus The choroid plexus epithelial cells are characteristically polarized: the apical membrane faces the lumen of the ventricles and the basolateral membrane faces the blood side where the capillaries are present (Fig. 2a). Numerous classes of ion channels and transporters are targeted specifically to each side of the cellular membrane, and they all work in concert to secrete CSF. The first step in this process is the filtration of plasma at the level of the choroidal capillary endothelium; the fenestrated structure oVers minimal restriction to the passage of ions and other molecules (Fig. 2b). The coordinated activity of ion channels and transporters localized in the apical and basolateral membranes of choroid plexus epithelial cells determines the vectorial transport of salt and water to CSF. The primary driving force for fluid secretion is the Naþ-Kþ-ATPase pump, which is unusual in choroid plexus in that it is located in the apical membrane (Masuzawa et al., 1984). The hydrolysis of ATP by the ATPase provides the energy for creating the Naþ gradient essential for primary and secondary transport processes to generate a net transcellular movement of ions in CSF secretion (Figs. 2c, 3). DiVerent populations of ion channels have been described in the choroid plexus epithelial cells, and have been suggested to play a role in CSF secretion as well as cell volume regulation. Two types of Kþ channels have been identified in the rat lateral and fourth ventricle choroid plexus:  The inward-rectifying Kþ channel, Kir7.1, was investigated with

electrophysiological and immunocytochemical techniques (Doring et al., 1998; Kotera and Brown, 1994c; Nakamura et al., 1999). The conductance is time-independent, blocked by Ba2þ and Csþ. It is expressed in the apical membrane, suggesting it might contribute to the resting potential of the cell as well as serving as an apical leak pathway for
90% of the Kþ ions pumped into the cell by the Naþ=Kþ-ATPase  Delayed-rectifying Kþ channels, Kv1.1 and Kv1.3, have also been investigated with electrophysiological and immunocytochemical techniques (Kotera and Brown, 1994c; Speake et al., 2004). They exhibit timedependent activation at depolarizing potentials and are blocked by Ba2þ, tetraethylammonium (TEA), Csþ, and 4-aminopyridine (4-AP). In addition, they are Ca2þ-insensitive and inhibited by serotonin (5-HT), probably via stimulation of protein kinase C (Speake et al., 2004). They also are expressed at the apical membrane, suggesting they might contribute to the recycling of Kþ ions pumped into the cell by the and processed 48 hours later for immunocytochemical detection of AQP1. Scale bar ¼ 10
m. Arrow indicates the position of the optical section perpendicular to the plane of the cell layer. (C) AQP1 localizes at apical surfaces.

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Figure 2 Pathways of cerebrospinal fluid (CSF) circulation. (A) CSF is secreted by the choroid plexus into the ventricles, circulates to the subarachnoid space, and is absorbed into the venous system by the arachnoid villi. (B) Cellular linings separate the CSF system from the brain. Inside the brain, ependymal cells separate the ventricular CSF from the nervous tissue. On the outside surfaces of the brain, cells of the pial–glial linings separate the CSF contained in the subarachnoid space from the nervous tissue. In the ventricles, the choroid plexus epithelium separates ventricular CSF from the blood supplied by the vascular plexus. (C) Immunostaining of cryostat-sectioned fourth ventricle choroid plexus of rat (postnatal day P5) for AQP1 (green, FITC-conjugated secondary antibody) and prealbumin (blue, from Cy5 fluorophore imaging). Scale bar ¼ 20
m.

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Figure 3 Hypothetical model of cGMP-induced regulation of CSF secretion in the choroid plexus barrier. Activation of guanylate cyclase and elevation of cGMP can influence ion channels and transporters in the choroid plexus, causing a decrease in CSF secretion. An increase in intracellular cGMP is correlated with inhibition of Naþ-Kþ-ATPase (Ellis et al., 2000), and activation of AQP1 ion channels (Anthony et al., 2000). Based on studies reported in other systems, elevation of cGMP inhibits Kþ channels (Hirsch and Schlatter, 2003; Shimoda et al., 2002), and activates the Naþ-Kþ-Cl cotransporter (Fujita et al., 1989).

Naþ=Kþ-ATPase, which is vital to the maintenance of cell volume. Also, they might influence CSF secretion as they aVect the resting membrane potential, which plays an important role in driving salt movement (Speake et al., 2001). Results from Speake and colleagues (2004) indicate that these channels are active in the physiological range of the resting membrane potential of the choroid plexus epithelial cells, between 25 and 65 mV, suggesting they contribute in keeping the intracellular negative membrane potential. Also, modulating the activity of these channels will change the resting membrane potential, and

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therefore might aVect the movement of anion at the apical side, resulting in a decrease in the rate of CSF secretion. Additional classes of Kþ channels might contribute to the whole-cell Kþ conductance in the choroid plexus epithelial cells at the basolateral membrane. Two types of anion channels have been characterized in choroid plexus:  Inwardly rectifying anion channels have been investigated using

electrophysiological techniques in rat, mouse, and pig choroid plexus (Kajita et al., 2000a,b; Kibble et al., 1996, 1997; Kotera and Brown, 1994a,b; Speake and Brown, 2004). They exhibit time-dependent activation at hyperpolarizing potentials, high permeability to HCO3, are blocked by Ba2þ, Cd2þ, Zn2þ, and Hþ, and are activated by protein kinase A and cAMP. They provide the major contribution to the wholecell conductance at the resting membrane potential, suggesting they have a role in CSF secretion as counterions enabling the electroneutral net export of Naþ into the ventricle. The molecular identity still remains to be identified. ClC-2 has been proposed as a possible candidate in pig choroid plexus (Kajita et al., 2000a), but studies done in ClC-2 knockout mice suggest that there might be a novel, not-yet-identified inward-rectifying anion channel (Speake et al., 2002). Studies on transgenic knockout mice lacking the cystic fibrosis (CFTR) gene suggest that CFTR is not the anion channel in choroid plexus either (Kibble et al., 1997).  Volume-sensitive anion channels also are present, and have been investigated using electrophysiological techniques in rat and mouse (Kibble et al., 1996, 1997; Speake and Brown, 2004). These channels are activated by cell swelling, exhibit outward rectification, and depend on intracellular ATP. The membrane localization of these channels remains to be determined. Their role in CSF secretion is not clear, since they do not contribute substantially to the whole-cell currents at normal cell volume. Comparison of the various ion channels conductances in the lateral and fourth ventricle choroid plexus in the rat revealed no significant diVerences in the cells from the two locations (Speake and Brown, 2004). However, species diVerences have been reported in ion channel expression between mammalian and amphibian choroid plexus (see review, Speake et al., 2001), which makes it diYcult to compare their potential regulatory roles in choroid plexus function. For example, large conductance Ca2þ-dependent Kþ channels are expressed in amphibia and not in mammalian choroid plexus (Brown et al., 1988; Loo et al., 1988).

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IV. Function of AQP1 as a Gated Cation Channel Data from studies of AQP1 suggest that it may also contribute as a gated ion channel, as well as a water channel, to the function of choroid plexus. Aquaporins are known for mediating transport of water and glycerol, but additional roles as regulated ion channels have become evident. Thus far, several members of the aquaporin family have been described as both water channels and ion channels: AQP0, AQP1, and AQP6 (Anthony et al., 2000; Ehring et al., 1990, 1992; Hazama et al., 2002; Ikeda et al., 2002; Saparov et al., 2001; Yasui et al., 1999; Yool et al., 1996; Zampighi et al., 1985). AQP1 has been reported to mediate water fluxes but also the transport of other solutes, such as CO2 (Cooper and Boron, 1998; Nakhoul et al., 1998; Prasad et al., 1998) and glycerol (Abrami et al., 1995, 1996). Conflicting data from Verkman and colleagues (Fang et al., 2002; Yang et al., 2000), who reported no change in CO2 permeability in erythrocytes, lung, and kidney of wild-type and transgenic mice lacking AQP1, suggested to these investigators that AQP1-mediated CO2 transport may not be physiologically relevant. However, it has been reported that the tobacco plasma membrane aquaporin NtAQP1 does facilitate CO2 membrane transport, which has a significant function in photosynthesis and stomatal opening (Uehlein et al., 2003). Thus, the capacity for various aquaporins to contribute to permeation of solutes other than water and glycerol remains a viable hypothesis and an area for continuing investigation. A current hypothesis is that AQP1 might have a five-pore structure (Fig. 4), with four constitutively active water-selective pores, located one in each of the individual subunits (Murata et al., 2000; Sui et al., 2001), and a putative ion-conducting pore in the center of the tetramer (Yool and Weinstein, 2002). The bacterial glycerol facilitator (GlpF), related to aquaporins in the MIP family, crystallizes as a symmetric tetramer of channel subunits; although GlpF is not known to be an ion channel, its central cavity has sites for coordinating ion binding that suggest that this pathway should be considered as a candidate ion pore in other regulated aquaporins (Fu et al., 2000). In support of the five-pore hypothesis, pharmacological data show that the water and ion conduction pathways in AQP1 are distinct; osmotic water permeability but not ionic conductance is blocked by tetraethylammonium (Brooks et al., 2000; Yool et al., 2002), whereas the ionic conductance but not the water permeability is blocked by cadmium (Yool, 2002). The water permeability of AQP1 is an apparently constitutive property that requires assembly of the channel as tetramers in the plasma membrane (Jung et al., 1994; Preston et al., 1993). In contrast to the ion conductance, water flux is suggested to be independent of signaling mediated through the C-terminus, since proteolytic removal of the terminus after

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Figure 4 Structure of AQP1. Transmembrane topology (A, side view) and tetrameric assembly (B, extracellular view) of AQP1 (generated from protein database information from (Murata et al., 2000). (A) Transmembrane domains are indicated by diVerent colors. Carboxyl (C) and amino (N) terminal domains are intracellular. (B) Five-pore hypothesis: four constitutively active water-selective pores located one in each of the individual subunits, and a putative ionconducting pore in the center of the tetramer. Permeating molecules are for illustration and not to scale. Pictures were generated with RASMOL from structural coordinates of AQP1 deposited in RCSB Protein Data Bank, PDB ID no. 1FQY.

AQP1 proteins are incorporated into red blood cell membranes does not alter the osmotic water permeability (Zeidel et al., 1994). In addition to the osmotic-driven transport of water, AQP1 has been proposed to have a role in GTP-induced rapid gating of water in secretory vesicles in exocrine pancreas (Abu-Hamdah et al., 2004; Cho et al., 2002). Based on these studies, it was proposed that a Gi3-PLA2 pathway and potassium channel might be involved in AQP1 regulation. The initial report of a regulated cationic conductance for AQP1 (Yool et al., 1996) was disputed (Agre et al., 1997). Subsequent work confirmed ion channel function for AQP1 in the oocyte expression system, provided evidence for direct activation by intracellular cGMP, and identified a

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putative binding domain in the carboxyl terminal domain of the channel (Anthony et al., 2000; Boassa and Yool, 2002, 2003). These findings were confirmed independently with AQP1 channels reconstituted in lipid bilayers (Saparov et al., 2001). AQP1 currents are cGMP-induced, cation-selective (Kþ ffi Csþ  Naþ > TEAþ), and sensitive to block by Cd2þ (Yool, 2002). In both the oocyte expression system and reconstituted bilayers, the proportions of active AQP1 ion channels (calculated from the measured ratios of ionic conductance and water flux) were found to be surprisingly low, calling into question the possible physiological relevance of an AQP1-mediated ionic conductance. Model-based calculations from proximal tubule suggested that even a relatively small subpopulation of active cation channels could have a measurable influence on Naþ reabsorption (Yool and Weinstein, 2002), but the question of whether native AQP1 can function as a gated ion channel remained to be demonstrated. An attempt to clarify the discrepancy in the findings in the diVerent expression systems (oocytes and bilayers) was done by expressing AQP1 in human embryonic kidney cells (Tsunoda et al., 2004), but whole-cell patch clamp experiments failed to reveal any cGMP-mediated ion channel activity. Surprisingly, the authors also were not able to show the presence of typical endogenous currents previously described in this cell type, and the recordings may have been compromised by a technical error.

V. Physiological Relevance of AQP1 Ion Channels in Choroid Plexus In primary cultures of rat choroid plexus, native AQP1 mediates a robust cyclic-GMP-gated conductance with channel properties similar to those described previously in the oocyte expression system (Boassa, Stamer, and Yool, unpublished data). Activation and block of the AQP1-mediated ionic current altered net fluid movement across the choroid plexus barrier in vitro, suggesting that the AQP1-mediated ionic conductance has physiological significance for the regulation of cerebrospinal fluid secretion in a manner that is consistent with the observed eVects of cGMP-linked signaling in vivo. Atrial natriuretic peptide (ANP) receptors in choroid plexus epithelia are receptor guanylate cyclases, that when bound by the peptide ligand, stimulate cGMP generation (Israel et al., 1988; Tsutsumi et al., 1987) and inhibit cerebrospinal fluid production (Steardo and Nathanson, 1987). Our experiments on water flux movements across confluent monolayers of choroid plexus in vitro showed that activation and block of the Aquaporin-1-mediated ionic current altered net fluid movement in a manner that is consistent with the observed eVects of cGMP-linked signaling in vivo. These data suggest that cGMP-mediated activation of AQP1 ion

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channels could be one of several mechanisms that contribute to the decreased CSF secretion in response to ANP. Several possible mechanisms involving activated AQP1 ion channels might contribute to the known regulatory response. For example, depolarization induced by AQP1 activation may decrease Cl export by decreasing the electrochemical driving force; changes in the AQP1 channel structure may modulate ion channels and transport proteins involved in CSF secretion that are associated with AQP1 by protein interaction domains or scaVolding proteins; the backleak of Naþ via open AQP1 ion channels may oVset the net eZux of Na driven by the apical Na-K-ATPase pump and thus reduce concomitant water flux; or changes in the AQP1 channel structure that enable the ion channel function might occlude water-conducting AQP1 pathways. Any or all of these possibilities could contribute to the observed reduction in net water movement across the apical membrane, with a net result of decreased fluid movement from the basolateral (blood side) to the apical (CSF side). It is clear that the inhibitory eVect of ANP on CSF production cannot be attributed to AQP1 channels alone. Published data document the role of the Na-K-ATPase pump as another important target of regulation by cGMP in choroid plexus. Sweadner and colleagues reported that activation of guanylate cyclase and elevation of cGMP is correlated with inhibition of Naþ-Kþ-ATPase (Ellis et al., 2000), a mechanism that is consistent with a decrease in CSF secretion. Figure 3 suggests a simplified hypothetical model for cGMP-induced modulation of CSF secretion.

VI. Regulation of Cerebrospinal Fluid Production Several signaling cascades in addition to that regulated by ANP have a direct eVect on transporters and ion channels present in the choroid plexus epithelium, altering their transport activity and therefore modulating the net transcellular movement of solutes and water. Receptors for serotonin (5-HT), arginine vasopressin (AVP), and norepinephrine have been localized to the choroid plexus epithelium (for review, see (Nilsson et al., 1992b). The receptor for serotonin (5-HT1C) is highly expressed at the apical membrane of the choroid plexus epithelial cells (Giordano and Hartig, 1987), and its density is 10-fold higher than in any other region in the brain (Molineaux et al., 1989). Serotoninergic fibers originating in the midbrain project to the ependyma of the four ventricles. It has been proposed that 5-HT is secreted into the CSF and can reach the choroid plexus by diVusion to modulate CSF secretion (Hartig et al., 1990; Pazos et al., 1984). Serotonin can decrease CSF production (Lindvall-Axelsson et al., 1988). It has been suggested that a possible mechanism for this modulation could be the regulation of ion channels in the apical membrane: serotonin via 5-HT1C

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þ

receptors activates Cl channels and inhibits K channels (Garner et al., 1993; Hung et al., 1993). Whereas inhibition of Kþ channels by serotonin is consistent with decreased CSF secretion, the eVect on the Cl channels is less clear, and it has been suggested that inhibition of Kþ channels is the main factor in influencing CSF production induced by serotonin (Hung et al., 1993). ANP receptors in choroid plexus epithelial cells couple to guanylate cyclase and stimulate cGMP production (Israel et al., 1988; Tsutsumi et al., 1987). Both natriuretic peptide receptors type A (NPR-A) and C (NPR-C) have been described in choroid plexus (Brown and Zuo, 1993; Herman et al., 1996). ANP increases blood flow to choroid plexus (Schalk et al., 1992) but inhibits CSF secretion (Steardo and Nathanson, 1987), suggesting that the ANP eVect on CSF production might be due to a direct action on transport mechanisms in the choroid plexus epithelium (see Fig. 3). Vasopressin receptors V1a are expressed in the choroid plexus cells (Barberis and Tribollet, 1996). Furthermore, vasopressin is also a polypeptide synthesized by the choroid plexus (Chodobski and SzmydyngerChodobska, 2001) and, when administered intravenously, reduces both blood flow to choroid plexus and CSF secretion (Faraci et al., 1988, 1990).

VII. Choroid Plexus ‘‘Dark Cells’’ An interesting morphological correlate of the functional state of choroid plexus cells has been inferred from the presence of ‘‘dark cells.’’ Wislocki and Ladman (1958) were the first to describe two diVerent cell types in the choroid plexus epithelium, called ‘‘light’’ and ‘‘dark’’ epithelial cells, based on their anatomical appearance at the microscopic level. The authors suggested that the diVerence in apparent cytoplasmic density between the two cell types could reflect diVerent phases in the secretory cycle of the epithelium, and represent two diVerent ‘‘phenotypes’’ of the same basic cell. Subsequently, they have been observed in adult rat choroid plexus (Johanson et al., 1999a,b; van Deurs et al., 1978) and in the fetal mouse choroid plexus (Sturrock, 1979). The dark cells compose 5 to 10% of the total cell number at all ages. Typically, they are distinguishable from the light cells in having a dark, electron-dense cytoplasm (visible with both light and electron microscopy), more narrow microvilli, dilated basolateral spaces, and a smaller size, probably as a consequence of water lost from the cytoplasm. Figure 5 represents a phase contrast image of rat primary choroid plexus cell culture showing the two diVerent phenotypes. The occurrence of dark cells increases reversibly in conditions associated with a downregulation of CSF secretion, as in the choroid plexus of dehydrated or hydrocephalic animals (Schultz et al., 1977; Shuman and

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Figure 5 Light and dark cells in the choroid plexus epithelium. Phase contrast image (left) of primary culture of rat choroid plexus processed for immunocytochemical detection of AQP1 (right). Scale bar ¼ 10
m.

Bryan, 1991). The number of dark epithelial cells increases by more than twofold after exposure of the choroid plexus to arginine vasopressin (AVP) (Johanson et al., 1999a; Liszczak et al., 1986; Schultz et al., 1977), which is associated with decreased CSF production. Similarly, dark cells can also be induced by other peptide hormones, such as ANP and basic fibroblast growth factor FGF-2 (Johanson et al., 1999b; Preston et al., 2003; Weaver et al., 2003), which decrease CSF formation rate. The current hypothesis is that these dark cells are associated with a state of enhanced reabsorption of excess CSF (or a state of downregulated secretion) involving ion channels and transporters located at the apical membrane of the choroid plexus epithelium. In favor of this idea is the stimulating eVect of FGF-2 and AVP on the Naþ-Kþ-2Cl cotransporter, which has been proposed to play a central role in the reabsorption of Kþ from the CSF to blood (Wu et al., 1998) and the AVP-induced decrease in Cl eZux (Johanson et al., 1999a). Considering that the movements of ions and water are tightly coupled in the epithelium, both mechanisms might possibly explain the inhibition of CSF secretion mediated by AVP. Further studies are needed to test this hypothesis.

VIII. Barrier Function of the Choroid Plexus Comparing the compositions of CSF and plasma (Table I), it is important to note in the CSF the lower levels of proteins and amino acids and the slightly lower pH. CSF ion homeostasis is vital and the choroid plexus is responsible for keeping a stable composition of solutes, buVering the system against the wide changes that can occur in blood plasma. For example, it is essential to

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Composition of CSF and Plasma in Humans*

Constituent Naþ Kþ Mg2þ Ca2þ Cl HCO 3 Glucose Amino acids pH Osmolality (mosmol=Kg H2O) Protein (mg=100 g) pCO2 (mm Hg)

Plasma

CSF

150–159 4.6 1.6 4.7 99 26.8 4–6 2.4–2.6 7.4 289 7000 41

147 2.9 2.2 2.3 113 23.3 0.8 0.7 7.3 289 25–42 50–51

*Concentrations of various solutes are reported as mM (unless otherwise indicated). Compiled from: (Davson and Segal, 1996; Davson et al., 1987).

maintain comparatively lower levels of Kþ and Ca2þ in CSF, since even small changes in their concentration can alter neuronal excitability. The structural organization of the choroid plexus is suitable for this function: the presence of the tight junctions between cells confers a barrier function to the epithelium, and the expression of selective transporters and ion channels enables the choroid plexus to regulate the passage of substances across the barrier. The CSF contributes to many functions in serving the brain (see review by Johanson, 2003), such as: (a) protection against shearing and tearing forces by creating a liquid suspension for the brain; (b) stabilization of intracranial pressure by adjusting the CSF volume in accordance with changes in blood or nervous tissue volume; (c) primary source for transport of micronutrients, growth factors, and hormones that are essential for brain development and metabolism; (d) protection against oscillation of concentration of certain ions (such as Kþ and Hþ) in the brain extracellular fluid; (e) removal of catabolites, protein products, and toxic substances (actively transported by choroid plexus); and (f) parasynaptic transmission by transporting neurotransmitters over long distances. The circulating CSF also serves as a route of drug delivery to CNS cells. Choroid plexus is present on the floor of each lateral ventricle. It passes into the interventricular Foramina of Monro and is present on the roof of the third ventricle. The cerebral (Sylvian) aqueduct connects the third with the fourth ventricle and represents the narrowest passage in the ventricular system. There is also choroid plexus on the roof of the fourth ventricle, organized in a T-shape. The ventricles are continuous with the central canal

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of the spinal cord. Figure 2a summarizes the pathways of CSF circulation. In adult humans, the total weight of the choroid plexus in the four ventricles is about 1 to 2 g, and it contains 108 epithelial cells organized in fingerlike projections (villi) that confer a total surface area of about 200 cm2 (Voetmann, 1949). The choroid plexus consists of many highly permeable (fenestrated) capillaries and choroid epithelial cells. Fluid is actively secreted through these cells from blood into the ventricular lumen to become CSF. The epithelium of the choroid plexus consists of a single layer of cuboidal frond-like cells, tightly packed together via tight junctions or zonula occludens. These tight junctions located near the apical region (CSF-facing side) of the epithelium form the blood–CSF barrier, providing a physical restriction to the passage of most solutes between plasma and CSF. The ultrastructural organization of organelles in choroid plexus epithelial cells consistently reflects their function in active transport associated with CSF secretion and ion homeostasis. In fact, they typically have a high density of mitochondria, a rich Golgi apparatus, and an extensive microvilli system on the apical membrane (Keep and Jones, 1990). They resemble the cells of the renal proximal tubule epithelium, which is also highly specialized to transport water, ions, and organic solutes. Several neurotransmitters, neuropeptides, and growth factors can influence CSF secretion. One mechanism responsible for modulating CSF production relies on a reduction of blood flow to the choroid plexus, limiting the delivery of water and ions to the epithelium and therefore reducing the rate of secretion. The blood supply to the choroid plexus is from small branches of the internal carotid arteries. Astrocytes are present to support both the neurons and the blood vessels. The ventricles, filled with CSF, are lined by ependymal cells, and are connected together by foramina. CSF circulation throughout the ventricles is aided by the cilia of ependymal cells. Ventricular CSF mixes with fluid in the subarachnoid space and then is conveyed to drainage sites, where the CSF is reabsorbed back into the venous bloodstream through the arachnoid villi. The total volume of CSF in adult humans is about 140 ml; about 600 ml are secreted daily to replenish the circulating CSF approximately every 5 to 6 hours. The mean volume of the ventricular system is close to 30 ml (Johanson, 2003). The rest of the CSF is distributed around the spinal cord (30 ml) and in the subarachnoid spaces and cisterns (80 ml) that envelop the cerebral and cerebellar hemispheres. Three diVerent cellular linings (each composed of a single layer of cells) separate the CSF system from the brain (Fig. 2b). Inside the brain, ependymal cells separate the ventricular CSF from the nervous tissue. On the outside surfaces of the brain, cells of the pial–glial linings separate the CSF contained in the subarachnoid space from the nervous tissue. In the ventricles, the choroid plexus epithelium separates ventricular CSF from

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the blood supplied by the vascular plexus. While cells of the ependymal and pial–glial linings possess discontinuous gap-junctions in the intercellular regions, allowing relatively free passage of solutes from and to CSF, the choroid plexus epithelium represents a barrier for many molecules and ions. As a consequence, the composition of the CSF is more similar to the brain interstitial fluid than to the plasma.

IX. Neuroendocrine Function The choroid plexus acts as both a target and a source for hormonal and neuroendocrine signals within the brain. The circulating CSF produced by choroid plexus conveys the signaling molecules that are selectively imported or generated by choroid plexus, such as hormones and growth factors, throughout the brain. Hormones moving from the blood to the CSF through the choroid plexus epithelium can reach their target area in the brain by diVusing through the brain interstitial fluid. Neuroendocrine factors produced in the brain or choroid plexus similarly can be transported through the CSF circulation. This volume transmission pathway is thought to represent an important route of intercellar communication in the brain (Agnati et al., 1995). The choroid plexus is also the main site of synthesis of the thyroid hormone transport protein transthyretin (TTR or prealbumin) in the brain (Dickson and Schreiber, 1986; Dickson et al., 1985, 1986; Herbert et al., 1986; Kato et al., 1986; Mita et al., 1986). TTR represents 20% of total choroid plexus protein synthesis (and is almost 50% of the protein secreted by choroid plexus), to function as the carrier protein for thyroid hormones in the brain. With higher aYnity for thyroxine (T4) than triiodothyronine (T3), TTR carries T4 throughout the CSF. In the brain, T4 is converted to T3 by deiodinases (Southwell et al., 1993). Secretion of TTR by the choroid plexus may provide a mechanism for the regulation of thyroid hormones levels in the brain (Schreiber and Richardson, 1997; Schreiber et al., 2001; Southwell et al., 1993). In addition, evidence indicates that the choroid plexus is a major site of synthesis of insulin-like growth factor-II (IGF-II) in the brain, which is known to have trophic and metabolic eVects on neurons and glial cells (Stylianopoulou et al., 1988). The mammalian choroid plexus receives innervation from noradrenergic sympathetic fibers, cholinergic, and peptidergic fibers which modulate CSF secretion (Nilsson et al., 1992b), although species-specific diVerences have been described. Adrenergic fibers project from the superior cervical ganglia to the choroid plexi, which express beta-adrenergic receptors (Lindvall et al., 1977b, 1985; Nathanson, 1980). Sympathetic denervation in rabbit significantly increased CSF secretion and Naþ=Kþ-ATPase activity, whereas

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electrical stimulation of superior cervical ganglia significantly reduced CSF formation (Lindvall et al., 1978a,b, 1982), suggesting that the sympathetic nervous system has a tonic inhibitory eVect on CSF secretion. Noradrenaline reduced CSF production in rabbit and cat (Haywood and Vogh, 1979; Lindvall et al., 1979), probably by stimulation of cAMP production with activation of beta-receptors (Lindvall et al., 1985). Findings in other species are not consistent. In the amphibian, choroid plexus (Saito and Wright, 1984, 1987) correlated cAMP production with elevated apical secretion of bicarbonate and Cl, predicted to increase CSF production. Sympathetic denervation of the choroid plexus in rats had an eVect opposite to that observed in rabbit, decreasing the Naþ=Kþ-ATPase activity (Lindvall et al., 1982). Although the mechanisms of sympathetic nervous system modulation of CSF secretion vary, it is clear that adrenergic innervation mediates a direct eVect on transport mechanisms of the epithelium. Cholinergic innervation of choroid plexus is less well characterized (Edvinsson et al., 1973; Lindvall et al., 1977a). Cholinergic agonists, such as carbachol and acetylcholine, inhibit CSF secretion and Naþ=Kþ-ATPase activity (Lindvall and Owman, 1981), probably via cGMP (Ellis et al., 2000). Muscarinic receptors were found in the choroid plexus of the rat lateral ventricles, but not of the third or fourth ventricles (Rotter et al., 1979). More studies are needed to investigate the role of cholinergic innervation of the choroid plexus. Peptidergic nerves, containing vasoactive intestinal polypeptide, are present but primarily associated only with the vascular bed (Lindvall and Owman, 1981; Nilsson et al., 1990a,b). The origin of these fibers is not clear. Based on experiments done in choroid plexus from rats, vasoactive intestinal polypeptide (VIP) was reported to inhibit CSF production (Nilsson et al., 1991). VIP could mediate a synergistic eVect with norepinephrine (Nilsson et al., 1992a,b), but species-specific diVerences present a more complicated picture and further investigations are needed to dissect out the various components.

X. Pathophysiology of the Choroid Plexus The net movement of water across the blood–brain and the blood–CSF barriers is important in the regulation of brain water content. Water imbalance in the brain can have serious and life-threatening implications, such as altered excitability and neurodegeneration, disruption of the supply of nutrients, loss of signaling molecules, and the accumulation of unwanted toxins and metabolites. Hydrocephalus is a hydrodynamic disorder that consists of an increase in CSF volume within the cranial cavity, and which can occur with or without an elevation of intracranial pressure (Johanson,

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2003). In general, it can be a consequence of overproduction of CSF, but most commonly is due to obstruction of outflow or impaired absorption of CSF. Overproduction of CSF is associated with choroid plexus tumors (papilloma or carcinoma), which typically induce increases in intracranial pressure (Milhorat et al., 1976; Rickert and Paulus, 2001). Obstruction of CSF flow is the most common cause of hydrocephalus; it can be due to congenital malformations or to other lesions (tumors, infections, hemorrhage) that can ultimately impair the CSF outflow pathway. Impairment of CSF absorption has been linked to venous hypertension, and it can cause pseudotumor cerebri (also called benign intracranial hypertension). This condition is associated with increased intracranial pressure without enlargement of the ventricles. Clinical interventions for hydrocephalus are generally limited to surgical procedures, such as shunting of CSF via catheter to other locations of the body. DiVerent pharmacological agents have been used with limited success to manipulate CSF secretion rate, but in general, their therapeutic value is handicapped by side eVects. Furosemide and acetazolamide, inhibitors of carbonic anhydrase, have been used to reduce water content in the CNS by decreasing CSF secretion rate
50 to 60%. Hypertonic osmotic agents such as mannitol or isosorbide that create an osmotic gradient between blood and brain are used to induce a movement of water from nervous tissue. However, all these drugs are only used as temporary treatment for mild hydrocephalus and are not eVective for long-term therapy.

XI. Conclusions Understanding the mechanisms involved in the modulation of CSF secretion is of fundamental importance. An appreciation of AQP1 as an ion channel in addition to its role as a water channel should oVer new targets for therapeutic strategies in diseases involving water imbalance in the brain. AQP1 is highly expressed in the choroid plexus, and understanding its functional properties might reveal it as a potential target for regulating water and salt movements in development, homeostasis, and pathological conditions.

References Abrami, L., Berthonaud, V., Deen, P. M., Rousselet, G., Tacnet, F., and Ripoche, P. (1996). Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: Inhibition studies. Pflugers. Arch. 431, 408–414. Abrami, L., Tacnet, F., and Ripoche, P. (1995). Evidence for a glycerol pathway through aquaporin 1 (CHIP28) channels. Pflugers. Arch. 430, 447–458.

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Control of Food Intake Through Regulation of cAMP Allan Z. Zhao Department of Cell Biology and Physiology University of Pittsburgh Pittsburgh, Pennsylvania 15261 I. II. III. IV. V. VI.

Introduction Regulation of Feeding at the Hypothalamus Hypothalamic EVects of cAMP on Food Intake cAMP as an Orexigenic Second Messenger The Anorectic EVects of cAMP in the PVN Regulation of Food Intake by Leptin Requires a PI3K-PDE3B-cAMP Signaling Pathway VII. What Elevates the Intracellular cAMP Levels in the NPY-Neurons in a Negative Energy Balance State? VIII. A Working Model for Hypothalamic Control of Food Intake Involving Regulation of cAMP—A Perspective from the NPY/AgrP Neurons IX. Dysregulation of cAMP in the Hypothalamus—Implication in Obesity References

The 30 , 50 -cyclic adenosine monophosphate (cAMP) is a classic second messenger that is intimately involved in the regulation of food intake at the hypothalamus. cAMP can mediate the orexigenic and anorectic eVects of various peripheral hormones or neuropeptides in a region-specific and neuron-specific manner. The importance of cAMP is particularly highlighted in a series of findings about cAMP transducing the anorectic signals of leptin and -msh. This chapter provides an overview of several studies on how regulation of food intake takes place with cAMP as the second messenger in the hypothalamus. C 2005, Elsevier Inc.

I. Introduction The 30 , 50 -cyclic adenosine monophosphate (cAMP) is a classic second messenger that mediates a wide range of cellular functions, such as hormone secretion, protein synthesis, gene expression, and long-term potentiation (LTP) (for a review, see Beavo and Brunton, 2002). The steady state of cAMP is controlled by the activities of adenylyl cyclases (AC) and phosphodiesterases (PDE) (Cooper, 2003; Soderling and Beavo, 2000). To date, 10 diVerent families of adenylyl cyclases (including a soluble one) have been Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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identified (Cooper, 2003). In contrast, the stories of cAMP-degrading PDEs are very complex. We now know that there are at least 12 PDE gene families composed of more than 40 diVerent isozymes (including various splice variants; Soderling and Beavo, 2000). The activities of ACs are primarily regulated by diVerent coupled G-proteins, Ca2þ/calmodulin (CaM), and direct phosphorylation by PKC (protein kinase C), PKA (cAMP-dependent protein kinase), and CaM-kinases (Cooper, 2003). Similarly, a wide array of physiological conditions, such as hormonal stimulation, glucose concentrations, and inflammation, can modulate the expression and activities of various cAMP-PDEs through a variety of molecular mechanisms, such as Ca2þ/calmodulin, Ser/Thr- and Tyr-phosphorylation, cGMP-inhibition, and direct interactions with other signaling proteins (Essayan, 2001; Houslay and Milligan, 1997; O’Connell et al., 1996; Soderling and Beavo, 2000). Together, these two types of enzymes regulate the duration and amplitude of intracellular cAMP signals in response to diVerent physiological stimuli. Along with PKA, guanine nucleotide exchange factors (GEFs), cyclic nucleotide-gated channels, and PKA-anchoring proteins (AKAPs), these two types of enzymes also define the local concentrations (‘‘microdomains’’) of cAMP that, in turn, control various cellular functions (Beavo and Brunton, 2002; Rich et al., 2001; Steinberg and Brunton, 2001). Because the broad topics of cAMP regulation and its cellular eVects have been reviewed extensively elsewhere, the primary focus of this chapter is to discuss progress in understanding the roles of cAMP in the regulation of appetite and body weight.

II. Regulation of Feeding at the Hypothalamus Abundant evidence has shown that the control of food intake is achieved primarily at the hypothalamus (Schwartz et al., 2000; Williams et al., 2001; Woods et al., 2000). Within the hypothalamus, several regions including the ARC (arcuate nucleus), VMH (ventromedial hypothalamus), DMH (dorsomedial hypothalamus), PVN (paraventricular nucleus), and the lateral hypothalamus are implicated in the regulation of food intake and body weight (Schwartz et al., 2000). A variety of peripheral hormones have been demonstrated to influence energy homeostasis at these sites, such as leptin, insulin, ghrelin, cholecystokinin (CCK), and glucagon-like peptide-1 (GLP-1). (Baskin et al., 1999; Moran, 2000; Schwartz et al., 2000; TangChristensen et al., 2004; Tschop et al., 2002; Zhang et al., 1994). Among these hormones, leptin represents the long-sought adipostat signal responsible for the tight control of feeding behavior and adiposity (Zhang et al., 1994). The null mutation of leptin or leptin receptor in both rodents and humans can cause severe overfeeding (hyperphagia), gross obesity, stunted linear growth,

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diabetes, and sterility (Lee et al., 1996; Montague et al., 1997; Zhang et al., 1994). The consistency of these phenotypes as a result of leptin or leptin receptor mutations in both rodents and humans highlights the importance of this hormone in the long-term maintenance of body weight and energy homeostasis. The aforementioned peripheral signals are received and integrated by the hypothalamic neural circuitry, which, in turn, regulates the feeding behavior through a wide array of orexigenic and anorectic neuropeptides (Williams et al., 2001; Woods et al., 1998, 2000), such as neuropeptide Y(NPY), agouti-related peptide (AgrP), melanin-concentration hormone (MCH), orexins, corticortrophin-releasing hormone (CRH), and -melanocyte-stimulating hormone (-msh). cAMP plays important roles in mediating either the expression or the actions of these neuropeptides. The hypothalamic neurons that synthesize and secrete both NPY and AgrP are traditionally called NPY/AgrP-neurons, and are almost exclusively localized in the ARC region (Bi et al., 2003; Wolden-Hanson, 2004). NPY and AgrP are orexigenic agents, whose expression can be rapidly induced in a negative energy state (low adiposity and low leptin) and, subsequently, can stimulate food intake (Bertile et al., 2003; Bi et al., 2003; Schwartz et al., 1996). In a positive energy state, however, the expression of NPY and AgrP in the ARC are suppressed, which eliminates the stimulatory signals of feeding (Elias et al., 1999; Schwartz et al., 1996; Swart et al., 2002). When repeatedly injected into the 3rd ventricle, NPY and AgrP can induce hyperphagia and obesity (McMinn et al., 1998; Paez and Myers, 1991; Paez et al., 1991; Vettor et al., 1994). Interestingly, gene targeting of NPY only caused mild seizure but did not perturb the feeding behavior. Such observations serve as a reminder that evolution has put multiple compensatory mechanisms in place to safeguard energy intake (Erickson et al., 1996). However, NPY-deletion in the ob/ob mice did lead to significant reduction in food intake and body weight, suggesting the importance of NPY in leptin-controlled energy homeostasis (Erickson et al., 1996). The POMC–neurons located in the ARC synthesize and release an anorectic peptide, -msh, a proteolytic product from proopiomelanocortin (POMC) protein (Cone et al., 2001; Ellacott and Cone, 2004). The anorectic function of -msh is mediated through two of its cognate receptors, melanocortin-3R and -4R), both of which are seven transmembrane G-protein coupled receptors and highly expressed in the PVN-region (Cone et al., 2001). Gene targeting of MC4R in mice produced hyperphagia and gross obesity (Huszar et al., 1997) that are phenotypically similar to the syndromes observed in the patients carrying homozygous mutations of MC4R (Tao and SegaloV, 2003; Vaisse et al., 2000). Both NPY/AgrP- and POMC-neurons project to the PVN (Cowley et al., 1999). The released AgrP will serve as a specific antagonist of -msh to compete for the binding to the melanocortin receptors, therefore blocking the anorectic action of -msh (Mizuno et al., 2003; Doghman et al., 2004).

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NPY/AgrP neurons also innervate the POMC neurons, which allows NPY to exert an inhibitory eVect on the activity of POMC neurons (Cowley et al., 2001). In the context of cAMP signaling, both lateral hypothalamus (LH) and perifornical hypothalamus (PFH) have also been implicated in the control of feeding due, at least in part, to the expression of orexins and MCH in this region (Shimada et al., 1998). The discussion here focuses primarily on the roles of cAMP in the expression and functions of these peripheral signals and neuropeptides in the hypothalamus.

III. Hypothalamic Effects of cAMP on Food Intake Like the complex hypothalamic neural network involved in the food-intake control, the roles of cAMP in the scheme of food-intake control are also multifaceted, and appear to be region-specific and neuron-specific. Here, this reviewer summarizes some of the major observations about the roles of cAMP in regulating food intake within the hypothalamic neural network. A graphic illustration of this summary is provided in Fig. 1.

IV. cAMP as an Orexigenic Second Messenger A direct line of evidence demonstrating the in vivo eVects of cAMP on the expression of orexigenic neuropeptides in the hypothalamus came from Akabayashi et al., 1994. In this case, a cAMP analog, N6-dibutyryl cAMP, was directly injected into the 3rd ventricles of the brain-cannulated rats. Through quantitative imaging analysis, the expression of NPY was found sharply increased only in the ARC and medial parvocellular portion of PVN (mPVN). This region-restricted eVect of cAMP on NPY expression appeared to be specific as the expression of another orexigenic peptide, galanin, was not aVected at all (Akabayashi et al., 1994). Although we cannot rule out the possibility of cAMP stimulating NPY expression via another type of neuron, these observations, when combined with other cellular studies (Liu et al., 1999; May et al., 1995; Pance et al., 1995), indicate a direct positive role of cAMP on the expression of NPY in the NPY/AgrP-neurons in the ARC. Some of the physiological and pharmacological evidence supporting the role of cAMP as an orexigenic second messenger came from a series of studies by Stanley and his colleagues (Gillard et al., 1997, 1998). Injection of 8-Bromo-cAMP or cAMP-elevating agents (e.g., forskolin and IBMX) in the PFH and LH produced strong appetite-enhancing eVects (Gillard et al., 1997, 1998). This orexigenic eVect could be alleviated by co-injection of a PKA-inhibitor, H-89. Injection of the same cAMP-elevating reagents

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Figure 1 The diVerential eVects of cAMP on food intake in diVerent regions of hypothalamus. The intracellular cAMP molecules that have a stimulatory eVect on food intake are labeled in red, whereas the cAMP molecules mediating the satiety action are labeled in black. In this case, an intracerebraventricular (icv) injection of cAMP or forskolin will elevate the phosphorylation and activities of CREB and, subsequently, the expression of NPY as well as AgrP. The anorectic peptide, -msh, released from the POMC-nerve fibers innervating to the PVNregion (note: the CRH-neuron is only used as an example), will stimulate the production of cAMP through its Gs-coupled melanocortin receptors (MC4R), the levels of phospho-CREB, and, consequently, the expression levels of anorectic neuropeptides (such as CRH). Similarly, GLP-1, released from the eVerent nerve fibers projecting to the PVN-region, also binds to the CRH-neurons and stimulates cAMP production, which will eventually lead to reduced food intake. Studies have shown that site-specific injection of cAMP-analogs in perifornical and lateral hypothalamus (PFH and LH) potently increases food intake, although the mechanisms of such actions in the PFH and LH are not yet clear. However, the expression of orexins in LH can be stimulated by cAMP.

in some other hypothalamic sites (e.g., amygdala, PVN, and anterior hypothalamus) did not enhance feeding, suggesting that the eVects of the cAMP analog or cAMP elevating agents were relatively region-specific in the hypothalamus (Gillard et al., 1997, 1998). Although it is still not clear how cAMP enhanced feeding in these site-specific injection studies, two potential explanations can be considered (without excluding other possibilities):

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(1) Injection of cAMP in the LH may have stimulated the expression and secretion of orexins that have been shown to be of orexigenic eVects. (2) It is possible that the cAMP-analog or cAMP-elevating agents were not confined just to the specific injection sites but, rather, were able to diVuse into some adjacent regions. For example, diVusion of these agents into the ARC would be expected to stimulate the expression of NPY and AgrP and, subsequently, food intake.

V. The Anorectic Effects of cAMP in the PVN A critical mechanism by which -msh exerts its satiety eVect is through the increase of cAMP production. One of its receptors, melanocortin-4R (MC4R), has been convincingly demonstrated to be intimately involved in energy balance and to be expressed in several parts of the CNS, including the PVN of the hypothalamus (Kishi et al., 2003). The melanocortin receptors are coupled to Gs (Srinivasan et al., 2003). The increase of cAMP synthesis, upon -msh binding, is critical to the physiological function of -msh. Mutations that destroy the ability of MC4R in cAMP synthesis abolish the satiety action of -msh and lead to eating disorders as well as early onset of obesity (Donohoue et al., 2003; Tao and SegaloV, 2003). GLP-1 is one of the extensively studied peripheral hormones that suppress food intake by stimulating cAMP production in the PVN region (McMahon and Wellman, 1998; Sarkar et al., 2003). It is a very potent insulinotropic hormone primarily made and secreted from both the open-type L-cells of the distal intestine and in the nucleus tractus solitarius neurons, whose eVerent nerve fibers project to the PVN of the hypothalamus (Habener, 1993; Holst, 1994). GLP-1, through its Gs-coupled receptors, elevates intracellular cAMP levels as well as Ca2þ concentrations (for a review, see Habener, 1993). Intracerebroventricular (icv) injection of GLP-1 induces potent inhibitory eVects on feeding as well as angiotensin II-stimulated drinking behavior (Turton et al., 1996). Icv co-injection of exendin-4, a specific GLP-1 antagonist, can not only block the satiety eVect of GLP-1 but also powerfully augment feeding in satiated rats (Schick et al., 2002). Although the mRNA of GLP-1 receptor has been mapped to the PVN, VMH, ARC, and SON (supraoptic nucleus) (Shughrue et al., 1996), icv injection of GLP-1 produces c-fos accumulation only in the PVN and amydaloid nucleus (Turton et al., 1996). A 2003 study has further identified that the termini of GLP-1 neurons densely innervate CRH neurons in the PVN (Sarkar et al., 2003). CRH is an anorectic neuropeptide, whose expression and release is stimulated by cAMP and cAMP-induced CREB activity (Helmreich et al., 2001; Itoi et al., 1999, 2004; Sarkar et al., 2002). Thus, an increase of cAMP production in the CRH neurons as a result of GLP-1 binding will be predicted to stimulate

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the expression and secretion of CRH, which, in turn, will lead to decreased food intake.

VI. Regulation of Food Intake by Leptin Requires a PI3K-PDE3B-cAMP Signaling Pathway Soon after the identification of the leptin receptors, it was realized that they belong to the Class I cytokine receptor family (Baumann et al., 1996; White and Tartaglia, 1996). Consequently, the JAK-STAT (Janus kinase-Signal transducer and activator of transcription) pathway should be an integral part of leptin’s signaling and functions, particularly for the so-called longform variant of the leptin receptors, OB-Rb. OB-Rb is expressed predominantly in the hypothalamus (Baskin et al., 1999; Baumann et al., 1996; Lee et al., 1996; Tartaglia et al., 1995). Indeed, the importance of the JAK-STAT pathway is well demonstrated by the phenotypes of db/db mice in which the leptin-induced JAK-STAT signaling is absent due to the lack of OB-Rb expression (Ghilardi et al., 1996; Vaisse et al., 1996). Perhaps as a further highlight of the importance of this pathway in leptin’s central actions, mutant mice carrying a knock-in mutation in OB-Rb that renders it incapable of activating STAT3 developed severe hyperphagia and gross obesity (Bates et al., 2003). However, the JAK-STAT pathway is not the only signaling mechanism for the physiological functions of leptin. The concept of cAMP regulation by leptin was initially established in the peripheral systems, including the liver and pancreatic -cells (Fehmann et al., 1997; Zhao et al., 1998, 2000). In these cases, leptin triggers a PI3-kinase dependent activation of a cAMPdegrading enzyme, phosphodiesterase 3B (PDE3B), which, in turn, causes reduction of intracellular cAMP (Zhao et al., 1998, 2000). The physiological significance of this signaling mechanism is reflected in the fact that leptin can suppress GLP-1-stimulated insulin secretion and glucagon-stimulated glycogenolysis (Aiston and Agius, 1999; Ceddia et al., 1999; Fehmann et al., 1997; Kulkarni et al., 1997; Nemecz et al., 1999; Zhao et al., 1998, 2000). Only since 2001 has evidence begun to emerge to show that such signaling scheme occurs not only in the periphery but also in the CNS, with a significant impact on the feeding behavior. With a transgenic mouse model carrying a cAMP-responsive element (CRE)-driven lacZ gene, Beavo and his colleagues found that the expression of the lacZ gene was drastically elevated in the NPY/AgrP neurons in the ARC during fasting, which was also concomitant with the increase of NPY expression (Shimizu-Albergine et al., 2001). Because elevated expression of the reporter lacZ requires increased cAMP levels, the intensity of its transcripts became a de facto indicator of cAMP levels in vivo (Shimizu-Albergine

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et al., 2001). Interestingly, the fasting-induced cAMP signal was restricted to the NPY/AgrP neurons and was not found in the POMC neurons (Shimizu-Albergine et al., 2001). Intracerebraventricular (icv) injection of leptin reduced lacZ expression during fasting (Shimizu-Albergine et al., 2001). Importantly, injection of a pharmacological inhibitor of PDE3 elevated the levels of lacZ transcript and such an increase was restricted only to the NPY/AgrP-neurons in the ARC (Shimizu-Albergine et al., 2001). These data are reminiscent of similar findings in the periphery, and suggest that leptin may also induce the PDE3B-cAMP signaling pathway in the hypothalamus. The definitive functional proof of this signaling mechanism in leptin-regulated energy homeostasis came from several studies. Our group has found that icv injection of leptin significantly stimulated the activities of PI3-kinase as well as PDE3B and reduced cAMP level in the hypothalamus (Zhao et al., 2002). Importantly, pharmacological blockade of the PDE3 activity with a specific inhibitor completely blunted the satiety and weightreducing eVects of leptin (Zhao et al., 2002). Schwartz and his colleagues examined the upstream component of this pathway by icv injecting the inhibitors of PI3-kinase, wortmannin, and LY294002. Both inhibitors of PI3K blocked the satiety actions of leptin (Niswender et al., 2001). Thus, the PI3K-PDE3B-cAMP signaling pathway is very likely to be an essential mechanism for leptin not only in the periphery but also in the hypothalamus. Interestingly, counter-regulation of cAMP-induced cellular functions by leptin has also been found outside the context of food-intake control. Quintela et al. (1997) have demonstrated that leptin treatment of fetal rat neurons could completely block cAMP-induced somatostatin release (Quintela et al., 1997). It is important to point out that the PI3K-PDE3B-cAMP pathway is not an isolated signaling mechanism for leptin. Rather, it is found to crossregulate the activity of JAK-STAT3 pathway as pharmacological blockade of PDE3B activity in the hypothalamus could eliminate leptin-stimulated phosphorylation and DNA-binding activity of STAT3 (Zhao et al., 2002). These findings, although primarily based on pharmacological evidence, suggest that the reduction of cAMP through PI3K-dependent activation of PDE3B is a prerequisite step for the satiety and weight-reducing actions of leptin in the hypothalamus. Potentially, regulation of cAMP through the PI3K-PDE3B pathway can be a broad signaling theme for food-intake control. Studies by Schwartz and his colleagues have found that the satiety eVect of insulin is also dependent on the activity of PI3-kinase (Niswender et al., 2003). In the periphery, activation of PDE3B and the subsequent reduction of cAMP by insulin have been well demonstrated to mediate the anti-lipolytic or anti-glycogenolytic eVects of insulin in the fat and liver, respectively (Degerman et al., 1997). Indeed, our recent studies have found that icv co-injection of a PDE3 inhibitor with insulin could completely block

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the satiety eVect of insulin (unpublished observation). The evidence gathered so far suggests that the PI3K-PDE3B-cAMP pathway is a critical signaling mechanism in the broad schemes of food-intake control. However, a complete validation of this concept should wait for more molecular genetic evidence, such as a genetic model with CNS specific knock-out of PDE3B gene. In addition, PDE3A, structurally related to PDE3B, is also highly expressed in the CNS (Reinhardt et al., 1995). It remains to be determined if PDE3A might also be involved in mediating the satiety actions of leptin.

VII. What Elevates the Intracellular cAMP Levels in the NPY-Neurons in a Negative Energy Balance State? A critical issue following these studies remains to be resolved. If fasting elevates cAMP levels in the NPY/AgrP-neurons, what is (are) the hormone(s) responsible for the increase of cAMP in the NP/AgrP neurons? Although at this stage there is no definitive evidence to pinpoint the specific candidate(s), this reviewer proposes, based on some recent evidence, that ghrelin is one such hormone responsible for the cAMP increase in the NPY/AgrP neurons, for the following reasons: (1) Ghrelin is a 28-aa peptide hormone secreted primarily from the stomach. Its expression has also been identified in the hypothalamus (Cowley et al., 1999). One of its primary functions was initially described as a secretagogue of growth hormone secretion in the pituitary (Date et al., 2000; Kojima et al., 1999). It was later realized that ghrelin actively participates in the regulation of energy intake. The plasma concentrations of ghrelin are high during fasting and fall as the ingested food passes through the stomach (Tschop et al., 2001). Peripheral infusion or central injection of ghrelin can potently increase food intake and adiposity (Tschop et al., 2000). (2) Ghrelin receptors are mapped to the NPY/AgrP neurons, but not to the POMC-neurons, which is similar to the distribution of PDE3B activity (Cowley, 2003; Kamegai, 2001; Willesen, 1999). (3) Icv injection of ghrelin aVects only the expression of orexigenic NPY and AgrP peptides, but not the anorexic -msh (Cowley et al., 2003; Ishii et al., 2003). (4) Based on sequence and structural analysis, ghrelin receptors are presumably coupled to the Gq, in which case Caþþ and phospholipase C will act as the primary second messengers (Kamegai et al., 2004; Luque et al., 2004; Malagon, 2003). However, a series of studies using the isolated primary hypothalamic neurons demonstrated that ghrelin stimulated Ca2þ-influx via a protein kinase Adependent pathway in the NPY/AgrP neurons, as co-incubation of H-89 could completely block ghrelin’s actions on Ca2þ-influx (Kohno et al., 2003). Interestingly, leptin was able to block the eVect of ghrelin on Caþþinflux in the NPY/AgrP neurons (Kohno et al., 2003; Zhao et al., 2000), which was consistent with the concept of leptin-reducing cAMP in the NPY-neurons

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Figure 2 A working model of hypothalamic regulation of food intake involving modulation of intracellular cAMP levels—a perspective from the arcuate nucleus (ARC). (A) In fasting, the increased plasma concentrations of ghrelin will be sensed by the GHS-receptors in the NPY/ AgrP-neurons. The ghrelin neurons within the hypothalamus also send out ghrelin to the NPY/AgrP-neurons. The subsequent increase of cAMP in the NPY-neurons will stimulate the expression of orexigenic NPY and AgrP. The increased release of NPY will inhibit the neuronal activity of POMC neurons through the Y1-receptors of NPY. Elevation of

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(Shimizu-Albergine et al., 2001; Zhao et al., 2002). In a separate study, ghrelin treatment of somatotropes sharply elevated the level of cAMP as well as the expression of a cAMP-responsive element (CRE)-driven reporter gene (Malagon et al., 2003). It is not unusual for a G-protein coupled receptor to form ‘‘promiscuous’’ partners with diVerent types of G-proteins. For example, MCH receptors have been shown to couple to both Gi and Go/Gq (Hawes et al., 2000). It is important to note that ghrelin may not be the only cAMPelevating hormone in the NPY/AgrP neurons, and that similar roles of other peripheral hormones, though remaining to be defined, should not be discounted.

VIII. A Working Model for Hypothalamic Control of Food Intake Involving Regulation of cAMP—A Perspective from the NPY/AgrP Neurons The evidence summarized so far strongly suggests that regulation of cAMP level at the hypothalamus is critical to the control of energy homeostasis. From the available physiological and molecular evidence, the following working model is proposed to illustrate how modulation of cAMP levels in the ARC of the hypothalamus dictates hormonal regulation of energy homeostasis (Fig. 2A,B). In a negative energy balance state (such as fasting), the plasma concentrations of peripheral hormones like ghrelin will elevate, which, in turn, will be sensed by the hypothalamus, particularly through the GHS-R (growth hormone secretagogue receptor) on the NPY/AgrP neurons in the ARC. Upon ghrelin binding, the levels of cAMP as well as the activity of PKA will increase and consequently stimulate the expression of these two orexigenic peptides. The increased release of NPY will enhance food intake both by directly acting in the PVN and by suppressing the activity of POMC neurons. On a separate front, the secreted AgrP will act as an antagonist of -msh on the melanocortin receptors-3/4 (MC3R and MC4R) to neutralize intracellular cAMP level will also have an inhibitory effect on the tyrosine-phosphorylation and activities of STAT3. (B) In refeeding, the rising leptin signals will be integrated into the NPY- as well as POMC-neurons by the leptin receptors (OBR). Upon leptin binding, PI3-kinase and PDE3B will be activated in the NPY/AgrP-neurons. The subsequent reduction of cAMP will not only shut down the expression of orexigenic NPY and AgrP but will also relieve the inhibitory pressure on STAT3-phosphorylation. The decreased expression of NPY should also diminish the negative effect of NPY on POMC neurons as well as the antagonistic effect of AgrP on melanocortin receptors in the target neurons of -msh. Leptin also induces tyrosinephosphorylation on STAT3 in the POMC neurons, which is expected to increase the expression of POMC gene.

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the anorectic eVect of -msh. As a result of these combined actions, food intake will be initiated. In a positive energy balance state, the circulating leptin and insulin signals will both rise, which, in turn, will be integrated by the neurons in several regions of the hypothalamus, including the ARC. Upon leptin or insulin binding, the PDE3B activity in the NPY/AgrP neurons will be stimulated following the activation of PI3-kinase in the same cell population. The subsequent reduction of cAMP will cause a decrease in PKA activity as well as the phosphorylation of CREB. This decline of cAMP will suppress the expression of NPY and AgrP and thus decrease the orexigenic signals in the PVN. The reduction of cAMP levels will also relieve the inhibitory pressure on the activation of transcription factor STAT3 in the NPY- and/ or POMC-neurons, which will then lead to increased production and release of the anorectic -msh. The binding of -msh to its PVN target neurons will increase the cAMP synthesis through Gs-coupled MC-R, further stimulating the expression of other anorectic neuropeptides, such as CRH.

IX. Dysregulation of cAMP in the Hypothalamus—Implication in Obesity Recent studies have made us realize the importance of modulating cAMP levels within the complex hypothalamic neural network to the overall scheme of food-intake control and body weight regulation. Future studies should be directed toward understanding whether cAMP regulation in the hypothalamus has gone awry in obesity and type-2 diabetes. Already, we know that mutations reducing or nullifying the ability of cAMP-production in the MC4R can cause early onset of obesity and eating disorder in humans (Donohoue et al., 2003). A 2004 study found that leptin’s ability to activate PDE3B is significantly diminished in the hypothalamic tissues of the dietinduced obese rats (Sahu, 2004). However, more genetic and physiological studies are needed, particularly those with neuron-specific knock-out models, to completely elucidate the impact of cAMP-regulating pathways to the long-term control of food intake and body weight.

References Aiston, S., and Agius, L. (1999). Leptin enhances glycogen storage in hepatocytes by inhibition of phosphorylase and exerts an additive eVect with insulin. Diabetes 48, 15–20. Akabayashi, A., Zaia, C. T., Gabriel, S. M., Silva, I., Cheung, W. K., and Leibowitz, S. F. (1994). Intracerebroventricular injection of dibutyryl cyclic adenosine 30 ,50 -monophosphate increases hypothalamic levels of neuropeptide Y. Brain Res. 660, 323–328.

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Factors Affecting Male Song Evolution in Drosophila montana Anneli Hoikkala,* Kirsten Klappert,{ and Dominique Mazzi* *Department of Biological and Environmental Science FIN-40014 University of Jyva¨skyla¨, Finland { Department of Evolutionary Biology, Dyer’s Brae House University of St. Andrews, Fife, KY16 9th, Scotland, United Kingdom

I. Background A. The Evolution of Species-Specific Signals B. Coevolution of Male Signals and Female Preferences Through Sexual Selection C. Why Study Factors AVecting Song Evolution in Drosophila montana? II. Male Song Variation in D. montana and Other Species of the virilis Group A. Species-Specificity of Male Courtship Songs B. Genetic Variation in the Male Courtship Song within the Species C. The Genetic Basis of Male Song Characters in the virilis Group Species III. Female Preferences for Male Song Characters A. Female Preferences for Male Song Traits and the Benefits Gained from Female Mate Choice B. Song Simulation Experiments C. Temperature-Dependence of Male Songs and Females Preferences D. Variation in Female Preference E. Dissecting the Underlying Genetics of Female Preference IV. Song as a Species-Recognition Signal V. Summary Acknowledgments References

D. montana (a species of the D. virilis group) has spread over the northern hemisphere, populations from diVerent areas showing both genetic and phenotypic divergence. The males of this species produce an elaborate courtship song, which plays a major role both in species recognition and in intraspecific mate choice. The genetic architecture and physical constraints, as well as the importance of the signal for species recognition, set boundaries within which this signal can vary. Within these limits, courtship song parameters may change, depending on the males’ physical condition and on the environment they inhabit. Females are likely to aVect song evolution by exerting directional selection toward higher carrier frequencies. Given this complexity, only a comprehensive, multidisciplinary approach, starting Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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with traditional field observation and combining controlled behavioral experiments, biometric measurements, and sophisticated molecular techniques, has the potential of shedding light on the past history and the evolution of this signal, and, eventually, adding to our understanding of the mechanisms, functions, and outcomes of sexual selection in acoustic communication systems. C 2005, Elsevier Inc.

I. Background A. The Evolution of Species-Specific Signals Animal species have evolved a stunning variety of signals. Signal diversity may have arisen as a by-product of genetic divergence in response to diVerent environmental conditions (Mayr, 1963). Alternatively, species-specific signals may have diverged to prevent costly matings between heterospecifics (Dobzhansky, 1951). However, in many species, individuals of one sex, typically males, exhibit spectacular ornaments and/or complex courtship displays that are diYcult to account for on the basis of natural selection (Darwin, 1871). Prime examples of displays that do not make immediate sense from a ‘‘survival of the fittest’’ viewpoint include the peacock’s (Pavo cristatus) train and the ornamental-only nuptial gift prey items provided by male hanging flies (Mecoptera) prior to copulation. It is now generally accepted that the driving force promoting the evolution and the maintenance of those extravagant (and of other less conspicuous) displays is mate choice imposing selection on conspecifics. The alternative pathways of signal evolution need not to be mutually exclusive. For example, one and the same signal can function in species recognition and simultaneously in mate choice, and thus be concurrently aVected by stabilizing natural and directional sexual selection. For the study of complex coevolved behavioral traits, such as male signals and female preferences, it is essential to know whether variation in the traits is qualitatively similar within and among species. The evolution of species-specific signals might involve novel genetic processes such as the fixation of alleles with a large eVect (Coyne et al., 1994) or changes in regulatory genes and genomic resetting (Rose and Doolittle, 1983). Stabilizing and directional selection leave diVerent signs on the genetic architecture of traits, allowing researchers to trace the relative importance of the key forces responsible for their evolution (see, e.g., Hankison and Morris, 2003; Wymann and Whiting, 2003). Moreover, most evolutionary theories assume that signal evolution requires coordinated changes in both sexes, i.e., coevolution of trait and preference, a matter that has proven hard to validate empirically (Pomiankowski and Sheridan, 1994).

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B. Coevolution of Male Signals and Female Preferences Through Sexual Selection Mate choice is the evolutionary process leading to the propensity of members of one sex to mate nonrandomly with respect to one or more varying traits in members of the opposite sex (Heisler et al., 1987). Mate choice (hereafter, used interchangeably with ‘‘female choice’’) often entails costs, which must be outweighed by advantages to choosy females. Choice can be costly, for example, in terms of the time and energy it takes to sample potential mates (Milinski and Bakker, 1992) or increased exposure to predation (Hedrick and Dill, 1993). Sometimes, the act of mating itself can impose direct costs, for example, the toxic compounds in the male Drosophila melanogaster seminal fluids (Chapman et al., 1995) and sexual harassment in the dung fly Sepsis cynipsea (Mu¨ hlha¨ user and Blanckenhorn, 2002). Potential benefits to choosy females fall into two distinct categories. Direct (‘‘material’’) benefits include often readily observable resources, such as parental care (Qvarnstro¨ m et al., 2000), territories (Alatalo et al., 1986), or nutrients (Wagner and Harper, 2003). Indirect (‘‘genetic’’) benefits are less straightforward to detect and, hence, more controversial. Here, the chief claim goes back to a verbal argument constructed by Sir R. A. Fisher (1930) and formalized by Lande (1981). Fisherian models of sexual selection assume an ‘‘arbitrary’’ female preference for a particular male trait that is, at least loosely, associated with male survival. The oVspring of attractive males and choosy females will inherit both the genes for the preferred trait and the genes for the preference, thus giving rise to a genetic correlation between trait and preference (Bakker, 1993; Wilkinson and Reillo, 1994). The feedback of trait exaggeration, even beyond the optimum for survival, and female preference for exaggerated traits has become known as the ‘‘runaway process.’’ It is predicted to come to a halt when the costs to males bearing the attractive trait eventually exceed the benefits through the mating advantage. Choosy females indirectly benefit from the enhanced reproductive success of their attractive male oVspring (‘‘sexy sons’’), which are successful because the spread of the attractive male trait genes is paralleled by the spread of the female preference genes. For a review of the evidence for enhanced fitness of females mated to attractive males, see Møller and Alatalo (1999). The origin and evolution of mate choice raise a series of questions, which have been the subject of vehement debates over the recent history of evolutionary biology (Kirkpatrick and Ryan, 1991). A plausible answer to the question of what prevents low-quality males from unfairly obtaining matings by advertising to the same extent as high-quality males was given by Amotz Zahavi with the crucial notion of the ‘‘handicap principle’’ (Zahavi, 1975, 1977; Zahavi and Zahavi, 1997). The handicap principle states that ornaments are costly or deleterious characters, with the marginal costs of

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producing and maintaining exaggerated ornaments being higher to lowquality males than to high-quality males. Provided the advantage through superior genes obtained from high-quality males outweighs the costs of the handicap, the net quality of choosy females’ oVspring will be higher than those of indiscriminate females. Therefore, the handicap acts as a reliable indicator of genetic quality and its costs ensure the honesty of the signal by preventing low-quality males from cheating (Andersson, 1982; Møller, 1989). Another question undermining the appreciation of good gene models concerns the expected rapid erosion of variation in traits closely associated to fitness. Persistent female preference for certain male traits should eventually drive the preferred trait to fixation, thus eliminating any further benefit of choice. The discrepancy between the expected depletion of variation in sexual traits in the face of strong directional selection and the often substantial variation observed in sexual traits, the so-called ‘‘lek paradox,’’ has long haunted the study of sexual selection (Pomiankowski and Møller, 1995; Rowe and Houle, 1996). Two main arguments have been proposed for its explanation (reviewed in Tomkins et al., 2004). First, fluctuating selection arguments hold that the best phenotype varies in time and space, such that the target of preference moves depending on environmental circumstances. Paramount for the study of sexual selection is the hypothesis put forward by Hamilton and Zuk (1982), suggesting that secondary sexual traits reliably convey heritable resistance to parasites and other pathogens. As only resistant males can produce and maintain exaggerated ornaments, females mating with showy males gain resistance genes for their oVspring (Milinski and Bakker, 1990; Møller, 1990). Maintenance of genetic variation in sexually selected traits is guaranteed by cyclically varying selection pressure, as diVerent host alleles for resistance are advantageous, depending on the current prevalent threat. Second, mutation-selection arguments propose that newly arising mutations supply enough variation to compensate for its erosion by constantly exerted directional selection. The recently resuscitated concept of ‘‘condition-dependence’’ suggests that the overall condition depends on a number of underlying traits, each of which is aVected by mutations to combine into a large mutational target (‘‘genic capture,’’ after Rowe and Houle, 1996) that can counteract the depletion of variation due to continuing directional selection (see Tomkins et al., 2004, for an extensive discussion of the genic capture hypothesis). Kotiaho et al. (2001) showed that in the dung beetle, Onthophagus taurus, male residual body mass (a proxy for condition) is genetically correlated to a sexually selected trait (courtship rate). Hence, in this species, the condition has a genetic basis and courtship rate is a condition-dependent trait preferred by females. While the theories already discussed stem from a functional approach attempting to clear the reasons for mate choice, how mate choice selects

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on sexual traits, and how the two traits (co)evolve, a mechanistic approach produced the concept of ‘‘sensory drive’’ (also known as ‘‘sensory exploitation,’’ ‘‘sensory trap,’’ or ‘‘receiver bias’’). The hypothesis assumes that the female nervous system is tuned to cues that may be important in certain contests such as foraging or predator avoidance. Males evolve traits to exploit such preexisting biases or preferences, and those males that match the signals females are predisposed to perceive as ‘‘attractive’’ trigger a heightened response. This approach notably diVers from ‘‘runaway’’ and ‘‘good genes’’ processes in that preference and trait do not evolve in concert. Rather, the evolution of female preferences precedes the male trait, the latter evolving accordingly to maximally stimulate the female sensory system. Experimental evidence for this hypothesis is provided, for example, by studies on tungara frogs (e.g., Ryan and Rand, 1990, 1995; Wilczynski et al., 2001), swordtail fish (Xiphophorus spp., e.g., Basolo, 1990), guppies (Poecilia reticulata; Rodd et al., 2002), and three-spined sticklebacks (Gasterosteus aculeatus; Smith et al., 2004). Even more radical with respect to the evolutionary dynamics, if less relevant for the evolution of signals, is the relatively recent ‘‘chase-away hypothesis.’’ It states that sexual conflict promotes antagonistic, rather than mutualistic, coevolution, whereby manipulative reproductive strategies in one sex are counteracted by the evolution of resistance to such strategies in the other sex. Experimental evidence for sexually antagonistic genes, which benefit one sex while simultaneously harming the other, is rapidly accumulating, primarily thanks to the work of Rice and coworkers on Drosophila melanogaster (e.g., Holland and Rice, 1999; Rice, 1996, 1998), and, on a phylogenetic level, by Arnqvist and Rowe’s work on water striders (Gerris spp.; e.g., Arnqvist and Rowe, 2002). A comprehensive review on sexual conflict with a particular emphasis on the chase-away hypothesis is found in Pizzari and Snook (2003). In spite of the wealth of available information on male ornaments and displays and on female preferences for them, empirical evidence for the mechanisms and the genetic basis of coevolution is still lacking (Butlin and Ritchie, 1989). Traits such as the male courtship song (produced by wing vibration), and the female preference for this song in various Drosophila species, oVer a valuable opportunity to study the evolution of traits requiring coordinated changes in both sexes. Acoustic signals are relatively easy to record and analyze, and the shape of the female preference can be determined by studying the relationship between parameters of the male song and the probability of female acceptance (Ritchie, 1996). Comparisons of male and female traits in diVerent species and strains with established phylogenies can also provide information as to which of the two traits is driving the coevolution. Therefore, acoustic communication systems may prove well suited to study the factors aVecting display evolution, as well as to

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evaluate the dynamics of coevolution between male displays and female preferences.

C. Why Study Factors Affecting Song Evolution in Drosophila montana? Most genetic studies on Drosophila songs have used D. melanogaster. In spite of the great merits of this species for genetic studies, it is not the best choice for investigating the evolution of male songs/female song preferences since the song of this species is very conserved compared, for example, to the songs of the virilis group species. Also, female preferences in this species are hard to measure and quantify (Ritchie et al., 1999). Sequence data on the song genes as well as on several other genes aVecting signal transmission in D. melanogaster can, however, be used to identify and study ‘‘candidate’’ genes in less well known Drosophila species (e.g., Huttunen et al., 2002a,b), thus providing the chance to combine the benefits of a good genetic model and a good behavioral model in one organism. The virilis group, to which D. montana belongs, is one of the major species groups in the subgenus Drosophila, and there are excellent reviews dedicated to the genetics, biology, and evolution of the group (e.g., Throckmorton, 1982). The virilis group species are good models for evolutionary research because of the high genetic and phenotypic variability between and within the species and of the availability of numerous molecular markers (Huttunen and Schlo¨ tterer, 2002; Orsini and Schlo¨ tterer, 2004; Pa¨ a¨ llysaho et al., 2001) and physical chromosome maps (e.g., Gubenko and Evgenev, 1984) for some species of the group. The virilis group consists of two subgroups, virilis and montana. In the species belonging to the virilis subgroup (D. virilis, D. lummei, D. americana americana, D. americana texana, and D. novamexicana), genetic change was most pronounced as the subgroup became established, and thereafter the species have changed conservatively for both chromosomes and proteins, occupying only one major larval substrate (Throckmorton, 1982). The pattern of evolution in montana subgroup species (D. kanekoi, D. ezoana, D. littoralis, D. borealis, D. flavomontana, and D. montana) has been somewhat diVerent, with definite ‘‘hot spots’’ of major change with ecological shifts and changes in chromosome structure and protein constitution (Throckmorton, 1982). Consequently, the crossability of diVerent species is much higher among the species of the virilis subgroup than among the species of the montana subgroup. Prezygotic reproductive isolation between sympatric virilis group species in Scandinavia (D. montana, D. littoralis, D. ezoana, and D. lummei) seems to be partly due to the diVerence in timing of the mating periods (Aspi et al., 1993) and partly to chemical clues aVecting female attractiveness and acoustic signals produced by individuals of both sexes (Liimatainen

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and Hoikkala, 1998). Females of the virilis group species spread their wings in an ‘‘acceptance posture’’ when they are ready to mate (Vuoristo et al., 1996), which facilitates elaborate studies on female song preferences with simulated male songs in the laboratory (Ritchie et al., 1998). A further advantage of D. montana in behavioral studies is that the females of this species always require hearing the song of the courting male before accepting it as a mating partner (Liimatainen et al., 1992).

II. Male Song Variation in D. montana and Other Species of the virilis Group A. Species-Specificity of Male Courtship Songs Males of diVerent Drosophila species produce a variety of species-specific songs during courtship. The two basic song types are trains of single or polycyclic sound pulses (‘‘pulse song’’) and modified sine waves (‘‘sine song’’; Cowling and Burnet, 1981). The division into pulse song and sine song is not clear, however, and the courtship songs may have features of both song types (Ewing and Miyan, 1986). Pulse songs consisting of single-cycle sound pulses and sine songs are typical for most species of the melanogaster group, while outside this group the sound pulses have become polycyclic and the pulses are arranged in dense pulse trains. Variation in, for example, the intervals between successive sound pulses, carrier frequency, and the length and rhythmic structure of the pulse trains makes the songs of the more than 100 species studied so far species-specific. In the virilis group of Drosophila, the songs of all species consist of polycyclic sound pulses arranged in trains with shorter or longer interpulse intervals (IPIs) between successive sound pulses (Fig. 1; Hoikkala and Lumme, 1987; Hoikkala et al., 1982). The division into the conserved virilis and the more evolved montana subgroup species can also be observed in the evolution of their songs. In the virilis subgroup, the courtship songs of all species consist of polycyclic sound pulses arranged in dense pulse trains with no pauses between successive sound pulses, within-species variation in most song characters exceeding the variation between species (Hoikkala and Lumme, 1987). In the montana subgroup, the songs of all species, except D. borealis, have clear pauses between successive sound pulses, leading to species-specific IPIs (Hoikkala and Lumme, 1987). Other song traits that vary between the species (see Fig. 1) are PN (the number of pulses in a pulse train), PTL (the length of a pulse train), PL (the length of a sound pulse), CN (the number of sound cycles in a pulse), and FRE (the carrier frequency of the song). In several Drosophila species, the song repertoire of males consists of two to four diVerent song types (Ewing, 1970).The flies of both sexes may also

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Figure 1 Oscillograms of the male courtship song in (A) D. virilis, (B) D. montana, and (C) D. littoralis. PL is the pulse length, IPI is the interpulse interval, and PTL is the pulse train length.

produce a specific type of song when they are courted. In males, these songs are called inhibitory songs, while in females, they may have a dual eVect. They may stimulate the males to continue courtship or they may repel males and cause them to stop courting (Satokangas et al., 1994). In the virilis group, the males of four species produce a ‘‘secondary’’ song in addition to the ‘‘primary’’ courtship song, and the males of all species also produce inhibitory songs (Suvanto et al., 1994). The inhibitory songs of males and the songs produced by females are less regular than the actual courtship songs, but they may nonetheless serve sex and species recognition. In D. montana, the male courtship song consists of trains of pulses with short interpulse intervals (Fig. 1). The song traits vary considerably within the species, but a distinctive IPI is retained. D. montana males do not produce a secondary courtship song, but their inhibitory song is species-specific, with

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short pauses between successive sound pulses (Suvanto et al., 1994). Lee (1986) has proposed that male courtship songs have evolved from male refusal signals. In D. montana, inhibitory and courtship songs do indeed have similar features, even though the inhibitory song is more irregular. Also, the songs produced by females of this species resemble the male inhibitory songs but for the absence of a clear pause (Satokangas et al., 1994). B. Genetic Variation in the Male Courtship Song within the Species Genetic drift, combined with adaptation to diVerent environments, can create geographical variation in male song traits, provided that the song is not aVected by stabilizing selection or by directional selection toward a uniform phenotype in diVerent populations. Among the virilis group species, within-species variation in the male courtship songs has been most thoroughly studied in D. littoralis, D. virilis, and D. montana, using both laboratory strains and the progenies of wild-caught females. These studies have revealed that the songs may change during laboratory maintenance and the process of inbreeding (Aspi, 2000). In D. littoralis, the songs of old laboratory strains from Europe and the Caucasus diVered from each other more than the songs of the fresh isofemale strains (progenies of wild-caught females) from three localities in Finland, but the study did not reveal geographic variation in any song trait (Hoikkala, 1985). In D. virilis, laboratory strains from diVerent continents showed divergence in some courtship song traits, but the divergence between old and fresh strains from Japan reached the level of geographical variation (S. Huttunen et al., unpublished data). Studies on song divergence in D. montana populations using both laboratory strains and the progenies of wild-caught females are under way. Preliminary results indicate significant divergence in some song traits among geographically isolated populations, with males from the United States singing at a higher carrier frequency than males from both Canada and Finland (D. Mazzi and A. Hoikkala, unpublished data). Larger geographic variation in male songs among D. montana populations compared to that among D. littoralis and D. virilis populations could be due to two reasons. First, D. montana has spread around the northern hemisphere and adapted to more variable environments than the other two species, especially more than that of D. virilis, which lives near human beings at market places and in breweries (Throckmorton, 1982). Second, among the three above mentioned species, the song is an obligatory prerequisite for mating only in D. montana (Hoikkala, 1988); hence, female preferences could have influenced song evolution diVerently in separate populations. Sexual selection theories assume that the selected male characters exhibit genetic variation. Aspi and Hoikkala (1993) have estimated the heritability

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of diVerent song traits in D. montana and D. littoralis using father–son regression for fathers and sons raised in the laboratory as well as for wildcaught fathers and their laboratory-reared sons. The heritability estimates based on the across-environment regression (the method developed by Riska et al., 1989) were generally lower than the estimates for laboratory-reared flies. In D. montana, significant heritability was found under laboratory conditions for PN (0.66), IPI (0.80), and PL (0.69). Heritability estimates across environments were, however, nonsignificant for all song traits except PN (0.43), mainly because of large phenotypic variability of song characters in the field and, in some cases, because of genotype–environment interactions (Aspi and Hoikkala, 1993). Variability and evolvability of song characters was studied further by Suvanto et al. (1999) in a wild D. montana population. Heritabilities were low and nonsignificant, largely because of high residual variation. Nevertheless, high coeYcients of additive variation (CVAs) implied that the male song still retains additive genetic variation. CVAs of sexually selected traits were even higher than those of more ‘‘neutral’’ song traits. The authors also found evidence of genotype–environment interactions increasing the amount of additive variation among males in sexually selected song traits. There is some evidence of condition-dependence of sexually selected song traits in D. montana. Aspi and Hoikkala (1995) found copulating D. montana males to have shorter PLs (in one study year, also higher PN) than randomly sampled males, suggesting that short and dense sound pulses (i.e., high carrier frequency) are favored by females in wild populations. The selection diVerentials for most song characters for males collected in the wild before and after overwintering were opposite in sign to the selection diVerentials of sexual selection. Here as well, PL and PN were the main targets of selection. Hoikkala and Isoherranen (1997) further showed that the pulse characters (PL, CN, and FRE) are more sensitive to a cold treatment than the pulse train characters, repeatability of these characters being higher among overwintered males than among males reared in the laboratory. However, male song characters are also sensitive to environmental temperature (Hoikkala, 1985; Ritchie et al., 2001); hence, the quality of the male song may vary from day to day and even within one given day in the wild, making sexual selection based on male song variation increasingly challenging for females.

C. The Genetic Basis of Male Song Characters in the virilis Group Species In the virilis subgroup, which includes species with structurally similar songs, the genetic basis of between-species song variation is mainly polygenic and autosomal, whereas in the montana subgroup, the species-specific characters

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of the male courtship song are largely determined by one or more X chromosomal gene(s) interacting with a few autosomal genes (Hoikkala and Lumme, 1987). Hoikkala and Lumme (1987) have suggested that a major change on the X chromosome has occurred during the separation of the virilis and montana subgroups, allowing variation in IPI, and, consequently, also in PL. A major gene (or a group of genes) largely responsible for song diVerences between D. virilis (IPI about 20 ms) and D. littoralis (IPI about 300 ms) has been localized on the proximal end of the X chromosome, but more thorough localization attempts have failed because of the species diVerences in the gene arrangement in this area (Hoikkala et al., 2000; Pa¨ a¨ llysaho et al., 2001). Crosses between D. virilis (a virilis subgroup species) and D. flavomontana females (a montana subgroup species) with D. virilis, D. flavomontana, D. montana, and D. littoralis males confirmed the central role of an X chromosomal gene in determining the length of the pause between successive sound pulses and, together with autosomal genes, in determining the PL also in other species of the montana subgroup (Pa¨ a¨ llysaho et al., 2003). In D. flavomontana, D. montana, and D. littoralis, the hybrid songs showed dominance for shorter sound pulses and longer pauses between the pulses, suggesting this to be the direction of song evolution in the studied species (assuming the direction of dominance indicates the direction of evolution; Wagner and Bu¨ rger, 1985). Diallelic crosses between four inbred D. montana strains revealed additive genetic variation in PN, IPI, CN, and FRE, but not in PTL (Suvanto et al., 2000). In this study, the carrier frequency of the song (FRE) showed heterosis and unidirectional dominance toward a higher song frequency. The pulse characters PL and FRE are highly correlated and, in these characters, the direction of song evolution in the species of the montana subgroup and within D. montana seems to be the same. Another study on some of those same inbred strains (Aspi, 2000) showed evidence of additivity for the pulse train characters (PN, PTL) and IPI, and additivity, dominance, and epistasis for the pulse characters (PL, CN, and FRE). In FRE, the inbreeding depression was 14%, suggesting that this song character is associated with fitness. Both these studies indicate that song variation among conspecific strains is mainly owed to autosomal genes. Thus, the X chromosomal gene responsible for interspecific song diVerences seems to matter less for intraspecific song variation. More thorough analyses (pedigree and QTL analysis) of the genetic basis of song variation within and among D. montana populations are in progress (K. Klappert and D. Mazzi, unpublished results). Several mutations are known to alter the patterns of male courtship songs in D. melanogaster, and the genes (e.g., per and nonA) where these mutations have occurred can be regarded as candidate genes aVecting natural variation (Peixoto and Hall, 1998). Candidate genes can be used as markers in QTL analyses to determine if any of them coincide with QTLs of large eVect

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(Gleason and Ritchie, 2004). Orthologous genes from other Drosophila species have also been transformed into D. melanogaster to investigate whether they carry species-specific information on song traits. Campesan et al. (2001) transformed nonA gene of D. virilis in D. melanogaster and found the courtship song of transformant males to have several features of the D. virilis song, which indicates that nonA may encode species-specific information on male song traits. Huttunen et al. (2002a) did not, however, corroborate the idea that the nonA coding region aVects interspecific song diVerences in the virilis group species when comparing the DNA sequences of the coding region of the nonA gene of D. littoralis, D. virilis, and D. melanogaster (Huttunen et al., 2002b) or when studying sequence variation in a repetitive region of the gene in all the species of the virilis group.

III. Female Preferences for Male Song Characters A. Female Preferences for Male Song Traits and the Benefits Gained from Female Mate Choice The virilis species group has a short mating period in spring, in which the flies gather on food patches of rotting plant material, where courtship and mating takes place (Aspi et al., 1993). Female choice can be relatively cheap on these sites, since females can sample among several males within a short period of time without the need to travel between locations. Hence, females are not likely to pay high costs of choice in terms of energy expenditure or enhanced exposure to predation risk. This form of ‘‘lekking,’’ as well as the comparatively large body size of D. montana, enable the study of their mating behavior not only in the laboratory, but also in the wild (Aspi et al., 1993). In their 2-year field study, Aspi and Hoikkala (1995; see the chapter on male songs) showed D. montana females to prefer males with short and dense sound pulses, resulting in a song with a high carrier frequency. In the field, male mating success depends largely on the phenotypic variation in courtship traits among the males and not solely on sexual selection exercised by the females. Significant diVerences in the songs of the males before and after hibernation may reflect phenotypic condition of the males, suggesting that the songs of inferior males might have changed over the winter more than the songs of relatively high-quality males. As the flies mate in spring, high variation in the pulse characters of overwintered males oVers the females a good opportunity for choice during the mating season, and condition-dependency of these characters enables the females to select a male in good condition as a mating partner (Zahavi, 1977). The magnitude of sexual selection on male characters can change over the years, as shown in the study by Aspi and Hoikkala (1995). Small males had a

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mating advantage in some years, but this was not a consistent pattern. Male size does not seem to play an important role for mate choice in D. montana, the advantage of males with a small body size in the field possibly being due to diVerent environmental conditions and not necessarily to active female choice or female preferences (small males have a larger wing-load index and are better able to fly in colder temperatures; Aspi and Hoikkala, 1995; Hoikkala and Suvanto, 1999). The real targets of sexual selection may thus be diYcult to identify, due to masking eVects of environmental conditions and phenotypic flexibility. Because of the large eVects of environmental conditions on male songs, the targets of sexual selection have been further dissected in lab studies. The finding that D. montana females pay attention to the pulse characters (PL, CN, FRE) of male songs (Aspi and Hoikkala, 1995) was confirmed by mate choice experiments with lab-reared males (Hoikkala and Suvanto, 1999), where the males producing high-frequency songs succeeded in their courtship more often than males that produced low-frequency signals. For this study, F2-oVspring of wild-caught females were kept for six months in ‘‘artificial winter’’ (cold and dark room, 4
C) prior to the experiment. Male songs were recorded both before and after the cold treatment, and the changes within the males were found to be significant (Hoikkala and Suvanto, 1999; Suvanto et al., 1999). Male mating success was measured as the number of successful trials in direct mate choice experiments, where each male competed in all possible pairwise combinations against all the other males (three replicates with nine males each, n ¼ 36). The mating success of the males correlated strongly with the carrier frequency of their song recorded after, but not before, the cold treatment. Females copulated more often with the male that began to court first, but even when male precedence was entered as a covariate, song frequency was significantly correlated with male mating success. The finding that female preference is based on a male song trait that changes considerably during hibernation suggests that this trait reflects the viability and condition of the males during the spring mating season, further supporting the claim that sexual selection in D. montana has evolved in concert with the predictions made by viability indicator hypotheses. What kinds of benefits do the females gain from choosing a good singer as their mating partner? Hoikkala et al. (1998) showed that D. montana females gain indirect benefits (better survival of the progeny; Fig. 2), but not direct benefits (e.g., increased fecundity) from mating with males producing highfrequency songs. In this experiment, the courtship songs of 100 wild-caught males were recorded, after which the males were mated with lab-raised females in single-pair matings for an analysis of fecundity and oVspring viability. Females mated with males producing a song with a comparatively higher carrier frequency gained indirect benefits in terms of enhanced

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Figure 2 Nonparametric fitness function (solid line) for male oVspring survival as a function of courtship song frequency. OVspring survival was estimated as a proportion of oVspring emerging from the eggs oviposited within the first 3 days after copulation. The fitness function was estimated using the cubic-spline approach (Schluter, 1988), which provides an univariate nonparametric estimate of fitness probabilities across the range of considered characters (from Hoikkala et al., 1998).

oVspring survival. Consequently, the carrier frequency of the song, which has been shown to be a target of female choice (Aspi and Hoikkala, 1995; Hoikkala et al., 1998), to be sensitive to environmental factors (Hoikkala and Isoherranen, 1997), and to reflect male sexual activity (Hoikkala et al., 1998), is a good candidate for a sexual trait that has evolved through a viability indicator process (Rowe and Houle, 1996).

B. Song Simulation Experiments Usually, prior to being mounted by a male, D. montana females show acceptance by spreading their wings (Vuoristo et al., 1996). The elegance of female choice experiments with D. montana lies in utilizing this wingspreading posture in playback experiments. This experimental setup allows determining female preference functions with great precision without the drawbacks of mass mating experiments. The experiments can be performed in the absence of males, but the presence of a muted male (whose wings have been amputated) can enhance female responsiveness by about 40% without altering the overall preference functions (Ritchie et al., 1998). A further advantage of playback experiments using synthetic songs is the possibility to create songs diVering only in the trait of interest. The relative importance of specific song characters can hence be analyzed separately, with the females’ choice being unbiased by unknown correlated characters. By using

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playback experiments, and thus uncoupling song from other male characteristics, it is possible to determine the eVects of sexual selection acting on specific song parameters rather than on correlated parameters. Ritchie et al. (1998) confirmed the correlational evidence for selection pressure exercised by the females on male song traits by playing synthesized song varying independently in pulse length and carrier frequency back to females. Songs consisting of short sound pulses with a high carrier frequency were the most attractive, with more than 60% of females responding to the stimuli, followed by songs with short, medium-frequency pulses and medium length, low-frequency pulses with a proportion of approximately 45% of females responding. The combination of both pulse length and carrier frequency was more important than either of the parameters alone for the eVectiveness of a song at evoking female responses. Variation in female response rate was, however, not due to the eVect of diVerent synthetic songs on the wingless males’ courtship intensity. The shape of the preference functions of D. montana females for pulse length and carrier frequency was further characterized by fitting parametric and nonparametric functions to the frequency of female acceptance responses to synthetic songs (Ritchie et al., 2001). The regression coeYcients derived by the model revealed that females prefer short pulse length (significant linear regression coeYcient) with medium carrier frequencies (significant quadratic regression coeYcient). This finding was confirmed by fitting a cubic spline (Ritchie, 1996; Schluter, 1988), a nonparametric estimation of the female preference function (Fig. 3). This result may, at first, seem to contradict earlier findings of field and lab studies in that in this experiment females do not exert a directional selection for higher song frequencies, but rather a stabilizing selection toward medium frequencies. However, the song models used in the playback experiment exceeded the range of carrier frequencies naturally produced by males, and females preferred frequencies peaking around 350 Hz. This is at the upper range of frequencies produced by males and would thus result in directional selection for higher carrier frequencies within the population. Therefore, the previous studies are confirmed in their finding that songs with short pulses and high carrier frequency are the most attractive.

C. Temperature-Dependence of Male Songs and Females Preferences Acoustic communication and signaling are widespread among poikilotherm animals. Sound production in these systems often relies on muscle contraction performance and is therefore severely aVected by environmental temperature (Bailey, 1991). This has implications for sexually selected male courtship signals, as most hypotheses on sexual selection rely on the honesty

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Figure 3 Mean cubic spline fitted to female responses when presented with song varying in (a) pulse length (smoothing parameter ¼ 16.69) and (b) carrier frequency (smoothing parameter ¼ 10.53). Dotted curves are 1 standard error and the shaded blocks on the abscissae indicate approximate 95% confidence intervals of the trait in males (from Ritchie et al., 2001).

of male signals as indicators of genetic quality (reviewed by Kokko et al., 2003). If the signals show environmentally influenced plasticity, their role in mate choice becomes questionable. One possible solution to this problem would be an adjusted temperature-dependent female preference function which fits to the altered male signaling in diVerent temperature regimes, the so-called ‘‘temperature coupling’’ (Gerhardt, 1978). This mechanism has indeed been found in many acoustic signaling species, mostly in frogs and insects (Pires and Hoy, 1992). D. montana females have to be able to choose a good singer even if the male song traits vary according to the environmental temperature. Ritchie et al. (2001) investigated the temperature-coupling hypothesis in D. montana with song simulation experiments. The courtship song of D. montana males changes quite substantially with temperature (approximately 15 Hz

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C;

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per Ritchie et al., 2001), and thus it is conceivable that female preference and male trait coordination is maintained by some sort of temperature coupling. This mechanism would reduce the chance of a female’s mating with a relatively low-quality male producing a high frequency song because it is warmer than others. Preference was investigated by female response to synthesized songs that varied in carrier frequency between 170 and 500 Hz in 30 Hz increments. Females were kept at the respective temperature overnight before the playback experiment to allow temperature adjustment. The preference function was found to be similar under diVerent temperature regimes (15, 20, and 25
C) with the response curve being less strongly peaked at 20
C than at the other temperatures. DiVerentiation of the fitted cubic splines allowed calculating the peak preference. However, the female peak preference only poorly matched that predicted from the distribution of carrier frequencies of males at the same temperature. Thus, in D. montana, male song and female preference function do not seem to be coupled with respect to temperature. Ritchie et al. (2001) suggested that temperature coupling often arises due to a common eVect of temperature on song and preference, rather than as an advantageous characteristic whose function is to maintain coordination in temperature-aVected communication systems. Females can adopt three diVerent, not mutually exclusive strategies in their mate choice: threshold, absolute, or relative mate choice (Lande, 1981), with the choice of strategy being critical in environments where the male signals vary according to the temperature. Threshold and absolute mate choice require a male signal which exceeds an internally determined and genetically fixed threshold value in the female or falls within an arrow preference window. Females using relative choice, however, can choose the best male from the available potential partners (‘‘best-of-n’’ rules; Jennions and Petrie, 1997), preferring a male with a character more extravagant than the mean. This flexible mate choice strategy thus allows females to choose attractive males even when the males’ courtship signals vary with environmental conditions, though even with this strategy problems occur when the choice is sequential rather than simultaneous. Hoikkala and Aspi (1993) have shown that D. montana females exert a relative mate choice with strong directional selection for higher song frequencies and become more selective when they can choose between two courting males, as long as the signals emitted by the courting males are within the range of acceptable cues. In the wild, this tactic is feasible, as D. montana flies gather on patches of decomposing masses of plant material where they court and mate (Aspi et al., 1993), allowing females to easily sample several potential mates. Hence, Drosophila females are able to remember the stimuli they received during courtship for some minutes (Kyriacou and Hall, 1984) and thus they are able to compare prospective mates within this time span. Costs of comparing several prospective mates are low and probably do not outweigh the benefits

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gained by such a strategy; therefore, ‘‘it is not surprising that D. montana females have adopted a relative mate choice strategy’’ (Aspi, 1992).

D. Variation in Female Preference As already mentioned, female preference does not only vary between, but also within, the species. Suvanto et al. (2000) conducted experiments to determine the genetic variation in mating signals and mate choice in crosses between and within inbred strains of D. montana. Male song characteristics and cuticular hydrocarbons of both sexes, as well as some behavioral traits, were found to diVer among these strains, which were derived from lab strains originating from Finland, Japan, and Alaska, and have been inbred for 20 generations. There was no sexual isolation between the strains, however, with the courtship being even shorter when females were courted by males of a foreign strain. Ritchie et al. (2004) analyzed the variance among F1-families from a natural population of D. montana in Finland for components of male songs and female preferences. All song traits varied to a great extent among families, with carrier frequency (the most important trait in sexual selection) showing the greatest variation. This gives additional support to the claim that carrier frequency is a condition-dependent song trait indicating male quality. There was also significant evidence for genetic variation both between sisters within families and among families for some components of female preference. The slope of the linear female preference function for carrier frequency did not vary, but females varied significantly in overall responsiveness indicating individual diVerences in choosiness. More choosy females could have been expected to preferably mate with males of higher quality, thus leading to assortative mating, but no covariance between female responsiveness and male carrier frequency across families was found.

E. Dissecting the Underlying Genetics of Female Preference The ultimate aim of studies on the evolution of male traits and female preferences is the investigation of coevolution of preference and traits. However, very few studies on covariance have been conducted so far (see review in Bakker and Pomiankowski, 1995). While the genetic basis of the male acoustic signal has been analyzed in many species (e.g., Gleason et al., 2002; Williams et al., 2001; see also paragraph on male song in this publication), it is far more diYcult to do the same for female preference (Doi et al., 2001; Ting et al., 2001). So far, most studies dissecting the genetic architecture of female preferences have relied on classical genetic approaches, such as that in D. montana by Isoherranen et al. (1999). In this study, the behavior of single pairs of

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D. virilis, D. montana, the hybrid F1, and the back-crosses (BC), with the males being normal or mute, were observed. DiVerences among the diVerent BC and their parental species suggested that female song requirements and female receptivity are determined by diVerent genetic factors. Only since 2000 have researchers begun to utilize molecular genetic techniques such as QTL, pedigree, or animal model analysis to answer questions about the genetic architecture of female preference and covariance between traits and preference (see review in Kyriacou, 2002).

IV. Song as a Species-Recognition Signal Virilis group species sharing the same habitat diVer in their songs, suggesting that the songs may be relevant for species recognition (Hoikkala et al., 1982). Species-recognition signals should show constancy over the species distribution range if they are under stabilizing selection (Henderson and Lambert, 1982). In D. montana, one song trait, IPI, fulfills this prerequisite, as it remains invariable (contrary to other song traits) in songs of males from natural populations in Europe and North America (D. Mazzi and A. Hoikkala, unpublished results). This song trait is thus a good candidate for a speciesrecognition signal, that is it seems to have, in the virilis group species, the same role as in some other Drosophila species (Tomaru et al., 1995). In Scandinavia, interspecific courtships are quite common among four partially sympatric species of the virilis group (D. montana, D. ezoana, D. littoralis, and D. lummei), but these courtships have never been observed to lead to copulation (Liimatainen and Hoikkala, 1998). A heterospecific male courting a D. montana female is rejected as soon as it vibrates its wings and produces a ‘‘wrong’’ song (Liimatainen and Hoikkala, 1998). Thus, the male song seems to play a major role in species recognition and in preventing interspecific matings. Saarikettu et al. (unpublished) showed that the sexual isolation between D. montana females and D. lummei males can be overcome by playing back to D. montana females simulated courtship songs with species-specific characters, while the female is being courted by a mute D. lummei male. Here, a change in a single character, IPI, significantly contributed to increasing female acceptance, thus providing convincing evidence for the importance of acoustic cues in maintaining sexual isolation between closely related species.

V. Summary D. montana is a unique species in several respects, diVering notably even from closely related species belonging to the same species group. It has spread over the northern hemisphere, populations from diVerent areas

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showing both genetic and phenotypic divergence. The obligatory song performed by males during courtship plays a major role in the mating rituals of this species, bearing relevance to both mate choice and species recognition. Geographic variation in male song does not necessarily mirror genetic divergence of populations, implying that song evolution has been aVected by diVerent selection pressures in diVerent populations. D. montana has a short mating period in spring, during which the flies gather on food patches, where courtship and mating take place. Here, females mate preferentially with males whose courtship song consists of short and dense (high frequency) sound pulses (Aspi and Hoikkala, 1995), gaining indirect benefits (better oVspring survival) from their choice. The carrier frequency of the song is sensitive to environmental factors and also reflects male sexual activity (Hoikkala and Suvanto, 1999) and is therefore a good candidate for a sexual trait that has evolved through a viability indicator process (Rowe and Houle, 1996). There is evidence of a major X chromosomal gene (or a group of closely linked genes) aVecting among-species diVerences in male song in the montana subgroup (Hoikkala and Lumme, 1987). Sex-linked genes do not, however, have a detectable eVect on song variation within D. montana (Aspi, 2000; Suvanto et al., 2000). Thus, the genetic basis of inter- and intraspecific song variation seems to be diVerent, interspecific variation being of ‘‘type I’’ architecture (one or a few genes of large eVect and a few genes of minor eVect), while intraspecific variation is of ‘‘type II’’ architecture (many genes of small eVect), after Templeton’s (1981) classification. The song traits subject to female choice have more additive variation (low h2, but high CVA) than the more neutral song traits in a wild population in Finland (Suvanto et al., 1999). As stabilizing and directional selection leave diVerent signs on the genetic architecture of the song traits, it is possible to trace the past selection pressures as well as to predict the direction of song evolution with biometrical methods. In D. montana, such studies have revealed significant directional dominance for higher carrier frequency, suggesting that the song of this species has evolved toward high frequency (Suvanto et al., 2000). The direction of dominance corresponds to the direction of female preferences for male songs, indicating that female preferences might be a driving force in song evolution (e.g., Ritchie et al., 1998). There is evidence of genetic variation among the progeny of wild-caught females in overall female responsiveness, but not for variation in the slope of the linear female preference function for carrier frequency (Ritchie et al., 2004). Thus, the issue of genetic correlation between male song traits and female preferences, which is central to sexual selection theories, remains open. In conclusion, the courtship song of the male D. montana is an elaborate signal, the evolution of which is subject to the influence of a number of

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internal and external factors. The genetic architecture and physical constraints set boundaries within which the signal can vary. A further restraint is imposed by the role of the signal as a species recognition mechanism. Within these limits, courtship song parameters may change depending on the male’s physical condition and on the environment the male inhabits. Females are likely to aVect song evolution by exerting directional selection on the carrier frequency of the song. Given this complexity, only a comprehensive, multidisciplinary approach starting with traditional field observation, and combining controlled behavioral experiments, biometric measurements, and sophisticated molecular techniques has the potential of shedding light on the past history and the evolution of this signal, and, eventually, adding to our understanding of the mechanisms, functions, and outcomes of sexual selection in acoustic communication systems.

Acknowledgments We are grateful to M. G. Ritchie for comments. The work was supported by the EU Research Training Network HPRN-CT-2002-00266 and a grant from the Academy of Finland (50591) to A. Hoikkala.

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Prostanoids and Phosphodiesterase Inhibitors in Experimental Pulmonary Hypertension Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, and Norbert Weissmann Department of Internal Medicine II, Justus-Liebig University Giessen, 35392 Giessen, Germany

I. Introduction II. Animal and Organ Models of Pulmonary Hypertension III. Prostanoids and PDE Inhibitors in Acute and Chronic Hypoxia A. Acute Hypoxia as a Model for Pulmonary Hypertension B. Chronic Hypoxia as a Model for Pulmonary Hypertension IV. Prostanoids and PDE Inhibitors in Monocrotaline-Induced Pulmonary Hypertension A. Monocrotaline as a Model of Pulmonary Hypertension V. Prostanoids and PDE Inhibitors in U46619-Induced Acute Pulmonary Hypertension A. U46619-Induced Vasoconstriction as a Model of Pulmonary Hypertension VI. Less Frequently Used Models of Experimental Pulmonary Hypertension A. Pulmonary Embolism B. Meconium Aspiration C. Shear Stress VII. Combination of PDE Inhibitors with Vasodilators VIII. Summary and Concluding Remarks Acknowledgments References

Pulmonary arterial hypertension (PAH) is a progressive disease with a poor prognosis, characterized by intimal lesions, medial hypertrophy, and adventitial thickening of precapillary pulmonary arteries. Several approved therapies are currently available for the treatment of PAH, of which intravenous epoprostenol is the best explored over the past decade. Newly available oral endothelin receptor antagonists, although clinically eYcacious, bear the risk of liver toxicity in a significant portion of patients. Substances that stimulate the formation of the second messengers cyclic adenosine monophosphate (cAMP) or guanosine monophosphate (cGMP) have proved useful in the treatment of various forms of pre-capillary pulmonary hypertension. These second messengers of the endogenous vasodilator mediators that include prostacyclin and nitric oxide (NO) are hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs), a class of Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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enzymes from which 11 isoforms have been characterized. This chapter highlights developments in the treatment of experimental pulmonary hypertension with special attention to prostanoids and PDE inhibitors. We summarize findings for the acute vasodilatory as well as chronic eVects of prostanoids, PDE inhibitors, or combinations of both, in animal models of pulmonary hypertension. C 2005, Elsevier Inc.

I. Introduction Pulmonary arterial hypertension (PAH) is an unresolved clinical challenge, linked with high mortality based on structural changes in the pulmonary arteries (D’Alonzo et al., 1991). These structural changes include distal extension and proliferation of smooth muscle cells as well as proliferation of fibroblasts and abnormal endothelial cell proliferation (for overviews, see Humbert et al., 2004; JeVery and Wanstall, 2001). The increased pulmonary vascular resistance increases right heart afterload, concomitant with right ventricular hypertrophy that may culminate in cor pulmonale. Imbalances in vasodilatory and vasoconstrictive mediators have been implicated in both the predominance of increased vasomotor tone and in the chronic remodeling of resistance vessels. The first evidence suggesting a role for prostanoids in pulmonary hypertension was reported by Christman et al. (1992), who demonstrated a reduced excretion of prostaglandin (PG)I2 and an enhanced excretion of thromboxane metabolites in patients with idiopathic pulmonary arterial hypertension (IPAH, formerly primary pulmonary hypertension (PPH)). Prostaglandin I2 (PGI2, prostacyclin) is a metabolite of arachidonic acid that is produced by the vascular endothelium. It stimulates the formation of the second messenger cyclic adenosine monophosphate (cAMP) through activation of the prostacyclin receptor, which inhibits the proliferation of smooth muscle cells and decreases platelet aggregation (Kothapalli et al., 2003; Li et al., 2004; Wharton et al., 2000) (Fig. 1). In line with the observations of Christman and colleagues, the pathophysiological role of the prostacyclin system was underscored by the observation that expression of prostacyclin synthase in patients with severe pulmonary arterial hypertension was decreased (Tuder et al., 1999). Treatment of patients suVering from PAH with prostacyclin or prostacyclin analogues has been shown to improve pulmonary hemodynamics and the clinical situation when applied either by infusion, inhalation, orally, or subcutaneously (for a review, see Badesch et al., 2004). However, the cAMP-mediated eVects of prostacyclin are limited by a group of specific nucleotide phosphodiesterases (PDEs), which hydrolyze the prostacyclin-induced second messenger cAMP. In addition, the second messenger cGMP, which is derived from activation of guanylate cyclases, either by nitric oxide or natriuretic peptides, is

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Figure 1 Signaling pathways of prostanoids, nitric oxide (NO), and natriuretic peptides. Ligands (i.e., prostacyclin, NO, atrial natriuretic peptide [ANP] or brain natriuretic peptide [BNP]) activate membrane-bound or soluble cyclases. Adenylate and guanylate cyclases generate cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) from ATP and GTP. The intracellular second messengers cAMP and cGMP activate protein kinases, which phosphorylate target proteins and induce cellular responses (i.e., vasodilation, inhibition of smooth muscle cell proliferation, platelet aggregation, gene transcription) and are thus key mediators to target for the treatment of pulmonary hypertension. Phosphodiesterases (PDEs) limit the eVects of the ligands by degradation of the second messengers cAMP and cGMP into inactive AMP and GMP (modified from Ghofrani et al., 2004b).

hydrolyzed by specific PDEs (Fig. 1). At present, mammalian PDEs are divided into 11 distinct families based on domain structure and catalytic and regulatory properties (Table I). By alternative mRNA splicing, and the use of alternative promoter initiation sites, more than 50 isoenzymes have been identified (Beavo, 1995; Conti and Jin, 1999; Conti et al., 1995; Maurice et al., 2003). In experimental pulmonary hypertension, enhanced activity of PDEs was observed (MacLean et al., 1997; Wagner et al., 1997), which suggested the potential of PDE inhibitors as therapeutic tools that could augment and prolong prostanoid-induced vascular eVects. Application of selective PDE inhibitors in patients with PAH has consistently resulted in potent pulmonary vasodilation (Ghofrani et al., 2002c, 2003a,b) and amplification of the eVects of inhaled iloprost, a stable prostacyclin analogue (Ghofrani et al., 2002a).

Table I Selective Inhibitors for Cyclic Nucleotide Phosphodiesterase Familiesa PDE family regulation

Substrate

PDE1: Ca2þ/CAM-stimulated PDE

cAMP and cGMP

PDE2: cGMP-stimulated PDE PDE3: cGMP-inhibited PDE

cAMP and cGMP cAMP > cGMP

PDE4: High aYnity, Rolipram-sensitive cAMP-specific PDE

cAMP Piclamilast (RP 73-401) Zardaverine Tolafentrine cGMP

PDE5: cGMP-specific PDE

IC50, mM

Inhibitor 8-MethoxymethylIBMX Vinpocetine EHNA Cilostamide Milrinone Amrinone Zardaverine Tolafentrine Enoximone Siguazodan Vesnarinone Motapizone Trequinsin (HL-725) Indolindan (LY195115) SCA40 Rolipram 0.001 0.2 0.09 Zaprinast (MB22948) Sildenafil Vardenafil

Reference

4

(Wells and Miller, 1988)

20 1

(Hagiwara et al., 1984) (Mery et al., 1995)

0.005 0.3 16.7 0.6 0.06 1 0.4 8.5 0.03-0.008 0.0003 0.08 0.8 2 (Jacobitz et al., 1996) (Schudt et al., 1991) (Schermuly et al., 1999) 0.76 0.003 0.001

(Hidaka and Endo, 1984) (Harrison et al., 1986) (Sudo et al., 2000) (Schudt et al., 1991) (Schermuly et al., 1999) (Kariya et al., 1982) (Torphy and Cieslinski, 1990) (Masuoka et al., 1993) (Rabe et al., 1993) (Ruppert and Weithmann, 1982) (KauVman et al., 1986) (Cortijo et al., 1996) (Sheppard et al., 1972)

(Gillespie and Beavo, 1989) (Ballard et al., 1998) (Saenz et al., 2001)

PDE6: Photoreceptor cGMPspecific PDE

cGMP

PDE7: High-aYnity, Rolipraminsensitive cAMP-specific PDE PDE8: High-aYnity and IBMXinsensitive cAMP-specific PDE PDE9: High-aYnity cGMPspecific PDE PDE10: cAMP-Inhibited cGMP PDE PDE11: Dual specificity cGMP-binding PDE

cAMP

a

Tadalafil Dipyridamole* E4010 E4021 UK343-664 DMPPO T1032 DA8159 Zaprinast Dipyridamole* Sildenafil IBMX Dipyridamole

cAMP

Dipyridamole*

cGMP

Zaprinast Sildenafil Dipyridamole* Zaprinast Zaprinast Dipyridamole* Tadalafil Vardenafil

cAMP < cGMP cAMP and cGMP

0.001–0.007 0.9 0.0005 0.004 <0.003 0.003 0.001 0.005 0.15 0.38 0.03 3 9 4.5–40

35 7 1.1 10.8 12 0.37 0.037 0.162

(Gresser and Gleiter, 2002) (Nemecek and Honeyman, 1982) (Watanabe et al., 2000) (Saeki et al., 1995) (Bonnell et al., 2004) (Coste and Grondin, 1995) (Kotera et al., 2000) (Doh et al., 2002) (Gillespie and Beavo, 1989) (Gillespie and Beavo, 1989) (Kotera et al., 2000) (Hetman et al., 2000) (Hetman et al., 2000) (Fisher et al., 1998) (Hayashi et al., 1998) (Soderling et al., 1998) (Fisher et al., 1998) (Soderling et al., 1998) (Soderling et al., 1999) (Soderling et al., 1999) (Fawcett et al., 2000) (Fawcett et al., 2000) (Gresser and Gleiter, 2002) (Gresser and Gleiter, 2002)

EHNA, erythro-9-(2-hydroxy-3-nonyl)adenine; IBMX, 3-isobutyl-1-methylxanthine; DMPPO, 1,3 dimethyl-6-(2-propoxy-5-methane sulphonylamidophenyl)-pyrazolo[3,4-d]pyrimidin-4-(5H)-one; Ca2þ/CAM, calcium/calmodulin; *, inhibits adenosine uptake.

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Thus, as summarized in Fig. 2, the increase of either cAMP or cGMP levels is a promising therapeutic approach for the treatment of pulmonary hypertension, since both of these agents have vasodilating and antiproliferative properties (Kothapalli et al., 2003; Li et al., 2004; Wharton et al., 2000). The levels of cAMP and cGMP can be increased by stimulation of their respective cyclases, or by inhibition of their degradation. Animal models of experimental pulmonary hypertension have been employed in the development of new therapeutic options for the treatment of pulmonary hypertension, specifically with respect to the use of prostanoids or PDE inhibitors. Furthermore, animal models have been used to evaluate and decrypt empirical observations from investigations conducted in patients treated with prostanoids or PDE inhibitors. Thus, the aim of this chapter is to summarize these investigations and the developments in experimental pulmonary hypertension, focusing on the eVects of prostanoids and PDE inhibitors as drugs for the treatment of pulmonary hypertension. Consequently, the overview given here is (a) restricted to investigations in intact lungs or in animal models, and therefore does not focus on studies conducted at cellular or vascular levels, and (b) does not focus on studies with nitric oxide.

Figure 2 Schematic depiction of synergism between agonists and phosphodiesterase inhibitors. DiVerent agonists, e.g., prostanoids, nitric oxide (NO), and natriuretic peptides (atrial natriuretic peptide [ANP] or brain natriuretic peptide [BNP]), increase the intracellular concentrations of the second messengers cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP). Concomitant application of cyclic phosphodiesterase (PDE) inhibitors, either intravascular or inhaled, stabilizes the second messenger and amplifies the eYcacy of the agonists.

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II. Animal and Organ Models of Pulmonary Hypertension The most common animal models used for investigations of pulmonary hypertension are (a) exposure to chronic (normobaric or hypobaric) hypoxia and (b) subcutaneous or intravenous application of monocrotaline or monocrotaline pyrrole. Additional stimuli, including increased shear stress (endarterectomy, thromboembolism, monocrotaline in combination with pneumectomy, ductus arteriosus ligation) or genetic alterations have been employed, albeit less frequently, to induce pulmonary hypertension in diVerent mammals (JeVery and Wanstall, 2001). Aside from these chronic approaches, which are accompanied by structural changes to the pulmonary vasculature, setups with acute induction of pulmonary hypertension have also been used. In these models, acute alveolar hypoxia or an intravenous infusion of the thromboxane mimetic U46619 has been used to increase vascular tone. These investigations can be performed in intact animals and also in explanted, artificially ventilated and perfused lungs, with the advantage that the pulmonary circulation can be directly investigated, independently of any confounding neural or humoral eVects. However, in these systems, any hemodynamic eVects of the prostanoids or PDE inhibitors cannot be assessed.

III. Prostanoids and PDE Inhibitors in Acute and Chronic Hypoxia A. Acute Hypoxia as a Model for Pulmonary Hypertension Acute alveolar hypoxia provokes a vasoconstrictor response (hypoxic pulmonary vasoconstriction, HPV), which reduces blood flow to hypoxic acini of the lung. By this physiological principle, also known as the von Euler-Liljestrand mechanism (Fishman, 1976; Staub, 1985; Voelkel, 1986; von Euler and Liljestrand, 1946), perfusion of well-ventilated regions of the lung is improved at the expense of poorly ventilated regions. Thus, HPV optimizes pulmonary gas exchange by adaptation of blood flow to alveolar ventilation. From a phylogenetic point of view, HPV may be termed normoxic pulmonary vasodilation, since it helps to prevent perfusion of the lungs in the fetus by a permanent vasoconstriction in the unborn. When ventilating the entire lung with a hypoxic gas, the beneficial properties of HPV are lost because it now causes a general increase in pulmonary vascular resistance (pulmonary hypertension) due to overall vasoconstriction. This may also contribute to the development of chronic hypoxia-induced pulmonary hypertension. Against this background, acute hypoxic challenges have been used as models for acute pulmonary hypertension in intact, anesthetized animals as well as in isolated perfused and ventilated lungs. Moreover,

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acute hypoxia is a reproducible, nondamaging vasoconstrictor stimulus that allows acute testing of the hemodynamic eVects of vasoactive agents.

B. Chronic Hypoxia as a Model for Pulmonary Hypertension Chronic alveolar hypoxia leads to the development of pulmonary hypertension. Aside from the possible contribution of HPV to this process (Weissmann et al., 2003), an additional major characteristic is a vascular remodeling process that occurs within weeks after exposure of animals to reduced oxygen concentrations. This remodeling process is characterized by de novo muscularization of formerly nonmuscularized precapillary vessels and a hypertrophy of the vessel media with increased muscularization of the pulmonary vasculature in general (Durmowicz and Stenmark, 1999; JeVery and Wanstall, 2001). These structural changes cause a narrowing of the vascular lumen, and are the major structural characteristics of hypoxiainduced pulmonary hypertension, in addition to right ventricular hypertrophy. However, some doubt exists concerning whether or not these structural changes in vessel wall thickness are suYcient to cause pulmonary hypertension, or if nonstructural eVects on vascular tone also play a role. These concerns notwithstanding, hypoxia-induced pulmonary hypertension has been widely used as a model to investigate hemodynamic and morphological eVects of PDE inhibitors on the pulmonary circulation (Table II). Aside from the obvious rationale for using vasodilating agents for the treatment of pulmonary hypertension, additional evidence exists that suggests that prostanoids in particular are good candidates for the treatment of this disease. Badesch and colleagues have demonstrated that in chronic hypoxia of the neonatal calf, there is a deficit in PGI2 and PGE2 production (Badesch et al., 1989). Along the same lines, Geraci and colleagues demonstrated that in PGI2 receptor-deficient mice, pulmonary hypertension is aggravated when mice are exposed to chronic hypoxia (Hoshikawa et al., 2001). Investigations in piglets have suggested that there is a shift toward vasoconstriction mediated by the release of cyclooxygenase-derived vasoactive mediators (Fike et al., 2003). These investigations favor the substitution of the vasodilating agent. On the other hand, investigations in chronically hypoxic rats indicate that PGI2 production is increased in the pulmonary vasculature, but sensitivity of adenylate cyclase to prostacyclin is decreased (Shaul et al., 1991). Moreover, mRNA levels of PGI2-synthase and the stable metabolite from PGI2, 6-keto-PGF1a, are increased in chronically hypoxic rats (Blumberg et al., 2002). These experiments support the conclusion that, at least in rats, endogenous prostanoid production may be up-regulated to antagonize pulmonary hypertension. In spite of

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Table II Studies with Chronic Phosphodiesterase Inhibitor Treatment in Experimental Pulmonary Hypertension Author

Year

Drug

Target

Model

Approach

Takahashi et al. Burch et al. Takahashi et al. Eddahibi et al. Kodama et al. Hanasoto et al. Zhao et al. Inoue et al. Zhao et al. Sebkhi et al. Sauzeau et al. Schermuly et al. Itoh et al. Kang et al. Schermuly et al. Phillips et al.

1996 1996 1998 1998 1999 1999 2001 2002 2003 2003 2003 2004b 2004 2003a 2004a 2004

PDE5 PDE3 PDE5 PDE5 PDE5 PDE5 PDE5 PDE5 PDE5 PDE5 PDE5 PDE5 PDE5 PDE5 PDE3/4 PDE3/4

MCT MCT MCT Hypoxia MCT Hypoxia Hypoxia MCT Hypoxia Hypoxia Hypoxia MCT MCT MCT MCT Hypoxia

preventive preventive preventive preventive preventive preventive preventive preventive preventive curative preventive curative preventive preventive curative preventive

Rondelet et al.

2004

E4021 Amrinone E4021 DMPPO E4010 E4010 Sildenafil T1032 Sildenafil Sildenafil Sildenafil Sildenafil Sildenafil DA8159 Tolafentrine Cilostamide/ Rolipram Sildenafil

PDE5

Shear stress

preventive

conflicting reports that describe either a lack of or an increased production of prostanoids in experimental pulmonary hypertension, it seems to make sense to either substitute (in the case of a lack) or to support (in the case of increased production) the concentration of prostanoids in the pulmonary vasculature. 1. Prostanoids in Acute Hypoxia a. Intravenous PGD2 and PGE1 in Acute Hypoxia. Few investigations have focused on the eVects of PGD2 and PGE1 on the acute hypoxic responses of the lung vasculature. As early as 1975, Weir and colleagues demonstrated in experimental pulmonary hypertension in dogs that intravenous infusion of PGE1 inhibits the pulmonary vascular pressor response to hypoxia (Weir et al., 1975). In acute hypoxic pulmonary hypertension, intravenous PGD2 caused a selective pulmonary vasodilation in neonatal lambs (Philips et al., 1983). In this model, systemic arterial pressure was increased. In fetal lambs rendered hypoxic with diaphragmatic hernia, PGD2 was more eVective in comparison to PGE2 and PGI2 (Ford et al., 1990). Anti-pulmonary hypertensive eVects were also described for an intravenous application of PGE1 in single lung ventilation in pigs (Hatori et al., 2000).

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b. Intravenous PGI2 in Acute Hypoxia. In experiments with PGI2 infusion, LeZer and colleagues concluded from experiments in dogs and cats that this agent may protect against pulmonary hypertension (LeZer and Passmore, 1979). Subsequently, PGI2, a PGI2-stimulating agent (Nafazatrom), or the stable PGI2 analogues (ZK 36-374, CL 115,347) have been investigated with respect to their anti-pulmonary hypertensive eVects in dogs, rats, and pigs maintained under acute hypoxia (Archer et al., 1986; Ensley and Rubin, 1985; Ford et al., 1990; Ishibe et al., 1989; Owall et al., 1991; Sprague et al., 1984). In all of these studies, a decrease in HPV or pulmonary vascular resistance was achieved. One of these investigations in dogs concluded that by attenuation of HPV, PGI2 increases pulmonary shunt flow (Sprague et al., 1984). When addressing the question of pulmonary selectivity of PGI2, systemic vasodilation became evident in a study in dogs (Ishibe et al., 1989) whereas in intact pigs, only minor systemic vasodilation was reported (Owall et al., 1991). Together, these investigations demonstrated that the intravenous application of PGD2, PGE2, and PGI2 can be used to antagonize acute hypoxic pulmonary hypertension; however, contradictory data exist concerning their pulmonary selectivity. 2. Phosphodiesterase Inhibitors in Acute Hypoxia a. Intravenous PDE 3 Inhibitors in Acute Hypoxia. In 1991, Haynes and colleagues conducted one of the first studies investigating PDE inhibitors with respect to their eVect on the vasoconstrictor response to acute alveolar hypoxia. This study demonstrated that the PDE 3 inhibitors indolidan and trequinsin reduced HPV in isolated rat lungs (Haynes et al., 1991). Similar results were obtained in an isolated rat lung preparation using the PDE 3 inhibitor SCA40 (6-bromo-8(methylamino)imidazol[1, 2-a]pyrazine2-carbonitrile) (Crilley et al., 1998). In anesthetized dogs, the PDE 3 inhibitor milrinone reduced lung vascular resistance with no eVect on cardiac output, although a decrease in systemic blood pressure was observed (Kato et al., 1998). In 2004, the PDE 3 inhibitor cilostamide was demonstrated to attenuate HPV in an in situ rat preparation (Phillips et al., 2004). b. Intravenous PDE 1, 2, 4 and Non-selective PDE Inhibitors in Acute Hypoxia. In the same study, the PDE 1 inhibitor vinpocetine, the PDE 2 inhibitor EHNA, and the PDE 4 inhibitor rolipram also attenuated HPV (Phillips et al., 2004). In these investigations, the nonselective PDE inhibitor IBMX was also eVective. The same eVect was found by Haynes et al. when applying the PDE 4 inhibitor rolipram (Haynes et al., 1991). Interestingly, to the best of our knowledge, these are the only studies investigating the eVect of PDE 1, 3, and 4 inhibitors with respect to their eVects on HPV.

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c. Intravenous PDE 5 Inhibitors in Acute Hypoxia. In 1991, Haynes and colleagues demonstrated that the PDE 5 inhibitor zaprinast caused a reduction in the vasoconstrictor response of isolated rat lungs to acute alveolar hypoxia (Haynes et al., 1991). The same compound (zaprinast, also called MB 22948) was shown 2 years later to induce a selective pulmonary vasodilation when compared to its eVects on the systemic circulation in intact anesthetized newborn lambs exposed to acute hypoxia (Braner et al., 1993). Similar results were achieved in intact rats and in isolated lungs from chronically hypoxic rats when applying the PDE 5 inhibitor zaprinast (Cohen et al., 1996). However, in this study, the PDE 5 inhibitor E4021 turned out to be more selective, without any dilating eVect in the systemic circulation at the doses applied (Cohen et al., 1996). The data of Nagamine et al. are consistent with these observations, showing an inhibition of HPV in the anesthetized normotensive rat by zaprinast (Nagamine et al., 2000). Inhibition of HPV was also achieved in isolated rabbit lungs by zaprinast (Weissmann et al., 2000). Investigations with the PDE 5 inhibitor sildenafil in isolated perfused mouse lungs revealed marked inhibition of HPV (Zhao et al., 2001, 2003), thus confirming that PDE 5 inhibitors act as pulmonary vasodilatory agents even in rats. In conclusion, PDE 5 inhibitors have pulmonary vasodilatory capacity in several diVerent experimental models (that include several species) of acute pulmonary hypertension induced by acute hypoxic ventilation. 3. Phosphodiesterase Inhibitors in Chronic Hypoxia a. Intravenous PDE 3 and PDE 4 Inhibitors in Chronic Hypoxia. The application of PDE 3 and PDE 4 inhibitors in animals to target hypoxiainduced pulmonary hypertension has only been investigated in one study to date (Phillips et al., 2004). However, in this investigation, the dosage applied was not eVective in contrast to combination with iloprost. Nevertheless, Wagner et al. found that PDE 3A mRNA transcripts are up-regulated in the pulmonary arteries of chronically hypoxic rats, suggesting that PDE 3A may also play a role in the development of this form of pulmonary hypertension (Wagner et al., 1997). These data are consistent with the investigations of MacLean et al. (1997) who reported increased cAMP-PDE activity in first branch and in intrapulmonary arteries, but not in the main pulmonary artery and resistance vessels of rats exposed to chronic hypoxia (MacLean et al., 1997). Interestingly, while changes in the expression and activity of the cAMP-dependent PDEs was found in the pulmonary circulation (MacLean et al., 1997; Wagner et al., 1997), no study, aside from the very recent investigation of Phillips et al. (2004), has addressed the eVect of PDE 3 and PDE 4 inhibitors in hypoxia-induced pulmonary hypertension.

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b. Intravenous PDE 5 Inhibitors in Chronic Hypoxia. With the exception of the studies of Phillips et al. of the PDE family, only PDE 5 inhibitors have been investigated in intact animals or in isolated lungs from animals exposed to chronic hypoxia with respect to their vascular eVects (Phillips et al., 2004). In 1998, Eddahibi et al. studied the eVects of the PDE 5 inhibitor DMPPO in intact chronic hypoxic rats. The DMPPO caused a dose-dependent decrease in pulmonary artery pressure when infused intravenously in chronic hypoxic rats with no eVects on cardiac output or systemic arterial pressure (Eddahibi et al., 1998). Continuous infusion during chronic hypoxic exposure reduced pulmonary artery pressure and the degree of muscularization of small pulmonary arterial vessels. Similar results were obtained in rats with oral application of the PDE 5 inhibitor E-4010 (Hanasato et al., 1999). In these experiments, E-4010 reduced pulmonary artery pressure with no eVect on systemic arterial pressure, right ventricular hypertrophy, medial wall thickness, and the degree of muscularization. A single-dose application selectively reduced pulmonary artery pressure. Treatment of chronically hypoxic mice with oral application of the PDE 5 inhibitor sildenafil also reduced development of pulmonary hypertension (Zhao et al., 2001). In these studies, Zhao and colleagues elegantly demonstrated that it was not only eNOS-derived NO that contributed to these eVects of the PDE 5 inhibitor (Zhao et al., 2001). These observations are in line with their study demonstrating that in natriuretic peptide (NPR-A) knockout mice, the anti-remodeling eVects of sildenafil on the pulmonary vasculature and on the right ventricle are reduced. This suggests that the natriuretic peptide pathway plays a major role in these eVects of sildenafil (Zhao et al., 2003). While all of the cited investigations initiated the treatment of chronically hypoxic animals at onset of hypoxia, Sebkhi et al. (2003) demonstrated that even curative application of sildenafil reduces pulmonary artery pressure and vascular muscularization in lungs from chronically hypoxic rats. In essence, these investigations demonstrate that PDE 5 inhibition has anti-pulmonary hypertensive eVects, with selective eVects on the pulmonary vascular resistance. The eVects of the PDE 5 inhibitors may only partially be explained by changes in the cGMP-dependent PDE activity, since MacLean et al. have demonstrated that the activity of these isoforms is only up-regulated in first branch and intrapulmonary arteries and not in pulmonary resistance arteries in the rat (MacLean et al., 1997). Thus, the selective pulmonary eVects of PDE 5 inhibitors are most probably attributable to (a) a generally high level of PDE 5 in the pulmonary circulation compared with the systemic circulation (Ahn et al., 1991; Giordano et al., 2001; Hanson et al., 1998) and (b) the fact that NO production in the lung is quite high, akin to the situation in the corpus cavernosum (Bloch et al., 1998; Grimminger et al., 1995; Nangle et al., 2003; Spriestersbach et al., 1995).

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c. PDEg in Chronic Hypoxia. Though not investigated with respect to a therapeutic approach aVecting PDE 6, it has been shown in the rat that the inhibitory subunit of PDE 6, PDEg, is expressed in the main, first, intrapulmonary, and resistance arteries of the lung and is up-regulated by exposure of the animals to chronic alveolar hypoxia (Murray et al., 2003). Further experiments are required to evaluate the role of PDEg as a possible target for treatment of hypoxia-induced pulmonary hypertension. 4. Pulmonary-Selective Delivery of Prostanoids in Acute and Chronic Hypoxia In an attempt to overcome the nonselective eVects of intravenous applications of prostanoids, Welte and colleagues applied PGI2 as an aerosol in dogs challenged with acute hypoxia (Welte et al., 1993). In this setup, pulmonary vasodilation was achieved without systemic side eVects. Furthermore, another report from the same laboratory proved that inhaled PGI2 is protective for the right ventricle of the heart (Zwissler et al., 1995). In intact sheep and pigs, HPV was also reduced by inhaled PGI2 (Booke et al., 1996; Max et al., 1999). Pulmonary vasodilatory selectivity was also evident when the stable prostacyclin analogue beraprost was delivered as an aerosol in rats challenged with acute hypoxia (Abe et al., 2001). Although not providing data concerning systemic eVects, Geraci and colleagues demonstrated in an elegant study that selective overexpression of PGI2 in the lung protects against hypoxia-induced pulmonary hypertension in mice (Geraci et al., 1999).

IV. Prostanoids and PDE Inhibitors in Monocrotaline-Induced Pulmonary Hypertension A. Monocrotaline as a Model of Pulmonary Hypertension The pyrrolizidine alkaloid monocrotaline (MCT) is extracted from plants of the genus Crotalaria (Huxtable, 1990), and it is used experimentally to elicit pulmonary hypertension in rats. The proposed mechanism of action includes the activation of MCT by the liver to the putative electrophile monocrotaline pyrrole (MCTP) (Huxtable, 1990; Rosenberg and Rabinovitch, 1988; Wilson et al., 1992), which causes endothelial injury in the pulmonary vasculature with subsequent remodeling of the precapillary vessels (medial thickening, de novo muscularization of small pulmonary arterioles) (Rosenberg and Rabinovitch, 1988). Due to this mimicking of clinical pulmonary arterial hypertension, the rat MCT model has repeatedly been employed

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for nvestigating acute hemodynamic and gas exchange eVects of vasodilators and chronic anti-remodeling eVects of anti-inflammatory and antiproliferative agents (Cowan et al., 2000; Ono and Voelkel, 1991; Prie et al., 1997; Sakuma et al., 1999) (Table II). In addition, right atrial injection of chemically synthesized monocrotaline pyrrole (MCTP) in dogs is another way to induce chronic pulmonary hypertension (Chen et al., 1997a,b, 1998).

1. Acute Hemodynamic Effects of Prostanoids in MCT-Induced Pulmonary Hypertension In 1986, Czer et al. investigated the eVects of a prostacyclin infusion on hemodynamics and thromboxane generation in a model of acute pulmonary hypertension induced by monocrotaline injection in dogs (Czer et al., 1986). Prostacyclin prevented lung platelet deposition, pulmonary hypertension, and decreased thromboxane generation. Similar results were obtained with inhaled prostacyclin, which dose-dependently reduced pulmonary arterial pressure in the anesthetized MCT-treated rat without altering systemic arterial pressure (Hill and Pearl, 1999). The eVect of inhaled prostacyclin was amplified when inhaled nitric oxide (20 ppm) was applied. In the same year, it was shown in a similar experimental setting that infused prostacyclin reduces the pulmonary artery pressure and that this eVect was stronger when inhaled nitric oxide was added (Aranda et al., 1999). In a 2004 report, the prostacyclin analogue iloprost was shown to be eVective in reduction of right ventricular systolic pressure in the anesthetized MCT-treated rat model (Schermuly et al., 2004a).

2. Acute Hemodynamic Effects of Phosphodiesterase Inhibitors in MCT-Induced Pulmonary Hypertension In a model of heart transplantation in MCTP-induced pulmonary hypertension in dogs, it was shown that the PDE 3 inhibitor milrinone increased right ventricular function with significant improvement in pulmonary vascular resistance (Chen et al., 1998). In another study, a dual selective inhibitor of PDE 3/4 was employed in the setting of monocrotaline-induced PAH in rats (Schermuly et al., 2004a). This drug, tolafentrine, reduced right ventricular systolic pressure (RVSP) in a dose-dependent manner when infused. Pulmonary vasodilation was accompanied by a decrease in the systemic arterial pressure and by an increase in cardiac index, a well-known phenomenon of PDE 3 inhibitors. In a similar experimental setting where intact anesthetized rats were injected with monocrotaline, the selective PDE 5 inhibitor T-1032 reduced mean arterial pressure (MAP) and right ventricular systolic pressure without a change in heart rate (Inoue et al., 2002). Interestingly, at the lower dose range, the change in RVSP was more potent than

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that in MAP, which indicates pulmonary selectivity even when applying the drug intravenously. 3. Long-Term Effects of Prostanoids in MCT-Induced Pulmonary Hypertension a. Prostacyclin (PGI2) and Analogues. The important role of prostacyclin and its analogues in the development of pulmonary hypertension was investigated in several studies. Yuki et al. (1994) treated MCT-injected rats with oral beraprost, a prostacyclin analogue and found a significant and dosedependent reduction of pulmonary artery pressure with no eVects on systemic arterial pressure after a 3-week treatment period. Similar results were obtained from Miyata et al. (1996) who demonstrated that oral beraprost reduced the right heart hypertrophy, as assessed by the right to left ventricle plus septum ratio. Further studies compared and combined oral beraprost with an endothelin receptor A antagonist and found additive eVects of beraprost in the presence of endothelin blockage on hemodynamics and vascular remodeling (Ueno et al., 2000, 2002). The combined approaches were continued with sildenafil plus oral beraprost (Itoh et al., 2004) and tolafentrine plus iloprost (Schermuly et al., 2004a). Both studies demonstrated beneficial eVects of systemically delivered prostacyclin analogues on remodeling and on hemodynamics which could be amplified in the presence of either PDE 5 or PDE 3/4 inhibition. b. Gene Transfer of Prostacyclin Synthase in MCT-Induced Pulmonary Hypertension. Another option to apply prostanoids is the gene transfer of the suitable enzyme. Nagaya et al. (2000) employed a viral vector to deliver the human prostacyclin synthase (PGIS) gene into rats with MCT-induced pulmonary hypertension. When compared with empty vector-transfected animals, the PGIS-transfected rats developed less severe pulmonary hypertension with lower pulmonary vascular resistance, smaller medial wall thickness, and improved mortality. Similar results were obtained by gene transfer of the human PGIS into the liver of MCT-treated rats (Suhara et al., 2002). Plasma levels of the prostacyclin metabolite 6-keto-prostaglandin F1alpha were significantly elevated and accompanied by a decrease in endothelin levels and by improved survival. c. Intravenous Prostaglandin E1 (PGE1) in MCT-Induced Pulmonary Hypertension. Prostaglandin E1 (PGE1) is a potent vasodilator and activates, in contrast to prostacyclin (which binds selectively to the prostacyclin (IP) receptor), the prostaglandin E1 (EP) receptor. The protective eVects of daily injections of PGE1 on the development of MCT-induced pulmonary hypertension have been demonstrated in several studies (Ono et al., 1995; Sakuma et al., 1999). Interestingly, in a diVerent study, PGE1 was injected

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daily one week after MCT application but this did not decrease the extent of pulmonary hypertension, suggesting that a significant portion of PGE1 eVects are exerted by anti-inflammatory properties in the initial phase of MCT-induced pulmonary hypertension (Miyata et al., 1996). d. Inhaled Prostaglandin E1 (PGE1) in MCT-Induced Pulmonary Hypertension. In 2002, Kato et al. investigated the suppressive eVects of long-term prostaglandin E1 aerosol application on hemodynamics and leukocyte sequestration and activation in the pulmonary microvasculature in two studies (Kato et al., 2002a,b). The main findings of these studies were an improvement in hemodynamics, right heart hypertrophy, as well as a decrease in leukocytes in the pulmonary microvasculature. However, the antileukocyte eVects of PGE1 in the early stage of progression of the disease may contribute to these protective eVects. 4. Long-Term EVects of Phosphodiesterase Inhibitors in MCT-Induced Pulmonary Hypertension a. Phosphodiesterase 3/4 Inhibitors in MCT-Induced Pulmonary Hypertension. In 1996, Burch et al. studied the eVects of the PDE 3 inhibitor amrinone in monocrotaline-induced pulmonary hypertension in rats (Burch et al., 1996). In these experiments, animals were treated with amrinone (100 mg/kg daily) from days 1 to 21, resulting in no significant changes in either right to left ventricular ratio or lung- to body-weight ratio. In contrast, a recent report demonstrated the protective eVects of tolafentrine, a dual selective PDE 3/4 inhibitor, when it was continuously infused from week 2 to 4 after MCT injection (Schermuly et al., 2004a). In a rescue treatment group, the combination of tolafentrine and iloprost reversed pulmonary vascular remodeling when applied from week 4 to 6 by infusion. There was a significant reduction of the expression and activity of the matrix metalloproteinases (MMP) 2 and 9 in the treatment groups when compared to placebo-treated animals. These MMPs play an important role in matrix remodeling and progression of the disease. The anti-remodeling eVects were demonstrated by reduction of right to left ventricular plus septum weight (RV/LV þ S), improved hemodynamics, and reduction of the amount of fully muscularized small pulmonary arteries. b. Intravenous PDE 5 Inhibitors in MCT-Induced Pulmonary Hypertension. The majority of studies investigating the eVects of PDE inhibitors on mortality, remodeling, and hemodynamics employed inhibitors of the cGMP-specific PDE 5. Phosphodiesterase 5 is abundantly expressed in lung tissue (Ahn et al., 1991) and therefore a likely candidate target for the treatment of pulmonary hypertension (Ghofrani et al., 2004a). In 1996, Takahashi et al. investigated the eVects of E4021, a PDE 5 inhibitor, on the

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development of MCT-induced pulmonary hypertension (Takahashi et al., 1996). This drug dose-dependently reduced medial wall thickness, RV/LV þ S ratio, and smooth muscle area (as a marker of pulmonary arterial muscularization), with 100 mg/kg/day being the most eVective dose. The same group demonstrated two years later that this concentration of E4021 reduces levels of plasma endothelin-1 (Takahashi et al., 1998), which is a potent vasoconstrictor and mitogen. In another study, the eVects of the PDE 5 inhibitor E4010 on mortality, plasma cGMP-, and atrial natriuretic peptide (ANP) levels were investigated (Kodama and Adachi, 1999). At a concentration of 0.1% in the food, this drug decreased mortality and increased plasma and tissue level of cGMP level, without aVecting plasma ANP levels. Similar results were obtained with T-1032, another orally bioavailable PDE 5 inhibitor. Chronic feeding of MCT-treated rats with T-1032 delayed mortality and suppressed right ventricular growth (Inoue et al., 2002). The protective eVect of the PDE 5 inhibitor DA-8159 on the development of MCT-induced pulmonary hypertension was investigated in studies from Kang et al. who investigated right ventricular hypertrophy and medial wall thickening (Kang et al., 2003a,b). This compound prevented myocardial fibrosis and amplified the level of lung cGMP and reduced remodeling parameters. In a 2004 publication, Itoh et al. investigated sildenafil, a PDE 5 inhibitor, alone or in combination with beraprost, a prostacyclin analogue, on the development of experimental PAH (Itoh et al., 2004). Sildenafil has been approved for the treatment of erectile dysfunction (Cheitlin et al., 1999) and acute hemodynamic data in patients with PAH are available (Ghofrani et al., 2002b). The long-term treatment of MCT-treated rats resulted in a decrease in RVSP, right heart hypertrophy, and medial wall thickness. The curative application of sildenafil after the development of pulmonary hypertension yielded similar results (Schermuly et al., 2004b). Sildenafil reduces pulmonary artery pressure and vascular muscularization in lungs from chronically ill rats and reduced MMP 2 and 9 expressions. Additionally, the degree of fully muscularized small (<50mm) pulmonary arteries was decreased.

V. Prostanoids and PDE Inhibitors in U46619-Induced Acute Pulmonary Hypertension A. U46619-Induced Vasoconstriction as a Model of Pulmonary Hypertension The thromboxane mimetic U46619 has repeatedly been used to induce acute pulmonary hypertension in intact animals as well as in isolated lungs. By binding to the thromboxane A2 receptor, U46619 increases intracellular calcium [Ca2þi] by activation of phospholipase A2 via Gq coupling (Kinsella

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et al., 1997). The U46619-induced vasoconstrictor response has, at least for acute hypoxia, been shown to act via hypoxia-independent pathways (Weissmann et al., 1999), favoring U46619-induced pulmonary hypertension as an independent approach to investigate PDE inhibitors toward their anti-hypertensive potential. 1. Prostanoids in U46619-Induced Pulmonary Hypertension a. Intravenous Prostanoids in U46619-Induced Pulmonary Hypertension. Several studies in sheep employing U46619-induced acute pulmonary hypertension demonstrated that PGE1 is a potent vasodilator of the pulmonary circulation (Kavanagh et al., 1996; Kleen et al., 1998; Pearl and Siegel, 1992; Prielipp et al., 1988). Similar data were obtained with infusion of the prostacyclin analogue ciloprost, which decreased the elevated pulmonary artery pressure comparable to inhaled nitric oxide, but was accompanied by systemic hypotension (Van Obbergh et al., 1996). However, another study employing U46619-preconstricted isolated rabbit lungs showed that the intravascular application of either PGE1 or prostacyclin lowered pulmonary artery pressure but did not improve gas exchange (Walmrath et al., 1997). In PGF2a-induced vasoconstrictive pulmonary hypertension in dogs, intravenous administered PGE1 dose-dependently reduced systemic and pulmonary vascular resistances with deterioration of gas exchange at the higher dose ranges (Dagher et al., 1993). b. Inhaled Prostanoids in U46619-Induced Pulmonary Hypertension. Inhaled prostacyclin was demonstrated to be superior compared to intravenous and inhaled PGE1 regarding reduction of the pulmonary vascular resistance (Kleen et al., 1998). Walmrath and coworkers compared inhaled versus intravenous PGE1 and PGI2 and found that aerosolized prostanoids reduce pulmonary artery pressure and improve ventilation-perfusion matching (comparable to inhalation of nitric oxide) while intravenous prostanoids reduce pulmonary artery pressure with no impairment of gas exchange (Walmrath et al., 1997). Again, the eYcacy of inhaled prostacyclin with respect to pressure reduction was greater when compared to that of PGE1. The selective pulmonary vasodilation of inhaled prostanoids (prostacyclin or iloprost) was demonstrated in several studies where acute pulmonary hypertension was induced either in intact rabbits or isolated rabbit lungs (Schermuly et al., 1999, 2000, 2001a,b, 2003). In summary, the improvement of ventilation-perfusion matching upon transbronchial administration of inhaled prostanoids further supports the concept that vasodilator application by inhalation promotes vasorelaxation in well-ventilated lung areas without aVecting systemic blood pressure.

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2. Phosphodiesterase Inhibitors in U46619-Induced Pulmonary Hypertension a. Intravenous PDE 3/4 Inhibitors in U46619-Induced Pulmonary Hypertension. In an in situ rabbit lung preparation, milrinone reduced U46619-induced pulmonary hypertension (Clarke et al., 1994). Similar results were obtained in isolated lungs preconstricted with U46619 from LPStreated rats with milrinone, while the PDE 2 inhibitor EHNA had no eVect (Holzmann et al., 2001). In PGF2a-induced vasoconstrictive pulmonary hypertension in dogs, amrinone reduced mean arterial and pulmonary arterial pressure (Dumont and Dagher, 1994). In later studies, milrinone was also shown to have vasodilating eVects on the systemic circulation in intact cats (Matot and Gozal, 2004), rabbits (Deb et al., 2000), and piglets (Foubert et al., 2002). Both the PDE 3 inhibitor motapizone and the PDE 4 inhibitor rolipram displayed pulmonary vasodilator activity in U46619preconstricted intact rabbits and in isolated rabbit lungs, suggesting that PDE 3 and 4 are the most prominent cAMP-hydrolyzing PDEs (Schermuly et al., 1999, 2000). In addition, dual-selective inhibition of PDE 3 and 4, either by zardaverine or tolafentrine, resulted in eVective pulmonary vasodilation (Schermuly et al., 1999, 2000, 2003). In isolated rabbit lungs with U46619-induced pulmonary hypertension, synergistic eVects of PDE 3 and PDE 4 inhibitors have been reported (Schermuly et al., 2000). b. Intravenous PDE 5 Inhibitors in U46619-Induced Pulmonary Hypertension. In an in situ rabbit lung preparation, Clarke et al. (1994) demonstrated that dipyridamole as well as zaprinast reduced U46619-induced pulmonary hypertension. They also demonstrated that a combination of each of these drugs with a PDE 3 inhibitor had synergistic eVects. Similar results with dipyridamole and zaprinast were obtained in intact rabbits with U46619-induced pulmonary hypertension (Schermuly et al., 1999, 2001c). The anti-pulmonary hypertensive eVect of zaprinast was also evident from investigations in an U46619-model of isolated rabbit lungs and in rat lungs from LPS-treated animals (Holzmann et al., 2001; Weissmann et al., 2000). As early as 1993, it was observed in newborn lambs that intravenously infused MB-22948 (zaprinast) selectively dilates the pulmonary vasculature when compared to its eVect on the systemic circulation (Braner et al., 1993). Similar results were obtained in spontaneously breathing cats (Matot and Gozal, 2004). Moreover, in isolated rat lungs precontracted with U46619, DMPPO amplified the vasodilator eVects of the ANP and sodium nitroprusside (Eddahibi et al., 1998). The PDE 5 inhibitor E4021 was shown to dose-dependently reduce the elevated pulmonary artery pressure in isolated rat lungs (Ohnishi et al., 1999). More recent studies in awake

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lambs demonstrated that sildenafil applied via a nasogastric tube acted as a selective pulmonary vasodilator (Weimann et al., 2000). Predominant pulmonary vasodilation was induced by a sildenafil analogue, UK 343–664, in a swine model (Bonnell et al., 2004). c. Inhaled PDE 3/4 Inhibitors in U46619-Induced Pulmonary Hypertension. In 2000, it was shown in an isolated rabbit lung preparation with U46619-induced pulmonary hypertension that both motapizone (PDE 3) and rolipram (PDE 4) synergistically reduced pulmonary artery pressure (Schermuly et al., 2000). The dual selective PDE 3/4 inhibitor tolafentrine displayed similar eYcacy and was shown to selectively dilate the pulmonary circulation when being applied as aerosol in an intact animal preparation (Schermuly et al., 2001a). d. Inhaled PDE 5 Inhibitors in U46619-Induced Pulmonary Hypertension. Inhaled PDE 5 inhibitors are pulmonary-selective vasodilators; the transbronchial route of administration delivers the drugs to well-ventilated areas of the lung with intrapulmonary vasodilation. This was shown by inhalation of sildenafil and zaprinast in awake lambs with U46619-induced pulmonary hypertension (Ichinose et al., 1998, 2001). Similar results were obtained in an intact rabbit preparation where nebulized dipyridamole was shown to selectively dilate the pulmonary vascular bed (Schermuly et al., 2001a,c).

VI. Less Frequently Used Models of Experimental Pulmonary Hypertension A. Pulmonary Embolism A less frequently used model to achieve experimental pulmonary hypertension is the microembolization of the pulmonary circulation. In this approach, pulmonary vascular resistance is increased by embolization of the pulmonary circulation with glass beads. In this category of pulmonary hypertension, intravenous infusion of PGE1 as well as PGI2 have been shown to decrease pulmonary artery pressure or vascular resistance in pigs and dogs (Dervin and Calvin, 1990; McLean et al., 1990; Prielipp et al., 1991). In the pig, the dosage used also decreased systemic blood pressure (McLean et al., 1990; Prielipp et al., 1991). To the best of our knowledge, only one investigation studied the eVect of a PDE inhibitor in glass bead-induced pulmonary hypertension (Tanaka et al., 1992). In this investigation, the PDE 3 inhibitor milrinone was shown to result in pulmonary vasodilation in dogs.

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B. Meconium Aspiration Perinatal aspiration of meconium is frequently accompanied by development of pulmonary hypertension. In animal models, application of meconium has been shown to induce an increase in pulmonary artery pressure and pulmonary vascular resistance (Shekerdemian et al., 2002, 2004). Intravenous infusion of sildenafil in a piglet model of meconium aspiration-induced pulmonary hypertension has been shown to reduce pulmonary artery pressure and pulmonary vascular resistance selectively without adverse eVects on systemic hemodynamics (Shekerdemian et al., 2002). Summarizing the data of more rarely used models of experimental pulmonary hypertension, it turned out that with sildenafil, again, selective pulmonary vasodilation is achieved.

C. Shear Stress Rondelet et al. (2004) induced pulmonary hypertension in piglets by anastomosis of the left subclavian artery to the pulmonary arterial trunk. After 3 months of chronic treatment with sildenafil, the overcirculation-induced PAH was partially prevented and expression of endothelin-1, angiotensin-2, inducible nitric oxide synthase, as well as other pro-inflammatory/proliferative genes, was normalized. Summarizing the data of more rarely used models of experimental pulmonary hypertension, it turned out that with sildenafil, again, selective pulmonary vasodilation is achieved.

VII. Combination of PDE Inhibitors with Vasodilators It is obvious to combine inhibitors of PDE with vasodilators to stabilize the second messengers cGMP and cAMP (Fig. 2). Studies were performed to combine zaprinast (Ichinose et al., 1998), E4021 (Ohnishi et al., 1999), sildenafil (Ichinose et al., 2001; Weimann et al., 2000) or dipyridamole (Foubert et al., 2002, 2003) with inhaled nitric oxide. The PDE 5 inhibitors have been shown to stabilize cGMP and to prolong and amplify the vasodilatory eVects of inhaled nitric oxide. In another study, the natriuretic peptide urodilatin was combined with dipyridamole, which increased plasma cGMP levels and eYcacy of a short-term urodilatin infusion in intact rabbits (Schermuly et al., 2001c). On the prostanoid/cAMP-axis, it was shown that inhibitors of PDE 3 and 4 (or dual selective PDE 3/4 inhibitors) amplify the vasodilatory eVects of prostacyclin (Schermuly et al., 1999, 2000) and

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Figure 3 Regulation of PDE activity by calcium/calmodulin, by phosphorylation via protein kinases, or by cGMP-binding. The cross-talk between diVerent second messenger signaling pathways includes modulation of phosphodiesterase (PDE) activity by calcium (Ca2þ)/calmodulin (Cam), by phosphorylation via protein kinases, or by cyclic guanosine monophosphate (cGMP) binding. Substrate specificity of the PDEs is indicated by the blue arrows. Stimulation is indicated by green arrows, inhibition by red arrows. PKA, protein kinase A; PKG, protein kinase B; cAMP, cyclic adenosine monophosphate.

iloprost (Schermuly et al., 2001a, 2003). When combining the PDE 4 inhibitor rolipram and the prostacylin analogue cicaprost, a higher attenuation of HPV was achieved in an in situ perfused rat lung compared to the eVects of these agents used alone (Phillips et al., 2004). The same group demonstrated that in chronic hypoxia, intravenous co-application of the prostacyclin analogues iloprost and the PDE 3 inhibitor cilostamide, or the PDE 4 inhibitor rolipram reduced pulmonary artery pressure, right ventricular hypertrophy, and distal vessel muscularization without a significant eVect on systemic blood pressure, whereas the sole application of these agents had no significant eVect on the development of pulmonary hypertension. The same eVect was true for the combination of cilostamide and rolipram (Phillips et al., 2004). Interestingly, the PDE 5 inhibitor zaprinast amplified and prolonged the eVects of inhaled prostacyclin (Schermuly et al., 1999), which demonstrates crosstalk between the signaling pathways. This crosstalk may occur via inhibition of either PDE 3 by cGMP or inhibition of other cAMP-specific PDEs by zaprinast (Fig. 3). However, the selective PDE 5 inhibitor sildenafil has been shown in clinical trials to act in tandem with inhaled iloprost, supporting the hypothesis of cGMP-inhibited PDE 3 (Ghofrani et al., 2002b; Wilkens et al., 2001). Strong eYcacy was also noted when combining clinically available nonselective PDE inhibitors, for example, theophylline or pentoxyfylline, with inhaled prostanoids (Schermuly et al., 2001a,b). These drugs amplify the vasodilatory eVects of prostacyclin or iloprost in experimental models of pulmonary hypertension.

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VIII. Summary and Concluding Remarks In several models of experimental pulmonary hypertension induced by hypoxia, monocrotaline, or other vasoconstrictors, both inhaled and intravenous vasodilators have demonstrated the potential to attenuate pulmonary vasoconstriction. Intravenous vasodilators, however, aside from being potent agents, lack pulmonary and intrapulmonary selectivity. Thus, when infused, these agents are operative in the systemic circulation as well as in the pulmonary circulation. Moreover, inside the lung, nonventilated areas are as eVectively dilated as well-ventilated areas, thereby impairing gas exchange. In contrast, inhaled vasodilators selectively reduce pulmonary artery pressure (leaving the systemic circulation unaVected) and improve gas exchange (due to intrapulmonary selectivity for well-ventilated areas). In contrast to infused prostanoids, oral PDE 5 inhibitors, despite systemic routes of administration, have demonstrated in several studies preferential selectively for the pulmonary circulation. The most plausible explanations are the high and abundant expression of PDE 5 in the pulmonary circulation (which may even increase under conditions of pulmonary hypertension; see McLean et al., 1990) and the high production of nitric oxide in the lung, preferentially in well-ventilated (and thus well-oxygenated) areas of the lung. Thus, inhibitors of PDE 5 may even amplify endogenous nitric oxide signaling, which is the key regulator in the adaptation of perfusion to ventilation in the lung. Despite the fact that 11 families of PDEs have been identified, the experimental work so far has focused primarily on PDEs 3 to 5, as no selective inhibitors of PDEs 1 and 2 are yet available. However, the latter PDEs are of major interest clinically, since isoforms of PDE 1 have been shown to be up-regulated in proliferating smooth muscle cells and therefore may be involved in pulmonary-vascular remodeling processes. On the other hand, PDE 2 is expressed in endothelial cells and might therefore have a role in mediation of endothelial functions (or dysfunction). Furthermore, PDE 2 is also expressed in the heart and may influence cardiac remodeling, which, at least in the case of the right ventricle, plays an important role in the course of pulmonary hypertension. The role of cAMP-specific PDEs 3 and 4 in acute and chronic experimental studies was also investigated in detail. Both PDEs are involved in the regulation of vascular tone and the remodeling processes of the pulmonary circulation. To prevent systemic eVects of PDE 3 and 4 inhibitors, both agents can also be delivered by aerosolization, as previously shown in clinical as well as experimental studies.

Acknowledgments We ask all authors to whose work we did not refer to accept our sincere apologies. We thank Rory Morty for linguistic editing of the manuscript.

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14-3-3 Protein Signaling in Development and Growth Factor Responses Daniel Thomas, Mark Guthridge, Jo Woodcock, and Angel Lopez Cytokine Receptor Laboratory, Division of Human Immunology Hanson Institute, Institute of Medical and Veterinary Science Adelaide SA 5000, Australia

I. Introduction II. 14-3-3: A Dimer with Phosphoserine-Binding Activity A. Biophysical Properties of 14-3-3 Proteins B. Isoforms and Dimers—A Clue to Regulation? C. Phosphorylation of 14-3-3 Binding Motifs III. 14-3-3 Pathways in Drosophila Development A. Tissue Growth and Cell Cycling B. Determining Cell Polarity C. Neuronal Signaling IV. V. VI. VII.

Interaction with the Ras-Raf Signaling Pathway 14-3-3 and Growth Factor Signaling Phosphorylation of 14-3-3 by Sphingosine-Dependent Kinase Conclusions References

Tyrosine and serine phosphorylation are central to cellular signaling in growth and development. 14-3-3 proteins function as dimeric phosphoserine-binding proteins with documented interactions throughout the eukaryotic proteome and are highly conserved in both the animal and plant kingdoms. Binding of 14-3-3 to a client protein can have a range of context-dependent eVects, including conformational change, enzyme inhibition, a shielding eVect, re-localization, and bridging between two molecules. Proteome-based strategies utilizing mass spectrometry have revealed an unprecedented central stage for 14-3-3 in signal transduction with interacting partners composing at least 0.6% of the cellular proteome. 14-3-3 has been shown to bind to the human GM-CSF, IL-3, and IL-5 receptors and is required for the transmission of cell survival. 14-3-3 is involved in survival-specific signals, acting not only at the receptor level but also at critical steps downstream of the receptor. This phosphoserinemediated pathway works independently of tyrosine kinases, highlighting an alternative mechanism of signaling for this receptor family. Other growth Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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factor receptors and their adaptors are also being shown to associate with 14-3-3 and/or have putative 14-3-3 interaction sequences, such as the prolactin receptor, IGF-1 receptor, and some G-protein coupled receptors. 14-3-3 proteins are remarkably conserved through eukaryotic organisms and in Drosophila are required for photoreceptor development, learning, timing of cell cycles, and maintenance of cellular polarity. These findings are elevating our initial description of biochemical interactions to a better understanding of 14-3-3 function at the level of the whole organism. Further study should explore the integration of phosphoserine and phosphotyrosine signaling by 14-3-3 proteins and the role of isoform-specific functions in higher organisms. The prevalence of functional 14-3-3 binding sites throughout the proteome, and especially among growth factor receptors and signaling molecules, reflects a global role for 14-3-3 in multiple cellular decision making. C 2005, Elsevier Inc.

I. Introduction One of the great mysteries of biology is how cells, both collectively and individually, make appropriate decisions in response to various signals at precise anatomical and developmental positions. Experiments in a number of eukaryotic species in the last two decades validate the existence of major conserved signaling pathways with multiple points of interconnection, serving to integrate an appropriate cellular response from wide sources of information (Hunter, 2004). These pathways include the canonical growth factor receptor/ Ras/Raf/MAPK cascade for proliferation (Campbell et al., 1998), TGF- / serine-threonine kinase receptor/SMAD for growth arrest and diVerentiation (Massague et al., 2000), and PI-3K/Akt/BAD pathway for growth factor-dependent survival (Datta et al., 1997). Phosphorylation of proteins on serine, threonine, and tyrosine residues by protein kinases is a central process in all of these pathways. Phosphorylation cascades have a number of unique properties ideally suited to the transmission of intracellular data including (a) reversibility, akin to an ‘‘on/oV’’ switch that is mediated by kinases and phosphatases (Alonso et al., 2004), (b) rapid amplification of a localized signal by recruitment, phosphorylation, and activation of other kinases, (c) integration of multiple signaling inputs into a single output, termed nodal signaling; examples include MAPK and retinoblastoma, which require phosphorylation by a number of kinases on multiple sites to achieve full kinase activity (Harbour et al., 1999), (d) and threshold setting which serves to increase the signal-to-noise ratio. These properties, working together in a global signaling network, enable cells to respond dynamically and rapidly to diverse environmental perturbations and yet maintain their normal cellular function. (For a comprehensive

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review regarding mathematical descriptions of phosphorylation pathways to explain cellular ‘‘robustness,’’ (see Stelling et al., 2004.) It has become clear that the 14-3-3 family of proteins is an integral component of these phosphoprotein networks by virtue of their biophysical property of binding phosphoserine in a recognizable sequence context (Aitken, 2002; Dougherty and Morrison, 2004; Tzivion et al., 1998). Indeed, 14-3-3 was the first protein discovered that had phosphoserine-binding ability (Muslin et al., 1996). Since then, other proteins have been discovered to have phosphoserinebinding domains, such as the WW domain, forkhead associated (FHA) domain, polo box domain (PBD), and the Brca1 c-terminal (BRCT) domain (Elia et al., 2003; Lu et al., 1999; Rodriguez et al., 2003; YaVe and Elia, 2001). These domains are duplicated and shuZed in various combinations, being found in a wide range of eukaryotic proteins. On the other hand, the 14-3-3 family of proteins appears not to be utilized as domains within larger proteins but remains as distinct and separate proteins. The 14-3-3 proteins are able to bind a wide variety of cellular proteins involved in cell signaling, scaVolding, and metabolism, with reports showing the number of interacting partners comprising at least 0.6% of the cellular proteome (Jin et al., 2004; Meek et al., 2004; Rubio et al., 2004). They have been shown to play an essential role in the regulation of a number of cellular processes including growth factor-mediated cell survival, cell cycle progression, polarity, cell migration, and apoptosis. The precise molecular mechanisms underpinning the requirement for 14-3-3 in all of these pathways is currently a matter of intense investigation, with various models having been proposed. This chapter aims to fill some of the gaps in our current understanding of signal transduction, with particular attention to the role of 14-3-3 as a global regulator involved at many points in growth factor signaling. 14-3-3 associations with growth factor receptors, including the GM-CSF/IL-3/IL-5 receptor and IGF-1 receptor and protein kinases including Raf, protein kinase A, and sphingosine-dependent kinase, are discussed.

II. 14-3-3: A Dimer with Phosphoserine-Binding Activity A. Biophysical Properties of 14-3-3 Proteins 14-3-3 proteins are 28 to 33kDa small acidic proteins first purified as abundant proteins inside neurons, their name based on nomenclature used for 2-dimensional gel electrophoresis (Moore and Perez, 1967). Seven isoforms of 14-3-3 have been described in humans, / , , , /, , , and , with  and  representing the phosphorylated versions of and , respectively. Each isoform is encoded by a separate gene and, in general, 14-3-3 is expressed at high levels in all tissues (with the exception of  and  which are predominantly

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expressed in epithelium and T-cells, respectively) (Toker et al., 1992). There are two well-recognized consensus sequences that are able to bind 14-3-3 when phosphorylated: Mode 1 RSXpS/TXP and Mode 2 RXXXpS/TXP, where X is any amino acid and pS/T represents phosphorylated serine or threonine. In most cases, the regulated phosphorylation of the associated protein controls its binding to 14-3-3 proteins. This is similar to src homology 2 (SH2) domains and phosphotyrosine-binding (PTB) domains which require phosphorylation of a specific tyrosine residue in order to facilitate high-aYnity binding. Many adaptor proteins discovered in the study of tyrosine kinase receptor pathways, such as Grb2 and Shc, contain modular SH2 or PTB domains that function to co-localize signaling proteins in an intimate multiprotein complex (Ravichandran, 2001). 14-3-3 proteins are naturally dimeric, being composed of two monomers that are each capable of binding a separate phosphoserine/threonine motif and which may, in a similar fashion, function as adaptor proteins that recruit and assemble phosphoserine-bearing proteins. In contrast to 14-3-3, SH2 adaptor proteins are not naturally found in a dimeric form, although their binding partners often undergo dimerization in the phosphorylated state. To date, none of the SH2-containing adaptor proteins appear to have additional domains that are either homologous to 14-3-3 or even possess phosphoserine-binding activity. These diVerences would suggest that the regulation of phosphoserine/threonine signaling pathways by 14-3-3 is independently regulated from phosphotyrosine-signaling pathways. It is conceivable that the two pathways may run parallel in certain scenarios with diVerent kinetics that aVect the final outcome in diVerent ways. While most 14-3-3-associated molecules bind via the motifs RSXpS/TXP or RXXXpS/TXP, exceptions do exist, as in the case of exoenzyme S, GPIb, and p190RhoGEF (Henriksson et al., 2002; Masters et al., 1999; Petosa et al., 1998; Wang et al., 1999; Zhai et al., 2001). In addition, 14-3-3 has been shown to bind expanded polyglutamine tracts in the protein ataxin1, thereby stabilizing the proteins and promoting neurodegeneration (Chen et al., 2003). In such cases, binding appears to be independent of phosphorylation, suggesting that an alternative means of regulating the 14-3-3 client protein interaction may be in operation. Each 14-3-3 monomer is composed of nine  helices arranged in antipar˚ amphipathic groove that mediates phosallel fashion to form a long
30 A phoserine/threonine target binding (Lui et al., 1995). The resolved crystal structures of 14-3-3 and 14-3-3 binding a phosphomimetic peptide demonstrate the structural similarity between isoforms and highlight the remarkable degree of structural conservation across the family (Lui et al., 1995; Xiao et al., 1995). The bulk of the conserved residues are seen to lie within the phosphoserine-binding groove (YaVe et al., 1997). Important

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interactions occur between the phosphoserine and the groove but also among other amino acids that form part of the 14-3-3 consensus binding site. This enables a high degree of specificity in 14-3-3 phosphoserine-binding interactions and would imply that regulation could alternatively be achieved by post-translational modification of the other residues that constitute a 143-3 binding site. This has been demonstrated in the case of p53 and Cdc25C, wherein phosphorylation of the serine at the 2 position prevented 14-3-3 association (Bulavin et al., 2003; Waterman et al., 1998). Obsil et al. (2001) reported the crystal structure of 14-3-3 together with a full-length client protein, serotonin N-acetyltransferase. The structure of 14-3-3, as in previous publications, was likened to a W-shaped clamp. Conformational changes were noted in the structure of 14-3-3 bound to the full length serotonin N-acetyltransferase compared to peptide binding, such that there was further opening up of the central channel with displacement of helix 8 away from the central binding groove (Obsil et al., 2001). This information has been useful in supporting the role of 14-3-3 as a dynamic clamp or ‘‘molecular anvil’’ that, upon binding its client protein, can assist in retaining an altered conformational state so that, upon phosphorylation, it is held in an active conformation that without the assistance of 14-3-3 would only be transient (YaVe, 2002; YaVe et al., 1997). B. Isoforms and Dimers—A Clue to Regulation? Varying numbers of 14-3-3 isoforms exist in diVerent organisms, yet each sequence retains a remarkable degree of homology. S. cerevisiae have two (BMH1 and BMH2), Drosophila has two (D14-3-3zeta/leonardo and D14-33epsilon), C. elegans has two (ftt-1 and ftt-2), while mammalian species have seven. Certain plant species such as Arabidopsis have nine isoforms and it has been postulated that isoform specificity may represent one level of regulation in 14-3-3 signaling. Interestingly, each individual isoform is more highly conserved among species than the conservation of isoforms within a single species. For example, 14-3-3 zeta in humans is 96 to 100% identical to zeta isoform in Drosophila and mouse, whereas the human epsilon and zeta isoforms show less than 60% identity (Rosenquist et al., 2000). This suggests strong selective pressure which may indeed reflect isoform-specific functions. The question of isoform-specific functions for the 14-3-3s has been contentious and diYcult to investigate, possibly because of the many and varied functions proposed for these proteins. Nevertheless, it has been postulated that isoform specificity may represent one level of regulation in 14-3-3 signaling. How such regulation occurs is currently unclear, especially given the high level of expression of all isoforms in cells and their high degree of homology.

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The most highly conserved residues between the 14-3-3 isoforms lie within the phosphopeptide-binding groove and so any diVerences in isoform function are presumably not related to phosphopeptide binding. When phosphopeptide libraries were tested on six of the human 14-3-3 isoforms as well as the two yeast isoforms, BMH1 and BMH2, all the isoforms were found to have very similar preferences for the individual residues within each consensus motif (Aitken et al., 2002). However, when similar experiments were performed for each of the nine Arabidopsis isoforms against a phosphopeptide based on the Hþ-ATPase C terminus, a wide range of binding aYnities was demonstrated by plasmon resonance spectroscopic analysis (Rosenquist et al., 2000). Binding of the HþATPase peptide ranged from very strong for some isoforms to extremely weak (Rosenquist et al., 2000). The Hþ-ATPase 14-3-3 binding motif is unique, being found only in plants (QQYpTV), and diVers from the usual mode 1 and mode 2 binding sequences found in most client proteins. This raises the notion that client proteins containing phosphopeptide sequences deviating from the usual consensus motifs may be more likely to show isoform specificity in regard to direct binding (Sehnke et al., 2002). It is clear that 14-3-3 isoforms are found as heterodimers in vivo and this may be more common than the occurrence of 14-3-3 homodimers. In the proteomics analysis by Jin et al. (2004), 14-3-3 target proteins were isolated for mass spectrometry by 14-3-3 antiFLAG immunoprecipitation. By far the most commonly associated molecules with 14-3-3 were the other 14-3-3 endogenous isoforms, indicating both their relative abundance and propensity for heterodimerization. Some studies suggest that a unique dimerization repertoire exists for each of the human isoforms (Aitken et al., 2002). When stably expressed in PC12 cells, 14-3-3 could form heterodimers with and . 14-3-3  could form heterodimers with all the other isoforms but no homodimers were detected, which suggests a basic interaction logic among members. These preliminary experiments highlight the need for further large-scale analyses of 14-3-3 isoform hetero/homodimerization in various subcellular compartments and for relating 14-3-3 isoforms to distinct subcellular functions. 14-3-3  is expressed primarily in epithelial cells and functionally appears to be the most specific of all human isoforms. Gene disruption studies of both 14-3-3 isoforms in Drosophila have shown major defects in cell cycle regulation that prevents normal embryogenesis (Su et al., 2001). Similarly in yeast, knockout of both isoforms alters cell budding and cell growth consistent with a cell cycle defect (van Heusden et al., 1995). The 14-3-3  isoform in humans appears to parallel the functions of 14-3-3 seen in these lower organisms. 14-3-3  expression is regulated by BRCA1 and p53 as part of the DNA-damage cell-cycle checkpoint (Aprelikova et al., 2001; Chan et al., 1999, 2000). Also, by interacting with Cdc2-Cyclin B complexes and sequestering them in the cytoplasm rather than nucleus, 14-3-3  can induce a G2/

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M arrest. Epithelial tumors, such as gastric cancer and breast cancer, have significantly reduced levels of 14-3-3 . Reduced 14-3-3  levels would presumably allow cells to override the G2-M checkpoint and would therefore aVord transformed cells a growth advantage. These functions correlate well with the defects in cell cycle control observed in the yeast and Drosophila gene disruption studies, implicating loss of G2-M control. C. Phosphorylation of 14-3-3 Binding Motifs In many cases, two or more 14-3-3 binding sites have been identified in target proteins. For example, 14-3-3 has been shown to bind serine 112, serine 136, and serine 155 in the pro-apoptotic bcl-2 family member, BAD (Datta et al., 2000; Lizcano et al., 2000; Tan et al., 2000; Zha et al., 1996). Similarly 14-3-3 has been shown to bind serine 259 and serine 621 in Raf-1 (Kuroda et al., 1996). It has been proposed that in such cases, two 14-3-3 binding sites in target proteins are important for establishing high-aYnity binding and a stable protein complex. It is also possible that 14-3-3 binding of two sites is important in the allosteric regulation of the target protein by promoting a conformational change in keeping with the ‘‘molecular anvil’’ model, such that 14-3-3 can clamp target proteins in a specific conformation (YaVe et al., 1997). While the 14-3-3 proteins have been shown to bind two sites in the same protein, they have also been shown to function as classical adaptors or scaVold proteins and bind two sites in two diVerent proteins. For example, 14-3-3 has been shown to bring together Raf-1 and several signaling proteins such as Bcr, protein kinase C, or the apoptosis regulator A20 (Braselmann and McCormick, 1995; van Der Hoeven et al., 2000; Vincenz and Dixit, 1996). The 14-3-3 proteins have also been suggested to act as adaptors linking glycogen synthase kinase-3 and Tau (Agarwal-Mawal et al., 2003) or the Ron receptor and the 6 4 integrins (Santoro et al., 2003). It has also been observed that where two or more 14-3-3 binding motifs are present on a client protein, there is often substantial variation in their binding aYnities for 14-3-3 when measured individually using phosphopeptide analysis (Aitken, 2002; YaVe et al., 1997). Regardless of the diVerence in binding aYnity, all sites appear to be required for optimal 14-3-3 binding and function of its client protein. It has been proposed that the high-aYnity motifs may act as the molecular gatekeepers, being required for the initial 14-3-3 interaction once in the phosphorylated state (YaVe et al., 1997). Once bound, other motifs become more accessible to kinases, or less susceptible to phosphatases, and can increase overall binding aYnity cooperatively, even though they are not high-aYnity binding sites themselves. This has been particularly well demonstrated in the case of Bad, in which serine 136

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appears to act as the initial attractor, whereas serine 112 helps to stabilize the complex (Chiang et al., 2003; Datta et al., 2000). The majority of 14-3-3 interactions are regulated by kinases and phosphatases acting on the phosphorylation sites of client proteins. A number of kinases have been shown to phosphorylate 14-3-3 binding sites, including Akt, p90Rsk, casein kinase 1, casein kinase II, protein kinase A (PKA), and PAK (for further references, see Aitken, 2002; van Hemert et al., 2001). It would appear that the substrate specificity of some of the kinases that phosphorylate 14-3-3 binding motifs overlaps with the consensus motif for 14-3-3 binding, a tantalizing concept in the study of global signaling. The basophilic kinases (e.g., PKA, Akt, checkpoint kinases Chk1 and Chk2/ Cds1) have been identified as being important in phosphorylating 14-3-3 binding sites and, similar to the motif for 14-3-3 binding, demonstrate a preference for basic residues at the 2 and/or 3 positions. This has revealed important clues as to the general biological functions of 14-3-3. For example, Akt/PKB phosphorylates targets that are important in the regulation of apoptosis, such as BAD and the transcription factor FKHRL1 (Durocher et al., 2000). Many of these targets are bound and inactivated by 14-3-3 binding, leading to cytoplasmic sequestration of Bad and Forkhead that prevents activation of apoptotic pathways at both the mitochondria and nucleus (Chiang et al., 2003; Datta et al., 2000; Durocher et al., 2000). In a similar fashion, ChK1 phosphorylates serine 216 of cdc25, leading to 14-3-3 binding and cell cycle arrest (Peng et al., 1997; Sanchez et al., 1997). It is likely that ongoing study of the proximal kinases responsible for activating 14-3-3 binding sites on client proteins will prove fruitful in our understanding of the global functions of 14-3-3.

III. 14-3-3 Pathways in Drosophila Development A. Tissue Growth and Cell Cycling Many important signaling molecules involved in cancer and developmental biology were first discovered in the fruit fly and later found to have homology to human and other vertebrate genes, including decapentaplegic, hedgehog, and patched (Lawrence and Struhl, 1996; Padgett et al., 1987). The canonical receptor tyrosine kinase Ras/Raf/MAPK signaling pathway, for instance, has all components represented in Drosophila and is active throughout development, especially in regard to cell proliferation and cell survival (Perrimon and Perkins, 1997). Temporal regulation and tissuespecific interpretation of such a universal signaling cassette appears to involve a number of mechanisms, including the existence of multiple tissuerestricted EGF-like ligands, tissue-specific negative and positive feedback

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loops induced by the Ras/Raf/MAPK pathway, and cooperation with other receptor tyrosine kinases (Perrimon and Perkins, 1997). Consistent with studies in human cells, 14-3-3 plays an important role in the intracellular components of this pathway, including the activation of Raf downstream of Ras (Chang and Rubin, 1997; Kockel et al., 1997; Li et al., 1997) and regulation of cyclin-dependent phosphastases/kinases. The two Drosophila 14-3-3 isoforms,  and  , show a change in localization from the cytoplasm to the nucleus as mitosis proceeds (Su et al., 2001). The phenotypes of Drosophila 14-3-3  and  mutants confirmed their conserved role in the cell cycle; 14-3-3  is required to time mitosis in undisturbed post-blastoderm cell cycles and to delay mitosis following irradiation and 14-3-3  is required for normal chromosomal separation in syncytial mitosis (Su et al., 2001). A technique using caged phosphopeptide activation has demonstrated further evidence for a role for 14-3-3 in cell cycle control (Nguyen et al., 2004). Human cell lines were incubated with phosphopeptides that contain a photolabile group protecting the phosphoserine so that release of uncaged phosphopeptide can be controlled in a rapid and temporally regulated manner inside cells on exposure to ultraviolet (UV) irradiation. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3 binding and caused premature cell cycle entry, release of G1 cells from interphase arrest, and loss of the S-phase checkpoint after DNA damage (Nguyen et al., 2004).

B. Determining Cell Polarity An intriguing observation by Benton and St. Johnston (2003) was the involvement of 14-3-3 in determining epithelial cell polarity in the developing Drosophila embryo. PAR-1, a serine/threonine kinase responsible for cell polarity, had been known to associate with 14-3-3 but in a phosphoserineindependent manner. Combination mutants of both Drosophila 14-3-3 isoforms showed the same defects in oocyte determination and A–P axis formation as a PAR-1 mutant embryo. In a yeast two-hybrid screen for 14-3-3 binding partners, a PDZ domain containing protein named Bazooka was identified that contained two consensus 14-3-3 sites required for defining apical complexes. A model is presented whereby PAR1 phosphorylates Bazooka on two 14-3-3 binding sites, allowing 14-3-3 binding and preventing Bazooka from forming laterally localized complexes. Since 14-3-3 can also associate directly with PAR-1 through a domain distinct from the phosphoserine-binding pocket (Benton et al., 2002), the authors concluded that 14-3-3 bound at one site could maintain PAR-1 in a transient complex with Bazooka, possibly to promote phosphorylation (and so 14-3-3 binding) at the

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other. Cooperative binding with additional phosphoserine-independent binding may be an emerging theme in other 14-3-3 kinase–substrate interactions.

C. Neuronal Signaling Interestingly, a major part of 14-3-3 function in Drosophila may be related to neuronal signal transduction. 14-3-3 expression is highest in the Drosophila brain, especially in the mushroom bodies, structures known to be important for learning and memory. In a screen for genes required in Drosophila olfactory learning behavior, 14-3-3  (leonardo) was isolated, implicating 14-3-3  as necessary for this important function (Broadie et al., 1997; Skoulakis and Davis, 1996; Philip et al., 2001). Most of the genes isolated thus far in Drosophila memory pathways have centered around the cyclic AMP pathway, many of which have been shown to interact with 14-3-3 in both humans and insects. Stimulation of protein kinases by cyclic AMP is required for longterm potentiation across the synaptic cleft (Waltereit and Weller, 2003). Both protein kinase A and many of its substrates including NFAT, neurofibromin, Raf, and MAPK have been shown to bind 14-3-3 (Chow and Davis, 2000; Dumaz and Marais, 2003; Feng et al., 2004). 14-3-3 also has highest expression in the brain of both humans and mice (1% of all soluble protein) and has been utilized as a clinical screen for patients suspected to have CNS prion disease. 14-3-3 function in neurons likely involves multiple binding partners, including members of the cAMP-stimulated pathway and the requirement for 14-3-3 in brain function may be a reflection of the complexity and multiplicity of neuronal signaling. Many human neurodegenerative diseases are highlighting the involvement of 14-3-3 in the modulation of a variety of pathological neuronal proteins, including ataxin-1 in spinocerebellar ataxia, Tau in Alzheimer’s disease, and LIS1 in Miller-Dieker syndrome (Agarwal-Mawal et al., 2003; Chen et al., 2003; Toyo-oka et al., 2003).

IV. Interaction with the Ras-Raf Signaling Pathway Raf serine/threonine kinase plays a critical role in transmission of growth factor-stimulated signaling via the Ras proto-oncogene (Campbell et al., 1998). Initial models suggested that the major function of Ras is in the activation of Raf by translocation to the plasma membrane (Leevers et al., 1994; Stokoe et al., 1994). However, the interaction between Ras and Raf-1 alone in vitro is insuYcient to activate Raf-1 kinase activity, suggesting that other factors are required. Raf-1 has been shown to bind 14-3-3 in the inactive conformation, as well as other factors such as heat shock protein 90 and p50 (Morrison and Cutler, 1997). Importantly, addition of 14-3-3 to

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membrane-free Raf-1 allows the activation of Raf by Ras (Kuroda et al., 1996). It is likely that 14-3-3 participates in a multisubunit protein/lipid complex responsible for the in vivo activation of Raf-1. Multiple binding sites on Raf-1 appear to interact with 14-3-3, including one in the cysteinerich domain (CRD) and two other sites with consensus 14-3-3 binding sequences at positions 259 and 621. Interestingly, the CRD of Raf can also bind anionic membrane phospholipid, phosphatidylserine, and the binding sites for phosphatidylserine and 14-3-3 overlap and show competitive binding (Campbell et al., 1998). Phospholipid interactions appear to be a common theme of 14-3-3 biology with some suggesting that the molecule functions to shield sites on proteins that would otherwise be involved with lipid moieties, thereby sequestering the molecule from a membrane-bound location. There are three functional Raf proteins in humans, A-Raf, B-Raf, and C-Raf, and all three depend on activation segment phosphorylation for proper activity. C-Raf and A-Raf require additional serine and tyrosine phosphorylation within the N region of the kinase domain for full activity (Mason et al., 1999). Serine 621, located within the catalytic domain of Raf, is required for 14-3-3 binding to Raf (RSXpSXPS). Mutation of this residue prevented activation of the Raf catalytic kinase domain by phorbol esters and Src, as well as disrupting 14-3-3 binding to Raf, confirming that 14-3-3 is required for both the stabilization of inactive Raf and also for proper kinase activity stimulated by growth factors (Yip-Schneider et al., 2000). The data summarized thus far highlight the subtle complexity of 14-3-3 in the regulation of Raf-1 in growth factor responses. It would seem that 14-3-3 is required in multiple steps of Raf activation and can interact via at least three diVerent binding sites, including a nonphosphorylated cysteine-rich domain. The mechanisms involved likely incorporate some of the concepts already discussed, including initial gatekeeping by a high-aYnity site, conformational changes in Raf, and re-localization.

V. 14-3-3 and Growth Factor Signaling Since the initial discovery of oncogenic growth factor receptors having constitutive tyrosine kinase activity (such as v-fms, v-mpl, and v-kit), much of the signaling literature to date has focused on tyrosine phosphorylation/ dephosphorylation as playing the major role in growth factor signaling (Schlessinger, 2000). Most hematopoietic growth factors are remarkable in their ability to stimulate a variety of functions (survival, proliferation diVerentiation, and eVector functions), with presumably all activities being transmitted via the one receptor complex. The spectrum of activities induced by a growth factor can depend on the developmental stage and lineage of the target cell being investigated, a common theme in ligand-receptor

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interactions in developmental biology (Perrimon and Perkins, 1997). The signaling pathways responsible for individual activities are not well understood but important concepts are beginning to emerge. First, it has been noted that receptors such as the common beta subunit of the GM-CSF, IL-3, and IL-5 receptors are phosphorylated not only on tyrosine but also on serine upon ligand binding, including serine 585. Second, while phosphorylation of diVerent tyrosines leads to the recruitment of several proteins containing SH2 or PTB domains such as STATs, Shc, or SHIP-2, the discrete phosphorylation of serine 585 promotes the association of 14-3-3 (Stomski et al., 1999). Third, while several phosphorylated tyrosines can support the recruitment of STATs, Shc, or SHIP-2, only serine 585 can recruit 14-3-3. The significance of these observations becomes clear when they are related to biological outcomes. In particular, two important observations have been made. First, there is a clear redundancy in terms of tyrosine phosphorylation whereby a mutated tyrosine can be compensated by other tyrosine residues. That is, no single tyrosine has been linked to a specific function. Second, and in contrast to the first point, serine 585 has been directly linked to cell survival. We have shown that the recruitment of 14-3-3 to serine 585 is essential for the survival signal (Guthridge et al., 2000) and couples the receptor to a downstream survival pathway (Guthridge et al., 2004). It is interesting to note that 14-3-3 plays a critical role in this survival pathway, acting at diVerent levels, from a membrane proximal adaptor linking the GM-CSF receptor to PI-3K, to a sequestering agent removing a pro-apoptotic factor and inactivating the Forkhead transcription factor (Fig. 1). Both activities appear to occur contemporaneously upon ligand binding and illustrate the mechanistic plasticity of 14-3-3, ultimately aVecting the same survival function. The extent of 14-3-3 binding and serine phosphorylation of the majority of growth factor receptors is unknown but, based on our preliminary findings, similar scenarios are likely to exist among other receptor family members. The insulin growth factor (IGF-1) receptor, for example, can also stimulate a robust survival response and is expressed on long-term repopulating stem cells (Zumkeller and Burdach, 1999). 14-3-3 has been shown to bind to IRS-1, an SH2-containing adaptor molecule important in IGF receptor signaling, as well as to the IGF-1 receptor itself (Craparo et al., 1997). Interaction occurs via three consensus phosphoserine motifs, one of which was within the phosphotyrosine binding domain of IRS-1 (Ogihara et al., 1997) but the functional consequences of these interactions remain to be elucidated. The prolactin receptor has been shown to bind 14-3-3 via a similar motif KCSTWP which occurs in the major functioning isoform of this receptor and is conserved among a wide variety of species (Olayioye et al., 2003) Similar to the GM-CSF/IL-3/IL-5 receptor, the prolactin receptor is also a member of the cytokine receptor superfamily. Interestingly, mutation of the

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Figure 1 Model illustrating the multiple roles of 14-3-3 in growth factor survival signaling as demonstrated for the GM-CSF/IL-3/IL-5 receptor. Growth factor stimulation results in phosphorylation of serine 585 of common beta subunit ( c) and recruitment of 14-3-3 zeta. This enables activation of PI3K and subsequent Akt/PKB phosphorylation of Bad and transcription factor Forkhead. 14-3-3 binding to phosphorylated residues on these molecules results in their cytoplasmic sequestration and prevention of default apoptotic signaling. 14-3-3 is also required for Raf activation in proliferation pathways.

critical threonine residue resulted in a receptor that exhibited increased basal and prolactin-induced tyrosine phosphorylation compared with the wildtype receptor, highlighting a possible role for 14-3-3 in modulating both phosphoserine and phosphotyrosine signaling pathways.

VI. Phosphorylation of 14-3-3 by Sphingosine-Dependent Kinase Sphingolipids and their metabolites, ceramide, sphingosine, and sphingosine-1-phosphate, are an emerging family of nonprotein signaling molecules involved in diVerentiation, apoptosis, and proliferation pathways in response to growth factors, G-protein coupled receptor agonists, and stressful stimuli. Sphingosine and sphingosine-1-phosphate derive from ceramide, a major secondary messenger which is produced by sphingomyelinase-induced hydrolysis of sphingomyelin (Ohanian and Ohanian, 2001). Woodcock et al. (2003) found that a sphingosine-dependent kinase could phosphorylate 14-3-3

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in vitro and in vivo on a serine residue (Serine 58) located within the dimer interface. By developing an antibody that specifically recognizes 14-3-3 zeta phosphorylated on serine 58 and employing native-PAGE and cross-linking techniques, it was shown that all 14-3-3 resorts to a monomeric form after serine 58 phosphorylation. Many growth factor receptors appear to be discretely localized to specialized lipid rafts, presumably hot spots for sphingosine signaling (Vamosi et al., 2004). Conceivably, sphingosinedependent kinase-induced monomerization represents another rapid regulatory mechanism of 14-3-3 signaling which may be involved in apoptosis induction by sphingosine (Suzuki et al., 2004).

VII. Conclusions Since 2000, 14-3-3 proteins have become increasingly relevant to signal transduction research with a range of interactions that impinge on virtually all aspects of cellular biology. Proteomic screens have revealed a multitude of novel binding partners, especially in the field of signal transduction, in addition to the hundreds of interactions already reported. 14-3-3 appears to have diverse roles ranging from cell metabolism, cell shape, scaVolding, adhesion, survival, proliferation, and mitosis. It is unlikely that one mechanistic model can account for all 14-3-3 functions; rather, specific molecular mechanisms are determined by the nature and conformation of 14-3-3 partners in a given interaction. It is tempting to speculate on the high frequency of 14-3-3 binding sequences and associated binding proteins in the proteome, reflecting a requirement for multiple cellular decision points for the proper coordination of signal transduction. Although the biochemistry is well characterized, a large gap remains in our understanding of 14-3-3 function at the level of the organism. Further study should explore the integration of phosphoserine and phosphotyrosine signaling by 14-3-3 proteins and the role of isoform-specific functions in higher organisms. The prevalence of functional 14-3-3 binding sites throughout the proteome, and especially among growth factor receptors and signaling molecules, may reflect multiple cellular decision points that together contribute to the high degree of plasticity evident in biological systems.

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Skeletal Stem Cells in Regenerative Medicine Wataru Sonoyama,* Carolyn Coppe,* Stan Gronthos,{ and Songtao Shi * *Craniofacial and Skeletal Diseases Branch National Institute of Dental and Craniofacial Research National Institutes of Health, Bethesda, Maryland 20892 { Mesenchymal Stem Cell Group, Division of Haematology Institute of Medical and Veterinary Science Frome Road Adelaide SA 5000, Australia

I. Introduction II. Isolation and Characterization of MSCs from Bone Marrow III. Niche Microenvironment of BMSSCs IV. Therapeutic Uses of BMSSCs A. Bone Regeneration B. Cartilage Regeneration C. Adipose Tissue Regeneration D. Neural Tissue Regeneration E. Potential to Regenerate Other Tissues V. Delivery of BMSSCs VI. Alternative Sources of MSCs VII. Future Direction References

Postnatal stem cells have been isolated from a variety of tissues and they are highly expected to have potentiality to be utilized for cell-based clinical therapies. Bone marrow stromal stem cells (BMSSCs) derived from bone marrow stromal tissue have been identified as a population of multipotent mesenchymal stem cells that are capable of diVerentiating into osteoblasts, adipocytes, chondrocytes, muscle cells, and neural cells. The most significant tissue regeneration trait of BMSSCs is their in vivo bone regeneration capability, which has been widely studied for understanding molecular and cellular mechanisms of osteogenesis, and, more importantly, developing into a stem-cell-based therapy. Recent studies further demonstrated that BMSSC-mediated bone regeneration is a promising approach for regenerative medicine in clinical trials. However, there are some fundamental questions that remain to be answered prior to successful utilization of BMSSCs in clinical therapy. For instance, how to maintain stemness of BMSSCs will be a critical issue for developing methodologies to propagate Current Topics in Developmental Biology, Vol. 67 Copyright 2005, Elsevier Inc. All rights reserved.

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multi-potential stem cells in vitro, in order to allow the development of eVective clinical therapies. # 2005, Elsevier Inc.

I. Introduction At present, tremendous research eVorts are being focused on developing stem-cell-based therapies for the purpose of regenerative medicine, utilizing stem cells derived from either embryonic or postnatal tissues. Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst and seem to hold great promise for regenerative and reparative medicine, depending on their pluripotent diVerentiation capacity (Audet, 2004; ReubinoV et al., 2000; Shamblott et al., 1998; Thomson et al., 1998). However, ethical controversy on the origin of ES cells, lack of underlying knowledge of how to regulate the diVerentiation of ES cells, and the question of immunotolerance of allogeneic preparations are all major obstacles for utilizing ES cells for cell-based therapies. On the other hand, postnatal stem cells can be isolated from tissue obtained from adults and children, and are thought to have the capacity to diVerentiate into multiple cell types. Currently, many kinds of tissue-specific stem cells have been discovered in a variety of organs including but not limited to bone marrow (Castro-Malaspina et al., 1980; Jiang et al., 2002a), neural tissue (Flax et al., 1998; Johansson et al., 1999), muscle (Chen and Goldhamer, 2003; Huard et al., 2003), skin (Janes et al., 2002; Lavker and Sun, 2003), intestine (Marshman et al., 2002), liver (Alison et al., 1997; Dabeva and Shafritz, 2003), dental pulp (Gronthos et al., 2000, 2002; Miura et al., 2003), and periodontal ligament (Seo et al., 2004). Notably, postnatal stem cells, such as hematopoietic stem cells (HSCs), have already proved to be eVective for the treatment of diVerent hematological conditions and malignancies in conjunction with various clinical therapies (Bacigalupo et al., 2000; Buckner, 1999). Therefore, expectations are high that other types of postnatal stem cells can also be used in cell-based therapies for various clinical applications in the near future (Korbling and Estrov, 2003). Bone marrow stromal tissue contains bone marrow stromal stem cells (BMSSCs), also described as mesenchymal stem cells (MSCs). The most important tissue regeneration trait of BMSSCs is their capacity to reconstruct bone and bone-associated marrow elements when implanted into immunocompromised animals (Ashton et al., 1980; Bab et al., 1986; Cassiede et al., 1996; Friedenstein et al., 1982; Goshima et al., 1991; Krebsbach et al., 1997; Mizuno et al., 1997). In the past few decades, many eVorts have been made to elucidate the characteristics of BMSSCs and to utilize them as vehicles for regenerative medicine. In this chapter, we

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discuss the cellular and molecular properties of BMSSCs and assess their potential for diVerent clinical applications.

II. Isolation and Characterization of MSCs from Bone Marrow In bone marrow aspirates, BMSSCs represent a small percentage of the total bone marrow mononuclear cell population (Castro-Malaspina et al., 1980; Falla et al., 1993; Pittenger et al., 1999; Simmons and Torok-Storb, 1991a,b; Waller et al., 1995). Thus far, there is no decisive method to isolate a pure homogeneous population of BMSSCs from bone marrow. A broadly utilized method to isolate BMSSCs is based on the colony forming unitfibroblast (CFU-F) assay, originally described by Friedenstein and colleagues (Friedenstein, 1976; Friedenstein et al., 1970). CFU-F are isolated from bone marrow aspirates based on their ability to adhere to a plastic surface and generate clonogenic colonies when plated at low cell densities in media containing fetal bovine serum (Friedenstein, 1976; Friedenstein et al., 1970). However, multipotential BMSSCs may represent only a small fraction of the total CFU-F population (Castro-Malaspina et al., 1980; Falla et al., 1993; Gronthos et al., 2003; Kuznetsov et al., 1997; Pittenger et al., 1999; Waller et al., 1995). Moreover, these plastic-adherent cells are not a homogenous population, but rather represent a continuum of stromal precursor cells composed of pluri-, multi-, bi-, and uni-potential progenitors (Owen and Friedenstein, 1988). Moreover, other cell types with ability to adhere to a plastic surface, such as endothelial cells, smooth muscle cells, and macrophages, are often mixed in among the CFU-F population in early culture. Although these contaminated cell populations eventually disappear following continuous ex vivo expansion, BMSSCs concurrently begin to gradually lose their ‘‘stemness’’ with successive cell passages. Therefore, many eVorts have been made to purify BMSSCs from bone marrow aspirates in order to determine the characteristics of pluri-potential BMSSC and establish eVective methods to identify and enrich these precursor cells. To date, only a handful of monoclonal antibody reagents have been identified that preferentially recognize cell surface molecules expressed by all human bone marrow CFU-F. The STRO-1 monoclonal antibody was first described as one potential reagent that reacted with a cell surface molecule highly expressed by human bone marrow CFU-F (Simmons and Torok-Storb, 1991b). Studies showed that STRO-1þ cells isolated from adult bone marrow contained all the CFU-F population and exhibited the ability to diVerentiate into multiple cell lineages including myelo-supportive stromal cells, osteoblasts, adipocytes, and

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chondrocytes (Dennis et al., 2002; Gronthos et al., 1994, 2003; Simmons and Torok-Storb, 1991a). Furthermore, the STRO-1þ fraction of bone marrow mononuclear cells showed a high clonogenic capacity when compared to unfractionated cells (Shi and Gronthos, 2003). In addition to STRO-1, two other antigens, CD106 (VCAM-1) and CD146 (MUC18), were also found to be candidate markers for the purification of BMSSCs (Gronthos et al., 2003; Shi and Gronthos, 2003). Their expression was found to be restricted to a minor fraction of STRO-1 high expressing bone marrow mononuclear cells, and showed little or no expression on bone marrow reticular cells, adipocytes, osteoblasts, and HSCs. These studies demonstrated that the CFU-F population could eVectively be purified to levels of up to 5000-fold more than unfractionated cells based on the immunophenotypes STRO1bright/CD106þ or STRO-1bright/CD146þ (Gronthos et al., 2003; Shi and Gronthos, 2003). A more in-depth understanding of the features of these cell surface antigens will allow us to purify BMSSCs and examine the characteristics of BMSSCs at diVerent stages of development (Gronthos et al., 1999). Other antigens including SH2, SH3, SH4, MAB-1470, and -smooth muscle actin were also reported to be positive for BMSSCs (Conget and Minguell, 1999; Galmiche et al., 1993; Pittenger et al., 1999; Simmons and Torok-Storb, 1991b). The fact that many of these markers are expressed by endothelial/perivascular cells suggests that the putative stem cell niche of BMSSCs may be closely associated with blood vessels (Shi and Gronthos, 2003). Despite the fact that the gene and protein expression profiles of cultured BMSSCs has been described, it is diYcult to determine the precise genetic signature of BMSSCs as they exist in the bone marrow microenvironment. Highly purified STRO-1bright/CD106þ BMSSCs were shown to lack the expression of osteogenic master gene (Cbfa-1) and adipogenic marker (PPAR 2) (Gronthos et al., 2003; Shi and Gronthos, 2003), consistent with the findings of Pittenger et al. (1999). Collectively, these studies suggest that at least a proportion of BMSSCs are not fully committed to any particular cell lineage. However, following ex vivo expansion and prolonged exposure to serum, many bone, adipocyte, and cartilage genes were notably upregulated by BMSSC (Castro-Malaspina et al., 1980; Gronthos et al., 2000, 2003; Rickard et al., 1996; Vilamitjana-Amedee et al., 1993). At the protein level, some markers expressed at early cell passages were subsequently down-regulated in later cell passages, in accord with the genetic profile of BMSSC assessed at the same culture periods (Abboud et al., 1986; Aye et al., 1992; Chen et al., 1991; Gronthos et al., 1999, 2000, 2003; Irlin and Peled, 1992; Long et al., 1990; Pittenger et al., 1999; Simmons et al., 1992; Soligo et al., 1990). These findings clearly indicate that the characteristics of BMSSCs begin to become unstable after prolonged cell culture. Functional data has also shown that extensive cell growth leads to a reduction in

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expression of the stem cell marker, STRO-1, cellular senescence, and a decrease in osteogenic potential by BMSSC (Shi et al., 2002; Simonsen et al., 2002). These results imply that it is necessary to develop optimal culture conditions to eVectively maintain BMSSCs in an uncommitted status during cell expansion. Existence of multipotent adult progenitor cell (MAPC) has been reported in both rodent and human bone marrow following selection under specific culture conditions (Jiang et al., 2002a,b). MAPCs showed a high proliferation capacity and could diVerentiate in vitro, at the single-cell level, into endothelial cells, neural cells, and hepatocytes, which surprisingly represents cells originating from all three germ layers. Moreover, when injected into early blastocysts, distribution of the cells originating from MAPCs was demonstrated in many organs using a chimeric mouse model (Jiang et al., 2002a,b). Additionally, the phenotype of cultured human MAPCs was negative for major human leukocyte antigen (HLA) class I and DR and cofractionated with MSC (Reyes et al., 2001). These results suggest that MAPCs may represent the pluripotential component of the BMSSC population and, as such, are an attractive stem cell source for designing cell-based therapies. However, it is necessary to conduct more studies to confirm the existence of MAPCs in human bone marrow and further elucidate their characteristics and full potential.

III. Niche Microenvironment of BMSSCs Postnatal stem cells are thought to reside in a specific microenvironment, or ‘‘stem cell niche.’’ It is proposed that stem cells cradling within their respective niches are quiescent and maintain basic stem cell characteristics, including a self-renewing capacity and an undiVerentiated or noncommitted status, until signaled to proliferate and diVerentiate into functional cells. There are considerable variations in diVerent putative stem cell niches that nurture a variety of postnatal stem cell types (Bianco and Robey, 2001; Fuchs and Segre, 2000; Fuchs et al., 2004; Shen et al., 2004). It has been revealed that osteoblasts play an important role in maintaining the HSC niche governed by bone morphogenic protein (BMP) (Calvi et al., 2003) and parathyroid hormone (PTH) signaling pathways (Zhang et al., 2003). These findings are the first to identify that osteoblasts derived from BMSSCs are functional regulators of their counterpart stem cells, HSCs. Although the BMSSC niche is located within the bone marrow stromal compartment, its precise location and interaction with other cells is poorly understood. There are a variety of cell types in the bone marrow, including adipocytes, fibroblasts, osteoblasts, endothelial cells, macrophages, reticular cells, and HSCs. BMSSCs express many diVerent kinds of growth factors and

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cytokines (Haynesworth et al., 1996; Majumdar et al., 1998; Pittenger et al., 1999) that may influence the development of diVerent cellular components within the bone marrow (Kaisho et al., 1994; Lorgeot et al., 1997). Conversely, other cell types in the bone marrow also produce growth factors and cytokines that would influence the development BMSSCs, helping to preserve the homeostasis of the bone marrow organ. A putative stem cell niche for BMSSCs has been identified, localized to the area surrounding the bone marrow microvasculature, based on immunohistochemical staining and extensive FACS analysis (Gronthos et al., 2003; Shi and Gronthos, 2003). Interestingly, the niche of dental pulp stem cells (DPSCs), another mesenchymal stem cell population, was also localized to the perivasculature area within dental pulp tissue, suggesting a potential common microenvironment for MSCs derived from diVerent tissues (Shi and Gronthos, 2003). However, whether other MSCs derived from adipose tissue (Gimble and Guilak, 2003) or periodontal ligament (Seo et al., 2004) share a similar perivasculature niche has not yet been determined. Continued eVorts to identify and understand the microenvironment that nurtures mesenchymal stem cells may lead to the establishment of successful isolation methods and the development of appropriate culture conditions for maintaining stem cell characteristics during ex vivo expansion.

IV. Therapeutic Uses of BMSSCs Clinically, HSCs have been successfully isolated and transplanted for the treatment of various hematological disorders and malignancies (Bacigalupo et al., 2000; Buckner, 1999). This long-term success of stem-cell-based therapy has fueled investigations into the potential therapeutic applications of other postnatal stem cell populations for the repair of damaged or diseased tissues. While BMSSCs have the ability to diVerentiate into a variety of cell types, they seem to readily default toward an osteogenic pathway, which may provide promising stem-cell-based therapies to treat nonunion fractures, congenital bone defects, bone disorders, and bone destruction caused by cancer. Surprisingly, some experimental data suggests that BMSSCs may also have the potential to be utilized in the treatment of non-bone-related diseases. These results give rise to new opportunities to explore the therapeutic potential of BMSSCs while raising new challenges for fully realizing the precise nature and characteristics of BMSSCs. A. Bone Regeneration The osteogenic diVerentiation capacity of BMSSCs has been well described in many previous reports. BMSSCs can diVerentiate into osteoblasts in vitro when cultured with dexamethasone, inorganic phosphate, and ascorbic acid

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(Gronthos et al., 1994; Pittenger et al., 1999). They are also capable of forming a mineralized matrix consistent with physiological hydroxyapatite correlating with an up-regulation in the expression of the osteoblastassociated marker, alkaline phosphatase (Gronthos et al., 1994; Pittenger et al., 1999). Clonal analysis has demonstrated that a large portion of BMSSCs-derived clonal cell lines exhibited some level of osteogenic potential above other diVerentiation pathways (Friedenstein, 1980; Gronthos et al., 2003; Kuznetsov et al., 1997; Muraglia et al., 2000). Importantly, BMSSCs were shown to be capable of regenerating a functional bone/marrow organ-like structure when transplanted either in diVusion chambers, or beneath the kidney capsule, or with an inductive biocompatible carrier into immunocompromised animals. (Ashton et al., 1980; Bab et al., 1986; Cassiede et al., 1996; Friedenstein et al., 1982; Goshima et al., 1991; Krebsbach et al., 1997; Mizuno et al., 1997). It has been proposed that the initiation of human osteogenesis triggers a series of responses from recipient cellular components, including up-regulated expression of bFGF and MMP9 leading to the establishment of a murine hematopoietic marrow chamber in open transplants (Batouli et al., 2003). These studies point to a practical potential to utilize BMSSCs for bone regeneration in the clinic. However, approximately 60% of single colonyderived BMSSC lines were able to form bone in vivo when transplanted into immunocompromised mice, with fewer than half having the potential to also support local hematopoiesis. The remaining 40% of single colony-derived BMSSC lines failed to generate any mineralized tissue in vivo (Gronthos et al., 2003; Kuznetsov et al., 1997). Thus, heterogeneity is a feature of BMSSCs, both in cell culture and in vivo, which is a practical reality that needs to be addressed in order to develop successful MSC-mediated therapies in the future. In preclinical animal models, there is a considerable amount of successful experimental data to indicate that BMSSCs can be applied for bone repair or regeneration. Ex vivo expanded mouse BMSSCs were applied to repair a critical-sized craniotomy defect in immunocompromised mice. Within 2 weeks, approximately 99% healing was achieved with a comparison of 17.7% healing in the control group comprised of spleen-derived stromal cells (Krebsbach et al., 1998). Human BMSSCs have also been found to increase bone formation in critical-sized segmental defects in the femurs of adult athymic rats. Those femurs that received BMSSCs proved to be biomechanically stronger than the controls treatments without BMSSCs (Bruder et al., 1998). Additional supporting data were obtained using larger animal models to further confirm the therapeutic potential of BMSSC-mediated bone regeneration (Chistolini et al., 1999; Kon et al., 2000; Petite et al., 2000; Shang et al., 2001). In order for BMSSC-mediated bone regeneration to become an eVective and accepted therapy, a successful method of delivery of adequate amounts

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of ex vivo expanded BMSSCs needs to be developed. Genetic manipulation is currently being used in certain groups as a way to maintain large amounts of committed osteogenic cells during ex vivo expansion. In one method, overexpression of BMP by BMSSCs was capable of causing constant delivery of these potent osteoinductive proteins, leading to an improved healing of large segmental femoral and calvarial defects in animal models (Gysin et al., 2002; Lieberman et al., 1998). Telomerase, a cellular ribonucleoprotein reverse transcriptase, has also been utilized to maintain large populations of osteoprogenitors. By preserving the telomere regions of the chromosomes, telomerase allows the cell to exceed the natural limit of cellular replication. Transduction of telomerase into BMSSCs caused an increase in both the lifespan and the bone-forming capacity of BMSSCs, resulting in enhanced osteogenic capacity in vitro and in vivo in mice (Shi et al., 2002; Simonsen et al., 2002). BMSSCs have been successfully used for human segmental bone defect treatment (Quarto et al., 2001). Following ex vivo expansion, autologous BMSSCs were transplanted with a macroporous hydroxyapatite scaVold into patients with nonhealing bone defects caused from unsuccessful surgical attempts, trauma, or plurifragmental fractures. The newly regenerated bone integrated with existing host bone and the healing period was shortened. However, the fate of the autologous BMSSC and their direct contribution to the new bone formation remains to be determined. In a 2004 study, a successful bone marrow stromal cell-mediated tissue regeneration case report was described (Warnke et al., 2004), in which a large part of surgically removed mandible was attempted to be reconstructed. The custom-made mandible-shaped scaVold was filled with autologous bone marrow cells, bovine bone mineral blocks, and BMP-7, and then incubated in the patient’s back for 7 weeks prior to surgical reconstruction of the mandible. The reconstructed mandible was found to be functionally remodeling, and masticatory function was partially recovered. These human clinical trials provide convincing evidence to encourage utilizing mesenchymal stem cells for cell-based therapies for a range of diVerent applications.

B. Cartilage Regeneration Studies have shown that BMSSCs can undergo chondrogenic diVerentiation when cultured under a 3-dimensional serum-free setting in the presence of TGF- s (Pittenger et al., 1999), confirmed by the expression of type II collagen and aggrecan (Gronthos et al., 2003; Pittenger et al., 1999). Moreover, ex vivo expanded BMSSCs were found to have the ability to generate ectopical cartilage in vivo (Ashton et al., 1980; Bab et al., 1986; Cassiede et al., 1996; Goshima et al., 1991). These results are well matched with the

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observation that most clones (60–80%) of human BMSSCs displayed an osteo/chondrogenic potential (Muraglia et al., 2000). Ovine BMSSCs implanted to fetal tracheas showed a chondrogenic diVerentiation and produced cartilage matrix, glycosaminoglycans, and type II collagen (Fuchs et al., 2003). In addition, improved cartilage repair in experimental defects made on the patellar groove in a rabbit model has also been reported (Im et al., 2001). Although the potential of using BMSSCs for cartilage repairing or regeneration shows much promise, the mechanisms of BMSSC-mediated cartilage regeneration are not yet fully elucidated, which may hinder the development of cell-based therapy for cartilage regeneration.

C. Adipose Tissue Regeneration Adipogenic diVerentiation of BMSSCs is observed after cultivation with a combination of various inductive agents such as dexamethasone, hydrocortisone, isobutilmethylxantine, indomethacin, and insulin (Gimble and Guilak, 2003; Gronthos et al., 2003; Pittenger et al., 1999). Under these conditions, a large number of BMSSCs changed morphology and became adipocytes, characterized with cytoplasmic lipid vacuoles. This diVerentiation was also genetically confirmed by the expression of the fat-associated markers, PPAR 2 and leptin (Gronthos et al., 2003). Although engineering adipose tissue has important clinical impacts for endocrinologic disorders and cosmetic surgery, there is no updated experimental evidence to demonstrate that BMSSCs can be used for adipose tissue engineering in large preclinical models or in humans.

D. Neural Tissue Regeneration Cultured BMSSCs have the ability to diVerentiate into neural cell lineages, such as astrocyte and oligodendrocyte, and this fact has led to many studies trying to utilize bone marrow cells for neural tissue regeneration or repair (Azizi et al., 1998; Chopp et al., 2000; Hofstetter et al., 2002; Jin et al., 2002; Li et al., 2001; Woodbury et al., 2000; Zhao et al., 2002). Human BMSSCs injected directly into rat brain were found to be able to survive for 72 days without any evidence of an inflammatory response or rejection (Azizi et al., 1998). Interestingly, adult bone marrow cells were found to be able to enter the brain and generate neurons in patients that had undergone bone marrow transplantation (Mezey et al., 2003). Moreover, it was also reported that bone marrow cells improve neural function in spinal cord injuries of rat (Chopp et al., 2000; Hofstetter et al., 2002). However, the exact identity and origin of these neural progenitors remains to be defined. Nevertheless, these

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results suggest a possibility for using BMSSCs to prevent neurodegenerative diseases or to repair damaged neural tissues.

E. Potential to Regenerate Other Tissues Further evidence for the multipotential of BMSSCs can be seen by their capacity to diVerentiate into cardiac muscle cell, skeletal muscle cell, endothelial cell, tenocyte, and hepatic cell (Korbling and Estrov, 2003; Minguell et al., 2001). Of the many therapeutic applications that have been suggested, the strategy to utilize BMSSCs for heart disease treatment has been considered one of the more important clinical advances due to the alarming increase in the incidence of cardiovascular disease throughout developed countries. Early studies reported that a demethylation compound, such as 5-azacytidine or amphotericin B, induced myogenic diVerentiation of BMSSCs in vitro (Makino et al., 1999; Phinney et al., 1999; Wakitani et al., 1995). Follow-up studies demonstrated that BMSSCs had varying capacities to diVerentiate into cardiomyocytes, endothelial cells, pericytes, or smooth muscle cells using various animal models (Airey et al., 2004; Davani et al., 2003; Gojo et al., 2003), and even demonstrating a potential functional improvement (Min et al., 2002). A randomized clinical trial demonstrated the eVectiveness of using autologous BMSSCs to treat acute myocardial infarction patients (Chen et al., 2004). A significantly improved functional recovery was identified in those patients that received autologous BMSSC injection when compared to the control group that received standard saline alone. Although all these studies encourage further investigations into the therapeutic potentials of BMSSCs, great care should be taken in assessing their eYcacy. Particularly, the precise functional mechanisms contributing to the transdiVerentiation of BMSSCs are still yet to be determined. Therefore, more research is required to elucidate whether BMSSCs are capable of regenerating these tissues, either directly through transdiVerentiation or indirectly by cell fusion or by stimulating the recruitment of local tissue-specific precursor populations.

V. Delivery of BMSSCs An interaction between BMSSCs and the carrier materials is critical in determining the survival and diVerentiation pathways of these cells for diVerent clinical applications. For example, human BMSSCs can diVerentiate into osteogenic cells only in association with the carrier material containing calcium phosphate when transplanted into immunocompromised

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mice. In contrast, mouse BMSSCs can form bone in vivo with several kinds of carrier materials that may not contain a calcium phosphate component (Krebsbach et al., 1997). This diVerence suggests that the biomaterials suitable for mouse or animal BMSSC-mediated tissue regeneration may not be applicable for human BMSSCs. Therefore, it is necessary to develop specific biocompatible scaVold/materials to promote human BMSSC diVerentiation in vivo for tissue regeneration and integration into the surrounding environment, in order to generate accurate reconstruction of functional complex organ systems. Systemic infusion is another option for the delivery of BMSSCs for therapeutic purposes. Systemic infusion is an attractive technique because of easy accessibility and reduced trauma compared to direct injection or surgical transplantation. Although it was suggested that systemically infused BMSSCs have the capacity to migrate into an injured site (Wang et al., 2002a,b), the eYciency of delivered BMSSCs to reach the desired sites remains a controversial issue. Previous reports have suggested that the monocyte chemoattractant protein-1, increased in injured rat brain, seemed to support the migration of BMSSCs to that site (Wang et al., 2002a,b). Moreover, genetically manipulated BMSSCs have been successfully used as vehicles for gene therapy to treat systemic diseases such as osteogenesis imperfecta (Horwitz et al., 1999, 2001, 2002), metachromatic leukodystrophy, and Hurler syndrome (Koc et al., 2002). If indeed BMSSCs can be targeted to the desired sites, it may provide insight for understanding the nature of BMSSC homing, which will greatly benefit systemic application of BMSSCs in the future.

VI. Alternative Sources of MSCs Although BMSSCs are the most well-studied MSC population, other tissues have also been found to contain tissue-specific MSCs. Adipose tissue is one of the tissues that has proved to contain MSCs, named adipose-derived adult stromal (ADAS) cell (Gimble and Guilak, 2003; Halvorsen et al., 2001; Hicok et al., 2004). ADAS cells were identified as a population of postnatal stem cells with multipotent diVerentiation potential (Gimble and Guilak, 2003; Halvorsen et al., 2001; Tholpady et al., 2003; Wickham et al., 2003; Zuk et al., 2001). More interestingly, ADAS cells were able to diVerentiate into osteoblast in vitro and in vivo (Cowan et al., 2004; Halvorsen et al., 2001; Hicok et al., 2004; Lee et al., 2003; Zuk et al., 2001), which suggest a great potentiality to use ADAS cells for bone regeneration (Cowan et al., 2004). Clinical availability of adipose tissue is an important advantage to utilize ADAS cells for therapies. However, MSCs isolated from diVerent tissues may have diVerent developmental potentials. Several studies have suggested that MSCs derived from craniofacial bone marrow stromal tissue may diVer from those derived from long bone marrow, with respect to their proliferation

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capacity and tissue regeneration capabilities (Akintoye et al., 2002). Therefore, great care should be taken in designing stem cell-based therapies that utilize the appropriate stem cell populations from the most favorable donor site. This will ensure that the right source of postnatal stem cell population is utilized for appropriate regenerative medicine applications. It has been reported that peripheral blood may contain circulating MSCs (Fernandez et al., 1997; Kuznetsov et al., 2001; Zvaifler et al., 2000). These MSCs demonstrated similar properties to BMSSCs, in respect to their capacity to form clonogenic adherent cell clusters, and by their ability for osteo/adipogenic diVerentiation in vitro and potential to generate bone when transplanted into immunocompromised mice (Kuznetsov et al., 2001). However, conflicting data from other studies suggests that peripheral blood MSCs were found only in patients receiving chemotherapy and G/GM-CSF administration, but not in blood from healthy donors (Fernandez et al., 1997). The existence and multipotency of mesenchymal cells in umbilical cord blood has also been demonstrated (Gang et al., 2004; Goodwin et al., 2001; Lee et al., 2004). This suggests that MSCs in circulation may have some function during development or in times of stress and disease in postnatal organisms. Further studies are clearly required to elucidate their characteristics and roles in tissue healing or regeneration.

VII. Future Direction Accumulating evidence strongly supports the notion that BMSSCs can be utilized not only for bone regeneration but also for the treatment of other non-bone-related diseases. One important question that remains to be answered is whether therapeutic outcomes from these non-bone-related disease treatments come from transdiVerentiation of BMSSCs or their fusion with the already existing cells or the recruitment of endogenous stem cell populations. It is clear that continuing studies are still required to properly identify the stem cell characteristics and full developmental potential of diVerent MSC populations and to develop methodologies to propagate multipotential stem cells in vitro, in order to allow the development of eVective clinical therapies.

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Index A Acceptance posture, D. virilis group female, 231 Adenylyl cyclases (ACs), cAMP steady state controlled by, 207–208 Adipose-derived adult stromal (ADAS) cells, 316 Aggrecan, in antler chondrocytes, 18, 20–21 Aging ethylene/leaf senescence v., 67 leaf senescence v. developmental, 62–64 Agouti-related peptide (AgrP), feeding behavior regulated by, 209–210 AGS. See Antler growth stimulus Alkaline phosphatase (ALP) activity in antler cartilage/bone, 16, 20, 21 in antler mesenchymal cells, 17 mesenchymal cell diVerentiation v., 11–12 Alzheimer’s disease, 14-3-3 in, 294 Androgen in antler development/regeneration regulation, 25–26 pedicle formation v., 5 ANP. See Atrial natriuretic peptide Antibodies, CR-1 v. monoclonal blocking, 117–118 Antler growth stimulus (AGS), IGF-I as, 27 Antlerogenic periosteum (AP) antler/pedicle development initiated by, 3–4 molecular characterization of, 5–6 Antlers, 2 annual loss/regrowth cycle of, 7 branch formation in, 4, 13–14 cell lineage studies in, 10 chondrogenesis in, 17–21 development of first set of, 3–6 development/regeneration regulation in, 24–37 early development of, 7–13 external/systemic factors regulating development/regeneration of, 24–29 factors influencing size of, 6–7

growth factors/hormone receptors/ signalling molecules in, 30–37 growth rates/sizes of, 13–14 human fascination with, 2 local mechanisms regulating development/ regeneration of, 29–37 longitudinal growth in, 15–17 as mammalian regeneration model, 2–37 molecules expressed in early regenerating, 36–37 ontogeny of, 13–15 ossification/remodeling in regeneration of, 21–23 pedicle height v. phylogeny of, 3 regeneration of, 6–7 AP. See Antlerogenic periosteum Apoptosis embryonic, 136–137 nucleus as primary target of, 158 AQP1 apical localization in developing choroid plexus, 183, 184–185 choroid plexus water permeability enabled by, 182 function as gated cation channel, 189–191 hypothetical five-pore structure, 189–190 Aquaporin water channels, 182–183 Aquaporins (AQPs) in choroid plexus, 182–199 as MIPs, 182 Arabidopsis autophagic genes in normal/embryonic mutant, 140, 142–143, 170 as leaf senescence model, 52 molecular/genetic control of leaf senescence/longevity in, 50–77 14-3-3 protein isoforms in, 289–290 reproductive structure v. leaf senescence, 76 zygotic embryogenesis, 139–145 Arginine vasopressin (AVP), CSF production decreased by, 193

325

326 Atrial natriuretic peptide (ANP), CSF secretion decreased in response to, 191–192, 193 Autophagosomes, 155 in cell dismantling, 160, 161 Autophagy control of metabolic/selective v. destructive, 171 in leaf senescence, 58 in PCD, 154–157 Autophagy proteins, death function of plant orthologues to yeast, 169–170 Avena sativa, subtilisin-like serine proteases in, 164–165 AVP. See Arginine vasopressin B Basal cell carcinomas, CR-1 expression in, 115 14-3-3 binding motifs phosphorylation of, 291–292 varying binding aVinities in, 291–292 Biology, antlers’ relevance to, 2–3 Bladder carcinomas, CR-1 expression in, 114–115 Blastemas, early antler regions described as, 9–10 Blood supply, shed antler/pedicle, 8 BMPs. See Bone morphogenic proteins BMSSCs. See Bone marrow stromal stem cells Bone antlers having compact/cancellous, 16, 22 regeneration/BMSSCs in animal models, 311 regeneration v. ADAS cells, 315–316 regeneration v. BMSSCs, 310–312 Bone marrow cell types in, 309 isolation/characterization of BMSSCs from, 307–309 Bone marrow stromal stem cells (BMSSCs) in adipose tissue regeneration, 313 antigens positive for, 308 in bone/bone-associated marrow reconstruction, 306–307 bone marrow microenvironment genetic signature of, 308–309 bone/marrow organlike structure regenerated by, 311 chondrogenic diVerentiation of, 312–313 delivery of, 314–315

Index human segmental bone defect treatment with, 312 isolation/characterization of, 307–309 MAPC as pluripotential component of, 309 in neural tissue regeneration, 313–314 niche localization for, 309 niche microenvironment of, 309–310 prolonged cell culture v. characteristics of, 308 regenerative medicine v. skeletal, 306–316 therapeutic delivery of ex vivo expanded, 311–312 therapeutic uses of, 310–314 Bone morphogenic proteins (BMPs) binding/signaling by, 93 HSC niche signaling pathway of, 309 Nodal inhibiting signaling by, 106 skeletal development/regeneration regulated by, 30, 33, 34 Breast cancer 14-3-3  v., 291 CR-1 expression in, 110, 112–113 Breast cancer cells, estrogen v. MCF-7, 100–101 Brocket deer, antler structure, 14 Buds anatomy/histology of antler, 10–12 antler branches visible at antler, 8 C Caþþ, in leaf senescence regulation, 59, 61 Caenorhabditis elegans developmental cell death in, 137 metabolic rate determined by genes, 63 14-3-3 protein isoforms in, 289 CAMP. See Cyclic adenosine monophosphate Cancer. See also specific types of cancer BMSSCs v. bone defects/disorders/ destruction from, 310 CR-1 v. therapy in human, 115–118 Carcinomas, CR-1 expression in human, 109–115 Cardiogenesis EGF-CFC gene expression/function in, 96–97 wnt/ -catenin signaling in, 102 Cartilage antler, 19–21 calcification of antler, 16, 21 regeneration with BMSSCs, 312–313

Index Caspases metacaspases as ancestors of canonical, 166, 167 in metazoan apoptosis, 164 CD106, as candidate marker for BMSSC purification, 308 CD146, as candidate marker for BMSSC purification, 308 Central nervous system (CNS), main types of extracellular fluids in, 182 Cerebrospinal fluid (CSF), 182 composition v. plasma, 194–195 neurotransmitters/neuropeptides/growth factors v. secretion of, 196 production regulation, 192–193 secretion by choroid plexus ion channels, 185–188 secretion downregulation v. dark cells, 193–194 secretion v. AQP1-mediated ionic conductance, 191–192 system separated from brain, 186, 196–197 Cervical cancer, CR-1 expression in, 110, 113–114 CFC domain. See Cripto-1/FRL-1/Cryptic domain CFU-Fs. See Colony forming unit-fibroblasts CGMP. See Cyclic guanosine monophosphate Chase-away hypothesis, 229 Chlorophyll, loss in leaf senescence, 52 Chloroplasts, senescence v. membrane structure in, 53 Chondroblasts, mesenchymal cells diVerentiating into, 12 Chondrocytes ALP/PTHrP v., 12 STRO-1þ cells diVerentiating into, 307–308 type X collage v. hypertrophy of, 19–20 Chondrogenesis, antler, 17–21 Choroid plexus, 182 barrier function, 194–197 ‘‘dark cells,’’ 193–194 development, 183, 184–185 innervation, 197–198 ion channels in, 185–188 localization, 195–196 neuroendocrine function, 197–198 pathophysiology, 198–199 physiological roles of aquaporins in, 182–199

327 structure, 196 CNS. See Central nervous system CNS prion disease, 14-3-3 in screening for, 294 Collagen antler cartilage having type X, 18, 19–20 antler chondrocytes v. type II, 18 antler mesenchymal cells v. type II, 17 chondrogenic diVerentiation v. type IIA, 19 Colon cancer, CR-1 expression blocking v., 117–118 Colon carcinoma cells, anti-CR-1 antisense oligonucleotides v., 116–117 Colony forming unit-fibroblasts (CFU-Fs) in BMSSC isolation, 307–308 homogeneity of, 307 Colorectal cancer, CR-1 expression in, 110, 111–112 Cripto-1 (CR-1), 86–120 adult tissues expressing isoforms of, 92 in EGF-CFC protein family, 88 expression in human carcinomas/ premalignant lesions, 109–115 FGFR-1 signaling pathway activated by, 107–108 Glypican-1/c-src/MAPK/AKT signaling pathway activated by, 104, 107–109 intracellular signaling pathways activated by, 103–109 mammalian cell Activin signaling v., 104, 105 in milk v. development, 98 mRNA encoded by, 92 Nodal/ALK4/ALK7/Smad-2 signaling pathway activated by, 103–106 protein size variation in, 90 as therapy target in human cancer, 115–118 in transformation/tumorigenesis, 99–102 Cripto-1/FRL-1/Cryptic (CFC) domain in EGF-CFC genes, 88 human CR-1, 89 CSF. See Cerebrospinal fluid Cyclic adenosine monophosphate (cAMP) food intake v. hypothalamic eVects of, 210, 211 in long-term potentiation across synaptic cleft, 294 in model of hypothalamic feeding control, 216–217, 217–218 in NPY neurons v. energy balance, 215–217

328 Cyclic adenosine monophosphate (cAMP) (cont.) obesity v. hypothalamic dysregulation of, 218 as orexigenic second messenger, 210–212 in PAH signaling pathway, 252, 253 PDE inhibitors/vasodilators stabilizing, 256, 271–272 in pulmonary hypertension treatment, 256 regulation/cellular eVects, 207–208 regulation v. food intake control, 207–218 Cyclic guanosine monophosphate (cGMP) AQP1 activated by intracellular, 190–191 in PAH signaling pathway, 252–253 PDE inhibitors/vasodilators stabilizing, 256, 271–272 in pulmonary hypertension treatment, 256 Cytokinins, leaf senescence delayed by, 65–66 Cytoskeleton, changes v. apoptotic/ autophagic cell death, 157 D Deer. See also specific types of deer antlers modeling mammalian regeneration, 2–37 Dental pulp stem cells (DPSCs), MSC common microenvironment v. niche of, 310 Developmental aging, Arabidopsis genes v., 62–64 DNA microarray, SAG investigation aided by, 56 Drosophila embryo epithelial cell polarity v. 14-3-3, 293–294 female preferences/mate choice benefits in, 236–238 metamorphosis v. developmental cell death, 164 14-3-3 pathways in development of, 292–294 14-3-3 protein isoforms in, 289 tissue growth/cell cycling v. 14-3-3 pathways, 292–293 Drosophila melanogaster direct costs of mating in, 227 male courtship songs v. mutations, 235–236 sexually antagonistic genes in, 229

Index Drosophila montana courtship song frequency v. oVspring survival, 237–238 courtship song v. temperature, 240–241 factors influencing male song evolution in, 226–245 female preference v. PL/carrier frequency, 239, 240 female preference variation in, 242 females preferring short/dense sound pulses, 236 genetic variation in courtship songs of, 233–234 genetics of female preference in, 242–243 male size v. mate choice in, 236–237 male song variation in, 231–236 relative mate choice strategy in, 241–242 as song evolution study subject, 230–231 song oscillogram, 232 song repertoire/traits, 232–233 song simulation experiments, 238–239, 240 Drosophila virilis, song oscillogram, 232 Drosophila virilis group, 230–231 female acceptance in, 231 male song character genetic basis in, 234–236 male song variation in, 231–236 song diVerences among, 243 E ECM. See Extracellular matrix EGF. See Epidermal growth factor EGF-CFC co-receptors, Activin receptor use modulated by, 104–105 EGF-CFC genes embryonic development v. expression of, 92–97 as example gene family in cancer pathogenesis, 120 genomic organization, 91–92 physiochemical properties of, 88–91 structure/genomic organization, 88–92 EGF-CFC proteins, Nodal-independent pathway cell motility induction by, 119–120 Elk. See Wapiti Embryogenesis 14-3-3 v. cell polarity in Drosophila, 293–294 Cr-1 expression during, 93–94

329

Index CR-1 v. MAPK/AKT signaling pathways in, 108 EGF-CFC gene expression in, 92–97 model plant embryonic systems in, 139–154 morphogens in, 87–88 oncogenesis v., 86–87 PCD in plant, 136–171 14-3-3 protein isoforms v. Drosophila, 290–291 somatic, 145 viviparous v. amphibian/invertebrate, 137 Embryogeny early Picea abies, 140, 146, 147 late Picea abies, 146, 147–148 Embryonic stem (ES) cells, ethical controversy v. medical use of, 306 Embryos early development of Picea/Arabidopsis, 140, 146, 148 elimination of subordinate Pinus sylvestris, 151–152 PCD trigger signal source in, 144–145 in seed removed by PCD, 138 EMT. See Epithelial mesenchymal transition End buds, ovarian hormones stimulating mouse mammary, 97 Endometrial cancer, CR-1 expression in, 110, 113 Endopeptidases, in stroma protein degradation, 55 Endoplasmic reticulum (ER), membranes as autophagosome precursors, 156–157 Enzymes, leaf senescence v. lipid-degrading, 53–54 Epidermal growth factor (EGF), in developing antlers, 30, 34–35 Epithelial mesenchymal transition (EMT) Cr-1 mRNA expression v., 93–94 in gastrulation, 94 induction v. Cr-1, 101–102 Epithelium CR-1 immunoreactivity in, 109–110 ‘‘light’’/‘‘dark’’ cells in choroid plexus, 193 proliferation/involution cycles in mammary gland, 100–101 regenerating wound, 8, 10 ER. See Endoplasmic reticulum ES cells. See Embryonic stem cells Estrogen in antler development/regeneration regulation, 27

levels v. antler regeneration, 7–8 Ethylene, leaf senescence accelerated by, 66–67 Expressed sequence tag (EST) analysis, SAGs identified using, 56 Extracellular matrix (ECM) adhesion v. EMT, 94 in prenatal mouse mammary cell interaction, 97 F Fallow deer, antler structure, 14 FGF-4, immunolocalization in early antler, 36, 37 Fibroblast growth factor (FGF), skeletal development/regeneration regulated by, 28, 30, 34 G GAGs. See Glycosaminoglycans Gall bladder carcinoma, CR-1 expression in, 110, 112 Gastric cancer 14-3-3  v., 291 CR-1 expression in, 109–110 Gastrulation, EGF-CFC gene expression v., 94–95 Genes. See also specific types of genes Cis-acting regulatory elements of senescence-induced, 72 potential senescence-regulatory, 69–72 senescence v. regulation by transcription factor, 69–70 viral infection/carcinogens activating, 86 Germ layer, formation v. EGF-CFC gene expression, 94–95 GH. See Growth hormone Ghrelin cAMP increase in NPY/AgrP neurons v., 215–217 in negative energy balance state, 217 Glucagonlike peptide-1 (GLP-1) energy homeostasis influenced by, 208 feeding/drinking behavior v., 212–213 Glycosaminoglycans (GAGs), in antler cartilage, 18, 20–21 Growth hormone (GH), IGF-I secretion regulated by, 28 Growth plates, growing antler tips v., 15–17, 18, 21

330 H Handicap principle, in mate choice, 227–228 Heart disease, treatment using BMSSCs, 314 Helicobacter pylori, infection v. CR-1 expression, 110 Hematopoietic stem cells (HSCs) hematological conditions/malignancies v., 306 osteoblasts in maintaining niche of, 309 Hormones hypothalamus energy homeostasis influenced by, 208 mouse mammary gland v. ovarian, 97–98 in pregnancy v. Cr-1 expression, 99–100 HPV. See Hypoxic pulmonary vasoconstriction HSCs. See Hematopoietic stem cells 5-HT. See Serotonin Hydrocephalus, CSF/choroid plexus tumors v., 198–199 Hyperphagia, NPY/AgrP inducing, 209 Hypothalamus feeding regulation at, 208–210 region-specific cAMP eVects in, 211–212 Hypoxia intravenous PDE 3/4 inhibitors in chronic, 261 intravenous PDE 3 inhibitors in acute, 260 intravenous PDE 5 inhibitors in acute, 261 intravenous PDE 5 inhibitors in chronic, 262 PDE 1/2/4/non-selective inhibitors in acute, 260 PDE in chronic, 263 prostanoids/PDE inhibitors in acute/ chronic, 257–263 pulmonary hypertension modeled by acute, 257–258 pulmonary hypertension modeled by chronic, 257, 258–263 Hypoxic pulmonary vasoconstriction (HPV), hypoxia/pulmonary gas exchange v., 257–258 I ICM. See Inner cell mass IGF. See Insulin-like growth factor IGF-I, antler cell proliferation v., 27–29 IGF-II antler cell proliferation v., 27–28

Index synthesis in choroid plexus, 197 Indian hedgehog (IHH), PTHrP/ chondrogenesis v., 30, 32–33 Inner cell mass (ICM), Cr-1 in blastocyst, 93 Insulin, in positive energy balance state, 218 Insulin-like growth factor (IGF), in antler development/regeneration regulation, 27–29 Interpulse intervals (IPIs) D. virilis/D. montana subgroup variation in, 234–235 Drosophila song, 231, 232 Interstitial fluid (ISF), CNS, 182 Invertebrates, EGF-CFC genes in, 88 Ion channels choroid plexus, 185–188 physiological relevance of choroid plexus AQP1, 191–192 IPIs. See Interpulse intervals ISF. See Interstitial fluid J JA. See Jasmonic acid JAK-STAT3 pathway, PI3K-PDE3B-cAMP pathway cross-regulating, 214–215 Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, leptin signaling/function v., 213 Jasmonic acid (JA), leaf senescence v., 67

K Kinases 14-3-3 phosphorylation by sphingosinedependent, 297–298 pathways v. protein phosphorylation by protein, 286 senescence regulation v. receptorlike, 70–71 KN1, leaf senescence v., 66 L L/R axis. See Left/right axis Leaf senescence Arabidopsis as model for studying, 52 conclusions/challenges in, 73–77 ethylene not essential to, 66–67 identification/analysis of genes associated with, 55–59

331

Index molecular/genetic control of Arabidopsis, 50–77 molecular states v. causal factors, 62 processes v. SAG expression, 60–61, 62–72 regulatory genes associated with, 59, 60–61 symptoms, 52–55 as topic for poets/artists, 50 Leaves development of, 51 molecular/genetic control of senescence/ longevity in, 50–77 as sugar sources/sinks, 64 Left/right (L/R) axis, EGF-CFC gene expression in formation of, 95–96 Leptin adipostat signal represented by, 208–210 food intake regulation v. PI3K-PDE3BcAMP pathway, 213–215 in positive energy balance state, 218 Longevity, molecular/genetic control of Arabadopsis leaf, 50–77 M Major intrinsic protein (MIP) family, distribution, 182 Mammals AQPs found in, 182 Atg proteins essential to autophagy in, 169–170 deer antlers modeling regeneration in, 2–37 Mammary glands, EGF-CFC proteins in development of, 97–98 MAPC. See Multipotent adult progenitor cell Mate choice cost/benefit/evolution of, 227–230 direct/indirect benefits of, 227 MCT. See Monocrotaline MCTP. See Monocrotaline pyrrole Meconium, in modeling pulmonary hypertension, 271 Membranes leaf senescence v. integrity of, 57–58 leaf senescence v. structure of chloroplast, 53 Mesenchymal cells antler growth v. proliferation/life span of, 17 chondrogenic diVerentiation v., 16, 18–19 in early antler development, 11–12 in growing antler, 15–17

Mesenchymal stem cells (MSCs) alternative sources of, 315–316 in peripheral blood, 316 potential common microenvironment for, 310 Metabolic rate, Arabidopsis leaf senescence v., 63–64 Metacaspases, caspase-like substrate specificity in, 165–168 Miller-Dieker syndrome, 14-3-3 in, 294 Minerals, leave senescence v. levels of, 55 MIP family. See Major intrinsic protein family Mitochondria, autophagic cell death activation v., 150, 160 Mitochondrial permeability, PCD v, 162–163 Monocrotaline (MCT) as pulmonary hypertension model, 263–267 rat PAH induced by pyrrolizidine alkaloid, 263–264 Monocrotaline pyrrole (MCTP) dog chronic pulmonary hypertension v., 264 MCT activation to, 263 Moose, antler span/structure, 13–14 Morphogens, in embryogenesis, 87–88 MSCs. See Mesenchymal stem cells Multipotent adult progenitor cell (MAPC), in XXX rodent/human bone marrow, 309 N Neural (N)-cadherin, aggressive malignant tumor phenotype v., 101 Neuropeptide Y (NPY) expression v. cAMP, 210–212 feeding behavior regulated by, 209–210 Neuropeptides, orexigenic/anorectic, 209–210 Nitrogen, status v. sugar signaling in leaves, 65 Nodal BMPs v., 93 CR-1 v., 89–91 EGF-CFC proteins as co-receptors for, 92–93 in L/R axis formation, 95–96 signaling mediated by EGF-CFC proteins, 103–105 signaling modulated by antagonists, 104, 105–106

332 Non-small-cell lung cancer (NSCLC), CR-1 expression in, 109, 110, 115 Notch receptor proteins, EGF-CFC proteins v., 89 NPY. See Neuropeptide Y NPY/AgrP neurons fasting-induced cAMP signal in, 213–214 in hypothalamic feeding control, 216–217, 217–218 NPY neurons, intracellular cAMP levels in, 215–217 NSCLC. See Non-small-cell lung cancer Nuclei, PCD v. changes in, 158–159 O Oligonucleotides, CR-1 expression v. antisense, 116–117 Oncogenesis, embryogenesis v., 86–87 Ontogeny, antler, 13–15 Ossification, in regenerating antlers, 21–23 Osteoblasts ADAS cells diVerentiating into, 315 BMSSCs diVerentiating into, 310–311 HSCs regulated by BMSSC-derived, 309 Osteoclasts, in antler ossification, 22–23 Ovarian cancer, CR-1 expression in, 110, 114 P PAH. See Pulmonary arterial hypertension Pancreatic cancer, CR-1 expression in, 110, 111 Paracaspaces, 166–168 cell-death function of, 168 Parathyroid hormone-like related protein (PTHrP) in antler osteoclast regulation, 22–23 in early antler development, 11–12 immunolocalization, 12, 36–37 skeletal development/regeneration regulated by, 30, 32–33 Parathyroid hormone (PTH), HSC niche signaling pathway of, 309 Paraventricular nucleus (PVN) anorectic eVects of cAMP in, 212–213 food intake/body weight v., 208 Pathogenesis-related (PR) proteins, SAGs encoding, 58 PCD. See Programmed cell death PDE inhibitors, 254–255

Index in acute/chronic hypoxia, 257–263 in acute hypoxia, 260–261 in chronic hypoxia, 261–263 in experimental PAH, 252–273 hypoxia-induced pulmonary hypertension v., 258, 259 MCT-induced pulmonary hypertension v., 266–267 in monocrotaline-induced pulmonary hypertension, 263–267 pulmonary hypertension v. vasodilators with, 256, 271–272 U46619-induced acute pulmonary hypertension v., 267–270 PDEs. See Phosphodiesterases Pedicles anlages of, 5 antler casting v., 8, 10–11 antler formation not requiring, 3 antler growth from, 3, 4 epithelialization of, 8 PEMs. See Proembryogenic masses Periosteum antler/pedicle development initiated by antlerogenic, 3–4 antler regeneration v. pedicle, 9–10 PGD2. See Prostaglandin D2 PGE1. See Prostaglandin E1 PGE2. See Prostaglandin E2 PGI2. See Prostaglandin I2 Phagocytes, in apoptosis, 154 Phosphodiesterases (PDEs) activity regulated by, 272 cAMP-mediated prostacyclin eVects limited by, 252–253 cAMP steady state controlled by, 207–208 mammalian families/inhibitors of, 253, 254–255 Phosphorylation cascades 14-3-3 proteins in, 287 intracellular data transmission v., 286–287 Phosphoserines 14-3-3 protein binding activity v., 287–292 14-3-3 protein structure v. binding of, 288–289 Photoperiods, antler growth cycle modified by, 24–25 Photosynthesis, sugar v. genes associated with, 64–65 Phytohormones, in leaf senescence, 65–68

Index Picea abies metacaspase cell-death function demonstrated in, 168–169 somatic embryogenesis in, 145–151 zygotic embryogenesis in, 140, 145–148 Pineal gland, photoperiods linked to antler growth by, 24–25 Pinus sylvestris monozygotic polyembryony of, 151–154 phenotypically distinct cell-death programs in, 153–154 PLs. See Pulse lengths Polyembryony, obligate/sporadic monozygotic, 151 POMC neurons. See Proopiomelanocortin neurons Postnatal stem cells ES cells v., 306 putative stem cell niches nurturing, 309 PPRs. See PTH/PTHrP receptors PR proteins. See Pathogenesis-related proteins Proembryogenic masses (PEMs) PCD pathway v. cells in, 160, 161 somatic embryos developing from, 148–149 Proembryogeny, in Picea abies, 146–147 Programmed cell death (PCD) animal, 136–137 autophagic v. apoptotic, 154 basal-to-apical gradient, 152, 154, 159–160 cell dismantling pathway, 150, 159–161 chromatin/nuclear envelope events in plant, 158–159 core machinery in plants, 162–164 cytoskeletal changes v., 150, 157–158 domains conserved in evolution, 163–164 evolution, 162 leaf senescence as type of, 50–51 mechanics, 154–161 metacaspases regulating, 168 molecular components regulating, 162–170 pharmacological manipulation of autophagic Picea abies, 150–151 phenotypically distinct Pinus sylvestris, 153–154 in plant embryogenesis, 136–171 plant v. animal, 136–138 practical applications of understanding, 171 signal triggering embryonic, 144–145 somatic embryos/PEMs v. autophagic, 149

333 stages of eukaryotic, 154 Prolactin receptors, 14-3-3 binding to, 296–297 Proopiomelanocortin (POMC) neurons fasting-induced cAMP signal in, 214 in regulating feeding behavior, 209–210 Prostacyclin synthase, gene transfer v. MCTinduced pulmonary hypertension, 265 Prostaglandin (PG) D2, acute hypoxia v. intravenous, 259 Prostaglandin (PG) E1 acute hypoxia v. intravenous, 259 MCT-induced pulmonary hypertension v. inhaled, 266 MCT-induced pulmonary hypertension v. intravenous, 265–266 Prostaglandin (PG) E2, deficit v. chronic hypoxia, 258 Prostaglandin (PG) I2 acute hypoxia v. intravenous, 260 deficit v. chronic hypoxia, 258 MCT-induced pulmonary hypertension v., 265 Prostanoids. See also specific prostanoids in acute/chronic hypoxia, 257–263 in acute hypoxia, 259–260 eVects in MCT-induced pulmonary hypertension, 265–266 in experimental PAH, 252–273 first suggested in PAH, 252 hypoxia v. pulmonary-selective delivery of, 263 in monocrotaline-induced pulmonary hypertension, 263–267 pulmonary hypertension v. up-regulated rat, 258–259 U46619-induced acute pulmonary hypertension v., 267–270 in U46619-induced pulmonary hypertension, 268 U46619-induced pulmonary hypertension v. inhaled, 268 U46619-induced pulmonary hypertension v. intravenous, 268 Proteins. See also Pathogenesis-related proteins 14-3-3 proteins v. phosphoserinebearing, 288 activities of caspase-like plant, 164–169 leaf senescence v. degradation of, 54, 68–69

334 Proteins (cont.) mammary gland development v. EGF-CFC, 97–98 transformation/tumorigenesis v. EGF-CFC, 99–102 14-3-3 proteins binding by, 288 biophysical properties of, 287–289 development/growth factor responses v. signaling by, 286–298 dimerism/phosphoserine-binding in, 287–292 growth factor receptor binding/serine phosphorylation by, 296–297 growth factor signaling v., 295–297 in growth factor survival signaling, 296, 297 human isoforms of, 287–288, 289–290 isoform homology in, 289–290 isoforms/dimers v. regulation of, 289–291 kinases/phosphatases regulating interactions by, 292 monomer structure in, 288–289 multiple models accounting for functions of, 298 in phosphoprotein networks, 287 phosphorylation of binding motifs in, 291–292 phosphoserine/phosphotyrosine signaling by, 298 sphingosine-dependent kinases in phosphorylation of, 297–298 target proteins having multiple binding sites for, 291 in vivo heterodimeric isoforms of, 290 PTH. See Parathyroid hormone PTH/PTHrP receptors (PPRs), skeletal development/regeneration regulated by, 30, 32–33 PTHrP. See Parathyroid hormone-like related protein PTLs. See Pulse train lengths Pulmonary arterial hypertension (PAH), 252 prostanoids/phosphodiesterase inhibitors in experimental, 252–273 Pulmonary hypertension animal models v. treatment options, 256 animal/organ models of, 257 inhaled PDE 3/4 inhibitors v. U46619-induced, 270

Index inhaled PDE 5 inhibitors v. U46619-induced, 270 intravenous PDE 3/4 inhibitors v. U46619-induced, 269 intravenous PDE 5 inhibitors v. MCT-induced, 266–267 intravenous PDE 5 inhibitors v. U46619-induced, 269–270 less frequently used models of, 270–271 modeled by acute hypoxia, 257–258 PDE 3/4 inhibitors v. MCT-induced, 266 PDE inhibitors v. hypoxia-induced, 258, 259 Pulmonary thromboembolism, as pulmonary hypertension model, 270 Pulse lengths (PLs) D. virilis/D. montana subgroup variation in, 234–235 Drosophila song, 231, 232 as sexual selection target, 234 Pulse train lengths (PTLs), Drosophila song, 231, 232 PVN. See Paraventricular nucleus R Raf-1, 14-3-3 in regulating, 294–295 RANKL. See Receptor activator of NFB ligand RARs. See Retinoic acid receptors RAs. See Retinoic acids Ras-Raf signaling pathway, 14-3-3 protein interaction with, 294–295 Reactive oxygen species (ROS), protein degradation in leaf senescence v., 54–55 Receptor activator of NFB ligand (RANKL), in antler osteoclast regulation, 22–23 Red deer antler growth v. photoperiod change, 24 antler structure, 4, 14 genes expressed in antler regeneration by, 29 Regeneration in antlers, 6–7 antlers’ limitations in investigating, 3 BMSSCs in cartilage, 312–313 BMSSCs in neural tissue, 313–314 BMSSCs v. adipose tissue, 313 BMSSCs v. bone, 310–312 deer antlers modeling mammalian, 2–37

Index healing wounds defining, 12–13 ossification/remodeling in antler, 21–23 regulation of antler, 24–37 territory of pedicles, 9 Regenerative medicine BMSSCs in, 306–316 natural regeneration examples v., 3 Retinoic acid receptors (RARs), velvet antler skin expressing, 31–32 Retinoic acids (RAs) in antler buds, 14 skeletal development/regeneration regulated by, 30, 31–32 Retinoic X receptors (RXRs), velvet antler skin expressing, 31–32 Retroviruses, cancer v. insertional mutagenesis by, 86 RNA, levels decreased by leaf senescence, 54 ROS. See Reactive oxygen species RXRs. See Retinoic X receptors S SA. See Salicylic acid Saccharomyces cerevisiae genes essential for autophagy identified in, 169 14-3-3 protein isoforms in, 289 SAGs. See Senescence-associated genes SAIDS. See Simian AIDS-like virus Salicylic acid (SA), SAG expression v., 67 SAR. See Systemic acquired resistance Saspases, 164–165 Senescence biotechnological application of, 72–73 components v. emergent properties, 76–77 eukaryotic translation factor 5A v. plant, 71 inducing/suppressing elements in process of, 75–76 mitotic/postmitotic, 51 molecular/genetic control of Arabadopsis leaf, 50–77 plant developmental aging v., 51 signal perception v. receptor kinases, 70–71 Senescence-associated genes (SAGs) earlier developmental stages v., 74–75 enhancer/promoter traps v., 56 functional categories of, 57–59 identification/functional analysis of, 55–59 investigative techniques v., 56

335 in macromolecule breakdown/recycling, 57–58 in pathogenesis/defense, 58–59 regulatory, 59, 60–61 regulatory factors v., 60–61, 62–72 regulatory mode of, 62 Sensory drive, in mate choice, 228–229 Serotonin (5-HT), CSF production decreased by, 192–193 Sex steroids. See also specific sex steroids in antler development/regeneration regulation, 25–27 Sexual selection animal signal diversity v., 226 male signal/female preference coevolution through, 227–230 variation in traits v., 228 Shh. See Sonic hedgehog Signals 14-3-3 in transduction of Drosophila neuronal, 294 14-3-3 proteins v. growth factor stimulated, 295–297 anatomy/development v. cell response to, 286 development/growth factor responses v. 14-3-3 protein, 286–298 evolution of species-specific animal, 226 female preferences coevolving with, 227–230 Raf serine/threonine kinase v. growth factor stimulated, 294–295 song as species-recognition, 243 temperature v. poikilotherm, 239 Sikka deer, estrogen inducing premature antler mineralization in, 27 Sildenafil, in modeling pulmonary hypertension, 271 Simian AIDS-like virus (SAIDS), infection v. monkey cripto-1 expression, 97 Skeletal stem cells, regenerative medicine v. skeletal, 306–316 Songs additive genetic variation in D. montana, 235 environmental conditions v. male D. montana, 237 evolution of male D. montana, 226–245 geographic variation in Drosophila courtship, 233

336 Songs (cont.) intra-species variation in Drosophila, 233–234 preference v. characters of male courtship, 236–243 pulse/sine types of, 231 repertoire of Drosophila, 231–232 simulation of D. montana, 238–239 as species-recognition signals, 243 species-specificity of male Drosophila courtship, 231–233 temperature v. D. montana courtship, 239–242 varying traits in Drosophila, 231 Sonic hedgehog (Shh), in antler chondrogenesis, 14–15 Sphingolipids, in signaling, 297–298 Spinocerebellar ataxia, 14-3-3 in, 294 Stem cells. See also specific types of stem cells malignant/normal/embryonic, 87 STRO-1 bone marrow CFU-F protein reacting with, 307 expression v. extensive BMSSC growth, 308–309 STRO-1þ CFU-F population contained in, 307–308 clonogenic capacity, 308 Sugars, as signaling molecules in leaf senescence, 64–65 Suspensor autophagosome origin in PCD of, 156–157 development in Arabidopsis mutants, 139–145 PCD eliminating plant embryo, 138 Picea abies PCD v. terminally diVerentiated, 149–150 transformation without viable secondary embryos, 140, 142–143, 144 Systemic acquired resistance (SAR), PR proteins associated with, 58 T Tartrate-resistant acid phosphatase (TRAP) cells in nonmineralized cartilage expressing, 22, 23 chondrocytes from nonmineralized cartilage expressing, 22–23 Telomeres, senescence v. structure of plant, 71–72

Index Testicular cancer, CR-1 expression blocking v., 117 Testicular carcinomas, CR-1 expression in, 110, 115 Testosterone antler mineralization/velvet shedding v., 5, 6 IGF-I concentrations associated with, 27–28 levels v. antler growth, 13, 24, 25–27 levels v. antler regeneration, 2, 7–8 TGF. See Transforming growth factor TGF EGF-CFC proteins as co-receptors for, 92 MAPK pathway crosstalk with, 108 proteins v. Nodal signaling, 104, 106 Tines, in antler growth, 14 Transforming growth factor (TGF), skeletal development/regeneration regulated by, 30, 33–34, 35 Transplantation, antler/pedicle development v. periosteal, 3–4 Transthyretin (TTR), synthesis in choroid plexus, 197 TRAP. See Tartrate-resistant acid phosphatase Tube cells in cell dismantling, 160, 161 in early v. late Picea abies embryogeny, 146, 147–148 Tween (twn) mutants, complete primary embryo development in Arabidopsis, 140, 144 Tyrosines, growth factor signaling v. phosphorylation/dephosphorylation of, 295–297 U U46619, pulmonary hypertension modeled using, 267–270 Urodeles, RAs v. skeletal development/ regeneration in, 31 V Vacuolar processing enzymes (VPEs), caspase-like enzymatic activity in, 165 Vacuoles Arabidopsis mutant suspensor cell, 140, 141–144

337

Index autophagic cell death v. lytic/protein storage, 155, 156 Vascular endothelial growth factor (VEGF), in red deer antlers, 30, 35 Vascular system, PCD in establishing plant, 138 Vasodilators, pulmonary hypertension v. PDE inhibitors with, 256, 271–272 VEGF. See Vascular endothelial growth factor VEIDases, embryogenesis v. pharmacological inhibition of, 165, 166 Velvet in antler development, 4, 5 loss in deer reproductive cycle, 25, 26 new antler, 7, 8 Vertebrates, EGF-CFC proteins conserved in, 88, 89

Vitronectin receptors (VNRs) cells in nonmineralized cartilage expressing, 22 chondrocytes from nonmineralized cartilage expressing, 22–23 VPEs. See Vacuolar processing enzymes W Wapiti, antler structure, 14 Y YODA, Arabidopsis suspensor development v., 140–141, 142–143 14-3-3 , Drosophila olfactory learning behavior implicating, 294