International Review of Cytology, Volume 185: A Survey of Cell Biology

VOLUME 185

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1 988 1949-1 984 19671984-1 992 1993-1 995

EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Rosa Beddington Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Charles J. Flickinger Hiroo Fukuda Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald

M. Melkonian Keith E. Mostov Andreas Oksche Vladimir R. Pantic L. Evans Roth Jozef St. Schell Manfred Schliwa Robert A. Smith Wilfred D. Stein Ralph M. Steinman M. Tazawa Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Biochemistry University of Tennessee Knoxville, Tennessee

VOLUME 185

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

Front cover photograph: Serum-free collagen gel culture of rat aortic and renal venous explants. (For more details, see Chapter 1, Figure 4.)

This book is printed on acid-free paper.

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Copyright 0 1999 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1999 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0074-7696199 $25.00

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CONTENTS

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Autoregulation of Angiogenesis by Cells of the Vessel Wall R. F. Nicosia and S. Villaschi I. I1. 111. IV. V.

Introduction ........................................................... Vasculogenesis and Angiogenesis ......................................... Evidence That Blood Vessels Can Autoregulate Angiogenesis: Rat Aorta Model . . . . . Interactions between Endothelial Cells and Fibroblasts or Smooth Muscle Cells . . . . . Growth Factors and Other Soluble Regulators of Angiogenesis Produced by Cells of the Vessel Wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Proteolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Extracellular Matrix and Cell Adhesion Molecules ............................. VIII. Vasoactive Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary and Future Directions ........................................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 5 7 9 17 19 23 26 31

Fibroblast Growth Factors as Multifunctional Signaling Factors Gyorgyi Szebenyi and John F. Fallon I. I1. Ill. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure. Regulation of Synthesis. Subcellular Localization. and Release of FGFs . . . . FGF-Binding Proteins and Signaling Pathways ................................ The Biological Activities of FGFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

45 47 56 72

vi

CONTENTS

V . Concluding Remarks ..................................................... References ............................................................

85 87

Structural and Functional Characteristics of the Centrosome in Gametogenesis and Early Embryogenesis of Animals Marina M. Krioutchkova and Galina E. Onishchenko I . Introduction ............................................................ II . Behavior of the Centrosome during Gametogenesis ............................

107 110

Ill. Behavior of Centrioles and the Centrosome in Cells in Early Development and during Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions ............................................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

122 143 144

Cell and Molecular Biology of the Pars Tuberalis of the Pituitary Werner Wittkowski. Jijrgen Bockmann. Michael R. Kreutz. and Tobias M. Bockers I . Introduction ............................................................ II . Characteristics of Pars Tuberalis ........................................... 111 . Gene Expression of Pars Tuberalis-Specific Cells .............................. IV. Biorhythmic Alterations ................................................... V. Physiological Significance: Pars Tuberalis as the Major "Zeitgeber" of Pars Distalis Activity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Concluding Remarks ..................................................... ............................................ References . . . . . . . . . . . .

157 158 165 177 182 186 187

Polarization of the Na+.K+-ATPasein Epithelia Derived from the Neuroepithelium Lawrence J . Rizzolo I. Introduction ........................................................... 195 II . Polarity and Transepithelial Transport ....................................... 197 111. Plasticity: Remodeling Cell Polarity ......................................... 202 IV. General Models for Epithelial Polarity ....................................... V . Diversity of Na+.K+-ATPase lsoforms and Tissue Specificity ..................... VI. Mechanisms That Polarize the Distribution of the Na+.K+-ATPase . . . . . . . . . . . . . . . .

205 210 214

CONTENTS

VII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii 226 228

Desmosomes: Intercellular Adhesive Junctions Specialized for Attachment of Intermediate Filaments Andrew P. Kowalczyk. Elayne A . Bornslaeger. Suzanne M. Norvell. Helena L. Palka. and Kathleen J. Green Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultrastructural Properties and Tissue Distribution of Desmosomes . . . . . . . . . . . . . . . . Transmembrane Components of the Desmosome: Desmosomal Cadherins . . . . . . . . . Plaque Components of the Desmosome .................................... Protein-Protein Interactions in the Desmosome: A Model for Desmosome Assembly .......................... VI . Regulation of Desmosome Assembly ....................................... VII . Desmosomal Components in Signal Transduction and Development . . . . . . . . . . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

268 270 278 282 283

Index ............................................................

303

I. I1. 111. IV. V.

237 238 241 248

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin

Tobias M. Bockers (157), lnsfifute of Anafomy, AG Molecular Neuroendocrinology, Wesiralische Wilhelms-Universitat, 0-48149 Miinster, Germany Jurgen Bockrnann (157), Institute of Anatomy, AG Molecular Neuroendocrinology, Wedfalische Wilhelms-Universitat, 0-48149 Munster, Germany Elayne A. Bornslaeger (237), Departmenfs of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 6061 1-3008 John Fallon (45), Department of Anatomy, University of Wisconsin Medical School, Madison, Wisconsin 53706- 1532 Kathleen J. Green (237),Departments of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 6061 1-3008 Andrew P. Kowalczyk (237), Department of Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, lllinois 606I 1-3008 Michael R. Kreutz (157), lnstitute of Medical Psychology, University of Magdeburg, 39120 Magdeburg, Germany Marina M. Krioutchkova (107), Department of Cytology and Histology, Moscow State University, Moscow- I 19899, Russia Roberto F. Nicosia (1), Department of Pathology and Laboratory Medicine, Al/egheny University of the Health Sciences, Philadebhia, Pennsylvania 19102 Suzanne M. Nowell (237),Departments of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611-3008 ix

X

CONTRIBUTORS

Galina E. Onishchenko (107),Department of Cflology and Histology, Moscow State Universiity, Moscow-119899, Russia Helena L. Palka (237),Departments of Pathology and Dermatology and the R.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611-3008 LawrenceJ. Rizzolo (195),Departmentof Surgery, Section ofAnatomy, Yale Universify School of Medicine, New Haven, Connecticut 06520 Gyorgyi Szebenyi (45),Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-91I 1 Sergio Villaschi (I), Department of Surgery, Universify of Rome "Tor Vergata," Rome, Italy Werner Wittkowski (157), Institute of Anatomy, AG Molecular Neuroendocrinology, Westfalische Wilhelms-Universitit, 0-48149 Munster, Germany

Autoregulation of Angiogenesis by Cells of the Vessel Wall R. F. Nicosia* and S. Villaschit *Department of Pathology and Laboratory Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102; and tDepartment of Surgery, University of Rome “Tor Vergata,” Rome, Italy

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~

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The cells of the vessel wall can regulate angiogenesis by producing growth factors, proteolytic enzymes, extracellular matrix components, cell adhesion molecules, and vasoactive factors. This properly enables preexisting blood vessels to generate new vessels in the absence of exogenous angiogenic stimuli. Vascular autoregulation of angiogenesis can be studied by culturing rat aortic or venous explants in collagen gels under serum-free conditions. In this system, the combined effect of injury and exposure of explants to collagen triggers a self-limited angiogenic response. Interactions among endothelial cells, smooth muscle cells, and fibroblasts play a critical role in the regulation of this process. This chapter reviews the literature on angiogenesis, focusing on the vessel wall as a highly specialized and plastic tissue capable of regenerating itself through autocrine, paracrine, and juxtacrine mechanisms. KEY WORDS: Angiogenesis, Endothelium, Smooth muscle cells, Pericytes, Fibroblasts, Growth factors, Matrix metalloproteinases, Plasminogen activators, Extracellular matrix.

1. Introduction Angiogenesis, the process by which blood vessels develop from the endothelium of a preexisting vasculature, plays a fundamental role in the growth, survival, and function of normal and pathologic tissues. The cardiovascular system is the first functioning organ system (Wilting er al., 1995a) and its developmental failure results in the early intrauterine death of the embryo (Shalaby er aZ., 1995). Once blood vessels have formed and remodeled during embryonal development and fetal growth, they become quiescent International Review of Cytology, VoL 185

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Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved.

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(Folkman and Cotran, 1976) but retain their vasoformative properties, which become manifest again in the adult during wound healing (Knighton et al., 1990) and the female menstrual cycle (Jakob et aL, 1977). Angiogenesis is also reactivated during pathologic processes such as cancer, rheumatoid arthritis, psoriasis, complicated atherosclerosis, diabetic retinopathy, and hemangiomas (Folkman, 1995). Because the neovasculature contributes to the progression of these disorders, antiangiogenic drugs have been proposed as possible therapeutic agents and are currently being tested in clinical trials (Folkman, 1996). There is also considerable interest in the pharmacologic stimulation of angiogenesis in patients suffering from delayed wound healing, venous stasis ulcers, and obliterative vascular disorders (Takeshita et al., 1994; Isner et al., 1996). New blood vessels develop as a result of complex interactions between endothelial cells and angiogenic regulators such as soluble growth factors (Moses et al., 1995),insoluble extracellular matrix (ECM) molecules (Ingber and Folkman, 1989), and matrix-degrading proteolytic enzymes (Ray and Stetler-Stevenson, 1994; Cornelius et al., 1995). Growth factors capable of stimulating angiogenesis are secreted by a variety of cell types, including embryonal cells (Drexler et al., 1992), cancer cells (Folkman and Cotran, 1976), epithelial cells (Brown et al., 1992), macrophages (Sunderkotter et al., 1994), and lymphocytes (Lutty et aL, 1983). Angiogenic factors are also produced by the cells of the vessel wall: endothelial cells (Hannan et al., 198Q smooth muscle cells (Brogi et al., 1994), and fibroblasts (Hlatky et al., 1994). Thus, blood vessels that have switched from quiescence to an activated state as a result of injury or other stimulatory conditions have the capacity to generate new vessels without the intervention of nonvascular cells. This autoregulation of the angiogenic process is observed when vascular explants are cultured in three-dimensional biomatrix gels in the absence of serum or exogenous growth factors (Kawasaki et al., 1989; Nicosia and Ottinetti, 1990; Brown et al., 1996). Understanding how blood vessels regulate their own growth may provide new insights into the angiogenic process and its mechanisms. The purpose of this chapter is to reappraise critically the literature on angiogenesis,focusing on the vessel wall as a highly specialized and plastic tissue capable of regenerating itself through a vasoformative process mediated by autocrine, paracrine, and juxtacrine mechanisms.

II. Vasculogenesis and Angiogenesis A. Formation of Blood Vessels during Embryonal Development Blood vessels form in the embryo as a result of two fundamental processes: vasculogenesis and angiogenesis (Risau et al., 1988). Vasculogenesis occurs

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when blood vessels emerge de n o w from a subpopulation of mesenchymal cells known as angioblasts, which differentiate into endothelial cells. After the first capillary tubes have developed, new vessels form as a result of angiogenesis, i.e., sprouting of new microvessels from preexisting vessels, vasculogenesis, or a combination of the two processes (Wilting et al., 1995a). Embryonal endothelial cells generate sprouts that branch, become canalized, and anastomose with each other giving rise to capillary loops and networks. The sprouts grow as a result of both endothelial cell migration and proliferation as well as recruitment of new angioblasts from the surrounding mesenchyme or from other blood vessels (Wilting et al., 1995b). Capillaries give rise to larger vessels by fusion of adjacent vessels and by lateral intercalation of proliferating endothelial cells resulting in luminal expansion. For example, the paired dorsal aortas of the chick embryo develop out of a close-meshed capillary plexus which is remodeled into two longitudinal tubes (Hirakow and Hiruma, 1983). The two aortas then fuse laterally, giving rise to the definitive aorta which eventually expands by intercalated endothelial proliferation (Wilting et al., 1995b). Vascular remodeling during embryonal development is also characterized by regression of capillaries and disappearance of endothelial cells through cell death, emigration, and transdifferentiation (Wilting et al., 1995b). Retraction of capillaries with incorporation of endothelial cells into the main vessels has also been described (Christ et al., 1990). The primary capillary network of the embryo is composed exclusively of endothelial cells. As arteries and veins develop, the mesenchyme around the endothelium differentiates into smooth muscle cells and fibroblasts. Embryonal differentiation of vascular smooth muscle cells proceeds centrifugally at first in the aorta and then in the main veins (Wilting et aL, 1995b). Differentiation of arteries and veins results in the gradual formation of three anatomic layers: the intima, the media, and the adventitia. Two main types of arteries develop: elastic arteries, which include the aorta and its main branches, and muscular arteries. In the newborn, the arterial intima is composed almost exclusively of endothelial cells which rest on a basement membrane. As they mature and eventually age, blood vessels thicken, acquiring more layers of smooth muscle cells. The intima of arteries and veins becomes populated in the adult by medialderived smooth muscle cells known as myointimal cells which produce collagen, elastic tissue, basement membrane molecules, and proteoglycans (Schwartz et al., 1990). The media of elastic arteries is composed of alternating layers of smooth muscle cells and elastic laminas (Dingemans et al., 1981). In contrast, muscular arteries have less elastic tissue which organizes into laminae only at the boundaries of the intima with the media and the adventitia. Veins develop thinner walls than arteries and have tunica medias composed primarily of smooth muscle cells with relatively low amounts of elastic tissue. The adventitia of both arteries and veins is composed primarily of fibroblasts and interstitial collagen. Microvessels known as vasa va-

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sorum develop in the adventitia and penetrate into the tunica media of large vessels to nourish the outer layers of smooth muscle cells (Barger et al., 1984). Even after the vascular system has fully differentiated, the boundaries between the adventitia of arteries or veins and the connective tissue around arterioles, capillaries, and venules remain ill-defined. Thus, the fibroblasts that surround the endothelium of arterioles, capillaries, and venules are essentially an anatomic extension of the adventitial fibroblasts of arteries and veins into the peripheral microvascular bed. The capillaries and postcapillary venules are coated by a single discontinuous layer of pericytes which resemble smooth muscle cells and surround the endothelium with dendritic processes (Sims, 1986). During vascular development, smooth muscle cells and pericytes are recruited by the endothelial cells from the surrounding mesenchyme (Schwartz et al., 1990). Pericytes can only be identified in the late fetal period (Donahue and Pappas, 1961). The basement membrane of capillary endothelial cells during embryonal development is poorly developed (Tonnesen et al., 1985). As the capillaries mature, endothelial cells lay down a continuous basement membrane which they share with the surrounding pericytes (Sims, 1986). They also differentiate into morphologically and functionally distinct endothelial subtypes based on interactions with the local environment. For example, capillary endothelial cells of the choroidal plexus become fenestrated in response to induction by the choroidal epithelial cells (Wilting and Christ, 1989), whereas brain capillary endothelial cells acquire tight junctions and bloodbrain features in response to stimuli by surrounding astrocytes (Stewart and Wiley, 1981).

B. Angiogenesis during Adult Life After the vascular system has developed and matured, blood vessels gradually stop growing and become quiescent. Angiogenesis is reactivated in the Adult at the level of the microcirculation, where physiologic or pathologic stimuli can induce endothelial sprouting from preexisting microvessels (Folkman and Cotran, 1976). New vessels can also originate from the endothelium of large or medium vessels during the neovascularization of atherosclerotic plaques, the recanalization of thrombi, and the development of collateral circulation (Takeshita et al., 1994; Sueishi et al., 1997). Formation of microvessels from a preexisting vessel is a multistep process (Folkman, 1985) characterized by an orderly sequence of events which can be summarized as follows: (i) vascular engorgement and dilatation with activation of endothelial cells; (ii) localized degradation of the subendothelial basement membrane; (iii) migration of endothelial cells into the interstitium through gaps in the basement membrane of the parent microvessels;

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(iv) formation of immature sprouts composed of activated endothelial cells; (v) proliferation of endothelial cells behind the migrating endothelium of the sprouts; (vi) remodeling/digestion of the preexisting ECM around the sprouting endothelium and deposition of a new ECM; (vii) canalization of the neovessels with formation of anastomoses and capillary loops followed by establishment of blood flow; (viii) incorporation of pericytes around the endothelium; (ix) formation of a continuous basement membrane; and (x) vascular regression, attributed to both lack of blood flow (Clark and Clark, 1939) and removal of angiogenic stimuli (Ausprunk et al., 1978).

111. Evidence That Blood Vessels Can Autoregulate Angiogenesis: Rat Aorta Model In 1959 Williams found that microscopic wounds of connective tissue produced without damaging blood vessels healed without angiogenesis and proposed that vascular injury was needed to stimulate neovascularization during wound healing. Subsequently, O’Donaghue and Zarem (1971) observed that mouse skin isografts implanted in a transparent chamber stimulated angiogenesis.They also found that purified collagen showed no angiogenic activity and argued that since the full-thickness skin graft consisted primarily of collagen and blood vessels, the failure of collagen to stimulate angiogenesis implicated the blood vessels of the graft as key factors in the stimulation of neovascularization. In 1982 we reported that explants of rat aorta cultured in plasma clot in the presence of fetal bovine serum gave rise to luxuriant outgrowths of branching microvessels (Nicosia et al., 1982). We later discovered that the angiogenic response of the rat aorta did not require exogenous stimuli and also occurred under serum-free conditions in a chemically defined growth medium without addition of angiogenic factors (Nicosia and Ottinetti, 199O)(Fig.1).On this basis, we hypothesized that the aortic wall was capable of regulating its own angiogenic response trough endogenous mechanisms activated by the injury of the dissection procedure. Similar observations were made concurrently by Kawasaki et al. (1989) and were confirmed later by Nissanov et al. (1995) and Akita et al. (1997). The autoregulation of angiogenesis by the vessel wall is not restricted to the rat aorta since rat venous explants (Fig. 2) and human chorionic vessels (Brown et al., 1996) are similarly capable of generating an angiogenic response under serumfree conditions. The outgrowth in the rat aorta cultures is composed of three main cell types: fibroblasts, endothelial cells, and pericytes (Nicosia and Ottinetti, 1990). Fibroblasts originate from the aortic adventitia and appear in the

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FIG. 1 Serum-free collagen gel culture of rat aorta. Microvessels (arrows) sprouted from the aortic explant (asterisk) are surrounded by fibroblasts (arrowheads). Scale bar = 150 p M .

FIG. 2 Serum-free collagen gel culture of rat renal vein (asterisk). The outgrowth is composed of branching microvessels (arrows) and fibroblasts (arrowheads). Scale bar = 100 pM.

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gel at Day 2 of culture. Endothelial cells migrate from the injured edges of the aortic intima at Days 3 or 4, giving rise to microvascular sprouts. The neovessels elongate, branch, develop lumina, anastomose, and eventually stop growing at Days 9 or 10. During the first days of culture, the neovascular sprouts are composed primarily of endothelial cells which closely interact with the surrounding fibroblasts. As the vascular outgrowth expands and eventually matures, pericytes migrate from the root to the tip of the microvessels, crawling along the endothelium, which they use as a surface for attachment, proliferation, and contact guidance. Pericytes originate at least in part from a subpopulation of smooth muscle cells located in the intima or the subintimal layers of the tunica media (Nicosia and Villaschi, 1995). It is also possible that fibroblasts become incorporated into the microvessels and differentiate into pericytes, as reported by Clark and Clark (1939) and Rhodin and Fujita (1989). The vascular remodeling that follows the growth phase is characterized by retraction of the small endothelial branches into the main stems of the microvessels. Regression and remodeling of the microvasculature is accompanied by an increase in the number of pericytes. As a result of endothelial retraction and periendothelial accumulation of pericytes, microvessels become shorter, thicker, and less branched during the regressionhemodeling phase (Nicosia and Villaschi, 1995). The aortic outgrowths are regulated by autocrine, paracrine, and juxtacrine interactions among endothelial cells, pericytes, and fibroblasts. The nature of these interactions can be studied by coculturing isolated cell strains in collagen gels.

IV. Interactions between Endothelial Cells and Fibroblasts or Smooth Muscle Cells Relatively few studies have explored the mechanisms that regulate the paracrine and juxtacrine interactions between the cells of the vessel wall. These studies have been limited by the use of fetal bovine serum (FBS), which is added to growth media in order to propagate and maintain cells in culture. FBS contains many growth and attachment factors which have profound effects on cell behavior and disrupt the paracrine cross-talk between different cell types. Advances in tissue culture have led to the development of serum-free media for endothelial cells (Knedler and Ham, 1987). These optimized media enable us to perform coculture experiments in the absence of FBS. Rat aortic endothelial cells grown in serum-free medium on a gel of interstitial collagen reorganize into a network of microvascular tubes when

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overlaid with a second layer of collagen (Nicosia et aL, 1994~).These microvessels resemble the microvascular outgrowths of the rat aortic cultures except that they are not surrounded by fibroblasts or pericytes and have a limited life span since they disintegrate within 3 to 4 days. Fibroblasts added to the collagen gel stabilize the microvessels which remain viable for at least 21 days (longest time studied). At variance with serum-stimulated cultures in which the endothelium is overgrown by fibroblasts, serum-free cocultures promote the coexistence of the two cell types in what becomes a symbiotic system of reciprocal paracrine stimulation (Villaschi and Nicosia, 1994).The stabilization of the microvessels requires the contiguous presence and juxtacrine stimulation of the fibroblasts. Fibroblasts have been shown by our laboratory and by others to produce soluble factors that stimulate angiogenesis (Sato et aL, 1987; Villaschi and Nicosia, 1994). Factors other than angiogenic molecules, however, are likely to contribute to the juxtacrine effect of fibroblasts on the microvasculature since fibroblastconditioned medium, which has angiogenic activity (Villaschi and Nicosia, 1994), prolongs the survival of isolated endothelial networks by only 4 to 5 days. These factors may include the insoluble components of the basement membrane which accumulate around the microvessels in endothelial/fibroblast cocultures and have stabilizing effects on neovessels (Nicosia et al., 1998). Fibroblasts may cooperate with the endothelium during microvascular sprouting because of their ability to activate proteolytic enzymes which digest the ECM (Gilles et aL, 1997). Endothelial cells and fibroblasts also stimulate each other’s capacity to contract the collagen gel. This phenomenon is mediated, at least in part, by soluble factors since collagen gel contraction by either isolated endothelial cells or fibroblasts is stimulated by growth medium conditioned by the other cell type. Endothelial cells promote the transformation of fibroblasts into myofibroblasts, specialized connective tissue cells with contractile ability (Gabbiani etal., 1971;Villaschi and Nicosia, 1994). Fibroblast-mediated collagen gel contraction is promoted, at least in part, by the endothelial-derived peptide endothelin-1 (ET-1). The fibroblast-derived factors that promote endothelial contraction of the collagen gel are unknown. The combined contraction of collagen in the cocultures is reminiscent of the contraction of granulation tissue which results in wound closure during tissue repair (Gabbiani et aL, 1971). The serum-free collagen gel overlay coculture method can also be used to investigate the juxtacrine and paracrine interactions between endothelial cells and smooth muscle cells. Smooth muscle cells isolated from the intimal aspect of the rat aorta (ISMCs) or the tunica media (MSMCs) and cultured in collagen gel without endothelial cells exhibit a round shape and tend to degenerate. In the coculture experiments, the ISMCs that are close to the endothelium become dendritic and migrate toward the microvessels, which are eventually surrounded by these cells (Nicosia and Villaschi, 1995). In

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contrast, MSMCs stay round, degenerate, and only rarely migrate toward the endothelium. ISMCs stabilize the microvessels, prolonging the life span of the endothelium. The morphology of the microvessels also suggests that the ISMCs promote endothelial cell differentiation. Ultrastructural studies demonstrate a striking similarity between the microvessels of the endothe1iaVISMCs cocultures and those of the primary aortic cultures. The ISMCs that have migrated around the microvessels of the cocultures are virtually indistinguishable from the pericytes of the primary aortic cultures. These experiments demonstrate that endothelial cells chemotactically attract a subpopulation of smooth muscle cells which transform themselves into pericytes establishing contacts and junctional communications with the endothelium. The behavior of ISMCs is similar to that of microvessel-derived pericytes which have been shown by others to adhere to capillaries in collagen gel culture (Minakawa et al., 1991). Pericytes, depending on their functional state, degree of differentiation, and spatial relation to the endothelium, may play different roles during the angiogenic process. The observation in the rat aorta model that the progressive increase in the number of pericytes coincides with the arrest of vascular proliferation is consistent with the idea that pericytes may function as negative regulators of angiogenesis and as inducers of endothelial differentiation and stabilization (Orlidge and D’Amore, 1987; Nicosia and Villaschi, 1995). Pericytes are believed to inhibit endothelial migration and proliferation by juxtacrine mechanisms mediated by physical contacts. On the other hand, it is possible that pericytes may promote microvascular sprouting during early stages of angiogenesis when they lose contact with the endothelium and produce endothelial growth factors in response to angiogenic stimuli (Diaz-Flores et al., 1992; Nehls et al., 1992). In the next sections we review the vascular-derived soluble factors, proteolytic enzymes, ECM molecules, cell adhesion molecules, and vasoactive substances that are likely to participate in the autocrine, paracrine, and juxtacrine mechanisms regulating the interactions among endothelial cells, fibroblasts, and smooth muscle cells/pericytes during the angiogenic response of the vessel wall.

V. Growth Factors and Other Soluble Regulators of Angiogenesis Produced by Cells of the Vessel Wall We describe here the soluble factors implicated in the autoregulation of angiogenesis by the vessel wall. This section discusses vascular-derived regulators of angiogenesis and does not include molecules such as transforming growth factor-a, angiogenin, and epidermal growth factor, which

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contribute primarily to epithelium- and cancer-related angiogenesis, and interleukins, which are mostly involved in inflammation-related and immune-mediated angiogenesis.

A. Growth Factors 1. Fibroblast Growth Factors Fibroblast growth factors (FGFs) are a family of heparin-binding polypeptides with potent angiogenic activity. To date, at least nine FGFs and four types of FGF receptors have been described (Moses et aL, 1995; Gorlin, 1997). Endothelial cells, smooth muscle cells, and fibroblasts produce acidic fibroblast growth factor (aFGF, FGF- 1) and basic fibroblast growth factor (bFGF, FGF-2) and express FGF receptors, which are transmembrane proteins with tyrosine kinase activity (Schweigerer et aL, 1987; Root and Shipley, 1991; Hughes et aL, 1993). The FGF-FGF receptor system represents an important endogenous stimulator of endothelial migration, proliferation, proteolytic activity, and blood vessel formation (Gospodarowicz and Neufeld, 1986; Montesano et al., 1986; Schweigerer et al., 1987; Mignatti et al., 1989). FGFs also promote the migration and proliferation of smooth muscle cells and fibroblasts (Root and Shipley, 1991; Lindner, 1995a). FGFs are stored intracellularly and in the basement membrane, where they are bound to heparan sulfate proteoglycans. The mRNA of bFGF and its receptor FGFR-1 is upregulated in endothelial cells and smooth muscle cells after vascular injury (Lindner and Reidy, 1993). aFGF and bFGF lack signal peptide sequences and are not secreted through the conventional endoplasmic reticulum/Golgi pathway. Injury plays an important role in the release of FGFs since the plasma membrane of transiently injured cells becomes permeable to large molecules, allowing free diffusion of FGFs into the extracellular space (Mutsukrishnan et aL, 1991). It has also been postulated that FGFs may be released through an exocytotic pathway independent of the endoplasmic reticuludGolgi complex (Mignatti and Rifkin, 1991). Basement membrane-bound FGFs are released by heparinases or plasmin (Vlodavsky et aL, 1987; Saskela and Rifkin, 1990). Once they are secreted in the extracellular space, the FGFs are free to interact with their receptors on the cell surface. After receptor binding, aFGF and bFGF are internalized and translocated to the nucleus just a few hours before the onset of DNA synthesis (Hawker and Granger, 1994). Although the mechanism by which nuclear-bound FGFs stimulate DNA synthesis is unknown, the process of FGF nuclear translocation is believed to be a requirement for quiescent cells to reenter the cell cycle.

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Immunohistochemical stain of the rat aorta reveals that bFGF is stored in the cytoplasm of endothelial and smooth muscle cells (Villaschi and Nicosia, 1993). In the rat aorta model, the injury of the dissection procedure causes a release of bFGF from its storage sites. bFGF levels in aortaconditioned medium are highest during the first days of culture and gradually decrease over time, becoming undetectable when microvessels stop growing. Treatment of rat aortic cultures with anti-bFGF antibodies causes a 40% inhibition of the angiogenic response. Anti-bFGF antibodies also inhibit the angiogenic effect of endogenous bFGF in cultures of human chorionic vessels (Brown et al., 1996) and bovine capillary endothelial cells (Sato et al., 1991).

2. Vascular Endothelid Growth Factor Vascular endothelial growth factor (VEGF), also known as vascular permeability factor because of its capacity to greatly increase vascular permeability (Dvorak et al., 1995),is a heparin-binding angiogenic factor with endothelial target specificity (Ferrara et al., 1992). VEGF belongs to a family of endothelial growth factors which includes the recently discovered VEGF-B and the lymphatic endothelium-specific VEGF-C (Enholm et al., 1997; Kukk et al., 1997). VEGF stimulates endothelial migration, proliferation, and proteolytic activity (Ferrara et al., 1992). At variance with aFGF and bFGF, VEGF has a signal peptide and is secreted through conventional pathways. VEGF is produced by a variety of cells, including endothelial cells (Ladoux and Frelin, 1993), smooth muscle cells (Ferrara et al., 1991), and fibroblasts (Minchenko et al., 1995). Secretion of VEGF is markedly stimulated by hypoxic conditions (Shweiki et al., 1992; Minchenko et al., 1995). Four VEGF isoforms are produced by alternative splicing of the VEGF gene (Ferrara et al., 1992). The shortest form, VEGFlZ1,is secreted and can be recovered in conditioned media. The two longer forms, VEGFlS9and VEGF206,are secreted but bind almost exclusively to the cell membrane or the ECM and are not found in the soluble phase. VEGF165 has an intermediate behavior. As for the FGFs, heparin, heparan sulfate, and heparanase all induce release of substrate-bound VEGF, suggesting that heparan sulfate proteoglycans are extracellular binding sites for VEGF (Houck et al., 1992; Ferrara et al., 1992). VEGF165and VEGFls9 can also be released from their binding sites by plasmin (Houck et al., 1992). VEGF binds to the tyrosinase kinase receptors flk-UKDR and flt-1, which are expressed in the endothelium of blood vessels (Terman et al., 1992;Millauer et al., 1993; Quinn et al., 1993). Knockout of the flk-1 receptor gene causes failure of vasculogenesis in the embryo (Shalaby et al., 1995). Knockout of pr-I results in endothelial disorganization daring vasculogenesis and formation of abnormally dilated vascular structures (Fong et al., 1995).

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Knockout of the VEGF gene, which is expressed during vasculogenesis in the mesenchyme surrounding developing blood vessels, causes failure of vasculogenesis not only in the homzygous animal but also in the heterozygous form (Ferrara et al., 1996). Thus, the VEGFNEGF receptor system is an absolute requirement for embryonal vasculogenesis. VEGF is also critical for angiogenesissince anti-VEGF antibodies (Borgstrom et al., 1996) or dominant-negative forms of the fik-1 receptor (Millauer et al., 1994) have potent antiangiogenic activity in vivo. In the rat aorta model, the explants and their outgrowths secrete VEGF in the culture medium. Inhibition of VEGF with a neutralizing antibody causes a 70% reduction of the angiogenic response (Nicosia et al., 1998). Inhibition of endogenous VEGF with a neutralizing antibody also reduces angiogenesis in cultures of human chorionic vessels. However, maximal inhibition of angiogenesis in these cultures is obtained with a combination of anti-bFGF and anti-VEGF antibodies (Brown et al., 1996), suggesting that vascular-derived bFGF and VEGF cooperate in promoting the angiogenic response of injured blood vessels. 3. Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF) is a potent mitogen and chemotactic factor for smooth muscle cells and fibroblasts (Ross et al., 1974; Antoniades et al., 1975). PDGF is composed of two polypeptides, A and B, which can associate forming homodimeric (AA and BB) or heterodimeric (AB) complexes (Hart et al., 1990). The PDGF receptor is composed of two subunits, CY and p chains. These two chains dimerize forming ma, pp, and Cup receptors (Seifert et al., 1988). PDGF AA binds only to the PDGF act receptor. PDGF BB binds to all three receptors. PDGF AB binds to the (Y(Y and receptors. PDGF is produced by endothelial cells (Kazlauskas and DiCorleto, 1985), smooth muscle cells (Majesky et al., 1988), and fibroblasts (Antoniades et al., 1991).Large vessel endothelial cells do not respond in vitro to PDGF (Kazlauskas and DiCorleto, 1985) but may express the PDGF aa receptor in vivo after injury (Lindner, 1995b). Microvascular endothelial cells apparently express the PDGF p/3 receptor (Smits et al., 1989) and proliferate in response to PDGF (Bar et al., 1989).The angiogenic activity of PDGF may be due to both a direct effect on the microvascular endothelial cells (Bar et al., 1989) and an indirect effect mediated by smooth muscle cells and fibroblasts, which in response to PDGF stimulation secrete endothelial cell growth factors such as bFGF and VEGF (Finkenzeller et al., 1992; Brogi et al., 1994). PDGF, which is upregulated in injured blood vessels (Schwartz et al., 1990), is a potent stimulator of angiogenesis in the rat aorta model (Nicosia et al., 1994b). The outgrowth of PDGF-stimulated aortic cultures contains a markedly increased number of spindly mesenchy-

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ma1 cells which probably mediate the angiogenic effect of PDGF in the system. The PDGF BIP ligandheceptor system also seems to play an important role in the recruitment of smooth muscle cells by the endothelium. The endothelium of injured rat carotid arteries expresses PDGF B, whereas the underlying smooth muscle cells express the PDGF /3 receptor (Lindner, 1995b). A similar mechanism may regulate the interactions between microvascular endothelial cells and pericytes because pericytes migrate and proliferate in response to PDGF B (D’Amore and Smith, 1993). PDGF B knockout mouse embryos have markedly dilated vessels and lack mesangial cells, which are glomerular capillary-supporting cells closely related to pericytes (Leveen et aZ., 1994). Abnormal development of the cardiovascular system is also observed in patch mice whose PDGF a receptor gene has been deleted. The vessels of patch mice are lined by a normal endothelium but contain a reduced number of smooth muscle cells and exhibit abnormal fragility (Schatteman et aZ., 1995).

4. Insulin-like Growth Factor Insulin-like growth factor-I (IGF-I) is produced by a variety of cell types, including endothelial cells (Bar, 1992), smooth muscle cells (Delafontaine et al., 1991), and fibroblasts (Pietrzkowski et aZ., 1992). IGF-I expression is observed in connective tissue mesenchyme during embryonal development and is upregulated in the vessel wall of the adult animal during vascular injury (Khorsandi et aZ., 1992) and angiogenesis (Hansson et al., 1989). IGFI production in smooth muscle cells is stimulated by PDGF and bFGF (Delafontaine et al., 1991). IGF-11, a peptide shorter than IGF-I, elicits similar growth responses as IGF-I (Baker et aZ., 1993). The activity of IGF-I and IGF-I1 is modulated by IGF-binding proteins which may either potentiate or inhibit the autocrine and paracrine effects of the IGFs (Bar, 1992). IGF-I and IGF-I1 are probably not required for vasculogenesis and angiogenesis since knockout of IGF-I, IGF-11, or the IGF-I receptor (IGFIR) gene, which both growth factors use to transduce their signals, has no effect on the development of the cardiovascular system (Baker et al., 1993; King et al., 1985). The small size of the embryos lacking IGF-IR, IGF-I, and IGF-11, alone or in combination, indicates that IGFs play an important role in maintaining normal growth rate and in maximizing the efficiency of growth in the various organ systems of the embryo including the cardiovascular system (Baker et aZ., 1993). In fact, addition of IGF-I to serumfree cultures of rat aorta causes a potentiation of the angiogenic response of the aortic explants (Nicosia et aZ., 1994b). 5. Scatter Factor Scatter factor (SF), also known as hepatocyte growth factor, is a secreted endothelial mitogen produced by fibroblasts (Coffer et aZ., 1991). SF binds

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to the c-met receptor, which is coded by a protooncogene (Rosen and Goldberg, 1997). SF, which was first characterized as a potent epithelial mitogen that caused scattering of cultured epithelial cells, was subsequently found to stimulate endothelial cell migration, proliferation, capillary tube formation, and angiogenesis (Rosen et al., 1991; Bussolino et al., 1992). Secretion of SF by fibroblasts is potentiated by bFGF (Roletto et al., 1996). Like other angiogenic factors, SF binds to the ECM (Lamszus et al., 1996).

6. Heparin-Binding Epidermal Growth Factor-like Factor Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family which, unlike EGF, has strong affinity for heparin (Moses et al., 1995). Cell surface heparan sulfates play a critical role for the binding and function of HB-EGF. HB-EGF is produced by a variety of inflammatory and noninflammatory cells, including endothelial cells and smooth muscle cells (Yoshizumi et al., 1992; Dluz et al., 1993). HB-EGF is a potent mitogen for smooth muscle cells and fibroblasts but not for endothelial cells (Moses et al., 1995). HB-EGF binds to the EGF receptor (Higashiyama et al., 1992) but it is not yet clear which of the EGF receptors is involved in the biological function of this growth factor (Moses et al., 1995). HB-EGF mRNA is overexpressed in injured blood vessels (Moses et al., 1995) and can be demonstrated in wound fluid 1 day following skin injury (Powell et al., 1993). HB-EGF is upregulated by bFGF and PDGF (Dluz et al., 1993). 7. Transforming Growth Factor+

Transforming growth factor+ (TGF-P) is a multifunctional peptide capable of regulating the migration, proliferation, and differentiation of a variety of cell types. Three related proteins with high structural similarities have been described in mammalian cells: TGF-Pl, TGF-p2, and TGF-p3 (Massague, 1990).The effects of TGFPs are critically dependent on the differentiation state of the target cells and the presence or absence of other growth factors (Sporn et al., 1987). TGF-Pl stimulates the proliferation and ECM production of fibroblasts (Fine and Goldstein, 1987) and promotes the transformation of these cells into myofibroblasts, which have the capacity to contract collagen (Desmouliere et al., 1993).TGF-Pl can either stimulate or inhibit smooth muscle cell proliferation depending on its concentration and on the density of the cultured cells (Majack et al., 1990). The effect of TGF-P1 on endothelial cells and angiogenesis appears to be contextual because TGF-P1 inhibits the migration and proliferation of endothelial cells in two-dimensional culture (RayChaudury and D’Amore, 1991) but promotes the organization of endothelial cells into capillary tubes in three-

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dimensional collagen gel culture (Madri et al., 1988). This phenomenon may be related to the capacity of TGF-P1 to stimulate production of ECM molecules that have morphogenetic and stabilizing effects on endothelial cells (Madri et al., 1988;Nicosia et aL, 1993) and to modulate the production of proteolytic enzymes and their inhibitors (RayChaudury and D’Amore, 1991). TGF-P1 can either promote or inhibit the in vitro angiogenic activity of bFGF or VEGF in collagen gel culture depending on its concentration in the culture medium (Pepper et al., 1993b). In vivo injection of TGF-P1 in the subcutaneous tissue induces formation of granulation tissue and fibrosis (Roberts et al., 1986). The in vivo angiogenic activity of TGF-P1 has been attributed to the capacity of this growth factor to recruit at the site of injectionhmplantation macrophages and other inflammatory cells which secrete direct-acting angiogenic factors (Roberts et al., 1986; Wahl et al., 1987; RayChaudury and D’Amore, 1991). TGF-P1 is secreted in an inactive form and is believed to be activated by plasmin or an acid microenvironment (Sporn et aL, 1987; RayChaudury and D’Amore, 1991). Active TGF-P1 in turn stimulates production of plasminogen activator inhibitor-1 (PAI-l), which blocks production of plasmin by inhibiting the activity of plasminogen activators (RayChaudury and D’Amore, 1991). During angiogenesis this self-regulating system may be triggered when endothelial cells and pericytes develop cell-cell contacts which activate TGF-P1 (RayChaudury and D’Amore, 1991).Endogenous TGF-P1 production is upregulated in response to vascular injury (Majesky et al., 1991) and during cutaneous wound healing (Cromack et al., 1987). The role of TGF01 in embryonal vasculogenesis and angiogenesis was initially unclear since mice that survived the knockout of TGF-P1 had a normal cardiovascular system and suffered from a lethal disorder of the immune system (Kulkarni et al., 1993). The lack of cardiovascular abnormalities in these mice was attributed to the replacement of TGF-P1 by other embryonal TGF-P isoforms or by maternal TGF-P1 (Kulkarni et al., 1993). Subsequent studies showed that TGF-P1 null mouse embryos that died in utero had defective yolk sac vasculogenesis due to inadequate endothelial differentiation and capillary tube formation (Dickson et aL, 1995).

B. Other Soluble Regulators of Angiogenesis

1. Tissue Factor Tissue factor (TF; thromboplastin) is a procoagulant protein secreted by endothelial cells, smooth muscle cells, and pericytes in response to injury and blood flow-mediated changes in shear stress (Taubman, 1993; Grabowski and Lam, 1995; Bouchard et al., 1997). TF is a chemoattractant for

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smooth muscle cells with the same potency as that of PDGF or bFGF (Sato et al., 1996). The observation that yolk sac blood vessels of mouse embryos lacking the TF gene have no pericytes and are prone to hemorrhage suggests that TF may also play a developmental role in the differentiation of the perivascular mesenchyme and the recruitment of mural cells by the endothelium (Carmeliet et al., 1996). The possibility that a similar mechanism may operate in the adult is supported by the observation that the endothelium of angiogenic microvessels expresses TF, which is otherwise not detectable in quiescent vessels (Contrino et al., 1996). 2. Tumor Necrosis Factor-a

Tumor necrosis factor-a (TNF-a), also known a cachectin for its ability to induce cachexia, plays a role in inflammation and hematopoiesis. Endothelial effects of TNF-a include the induction of class I major histocompatibility antigens, procoagulant activity, interleukin-1, and leukocyte adhesiveness. TNF-a has been shown to stimulate endothelial cell migration and capillary tube formation in vitro (Leibovich etal., 1987).Like TGF-P, however, TNFa inhibits endothelial cell proliferation in vitro (Frater-Schroder et al., 1987). The in vivo angiogenic activity of TNF-a (Leibovich et al., 1987) may be attributed to the molecule’s ability to attract macrophages and mast cells, which in turn secrete direct-acting angiogenic factors (D’Amore and Smith, 1993; Leibovich et al., 1987). TNF-ais also produced by smooth muscle cells (Warner and Libby, 1989) and may contribute to the mechanisms that regulate vascular-related angiogenesis during wound healing.

3. Angiopoietins The angiopoietins are a newly discovered family of peptides which play a critical role in the development of the vessel wall during embryonal vasculogenesis and angiogenesis.Angiopoietin-1 (Ang-1) and angiopoietin2 (Ang-2) bind to the Tie-2 tyrosine kinase receptor which is expressed in endothelial cells (Sato et al., 1995; Davis et al., 1996; Maisonpierre et al., 1997). Ang-1 and Ang-2 are produced by the perivascular mesenchyme and smooth muscle cells during embryonal development (Davis et al., 1996). Genetic ablation of Ang-1 or the Tie-2 receptor results in abnormal vasculogenesis with failure of the newly formed vasculature to differentiate (Sato et al., 1995; Suri et al., 1996). Vessels formed in the absence of a functioning Ang-l/Tie-2 system do not acquire a properly assembled mural layer of smooth muscle cells or pericytes and become dilated (Puri et al., 1995; Sat0 et al., 1995;Suri et al., 1996).Ang-1 is unable to induce endothelial migration or proliferation even though it induces phosphorylation of the endothelial Tie-2 receptor (Sun et al., 1996). These findings suggest that endothelial

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cells respond to stimulation by Ang-1 by secreting factors that recruit mural cells and contribute to the harmonious differentiation of the vascular wall (Suri et al., 1996). Interestingly, patients with arteriovenous malformations, which exhibit abnormal layering of smooth muscle cells around the endothehum, have point mutations of the Tie-2 receptor (Vikkula et al., 1996). Ang-2 competes with Ang-1 for the binding to the Tie-2 receptor. The overexpression of Ang-2 in transgenic mice causes abnormal vascular development similar to that observed in Ang-1 and Tie-2 knockout mice, which suggests that Ang-2 is a natural inhibitor of Ang-1 (Maisonpierre et al., 1997). Abnormal vasculogenesis is also observed in mouse embryos with Tie-1 receptor knockout, but the ligand for the Tie-1 receptor has not yet been identified (Sato et al., 1995).

VI. Proteolytic Enzymes During the early stages of angiogenesis, activated endothelial cells create localized gaps in the basement membrane through which they sprout into the surrounding tissue. As they migrate through the interstitial collagen, endothelial cells generate additional defects in the ECM for the sprouting neovessels. These processes are regulated by proteolytic enzymes and enzyme inhibitors which act in concert to ensure a balanced degradation of the perivascular matrix. Two major classes of proteolytic enzymes and respective inhibitors have been implicated in angiogenesis: plasminogen activators (PAS) and matrix metalloproteinases (MMPs).

A. Plasminogen Activators The PAS hydrolyze plasminogen, a zymogen present in plasma, to form plasmin, a broad spectrum serine protease which breaks down a variety of protein substrates (Saskela, 1985). Endothelial cells can directly synthesize tissue-type PA (t-PA) and urokinase-type PA activator (u-PA) (Loskutoff and Edgington, 1979; Pepper et al., 1987). t-PA is a secreted enzyme which plays an important role in the degradation of fibrin by both arterial and venous endothelial cells. Localized production of plasmin, unaffected by inhibitors of fibrinolysis, is mediated by uPA which has been demonstrated at the tip of growing microvessels (Bacharach and Keshet, 1992). The uPA receptor (uPAr) binds and localizes uPA at critical sites of proteolysis during angiogenesis (Loskutoff and Edgington, 1979; Barnathan et al., 1990). Receptor-bound uPA generates plasmin in close proximity of the sprouting endothelial cell. Endothelial cells also produce PAI-1 which inhib-

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its PA activity and ensures a balanced degradation of the ECM (Bacharach and Keshet, 1992). Angiogenic factors, such as bFGF and VEGF, induce upregulation of both uPA and PAI-1 (Moscatelli and Rifkin, 1988; Pepper et al., 1987,1991). Vascular injury causes upregulation of tPA, uPA, uPAr, and PAI-1 in both endothelial cells and smooth muscle cells (Reidy et aL, 1996). The expression of uPA, uPAr, and PAI-1 is restricted to the site of injury and at the wound edge, implicating these molecules in endothelial and smooth muscle cells migration. Capillary tube formation in vitro and angiogenesis in vivo can be inhibited with uPAr antagonists (Min et aL, 1996).Plasmin functions either directly by digesting noncollagenous components of the ECM (Liotta et al., 1981) or indirectly by activating MMP-1, which in turn digests interstitial collagen (Gross et aL, 1982). Gene knockout experiments indicate that the PA/PAI-1 system is not required for embryonal vasculogenesis and angiogenesis since mice that lack the uPA, tPA, or PAI-1 genes have marked defects in fibrinolysis and coagulation but exhibit a normal cardiovascular system (Carmeliet et aL, 1997a). The PA/PAI-1 system may, however, play a role in reactive and pathologic processes. For example, in vivo intimal migration of smooth muscle cells in response to injury is reduced in mice having a disruption of the plasminogen gene (Carmeliet et al., 1997b).

B. Matrix Metalloproteinases The MMPs are a family of zinc-dependent endopeptidases which are secreted as zymogens and activated extracellularly. MMPs are classified into three groups depending on their substrate specificity: interstitial collagenases (substrate: interstitial collagen), stromelysins (substrates: laminin and fibronectin), and gelatinases (substrate: type IV collagen) (Ray and StetlerStevenson, 1994). There is some functional overlap between the groups since some gelatinases (MMP-2) can digest fibrillar collagen, whereas stromelysins can digest type IV collagen. The function of the MMPs is regulated at multiple levels, including gene activation, transcription, mRNA stability, translation, secretion, binding to ECM components, proenzyme activation, and inactivation by tissue inhibitors of metalloproteinases (TIMPs) (Ray and Stetler-Stevenson, 1994). Endothelial cells, smooth muscle cells, and fibroblasts produce both MMPs and TIMPs (Cornelius et aL, 1995; Forough et aL, 1996; Gilles et aL, 1997). The function of MMPs is modulated by growth factors. For example, expression of interstitial collagenase (MMP1) is induced by both bFGF and VEGF (Unemori et aL, 1992; Kennedy et aL, 1997). These growth factors also stimulate the plasminogen system promoting the formation of plasmin which in turn transforms proMMP-1 into its active form (Unemori et d.,1992). The regulation of MMP synthesis and secretion is cell and tissue specific and the capacity of differ-

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ent growth factors to induce these enzymes varies with the cell type. Stimuli other than growth factors such as cell shape changes, as well cell-cell and cell-matrix interactions, can influence MMPs production (Woessner, 1994). For example, gelatinase B (MMP-9) is induced by the ECM molecule thrombospondin-1 (Quian et aZ., 1997). Gelatinase A (MMP-2) exhibits only a slight response to growth factors and its synthesis is modulated by the ECM and intracellular calcium influx (Woessner, 1994). A cell-associated enzyme known as membrane-type metalloproteinase (MT-MMP-1) has been shown to activate MMP-2 by proteolytic mechanisms (Sato et aZ., 1994). MT-MMP-1 belongs to a family of cell surface enzymes which are believed to play an important role in the activation of MMPs by cell-cell contact. Recent studies indicate that MT-MMP-1 expression by fibroblasts, which results in the activation of MMP-2, is upregulated by interstitial collagen (Gilles et al., 1997). The activity of the MMPs is neutralized by a group of endogenous inhibitors known as TIMPs (Ray and StetlerStevenson, 1994; Woessner, 1994). TIMP-1 forms a complex with activated MMP-1, stromelysin, or MMP-9, whereas TIMP-2 binds to MMP-2. Based on their capacity to inhibit MMP activity,TIMPs can regulate the angiogenic process by modulating the degradation of the ECM. Thus, while they can inhibit angiogenesis by suppressing MMP activity, TIMPs can also promote angiogenesis by preventing excessive proteolytic degradation of the ECM. Up to a certain level, MMPs promote capillary tube formation in vitro, but they have the opposite effect when their concentration is too high (Schnaper et aZ., 1993; Qian et al., 1997). The promoting or inhibitory effects of MMP2 can be regulated by an adequate concentration of TIMP-2. TIMP-2 also has the capacity to inhibit endothelial cell proliferation independently of its anti-MMP activity (Ray and Stetler-Stevenson, 1994). MMPs, which have been localized by in situ labeling techniques in developing microvessels (Galis et al., 1994; Brooks et al., 1996), are believed to be necessary for angiogenesis since neutralization of MMP activity inhibits capillary tube formation in vitro (Schnaper et aZ., 1993; Qian et al., 1997). MMP-2-deficient mice develop normally without anatomical abnormalities but have a reduced capacity to generate new vessels during pathologic processes such as tumor growth (Itoh et aZ., 1998). The absence of cardiovascular defects in these mice has been attributed to the functional redundancy of matrix metalloproteinases (Itoh et al., 1998).

VII. Extracellular Matrix and Cell Adhesion Molecules A. Extracellular Matrix Molecules The ECM that surrounds the endothelium of microvessels is both a mechanical barrier and a substrate requirement for the angiogenic process. The

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morphogenetic behavior of endothelial cells and the response of these cells to soluble growth factors and proteolytic enzymes is tightly linked to the insoluble molecules of the ECM. Endothelial cells are anchorage-dependent cells which must attach to adhesive substrates in order to survive and grow. This process is mediated by a family of cell surface receptors known as integrins (Hynes, 1992) which transduce ECM cues to the nucleus through intracellular pathways of biochemical and mechanochemical signals (Ingber and Folkman, 1989). The importance of the ECM in angiogenesis is underscored by experimental evidence indicating that the stimulatory effect of growth factors and other soluble regulators of angiogenesis is not sufficient to explain how blood vessels form (Ingber, 1991).

1. Developmental Changes in the Extracellular Matrix during Angiogenesis The endothelium of quiescent microvessels is ensheathed by a basement membrane and fibrils of interstitial collagen admixed with proteoglycans. During angiogenesis, the microvascular ECM undergoes complex maturational changes due to the proteolytic degradation of the preexisting matrix and the deposition of a new matrix (Nicosia and Madri, 1987). At the ultrastructural level, developing neovessels are surrounded by a tenuous and discontinuous basement membrane (Schoefl, 1963). Because of the high permeability of the sprouting endothelium, the perivascular space is edematous and may be permeated by plasma-derived large molecules such as fibrinogen, fibronectin, vitronectin, von-Willebrand factor, and plasminogen. The extravascular activation of the coagulation cascade results in the formation of fibrin which provides a permissive ECM scaffold for endothelial cell migration, proliferation, and tube formation. Vitronectin, fibronectin, and von-Willebrand factor promote endothelial migration by acting as substrates for the integrin receptors expressed by sprouting endothelial cells (Hynes, 1992). Plasminogen functions as a zymogen for the formation of plasmin, which endothelial cells use to break down fibrin and other ECM molecules and to generate active MMP-1 (Liotta et al., 1981; Gross et al., 1982). The provisional ECM secreted by endothelial cells contains fibronectin, type V collagen, and small amounts of laminin and type IV collagen (Tonnesen et aL, 1985; Nicosia and Madri, 1987). As the microvessels mature, laminin and type IV collagen accumulate in the subendothelial space forming a basement membrane (Nicosia and Madri, 1987). Additional ECM molecules produced by vascular cells which may modulate angiogenesis include the glycoproteins entactin (Nicosia et al., 1994a), thrombospondin-1 (Koch et aL, 1993), osteopontin (O’Brien et al., 1994), and tenascin (Zagzag et al., 1996) as well as the heparan sulfate proteoglycans perlecan (Aviezer et al., 1994) and syndecan-1 (Kainulainen et aL, 1996). As the basement membrane matures, fibrils of the interstitial colla-

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gens type I and I11 are deposited by the endothelial cells, pericytes, and surrounding fibroblasts around the newly formed microvessels (Nicosia and Madri, 1987).

2. Role of the Extracellular Matrix in Angiogenesis The ECM plays a fundamental morphogenetic role in angiogenesis because it promotes the organization of endothelial cells into capillary tubes. As endothelial cells migrate into the connective tissue, collagen and the other ECM molecules that surround the endothelium induce capillary tube morphogenesis. This phenomenon can be reproduced in vitro by culturing endothelial cells on basement membrane-like matrices (Kubota et aL, 1988) and in gels of interstitial collagen (Montesano et aL, 1983)or fibrin (Fournier and Doillon, 1992). Destabilization of endothelial cell-ECM contacts causes detachment of endothelial cells from the basement membrane and disruption of microvessels. Detached endothelial cells round up, cease to proliferate, and undergo apoptosis. This effect can be achieved by treating developing microvessels with angiostatic steroids, inhibitors of collagen synthesis, inhibitors of basement membrane synthesis, or fungal-derived antibiotics which destabilize cell-matrix interactions (Ingber and Folkman, 1989). Angiogenesis can also be inhibited by interfering with the binding of integrin receptors to RGD (Arg-Gly-Asp)-containingcomponents of the perivascular matrix (Nicosia and Bonanno, 1991). The RGD-sensitive a& and avosintegrin receptors have been proposed as potential targets for antiangiogenic therapy because they are expressed by the microvascular endothelium during angiogenesis. Expression of the avosor avosreceptors in endothelial cells appears to be regulated by separate growth factor pathways since angiogenesis induced by bFGF apparently depends on aVP3, whereas angiogenesis initiated by VEGF requires avPs(Brooks et aL, 1994; Friedlander et al., 1995). RGD-containing synthetic peptides inhibit angiogenesis by interfering with the binding of RGD-sensitive receptors to RGD-containing ECM molecules (Haymann et aL, 1985;. Nicosia and Bonanno, 1991). Because the perivascular matrix is particularly rich in RGD-containing molecules and the avP3and avPsintegrin receptors can bind to a variety of ECM molecules (Hynes, 1992), it is likely that sprouting endothelial cells utilize a redundant system of RGD-containing substrates for attachment and migration. It is clear, however, that some of these matrix molecules are required for proper vascular morphogenesis. Gene knockout studies have shown that ablation of the fibronectin gene causes malformations of the cardiovascular system (George et al., 1993). Similar results have been observed in mice lacking the as chain of the fibronectin receptor (Yang et aL, 1992).

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Based on their capacity to influence endothelial cell adhesion and shape (Ingber and Folkman, 1989) and to bind angiogenic factors (Moses et al., 1995), ECM molecules can modulate the migratory and proliferative response of endothelial cells. Thus, fibronectin, laminin, and entactin added to a basal gel of interstitial collagen promote the elongation of microvessels sprouting from explants of rat aorta (Nicosia et al., 1993, 1994a). ECM molecules may, however, have opposite effects on angiogenesis depending on their concentration. These effects can be observed in the rat aorta model by varying the amounts of ECM molecules added to the collagen gel. For example, the basement membrane-derived laminin-entactin complex added to a basal gel of interstitial collagen stimulates rat aorta angiogenesis at 30-300 pglml, whereas it has inhibitory effects at 3000 pglml. High concentrations of laminin-entactin also have stabilizing effects and prevent the spontaneous regression of microvessels (Nicosia et al., 1994a). A similar concentration of laminin-entactin is found in native basement membranelike matrices (Kleinmann et al., 1986). This suggests that the mature basement membrane that surrounds the endothelium during the late stages of blood vessel formation may function as a stop signal that turns angiogenesis off and stabilizes the newly formed microvasculature. TGFP-1, which is presumably activated when the endothelial cells of mature microvessels are surrounded by pericytes, may contribute to this late stage of the angiogenic process by stimulating the production and deposition of basement membrane molecules (RayChaudury and D’Amore, 1991). Heparan sulfate proteoglycans, which are present in the basement membranes and on the cell surface, can modulate the response of endothelial cells and smooth muscle cells/pericytesto bFGF, VEGF, and other heparinbinding angiogenic factors by sequestering these molecules in the ECM (Ferrara et al., 1992; Moses et al., 1995). Heparitinases secreted in response to injury or pathologic stimuli may release the growth factors from the ECM making them available for angiogenic stimulation (Vlodavsky et al., 1987; Houck et al., 1992; Ferrara et al., 1992). Heparin, a highly sulfate glycosaminoglycan,is stored in the cytoplasmicgranules of mast cells, which are believed to modulate angiogenesis by releasing this molecule in the extracellular space (Meininger and Zetter, 1992). Heparan sulfate proteoglycans may also act as low-affinity receptors for FGFs, which are more soluble and stable when complexed with the sulfate groups of these molecules (Moses et al., 1995). Conformational changes induced by the heparan proteoglycans may allow the FGFs to better interact with their high-affinity receptors on the endothelial cell surface. Perlecan, a basement membrane proteoglycan, is a potent inducer of bFGF-mediated angiogenesis (Aviezer et al., 1994). Similarly, syndecan-1, a cell surface proteoglycan, is believed to play a role in angiogenesis because it is transiently expressed by endothelial cells during formation of microvessels (Kainulainen et. al., 1996). Be-

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cause of its ability to bind angiogenic factors and extract them from tissues, heparin may also exhibit antiangiogenic activity if administered at pharmacologic doses together with hydrocortisone or other angiostatic steroids (Moses et al., 1995). The enzymatic digestion of the ECM by vascular cells generates fragments which may have opposite effects of the intact molecule. For example, hyaluronan, a large glycosaminoglycan present in the interstitial ECM, has antiangiogenic activity,whereas its oligosaccharidefragments potentiate the action of angiogenic factors (Rooney et aL, 1995). Conversely, proteolytic digestion of fibronectin (Homandberg et al., 1985), plasminogen (O’Reilly et al., 1996), or collagen type XVIII (O’Reilly et al., 1997) generates fragments with antiangiogenic activity.

6.Cell Adhesion Molecules The organization of endothelial cells into capillary tubes is mediated not only by cell-matrix adhesive events but also by cell-cell interactions. Cytoadhesive proteins such as the selectins and cadherins are believed to play a role in regulating cell-cell interactions that take place during capillary tube morphogenesis (Koch et al., 1995; Dejana, 1996; Dejana et al., 1997). Cell-cell interactions during capillary morphogenesis are also regulated by integrins, which in addition to engaging ECM molecules have the capacity to bind to each other (Dejana et al., 1997), and by molecules of the Ig superfamily such as platelet-endothelial cell adhesion molecule-1 (Lu et al., 1996; DeLissler et al., 1997). E-selectin, a surface molecule that localizes in adherent junctions, contributes to the formation and maintenance of capillary tubes by interacting with sialyl Lewis-X and sialyl Lewis-A carbohydrate moieties on adjacent cells (Nguyen et al., 1993). Similarly, vascular endothelial cadherin, which is localized in endothelial adherent junctions, is required for capillary tube formation and maintenance (Dejana et al., 1997; Matsuma et al., 1997).

VIII. Vasoactive Factors

Vasoactive events may play a role in angiogenesis because vasodilatation and increased blood flow promote capillary growth (Ziada et al., 1984). During angiogenesis in vivo capillaries with higher flow generate more sprouts and change gradually into arterioles and venules (Thoma, 1911; Clark and Clark, 1939). Conversely, neovessels that are not supplied with blood tend to regress (Clark and Clark, 1939). Vascular regression is associ-

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ated with contraction of the neovessels and endothelial retraction (Clark and Clark, 1939; Nicosia and Villaschi, 1995). Vasodilating agents have been reported to have angiogenic activity (Ziche et al., 1982; Ziada et al., 1984; Masferrer et aZ., 1989). Vasoactive events may regulate the angioformative behavior of endothelial cells by activating through mechanochemical mechanisms endothelial genes involved in angiogenesis (Ingber, 1991;Gimbrone et al., 1997). Angiogenic factors may modulate vascular tone and blood flow. For example, aFGF, bFGF, and VEGF cause vasodilation (Ku et al., 1993; Wu et al., 1996b), whereas PDGF is a potent vasoconstrictor (Berk et aL, 1986). Vascular cells produce a variety of vasoactive molecules, including nitric oxide, prostaglandins, and endothelins, all of which may contribute to the mechanisms by which the vessel wall regulates angiogenesis. Flow-dependent vasodilation is mediated by intracellular influx of calcium, calcium-mediated activation of nitric oxide (NO) synthase, and synthesis of NO, also known as endothelial-derived relaxing factor (Moncada, 1997).Endothelial cells express constitutively a form of nitric oxide synthase which generates NO from L-arginine. Endothelial cell-derived NO maintains a vasodilator tone that is essential for the regulation of normal blood flow and pressure. Smooth muscle cells, like many other cell types, can express an inducible form of nitric oxide synthase (iNOS) in response to cytokines such as IL-1 or TNF-a. Small arteries and arterioles are a major site of NO-mediated vasodilation in response to increase blood flow and shear stress. NO mediates the vasodilator effect of ADP, histamine, serotonine (Moncada, 1997), aFGF, bFGF, and VEGF (Ku et aZ., 1993; Wu et aZ., 1996b). NO apparently also regulates the increased vascular permeability caused by VEGF at the level of postcapillary venules via a signaling cascade involving guanylate cyclase stimulation and guanosine 3'3'-cyclic monophosphate-dependent protein kinase (Wu et al., 1996b). It has also been proposed that NO mediates the endothelial-specific mitogenic effect of VEGF (Morbidelli et aZ., 1996). NO promotes the proliferation of and PA production by endothelial cells through upregulation of endogenous bFGF (Ziche et aZ., 1997). Expression of iNOS by nonendothelial cells has been implicated in angiogenesis since the angiogenic activity of monocytes requires a functioning NOS system (Leibovich et aZ., 1994). Interestingly, NO inhibits the migration and proliferation of smooth muscle cells (Garg and Hassid, 1989). Thus, increased levels of NO during the early stages of angiogenesis may explain not only the vasodilation and increased blood flow but also the finding of immature endothelial sprouts without pericytes or smooth muscle cells. The role of NO in angiogenesis,however, is debated since inhibitors of NO synthase have antiangiogenic activity in the rabbit cornea model (Ziche et aZ., 1994) but increase vascular density in the chorioallantoic membrane of the chick embryo (Pipili-Synetos et aZ., 1993).

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The ETs are a family of small peptides secreted by both vascular endothelial and smooth muscle cells. Three different isoforms have been described (ET-1, ET-2, and ET-3). Endothelial cells produce exclusively ET-1, whereas other tissues produce ET-2 and ET-3. ET-1 production is induced by a variety of factors, including TGFP-1 and hypoxia (Luscher and Wenzel, 1995). ET-1 causes transient vasodilation followed by profound and sustained vasoconstriction. ET-1 also stimulates smooth muscle cell migration and proliferation (Bobik et aZ., 1990) and potentiates the effect of PDGF BB on these cells (Weissberg et aZ., 1990). The mitogenic effect of ET-1 is mediated by the ETA and ETB receptors (Luscher and Wenzel, 1995). The ETA receptor is responsible for vasoconstriction, whereas the ETB receptor, which is also expressed in endothelial cells, is linked to NO and prostacyclin release (Luscher and Wenzel, 1995). During embryonal development, ET-1 is expressed intensely in the endocardium of the outflow tract and in the endothelium of arch arteries and dorsal aorta (Kurihara et al., 1997). Neural crest-derived ectomesenchymal cells migrate from the pharyngeal arches to the heart outflow tract and arch arteries where they contribute to the formation of great vessels. Ectomesenchymal cells, which provide support to the endothelial cells and give rise to smooth muscle cells, may be recruited to their final destination sites by ET-1. In fact, ET-1 knockout mouse embryos exhibit a variety of cardiovascular malformations, including interrupted aortic arch, hypoplasia of the aortic arch, aberrant subclavian artery, and ventricular septa1 defects with abnormalities of the outflow tract (Kurihara et aZ., 1997). The frequency of these abnormalities increases by treatment with neutralizing anti-ET-1 monoclonal antibodies or selective ETA receptor antagonists (Kurihara et al., 1997). Prostaglandin El(PGEJ and, to a lesser extent PGE2 and PGFh, stimulate angiogenesis in vivo (Ben-Ezra, 1978). The angiogenic activity of prostaglandins is probably mediated by cytokines and growth factors secreted by inflammatory cells which are chemotactically attracted to the site of angiogenesis (Ziche et al., 1982; Odedra and Weiss, 1991) and by fibroblasts which secrete VEGF in response to prostaglandin stimulation (Ben-Av et al., 1995). Prostaglandins such as PGEl may also facilitate angiogenesis by causing vasodilation and increased blood flow. Adenosine, a vasodilator metabolite which accumulates in tissues during hypoxia and anaerobic metabolism, has been shown to stimulate endothelial cell migration and proliferation in vitro as well as angiogenesis in vivo (Ziada et al., 1984).Although the relatively high doses of adenosine required to stimulate angiogenesis have raised doubts about its physiologic role, these observations suggest that small molecules of nonproteic nature released during wound healing or other processes associated with hypoxia may influence the angiogenicresponse of blood vessels. For example, inosine-a metabolic derivative of adenosine-and nicotinamide, both of which accu-

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mulate in tissues during anaerobic metabolism, have been shown to have angiogenic activity (Odedra and Weiss, 1991).

IX. Summary and Future Directions Our current knowledge of the endogenous angiogenic properties of blood vessels can be summarized as follows: (i) Preexisting blood vessels have the capacity to generate new vessels in the absence of exogenous angiogenic stimuli; (ii) vascular autoregulation of angiogenesiscan be studied by culturing arterial or venous explants in vitro; (iii) angiogenesis in vascular organ culture is induced by the combined effect of injury and exposure of the explants to collagen; (iv) the angiogenic response of the vessel wall is mediated by autocrine, paracrine, and juxtacrine interactions among endothelial cells, smooth muscle cells/pericytes, and fibroblasts; (v) the cells of the vessel wall regulate angiogenesis by producing growth factors, proteolytic enzymes, components of the ECM, cell adhesion molecules, and vasoactive factors; and (vi) molecules involved in the regulation of angiogenesis are expressed by blood vessels during embryonal development, the female menstrual cycle, wound healing, hypoxic conditions, and pathologic processes. The cascade of cellular events occurring during the angiogenic response of the vessel wall to injury and the putative endogenous regulators of this process are schematically represented in Fig. 3. Observations made with models of vascular organ culture pose intriguing questions for future research. For example, can formation of blood vessels be inhibited by targeting a single angiogenic factor? Gene knockout experiments indicate that this is possible when blood vessels develop from the undifferentiated mesenchyme during vasculogenesis in the embryo (Ferrara et al., 1996). Vascular organ culture experiments, however, suggest that formation of neovessels from preexisting blood vessels is only partially inhibited with antibodies against individual growth factors (Villaschi and Nicosia, 1993; Brown et al., 1996; Nicosia et al., 1998). This observation, which may also apply to pathologic angiogenesis, raises the possibility that antiangiogenic factor therapy in the adult may not succeed until the combinations of growth factors regulating angiogenesis in different pathologic conditions have been characterized. Alternatively, drugs capable of blocking signal transduction pathways possibly shared by different angiogenic factors may overcome this limitation. What molecular events initiate angiogenesis? In vivo and in vitro experiments suggest that endothelial growth factors, such as bFGF and VEGF, can directly stimulate angiogenesiswithout the involvement of other molecules or cell types. During in vivo experiments, however, the local delivery

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AUTOREGULATION OF ANGIOGENESIS INJURY

ACTIVATION OF ENDOTHELIAL CELLS, SMOOTH MUSCLE CELLSPERICYTESAND FIBROBLASTS

LOCALIZED PROTEOLYSIS OF TEEBASEMENTMEMBRANJ?

GROWTH FACTORS NITRIC OXIDE

INTERSTITIALCOLLAGEN

1

b ENDOTEELIALMIGRATION AND PROLIFERATION

CAPILLARY TUBE MORPHOGENESIS

I

CELL ADHESION MOLECULES

.c BALANCED DEGRADATION OF THEINTERSTITIALECM

CHEMOTACTICFACTORS (?) VASOACTIVE FACTORS

FORMATION OF ANASTOMOSES AND CAPILLARY LOOPS AND ESTABLISHMENTOF BLOOD FLOW

1

RECRUITMENTOF PERICYTFS SMOOTH MUSCLE CELLS

TGFbeta-1

I

+

b ENDOTEELIAL DIFFERENTIATION/ QUIESCENCE

1 T

STABILIZATION AND SURWAL OF NEOVESSELS

FIG. 3 Autoregulation of angiogenesis by the vessel wall: putative cascade of cellular and molecular events regulating formation of new vessels in response to injury.

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of growth factors causes connective tissue injury which may activate fibroblasts or inflammatory cells. During in vitro experiments, fetal bovine serum may influence the response of isolated endothelial cells to bFGF and VEGF because it contains a variety of platelet-derived factors capable of activating the endothelium (Pepper et al., 1993a;Montesano et al., 1996). In the serumfree environment of the rat aorta model, bFGF functions as an angiogenic factor during the vasoproliferative response of the explants, but it behaves as a trophic factor after the angiogenic growth phase (Villaschi and Nicosia, 1997). Once they have stopped growing, neovessels survive but no longer proliferate in response to bFGF. This suggests that the angiogenic activity of bFGF manifests itself only when the aortic endothelium has been injured. The observation that bFGF and VEGF may be expressed by the vessel wall in the absence of angiogenesis (Cordon-Cardo et al., 1990; Couffinhal et al., 1997) further supports the idea that the mere presence of angiogenic factors is not sufficient to initiate angiogenesis. If this hypothesis is correct, what molecular events in addition to secretion of angiogenic factors are required for the induction of angiogenesis? Conditions that promote the angiogenic activity of growth factors may vary depending on the context in which angiogenesis takes place. In the rat aorta model, injury and exposure of the endothelium to collagen create a permissive environment for angiogenesis (Nicosia and Ottinetti, 1990; Nicosia et al., 1998). Similar conditions may occur in vivo during wound healing or the recanalization of thrombi when the apical surface of injury-activated endothelial cells is exposed to fibrin, collagen, and other ECM molecules (Sueishi et al., 1997). Injury of vascular cells may also release lysosomal enzymes, such the cathepsins B and L, which are capable of degrading the main components of the basement membrane (Guinec et al., 1993). In tumor-related angiogenesis, cancer cells may stimulate fibroblasts to produce collagen, which has been shown to indirectly activate MMP-2 by upregulating fibroblast MT-MMP1 (Gilles et al., 1997). Fibroblast-derived MMP-2 may in turn digest the microvascular basement membrane and bind to the a& receptor of endothelial cells, thereby promoting endothelial sprouting (Brooks et al., 1996). In inflammation-related angiogenesis, TNF-a produced by macrophages (Leibovich et al., 1987) may contribute to the induction of angiogenesis by activating endothelial proteolytic enzymes (Ray and Stetler-Stevenson, 1994). In fact, human capillary endothelial cells are unable to penetrate fibrin and form capillary tubes in response to bFGF or VEGF unless TNFa is added to the culture medium (van Hinsbergh et al., 1997). What mechanisms regulate the formation of vascular anastomoses? In the rat aorta model, the neovessels grow toward each other forming loops as seen during angiogenesis in vivo. The tendency of neovessels to form anastomotic connections becomes apparent when two or more vascular

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explants are embedded in the same collagen gel (Figs. 4 and 5). A possible explanation is that the endothelial tips of the sprouting neovessels secrete endothelial-specificchemoattractants. In addition, endothelial cells, by contracting collagen fibrils, create traction forces which may guide opposing sprouts toward each other (Vernon et al., 1995). How do endothelial cells recruit smooth muscle cells/pericytes? The rat aorta model and the endothelial-smooth muscle cells coculture experiments strongly indicate that these cells interact by paracrine mechanisms. Gene knockout experiments suggest that Ang-1 produced by the perivascular mesenchyme signals the endothelium to secrete pericyte-chemotactic factors (Sun et al., 1996). Does Ang-1 promote the expression of PDGF B, HB-EGF, TF, ET-1 (D’Amore and Smith, 1993; Lindner, 1995a; Luscher and Wenzel, 1995;Moses et al., 1995; Contrino et al., 1996), or other factors capable of attracting pericytes? Does TGF-P1, which has been implicated in the differentiation of microvessels (D’Amore and Smith, 1993),modulate the production of Ang-l? Is TGF-Pl the signal that turns angiogenesis off? After they have established contact with the microvessels, pericytes form junctions with the endothelial cells (Nicosia and Villaschi, 1995). What cell adhesion molecules regulate the interaction between endothelial cells and pericytes?

FIG. 4 Serum-free collagen gel culture of rat aortic (A) and renal venous (V) explants. The microvessels of adjacent outgrowths have migrated toward each other forming anastomotic connections (arrows). Scale bar = 450 p M .

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FIG. 5 Anastomotic connections (arrows) between microvascular outgrowths of adjacent rat aortic explants in serum-free collagen gel culture. Scale bar = 300 p M .

What are the mechanisms responsible for the regression of neovessels? In the rat aorta model of angiogenesis,the neovasculature regresses through a process that resembles vascular regression during angiogenesis in vivo. Endothelial cells retract, becoming reabsorbed into the main stems of the neovessels which become thicker and less branched. Changes in cell-matrix interactions may be involved in this process. Do endothelial cells downregulate critical integrin receptors such as a,& or a,&? What is the role of proteolytic enzymes during vascular regression? Does the lack of blood flow affect the survival of the neovessels through the action (or lack of action) of vasoactive factors? What factors mediate the contraction of the endothelium responsible for the retraction of the neovessels? Finally, do blood vessels vary in their capacity to autoregulate angiogenesis? The observation that rat veins and human chorionic vessels are capable of generating new vessels indicates that autoregulation of angiogenesis is not restricted to the rat aorta. As more experiments with vascular organ models are carried out, we will learn whether there is heterogeneity of angiogenic regulation among blood vessels isolated from different vascular beds and animal species. These studies may ultimately lead to the development of novel therapeutic strategies for the pharmacologic stimulation or inhibition of the angiogenic process.

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Acknowledgments This work was supported by NIH Grant HL52585.

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Fibroblast Growth Factors as Multifunctional Signaling Factors Gyorgyi Szebenyi and John F. Fallon Anatomy Department, University of Wisconsin, Madison, Wisconsin 53706

~~

The fibroblast growth factor (FGF) family consists of at least 15 structurally related polypeptide growth factors. Their expression is controlled at the levels of transcription, mRNA stability, and translation. The bioavailability of FGFs is further modulated by posttranslational processing and regulated protein trafficking. FGFs bind to receptor tyrosine kinases (FGFRs), heparan sulfate proteoglycans (HSPG), and a cysteine-rich FGF receptor (CFR). FGFRs are required for most biological activities of FGFs. HSPGs alter FGF-FGFR interactions and CFR participates in FGF intracellular transport. FGF signaling pathways are intricate and are intertwined with insulin-like growth factor, transforming growth factor+, bone morphogenetic protein, and vertebrate homologs of Drosopbih wingless activated pathways. FGFs are major regulators of embryonic development: They influence the formation of the primary body axis, neural axis, limbs, and other structures. The activities of FGFs depend on their coordination of fundamental cellular functions, such as survival, replication, differentiation, adhesion, and motility, through effects on gene expression and the cytoskeleton. KEY WORDS: FGF, FGF receptor, Gene expression, Signaling, Patterning, Development, Cytoskeleton.

1. Introduction Fibroblast growth factors (FGFs) were first isolated in the 1970s from bovine brain extracts based on their mitogenic and angiogenic activities. Subsequent research established that FGFs form a family of structurally related polypeptide growth factors, have diverse activities, and are produced at some point during the development of each of the four histological tissue types (epithelia, muscle, connective, and nervous tissues). Even though Internntional Review of Cyrology, Vol. 185 0074-76%/99 $25.00

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Copyright 0 1999 by Academic Press. All rights of reproduction in any form reserved.

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GYORGYI SZEBENYI AND JOHN F. FALLON

FGFs and their target cells are widely distributed throughout the developing and adult body, each FGF and FGF receptor shows a restricted, albeit overlapping, spatial and temporal expression pattern. The regulation of synthesis and activity of FGFs and their receptors is complex and occurs at all levels of processing, including activation of transcription, posttranscriptional modifications (splicing, polyadenylation, and mRNA stability), translation initiation, posttranslational modifications (glycosylation, phosphorylation, and ribosylation), intracellular trafficking, secretion, bioavailability, and ligand-receptor interactions (see Sections I1 and III,A,3). In addition, postreceptor FGF signaling pathways are intricate: Several biochemical cascades interact and are integrated into a unique response to the multiple, and sometimes antagonistic, signals constantly bombarding cells (see Section 111,D). The effects of FGFs on cellular functions depend on the biochemical state and environment of their target cells. In addition to their initially observed effects on cell replication and angiogenesis, FGFs regulate cell survival and apoptosis, adhesion, motility, and differentiation (see Section IV,B). FGF modulation of complex biological events, such as tumor formation, the remodeling of blood vessels, and effects on morphogenesis, is likely the result of FGF regulation of several cellular functions. Progress in understanding the roles of FGFs in embryonic development has greatly accelerated since clones of fgfs, large amounts of pure recombinant proteins, and novel methods to test these reagents in vivo have become widely available. There is now a massive amount of data that demonstrate FGFs’ involvement in gastrulation, neurulation, the anteroposterior specification of body segments, and organ morphogenesis in both invertebrates and vertebrates (see Section IV,A). This review is organized as follows: first, we discuss how FGFs are synthesized and released, with an emphasis on the formidable variety in their structure; next, we give a description of FGF-binding proteins and the signaling pathways that mediate the actions of FGFs; then, we discuss the biological activities of FGFs in embryonic development and relate these activities to their effects on fundamental cellular activities, such as cell survival,apoptosis, replication rates, cell-matrix adhesion, cell-cell interactions, cell motility, and differentiation. The aim is to summarize the major findings in FGF biology, to indicate the complexity of FGF signaling pathways and ways they integrate with other cellular activities, and to point out areas where research is lacking. We attempt to explore most of the major issues in FGF biology but can cite only a limited number of specific experiments to illustrate each point. Medline currently contains about 3000 references on the subject, over 75% of which were published in the 1990s. It is impossible and, given the accessibility of electronic databases, unnecessary to provide an exhaustive

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47

bibliography. We emphasize primary research results reported in 1995-1997 and only cite earlier work that has not been reviewed by others. The list of reviews in the Appendix will help to locate additional primary resources in special fields of interest and provides a variety of perspectives. We hope that our survey of the FGF field will provide a framework to evaluate the mass of information available on this topic.

II. Structure, Regulation of Synthesis, Subcellular Localization, and Release of FGFs FGFs belong to a family of structurally related polypeptide growth factors, designated by the acronym FGF and a number (FGF-n). See Table I for a list of other names for FGFs still encountered in the literature. FGFs also belong to the larger heparin binding growth factor family (HBGF). The fgfihave been cloned from many species, including mammals, birds, fish, amphibians, fruit flies, and worms. The most conserved sequence within the predicted protein sequence of FGFs is within a core of 120 amino acids (Fig. 1). Within this region, fgf orthologs (divergence resulting from speciation) are 71-100% identical in aa composition, whereas the protein sequences of fgf paralogs (divergence due to gene duplication resulting in several isotypes within a single species) are 22-66% identical. Phylogenetic analyses indicate that there were at least two phases of gene duplications that gave rise to the present diversity in fgfs: The first series of duplications resulted in separate genes forfgf-3, -5, -7, -8, and -9 and three ancestor genes for fgf-l/fgf-2, f g f 4 f g f - 6 , and fgf-ll/fgf--12; the second series of duplications gave rise to separate fgf-1, -2,-4, -6, -11,and -12 genes. The first phase of duplications probably occurred at the time of emergence of the vertebrates and the second phase at the time of fin-to-limb transition (Coulier et al., 1997), consistent with the observations that FGFs contribute to the patterning of the body and the development of the limbs (see Section IV,A). It is expected that more homologous sequences will be found as the interest in FGFs grows and the molecular databases become larger. A. Structure and Regulation of fgfGenes

Most fgf genes have a similar exonhtron organization (Table I). There are three coding exons in fgf-1 to -6 and in fgf-1.5 and in the invertebrate fgf genes, egl-17and bnl (Burdine et al., 1997; Sutherland et al., 1996). Each of the exons encode parallel p strands which fold into a distinct structural domain, called the p trefoil, that has the geometry of a trigonal pyramid

TABLE I Nomenclature of FGF and Some Features of FGF Genes

Name

Alternative names

Chromosomal location of genes (humany

Number of coding exons

FGF-1

Acidic FGF (aFGF)

5q31.3-33.2

3

FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF-10 FGF-11 FGF-12 FGF-13 FGF-14 FGF-15 EGL-17 BNL

Basic FGF (bFGF) INT-2 HST-1, k-FGF (Kaposi)

4q26-27 llq13 llq13.3 4q21 12~13 15 10q24 13q12-ql3 5~12-13 17~12 3q28 xq21 13

3 3 3 3 3

HST-2 KGF (keratinocyte GF) AIGF (androgen induced) GGF (glial) FHF-3 FHF-1 FHF-2 FHF-4

Branchless

Based on Emoto et al. (1997). Based on searches of Genbank through September 1997.

6 (1A-D,2,3)

5 3 3 3

Genes cloned fromb Human, hamster, bovine, rat, pig, chick, mouse Human, opossum, bovine, rat, chick, mouse, sheep, Xenopus, newt Human, chick, fish, mouse, Xenopus Human, chick, bovine, mouse, Xenopus Human, mouse, rat Human, mouse Human, mouse, rat, sheep, dog Human, mouse, chick, Xenopus Human, rat, mouse, Xenopus Human, rat, chick, mouse Human, mouse Human, mouse, chick Human, mouse, chick Mouse

Mouse C. elegans Drosophila

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS

49

cuc GIR repeats nuclear localization signals

AUG sigm sequencl

lation site C

0

a, a,

1PKC phosphorylatioin

site

elycosylation site ADP-ribosylation site

C

CUG: AUG: signal sequence: FGFR 8 HEPARIN

binding regions: glycosylation sites: ADP-ribosylation site:

osphorylation site $::$:$$:

$;@+$ *iii
1” ysteine

FIG. 1 The structure of a generic FGF protein is shown. The four translation initiation sites (three CUGs and one AUG), sites of posttranslational modifications, and the conserved core region that contains receptor-binding sites are indicated. The scale, in amino acids, is marked in the boxes. References and a description of the functional significanceof the sites indicated in this figure are provided in Section I1,C.

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GYORGYI SZEBENYI AND JOHN F. FALLON

(McWhirter et al., 1997; Murzin et al., 1992). The 3-D structure of some wild-type and mutant FGFs are in the molecular database, accessible at www.ncbi.nlm.nih.gov. The structures of the fgf--13 and fgf-8 genes diverge from the three-exon patterns but are clearly related to the other fgfgenes. In the fgf--13 gene, which has five exons, exons 1 and 2 correspond to exon 1 in the three-exon gene pattern, exon 3 to exon 2, and exons 4 and 5 to exon 3 (Smallwood et al., 1996). In fd-8, which is the most divergent member of the FGF family with six exons in the mouse, the first four exons (exons 1A-1D) are variants of exon 1 of the three-exonfgfgenes, whereas exons 2 and 3 conform to the conserved pattern in size and position (MacArthur et al., 1995b). In addition to the conservation of sequences and the positions of exon/ intron junctions, the genomic loci of some of the fgf genes also provide evidence for the common evolutionary history of this gene family (Table I). The f g f 3 and -4 genes are genetically linked in both mice and humans, fgf-2and fgf-5 are on the same chromosome in humans, and fgf4/fd-6and fgf-l/fgf-2 are on phylogenetically related chromosomes that have other common markers (Verdier et al., 1997). Cis-regulatory elements in the fgf--1, -2, -3, -4, and -15 genes have been studied and some of these were shown to bind regulatory proteins. In the fgf-l gene, there are four 5' untranslated exons, each under the control of a separate promoter (1A-1D). Transcription from each of these promoters is regulated by distinct mechanisms (Chotani and Chiu, 1997). In one of the two regulatory regions within the brain-specific 1B promoter, an 18nucleotide minimal cis sequence interacts with a protein complex that contains two DNA-binding proteins (Myers et al., 1995; Ray et al., 1997b; Voulgaropoulou et al., 1994). There are four promoters in the fgf-2 gene (PO,P I , Pz, Pi,);they lack a TATA or CCAAT box but have GC-rich regions and consensus binding sites for the transcription factors SP1 and AP1 and for negative regulatory proteins (Basilico and Moscatelli, (1992). A consensus site for EGR-1, a zinc finger DNA-binding protein, is located in the POpromoter and is sufficient to direct transcription in C6 cells in response to stimulation with phorbol esters (Pasumarthi et al., 1997). In the fgf-3 gene, three promoters have been mapped in the mouse and two in the Zebrafish. One of these promoters is conserved across species, both in sequence and location (Kiefer et al., 1996). As in the fgf-2 gene, the fgf-3 promoters contain both positive and negative regulatory elements (Murakami et al., 1993). The mouse fgf-4 promoter does have TATA, CCAAT, and Spl consensus sequences and a 3' enhancer region that contains SP1, OCT,and high-mobility group (HMG/fx) consensus binding sites. The CCAAT sequence is recognized by the NF-Y and NF-YB transcription factors (Bryans et al., 1995; Lamb et al., 1996). The regulatory proteins SP1 and SP3 were shown to bind the Spl DNA motif; the human enhancer lacks the Spl region and is weaker than its mouse equivalent

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS

51

(Lamb et al., 1996). The OCT and the HMG/fx sites have been shown to be essential for fgf-4 expression in undifferentiated embryonic carcinoma cells (Rizzino and Rosfjord, 1994; Yuan et al., 1995). The DNA-binding proteins OCT-3 (recognizes the o m 2 element) and SOX-2 (binds to HMG/ fx region) act as transcriptional activators of the fgf-4 gene, but only when both are present (Yuan et al., 1995). The DNA binding of OCT and SP1related proteins is influenced by the redox state of these proteins (Lickteig et al., 1996). The fgf--15 promoter also contains a TATA box and consensus HOX and PBXl binding sites and it is a direct target of an E2A-PBX1 fusion protein (McWhirter et aL, 1997). The activity of each fgfgene is cell type specific and developmentally regulated; some of the expression patterns are described later. A number of proteins were shown to regulate the expression of FGFs, though their mechanisms of action are mostly unknown and they may not all be at the level of transcription. The expression of fgf-2 is stimulated by glucocorticoids, CAMP,forskolin (via the stimulation of adenylate cyclase), and phorbol esters and is inhibited by p53 (Halaban, 1996; Meisinger et al., 1996; Riva et al., 1996). Stimulation by phorbol esters is known to be mediated through the Po and Pi promoters and involves the erg-l motif (Pasumarthi et al., 1997). The activity of the fgf-3 promoter is modulated by CAMPand retinoic acid (Grinberg et al., 1991; Murakami et al., 1993). The repression of fgf-4expression in embryonic carcinoma and embryonic stem cells upon retinoic acid-induced differentiation involves a change in the binding of nuclear factors to the OCT and HMG regions (Lamb et aL, 1996). The fgf4 andfgf-6 mRNA expression is absent in mice that carry a targeted deletion in the myf-5 gene, suggesting that this myogenic factor is within the pathway that regulates the fgf-4 and fgf-6 genes (Grass et al., 1996). The fgf-7 is interleukin inducible and fgf-7mRNA levels are also influencedby estradiol and progesterone in the female reproductive tract (Rubin et al., 1995). The fgf-7 promoter is steroid inducible (Fasciana et al., 1996). The expression of f g f - l and -8 is also steroid dependent (Okamoto et aL, 1996; Payson et al., 1996). In addition, f g f l mRNA is induced by WNT-1 in the developing brain (Lee et al., 1997). Cross-regulation between growth factors is described in Section III,D. The recent explosion in the identification and isolation of fgf genes has led to a better understanding of their evolutionary history and expression patterns, and it opens the way for studies on the mechanism of their regulation, a field that has lagged behind cloning and descriptive developmental work.

6 . FGF Transcripts Not only are there several fgfgenes but there is also diversity in fgfmRNA structure. Diversity can arise from alternative promoter usage (as in fgf-I),

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GYORGYI SZEBENYI AND JOHN F. FALLON

alternative splicing (in fgf-1, -2, -3, -8,and -12), and the use of alternative polyadenylation sites (in fgf-2,-2, -3, and -5) (Blunt et aL, 1997; Crossley and Martin, 1995; Gemel et al., 1996; Hartung et al., 1997; Hattori et al., 1996;Basilico and Moscatelli, 1992). The potential mRNA variants present in different species may vary. For example, there are only four predicted fgf-8 mRNAs in humans but at least eight in mice (Gemel et al., 1996). In addition, different cell types can control the number and types of fgfmRNA variants they express. The variants may encode proteins with different biological activities, as is the case forfgf-8 (Blunt et al., 1997). On the other hand, the four fgf-3 transcripts all contain the same open reading frame (Mansour, 1994). The four fgf-1 mRNAs are the result of tissue-specific alternative promoter usage: The 1B mRNA is transcribed in the brain, whereas 1C is preferentially expressed in vascular smooth muscle (Chotani et al., 1995; Ray et al., 1997b). Eight types of fgf-2 transcripts were found in chick embryos: Three of these have the canonical sequence, three use an alternative exon 1 (alt-fgf-2), and two are antisense mRNAs (Borja et al., 1996; Li et al., 1996a; Savage and Fallon, 1995). In Xenopus, an excess of antisense efdmRNA depresses sense fgf mRNA levels, both by targeting fgftranscripts for degradation, as was initially proposed, and also by inhibiting the translation of efgf (Lombard0 and Slack, 1997). Antisense fgf-2 can be translated into protein products in both amphibians and rodents (Knee et al., 1997; Li et al., 1996b). Finally, the rat fgf-5 mRNA has a truncated variant that lacks sequences encoded by exon 2 (Hattori et al., 1996). Therefore, when evaluating the effects of FGFs in a particular system, one should consider the presence of various FGF isoforms and the possible functional consequences of this variety. C. Translation, Structure, and Trafficking of FGFs

The use of alternative 5' CUG translation initiation sites in fgf-2 (three CUGs) and fgf-3 (one CUG) yields high-molecular-weight ( H M W ) isoforms of FGF proteins in addition to the lower-molecular-weight (LMW) form that is initiated at the canonical AUG codon (Fig. 1). The HMW forms of FGF-2 (20-22.5 kDa) contain nuclear localization sequences, whereas the LMW (18 kDa) form lacks this signal and is predominantly cytoplasmic (Table 11). The HMW and LMW isoforms of FGF-2 associate with distinct sets of proteins and have a different range of biological activities (Bikfalvi et al., 1995; Patry et al., 1997). The translation of the different isoforms of the FGF-2 protein can be regulated separately, based on the differential tissue expression of the isoforms in fgf-2 overexpressing transgenic mice (Coffin et al., 1995). HMW forms of FGF-2 are not found in every species; in fish, for example, there is a translation stop codon immediately 5' of the

53

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS TABLE II Nuclear Localizationor Signal Sequences in FGFs

FGF type FGF-1 FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF-10 FGF-11 FGF-12 FGF-13 FGF-14 FGF-15 EGL-17 BNL

Nuclear localization sequence

+ + +

Putative signal sequence

+ (nonconsensus) + + + + +

+ + + +

+

AUG (Hata et al., 1997). Several cis-regulatory sequences have been found that contribute to the translational control of fgf-2 mRNAs (Prats et al., 1992). In the 5’ untranslated region of FGF-2, there are two unusual folding regions that form a Y-stem-loop, which is characteristic of RNAs with internal ribosome entry sites; one of these regions is 5‘ of the first CUG and the other one is 5’ of the initiator AUG (Le and Maize], 1997). The use of internal ribosome entry sites may allow for the translation of fgf-2 mRNA specifically, independent of the low-abundance cap-binding initiation factor, iIF-4E (Vagner et aL, 1995). The translation of thefgf-5 mRNA is negatively regulated by a short 5’ sequence that contains an open reading frame (Basilico and Moscatelli, 1992). FGF-3 to -8 and -10 have consensus signal sequences and are targeted for secretion through the endoplasmic reticulum (ER)-Golgi pathway (Table 11) (Emoto et al., 1997; Fernig and Gallagher, 1994; Yamasaki et al., 1996). The efficiency of FGF secretion can be cell-type dependent; FGF3, which has an atypical signal peptide, is secreted efficiently from transfected NIH 3T3 cells, but it is retained in the Golgi of COS cells (Kiefer et al., 1993).FGF-1, -2, -9, -11, -12, -13, and -14 do not have consensus secretory signal sequences but can be secreted from cells (Table 11). FGF-1 and the 18-kDa LMW isoform of FGF-2 were shown to be released by a novel,

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GYORGYI SZEBENYI AND JOHN F. FALLON

energy-dependent secretory pathway that is not blocked by brefelden A, methylamine, or verapamil and thus is independent of the ER-Golgiexocytosis pathway (Florkiewicz et al., 1995; Friesel and Maciag, 1995). FGF-1 release is dependent on a cytosolic retention domain in a region of its carboxyl terminal that had previously been shown to associate with phosphatidyl serine (Shi et al., 1997; Tarantini et al., 1995). FGF-9 has an internal hydrophobic domain that may allow for insertion into the E R membrane (Song and Slack, 1996). While FGF isoforms containing a signal peptide (Table 11) are released constitutively, the secretion of isotypes that lack a leader sequence is from regulated intracellular pools; release can be triggered by heat shock, injuries, and normal mechanical stress such as distortions of the plasma membrane associated with cell movement (Cheng et al., 1997). At least in pathological conditions, oxidized low-density lipoprotein (LDL) can also induce FGF-1 release from fibroblasts and vascular smooth muscle by transiently altering membrane permeability (Ananyeva et al., 1997). The availability of secreted FGFs for receptor binding and biological activitiesmay be further regulated by components of the extracellular matrix, mostly basement membrane heparan sulfate proteoglycans, that bind, retain, and modulate the activities of FGFs (see Section II1,B) (Fernig and Gallagher, 1994). The release of matrix-stored FGFs can be facilitated by thrombin and plasmin (Gospodarowicz et al., 1978). Extracellular FGFs can be endocytosed and transported within cells. Tyrosine Kinase FGF receptor-4 (FGFR4)-mediated uptake of FGFs requires part of the cytoplasmic portion of FGFR4 but is independent of kinase activity (Munoz et al., 1997). In contrast, mutations in the autophosphorylation site of FGFRl reduce ligand internalization, suggesting that different FGFRs internalize ligands by distinct mechanisms (Munoz et al., 1997;Sorokin et al., 1994). Retrograde axonal transport of FGF-2 is blocked by colchicine, suggesting that it is dependent on fast axonal transport involving microtubules (Blottner et al., 1997). The FGF-2 endocytic pathway involves caveolae, tubulovesicular early endosomes, multivesicular late endosomes, and lysosomes (Gleizes et al., 1996). Several FGFs (Table 11) contain functional nuclear localization consensus sequences (Basilico and Moscatelli, 1992; Smallwood et al., 1996;Yamasaki et aL, 1996). FGF-3 has a nucleolar targeting signal in the COOH terminal of the peptide, and it can accumulate in the nucleolus (Kiefer et al., 1993). Both the HMW and LMW isoforms of FGFs have been found in the nucleus of various cells, even though the LMW form is predominantly in the cytoplasm. Besides being targeted into the nucleus of cells that endogenously produce FGFs, exogenously applied FGFs can also move into the nuclei of nonexpressing cells. However, endogenous expression is required for some of the biological activities of FGF-2. For example, the proliferation of glioma cells is enhanced only if FGF-2 is transfected into these cells but

FGFS AS MULTIFUNCTIONAL SIGNALING FACTORS

55

not in response to bath-applied FGF-2 (Joy et al., 1997). The subcellular localization of FGF-2 varies during the cell cycle and may be regulated by cell-cell contact (Joy et al., 1997); upon mitotic activation, FGFs can translocate into the nucleus in the GI phase (Friesel and Maciag, (1995). Deletion of the nuclear localization sequence from FGF-1 yields a mitogenically inactive molecule, emphasizing the functional significance of nuclear FGFs (Lin et aL, 1996). FGFRs and heparan sulfate proteoglycans (HSPGs) may normally be required for nuclear transport (see Sections II1,A and II1,B) (Kilkenny and Hill, 1996;Maher, 1996;Prudovsky et al., 1996;Wiqdlocha et al., 1995). Many current studies are attempting to gain a better understanding of the biological role of nuclear FGFs. Posttranslational modifications of FGFs include glycosylation,phosphorylation, and ADP-ribosylation (Fig. 1). FGF-3 to -9 are glycosylated, even though FGF-9 lacks a signal sequence, and it is unknown how it is targeted to the Golgi (Song and Slack, 1996). Some FGFs heterologously expressed in bacteria, and thus lacking glycosylation, are biologically active, putting into question the functional significance of this modification. Glycosylation may, however, have a role in modulating interactions between proteases and FGFs since mutating the glycosylation site in FGF-4 results in the cleavage of FGF-4 prior to secretion, yielding peptides that are biologically more active than the full-length protein (Bellosta et aL, 1993). In FGF-2, the state of phosphorylation of threonine 121 [probably by protein Kinase A(PKA)] (Fig. 1) alters binding affinity for FGFRs (Baird, 1994). ADP ribosylation requires an arginine-specific glycosylphosphatidylinositollinked ADP ribosyltransferase and is inhibited by heparin (Jones and Baird, 1997).Finally, protein processing can continue after FGF release from cells. FGF-1, for example, can be released as a biologically inactive homodimer that requires activation in the extracellular matrix before it is able to bind heparin (Jackson et al., 1995). Structure-function studies, using FGF-1, -2, -4, and -7, have identified amino acids in FGFs that are required for receptor binding and biological activities (Fig. 1) (Ray et al., 1997b; Wong et al., 1995; earlier work is reviewed in Miller and Rizzino, 1994). A 10-amino acid sequence within the first putative receptor binding site is sufficient to elicit DNA synthesis in neural progenitor cells (Ray et al., 1997a). Crystallographic data confirmed that receptor binding sites are complex and consist of several noncontiguous residues (Springer et aL, 1994). Receptor-ligand interactions are further described in Section 111. The two conserved cysteines (Fig. 1)contribute to maintain the heat and acid stability of FGFs; however, if these cysteines are forced to form an intramolecular disulfide bond, FGFs lose their activities, perhaps due to changes in the conformation of the peptide (Basilico and Moscatelli, 1992). There is a serine-rich region in FGF-5 and FGF-10, but its significance is unknown (Yamasaki et al., 1996). There are

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GYORGYI SZEBENYI AND JOHN F. FALLON

12 amino acids conserved in all FGFs, located at sites thought to be important in maintaining the 3-D structure of the proteins and to be part of the binding sites for sugars (Coulier et al., 1997). In summary, in addition to the regulated expression of several distinct fg-f genes, mRNA and protein processing generate many FGF peptide variants from individual fgf genes. All FGFs can be released, but some forms also accumulate in the nucleus or cytoplasm of both producing and target cells. Secreted FGFs can be stored in the extracellular matrix, and their distribution may be controlled by proteases. There is still much to be learned about the regulation of expression, processing, intracellular trafficking, release, and activation of fgf gene products.

111. FGF-Binding Proteins and Signaling Pathways FGFs bind to at least three distinct types of receptors: FGFR, HSPG, and a cysteine-rich FGF receptor (CFR) (Table 111).

A. FGFRs

L Fgfr Genes and Regulation of Receptor Expression As their ligands, fgfrs also form a gene family. The fgf. genes probably evolved from a single ancestral gene that expanded in a single phase to the four forms present in all contemporary vertebrates, including amphibians (Coulier et al., 1997; Shiozaki et al., 1995). In Table IV we list alternative names still used for FGFRs and in Section III,A,3 describe the current nomenclature used for the splice variants. The aa sequence identity between the four receptors is 60-95%; the most highly conserved regions are within domains active in signaling. As expected, based on the presence of FGFs in invertebrates, f& were found in fruit flies, worms, and sea urchins, in addition to vertebrates (Table 111) (Beiman et al., 1996 DeVore et al., 1995; Gisselbrecht et aL, 1996; McCoon et al., 1996; Reichman-Fried and Shilo, 1995). While the predicted protein sequences of invertebrate FGFRs contain the 72 aa that are conserved in all fgfrs, their overall sequence identity with the vertebrate genes is only about 40%. Thus, the invertebrate fgfi genes are not considered orthologs of any of the vertebrate genes, but form a group of their own (Coulier et al., 1997). The organization of fgfr is conserved (Fig. 2). Currently, the genomic structure of fe.3 is the most completely characterized among the fgfr genes. It is contained within 16 kb and has 19 exons with exon sizes between 54

TABLE 111 Nomenclatureof Genes Encoding FGF-Binding Proteins

FGFbinding proteins

Types of FGF-binding proteins

Alternative names

Genes cloned from

fgfr FGFRl FGFR2 FGFR3 FGFR4 C. elegans FGFR Drosophila FGFRa Drosophila FGFRb Sea urchin FGFR

Pg, fms, cek-1 bek, K-sam, kgfr, cek-3 cek-2 frek egl-15 dfrl, breathless (btl) dfrl/dfgf-R2, heartless spfgr

Human, newt, Human, newt, Human, newt, Human, newt, C. elegans Drosophila Drosophila Sea urchin

rat, mouse, Xenopus, chick, bovine, fish rat, fish, Xenopus, chick, mouse chick, mouse, fish Xenopus, quail, fish, monkey

Syndecans-1, -2, -3, -4

2 = Fibroglycan 3 = N-syndecan 4 = Ryodocan, amphiglycan 2 = Cerbroglycan 3 = OCI-5 4 = K-glypican dDLY (Drosophila gly)

Drosophila, mouse, chick, Xenopus, human, rat

hspg

Glypicans-1, -2, -3, -4, -5

Perlecans Betaglycans cfr

MG-160, E-selectin ligand (ESL-1)

Rat, human, mouse, chick

Mouse, human, rat, C. elegans Rat, human, chick, pig Chick, mouse, human, rat, bovine

TABLE IV Specificity of FGFR-FGF Interactions

FGF type FGFR Variant FGFRl B varianta C variant FGFR2 B variant C variant FGFR3 B variant C variant FGFR4 C only

FGF-1

FGF-2

FGF-3

FGF-4

+++ +++ +++ +++

++ +++

+ -

+ +++ + +++

-

++

++ -

FGF-5

FGF-6

FGF-7

FGF-8

FGF-9

-

-

-

-

-

++

++

-

-

+

-

-

-

++

+++

-

+

+

+++ ++ +++ +++

+++ +++

-

-

-

-

+++

-

+++

+

-

+++

+++

-

+++

-

+++

-

-

-

++ +++

Note. Mitogenic activities of FGF paralogs were measured in BaF3 cells expressing various FGFR variants. FGF-1 showed a maximal response in these assay and the activities of other FGFs were expressed as a percentage of FGF-1 activity: -, 4 0 % ; +, 10-40%; + +, 40-75%; and + + +, 75-100% (based on Ornitz et aL, 1996). See Fig. 2 for the location of alternatively spliced exons in the FGFR gene in relation to protein structure.

59

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS

GENE OURUGUURE exon X 1

m

m

m

m

(promoter)

2

signal peptide

I

1\91

3

acidic box IBI CAM-bindin domain heparin-biding region E

4

5 and 6

ligand binding domain

7 8 (B) or 9 (C)

-1

(confers ligand specificity)

10

transmembrane region jwtamembrane region

11-14

14-18

19 COOH FIG. 2 The structure of a generic FGFR gene and protein. Note that the carboxyl half of the Ig-3 loop is encoded alternatively by exons 8 or 9, yielding the B or C splice variants (present in FGFR1-3 but not in FGFR4) that differ in their ligand-binding properties. The major structural features of FGFRs, an acidic box, CAM-binding domain, heparin-binding region, Ig loops, transmembrane, juxtamembrane, kinase, and kinase insert regions, are indicated by different filling patterns (indicated in the boxes on the right). References and a description of the functional significanceof each of the domains are provided in Section II1,A.

and 270 nt, except for exon 19, which is 1644 nt long and contains a single consensuspolyadenylation signal and untranslated sequences (Perez-Castro et al., 1995). Similarly,fgfrl and fgfr4 have at least 17 exons and the Caenor-

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GYORGYI SZEBENYI AND JOHN F. FALLON

habditis elegansfgfr 18coding exons (in addition to 3 3’ untranslated exons). The positions of the exonlintron boundaries are similar in each of the fgfr genes, with the splice sites conforming to the gt/ag rule (DeVore et aZ., 1995; Shiozaki et aZ., 1995;fgfil structure is reviewed in Givol and Yayon, 1992). Little is known about the mechanisms that regulate the expression of fgfrs, even though changes in the distribution of fgfi mRNAs during both developmental and physiological changes are well documented (see Section IV). Consensus transcription factor binding sites were identified in the 5’ flanking sequences of the fgfr3 and the Drosophila fgfra genes. While the 5’ flanking region of the fgfr3 gene has no TATA or CCAAT sequences, it is GC rich and has spl, ap2, krox24, and zeste consensus sites. Similarly, the Drosophila f&u gene has zeste sites in addition to fushitarazu, evenskipped, engrailed, abdominal B, paired, zerknullt, and myoD consensus sites; these sites have not been evaluated functionally (Perez-Castro et al., 1995). Also, there are several binding sites for a POU-containing transcription factor, Drifter, infgfra. Drifter regulates fgfra mRNA levels but is not required for the transcriptional activation of this gene (Anderson et al., 1996). Another Drosophila transcription factor, CEBP, binds directly to multiple sites in the 5’ flanking region of the fgfra gene (Murphy et al., 1995). The fgfrl 5’ flanking region lacks known consensus sites but an unidentified 100-kDa protein binds to a 20-nt sequence within its functional promoter (Saito et al., 1994). Similar to other fgf and fgfr genes, the 5‘ region of fgfr-2 lacks TATA or CCAAT elements but is GC rich. In general, CpG islands are found in the promoters of several growth factor and growth factor receptor genes and are targets for CpG methylases. Two transcripts were mapped in fgfru, each initiated at a distinct transcription start point (It0 et al., 1994). The overproduction of the 22.5-kDa isoform of FGF-2 in the rodent AR4-2.l pancreatic acinar cells leads to an increase in f g f l mRNA levels by increasing the half-life of this receptor mRNA, indicating post transcriptional regulation of this message (Estival et aL, 1996). Steroids, which regulate the production of some FGFs (see Section II,A), can also modulate fgfr mRNA levels (Ruohola et aL, 1995). Interestingly, some of the consensus binding sites for transcriptional activator proteins in fgfrs and in their ligands are the same, providing a potential mechanism for their coordinated regulation. In addition to the regulation of transcript levels, the expression of FGFRs is translationally regulated. Thefgpl mRNA contains a translational inhibitory element in its 3‘ untranslated region that interacts with regulatory proteins (Robbie et aZ., 1995). The ligand-dependent downregulation of FGFR2 also involves posttranscriptional mechanisms (Ali et al., 1995). Treatment of various cell lines with phorbol esters, activators of protein kinase C (PKC), results in a decrease in cell surface FGFRs due to an

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS

61

increase in receptor internalization (Asakai et al., 1995; Liuzzo and Moscatelli, 1996). In summary, there are four fgfr genes in vertebrates whose expression and activities are regulated at the level of transcription, translation, posttranslational modifications, and targeting to the proper membranous compartments. In comparison to the mass of data on the expression patterns of FGFs and FGFRs, our understanding of the mechanisms that bring about their unique expression patterns is cursory.

2. Conserved Structural Features of FGFRs FGFRs share a common structural plan (Fig. 2). They are single transmembrane proteins. Their extracellular domain consists of cysteine-flanked immunoglobulin-likemotifs (Ig loop), an acidic box, a heparin-binding region, and a cell adhesion molecule (CAM) homology domain. In most FGFRs there are two or three Ig loops, but the Zebrafish FGFR4 has four and the Drosophila FGFR2 has five Ig loops. The intracellular region of FGFRs includes a juxtamembrane region, two conserved kinase domains, a kinase insert region, and a carboxy tail that has several potential autophosphorylation sites that interact with intracellular substrates (Doherty and Walsh, 1996;Friesel and Maciag, 1995;Mohammadi et al., 1996). The FGF binding site is contained within 139 aa that includes parts of Ig-2 and -3 loops (Wang et al., 1995). Ligand selectivity is determined by the Ig-3 loop (Fig. 2 and Table IV) (Ornitz et al., 1996). In addition to FGFs, there are other structurally unrelated small secreted proteins that bind to the extracellular region of FGFRs (Kinoshita etal., 1995).For a discussion of receptor-ligand interactions see Section III,B,3. The Ig-1 loop is the least conserved region in FGFRs and is not required for ligand binding; however, it may regulate binding affinity quantitatively since its removal results in a receptor with an increased binding affinity toward FGFs (Shi et al., 1993). The acidic box binds divalent cations, including calcium, copper, manganese, and magnesium. These ions are thought to support an optimal conformation of FGFRs that is required for liigh-affinity interactions between FGFRs and HSPGs and consequently promotes receptor-ligand binding (Kan et aZ., 1996; Patstone and Maher, 1996). The role of HSPGs in FGF signaling is further discussed in Section II1,B. The CAM homology region was shown to be a binding site for L1, NCAM, and N-cadherin. Each of these transmembrane cell-adhesion molecules has a homophilic CAM site that mediates their interactions with FGFRs (Doherty et al., 1996). The juxtamembrane region of FGFRl contains a binding site for PKC; FGFR signaling and receptor numbers on the cell surface are modulated by phosphorylation in this region (Asakai et al., 1995;Gillespie et al., 1995). The FGFR protein is also glycosylated: Nascent

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GYORGYI SZEBENYI AND JOHN F. FALLON

FGFRs are -130 kDa, yet they migrate in protein gels at apparent weights of -150 kDa due to posttranslational modifications. The kinase domains and several of the phosphorylation sites in FGFRs are required for FGFinitiated signal transduction (Fig. 3 and Section 111,D). Besides their localization to the plasma membrane, FGFRs have also been detected in the nuclear envelope and in the nuclear mark of GI mitotic cells (Fig. 3) (Kilkenny and Hill, 1996; Stachowiak et al., 1997b). Only the 3-loop, not the 2-l00p, form of FGFRl appeared in the nucleus,

FIG. 3 A schematic diagram of FGF signaling pathways. For simplicity, only those proteins are indicated that are discussed in the text; many others were omitted. This figure accompanies and summarizes the data discussed in Section II1,D.

FGFs AS MULTIFUNCTIONAL SIGNALING FACTORS

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suggesting that the first loop may also be required for the nuclear targeting of this receptor (Prudovsky et aL, 1996). A splice variant of FGFR3, which contains the kinase domains but lacks the transmembrane region, has also been seen in the nucleus of breast epithelial cells by immunocytochemistry (Johnston et al., 1995). In addition, some forms of FGFRs can be secreted. The enzymatic hydrolysis of the extracellular region of FGFRl by 72-kDa gelatinase A (matrix metalloproteinase type 2) was observed. Released receptors may retain their ligand-binding activities (Levi et aL, 1996).

3. Receptor Variants and Ligand-Binding Specificity of FGFRs Alternative mRNA splicing of fgfn generates receptor variants, some of which have distinct ligand-binding and signaling properties (Friesel and Maciag, 1995; Miller and Rizzino, 1994; Ornitz et aL, 1996) (Fig. 2). The carboxy-terminal half of the Ig-3 loop, in all but the fgfr-4gene, is encoded by three alternatively spliced exons designated as A-C. Receptors containing exon A are secreted and these secreted forms of FGFRs may interfere with FGF signal transduction initiated at the plasma membrane. The B and C splice variants of each receptor isoform differ in their ligand binding (Fig. 2 and Table IV) (Ornitz et al., 1996). For example, FGFR2B binds specifically FGF-7, whereas FGFR2C binds preferentially FGF-1. Note, however, that FGFR3B binds to and is activated by FGF-9 and FGF-1 but not by FGF-7 (Santos-Ocampo et aL, 1996). Some of the regulatory regions in the fgfr2 gene that control splicing have been identified. Besides the three Ig-loop variants, each FGFR type also has isoforms that lack the Ig-1 loop (two-loop forms); these have altered ligand-bindingaffinities and, as discussed previously may acumulate in different subcellular compartments but do not affect the specificity of FGF-FGFR interactions (Shi et. al., 1993). Also, there are variants within the juxtamembrane region which contain a phosphorylation site for PKC, a negative regulator of FGFRl in Xenopus oocytes (Gillespie et al., 1995). Other potential regulators of FGFR activity are signaling-defectivevariants. An FGFR2 variant that lacks the phospholipase (PLC-7)-binding region and a kinase-deficient FGFRl have been cloned (see Section III,D)( Wang et al., 1996; Yan et al., 1993). B. HSPGs All FGFs tested bind heparin with a kDa of -1-2 nM. Heparin is commonly used in experiments as a substitute for HSPGs, the physiological substrates for FGFs and FGFRs. HSPGs are sulfated glycosaminoglycans (GAGS) covalently bound to a core protein. Heparin is composed of the same sugar

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residues as HSPGs but is more highly and uniformly sulfated. FGF binding to HSPGs depends on the length and on the state of sulfation of the HS chains, even though small synthetic nonsulfated oligosaccharides can activate the FGF signaling pathway in vitro (Ishihara et al., 1997; Ornitz et al., 1995). FGF-2 has been cocrystallized with heparin fragments and in complexes with synthetic oligosaccharides,and the atomic structure of these interactions has been resolved (Ornitz et al., 1995; Faham et al., 1996). The amino acids that participate in these interactions are not conserved in various FGFs, consistent with previous observations showing that changes in specific saccharide sequences and their sulfation in HSPGs alter their FGF binding (Rapraeger, 1993). Based on nuclear magnetic resonance structural analysis, an active form of FGF-2 can be a cis tetramer, held by a decasaccharide in a conformation in which the receptor-binding sites are exposed on the surfaces (Moy et al., 1997). Not only FGFs but also FGFRs have conserved heparin binding sites and bind HSPGs (Kan et al., 1996). While some models propose that HSPGs are cofactors that trigger conformational changes in FGFs and FGFRs allowing each to dimerize and act to facilitate and stabilize FGFFGFR binding, other models suggest that HSPGs are part of the active signaling complex (Fernig and Gallagher, 1994). Also, FGF-independent activation of signaling pathways through FGFR4, but not FGFRl, was observed using heparin (Gao and Goldfarb, 1995). Even though the heparin-binding property of FGFs has been recognized since their discovery and was utilized in their initial purification, it was demonstrated only recently that HSPGs modulate many activities of FGFs, not only in culture systems but also in vivo (Brickman and Gerhart, 1994; Itoh and Sokol, 1994; Walz et al., 1997). While numerous studies indicate a HSPG requirement for FGF signaling, some argue that HSPGs are not absolutely required for FGF-induced biological activities, but only enhance these by increasing the affinity of FGFs for FGFRs (Fannon and Nugent, 1996; Roghani et al., 1994). According to studies on FGF-induced gene expressions (see Section III,D,2), FGF-1 can activate early response genes in the absence of exogenous heparin, but for sustained cellular responses HSPGs are required (Donohue et al., 1997). In addition, HSPGs protect FGF-2 in the extracellular matrix from degradation: The half-life and distribution of extracellular FGF reserves could be controlled by HSPGs which could lead to sustained responses to FGFs (Donohue et al., 1997; Vlodavsky et al., 1996; Yeoman 1993). Also, HSPGs may participate in the internalization of FGFs (Quarto and Amalric, 1994). The HSPGs implicated in FGF action include syndecans 1-4 (cellassociated, transmembrane proteoglycans) (Dealy et al., 1997; Rapraeger, 1993; Steinfeld et al., 1996), glypican (anchored to the membrane by a glycosylphosphatidylinositol group) (Bonneh-Barkay et aL, 1997; Song et

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al., 1997),and perlecan (an extracellular, basal lamina proteoglyean) (Table III)(Aviezer et al., 1997). FGFs may require different HSPGs with distinct GAG chains for their activities. Glypican, for example, promotes the mitogenic activity of FGF-2 while it inhibits responses to FGF-7 (BonnehBarkay et al., 1997). Therefore, HSPGs may be among the factors that regulate specific FGF-FGFR interactions and determine which biological activity of an FGF prevails.

C. CFR CFR is a 150-160-kDa integral membrane sialoglycoprotein that lacks HS chains and binds FGFs. This single transmembrane protein has an extracellular region with 16 cysteine-rich repeats and a short intracellular tail. FGF binding to CFR and to FGFRs is mutually exclusive, suggesting that the binding sites in FGFs for these two receptors are overlapping, and CFR may regulate intracellular FGF levels (Zhou et al., 1997). Indeed, CFR overproduction reduces intracellular accumulation of FGF-1 and FGF-2 without affecting their rates of degradation (Zuber et al., 1997). Consistent with its proposed role in protein targeting, CFR was also isolated from the medial Golgi membrane (Gonatas et al., 1995). In addition, a CFR-related protein is also found on the surface of leukocytes and myeloid cells and can mediate adhesion to endothelial cells, functioning as a ligand for the endothelial cell-adhesion molecule, E-selectin (Steegmaier et al., 1997). Currently, only a few laboratories work on this interesting protein. Further insight into its function may provide a better understanding of the intracellular trafficking of FGFs.

D. FGF Signaling Pathways 1. Intracellular FGFR-Depencnt Biochemica Cascades FGFRs are structurally similar to other transmembrane receptor tyrosine kinases (Fig. 2), and some of the signaling pathways activated by them are shared among several receptors of this type (Fig. 3). Signal transduction is initiated by ligand binding that results in receptor dimerization and subsequent intermolecular autophosphorylation of tyrosines within the dimer. Seven phosphorylation sites have been mapped in FGFRl (Mohammadi et al., 1996). Mutations in cysteines in the extracellular region of FGFRs, if they result in the formation of intermolecular disulfide bonds, result in constitutively signaling receptors with pronounced biological effects, demonstrating that dimerization can activate these receptors (Neilson

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and Friesel, 1996). Many of the biological activities of FGFs are blocked by the overexpression of kinase-deficient receptors (dnFGFRs), indicating the functional importance of intermolecular phosphorylation in FGFR complexes (Itoh et al., 1996;Launay et al., 1996;McFarlane et al., 1996;Roghani et al., 1996; Saffell et aL, 1997). Note, however, that in some cells mitogenic responses may be normal while receptor transphosphorylation is much reduced (Krufka et al., 1996). Also, recall that FGFs have nuclear localization sequences, and there may be signaling pathways that are distinct from those activated at the cell surface. FGFRs have been implicated in the translocation of FGF-2 into the nucleus in GI in mitotic cells (Kilkenny and Hill, 1996). For this function of FGFRl, the Ig-1 loop is required (Prudovsky et al., 1996). The signaling pathways activated by FGFRl have been studied the most extensively. Other FGFR isoforms signal somewhat differently (Kanai et aI., 1997; Reichman-Fried and Shilo, 1995; Vainikka et al., 1996; Wang and Goldfarb, 1997). The elucidation of FGF signaling pathways has greatly accelerated after the identification, characterization, and experimental reproduction of FGFR mutations in human skeletal disorders. Many of these are in the ligand-binding domain of FGFRs and result in receptors that are constitutively active (Bellus et al., 1996; Lewanda et al., 1996; Malcolm and Reardon, 1996; Muenke and Schell, 1995; Neilson and Friesel, 1996; Webster and Donoghue, 1997; Wilkie et al., 1995).

a. FGFR Activation of the SHC/FRS2-RAF/MAPKKK-MAPKKMAPK Pathway Subsequent to receptor phosphorylation, SH-2 (src homology) domain-containing proteins and phosphotyrosine-binding domain (PTB) docking proteins bind to specific phosphotyrosines. For example PLC--y is a SH-2 domain protein and SHC and FRS2 (FGFR substrate 2) are PTB docking proteins. In FGFRl, Y766 was shown to be a PLC-7 binding site and Y653 and Y654 interact with SHC. Y730 is part of a consensus binding site for the regulatory p85 subunit of phosphatidyl inosito1 3 kinase (P13K),but no physical association between PI3K and FGFRs has been demonstrated (Kouhara et al., 1997; Mohammadi et al., 1996). SHC and FRS2 function as docking molecules for FGFRl; FRS2 is more specific to FGFRs, whereas SHC associates promiscuously with several receptor kinases. The membrane localization of at least FRS2 is dependent on myristylation (Kouhara et al., 1997). When phosphorylated, docking proteins bind directly to the GREi2-SOS complex which functions as an adapter to RAS. Some of the adapteddocking proteins for different FGFR isoforms are distinct: FGFR4 was shown to associate with a p85 serine kinase, and the activated FGFR3-GRB2-SOS complex contains either a novel 66-kDa protein or SHC (Kanai et al., 1997; Vainikka et al., 1996). Then, membrane-associated RAS recruits RAF-1, a serine-threonine MAP

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kinase kinase kinase (MAPKKK), an activator of MAPKK (MEK). In turn, MAPKK activates MAPK (note that ERK2 = p42 and ERKl = p44). MAPK can signal directly into the nucleus by phosphorylating transcription factors, such as JUN, FOS, and the ribosomal S6 kinase. FGFRl mediates p70 S6 kinase activation associated with FGF-2-induced proliferation of endothelial cells (Kanda et al., 1997). While this is a well-characterized pathway, it is becoming clear that there are several proteins acting in parallel with RAS, RAF, and MAPK. For example, several mammalian isoforms of MAPK (at least five groups) and parallel MAPK pathways have been found (Ferrell, 1996). In addition, besides nuclear substrates, MAPK can also phosphorylate cytoskeletal proteins, phospholipase, and protein kinases. The SHC/pp90 binding sites are required for FGF-induced mitogenic effects and induction of neuronal differentiation of PC12 cells (Mohammadi et al., 1996).The RAS/RAF/MAPK pathway has to be functional for FGFRmediated responses during gastrulation, migration of selected cell types in Drosophila, and activation of the urokinase plasminogen activator (uPA) gene (Besser et al., 1995; LaBonne and Whitman, 1997; Reichman-Fried and Shilo, 1995). ERK2 can be activated by FGF-2 in smooth muscle cells (Karim et al., 1997).FGF-Zstimulated endothelial cell motility is dependent on MAPK-activated translocation of phospholipase A2 (cPLA2) to membranes and subsequent arachidonate release (Sa et al., 1995). Each of the kinases in the MAPK pathway can be regulated by multiple signals; for example, PKC and PKA are both regulators of RAF-1 kinase. FGF-2 stimulation of MAPK and S6 kinase activity depends strongly on MAPK phosphatase levels (Campbell et al., 1995). The induction of uPA gene by FGF-2 is dependent on an AP-1 consensus sequence in the uPA gene (recognized by JUNFOS dimers) and blocking AP-1 activity inhibits FGF's effects in Xenopus embryos on mesoderm formation (Besser et al., 1995; Dong et al., 1996). While activation of MAPK and elevated jun B and cfos transcript levels are required for FGF activity, these are not sufficient for the repression of myogenic differentiation, the induction of neuronal differentiation, or cell scattering by FGF-2 (Kudla et al., 1995; Renaud et al., 1996;van Puijenbroek et al., 1997). Also, a mutant FGF-1 (L132Q) that retains the capacity for receptor binding, receptor phosphorylation, and induction of immediate early genes does not have mitogenic or oncogenic effects (Burgess et al., 1994), suggesting that these biochemical events are not sufficient in themselves to bring about a biological response.

b. FGFR Stimulation of PLC-y,PKC, IP3,and Cd' Release PLC-y hydrolyses PI to inositol1,4,5 triphosphate (IP3) and diacylglycerol (DAG). IP3 induces Ca2+ release from intracellular stores, whereas DAG is an activator of PKC, a serinekhreonine-specific kinase. FGFs have been shown

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to induce Ca2+release and hydrolysis of phosphoinositides (Merle et al., 1995;Shitaka et al., 1996).FGFR-promoted neurite extension involves PLCy, DAG lipase, arachidonic acid, possibly a G-protein, L-type Ca2+channels, and a Ca2+/ca1modulin-dependentkinase (CaM) and can be modulated by PKA and PKC (Doherty and Walsh, 1996; Hall et al., 1996; Shitaka et al., 1996; Williams et al., 1995). CaM phosphorylates microtubule-associated protein 2 (MAP2), tubulin, and other neuronal proteins and thus can modulate the cytoskeletal dynamics of neurons (Williams et al., 1995).In contrast, induction of DNA synthesis, activation of RAS, upregulation of uPA, and mesoderm induction are independent of PLC-y activation (Huang et al., 1995; Roghani et al., 1996; Gotoh and Nishida, 1996). RAF-1 can be phosphorylated by RAS-dependent or PLC-y-dependent mechanisms or both (Huang et al., 1995). Several signaling pathways downstream of FGFRs were revealed by genetic analysis in C. elegans. Some of these are RAS dependent, whereas others are not (DeVore et al., 1995). FGFR isoforms are likely to interact with different intracellular substrates, which may lead to different biological effects (Reichman-Fried and Shilo, 1995). In conjunction with the FGF and CAM homology binding domains and the kinase regions, other domains of FGFRs also play roles in signaling (Neilson and Friesel, 1996). The transmembrane region is essential for maintaining a conformation of FGFRs that regulates ligand-dependent autophosphorylation since FGFRs with specific mutations (G380R) in this region are constitutively active (Li et al., 1997; Webster and Donoghue, 1996). The juxtamembrane region contains phosphorylation consensus sequences which allow for PKC regulation of FGFR signaling (Gillespie et al., 1995). In chimeras in which the interkinase regions of FGFRl and FGFR4 were interchanged, mitogenic responses were altered, indicating the importance of this region in FGF signaling (Wang and Goldfarb, 1997). Tyrosines in the carboxy terminus (from aa 764 to the end) of FGFR2 can regulate negatively the transforming activity (colony formation in soft agar) of this receptor (Lorenzi et al., 1997). In summary, FGFRs share signaling pathways with other tyrosine kinases. The biological outcome of FGF stimulation depends on the exact quantities and combinations of ligand, receptor, HSPG types, and signaling intermediates present in a particular cell. There is accumulating evidence that effects on different cellular functions are mediated by distinct combinations of signaling pathways, some of which may involve nuclear forms of FGFs and FGFRs. 2. FGF Target Genes

FGF treatment results in changes in the steady-state levels of many different mRNAs. The mechanism of these FGF-induced alterations has only been

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studied for a few genes. While in many cases the effects are indirect, FGFs were shown to affect the transcription of several genes directly, independent of protein synthesis, presumably acting on nuclear proteins present in the cell before the FGF challenge (Besser et aZ., 1995). FGFs can be targeted into the nucleus and are found in the nuclear matrix, the nucleolus, and in association with chromatin (see Section I1,C) (Stachowiak et aL, 1997b). The observation that treatment of cell extracts with DNase I or RNase A does not result in FGF-2 release, but washes with 0.5 M NaCl do, suggests that FGF-2 does not directly bind to nucleic acids but is associated with nuclear proteins (Hawker and Granger, 1994). FGF-2 inhibits MyoDenhanced myogenin transcription by blocking the opening in chromatin structure that is required for the induction of this gene (Gerber et aL, 1997). FGFs regulate the transcription of a number of genes in a cell-free system. Furthermore, an FGF-inducible response element (Fire) was mapped in the syndecan 1 gene: It consists of five elements, bound by at least five nuclear proteins, three of which are regulated specifically by FGF-2 (Jaakkola et al., 1997). Even though FGFs can enhance transcription through the activation of transcription factors that bind to ap-1 sites, not all FGFresponsive DNA-protein complexes contain c-JUN or c-FOS (Boudreaux and Towler, 1996;Dong et aL, 1996).The effect of FGFs on gene expression is cell-type dependent. For example, while FGFs increase collagenase 1 mRNA levels in fibroblasts, the same FGFs inhibit the expression of this metalloprotease in keratinocytes (Pilcher et al., 1997). In addition to transcriptional activation of genes, FGFs can also maintain the expression of genes whose initial induction depends on other factors (Tada et aL, 1997). Among the FGF-regulated genes (including direct and indirect targets) are immediate early response genes (c-fos, c-jun, c-myc, egr-I, thrombospondin-1, fnk, and AP-2), delayed early response genes (proliferin, ornithine decarboxylase, glyceraldehyde 3-phosphate dehydrogenase, and an aldose reductase-related protein), homeobox genes (msx-I, evx-I, xnot, xcad, xhox, evel, hoxb9, hox-dll-dl3, and Zim-I),patterning genes ( X b r d nrl, en-2, cadl, and Shh), growth factors and their receptors (nerve growth factor, human chorionic gonadotropin, PDGF-A, IGF-11, FGFs, IGFbinding protein 6, and FGFR2), skeletal muscle regulatory factors (myoD1 and myogenin), matrix proteins (integrins; collagens I, 111, and IV; tenascinC; NCAM; and actin), proteases, protease activators and inhibitors (plasminogen activators, nexin-1, collagenases, and metalloproteinase inhibitors), and the genes for LDL receptor, fatty acid synthase, phosphofructose, an ER Ca2+ATPase, osteocalcin and proenkephalin (Barlow and FrancisWest, 1997; Boudreaux and Towler, 1996; Cohn et aL, 1997; Delbridge and Khachigian, 1997; Deng, 1997; Donohue et al., 1995, 1996; Gabbitas and Canalis, 1997; Iseki et al., 1997; Klein et aL, 1996; Kury et al., 1997; Mahler et al., 1996; Miyake et al., 1997; Pickering et al., 1997a,b; Pilcher et al., 1997;

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Rusnati et al., 1996; Shen et al., 1997; Taira et al., 1997). Some of the FGFinduced genes encode transcription factors (EGR-1, HOX proteins, the zinc finger proteins SLUG, STAT-l), whereas others, such as proteases and matrix proteins, effect cell behavior more directly and rapidly (Donohue et al., 1995; Su et al., 1997). While heparin is required for the sustained expression of delayed early genes, the expression of immediate early genes is independent of heparin (Donohue et al., 1997). In addition to transcriptional regulation, FGFs also influence mRNA stability and the translation and posttranslational modification of proteins. For example, FGF-2 increases the stability of the mRNA that encodes the retinoic acid-binding protein, CRABP I (Means and Gudas, 1996). Regulation at the protein level may be achieved by the phosphorylation of transcription factors in their DNA-binding domains upon FGF treatment. While FGFs may induce the phosphorylation of myogenic proteins through the activation of PKC and PKA, the functional significance of this modification has been questioned (Olwin et al., 1994). FGF-2 modulates biglycan at the level of transcription, translation, and sulfation (Kinsella et al., 1997). While this list of FGF-modulated proteins is certainly incomplete and focuses mostly on FGF-2 targets rather than on those of other FGF types, it serves to make the point that FGFs affect the expression and functioning of many diverse groups of proteins, as might be expected based on their broad range of actions. While not all these FGF regulated proteins are expressed at the same time and in the same cell type, many of them have been observed to be activated simultaneously or sequentially. 3. Interactions with Other Growth Factor Signaling Pathways

FGFs can work either synergistically or antagonistically with other growth factors. Some of the growth factors that often colocalize and act in cooperation with FGFs are members of the transforming growth factor+ (TGFp),insulin-likegrowth factor (IGF), and vertebrate homologs of Drosophilu wingless (WNT) families (Means and Gudas, 1996; Thesleff and Sahlberg, 1996). Interactions between these factors are at the level of transcriptional cross-regulation, the modulation of shared signaling pathways, and common target genes. Several members of the TGF-P family interact with FGFs to bring about complex biological responses such as angiogenesisand mesoderm induction, each of which require the activity of multiple genes and balanced signaling through several pathways operating in groups of heterogeneous cell communities. TGF-P potentiates the mitogenic effect of FGF-2 on endothelial cells but modulates the effects of FGF-2 on the synthesis of proteases both positively and negatively. As a result, TGF-/3 can inhibit FGF-induced formation of vascular tubes, even though normally both these factors are

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needed for proper proteolytic balance during angiogenesis (Wright and Huang, 1996). Similarly, FGF-2, in combination with TGF-61, can act as a positive myogenic signal (Stern et aZ., 1997). For mesoderm induction in Xenopus, both activin and FGF signaling pathways are required. Furthermore, the transcriptional induction of some activin-responsive genes depends on both a functional serine-threonine TGF-fi receptor and the FGFR-mediated activation of RAS (Gotoh and Nishida, 1996). Bone morphogenic proteins (BMPs), which also belong to the TGF-fi family, often colocalize with FGF-2, -4, and -8 during the embryonic development of various structures, and they target the same cell types. The interactions between BMPs and FGFs can be either synergistic (Lough et aL, 1996; Northrop et al., 1995) or antagosnistic (Neubuser et aL, 1997). IGF-I is required for FGFs’ effects on the survival of cultured Schwann cell precursors (Gavrilovic, 1995). Likewise, FGF-2 stimulation of cell proliferation in limb mesoderm explants is dependent on IGF-I activity. In this same system, however, FGF-2 maintains msx-l expression independent of IGF-I levels (Dealy and Kosher, 1996a). FGF-2 and IGF-I or IGF-I1 enhance synergistically the proliferation and differentiation of lens fiber cells (Liu et aL, 1996). Similarly, FGF-2 and IGF-I together have a more pronounced effect on DNA synthesis in inner ear epithelial cell cultures than either of these factors has on its own (Zheng et aL, 1997). In addition to BMPs and IGFs, members of the WNT family are also found frequently in the proximity of high FGF synthesis domains during development, and they act in cooperation with FGFs. In the Xenopus gastrula, for example, the presence of Xwnt-8 modulates the tissue type induced by FGF-2 (Isaacs, 1997). Also in Xenopus embryos, FGF-2, in combination with noggin (which binds to and neutralizes the activity of BMP-4) and WNT3a, induces posterior neuronal markers (Lamb and Harland, 1995; Sasai and De Robertis, 1997). WNT-1, -2, and -3 and FGF-3, -4, and -8 are often coactivated during the progression of malignant tumors and interact to promote mammary tumor formation (MacArthur et aL, 1995a; van Leeuwen and Nusse, 1995). In addition to pathways activated by IGFs, TGF-fis, and WNTs, other factors also alter the outcome of FGF signaling. For example, low concentrations of interleukin-1j3and FGF-2 act synergisticallyto enhance the proliferation of cultured vascular smooth muscle (Bourcier, 1995).Leukemia inhibitory factor (LIF) and FGF-2 cooperate during vasculogenesis and neural crest development to bring about more complete responses than either of these factors does on its own. LIF and FGF-2 must be present simultaneously to induce and maintain colony formation by embryonic endothelial cells in culture, whereas they act sequentially to promote a larger number of neuronal crest cells to differentiate as neuronal cells (Gendron et al., 1996; Murphy et aZ., 1994). In neural cells, the initiation of tyrosine hydroxylase

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expression requires the combined action of PKC activators and FGF-1 (Du and Iacovitti, 1997). The oncogenic transforming effect of FGF-2 on retinal pigment endothelial cells is enhanced by vascular endothelial growth factor (VEGF) due to the upregulation of FGFRs by VEGF (Guerrin et al., 1997). Based on these documented interactions between various growth factors, much information can be gained by screening cell types of interest for responses to a variety of factors and evaluating whether these act synergistically with FGFs.

IV. The BiologicalActivities of FGFs

FGFs regulate cell survival, apoptosis, proliferation, differentiation, matrix composition, chemotaxis, cell adhesion, migration, and growth of cell processes. Different cell types, or even the same cell, may display alternate, sometimes opposite responses to FGFs, depending on the state of differentiation, biochemical status, and the cellular, physical, and chemical environment of the cell. Besides the multiple effects of each FGF isotype, different FGFs and their splice variants may have distinct actions. The analysis of the expression patterns and activities of FGFs during embryonic development has provided further insight into their normal biological functions. In the vertebrate embryo, the activities of FGFs are required from the earliest stages of development through the detailed patterning of organs. At least one FGF paralog is expressed at some time during the development of many, if not all, of the organ systems. Specific FGFs and FGFRs are expressed in all four of the basic histological tissue types: They are found in various epithelia, loose connective tissue stroma, skeletal connective tissues, all types of muscle, and neurons and glia. The expression patterns of FGFs and FGFRs are dynamic during organ development and vary with the stage of tissue differentiation. As expected, based on their wide tissue distribution, FGF signaling contributes to the development of all the organ systems. During organ development, FGFs affect the survival, replication, differentiation, and migration of various cell types and modulate cellular and tissue contacts, especially epithelial-mesenchymal interactions. They are among the determinants for the anteroposterior arrangement of substructures within organs and also for the fine patterning of cell and tissue morphology. As a consequence of these various activities, FGFs are major modulators of organ morphogenesis. In the following sections we first provide a survey of the expression patterns and activities of FGFs in the embryo through neurulation, then focus on their effects on cellular functions in

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selected developing organs, choosing those in which the effects have been studied most extensively or conclusively.

A. FGFs and the Patterning of the Body 1. FGFs in the Oocyte, Blastula, and Gastrula and Their Roles in Mesoderm Formation and the Patterning of the Primary Body h i s FGFs are expressed from the earliest stages of embryonic development. The efgf (embryonic fgfl found in Xenopus and related to mammalian fgf4 and -6),fgf-2,andfgf-9 mRNAs are present as maternal transcripts before their expression becomes zygotic (Slack et al., 1996; Song and Slack, 1996; Isaacs, 1997). The fgf-4 mRNA is first detected at the four-cell stage in the mouse embryo, then in the inner cell mass (ICM) of the blastocyst. The fgf-4, but not fgf-3 or fgf-5, gene is required for early embryonic survival, based on results of targeted gene mutations (Feldman et al., 1995;Goldfarb, 1996). ICM cells cultured from the blastocysts of fqj-4-'- mouse embryos proliferate at much reduced rates compared to those from normal embryos (Feldman et al., 1995). In the mouse gastrula, fgf-2, -3, -4, -5, -8, and -9 are all expressed at the time of mesoderm induction. The fgf-2 and fgf-9 expression is initiated before gastrulation, whereas fgf-3 is activated in migrating mesodermal cells, fgf-4 in the anterior primitive streak, fgf-5 in the ectoderm, and fgf-8 in epiblast cells that will move through the primitive streak to form mesoderm and endoderm (Crossley and Martin, 1995;Yamaguchi and Rossant, 1995). In the chick prestreak embryos, several FGF-2 isoforms accumulate predominantly in the nucleus, then later, during primitive streak formation, in the cytoplasm and basal lamina of epiblast cells (Riese et al., 1995). Like some fgfs, some fgfr mRNAs are maternal transcripts. The f g f l mRNA is present in immature oocytes but is only translated after the initiation of meiotic maturation (Robbie et al., 1995). In the embryo, fgfrl, -2, and -4 mRNAs are in the primitive ectoderm of the blastocyst prior to gastrulation (Thisse et al., 1995). During gastrulation, f g f r l expression follows the anterolateral migration of the forming mesoderm and f g f 2 expression becomes limited to the ectoderm. Also, fgfr3 is expressed primarily in the ectoderm. The fgfr4 mRNA is present in both the ectoderm and the ventral mesoderm (Boucaut et aL, 1996). HSPGs are synthesized in the blastula as well, mostly in ectodermal cells (Brickman and Gerhart, 1994). Syndecan-1 is expressed at the time of axial mesoderm formation (Itoh and Sokol, 1994). The third FGF-binding protein, CFR, is not found in the

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gastrula (Fayein et aL, 1996), and it remains unknown whether it plays a role during early development. The influence of FGFs on mesoderm formation was first demonstrated in Xenopus (Isaacs, 1997). FGF-1-4 and eFGF or activin can replace the activity of the presumptive endoderm (vegetal pole) to induce animal cap elongation and the expression of mesodermal markers in vitro. In addition, FGF overexpression suppresses anterior development, and dnFGFR inhibits the formation of posterior and lateral mesoderm and disrupts the normal dorsoventral pattern (Griffin et aL, 1995; Slack et aL, 1996). Dorsal and ventral mesoderm are differentially affected by FGFs. It was suggested that this asymmetry is due to the ventral expression of BMP4 and the dorsal expression of WNT genes that work in synergy with FGFs (Gotoh and Nishida, 1996). The disruption of fgfrl results in severe growth retardation, and the misshapen embryos die at the gastrula stage. The results of FGF overexpression and inhibition of FGF signaling pathways in different species, including Xenopus, Zebrafih, and mouse, are strikingly similar. In each of these experimental systems, FGFs alter early morphogenetic movements and cell specification: Ectopic eFGF production leads to abnormal cell accumulation at improper locations and to the formation of axis-like extensions (Griffin et al., 1995) and fgfr-l- cells fail to migrate from the posterior primitive streak (Ciruna et al., 1997). A description of the morphogenetic movements that occur during gastrulation and their dependence on cell-matrix interactions, especially on properly assembled and oriented fibronectin fibrils and laminin, is provided in Boucaut et al. (1996). The role of FGFs in the regulation of cell shape and motility is further discussed in Section IV,B. It is noteworthy that some matrix molecules are not only regulated by FGFs but also may bind to and directly activate FGFRs. In addition, the fact that some mesodermal markers are expressed in f@l-’- cells indicates that FGFs regulate mesoderm patterning rather than its induction (Ciruna et al., 1997; Deng et al., 1997). Similarly, in Drosophila with defective FGFRb, abnormalities in the patterning of the mesoderm are the result of a failure in cell migration (Gisselbrecht et aL, 1996; Shishido et aL, 1997). Experiments in which dnFGFRs were expressed after the induction of the mesoderm had already occured further demonstrate that FGFR-activated signaling pathways are important in maintaining the expression of mesodermal markers (Kroll and Amaya, 1996). In newly emerging models, FGFs are presented as competence factors that are required for mesoderm induction by activin and for the continued expression of a number of mesodermal genes, replacing previous ideas of FGFs being among the mesoderminducing factors (Dyson and Gurdon, 1997; Isaacs, 1997). FGFs control the expression of a subset of genes that mark the mesoderm. Some of the mesodermal genes that are strongly FGF responsive are Xcad-

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3 (a member of the caudal gene family) and the early pan-mesodermal markers Xbra and tbx6 (the Xenopus and Zebrafish brachyury homologs, respectively) (Hug et al., 1997; Schulte-Merker and Smith, 1995). Another mesodermal gene, sna-1, on the other hand, is not affected by dnFGFRs. FGFs have biological effects in brachyury null mutants (ntl), indicating that FGFs use several alternative signaling pathways in the embryo rather than all their effects being funneled into a simple, linear, hierarchical gene cascade (see Section 111,DJ) (Griffin et aL, 1995). Similarly, FGFs affect only a subset of dorsal (gsc), posterior (eve-I and cad-I), and anterior markers (pax-2).The expression of the anterior gene, id-1, or several dorsal mesoderm markers (goosecoid, noggin, and LZM)is not altered by eFGF overexpression (Griffin et. al., 1995; Isaacs, 1997).The expression of the lateroventral markers Xwnt8 and Xsna is not dependent on FGF signaling either (Isaacs, 1997). FGFs also selectively induce the expression of homeobox genes: For example, FGF-2 treatment leads to an increase in the expression of hoxdl but not of hoxal (Kolm and Sive, 1995). The FGF signaling pathway known to be required for proper early embryonic development includes FGFRs (Yamaguchi and Rossant, 1995),HSPGs (Brickman and Gerhart, 1994; Itoh and Sokol, 1994), RAS, RAF (Isaacs, 1997; Slack et aL, 1996), MAPKK (MEK), MAPK (Gotoh and Nishida, 1996; LaBonne et aL, 1995; LaBonne and Whitman, 1997; Umbhauer et al., 1995), and AP-1-dependent transcriptional regulation (Dong et aL, 1996). The inactivation of any one of these molecules leads to similar developmental defects (posterior truncations), and their overexpression confers phenotypes similar to those seen with elevated FGF levels (suppression of anterior development and enlargment of posterior structures). Since each FGFR binds multiple ligands, including cell adhesion molecules (I. Mason, 1994), the inactivation of receptors and signaling intermediates may result in more drastic changes than the inhibition of expression of selected ligands. While the MAPK pathway is essential for gastrulation, PLC-.)I activation and Ca2+efflux are not necessary for FGFs' effects on mesoderm formation (Muslin et al., 1994). Mesoderm induction requires an interplay between several growth factors. For some of the effects of activins, WNTs, and retinoic acid, activated FGF signaling pathways are required. For example, dnFGFRs block activinmediated mesoderm induction and the activation of some mesodermspecific genes, such as Xbra, Xnot, MyoD, and mix-I. Some other activinregulated genes are not affected by dnFGFRs (Gotoh and Nishida, 1996). FGF and activin' signaling share RAS as an intermediate, whereas RAF and AP-1 activation are specific to FGF induction (Dong et al., 1996). The regional effects of FGFs on mesoderm induction also suggest that the appropriate localization of other factors besides FGFs is required for the establishment of the proper pattern along all three axes of the embryo.

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2. FGFs Affect Neural Induction and the Rostrocaudal Patterning of the Brain The first step in vertebrate neural development is initiated by a signal from the dorsal mesoderm that inhibits epidermal development. The fgf-3, -8, and -15 mRNAs are expressed in the neural plate in a non overlapping pattern (McWhirter et al., 1997). While FGFs on their own do not induce neuronal ectoderm in Xenopus animal caps, FGF signaling pathways are required for neuronal induction by primary neuronal inducers, such as noggin, dominant-negative activin receptor, and the endogenous mesoderm (Launay et al., 1996; Mayor et al., 1997). Thus, in neural induction, as in mesoderm induction, FGFs have a permissive role. In addition, FGFs are also part of a second, posteriorizing signal that is required for the anteroposterior specification of neuronal structures (Doniach, 1995; Mason, 1996; Sasai and De Robertis, 1997). The fgf-8 mRNA is expressed locally in signaling centers operational during early brain development, such as the anterior neural ridge, which patterns the anterolateral neural plate, and the isthmus region, which patterns the midbrain and cerebellum (Crossley et al., 1996a; Shimamura and Rubenstein, 1997). Ectopic FGF-8 or -4 transform chick forebrain to midbrain; the tissue closest to the source of FGFs is always caudal (posterior) (Crossley et al., 1996a).At the met-mesencephalic junction, fgf-8colocalizes with the transcript for protease nexin-1 and regulates the expression of this gene (Kury et al., 1997). In chimeric embryos that constitutively overexpress FGF-4, the development of anterior structures, most notably that of the eyes, is impaired (Abud et al., 1996b). Posteriorization by XBRA involves the activation of efgf and requires an intact FGF signaling pathway and thus is blocked in the presence of a dnFGFR (Taira et al., 1997). However, neural patterning is normal in transgenic frogs that express dnFGFRs (Kroll and Amaya, 1996). Some of the genes that are induced by FGFs in other systems are also activated in the nervous system in response to FGFs; these include en-2 and wnt-1 (Crossley et al., 1996a). WNT1, in turn, is a regulator of fgf-8 expression; en-1 activation in itself was shown to be sufficient to induce changes in anteroposterior polarity (Lee et al., 1997). Also, FGFs can induce the expression of posterior neuronal markers such as cash4, a vertebrate achaete-scute homolog found in the presumptive posterior nervous system, krox-20 in the brain, and hoxb-9 in the spinal cord, whereas it represses the anterior markers otx2 and xug-1 (Henrique et al., 1997; Lamb and Harland, 1995). The signaling pathways required for FGFs" effects on early development are currently being worked out. In the mouse, the targeted disruption of f g f 3 has no effect on neural induction, whereas there are abnormalitiesin the neural tubes of chimeras (spina bifida and duplications) that contain fgf1-'- cells (Deng et al., 1996, 1997). HSPGs modulate the

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responses of developing neurons to FGFs and syndecan-3 is found in the developing brain and neural tube with high expression in the floor plate (Gould et al., 1995). The unique roles of specific HSPGs in neuronal development need to be explored.

3. FGFs and the Patterning of the Limbs In addition to patterning along the primary body axis and brain, FGFs also contribute to the establishment of the secondary body axis. The fgf-2 and fgf-10 and fgfr1 and f g f 2 mRNAs are present in the limb field mesoderm prior to limb budding (Cohn and Tickle, 1996). Application of FGF-1, -2, -4, -8, or -10 to the flank induces the formation of extra limbs, indicating a role for FGFs in the initiation of limb outgrowth (Abud et al., 1996b;Cohn et al., 1995; Crossley and Martin, 1995; Crossley et al., 1996b; Mahmood et al., 1995; Ohuchi et al., 1997; Tanaka and Gann, 1995; Vogel et al., 1996). In the ectoderm of the emerging limbs,fgf2 and -8 are expressed in distinct spatial and temporal patterns: f g f 2 has the earliest and broadest expression pattern (Tickle and Eichele, 1994), whereas fgf-8 premarks the apical ectodermal ridge, a structure that is required for normal limb growth (Michaud et al., 1997). The expression of fgf-10 in the limb field mesoderm is required for the induction of fgf-8 in the ectoderm (Ohuchi, et al., 1997). The fgf-4 mRNA appears later and is restricted to the posterior (postaxial) region of the Limb apical ectodermal ridge that is close to the SHH-containing polarizer (Marigo et al., 1996). During the ridge-dependent stages of limb development, ectopic application of FGFs can prevent apoptotic cell death (Fallon et al., 1994) and truncations that otherwise follow the surgical removal of the ridge, indicating that FGFs can support the realization of the proximodistal (shoulder to fingers) axis of the limbs (Goldfarb, 1996; Mahmood et al., 1995). Ectopic expression of FGF-2 in anterior limb bud mesoderm results in the parallel duplications of skeletal elements along the proximodistal axis (Riley et al., 1993). While it was proposed that this was due to stimulation of mesodermal proliferation, further studies are needed to understand the mechanisms involved. There is good evidence that the expression offgf-4, shh, and wnt-7a is interdependent since loss or reduction of any one of these results in downregulation of the others and leads to defects in limb patterning along all three axes (Johnson and Tabin, 1997). Therefore, the biological activities of FGFs in the limb, as in other systems, depend on the balance between their expression and the presence of other signaling molecules, especially IGF-I, BMPs, WNTs, and SHH (see Section III,D,3). The expression of these factors is FGF dependent and each of them in turn modulates FGF expression levels (Akita et al., 1996; Dealy and Kosher, 1996b; Duprez et al., 1996; Ganan et al., 1996; Grieshammer et al., 1996; Logan et al., 1997; Noramly et al., 1996; Ros et

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al., 1996; Zeller and Duboule, 1997). Other FGF-regulated genes in the limb include various hox genes (hoxd-11, -d12, -d13, msx-1, and evx) (Johnson and Tabin, 1997). The signaling pathways mediating FGFs actions in the limb have not been characterized but the expression patterns of fgfrs correspond to distinct stages of differentiation of cartilage nodules, and changes in receptor activities result in dramatic alterations of the limb skeletal pattern (Deng et al., 1996; Szebenyi et al., 1995; Yamaguchi and Rossant, 1995). In addition to FGFRs, the distribution of syndecans also correlates with chondrogenic development in the limbs. Syndecan-3 has been shown to be regulated by FGFs in vitro. An anti-syndecan antibody blocks FGF-2-induced proliferation and outgrowth of cultured limb mesodermal cells (Dealy el al., 1997). FGFs continue to influence the development of the limbs until they attain their adult form and size by modulating bone, muscle, and nerve growth (Coffin et al., 1995; Deng et al., 1996; Itoh et al., 1996). In summary, FGFs are among the major regulators of the patterning of the developing embryo. Some cells in all tissue types and organs express and respond to FGFs. The exact responses depend on both the type and amount of FGF applied and on the nature of the responding cells and their environment. Currently, the effects of FGFs on a cell type cannot be predicted apriori, nor can they be explained by a single mechanism. Rather, the activities of FGFs during embryonic development appear to depend on FGF regulation of several fundamental life functions, such as the ability to survive, replicate, make attachments, move, and attain a characteristic form.

6. FGFs and Cellular Economy during Organ Development 1. FGFs Support Cell Survival during Organogenesis

Cells whose survival was shown to be dependent on FGF-1 or -2 include neurons (both central and peripheral), glia, various mesenchymal cells, lens epithelia, endothelial cells, vascular smooth muscle cells, and the neural crest-derived glomus cells of the carotid body (Campochiaro et al., 1996; Chow et al., 1995; Fox and Shanley, 1996; Grothe and Wewetzer, 1996; MaciaS et al., 1996; Mia0 et al., 1997; Nurse and Vollmer, 1997). In the nervous system, F G F J acts as a target-derived motor neuron trophic factor (Grothe and Wewetzer, 1996). In various developing organ systems, FGFs act as ectoderm-derived factors that maintain the viability of underlying mesenchymal cells (see Section IV,B,5). For example, the apoptosis of dental mesenchymal cells is prevented by FGF-4, which is thought to substitute for a survival factor that is normally supplied by the epithelium

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(Vaahtokari et al., 1996). FGF-4 and FGF-2 are found in the ectoderm at the tip of growing limb buds and can maintain the viability of distal limb and interdigital mesenchyme cells (Macias et al., 1996; Fallon et al., 1994). FGFs can support cell survival without affecting cell replication rate. Inhibition of endogenous FGF synthesis by antisense fgf-2 mRNA induces inappropriate entry into S phase, followed by apoptosis (Fox and Shanley, 1996). FGF-2 overexpression can rescue lens cells from apoptosis (Stolen et al., 1997). Functional FGFRs are required to mediate the effects of FGFs on cell survival: overexpression of a dnFGFR induces apoptosis in the lens epithelium and fiber cells (Chow et al., 1995; Robinson et al., 1995a), and activation of FGFRs by either FGF or N-cadherin blocks apoptosis of cultured ovarian surface epithelial and follicular granulosa cells (Trolice et al., 1997). The ability of FGFs to regulate cell survival may relate to their regulation of cell-matrix interactions. FGF-2 downregulation of integrin p4 synthesis correlates with its ability to prevent apoptosis in vascular endothelial cells (Miao et al., 1997). While FGFs affect survival on their own in many culture systems, normally several factors cooperate to keep cells alive; for example, FGF-2 and TGF-P3 show strong synergy in maintaining the viability of motorneurons (Gouin et al., 1996). 2. FGFs Modulate Cell Proliferation during Tissue Formation

FGF-1, -2, -7, and -9 were initially isolated based on their proliferative effects. FGF-2 induces DNA synthesis in many cell types (Gospodarowicz et al., 1978) but some FGFs, most strikingly FGF-7 and FGF-10, act tissuetype specifically (Emoto et al., 1997; Rubin et al., 1995). Besides varying responses depending on the particular cell type, FGFs' effects also depend on the state of development of the cell. For example, whereas FGF-1, -2, and -4, in combination with IGF-I, promote the survival of Schwann cell precursors without inducing a change in DNA synthesis, these factors stimulate the proliferation of mature Schwann cells (Gavrilovic, 1995).In addition to their better known roles as mitogens, FGFs can also regulate cell replication negatively: Infgf-5 null mutant mice (long hair and angora phenotype), the proliferation of hair follicles is extended, indicating that normally FGF5 inhibits the proliferation of matrix epithelial cells (Goldfarb, 1996). Similarily, low concentrations of FGF-1 and -2 are growth inhibitory for breast carcinoma cell lines that express several functional FGFRs (McLeskey et al., 1994). Several forms of achondroplasia are the result of the overactivity of FGFRs and in fgfr3-'- mice the skeleton is enlarged, leading to the interpretation that FGFs restrain, rather than promote, chondrocyte proliferation (Deng et al., 1996; Li et al., 1997). At the same time, FGFs promote the proliferation and inhibit the differentiation of cultured osteoblasts (Tang et al., 1996).

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FGFs stimulate DNA synthesis only if present in the Go to GI phase of the cell cycle (Ingber et al., 1995). Some of the intracellular FGF signaling pathways operational in cell replication have been investigated. FGF induction of DNA synthesis does not depend on PLC-.)Iactivation. While activation of DNA synthesis is observed in cells lacking FGFRs, both the nuclear translocation of FGFs and the activation of FGFRs are required for cell division to occur (Wigdlocha et al., 1996). FGF-2 and FGFRl can coordinately accumulate in the nucleus of proliferating glial cells (Stachowiak et al., 1997a). FGF-2-stimulated BrdU incorporation into cultured precardiac myocytes is reduced by an anti-FGFR antibody, further implicating FGFRs as mediators of FGF signaling for DNA synthesis (Sugi et al., 1995). The signaling pathways that mediate the proliferative effects of FGFs (FGFW SHC/pp90/FRS-GRB-2-SOS-RAS-RAF-MEK-MAPK-RSK-S6) are described in Section 111,DJ and illustrated in Fig. 3. Presumably FGFs, as other growth factors, alter the levels and activities of cell cycle regulators. The FGF-2 promoter, in turn, is a target for p53 (Halaban, 1996). Little information is available on the molecular interactions between FGFs and cell cycle regulatory proteins. Even though the observation that there is a correlation between cell shape, polarity, and adhesive properties is not recent, only a few investigators are devoted to understanding the relationship between cell geometry and the effects of growth factors (Gospodarowicz et al., 1978; Ingber et al., 1995). It was suggested that the FGF-promoted proliferation of neuroepithelial cells depends on FGF-enhanced cell spreading and is only realized on certain substrates (Kinoshita et al., 1993). Increasing the concentration of cell substrates that allow for more extensive cell spreading, while keeping FGF levels constant, results in a directly proportional stimulation of DNA synthesis; this response is primarily dependent on the actin cytoskeleton and nuclear skeleton, but microtubules can partially substitute in case the actin framework is disrupted (Ingber et al., 1995). As with other FGF acivities, FGF-induced proliferation can be enhanced by other factors, for example, by NGF in the neuroectoderm (Temple and Qian, 1995). 3. FGFs Regulate Differentiation Pathways

As the effects of FGFs on cell proliferation may be either positive or negative, their influence on cell differentation can also be either stimulatory or inhibitory. Even though mitogenic and differentiation effects are often thought of, and indeed often are, mutually exclusive, this is not always the case because some differentiated cell types retain their proliferative properties. One of the most extensively characterized systems for the negative effects of FGFs on cell differentiation is skeletal muscle. The expression of several FGFs cfgf-I, -2, -5, -6, -7, and -8 mRNAs) was demonstrated

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both in vitro and in vivo at various stages of muscle development (Hannon et aL, 1996; Olwin et al., 1994). FGFs can maintain the early proliferation of myocytes by delaying their terminal differentiation and fusion to form myotubes. Constitutive production of FGFRl in the myotome blocks muscle differentiation, and the production of a dnFGFR induces premature differentiation and prevents the migration of myotomal myoblasts into the limbs (Itoh et aL, 1996). Paradoxically, an anti-FGF-2 antibody blocks the myogenic signal from the neural tube, suggesting that FGFs’ effects are strongly dependent on the exact state of differentiation and environment of a cell (Stern et al., 1997). Also, the targeted inactivation of the fgf-6 gene impairs muscle regeneration, and infgf-6-’- mice myoD and myogenin expression are reduced, compared to fgf-6+’+ controls, following crushinduced injuries (Floss et al., 1997). The operational signaling pathways during muscle differentiation have been studied. Commitment to terminal differentiation occurs in GI and FGF has to be present in the cells at this phase to block it. If FGF-2 is removed for 2-3 h in GI, the differentation process is initiated (Olwin et aZ., 1994). FGFs regulate the transcription and posttranslational processing of myogenic regulatory factors (Gerber et al., 1997).The MAPK pathway is activated in response to FGF treatment of myoblasts; however, this is not sufficient for the repression of differentiation (Campbell et aZ., 1995; Kudla et d.,1995). As with muscle, FGF-1 inhibits osteoblast differentiation in vitro (see Section IV,B2), whereas it promotes bone development in vivo (Iseki et al., 1997; Tang et al., 1996). In contrast to the repressive effect of FGFs on some aspects of skeletal muscle differentiation, FGFs promote cardiac myogenesis, lens fiber differentiation, and the neuronal differentiation of cultured adrenal chromaffin cells (Bosco et al., 1997; Grothe and Meisinger, 1997;Robinson et al., 1995b; Sugi and Lough, 1995). FGF-2 induces the transdifferentiation of retinal pigment epithelium into neural retina (Sakaguchi et aL, 1997). FGF-2 antibodies inhibit the differentiation of neuronal retina without an effect on apoptosis or proliferation (Pittack et al., 1997). In cultures of hippocampal cells, a distinct subpopulation of neurons differentiates in response to FGF2 (Vicario-Abej6n et aL, 1995). In cultures of cortical progenitor cells, FGF2 can promote the differentiation of the oligodendrocyte lineage, whereas astrocyte differentiation depends on other cytokines. These observations demonstrate again that FGFs’ effects are cell-type specific (Bartlett et aL, 1995; Qian et al., 1997). The production of different FGFR variants and differences in the structure of HSPGs may contribute to some of these differences (Grothe and Wewetzer, 1996).The FGF-induced signaling pathway required for neural differentiation involves PLC-.)I and thus is similar to the pathway involved in neurite outgrowth (see Section 111,DJ; Fig. 3). While the MAPK pathway is activated by FGF-1 in PC12 cells, it is not sufficient to promote the neuronal differentiation of these cells (Renaud

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et aL, 1996). Interestingly, in the presence of dnFGFRs, lens fiber cells initiate the differentiation program properly but then they fail to elongate (Robinson et al., 1995a). While elevated levels of secreted FGF-2 also inhibit fiber cell elongation (Stolen et aL, 1997), FGF-1 synthesis promotes differentiation (Robinson et aL, 1995b).These data indicate that FGF signaling pathways are finely tuned modulators of cell behavior and FGFs have the ability to alter cytoskeletal rearrangments, which is a requirement for changes in cell morphology that accompany and mark cell differentiation.

4. FGFs Affect Cell Morphology and Motility

Consistent with data showing FGFs as regulators of the synthesis of extracellular matrix molecules and FGFRs as matrix-binding proteins, FGFs modulate the shape and motility of many cell types (Manske and Bade, 1994). For example, vascular smooth muscle cells become more elongated and migratory in response to FGF-2. This behavior is associated with FGFinduced changes in integrin expression and disassembly of the actin stressfiber cytoskeleton. The effect of FGF on the migration of these cells is synergistic with the chemotactic influence of PDGF (Pickering et al., 1997b). Both FGF-2- and PDGF-BB-stimulated migration of smooth muscle cells are blocked by anti-uPA antibodies (Stepanova et aL, 1997). In NIH 3T3 cells, the 18-kDa form of FGF-2 specifically promotes migratory behavior, whereas the HMW isoforms are ineffective in eliciting this response (Bikfalvi et aL, 1995). Also, only the 18-kDa form (see Section I1,C) modulates the synthesis of integrins in these cells (Klein et al., 1996). Both FGF-2 and FGF-4 are chemotactic for limb myogenic cells (Li et al., 1996c; Webb et al., 1997). Growth cone motility is a specializedform of cell movement that requires a dynamic remodeling of the membrane, cytoplasmic contents, and the cytoskeleton and rapid changes in cell-matrix adhesion to allow for their exploratory behavior and the generation of tracking forces. The ability of some cell surface adhesion molecules, such as L1, N-CAM, and N-cadherin, to promote neurite outgrowth depends on their activation of FGFRs (Saffell et al., 1997). The FGF signaling pathways mediating the CAM-stimulated extension of neuronal processes have been described in Section 111,DJ (Doherty and Walsh, 1996; Hall et aL, 1996). FGF-l-enhanced neurite outgrowth is accompanied by an increase in the transcription of GAP-43 and T a-tubulin, two growth-associated molecules (Mohiuddin et aZ., 1996). After the initiation of neurite outgrowth, the final elaboration of neuronal shape can also be regulated by FGFs. By acting as neurotropic factors and by promoting sprouting and neurite branching, FGFs contribute to the establishment and maintenance of target innervation (Aoyagi et al., 1994; Fagan et aL, 1997; Shitaka et al., 1996).

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The importance of FGF signaling in the gastrula during morphogenetic movements is described in Section IVJ. FGFs are also major regulators of cell movement during organ formation (Montell, 1994; Skaer, 1997). In C. elegans, FGF (EGL-17) and FGFRs (EGL-15) are required for the proper directional migration of sex muscle myoblasts (Burdine et al., 1997; DeVore et al., 1995). In Drosophila, FGFRl (breathless) and BNL (branchless; Drosophila FGF) are produced in migrating tracheal cells and midline glial cells and are required at the onset of migration of these cells. The migratory response to FGF is rapid (<1 h), suggesting that the initiation of these effects does not require changes in protein synthesis. The failure of tracheal cells to migrate and elongate in fruit flies with a nonfunctional FGF signaling system results in a defective branching pattern of the respiratory system (Reichman-Fried and Shilo, 1995;Sutherland et al., 1996).These observations in fruit flies are highly reminiscent of the effects of FGFs on the patterning of epithelial tubes in vertebrates. 5. FGFs Mediate Epithelial-Mesenchymal Interactions, Induce the Transdifferentiation of These Tissue Types, and Contribute to the Patterning of Epithelial Tubes FGFs and FGFRs are produced at the interfaces between epithelia and the underlying connective tissue mesenchyme in various developing organs and were shown to regulate reciprocal interactions between these tissues (Boyer et al., 1996;Markwald et al., 1996). For example, FGF-2 and FGFR2 are highly produced in the prostate epithelium, whereas FGF-7 is present in the supporting stroma (Culig et al., 1996; Thomson et al., 1997). FGF-7 is also present in skin, lung, and salivary gland mesenchyme, where it supports the survival and maintains the structure and functioning of the overlying epithelia (Rubin et al., 1995).Mice that overproduce FGF-7 under the control of a keratin promoter have thick, wrinkled, sparsely hairy skin, lack a fatty hypodermis, and salivate profusely due to defects in the differentiation of skin and the salivary glands. A dnFGFR2B blocks the elaboration of the characteristic branching of bronchi in the airway epithelium (Skaer, 1997). Similarly,lung branching morphogenesis is controlled by FGFs (Cardoso et d., 1997; Toriyama et al., 1997). In addition, FGFR2B, but not FGF-7, based on results infgf-7-’- mice, is required for wound reepithelialization, a process that involves enhanced proliferation and migration of keratinocytes (Guo et al., 1996; Yamaguchi and Rossant, 1995). Interestingly, keratinocytes produce FGFR2B only while they reside in the spinous layer of the epidermis, a suprabasarlayer that is characterized by a high density of desmosomes (Marchese et al., 1997). During the development of malignant prostate tumors, increased production of FGF-2, -3, and -5 and exon switching infgfr-2 from the B to the C isotype (see Section III,A,3; Fig. 2) correlate

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with the level of malignancy of prostate epithelial cells, indicating that specific FGFs and FGFRs affect epithelial morphology differently (Rubin et al., 1995). In contrast to the induction of epithelialization by FGF-7, FGF-1 can induce epithelia to transdifferentiate into mesenchyme. This FGF-1induced transformation is achieved by increasing the dissociation and motility of epithelial cells which involves a loss of normal cell polarity and changes in cell-cell interactions (Boyer et al., 1996; Fialka et al., 1996). Upon FGF-1 treatment, adherent junctions (desmosomes) disassemble, marked by a decrease in desmoplakin and desmoglein and the redistribution of E-cadherin and p-catenin (Boyer et al., 1996). Some of these effects require the transcriptional activation of AP-1 target genes, consistent with previous observations that implicated c-fos in the regulation of epithelial polarity (Fialka, et aL, 1996). In a rat bladder cancer cell line, these changes are mediated by FGF-1-induced activation of slug, a Zinc finger transcription factor (Savagner et al., 1997). FGF-2 can also modulate the properties of epithelia by regulating the levels of gap-junctional proteins, such as connexin 43 (Pepper and Meda, 1992). As indicated previously, FGFR2B mediates FGFs’ effects on epithelia in several systems. In the gastrula, FGFRl is required for epithelialmesenchymal transformations since f@-’- cells remain epithelial (Ciruna et al., 1997). Among HSPGs, syndecan-3 is implicated most strongly in epithelial-mesenchymal interactions based on its production pattern in the developing lens, otic vesicle, genital ridge, sclerotome, feather buds, and limb bud (Dealy et al., 1997; Gould et al., 1995). Several FGFs participate in tooth morphogenesis, a process that involves an interplay between tooth epithelia and mesenchyme (Stock et al., 1997). The production of FGFs and other signaling molecules parallels the induction of the mesenchyme by the epithelium and the period of mesenchymeregulated epithelial morphogenesis. Initially, the epithelium produces FGF-8 and BMP-2 and -4,which together determine the positions of the prospective teeth (Neubiiser et al., 1997). Later, the induced mesenchyme synthesizes FGF-3, BMP-3 and -4,and activin which are thought to signal back to the epithelium and induce it to differentiate as tooth enamel. Then, the enamel produces FGF-4, BMP-2, -4, and -7, and Shh (Thesleff and Sahlberg, 1996). An analogous sequence of gene expresions and inductions takes place during feather development. Ectodermal FGF-2 (in the epidermal placodes of feather germs) induces the transcription of fgfrl in the mesoderm (in dermal condensations). Based on studies in a scaleless and featherless chick mutant (sclsc) and FGF-induced feather formation in normally apteric regions of the skin, FGF signaling between epithelium and mesenchyme is required for feather formation (Song et al., 1996; Widelitz

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et al., 1996). During feather development, wnt7a, shh, msx-1, and msx-2 are activated (Chuong et al., 1996). Another example for epithelialmesenchymal cross-signalingduring organ development is the dependence of the outgrowth of the facial mesenchyme, called prominences, on FGFs synthesized in the facial ectoderm (Richman et al., 1997;Wilke et al., 1997). A description of epithelial-mesenchymal interactions and accompanying gene expression in limb development is provided in Section IV,A,3. An example for FGF-Zregulated transdifferentiation of an epithelium to mesenchyme during organogenesis is the formation of the cardiac cushion mesenchyme from the cardiac inner epithelium. This tissue transdifferentiation is marked by loss of cell-cell interactions and increased migratory behavior of cells (Markwald et al., 1996). Therefore, FGFs affect tissue morphology both by the regulation of cell numbers and by altering cell shape and cell-cell interactions.

V. Concluding Remarks Studies on the distribution of fgf and fgfr mRNAs and results of in vivo and in vitro manipulations of their expression suggest that FGF paralogs have unique, though perhaps overlapping, functions during organ development. The conservation of the expression patterns and general functions of FGFs in different species is remarkable. Since FGFs affect complex biological processes by modulating several cellular functions simultaneously, it is often unclear whether their effects on the survival, proliferation, motility, or other aspects of cellular activities are most relevant in the process one chooses to study. Unfortunately, it is rare that all these parameters are evaluated in a single experimental system. The discovery of FGF variants and the identification of a large assortment of target genes have considerably enriched the field. In the future, there surely will be many reports on the patterns of expression, biological effects, and results of inactivation of specificparalogs and their isoforms in various developmental systems. Since it is a standard practice to test for potential changes in f g f expression following experimental manipulations of embryos, we may also anticipate progress in the identification of both modulators of fgf expression and the cloning of target genes. There is much work to be done on characterizing the molecular regulation of f g f genes and this field is expected to expand. Finally, there are only a modest number of studies in cell biology and biophysics that address the physical mechanisms of FGF action. It is unclear if there is a unifying theme that links all the apparently different actions of FGFs.

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Appendix Selected Reviews on FGF Biology 0 The structure and function of FGFs and their receptors are discussed in Fernig and Gallagher (1994), Friesel and Maciag (1995), Johnson and Williams (1993), J. I. Mason (1994), Miller and Rizzino (1994), Yeoman (1993), and Basilico and Moscatelli (1992). 0 The evolution of fgf and fgfr genes is detailed in Coulier et al. (1997). 0 The structure of FGFR is described in Bateman and Chothia (1995), Coutts and Gallagher (1995), Givol and Yayon (1992), and Green et al. (1996). 0 The role of HSPGs in FGF action is the subject for the reviews by Coutts and Gallagher (1995), Rapraeger (1993), and Vlodavsky et al. (1996). 0 FGFB structure and biology is the focus of the paper by Coulier et al. (1994). 0 FGF-7 biology is detailed in Rubin et al. (1995). 0 FGF signaling pathways are outlined in Friesel and Maciag (1995), Gotoh and Nishida (1996), and Isaacs (1997). 0 Interactions between CAMSand FGFRs are described in Doherty and Walsh (1996) and I. Mason (1994). 0 The role of FGFs in cell cycle regulation is discussed in Halaban (1996). 0 The effects of FGFs on cell proliferation are discussed in a classic paper by Gospodarowicz et al. (1978). 0 The functional significance of nuclear FGFs and FGFRs, with a focus on the nervous system, is summarized in Stachowiak et al. (1997b). 0 The role of growth factors, including FGFs, on cell motility and migration was reviewed by Manske and Bade (1994). The role of FGFs in cell migration in invertebrates is discussed by Monte11 (1994) and Skaer (1997).

The following reviews focus on the role of FGFs in development: 0 The role of FGFs in gastrulatiodmesoderm formation in Xenopus is detailed in Gotoh and Nishida (1996), Grunz (1996), Isaacs (1997), SchulteMerker and Smith (1995), and Slack et al. (1996). 0 The developmental roles of FGFs and FGFRs in humans, mouse, and chick are described by Abud et al. (1996a), Goldfarb (1996), and Yamaguchi and Rossant (1995). 0 The role of FGFs in neural induction is described in Doniach (1995) and Mason (1996), and the role of FGFs in the later development and maintenance of the nervous system is addressed by Bartlett et al. (1995), Doherty and Walsh (1996), Eckenstein (1994), Grothe and Meisinger

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(1997), Grothe and Wewetzer (1996), Sensenbrenner (1993), Stachowiak et al. (1997b), and Temple and Qian (1995). 0 The influence of FGFs on epithelial-mesenchymal interactions is reviewed by Boyer et al. (1996), Culig et al. (1996), Markwald et al. (1996), and Thesleff and Sahlberg (1996). 0 Data on the role of FGFs in limb development are summarized in Johnson and Tabin (1997), Tanaka and Gann (1995), Cohn and Tickle (1996), and Tickle and Eichele (1994): 0 On tooth development (Thesleff and Sahlberg 1996); 0 On heart development (Markwald et al., 1996); 0. In hematopoiesis (Allouche and Bikfalvi 1995; Bikfalvi and Han 1994); 0 In angiogenesis (Gospodarowicz et al., 1978) 0 In vascular modeling by (Chabrier, 1996); 0 In myogenesis (Coulier et al., 1994; Olwin et al., 1994); 0 In prostate development (Culig et al., 1996); 0 In the formation of the skeleton (Bellus et al., 1996; Erlebacher et al., 1995; Malcolm and Reardon, 1996; Webster and Donoghue, 1997; Wilkie et al., 1995); 0 In tumor progression (van Leeuwen and Nusse 1995; Wright et al., 1993). In addition, there are book chapters (Pimentel, 1994) and special journal issues (Mol. Reprod. Dev. 39,1994) that are entirely devoted to FGFs and cover a variety of topics.

Acknowledgments The following people have read and commented on the manuscript: John Callaway, Nick Caruccio, Randall Dahn, Erik Dent, Ron Dirks, Karen Downs, Lotfi Ferhat, Paul F. Goetinck, Scott Guimond, Matthew Harris, Rose Kinvin, Angie Rizzino, Maria A. Ros, B. Kay Simandl, David Stocum, and Craig Stolen. Their input and encouragement is appreciated. This work was supported in part by NIH Fellowship AR08270 (G.S.) and NIH Grant HD32551 and the University of Wisconsin Vilas Associates Program ( J.F.F.).

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Vogel, A,, Roberts-Clarke, D., and Niswander, L. (1995). Effect of FGF on gene expression in chick limb bud cells in vivo and in vitro. Dev. Biol. 171, 507-520. Vogel, A., Rodriguez, C., and Izpisua-Belmonte, J. C. (1996). Involvement of Fgf-8 in initiation, outgrowth and patterning of the vertebrate limb. Development 122,1737-1750, Voulgaropoulou, F., Myers, R. L., and Chiu, I. M. (1994). Alternative splicing of fibroblast growth factor 1 (FGF-I) transcripts: A cellular dilemma in determining exon selection and exclusion. D N A Cell B i d . 13, 1001-1009. Walz, A,. McFarlane, S., Brickman, Y. G., Nurcombe, V., Bartlett, P. F., and Holt, C. E. (1997). Essential role of heparan sulfates in axon navigation and targeting in the developing visual system. Development l24,2421-2430. Wang, F.. Kan, M., Xu, J., Yan, G., and McKeehan, W. L. (1995). Ligand-specific structural domains in the fibroblast growth factor receptor. J. Biol. Chem. 270,10222-10230. Wang, J. K., and Goldfarb, M. (1997). Amino acid residues which distinguish the mitogenic potentials of two FGF receptors. Oncogene 14,1767-1778. Wang, L. Y., Edenson, S. P., Yu, Y. L., Senderowicz, L., and Turck, C. W. (1996). A natural kinase-deficient variant of fibroblast growth factor receptor 1. Biochemistry 35,10134-10142. Webb, S. E., Lee, K. K. H., Tang, M. K., and Ede, D. A. (1997). Fibroblast growth factors 2 and 4 stimulate migration of mouse embryonic limb myogenic cells. Dev. Dyn. 209,206-216. Webster, M. K., and Donoghue, D. J. (1996). Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J. 15,520-527. Webster, M. K., and Donoghue, D. J. (1997). FGFR activation in skeletal disorders: Too much of a good thing. Trends Genet. 13,178-182. Widelitz, R. B., Jiang, T. X., Noveen, A., Chen, C. W., and Chuong, C. M. (1996). FGF induces new feather buds from developing avian skin. J. Invest. Dermatol. 107,797-803. Wiqdlocha, A., Falnes, P. O., Rapak, A., Klingenberg, O., Munoz, R., and Olsnes, S. (1995). Translocation of cytosol of exogenous, CAAX-tagged acidic fibroblast growth factor. J. Biol. Chem. 270,30680-30685. Wiedlocha, A., Falnes, P. O., Rapak, A., Munoz, R., Klingenberg, O., and Olsnes, S. (1996). Stimulation of proliferation of a human osteosarcoma cell line by exogenous acidic fibroblast growth factor requires both activation of receptor tyrosine kinase and growth factor internalization. Mol. Cell. Biol. 16, 270-280. Wilke, T. A., Gubbels, S., Schwartz, J., and Richman, J. M. (1997). Expression of fibroblast growth factor receptors (Fgfrl, Fgfr2, Fgfr3) in the developing head and face. Dev. Dyn. 210,41-52. Wilkie, A. O.,Morriss-Kay, G. M., Jones, E. Y., and Heath, J. K. (1995). Functions of fibroblast growth factors and their receptors. Curr. Biol. 5, 500-507. Williams, E. J., Mittal, B., Walsh, F. S., and Doherty, P. (1995). A Ca2+/calmodulin kinase inhibitor, KN-62, inhibits neurite outgrowth stimulated by CAMS and FGF. Mol. Cell. Neurosci. 6,69-79. Wong, P., Hampton, B., Szylobryt, E., Gallagher, A. M., Jaye, M., and Burgess, W. H. (1995). Analysis of putative heparin-binding domains of fibroblast growth factor-1. Using sitedirected mutagenesis and peptide analogues. J. Biol. Chem. 270,25805-25811. Wright, J. A,, and Huang, A. (1996). Growth factors in mechanisms of malignancy: Roles for TGF-P; and FGF. Histol. Histopathol. 11,521-536. Wright, J. A,, Turley, E. A,, and Greenberg, A. H. (1993). Transforming growth factor beta and fibroblast growth factor as promoters of tumor progression to malignancy. Crit. Rev. Oncogenes 4,473-492. Yamaguchi, T. P., and Rossant, J. (1995). Fibroblast growth factors in mammalian development. Curr. Opin. Genet. Dev. 5, 485-491.

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Structural and Functional Characteristics of the Centrosome in Gametogenesis and Early Embryogenesis of Animals Marina M. Krioutchkovaand Galina E. Onishchenko Department of Cytology and Histology, Moscow State University, Moscow-119899, Russia

We present a description of the wide spectrum of centrosome behavior during gametogenesis, early development, and cell differentiation. During meiosis and terminal differentiationof gametes there occurs a process of centrosome maturation which includes alterations in characteristics such as the number of centriolar cylinders and their structure if the basal body is formed and ability to function as MTOC, reduplicate, split, and serve as a polar organizer. Such centrosome properties require modificationsof the molecular composition. Maturation of the centrosome in gametes may be compared to transformation of centrosome characteristics during terminal differentiation of other cells. After fertilization different properties of maternal and paternal centrosomes are supposed to combine, adding to each other in the fused (hybrid) centrosome of a zygote. Restoration of centrosome features typical in diploid somatic cells takes place in cells of a developing embryo in the course of early cell cycles. KEY WORDS: Centriole, Centrosome, Basal body, Embryogenesis, Differentiation, Meiosis, Mitosis.

1. Introduction One of the major problems in modern cell biology is understanding the characteristicsof centrosomes,including their molecularcomposition, structural features, and functions (Kuriyama, 1992; Kalt and Schliwa, 1993; Schatten, 1994). A remarkable discovery in this field has been the presence of y-tubulin, which is an invariant constituent of the centrosome and nucleInternationul Review of Cytology, Vol. 185 0074-76%/99$25.00

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ates microtubules (MTs) in many organisms (from yeasts to mammals) (Stearns et al., 1991; Joshi et al., 1992; Horio and Oakley, 1994; Joshi, 1994; Steams and Kirschner, 1994; Fuller et al., 1995; Zheng et al., 1995; Moritz et al., 1995; Li and Joshi, 1995; Murphy and Stems, 1996). The y-tubulin remains a single protein with a well-known function among a huge list of proteins and antigens found in association with the centrosome, and we are far from knowing the complete molecular composition of centrosomes. In spite of the long history of morphological investigations (Brinkley, 1985; Vorobjev and Nadezhdina, 1987; Bornens, 1992), the study of structural features of this complex organelle is not finished, due to the considerable attention of research devoted to the centrosome of cultured cells as the most convenient model for electron microscopy. There have been numerous theories on the structure and functions of the centrosome (Mazia, 1984, 1987, 1993; Brinkley, 1985; Vorobjev and Nadezhdina, 1987), and among them the extremely wide and detailed definition of centrosome functions belongs to Schatten (1994). Microtubule (MT) organization and nucleation are the best understood functions of the centrosome. In animal cells the microtubule organizing centers (MTOCs) exist as fibrillar material of the centrosome that is organized in different patterns depending on the cell type or phase of the cell cycle. An ability of the centrosome to form MT cytoskeleton plays an important role in many cellular processes: genome segregation at mitosis and meiosis (Pickett-Heaps, 1969; Nicklas, 1971, 1975; Roos, 1976; McIntosh, 1983), support of the cell form and polarity, and different kinds of motility of the cell and its organelles (Byers and Porter, 1964; Vasiliev and Gelfand, 1976; Byers and Porter, 1977; Albrecht-Buehler and Bushnell, 1979; Gotlieb et al., 1981; Hamaguchi et al., 1986; Bornens, 1991; Kellogg et al., 1994). A centriole is a component of the centrosome whose structure has been examined in great detail (Vorobjev and Nadezhdina, 1987; AlbrechtBuehler, 1990,1992,1994;Bornens, 1992).The functional role of the centriole is less clear than that of the centrosome. In animal cells, centrioles are located inside the centrosome, but their absence is considered to have no effect on centrosome function as MTOC. There is an opinion that centrioles are associated with the fibrillar material of the centrosome solely their correct segregation in mitosis (Pickett-Heaps, 1969; Sluder, 1990,1992). In this case a well-known function of the centriole is assembly of the axoneme of a cilium or a flagellum (Wheatley, 1982). Sluder suggested that the centrosome possesses some reproductive elements and serves as a polar organizer (Sluder, 1990, 1992). Existence of this polar organizer, whose structure is not yet known, determines abilities of the centrosome to reproduce, split, and assemble a bipolar spindle in

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mitosis. If the centrosome contains centrioles, their number correlates with the reproductive capacity of the centrosome (Sluder and Rieder, 1985a) and it may show doubling and separation of the polar organizers during preparation for mitosis. This hypothesis was based on a functional analysis of coincidence of centrosomal and centriolar cycles and developed Mazia’s concept of centrosome properties (Mazia, 1987).In many cases the behavior of the centriole may follow that of the centrosome but this is not always correct. A centrosome of a diploid somatic cell contains two centrioles (Vorobjev and Chentsov, 1982), and a dividing cell has double the quantity of DNA and a double set of centrioles. Coordinated events of the nuclear and centriolar cycles maintain the correlation between the numbers of chromosomes, and the centriole during mitosis. That situation may be observed in the cell cycle of polyploid somatic cells as well (Onishchenko, 1978; Onishchenko and Chentsov, 1986). However, the coordination of nuclear and centriolar cycles may be disturbed when the cell loses its proliferative ability during terminal differentiation. Sometimes, the centrioles disappear all together (Onishchenko, 1993). Contrary to polyploidization, a natural decrease of ploidy occurs in gametogenesis. Reduction from the diploid content of DNA to one-half occurs due to the absence of duplication of genetic material between meiosis I and meiosis 11. As a result, four haploid cells are formed from one diploid cell. Fusion of the spermatozoon with the egg restores the diploid state of the cell and its proliferative capacity. Analysis of the behavior of the centrosome and its centrioles at key moments of preparation for forming a new organism and during early development raises a lot of questions. HOW does coordination of the nuclear and centriolar cycles in gametogenesis and early embryogenesis differ from that of the same events in somatic cells? Is the centriole an integral part of the centrosome or its derivative like MTs? Are there common principles of inheritance for centrioles and the centrosome, and, if so, what mechanism is used? Is the centrosome inherited through the maternal lineage or the paternal one, and in cases of hybrid centrosome, are there any qualities unique to the maternal or paternal part of the centrosome? Are there any common rules of centrosome behavior in gametogenesis and early development of different species of animals? Is there any resemblance between centrosomes of gametes and of other cells terminally differentiated? In this review we discuss these and other questions. We attempt to represent a wide spectrum of centrosome modes, depending on the behavior of centrioles in gametogenesis, in early embryogenesis, and during cell differentiation. We hope to reanimate the interest in the centriole to centrosome biologists once again.

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II. Behavior of the Centrosome during Gametogenesis A. The Centrosome in Spermatogenesis According to the principle of correlation between the nuclear and centriolar cycles typical for somatic cells, the spindle poles of meiosis I should contain two centrioles and of meiosis I1 one in gametogenesis. Indeed, the correlation between cellular ploidy and the number of centrioles is maintained at maturation of male gametes of some species. Thus, two centrioles at the spindle poles of meiosis I and a single centriole at the spindle poles of meiosis I1 were detected in Bombyx mori (Friedlander and Wahrman, 1970), Sciara coprophila (Phillips, 1966), Chrysopa carnea (Friedlander and Wahrman, 1966), and Dermatobia hominis (Quagio-Grassiotto and de Lello, 1996). A single centriole was found in early spermatids of some insects (Friedlander and Wahrman, 1971;Szollosi, 1975;Baccetti and Dallai, 1977),proboscises (Marchand and Mattei, 1978),nematodes (Favord, 1961), and earthworms (Anderson et al., 1967) (Fig. 1, right column). Many animals, however, have two centrioles in their mature spermatozoa, e.g., coelenterates (Szollosi, 1964; Summers, 1970,1972;Stagni and Lucchi, 1970; Afzelius, 1971; Lunger, 1971; Afzelius and Franzen, 1971a; Hinsch and Clark, 1973; Chapman, 1974), jellyfish (Frank, 1973), flatworms (Hendelberg, 1970;Justine et al., 1985; Swiderski, 1986), aschelminthes (Afzelius and Ferraguti, 1978a), annelids (Colwin and Colwin, 1961; Potswald, 1967; Reger, 1967; Anderson et al., 1967; Fallon and Austin, 1967; Defretini and Wissocq, 1974; Bertout, 1976; Bondi and Farnesi, 1976; Kubo, 1977; Kubo and Sawada, 1977; Troyer and Cameron, 1980), brachiopods (Afzelius and Ferraguti, 1978b),mollusks (Gall, 1961;Longo and Dornfeld, 1967;Walker and McGregor, 1968; Loubress, 1971; Popham, 1974; Buckland-Nick and Chia, 1976; Fields and Thompson, 1976; Afzelius, 1979; Gracia er al., 1993; Johnson et al., 1996), echinoderms (Longo and Anderson, 1969a; Dan and Sirakami, 1971; Chia et aL, 1975; Summers et al., 1975; Fontaine and Lambert, 1976; Afzelius, 1977; Amemiga et al., 1980; Kuriyama and Kanatani, 1981), and arthropods (Ito, 1966; Boissin and Manier, 1967; Breucker and Horstman, 1968; Pochon-Masson, 1968; Baccetti et al., 1969; Fain-Maurel, 1970 Phillips, 1970a,b; 1974; Baccetti and Dallai, 1976, 1978) as well as in some vertebrate animals such as ascidians (Cotelli et aL, 1980; Fukumoto, 19Sl), fishes (Billard, 1970; Afzelius, 1978; Boisson er al., 1967, 1968, 1969; Mattei, 1970; Mattei and Mattei, 1976; Stanley, 1965, 1969, 1971a,b), amphibians (Poirier and Spink, 1971; Rappoport, 1975; Folliot, 1979; Picheral, 1979), reptiles (Austin, 1967; Furieri, 1970), birds (Nagano, 1962; McIntosh and Porter, 1967; Luke et al., 1968;Maretta, 1977; Cheryl and Phillips, 1986; Thurston and Hess, 1987; Soley, 1994), and mammals (Fawcett, 1958,1970, 1975; Fawcett and Ito, 1963; Fawcett and Phillips, 1969, 1970).

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Rattner (1972) studied the structure of meiotic poles during spermatogenesis of the mammals Heterohyrux syriacus and Memetes berdmorei by electron microscopy and found two centrioles in spermatozoa. Each spindle pole of the first and second meiotic divisions had two centrioles (Fig. 1, left column). Similar results were obtained for the mollusk Lymnaea stagnalis (Krioutchkova et al., 1994a). The fact that the poles of both meiosis I and meiosis I1 spindles contain a pair of centrioles favors the suggestion that the correlation between nuclear and centriolar cycles in these cases is disturbed. Such disturbance occurs before meiosis I1 when centrioles are duplicated while DNA synthesis is blocked. During meiosis I1 the haploid sets of chromosomes move toward spindle poles which contain a pair of centrioles. As a result each spermatid gets two centrioles. Although similar ultrastructural investigations of the meiotic centrosome in spermatogenesis are rare, the presence of two centrioles in mature spermatozoa serves as indirect evidence for a similar course of events in the majority of animals. Spermatozoa of most animals contain a pair of centrioles. However, both centrioles organize flagellar axoneme in spermiogenesis of a particular species. In those cases centriolar cylinders are oriented in parallel to each other and serve as the base for two flagella (Fig. 1,left column, a) (Baccetti et al., 1969;Hendelberg, 1970; Justine and Mattei, 1984; Mattei, 1970; Swiderski, 1986). As a rule, only one centriole from the pair forms an axoneme of singular flagellum (Fig. 1, left column, b). This centriole is termed the distal one, since it is located distant from the nucleus. The second centriole, which lies near the nucleus, is noted the proximal one. As a rule, the mother centriole becomes the distal one and the daughter centriole the proximal one. The role of the proximal centriole is not clear, because it has no obvious function and it may completely disappear sometimes in spermiogenesis. Thus, two centrioles located in parallel, are found in early spermatids of the Cestoda Aporina delafondi, but only one of them forms the basal body of spermal axoneme; the other disappears in spermiogenesis (Fig. 1, left column, d) (Ba and Marchand, 1994). Two centrioles that were equal in size and reciprocally perpendicular were described in early spermatids of the ascidia Ciona intestinalis (Cotelli et al., 1980). In spermiogenesis the daughter centriole occupies a distal position and forms an axoneme of the flagellum. The mother centriole migrates toward the nucleus and orients in parallel to the daughter centriole. The length of the mother centriole, which is proximal in this special case, diminishes during spermiogenesis. Similar changes of the proximal centriole were described for spermiogenesis in the fish Squalus suckleyi (Stanley, 1971a,b). A lack of final stages of daughter centriole elongation in secondary spermatocytes may partly cause a reduced size of the proximal centriole. For example, the daughter centriole was shorter than the mother at telophase of meiosis I1 in spermatocytes of the fish Oligocottus maculosus. The angle

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MEIOSIS I

ntriole reproduction

MEIOSIS II

MEIOSIS II

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between these centrioles differs from 90". The size and localization of centrioles remains the same during spermiogenesis and in mature spermatozoa (Stanley, 1969). A short daughter centriole was observed in early spermatids of some annelids, starfishes, and mollusks (Potswald, 1967; Buckland-Nick and Chia, 1976; Dan and Sirakami, 1971). It should be noted that the proximal centriole is smaller than the distal one in mature spermatozoa of numerous animals. In spermiogenesis of mollusks there are some cases where the short daughter centriole (proximal) completely disappears (Gall, 1961; Walker and McGregor, 1968;Loubress, 1971) or together with the mother centriole (distal) it participates in the formation of a basal body (Fig. 1, left column, c and d). The latter has been described for spermiogenesis of the brachiopod mollusk Littorina sitkana (Buckland-Nick and Chia, 1976). In the early spermatid of this mollusk, the procentriole (proximal centriole) is located at a right angle to the distal centriole. In spermiogenesis the small daughter centriole migrates toward the proximal end of the mother centriole, and they orient end-to-end. Finally, the daughter centriole changes into an electron-dense plate. So, in the mature spermatozoon of this mollusk, there is only one basal body which is formed from two centrioles. The basal body cylinder originates from the distal centriole, and the electron-dense plate at its proximal end from the proximal centriole. A similar basal body morphogenesis was described in spermiogenesis of the brachiopod mollusk L. stugnalis (Krioutchkova et ul., 1994a). Both variants of centriole morphogenesis lead to the appearance of only one basal body in the mature spermatozoon in spite of the centriole replication before the second meiotic division. Therefore, the occurrence of a single basal body in mature spermatozoa that is described for some invertebrates [arthropods (Fahrenbach, 1973; Baccetti and Dallai, 1978), coelenterates

FIG. 1 Scheme for centriole behavior during spermatogenesis. The left column presents a variant of a male meiosis when the centrioles are reduplicated two times: before the onset of meiosis and between the first and the second meiotic divisions. As a result, each spindle pole of meiosis I and meiosis I1 includes two centrioles. The right column presents a variant of a male meiosiswhen the centrioles are reduplicated only before the start of meiotic divisions. So, each spindle pole contains two centrioles in meiosis I and one centriole in meiosis 11. The fate of centriolar cylinders may be different during sperm maturation. They may transform into one or two basal bodies and organize flagella in the motile sperms (left column, a-d; right column, a), may remain unchanged and not assemble axial elements (left column, e; right column, b), or completely disappear in immobile sperms (left column, f; right column, c). In certain cases numerous basal bodies are formed de n o w near fibrogranular material in spermiogenesis, and as a result a spermatozoon has a great number of flagella (left column, g), but the motile capacity of such a cell is limited.

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(Afzelius, 1979),annelids (Bondi and Farnesi, 1976)]and vertebrates [ascidians (Woollacott, 1977;Flood and Afzelius, 1978;Fukumoto, 1985;Lambert and Koch, 1988), fish (Jamieson, 1989), birds (Thurston and Hess, 1987)] cannot serve as evidence that centriole replication is absent in interphase between two meiotic divisions. Detailed investigation of spermatocytes in the second meiotic division or the earliest spermatids is required to solve this problem. The absence of centriole replication before the second meiotic division of spermatocytes was shown for some species of insects and invertebrates (Friedlander and Wahrman, 1966,1971;Phillips, 1966,1967).In those cases a spermatid contains only one centriole which changes into a single basal body during spermiogenesis (Fig. 1, right column, a). So, in these animals a correlation between DNA content and the number of centrioles is observed in primary and secondary spermatocytes. Meiosis was investigated in detail for spermatocytes of the neuropteran insect C. carnea (Friedlander and Wahrman, 1966). In primary spermatocytes, procentrioles appeared next to the centriolar cylinders in prophase. Growing daughter centrioles reached the size of the mother centrioles, and then they both continued to elongate up to 8 pm in length. As a result, each spindle pole of the first meiotic division contained a V-shaped structure formed by giant centrioles, located at an angle slightly larger than 90". During the second meiotic division, the two halves of the V-shaped structure, which corresponds to a giant centriole, occupied a spindle pole each. The authors explained the additional elongation of the centrioles by their function as basal bodies of flagella. The increase in the basal body length often occurred in spermiogenesis of animals but not in meiosis as described above (Nagano, 1962; McIntosh and Porter, 1967; Longo and Dornfeld, 1967;Luke et aZ., 1968;Mattei and Mattei, 1976;Maretta, 1977; Soley, 1994). In spermiogenesis modification of the basal body may be more complex. For example, in mammals the distal centriole gets two additional elements: an internal homogeneous cylinder of increased electron density and an external cylinder, formed by nine amorphous columns. During such changes, the distal centriole may increase in size and then MT triplets inside the centriolar cylinder disappear. At the same time the additional internal cylinder disintegrates and in the mature spermatozoon only the external cylinder retains its electron-dense columns, gaining a cross striation (Fawcett and Phillips, 1969; Fawcett, 1975). Similar structural changes of the basal body were described in spermiogenesis of the gastropod mollusk L. stagnalis (Krioutchkova et aZ., 1994b). In insects, morphogenesis of the basal body may lead to the total disappearance of the centriolar composition. In mature spermatozoa the remnants of the centriolar cylinder may look like an electron-dense ring, as in B. rnori (Friedlander and Wahrman, 1970),

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or a plate of the irregular form, as in Drosophilu melunoguster (Bairati and Perotti, 1970). It should be noted that complex and variable reorganizations of the basal body in spermiogenesis are typical only for animals with internal fertilization. Such processes are thought to be related to the formation of additional axial elements around the flagellar axoneme (Fawcett, 1970, 1975; Phillips, 1970a,b;Dallai, 1979). These basal body reorganizations are needed for anchorage of the sperm tail. Additional axial elements are proposed to appear for protection of axonemal microtubules during tail motility, when a spermatozoon is moving a significant distance up to the egg inside female ducts that may contain a fluid with different viscosity. Indeed, in different animal groups there are organisms with external fertilization which occurs when gametes fuse in a water environment. Their spermatozoa have tails without additional axial structures and basal bodies which retain the standard centriolar structure during spermiogenesis. Thus, among animals with internal fertilization differentiation of the centriolar cylinder during spermiogenesis is related to the role that the centriole serves as a basal body of the tail, with a complex arrangement of axial elements. The fact that during spermiogenesis the proximal centriole (not forming axial elements) remains intact or is subjected to destruction partially or fully supports this conjecture (Fawcett, 1958; Fawcett and Ito, 1963; Longo and Dornfeld, 1967; Loubress, 1971; Stanley, 1971a,b; Cotelli et ul., 1980). The exceptional cases are those where the proximal centriole unites with the distal one and together they form a singular basal body (Breucker and Horstman, 1968; Krioutchkova et ul., 1994a). Thus, among the animals with internal fertilization, opposite to the animals with external fertilization, the definition of basal body varies between early and late spermiogenesis. In the early spermatid there is a standard centriole lying at the base of the axoneme, but in the mature spermatozoon the structure of the basal body may be completely different from the centriolar one. In some species of animals with internal fertilization centrioles of spermal cells lose their capability to form an axoneme. As a rule, aflagellate spermatozoa are seen only when, during fertilization, they immediately get close to or at a short distance from the egg. In these cases the aflagellate spermatozoa either are immotile or acquire aflagellate forms of motility which allow them to move a short distance. The centrioles of aflagellate spermatozoa having lost their axoneme formation function may retain intact (Fig. 1: left column, e; right column, b) (Boisson et al., 1967; Breucker and Horstman, 1968; Pochon-Masson, 1968; Dallai et al., 1970; Fain-Maurel, 1970; Baccetti et uL, 1974; Dallai, 1979;) or they completely disappear (Favord, 1961; Robison, 1966, 1972; Justine et uL, 1985). In the latter case, the mature spermatozoa are acentriolar (Fig. 1: left column, f; right column, c).

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It should be stressed that some animals have spermatozoa with restricted motility. These species are considered to be transitional between those with flagellate spermatozoa and aflagellate ones. Different variants of atypical centriolar and axonemal morphogenesis have been described during differentiation of spermatozoa with restricted motility (Dallai, 1979). For example, the termite Mustotermes durviniensis has multiflagellate spermatozoa. Those sperm cells move very slowly in spite of the existence of numerous flagella. It has been shown that their basal bodies (about 100 centriolar cylinders in every cell) consist of the nine mictotubular doublets but not of the triplets (Baccetti and Dallai, 1978). Formation of the basal bodies is connected with unique features of later stages of spermiogenesis in M. durviniensis. After the meiotic divisions are finished, a haploid spermatid contains only two centrioles. Formation of multiple centrioles de n o w occurs later (Fig 1, left column, g), and this process is similar to that during ciliogenesis in different epithelial tissues (Anderson and Brenner, 1971; Dustin et ul., 1975). The other example is the special centrioles formed during spermatogenesis of the insect S. coprophilu. Two giant centrioles were observed at each spindle pole of the first meiotic division, and one of them was seen at the spindle poles of the second meiotic division. These giant centrioles consist of 70 pairs of microtubules. All of them serve as a template for the axoneme doublets (Phillips, 1966, 1967). Other examples of atypical spermato- and spermiogenesis have been described in reviews by Afzelius (1979) and Dallai (1979). Thus, during later stages of differentiation the centrosome preserves reproductive competence in most animals. Moreover, its reproduction become autonomic in relation to the nuclear cycle and the centrosome reduplicates between two meiotic divisions, whereas DNA replication is absent. As a result, in most cases, the mature spermatozoon contains two polar organizers apart from the number of basal bodies in it. It is important to note, once again, that during spenniogenesis of animals with inner fertilization there is often deep transformation of centriolar cylinders. As a result, an appearance of the basal body may greatly differ from the classic centriolar structure, or in certain cases a mature spermatozoon may be devoid of any visible basal bodies. Therefore, penetration of the basal body into the ooplasm does not always indicate penetration of the male centrioles and, consequently, their paternal inheritance. On the contrary, in animals with outer fertilization, the centriolar structure remains unchanged in the mature spermatozoon and may be used to trace the number of polar organizers in it. These male centrioles are considered to remain in the zygote after fertilization, supporting paternal origin of centrioles of the developing embryo. However, in the future, different molecular markers should be used to identify a mode of inheritance of both the centrosome and the centrioles.

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Analysis of the molecular characteristics of centrosomes and centrioles during spermatogenesis for animals with any type of fertilization is a primary task for centrosome biologists.

B. The Centrosome in Oogenesis The structure of centrosomes in egg meiosis has been analyzed for different species of animals using light microscopy (Boveri, 1887, 1901; Wilson, 1925; Raven et al., 1958; Raven, 1959; Schatten, 1994). Similar electronmicroscopic investigations are very rare although they are needed for identification and characterization of centrioles within the centrosome. Even the scarce data available permit us to understand enormous variations in centrosome structure and size of the first and second meiotic divisions compared to the centrosome in mitotic divisions of most somatic cells. It should be noted that in contrast to the main stages of spermal differentiation, egg meiosis occurs in completely differentiated cells, which have distinct compartmentalization of the cytoplasm. It is well known that in different cells, the centrosome seems to be the particular organelle which organizes the internal space of the cell. Therefore, close correlation between the complex organization of the centrosome and egg cytoplasm seems to be evident. Typical for somatic cells, there is correlation between the DNA content and the centrosome number retained during egg meiosis in some animal species: each spindle pole contains a pair of centrioles at the first meiotic division and a single centriole at the second (Fig. 2, la-ld). These spindle poles have a conspicuous astral structure. Those data were obtained for egg meiosis of the pulmonary mollusks Agriolimax reticulatus, Limaxflavus (Raven et al., 1958), Succineaputris, and Physa acuta (Raven, 1959);the sea flatworms Polychoerus carmelensis (Costello, 1961),using light microscopy; and the mollusk Mytilus edulis (Longo and Anderson, 1969b) and the crawfish (Ruthmann, 1959),using electron microscopy. Therefore, centriole reproduction and DNA replication are absent between two meiotic divisions of the eggs. Remarkably unlike spermatogenesis, centriole reduplication in oogenesis may be inhibited even up to the onset of meiosis. For example, by light microscopy investigation only one centriole has been observed within the centrosome of the inner spindle pole during the first meiosis of the pulmonary mollusks Planorbis planorbis, P. carneus, and L. stagnalis (Raven et ab, 1958; Raven, 1959). During the second meiosis, the same centriole participates in the organization of the outer spindle pole, whereas the spermal aster forms the inner one. In these organisms, fertilization occurs when the spindle of the first meiotic division is not yet formed.

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I

FIG. 2 Scheme for centriole and centrosome behavior during oogenesis and two early cellular cycles of the embryo. (1)There are two variants of the centriole cycle in egg meiosis. In some cases the centrioles are duplicated before the onset of meiosis; therefore, each spindle pole contains two centrioles in meiosis I (la, lb) and one centriole in meiosis I1 (lc, Id). After finishingmeiotic divisions the maternal centriole of the egg disappears. Each spindle pole of the first mitosis may include two centrioles that are considered to be of sperm origin,reduplicated in a zygote. In other cases the centrioles are not duplicated before the onset of meiosis; therefore, each spindle pole includes only one centriole in meiosis I (2a, 2b). Spindle poles of meiosis I1 are different: the outer pole contains a single centriole of the egg (2d,2e, 2d*, 2e*), and the inner pole may contain a basal body of the sperm (2d, 2e), penetrating the egg during fertilization or two small MT asters (2d*, 2e*) which are, probably, of sperm origin, too. The outer part of such a pole seems to consist of the centrosomal material of maternal origin. As a result, in both cases after meiotic divisions of the egg there is a hybrid centrosome of maternal and paternal origin devoid of any centriolar structkes in the zygote. The fate of the sperm basal bodies after the end of egg meiosis is not clear. New centrioles are assembled de novo in the first cell cycle and each spindle pole of the first mitosis has only one centriole immersed in the hybrid centrosome. Each spindle pole of the second mitosis contains two centrioles. These centrioles are likely to result from two rounds of centriolar duplication in the first blastomeres.

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At the moment of spindle formation of second meiotic division, the spermal aster of P. planorbis or P. curneus completes division into two asters, containing centrosomes with singular centrioles. A connection between such asters is retained until the second meiotic division. The inner pole of the spindle contains either both asters or one of them (Raven, 1959). Meiosis in the mollusk L. sragnalis has similar features but differs in details (Krioutchkova et al., 1994b). During the second division of egg maturation, the asymmetrical spindle with the outer pole, smaller than the inner one, is formed. The outer pole is astral and contains a singular centriole, and the inner pole is anastral and is acentriolar. Next, the spermal aster fuses with the inner pole and this pole gets an astral arrangement. According to light microscope investigation, the centrosome of the spermal aster does not embody any centriolar structure (Raven et al., 1958).Reinvestigation of this system by electron microscope has revealed that both spindle poles of the first maturation division and the outer pole of the second one evidently contain the single centriole (Fig. 2,2a-2e). On the contrary, the aster of the inner pole of the second maturation division has an unusual composition. There are two areas in the polar centrosome: the central area has the typical organization and contains the spermal basal body (which was not discovered by light microscopy) and the peripheral one, which includes different cytoplasmic organelles but has no centriolar structures (Fig. 2, 2d). Formation of such a complex structure begins in anaphase I, when the compact centrosome of the inner pole transforms into the “hollow” sphere with a centriole precisely in the center of it (Fig. 2,2b). Later, at telophase I, the fibrillar material of the “spherical centrosome” of the inner spindle pole includes different cytoplasmic organelles. The centrosome of the inner pole of prophase I1 has a similar structure, but unlike that in telophase I it does not contain a centriole (Fig.2, 2c). The single egg centriole surrounded by a small fibrillar halo migrates to the plasma membrane and contributes to the organization of the meiosis I1 outer spindle pole. During anaphase 11, the layer of fibrillar material, containing different cytoplasmic organelles, is preserved and forms the periphery of the inner pole centrosome. However, the fibrillar material of its central part is associated with the sperm basal body (Fig. 2, 2d). The sperm basal body in the inner pole of meiosis I1 can be easily identified because its structure and size differ considerably from the centriole cylinder (Krioutchkova et aZ., 1994a). Remarkably, the spermal aster loses a connection with the male pronucleus and migrates autonomously toward the zone of the acentriolar inner pole of the meiosis I1 spindle, formed by the female centrosome. Fusion of the sperm aster and female pole centrosome leads to the formation of the complex hybrid centrosome in the inner spindle pole of the second maturation division. Ultimately, the same hybrid centrosome functions in the developing L. sragnalis organism.

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The biological significance of this process can be explained in the following way. According to the ideas of Sluder (1992), the centrosome should contain two polar organizers for normal functioning in the cell cycle. Reduplication and separation of these centrosomal elements, before they enter mitosis, provide formation of the normal bipolar spindle. In meiosis of L. stagnalis, the centrosome contains only two polar organizers (two centrioles) instead of four (four centrioles). This fact indicates that the centrosome of L. stagnalis oocytes loses its ability to duplicate before the onset of meiosis. During formation of the meiosis I spindle poles, the egg centrosome divides precisely into two, so that each of the poles contains just one polar organizer (one centriole)..When the first meiotic division is completed, the centrosome possessing one polar organizer remains in the egg. This centrosome, being incapable to reduplicate, can organize only one spindle pole (the outer one) in the second maturation division. In these circumstances, the male centrosome containing the sperm basal body participates in organization of the inner spindle pole. The behavior of the centrosome of the first polar body represents more evidence that the female centrosome of L. stagnalis is unable to reproduce. The centrosome of the first polar body does not duplicate and split, as well as the centrosome that remains in the egg after the first meiotic division. Consequently, it has a single centriole during the second meiotic division. Being incapable to form a spindle, the centrosome of the first polar body forms a monaster. Our results confirm the idea that the egg centrosome in animals can lose its reproductive ability during oogenesis. This hypothesis was advanced by Sluder in his review about the problem of centrosome inheritance in the echinodermata (Sluder, 1992). According to the author, the reproductive disability of the egg centrosome appears just in meiosis. If starfish egg centrosomes of meiosis I or I1 are injected into a zygote in mitosis, only the egg centrosome of meiosis I preserves its capacity to duplicate and to organize bipolar spindles in several mitotic cycles of embryos. The replaced centrosome of meiosis I1 is capable of forming only a monaster. It should be emphasized that in L. stagnalis, the basal body centrosome which participates in organization of the inner spindle pole of female meiosis I1 probably includes two polar organizers, since at spermiogenesis a single basal body was formed from two centriolar cylinders (Krioutchkova et d., 1994a). The cited results that in the mollusks P. planorbis and P. corneus there is a split of a sperm aster before the onset of female meiosis I1 (Raven, 1959) provide indirect support of the existence of a bivalent sperm centrosome in this animal group. We have conducted an ultrastructural investigation of female meiosis in the gastropod mollusk Physa rubra that demonstrated new fascinating features in the behavior of male and female centrosomes. The behavior of female centrosome in egg meiosis I and I1

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is the same as that in oogenesis of L. stugnulis (Fig.2, 2a-2c). As for the sperm centrosome we suppose that it participates in organization of the inner pole of the meiosis I1 spindle, like the sperm centrosome of L. stugnulis does. Opposite to the last case in Ph. rubru, there are two small asters inside the inner pole (Fig. 2, 2d* and 2e*) (Vinogradova et ul., 1998). We believe that they derive from the sperm aster centrosome that has lost its basal body and has divided into two. Instead of the basal body, there is an electron-dense globule with a diameter of about 0.15 pm in the center of each small aster. So, in Ph. rubru eggs, an asymmetrical spindle of meiosis I1 has an outer centriolar pole with the typical aster organization, and an inner acentriolar pole with an atypical complex composition. A mature spermatozoon of Ph. rubru contains a basal body with the same structure and size as those in L. stugnalis. After fertilization, the basal body gets into the ooplasm but does not participate in the formation of the inner spindle pole of meiosis 11.At the same time, the male centrosome organizes the sperm aster that divides into two. This capability of the sperm aster may indicate that it contains two polar organizers like the male centrosome of L. stugnulis. Besides, the sperm centrosome of Ph. rubru loses the connection to the male pronucleus and therefore displays autonomous behavior as well as the male centrosome in L. stugnulis egg. But, in contrast to the last one, the polar organizers of Ph. rubru lose their connection with the basal body. Considering our data, we speculate that the structure of the polar organizers of the male centrosome corresponds to the electrondense globules that we have found in the inner part of the inner spindle pole of meiosis 11.After meiosis has been completed the hybrid acentriolar centrosome remains in the zygote of Ph. rubru. The centrioles can disappear from the centrosome of the oocyte even before the onset of spindle organization in meiosis I. However, in this case, the centrosome does not lose its ability to split and forms the meiotic spindle that resembles an acentriolar barrel-like spindle of high plants. These results were obtained for mice, rats, and monkeys using detailed electron-microscopic investigations of egg maturation divisions (Szollosi et al., 1972; Zamboni, 1970; Szollosi, 1975). Additionally, the centrioles were not found in mature eggs of some species of animals (Sonneblick, 1950 Paweletz and Mazia, 1978; Gard et ul., 1990; Krioutchkova et ul., 1994b; Vinogradova et d.,1998). Egg differentiation may proceed in a very specific manner. For example, in Drosophilu eggs the spindle poles of meiosis I and 11 do not contain any centrioles (Sonneblick, 1950). After meiosis has been completed, the centrioles of nutritional follicle cells migrate to the mature acentriolar egg, where they primarily form aggregates and then disintegrate (Mahowald et ul., 1979). The biological significance of such centriole behavior is not yet

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clear. One can speculate that material of disintegrated centrioles will be utilized during early cleavages of an embryo. So, in contrast to spermatogenesis in most species of animals, there are no investigated cases of oogenesis when the centrosome duplicates between two meiotic divisions. At variance in some species, the centrosome of meiosis I and I1 can include centrioles which “signalize” about the existence of polar organizers in it, and therefore it can form polar spindles in both meiotic divisions without the assistance of a male centrosome. Female centrosomes often lose reproductive ability even before the onset of meiosis. In such cases the centrosome does not split and cannot itself organize both spindle poles of meiosis 11. Consequently, sperm centrosomes have to be involved in egg maturation. It is more difficult to investigate the behavior of the acentriolar centrosome in meiotic divisions of mammalian and DrosophiZu species. That type of centrosome has no visible features that can be used to estimate its reproductive ability or the existence of polar organizers in it. Various approaches to analysis of the molecular composition of the centrosome during oogenesis in different animals will solve the problem of the centrosome’s existence and its properties in mature oocytes.

111. Behavior of Centrioles and the Centrosome in Cells in Early Development and during Differentiation A. Centrioles and the Centrosome in Early Embryogenesis Fusion of gametes leads to the association of two cellular systems, and their elements, including the centrosome, have different structural and functional features. It is noteworthy that the centrosome itself is a complex organelle with specific characteristics determined by its different structural components. As a result of gamete differentiation, some of these components may be incorporated into the male or female centrosome. After fertilization, the paternal and maternal parts of the centrosome may fuse within the zygote to form a hybrid centrosome of the animal embryo (Krioutchkova et uZ., 1994b; Schatten, 1994) However, until now, the version of the paternal origin of the zygote centrosome prevails in cell biology. That point of view grew up from the classic studies of the XIX century, when the eggs of invertebrates were used as a favorite material for cytological investigations (Boveri, 1887,1901; Wilson, 1925). The eggs of sea urchins are the best example of such an object and until recently they have remained the most popular object of cell biology. In sea urchins, fertilization occurs after egg meiosis. The egg

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centrosome disintegrates and the microtubule system is not detected in the ooplasm up to sperm penetration. The egg centriole that was included in the pole centrosome of the meiosis I1 spindle also disappears from the ooplasm when maturation divisions have been completed. After fertilization, the earliest activity of the centrosome as MTOC begins from organization of a radial system of microtubules around the paternal centrosome lying next to the male pronucleus and named the sperm aster. The microtubules of the sperm aster play a key role in the “dance” of both male and female pronuclei. When pronuclei contacted each other, a syngamy began. Some authors believe that during syngamy only the sperm centrosome is dispersed above the nuclear surface of both pronuclei, forming an asymmetrical monaster. At the same time the microtubule system of the sperm aster is destroyed. Then the monaster microtubules disappear, whereas two compact centrosomes of the first mitosis are formed and a mitotic I spindle with astral poles begins to organize (Harris et al., 1980;Schatten et al., 1986). A paternal origin of the zygote centrosome in sea urchins has been confirmed by some electron-microscopic investigations of the sperm centrosome from fertilization to first cleavage (Longo and Anderson, 1968;Paweletz et al., 1987a,b). Two paternal centrosomes with a single centriole in each of them were observed next to the male pronucleus before the male pronucleus contacts the female one. In this case, one of the centrosomes is usually lying in the invagination of the nuclear envelope of the male pronucleus, and it remains in such a position just after syngamy. At the beginning, another paternal centrosome is situated close to the first, but after syngamy it migrates along the nuclear envelope to the opposite side of the fused nuclei. Spindle pole centrosomes have already been duplicated at the onset of the first mitosis. Each of them contains a diplosome. Thus, the continuity of centrioles as well as of the centrosomal material is maintained in the zygote of sea urchins by the paternal lineage. Beside sea urchins, the sperm aster was found in fertilized eggs of many other invertebrates, such as echinoderms (Wilson, 1925; Hamaguchi and Hiramoto, 1980;Hamaguchi and Kuriyama, 1982;Sluder et al., 1989;Sluder, 1990), nematodes (Boveri, 1887; Wilson, 1925; Albertson, 1984), mollusks (Raven, 1959;Raven et al., 1958;Longo and Anderson, 1969c),and annelids (Fernandez et al., 1994), and of vertebrates, including the following mammals: ascidians (Sawada and Shatten, 1988), amphibians (Heidemann and Kirschner, 1975; Stearns and Kirschmer, 1994), marsupials (Breed et aL, 1994); rabbit (Longo, 1976; Yllera-Fernandez et el., 1992, cow (Long et al., 1993; Navara et al., 1994,1996), sheep (Le Guen and Crozet, 1989; Crozet, 1990), rhesus monkeys (Wu et al., 1996), and humans (Van Blerkom et al., 1995; Sathananthan et al., 1996) (a more detailed list of the investigated objects is found in Schatten, 1994).

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The evidence presented above led to the assumption that the spermatozoon introduced the active centrosome into the zygote. That paternal centrosome is capable of organizing a microtubule system, to reduplicate and to split into two. Contrary to the sperm centrosome, retaining its reproductive ability in spermatogenesis of most animal species, the egg centrosome loses this ability during oogenesis, but after fertilization it is again capable of organizing the microtubule system. This is evident from several experiments of Sluder and coauthors, made on the eggs of sea urchins and starfishes, particularly from those where migration of pronuclei toward the center of fertilized eggs was blocked (Sluder and Rieder, 1985b; Sluder, 1992). In those experiments, unfertilized eggs were briefly treated with colcemid. That treatment did not prevent fertilization but led to the absence of the sperm aster and other microtubules. As a result, pronuclei were separated far from each other and entered mitosis independently. After photochemical inactivation of the colcemid, a functional bipolar spindle appeared in association with the male centrosomes and chromosomes. However, the female centrosome formed a monaster that persisted until the zygote entered telophase (Sluder and Rieder, 1985b). The same result was obtained if the starfish zygotes were slightly flattened (Sluder et al., 1989). In all cases, the male pronucleus and centrosomes formed the bipolar spindle, whereas the female pronucleus and centrosome could only organize the monaster. Prolonged observation showed that between subsequent mitoses, sperm centrosomes doubled and formed a corresponding number of bipolar spindles. At the same time the female centrosome did not seem to be reduplicated but it was not obviously split and was not able to organize the monaster once again. These results and those in which unfertilized eggs were activated artificially (Mazia, 1984, 1987; Paweletz et aZ., 1984; Schatten et aZ., 1992) have shown that the maternal centrosome is able to function as an MTOC but it loses its capability to split and to form a bipolar spindle. On the contrary, the paternal centrosome retains all these functional qualities during spermatogenesis. After fertilization the male centrosome introduces its properties into the ooplasm and plays the key role in organization of the mitosis I spindle (Sluder, 1992). In this case, the paternal centrosome brings partly its own MTOC into the ooplasm and accepts the maternal centrosome as the additional MTOC. There are several other proofs of this point of view in the late investigations. On the one hand, the pericentrin known as a highly conserved integral protein of the centrosome in different animal cells (Doxsey et al., 1994) was detected in the spermatozoa of Xenopus laevis. As a part of sperm centrosome it was involved in the initial establishment of organized microtubule arrays (Stearns and Kirschner, 1994). On the other hand, it was shown that the sperm centrosome had recruited y-

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tubulin from the ooplasm extracts of X.laevis before it began to function as an MTOC of the sperm aster in vitro (Doxsey et al., 1994). So, the male centrosome may accept female centrosomal proteins. Besides, structural reorganization of the centrosome at early cleavages of L. stagnalis was shown to proceed through the same steps as those in female meiosis (Krioutchkova et aL, 1994b). This observation may be considered indirect evidence of a maternal origin for a particular part of the centrosome that serves not only as microtubule nucleating sites but also as an organizer of the whole microtubule system. In general our electron-microscopic investigations of fertilization in L. stagnalis and Ph. rubra may be considered a confirmation that a hybrid centrosome can be formed from two parts-the paternal polar organizers that provide centrosome reproduction and its splitting, and the maternal organizers of the microtubule system. Thus, as was earlier mentioned, the female centrosome of L. stagnalis had lost its capability to reproduce before an egg entered meiosis. So, divided into two in meiosis I, it cannot accomplish these processes in meiosis 11. As a result, organization of the complete inner pole of the meiosis I1 spindle requires fusion of the egg centrosome and the sperm one, which includes a basal body (Krioutchkova et al., 1994b). We have not observed any basal body inside the inner part of the pole centrosome of the meiosis I1 spindle in the allied species Ph. rubra. As has been mentioned above, this component of the pole centrosome contains two small asters of microtubules. Therefore, we have assumed that it originates from the sperm centrosome, which had succeeded in dividing into two before it was included in the inner pole of the egg meiosis I1 spindle. Two active centrosomes of different origin are functioning in the fertilized egg. One is the female centrosome, which has lost its reproductive and splitting abilities before the onset of meiosis 11, but has remained capable of forming both the outer pole and the peripheral part of the inner pole and of organizing the microtubules of the meiosis I1 spindle. The other is the male centrosome, which is capable of dividing into two and forming two equal stars situated inside the central part of the inner pole (Vinogradova et al., 1998). Division of the sperm aster and its participation in the arrangement of the egg meiosis I1 spindle has been described for several other representatives of this group of mollusks (Raven et aL, 1958; Raven, 1959). The egg centrosome is evidently able to split and to organize the bipolar spindle in parthenogenesis of species of animals where the male centrosomes possess this function in normal development. For example, in cow eggs, events run according to the classical scheme after fertilization, but in parthenogenetic activated eggs, the male centrosome is absent and just the female centrosome organizes the mitotic spindle in a series of cell divisions (Navara et al., 1994). This means that activation of the maternal polar organizers is possible in those eggs where the centrosome keeps such a

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component after the completion of meiosis. Besides, polar organizers can be formed de novo during the formation of numerous cytasters in artificially activated eggs (Dirksen, 1961; Kuriyama and Borisy, 1983). Consequently, during fertilization the paternal polar organizers have to dominate the maternal ones (Sluder, 1992). It is well known that the cell usually needs two polar organizers for the normal course of events. Most of the animal spermatozoa contain just two polar organizers, which, being carried in the egg at fertilization, provide its normal development. The additional polar organizers can only induce multipolar divisions. Therefore, during egg maturation those components either move into polar bodies, as occurs in L. stugnulis (Krioutchkova et al., 1994b), or disintegrate after the end of meiosis, as takes place in sea urchins or starfishes (Sluder, 1992). Modern research methods make it possible to determine the steps of the fertilization system in the animal kingdom where centrosome inheritance of paternal lineage seems to be completely interrupted. The system of fertilization with maternal lineage of centrosome inheritance has been described for the mouse. It was shown that in cultured eggs, just the mature eggs contain the active centrosome, which organizes a complex system of microtubules in the ooplasm, whereas the spermatozoon does not have any centrosomal material (Schatten et aZ., 1986). In unfertilized mouse eggs, in addition to two diffused centrosomes, which are situated at the poles of the divergent spindle of meiosis 11,there are 16 spot centrosomes, organizing miniasters, in the ooplasm. None of the centrosomes contains any centrioles, which have disappeared from the ooplasm before the beginning of meiosis I. At fertilization the sperm aster is not formed and after the end of meiosis I1 division, the number of spot centrosomes increases, and some of them associate with the surface of both pronuclei (Schatten et al., 1985, 1986). By the time spindle organization at first cleavage begins, all spot centrosomes are associated with the surface of both pronuclei, and at prophase they gather to two clusters on opposite sides of the pronuclei, which come in tight contact with each other. The diffuse poles of the divergent or barrel-like spindles of mitosis I are formed just at those places. Electronmicroscopic investigations of such spindle poles have revealed abundant foci of convergence of the microtubules. These pole components consist of the osmiofilicthin-fibrillar material, which includes numerous membrane vesicles. Two basal bodies of the spermatozoon do not take part in organization of the acentriolar spindle poles, in spite of the fact that they penetrate into the ooplasm at fertilization. Moreover, the centrioles appear in the cells of mouse embryo only at the blastocyste stage (Gueth-Hallonet et al., 1993.) Abumuslimov et al., 1994, 1996). On the basis of these results, a conclusion has been made that the centrosome of the mouse is exclusively of maternal origin. Mentioned earlier, our data on L. stugnalis and Ph. rubru have shown that the basal body may move in the ooplasm separate

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from the male pronucleus to be incorporated in the meiotic spindle pole of the oocyte; additionally, for this purpose, in turn, the polar organizers may leave the basal body. The mouse mature spermatozoon may contain two polar organizers, according to the number of sperm basal bodies. Comparison of the results obtained for the mouse and for two species of mollusks led to the assumption that in the mouse, after fertilization, the polar organizers can lose their association with the basal bodies and couple with the egg centrosome to form a hybrid centrosome of the zygote. It seems that modern methods are not efficient at following the behavior of the paternal centrosome after its penetration into ooplasm at fertilization of the mouse. Besides, there are a lot of cytasters in the mouse ooplasm, and a sperm aster, if it exists in the mouse, may be easily lost among these cytasters after the basal bodies disappear. There is a group of organisms with a centrosome of maternal origin, such as animals with parthenogenetic development (Wilson, 1925; Beatty, 1967; Cuellar, 1974). This type of centrosome behavior and its inheritance, which is independent of sperm behavior, has not been completely investigated to date. However, it is clear that the centrosome of mature eggs in those organisms has to retain all the characteristics of the active one. We have already noted that there are spermatozoa in the animal kingdom containing a single basal body associated with only one polar organizer. This may be seen in several species of insects (see section I). In this case, the second active polar organizer has to remain in the egg at fertilization. Indeed, using electron microscopy it has been shown that in two species of insects, C. carnea and D. melanogaster, the basal body has a structure that is quite different from the centriolar one, and in the zygote it participates in the formation of one mitotic spindle pole (Perotti, 1975;Friedlander, 1980). In the same spindle, another pole contains the standard centrioles that, according to the author’s opinion, may be of maternal origin (Friedlander, 1980). After cleavage of this zygote the centrosome of one blastomere will get the paternal polar organizers, but of another blastomere-the maternal ones. 6 . Origin of Centrioles in Cells in Early Development

It is likely that the centrioles remain associated with the polar organizers and are inherited together by paternal lineage in the animal with outer fertilization. In this case, basal bodies retain their structural composition (normal centrioles) and replication ability, as occurs in sea urchins (Sluder, 1992). However, among animals with inner fertilization, whose basal bodies lose their centriolar structure and, as a result, do not keep their connection

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with the polar organizers and their reproductive ability, the centrioles do not seem to be inherited thereafter, they have to be formed de novo in embryos. As such, in L. stagnalis, in spite of the earliest presence of the basal body inside the hybrid centrosome of the mature egg, we have failed to find the sperm basal body in any pole of the first mitosis spindle and have found one centriole with the usual structure in each pole (Fig. 2, top right). These facts suggest an absence of continuity of centriolar structures, unlike the centrosome during fertilization in these mollusks and de novo formation of centrioles in interphase of the first mitosis. Simultaneous de novo formation of two centriolar cylinders can be observed in the zygote (Fig. 3). Amorphous electron-dense globules resembling the deuterosomes are found near each centriole. Additionally, a structure called the cartwheel, typical with the daughter centriole or with centrioles forming de novo (Robbins et aZ., 1968;Anderson and Brenner, 1971;Vorobjev and Chentsov, 1982), is always seen inside the centriolar cylinder at the end facing the electron-dense globule. These features indicate the new start of the centrio-

assembly of centriole de novo

I

first centriole cycle

second cell cycle

L

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centriole segregation in the first mitosis

. I I .

second centriole cycle

f centriole

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segregation in the second mitosis

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- deuterosome,

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- procentriole,

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FIG. 3 Diagram showing possible centriolarevents during two cell cycles in early development. There is an assembly of the centrioles de novo in the zygote during the first cell cycle and two rounds of centriole duplication during the second cell cycle.

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lar existence in the first cell cycle of the developing organism. Two centrioles in the centrosome composition appear in blastomeres of L. stagnulis during the second cell cycle, and, as a result, the spindle poles of the second mitosis contain diplosomes (Fig. 2, top right; Fig. 3). Afterwards, this state is preserved, since the prophase poles of the third mitosis contain two centrioles each (Krioutchkova et al., 1994b). Thus, in L. stagnalis two rounds of centriole duplication in the second cell cycle seem to occur in parallel with one round of DNA duplication. The other possibility should not be excluded: at the stage of two blastomeres, two centrioles are formed de n o w once more, and only after those events, one round of centriole replication, as well as DNA replication, follows. In allied species of the gastropod mollusk Ph. rubru the basal body has been shown to disappear from the sperm centrosome until formation of the meiotic I1 spindle of the oocyte, so the hybrid centrosome of the inner spindle pole is the acentriolar one. All the following events of reappearance of centrioles and reoccurrence of the centriolar cycle are the same in early embryogenesis of Ph. rubra and L. stagnalis (Vinogradova et al., 1998). It has been already mentioned that in mice the sperm basal body does not participate in organization of the spindle poles at the first meiosis of the zygote. The centrioles are formed de novo only after 4 days of development (at the stage of the blastocyst). Centriole formation occurs asynchronously in different cells and independently on centrosomes of mitotic spindles (Abumuslimov et al., 1994). Incorporation of centrioles into the MTOC takes place later (Abumuslimov et al., 1996), and finally the centrosome, containing two centrioles and functioning properly, is formed. At the same time, as we have already mentioned, there are cases when the spermatozoon of animals with inner fertilization has basal bodies that keep their triplet organization. In these cases the basal body is able to reproduce, providing in this way the paternal origin of centrioles in the developing organism. For instance, in C. carnea the single basal body of the spermatozoon may be seen as the mother centriole in one of the blastomeres during several cell cycles (Friedlander, 1980). In human the spermatozoon has two basal bodies with different structures. One of them may lose its centriolar structure during spermiogenesis and really serves as the basal body of an axoneme. The other does not form the axoneme and keeps the centriolar structure. After fertilization this centriole may serve as an ancestor of the centrioles of embryos (Sathananthan et al., 1996). Taken together, the results on the centrosome state after fertilization have shown that the problem of centrosome origin in the developing organism is still far from clear. At present, the results indicate that the centrosome of a zygote may incorporate paternal and maternal components possessing different properties and having a hybrid nature, like the genetic material of the cell nucleus. The question is how widespread is the hybrid centrosome

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in developing organisms of different species? A combination of more sophisticated assays and greater knowledge of the molecules involved should lead to an understanding of the modes and mechanisms of centrosome maturation in gametes and principles of its inheritance. More work is needed to appreciate the result of the cooperative contribution of the centrosome and the centriole in the development program of a new organism. C. The Centriolar Cycle of Proliferating Cells The appearance of procentrioles in the acentriolar cells of the embryo indicates the onset of centriolar ontogenesis for these organisms. As such, assembly of the procentriole is the starting point of centriole formation. The next steps in this process include the formation of nine sets of MT singlets,then duplets, and finally triplets at the periphery of the procentriole. Growth of the centriole is terminated when this organelle achieves its final length and diameter. Continuous existence of the centrioles is provided by the centriolar cycle within the cell cycle. From the beginning each centriolar cycle includes four stages: (1) assembly of a procentriole next to and at a right angle to the wall of the mature centriole termed a mother from this moment; (2) elongation of the daughter centriole, as a rule, up to the length equal to the mother centriole; ( 3 ) termination of centriolar growth; and (4) subsequent separation of two centrioles from each other (Kuriyama and Borisy, 1981; Vorobjev and Chentsov, 1982). Events of the centriolar cycle throughout the cell cycle have been investigated in detail for cultured somatic cells (Brinkley, 1985; Vorobjev and Nadezhdina, 1987; Vandre and Borisy, 1989; Lange and Gull, 1995) and for cells in situ (Zeligs and Wollman, 1979; Onishchenko and Chentsov, 1986). Different stages of the centriolar cycle being synchronized to specific stages of the cell cycle never fully coincide with them and are somewhat shifted in time in a certain range of the cell cycle. Ultrastructural studies showed that centriole replication began in the range from the GllS boundary to the middle of S phase (first stage), then continued through S and G2 phases (second stage), and was completed in the interval from the end of G2 phase to the metaphase of mitosis (third stage). Separation of two centrioles may occur from anaphase to the early G1 phase of the next cell cycle (fourth stage). Shifting of certain stages of the centriolar cycle from one part of the cell cycle to another, dissociation of centriolar replication from other events of the cell cycle, or full termination of the centriolar cycle within the cell cycle may occur in different circumstances-during cell differentiation, in cases of cell pathology, or under various experimental treatments (Onishchenko, 1993).

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Embryonic cells have been used by many investigators as experimental systems for the study of centrosome duplication (Sluder, 1992; Schatten, 1994), and centriolar duplication has sometimes been used as a landmark of such process. The cell cycle in somatic cells is much more complex than the embryonic cell cycle. It is not yet clear whether the ultrastructural features of the centriolar cycle in embryonic cells are the same as those in somatic cells. The degree of coincidence in time of certain stages of the centriolar cycle with certain phases of normal embryonic cell cycle is absolutely unknown. However, there is enough evidence that centriole duplication can be uncoupled from other cell cycle events in embryonic cells after various experimental treatments (Mazia, 1987; Sluder and Lewis, 1987; Sluder, 1992). Study of the regulatory mechanisms that are at work to limit certain events of the centriolar cycle to different phases of the cell cycle in both embryonic and somatic cells is proceeding vigorously at this moment and represents one of the most challenging fields of modern cell and developmental biology. In any case, it is obvious that after the first centriolar cycle an embryonic cell acquires a set of two centrioles: the mother centriole and the daughter centriole. It has been shown for somatic cells that these centrioles are different in their structure, function (Vorobjev and Nadezhdina, 1987), and molecular composition (Lange and Gull, 1995).The centrioles of embryonic cells are not carefully investigated, but according to the results obtained for L. stagnalis (Krioutchkova et al., 1994b), Ph. rubra (Vinogradova et al., 1998), and the mouse (Abumuslimov et al., 1994), the main difference that appears at the first centriolar cycle is the presence of a structure termed the cartwheel, located inside the proximal part of the daughter centriole only. This difference remains in the next centriolar cycles of dividing cells. Until now it has not been clear when in early embryogenesis the mother centriole starts functioning as an MTOC for the interphase MT system (Vorobjev and Nadezhdina, 1987). D. Why Does Every Diploid Somatic Cell Have Two Centrioles? The existence of two different centrioles in the cell raises the question of whether their variance reflects the particularities of their functions. Unfortunately, the problem of centriole functions as a whole has not yet been solved. There are some speculations in the literature. In interphase, centrioles are capable of playing an essential role in organizing the cytoplasmic MT system (Luykx, 1970; Fulton, 1971; Wheatley, 1982; Brinkley, 1985; Vorobjev and Nadezhdina, 1987), which creates cell polarity (Bornens, 1991). It is well known that the mature centriole (the mother) can nucleate a primary

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cilium and is able to serve as the main organizing structure for microtubule nucleating material at interphase and mitosis (Brinkley, 1985; Vorobjev and Nadezhdina, 1987). Although centrioles are not a formal prerequisite for normal function of a spindle at mitosis and meiosis, they can provide correct congression of the centrosomal material between daughter cells (Calarco-Gillam et al., 1983). Moreover, the orientation of a cytoskeleton in relation to the extracellular substrate may depend on the position and orientation of the centriole (Malech et al., 1977; Albrecht-Buehler and Bushnell, 1979; Wright and Moisand, 1982; Rogers et al., 1985). Such a special orientation has been shown just for the mother centriole in certain phases of the cell cycle and under some treatments (Vorobjev and Chentsov, 1982; Gudima et al., 1988; Alieva et al., 1992). Thus, the fundamental functions of the mature mother centriole are likely to comprise the following processes: (1) participation in congression of the centrosomal material that nucleates assembly of the cytoplasmic and spindle microtubules, (2) polar orientation of the cytoplasmic MT system and via it polar transport of membrane organelles, (3) orientation of the cell in a surrounding space via the cytoskeleton, and (4) formation of cilia or flagella. A mature mother centriole has been shown to possess a molecular marker, cenexin (Lange and Gull, 1995). A functional role of the daughter centriole is absolutely unknown. It has been shown that as a structure for the collection of pericentriolar material (PCM) it is less active than the mother centriole. Therefore, the daughter centriole is considered immature (Vorobjev and Chentsov, 1982). However, there may be a functional property of the daughter centriole that differs from those of the mother centriole and does not concern organization of the PCM and through it of the whole MT system. The case in point is the contribution of the daughter centriole in the process of accomplishment of the centriolar cycle. There are some facts worth discussing. Since first assembly de novo at early embryogenesis a component of the centriole with the cartwheel structure corresponding to the procentriole may occupy only the newly formed centriole (Fig. 3). After the start of the first centriolar cycle, such a centriole is the daughter one but never the mother one in the dividing cells. By the results obtained for cultured PE cells (Vorobjev and Chentsov, 1982) and for hepatocytes of mice (Onishchenko and Chentsov, 1986), the procentrioles are not more than 0.13-0.15 pm in length in the first steps of the daughter centriole assembling in S phase of the cell cycle. Later, during G2 phase, the cartwheels grow in length together with the triplets (Vorobjev and Chentsov, 1982), and these components of fulllength daughter centrioles achieve a 0.25-pm value (Onishchenko and Chentsov, 1986). This means that growth of a cartwheel coincides with elongation of MT triplets of the daughter centriole in the cell cycle (S-G2). At mitosis the

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cartwheels are distributed between daughter cells inside daughter centrioles. As a result, after mitosis each daughter cell possesses a single cartwheel situated inside the daughter centriole. We hypothesize that for the new cycle of centriolar duplication the cartwheel should leave the daughter centriole and divide to form two new procentrioles next to the centrioles existing in the cell. Thus, we assume that the essential function of the daughter centriole is duplication and distribution of the procentriole material (cartwheel) during the cell cycle. This component is assembled once, at a certain moment of animal ontogenesis near fibrillar material similar to deuterosomes. From that moment it becomes an element existing as a part of the centriole from generation to generation of cells, to support the centriolar cycles in cells capable of proliferating. We believe that the daughter centriole functions as a carrier of the procentriole during the cell cycle (Fig. 3). This assumption may explain the cause of existence of a set of two centrioles within a diploid somatic cell of animal origin that have different structural and functional properties. However, it should be noted that the cartwheel has not always been seen inside the daughter centriole. This structure is absent in any centrioles of the cell during Go phase (Albrecht-Buehler and Bushnell, 1980;Bystrevskaya et al., 1988). There is a hub inside the distal part of the daughter centriole, similar to that within the mother centriole. Behavior of the cartwheels is not clear in those cases, but the possibility must not be ruled out that the material of the procentriole outside the daughter centriole temporarily loses its cartwheel structure and later restores it at GUS or S phase when replication of centrioles starts again.

E. Is the Centriole a Part of the Centrosome? The problem of genesis of the centriole will be solved when the relationship of all the components of the centriolar cylinder to the centrosome is understood. It has been well known that the centriolar cylinder consists of nine sets of MT triplets included in the wall composed of fibrillar material (centriolar matrix-Fulton, 1971). A question is whether the centriolar matrix is a component of the centrosome. Using anticentrosomal antibodies is the best tool for investigation of this question. A lot of antibodies have been obtained to different antigens of centrosomes of different animal cells (Kimble and Kuriama, 1992; Kuriyama, 1992; Kalt and Schliwa, 1993) and a few of them to some antigens of the centriolar cylinder (Lange and Gull, 1995). To date, an answer has not been found regarding wether the centriolar matrix is a part of the centrosome or under certain conditions these components, usually coupled, can exist separately. The ultrastrutures

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of the centrtiolar matrix and the centrosome look identical, since both of them consist of fine fibrillar material. To understand whether any parts of the centriolar cylinder may function as a centrosome, an estimation of MT organizing activity may be used as a functional test. The wall of a centriole usually does not organize the main pool of MTs, but in some cases numerous MTs contact fibrillar material of the centriolar cylinders of the daughter centriole as well (recovery after colcemid or ionophore treatment-Alieva et al., 1992; under cytochalasin B treatment-Onishchenko, 1993; in cultured cells of Go phase-Albrecht-Buehler and Bushnell, 1980). We consider two possibilities: (1) the centriolar walls themselves may bind ytubulin submolecular complexes; (2) the centriolar walls first acquire the centrosome material, which, in turn, binds y-tubulin. In fact, a general problem arises whether a centriole is a derivative of the centrosome or its creation depends on the function of the cell component that differs from the centrosome. The most well known proposal belongs to Mazia and has been developed by researchers of his school. According to Mazia’s ideas, formation of the centriole results from centrosome activity connected with the assembly of centriolar microtubules (Mazia, 1984, 1987). However, it is not clear in this case which one of the centriolar parts corresponds to the centrosomal material and which of them (except of the MT triplets) is a product of centrosome activity. The following possibilities will be considered here. First, the centriolar matrix is a part of the centrosome that nucleates microtubules and sets them in a regular pattern of the centriolar cylinder. Second, the procentriole (centriolar cartwheel) is a part of the centrosome that contains microtubule initiating sites. If the latter is true, the centriolar matrix may serve simply to support a cylindrical form previously created by the procentriole, and if so its molecular composition will differ significantly from the centrosomal one. In that case the centriole may be considered to be a derivative but not a part of the centrosome, like the other microtubules of the cell (a cytoplasmic system, a mitotic spindle, cilia, and flagella). Except for the centriolar matrix and the procentriole, there is one more component in the cell that may relate to the centrosome. Here we are dealing with the fibrillar or fibrogranular masses organized in different manners, which appear in a cell to assemble centrioles de now. These fibrogranular complexes, termed for different tissues and organisms differently, are regarded as functioning as centriolar precursor structures. They have been observed during organization of numerous centrioles, primarily in multiciliated cells of vertebrates or invertebrates (Dirksen, 1991). Condensed structures that resemble fibrogranular complexes or some of their parts have been shown for centriole formation de n o w in a zygote during normal development (Krioutchkova et al., 1994b) or in numerous cytasters .when see urchin eggs are artificially activated (Dirksen, 1961; Kallenbach, 1985).

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When a problem of centriolar or centrosomal genesis is discussed in the literature, a question arises as to whether these cell components include any nucleic acids. The presence of RNA in the centriole, the basal bodies, or the centrosome is well documented (Hartman et aL, 1974; Heidemann et aL, 1977; Went, 1977; Peterson and Berns, 1980; Bornens and Karsenti, 1984; Brinkley, 1985; Vorobjev and Nadezhdina, 1987; Rattner, 1992), but neither the chemical structure nor any functions of such RNA have yet been cleared. Moreover, there are no results on the precise localization of RNA inside the centrosome or the centriole. It is not clear whether RNA is responsible for duplication of a whole centrosome, a centriole, or an alleged polar organizer. There have been many attempts to reveal DNA in the centrosome (Vorobjev and Nadezhdina, 1987;Dirksen, 1991;Rattner, 1992).At first, some of them seemed successful (Smith-Sonnenborn and Plaut, 1967; Goodenough, 1989; Hall et al., 1989), but later they had no confirmation (Johnson and Rosenbaum, 1990;Kuroiwa et al., 1990;Johnson and Dutcher, 1991). Now, a negative point of view on this subject is dominant (Schatten, 1994). In any case, an analysis of centriolar behavior has shown that a duplicating template should exist in the centriole that provides a semiconservative pattern of centriole duplication (Kochanski and Borisy, 1990). If a centriole is a part of the centrosome, the same mechanism may control the duplicating process of the latter. However, there are some results obtained for unicellular organisms that may be considered contradictory to an assumption that RNA contributes to the process of centrosome duplication. So, the basal bodies of Paramecium have been shown to contain RNA as well (Dippell, 1968, 1976). A mitotic spindle of ciliated Protozoa is known to be intranuclear and organized by polar centrosomes (termed in the literature differently), which are inserted into the nuclear envelope and do not contain any centrioles (Jurand and Selman, 1970; Raikov, 1994). The basal bodies of the ciliated Protozoa also have no relation to the polar structures of the mitotic spindle. The number of basal bodies in these unicellular organisms increases during interphase due to the activity of the fibrogranular material (Tamm and Tamm, 1980), as in multiciliated cells of mammals (Dirksen, 1991). There may be several explanations for these results. On one hand, nucleic acid molecules of basal bodies may have no relation to the polar organizers of the mitotic spindles, and their presence is needed just to provide formation of centrioles or basal bodies. On the other hand, different variants of nucleic acids are probably in the polar centrosomes and in the basal bodies/ centrioles simultaneously to establish any processes of centrosome duplication, including duplication of the polar organizers, the centrioles, or the basal bodies. At least an existence of different molecules acting as a dupli-

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cating template should not be excluded. They may be involved in different forms of centrosome activity connected with duplication and splitting.

F. Patterns of the Centrosome Acting as MTOC in Differentiated Cells The activity of the centrosome as MTOC is its only function that is not related to the duplicating model of centrosome behavior. Realization of MTOC function of centrosome material involves different proteins. Some of them play a structural role (centractin, centrin); others (y-tubulin) are involved in MT nucleation (Kimble and Kuriyama, 1992; Kuriyama 1992; Schatten, 1994). The centrosome, acting as an MTOC, has several forms of organization and may be of different localizations in cells. These features of the centrosome depend on the proliferative activity of the cell and on the pattern of cell differentiation. The centrosome may contain centrioles and have features of centriolar satellites (sometimes termed the subdistal appendages-Bornens, 1992) or of MT convergent centers. These structures are termed the pericentriolar material (Brinkley, 1985; Vorobjev and Nadezhdina, 1987; Rattner, 1992). The ultrastructure and chemical composition of such a centriolar centrosome are well known, Numerous results have shown that this pattern of the centrosome is most typical to somatic cells capable of proliferating. If a cell retains its mitotic activity, the features of the centrosome do not depend on the cell ploidy, but the number of centrioles correlates with the number of chromosome sets in both diploid (Robbins et aL, 1968; Vorobjev and Chentsov, 1982; Vorobjev and Nadezdina, 1987) and polyploid cells (Fawcett, 1966; Matthews et aZ., 1967; Onishchenko, 1978, 1993). Maintenance of this correlation is provided by two options. First, centrosome replication should be coordinated with other events of cell cycle progression. It may be controlled by the activation and subsequent inactivation of the centrosome replication machinery at the appropriate time during the cell cycle (Balczon et aZ., 1995). Second, the centrosomes and their components, such as the centrioles, should be distributed correctly between both poles of the bipolar spindle. This process is needed to support a proper centrosome composition in daughter cells (Fig. 4). When a C mitosis or a multipolar one are the steps of cell differentiation, as, for example, has been shown for megakaryocytes (Ode11 et aL, 1968; Boll and Domever, 1980), the absence of cytokinesis helps to retain the normal structure of the centrosome containing as many centrioles as the cell ploidy is (Fig. 4) (Moskvin-Tarkhanov and Onishchenko, 1978; Radley and Scurtfield, 1980). On the contrary, cytokinesis in multipolar mitosis leads to the formation of atypical cells

-

DNA and centriole reproduction absence of cytokinesis

T

I

bipolar polyploid mitosis

I

bipolar

cytokinesis

--

immature somatic cell

I

c-mitosis 1

-

t

._..i_i. .....-

of cytokinesis

t

I

J ...

i

hypcdiploid nucleus

-i

polyploid nucleus

FIG. 4 Scheme for centriole behavior in dividing cells of different ploidy. Variants are presented where cell ploidy and centriole number may increase as a result of bipolar or multipolar mitosis without cytokinesis or of nuclear reconstruction after c-mitosis. Differentiation of different cells is known to include some of these modes of centrosome behavior (hepatocytes, cardiomyocytes, megakaryocytes). Besides, cytokinesis after multipolar mitosis often leads to the formation of atypical cells (aneuploid nuclei or an abnormal number of centrioles).

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with an abnormal number of chromosomes or centrioles (Fig. 4) (Onishchenko, 1993). The mechanisms and pathways used by somatic cells to establish bipolarity of the mitotic spindle are not clear at present. There are different points of view in the literature (Luykx, 1970; Nicklas, 1971). Some authors relate the spindle bipolarity to the number of centrosomes in dividing cells (Sluder and Rieder, 1985a; Rieder and Alexander; 1990; Sluder, 1992; Zhang and Nicklas, 1995a,b). This might be true because in diploid cells each spindle pole of the bipolar figure contains one polar centrosome, including two centrioles. However, cells with numerous centrosomes do not necessarily divide with multipolar mitosis. For instance, in cultured cells of the neuroblastoma containing a lot of centrioles, the multipolar figures appear at the first phases of mitosis, but they transform into bipolar ones later (Ring et al., 1982). The presence of numerous centrioles in the poles of bipolar spindles (Fig. 4) has been shown for dividing polyploid hepatocytes as well (Onishchenko, 1978). This means that the bipolarity of the spindle does not depend on the number of centrosomes existing in the cell. Independent of the cell ploidy and the number of centrosomes in the cell, it is established with congression of all chromosomes in the unique metaphase plate and with correct segregation of all centrosomes between two poles (Fig. 4) by mechanisms remaining unknown. An understanding at the molecular level of the mechanisms involved in the establishment of spindle bipolarity and in correct segregation of the centrosomes will be of considerable interest in the future. A growing body of evidence suggests an important role of motor proteins such as dynein’s and kinesin’s family in molecular strategies used by somatic cells to establish bipolarity of the spindle and to segregate chromosomes and centrosomes properly (Hyman and Karsenti, 1996;Heald et al., 1997). Often terminal differentiation of cells leads to an irreversible loss of proliferative capacity. It may concern both diploid and polyploid cells. There is a wide spectrum of centrosome patterns in cells of terminal differentiation. Variations concern the composition of the centrosome, the presence and number of centrioles, the position of the centrosomal material functioning as an MT nucleating site, and the level and character of centrosome activity. Variants of the centrosome in cells terminally differentiated may be represented as the following: 1. The centrosome contains centrioles. The main features of organization and activity of the pericentriolar material are in its entity, like in proliferating cells (Fig. 4). This model of the centrosome is typical of cells with a prominent inner polarity, observed as an asymmetric distribution of cytoplasmic organelles (polymorphonuclear leukocytes, monocytes, lymphocytes, osteoclasts) or to cells with a stable polarized morphology (neuronal

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cells) (Bornens, 1991). This pattern of the centrosome may be seen in differentiated cells with a different number of nuclei (hepatocytes, osteoclasts) (Figs. 4 and 6). 2. Centrosomal MT nucleating material loses its association with centrioles and relocalizes to other cell compartments. The final steps of centrosome reorganization involve the elimination of centrioles. Such a method of centrosome transformation has been observed in polarized and nonpolarized cells. Several but not all examples are represented. The first case is a transition of epithelial cells from the bottom of the crypt to the top of the villus. In terminally differentiated enterocytes, at first, centrioles free from the pericentriolar material are located underneath the plasmic membrane of the apical part of the cells (Fig. 5 ) and then they disappear (Komarova and Vorobjev, 1994). MT nucleating material containing y-tubulin molecules is assumed to be associated with the apical part of the plasmic membrane that results in the unique orientation of cytoplasmic microtubules with respect to their structural polarity, the (+) end being distal from the apical part of the cell (Joshi, 1994). It may be supposed that such a model of centrosome organization is required to form a particular MT system that transports endocytotic vesicles from the apical compartment to the baso-lateral one. The second case is a transformation of immature megakaryocytes into mature ones. The immature megakaryocytes increase their ploidy at early stages of differentiation (Ode11 et aZ., 1968; Paulus, 1970 Penington and Olsen, 1970).This process includes parallel duplication of DNA and centrosomes and consequent multipolar mitosis without cytokinesis. As a result, an immature megakaryocyte contains a polyploid polymorphous nucleus or several nuclei and numerous centrioles grouped together in a common cell center (Fig. 4). Their pericentriolar material is functioning as a unique MTOC. During maturation of the megakaryocyte, the cell center is disrupted and singular centrioles are dispersed in the cytoplasm or in platelets formed by cytoplasmic fragmentation (Moskvin-Tarkhanov and Onishchenko, 1978; Radley and Scurfield, 1980). The mature megakaryocytes have no inner polarity. Their function of platelet release is accomplished by a complex demarcation membrane system, which is in continuity with the cell membrane and has the function of delineating developing platelets’ fields over all cytoplasmic periphery (Shaklai and Tavassoli, 1978). The cytoskeleton of mature megakaryocytes has sometimes attracted the attention of researchers (Baatout, 1996). Unfortunately, there are no investigations on the state of the centrosome and the MT system during megakaryocyte maturation. Nevertheless, the cited results show that such a step of terminal differentiation as damage to inner polarity is likely to require a preliminary disintegration of the common centriolar MTOC.

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?+-

+.7

4

diploid immature cell FIG. 5 Scheme for centrosome transformation (changes in the position of MTOCs, or of centriole number) at terminal differentiation of epithelial cells in different tissues or organisms. (a) Centrioles of enterocytes in intestine villus are located beneath the apical part of the cell membrane. They are not associatedwith any PCM and they sometimesdisappear. MT nucleating material is, probably, dispersed in the apical part of the cytoplasm. (b) In multiciliated epithelial cells, basal bodies, which are formed de novo near fibrogranularmaterial, are situated at the subapical cortex. They appear to assemble the MTOCs responsible for organization of the cytoplasmic MT, in addition to their role in the morphogenesis of cilia. The fate of the cell's own centrioles is not yet clear. (c) Differentiation of secretory cells of the salivary glands in Diptera comprises a high increase of cell ploidy as a result of multiple replications of DNA without subsequent mitosis. In these cells there is no parallel reproduction of centrioles. MTOCs abandon their pericentriolar position and move to the surface of the giant nucleus, containing polytene chromosomes.

The third case is the cells of salivary glands of Diptera. They contain nuclei with polytene chromosomes of high ploidy (Berendes, 1973).Numerous cycles of DNA replication occur without parallel duplication of centrioles during polytenization of these cells (Onishchenko, 1993). MT nucleating sites are relocalized from the pericentriolar position to the perinuclear one (Abumuslimov et al., 1991). Thus, in these cells the polarity of the giant cytoplasm is established by the MT system radiating from the large nuclear surface instead of the small centriolar structures (Fig. 5). The fourth case is a myoblast-myotube transition during myogenesis, when centrosome material redistributes from the pericentriolar position to a pennuclear one, and centrioles are eliminated (Konishi et al., 1973;Tassin

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et aL, 1985a).Myotubes produced by fusion of competent myoblasts display no antero-posterior polarity. Their MT system is assembled by MT organizers, associated with the nuclear envelope of numerous nuclei laying in the middle part of the myotubes (Fig. 6). Golgi apparatus surrounds each nucleus (Tassin et aL, 1985b). So, a radial system of microtubules around each nucleus together with the Golgi apparatus makes it possible to accumulate different membrane components with an even distribution over the membrane surface (Jasmin et aL, 1989). Thus, the absence of inner polarity in myotubes seems to be necessary for equal distribution of acetylcholine receptors in the plasma membrane and, in turn, to be an important option for the development of the neuromuscular junction at the stage of muscle differentiation.

3. One more type of centrosome behavior during terminal differentiation is centriole- and ciliogenesis. As has been mentioned already, during ciliated cell differentiation in epithelial cell types, cilium formation is preceded by organization of centrioles from precursor structures such as fibrogranular complexes (Anderson and Brenner, 1971; Dirksen, 1971, 1991; Lemullois

a

fusion

osteoblast

osteoclast

myoblast

myotube

FIG. 6 Scheme for centrosome transformation at terminal differentiation of multinuclear cells. (a) Fusion of mononuclear osteoblasts results in formation of multinuclear osteoclasts. Usually, the multinuclear osteoclast has a united cell center that includes numerous centrioles and functions as MTOC. (b) Fusion of myoblasts leads to formation of multinuclear myotubes where MTOCs surround the nuclei. The centrioles often disintegrate during differentiation of muscle fibers.

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et ab, 1988). Centriole multiplication in differentiated multiciliated cells differs from the numerous cyclic duplications of preexisting centrioles by arresting cells at the Gl/S boundary of the cell cycle, using either hydroxyurea or aphidicolin (Balczon et al., 1995). Formed de novo, centrioles migrate toward the apical portion of the cell to start ciliogenesis. Apart from functioning as basal bodies, the centriolar cylinders are covered with MT nucleating sites termed the basal feet and corresponding to the satellites of the mature centriole in somatic dividing cells. Microtubules nucleated on the walls of basal bodies extend underneath the plasmic membrane and toward the nucleus (Fig. 5). y-Tubulin has been found at the base of the cilia (Muresan et aL, 1993). The microtubules appeared to emanate from areas that stained for y-tubulin. So, the MT system fills up the whole apical portion of the cell. This pattern of y-tubulin distribution in ciliated cells may explain the unidirectional orientation of microtubule arrays toward the nucleus in these epithelial cells. The fate of two own centrioles of differentiated cells is not yet fully clear. According to some data they may remain in the cell. Other researchers have shown that own centrioles were eliminated when the process of de novo centriolegenesis occurred (Dirksen, 1991). In any case, preexisting own centrioles of ciliated cells do not participate in assembly of the MT system, losing their association with the centrosoma1 material. The bulk of such material is likely to increase and is dispersed between numerous newly formed basal bodies.

Thus, some of the types of differentiation include coordination of centriolar, centrosomal, and chromosomal cycles. Formation of other differentiated cells involves different variants of the uncoupling between these cycles, and those shifting are reversible for some cellular types or irreversible for others. Similar models of the interrelationship between centriolar, centrosomal, and chromosomal cycles may be seen in diploid and polyploid cells. The pattern of centrosome behavior is likely to depend on the proliferative competence of the cell and on the direction of differentiation. It in turn determines an existence of inner polarity through organization of the microtubule system and the system of intermediate filaments. Gametes are a special group of terminally differentiated cells. Different models of centrosome behavior and a wide spectrum of centrosome organization may be seen during differentiation of male and female germ cells of different animals (see section I). As a result, the male and female gametes of every animal species contain centrosomes possessing particular features that may complement each other after fertilization. Thus, fertilization leads to fusion not only of the chromosomal sets but of the centrosomal materials. In early embryogenesis there is renewal of the centriolar structure of the centrosome and restoration of coordination between the centriolar, centrosomal, and chromosomal cycles.

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IV. Conclusions After a discovery of the centrosome and the centriole at the end of 19th century, the organization, functions, and chemistry of these organelles have been attracting the most brilliant minds in cellular biology up to our days. Great contributions to the investigation of the centrosome have been made by Boveri (1887, 1901) and Mazia (1984, 1987, 1993). These days, those researchers who are developing their ideas, and are dealing with the problem of the centrosome, raise more and more questions, since the enigma of both the centrosome and the centriole has not yet been solved and continues to challenge biologists today. Schatten (1994), in his article dedicated to Mazia, has clearly defined the main questions for centrosome biologists of the 21st century. In our review we would like to inspire interest in several other aspects of the centrosome problem. As such, the analysis of the molecular content of centrosomes with different features or of different origin (active or inactive, interphase or mitotic, centriolar or acentriolar, pericentriolar or peri basal body or perinuclear, male or female, etc.) will make it clear what kind of molecular events establish the distinct properties of the centrosome. To understand the nature of the centriole and its relationship with the centrosome, it is important to realize whether the centriole is part of the centrosome or is its derivative. Answers to the next questions may help reveal still unknown functions of the centriole: why in the diploid cell there should be two centrioles, how the features and properties of the mother centriole differ from the daughter one, why numerous centrioles exist in some kinds of cells, and why there are no centrioles in the others. What molecular events occur during maturation of the centriole and the centrosome as well? Investigation of the cells containing different patterns of the centrosome will clear up the problem of how definite properties of the centrosome influence cell physiology (organization of the cytoskeleton, integrity of the cell center, cell motility, cell adhesion to the extracellular matrix and to each other, integrity of the Golgi apparatus, secretory ability, etc.). These and many other problems may be solved using cells during gametogenesis and early development. Furthermore, there are some important questions concerning the centrosome within these models. What are the mechanisms ruling centrosome cycles in different gametes? How do the molecular composition of the centrosome and its properties transform during maturation of the oocyte or the spermatozoon? Investigation of the different modes of centrosome maturation during gametogenesis has the principal significance of understanding centrosome behavior during fertilization and following development of the embryo.

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Acknowledgments We thank our colleague and friend Dr. Elena Smirnova for help in preparing the manuscript. The centrosome is a large field to condense into a limited number of references, and we apologize for the references not included. This work was supported by a grant from the Russian Basic Research Foundation under Project N-97-04-48171.

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Cell and Molecular Biology of the Pars Tuberalis of the Pituitary Werner Wittkowski,* Jijrgen Bockmann,* Michael R. Kreutz,t and Tobias M. Bockers* *Institute of Anatomy, AG Molecular Neuroendocrinology, Westfalische Wilhelms-Universitat, D-48149 Miinster, Germany; and ?Institute of Medical Psychology, University of Magdeburg, 39120 Magdeburg, Germany

The pars tuberalis of the adenohypophysis is mainly composed of a special type of endocrine cells, pars tuberalis-specific cells, lining the primary capillary plexus of the hypophysial portal system. Dense expression of melatonin receptors and marked changes in morphological appearance, production pattern, and secretory activity during annual cycle show that these cells are highly sensitive to changes in photoperiod. This leads to the hypothesis that the pars tuberalis is involved in the transmission of photoperiodic stimuli to endocrine targets. Several investigations support the theory that pars tuberalisspecific cells are multipotential cells exerting a modulatory influence on the secretory activity of the pars distalis. Specifically, there is accumulating evidence that seasonal modulation of prolactin secretion, independent of hypothalamic input, is due to melatoninregulated activity of pars tuberalis-specific cells. The exact nature of secretory products and their effects within neuroendocrine regulation, however, remain rather enigmatic. Accordingly, molecular mechanisms regulating gene expression under the influence of photoperiod, respectively, circulating melatonin levels are still incomplete. Recent cloning of melatonin receptor genes and new data on intracellularsignal transduction will probably lead to new insights on melatonin action and pars tuberalis-specific cell physiology. KEY WORDS: Pituitary, Pars tuberalis, Photoperiod, Melatonin, Tuberalin, Prolactin.

1. Introduction

The pars tuberalis (PT) beside the pars distalis (PD) and the pars intermedia (PI) is a clearly defined part of the adenohypophysis. In most studies Inlernalional Review of Cyrology,

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concerning the pituitary gland the morphology and possible role of the PT have received only cursory treatment and little investigative attention in comparison to the PD and PI. Findings reported in the last few years, however, have provided data which open several new and exciting aspects. Based on these results it may be suitable to outline a framework for a functional role of the PT which is already implied by its strategic position at the neurohemal contact area of the median eminence.

II. Characteristics of Pars Tuberalis A. Topography The PT constitutes a well-defined part of the vertebrate adenohypophysis (Wingstrand, 1966;Fitzgerald, 1979; Gross, 1984; Stoeckel and Porte, 1984). In mammals it consists of strains of a bi- to multilayered glandular epithelium surrounding the hypophysial stalk and extending along the ventral surface of the median eminence (Fig. 1). In its distal extension the PT is continuous with the PD and the PI so that no clear-cut boundaries can be determined. The secretory cells of the PT are in close contact with capillaries of the primary plexus of the portal vessel system together with nerve endings of the median eminence. Due to these spatial relations, secretory products of the PT, as well as peptide hormones of the hypothalamic nerve endings, are conveyed to the PD by a vascular chain. By this strategic position a modulatory influence of PT-specific cells on PD cells, as suggested by a number of authors, is conceivable.

8. Phylogenetic and Comparative Aspects The PT as part of the pituitary gland is present in nearly all tetrapods, whereas it is absent in fish (Fitzgerald, 1979). As in mammalian species, the PT of Amphibia, Reptilia, and Aves appears as a distinct part of the hypophysial gland associated with median eminence and encircling the hypophysial stalk. Whereas in other vertebrate classes the PT is in continuity with the PD, in some amphibia-namely, Anura-the PT is found completely separated from the PD. From these comparative observations it has been suggested that the PT may be “regarded as an exclusive tetrapod novelty” which could have developed in the course of adaptation to “terrestrial modes of living” (Fitzgerald, 1979; Fig. 2).

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FIG. 1 Schematicdrawing of a midsagittal section of hypothalamusand hypophysiswith special reference to topographicrelations of pars tuberalis and pars distalis of the adenohypophysisand their connection by the portal vessel system.

Further description and synopsis of data in this review are focussed on mammalian species.

C. Development and Differentiation As a subdivision of the adenohypophysis, the PT originates from the anteroventral part of Rathke’s pouch. During early development, bilateral paramedian processes of the PT anlage grow rostrally along the median eminence, fuse, and surround the hypophysial stalk. The processes are

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Rodentia (Rat)

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Chelonia (Testudo)

Crocodilia (Alligator)

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Pars tuberalis Pars distalis Hypothalamus, Neumhypophysis

FIG. 2 Comparative aspects of pars tuberalis extension and topography in different vertebrate classes. Drawing modified from Fitzgerald (1979).

obviously separated from the anlage of the PD by the so-called Atwell’s recess, a connective tissue space, through which, later on, portal vessels penetrate into the PD. As pointed out, especially by the investigations of Stoeckel and Porte (1979, 1984), developmental characteristics of the PT are original and distinct. An early sign of differentiation in rat and mouse is the occurrence of glycogen, which is not observed in other parts of the adenohypophysis. Glycogen storage is accompanied by a secretory differentiation as early as on Embryonic Day 15 in the rat with a welldeveloped Golgi apparatus, rough endoplasmic reticulum (RER), and secretory granules. At this time no similar differentiation is observed in the PD. Late fetal PT in the rat is found to consist of two cell types: PT-specific cells with all signs of secretory activity and interspersed follicular cells

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(Holbeck et al., 1993; Fig. 3). In the course of human embryonic development the secretory differentiation of PT-specific cells precedes that of gland cells in PD and PI (Neumann, 1997).

D. Morphological Characteristics of Cell Types Morphological and immunohistochemical investigations show that the pars tuberalis consists of different, clearly distinguishable types of cells (Fitzgerald, 1979; Stoeckel and Porte, 1984): PT-specific cells, follicular cells, and PD cell types. The distribution of the different types of cells in the PT varies from species to species. In addition, their percentage of the whole population is very inconstant (Gross, 1984). Presently, the most reliable data concern rat, hamster, and sheep. In the rostrocaudal direction the percentage of PD cells increases gradually and from a cytological point of view there is some difficulty in determining the distal borders of the PT. In the rat Rudolf et al. (1993) found not only that PD cells are spreading in the PT, but also PT-specific cells extend on the ventral surface of the PD during perinatal development. Postnatally, they persist in the PD as

FIG. 3 (A) Semithin frontal section through the median eminence of the fetal rat (20 days). The cell cord of the pars tuberalis (PT) extends along the surface of the median eminence and consists of specific secretory cells and follicular cells. CT,connective tissue with capillaries of the primary portal plexus; EL, external layer of the median eminence. Bar, 50 pm. (B) Ultrastructure of specific secretory cells of the pars tuberalis displaying signs of active secretion. Arrows, secretory granules; fetal rat, 20 days. Bar, 2 pm. Reproduced from Bockers et al. (1990) with permission from Springer-Verlag.

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smaller clusters in the vicinity of large portal vessels. Investigations in other species, including human, are still incomplete.

1. Pars Tuberalis-Specific Cells PT-specific cells exhibit all the characteristics of endocrine peptide secreting cells but are not comparable with any cell type of the PD (Figs. 4 and 5). As in the late fetal stage these cells contain very low numbers of secretory granules-with a diameter of 100-150 nm-and are further characterized by groups of peculiar cup-shaped lysosomes and long ribbons of the endoplasmic reticulum (Stoeckel and Porte, 1984; Wittkowski et al., 1984; Schulze-Bonhage and Wittkowski, 1991). Release of secretory products by exocytosis into the perivascular space of portal vessels has been shown by Merks et al. (1993). Slight species-specific differences in morphology have been observed such as a higher number of secretory granules-with a diameter up to 300 nm-in the mouse (Stoeckel et al., 1979) or ample storage sites of glycogen granules especially in photoperiod-dependent animals such as hamster, hedgehog, or even mouse (Wittkowski et al., 1984; Rutten et al., 1988; Bockmann et aZ., 1998; Fig. 5).

2. Follicular Cells Follicular cells are small, contain irregularly shaped nuclei, and are often found in groups around central cavities filled with electron-dense material. Ultrastructural signs of secretory activity are missing (Stoeckel and Porte, 1984). In the guinea pig, Kameda (1996a,b) has described two separate populations of cells, one type exhibiting long cytoplasmic processes partly surrounding PT-specific cells and containing bundles of vimentin-immunoreactive intermediate filaments. The other type was found arranged as follicles, especially in the ventrocaudal region, and displayed S-100 immunoreactivity. S-100 protein is known as a marker protein of glial cells suggested to play a role in development and maintenance of the nervous system (van Eldik et al., 1992). Similar ultrastructural observations were made in the ovine PT (Brandt, unpublished results; Fig. 4). Heterogeneity of quite similar S-100 protein-positive cells in the anterior pituitary and a partly myeloid origin of the folliculo-stellate cell group are discussed by Allaerts et al. (1996). Data are reported that stellate cells are involved in interleukin6 production and may function as immune accessory cells (Allaerts et al., 1997).

3. Pars Distalis Cells PD cells are especially found in the distal part of the mammalian PT as, on the other hand, PT-specific cells spread within the rostra1 part of the

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FIG. 4 (A) FT-specific cells and follicular cells of the ovine pars tuberalis. Bar, 10 pm. (B) Ovine PT-specific cell with small secretory granules. Bar, 2 pm. Photographs by C . Brandt.

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FIG. 5 (A) Characteristic FT-specific cells in the mouse with high numbers of large secretory granules; neighboring capillaries of the portal plexus and nerve endings (N) of the median eminence. Bar, 2 pm. (B) Detail of a PT-specific cell displaying an accumulation of glycogen deposits often associated with secretory granules. Bar, 1 pm.

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PD (Rudolf et al., 1993). PD cells specifically differ from PT-specific cells by their high number of secretory granules. According to ultrastructural and immunocytochemicalfeatures, most of them can be identified as gonadotropes. Also, low numbers of thyrotropes may occur in the caudal part of the pars tuberalis (Stoeckel and Porte, 1984), whereas other secretory cell types of the PD are met very seldomly (Breitkopf, 1993).

111. Gene Expression of Pars TuberalisSpecific Cells

A. Secretory Products PT-specific cells with their characteristic appearance and their dominating number are especially interesting as their function still poses many questions. Chromophobic in conventionally stained sections, they were widely regarded in the past as undifferentiated or secretory inactive cells, whereas the more active, clearly immunopositive PD cells of the pars tuberalis were evaluated under various experimental conditions (Dellmann el al., 1974; Gross, 1984; Stoeckel and Porte, 1984). Characterization of secretory products of PT-specific cells is rather difficult because the levels of stored secretory proteins in these cells in many species are very low and hardly detectable. Despite this feature it became evident that hormonal subunits, already known as secretory products of PD cells, are expressed in PT-specific cells.

1. Common a Chain and p Subunits of TSH It is well established that PT-specific cells in rat, Djungarian hamster, and mouse react with antibodies against P-TSH (Gross, 1984; Wittkowski et al., 1988; Schulze-Bonhage and Wittkowski., 1990; Sakai et al., 1992; Rudolf et al., 1993, Bockmann et al., 1998). A characteristic spotlike distribution of reaction products is found in the cytoplasm (Fig. 7B). Electron-microscopic examination shows that immunolabeling is not restricted to secretory granules but is also present in the RER and Golgi apparatus (Bergmann et d., 1989; Stoeckel et al., 1994). Stoeckel et al. (1994) and Bockers et al. (1994) also detected expression of the (Y chain of glycoprotein hormones within these cells. Demonstration of messenger RNAs of both subunits emphasized the fact that PT-specific cells express the same hormonal subunits as thyrotropes (Figs. 6 and 7). Thorough examination of PT-specific cells of the ovine pituitary underlines these results: they react with antibodies against the common (Y chain of glycoprotein hormones while antibodies directed against the /3 chains of

FIG. 6 (A-D) In siru hybridization for different hormonal subunits in subsequent parallel sections of the ovine adenohypophysis. Note that the expression of p-FSH (A) and p-LH (B) subunits is restricted to the caudal part of the pars tuberalis (arrowhead), while 0-TSH (C) and the common (Y chain (D) are found throughout the whole extent of the pars tuberalis (triangles). In the pars distalis (PD) hormonal subunits display strong signals in cells or cell clusters in a characteristic manner.

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FIG. 7 Immunocytochemistry and in situ hybridization of the ovine pars tuberalis. (A) Immunoreaction with anti-LHP-antibody only shows clusters of intensely stained gonadotropes in the distal portions of the pars tuberalis. PT-specific cells do not react. ME, median eminence. Bar, SO pm. (B) Immunoreaction with anti-LHcu-antibody reveals staining of FT-specific cells in the rostra1 portion of the pars tuberalis where gonadotropes are not present. Typically, in PT-specific cells the reaction product is mostly found as a spot-like precipitate. ME, median eminence. Bar, 50 pm. (C) Section of the pars tuberalis hybridized with a3’S-labeled oligonucleotide complementary to the mRNA of the common 01 chain. There is a well-defined signal throughout the whole pars tuberalis with higher intensity in regions with gonadotropes (arrows) and lower intensity in regions with PT-specific cells, corresponding to the cu-subunitimmunocytochemical reaction. No labeling is observable in the median eminence (ME). Bar, 150 pm. Reproduced from Wittkowski el al. (1995) with permission from Universitatsverlag Ulm.

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LH, FSH, and P-lipotropin failed to detect antigens in these cells. Interestingly, in contrast to rat and hamster, the presence of TSHP could not be demonstrated in PT-specific cells. By in situ hybridization and Northern blot analysis messenger RNAs of the common a chain of P-TSH (!) and to a far lower extent of PRL and POMC could be attributed to ovine PT-specific cells (Bockers et aL, 1996). These findings suggest that ovine PT-specific cells are capable of expressing different mRNAs of hypophyseal hormones, including TSHP. However, the absence of corresponding proteins raises the question of how gene transcription and translation are related in this cell type. Hormone synthesis might be regulated primarily at the translational level and be followed by a constitutive secretion. A disproportion between mRNA and protein levels of a subunits on the one and that of /3 subunits on the other side is also apparent in rat, hamster, and mouse but considerably more pronounced in sheep. Differential and independent regulatory mechanisms of expression are therefore likely, and, consequently, TSH cannot be regarded as the major product of PT-specific cells. Stoeckel et al. (1994) have discussed the possibility that synthesis and release of the a subunit could occur independent of that of P subunits. Recent studies on molecular characteristics and on the trophic and stimulatory action of the free a chain on PRL cell differentiation and secretion (Blithe et aZ., 1991;Thokatura and Blithe, 1995;Van Bael and Denef, 1996) support earlier results by Begeot et al. (1984) that the a chain alone is capable of inducing differentiation of PRL cells. In this connection the ontogenetic aspects of gene expression are of interest. As described, differentiation of PT-specific cells precedes that of PD cells. In the rat the achain protein could be demonstrated by immunocytochemistry on Embryonic Day 14 followed by P-TSH protein on Day 15 (Stoeckel et aL, 1993; Wittkowski et al., 1997). The onset of expression of both subunits in the PD, however, was about 4-5 days later. These data would fit with a precursor function of the PT and a special trophic action of the common a chain (and other secretory products) in the fetal differentiation of PRL cells. With others, Van Bael and Denef (1996) suggest that the common a chain also stimulates rapid expansion of PRL cells during postnatal life in the rat. This may be consistent with the observation of a characteristic increase of TSH immunoreactivity of PT-specific cells after birth and a reduction in adulthood (Schulze-Bonhage and Wittkowski, 1990). Immunocytochemical studies of the PT, in addition to PT-specific cells, exhibit species-dependent varying numbers of PD cells, predominantly in the distal extension of the PT (Gross, 1984). Therefore, results of immunohistochemical investigations or of in situ hybridization studies caused some confusion concerning classification of positive cells as PD cells or PT-specific cells. In the ovine, PT beside few TSH p-immunoreactive thyrotropic cells

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clusters of LHB and FSHB gonadotropic cells are found (Gross et al., 1984; Tillet et al., 1990; Fig. 7). Estrogen receptors could be identified in the cell nuclei of pars tuberalis gonadotropes (Skinner et al., 1992). According to the small number of immunoreactive cells, the secretion rate of L W S H is not very high, so that it is very unlikely that PT gonadotropes are involved in photoperiod-dependent changes of reproductive functions (Gross et al., 1984; Nakazawa et al., 1991). Pelletier et al. (1992) described the fact that B-LH expression in sheep PT might comprize PT-specific cells, a result which is contradictory to own observations. The finding of 0-lipotropin immunoreactivity also points to PD cells clustering in the distal part of the pars tuberalis. ACTH, however, which is processed from the same preprohormone POMC as lipotropin, was not detectable by immunocytochemistry. 2. The Unidentified Factor: “Tuber&’’A PRL-Releasing Factor?

So far, the only hormonal subunits which have been detected in PT-specific cells by immunocytochemistry are glycoproteins, which are already known from the PD. The search, however, for a PT-specific hormonal product is still continuing. Investigations by Morgan and co-workers for PT-specific secretory products, the so-called “tuberalin” (Stoeckel et al., 1994), in primary cultures of sheep pars tuberalis led to puzzling results (Morgan et al., 1992,1994a). Using 35S-labeledmethionin, they studied synthesis and secretion of proteins after various stimuli (melatonin, forskolin). The only protein which could be identified for sure was prolactin. It could, however, not be attributed to PT-specific cells conclusively and its secretion pattern was not influenced by melatonin. In further studies, Morgan et al. (1994a) reported synthesis and secretion of a number of labeled proteins and their increase by forskolin stimulation and inhibition by melatonin. Specifically, a p72 protein was identified as a useful marker of melatonin effects in the PT. P72 is supposed to be secreted in a calcium-independent manner. Structural identification of the protein, however, has not been successful. Newly published experiments by the same group give evidence for a PTsecreted factor(s) that acts upon PRL cells of the PD by increasing c-fos gene expression of tumor cell lines of the PD. Both conditioned medium from ovine PT cells and coculture of PT with PD cells increased PRL secretion by PD cells (Morgan et al., 1996). By ultrafiltration of conditioned medium of PT cells the activity of the released products could be attributed to two peptide factors (tuberalin), one with a molecular sue greater than 10 kDa, and the other with a size between 1 and 10 kDa. The chemical

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identity of the factors is not yet known. Their secretion by PT cells is enhanced by forskolin and inhibited by melatonin. Studying biological activity of the common a chain the effects of the described peptide factors on PD cells could not be mimicked (Morgan, unpublished observations cited in Morgan et al., 1996). Several secretory products and hypotheses may be considered: First, it is uncertain whether the TSH subunits only act as heterodimers (TSHa and -0) on specific TSH receptors in the PD or whether a subunit alone can act as a hormonal messenger. The possibility of a free a subunit should be considered, with its own biological functions, which are not yet outlined. Presumed effects concern differentiation and stimulation of lactotrophs. Last but not least, an unknown PT-specific factor(s)-according to a proposal by Stoeckel et al. (1994) called “tubera1in”-has to be taken into consideration. A less speculative proposal for such a factor is made by Morgan et al. (1996). The autors describe PT-released peptide factors which exert stimulatory effects on PD cells and might mediate photoperiodic effects, especially on lactotrophs. Altogether, PT-specific cells might be able to coexpress and release several active agents. This would be no unique phenomenon, as in the PD multipotential cells have also been described storing ACTH together with LH, FSH, TSH, or PRL and resembling corticotropes but also other cell types (Childs, 1991).

6 . Regulatory Aspects of Gene Expression

1. Thyrotropes and PT-Specific Cells: Common Characteristics and Differences In the last 10 years some of the numerous questions concerning regulation of gene expression in this enigmatic cell type have been answered: Transcripts of both TSH subunits ( a and p) reveal identical sizes and sequences in both PD and PT (Bockmann et al., 1997). Molecular anal-

ysis of PT TSH revealed no significant differences compared with TSH of PD thyrotropes. For example, it has been demonstrated that P-TSH expressed by PD or PT-specific cells does not differ in the 5’-3’-untranslated region, poly(A) tract length, and transcriptional start sites (Bockmann et al., 1997). Regulation of cellular activity and gene expression is, however, completely different and seems to be closely related to photoperiodic stimuli: 0 Absence of classical receptors: In contrast to thyrotropes of the PD, PT-specific cells do not express T3 or TRH receptors (Bockmann et al.,

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1997; Fig. 8). Hence, TSH synthesis in the PT is not under control of the classic regulatory factors T3 and TRH. 0 Absence of the transcription factor Pit-1: Pit-1 possibly links TSHP transcription to two major second messenger pathways in PD thyrotropes. In PT-specific cells this transcription factor could not be demonstrated (Bockmann et al., 1997). Lin et al. (1994) report that Snell dwarf mice which are known to be GH-, PRL-, and TSH-deficient because of a mutation in the POU domaine gene, coding for the transcription factor Pit-1, express TSHP in cells on the tip of the pars distalis during embryogenesis. They conclude that PD thyrotropes might arise from two different stem cells. Results by Bockmann et al. (1997), however, indicate that early-differentiating TSH-expressing PT-specific cells were described in this paper that are not concerned by the lack of Pit-1. Drolet et al. (1991) characterized a transcription factor which is expressed in the anterior pituitary during prenatal development. They observed that the pattern of gene expression of this factor corresponds to the onset and distribution of TSHP gene expression. Further description of the so-called thyrotroph embryonic factor (TEF) revealed that it binds to and transactivates the TSHP promotor. TEF is member of the leucine zipper (bZIP) gene family and shows homology with the albumin D box-binding protein (DBP). Coexpressed in a pituitary cell line, TEF and DBP can readily form heterodimers. Our own observations on the localization of TEF gene expression in the fetal pituitary showed clear signals only in the PT area and not attributable to PD thyrotropes (Bockers et al., unpublished observations). This pattern is also valid for postnatal development. In the mature organism, however, TEF is distributed in several areas of the rat brain. These combined data lead to the conclusion that not only the expression of receptors but also intracellular signaling cascades in PT-specific cells are largely different from those in PD thyrotropes. In vivo experiments in rats (Bockmann et al., 1997) confirmed this difference: following treatment with T4 or with TRH, only thyrotropes of the PD reacted with reduced (T4 treatment) or enhanced (TRH treatment) formation of TSHP mRNA; mRNA expression in PT-specific cells was not altered.

2. Melatonin Receptors: A Peculiarity of PT-Specific Cells With the demonstration of “high-affinity binding sites” for [1251]iodomelatonin in the pars tuberalis in the rat by Williams and Morgan (1988), the functional significance of this part of the adenohypophysis has attracted high interest. Further autoradiographic studies have confirmed this result

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in other species (Fig. 9) and have shown that the characterized binding sites belong to the group of membrane-bound receptors which are coupled to adenylate cyclase by Gi proteins (Morgan et al., 1989a-c; Reviers et al., 1989; Weaver and Reppert, 1990; Stankov et al., 1991a,b). Undoubtedly PT-specific cells constitute the melatonin-responsive cell group (Morgan et al., 1991a, 1994b). Apparently, the intense labeling of the pars tuberalis with iodomelatonin is a general feature of mammals and not restricted to photoperiod-sensitive species. Only the existence of melatonin receptors in the human pars tuberalis is still controversial (Weaver et al., 1993). Further specific binding sites have been detected in numerous regions of the brain and also in peripheral organs. The distribution pattern, however, shows a wide species variability probably posing more questions than answers (Morgan et al., 1994b). The development of photosensitivity and circadian rhythmicity is already observable during the fetal period. This differentiation depends on the materno-fetal transfer of melatonin as a photoperiodic signal which is known to readily cross the placental barrier. The fetal pituitary has been shown to respond earlier than the brain (Williams et al., 1991) and specific iodomelatonin binding sites have been identified in the fetal pituitary of several species. In the fetal rat, for example, melatonin binding sites are already present at Gestational Day 15 (Williams et al., 1991). The authors therefore conclude that the fetal pituitary may have the potential to respond to maternal melatonin signals from Day 15 of gestation. Similar results are reported from the Djungarian hamster Phodopus sungorus (Carlson et al., 1991; Rivkees and Reppert, 1991) and the Syrian hamster (Duncan and Davis, 1993). In the ovine fetus, a mature distribution of melatonin binding sites including PT is attained toward the end of gestation (Morgan et al., 1994b). The fetal development of melatonin binding sites coincides with

FIG. 8 (A) By Northern blot analysis,TSHa and -p mRNA can be detected in the hypophyseal pars distalis and pars tuberalis. TRH- and T3receptor mRNA, however, are solely expressed in the pars distalis. The sizes of the detected mRNAs are in agreement with published cDNA clones. A 0.2- to 9.5-kb RNA ladder was used as a molecular weight standard. (B) Comparison of TSHB, and TRH- and T3-receptor expression in pars distalis and pars tuberalis by PCR (p-actin was used as a control). The template concentration of pars tuberalis and pars distalis cDNA ranged from 1 pg to 1 ng (1 pg, 0.5 pg, 0.1 pg, 50 ng, 10 ng, and 1 ng). P-Actin (309 bp) and TSHP fragments (378 bp) could be amplified in pars distalis and pars tuberalis down to a template concentration of 5 ng. TRH-receptor (732 bp) and T3-receptor (428 bp) fragments could only be detected using pars distalis cDNA as a template down to a concentration of 50 ng. In the pars tuberalis, no PCR products could be amplified at any template concentration. Reproduced from J. Bockmann et al. Thyrotropin expression in hypophyseal thyrotropin-releasing hormone, and Pitpars tuberalis-specificcells is 3,5,3’-triiodothyronine, 1 independent. Endocrinology 138(3), 1019-1028 (1997) 0 The Endocrine Society.

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FIG. 9 Melatonin binding in the pars tuberalis of the Mongolian gerbil (Meriones unguiculatus). (A) Frontal section of basal hypothalamus with median eminence and pars tuberalis I-melatonin binding stained with Cresyl violet. Bar, 160 pm. (B) Localization of specific as a dark zone over the pars tuberalis in a frontal section adjacent to A. Bar, 160 pm. Reproduced from Wittkowski et al. (1995) with permission from Universitatsverlag Ulm.

the secretory differentiation of PT-specific cells and their expression of common a chain and P-TSH in the rat (Wittkowski et al., 1997). The expression of binding sites of melatonin is affected by the developmental stage. Concerning the pituitary, a characteristic decline or disappearance of receptor expression in the PD during postnatal development has been observed. Melatonin receptors localized in the PD in the early postnatal period of the rat are not present in adult animals (Williams et al., 1991). Similar results have been elaborated using binding assays (Vanecek, 1988). As melatonin receptors of the PD are supposed to be expressed by gonadotropes, it has been proposed that a postnatal decline of melatonin receptors is related to a loss of inhibitory effect of melatonin on LHRH-

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induced LH release by the pars distalis (Vanecek; 1988,1991;Vanecek and Vollrath, 1990; Vanecek and Klein, 1992). As demonstrated by several studies, the number or affinity of melatonin receptors can be regulated by melatonin itself. Thus, suppression of melatonin secretion by constant light or pinealectomy causes an increase in the number of melatonin receptors (Pelletier et al., 1990;Gauer et al., 1992) and an inverse effect is attained in these animals by single melatonin injections (Gauer et al., 1993a,b). Contrary data are reported by Duncan et al. (1993): in quantitative studies in the pars tuberalis and suprachiasmatic nucleus of pinealectomized Djungarian hamsters exposed to constant illumination or injected with melatonin, affinity or density of receptors was not significantly affected. Diurnal variation of receptor density in the PT inversely related to plasma levels of melatonin has been reported from Piketty and Pelletier (1993), Masson-Pevet et al. (1993), and Gauer et al. (1993a,b, 1994). Testosterone implantation had no effect on melatonin receptor density (Skene et al., 1993). An important step forward has been made through molecular cloning and identification of three G-protein-coupled melatonin receptor subtypes in vertebrates. Initially cloned from Xenopus dermal melanopheres (Ebisawa et al., 1994), Reppert et al. (1994) finally succeeded in cloning the first mammalian-derived melatonin receptor from ovine F T mRNA. Until now, cDNAs for a Me1 l a receptor and Me1 l b receptor, which are both present in mammals, and the Me1 l c receptor, which has not yet been found in mammals, are sequenced (Ebisawa et al., 1994;Reppert et al., l994,1995a,b, 1996a). Ligand-binding properties and signaling mechanisms are very similar, though they show structural dissimilarity.High-affinity binding of melatonin and coupling to Gi protein appears to be a common signaling pathway for all three subtypes. Only the mammalian Me1 l a melatonin receptor is expressed in the pars tuberalis and hypothalamic suprachiasmatic nucleus, the presumed sites of seasonal and circadian actions of melatonin. The cited circadian and seasonal changes of melatonin receptor density raise the question of which molecular mechanisms are involved in regulating their expression. Barrett et al. (1996) have addressed melatonin receptor regulation in the ovine pars tuberalis. They showed that Me1 l a mRNA expression is induced rapidly through changes of cAMP levels. Forskolin enhances mRNA expression and melatonin reduces this effect. Expression of functional receptors, however, may include other factors, because there is a delay between induction of mRNA and protein formation. The observation of a spontaneous increase of Me1 l a mRNA and protein without corresponding changes of cAMP levels indicates an autoregulatory mechanism. Further investigations are necessary for the understanding of the dynamics of Me1 l a receptor regulation. Newly, Reppert et al. (1996b) cloned and characterized a G-protein-coupled receptor from human pitu-

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itary which is closely related to the melatonin receptor family but does not bind melatonin. The pattern of expression in human hypothalamus and pituitary suggested a neuroendocrine function.

3. Possible Mechanisms of Melatonin Action in PT-Specific Cells From a number of studies in sheep as well as in other species (rat, hamster), there is evidence that melatonin in PT-specific cells inhibits forskolinstimulated cyclic AMP production but has no significant effect on basal cyclic AMP levels (Carlson et aL, 1989;Vanecek and Vollrath, 1989;Morgan et al., 1989b,c, 1991a,b; Weaver et al., 1990). Therefore, it was concluded that the physiological effects of melatonin in the pars tuberalis are mediated by coupling to intracellular signal cascades which are positively linked to adenylate cyclase; in detail, melatonin binds to a Me1 l a receptor which is coupled to a member of the Gi family of G proteins. The mechanisms by which melatonin alters intracellular signal cascades have not yet been elucidated. Morgan and co-workers have introduced primary culture of ovine PT-specific cells as a model system for studying the molecular and cellular effects of melatonin action. In this culture system it was shown that melatonin inhibits the cascade of forskolin-stimulated elevation of intracellular cyclic AMP, protein kinase A activation, and cyclic AMP response element binding protein phosphorylation (Hazlerigg et al., 1991; McNulty et al., 1994a,b). Thus, a translational control of protein synthesis is assumed and, also, interference with transcriptional regulation cannot be excluded (Morgan et aL, 1994b). Whereas in other cases pertussis toxin is known to disrupt receptor G protein coupling, Morgan et al. (1995) showed that in the ovine PT inhibition of CAMPby the melatonin receptor is choleratoxin-sensitive. In a recent study Ross et al. (1996) investigated the expression of the primary response genes of the AP-1 complex that is coupled to different intracellular second messenger systems including CAMP,MAP kinase, and PKC. Again, melatonin only inhibited forskolin-stimulated gene expression but had no effect on basal levels of transcription of any of the AP-1 genes (junB, C-jun, junD) in ovine PT primary culture. Thus, these data indicate that the major function of melatonin within the PT is probably to attenuate gene transcription. 4. Possible Antagonists of Melatonin

A central question remains regarding which endogenous factors, apart from melatonin, regulate PT function at the cellular level, i.e., act(s) as antagonists of melatonin with stimulatory effects on adenylate cyclase. Recently, the expression of receptors for insulin-like growth factor

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(IGF1)-members of the RTK family-was reported from the sheep PT (Williams et al., 1995).Their distribution is comparable to that of melatonin receptors. With respect to controversial results in the rat, species differences of localization of IGFl receptors cannot be excluded (Aguado et aZ., 1994; Williams et al., 1995). Functional aspects of IGFl receptors were investigated by Hazlerigg et al. (1996) by testing the effect of IGF on mitogen-activated protein kinase (MAPK) in primary cultures of F'T cells. MAPK may be stimulated by py subunits of Gi proteins (Alblas et aZ., 1994). As is known from forskolin, IGFl was also able to strongly activate MAPK. Melatonin, in contrast, only acts inhibitory, when MAPK was previously activated by forskolin. Effects of IGF and melatonin may be linked with each other or occur independently (Hazlerigg et al., 1996). The origin of IGF-local or systemic-has to be investigated. Morgan et al. (1994b) have tested a number of compounds for their ability to increase adenylate cyclase activity in ovine PT-specific cells. Stimulatory activity was found after application of prostaglandin El and E2 or adenosine. The latter has to be emphasized because Stehle et al. (1992) were able to clone an A2-like adenosine receptor expressed in the F'T. A functional antagonism between these compounds and melatonin, however, was lacking because melatonin failed to inhibit their stimulatory effects. A new member of the G-protein-coupled receptor family has also been isolated from the ovine PT by Barrett et al. (1994). This receptor shows high affinity to analogues of a-MSH but is present only in very low numbers. A possible role of MSH-related peptides in the PT or interaction with melatonin is quite uncertain. Hazlerigg et al. (1993) have proposed an autoregulatory mechanism by which melatonin influences activity of PT-specific cells. In PT primary cultures, they found a sensitization of forskolin-stimulated CAMPproduction following 16 h of preincubation with physiological concentrations of melatonin. This effect may be mediated by uncoupling of the melatonin receptor from the inhibitory G protein. The authors suggest that under physiological conditions PT-specificcells are sensitized to hitherto unknown stimuli by nightly melatonin exposition. Under long photoperiods this effect would be lower than that under short photoperiods. The significance of such a mechanism, however, can only be estimated after the identification of endogenous ligands of receptors expressed by PT-specific cells.

IV. Biorhythmic Alterations A. Photoperiod-Induced Seasonal Alterations Various experimental investigations with marked functional changes such as thyroidectomy, hypophysectomy, gonadectomy, or adrenalectomy could

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not establish a convincing functional concept for the PT (Oota and Kurosumi, 1966; Dellmann et al., 1974; Gross, 1984; Stoeckel and Porte, 1984). Only gonadotropic cells, which may be intermingled between PT-specific cells, have been shown to react with higher activity following castration or hypophysectomy (Dellmann et al., 1974). Signs of specific functional significance of the PT were only found in connection with photoperiodinduced seasonal alterations of the endocrine system. In submammalian species changes in the morphology of the PT cells have been observed to be related to the periods of the annual cycle (Vandenberghe et al., 1973). The comparative study of Dellmann et al. (1974), which included the hibernating garden dormouse, supplied the first hint that a seasonal influence on the PT may also exist in mammals. Investigations in the Djungarian hamster have revealed characteristic differences of morphology, exocytotic activity, and immunoreactivity of PT-specific cells between animals exposed to long and to short photoperiods (Merks et al., 1993; Wittkowski et al., 1984, 1988). In detail, PT-specific cells of hamsters held under short-day conditions show cytological signs of inactivation as regression of endoplasmic reticulum and Golgi apparatus or clusters of glycogen particles and secretory granules with a marked decrease of exocytotic activity (Wittkowski et al., 1984; Merks et al., 1993). Similar cytological differences were found when comparing active and hibernating hedgehogs (Rutten et d.,1988).A distinct TSH-like immunoreactivity was observed in PT-specific cells of hamsters exposed to long photoperiods, whereas under short photoperiods immunoreactivity was nearly absent (Fig. 10). Interestingly, in the PD the number and distribution of TSH-positive cells did not differ significantly between both groups (Wittkowski et al., 1988). These results together with the observation of seasonal changes of melatonin receptor density in hamster and hedgehog PT (Masson-Pevet and Gauer, 1994) substantiated the suggestion of characteristic circannual variations in the activity of the mammalian PT, at least, in species with marked seasonal rhythmicity of gonadal and body functions (Hoffmann, 1985). A control function of the PT in seasonal reproduction with “antigonadotropic activity” has been postulated (Bittman, 1993) and confirmed by a number of experiments. Stanton et al. (1991) have found a significant decrease in melatonin binding sites in the PT of hibernating ground squirrels compared to those of awake, euthermic animals. In hamsters exposed to short photoperiods, the number of melatonin binding sites in the PT was also markedly reduced (Vanecek and Jansky, 1989). In different long-day breeders (hedgehog, hamster) the density of melatonin receptors in the pars tuberalis shows high levels in spring-summer and low values in autumn-winter (Masson-Pevet and Gauer, 1994). The decrease of receptor density was associated with gonadal regression (and short photoperiod). During the estrus cycle, however, no differences exist concerning

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C“

YI

D SP

B

E

FIG. 10 (A-F) Immunocytochemical stainings of frontal sections of the rostra1 pars tuberalis (TSHP, A-C; a chain, D-F) in long-photoperiod (LP) (A and D), short-photoperiod (SP)(B

and E), and pinealectomized (Px) animals kept in short photoperiod (C and F). Note that the TSHP and a-chain immunoreactivity is significantly reduced or nearly absent in SP in FT-specific cells (B and E). These alterations are absent in Px animals (C and F). Original magnification, X270; scale bar, 0.05 111111. Reproduced from J. Bockmann et at. Short photoperiod-dependent down-regulation of thyrotropin-a and -0 in hamster pars tuberalis-specificcells is prevented by pinealectomy. Endocrinology 137(5), 1804-1813 (1996) 0 The Endocrine Society.

affinity and concentration of melatonin binding in sheep (Helliwell and Williams, 1992). From their studies with timed infusion paradigms for delivery of melatonin Bartness et al. (1993) conclude that the duration of the melatonin signal is the critical point for both inhibitory and stimulatory effects on seasonal reproductive and metabolic functions in hamsters and sheep. This hypothesis does not exclude the forementioned variability of melatonin receptor density. In order to examine whether melatonin actually is “Zeitgeber” for translation of photoperiodic information into secretory activity of PT-specific cells several experiments have been carried out:

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0 In hamsters held under long-photoperiod conditions, the duration of the melatonin surge was prolonged by melatonin application in late afternoon (Bockers et al., 1995). This treatment was followed by cytological alterations of PT-specific cells which are typically observed in animals living under short photoperiods. Specifically, immunoreactivity of achain and pTSH was reduced significantly. 0 Another study was designed to investigate the effects of pinealectomy in hamsters. Animals kept in long photoperiods were pinealectomized and subsequently exposed to short photoperiods. All adaptive changes, which are usually a consequence of short photoperiods, could not be observed. PT-specific cells exhibited the morphological appearance of cells, as well as mRNA and protein expression (achain and 6-TSH), which are typical for long-photoperiod light regimes (Fig. 10).

Data of both experiments emphasize the hypothesis that melatonin levels regulate the secretory activity of FT-specific cells. In addition to seasonal and circadian rhythms, antithyrotropic effects of pineal melatonin are discussed to be mediated by the pars tuberalis. A coupling between pars tuberalis function and thyroid activity was concluded from experiments showing marked cytological and immunohistochemical alterations of fetal pars tuberalis cells following maternal application of T4 and propylthiouracil (Bockers et al., 1990; Holbeck et al., 1993). In adult animals no corresponding changes were observed. As postulated by Vriend (1983), Hoffmann (1985), and W e n d and Wasserman (1986), short photoperiods may exert inhibitory influences on the neuroendocrine thyroid axis. Ottenweller et al. (1987) found lowered plasma levels as well as suppressed rhythms of testicular hormones, thyroid hormones, and adrenal hormones.

B. Photoperiod-Induced Circadian Alterations Circadian variations of density of melatonin receptors, which are inversely related to plasma melatonin levels, were observed in the sheep PT (MassonPevet et al., 1993; Piketty and Pelletier, 1993). Recently, Gauer et al. (1994) could show identical daily rhythms of melatonin receptor density in the PT and suprachiasmatic nucleus of the rat. The regulation of both these receptor sites, however, seems to be different. Whereas in the suprachiasmatic nucleus the light-dark transition induces variation of receptor density, pars tuberalis receptor density depends on variations of plasma melatonin levels. A light-induced stimulation of immediate early genes in the photorecipient zone of the suprachiasmatic nucleus has been reported by Sumov6 and Illnerov6 (1996). These results also

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suggest direct functional changes of the circadian pacemaker following photic induction.

C. Photoperiodic Influences in Men Analogous reactions to photoperiodic influences are also recorded in men: prolongation of nightly periods in young adult volunteers together with a longer duration of nocturnal melatonin secretion also extends the typical night periods of prolactin, cortisol, or T3 concentrations and increases duration of sleep or nocturnal period of low temperature (Wehr, 1991; Wehr et al., 1993). In chronic exposure to long nights the authors found a reduction of hypophysial volume, and under following short nights an increase to initial values took place. Return to short nights was also accompanied by the subjective feeling of an increase in fatigue and a decrease in energy. Results emphasize that in humans there is the capacity to detect and to respond to changes in photoperiod. From other experiments it has been reported that melatonin may amplify the nocturnal increase of PRL (Waldhauser et al., 1987; Okatani and Sagara, 1993) and probably that of TSH (Melis et aZ., 1995). There has long been evidence of an antigonadotropic action of melatonin. Several case reports demonstrate the fact that melatonin deficiency, for instance, following tumor destruction of the pineal, activates the pituitarygonadal axis and in juvenile patients may be followed by precocious puberty (Fraschini et aZ., 1971; Brzezinski, 1997). Vice versa, pubertas tarda is combined with high melatonin concentrations. Melatonin levels are also increased in women with hypothalamic amenorrhea and in men with hypogonadotropic hypogonadism (Brzezinski, 1997).Elevated melatonin secretion, however, in women with secondary amenorrhea may be caused by low estrogen levels, because administration of estrogen significantly reduces nocturnal melatonin secretion (Okatani and Sagara, 1995). Voordouw et aZ. (1992), in a study with young normal women medicated with large daily doses of melatonin for 4 months, found inhibitory effects on ovarian function with a suppressed midcycle surge of LH secretion. Epidemiologic studies in different geographic regions point to photoperiod-influenced seasonal differences also in humans (Kauppila et aZ., 1987). Among people living in the arctic, gonadal function and conception rates were found to be lower in the dark winter months than those in summer. The seasonality of conceptions seems to be influenced by environmental factors, especially by photoperiod and temperature (Ronneberg and Aschoff, 1990). In further studies of the melatonin rhythm in arctic urban residents, Stokkan and Reiter (1994) demonstrated a consistent pattern

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with significantly elevated melatonin levels-both peak and duration-in January. Measurement of reproductive hormone concentrations in normal men provided evidence that there exists a seasonal rhythm of gonadotropins and testicular hormones with a significant increase in June and a nadir in August (Meriggiola et aL, 1996). In summary, a mediating and modulating function of the PT-besides hypothalamic control-in the photoperiod/melatonin-dependentchanges of endocrine functions also seems likely in men.

V. Physiological Significance: Pars Tuberalis as the Major “Zeitgeber” of Pars Distalis Activity? The PT is a unique structure. It is the only region which consistently shows a high density of melatonin receptors in all mammals, with the possible exception of the human pituitary. As the target .of melatonin, the PT is likely to act as the major interface in mediating this photoperiodic message to the endocrine system (Wittkowski et al., 1992,1995). The demonstrated sequence of secretory differentiation and expression of TSH subunits in fetal rats together with the occurrence of melatonin receptors accentuates the hypothesis that the PT has a precursor function for the PD and may be necessary for entrainment of biorhythmic alterations of the endocrine system (Stoeckel et aL, 1994; Wittkowski et aZ., 1997). Melatonin blocks secretion of putative PT-specific secretion products which are distributed in the PD by the portal vessel system. Such an intrahypophysial regulatory concept may explain the widespread actions of melatonin on endocrine targets. A. Participation in Regulation of Reproductive Function

Secretory activity of lactotrophs-under basal conditions-is inhibited by dopaminergic neurons projecting from the arcuate nucleus to the median eminence, the neural and the intermediate lobe (Fig. 11). Dopamine, the prolactin-inhibiting factor, binds to Gi-protein-coupled D2 receptors and acts on different intracellular signaling cascades (Ben Jonathan, 1996). In several species, melatonin has been found to exert an inhibitory effect on PRL secretion. Changes of PRL concentration or cytological alterations of lactotrophs reflect alterations in photoperiod or the administration of melatonin (Wang et al., 1991;Lincoln and Clarke, 1994). With their studies in hypothalamo-pituitary-disconnected (HPD) rams, Lincoln and Clarke

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(1994) proved that the melatonin signal is translated primarily within the pituitary gland and not by the hypothalamus. HPD rams as the controls show an increase of prolactin under long photoperiods and a decrease under short ones. Furthermore, under long days they react to melatonin treatment with a reduced prolactin secretion. These results favor the hypothesis that the PT is the melatonin target for photoperiodic modulation of this hormone. A similar conclusion may be drawn from lesion experiments (Maywood and Hastings, 1995)of the melatonin binding sites of mediobasal hypothalamus with the result that these sites are not essential for photoperiodic control of the lactotropic axis. On the other hand, the authors found evidence that an intact mediobasal hypothalamus is necessary for photoperiodic control of gonadotropic activity. The generation of PRL surges requires a stimulatory action by local or systemic agents. Numerous compounds have been proposed for such a releasing action on lactotrophs such as, for instance, oxytocin or TRH. The identity of a PRL-releasing factor (PRF) is still unknown. In light of recent data, PRF might be a product of PT-specific cells (Fig. 11). Among the spectrum of their secretory products, trophic and stimulatory action on lactotrophs is especially ascribed to the free a chain (see section 1II.A; Begeot et aZ., 1984; Stoeckel et aZ., 1994; Van Bael and Denef, 1996). A significantly increased PRL secretion triggered by two PT-derived peptide factors of (different size but) unknown chemical identity was reported by Morgan et al. (1996). Also, experimental data by Bockers et al. (1997) support the hypothesis of a link between PT-specific cells and PD cells, especially lactotrophs: When exposed to short photoperiods, hamsters and other long-day breeders undergo gonadal regression. With chronic exposure to short days, however, the animals become photorefractory and gonadal recrudescence occurs. Among hypophysial hormones, cellular mRNA and protein levels only of PRL (not LH or FSH) were significantly reduced during gonadal regression and returned to long-photoperiod levels with the beginning of recrudescence. A quite similar down- and upregulation concerned a-chain mRNA and protein of PT-specific cells. Interestingly, the increase of a chain precedes the surge of PRL secretion in the PD. This mechanism is apparently dopamine-independent, because tyrosine hydroxylase-as a key enzyme of dopamine synthesis-is also downregulated in the phase of regression and increased with beginning recrudescence. In another study with HPD rams, Lincoln and Clarke (1995) examined whether dopamine plays a role in relaying the influence of melatonin. Following surgical disconnection, brornocriptine, a dopamine agonist, sulpirid, a dopamine antagonist, and TRH, were applied, and their effects on PRL secretion measured. Bromocriptine caused a decrease in the plasma prolactin level in HPD rams indicative for dopamine D2 receptors on lactotrophs in the isolated pituitary gland. Increased PRL secretion was

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measured following TRH application, whereas sulpirid, in contrast to a stimulatory effect in controls, showed no effects in HPD rams. This lack of response to dopamine antagonist underlines the absence of endogenous dopamine mechanisms regulating the secretion of PRL in experimental disconnection. Sulpirid does not increase PRL level by antagonizing suppressive action of melatonin. Thus, there is evidence that melatonin acts in the pituitary gland through a dopamine-independent mechanism. Direct effects of melatonin on PD lactotrophs are improbable because these cells are devoid of the known membrane receptors. Alternate pathways by binding of melatonin to nuclear receptors may be excluded in the PD as in the PT (Hazlerigg et al., 1996). In conclusion, expression and release of free a chain and/or other secretory products by PT-specific cells cause stimulation of PRL expression in the PD. Both a chain and PRL have been shown as sensitive indicators (precursors?) of photoperiod-dependent changes. Seasonal as well as nutritional factors may be relevant at this level of the hypothalamo-pituitary axis. Studies on functional receptors of IGFl in the PT tentatively indicate another role for this region. As melatonin is the target of photoperiodic signals, circulating levels of IGFl may mediate the effects of nutrition at the level of the PT and influence reproductive functions (Hazlerigg et al., 1996; Williams et al., 1995).

B. Nonspecific Influences on Pars Distalis Activity Morgan et al. (1996) demonstrated that an increased secretion of PRL from PD primary culture cells is inducible by a PT-conditioned medium. Also, stimulatory effects could be observed in experiments with different tumor cell lines of the PD (including GH3 and NIH 3T3 cells). These results indicate that PT-specific factors-so-called tuberalin (s)-influence PD functions in a more comprehensive manner and mediate temporal programs to another endocrine axis as well. Possible regulatory control of the release

FIG. 11 Diagrammatic synopsis of regulatory mechanisms which enclose PT-specific cells as target and effector cells. PT-specific cells are under inhibitory control of pineal melatonin, whereas the nature of stimulatory influences is still a matter of speculation. Numerous recent data support a link between PT-specific cells and PD cells, especially lactotrophs. A secretory product of PT-specific cells, the so-called “tuberalin” is likely to act as a releasing factor on the secretory activity of lactotrophs or possibly other PD cells. Mel, melatonin; SCN, suprachiasmatic nucleus; DA, dopamine; RH/IH, releasindinhibiting hormones; Rf, releasing factor; Lact, lactotroph; Gon, gonadotroph; Thyr, thyrotroph; Oth, other PD cells; dotted lines, hypothetical connections.

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of neurosecretory neurons by PT-specific cells also has to be taken into account. Discussion of a primary role of the pars tuberalis in seasonality without a “circannual timer” in the hypothalamus has to include peculiarities of intracellular signal transduction. Duration of melatonin signal has been shown to influence sensitivity of PT-specific cells. Such a mechanism may account for quite different effects of long and short photoperiods.

VI. Concluding Remarks In the last 10 years the PT has evolved from a neglected appendix of the adenohypophysis to an important structure that is very likely to take part in chronobiological regulation of the endocrine system. This progress was mainly triggered by the finding of seasonal variations of cellular activity and the proof of dense expression of melatonin receptors solely on hypophysial PT-specificcells. Further studies may establish the hypothesis of the PT as a receiver for photoperiodic signals and concentrated on biorhythmic alterations, secretory products, and regulatory aspects of gene expression in PT-specific cells. As described in this review, despite numerous confirming data the concept of the PT as a photoperiod-dependent target which substantially influences endocrine activities in the PD is still full of gaps and poses the following essential questions: 0 Assuming that (Y chain and P-TSH are not the only active substances secreted by PT-specific cells, what is the identity of “tuberalin(s)”? 0 How is the activity of PT-specific cells regulated? Specifically, which factor exerts a stimulating influence on gene expression and by which intracellular mechanisms? 0 Are the target cells of PT-specific secretory substances only lactotrophs or also other cells within the PD (intrahypophyseal regulatory mechanism) or outside this organ? 0 Whereas especially in photoperiod-sensitive species numerous results confirm the concept of a special role of this part of the hypophysis, it still remains uncertain whether this holds true for other species. This question also concerns regulation of human circadian rhythms which are apparently influenced by melatonin.

Acknowledgments The authors thank Mrs. A. Lewedag, Mrs. S. Loheide, Mrs. J. Mollers, and Mrs. I. Winkelhus for their skillful assistance in preparing, and Mrs. C. Bramswig for critically reading the

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manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFGiWi 558/5-2).

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Polarization of the Na+,K+-ATPasein Epithelia Derived from the Neuroepithelium Lawrence J. Rizzolo Section of Anatomy, Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06520

The neuroepithelium generates a fascinating group of epithelia. One of their intriguing properties is how they polarize the distribution of the Na+,K'-ATPase. Typically, this ion pump is concentrated in the basolateral membrane, but it is concentrated in the apical membranes of the retinal pigment epithelium and the epithelium of the choroid plexus. A comparison of their development with that of systemic epithelia yields insights into how cells polarize the distribution of this and other membrane proteins. The polarization of the Na',KC-ATPase depends upon the interplay between different sorting signals and different types of polarity mechanisms. These include intracellular targeting signals that direct the delivery of newly synthesized proteins, and maintenance signals that stabilize proteins in the proper membrane domain. Conflicting signals appear to be arranged in a hierarchy that can be rearranged as cells respond to certain environmentalstimuli. Part of this response is mediated by changes in the distribution and composition of the cortical cytoskeleton. KEY WORDS: Cell polarity, Na+,K'-ATPase, Pigment epithelium, Choroid plexus, Ciliary body, Development.

1. Introduction The neuroepithelium lines the neurotube. During development the neuroepithelium proliferates and thickens to form the central nervous system, but the lumen persists as the lumen of the ventricular system. Generally, the lumen is lined by the ependyma, a permeable and epitheliod monolayer. In several exceptions to this general scheme, the neuroepithelium stops Infernofional Review of Cytology, Vol. 185

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proliferating and forms a relatively impermeable epithelial monolayer. Like all simple epithelia, these monolayers are polarized. Their apical and basal surfaces interact with different environments, have different functions, and consequently have different protein compositions. A central question in cell biology is how the polarized protein distribution is generated and maintained. Epithelial polarity varies with the environment and function of the epithelium. Because epithelia derived from the neuroepithelium border a fenestrated capillary bed, they function as part of the blood-brain barrier. Three regions are illustrated in Fig. 1. Despite their common origin and function as the blood-brain barrier, these epithelia have unique transport properties. A striking difference is how they transport water. The simplest structure is the choroid plexus found in the ventricles. The epithelium of the choroid plexus is corrugated to form villi that encase a tuft of capillaries. The epithelium continuously secretes cerebral spinal fluid, which circulates through the ventricular system. Two other epithelia form from the optic vesicle. The optic vesicle is a diverticulum of the neurotube that invaginates to form a double layered cup. Most of the inner layer proliferates to form the neural retina, but most of the outer layer forms a monolayer, the retinal pigment epithelium (WE). This creates an unusual phenomenon where the apical membrane of the RPE interacts with a solid tissue. The space between the W E and the neural retina remains continuous with the lumen of the third ventricle, but the RPE helps maintain contact with the neural retina by absorbing fluid. Failure to absorb fluid results in a detached retina. The most unusual region is the epithelial lining of the ciliary body, which forms the rim of the optic cup. Here, both the outer and the inner layers of the cup form monolayers. The apical membranes of the two monolayers are apposed and joined by gap junctions. This metabolic and electrical link allows neighboring cells from the two layers to function as a unit. Like the RPE and continuous with it, the outer layer contains pigment granules and is named the pigmented epithelium. The inner monolayer is the nonpigmented epithelium. As a unit, the ciliary body secretes the aqueous humor that nourishes the lens and cornea. A driving force for transepithelial transport is the Na+,K+-ATPase.By regulating the transepithelial flux of ions, it helps control water movement. Most epithelia distribute this pump to the basolateral membrane, but the RPE and epithelium of the choroid plexus distribute the Na+,K+-ATPase to the apical membrane (Wright, 1972; Bok, 1982; Masuzawa et aL, 1985; Ernst et aL, 1986; Gundersen et al., 1991; Rizzolo and Zhou, 1995). At first glance the epithelium of the ciliary body appears more typical, because the Na+,K+-ATPaseis basolateral (Ghosh et al., 1990;Coca-Prados et al., 1995). However, when one considers the fact that pigmented and nonpigmented cells function as a unit, with tight junctions principally in the nonpigmented

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layer, the Na+,K+-ATPaseis essentially nonpolarized (Raviola and Raviola, 1978; Wiederholt et al., 1991;Edelman et al., 1994). There are many reviews on the structure, function, and isoforms of the Na+,K+-ATPase(Schneider, 1992; Mercer, 1993; Fambrough et al., 1994; Levenson, 1994). This review examines mechanisms that regulate the distribution of the Na+,K+-ATPase with emphasis on epithelia derived from the neuroepithelium. Examination of these unique epithelia lends insight into the diversity of mechanisms that can regulate the distribution of this enzyme.

II. Polarity and Transepithelial Transport

The Na+,K+-ATPaseis an ion pump found in the plasma membrane of many cells. It performs an essential function by maintaining low intracellular concentrations of sodium and high intracellular concentrations of potassium. The pump is electrogenic, because each pump cycle exchanges three Na+ ions for two K+ions. The energy stored in these chemical and electrical gradients drives the transport of other ions and solutes across the plasma membrane and regulates cell volume. Besides these basic housekeeping functions, certain cells exploit these gradients for specialized functions, such as the transmission of action potentials. Simple transporting epithelia use the Na+,K+-ATPaseto provide energy for transepithelial transport. Transepithelial transport requires an asymmetric or polarized distribution of pumps, channels, and transporters (Almers and Stirling, 1984). To maintain the chemical gradients that are established by vectorial transport, tight junctions join the lateral membranes of neighboring cells and retard diffusion through the paracellular space (Anderson and Van Itallie, 1995). It is unimportant whether the Na+,K+-ATPaseis distributed to the apical or basal side of the tight junctions as long as its distribution is polarized. This is because a rich diversity of transport mechanisms complements the Na+,K+-ATPaseto transport any given solute in either direction across the epithelial monolayer. This can be illustrated with sodium absorption and secretion. Various epithelia do one or the other even though they all distribute the Na+,K+-ATPaseto the basolateral membrane. In absorptive epithelia such as the proximal kidney tubules, Na+ transporters are present in the apical membrane, such as the Na+/H+antiporter and various Na+coporters for glucose or amino acids (Giebisch and Wang, 1996). As sodium is actively pumped across the basal membrane against a concentration gradient, it passively diffuses into the cell across the apical membrane (Fig. 2A). In secretory cells such as in the respiratory tract, pancreatic, or glandular epithelia, a different arrangement of channels and transporters leads to secretion of NaC1, even with a basolateral Na+,K+-ATPase(Greger, 1996).

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B

C

Neural Retina

Aqueous Humor

Pars Plica FIG. 1 A schematic drawing of epithelia derived from the neuroepithelium. (A) The eye formed from a diverticulum of the third ventricle. The lining of the ventricle (ependyma and epithelium of the choroid plexus) is continuous with the RPE and ciliary epithelium. The lumen of the ventricle is continuous with the interphotoreceptor space that lies between the RPE and the neural retina (dark gray). The width of the lateral spaces between ependymal cells is exaggerated to emphasize that there are no tight junctions to limit diffusion between the lumen and the neural tissue. The choroid plexus is an invaginated structure that secretes cerebral spinal fluid into the lumen. Tight junctions present in the epithelium of the choroid plexus limit diffusion between the lumen and the fenestrated capillaries (dashed line). The

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(+) TEP

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(-)TEP

2K+

Na' Absorption

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FIG. 2 Comparison of absorptive and secretory epithelium. (A) Sodium absorption is illustrated for the proximal kidney tubule (modified from Giebisch and Wang, 1996). Sodiumxolute (S) coporters and a sodium:hydrogen antiporter in the apical membrane allow sodium to enter the cell from the apical membrane as the Na',K+-ATPase pumps sodium out the basolateral membrane. The transepithelial electrical potential (TEP)is positive on the apical side, which favors the diffusion of cations across the tight junctions in the apical to basal direction. (B) Sodium secretion is illustrated by respiratory epithelia (modified with permission from Greger, 1996).The Nat,Kt-ATPase provides the energy to drive C1- into the cell from the basal side, as chloride channels allow C1- to diffuse out the apical side. This creates a TEP that is negative apically and favors the basal to apical diffusion of sodium across the tight junctions.

The apical Na+ transporters are replaced by C1- channels, and the high concentration of extracellular Na+ on the basal side is used to drive C1into the cell (Fig. 2B). As C1- passively diffuses out through apical channels, Na+ passively diffuses across through tight junctions to maintain electrical neutrality.

iris and retinal capillary bed have been omitted for clarity. (B) An enlarged region from panel A to illustrate the relationship between the outer segments of photoreceptors (a) and microvilli of the RPE (b). Lateral infoldings (c) appose a basement membrane and fenestrated capillary bed. An apical junctional complex (d) includes tight junctions that limit diffusion between fenestrated capillaries and the interphotoreceptor space. The epithelium is laden with pigment granules (e) that give the epithelium its name and with mitochondria (f). Modified from Zhao et al. (1997). (C) An enlarged region from panel A to illustrate the structure of the ciliary epithelium, which secretes aqueous humor into the anterior chamber of the eye. A pigmented monolayer lacks tight junctions and apposes a nonpigmented monolayer that is linked by tight junctions. The apical membranes of two monolayers are joined by gap junctions (not shown). The epithelium is flattened (pars plana) proximal to the neural retina but infolded (pars plica) distally.

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The only epithelia that distribute the Na+,K+-ATPaseto the apical membrane are derived from the neuroepithelium. As noted earlier, these epithelia either secrete or absorb fluid. The need for an apical Na+,K+-ATPase reflects the special needs of neuronal tissue. For example, photoreceptors require high extracellular sodium concentrations to maintain the dark current and maintain the volume and composition of the interphotoreceptor space (Gallemore et al., 1997).The problem is the mechanisms that maintain the interphotoreceptor space deplete it of sodium. High apical Naf concentrations drive cotransporters and antiporters in the apical membrane of RPE (Fig. 3A). Depletion of Na+ is exacerbated by secretion of C1- across the basal membrane and an apical-positive transepithelial electrical potential. This favors an apical to basal leak of NaC across the tight junction. Consequently, the mechanisms for sodium secretion described in Fig. 2B cannot apply. An apical Na+,K+-ATPaseis the only way to support this constellation of transport functions and maintain high extracellular-sodium concentrations in the subretinal space. In contrast to the RPE, which is both secretory and absorptive, the choroid plexus is a secretory epithelium (Fig. 3B). Nonetheless, analogous constraints to secrete the proper balance of ions require an apical Na+,K+-ATPase(Saito and Wright, 1987; Keep et al., 1994). It is important to emphasize that these are not simply upsidedown epithelia. Although they share an apical Na+,K+-ATPase,the RPE and choroid plexus polarize the K+ and C1- channels and the Na+/H+ antiporter differently. Furthermore, the RPE polarizes other proteins in a fashion similar to other epithelia (Gundersen et al., 1991; Bok et al., 1992; Rizzolo and Joshi, 1993; Rizzolo et al., 1994; Huotari et al., 1995; Rizzolo and Zhou, 1995). Less well understood is the epithelium of the ciliary body (Fig. 3C). Despite their derivation from the neuroepithelium, the pigmented (PE) and nonpigmented (NPE) epithelia polarize the Na+,K+-ATPaseto the basolateral membranes (Okami et al., 1989).The unique features are (1)the apical membranes of the two epithelial layers are apposed and connected by gap junctions and (2) the pigmented layer lacks tight junctions (Raviola and Raviola, 1978). Consequently, the two layers act like a syncytium that couples an absorptive (PE) cell with a secretory (NPE) cell to secrete aqueous humor (Wiederholt et al., 1991; Edelman et al., 1994). Secretion occurs even though the distribution of the Na+,K+-ATPase about a PE :NPE cell pair is minimally polarized. Okami et al. (1989, 1990) determined the relative distribution of the Na+,K+-ATPasebetween the PE and the NPE cells and between the apical and the basolateral membranes of WE. The polarity in the PE :NPE pair was 2 : 1 compared with 6 :1in the RPE. The reason for this minimal polarity may lie in the expression of different Na+,K+-ATPaseisoforms in the PE and the NPE (Ghosh et al.,

B

A Interphotoreceptor Space Na+Anion

C

Aqueous Humor

Cerebral Spinal Fluid

2K+ ,Na'

Fenestrated Capillary Beds FIG. 3 Ion transport in derivatives of the neuroepithelium. (A) W E (modified from Prog. Ret. Eye Rex 16, Gallemore el al., Retinal pigment epithelial transport mechanismsand their contributions to the electroretinogram,509-566, copyright (1997) with permission from Elsevier Science). (B) Choroid plexus (modified from Keeper al., 1994). (C) Ciliary epithelium (modified from Edelman et al., 1994). See text for details.

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1990). In principal, differential regulation of the isoforms could generate a functional polarity that could be regulated on a short time scale. These epithelia illustrate the diversity of epithelial polarity that is required to accomplish tissue-specific functions. This diversity implies that multiple mechanisms regulate polarity and that alternative mechanisms may regulate any given protein. The diversity of mechanisms also allows plasticity, the ability of a cell to change its polarity. The plasticity of Na+,K+ATPase polarity is discussed below.

111. Plasticity: Remodeling Cell Polarity Epithelia continuously remodel their polarity during development and in response to physiologic or pathologic change. This plasticity is induced by the environment (Rodriguez-Boulan and Powell, 1992; Eaton and Simons, 1995; Drubin and Nelson, 1996). Interactions with the basal membrane induce the polarization of some apical membrane proteins. Additional interactions along the lateral membranes induce the polarization of many basolateral proteins. The effects of physiological change were illustrated by kidney tubules in vivo and in culture (Al-Awqati, 1996). An altered acid load caused intercalated cells to switch the polarity of the H+-ATPase and a Cl-/HC03- exchanger. Extracellular interactions do not precipitate wholesale reversals of polarity. Instead, they fine-tune polarity to enable the epithelium to function properly in altered circumstances.

A. Plasticity during Development The plasticity of Na+,K+-ATPasepolarity was examined during the development of kidney tubules. The initial event involved the conversion of mesenchyme into epithelium. Studies in organ culture demonstrated that the process requires laminin and its receptor, a6pl integrin (Klein et al., 1988; Sorokin et al., 1990). In vivo studies showed that the Na+,K+-ATPasewas expressed after epithelial formation and that its initial distribution was apicolateral or nonpolarized (Holthofer, 1987; Minuth et al., 1987; Avner et al., 1992). The lack of polarity was evident despite the presence tight junctions that separate the apical from the lateral membrane. The number of tight junctional strands increased as the Na+,K+-ATPasebecame basolaterally polarized, but there was no evidence that the two events were causally related. These studies did not address rearrangements of the cortical cytoskeleton. Such rearrangements have attracted interest, because of an

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association between the Na+,K+-ATPaseand the cytoskeleton that will be discussed in section VI. Studies of intestinal development illustrate the specificity of basal interactions and distinguish polarity of the Na+,K+-ATPasefrom other aspects of polarity (Neutra and Louvard, 1989). Early in development, the intestinal epithelium of mammals is stratified. Like kidney and mammary epithelia, interactions with the underlying substrate induce differentiation into a monolayer. This was demonstrated in culture where enterocytes required a feeder layer of fibroblasts (Kedinger et al., 1987a,b). Both cell types contributed to the basement membrane that formed beneath the epithelium. Although anti-laminin antibodies inhibited this process, purified laminin could not substitute for the feeder layer. Other laminin-rich, basement membrane preparations (e.g., Matrigel) were also ineffective. This contrasts with the ability of Matrigel to promote the differentiation of mammary epithelial cells (Stoker et al., 1990) and indicates the importance of tissuespecific interactions between the enterocytes and the feeder cell layer. Additional studies of intestinal development illustrate the importance of lateral interactions and the cortical cytoskeleton (Amerongen et aL, 1989). Spectrin and E-cadherin were evident during the stratified epithelial stage before the Na+,K+-ATPase.E-cadherin is a transmembrane protein whose extracellular domain binds the E-cadherin of an adjacent cell, while its cytoplasmic domain binds a protein complex that includes spectrin. Spectrin and E-cadherin colocalized in regions of cell-cell contact. Both were absent from the free, apical surface and basal membranes that contacted the basal lamina. When expression of the Na+,K+-ATPasewas upregulated, it colocalized with spectrin and E-cadherin. As a monolayer formed, the Na+,K+ATPase and spectrin colocalized along the lateral membranes. When basal interactions were disrupted by dissociating epithelial sheets from the basal lamina, the Na+,K+-ATPaseand spectrin also appeared in the basal membrane. As the number and length of the microvilli increased to form a brush border, a pool of spectrin and actin formed a terminal web at the base of the microvilli. The distinction between the terminal web and the cytoskeleton of the microvilli becomes important when we consider the polarization of neuroepithelial derivatives. In intestine, the cytoskeleton within the microvilli affected the polarization of a subset of apical membrane proteins (Costa de Beauregard et al., 1995). By inhibiting the formation of microvilli, these authors prevented the polarization of sucraseisomaltase, but not several other apical membrane proteins. These data suggest three types of mechanisms that polarize membrane proteins: those that use the microvillar or lateral membrane cytoskeleton, and those that are independent. Multiple mechanisms of polarity were also evident during the development of RPE (Rizzolo, 1997). Many polarized features appeared before

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the Na+,K+-ATPasebecame apically polarized. Gross structural features were present when the epithelial monolayer was formed. Apical junctional complexes that contained ZO-1 and occludin were evident near the apical end of the lateral membranes (Fujisawa et al., 1976; Williams and Rizzolo, 1997). The apical membrane was formed by microvilli, and centrosomes were subjacent (Rizzolo and Joshi, 1993). A microtubule organizing center was associated with the centrosomes and bundles of microtubules paralleled the apicobasal axis, as observed in other simple, transporting epithelia. The integrin a 3 p l was polarized to the basal membrane (Rizzolo et aZ., 1994). Other proteins shifted their distribution as development proceeded. Different shifts occurred during early, intermediate, and late stages of development. Initially, the Na+,K+-ATPasewas found in the apicolateral membranes, along with spectrin and ankyrin. In the early stage, the Na+,K+ATPase became apically polarized without a shift in the distribution of spectrin or ankyrin (Rizzolo and Zhou, 1995). At the end of the early stage, the integrin a6pl shifted from nonpolarized to basally polarized (Rizzolo et aZ., 1994). In the intermediate stage, infoldings appeared in the basal membrane, and microvilli began to elongate. In rat, the elongation of microvilli was accompanied by an increase in the apical pool of the protein PE2/CE9 (Marmorstein et aZ,, 1996, 1997). In chick, the REMP protein became basally polarized (Philp et aZ., 1995). With the formation of infoldings, a number of proteins shifted their distribution to include a pool in the basal membrane. These proteins included spectrin, ankyrin, and the 5 A l l antigen. The final distribution of these proteins was nonpolarized. Notably, the apical pool of spectrin was confined to the terminal web region and Zhou, 1995). at the base of the microvilli (Huotari et aZ., 1995; -010 Therefore, in RPE the Na+,K+-ATPaseis segregated from spectrin. The role of tight junctions in polarization was unclear. Throughout development, there were structural and compositional changes in the tight and adherens junctions (Sandig and Kalnins, 1990; Williams and Rizzolo, 1997).

B. Plasticity during Pathology: Polycystic Kidney Disease The modulation of Na+,K+-ATPasepolarity has been examined in autosoma1 dominant polycystic kidney disease (Grantham, 1996; Bacallao and Carone, 1997). Cysts arise from diverticula that form along the nephron and eventually lose their connection to the nephron. Within these cysts, some investigators observed a shift in the distribution of the Na+,K+ATPase and a corresponding shift in the distribution of spectrin and ankyrin (Wilson et al., 1991; Avner et aZ., 1992; Carone et aZ., 1994; Barisoni et aL, 1995; Ogborn et aL, 1995). The lack of Na+,K+-ATPasepolarity was reminiscent of early kidney development. The shift in polarity was selective, because tight junctions remained intact and the polarity of other proteins

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was unaffected. These included GP330, an apical membrane antigen, and Band 111, a basolateral transport protein (Wilson et al., 1991; Wilson, 1997). A further indication that polarity was retained was that the cysts secreted fluid and concentrated NaC1. It is controversial whether the depolarization of the Na+,K+-ATF'asecaused sodium secretion. Studies of humans and mice showed prominent staining of apical Na+,K+-ATPasein early cysts that was diminished in late-stage cysts (Wilson et aZ., 1991; Barisoni et al., 1995). In other studies, only 25% of the cystic epithelium was depolarized. In rat, altered polarity was observed in late, but not early, cysts (Gretz et aL, 1996). In mouse and cat models, the expression was variable with basolateral, nonpolarized, and low-expressingcells present (Barisoni et al., 1995; Eaton et aL, 1997). They observed no correlation between polarity and the age or size of the cyst. Subsequent human studies examined cysts with a basolateral distribution of the Na+,K+-ATPaseand observed the expression of an apical cystic fibrosis transmembrane conductance regulator-like chloride channel (Brill et al., 1996; Grantham, 1996). These studies support a model similar to NaCl secretion in the respiratory system or intestinal crypts (Fig. 2B). The take-home lesson is that wherever a shift in protein distribution was observed, it was restricted to a subset of proteins. This contrasts with models of renal ischemia where tight junctions dissociated and cells lost all polarity and transport function (Bacallao et al., 1994; Fish and Molitoris, 1994; Mandel et al., 1994). Genetic evidence supports the suggestion that some cysts resemble early stages of tubule development, where the Na+,K+-ATPasehas an apicolatera1 or nonpolar distribution. One genetic defect for autosomal dominant polycystic disease is in the polycystin gene (Grantham, 1996; Bacallao and Carone, 1997). Polycystin is developmentally regulated. It is present in low levels in adult tissue, but levels are elevated in fetal or diseased tissue. Structure predictions based on the primary sequence indicate that polycystin is a transmembrane protein whose extracellular domain contains dileucine motifs, lectin binding domains, and immunoglobulin repeats. These features suggest that polycystin mediates cell-cell or cell-matrix interactions that potentially regulate cell polarity or gene expression. For example, preliminary reports suggest that the expression of fetal isoforms of Na+,K+ATPase might account for the shift in polarity, because different isoforms may encode different polarization signals (Wilson, 1997). Although not definitively demonstrated in polycystic disease, I will examine the evidence for this hypothesis, as it regards derivatives of the neuroepithelium.

IV. General Models for Epithelial Polarity The preceding discussion emphasized the diversity of epithelial polarity and its plasticity. To allow this flexibility,multiple mechanisms have evolved

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to target and maintain proteins in the apical, lateral, or basal membranes. Early studies focused on targeting signals that are encoded within each protein to direct it to its proper location. It now appears that a protein may express a hierarchy of signals that can be interpreted differently by different epithelia or by the same cell in different circumstances. This section briefly reviews extracellular interactions that regulate cell polarity and intracellular mechanisms that target and maintain a protein in its proper location. Mechanisms specifically related to the Na+,K+-ATPasewill be discussed in greater detail in section VI.

A. Extracellular Interactions That Regulate Polarity The role of the extracellular environment has been studied in culture. Single-cell suspensions of Madin Darby canine kidney (MDCK) cells lacked polarity until the cells formed cysts (Wang et aL, 1990a,b).The cysts secreted basement membrane components into the lumen and formed apical microvilli on the ablumenal surface. To test whether extracellular matrix could modulate polarity, cysts were formed in collagen gels. These cysts were also polarized, but the apical membrane now faced the lumen. Further, when preformed cysts were transfered from aqueous solution to collagen gels, apical markers and microvilli relocated to the lumenal surface. This reversal of polarity was mediated by integrins (Schwimmer and Ojakian, 1995). Integrins are a family of cell adhesion molecules formed by Cllp dimers that span the plasma membrane. Beyond cell adhesion, these are signalingmolecules that transmit information about the environment (Haas and Plow, 1994; Dedhar and Hannigan, 1996). Integrins exert their effect through cytoskeletal rearrangements and tyrosine kinases that affect cellular organization and gene expression. Individual members of the /3l family can bind several different ligands, and many ligands can bind to different family members. This apparent redundancy actually leads to a refined cellular response. How a cell responds depends upon the composition of the extracellular matrix or basement membrane, the complement of integrins (and other receptors) that the cell expresses, and whether those integrins are in an activated state. Modulation of these factors likely regulates differentiation and polarity. The effects of lateral, cell-cell interactions were distinguished from cellbasement membrane interactions by culturing MDCK in low-calcium medium (Rodriguez-Boulan and Nelson, 1989). These conditions prevented cell-cell interactions principally by interfering with a calcium-dependent cell adhesion protein, E-cadherin. The remaining interaction with the culture substrate defined basal and free (or apical) surfaces and induced the polarization of an apical marker and apical microvilli. Polarization was

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further examined with membrane envelope viruses that bud specifically from the apical or basolateral membranes of polarized epithelia. When infected with influenza virus, virus budded from the apical surface as it would in normal epithelia. Despite apical polarity, basolateral markers were randomly distributed. Basolateral polarity was restored when calcium was added to the medium and cell-cell contacts were reestablished. E-cadherin induces basolateral polarity, but the role of other cell-cell adhesion proteins is less well studied (Drubin and Nelson, 1996; Marrs and Nelson, 1996). Like the integrins, cell-cell adhesion proteins interact with cytoplasmic complexes that affect the distribution of the cortical cytoskeleton. These complexes include many proteins that potentially mediate or regulate signaling,including Src and Yes kinases, protein-tyrosine phosphatase, small GTP-binding proteins, and catenins (Tsukita et al., 1991; Adamson et al., 1992; Behrens et al., 1993; Brady-Kalnay et al., 1995; Fagotto and Gumbiner, 1996). Recent attention has focused on P-catenin and plakoglobin, because their homologues are part of the WNT signaling pathway found in Drosophila, Xenopus, and mammals (Fagotto and Gumbiner, 1996; Peifer, 1997). Although E-cadherin is not expressed in the neuroepithelium, other family members are present (Grunwald, 1996). During the development of chick RPE, N-, R-, and B-cadherins are expressed at different times. Reagents to study cadherins in other species are not as well developed. In rodents, P-cadherin, a likely homologue of B-cadherin, and N-cadherin are present in RPE. N-cadherin has been described in cultured human RPE (Burke et al., 1996),but the presence of other cadherins cannot be formally eliminated. Undoubtedly, these cadherins regulate differentiation, and they likely regulate polarity.

6. lntracellular Mechanisms That Generate or Maintain Polarity Many of the mechanisms that regulate polarity have been reviewed extensively but will be summarized here (Rodriguez-Boulan and Powell, 1992; Le Gall et al., 1995; Mostov and Cardone, 1995; Drubin and Nelson, 1996; Keller and Simons, 1997). Mechanisms of polarity fall into two groups that are not mutually exclusive. One group transports or targets proteins to the apical or basolateral membranes, while the other maintains them in that location. For targeting, cells use vesicles to shuttle membrane proteins between intracellular compartments. I will ignore how different vesicles are transported specifically to the apical or basal membrane and focus on features of the transported protein that determine which type of vesicle it enters. One feature is an address label or sorting signal. Sorting signals are recognized by either of

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two sorting centers. The first center sends newly synthesized proteins directly to the apical or basolateral membrane. After synthesis in the endoplasmic reticulum and posttranslational modification in the Golgi apparatus, membrane and secretory proteins are delivered to the trans-Golgi network for sorting. The second center can either maintain polarity or mediate an indirect sorting pathway. For example, some proteins of the basolateral membrane are returned to an internal compartment by endocytosis. Proteins that enter this endosomal compartment follow one of three fates. They can be returned to the original plasma membrane, transcytosed to the opposite pole of the cell, or delivered to lysosomes for degradation. Many surface receptors are returned to the basolateral membrane, maintaining polarity. By contrast, some endocytosed proteins were intended for the apical membrane and are transcytosed. The process has been termed indirect sorting. The direct sorting pathway is favored by certain strains of MDCK (Gottardi and Caplan, 1993a; Mays et al., 1995). For apical proteins, only the indirect pathway is used by liver (Bartles et al., 1987). Other epithelia (e.g., intestinal cell lines, WE-J) use both pathways (RodriguezBoulan and Powell, 1992; Bonilha et al., 1997; Marmorstein et al., 1997). Both sorting centers apparently use similar signals and mechanisms (Keller and Simons, 1997). Other polarity mechanisms maintain proteins in the proper location. Tight junctions act as a fence that prevents the proteins and lipids of the apical and basolateral membranes from intermixing (van Meer and Simons, 1986). This barrier is insufficient to explain polarity for several reasons. As noted above, endocytic pathways can circumvent tight junctions. In culture models where junctions are not allowed to form (e.g., low-calcium medium), a certain degree of polarity is maintained (Rodriguez-Boulan and Nelson, 1989).In vivo, there is a difference in the composition of the basal and lateral membranes even though they are not separated by a bamer (Amerongen et al., 1989). A second maintenance mechanism anchors membrane proteins in place (Nelson and Veshnock, 1987; Morrow et al., 1989; Nelson and Hammerton, 1989).Linker proteins (e.g., ankyrin) bind membrane proteins to the cortical cytoskeleton that underlies the plasma membrane. Along the basal or lateral membranes, this cytoskeleton is formed by a lattice of spectrin and actin. The diversity of spectrin and ankyrin isoforms suggests that regional variations in the cytoskeleton might define and maintain subdomains of the basolateral membrane. A third alternative to maintain polarity is protein turnover. In some circumstances in some strains of MDCK cells, the Na+,K+-ATPaseis randomly distributed to the apical and basolateral membranes (Hammerton et al., 1991). Polarity is achieved, because the apical pool of the enzyme turns over more rapidly than the basolateral pool. The difference in half-life may be related to the association with the cytoskeleton.

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Targeting and maintenance mechanisms use signals encoded within the protein. Signals can involve protein structure, carbohydrate or lipid modifications, or physical properties. A protein may encode several signals. Recent progress has partially characterized signals that direct proteins to the basolateral membranes (Keller and Simons, 1997). These were identified in the cytoplasmic domains of transmembrane proteins. Deletion of these signals prevented basolateral targeting, and addition of the signals to nonsorted proteins conferred basolateral sorting. For many proteins, these signals overlap with, but are not identical to, the endocytosis signal that concentrates plasma membrane proteins into clathrin-coated pits. In each case, a tyrosine residue is critical. Other signals do not involve tyrosine or clathrinmediated endocytosis; in one instance a dileucine motif was implicated. Surprising, removal of basolateral targeting signals often resulted in apical targeting. Either transport to the apical membrane is a default or signaless, pathway, or weaker apical targeting signals were utilized. Accumulating evidence indicates that apical transport is mediated by signals. One mechanism is illustrated by lipid-linked proteins. These proteins are posttranslationally modified to replace the cytoplasmic and transmembrane domains with a glycosylphosphatidylinositol (GPI) linkage to a lipid anchor (Lisanti and Rodriguez-Boulan, 1990). These GPI-linked proteins, along with glycosphingolipids (sphingomyelin and glucosyl ceramide), are targeted to the apical membranes (Simons and Ikonen, 1997). The targeting of the glycosphingolipids and GPI-linked proteins may be related. The glycosphingolipids partition within the lipid bilayer perhaps because of their capacity to form hydrogen bonds. The hypothesis is that GPI-linked proteins partition into rafts of glycosphingolipid and that these rafts are excluded from the basolateral pathway. Evidence for the hypothesis is based on the inability to solubilize glycosphingolipids with Triton X100. This functional test was used to demonstrate that the influenza HA protein partitions into lipid rafts (Brown and Rose, 1992). This mechanism would segregate apical from basolateral membrane proteins in the Golgi, before packaging into transport vesicles. In this way, a physical property rather than amino acid sequence or higher ordered structure would form the sorting signal. A protein would be targeted to the apical membrane if it partitioned into the lipid raft or associated with one that did. This mechanism cannot operate in Fischer rat thyroid cells. In this cell line many GPIlinked proteins and glycolipids are basolateral, but other apical-specific proteins remain in the apical membrane (Zurzolo et aZ., 1993a). A second apical signal may include N-linked carbohydrate chains (Fiedler and Simons, 1995). This modification is required for many proteins to exit the endoplasmic reticulum, but early experiments indicated that it was not required for sorting. Two clues suggested that N-linked carbohydrates are actually involved. First, secretory proteins were secreted from MDCK cells

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in both directions if they lacked this modification, but apically secreted proteins were modified by N-linked sugars. Second, viral glycoproteins were often secreted apically if their cytoplasmic and transmembrane domains were removed. This may be because the N-linked carbohydrates constitute a weak apical signal that cannot compete with a dominant basolateral signal. Consonant with this view is the presence of a lectin homologue, VIP36, in apical transport vesicles. Although glycan ligands are yet to be identified for this putative carbohydrate binding protein, it is intriguing that it is present in a detergent-insoluble complex along with the apical marker, influenza HA. However, there are counterexamples. Gonzilez et al. (1987) found that some truncated viral glycoproteins can be secreted basolaterally as well as apically. Further, the hepatitis surface antigen lacks a cytoplasmic domain but is secreted basolaterally despite the presence of N-linked carbohydrate (Marzolo et al., 1997). Nonetheless, a hierarchy of signals that is interpreted differently in different cells may explain some of the controversy in this field. Much of the controversy comes from examining different experimental models, because different epithelia can use different mechanisms to achieve the same polarity. As noted above, different epithelia use direct, indirect, or a combination of these two sorting pathways. Even different strains of the same cell line can exhibit different mechanisms. For example, different strains of MDCK cells use different mechanisms to polarize the distribution of the Na+,K+-ATPase(Mays et al., 1995). Further confusion arises because different epithelia can interpret the same set of signals differently. This was most elegantly illustrated by expressing the human low-density lipoprotein receptor in a transgenic mouse (Pathak et al., 1990). The distribution of the receptor was basal in hepatocytes, basolateral in enterocytes, and apical in kidney tubules. A second example is the p subunit of the H+,K+-ATPase. When expressed in MDCK, this protein is polarized to the basolateral membrane, but when expressed in LLC-PK1, (a porcine kidney cell line) it is polarized to the apical membrane (Caplan, 1997). The diversity of signals and the examples and counterexamples cited above are likely related to the diversity of epithelia. Even though the basolateral membranes share many properties, the apical membranes are specialized to function in many different milieu. The need for diversity and plasticity defies attempts to overgeneralize mechanisms of polarity.

V. Diversity of Na+,K+-ATPaselsoforms and Tissue Specificity The Na+,K+-ATPaseis composed of a! and /3 subunits. A y subunit was also demonstrated in many tissues and lies near the ouabain binding site.

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Isoforms of the y subunit have not been described, and it has been ignored in studies of protein sorting. Multiple isoforms have been described for the a and 0 subunits, and these are expressed with different tissue specificity. To understand sorting, we must consider potential signals in the different isoforms of the a and polypeptide chains and their carbohydrate chains. This section focuses on structure and variability; the next focuses on mechanism. A. Structure and Topology The a and p subunits form a heterodimer, but it is unclear if dimers combine to form higher order complexes in v i v a The evidence supporting oligomers of dimers is controversial, because the a@dimer is fully functional (Blanco et al., 1994; Boldyrev and Quinn, 1994). In vertebrates, dimers must form in the ER for either subunit to be transported to the cell surface (Geering et al., 1989; Gottardi and Caplan, 1993c; Chow and Forte, 1995). This is typical of many oligomeric proteins and may be part of a quality control mechanism that leads to the degradation of misfolded proteins (Hammond and Helenius, 1995). In insects, dimerization is unnecessary for transport to the cell surface. This allowed Mercer and colleagues to exploit the bacculovirus system to demonstrate a specific association between a subunits (Blanco et al., 1994; Koster et al., 1995). Alpha subunits expressed alone exhibited an ATPase activity that was uninhibited by ouabain and independent of sodium or potassium. When any two isoforms of the a subunit were coexpressed, each isoform could be coprecipitated with antibodies that were specific for the other isoform. This suggests that (ap), tetramers may exist. The ability to form tetramers could be important for polarity in epithelia that express multiple isoforms. The putative association between a subunits was further examined by exploiting the similarity of the Na+,K+-ATPase and other P-type pump proteins. Pumps of this type (including the H+,K+-ATPase and Ca2+ATPase) form a phosphorylated intermediate and have a large ( a )subunit of approximately 100 kDa. They also share a similar membrane topology (Mercer, 1993). Hydropathy plots of their primary sequence predict that each large subunit spans the lipid bilayer 10 times. The large subunits are not glycosylated.The overall similarity in structure allows chimeric proteins to be constructed by swapping topologically equivalent segments. The rationale is that domain substitution is more likely to preserve tertiary and quaternary structure than deletion or point mutations. This and other recombinant DNA approaches allowed different functions to be assigned to different regions of the polypeptide chain (Fig. 4). The third cytoplasmic domain was responsible for specific a-a association (Koster et al., 1995).

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a-subunit I

P-subunit - 1 COOH

Catalytic Phospolylation Ankyrin a-subunit FIG. 4 Topological map of the Na+,K+-ATPase.The larger, a subunit with 10 transmembrane domains is shown to the left; the smaller p subunit with N-linked carbohydrate (CHO) is to the right. The membrane-spanning domains are numbered. The binding sites for ankyrin and other subunits of the Na+,K+-ATPaseare indicated next to the appropriate extramembranous domain. A basolateral sorting signal that has been localized to the fourth transmembrane domain is indicated by a gray bar. See text for details.

The forth extracellular domain and the extracellular domain of the p subunit were responsible for the a-fl association (Fambrough et al., 1994; Lemas et al., 1994a,b; Colonna et al., 1997). Ankyrin binding sites were identified in the second (major site) and third (minor site) cytoplasmic domains (Devarajan et al., 1994; Jordan et aL, 1995). As discussed in section VI, the technique of domain swapping was used by Caplan and colleagues to identify sorting signals. The Na+,K+-ATF'ase and H+,K+-ATPaseinclude fl subunits that also share several features. These subunits span the lipid bilayer one time with a cytoplasmic N terminus and an extracellular C terminus (Fig. 4). These subunits are glycosylated. Consistent with studies of the a! subunit, the site for u-fl association is in the extracellular domain (Geering et al., 1993; Hamrick et al., 1993).For the Na+,K+-ATPase,each isoform of the /3 subunit can bind each isoform of the a! subunit. There is evidence that the p subunit of the H+,K+-ATPasecan substitute for the p subunit of the Na+,K+ATPase, but this interaction must be weak in vivo (Gottardi and Caplan, 1993b,c; Lemas et al., 1994b).

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6.lsoforms and Their Distribution Three isoforms each have been described for the a and p subunits. The alp1 isoform is fairly ubiquitous, but expression of the others is more restricted (Schneider, 1992;Levenson, 1994).Besides cells that express only a l p l , multiple isoforms tend to be expressed by single cells. This makes it difficult to distinguish functional differences between the isoforms. Variation exists in the polarity and expression of individual isoforms within different regions of the neuroepithelium. The RPE and choroid plexus express the same isoforms. The only a subunit that has been detected is al, but the pl and p2 subunits are present (Zlokovic et aL, 1993; Gonzdez-Martinez et aZ., 1994; Ruiz et al., 1995, 1996). Quantitative PCR analysis of human RPE found equal amounts of pl and 62 mRNA. Therefore, each epithelium must express the alPl Na+,K+-ATPase,which is distributed to the basolateral membranes of kidney epithelium. Bok and colleagues cloned and sequenced the DNAs from human RPE to verify that different sbrting signals could not be encoded by minor sequence variation. Nonetheless, the data also suggest that a l p 2 is present, and p2 could express an apical targeting signal. As noted earlier, the a subunits may be able to self-associateto form higher order oligomers. A putative signal in 02 could then target an (al)filp2 complex to the apical membrane. This issue will be revisited in section V1.A. The distribution of the Na+,K+-ATPasein the ciliary epithelium is more complicated (Martin-Vasallo et aZ., 1989; Ghosh et al., 1990, 1991; CocaPrados et al., 1995). The pigmented layer that abuts the choroid expresses only the a1 and 01 subunits. There is a gradient of expression in the nonpigmented layer that abuts the aqueous humor. Expression of the a1 and pl subunits was undetectable by immunofluorescence in the pars plana (Fig. 1C) but increased distally in the proximal and distal regions of the pars plica. The a3 subunit was only observed in the distal region of the pars plica. The a 2 and p2 subunits were observed throughout the nonpigmented layer. These impressions were supported by Northern blots and immunoblots of the RNA and protein isolated from these regions. The remarkable observation was that each subunit was distributed to the basolateral membranes. Because the apical membranes of the pigmented and nonpigmented layers are apposed and linked by gap junctions, neighboring cells are metabolically and electrically coupled to form a functional unit (Fig. 3C). Although the functional differences between isoforms are not well understood, it appears that they may be differentially regulated (McDonough et al., 1992). Even though the distribution of total Na+,K+ATPase about an NPE:PE pair is nonpolarized, the asymmetric distribution of isoforms may lead to a functional polarity that can be regulated. From

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the perspective of this review, the notable finding is that the different derivatives of the neural epithelium interpret the sorting signals present in the isoforms in different ways. The al,pl, and /32 subunits can be maintained in either the apical or the basolateral membrane. However, there is a caveat to this argument. There is a difference between the p2 subunits isolated from retina and RPE. In WE, there is a leucine residue in place of proline at position 51 (Hernando et al., 1994; Ruiz et aZ., 1996). This could have a global effect on the structure of the cytoplasmic domain. It is unknown if the isoform expressed in the RPE is the same as that in the choroid plexus, and whether these differ from the isoform expressed in the ciliary epithelium.

W. Mechanisms That Polarize the Distribution of the Na+,K+-ATPase The preceding discussion illustrates the fact that the polarity of the Na+,K+ATPase is plastic and can be independent of the polarization of other membrane proteins. It also demonstrates that a variety of mechanisms can tailor epithelial polarity to meet the needs of different developmental or physiological states. The mechanism that localizes the Na+,K+-ATPaseto the basolateral membrane is likely shared by many epithelia and likely polarizes a subset of membrane proteins. If so, there must be a way to selectively modify or supersede this mechanism to selectively modify the distribution of the Na+,K+-ATPase.The various isoforms of the Na+,K+ATPase potentially express different sorting signals. But the observation that different epithelia can polarize the same isoform differently indicates that these signals can be interpreted differently. I will focus on three questions. Is a hierarchy of apical and basolateral sorting signals encoded in the Na+,K+-ATPase and interpreted differently by various epithelia? Do variations in the cortical cytoskeleton affect the polarity of different epithelia? What is the effect of the apical environment on polarity? A. lntracellular Sorting Signals Intracellular sorting signals are not absolute requirements for generating polarity. As noted earlier, misdirected protein can be degraded or inactivated after it arrives in the inappropriate membrane. Many studies indicate that each of these mechanisms can be used to regulate the distribution of the Na+,K+-ATPase.Some data suggest that intracellular transport is linked to the polarization of glycolipids, but this issue remains unresolved.

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To distinguish direct sorting from indirect sorting or selective protein degradation, several techniques were devised to label proteins when they first arrive at the apical or basolateral membranes. These combine pulseradiolabeling of nascent proteins with a second labeling technique that is specific for the apical or basolateral membrane (Zurzolo et al., 1993b). For example, biotinylation reagents will only label proteins on the cell surface. When polarized cells are grown on permeable filters, NHS-biotin can be added to the apical or basal sides. Diffusion of the labeling reagent to the opposite side is prevented by tight junctions, and the biotinylated proteins can be isolated with immobilized avidin. This approach was used with several clones of MDCK. The II/J clone delivered roughly equal amounts of Na+,K+-ATPaseto the apical and basolateral membranes. However, the half-life of the apical Na+,K+-ATPasewas less than lh, whereas the basolateral pool was much more stable (Hammerton et al., 1991; Zurzolo et al., 1993b). It was proposed that the interaction with the basolateral cytoskeleton stabilized the protein. Notably, the II/J clone failed to polarize the distribution of glycolipids (Mays et al., 1995). Using a different clone of MDCK that does polarize glycolipids, two studies demonstrated that it is possible to transport the Na+,K+-ATPase from the trans-Golgi network preferentially to the basolateral membrane (Zurzolo et al., 1993b). The first study used the inhibitor ouabain as the second label. A photoactivatable derivative of ouabain was added to either the apical or the basal side of MDCK. Exposure to UV light crosslinked ouabain to the Na+,K+-ATPasepresent in the corresponding membrane domain. There was no evidence of a transient apical pool. However, if the Na+,K+-ATPasedelivered to the apical membrane was inactive, it would not bind ouabain and would be undetected. Therefore, the experiment was repeated with the biotinylation technique. Neither technique detected the Na+,K+-ATPasein the apical membrane. The biotinylation technique was also applied to Fisher rat thyroid cells. Like the MDCK II/J clone, these cells do not polarize glycolipids. In contrast to the II/J clone, the Na+,K+ATPase was delivered preferentially to the basolateral membrane. These results inspired a search for an intracellular sorting signal. An elegant series of experiments identified a basolateral sorting signal in the a subunit (Caplan, 1997). These studies exploited the subdomainswapping strategy described earlier to identify binding domains in the Ptype ATPases (Fig. 4).The H+,K+-ATPaseis normally found in the apical membrane of gastric epithelial cells and retains this polarity when it is exogenously expressed in the LLC-PKlporcine kidney cell line. The H+,K+and Na+,K+-ATPaseshave been cloned and share 65% sequence identity. Recall that the strategy was to construct chimeras in which a transmembrane or an extra membranous, subdomain of one protein substituted for the other. Regions of minimal homology were selected as potential sorting

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signals. The first chimeras exchanged the N-terminal halves of each protein (Gottardi and Caplan, 1993~).Binding of the p subunits to the chimeras was selective and was determined by the C-terminal half of the chimera. For example, the H+,K+-j3subunit bound the chimera with the H+,K+-a C terminus. This finding corroborated earlier studies that placed the binding site in the extracellular loop between membrane domains 7 and 8 (Fig. 4; Lemas et aL, 1994a). By contrast, the N-terminal half of the a subunit determined sorting. The H+,K+-aN terminus directed the chimera to the apical membrane, but the Na+,K+-aN terminus directed the chimera to the basolateral membrane. More refined substitutions narrowed the sorting signal to seven amino acids within the predicted fourth transmembrane domain of the a subunit (Caplan, 1997). This is the first basolateral sorting signal to be identified in a transmembrane domain. It is distinct from another potential targeting site-the ankyrin binding site that is located in the first and second intracellular loops (Fig. 4). Recall that the lipid anchor of GPI-linked proteins served as an apical sorting signal by partitioning into glycosphingolipid rafts. Conceivably, the Na+,K+-ATPasebasolateral signal prevents partitioning into these rafts, while the homologous sequence in the H+,K+ATPase is soluble in glycosphingolipid. Notably, the II/J clone of MDCK, which was’unable to target the Na+,K+-ATPasedirectly to the basolateral membrane, also failed to target glycosphingolipids specifically to the apical domain. Support for this hypothesis comes from studies of the II/G clone of MDCK that does target the Na+,K+-ATPasedirectly to the basolateral membrane. When treated with fumonisin to inhibit the synthesis of sphingolipids, II/G no longer targeted the Na+,K+-ATPasebasolaterally. Targeting was restored when the drug was removed (Mays et al., 1995). The hypothesis fails to explain how Fisher rat thyroid cells polarize the Na+,K+ATPase by direct targeting. Further study will be required to determine the properties and mechanisms of this unique sorting signal. The presence of a dominant sorting signal in the a subunit does not necessarily mean that the p subunit is devoid of sorting information. When expressed alone, the H+,K+-Psubunit was transported to the cell surface and targeted to the apical membrane of LLC-PKlcells. When dimerized with an a subunit chimera, this apical sorting signal was masked or was subordinate to the sorting information of the a subunit. As postulated for other N-glycosylated membrane proteins, the carbohydrate on the H+,K+p subunit may be a weak apical targeting signal. However, in MDCK cells the H+,K+-Psubunit was targeted to the basolateral membrane (Caplan, 1997).Whatever the signal may be, this presents a problem for the hypothesis of the carbohydrate as an apical targeting signal, because much of the evidence for the hypothesis was developed in MDCK. Nonetheless, these

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data are consistent with the hypothesis of a hierarchy of signals that can be interpreted by different epithelia in different ways. Could a stronger apical signal be present in the Na+,K+-p2subunit? The only difference between the Na+,K+-ATPaseof kidney and the RPE or epithelium of the choroid plexus was the presence of the p2 isoform. A model based on a p2 signal would have to account for the observations that an equal amount of the pl subunit is expressed, and the pl subunit is predominantly in the apical membrane (Rizzolo and Zhou, 1995; Ruiz et al., 1995, 1996). The formation of tetramers might allow a dominant signal in the p2 subunit to govern targeting. There are several ways to explain the observation that the p2 subunit is basolateral in the epithelium of the ciliary body. First, like the H+,K+-Psignal the Na+,K+-p2signal may be interpreted differently in the various neuroepithelial derivatives. Second, as discussed earlier, it is conceivable that a leucine/proline substitution at position 51 could account for different sorting of closely related p2 isoforms. There is an alternate function of the p2 subunit to consider. This protein was also identified as the adhesion molecule on glia (AMOG) that mediates glialheuronal interactions (Muller-Husmann et al., 1993). It is unclear if AMOG serves this role as a component of the Na+,K+-ATPaseor as an independent protein. By analogy with the H+,K+-Psubunit, the Na+,K+p2 subunit may be able to reach the cell surface independent of the a subunit. In preliminary studies, the chick Na+,K+-P2subunit was expressed in MDCK cells where it was apically polarized (Robinson et al., 1995). Although the authors could not role out the possibility, they were unable to demonstrate an association with the endogenous a subunit. If the p2 subunit functions as an independent protein, another mechanism would be required for the apical polarization of the Na+,K+-ATPasein neuroepithelia. Although this alternate function may be important for the RPE whose apical membrane interacts with the neural retina, it cannot readily explain the apical distribution of the p2 subunit by the epithelia of the choroid plexus. Here, the 02 subunit is most likely part of the Na+,K+ATPase. Even if the p2 subunit expresses a dominant sorting signal, there must be a mechanism in place to interpret the signal. The p2 subunit was expressed during kidney development and in polycystic disease when the Na+,K+-ATPase is nonpolarized (Holthofer, 1987; Minuth et al., 1987; Avner et al., 1992; Wilson, 1997). A similar lack of polarity in the early stages of RPE development was discussed earlier (section 1II.A). Those studies examined the a1 and pl subunits, but antibodies were unavailable to examine the 0 2 subunit. To circumvent this problem, mRNA for the 01 and p2 subunits was amplified by the reverse transcriptase-polymerase chain reaction (Fig. 5). Primer pairs were synthesized to amplify mRNA from nucleotides 236 to 585 for pl message and 945 to 1474 for p2 message

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fi1 Primers

Pst I

240 330

(94) 99

fi2 Primers

Hind Ill

-590 (530) -300 (310) 250 (220)

-

-

FIG. 5 PCR analysis of &subunit mRNAs during W E development. Messenger RNA was isolated from the periphery of E5 or El2 RPE and amplified using subunit-specific primers. Control lanes show the amplification of cDNA clones that encode the /3l or p2 subunit as indicated. Additional controls demonstrated that the primers were specificfor the corresponding cDNA (not shown). To confirm the identify of the amplification products, the indicated lanes contain amplified DNA that was digested with PstI or Hind111 restriction enzymes. The measured sizes of the amplification and digestion products are indicated in base pairs. The predicted sizes are indicated in parentheses. Standard lanes contain a Hue111 digest of 4x174 DNA.

(Takeyasu et al., 1987; Lemas and Fambrough, 1993). The specificity of the primers was demonstrated by amplifying either pl or p2 sequences from cDNA clones (a gift of D. M. Fambrough, Johns Hopkins University). To determine which subunits were present when the Na+,K+-ATPasewas nonpolarized, WE was isolated from the peripheral regions of Embryonic Day 5 (E5) and E6 WE. Messenger RNA was isolated using dynabeads according to the manufacturer’s instructions (Dynabeads, Inc., Lake Success, NY). In each case, pl and p2 subunits were readily detected. Both subunits were also detected on E12, when the Na+,K+-ATPasewas apically polarized in all regions of the RPE. Although it was consistently more difficult to detect the p2 mRNA on E12, this might reflect differences in secondary structure that could affect the reverse transcriptase or amplification steps. Assuming that the p2 mRNA was translated and the p2 subunit reached the plasma membrane, these data suggest that the p2 subunit

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was insufficient to polarize the Na+,K+-ATPaseearly in development. The question becomes what mechanism is expressed later in development that can recognize a p2, or some other, signal? Establishing such a mechanism during the development of the RPE may be an early step in remodeling the apical microvilli. Because the microvilli function in diverse environments, the microvilli of each epithelium must be tailor-made. RPE microvilli are present when the RPE begins to differentiate but are continuously remodeled throughout development to form a highly specialized structure (Rizzolo, 1997). The importance of microvillar structure in apical polarity was illustrated by studies of intestinal epithelia (Costa de Beauregard et af., 1995). Disruption of microvilli affected the polarity of a subset of microvillar proteins. To understand the importance of the microvillar cytoskeleton for polarity, we must first consider the role the cortical cytoskeleton plays in maintaining basolateral polarity.

B. Interaction with the Cytoskeleton The cortical cytoskeleton can contribute to membrane polarity, because the cytoskeleton itself is polarized. The skeletal elements underlying the apical membrane are distinct from those of the basolateral membrane. They generally include ezrin and bundled actin, but other components vary from epithelium to epithelium. For example, the RPE contrasts with the intestinal epithelium by using myosin VIIa and a bundling protein other than villin (Hofer and Drenckhahn, 1993; Hasson et af., 1995). Other differences likely exist. The cytoskeleton underlying the basolateral membranes is formed by a lattice of actin and spectrin. Proteins such as ankyrin link membrane proteins to this cytoskeleton. There are many isoforms of ankyrin and spectrin (Lambert and Bennett, 1993; Beck and Nelson, 1996). An asymmetric distribution of these isoforms could lead to further polarity. For example, the isoform of spectrin in the terminal web of chick enterocytes differs from the isoform found along the lateral membranes (Glenney et al., 1983). A similar observation was made with Drosophila S2 cells (Dubreuil et al., 1997). The only demonstrated interaction between the Na+,K+-ATPaseand the cortical cytoskeleton is the ternary complex it forms with ankyrin and spectrin (Nelson and Veshnock, 1987; Morrow et af., 1989; Nelson and Hammerton, 1989).The functional significance of this association is unclear. Although binding to the cytoskeleton was proposed to limit diffusion, endocytosis, and degradation of the protein, these roles were questioned by gene knockout experiments in Drosophila (Lee et al., 1993). Spectrin translated from maternal mRNA allowed larvae to develop, but as the maternal mRNA was degraded a-spectrin disappeared. Despite the loss of a-spectrin,

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the basolateral polarity of the Na+,K+-ATPasewas preserved. The evidence in favor of a role for spectrin is reviewed below. Studies with the choroid plexus and RPE suggest that additional interactions should be explored. The hypothesis that the cortical cytoskeleton regulates the distribution of the Na+,K+-ATPasewas tested by inducing rearrangements of the cytoskeleton. Rearrangements were induced by the exogenous gene expression of E-cadherin. Besides mediating cell-cell adhesion, E-cadherin participates in a complex that includes spectrin. When expressed in fibroblasts,the cells formed intercellular contacts, and the cultures adopted the cobblestone appearance typical of epithelia (McNeill et al., 1990). Spectrin, ankyrin, and the Na+,K+-ATPasewere recruited to the regions of cell-cell contact. The implication was that E-cadherin effected the redistribution of the Na+,K+-ATPaseby reorganizing the cytoskeleton, but this interpretation must be viewed with caution. The observation that E-cadherin may be part of a signaling cascade means that changes in gene expression could also be important. This was illustrated by studies with the RPE-J cell line. This line was established by the transformation of rat RPE (Nabi et al., 1993). It displays some aspects of polarity, but the distribution of the Na+,K+ATPase is nonpolarized. Recall that neuroepithelia do not express Ecadherin. When E-cadherin was expressed in WE-J, the Na+,K+-ATPase was redistributed to the lateral membranes (Marrs et al., 1995). The most striking observation was that E-cadherin induced the expression of a number of junctional proteins. This remodeled, lateral membrane more closely resembled that of kidney epithelia than RPE. Given this constellation of changes, one can envision other effects such as altered expression of ankyrin isoforms, intracellular transport mechanisms, or protein degradation pathways. Many studies indicate that cadherins participate in outside-in and insideout signaling pathways (Grunwald, 1996). These functions would account for the diversity and developmental regulation of this protein family. In chick RPE, three cadherins were expressed, but their level of expression changed throughout development. B-cadherin was always expressed at high levels, before and after the Na+,K+-ATPaseshifts its polarity. Initially, Ncadherin expression was high, but it decreased to low levels beginning on E5. R-cadherin expression rose and then fell with a peak near E10-14. Therefore, N-cadherin expression is decreasing and R-cadherin increasing when the Na+,K+-ATPasebecomes polarized (Rizzolo and Zhou, 1995; Grunwald, 1996). On, E6 the Na+,K+-ATPasecolocalized with spectrin and ankyrin along the apical and lateral membranes. None of these proteins were evident in the basal membranes by immunofluorescence.As development proceeded, the Na+,K+-ATPasebecame more concentrated in the apical membranes and was undetected in the lateral membrane. There was no corresponding shift in the distribution of spectrin or ankyrin. Together,

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these observations indicated a genuine shift in the distribution of the Na+,K+-ATPase.Later, between E l 0 and E16, a pool of spectrin and ankyrin increased in the basal membranes, but the Na+,K+-ATPaseremained apically polarized. Accordingly, these proteins were independently redistributed at different times of development, presumably by different mechanisms. The final distribution of spectrin resembled its distribution in the kidney and intestine. It remains to be determined if there is a causal link between the correlation of cadherin expression and the redistribution of these proteins. The studies of E-cadherin in RPE-J cells suggest that the idea is worth exploring. Three hypotheses have been advanced to account for the apical distribution of the Na+,K+-ATPase.The first was that the cytoskeleton was reversed with spectrin and ankyrin concentrated in the apical membrane. The difficulty with this hypothesis is that the absence of the Na+,K+-ATPasedoes not relieve the cytoskeleton of other functions along the basolateral membrane. As emphasized in section 11, many proteins have a “typical” polarity, and many membrane proteins are linked to the cortical cytoskeleton. Spectrin immunoreactivity was low, but evident, in the lateral membranes of rat W E and epithelium of the choroid plexus (Gundersen et al., 1991; Alper et aZ., 1994). By contrast, lateral pools of spectrin and ankyrin were prominent in chick W E and choroid plexus (Marrs et aZ., 1993; Huotari et aZ., 1995; Rizzolo and Zhou, 1995). These observations lead to a second hypothesis that a specialized ankyrin links the Na+,K+-ATPaseto the apical pool of spectrin. Of the many spectrin and ankyrin isoforms, some have polarized or restricted intracellular distributions (Beck and Nelson, 1996). There is biochemical evidence for a ternary complex of Na+,K+-ATPase,ankyrin, and spectrin in neuroepithelia. In rat RPE, the three proteins could be crosslinked with bifunctional reagents (Gundersen et al., 1991). In chick choroid plexus, the complex was demonstrated on sucrose gradients and by electrophoresis on nondenaturing polyacrylamide gels (Marrs et aL, 1993). If the Na+,K+-ATPasewas uniformly distributed with spectrin, each of the studies noted above should have detected a lateral pool of the Na+,K+-ATPase.The suggestion that an isoform of ankyrin limits the distribution is based on the observation that the anion exchanger AE2 is basolateral in the choroid plexus (Marrs et al., 1993; Alper et al., 1994). The erythrocyte isoform, AE1, binds ankyrin and is highly homologous to AE2. Although an AE2/ankyrin complex has not been demonstrated, the possibility has not been eliminated. In this view, one ankyrin isoform would localize AE2 to the basolateral membranes, while a second ankyrin isoform localized the Na+,K+-ATPaseto the apical membrane. In chick RPE there is preliminary evidence that RPE expresses ankyrin isoforms not found in intestinal epithelia (Rizzolo and Zhou, 1995). However, there is an alternative explanation for a ternary

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complex. Consider the efficiency of polarity and the limitations of immunofluorescence. The proximity of adjacent microvilli and differences in the penetration of antibody could lead to a misleading amplification of fluorescent signal in the microvilli. Okami et al. (1990) addressed these issues in an immunoelectron microscopy study of rat RPE. Antibody labeled only the surface of the plastic sections, which allowed the density of the Na+,K+ATPase in different membranes to be quantified. Surprisingly,the Nat,K+ATPase was only 50% more concentrated in the apical membrane. Because there was 4X more apical membrane, this placed 85% of the Na+,K+ATPase in the apical membrane. Although the net result is apically polarity, sufficient Na+,K+-ATPaseis in the basolateral membrane to be detected biochemically as part of a ternary complex. The ambiguity that results from incomplete polarity leads to a third hypothesis that the Na+,K+-ATPasecan also bind cytoskeletal elements other than spectrin. Although immunofluorescence indicated apical pools of spectrin, ankyrin, and Na+,Kf-ATPase,these apical pools did not necessarily overlap. Because the apical microvilli collapsed along the apical membrane, they were unresolved from the terminal web region. Fortuitously, the microvilli could be resolved from the terminal web region in preparations of chick W E (Huotari et al., 1995; Rizzolo and Zhou, 1995). Elongated microvilli extended for several micrometers beyond the cell bodies. Immunoreactivity for the Na+,K+-ATPaseand ankyrin were observed throughout the microvilli. Nonetheless, the apical pool of a-spectrin was confined to the region at the base of the microvilli. This is consistent with Gundersen et al. (1991) who observed that the Na+,K'-ATPase could be efficiently crosslinked to ankyrin, but only small amounts of a-spectrin were in the complex. Despite the absence of a-spectrin, there was evidence that the Na+,K+-ATPasewas part of a macromolecular complex in the microvilli (Rizzolo and Zhou, 1995). Approximately 40% of the Na+,K+ATPase was insoluble in the detergent Triton X-100, which solubilizes membrane proteins unlinked to the cytoskeleton. Elements of the cytoskeleton were dissociated in the presence of EDTA. Under these conditions, nearly all the Na+,K+-ATPasewas solubilized. Given the fact that a-spectrin was absent from the microvilli, the question becomes what mechanism competes with spectrin to divert most of the Na+,K+-ATPaseinto the microvilli? One possibility is P-spectrin. Generally, spectrin is a heterodimer of intertwined a and P subunits. It is 0-spectrin that binds ankyrin (Beck and Nelson, 1996). Notably, P-spectrin exists independent of a-spectrin in the Golgi apparatus and the neuromuscular junction (Bloch and Morrow, 1989;Beck et al., 1994).Conceivably, P-spectrin exists alone in the microvilli. Alternatively, ankyrin or an ankyrin-like protein links the Na+,K+-ATPase to some other component of the microvilli.

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C. Apical Interactions during Development The presumptive neural retina apposes the apical membrane of RPE and regulates its development (Rizzolo, 1997). Regulation is mediated by soluble and insoluble factors. It is difficult to envision analogous interactions in the mature the choroid plexus for two reasons. The epithelium faces a lumen, and the flow of cerebral spinal fluid would oppose the diffusion of neuronal secretions. However, during early development neuronal secretions can bathe the apical membrane of the epithelium. Apical interactions are readily explored in the RPE, because the RPE and neural retina are easily isolated and cultured. Basal and lateral interactions were insufficientto fully polarize embryonic RPE. Some polarity was evident when E7 RPE was cultured on laminin, collagen IV, or its native basement membrane (Rizzolo, 1991). There were functional tight junctions, apical microvilli, and a basal distribution of integrins. Nonetheless, elements that were polarized on E7 in vivo were not polarized in culture. The distribution of talin, a cytoskeletal associated protein, became disorganized (Philp et al., 1990). Centrosomes and ytubulin were perinuclear rather than subjacent to the apical membrane, and the Na+,K'-ATPase was not polarized (Rizzolo, 1991; Rizzolo and Joshi, 1993). Furthermore, certain integrins (e.g., (r6Pl) had an apical distribution on E7 but were basally polarized in culture (Rizzolo et al., 1994). An organ culture model was developed to determine whether apical interactions could modulate the polarity that was induced by basal and lateral interactions. Eye cups were prepared by removing the anterior structures and the vitreous body. A tissue punch was used to remove a 6-mm disc of retina, choroid, and sclera. The neural retina was peeled off and stabilized on a cellulose acetate filter in one of two orientations. Either the inner (ganglion cell) or the outer (photoreceptor) surface of the neural retina was applied to the filter. The RPE and choroid were isolated as a unit by using a drawn glass pipette to tease away the sclera. The tissue was stabilized on a filter with the choroid against the filter. The RPEheural retina interface could be reconstituted by apposing the two filters such that the RPE contacted the inner or outer surface of the neural retina. The reconstituted tissue was held together near the air:medium interface by nylon mesh supports (Fig. 6). The tissue was cultured 18 h in SF2 medium (Rizzolo and Li, 1993) in an atmosphere of 10% C 0 2 and 40% 02.Alternatively, the RPE-choroid was cultured alone. This experiment was first performed with E7 tissue from chick to examine the effect of the neural retina on the distribution of integrins (Rizzolo et al., 1994). In normal development, integrin a6Pl was present in the apical and basal membranes on E7, but the apical pool disappeared after the photoreceptor layer began to differentiate. The apical pool of a6pl was observed in organ culture of

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Top View

Side View

Nylon mesh held in place by 0 ring

Teflon supporlS

T-

__

RPWChoroid supportedby cellulose acetate

I

\

Neural retina supportedby cellulose acetate

by ring insert

I

Culture dish

L

FIG. 6 Tissue holder for reconstitution experiments. The RPEneural retina interface was reconstituted in organ culture by holding the tissues together in a sandwich of nylon mesh. The apparatus was modified from the prototype described in Rizzolo et al. (1994). Nylon mesh was stretched over the opening of the top support and held in place by an 0 ring. A second sheet of nylon was draped over a small, tight-fitting ring and pressed into the center of the bottom support until the mesh was even with the surface. W E choroid was isolated in SF2 culture medium and placed choroid down on a cellulose acetate filter. The filter was lifted out of medium and placed on the nylon mesh of the bottom support. The neural retina was isolated and spread on a filter. It was lifted out of the medium, inverted, and placed on the W E . The top support was gently lowered between the posts of the bottom support. A pin on the bottom support prevented the supports from rotating relative to one another. The weight of the top support and elasticity of the nylon mesh held the tissues together without compressing them. The assembly rested on posts that raised it above the bottom of the culture dish. Culture medium was added to just cover the tissues. Incubations were performed with gentle rocking at 37°C under an atmosphere of 10% C 0 2 and 40% 02.

E7 RPE only if the outer surface of the primordial neural retina was apposed to the apical membrane of the W E . Detached retinas, retinal conditioned medium, and the inner surface of the retina were unable to maintain the apical pool of a6/3l. Direct contact with the presumptive photoreceptor layer modified the basal polarity that was induced by basement membrane components.

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This model was used to determine if the neural retina promotes the apical polarization of the Na+,K+-ATPase.After E l l , the Na+,K+-ATPase is apically polarized in all regions of the RPE. When the neural retina was removed from organ cultures of E l 4 RPE, the distribution of the Na+,K+ATPase became nonpolar (Fig. 7 and Table I). If the neural retina/RPE interface was reconstituted, the apical polarity of the Na+,K+-ATPasewas retained. Surprisingly, the inner surface of the retina was nearly as effective as the outer surface. This suggests that apical polarity was promoted by a diffusible factor rather than a specific surface interaction. If so, this factor was present in low concentration, because medium conditioned by the neural retina was unable to maintain polarity. Notably, in the cat retinal detachment results in the retraction of apical microvilli (Anderson el al., 1983). Conceivably, the effect on the Na+,K+-ATPaseis a consequence of effects on the structure or composition of microvilli. Countering this view is the observation that RPE cultured under certain conditions retain lush microvilli and apically polarized Na+,K+-ATPase(Mircheff et al., 1990; Hu

No Retina

Normal Retina

FIG. 7 The polarity of Na+,K+-ATPaseis maintained in culture only in the presence of the neural retina. The RF'Emeural retina interface was reconstituted as described in Fig. 6 and cultured for 18 h. (A) No neural retina. (B) The outer surface of the neural retina was reconstituted with the W E . (C) The inner surface of the neural retina was reconstituted with the W E .Bar, 5 pm.

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LAWRENCE J. RIZOLO TABLE I In ReconstitutionExperiments, the Neural Retina Maintains the Na+,K+-ATPasein the Apical Membrane Regardless of Its Orientation

Orientation of neural retina

Polarity index"

No retina Normal Reversed

1.5 2 0.9 7.5 ? 0.8 5.3 ? 1.3

a Fluorescence was quantified from the experiments described in Fig. 7. The polarity index was determined by a modification of the method of Gottardi and Caplan (1993b). Briefly, a 4-pm-square box was slid along the apical and lateral membranes and the intensity of the fluorescence within the box was averaged. Blank values were averaged from the cell bodies and subtracted. Because the lateral membranes of two cells were included in the box, the index was determined by dividing the apical intensity by one-half the lateral intensity. Because the surface area of the apical and lateral membranes was not determined, this ratio is not a true measure of polarity. However, the index does reflect changes in protein distribution. Measurements were averaged from 5-6 fields of two independent experiments. The standard error is indicated.

et al., 1994). Generally, this polarity was attained only after prolonged times in culture. It was accompanied by changes in gene expression and the redistribution of cytoskeletal elements (McKay et al., 1997). Conceivably, the differentiation of early embryonic W E regresses in culture and needs apical stimuli to repolarize the Na+,K+-ATPase. More mature RPE may be able to gradually remodel its apical membrane proteins without the benefit of apical stimuli if given the proper culture conditions. In other words, retinal interactions may promote a process that occurs more gradually on its own in culture.

VII. Concluding Remarks

The polarization of the Na+,K+-ATPaseillustrates the plasticity of epithelia and the need to understand how the environment regulates polarity. Most studies of polarity have focused on intracellular mechanisms such as sorting signals and protein stabilization. Polarity depends upon a hierarchy of

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signals that can be interpreted differently by different epithelia or differently by the same epithelium in varying circumstances. There is strong evidence for a novel signal found in a transmembrane domain of the a1 subunit. There is suggestive evidence for a second, conflicting signal that resides in the j32 subunit. Although different sorting information may be encoded in each of the different isoforms of the Na+,K+-ATPasesubunits, the al, j31, and j32 subunits can reside in either the apical or the basolateral membranes. Therefore, the manner in which their sorting information is interpreted depends upon the sorting machinery found within each epithelium. The challenge of future investigations will be to reveal the mechanisms that interpret these signals. Some of these mechanisms will involve ankyrin. By binding the Na+,K+-ATPaseto the spectrin-based cytoskeleton, ankyrin may retard endocytosis or degradation of the enzyme. This simple model must be modified, because the Na+,K+-ATPasedoes not always colocalize with spectrin, as demonstrated in the RPE. The existence of different ankyrin isoforms and ankyrin-like proteins may provide a means to limit the distribution of the Na+,K+-ATPaseto subdomains of the cortical cytoskeleton. RPE is the first epithelium in which ankyrin, or an ankyrin immunolike protein, has been described in microvilli. This is consonant with the view that the composition of microvilli is epithelium-specific and that specializations of the microvillar cytoskeleton maintain a unique apical membrane composition. The challenge of future investigations will be to determine the different isoforms of ankyrin that bind the Na+,K+-ATPase and the different cytoskeletal elements with which these ankyrins interact. Finally, it becomes important to understand how signals from the extracellular environment regulate polarity. Clearly, E-cadherin can regulate polarity. In the neuroepithelium, N-, B-, and R-cadherins are developmentally regulated. By analogy to E-cadherin, changes in the expression of these cadherins likely alter RPE polarity. Second, developmental interactions with the neural retina are important for the development of the microvilli and for the initial polarization of the Na+,K+-ATPaseto the apical membrane. Because diffusible products of the neural retina appear to be involved, it is possible that neuronal secretions could also effect this initial polarization in the epithelium of the choroid plexus. The Na+,K+ATPase illustrates that epithelial polarity is a multifaceted problem. Resolving it will require understanding how extracellular signals modify intracellular sorting pathways and the organization of the cytoskeleton.

Acknowledgments I thank J. Collins for expert technical assistance; Dr. D. M. Farnbrough for helpful discussion Drs. D. Bok, M. Cocaand providing cDNA clones of the subunit of the Na+,K+-ATPase;

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Prados, J. A. Marrs, A. D. Marmorstein, S. S. Miller, and E. Rodriguez-Boulan for helpful discussion; and Drs. M. J. Caplan and M. Kashgarian for critically reviewing the manuscript. Work from the author’s laboratory described in this chapter has been supported by NIH Grant EY 08694. The monoclonal antibodies to the Na+,K+-ATPasewere provided by the Developmental Studies Hybridoma Bank (Johns Hopkins University, Baltimore, MD) under NICDN Contract N01-HD-6-2915.

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Desmosomes: Intercellular Adhesive Junctions Specialized for Attachment of Intermediate Filaments Andrew P. Kowalczyk,*st Elayne A. Bornslaeger,S Suzanne M. Norvell,S Helena L. Palka,S and Kathleen J. Green**tS *Department of Pathology, *Department of Dermatology, and the TR.H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611

Cell-cell adhesion is thought to play important roles in development, in tissue morphogenesis, and in the regulation of cell migration and proliferation. Desmosomes are adhesive intercellular junctions that anchor the intermediate filament network to the plasma membrane. By functioning both as an adhesive complex and as a cell-surface attachment site for intermediate filaments, desmosomes integrate the intermediate filament cytoskeleton between cells and play an important role in maintaining tissue integrity. Recent observations indicate that tissue integrity is severely compromised in autoimmune and genetic diseases in which the function of desmosomal molecules is impaired. In addition, the structure and function of many of the desmosomal molecules have been determined, and a number of the molecular interactions between desmosomal proteins have now been elucidated. Finally, the molecular constituents of desmosomes and other adhesive complexes are now known to function not only in cell adhesion, but also in the transduction of intracellular signals that regulate cell behavior. KEY WORDS: Cell adhesion, Cytoskeleton, Cadherins, Desmoplakin, Catenins, Plakoglobin.

I. Introduction Cell-cell interactions play an important role in the development and maintenance of tissue structure and function in multicellular organisms. One of the most prominent cell-cell adhesive structures assembled by cells of InlemariunaI Review of Cyrulogy, Vul. 185 0074-76%/99 $25.00

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epithelial origin is the desmosome. Desmosomes are adhesive intercellular junctions that couple the intermediate filament cytoskeleton to regions of tight cell-cell adhesion (Bock and Clark, 1987; Cowin et al., 1985; Franke et al., 1987a;Green and Jones, 1996; Green and Stappenbeck, 1994;Schmidt et al., 1994; Schwarz et al., 1990; Steinberg et al., 1987). By linking the intermediate filament (IF) cytoskeleton to sites of cell-cell adhesion, desmosomes function as a bridge between cells to create a transcellular network of intermediate filaments that is critical for tissue integrity (Coulombe and Fuchs, 1994; Fuchs, 1992; Steinert and Bale, 1993). Emerging evidence that desmosomes are critical to tissue function has been provided recently by the characterization of human skin disorders and animal models in which desmosomal components have been mutated (Allen et al., 1996; Bierkamp et al., 1996; Koch et al., 1997; McGrath et al., 1997; Ruiz et al., 1996). The ability of desmosomes to withstand the harsh denaturing conditions used during desmosome purification from tissues is well documented. In addition, the report that desmosomes can be identified at the ultrastructural level in the 2000-year-old skin of Egyptian mummies firmly establishes the status of the desmosomes as one of the most stable cellular organelles (Perrin et al., 1994). However, recent work has changed the perception that desmosomes-and the IF cytoskeleton to which they attach-are merely static structures in living tissues (Skalli et al., 1992,1995). This review is intended to give a broad overview of the expanding area of desmosome research and to provide the most recent and detailed information on protein interactions that occur within the desmosome. In addition, emphasis will be placed on the dynamic properties of desmosomes and how these structures respond to changes in the extracellular environment and to intracellular signaling pathways.

II. Ultrastructural Properties and Tissue Distribution of Desmosomes At the ultrastructural level, desmosomes appear as “spot welds” between adjacent cells, with a central core region sandwiched between two symmetrical electron-dense cytoplasmicplaques (Fig. 1) (Farquhar and Palade, 1963; Kelly, 1966; Kelly and Kuda, 1981; Kelly and Shienvold, 1976; Pirbazari and Kelly, 1985; Staehelin, 1974).The central core region of the desmosome includes the plasma membranes of adjacent cells, which are separated by a 30-nm space. In the center of the core region, between plasma membranes, is an electron-dense region termed the central dense stratum. On the cytoplasmic face of the desmosome, bundles of IFs extend toward the plasma membrane and attach to electron-dense plaques subjacent to the cell mem-

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A

B

Plakoglobin

Desmoplakin

Intermediate filaments

FIG. 1 Ultrastructure and schematic of desmosomal components. (A) The desmosome is composed of a central core region which includes the intercellular space between adjacent cells and a submembraneplaque region that functionsto anchor IF networks to the desmosome. (B) The desmosomal cadherins, desmogleins and desmocollins,are the transmembrane components of the desmosome. Plakoglobin and desmoplakin form complexes with the desmosomal cadherins that are thought to attach IFs. A number of other plaque components are also listed, but the proteins to which they bind are not currently known.

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brane. The plaque is a tripartite structure, with an electron-dense outer region adjacent to the membrane followed by an electron-lucent region, and a second electron-dense region deeper in the cytoplasm. Intermediate filaments do not appear to terminate at the plasma membrane, but instead, the filaments appear to loop through the plaque. Furthermore, in certain cells such as epidermal keratinocytes, bundles of IFs, termed tonofilaments, appear to disperse into individual filaments that extend to the plasma membrane. The ultrastructural characterization of desmosomes was followed by the development of procedures to isolate intact desmosomes from tissues (Drochmans et al., 1978; Gorbsky and Steinberg, 1981; Skerrow and Matoltsy, 1974). This led to the biochemical characterization of the major protein components of the desmosome and to the identification of these proteins as either transmembrane or plaque proteins (Fig. 1B) (Miller et al., 1987; Steinberg et al., 1987). These proteins include the large proteins desmoplakin I (332 kDa) and I1 (260 kDa) (Gorbsky et al., 1985; Green et al., 1990), which are thought to be the most abundant components of the desmosome (Mueller and Franke, 1983). Additional polypeptides include the transmembrane desmosomal glycoproteins, desmogleins (150 kDa) and desmocollins (110-115 kDa) (Blaschuk et al., 1986; Cohen et al., 1983; Gorbsky and Steinberg, 1981). Plakoglobin (83 kDa) is another major component in desmosome preparations, and this protein is known to be present in other types of adhesive intercellular junctions in addition to desmosomes (Cowin, 1994a; Cowin et al., 1986; Kapprell et al., 1987). In stratified tissues, a 75-kDa protein originally termed Band 6 is also prominent (Kapprell et al., 1988). This protein has been renamed plakophilin 1 and is part of the armadillo gene family as discussed below (Hatzfeld et al., 1994; Heid et al., 1994). The development of procedures to isolate desmosomal proteins was a significant advance, as this led to the development of antibody reagents and more recently the identification of DNA sequences encoding the desmosomal proteins (Collins and Garrod, 1994; Garrod, 1993; Schmidt et aZ., 1994). Desmosomes are assembled by epithelia of various tissues and are most prominent in tissues that are subjected to mechanical stress, such as the epidermis. However, desmosomes are not restricted to cells expressing the keratin class of IFs, such as epithelial cells, but are also found in tissues in which vimentin or desmin is the predominant IF protein expressed. For example, desmosomes are present within the intercalated discs that form between adjacent myocytes of the heart. In addition, desmosomes are assembled by follicular dendritic cells of the lymph system and are present in the arachnoid plexus of the meninges (Achstatter et al., 1989; Franke et al., 1981,1982;Kartenbeck et al., 1983,1984;Rungger-Brandle et al., 1989). Desmosome-related structures, termed complexus adhaerentes, have more

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recently been described in lymphatic endothelium and in the sinus of lymph nodes (Schmelz and Franke, 1993; Schmelz et al., 1994). The importance of desmosomes in resisting the forces of mechanical stress has been underscored by the observation that mutations in desmosomal genes, both in humans and in animal model systems, have profound impacts on tissue morphogenesis and integrity (Allen et aL, 1996; Bierkamp et aZ., 1996; Koch et aL, 1997; McGrath et al., 1997; Ruiz et aL, 1996).

111. Transmembrane Components of the Desmosome: Desmosomal Cadherins A. Structure of the Desmosomal Cadherins Work over the last 20 years has revealed that a family of calcium-dependent cell-cell adhesion molecules, termed cadherins, mediate the intercellular adhesion that is critical to tissue morphogenesis (Larue et aL, 1994;Takeichi, 1991, 1995). The classical cadherins, such as the epithelial cadherin Ecadherin, are assembled into structures closely related to desmosomes, termed adherens junctions (Boller et aZ., 1985). An important difference between adherens junctions and desmosomes is that the classical cadherins in adherens junctions are coupled to the actin cytoskeleton, whereas the desmosomal cadherins are linked to the IF network (Cowin, 1994b; Cowin and Burke, 1996). The adhesive function of cadherins was first demonstrated when Ecadherin was expressed exogenously in fibroblasts. Expression of Ecadherin in these normally nonadherent cells resulted in calcium-dependent cell-cell adhesion (Nagafuchi et aL, 1987). It is now known that cadherins are a large family of calcium-dependent adhesive glycoproteins. The transmembrane components of the desmosome, the desmogleins and desmocollins, are members of the cadherin gene superfamily and are thought to play a major role in epithelial cell-cell adhesion (Buxton and Magee, 1992; Koch and Franke, 1994; Magee and Buxton, 1991). Structural analyses of E-cadherin cDNA and other “classical cadherins,” such as N-cadherin and P-cadherin, indicate that these proteins comprise repeated domains in the extracellular portion of the molecules, termed cadherin repeats (EI-EIV) (Geiger and Ayalon, 1992; Kemler et aL, 1989; Takeichi, 1991) (Fig. 2). These repeats are approximately 110 amino acids each and contain calcium binding motifs that are thought to play important roles in maintaining cadherin tertiary structure. Flanking both the extracellular and the cytoplasmic sides of the membrane spanning domain are loosely conserved membrane anchoring domains termed E A and IA, respectively. Several studies have suggested that the cytoplasmic membrane anchoring domain

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242 Desmoglein

A

m

B

E-cadherin

FIG. 2 Structure of the desmosomal cadherins and the classical cadherin E-cadherin. Cadherins are type I transmembrane proteins with an amino-terminal extracellular domain and a carboxyl-terminal cytoplasmic domain. The extracellular domain includes repeated motifs, EI-EIV, that are found in all known cadherins. Extracellular (EA) and intracellular (IA) anchor domains flank a single pass transmembrane domain. The ICS domain in the cytoplasmic tails of the cadherins binds to both plakoglobin and p-catenin in the case of the classical cadherins, whereas the ICS domain of the desmosomal cadherins binds to plakoglobin. The desmocollins are alternatively spliced to yield an “a” or “b” form. The desmoglein cytoplasmic domain also includes a proline-rich linker domain (IPL),a desmoglein-specificrepeated unit domain (RUD), and a terminal domain (DTD).Nomenclature of domains is adapted from Koch ef al. (1991a).

plays an important role in the adhesive function of cadherins (see below), but specific functions for this region have not been defined. Extending further into the cytoplasm is a highly conserved domain, termed the intracellular cadherin segment (ICS), which binds to the plaque proteins P-catenin and y-catenin (Ozawa et al., 1990b),which is now known to be plakoglobin (Knudsen and Wheelock, 1992; Peifer et al., 1992). Both the desmogleins (Amagai et al., 1991; Koch et al., 1990; Nilles et al., 1991; Wheeler et aL, 1991) and the desmocollins (Collins et al., 1991; Holton et al., 1990; Kawamura et al., 1994; King et al., 1993; Koch et al., 1991b; Mechanic et al., 1991; Parker et al., 1991) form subclasses of the cadherin gene superfamily. The structural organization of the extracellular domains of the desmosomal cadherins closely parallels that of classical cadherins, although there is considerable divergence in the sequences of the cadherin repeats (Magee and Buxton, 1991). In contrast, the cytoplasmic domains of desmosomal cadherins diverge significantlyfrom those of classical cadherins and bind specificallyto plakoglobin but not to P-catenin (Plott et al., 1994). Desmogleins are particularly unusual in that these cadherins contain an extended cytoplasmic domain. In addition to the ICS domain, desmogleins contain a proline-rich linking region that is followed by a series

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of 29 amino acid repeats specific to the desmogleins, termed repeated unit domains (RUD). At the very carboxyl-terminal region is a glycine-rich domain, or desmoglein-terminal domain (DTD). The function of these desmoglein-specificdomains remains to be determined, although it is likely that this extended cytoplasmic tail of desmogleins plays a role in the organization of the desmosomal plaque. The cytoplasmic domains of desmocollinsare structurally similar to classical cadherins, and recent analysis of the desmocollin 2 gene (DSC2) indicates that desmocollins may be more closely related to classical cadherins than to desmogleins (Greenwood et aL, 1997). However, an interesting and unique aspect of desmocollins is that these desmosomal cadherins are alternatively spliced, yielding an “a” form with the typical ICS domain, and a “b” form that is unique to desmocollins.The “b” form of desmocollins results from the insertion of an exon encoding 11 amino acids that are not present in the “a” form followed by an in-frame stop codon, resulting in a truncated cytoplasmic domain lacking the ICS region. The “b” form of desmocollins does not appear to interact with plakoglobin (Troyanovsky et aL, 1994b), and the functional significance of this alternatively spliced form of desmocollin remains unknown.

6.Tissue Distribution Early studies of the biochemical properties of desmosomes indicated that heterogeneity exists between desmosomal glycoproteins isolated from various tissue sources (Giudice et aL, 1984; Jones et aL, 1986a, 1987; King et aL, 1991; Parrish et aL, 1990; Suhrbier and Garrod, 1986). It is now known that there are three different desmogleins (Dsgl, Dsg2, and Dsg3) and three different desmocollins (Dscl, Dsc2, and D s c ~ ) ,each encoded by a separate gene (Buxton et aL, 1993, 1994a). The genes for desmogleins and desmocollins are expressed in a tissue-specific and differentiationdependent manner. The most widely expressed desmosomal cadherins are Dsg2 (Koch et aL, 1992; Schafer et aL, 1994) and Dsc2 (Arnemann et aL, 1993; Nuber et al., 1995; Theis et al., 1993), which are found in simple epithelia, in the myocardium of the heart, and in the basal layer of the epidermis. Dsg3 is expressed in the basal and suprabasal layers of stratifying epithelia of the esophagus and cervix, and in the basal and spinous layers of the epidermis (Amagai et aL, 1996b; Arnemann et aL, 1993; Shimizu et aL, 1995; Shirakata et al., 1997). Dsc3 is expressed more widely in the epidermis, as well as in other stratifying epithelia (King et aL, 1995). In contrast, Dsgl and Dscl are restricted to the highly differentiated, uppermost layers of the epidermis (Amagai et aL, 1996b; Arnemann et aL, 1993; King et aL, 1995;Nuber et aL, 1995). Expression patterns of the desmosomal

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cadherins indicate that there is considerable overlap between the isoforms, and individual desmosomes can actually contain more than one isoform (North et al., 1996). Interestingly, genes for the desmosomal cadherins are located in a cluster on chromosome 18 (18q12.1) (Buxton et al., 1994b; Cowley et al., 1997a; Ishikawa et al., 1994; Simrak et al., 1995; Wang et al., 1994). The clustered arrangement of these genes suggests the presence of a shared regulatory region that might regulate the coordinated, differentiation-dependent expression of desmosomal cadherin genes in the epidermis. The existence of an overall regulatory mechanism to control desmosomal cadherin gene expression is supported further by the spatial patterns of expression of desmosomal cadherin genes in stratifymg epithelial morphogenesis in mouse tissues (King et al., 1997). The highly ordered expression patterns of desmosomal cadherins during tissue differentiation also suggests that tissue- and differentiation-specificexpression of desmosomal cadherins may play a role in tissue patterning (Chidgey et al., 1997). To date, however, very little is known about the mechanisms controlling desmosomal cadherin gene expression, although recent work indicates that protein kinase C and components of serum regulate Dsgl expression in cultured keratinocytes (Denning et al., 1998). The recent identification of the promoter region of the DSC2 gene (Marsden et al., 1997) and characterization of the genes and regulatory regions of DSGl and DSG3 (Adams et al., 1997; Puttagunta et al., 1994; Silos et al., 1996) should provide significant insights into the regulation of desmosomal cadherin gene expression.

C. Function of the Desmosomal Cadherins in Adhesion One of the most puzzling aspects of desmosome research has been the lack of empirical evidence that desmosomal cadherins mediate calciumdependent adhesion, a property that typifies the other members of the cadherin gene family. The classical cadherins have been found to mediate calcium-dependent, homophilic adhesion (Nagafuchi et al., 1987). In addition, recent structural (Nagar et al., 1996; Overduin et al., 1995; Shapiro et al., 1995) and biochemical studies (Brieher et al., 1996; Yap et al., 1997) suggest that classical cadherins form lateral dimers, which then form an adhesive unit that binds to cadherin dimers on opposing cells. To date, evidence that desmosomal cadherins mediate adhesion has come from relatively indirect approaches, such as antibody inhibition experiments in which IgG and Fab fragments directed against desmocollinswere shown to inhibit desmosome formation in cultured cells (Cowin et aZ., 1984). Likewise, in human patients suffering from the autoimmune blistering diseases pemphigus foliaceus and pemphigus vulgaris, autoantibodies directed against Dsgl

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and Dsg3, respectively, have been demonstrated to be the causative agents in these devastating skin diseases (Amagai et aZ., 1992, 1995b; Rock et aZ., 1990). The fact that these diseases are characterized by the loss of intercellular adhesion, or acantholysis,rather than cytolysis, has led to the conclusion that pemphigus antibodies interfere with cell-cell adhesion. This is supported by the recent observation that blistering occurs in the oral epithelium and epidermis of mice in which the Dsg3 gene was ablated (Koch et al., 1997). Although these studies support the hypothesis that the desmosomal cadherins mediate adhesion, direct evidence demonstrating that expression of a desmosomal cadherin in a normally nonadherent cell can promote intercellular adhesion is still lacking. A chimeric cadherin comprising the extracellular domain of Dsg3 and the cytoplasmic domain of E-cadherin was shown to mediate only weak homophilic adhesion when expressed in mouse L cell fibroblasts (Amagai et al., 1994b). Similarly, neither full-length Dsc2a nor a chimera of the Dsc2 extracellular domain and the E-cadherin cytoplasmic domain were able to mediate adhesion in L cells (Chidgey et al., 1996). Even coexpression of Dsgl and Dsc2a along with plakoglobin failed to mediate adhesion in the L cell system (Kowalczyk et aZ., 1996). Numerous factors may explain why these cadherins do not function optimally in L cells. One possibility is that cytoplasmic components required for desmosomal cadherin function may be needed for adhesion. Alternatively, it is possible that the correct pairs of cadherins need to be coexpressed at the proper stoichiometric ratio in order to function in adhesion. In addition, numerous studies have indicated that E-cadherin may be required for the assembly of desmosomes (see below), suggesting that the adhesive interactions of the desmosomal cadherins may require the presence of a functional classical cadherin. Although it remains to be demonstrated that the desmosomal cadherins do in fact mediate adhesion, recent studies in HT1080 cells demonstrated that the desmosomal cadherins may interact heterophilically. In experiments by Troyanovsky and colleagues (Chitaev and Troyanovsky, 1997), HT1080 cells were found to express both N-cadherin and Dsg2, but not plakoglobin or desmocollin. When these cells were transfected with exogenous Dscla and plakoglobin, Dsg2 was redistributed from a diffuse to a more clustered pattern. In addition, Dsg2 and Dscl were found to form heterodimeric complexes that could be immunoprecipitated from cell lysates. In addition, these heterodimers appeared to result from interactions between adjacent cells, rather than from lateral interactions within the same cell. These data suggest that the desmosomal cadherins may associate heterophilically and that these interactions may contribute to intercellular adhesion, although this remains to be demonstrated through direct experimentation.

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D. Role in Autoimmune Blistering Diseases

As discussed above, some of the best evidence that desmosomal cadherins mediate adhesion comes from the observation that these cadherins are targets of autoimmune antibodies in patients suffering from the pemphigus class of epidermal blistering diseases. In the two major classes of pemphigus, different desmosomal cadherins are the autoantigens (Amagai, 1994; Lin et al., 1997; Stanley, 1995). In pemphigus foliaceus, Dsgl is recognized by the autoantibodies, and blistering in these patients occurs in the uppermost layers of the skin where Dsgl is expressed. In pemphigus vulgaris, Dsg3 is the target antigen and patients develop blisters deeper in the epidermis, in the basal and suprabasal layers. In fact, patients with these diseases can now be diagnosed using an ELISA system to determine if the autoantibodies are directed against Dsgl or Dsg3 (Ishii et aL, 1997). Interestingly, a hallmark clinical feature of pemphigus vulgaris is the presence of blistering in the membranes of the oral mucosa. These lesions are not present in pemphigus foliaceus, and this appears to be due to the fact that Dsgl is highly expressed in the upper layers of the epidermis but is expressed at low levels in the oral mucosa (Shirakata et al., 1997). Thus, the loss of cell-cell adhesion between epithelial cells, or acantholysis,occurs in the epithelial layer in which the desmoglein gene is expressed. In several cases, desmocollins have been found to be targeted by autoantibodies, although the presence of autoantibodies directed against desmocollins is not observed as frequently as that for desmogleins (Dmochowski et al., 1993; Hashimoto et al., 1997). Most pemphigus sera bind to conformationally sensitive epitopes within the amino-terminal portion of the desmoglein extracellular domain (Amagai et al., 1992, 1995c, 1996a; Kowalczyk et al., 1995; Olague-Alcala et al., 1994). Intriguingly, structural and functional studies of classical cadherins suggest that this region of cadherins plays an important role in homophilic binding and in cadherin lateral interactions (Nose et al., 1990; Overduin et al., 1995; Ozawa et al., 1990a; Shapiro el al., 1995). Early studies of the pemphigus diseases demonstrated that sera from human pemphigus patients could cause epidermal blistering when injected into neonatal mice (Anhalt et aL, 1982; Rock et al., 1990). More recently, IgG directed against the desmosomal cadherins has been shown to cause blistering. Affinity-purified IgG from pemphigus vulgaris patients specific for Dsg3 was isolated using recombinant Dsg3 polypeptides and passively transferred into neonatal mice (Amagai et aL, 1992). Antibodies that bound to Dsg3 were found to cause blistering in the mice, providing direct evidence that these antibodies cause the blistering seen in pemphigus vulgaris patients. Another important observation was made when recombinant Dsg3 or Dsgl polypeptides generated using baculovirus were used to adsorb

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desmoglein-specificIgG from pemphigus sera (Amagai et al., 1994a, 1995b). The depleted sera were no longer capable of causing blistering in mice. These observations provide compelling evidence that the autoantibodies directed against Dsgl and Dsg3 cause the epidermal blistering seen in pemphigus patients, presumably by disrupting keratinocyte cell-cell adhesion. Further evidence that the blistering in pemphigus patients is due to the loss of desmosomal cadherin adhesive function was provided by experiments in which a dominant negative mutant of Dsg3 was expressed in the basal layer of the epidermis (Allen et al., 1996).Desmosomes were disrupted in the epidermis of mice expressing this mutant, and epidermal integrity was compromised. More recently, the function of Dsg3 was analyzed in mouse mutants in which the Dsg3 gene was ablated (Koch et al., 1997). These mice exhibited many of the classic features of human patients suffering from pemphigus vulgaris, including oral erosions and the loss of intercellular adhesion in the suprabasal layers of the mucosal epithelium and in the epidermis. In addition, the Dsg3 -/- mice also exhibited a balding phenotype that was identical to a previously described mouse mutant bal (Davisson et al., 1994; Sundberg, 1994), suggesting that Dsg3 also plays a role in hair follicle development. Although these observations have clearly demonstrated that there is a strict relationship between the loss of desmosoma1cadherin function and blistering, the mechanisms by which blistering occurs are not fully understood. It is likely that the autoantibodies from pemphigus patients interfere with adhesion and thereby disrupt the mechanical integrity of the epidermis. However, several reports have demonstrated a role for protease activation in response to antibody binding to keratinocyte desmogleins. For example, protease inhibitors attenuate the acantholysis induced in skin organ cultures in response to pemphigus IgG (Hashimoto et al., 1989; Wilkinson et al., 1989). Other studies have demonstrated that treating cells with antibodies against E-cadherin upregulates the expression of urokinase-type plasminogen activator (Frixen and Nagamine, 1993), suggesting that antibody interactions with cadherins can lead to protease production. Recently, changes in intracellular calcium levels and activation of protein kinase C have been observed in cells treated with pemphigus sera (Esaki et al., 1995; Seishima et al., 1995). These observations suggest that in addition to directly interfering with adhesion, other events downstream of antibody binding to desmosomal cadherins may play important roles in the clinical manifestations of the pemphigus diseases.

E. Other Extracellular Components of the Desmosome The observation that desmosomal cadherins do not mediate homophilic adhesion in the fibroblast model system suggests that other proteins may be

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involved in facilitating desmosome-mediated adhesion. A 20-kDa protein, termed E48 antigen, is a glycosyl phosphatidyl inositol linked protein that is related to the T cell protein Ly6. The E48 protein may be similar or identical to a 22-kDa protein (Gorbsky and Steinberg, 1981), also termed desmoglein I11 or dg 4, which is present in protein preparations enriched for desmosomal molecules (Schwarz et al., 1990). When expressed in fibroblasts, the E48 antigen mediates adhesion, suggesting that it may play a role in mediating keratinocyte cell-cell interactions (Brakenhoff et al., 1995). However, the protein is predominantly expressed by keratinocytes induced to differentiate. Therefore, to suggest a role for E48 antigen in desmosome assembly in simple epithelial cells, a homologue to E48 antigen would need to be found in simple epithelia.

IV. Plaque Components of the Desmosome A. Cadherin Cytoplasmic Domains

The cytoplasmic domains of cadherins are perhaps the most important component in the series of linkages that occur between the adhesive interactions of the cadherin extracellular domains and the cytoskeleton. Sequences in the cytoplasmic domains of cadherins appear to direct these proteins to associate specifically with either the actin or the intermediate filament cytoskeleton (Cowin, 1994b; Cowin and Mechanic, 1994). For example, classical cadherins such as E-cadherin form intercellular junctions termed adherens junctions. These are junctions in which the actin microfilament network is anchored to sites of adhesion mediated by classical cadherins. The cytoplasmic domains of classical cadherins associate with three cytoplasmic proteins termed catenins (Ozawa et al., 1989; Ozawa and Kemler, 1992). P-Catenin and plakoglobin (formerly termed y-catenin) bind directly to the cytoplasmic domain of cadherins (Aberle ef al., 1994; Jou et aZ., 1995). In addition, it appears that one cadherin molecule associates with either plakoglobin or P-catenin, but not both proteins simultaneously (Aberle et al., 1994; Nathke et al., 1994). Both P-catenin and plakoglobin also bind directly to a-catenin (Aberle et al., 1994; 0. Huber et al., 1997; Jou et al., 1995; Nieset et al., 1997; Obama and Ozawa, 1997), a vinculinrelated protein that promotes association of the cadherin-catenin complex with the actin network (Herrenknecht et al., 1991; Nagafuchi et al., 1991). a-catenin, in turn, binds directly to actin (Rimm et al., 1995), and to the actin binding protein a-actinin (Knudson et al., 1995; Nieset et al., 1997). Through this series of interactions, classical cadherins become specifically attached to the actin microfilament network. In contrast, the desmosomal cadherins bind directly to plakoglobin, but do not appear to interact with

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P-catenin (Plott et al., 1994). In addition, several studies have demonstrated that the desmosomal cadherins interact with plakoglobin in such a manner as to prevent plakoglobin from subsequently binding to a-catenin (Kowalczyk et al., 1997; Plott et al., 1994; Wahl et al., 1996; Witcher et al., 1996). In this manner, the desmosomal cadherin cytoplasmic domains appear to prevent plakoglobin from interacting with actin-associated proteins. This unique feature of desmosomal cadherin cytoplasmictails dictates that subsequent plaque interactions in the desmosome are targeted toward interactions with the intermediate filament cytoskeleton rather than the actin cytoskeleton. Numerous studies have demonstrated that both classes of desmosomal cadherins associate with plakoglobin. Interactions between desmoglein and plakoglobin were originally identified in extracts from human epidermis (Korman et al., 1989).For both desmocollinsand desmogleins,the carboxylterminal region of the ICS domain is now known to bind directly to plakoglobin. In desmoglein, a 19-amino-acid stretch at the carboxyl-terminal region of the ICS domain appears critical for plakoglobin binding (Mathur et al., 1994; Troyanovsky et al., 1994a). Direct interactions between these proteins were demonstrated using blot overlay assays in which radioiodinated plakoglobin was shown to bind directly to purified Dsgl (Mathur et al., 1994) and using the yeast two-hybrid system (Witcher et aL, 1996). In addition, similar results were reported using coimmunoprecipitation of in vitro translated Dsg3 and plakoglobin (Roh and Stanley, 1995b). The carboxyl-terminal domains of the desmogleins exhibit sequence similarity to the p-catenin binding domain of E-cadherin, suggesting that similar sequences mediate cadherin binding to plakoglobin. However, as discussed above, classical cadherins bind both plakoglobin and /3-catenin, but desmosoma1 cadherins do not interact with &catenin. The cytoplasmic domains of desmocollins also associate with plakoglobin (Kowalczyk et aL, 1994;Troyanovsky et al., 1994b). However, the “b” forms of desmocollins that are generated by alternative mRNA splicing lack the carboxyl-terminal region of the ICS domain and do not bind to plakoglobin (Troyanovsky et al., 1994b). The functional significance of the alternative splicing of the desmocollins with respect to desmosome assembly is not known. Another interesting motif is the RUD domain of the desmogleins. To date, this 29-amino-acid repeated unit domain has been found only in desmogleins. Again, the function of this domain, and the extended cytoplasmic tail of desmoglein in general, is not known. The RUD of Dsgl has been expressed in bacteria and purified and visualized by rotary shadowed electron microscopy (Rutman et al., 1994). Similar to predictions based on amino acid sequence analysis (Nilles et al., 1991), the RUD region was found to be globular in nature with a thin tail, perhaps corresponding to the glycine-rich terminal domain at the carboxyl terminus of the protein.

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Based on this structure and the finding that tail-tail structures were observed, it was suggested that this domain of desmoglein may be involved in the formation of dimers or oligomers (Rutman et al., 1994). An interesting property of the desmoglein cytoplasmic domain is that it appears to bind plakoglobin with high affinity (Chitaev et aZ., 1996; Mathur et aZ., 1994). In addition, the complex between Dsgl and plakoglobin forms with an apparent stoichiometry of six plakoglobin molecules for each desmoglein tail (Kowalczyk et aZ., 1996; Witcher et aZ., 1996). This is in contrast to the interactions between desmocollins and plakoglobin, and other cadherin-catenin interactions, where a 1:1 stoichiometry has been reported (Hinck et al., 1994a; Kowalczyk et al., 1996; Nathke et aZ., 1994; Ozawa and Kemler, 1992). One possible function of the desmoglein tail, and the RUD region in particular, may be to function as a scaffold for numerous plakoglobin proteins to cluster around the desmoglein tail. It will be interesting to define the function of the RUD region of desmoglein and the role of the unusual stoichiometry of binding between plakoglobin and desmoglein. A series of studies by Troyanovsky and colleagues have provided insights into the roles of the desmosomal cadherin cytoplasmic domains in desmosome assembly (Troyanovsky et al., 1993, 1994a,b). In these studies, the desmoglein or desmocollin cytoplasmic domains were fused to the gap junction protein connexin 45. These chimeras were then expressed in A431 epithelial cells, using the connexin domains to cluster the cadherin tails at the plasma membrane. One of the most interesting, and perhaps puzzling, findings was that the desmocollin cytoplasmic domain recruited desmosoma1 plaque components to the membrane, but the desmoglein cytoplasmic domain functioned as a dominant negative mutant and disrupted desmosome assembly. The desmocollincytoplasmicdomain recruited both plakoglobin and desmoplakin to the membrane where the connexins had formed gap junction-like structures. In addition, IFs were found to attach to regions of the membrane containing the desmocollin cytoplasmic tail, even though desmogleins were not present at these sites. In contrast, the desmoglein cytoplasmic tail not only failed to recruit desmoplakin and intermediate filaments, this chimera also disrupted endogenous desmosome assembly. Furthermore, the dominant negative activity of the desmoglein tail was abrogated by removal of the plakoglobin-binding domain. These data suggest that the interaction between desmoglein and plakoglobin is critical for normal desmosome assembly. Another interesting finding from these studies was that a truncation mutant of desmocollin lacking the plakoglobin binding domain could recruit desmoplakin, but not IFs, to the gap junction structures (Troyanovsky et aZ., 1994b). These results suggest that the IA domain of the desmocollins may play an important role in desmosome assembly. Furthermore, based on these results, it is possible that desmoplakin, either directly or indirectly,

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interacts with the IA domain of desmocollin during desmosome assembly. In contrast to the desmocollin IA domain, this region of desmoglein did not recruit desmoplakin or other desmosomal proteins to the membrane, suggesting that the cytoplasmic domains of the desmogleins and desmocollins perform very different functions in the assembly of the desmosomal plaque. However, the IA domain of Dsg3 was found to support the adhesive function of the E-cadherin extracellular domain, even without any apparent association with plakoglobin or the actin cytoskeleton (Roh and Stanley, 1995a). These results, along with previous studies of the IA domain of classical cadherins (Chen et al., 1997;Kintner, 1992), suggest that this region of the desmosomal cadherins may play an important role in the adhesive function of desmosomal cadherins and in the assembly of the cytoplasmic plaque of the desmosome.

B. Plakoglobin Plakoglobin was originally identified as a protein present in the cytoplasmic plaques of both adherens junctions and desmosomes (Cowin et al., 1986; Franke et al., 1987b). Subsequent analysis demonstrated that plakoglobin was related to a family of proteins that includes the adherens junction protein p-catenin and the Drosophilu segment polarity protein armadillo (Peifer, 1993; Peifer et al., 1994). The defining structural motif of this gene family is the presence of homologous repeated amino acid sequences, termed arm repeats. These repeats have now been found in a wide range of proteins, including the protein product of the tumor suppressor gene APC, protein components of the nuclear pore, as well as other junctionassociated proteins such as p120 and the plakophilins, which are discussed below (Peifer et al., 1994).The arm repeats appear to be involved in proteinprotein interactions. The central repeat domain of P-catenin has recently been crystallized and the three-dimensional structure determined by X-ray crystallography (A. Huber et al., 1997). The central repeat region forms a superhelix of helices that creates a shallow, positively charged groove. This positively charged groove is predicted to be an important interface for protein-protein interactions mediated by the arm repeats. Several P-catenin binding partners such as E-cadherin, APC, and the Tcf family of transcription factors exhibit long stretches of acidic amino acids, which would be negatively charged at physiological pH ranges and might interact with the basic groove of p-catenin. Although the three-dimensional structure of plakoglobin has not yet been determined, the central repeats of both plakoglobin and p120 are also highly basic, suggesting that these related arm proteins may interact with cadherin cytoplasmic domains through similar mechanisms (A. Huber et aZ., 1997).

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Plakoglobin appears to play an important role in linking cadherin cytoplasmic domains to cytoskeletal binding proteins. As discussed above, plakoglobin exhibits the unique ability among junctional proteins to associate with cadherins that are anchored to either the actin cytoskeleton or the IF cytoskeleton. Since plakoglobin binds directly to the cytoplasmic tails of the desmosomal cadherins, it is likely that plakoglobin plays an important role in linking the desmosomal cadherins to the IF cytoskeleton. As the multitude of protein-protein interactions in which plakoglobin engages in both adherens junctions and desmosomes continues to be examined, an elegant picture is emerging in which various domains of plakoglobin mediate specific binding events which are arranged in a precise hierarchy (Fig. 3). For example, the central domain of plakoglobin binds directly to the classical cadherins (Troyanovsky et al., 1996; Wahl et al., 1996; Witcher et aZ., 1996). In contrast, a-catenin binds to the amino-terminal domain of plakoglobin (Sacco et al., 1995) in the region encoded by exon 3 of the plakoglobin gene and including part of the first armadillo repeat (Aberle et al., 1996). In this manner, it appears that the central arm repeats form a functional domain that binds to the classical cadherins, thereby leaving the amino-terminal domain free to associate with a-catenin and the actin cytoskeleton. In striking contrast, the amino-terminal arm repeats of plakoglobin, including the region interacting with a-catenin, plays an important role in binding to desmosomal cadherins (Troyanovsky et al., 1996; Wahl et aZ., 1996; Witcher et al., 1996). In fact, plakoglobin that is bound to a desmosomal cadherin cytoplasmic domain does not form complexes with a-catenin (Kowalczyk et al., 1997; Plott et al., 1994). This may occur due to either steric hindrance or direct competition for binding. In contrast, desmoplakin does appear to bind to plakoglobin that is associated with the desmosomal cadherins and clusters these cadherin-plakoglobin complexes

Dsc

DSP N

........................................ -

a-catenin

APC

Dsg

............................

C

..............

Classical Cadherins

Desmoplakin FIG. 3 Schematic of the armadillo family member plakoglobin. Plakoglobin is a multifunctional protein with a series of conserved repeats, termed arm repeats, that mediate binding to numerous cytoplasmic proteins. The relative position along the repeats that each protein binds to is outlined.The asterisk in the amino-terminaldomain represents the GSK-3 consensus phosphorylation site. Refer to text for details and references.

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(Kowalczyk et aL, 1997) (see discussion below and Fig. 6). Thus, by inhibiting interactions between plakoglobin and a-catenin but allowing for interactions with desmoplakin, the cytoplasmic tails of the desmosomal cadherins dictate a conditional series of binding events. Through these initial interactions, the desmosomal cadherins ultimately become coupled to the IF cytoskeleton, whereas desmosomal cadherin interactions with the actin cytoskeleton are precluded. In contrast to the central arm repeats, which are highly conserved between plakoglobin, P-catenin, and armadillo, the amino- and carboxyl-terminal end domains of these proteins are significantly divergent. This suggests that these domains flanking the central arm repeats may perform functions specific to each arm family member. The function of the end domains of plakoglobin was recently analyzed in A431 epithelial cells by expressing deletion mutants of plakoglobin lacking either the amino- or the carboxylterminal domains and monitoring the effects on desmosome formation (Palka and Green, 1997). Interestingly, the carboxyl-terminal domain of plakoglobin appears to play an important role in junction assembly. Plakoglobin mutants lacking the carboxyl-terminal domain caused dramatic changes in desmosome organization, including the formation of fused desmosomes and the assembly of long desmosomes that occupied continuous regions of the plasma membrane. These results suggest that the carboxylterminal domain of plakoglobin plays an important role in limiting the overall size of the desmosome, perhaps by regulating lateral associations within the plaque. Consistent with this, Troyanovsky and colleagues have demonstrated that the carboxyl-terminal region of plakoglobin contains a self-interactive site that binds to carboxyl-terminal arm repeats (Troyanovsky et al., 1996), potentially acting to control the availability of these repeats for interactions with other desmosomal proteins. The role of plakoglobin in desmosome assembly and embryonic development has been studied in mouse mutants in which the plakoglobin gene was ablated. The most striking phenotype exhibited by plakoglobin knockout mice was the mechanical failure of the heart tissue around embryonic day 12 (Bierkamp et al., 1996; Ruiz et al., 1996). Intercellular adhesive interactions are mediated in the heart by specialized junctions termed intercalated discs, which include desmosomes, adherens junctions, and gap junctions. During embryonic development in plakoglobin null mice, the hearts of the animals failed due to a loss of cell-cell adhesion and subsequent bleeding as the hydrostatic pressures led to a loss of tissue integrity. In addition, the normal organization of intercellular junctions in the hearts of these mice was also perturbed. For example, desmoglein was present on the cell surface but was not clustered into desmosomes. Desmoplakin was also at cell borders along with desmocollin, but adherens junction components and desmosomal components were intermingled. Presumably,

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the loss of tissue integrity in these mice is due to the disorganization of the intercellular junctions and poor cytoskeletal interactions. Interestingly, desmosomes in other organs, such as the skin, appear to assemble. In the skin, the majority of desmosomes form normally, although the structures are occasionally poorly organized. In addition, in the small fraction of mice that escape the embryonic lethality, skin fragility and blistering are observed (Bierkamp et aL, 1996). However, due to the relatively normal appearance of desmosomes in most tissues, it is likely that other proteins, particularly the plakophilins (see below), can compensate for some functions of plakoglobin in desmosome assembly.

C. Plakophilins In addition to plakoglobin, another armadillo gene family member is now thought to be a major desmosomal plaque protein. A 75-kDa protein was isolated from desmosomes as a keratin binding protein and originally termed band 6 protein (Kapprell et aL, 1988). This protein is now known to be plakophilin 1 (Hatzfeld et al., 1994; Heid et aL, 1994) and is one member of a growing subfamily of the armadillo gene superfamily, which includes the closely related protein termed plakophilin 2 (Mertens et aL, 1996).The plakophilin subfamily also includes ~120"".a tyrosine phosphorylated protein that binds to E-cadherin and which is found in adherens junctions (Daniel and Reynolds, 1997). ~120'~" was originally identified as a src kinase substrate, suggesting that this family of proteins may play a role in regulating cadherin function in response to intracellular signaling pathways. Recently, another related protein termed p0071 was described (Hatzfeld and Nachtsheim, 1996). The mRNA for p0071 was found in all tissues examined, and the protein was often found at cell-cell borders and in the desmosomal plaques of cultured epithelial cells. Like pl2OCt",p0071 is proposed to play a role in the integration of cell signaling through cell-cell junctions. Several other proteins with homology to p0071 and p120''" have also been discovered, although no role for these proteins in intercellular junction assembly has been reported (Paffenholz and Franke, 1997;Sirotkin et al., 1997; Zhou et al., 1997). Interestingly, plakophilin 1expression also correlates with the differentiation state of tumors, with poorly differentiating cells typically lacking extensive plakophilin 1expression (Moll et al., 1997). The gene encoding human plakophilin 1 is found on the distal region of chromosome l q (Cowley et al., 1997b; Schmidt et al., 1997). A human patient with a skin blistering disease and plakophilin 1mutations was recently reported (McGrath et al., 1997). Both alleles of the plakophilin 1 gene were found to have defects that generated premature termination codons, resulting in a corresponding

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absence of plakophilin expression in the epidermis. The clinical manifestations of the lack of plakophilin expression included skin fragility and dysplasia, indicating a potential role for plakophilin in both keratinocyte cell-cell adhesion and epidermal morphogenesis. Interestingly, biopsies and immunofluorescence analysis demonstrated that although plakoglobin was present at keratinocyte cell-cell interfaces, desmoplakin was often found diffusely distributed in the cytoplasm. These findings suggest that plakophilin 1 may play an important role in the recruitment of desmoplakin to desmosomes and in anchoring keratin filaments to sites of keratinocyte adhesion. The plakophilins appear to be ubiquitously expressed and are found in cells that do not assemble desmosomes (Mertens et aL, 1996). In addition, both plakophilin 1 and plakophilin 2 are present in the nucleus, suggesting that these proteins have dual functions in the nucleus and at intercellular junctions (Mertens et aL, 1996; Schmidt et aL, 1997). Furthermore, both plakophilin 1 and plakophilin 2 occur as two isoforms due to alternative splicing. Plakophilin 2a is a protein predicted from cDNA analysis to comprise 837 amino acids, whereas an exon encoding an additional 44 amino acids is included in plakophilin 2b (Mertens et aZ., 1996). Similarly,plakophilin l a is a protein with a predicted 726 amino acids, while plakophilin l b has 747 amino acids, resulting from the splicing of exon 7 of the human plakophilin 1 gene. Interestingly, plakophilin l a is present in both nuclei and desmosomes of stratifying epithelia, whereas plakophilin l b is restricted to nuclei. Furthermore, although plakophilin 1 was found to be expressed in both epithelial and nonepithelial cells and was detected in nuclei, the recruitment of plakophilin l a to desmosomes was dependent on terminal differentiation (Schmidt et aL, 1997). Thus, the plakophilins might be regarded as constitutive nuclear proteins with desmosome localization being subject to some type of regulation. While the precise function of plakophilins in the desmosome is not yet known, band 6 (i.e., plakophilin 1) was found to bind directly to keratins (Hatzfeld et aL, 1994; Kapprell et al., 1988). More recent data suggest that band 6 may also interact with the desmoglein cytoplasmic domain (Mathur et aL, 1994). Thus, it is quite likely that plakophilins play a role in linking the desmosomal cadherins to the IF cytoskeleton, although the precise function of this emerging family of dual-function proteins remains to be determined.

D. Desrnoplakin 1. Structure

Desmoplakin is the most abundant of the desmosomal plaque components (Mueller and Franke, 1983). Comparisons between cDNAs for desmoplakin

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and several recently described proteins that mediate cytoskeletonmembrane interactions have revealed that desmoplakin is one member of a growing gene family, termed the plakins (Ruhrberg and Watt, 1997) (see below). Analysis of the predicted amino acid sequence from the desmoplakin cDNA suggests that desmoplakin is a homodimer consisting of two globular end domains joined by a central a-helical coiled-coil rod domain (Green et al., 1990; Virata et al., 1992) (Fig. 4). This prediction is supported by rotary shadowing electron microscopy of desmoplakin purified from bovine tissue (O’Keefe et al., 1989). Desmoplakin RNA is alternatively spliced in the region encoding the rod domain to generate two separate proteins, termed desmoplakin I and desmoplakin 11. The splicing is thought to result from the utilization of two alternative donor sites that compete for a single acceptor site during pre-mRNA splicing (Virata et al., 1992). The resulting proteins are thought to differ only in the rod domain and are derived from the same gene (Green et aZ., 1988). Both desmoplakin I and desmoplakin I1 exhibit a widespread tissue distribution, although desmoplakin I1 is less abundant in simple epithelia and is absent in heart (Angst et aL, 1990). The functional consequence of the alternative splicing is not known. The amino-terminal domain of desmoplakin is composed of heptad repeats that are interrupted by nonheptad regions (Virata et d., 1992). This structural organization is thought to allow for a-helical stretches to fold onto each other, resulting in antiparallel bundles that are stabilized by apolar interactions, resulting in a series of subdomains, termed Z, Y, X, W, and V. The central domain of desmoplakin is thought to be an a-

N-Terminal Globular Domain

1056aa

Desmosome Targeting Plakoglobin Binding

a-helical Coiled-Coil Rod Domain

889aa

Higher Order Aggregation

C-Terminal Globular Domain 926aa

Intermediate Filament Binding

FIG. 4 Schematic of desmoplakin I. Desmoplakin is a modular protein, with a central ahelical coiled-coil rod domain and globular amino- and carboxyl-terminal domains. The carboxyl-terminal domain binds directly to IF proteins and the rod domain is thought to promote the formation of higher order, multimeric structures important in plaque formation. The amino-terminaldomain binds directly to plakoglobin and directs desmoplakinrecruitment to the desmosomal plaque. Desmoplakin I1 (see Fig. 7) is generated by alternative RNA splicing to yield a shorter central rod domain. The asterisk in the carboxyl-terminal domain represents the CAMP consensus phosphorylation site at SerC23.

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helical coiled-coil rod-like structure with an estimated length of 130 nm in desmoplakin I. Approximately 87% of the central domain of desmoplakin is composed of a heptad substructure with a regularity that strongly predicts that the molecule is a two-stranded coiled-coil. Based on comparisons between the number of possible interchain ionic interactions that could occur between two desmoplakin monomers, it was predicted that the molecule is likely to be arranged as a dimer in which the monomer subunits are arranged in a parallel orientation (Green et al., 1990). Analysis of the predicted amino acid sequence suggests that each chain of the desmoplakin carboxyl-terminal domain also comprises a-helical subdomains, termed A, B, and C . These groups of 38 residue repeats are believed to be stabilized by ionic interactions. Interestingly, the periodicity of charged amino acids present in the desmoplakin carboxyl-terminal domain precisely matches the periodicity of charged residues in the a-helical coiled-coil rod domain of IF subunits (Green et al., 1990). These observations raised the possibility that interactions between the carboxyl-terminal domain of desmoplakin and IFs might be facilitated by the periodicity of these residues. 2. Interactions between Desmoplakin and Intermediate Filaments

Functional analysis of the various desmoplakin domains has demonstrated that desmoplakin is a modular protein, with the carboxyl-terminal, aminoterminal, and rod domains each performing specific functions (Bornslaeger et al., 1994; Kowalczyk and Green, 1996). To test directly the ability of desmoplakin to associate with IFs cDNA constructs encoding the carboxylterminal region of desmoplakin were transiently transfected into cultured cells (Stappenbeck and Green, 1992). The result was a striking alignment of desmoplakin along the IF network, followed by the disruption of normal filament architecture. The presence of the rod domain augmented the ability of the carboxyl-terminal domain to interact with filaments.Deletion analysis suggests that all three of the carboxyl-terminal subdomains were required for efficient alignment of desmoplakin with IF networks. Furthermore, the carboxyl-terminal68 amino acids were required for association with keratin but not vimentin networks (Stappenbeck et al., 1993). This region of desmoplakin is closely related to the carboxyl-terminal region of both BPAG 1 and plectin, suggesting that conserved mechanisms may dictate interactions between these proteins and the keratin cytoskeleton. The rod domain of desmoplakin was found to be important for efficient interactions between the desmoplakin carboxyl-terminal domain and the IF networks (Stappenbeck and Green, 1992). This is presumed to be due to the ability of the rod to mediate dimer formation, which might thereby promote proper folding of the carboxyl-terminal domain of desmoplakin

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and concentrate the number of subdomains available for interactions with IFs. In addition, expression of constructs encoding both the carboxylterminal and rod domains of desmoplakin led to remodeling of IF networks into filamentous structures that were composed of both desmoplakin polypeptides and IF subunits (Stappenbeck and Green, 1992). These structures consisted of a fine meshwork of filamentous material that resembled the inner plaque of the desmosome, where IFs loop into the desmosomal plaque. The observation that the formation of this meshwork required the desmoplakin rod suggests that the rod plays an important role in regulating the formation of higher order structures within the plaque. Recent studies have now demonstrated that the carboxyl-terminal domain of desmoplakin binds directly to a number of IF family members. Initially, in vitro studies with purified domains of desmoplakin and keratins indicated that desmoplakin binds to the type I1 subunits of the epidermal keratins, including K1, K2, K5, and K6. However, binding to vimentin, the type I keratin subunits expressed in epidermis, or the keratin pairs from simple epithelium (i.e., K8 and K18) was not detected (Kouklis et al., 1994). Desmoplakin was found to bind to an 18-amino-acidregion within the head domain of K5. It is likely that the binding of desmoplakin to the epidermal keratins may be particularly tight, since the desmoplakin-IF linkages in keratinocytes would need to withstand the particularly harsh mechanical stresses encountered in the epidermis. Nonetheless, while the linkage of desmoplakin to keratin filaments is thought to be critical to the mechanical integrity of the epidermis, desmoplakin is also thought to play an important role in linking other keratins and IF networks to desmosomes in a variety of tissues. The ability of the desmoplakin carboxyl terminus to bind directly to other IF proteins was recently observed using the yeast two-hybrid system and blot overlay assays (Meng et al., 1997). These experiments confirmed that the desmoplakin carboxyl terminus binds to the head domain of the type I1 epidermal keratin K1, similar to the in vitro binding reported in earlier studies for K5 (Kouklis et al., 1994). In addition, a direct interaction between desmoplakin and the simple epithelial keratins K8 and K18 was detected in the yeast two-hybrid system. However, this interaction required both keratin subunits, in contrast to the interaction between desmoplakin and K1 which did not require the K10 partner. Furthermore, the two-hybrid experiments demonstrated that both vimentin and desmin, which are type I11 intermediate filaments, also bind directly to desmoplakin. Together, these studies demonstrate that desmoplakin is an IF binding protein and provide further evidence that desmoplakin plays an important role in directly linking various types of IF networks to the plasma membrane.

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3. Role in Desmosome Assembly and Intermediate Filament Anchorage The desmoplakin amino-terminal domain is thought to be necessary for targeting desmoplakin to the desmosomal plaque. This is based on the finding that desmoplakin polypeptides expressed in cultured cells are not recruited to the desmosomal plaque unless the proteins contain the desmoplakin amino-terminal domain (Stappenbeck et el., 1993). Although deletion of the carboxyl terminus did not compromise incorporation into desmosomes, deletion of the first 194 amino acids of desmoplakin severely compromised the recruitment of desmoplakin polypeptides to desmosomes. Based on the observation that the amino-terminal domain of desmoplakin was required for desmosome incorporation, and on studies indicating that the carboxyl-terminal region of desmoplakin binds to IFs, it was hypothesized that IF could be uncoupled from desmosomes by expression of a dominant negative polypeptide comprising the desmoplakin aminoterminal domain but lacking carboxyl-terminal sequences needed for IF attachment. In fact, expression of such a polypeptide, DP-NTP, disrupted the assembly of endogenous desmoplakin into desmosomes (Fig. 5 ) (Bornslaeger et el., 1996). The mutant desmoplakin amino-terminal polypeptide incorporated into junctions that were ultrastructurally similar to the outer plaque of the desmosome. However, the displacement of endogenous desmoplakin from desmosomes inhibited the attachment of IFs to these cellcell junctions. These observations were the first direct evidence demonstrating that desmoplakin plays a critical role in coupling the IF network to the desmosome. Furthermore, the expression of DP-NTP and the loss of keratin attachment to the plasma membrane also led to the mixing of desmosomal and adherens junction components (Bornslaeger et eL, 1996). The fact that these two junctions, which are normally segregated into highly organized and separated membrane domains, were intermingled in cells expressing DP-NTP suggests that desmoplakin plays an important role in maintaining the organization of plasma membrane domains in cell types that assemble both adherens junctions and desmosomes.

4. Interactions between Desmoplakin and the Desmosomal Cadherin-Plakoglobin Complex

The observation that the amino-terminal domain of desmoplakin targeted the protein to the desmosomal plaque, along with the observation that DPNTP could apparently compete with endogenous desmoplakin for binding sites at the plasma membrane, suggested that the amino-terminal domain

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G

Desmosomal FIG. 5 Expression of an amino-terminal desmoplakin polypeptide (DP-NTP) causes keratin filament detachment from the plasma membrane of A431 cells. Immunofluorescence analysis of normal A431 cells (A and B) and cell lines expressing DP-NTP (C-F). Endogenous desmoplakin is detected using an antibody directed against the desmoplakin carboxyl-terminal domain (A and C). DP-NTP is monitored using a polyclonal antibody directed against the desmoplakin amino-terminal domain (E). Staining for keratin 18 is shown in B, D, and F. In cells expressing DP-NTF', endogenous desmoplakin is displaced from cell-cell borders except in occasional areas where large aggregates accumulate (C). In addition, keratin tonofilaments

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FIG. 6 DP-NTP clusters desmosomal cadherin-plakoglobin complexes. L cell lines coexpressing Dsgl and plakoglobin (A): Dsgl, plakoglobin, and DP-NTP (B): Dsc2a, plakoglobin, and DP-NTP (C): or an E-cadherin-Dsgl chimera,plakoglobin,and DP-NTP (D) were established. The chimeric cadherin comprised the E-cadherin extracellular domain and the Dsgl cytoplasmic domain. Immunofluorescence staining was used to detect the cadherin in each cell line. In the absence of DP-NTP,Dsgl stainingwas diffuse. In contrast, cadherins were clustered in L cell lines coexpressing DP-NTP. In addition, the E-cadherin-Dsgl chimera recruited DP-NTP to cell-cell interfaces. In each case, plakoglobin colocalized with the cadherin and DP-NTP. Reproduced from The Journal of Cell Biology (Kowalczyk et al.) 1997,139,773-784, by copyright permission of The Rockefeller University Press.

of the protein may interact with the desmosomal cadherin-plakoglobin complex. The ability of DP-NTP to associate with the desmosomal cadherin-plakoglobin complex was tested by coexpressing these proteins in L cell fibroblasts. L cells do not assemble desmosomes and do not normally express desmosomal or adherens junction proteins. In the absence of DPNTP, the desmosomal cadherins and plakoglobin were distributed in a

do not attach to the membrane except in areas where endogenous desmoplakin is present (D). DP-NTP i s largely in a continuous pattern at cell-cell interfaces (E), although keratin bundles are not attached to these junctions (F). A schematic depicting how DP-NTP might cause keratin filament detachment by competing with endogenous desmoplakin for binding to plakoglobin is shown in G. Reproduced from The Journal of Cell Biology (Bornslaeger et aL), 1996, l34, 985-1001, by copyright permission of The Rockefeller University Press.

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diffuse pattern along the plasma membrane (Kowalczyk et aZ., 1997) (Fig. 6). However, in cells coexpressing DP-NTP, the cadherins and plakoglobin were clustered into punctate regions of staining that colocalized with DPNTP. Confocal microscopy and trypsinization experiments demonstrated that the desmosomal cadherins were present in clustered complexes at the cell surface. Due to the inability of desmosomal cadherins to mediate homophilic adhesion in the L cell system, these complexes were distributed at the basal and apical aspects of the cell surface and not at cell-cell borders. However, a chimeric cadherin comprising the E-cadherin extracellular domain and the Dsgl cytoplasmic domain accumulated at cell-cell borders and recruited plakoglobin and DP-NTP to cell-cell borders (Fig. 6D). These proteins accumulated in a punctate pattern that was reminiscent of the distribution of desmosomal proteins in epithelial cells, and electron microscopy demonstrated the formation of an electron-dense submembrane structure similar in appearance to the outer region of the desmosomal plaque. Interestingly, coexpression of plakoglobin was required in order for DPNTP to cluster the desmosomal cadherins (Kowalczyk et aL, 1997). Dsgl was not clustered by DP-NTP in the absence of exogenously expressed plakoglobin. However, in addition to full-length plakoglobin, two plakoglobin mutants lacking either the amino- or the carboxyl-terminal domains could also support DP-NTP--mediated clustering of Dsgl. In addition, plakoglobin also coimmunoprecipitated with DP-NTP from lysates of L cells coexpressing Dsgl, plakoglobin, and DP-NTP. To determine if the interaction between DP-NTP and plakoglobin was direct, these proteins were tested for direct interactions in the yeast two-hybrid system. DP-NTP was found to bind directly to plakoglobin, but DP-NTP does not interact directly with the desmosomal cadherins, either Dsgl (Kowalczyk et aZ., 1997) or Dsc2a (Bornslaeger and Green, unpublished observations). In addition, although DP-NTP binds directly to plakoglobin, DP-NTP does not bind to 0-catenin (Kowalczyk and Green, unpublished observations). Thus, the desmoplakin amino-terminal domain appears to be linked to the desmosomal cadherins through plakoglobin. This is similar to the mechanism by which plakoglobin or 0-catenin link a-catenin indirectly to E-cadherin in adherens junctions (Aberle et aL, 1994; Jou et aZ., 1995). Together with the studies discussed above, these data indicate that desmoplakin binds directly both to the IF network and to the cadherinplakoglobin complex. These properties of desmoplakin indicate that this protein plays an important role in coupling desmosomal adhesive interactions to the IF cytoskeleton, thereby integrating the filament networks between adjacent cells.

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E. Desmoplakin Is a Member of a Family of Intermediate Filament Linking Proteins: The Plakin Family Structural comparisons between desmoplakin and other cytoskeleton-associating proteins have revealed that desmoplakin is one member of a growing gene family that has been termed the plakins (Fig. 7 and Ruhrberg and Watt, 1997). In addition to desmoplakin, the original members of this family include the 230-kDa bullous pemphigoid antigen (BPAG1) and plectin (Green et aZ., 1990,1992). Both BPAGl and plectin localize to the plaques of hemidesmosomes,cell-extracellular matrix adhesion structures that typically mediate epithelial cell adhesion to a basement membrane. Although both desmosomes and hemidesmosomes anchor the IF network to sites of cell adhesion, these adhesion structures are biochemically distinct (Green and Jones, 1996;Jones et al., 1986b). Adding to the complexity of the plakin gene family is the fact that several forms of these proteins are generated by alternative splicing of the RNA. In addition, some forms of BPAGl

FIG. 7 Plakin family of cytoskeletal linking proteins. A schematic diagram depicting the structural characteristics of the plakin family members is shown. See text for a detailed discussion of protein domains (see also Ruhrberg and Watt, 1997).

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and plectin contain an amino-terminal actin-binding domain that is also found in ACF7, a mouse protein with homology to both BPAGl and dystonin (Bernier el al., 1996; Byers et al., 1995), as well as desmoplakin (S. Guy and K. Green, unpublished observations). 1. Bullous Pemphigoid Antigen 1

The importance of BPAGl in cellular integrity is underscored by the results obtained in a mouse model in which the BPAGl gene was ablated. BPAGl null mice exhibited blistering characteristic of epidermolysis bullosa patients (Guo et d., 1995). In addition, these mice exhibited a striking and unexpected neurological disorder characterized by neurodegeneration and subsequent loss of motor function. Interestingly, the BPAGl -/- mice exhibited a phenotype of mice with a previously described recessive neurological disorder termed dystonia musculorum (Bernier et al., 1995). In addition to the epidermal forms of BPAGl (BPAGle), neuronal forms of BPAGl termed BPAGlnl and BPAGln2, or dystoninl and dystonin2, have also been described (Brown et al., 1995a,b; Yang et al., 1996). Both neuronal forms contain additional amino-terminal sequences, including a region with similarity to a family of actin binding proteins. Other splice forms of the epidermal BPAGl lacking the carboxyl terminus have also been reported (Hopkinson and Jones, 1994), suggesting that additional forms of BPAGl remain uncharacterized. The phenotype of BPAGl - - mice is thought to be due to the lack of BPAGl protein expression in the epidermis, as well as loss of expression of the neuronal forms of BPAGl. These studies have focused attention on the role of the BPAGl protein not only in IF-cell surface interactions, but also the role of this protein in providing a link between the IF and the microfilament networks (Dowling et al., 1997; Fuchs et al., 1997). 2. Plectin/IFAP 300

The analysis of adhesive complexes, from focal contacts and hemidesmosomes to adherens junctions, indicates that the linkage between the adhesive glycoproteins and the cytoskeleton is indirect and involves numerous adapter proteins. Desmosomes appear to be no exception to this rule, and, as discussed above, desmoplakin appears to be only one of several proteins that couple LFs to the desmosomal plaque. Another protein that may play a role in linking IF networks to the desmosome is IFAP 300, a large IF binding protein isolated from BHK fibroblasts (Lieska et al., 1985; Skalli et al., 1994; Yang et al., 1985). Immunofluorescence indicates that IFAP 300 is present in epithelial cells and aligns along IF networks. In addition, IFAP 300 was localized to the desmosomal and hemidesmosomal plaques,

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and immunoelectron microscopy demonstrated that the protein was localized deeper in the cytoplasmic plaque than desmoplakin. Interestingly, IFAP 300 isolated from BHK cells binds both to desmoplakin and to keratins, and IFAP 300 appears to enhance interactions between desmoplakin and keratins (Skalli et aL, 1995). These data suggest that IFAP 300 may function cooperatively with desmoplakin to couple IFs to desmosomes. Although the complete cDNA sequence encoding IFAP 300 has not yet been identified, this protein may be closely related or identical to plectin. Plectin is expressed widely throughout adult tissues and is thought to be important both in linking IF networks to the cell surface and in mediating interactions between various classes of cytoskeletal networks (Elliott et aL, 1997; Svitkina et al., 1997). Plectin was originally found to bind to microtubules and actin, as well as IFs (Wiche, 1989), and was recently visualized forming cross bridges among microfilament, microtubule, and IF networks (Svitkina et aL, 1997). Like desmoplakin, the plectin carboxylterminal domain interacts with IF networks (Wiche et aL, 1993), and the overall structural organization of plectin is very similar to that of desmoplakin (Green et aL, 1992). The interaction between the carboxylterminal domain of plectin and vimentin networks was found to occur within the fifth repeat domain in a stretch of 50 amino acids that is responsible for interactions with both vimentin and keratins (Nikolic et al., 1996). Recently, desmoplakin and plectin were found to interact in MDCK cells. Interestingly, desmoplakin interactions with plectin were found to occur in polarized MDCK cells, indicating that these proteins interact in a regulated manner (Eger et aZ., 1997). However, in human diseases in which plectin gene mutations have been described (Uitto et al., 1996) (see below) and in the mouse model system in which plectin was ablated (Andra et al., 1997), no changes in the ultrastructural organization of desmosomes have been noted. Thus, if plectin does play a role in desmosome assembly and the anchoring of IFs to the desmosome, it is likely to be a more auxiliary role than that described for desmoplakin. Growing interest in the plakin family of proteins has coincided with the discovery that a form of the human epidermal blistering disease epidermolysis bullosa associated with a late-onset muscular dystrophy is caused by mutations in plectin (Uitto et al., 1996). This form of the skin blistering disease in humans is very similar to the phenotype of knockout mice in which the plectin gene was inactivated (Andra et al., 1997). Mice lacking plectin exhibited skin blistering similar to that in epidermolysis bullosa patients as well as skeletal and cardiac muscle abnormalities. The plectin knockout mice typically died 2-3 days after birth. Like BPAG1, plectin is associated with hemidesmosomes, and its loss from these structures could explain the skin phenotype. It is not yet clear how defects in plectin lead to the development of muscular dystrophy, but this may result from compro-

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mised cytoskeletal linkage to the sarcolemmal membrane. Similar to BPAG1, isoforms of plectin have also been reported, although the forms isolated from both epidermis (plectin-e) and placenta (plectin-p) have an amino-terminal actin-binding domain. The actin binding domain of plectin is related to the actin binding domain in the neuronal forms of BPAGl (Ruhrberg and Watt, 1997). 3. Envoplakin and Periplakin

Two newly described proteins, envoplakin (Ruhrberg et al., 1996) and periplakin (Ruhrberg et al., 1997), have been added to the plakin family of cytoskeleton-associating proteins. Although structurally related to desmoplakin and present near desmosomes, these proteins exhibit different subcellular distributions, and it is likely that these proteins perform very different functions in cells. Envoplakin is a 210-kDa protein expressed in stratifymg epithelia where it colocalizes with desmoplakin both in the inner plaque region of the desmosome and along IF networks. Periplakin is a 1995-kDa protein structurally similar to desmoplakin and envoplakin in the amino-terminal domain and rod, but lacking a carboxyl-terminaldomain homologous to the IF binding region of desmoplakin. Induction of terminal differentiation by culturing keratinocytes in suspension increases envoplakin expression, and envoplakin is crosslinked into the cornified envelope during keratinocyte differentiation. Several other desmosomal components have also been reported to incorporate into the transglutaminase-crosslinked envelope, including desmoplakin and plakoglobin (Robinson et aL, 1997; Steinert and Marekov, 1997). The observation that envoplakin exhibits colocalization with desmoplakin and contains carboxyl-tenninal sequences homologous to the IF binding domain of desmoplakin suggests that envoplakin may also play a role in linking IFs to the plasma membrane. However, envoplakin has only one of the carboxyl-terminal homology domains found in desmoplakin, and periplakin lacks these domains altogether (Fig. 7). Both periplakin and envoplakin have a carboxyl-terminal linker domain that is also present in desmoplakin and plectin, which may mediate interactions with IFs (Ruhrberg and Watt, 1997). However, the ability of periplakin and envoplakin to bind directly to IFs and to mediate filament-membrane interactions remains to be demonstrated. Interestingly, periplakin and envoplakin form complexes that can be coimmunoprecipitated from keratinocyte lysates, perhaps via interactions mediated by the homologous rod domains of the two proteins (Ruhrberg et al., 1997). However, interactions between desmoplakin and envoplakin or periplakin were not observed. Periplakin and envoplakin colocalize in networks radiating out of the desmosomal plaque, and, like envoplakin, periplakin is incorporated into the transglutaminase-

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crosslinked cornified envelope. For this reason, it is postulated that in addition to playing a role in coupling IFs to the cell surface, envoplakin and periplakin may function to provide a scaffold for cornified envelope assembly. F. Other Desmosomal Plaque Proteins Another protein found deeper in the desmosomalplaque was originally identified by a monoclonal antibody and termed the 08L antigen (Ouyang and Sugrue, 1992).The cDNA encoding this 140-kDaprotein has been identified and the protein renamed pinin (Ouyang and Sugrue, 1996). The predicted amino acid sequence indicates the presence of three unique domains, including a serine-rich domain, a glutamine-proline/glutamine-leucinerepeat domain, and an acidic domain rich in glutamic acid. Pinin was found to be expressed in a variety of tissues including heart, brain, placenta, lung, liver, and pancreas. Pinin appears to associate with mature desmosomes and may play a role in stabilizing the attachment of IFs to the desmosomal plaque. Transfection of cDNA encoding the full-length protein into transformed 293 cells resulted in enhanced cell-cell adhesion which was manifest in a dramatic change in morphology. Control 293 cells fail to form tight associations, whereas cell lines expressing pinin formed well-organized colonies. The increase in cell-cell interactions was also evident in the increased numbers of desmosomes that formed in cells expressing pinin. Given these findings, it was suggested that pinin may play a role in enhancing cell-cell interactions by stabilizing IF attachment to desmosomes. Several other proteins have also been localized to the plaque region of the desmosome. Desmoyokin is a large protein originally thought to be associated with the desmosome (Hieda et al., 1989; Hashimoto et al., 1993). Subsequent analysis indicated that desmoyokin is homologous to the protein encoded by the human gene AHNAK (Hashimoto et al., 1993) and is localized to nondesmosomal regions of the keratinocyte plasma membrane (Masunaga et aL, 1995). A microtubule-binding protein termed pp170 has also been found in the plaque of desmosomes in confluent MDCK and Caco-2 cells, although the function of this protein has not been determined (Wacker et al., 1992). Two independently identified high-molecular-weight (240-250 kDa) proteins have been found to localize to the desmosomal plaque. The relationship between these proteins, termed desmocalmin and keratocalmin, is unclear. Desmocalmin was isolated from bovine muzzle epidermis by calmodulin-affinity chromatography and was also shown to bind to keratin filaments in a Mg2+-dependent manner (Tsukita, 1985). Keratocalmin is a calmodulin-bindingprotein isolated from human epidermis (Fairley et aL, 1991). Antibodies directed against keratocalmin localize

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to cell-cell borders of keratinocytes and recognize a cytoplasmic antigen localized to desmosomes. Although these proteins appear related, the primary structure and the function of these proteins in desmosome assembly have not yet been reported.

V. Protein-Protein Interactions in the Desmosome: A Model for Desmosome Assembly While our understanding of the genes that encode junctional proteins continues to grow, progress has also been made in the analysis of the molecular interactions between junctional components. Recent work from a number of laboratories has provided the basis for how desmosomal components may interact during the formation of these complex macromolecular structures. While several key questions still remain, the hierarchy of binding events in the plaque leading to attachment of IFs is being resolved. In addition, it is possible to speculate on the function of some of the newly described plaque components that exhibit structural similarity with previously identified and functionally characterized plaque proteins, as summarized in Fig. 8. As described above, recent work has demonstrated that desmoplakin binds directly both to IFs and to plakoglobin (Kouklis et aZ., 1994; Kowalczyk el aZ., 1997;Meng et aZ., 1997). These findings, along with the results from dominant negative experiments (Bornslaeger et aZ., 1996), firmly establish desmoplakin as a key player in coupling IFs to desmosomal cadherins. However, a common observation made in the analysis of adhesion structures is that multiple interactions act cooperatively to perform linking functions at

FIG. 8 Speculative model depicting the possible arrangement and protein interactions that may be occumng in the desmosome. See text for detailed discussion.

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the cytoplasmic face of the junction, and interactions within the desmosome appear to follow this pattern. In addition to plakoglobin playing an important role as a linking component in the plaque, plakophilin may also contribute to the linking of IF binding proteins to the desmosomal plaque. This idea is supported by the recent observation that plakophilin 1 mutations in humans cause defects in desmoplakin recruitment to desmosomes in keratinocytes (McGrath et al., 1997). Likewise, in plakoglobin knockout mice, desmoplakin is still recruited to desmosomes in most tissues (Bierkamp et al., 1996; Ruiz et al., 1996). Although speculative, it is likely that plakophilins may also bind to desmoplakin and function as additional linking proteins in the attachment of desmoplakins to desmosomal cadherins. This hypothesis is supported further by the fact that band 6/plakophilin 1 also appears to interact directly with desmoglein in vitro (Mathur et al., 1994). As discussed above, the desmoglein cytoplasmic tail appears to have the capacity to bind approximately six plakoglobin proteins, whereas the stoichiometry of the plakog1obin:desmocollin complex is closer to 1:1 (Kowalczyk et al., 1996; Witcher et al., 1996). It will be important to test the functional signficance of the stoichiometry of various plaque interactions and to determine the relative contributions of plakoglobin and plakophilin to the attachment of desmoplakin to the desmosomal cadherins. Moving deeper into the plaque, studies at the interface between desmoplakin and IFs suggest that the interaction between desmoplakin and the IF network is augmented by other IF binding proteins. For example, desmoplakin appears to form complexes with plectin/IFAP 300 (Eger et al., 1997), which also binds to keratin IFs. Envoplakin and periplakin may also interact with each other and with IFs. Although the precise interactions between components are not known, such interactions may strengthen the association of filaments with the desmosomal plaque. The idea that desmosomal plaque components may act cooperatively to provide strong IF attachment to the desmosome is supported further by the observation that pinin expression stabilizes filament interactions with the desmosomal plaque (Ouyang and Sugrue, 1996). Together, these observations suggest that multiple interactions function cooperatively to enhance filament attachment to the plaque and ultimately stregthen cell-cell adhesion. Last, perhaps the most important missing link in our understanding of the desmosome is the current lack of information regarding the protein interactions that occur between desmosomal cadherins during adhesion. Some insights into this problem have recently been provided by Troyanovsky and colleagues who demonstrated that desmogleins and desmocollins on opposing cells form complexes that can be coimmunoprecipitated (Chitaev and Troyanovsky, 1997). But it remains unclear precisely how the desmosomal cadherins form adhesive complexes. It will be important to understand how such interactions form, particularly in the context of the

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information from structural studies on dimer formation by classical cadherins discussed above. For example, do the desmosomal cadherins also interact laterally? Do such interactions lead to the formation of heterodimers that are the basic units in adhesion, or do the desmosomal cadherins form lateral homodimers that interact heterophilically? Certainly, resolving the crystal structure of the desmosomal cadherin extracellular domains would provide a significant leap forward in our understanding of these unique adhesion molecules. Ultimately, however, the adhesive function of desmosoma1 cadherins will need to be demonstrated in a cell type lacking other cell-cell adhesion molecules.

VI. Regulation of Desmosome Assembly

A. Calcium Switch Model of Assembly The most commonly used method to rapidly control the assembly and dissolution of intercellular junctions is the calcium switch model. Under normal culture conditions, which include calcium ions at millimolar levels, epithelial cells assemble extensive intercellular junctions. However, when calcium levels are lowered to approximately 75 nM, intercellular junctions are disassembled and the actin and IF cytoskeletal systems retract from the plasma membrane (Jones and Goldman, 1985; Mattey et al., 1990; Mattey and Garrod, 1986a,b; Watt et al., 1984). Due to the fact that both desmosomal and classical cadherins are thought to be dependent on calcium for function, the inability to assemble junctions in low calcium is thought to be due to the loss of cadherin-mediated adhesion. The lack of intercellular junction assembly in the absence of calcium, and therefore cadherin-mediated adhesion, suggests that cadherincadherin interactions trigger subsequent junction assembly. In the assembly of focal contacts, cell-extracellular matrix junctions containing adhesive glycoproteins termed integrins, ligation and clustering of integrins generate intracellular signals that trigger localized actin microfilament assembly at the cytoplasmic face of the plasma membrane in response to integrin adhesion (Yamada and Miyamoto, 1995). It is likely that cadherin-cadherin interactions trigger similar signals. In early studies of junction assembly in MDCK cells, a dramatic increase in the metabolic stability of the desmosoma1 cadherins and desmoplakin was observed when the cells were switched from low to normal calcium (Pasdar et al., 1995b; Pasdar and Nelson, 1988a,b, 1989; Penn et al., 1987a,b). In low-calcium medium, pulse-chase experiments demonstrated that desmogleins were turned over with a TIl2 of approximately 2-4 h, while desmoplakin exhibited a TIEof about 20 h.

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In contrast, desmoglein stability was increased to 20-24 h when MDCK cells were transferred to normal levels of calcium and desmoplakin stability was increased to 50-70 h. In addition to changes in half-life, switching cells from low calcium to normal calcium results in the transfer of junctional proteins from a detergent-soluble to a detergent-insoluble pool. In fact, junctional proteins appear to exist in different subcellular pools at different times during assembly. These studies suggest that the engagement of the cadherins in intercellular adhesion results in downstream signals that promote attachment of cadherins to plaque components which subsequently couple the complexes to the cytoskeleton.

B. Regulation of Intercellular Junctions by Tumorigenic Transformation and Tyrosine Phosphorylation The steps involved in regulating intercellular adhesion during development, wound healing, or metastasis are not well understood, although modulation of adhesive junctions is thought to play an important role in these events (Bryant, 1997;Garrod et al., 1996;Garrod, 1995;Hynes, 1996).For example, in the progression of cancers, the ability of cells to downregulate or alter the adhesive capabilities of their junctions is a contributing factor to tumor metastasis (Ben-Ze’ev, 1997). Furthermore, E-cadherin has been shown to function as a suppressor of tumor invasion (Vleminckx et al., 1991). In addition, a number of junctional proteins are altered or mutated in cells derived from tumors, including a-catenin (Bullions et al., 1997; Shimoyama et al., 1992), P-catenin (Kawanishi et al., 1995; Oyama et al., 1994; Sommers et al., 1994), and E-cadherin (Bohm et al., 1994; Oka et al., 1993; Vleminckx et al., 1991). Similarly,cells from tumors also fail to assemble normal desmosomes, and the expression of desmosomal components is often altered in tumor cells (Collins et al., 1990; Green et al., 1991). In breast and ovarian cancers, the loss of plakoglobin heterozygosity correlates with tumor progression, suggesting a role for plakoglobin as a tumor suppressor (Aberle et al., 1995). The loss of E-cadherin and desmosomal protein expression correlates with the metastatic behavior of tumors (Shinohara et al., 1998), and the loss of desmosomes may be an important marker for the evaluation of human tumors (Krunic et al., 1998). In addition to the alterations in junctional proteins observed in tumorigenesis and metastasis, junctional components are also integrated into growth factor signaling pathways. Recently, the zinc finger protein slug was shown to be an important intermediary in the dissolution of desmosomes during epithelial-mesenchymal transformation (Savagner et al., 1997). Tyrosine phosphorylation of junctional components, and in particular phosphorylation of members of the armadillo family of proteins, may be one

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of the critical steps involved in the modulation of cell-cell adhesion during development and wound healing. Several lines of evidence suggest that tyrosine phosphorylation of the cadherin-catenin complex affects cell adhesion and possibly the association of cadherin-catenin complexes with the cytoskeleton (Behrens et al., 1993; Fujii et al., 1996; Hamaguchi et al., 1993; Kinch et aL, 1995; Matsuyoshi et al., 1992). Recently, both plakoglobin and p-catenin have been shown to associate with the tyrosine kinase c-erbB-2 (Kanai et al., 1995), and the EGF receptor has been found to directly phosphorylate p-catenin in vifro (Hoschuetzky et aL, 1994). In addition to the apparent modulation by tyrosine kinases, the cadherin-catenin complex can also associate with protein tyrosine phosphatases (Aicher et aZ., 1997; Balsam0 et aZ., 1996; Brady-Kalnay et al., 1995; Fuchs et aL, 1996; Kypta et al., 1996). The observation that plakoglobin and P-catenin associate with both tyrosine kinases and phosphatases strongly suggests that the balance between phosphorylation and dephosphorylation of armadillo family proteins is a key element in the modulation of cell-cell adhesion (Fuchs et al., 1996; Hoschuetzky et al., 1994; Kanai et al., 1995; Kypta et al., 1996; Ochiai et al., 1994). Although tyrosine phosphorylation of p-catenin has been correlated with a decrease in classical cadherin-mediated adhesion in certain cells (Behrens et aL, 1993;Hamaguchi et aL, 1993;Matsuyoshi et al., 1992), the mechanisms by which phosphorylation of armadillo family members alters cell adhesion is not known. Results from several laboratories suggests that tyrosine phosphorylation of cadheridcatenin complexes increases the detergent solubility of cadherins and catenins, suggesting decreased association of the cadherinkatenin complex with the cytoskeleton (Fujii et al., 1996; Kinch et aL, 1995; Skoudy et al., 1996). In ras transformed MCF-1OA human breast epithelial cells, tyrosine phosphorylation of p-catenin correlates with increased solubility of both p-catenin and E-cadherin (Kinch et al., 1995). Tyrosine phosphorylation of @-cateninin EGF-treated HSC-1 squamous carcinoma cells also correlates with an increased detergent solubility of E-cadherin (Fujii et aL, 1996). Furthermore, tyrosine-phosphorylated pcatenin is predominantly in a Triton-soluble pool of proteins in EGFtreated A-431 cells and ras transformed MCF-1OA cells (Hoschuetzky et al., 1994; Kinch et al., 1995). Several mechanisms have been proposed to explain the adhesive changes associated with cadherin-catenin tyrosine phosphorylation (Daniel and Reynolds, 1997). One model suggests that tyrosine phosphorylation of the cadherin-catenin complex leads to decreased association with the cytoskeleton, perhaps by modulating the affinity of interactions between the catenins and the cadherin. Alternatively, it is possible that the catenin-cadherin complex remains intact but that the conformation of the catenins might be altered in such a manner that association with the cytoskeleton is prevented.

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Although it is still not clear how these phosphorylation events regulate intercellular junction assembly, the bulk of the observations are consistent with the idea that decreased association of cadherin-catenin complexes with the cytoskeleton in response to tyrosine phosphorylation leads to decreased intercellular adhesion. Given the potential importance of growth factor-regulated changes in intercellular adhesion during wound healing and tumorigenesis, it will be important to understand precisely how intercellular junctions are altered by growth factor signaling pathways. This type of detailed information will be critical for applying therapeutic interventions to regulate cell adhesion in pathologies such as chronic wounds and epithelialderived tumors. Although there is considerable evidence that tyrosine phosphorylation regulates the subcellular distribution of junction components, it is not yet clear if tyrosine phosphorylation of plakoglobin and P-catenin is the key event leading to the destabilization of intercellular junctions. In fact, some evidence suggests that phosphorylation of plakoglobin and P-catenin is not required for the modulation of cadherin-mediated adhesion. This was demonstrated by using a chimeric protein comprising the E-cadherin extracellular and transmembrane domain fused to a-catenin (Takeda et al., 1995). This protein, although unable to bind to plakoglobin or P-catenin, mediated adhesion when expressed in L cell lines. In addition, the adhesive function of the E-cadherin-a-catenin chimera was inhibited by transformation of cells with v-src. These results suggest that cadherin-mediated adhesion may be modulated by regulatory events distal to the cadherin-plakoglobidflcatenin complex, perhaps at the level of cytoskeletal attachment to acatenin. Growth factor stimulation and tyrosine phosphorylation may modulate not only classical cadherin-mediated adhesion but also desmosomal adhesion. As discussed above, the desmosomal cadherins interact with plakoglobin (Korman et al., 1989; Kowalczyk el al., 1994; Mathur et al., 1994; Peifer et al., 1992; Troyanovsky et al., 1994a,b). In addition, plakoglobin binds directly to the desmoplakin amino-terminal domain and thereby links the desmosomal cadherins to desmoplakin (Kowalczyk et al., 1997). Since desmoplakin plays an important role in anchoring IF networks to the cell surface and the desmosomal cadherins (Bornslaeger et al., 1996),it is attractive to speculate that this link might be disrupted by tyrosine phosphorylation of plakoglobin. Again, it may be that the binding of plakoglobin to the cadherins or plakoglobin to desmoplakin is altered, but in either scenario, the linkage of desmosomal cadherins to the IF network would be compromised. This would then be predicted to decrease desmosomal cadherin-mediated adhesion, leading to cellular shape change and increased cell motility. Although this particular model is speculative, the bulk of the evidence certainly suggests that phosphorylation of junctional proteins

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represents an important regulatory event in junction assembly and function. In addition, the members of the armadillo family of proteins appear to integrate information between growth factor signaling pathways and adhesive complexes, thereby allowing for rapid responses to intracellular or extracellular signals.

C. Regulation of Junction Assembly by Serine and Threonine Phosphorylation In addition to tyrosine phosphorylation, serine and threonine phosphorylation events are also thought to regulate intercellular junction assembly. Plakoglobin, desmoplakin, and the desmosomal cadherins are all thought to be phosphorylated on serine and threonine residues (Parrish et aZ., 1990; Pasdar et al., 1995a,b). The E-cadherin cytoplasmic domain contains a cluster of serine residues that are important for binding to catenins (Stappert and Kemler, 1994). In MDCK cells, desmosome assembly is inhibited by okadaic acid, suggesting that phosphatase activity is required for desmosome formation. Furthermore, the dissolution of desmosomes is inhibited by the protein kinase inhibitor H7 when MDCK cells are switched to lowcalcium medium (Pasdar et aZ., 1995a). However, protein kinase C activity may also be required for desmosome assembly. Desmoplakin translocation to the membrane can be induced in low-calcium medium through the activation of protein kinase C (Sheu et aZ., 1989). Although desmosome assembly appears to be regulated by a balance between kinase and phosphatase activity, the specific mechanisms by which these phosphorylation events modify the function of desmosomal proteins are not well characterized. However, the phosphorylation of the carboxylterminal domain of desmoplakin has been found to disrupt the association of desmoplakin with IF networks (Stappenbeck et al., 1994). As discussed above, the carboxyl-terminal domain of desmoplakin binds directly to IFs. Stappenbeck and co-workers found that this domain of desmoplakin is phosphorylated at a serine residue 23 amino acids (SerC23) from the carboxyl terminus. The serine residue was nested in a CAMP-dependent protein kinase consensus site. Furthermore, treating cells with forskolin to activate protein kinase A inhibited the alignment of desmoplakin polypeptides along keratin filament networks. However, in a desmoplakin mutant in which the serine residue was substituted with a glycine residue, forskolin had no effect on desmoplakin alignment along filaments. These data suggest that phosphorylation of SerC23 on desmoplakin inhibits desmoplakin binding to IFs. This is supported by more recent observations using the yeast two-hybrid system (Meng et al., 1997). Direct bind-

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ing between the desmoplakin carboxyl terminus and keratin was detected using wild-type desmoplakin. However, this binding was dramatically enhanced when the desmoplakin mutant containing the glycine in place of SerC23 was tested for direct interactions. Taken together, these data indicate that phosphorylation of the carboxyl-terminal domain of desmoplakin plays an important role in the association of this protein with IF networks, perhaps by modulating the affinity of these direct binding interactions.

D. Regulation of Desmosome Assembly by Adherens Junctions Desmosome assembly is regulated not only by extracellular calcium and growth factors, but desmosome formation also appears to require adherens junction formation. Early calcium switch experiments indicated that there is a close spatial and temporal coordination of desmosome and adherens junction formation (Green et al., 1987; O’Keefe et al., 1987). Also, antibody inhibition experiments in keratinocytes reveal that inhibition of the adhesive function of E-cadherin also inhibits or significantly delays desmosome assembly (Gumbiner et al., 1988; Lewis et al., 1994; Wheelock and Jensen, 1992). Furthermore, aggregation of epithelial cells in suspension is inhibited by antibodies to E-cadherin, even though these cells express desmosomal cadherins (Watabe et al., 1993). In fact, to our knowledge, no cell line or experimental model system has been established in which desmosomes form in the absence of a functional classical cadherin, such as E-cadherin. Furthermore, not only is a classical cadherin required, but the cadherin must be properly coupled to the cytoskeleton in order for desmosome assembly to proceed (Watabe et al., 1994). The observation that E-cadherin-mediated adhesion is required for desmosome assembly raises interesting questions regarding how the signaling mechanisms between these two separate junctions might operate. In retinal pigment epithelial cells, expression of E-cadherin induces desmosome assembly via the regulation of desmoglein expression (Marrs et al., 1995), indicating that in some systems the expression of E-cadherin may regulate expression of desmosomal genes. However, there appear to be other mechanisms by which E-cadherin regulates desmosome assembly. For example, colon carcinoma cell lines lacking expression of a-catenin fail to assemble desmosomes. However, desmosomes are assembled when these cells are treated with TPA to activate protein kinase C, suggesting that PKCmediated signals downstream of E-cadherin-mediated adhesion may regulate desmosome formation (van Hengel et al., 1997).

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Recent evidence also suggests that E-cadherin association with plakoglobin is required for desmosome assembly in an A431 cell model system (Lewis et al., 1997). A431 cells lacking E-cadherin were isolated and found to express normal levels of desmosomal proteins but did not assemble desmosomes. Interestingly, desmosome assembly was not rescued by expression of E-cadherin alone, but desmosome assembly was rescued in these cells by coexpressing exogenous E-cadherin together with plakoglobin. These results suggested that E-cadherin-plakoglobin complexes may play an important role in desmosome assembly. This hypothesis was supported further by the analysis of cell lines expressing a chimeric protein comprising the E-cadherin extracellular domain and plakoglobin as the cytoplasmic domain. Expression of this chimeric protein in A431 cells lacking endogenous E-cadherin rescued desmosome assembly. Given the potential for plakoglobin to function as a signaling molecule, it is possible that plakoglobin binding to E-cadherin might generate intracellular signals that modulate desmosome assembly. However, in addition to generating intracellular signals, E-cadherin-plakoglobin complexesmight function to nucleate desmosome assembly by providing a docking point for other desmosome proteins. In fact, E-cadherin has been detected in desmosomes by immunoelectron microscopy (Horiguchi et aZ., 1994; Jones, 1988). In addition, the amino-terminal domain of desmoplakin associates with classical cadherin-plakoglobin complexes when coexpressed in L cell fibroblasts (Kowalczyk, Bornslaeger, and Green, unpublished observations). Thus, it is possible that the initial recruitment of desmoplakin to the membrane may occur via E-cadherin-plakoglobin complexes. The recruitment of desmoplakin to the membrane by E-cadherin might be followed by recruitment and clustering of desmosomal cadherins and other desmosomal plaque components to areas of assembly that are nucleated around the initial E-cadherindesmoplakin complexes. Certainly, much more work is needed to clarify the mechanisms of cross talk between these two adhesive structures, with respect to both intracellular signaling and structural interactions between junctional proteins. Other insights into the relationship between desmosome assembly and adherens junction assembly have been derived from experiments in which mutant classical cadherins lacking a functional extracellular domain were expressed in cultured cells and developing embryos (Amagai et al., 1995a; Fujimori and Takeichi, 1993; Kintner, 1992; Zhu and Watt, 1996). These mutants not only disrupted the assembly of adherens junctions, they also disrupted desmosome assembly. The mutant cadherins are thought to act in a dominant negative manner by inhibiting the adhesive function of the endogenous classical cadherins, although the mechanisms by which this

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occurs are not fully understood (Fig. 9). One possibility is that the truncated cadherins deplete the cells of @-cateninand plakoglobin, thereby limiting the ability of the endogenous cadherins to associate with @-catenin and other cytoskeletal binding proteins. This may not be the case, however, because @-catenin and plakoglobin are still associated with endogenous cadherins in cells expressing mutant cadherins (Fujimori and Takeichi, 1993; Troyanovsky et al., 1993) (Norvell and Green, 1998). Another possibility is that the mutant cadherin cytoplasmic domains and the associated @- and a-catenin complexes compete with endogenous cadherins for binding sites on the actin cytoskeleton that are necessary for cadherin-mediated adhesion. In addition, the expression of dominant negative cadherins appears to cause decreased expression or accumulation of the endogenous cadherins (Zhu and Watt, 1996) (Norvell and Green, 1998). However, perturbation of cytoskeletal associations and cadherin expression may not be the only mechanisms by which cadherin mutants might function in a dominant negative manner. Recent observations indicate that cadherins and catenins associate with transmembrane phosphatases (Aicher et al., 1997; Balsam0 et al., 1996; Brady-Kalnay et al., 1995; Fuchs et al., 1996; Kypta et al., 1996) and other signaling proteins such as the adapter protein shc (Xu et al., 1997). These observations raise the intriguing possibility that mutant cadherins

A

Normal

B

Competition for Cytoskeletal Binding Sites

C

Competition for Signaling Molecules

FIG. 9 Model for how truncated cadherins lacking a functional extracellular domain might act as dominant negative mutants. (A) Normal cadherins associate with the cytoskeleton via the catenins. (B) Mutants may compete with endogenous cadherins for cytoskeletal binding sites. Full-length cadherins lacking cytoskeletal attachment would be unable to mediate strong intercellular adhesion. (C) Mutants may bind to and sequester signaling molecules “S” that are required for junction assembly. Note that B and C are not mutually exclusive.

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compete with endogenous cadherins for binding to such signaling molecules, which may be needed for normal junction assembly.

VII. Desmosomal Components in Signal Transduction and Development A. Plakoglobin and P-Catenin as lntracellular Signaling Proteins during Development The correct spatial and temporal establishment of cell adhesion is thought to be critical to tissue patterning during embryonic development. The adhesive properties of some cells are controlled, at least in part, by the timing at which adhesion molecules are expressed. The expression of desmosome proteins during mouse development has been characterized by Fleming et al. (Collins et aZ., 1995; Fleming et aZ., 1991; Garrod and Collins, 1994). Plakoglobin (DP 3) was found to be expressed earliest, with synthesis detected at the 8-cell stage. Desmoplakin was expressed by the 16-cell stage, although the accumulation of desmoplakin and plakoglobin in a punctate, desmosomal pattern was not detected until desmosomal cadherins were expressed at the early blastocyst stage. Adherens junction components are expressed very early, and E-cadherin, or uvomorulin, was originally defined as a calcium-dependent adhesion protein necessary for compaction (Damsky et al., 1983). More recently, it has become clear that intercellular junction components play an important role not only in cell-cell adhesion, but in the transduction of intracellular signals that ultimately regulate gene expression and tissue patterning during embryonic development. Investigation into the role of intercellular junction components in several developmental model systems has led to the discovery that junctional proteins play important roles in embryonic development. The realization that junctional proteins might be involved in signal transduction resulted from the observation that the Drosophila protein armadillo, a component of the wingless signaling pathway that governs segment polarity in the fly, is closely related to the vertebrate intercellular junction proteins plakoglobin and P-catenin (Peifer et al., 1992; Peifer and Wieschaus, 1990). In mammals, the armadillo family members have been implicated in a similar pathway involving growth factors of the wnt family (Heasman, 1997; Huber et aL, 1996a;Miller and Moon, 1996). In addition to their localization in intercellular junctions, plakoglobin and P-catenin also exist in non-cadherinassociated pools in the cytoplasm and in the nucleus. Although it is likely

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that the signaling functions of plakoglobin and P-catenin are not interchangeable, the non-cadherin-associated forms of these arm proteins appear to play important roles in the transduction of Wnt-1 signals (Funayama et al., 1995; Karnovsky and Klymkowsky, 1995). Consistent with their role in signaling, several reports have indicated that the non-cadherin-associated forms of catenins are targeted for rapid degradation (Aberle et al., 1997; Papkoff, 1997). In a fibroblast model system, the half-life of non-cadherinbound plakoglobin was found to be approximately 10 min (Kowalczyk et al., 1994), indicating a very tight control over the accumulation of cytosolic plakoglobin. It now appears that the key to Wnt-1 signaling is the regulation of the non-cadherin-associated pools of plakoglobin and P-catenin in the cytoplasm. The first indication that the regulation of catenin turnover might play an important role in signaling came from work in Drosophila, where the accumulation of armadillo was found to be regulated by wingless (Riggleman et al., 1990). As this signaling pathway is currently understood in vertebrates, the mammalian growth factor Wnt-1 regulates accumulation of the non-cadherin-associated plakoglobin and P-catenin by regulating the metabolic stability of these proteins. This occurs through the inactivation of glycogen synthase kinase (GSK-3), which is thought to phosphorylate plakoglobin and P-catenin on an amino-terminal serine residue with homology to a phosphorylation site in IKB.The phosphorylation of noncadherin-associated plakoglobin and P-catenin by glycogen synthase kinase is then thought to target the catenins for degradation via a ubiquitinmediated pathway (Aberle et al., 1997; Yost et al., 1996). In the absence of a Wnt-1 signal, plakoglobin and P-catenin are phosphorylated and the accumulation of these proteins in the cytoplasm is limited. In the presence of Wnt-1, there is increased cytoplasmic accumulation of P-catenin and plakoglobin due to decreased turnover of the non-cadherin-bound pools. Stabilization and accumulation of the cytoplasmic forms of plakoglobin and p-catenin appear to have several functions. Wnt-1 upregulates cell-cell adhesion, presumably by providing increased availability of catenins for linking cadherins to the cytoskeleton. In mammalian cells constitutively expressing Wnt-1, there is an increase in the accumulation of cadherins and both plakoglobin and p-catenin, with a concomitant increase in cell-cell adhesion (Bradley et al., 1993; Hinck et al., 1994b). However, the precise relationship between the junctional bound pools of the catenins, intercellular adhesion, and signaling is not fully understood. In Xenopus, overexpression of P-catenin or plakoglobin causes neural axis duplication. However, overexpression of cadherins inhibits the induction of a dorsal axis in Xenopus, presumably by binding free P-catenin and thereby depleting the cytoplasmic pool of p-catenin (Heasman et al., 1994). In addition, neural axis

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duplication in Xenopus induced by overexpression of plakoglobin or pcatenin is inhibited by overexpression of a cadherin (Fagotto et aL, 1996; Karnovsky and Klymkowsky, 1995), consistent with a role for cadherins in titrating the signaling function of catenins. A number of reports have demonstrated that both plakoglobin and pcatenin are translocated to the nucleus, and entry into the nucleus is thought to be required for signaling. p-Catenin and plakoglobin have been shown to bind to transcription factors of the TCFLEF family (Behrens et al., 1996; Huber et aL, 1996b;Molenaar et aL, 1996), and these interactions are thought to regulate transcription of Wnt-1-responsive genes. However, forms of plakoglobin and p-catenin that are tethered to the plasma membrane, and therefore unable to translocate to the nucleus, also function in signaling and induce axis duplication in Xenopus (Merriam et aL, 1997; Miller and Moon, 1997). These membrane-tethered forms may signal in one of two ways. It is possible that the membrane-bound forms compete with endogenous cadherins for binding to cytoplasmic binding partners, such as catenins or APC (see below). This would release endogenous plakoglobin and p-catenin for entry into the nucleus via interactions with transcription factors (i.e., XTcf). These interactions in the nucleus would then activate genes that signal neural axis development (Brannon et aL, 1997; Miller and Moon, 1997). An alternative explanation is that XTcf normally functions to inhibit the transcription of Wnt-1-responsive genes (Klymkowsky, 1997). Plakoglobin and 0-catenin that are anchored in the cytoplasm would be expected to bind XTcf at the plasma membrane, inhibit the translocation of the transcription factor to the nucleus, and thereby remove the inhibitory signal that prevents transcription of genes that induce neural axis development (Merriam et aL, 1997). In fact, recent work in the Cuenorhabditis elegans model system suggests that arm protein family members may have different roles as suppressors or activators of transcription in different developmental circumstances (Rocheleau et al., 1997; Thorpe et aL, 1997). While the precise mechanisms by which the catenins regulate development are not fully resolved, it is clear that these proteins play important roles not only in the assembly of intercellular junctions but also in tissue patterning during development.

8. Plakoglobin and P-Catenin Form Complexes with the Tumor Suppressor APC As discussed above, the cytoplasmic forms of plakoglobin and p-catenin are subject to rapid degradation through a proteosome-dependent pathway (Aberle et aL, 1997). Both plakoglobin and @-cateninform complexes with the protein product of a tumor suppressor termed the adenomatous polypo-

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sis coli gene (APC) (Rubinfeld et al., 1993; Su et al., 1993). The APC gene is mutated in families with recurrent colon polyps and a predisposition for developing hereditary colorectal cancer (Kinzler and Vogelstein, 1996; Polakis, 1995). The association of the APC protein with plakoglobin or pcatenin appears to target these complexes for degradation. In families with mutations in the APC gene leading to a truncated APC protein, pcatenin-Tcf complexes accumulate due to decreased turnover (Korinek et al., 1997;Morin et al., 1997;Rubinfeld et al., 1997). In addition, these studies suggest that the accumulation of P-catenin that is active in signaling plays a causative role in the development of colorectal cancers. This was further supported by the observation that mutations in 0-catenin that impair the turnover of these proteins can also occur in the development of colon carcinomas (Oyama et al., 1994; Ilyas et al., 1997; Shibata et al., 1996). As discussed above, phosphorylation of the amino-terminal domain of plakoglobin and P-catenin by GSK is thought to target these proteins for degradation. In experiments in which the amino-terminal domain of plakoglobin or p-catenin and the phosphorylation site are removed, there is a dramatic increase in cytosolic pools of these proteins (Palka and Green, 1997; Rubinfeld et al., 1997; Yost et al., 1996). Cadherins also modulate the turnover of catenins by competing with APC. Cadherins and APC form separate complexes with catenins and compete for binding to plakoglobin and p-catenin (Rubinfeld et al., 1995). Through these interactions, cytosolic levels of P-catenin and plakoglobin are tightly regulated, thereby providing a high degree of control over the signaling activity of these proteins. Seldom is it that the biological activity of a protein is restricted to one type of cellular process. Rather, proteins are often multifunctional, with various biological activities that are interwoven into a comprehensive scheme. The catenins and APC are no exception. In addition to signaling activities that are fundamental during development and carcinogenesis, interactions between P-cateninlplakoglobin and APC are also thought to regulate cell motility. APC associates with microtubules in cells and promotes microtubule assembly in vitro (Munemitsu et d., 1994; Smith et d., 1994). Recent evidence suggests that APC and &catenin form complexes that regulate microtubule polymerization. A truncated p-catenin lacking the amino-terminal domain accumulates in complexeswith APC that cluster at the tips of membrane extensions (Barth et al., 1997). In addition, the formation of complexes between APC and an amino-terminal truncated p-catenin was found to inhibit epithelial morphogenesis in a model system using MDCK cells in which growth factor-induced tubule formation was monitored (Pollack et al., 1997). A comprehensive understanding of how catenins and APC interact with the microtubule cytoskeleton to regulate tissue morphogenesis is still lacking. However, these studies have demonstrated that a complex interplay between intercellular junctions, the cy-

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toskeleton, and the nucleus guides tissue morphogenesis in a highly integrated manner. In addition, this system appears to be susceptible to gene mutations that ultimately lead to some of the most common cancers that occur in humans (Gumbiner, 1997).

VIII. Concluding Remarks The analysis of the desmosome as an adhesive structure and macromolecular complex has progressed in several stages. These stages include the initial morphological characterization, followed by the isolation of the major protein components, and more recently the identification of cDNA clones encoding many of the desmosomal proteins. When summarizing our current understanding of the interactions between desmosomal components, it becomes clear that the formation of a desmosome results from a series of highly ordered and complementary interactions. In this regard, the desmosome appears similar to other adhesion structures such as focal contacts and adherens junctions. In each of these structures, a multitude of cytoplasmic plaque interactions act cooperatively in the assembly and function of the adhesive complex. Presumably, these cooperative interactions impart mechanical strength to the junction. In addition, by employing numerous interacting proteins to attach filaments to the membrane, multiple points of regulation are provided. As discussed above, future studies will be aimed at furthering our understanding of the direct interactions that occur within desmosomes and how these interactions might be modulated. In addition, the function of these proteins at the tissue level will need to be addressed to understand how alterations in genes encoding desmosomal proteins might contribute to human diseases. Such studies, in parallel with biochemical analyses, should lead to a more sophisticated and comprehensive understanding of the role of the desmosome in cell and tissue function. As discussed above, intercellular junction proteins such as p-catenin and plakoglobin are now thought to modulate gene transcription through interactions with the TCF family of transcription factors. Emerging evidence indicates that p-catenin is also targeted by caspases during apoptosis, suggesting that the dismantling of intercellular junctions may be an important step in programmed cell death (Brancolini et al., 1997). It will be interesting to determine if desmosomal components are also targeted for proteolysis, and whether these events are simply occurring as cell death proceeds or are required for the progression of apoptosis. Unraveling the multitude of interactions that occur within intercellular junctions will be a major focus of investigators in this field as attempts are made to define the basic hierarchy of interactions within the desmosome. In addition, understanding how

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these interactions are woven into intracellular signaling pathways will be a continuing challenge for investigators working in the field of adhesive junction assembly.

Acknowledgments The authors thank the numerous laboratories that provided us with manuscripts prior to publication. We also thank members of the Green lab, past and present, for insights that have helped shape this review. Work in our lab is supported by grants from the National Institutes of Health ( R 0 1 AR41836, R 0 1 AR43380, and PO1 DE12328) and the March of Dimes (FY 97-0202) to K.J.G. and by National Institutes of Health Grant KO1 AR02039-014 to A.P.K. S.M.N. and H.L.P. were supported in part by T32ES07284 and T32CA09560-10, respectively. K.J.G. is the recipient of a Faculty Research Award from the American Cancer Society.

Note Added in Proof While this manuscript was being prepared for publication, Magee and co-workers reported that Dsgl and Dsc2 mediate adhesion when coexpressed with plakoglobin in L-cell fibroblasts (Marcozzi et al., 1998. J. Cell Sci. 111,495-509).

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A Adenohypophysis, see Pars tuberalis Adenosine, angiogenesis and, 25 Adherens junctions, desmosomes assembly regulation, 275-278 Adhesion, desmosomal cadherin function, 244-245,269-270 Amino acids, fibroblast growth factors, 55-56 Angiogenesis, 1-30 adults, 4-5 autoregulation angiopoietins, 16-17 by blood vessels, 5-7 fibroblast growth factors, 10-11 heparin-binding epidermal growth factor-like factor, 14 insulin-like growth factor, 13 metalloproteinases, 18-19 plasminogen activators, 17-18 platelet-derived growth factor, 12-13 scatter factor, 13-14 tissue factor, 15-16 transforming growth factor-& 14-15 tumor necrosis factor-a, 16 vascular endothelial growth factor, 11-12 by vessel wall, 26-27 cell adhesion molecules, 23 definition, 1 extracellular matrix developmental changes during, 20-21 role, 21-23 future directions, 26-30

initiation, 26, 28 vasocative factor role, 23-26 Angiopoietins, angiogenesis autoregulation, 16-17 Ankyrin, 219-222 APC, complex formation with plakoglobin and p-catenin, 281-282 Apical interactions, Na+,K+-ATPase, during development, 223-226 Apical transport, mediation, 209 Artery, development, 3 Asters, 119 sperm, division, 121 Autoimmune blistering diseases, desmosomal cadherin role, 246-247

B Basal body, reorganizations in spermiogenesis, 114-115 Basolateral sorting signal, Na+,K+-ATPase a subunit, 215-216 Biotinylation technique, 215 Bipolar spindle, 126 formation, 120, 124 Blastula, fibroblast growth factors in, 73-75 Blood vessels angiogenesis autoregulation, 5-7 formation during embryonal development, 2-4 Bone morphogenic proteins, interaction with fibroblast growth factors, 71 Brain, rostrocaudal patterning, effect of fibroblast growth factors, 76-77 Bullous pemphigoid antigen 1, 263-264

303

304

INDEX

C Ca2', FGFR stimulation of release, 67-68 E-Cadherin, 203 association with plakoglobin, 276 basolateral polarity and, 206-207 desmosomes assembly regulation, 275-276 as part of signaling cascade, 220 structure, 241-242 Cadherin-catenin complex protein tyrosine phosphatases and, 272 tyrosine phosphorylation, 272-273 Cadherin gene superfamily, subclasses, 242-243 Cadherins, 206-207 adhesive function, 241 cytoplasmic domains, 248-251 desmosomal binding to plakoglobin, 248-250 function in adhesion, 244-245, 269-270 role in autoimmune blistering diseases, 246-247 structure, 241-243 tissue distribution, 243-244 mutant, 277-278 in signaling pathways, 220 Calcium switch model, 270-271 CAM homology region, fibroblast growth factor receptor, 61 Carbohydrate chains, N-linked, 209-210 p-Catenin central repeat domain, 251 complex formation with APC, 281-282 as intracellular signaling protein during development, 278-280 stabilization and accumulation of cytoplasmic forms, 279-280 tyrosine phosphorylation, 272 Catenins, 248-249 Cell, morphology and motility, fibroblast growth factor effect, 82-83 Cell adhesion molecules, angiogenesis, 23 Cell-cell adhesion, 237-238 Cell-cell adhesion proteins, 207 Centriolar cycle, proliferating cells, 130-131 Centriole, 108-109 behavior in dividing cells of different ploidy, 136-138 with cartwheel structure, 132-133

in centrosome, 138-139 daughter, 111, 113 role, 132 de n o w assembly, 128-129 diploid somatic cell, 131-133 early embryogenesis, 122-127 loss of association with microtubule nucleating material, 139 in mature spermatozoa, 110-112 mother, role, 132 multiplication in differentiated multiciliated cells, 142 origin, in early development, 127-130 as part of centrosome, 133-136 reduplication in oogenesis, 117 roles, 131-132 Centrosome, 107-143 acting as microtubule organizing center, 136-142 centriole as part of, 108-109,133-136, 138-139 early embryogenesis, 122-127 egg, loss of reproductive ability, 120 in fertilized egg, 125 maternal, 124 lineage of inheritance, 126 origin, mouse, 126-127 paternal, 124 polar, 119-120 as polar organizer, 108-109 question of origin, 129-130 RNA in, 135 spot, 126 transformation at terminal differentiation, 139-140 y-tubulin in, 107-108 zygote, paternal origin, 122-123 Choroid plexus, Na+,K+-ATF'aseisoform expression, 213 Ciliary epithelium, 200-201 lining, 196 Na+,K'-ATPase distribution, 213 Cilium, formation, 141-142 Circadian alterations, photoperiod-induced, pars tuberalis, 180-181 Cis-regulatory elements, fgf genes, 50-51, 53 Collagen gel, fibroblast-mediated contraction, 8 Cysteine-rich fibroblast growth factor receptor, binding to fibroblast growth factors, 65

305

INDEX Cytokinesis, in multipolar mitosis, 136-138 Cytoskeleton, interation with Na+,K+ATPase. 219-222

D Desmocalmin, 267 Desmocollins, 242-243 autoantibodies against, 246 binding to plakoglobin, 249-250 IA domain, 250-251 Desmogleins, 242-243 autoantibodies directed against, 244-245 binding to plakoglobin, 249-250 Desmoplakin, 255-262 carboxyl-terminal domain, 258 interactions with desmosomal cadherin-plakoglobin complex, 259,261-262 intermediate filaments, 257-258 plakin family member, 263 rod domain, 257-258 role in desmosome assembly and intermediate filament anchorage, 259-260,268-269 structure, 255-257 Desmosomal cadherin-plakoglobin complex, interactions with desmoplakin, 259,261-262 Desmosomes, 237-283 assembly desmoplakin role, 259-260, 268-269 plakoglobin role, 253-254 protein-protein interactions, 268-270 regulation, 270-278 by adherens junctions, 275-278 calcium switch model, 270-271 intercellular junction regulation by tumorigenic transformation and tyrosine phosphorylation, 271-274 junction assembly regulation by serine and threonine phosphorylation, 274-275 roles of desmosomal cadherin cytoplasmic domains, 250-251 cadherins, see Cadherins, desmosomal components in signal transduction and development, 278-282 complex formation with APC, 281-282

intracellular signaling proteins, 278-280 desmocalmin, 267 desmoplakin, 255-262 desmoyokin, 267 keratocalmin, 267-268 other extracellular components, 247-248 pinin, 267 plakin family, 263-267 plakoglobin, 251-254 plakophilins, 254-255 tissue distribution, 240-241 ultrastructural properties, 238-240 Desmoyokin, 267 Differentiated cells, centrosome acting as microtubule organizing center, 136-142 Differentiation pars tuberalis, 159-161 pathways, regulation by fibroblast growth factors, 80-82 Diploid somatic cell, centrioles, 131-133 Diptera, cells of salivary glands, 140 Dopamine, role in relaying melatonin effects, 183, 185

E E48 antigen, 248 Egg, differentiation, 121 Embryo blood vessel formation, 2-4 capillaries, 3 Embryogenesis, early, centrioles and centrosome, 122-127 Endosomal compartments, fate of proteins entering, 208 Endothelial cells, interactions with fibroblasts or smooth muscle cells, 7-9 Enterocytes, terminally differentiated, 139-140 Envoplakin, 266-267, 269 Epidermolysis bullosa, 265 Epithelial-mesenchymal interactions, mediation by fibroblast growth factors, 83-85 Epithelial tubes, patterning, fibroblast growth factor effect, 83-85 Epithelium absorptive and secretory, 197, 199 ciliary body, 200-201

306

INDEX

Epithelium (continued) from neuroepithelium, 195-196, 198 unique transport properties, 196 Extracellular matrix, molecules, 19-23 developmental changes, during angiogenesis, 20-21 role in angiogenesis, 21-23

F Fertilization, animals with internal, centrioles, 115-116 fgf genes nomenclature, 48 protein, structure, 49 structure and regulation, 47-51 fgfr genes expression, regulation, 56, 58-61 structure, 56, 59 Fibroblast growth factor-binding proteins, nomenclature of genes encoding, 57 Fibroblast growth factor receptor CAM homology region, 61 conserved structural features, 61-63 expression, regulation, 60-61 intracellular dependent biochemical cascades, 65-68 variants and ligand-binding specificity, 63 Fibroblast growth factor receptor-fibroblast growth factor interactions, specificity, 58 Fibroblast growth factors, 45-85 amino acids, 55-56 angiogenesis autoregulation, 10-11 binding to CFR, 65 HSPGS, 63-65 biological activities, 72-85 cell morphology and motility, 82-83 cell proliferation during tissue formation, 79-80 cell survival during organogenesis, 78-79 differentiation pathway regulation, 80-82 epithelial-mesenchymal interactions, 83-85 neural induction and rostrocaudal patterning of brain, 76-77 patterning of limbs, 77-78 patterning of primary body axis, 73-75

biology, reviews on, 86-87 effects on cellular functions, 46 expression, regulation, 51 nuclear localization, 53-55 posttranslational modifications, 55 signaling pathways, 62, 65-72 interactions with growth factor signaling pathways, 70-72 intracellular FGFR-dependent biochemical cascades, 65-68 PLC-y, IP3, and Ca2+release, 67-68

SHC/FRS2-RAF/MAPKKK-MAPKKMAPK pathway, 66-67 target genes, 68-70 signal sequences, 535-54 target genes, 68-70 transcripts, 51-52 translation, 52-53 Fibroblasts aortal, 5, 7 interactions with endothelial cells, 7-9 Follicular cells, pars tuberalis, 162

G Gametogenesis, 109 centrosome behavior during, 110-122 oogenesis, 117-122 spermatogenesis, 110-1 17 Gastrula, fibroblast growth factors in, 73-75 G-protein-coupled melatonin receptor, 175 Growth cone motility, 82 Growth factors angiogenesis autoregulation, 10-15 fibroblast, 10-11 heparin-binding epidermal growth factor-like factor, 14 insulin-like, 13 platelet-derived, 12-13 scatter factor, 13-14 transforming growth factor-& 14-15 vascular cndothelial, 11-12 capable of stimulating angiogencsis, 2 Growth factor signaling pathways, interaction with FGF signaling pathway, 70-72

H Heparan sulfate proteoglycans, 22-23 fibroblast growth factor binding to, 63-65

307

INDEX Heparin-binding epidermal growth factor-like factor, angiogenesis autoregulation, 14 Hypothalmo-pituitary-disconnected rams, 182-183, 185

I Inositol 1,4,5-triphosphate, FGFR stimulation of release, 67-68 Insulin-like growth factor, angiogenesis autoregulation, 13 Insulin-like growth factor receptors, 176-177 Insulin-like growth factor I, interaction Mrith fibroblast growth factors, 71 Integrin receptors, 21 Integrins, epithelial polarity and, 206 Intercellular junction, regulation by serine and threonine phosphorylation, 274-275 by tyrosine phosphorylation, 271-274 Intermediate filaments anchorage, desmoplakin role, 259 interactions with desmoplakin, 257-258 Intracellular signaling proteins, plakoglobin and p-catenin, 278-280 Intracellular sorting signals, 214-219

Melatonin action in pars tuberalis-specific cells, 176 antagonists, 176-177 Melatonin receptors, 171, 173-176 Membrane proteins, anchoring, 208 Men, photoperiodic influences, pars tuberalis, 181-182 Mesoderm, formation, fibroblast growth factor role, 73-75 Metalloproteinases, angiogenesis autoregulation, 18-19 Microtubule nucleating material, loss of association with centrioles, 139 organizing activity, 134 organizing center, centrosome acting as, 136-142 Microtubule system, organizing, 124 Microvessels, formation, 4-5 Microvilli, immunoreactivity for Na+,K'ATPase and ankyrin, 222 Mollusks, spermatogenesis, 113 Myoblast-myotube transition myogenesis, 140-141 Myogenesis, myoblast-myotube transition, 140-141

N K Keratocalmin, 267-268

L Laminin-entactin complex, 22 Leukemia inhibitory factor, interaction with fibroblast growth factors, 71 Limbs, patterning, fibroblast growth factors and, 77-78 Lymnaea stagnalis centriole duplication, 129 female centrosome, 120 meiosis, 119 spermatogenesis, 111

M Megakaryocyte, maturation, 139 Meiosis, spermatocytes, 110-117

Na +,K+-ATPase,195-227 apical, half-life, 215 distribution polarization, 214-226 apical interactions during development, 223-226 interaction with cytoskeleton, 219-222 intracellular sorting signals, 214-219 isoform diversity, 210-214 distribution, 213-214 structure and topology, 211-212 models for epithelial polarity, 205-210 extracellular interactions regulating, 206-207 intracellular mechanisms, 207-210 plasticity during development, 202-204 during pathology, 205-206 polarity, transepithelial transport and, 197-202 role in transepithelial transport, 196-197 Na+,K+-ATPasep 2 subunit, function, 217-218

308

INDEX

Na',K'-P2 subunit, 217 Neovessels anastomotic connections, 29-30 regression, 30 Neural induction, effect of fibroblast growth factors, 76-77 Neuroepithelium, 195-196,198 ion transport, 200-201 ternery complex of Na',K'-ATPase, ankyrin, and spectrin, 221

0 Oocyte, fibroblast growth factors in, 73-75 Oogenesis, centrosome in, 117-122 Organogenesis, cell survival during, fibroblast growth factors and, 78-79

P Pars distalis cells, 162, 165 pars tuberalis, effects on activity, 185-1 86 Pars tuberalis, 157-186 cell types, morphological characteristics, 161-1 65 Circadian alterations, photoperiodinduced, 180-181 development and differentiation, 159-161 nonspecific influences on pars distalis activity, 185-186 participation in regulation of reproductive function, 182-185 photoperiodic influences in men, 181-182 phylogenetic and comparative aspects, 158-160 seasonal alterations, photoperiodinduced, 177-180 topography, 158-159 Pars tuberalis-specific cells gene expression, 165-177 (Y chain and p subunits of TSH, 165-169 melatonin action mechanisms, 176 melatonin antagonists, 176-177 melatonin receptors, 171, 173-176

thyrotropes and, 170-172 tuberalin, 169-170 morphological characteristics, 162-164 Pemphigus, desmosomal cadherins as autoantigens, 246-247 Pericentriolar material, 132, 136, 138 Pericytes, role in angiogenesis, 9 Periplakin, 266-267,269 Photoperiodic influences, pars tuberalis, in men, 181-182 Photoreceptors, sodium requirements, 200 Physa rubra, 120 basal body, 121 Pinin, 267 Pit-1, 171 Plakin family, 263-267 bullous pemphigoid antigen 1, 263-264 envoplakin, 266-267, 269 periplakin, 266-267, 269 plectin, 263-266 Plakoglobin, 248-249, 251-254 association with E-cadherin, 276 coexpression, 262 complex formation with APC, 281-282 as intracellular signaling protein during development, 278-280 role, 252, 269 in desmosomes, 253-254 stabilization and accumulation of cytoplasmic forms, 279-280 Plakophilins, 254-255 Plasminogen activators, angiogenesis autoreguation, 17-18 Platelet-derived growth factor, angiogenesis autoregulation, 12-13 PLC-y, fibroblast growth factor receptor stimulation of release, 67-68 Plectin, 263-266 Polarity, extracellular interactions that regulate, 206-207 Polar organizers, 126 loosing association with basal body, 127 Polycystic kidney disease, Na+,K+-ATPase plasticity, 205-206 Procentriole, 130,133 Prolactin cell differentiation and secretion, 168 surges, stimulation, 183 Prolactin-releasing factor, 169-170 Proliferating cells, centriolar cycle, 130-131 Prostaglandins, angiogenic activity, 25

309

INDEX Protein kinase C, FGFR stimulation of release, 67-68 Protein-protein interactions, desmosome, 268-270 Proteins lipid-linked, 209 signals encoded within, 209 turnover, 208 Proteolytic enzymes metalloproteinases, 18-19 plasminogen activators, 17-18

R Rat aorta model, angiogenesis, 5-7 Reproductive function, regulation, pars tuberalis role, 182-185 Retinal pigment epithelium, 196, 200 apical membrane, 223-224 P-subunit genes, 217-219 development, 203-204 Na+,K+-ATPaseisoform expression, 213 RNA, in centrosome, 135

Salivary glands, cells, Diptera, 140 Scatter factor, angiogenesis autoregulation, 13-14 SCH/FRS2-RAF/MAPKKK-MAPKKMAPK pathway, FGFR activation, 66-67 Seasonal alterations, photoperiod-induced, pars tuberalis, 177-180 Sea urchins, paternal origin of zygote, centrosome, 122-123 Serine, phosphorylation, junction assembly regulation, 274-275 Signaling pathways, fibroblast growth factors, 62, 65-72 Smooth muscle cells, interactions with endothelial cells, 7-9 Spectrin, 219-222 Sperm aster, in fertilized eggs, 123 Spermatogenesis,centrosome in, 110-117 Spindle, bipolar, 126 formation, 120, 124

Spindle poles, 126 Syngamy, 123

T Threonine, phosphorylation, junction assembly regulation, 274-275 Thyroid stimulating hormone, a! chain and p subunits, expression of pars tuberalis-specificcells, 165-169 Thyrotropes, pars tuberalis-specific cells and, 170-171 Thyrotroph embryonic factor, 171 Tissue, formation, cell proliferation during, fibroblast growth factor role, 79-80 Tissue factor, angiogenesis autoregulation, 15-16 Transepithelial transport, Na+,K+-ATPase role, 196-197 polarity and, 197-202 Transforming growth factor-p angiogenesis autoregulation, 14-15 interaction with fibroblast growth factors, 70-71 Tuberalin, 169-170 y-Tubulin in centrosome, 107-108 binding, 134 distribution in ciliated cells, 142 Tumorigenic transformation, intercellular junction regulation, 271-274 Tumor necrosis factor-a, angiogenesis autoregulation, 16 Tumor suppression, APC, complex formation with plakoglobin and p-catenin, 281-282 Tyrosine, phosphorylation, intercellular junction regulation, 271-274

v Vascular endothelial growth factor, angiogenesis autoregulation, 11-12 Vascular permeability factor, 11 Vasculogenesis, 2-4 Vasocative factors, role in angiogenesis, 23-26 Vasodilation, flow-dependent, mediation, 24

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