Epithelial Morphogenesis in Development and Disease (Cell Adhesion and Communication)

EPITHELIAL MORPHOGENESIS IN DEVELOPMENT AND DISEASE Cell Adhesion and Communication A series of books encompassing mo...

Author: Walter Birchmeier | Carmen Birchmeier

29 downloads 953 Views 24MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

EPITHELIAL MORPHOGENESIS IN DEVELOPMENT AND DISEASE

Cell Adhesion and Communication A series of books encompassing monographs on classes of adhesion molecules and monographs giving a broader functional synopsis on adhesion molecules of a particular system. Edited by Christo Goridis Volume 1Cell Adhesion Molecules in Cancer and Inflammationedited by A.A.Epenetos and M.Pignatelli Volume 2The Lamininsedited by P.Ekblom and R.Timpl Volume 3Tenascin and Counteradhesive Molecules of the Extracellular Matrixedited by K.L.Crossin Volume 4Adhesion Molecules and Chemokines in Lymphocyte Traffickingedited by A.Hamann Volume 5Cell Adhesion and Communication Mediated by the CEA FamilyBasic and Clinical Perspectivesedited by C.P.Stanners Volume 6Ig Superfamily Molecules in the Nervous Systemedited by P.Sonderegger Volume 7Epithelial Morphogenesis in Development and Diseaseedited by W.Birchmeier and C.Birchmeier Other volumes in preparation Cell Adhesion and Migration in Skin Diseaseedited by J.Barker and J.McGrath This book is part of a series. The publisher will accept continuation orders which may be cancelled at any time and which provide for automatic billing and shipping of each title in the series upon publication. Please write for details.

EPITHELIAL MORPHOGENESIS IN DEVELOPMENT AND DISEASE Edited by

Walter Birchmeier and Carmen Birchmeier Max-Delbrück-Center for Molecular Medicine Berlin, Germany

harwood academic publishers

Australia • Canada • China • France • Germany • India • Japan • Luxembourg Malaysia • The Netherlands • Russia • Singapore • Switzerland

This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Printed in Singapore. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-203-30376-8 Master e-book ISBN

ISBN 0-203-34322-0 (Adobe eReader Format) ISBN: 90-5702-419-5 (Print Edition) ISSN: 1023-7046 (Print Edition)

CONTENTS

Preface to the Series

vii

Preface

ix

Contributors

xi

A) Structural and Functional Characteristics of Epithelia 1.

Molecular Dissection of Tight Junctions S.Tsukita, M.Furuse and M.Itoh

2

2.

Hemidesmosomes D.R.Garrod

16

3.

Epithelial Polarity and Sorting P.Scheiffele and K.Simons

36

4.

Epithelial Gene Regulation R.M.Watts and H.-U.Bernard

63

5.

Cross-talk between Apical and Basolateral Domains of Epithelial Cells Regulates Microvillus Assembly M.Arpin, T.Crepaldi and D.Louvard

81

6.

Basement Membranes N.R.Smyth and M.Paulsson

99

B) Epithelia in Development 7.

Development of Epithelial Cell Polarity in Drosophila E.Knust

121

8.

Molecular Aspects of Early Mesoderm Development in Amphibian Embryos C.Niehrs and B.Ferreiro

134

9.

Cell Adhesion and Signalling in Early Xenopus Development D.Wedlich and M.Kühl

167

Tyrosine Kinase Receptors as Driving Forces for Growth, Motility and Differentiation of Epithelial Cells W.Birchmeier, C.Birchmeier, V.Brinkmann and C.Niemann

182

10.

vi

11.

The Function of Fibroblast Growth Factors in Skin Morphogenesis and Wound Repair S.Werner

194

12.

Laminin Isoforms and Epithelial Development P.Ekblom, E.Forsberg, D.Gullberg and M.Ekblom

218

13.

The Role of Ret Receptor Tyrosine Kinase and Glial Derived Neurotrophic Factor in Development of the Excretory and Peripheral Nervous Systems A.Schuchardt, P.Durbec, C.V.Marcos-Gutierrez, V.Pachnis and F.Costantini

239

14.

Epithelial-Mesenchymal Signaling in the Regulation of Epithelial Morphogenesis and Apoptosis during Tooth Development A.Vaahtokari and I.Thesleff

256

15.

Biological Functions of GAP Junction Channels Revealed by Targeted Connexin Deficient Mice K.Willecke and T.Ott

281

C) Epithelia in Disease 16.

Beauty is Skin Deep: Biology and Genetics of the Epidermal Keratins E.Fuchs

293

17.

Cultured Keratinocyte Grafts as Prototypes for Epithelial Morphogenesis during Wound Healing C.C.Compton

317

18.

Modulation of Epithelial Traits during the Progression of Carcinomas M.Grassi, J.P.Thiery and j.Jouanmau

345

19.

Multiple Mechanisms for Inactivation of E-Cadherin Cell Adhesion System in Cancer A.Ochiai, Y.Kanai and S.Hirohashi

356

Index

372

PREFACE TO THE SERIES

The development and normal functioning of all multicellular organisms is governed to a large part by the interactions cells undergo with neighbouring cells and with their acellular environment. Many of these interactions are mediated by cell-cell adhesion molecules and by extracellular matrix components and their cellular receptors, that is by molecules which establish direct cell-cell and cell-matrix contacts. These molecules are particularly important for determining whether a cell remains where it is or moves elsewhere and, if a cell moves, where it goes and when it stops migrating. These are of course key events during normal development, but they play equally crucial roles in adult physiology and pathology, such as the extravasation of white blood cells, inflammatory processes and wound healing, tumour invasiveness and metastasis. Moreover, recent advances in the field show that most, if not all, cell adhesion molecules are capable of triggering intracellular events, in the same way as diffusible growth and differentiation factors and their cellular receptors do. It is thus hardly surprising that clinicians are devoting increasing attention to the molecular mechanisms underlying cell adhesion, and that cell adhesion molecules are now being considered as suitable targets for drug development. This book series is aimed at scientists, both in academia and in industry, at graduate students planning to move into the area, at the clinician, who wants to become familiar with a field with many clinical implications, and at scientists already working in the field, who want to keep abreast with the recent developments outside their own speciality. Hence, each volume of the series reviews a particular segment of the field and provides a critical assessment of recent discoveries and future developments. Each volume has a volume editor, who is an expert in the field and invites contributors to cover the different aspects of the topic. By keeping the number of contributors to each volume small, we hope to avoid overlaps and redundancies, common pitfalls of multi-author volumes. By looking at the previous volumes, I have the impression that we have been successful. Previous volumes of the series addressed the role of cell adhesion in selected physiological and pathological phenomena or concentrated on important structural families. The volumes on Cell Adhesion Molecules in Cancer and Inflammation and on Adhesion Molecules and Chemokines in Lymphocyte Trafficking are examples of the first, those on The Laminins and on the CEA Family are examples of the second kind. The scope of the present volume is slightly different: it focuses on one type of tissue, epithelia, whose development and integrity relies heavily on cell adhesion phenomena. As such, this book integrates information on epithelial biology and pathology not available elsewhere in such a condensed fashion. Starting from the different kinds of adhesion junctions and cell-matrix interactions peculiar to epithelia, it moves on to present a state-of-the-art account of epithelial morphogenesis and of selected pathophysiological phenomena, in particular wound healing and cancerisation. A forthcoming volume will deal with adhesion phenomena underlying the biology and pathology of a particularly important epithelium, the

viii

epidermis of the skin; together, the two books will constitute an invaluable source of information for scientists and clinicians interested in skin biology and diseases. I am indebted to the staff at Harwood Academic Publishers, who made this enterprise possible, to the volume editors, each one an authority in his/her field, and last but not least to the many competent contributors. Christo Goridis

PREFACE

In this book we bring together a series of articles on the structural, functional and developmental characteristics of epithelia. The aim is to explain the characteristic features of epithelia in molecular terms. Typically, epithelia form continuous monoand multilayered sheets of tightly adhered cells. Characteristic are specialized organelle-like adhesive structures responsible for intercellular contacts (tight junctions, adherens junctions, desmosomes and gap junctions) and for the contacts with the a cellular basement membrane (hemidesmosomes). An additional characteristic of many epithelial cells is their polarization, that is, the distinct morphological appearance of the basal, lateral and apical surfaces. In the first part of the book, molecular components and the functions of adhesive structures; the mechanisms that underlie epithelial cell polarity and sorting; microvilli assembly in the apical membrane; and epithelial-specific gene expression are discussed. The second part of the book discusses various aspects of epithelial differentiation and development. The first cells that emerge during development are non-polarized, and differentiate into polarized cells with epithelial characteristics, i.e. ecto- and endoderm. Genetic research in a simple organism, the fruit fly, was used to analyze the genes and mechanisms by which the first polarized cells are formed in development. Subsequently, epithelial-mesenchymal conversions take place to generate mesodermal cells during gastrulation. These early steps in development are discussed in two chapters that report recent advances using the chick and the frog as model organisms. During further development, the majority of epithelial cells arise by growth and differentiation of existing epithelia. In most organs, mesenchymal cells are found below the epithelia. Exchange of signals between the epithelial and mesenchymal cell compartments drive growth and differentiation during organ development. Several chapters discuss the molecular nature of signals used to exchange information between the mesenchymal and epithelial cell compartments that direct the development of the kidneys, teeth or the mammary glands. Such signals are also important in epidermal development and regeneration of the epidermis, i.e. in wound repair. Defects in epithelial function and growth control play a major role in human diseases and are discussed in the third part of the book. We became interested in the function of the epithelial cell adhesion molecule Ecadherin in cancer during the late ’80s, when we realized that a monoclonal antibody that interferes with Ecadherin-mediated adhesion induced invasiveness, and that invasive carcinoma cells have frequently lost the E-cadherin protein. Today we know that E-cadherin is often mutated in sporadic cancers and germ-line mutations have recently been identified in inherited carcinomas. The cadherin-associated beta-catenin can induce transformation when mutated, and directly affects gene expression in colon carcinomas and melanomas by interacting with transcription factors of the TCF/LEF-1 family. Many other cell adhesion molecules have been implicated in cancer, like integrins, CEA (a member of the Ig-family), syndecan, EpCAM, CD44 and others. Severe blistering diseases can be caused by auto-immune antibodies that are directed against cadherins located in the desmosomes, and mouse-models that develop blistering diseases

x

have recently been produced. Genetically inherited disorders of keratins in the human epidermis have been identified and examined in detail, and transgenic mice that carry the equivalent mutations and that serve as models for the human diseases have been created. Keratinocytes can be kept in culture for biological studies, and such cultured keratinocytes have been used as grafts to treat severe wounds. Thus, insight into malignant carcinomas and skin diseases was gained through prior knowledge of the biological characteristics of epithelia. The improved understanding might in the future allow more directed and efficient therapy. This book will not only give newcomers all the information they need to gain access into this increasingly complex area of biology but allows experts to update their knowledge of the field with the latest developments. Walter Birchmeier Carmen Birchmeier

CONTRIBUTORS

Monique Arpin Laboratoire de Morphogenèse et Signalisation Cellulaires UMR 144 CNRS Institut Curie 26, rue d’Ulm 75231 Paris Cedex 05 France Hans-Ulrich Bernard Institute of Molecular and Cell Biology National University of Singapore 10 Kent Ridge Crescent Singapore 117609 Carmen Birchmeier Department of Genetics and Structural Biology Max-Delbrück-Centrum fur Molekulare Medizin Robert-Rössle-Straße 10 13125 Berlin Germany Walter Birchmeier Department of Growth and Differentiation Max-Delbrück-Centrum für Molekulare Medizin Robert-Rössle-StraBe 10 13125 Berlin Germany Volker Brinkmann Department of Growth and Differentiation Max-Delbrück-Centrum fur Molekulare Medizin Robert-Rössle-Straβe 10 13125 Berlin Germany Carolyn Compton Gastrointestinal Pathology Department of Pathology Harvard Medical School

xii

Massachusetts General Hospital Boston, MA 02114 USA Frank Costantini Department of Genetics and Development College of Physicians and Surgeons Columbia University New York, NY 10032 USA Tiziana Crepaldi Laboratoire de Morphogenèse et Signalisation Cellulaires UMR 144 CNRS Institut Curie 26, rue d’Ulm 75231 Paris Cedex 05 France Pascale Durbec Division of Development Neurobiology National Institute for Medical Research London NW7 1AA UK Peter Ekblom Department of Animal Physiology Uppsala University Biomedical Center Box 596 SE-751 24 Uppsala Sweden Marja Ekblom Department of Animal Physiology Uppsala University Biomedical Center Box 596 SE-751 24 Uppsala Sweden Beatriz Ferreiro Division of Molecular Embryology DKFZ Im Neuenheimer Feld 280 69120 Heidelberg Germany Erik Forsberg Department of Animal Physiology Uppsala University Biomedical Center

xiii

Box 596 SE-751 24 Uppsala Sweden Elaine Fuchs Department of Molecular Genetics and Cell Biology Howard Hughes Medical Institute The University of Chicago 5841 S. Maryland Chicago, IL 60637 USA M.Furuse Department of Cell Biology Faculty of Medicine Kyoto University Yoshida-Konoe-Sakyo-ku Kyoto 606 Japan D.R.Garrod Department of Cell and Structural Biology Stopford Building University of Manchester Oxford Road Manchester M13 9PT UK M.Grassi Laboratoire de Morphogenèse Cellulaire et de Progression Tumorale UMR 144 CNRS Institut Curie 26, rue d’Ulm 75248 Paris Cedex 05 France Donald Gullberg Department of Animal Physiology Uppsala University Biomedical Center Box 596 SE-751 24 Uppsala Sweden S.Hirohashi Pathology Division National Cancer Center Research Institute 5–1–1, Tsukiji, Chuo-ku Tokyo 104 Japan M.Itoh

xiv

Department of Cell Biology Faculty of Medicine Kyoto University Yoshida-Konoe-Sakyo-ku Kyoto 606 Japan J.Jouanneau Laboratoire de Morphogenèse Cellulaire et de Progression Tumorale UMR 144 CNRS Institut Curie 26, rue d’Ulm 75248 Paris Cedex 05 France Yae Kanai Pathology Division National Cancer Center Research Institute 5–1–1, Tsukiji, Chuo-ku Tokyo 104 Japan E.Knust Institut fur Genetik Heinrich-Heine Universität Düsseldorf Universitätsstraße 1 40225 Düsseldorf Germany M.Kühl Abteilung Biochemie Universität Ulm Albert Einstein Alice 11 89081 Ulm Germany Daniel Louvard Laboratoire de Morphogenèse et Signalisation Cellulaires UMR 144 CNRS Institut Curie 26, rue d’Ulm 75231 Paris Cedex 05 France Camelia V.Marcos-Gutierrez Divsion of Development Neurobiology National Institute for Medical Research London NW7 1AA UK Christoph Niehrs Division of Molecular Embryology DKFZ

xv

Im Neuenheimer Feld 280 69120 Heidelberg Germany Catherin Niemann Department of Genetics and Structural Biology Max-Delbrück-Centrum fur Molekulare Medizin Robert-Rössle-Straße 10 13125 Berlin Germany Atsushi Ochiai Pathology Division National Cancer Center Research Institute 5–1–1, Tsukiji, Chuo-ku Tokyo 104 Japan T.Ott Abteilung Molekulargenetik Institut fur Genetik Universität Bonn Römerstraße 164 53117 Bonn Germany Vasillis Pachnis Divsion of Development Neurobiology National Institute for Medical Research London NW7 1AA UK Mats Paulsson Institut fur Biochemie der Medische Fakultat Universität zu Köln Joseph-Stelzmann-Straße 52 50931 Köln Germany Peter Scheiffele European Molecular Biology Laboratory Postfach 1022.40 Meyerhofstraße 1 69117 Heidelberg Germany A.Schuchardt Department of Genetics and Development College of Physicians and Surgeons Columbia University New York, NY 10032

xvi

USA Kai Simons European Molecular Biology Laboratory Postfach 1022.40 Meyerhofstraße 1 69117 Heidelberg Germany Neil R.Smyth Institut fur Biochemie der Medische Fakultät Universität zu Köln Joseph-Stelzmann-Straße 52 50931 Köln Germany Irma Thesleff Developmental Biology Programme Institute of Biotechnology P.O. Box 56 University of Helsinki FIN-00014 Helsinki Finland Jean-Paul Thiery Laboratoire de Morphogenèse Cellulaire et de Progression Tumorale UMR 144 CNRS Institut Curie 26, rue d’Ulm 75248 Paris Cedex 05 France Shoichiro Tsukita Department of Cell Biology Faculty of Medicine Kyoto University Yoshida-Konoe-Sakyo-ku Kyoto 606 Japan Anne Vaahtokari Developmental Biology Programme Institute of Biotechnology P.O. Box 56 University of Helsinki FIN-00014 Helsinki Finland Robin M.Watts Institute of Molecular and Cell Biology National University of Singapore 10 Kent Ridge Crescent

xvii

Singapore 117609 Doris Wedlich Abteilung Biochemie Universität Ulm Albert Einstein Alice 11 89081 Ulm Germany Sabine Werner Max-Planck-Institut fur Biochemie Am Klopferspitz 18A 82152 Martinsried/München Germany Klaus Willecke Abteilung Molekulargenetik Institut fur Genetik Universität Bonn Römerstraße 164 53117 Bonn Germany

STRUCTURAL AND FUNCTIONAL CHARACTERISTICS OF EPITHELIA

1. MOLECULAR DISSECTION OF TIGHT JUNCTIONS SHOICHIRO TSUKITA, MIKIO FURUSE and MASAHIKO ITOH Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan

The establishment of compositionally distinct fluid compartments by the epithelium is crucial for the development and function of most organs in multicellular systems. The tight junction, an element of the epithelial junctional complex, is directly involved in this compartmentalization. Recent studies have identified various peripheral and integral membrane proteins which are specifically concentrated at tight junctions, allowing the dissection of tight junctions both structurally and functionally. Here, we present an overview of recent progress in the identification of tight junction components and discuss the possible molecular architecture of tight junctions. INTRODUCTION: STRUCTURE AND FUNCTIONS OF TIGHT JUNCTIONS In epithelial and endothelial cells, the tight junction (TJ) seals cells to create a primary barrier to the diffusion of solutes across the cell sheet, and it also functions as a boundary between the apical and basolateral membrane domains to maintain polarization (Gumbiner, 1987, 1993; Schneeberger and Lynch, 1992; Anderson and Van Itallie, 1995). In thin-section electron microscopy, TJs appear as a series of discrete sites of apparent fusion, involving the outer leaflet of the plasma membranes of adjacent cells (Farquhar and Palade, 1963). In freeze-fracture electron microscopy, these junctions appear as a set of continuous, anastomosing intramembrane strands or fibrils in the P-face (the outwardly facing cytoplasmic leaflet) with complementary grooves in the E-face (the inwardly facing extracytoplasmic leaflets) (Figure 1; Staehelin, 1973). It remains unclear whether the strands are predominantly lipid in nature, i.e. cylindrical lipid micelles, or represent linearly aggregated integral membrane proteins (Pinto da Silva and Kachar, 1982; Kachar and Reese, 1982). However, given the detergent stability of TJ strands visualized by negative staining (Stevenson and Goodenough, 1984) and freeze fracture (Stevenson et al., 1988), it is unlikely that these elements are composed solely of lipids. TIGHT JUNCTION-ASSOCIATED PERIPHERAL MEMBRANE PROTEINS Accumulating evidence has shown that some unique proteins constitute TJ. These TJ-specific proteins are subclassified into peripheral and integral membrane proteins.

Correspondence and Proofs: Shoichiro Tsukita, MD, Department of Cell Biology, Kyoto University Faculty of Medicine, Konoe-Yoshida, Sakyo-ku, Kyoto 606, Japan. Tel: 81–75–753–4372; Fax: 81–75–753–4660

CHARACTERISTICS OF EPITHELIA

3

Figure 1 Freeze-fracture replica image of mouse intestinal epithelial cells. Tight junctions appear as a set of continuous, anastomosing intramembranous strands ~10 nm in thickness in the P-face (arrows) with complementary grooves in the E-face (arrowheads). Bar, 0.1 µm.

ZO-1: ZO-1 with a molecular mass of 220kD was first identified by monoclonal antibody production against the membrane fraction from mouse liver, and it is localized in the immediate vicinity of the plasma membrane of TJ in epithelial and endothelial cells (Stevenson et al., 1986; Anderson et al., 1988). However, the distribution of ZO-1 is not necessarily restricted to TJ. ZO-1 occurs also in the junctional specializations of Sertoli cells of the testis (Stevenson et al., 1986) and in the small junctions of the slit diaphragm in podocytes of kidney glomeruli (Schnabel et al., 1990). Furthermore, as discussed below, ZO-1 is precisely colocalized with cadherins in cells lacking TJ, such as fibroblasts and cardiac muscle cells (Itoh et al., 1991, 1993). ZO-1 shows sequence similarity to the product of lethal (1) discs large-1 (dig), one of the tumor suppresser molecules in Drosophila (Itoh et al., 1993; Tsukita et al., 1993; Willott et al., 1993), and there are at least two isotypes of ZO-1 generated by alternative splicing (α+ and α−) (Willot et al., 1992). Recently, in addition to these proteins, many dig-like proteins have been identified, indicating the existence of a novel gene family named MAGUK (membrane-associated guanylate kinase homologues) (Figure 2; Woods and Bryant, 1993; Kim, 1995; Anderson, 1995). ZO-2: ZO-2, with a molecular mass of l60kD was identified as a ZO-1-binding protein by immunoprecipitation (Gumbiner et al., 1991). ZO-2 is also localized at TJ in both epithelial and endothelial cells, and belongs to the MAGUK family (Jesaitis and Goodenough, 1994). Detailed analyses of the subcellular distribution of ZO-2 in cells lacking TJ have not yet been performed.

4

EPITHELIAL MORPHOGENESIS

Figure 2 MAGUK (membrane-associated guanylate kinase homologue) family. These proteins have PDZ (PDZ), SH3 (SH3) and guanylate kinase (GUK) domains.

p130 (ZO-3): A phosphorylated 130kD protein was identified in the ZO-1/ ZO-2 complex by immunoprecipitation (Balda et al., 1993). Recent cDNA cloning revealed that this protein also belongs to the MAGUK family, and so it was designated as ZO-3 (B.R. Stevenson, personal communication). cingulin: Cingulin was identified by monoclonal antibody production using avian intestinal brush borders as antigens (Citi et al., 1988). Two forms with molecular masses of 108kD and l40kD are localized exclusively at TJ of both epithelial and endothelial cells (Citi et al., 1989). Purified cingulin is heat-stable, and has a rod-like appearance when observed by low-angle rotary shadowing. Its full-length cDNA has not yet been cloned, but analysis of partial cDNA clones suggested that cingulin exists as a coiled-coil dimer, which may allow cingulin to associate into supramolecular assemblies (Citi et al., 1993). 7H6 antigen: Monoclonal antibodies were raised against the bile canaliculi fractions obtained from rat liver, and one mAb (7H6) recognized an antigen of around 155kD which is localized at TJ of epithelial and endothelial cells (Zhong et al., 1993). 7H6 antigen has been proposed to be involved in the regulation of TJ permeability (Zhong et al., 1994). symplekin: Symplekin with a molecular mass of 130kD was identified by monoclonal antibody production (Keon et al., 1996). Both symplekin mRNA and protein occur in a wide range of cell types that do not form TJ. Symplekin is localized in all of these diverse cell types in the nucleoplasm, and only in those cells forming TJ is it recruited, partially but specifically, to TJ. rab3B, rabl3: Various small GTP-binding rab proteins have been shown to be involved in the regulation of membrane traffic along biosynthetic and endocytic pathways in eukaryotic cells. Among them, rab3B and rab 13 have been immunolocalized at TJ in polarized epithelial cells (Weber et al., 1994: Zahraoui et al., 1994). The participation of these rab proteins in polarized transport, in the assembly and/or the activity of TJ has been sugggested, but as yet there is no experimental support for this hypothesis.


5

OCCLUDIN: A TIGHT JUNCTION-ASSOCIATED INTEGRAL MEMBRANE PROTEIN To clarify the structure and function of TJ at the molecular level, integral membrane proteins present at TJ should be identified. However, such integral membrane components remained elusive for quite some time. This goal was recently achieved by identification of occludin, an integral membrane protein localized exclusively at TJ (Furuse et al., 1993). Molecular structure of occludin: Occludin with a molecular mass of ~65kD was first isolated from the chick liver using monoclonal antibody production, and its cDNA was cloned and sequenced (Furuse et al., 1993). Recently, mammalian homologues of occludin were also identified (Ando-Akatsuka et al., 1996). The amino acid sequences of human, murine, and canine occludins are very closely related (~90% identity), whereas they diverge considerably from those of the chicken. Regardless of this interspecies diversity in the occludin sequence, the basic structure appears to have been conserved during phylogenetic evolution (Figure 3). Both mammalian and chicken occludins are comprised of four transmembrane domains, a long carboxyl-terminal cytoplasmic domain, a short amino-terminal cytoplasmic domain, two extracellular loops and one intracellular turn. Characteristic conserved aspects are the high content of tyrosine and glycine residues in the first extracellular loop (~60%), which may be directly involved in cell adhesion, and the αhelical coiled-coil structure in the long carboxyl-terminal cytoplasmic domain. Subcellular distribution of occludin: Northern blotting analyses identified occludin mRNA in various mouse tissues. In testis, kidney, liver, lung, and brain, an intense 2.9kb band and two additional faint bands of 3.6 and 2kb were detected, but these were not detected in skeletal muscle, spleen, or heart (Saitou et al., 1997) (Figure 4A). Anti-mouse occludin mAb and pAb recognize several bands of around 60kD in the cultured mouse epithelial cell line, MTD-1A, the smallest of which is the most intense. This multi-banding pattern of occludin is also observed in other species such as chicken, pig, dog, and human (Figure 4B). In contrast, occludin is hardly detectable in cultured fibroblasts at the protein or mRNA level. Immunofluorescence microscopy revealed that in most simple epithelial cells, occludin is precisely colocalized with ZO-1 at the most apical part of their lateral membranes. For example, in the liver, the occludin signal is exclusively detected in the ZO-1-positive junctional complex regions of hepatocytes (Figure 5). In the longitudinal-section view of each bile canaliculus, a pair of continuous lines is visible both with anti-occludin and and-ZO-1 mAbs. In the intestinal epithelial cells, both occludin and ZO-1 are highly concentrated at the junctional complex regions (Furuse et al., 1993; Saitou et al., 1997). At the electron microscopic level, occludin signals are detected directly over the points of membrane contact in TJ of epithelial cells (Furuse et al., 1993). Information regarding occludin in endothelial cells is still fragmentary. Occludin is highly expressed and concentrated at cell-cell borders of brain endothelial cells, where the TJ is highly developed (Furuse et al., 1993; Hirase et al., manuscript to be submitted). In contrast, the occludin signal is very weak or undetectable in endothelial cells from non-neuronal tissues, which are relatively poor in TJ. In good agreement with these immunofluorescence observations, cultured brain endothelial cells express large amounts of occludin, whereas only small amounts of occludin have been detected in cultured aortic endothelial cells (Hirase et al., manuscript to be submitted). Possible functions of occludin: Some functions of chicken occludin have been elucidated. When chicken occludin was overexpressed in insect cells by recombinant baculovirus infection, peculiar occludin-enriched multilamellar structures accumulated in the cytoplasm. Thin section electron microscopy revealed that each lamella was transformed from intracellular membranous cisternae whose luminal space was completely collapsed, and that in each lamella, outer leaflets of opposing membranes appeared to be fused with no

6


Figure 3 Molecular structure of occludin. (A) Hydrophilicity plots of chicken and human occludins. Hydrophilic and hydrophobic residues are in the lower and upper parts of the frames, respectively. The axis is numbered in amino acid residues. There are four major hydrophobic, potentially membrane-spanning regions in the amino-terminal halves of both occludins (arrows). (B) Folding model for chicken occludin. See details in the text. #, glycine residue; *, glycine residue; •, positively charged residue; , negatively charged residue.


7

Figure 4 Expression of occludin. (left) Northern blot of poly(A)+RNA isolated from mouse tissues, (right) Western blot of cultured epithelial cells of various species with anti-occludin antibodies. Note the characteristic multi-banding pattern of occludin.

Figure 5 Double immunostaining of a frozen section of guinea pig liver with rat antioccludin mAb (left) and mouse anti-ZO-1 mAb (right). Bile canaliculi are occludin/ZO-1-double positive (arrows), whereas only the ZO-1 signal was detected in sinusoidal endothelial cells (arrowheads). Bar, 20 µm.

gaps, resembling TJ (Furuse et al., 1996) (Figure 6). These findings suggest that occludin can obliterate the extracellular space at TJ, and that this molecule is directly involved in the barrier function of TJ. When full-length chicken occludin was overexpressed in MDCK cells, the barrier function (transepithelial electrical resistance, TER) was up-regulated with a concomitant increase in the number of TJ strands (McCarthy et al., 1996). On the other hand, in MDCK cells, the expression of carboxyl-terminally truncated occludin led to a several fold increase in paracellular flux of small molecular weight tracers and destruction of the fence function of TJ as shown by the inability to maintain a fluorescent lipid in a specifically labeled cell-surface domain (Balda et al., 1996). Recently, a synthetic peptide corresponding to the second

8


Figure 6 Ultrastructure of the occludin-enriched multilamellar structures in insect Sf9 cells. Samples were fixed with tannic acid to well contrast proteinous structures. Each lamella has a membrane loop at both ends (arrows). In each lamella, the outer leaflets of opposing membranes are fused with no gaps, as observed at tight junctions in situ. Bar, 0.1 µm.

extracellular domain of chicken occludin was found to perturb the TJ permeability barrier in epithelial cells (Wong et al., 1997). Taken together, these observations indicate that occludin plays a central role in the barrier as well as the fence functions of TJ. Recently, the mouse occludin gene was knocked-out by homologous recombination (Saitou et al., unpublished data). Studies along this line should allow elucidation of the functions of occludin in more detail. MOLECULAR ARCHITECTURE OF TIGHT JUNCTIONS TJ strand: As discussed above, anti-occludin antibodies have been seen to exclusively label TJ directly over the points of membrane contact, the so-called kissing points, at the thin-section electron microscopic level (Furuse et al., 1993). Recent progress in immuno-freeze-fracture techniques allowed us to immunolabel freezefracture replicas with antibodies specific for the cytoplasmic domains of integral membrane proteins (Fujimoto, 1995). Using this technique, anti-occludin mAb specifically labeled the TJ strand proper in the freeze-fracture replicas of epithelial cells, indicating that occludin is at least one of the major components of the TJ strand itself (Fujimoto, 1995; Furuse et al., 1996) (Figure 7A). The question then arises as to whether TJ strands ~10nm in thickness can be reconstituted by occludin molecules. As described above, when occludin was overexpressed in insect Sf9 cells, TJ-like structures were formed. The freeze-fracture plane of these structures was characterized by a large number of particles of about 10nm in diameter, and very interestingly these particles tended to align to give a short TJ strandlike appearance (Furuse et al., 1996) (Figure 7B). A connexon consisting of 6 connexin molecules (gap


9

Figure 7 Localization of occludin on freeze-fracture replicas obtained from chicken liver (left) and occludin-enriched multilamellar structures in occludin-overexpressing Sf9 cells (right). Anti-occludin mAb exclusively labeled in situ tight junction strands (arrows) as well as short tight junction strand-like structures formed in multilamellar structures (arrowheads). Bar, 0.1 µm.

junction channel molecules), which also bear four transmembrane domains of a similar molecular size to those of occludin, is visualized as an intramembranous particle about 10nm in diameter in freezefracture images (Yancey et al., 1979). Therefore, it is tempting to speculate that several (~6) occludin molecules constitute a 10nm particle, which ‘polymerizes’ into a TJ strand within lipid bilayers. Of course, this hypothesis does not exclude the possibility that other integral membrane proteins and/or lipids are involved in the formation of TJ strands. TJ plaque: As compared to adherens junctions and desmosomes, the plaque structure (or plasmalemmal undercoat) was reported to be poorly developed at TJ. However, recent electron microscopic analyses clearly identified the fairly developed plaque structure just beneath the TJ plasma membranes (Keon et al., 1996). As discussed above, many TJ plaque-specific proteins have been identified. The molecular mechanisms of how these molecules are integrated into the plaque structure mostly remain unclear, but information regarding the molecular architecture of the TJ plaque is gradually accumulating. ZO-1 forms a molecular complex with ZO-2 and ZO-3 (p130) as revealed by immunoprecipitation (Gumbiner et al., 1991; Balda et al., 1993). The aminoterminal halves of both ZO-1 and ZO-2 directly bind to the carboxyl-terminal 150a.a. cytoplasmic domain of occludin in a non-competitive manner (Furuse et al., 1994; Itoh et al., unpublished data). Furthermore, ZO-1 directly binds to spectrin molecules and/or actin filaments (Itoh et al., 1991; Itoh et al., submitted). Therefore, at present the following molecular architecture of the TJ plaque is likely; occludin→ZO-l /ZO-2/ZO-3→actin filament/spectrin (Figure 8). The next question to be addressed is how other TJ plaque proteins such as cingulin, 7H6 antigen, symplekin etc. are integrated in the above molecular backbone of TJ.

10


Figure 8 Schematic drawing of the possible molecular architecture of tight junctions.

REGULATION OF TIGHT JUNCTION FUNCTIONS TJ function is known to be dynamically regulated in vivo (Schneeberger and Lynch, 1992; Anderson and Van Itallie, 1995). For example, the permeability of intestinal epithelial cells is controlled during absorption of nutrients (Atisook et al., 1990; Ballard et al., 1995), and TJ in endothelial cells is repeatedly destroyed and formed during leukocyte transmigration across the endothelium (Milks et al., 1986). Furthermore, in some pathological conditions such as Crohn’s disease (Hollander, 1988), the barrier function of TJ is downregulated. Although the precise regulatory mechanism of the permeability of endothelial and epithelial cells is an area of current active investigation, information is still limited. Some hormones such as glucocorticoids (Zettl et al., 1992) and TGF a and p (Buse et al., 1995; Woo et al., 1996) have been reported to affect the TJ function, and the correlation between the modulation of ZO-1/ZO-2/ZO-3/cingulin and the TJ functions has been intensively investigated (Balda et al., 1993; Citi and Denisenko, 1995), but no clear conclusions have yet been drawn. Occludin is heavily phosphorylated on its serine/threonine residues, and the multi-banding pattern of occludin in SDS-PAGE has been attributed to phosphorylation (Sakakibara et al., manuscript to be submitted). Detailed analyses of occludin phosphorylation led to the conclusion that slightly- or nonphosphorylated occludin is distributed on the basolateral membranes, and that highly-phosphorylated occludin is selectively concentrated at TJ. These findings suggest that the phosphorylation of occludin is a key mechanism in the regulation of TJ assembly.


11

RELATIONSHIP BETWEEN TIGHT AND ADHERENS JUNCTIONS The adherens junction (AJ) is a specialized region of the plasma membrane, where cadherin molecules (uvomolurin, L-CAM, A-CAM etc.) function as adhesion molecules and actin filaments are densely associated with the plasma membrane through a well-developed plasmalemmal undercoat (Farquhar and Palade, 1963; Volk and Geiger, 1984; Boiler et al., 1985; Takeichi, 1988, 1991). This type of junction is important for the formation and maintenance of tissues. AJ appear as belt-like in simple epithelial cells and endothelial cells, while they are spot-like in the intercalated discs of cardiac muscle cells. Small spot-like AJ have also been seen in cultured fibroblasts (Yonemura et al., 1995). At least two cytoplasmic proteins, α and β catenins, are tightly associated with the cytoplasmic domains of cadherins (Ozawa et al., 1989, 1990; Nagafuchi and Takeichi, 1989). These catenins show similarity to vinculin and the Drosophila armadillo gene product, respectively (Herrenknecht et al., 1991; Nagafuchi et al., 1991; McCrea et al., 1991), and constitute the undercoat of AJ together with other cytoplasmic proteins such as vinculin and α-actinin (Tsukita et al., 1992). In most types of simple epithelial cells, TJ are not clearly separated from AJ, but the two types of junctions are intermingled. In some epithelial cells such as intestinal epithelial cells, however, TJ and AJ are completely separated. In this connection, the behavior of ZO-1 is of interest. Although ZO-1 was first identified as a component of TJ plaques (Stevenson et al., 1986), it is also expressed in non-epithelial cells lacking TJ (Itoh et al., 1991, 1993). In cardiac muscle cells, ZO-1 is highly concentrated at the intercalated discs where N-cadherin functions as an adhesion molecule. In fibroblasts, ZO-1 is precisely co-localized with P-cadherin at cell-cell contact sites (Figure 9). In these non-epithelial cells, ZO-1 was recently found to be directly associated with a catenin (Itoh et al., manuscript to be submitted). In contrast, in intestinal epithelial cells, ZO-1 is exclusively localized at TJ probably through association with occludin, and this molecule has not seen at AJ in these cells (Itoh et al., 1993). Interestingly, at the initial phase of the polarization of epithelial cells, small spot-like AJ are formed, where ZO-1 is co-concentrated with E-cadherin (Yonemura et al., 1995). Then, in the mature belt-like AJ of fully-polarized epithelial cells, ZO-1 may be transferred from AJ to TJ, i.e. from a catenin to occludin. In good agreement with this, α and β catenins were reported to be co-immunoprecipitated with ZO-1 only at the initial stage of junction formation in MDCK cells, although cadherins were not identified in the immunoprecipitate (Rajasekaran et al., 1996). From the viewpoint of epithelial polarization and TJ differentiation, the molecular mechanism of this peculiar behavior of ZO-1 is one of the most interesting issues for future studies. CONCLUSION TJ is an essential structure for epithelial and endothelial cells to exert their physiological functions. The lack of information about the molecular architecture of TJ has made it difficult to analyze the structure and functions of TJ. However, now that a large number of TJ-associated peripheral and integral membrane proteins have been identified, it should now be possible to begin dissecting the structure of TJ at the molecular level and to modulate the functions of TJ at the cellular as well as the whole body level. REFERENCES Anderson, J.M. (1995) Zonula occludens (ZO)-1 and ZO-2: Membrane-associated guanylate kinase homologues (MAGUKs) of the tight junction. Biochem. Soc. Trans., 23, 470–475.

12


Figure 9 Colocalization of ZO-1 with P-cadherin in fibroblasts. ZO-1 (bottom) is highly concentrated at the P-cadherinbased cell-cell adhesion sites (top). Anderson, J.M., B.R.Stevenson, L.A.Jesaitis, D.A.Goodenough, and M.S.Mooseker (1988) Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells. J. Cell Biol., 106, 1141–1149 Anderson, J.M. and C.M.Van Itallie (1995) Tight junctions and the molecular basis for regulation of paracellular permeability. Am. J. Physiol.,269, G467–475. Ando-Akatsuka, Y., M.Saitou, T.Hirase, M.Kishi, A.Sakakibara, M.Itoh, S.Yonemura, M. Furuse, and Sh.Tsukita (1996) Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues. J. Cell Biol., 133, 43–47. Atisook, K., S.Carlson and J.L.Madara. (1990) Effects of phlorizin and sodium on glucose-elicited alterations of cell junctions in intestinal epithelia. Am.J. Physiol., 258, C77–C85. Balda.M.S., L.Gonzalez-Mariscal, K.Matter, M.Cereijido and J.M.Anderson. (1993) Assembly of the tight junction: the role of diacylglycerol. J. Cell Biol., 123, 293–302. Balda, M.S., J.A.Whitney, C.Flores, S.González, M.Cereijido and K.Matter. (1996) Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J. Cell Biol., 134, 1031–1049. Ballard, S.T., J.H.Hunter and A.E.Taylor. (1995) Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium. Ann. Rev. Nutr., 15, 35–55.


13

Boiler, K., D.Vestweber and R.Kemler (1985) Cell-adhesion molecule uvomorulin is localized in the intermediate junctions of adult intestinal epithelial cells. J. Cell Biol., 100, 327– 332. Buse, P., P.L.Woo, D.B.Alexander, H.H.Cha, A.Reza, N.D.Sirota and G.L.Firestone (1995) Transforming growth factor-a abrogates glucocorticoid-stimulated tight junction formation and growth suppression in rat mammary epithelial tumor cells. J. Biol. Chem., 270, 6505–6514. Citi, S. (1993) The molecular organization of tight junctions. J. Cell Biol., 121, 485–489. Citi, S. and N.Denisenko (1995) Phosphorylation of the tight junction protein cingulin and the effects of protein kinase inhibitors and activators in MDCK epithelial cells. J. Cell Sci., 108 , 2917–2926. Citi, S., H.Sabanay, R.Jakes, B.Geiger and J.Kendrick-Jones (1988) Cingulin, a new peripheral component of tight junctions. Nature (Lond), 33, 272–276. Citi, S., H.Sabanay, J.Kendrick-Jones and B.Geiger (1989) Cingulin: characterization and localization. J. Cell Sci., 93, 107–122. Farquhar, M.G. and G.E.Palade (1963) Junctional complexes in various epithelia. J. Cell Biol., 17, 375–409. Fujimoto, K. (1995) Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. J. Cell Sci., 108, 3443–3449. Furuse, M., K.Fujimoto, N.Sato, T.Hirase, Sa.Tsukita and Sh.Tsukita (1996) Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures. 109,429–35. Furuse, M., T.Hirase, M.Itoh, A.Nagafuchi, S.Yonemura, Sa.Tsukita and Sh.Tsukita (1993) Occludin: a novel integral membrane protein localizing at tight junctions. J. Cell Biol., 123, 1777–1788. Furuse, M., M.Itoh, T.Hirase, A.Nagafuchi, S.Yonemura, Sa.Tsukita and Sh.Tsukita (1994) Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J. Cell Biol., 127, 1617–1626. Gumbiner, B. (1987) Structure, biochemistry and assembly of epithelial tight junctions. Am. J. Physiol., 253, C749–C758. Gumbiner, B. (1993) Breaking through the tight junction barrier. J. Cell Biol., 123, 1631– 1633. Gumbiner, B., T.Lowenkopf and D.Apatira (1991) Identification of a 160-kDa polypeptide that binds to the tight junction protein ZO-1. Proc. Natl. Acad Sci. USA, 88, 3460–3464. Herrenknecht, K., M.Ozawa, C.Eckerskorn, F.Lottspeich, M.Lenter and R.Kemler (1991) The uvomorulin-anchorage protein a-catenin is a vinculin homologue.Proc. Natl. Acad. Sci. USA, 88, 9156–9160. Hollander, D. (1988) Crohn’s disease—a permeability disorder of the tight junction? Gut, 29, 1621–1624. Itoh, M., A.Nagafuchi, S.Yonemura, T.Kitani-Yasuda, Sa.Tsukita and Sh.Tsukita (1993) The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J. Cell Biol., 121, 491–502. Itoh, M., S.Yonemura, A.Nagafuchi, Sa.Tsukita and Sh.Tsukita (1991) A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites. J. Cell Biol., 115, 1449–1462. Jesaitis, L.A. and D.A.Goodenough (1994) Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppresser protein. J. Cell Biol., 124, 949–961. Kachar. B. and T.S.Reese (1982) Evidence for the lipidic nature of tight junction strands. Nature (Lond.), 296, 464–466. Keon, B.H., S.Schäfer, C.Kuhn, C.Grund and W.W. Franke (1996) Symplekin, a novel type of tight junction plaque protein. J. Cell Biol., 134, 1003–1018. Kim, S.K. (1995) Tight junctions, membrane-associated guanylate kinases and cell signaling. Curr. Opin. Cell Biol., 7, 641–649. McCarthy, K.M., I.B.Skare, M.C.Stankewich, M.Furuse, Sh.Tsukita, R.A.Rogers, R.D. Lynch and E.E.Schneeberger (1996) Occludin is a functional component of the tight junction. J. Cell Sci., 109, 2287–2298. McCrea, P.D., C.W.Turck and B.Gumbiner (1991) A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. Science, 254, 1359–1361.

14


Milks, L.C., G.P.Conyers and E.B.Cramer (1986) The effect of neutrophil migration on epithelial permeability. J. Cell Biol., 103, 2729–2738. Nagafuchi, A. and M.Takeichi (1989) Transmembrane control of cadherin-mediated cell adhesion: a 94 kDa protein functionally associated with a specific region of the cytoplasmic domain of E-cadherin. Cell Regul., 1, 37–44. Nagafuchi, A., M.Takeichi and Sh.Tsukita (1991) The 102 kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulation of expression. Cell, 65, 849–857. Ozawa, M., H.Baribault and R.Kemler (1989) The cytoplasmic domain of the cell adhesion molecule uvomorulin associates with three independent proteins structurally related in different species. EMBO J., 8, 1711–1717. Ozawa, M., M.Ringwald and R.Kemler (1990) Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. Proc. Natl. Acad. Set. USA, 87, 4246–4250. Pinto da Silva, P. and B.Kachar (1982) On tight junction structure. Cell, 28, 441–450. Rajasekaran, A.K., M.Hojo, T.Huima and E.Rodriguez-Boulan (1996) Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions. J. Cell Biol., 132, 451–463. Saitou, M., Y.Ando-Akatsuka, M.Itoh, M.Furuse, J.Inazawa, K.Fujumoto and Sh.Tsukita (1997) Mammalian occludin in epithelial cells: Its expression and subcellular distribution. Eur. J Cell Biol., in press. Schneeberger, E.E. and R.D.Lynch (1992) Structure, function, and regulation of cellular tight junctions. Am. J. Physiol., 262, L647–L661. Staehelin, L.A. (1973) Further observations on the fine structure of freeze-cleaved tight junctions. J. Cell Sci., 13, 763–786. Stevenson, B.R., J.M.Anderson and S.Bullivant (1988) The epithelial tight junction: Structure, function and preliminary biochemical characterization. Mol. Cell. Biochem., 83, 129– 145. Stevenson, B.R. and D.Goodenough (1984) Zonula occludentes in junctional complexenriched fractions from mouse liver: preliminary morphological and biochemical characterization. J. Cell Biol., 98, 1209–1221. Stevenson, B.R., J.D.Siliciano, M.S.Mooseker and D.A.Goodenough (1986) Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia. J. Cell Biol., 103, 755–766. Takeichi, M. (1988) The cadherins: cell-cell adhesion molecules controlling animal morphogenesis. Development, 102, 639–655. Takeichi, M. (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science, 251, 1451–1455. Tsukita, Sh., M.Itoh, A.Nagafuchi, S.Yonemura and Sa.Tsukita (1993) Submembranous junctional plaque proteins include potential tumor suppresser molecules. J. Cell Biol., 123, 1049–1053. Tsukita, Sh., Sa.Tsukita, A.Nagafuchi and S.Yonemura (1992) Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions. Curr. Opin. Cell Biol., 4, 834– 839. Volk, T. and B.Geiger (1984) A 135–kd membrane protein of intercellular adherens junctions. EMBO J., 3, 2249–2260. Weber, E., G.Berta, A.Tousson, P.St.John, M.W.Green, U.Gopalokrishnan, T.Jilling, E.J. Sorscher, T.S.Elton, D.R.Abrahamson and K.L.Kirk (1994) Expression and polarized targeting of a Rab3 isoform in epithelial cells. J. Cell Biol., 125, 583–594. Willott, E., M.S.Balda, A.S.Fanning, B.Jameson, C.Van Itallie and J.M.Anderson (1993) The tight junction protein ZO-1 is homologous to the Drosophila discs-large tumor suppresser protein of septate junctions. Proc. Natl. I. Acad. Sci. USA, 90, 7834–7831. Willott, E., M.S.Balda, M.Heintzelman, B.Jameson and J.M.Anderson (1992) Localization and differential expression of two isoforms of the tight junction protein ZO-1. Am. J. Physiol., 262, C1119–1124. Wong, V. and B.M.Gumbiner (1997) A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier. J. Cell Biol., 136, 399–409. Woo, L.P., H.H.Cha, K.L.Singer and G.L.Firestone (1996) Antagonistic regulation of tight junction dynamics by glucocorticoids and transforming growth factor-β in mouse mammary epithelial cells. J. Biol. Chem., 271, 404–412.


15

Woods, D.A. and Bryant, P.J. (1993) ZO-1, DlgA and PSD95/SAP90: Homologous proteins in tight, septate and synaptic cell junctions. Mech. Dev., 44, 85–89. Yancey, S.B., D.Easter and J.-P.Revel(1979) Cytological changes in gap junctions during liver regeneration. J. Ultrastruct. Res., 67, 229–242. Yonemura, S., M.Itoh, A.Nagafuchi and Sh.Tsukita (1995) Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells. J. Cell Sci., 108, 127–142. Zahraoui, A., G.Joberty, M.Arpin, J.J.Fontaine, R.Hellio, A.Tavitian and D.Louvard (1994) A small rab GTPase is distributed in cytoplasmic vesicles in non-polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells. J. Cell Biol., 124, 101–115. Zettl, K.S., M.D.Sjaastad, P.M.Riskin, G.Parry, T.E.Machen and G.L.Firestone (1992) Glucocorticoid-induced formation of tight junctions in mouse mammary epithelial cells in vitro. Proc. Natl. Acad. Sci. USA, 89, 9064–9073. Zhong, Y., K.Enomoto, H.Isomura, N.Sawada, T.Minase, M.Oyamada, Y.Konishi and M.Mori (1994) Localization of the 7H6 antigen at tight junctions correlates with the paracellular barrier function of MDCK cells. Exp. Cell Res., 214, 614–620. Zhong, Y., T.Saitoh, T.Minase, N.Sawada, K.Enomoto and M.Mori (1993) Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2. J. Cell Biol., 120, 477–483.

2. HEMIDESMOSOMES D.R.GARROD Department of Cell and Structural Biology, Stopford Building, The University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom

Epithelial cell sheets must be firmly attached to the underlying extracellular matrix. Certain epithelia are equipped with specialized junctional structures called hemidesmosomes that are primarily responsible for attachment. These epithelia include the epidermis, the cornea, the mammary epithelium, parts of the intestinal and respiratory tracts, and the amnion. Hemidesmosomes are recognised by their ultrastructural appearance (Weiss and Ferris, 1954; Kelly, 1966) (Figure 1). Located at the basal cell surface, their most prominent feature in cross section is a dense outer plaque closely applied to the inner surface of the plasma membrane. The outer plaque is separated by a less dense region from an inner dense plaque from which intermediate filaments extend into the cytoplasm. The whole structure forms a circular membrane domain of no more than 0.5 micrometres in diameter and approximately 150 nm deep from the plasma membrane to the inner surface of the inner plaque. The extracellular face of the hemidesmosome is in contact with the lamina lucida of the basement membrane. This region is characterised by the presence of fine anchoring filaments that extend

Figure 1 Drawing of an epidermal hemidesmosome as seen be electron microscopy. AFib =anchoring fibril; AFil=anchoring filament; Col=dermal collagen fibre; IP=inner plaque; KF=keratin filaments; LD=lamina densa; LL=lamina lucida; SBDP=sub-basal dense plate.


17

from the plasma membrane across the lamina lucida to the lamina densa. In some sections a linear density called the sub-basal dense plate is seen in the lamina lucida, closely associated with the extracellular face of the hemidesmosomal plasma membrane. Particularly in the epidermis and amnion, the matrix beneath the basement membrane contains numerous banded structures called anchoring fibrils which insert into the lamina densa. In favourable sections filamentous continuity between the anchoring fibrils and the anchoring filaments can be resolved (Ellison and Garrod, 1987). This means that there is effective continuity of structure in epidermis from the dermis right through to the intermediate filament cytoskeleton of the epidermal cells in which the hemidesmosome itself provides the transmembrane connection. Such continuity appears to be essential for strong adhesive binding of the epidermis to the dermis since genetic defects leading to absence or abnormality of anchoring fibrils, anchoring filaments, hemidesmosomes, or basal cell keratin filaments give rise to blistering diseases involving loss of adhesion between the epidermis and the dermis (Table 1). Elucidation of the nature of these diseases and the molecular composition of hemidesmosomes has progressed dramatically over the last decade, providing clear confirmation of the immense structural importance of hemidesmosomes. As this molecular composition becomes clearer several important questions can be approached experimentally:- How does their composition relate to their structure? How are they assembled and disassembled? What role do they play in signal transduction? This article will review the progress, hopefully adding a little new material to that contained in several other recent reviews (Schwartz et al., 1990; Legan et al., 1992; Garrod, 1993; Jones et al., 1994; Langhofer et al., 1994; Aplin and Church, 1994; Stanley, 1995; Green and Jones, 1996; Christiano and Uitto, 1996; Barradori and Sonnenberg, 1996; Eady and Dunhill, 1996). Before proceeding we should note that no genetic disease involving hemidesmosomes has also been found to involve desmosomes, the intercellular junctions that hemidesmosomes resemble and from which their name derives. This supports much evidence showing that the molecular composition of desmosomes and hemidesmosomes are entirely distinct (Legan et al., 1992; Garrod, 1993; Green and Jones, 1996). THE MOLECULAR COMPOSITION OF HEMIDESMOSOMES Figure 2 represents an attempt to assemble recent data on the molecular components of hemidesmosomes into a model of hemidesmosome structure. Each component will be discussed in some detail below; in brief the model is as follows: The membrane zone contains three transmembrane proteins, the α6 β4 integrin heterodimer and the 180kDa bullous pemphigoid antigen (BP180), whose cytoplasmic domains participate in the structure of the plaque region. Also in this region are two major cytoplasmic proteins, the 230kDa bullous pemphigoid antigen (BP230) and plectin, which seem to be involved in linking, directly or indirectly, between the membrane zone and the keratin intermediate filaments. The extracellular domains of the transmembrane proteins extend into the basement membrane where they mediate attachment to the extracellular matrix. Within the basement membrane, laminin 5 (LN5) and possibly also the extracellular domain of BP180 Table 1 Human Diseases Involving Hemidesmosomes Name of Disease

Type of Disease HD Component Involved

Structural Defect

Clinicopathologic al features

References

Epidermolysis bullosa sipmlex

Genetic

Split through basal epidermal cells above Hds

Epidermal blistering — more or less severe

McLean & Lane (1985)

Keratin 5 or 14

18


Name of Disease

Type of Disease HD Component Involved

Structural Defect

Epidermolysis bullosa-junctional (EBJ)

Genetic

α, β or γ chain of LN5 - anchoring fibrils

EBJ with pyloric atresia (PA-EBJ)

Genetic

α6 or β4 integrin

Split skin at level of lamina lucida few abnormal Hds Split at level of lamina lucida

EBS with muscular dystrophy (MDEBS)

Genetic

Plectin

Epidermolysis bullosa dystrophica Bart’s Syndrome Generalised benign atrophic epidermolysis bullosa (GABAB) Bullous pemphigoid Cicatricial pemphigoid

Genetic

Collegen VII anchoring fibrils

Split through basal epidermal cells; abnormal HDs, detached keratin filaments, atrophy of muscle fibres Absence of anchoring fibrils

Genetic

BP 180 LN5

Abnormal HDs

Autoimmune

BP 180 (BP 230)

Auotimmune

BP 180 LN5

Herpes gestationsis (HG)

Autoimmune

BP 180 (BP 230)

Clinicopathologic References al features depending on nature of mutation Severe epidermal Eady & Dunhill blistering (1996)

Severe epidermal blistering and pyloric stenosiis Epidermal blistering - severe muscular weakness

Vidal et al. (1995) Shimizu et al. (1996) Smith et al. (1996) McLean et al. (1996) Pulkkinen et al. (1996)

Epidermal blistering

Christiano et al. (1996)

Split at level of lamina lucida Split at level of basement membrane

Epidermal blistering, baldness, nail abnormalities Epidermal blistering Mucocutaneous blistering, healing with scaring

Split at level of lamina lucida

Epidermal blistering

McGrath et al. (1996) Jonkman et al. (1996) Stanley et al. (1988) Balding et al. (1996) Domloge-Hultsch N. et al. (1994) Diaz et al. (1990)

are components of the anchoring filaments. LN5 is covalently complexed with LN6 or LN7 through which it interacts in the lamina densa with other basement membrane components such as LN1 and nidogen, and, directly or indirectly, with collagen VII of the anchoring fibrils. In many respects this model is highly speculative: some of the suggested interactions have not been directly demonstrated and there are almost certainly several more components to be identified. However, the model begins to suggest how structural continuity between the intermediate filament cytoskeleton of basal epithelial cells and the extracellular matrix, and thus how strong binding of the epithelium to the matrix is maintained. The individual components and their interactions will now be considered in more detail.


19

Figure 2 Diagrammatic impression of the molecular composition of a hemidemodome employing information from a number of sources. α6=α6 integrin subunit; β4=β4 integrin subunit; BP180=180 kDa bullous pemphigoid antigen (BPAG2); BP230=230kDa bullous pemphigoid antigen (BPAG1); FN=fibronectin; IF=intermediate filaments; LN 1, 5, 6, 7=laminins 1, 5, 6 and 7.; TAM=tyrosine activation motif. The following papers are particularly acknowledged: Champiaud et al. (1995); Hirako et al. (1996); Tang et al. (1996); McLean et al. (1996); Legan et al. (1992); Garrod (1993); Barrodori and Sonneberg (1996) For further information see text and Figure 1.

20


TRANSMEMBRANE COMPONENTS α6 β4 integrin The α6 β4 integrin is the predominant integrin on the basal surface of epidermal cells where it is located mainly or exclusively in hemidesmosomes (Stepp et al., 1990; Sonnenberg et al., 1991). α6 is a conventional integrin a subunit showing 18–20% amino acid identity to other β integrins (Tamura et al., 1990). It has two subunits linked by a disulphide bond in the extracellular membrane-proximal region with eight internal repeats and four calcium binding sites towards the NH2-terminus. Extracellularly, the β4 subunit shows 36–38% amino acid identity to other β integrins (Tamura et al., 1990; Suzuki and Naito, 1990; Hogervorst et al., 1990). However, β4 integrin has a unique cytoplasmic domain consisting of over 1000 amino acids and containing four Type III fibronectin repeats, two on either side of a linker region which has a tyrosine activation motif (TAM) (Mainiero et al., 1995). Both α6 and β4 have alternativelyspliced variants of their cytoplasmic domains. Only the predominant A forms are expressed in epidermis, but there is no clear association between tissue expression of splice varients and presence or absence of hemidesmosomes (Hogervorst et al., 1993; Niessen et al., 1994; Uematsu et al., 1994) A major function of α6 β4 integrin is to mediate adhesion to the extracellular matrix. Evidence for this comes from both human genetic disease and null mutations for the α6 and β4 genes in mice. A patient with junctional epidermolysis bullosa with pyloric atresia was found to have greatly reduced expression of α6 β4 integrin by immunocytochemistry of epidermis, and abnormal hemidesmosomes. Mutations in both alleles of the β4 integrin gene were demonstrated (Vidal et al., 1995). These resulted in stomach constriction and severe epidermal blistering causing death at 8 months of age. Absence of β4 in PA-EBJ was also reported by Niessens et al. (1996) and absence of α6 in the same conditon by Shimizu et al. (1996). Such patients have incomplete hemidesmosomes (Niessens et al., 1996). Targeted inactivation of the α6 gene in mice resulted in a phenotype remarkably similar to human EBJ. The mice died at birth with severe epidermal blistering at the level of the dermo-epidermal junction. β4 staining was retained in the basal epidermis but was abnormally distributed, LN5 staining was normal but hemidesmosomes were absent (Georges-Labouse et al., 1996). It was surprising that absence of α6 was compatible with fetal development because of its reported function in kidney tuble morphogenesis and endoderm migration (Sutherland et al., 1993; Sorokin et al., 1990). Null mutations of the β4 integrin gene also resulted in a phenotype resembling EBJ (Dowling et al., 1996; van der Neut et al., 1996). Mice died shortly after birth with severe epidermal blistering. Hemidesmosomes were absent. Blistering occurred at the dermo-epidermal junction although there was some breakage of basal cells as in EBS because of disorganisation of basal keratin filaments. Expression of α6 but not other integrins was disturbed in basal epidermal cells, while BP180, the other major transmembrane component of hemidesmosomes, was distributed over the entire surface of basal cells as in wild type epidermis. This appears to indicate that neither BP180 nor other integrins, including α3 β1 which binds LN5 (Carter et al., 1990), can compensate for α6 β4 in epidermal adhesion. βl integrins and fibronectin have been implicated in the regulation of epidermal differentiation (Adams and Watt, 1993). Does α6 β4 contribute to this process? Dowling et al. (1996) describe several differences between the organisation of the epidermis in β4 —/— mice. “Pearls” of cells with closely spaced, different sized nuclei, little cytoplasm and keratin expression of basal cells were found suprabasally in epidermis and other stratified epithelia. Spinous layer cells did not flatten normally during terminal differentiation. Basal layer degeneration apparently mediated by apoptosis was found and mitotic cells were present above the


21

basal layer. These results suggest that β4 integrin may contribute to the regulation of differentiation and cell survival in epidermis and other stratified epithelia. Signal transduction is an important property of integrins, functioning in the regulation of cell phenotype and cell behaviour (Dedhar and Hannigan, 1996; Humphries, 1996). Recent work has demonstrated a mechanism for signal transduction by β4 integrin which potentially links ligand binding by the integrin to activation of the ras pathway and thus regulation of gene activity and growth control (Mainiero et al., 1995). Adhesion of A431 cells to LN5 or ligation with a specific monoclonal antibody resulted in multiple tyrosine phosphorylation of the β4 subunit by an unknown tyrosine kinase that is associated with the integrin. Phosphorylation mediated recruitment of the adaptor proteins She and Grb2 thus generating a potential link to the ras pathway. Some of the β4 tyrosine phosphorylation took place in the tyrosine-based activation motif (TAM). Mutation of the tyrosine residues in this motif did not inhibit recruitment of Shc/Grb2 but disrupted association of α6 β4 with hemidesmosomes, indicating that distinct tyrosine phosphorylation sites in the β4 tail are involved in these functions (Mainiero et al., 1995). Other signals involving α6 β4 integrin also appear to be involved in regulating hemidesmosome adhesion. Xia et al. (1996) identified a protein, p80, in human keratinocytes and colon carcinoma cells that is tyrosine phosphorylated but becomes dephosphorylated specifically on ligation of α6 β4 by LN5. The phosphatase inhibitor sodium vanadate blocked both dephosphorylation of p80 and α6 β4-mediated adhesion to laminin. P80 kinase activity was also shown to be associated with α6 β4 in the Triton-insoluble fraction. Thus p80 may be involved in either inside-out signalling that regulates α6 β4 adhesion or in α6 β4mediated signal transduction from the matrix. Mainiero and Pepe et al. (1996) investigated the regulation of α6 β4 adhesion by EGF. They showed that activation of the EGFR resulted in multiple tyrosine phosphorylation of the β4 subunit but partially suppressed She recruitment. In addition EGF caused disruption of hemidesmosomes and promoted α6 β4-dependent migration of cells towards laminins, suggesting a possible role for this mechanism in wound healing and tumour invasion. An entirely different signalling role for β4 integrin was suggested by Clarke et al. (1995). They found that transfection the β4 subunit into rectal carcinoma cells that expressed α6 β1 but not β4 induced partial cell cycle arrest and apoptosis. Furthermore, expression of the p21 protein (also called WAF1 or CiP1), an inhibitor of cyclin-dependent kinases, was induced. These results suggest a role for β4 in cell cycle regulation in these cells and are intriguing in relation to the effects on cell division and apoptosis found in the epidermis of 04 knockout mice (see above). The function of β4 integrin has been further examined by expression of mutant forms in hemidesmosomeforming 804G cells. Spinardi et al. (1993) showed that the cytoplasmic domain was involved in mediating the association of the integrin with the hemidesmosomal cytoskeleton. The region involved hemidesmosomal localization consists of 303 amino acids and includes the FN repeats and the intervening segment. Utilising mutants lacking either the extracellular or the cytoplasmic domain, so-called “head-less” and “tail-less” mutants, they showed further that over expression of the tail-less form disrupted hemidesmosome formation without significantly affecting specific cell-matrix adhesion, a type of structural dominant negative effect (Spinardi et al., 1996). By contrast, the head-less mutant was without effect on hemidesmosome assembly. In epidermis as well as cultured cells β4 integrin is subject to proteolytic processing and this may be important in modulating its activity (Giancotti et al., 1992). When stratifying epidermal cells move out of the basal layer they lose their hemidesmosomes and cease to express α6 β4. There is a continual requirement for controlled disassembly of hemidesmosomes in this situation and proteolysis of a major component could provide a contributary mechanism.

22


BP180 The other major transmembrane component of hemidesmosomes is the 180kDa bullous pemphigoid antigen (Klatte et al., 1988). This is also known as bullous pemphigoid antigen 2 (BPAG2) (BP230 is BPAG1) and collagen type XVII. It is a type 2 transmembrane protein (it has a single transmembrane domain and its NH2-terminus is intracellular) with a series of collagen domains in its extracellular region (Giudice et al., 1992; Hopkinson et al., 1992; Li et al., 1993). Several other molecules that are not classed as collagens show this chimeric or hybrid structure and it has been suggested that BP180 could be grouped with these proteins which include the C1q complement subunit, the collectins, the asymmetric form of acetylcholinesterase, ficolin and the macrophage scavenger receptor (Hopkinson and Jones, 1996). Two types human diseases involve BP180, the autoimmune diseases bullous pemphigoid (BP) and herpes gestationis (HG) and the genetic disease generalized atrophic benign epidermolysis bullosa (GABEB). In BP and HG complement-fixing autoimmune IgG binds to the basement membrane zone of epidermis causing formation of tense fluid-filled sub-epidermal blisters. Epitope mapping showed that BP and HG IgGs bind to a 14 amino acid squence in the non-collagenous mem brane-proximal domain of BP180 (Giudice et al., 1993). Passive transfer of the disease by injecting purified IgG from patients into newborn mice was unsuccessful because the epitope is not highly conserved so the human IgGs did not react with the murine protein. However, a rabbit antibody raised to recombinant murine BP180 caused blistering resembling that found in BP when administered to new-born mice (Liu et al., 1993). It appears that BP and HG autoantibodies exert a complement-mediated effect rather than directly inhibiting hemidesmosomal adhesion, because the rabbit antibody was ineffective in complement-deficient or complement-depleted mice (Liu et al., 1995). It has recently also been shown that autoantibodies from patients with cicatricial pemphigoid, a blistering disease primarily of mucosal tissue, also react with BP180 (Balding et al., 1996). These antibodies react with one or both of two sites on the BP180 molecule, one at or adjacent to the BP/HG epitope and the other at the COOH-terminal region. GABEB is an inherited non-lethal disease involving epidermal blistering from birth and baldness resulting from follicular atrophy in early childhood. It appears to result from deficiency in either LN5 or BP180 (Jonkman et al., 1996). Mutations in the BP180 gene (termed COL17A1) have been demonstrated in patients with this disease (McGrath et al., 1995, 1996). These patients have rudimentary hemidesmosomes and blister formation takes place at the dermo-epidermal junction. This evidence appears to indicate a role for BP180 in hemidesmosomal adhesion. There has so far been no BP180 null mutation in mice. Since the consequences of BP180 mutations are less severe than those of β4 intergrin in PAEBJ, it may be suggested that the adhesive contritribtion made by BP180 in hemidesmosomes is significant but less than that made by α6 β4 integrin. That two adhesion receptors are apparently required indicates the necessity for very strong adhesive binding to maintain cell matrix interactions at hemidesmosomes. Hemidesmosomes are highly insoluble structures. It was recently found that a proportion of the BP180 in basal corneal cells could be solubilised with non-ionic deteregent (Hirako et al., 1996). BP180 could then be purified from this soluble fraction and its structure studied. Hirako et al. showed by rotary shadowing that BP180 is a quaver-shaped molecule. It has a globular NH2-terminus, a central rod domain that extends from the cytoplasmic region through the membrane and into the collagenous region, and a flexible collagenous tail (Figure 2). Its overall length is bewteen 190 and 230 nm. Hirako et al. speculate that the globular domain lies in the outer plaque of the hemidesmosome, the rod domain traverses the lamina lucida forming an important component of the anchoring fibrils and that the flexible tail resides in the lamina densa where it may participate in ligand binding. Furthermore, crosslinking experiments indicated that BP180 forms a collegen-like trimer (Hirako et al., 1996).


23

It seems that when the α6 β4 integrin is disrupted BP180 cannot alone form hemidesmosomal plaques since hemidesmosomes are absent in α6 and β4 knockout mice while the distribution of BP180 remains undisturbed (see above). So how does BP180 participate in hemidesmosomal structure? Hopkinson et al. (1995) expressed deleted BP180 in rat blabber 804G cells that form hemidesmosomes in culture (Ridelle et al., 1991). They showed that a protein lacking the collagenous extracellular domain became colocalised with other hemidesmosomal components on the basal surface of the cells. Deletion of 36 amino acids from the NH2-terminus caused the protein to localise instead to the apical cell surface, while deletion of 27 amino acids from the extracellular membrane-proximal region allowed basal, but nonhemidesmosomal, location. By transfecting the largest of these deleted proteins into cells that express α6 integrin but not other hemidesmosomal components, Hopkinson et al. (1995) obtained evidence that this truncated BP180 protein binds to α6 integrin but that this interaction is prevented by both the 36 amino acid NH2 terminal deletion and the 27 amino acid membrane-proximal deletions. Borradori and Sonnenberg (1996) report some unpublished experiments that appear to question this association in hemidesmosome-containing cells. However, the sheer proximity of α6 β4 and BP180 makes some kind of interaction, direct or indirect, almost inevitable. BP180 may partcipate in regulation of hemidesmosome formation since phorbol ester treatment of DEJ-1 keratinocyes causes simultaneous serine phosphorylation of BP180 and hemidesmosome loss (Kitajima et al., 1995). THE BASEMENT MEMBRANE LN5 Previously called BM600, kalinin, nicein or epiligrin, LN5 is now known to be a member of the expanding family of laminins. It is a heterotrimer consisting of laminin α3, β3 and γ2 chains. The mature forms of these chains have molecular weights of 165, 140 and 105 kDa, respectively, considerably smaller than other laminin chains. LN5 has a rod-like structure with globular ends rather than the cross-like structure of the substantially larger LN1 (Burgeson, 1993). Antibodies to LN5 localise to the anchoring filaments of the lamina lucida and disrupt keratinocyte-basement membrane adhesion (Rouselle et al., 1991). As with α6 β4 integrin, human genetic disease provides direct evidence for the function of LN5 in adhesion at the dermo-epidermal junction (Table 1). First it was shown that the lethal form of junctional epidermolysis bullosa was characterised by abnormalities of hemidesmosomes and greatly reduced staining with antibody to LN5 (Tidman and Eady, 1986; Heagerty et al., 1987). Subsequently all three LN5 subunits have been cloned (Gerecke et al., 1994; Kallunki et al., 1992; Ryan et al., 1994) and mutations in the laminin β3 (Pulkkinen et al., 1995; Vailly et al., 1995), the γ2 (Pulkkinen et al., 1994; Aberdam et al., 1994; Vailly et al., 1995) and, most recently, the α3 (Kivirikko et al., 1995) genes have been found in patients with lethal (Herlitz-type) EBJ. This disease invovles severe skin blistering at the level of the dermoepidermal junction from birth resulting in death within the first year. Reduced expression of LN5 can also occur in the non-lethal blistering disease GABEB (Jonkman et al., 1996) (see above). Experimental evidence indicates that α6 β4 integrin binds to LN5 (Sonnenberg et al., 1993; Niessen et al., 1994b). Although new studies suggest that this integrin also binds to other forms of laminin (Spinardi et al., 1996), its interaction with LN5 appears to be of major importance for hemidesmosomal adhesion. A monoclonal antibody that disrupts epidermal-basement membrane adhesion has been reported to bind to the G domain of the LN5 α3 chain (Champliaud et al., 1995). This would position the COOH-terminus of the molecule in the lamina lucida adjacent to the basal surfaces of the epithelial cells.

24


All three chains of LN5 are substantially truncated compared with those of other laminins, the α3 and β3 chains being proteolytically cleaved extracellularly. Truncation of its short arms prevents self polymerisation which is mediated by domain VI of the β1 or β2 chains of other laminins. Furthermore, in contrast to the EGFlike domain of the yl chain, the EGF-like domain in the γ2 chain of LN5 has very low affinity for nidogen (Mayer et al., 1995) which mediates interaction between other laminins and the basement membrane components type IV collagen, perlecan and fibulins. So how does LN5 associate with the basement membrane? Human amnion contains two other forms of laminin, LN6 and LN7, that form covalent complexes by disulphide bonding with LN5 (Marinkovich et al., 1992; Champliaud et al., 1995). LN 6 is also present in human skin but LN7 is virtually absent. LN6 (also known as K-laminin) consists of α3, β1 and γ1 chains and LN7 (also known as K-S-laminin) of α3, β2 and γ1 chains. Since these β chains can link to other basement membrane components, the association of these laminins with LN5 presumably mediates its linkage to the basement membrane (Champliaud et al., 1995). In view of the structural continuity between anchoring fibrils and anchoring filaments that can be demonstrated in some situations (Ellison and Garrod, 1987), it is reasonable to speculate that binding may take place between type VII collagen, the principal component of anchoring fibrils (Bachinger et al., 1990; Rouselle et al., 1991), and LN5, 6 and/or 7, although no such binding has yet actually been demonstrated (Champliaud et al., 1995). THE PLAQUE BP230 Also known as bullous pemphigoid antigen 1 (BPAG1), BP230 is the antigen recognised by the majority of sera from patients with the autoimmune epidermal blistering disease bullous pemhpigoid. It is located in the plaque region of hemidesmosomes (Mutasim et al., 1985; Klatte et al., 1989; Tanaka et al., 1990). Since BP230 is a cytoplasmic protein it is difficult to envisage how antibodies could gain access to it in order to cause loss of hemidesmosomal adhesion and epidermal blistering. Cloning and sequencing of BP230 cDNA showed it to have homology to desmoplakin, a major component of the desmosomal plaque that is involved in linking desmosomes to intermediate filaments (Tanaka et al., 1991; Sawamura et al., 1991). Both molecules consist of globular end domains with an intervening region having the basic structural arrangement of a coiled-coil rod domain. The COOHterminal domains of these molecules are characterised by subdomains of 176 amino acids containing 38 amino acid repeats. Desmoplakin has three such subdomains and BP230 two (Green et al., 1992). Tang et al. (1996) showed that the NH2terminal globular domain of BP230 is similar in size to those of desmoplakin and the third member of this protein family, plectin (see below). Similarity of structure between desmoplakin and BP230 suggests possible similarity of function. The role of BP230 in hemidesmosome structure and function has been studied by targeted gene inactivation in mice (Guo et al., 1995). Such mice showed normal numbers of hemidesmosomes in basal epidermal cells, but these structures lacked inner plaques and had no attached intermediate filaments. The epidermis was fragile particularly in hairless regions such as the tail; epidermal detachment occurred by breakage of the basal cell above the level of the hemidesmosomes in manner reminiscent of but distinct from that seen in EBS. The mice also showed delayed wound re-epithelialisation indicating a role for hemidesmosome-IF interaction in epidermal cell migration. No effects on epidermal differentiation were reported and basal cell proliferation was apparently normal. During the first few weeks of life BP230 —/— mice developed severe dystonia involving abnormal limb posture, and muscle and nerve degeneration culminating in inability to move. This condition was


25

reminiscent of a hereditary neurodegenerative disease of mice called dystonia musculorum (dt/dt). dt was shown to be allelic with BP230 and one homozygous dt strain was shown to lack BP230 in the epidermis (Gou et al., 1995). Subsequently a candidate dt gene, Dystonin, has been cloned (Brown et al., 1996). Dystonin was shown to be predominantly expressed in sites of neurodegeneration. It encodes an NH2terminal region containing an actin binding domain and a COOH-region containing the majority of BP230. dt and bpagl are contained in the same transcription unit which has both neural and keratinocyte promotors. The transcript expressed in epidermis is smaller than those in nerve and encodes the full BP230 protein which lacks the actin binding domain. The two neural transcripts encode unique alternative NH2-termini, the actin binding region and the BP230 protein except for a small NH2-terminal region encoded by the first exon of the bpagl gene. The actin binding domain shows homology with those of spectrin and dystrophin. These results are consistent with a cytoskeletal cross linking function for dystonin in nerve, possibly a connector between actin and intermediate filaments. Direct evidence for this novel connecting role between actin and neurofilaments has been provided by Yang et al. (1996). They demonstrated by transfection that the NH2-terminus of the neuronal form, BPAG1n, associates with actin filaments, the COOH-terminus associates with neurofilaments, and the intact molecule associates with both types of filaments. Yang et al. also demonstrated that BAPG1 is expressed in sensory neurons and that the neurofilament cytoskeletons of these in BAPG —/— mice are disorganised, probably resulting in defective axonal transport. In figure 2 BP230 is shown with its NH2-terminus associated with the outer plaque and its COOHterminus adjacent to the inner plaque where the IFs attach. This orientation appears consistent with its role in linking between the hemidesmosomal plaque and the IFs, and also by analogy with desmoplakin for which both molecular (Stappenbeck et al., 1993; Bornslaeger et al., 1996) and ultrastructural (North, Bornslaeger, Green and Garrod: unpublished) evidence indicate such interactions. Plectin The importance of plectin in hemidesmosome structure has emerged recently in association with the human genetic disease epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) (Smith et al., 1996; McLean et al., 1996; Pulkkinen et al., 1996). This rare autosomal recessive disorder is characterised by the occurrence of epidermal blistering from birth with a later onset of symptoms of muscular dystrophy (Niem et al., 1988; Fine et al., 1989; Kletter et al., 1989). Epidermal lesions are characterised by splitting of the basal cells through the hemidesmosomal plaque region. Poorly formed hemidesmosomes remain attached to the basement membrane but the inner plaque is lacking and the keratin filaments are detached (McLean et al., 1996; Pulkkinen et al., 1996). The structure of these hemidesmosomes resembles that seen in BP230 —/ — mice (see above). Staining of epidermis from MDEBS patients revealed absence of plectin and the large hemidesmosomal protein known as HD1 (Orawibe et al., 1991) that may be identical to plectin (see below), and staining was also lost from the sarcolemma of skeletal muscle (Gache et al., 1996; Smith et al. 1996; McLean et al., 1996; Pulkkinen et al., 1996). Homozygous deletion mutations is the plectin gene (PLEC1) have been demonstrated in three patients with MD-EBS (McLean et al., 1996; Pulkkinen et al, 1996). Plectin is a member of the same family of proteins as BP230 and desmoplakin (Wiche et al., 1991; Green et al., 1992). It has a long central rod domain flanked by two globular domains (Foisner and Wiche, 1992). The COOH-terminal region has six homologous repeats compared with three for desmoplakin and two for BP230 (see above and figure 2). The recent cloning of human plectin has demonstrated the presence of a NH2-terminal spectrin-like actin binding domain (McLean et al., 1996). A widely distributed protein, plectin is believed to have a multifunctional role in cytoskeletal crosslinking. It has affinities for IF proteins, including lamin B of the nuclear lamina, microtubule-associated

26


proteins, spectrin and the plasma membrane skeleton (Foisner et al., 1988; Seiffert et al., 1992; Errante et al., 1994). Svitkina et al. (1996) have provided ultrastructural evidence for these various interactions. In view of the multiplicity of its affinities it is surprising that the consequences of plectin mutations are not even more far reaching than those seen in MD-EBS. Clearly plectin’s role in the functional organisation of the membrane skeleton in skeletal muscle is crucial: its actin binding domain is probably important here. In hemidesmosomes, the similarity between the defects in MD-EBS and the BP230 —/— mouse (lack of the hemidesmosomal inner plaque and detachment of IFs) suggests that these two proteins have a similar function, that of linking between the hemidesmosome and the keratin filaments (Figure 2). This is the second example of the “belt and braces” design of hemidesmosomes: there are two adhesion mechanisms, α6 β4 integrin and BP180, and two plaque-IF linkers, BP230 and plectin. In each case duplicity is required for normal function since mutations show that none of these molecules is sufficient alone. The organization of the human plectin gene (chromosomal location 8q24) has been described recently by Liu et al. (1996) and McLean et al. (1996). Two very large exons code for the central rod domain and the COOH-terminal globular domain. The NH2-terminal region is encoded by a large number of small exons. There are slight discrepancies regarding the precise number of exons (Liu et al. –32; McLean et al. –33) and size of the gene (Liu et al. –32 kb; McLean et al. →26 kb), and the size and amino acid seqence predicted for the NH2-termini of the protein differ between the two studies. The presence of many small exons raises the possibility of a number of alternatively spliced NH2-terminal variants of plectin, which may account for its apparent diversity of binding specificities. McLean et al. (1996) report that monoclonal antibodies to both HD1 and plectin give multiple bands of roughly similar size on immunoprecipitation, possible support for the existence of several alternative forms. Alternative splicing may also be one explanation of the puzzle surrounding the identity of HD1, plectin and a third protein, Intermediate Filament Associated Protein (IFAP) 300 (Skalli et al., 1994). Are they one and the same protein or three distinct molecular species? HD1 was described by Orawibe et al. (1991) as a 500 kDa component of hemidesmosomes, while both plectin and IFAP 300 have been reported to be associated with desmosomes as well as hemidesmosomes (Wiche et al., 1983, 1984; Skalli et al., 1994). A trivial explanation for this may be crossreactivity of antibodies between the related proteins in the two types of junctions. If the proteins are indeed present in both junctions, this represents an overlap in composition between otherwise distinct structures. It should also be noted that plectin mutations affect hemidesmosomes but not desmosomes, so if the latter contain plectin, its role must be less critical than in hemidesmosomes. McLean et al. (1996) found that monoclonal antibodies for HD1 and plectin gave slightly different staining patterns in normal epidermis, but that staining with both was absent in MD-EBS patients. This indicates either that the antibodies both recognise plectin or that the stability of HD1 is entirely dependant on plectin. Skalli et al. (1994) describe petide maps and sequence data that seem to indicate that plectin and IFAP 300 are distinct, but other evidence indicates close similarity between IFAP 300 and HD1 (Heida et al., 1992; Green and Jones, 1996). If IFAP 300 is distinct from plectin/HD1, its keratin binding properties (Skalli et al., 1994) do not enable it to compensate for the absence of either plectin/HD1 or BP230. OTHER HEMIDESMOSOMAL COMPONENTS At least two additional extracellular and one plaque component of hemidesmosomes have been described but not so far further characterised. Kurpakus et al. (1990) reported a 125 kDa extracellular component antibodies to which caused detachment of epithelial cells from dermis in organ culture. More recently, Marinkovich et al. (1996) reported that the autoantigen in epidermal linear IgA disease (LAD-1) is a 120 kD component of anchoring filaments. A 200 kDa protein of the hemidesmosomal plaque has been described


27

by Kurpakus and Jones (1991). Further elucidation of the contributions of these molecules to hemidesmosomal structure and function is awaited. TYPE II HEMIDESMOSOMES Strictly speaking hemidesmosomes are defined by their ultrastructural appearance. However, some of their molecular components are expreesed together in cells that do not possess bona fide hemidesmosomes. Thus a6β4 integrin and HD1 are expressed in Schwann cells and astrocytes of peripheral nerve (Sonnenberg and Linders, 1990; Heida et al., 1992). The latter author have therefore suggested that hemidesmosmoes having a recognisable plaque structure and containing the full complement of known hemidesmosomal components should be referred to as Type I hemidesmosomes while less well-defined stuctures containing a6β4 integrin and HD1 but lacking the BP antigens should be referred to as Type II hemidesmosomes. CONCLUSION These are exciting times in the study of hemidemosomes. We know a lot but there is much more to learn. Immediate important questions seem to be: How are the different components arranged within the structure of hemidesmosomes? How do the different components interact with each other? What other components are present and what is the relationship between HD1, IFAP 300 and plectin? How is the assembly and disassembly of the junctions regulated in wound healing and characterisation? How are signalling processes involved in regulating assembly/ disassembly? Do signalling systems regulate affinity of hemidesmosomal adhesion? How are signals transduced by hemidesmosomes and what are the functions of these signals in regulating cell behaviour and gene activity? Can suitable therapies be devised for genetic and autoimmune diseases involving hemidesmosomes? Given the current rate of progress, the answers to at least one of these questions could soon be available. ACKNOWLEDGEMENTS I thank Professor Robin Eady for advice and Denise Smith for artwork. REFERENCES Aberdam, D., Galliano, M.-F., Vailly, J., Pulkkinen, L., Bonifas, J., Christiano, A.M., Tryggvason, K., Vitto, J., Epetein, E.H., Ortonne, J.-P. And Meneguzzi, G. (1994) Herlitz’s junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the γ2 subunit of nice/kalinin (LAMININ 5). Nature Genet., 6, 299–304. Adams, J.C. and Watt, F.M. (1993) Regulation of differentiation and development by extracellular matrix. Development, 117, 1183–1198. Aplin, J.D. and Church, H.J. (1994) Basement membrane-hemidesmosome interactions. In “Molecular Biology of Desmosomes and Hemidesmosomes” (ed. J.E.Collins and D.R. Garrod) R.G. Landes Company, Austin, pp. 87–106. Bächinger, H.P., Morris, N.P., Lunstrum G.P., Keene, D.R., Rosenbaum, L.M., Compton, L.A. and Burgeson, R.E. (1990) The relationship of biophysical and biochemical characteristics of type VII collagen to the function of anchoring fibrils. J. Biol. Chem., 265,10095–10101. Balding, S.D., Prost, C., Diaz, L.A., Bernard, P., Bedane, C., Aberdam, D. and Giudice, G.V. (1996) Cicatricial pemphigoid autoantibodies react with multiple sites on the BP180 extracellular domain. J. Invest. Dermatol., 106, 141–146.

28


Barradori, L. and Sonnenberg, A. (1996) Hemidesmesomes: role in adhesion, signalling and human disease. Current Opinion in Cell Biology, 8, 647–656. Bornslaeger, E., Corcoran, C.M., Stappenbeck, T.S. and Green, K.J. (1996) Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly. J. Cell Biol., 134, 985–1001. Brown, A., Bernier, G., Mathieu, M., Rossant, J. and Kothary, R. (1995) The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen. Nat. Genet., 10, 301–306. Burgeson, R. (1993) Dermal-epidermal adhesion in skin in “Molecular and cellular aspects of basement membranes”ed. D.H.Rohrbach and R.Timpl. New York:Academic Press, pp. 49–66. Carter, W.G., Ryan, M.C. and Gahr P.J. (1991) Epiligrin, a new cell adhesion ligand for integrin α3β1 in epithelial basement membranes. Cell, 65, 599–610. Champliaud, M.F., Lunstrum, G.P., Rousselle, P., Nishiyama, T., Keene, D.R. and Burgeson, R.E. (1996) Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal adhesion. J. Cell Biol., 132, 1189–1198. Christiano, A.M. and Uitto, J. (1996) Molecular complexity of the cutaneous basement membrane zone. Revelations from the paradigms of epidermolysis bullosa. Exp. Dermatol., 5, 1–11. Clarke, A.S., Lotz, M.M., Chao, C. and Mercurio, A.M. (1995) Activation of the p21 pathway of growth arrest and apoptosis by the β4 integrin cytoplasmic domain. J. Biol. Chem., 270, 22673–22676. Dedhar, S. and Hannigan, G.E. (1996) Integrin cytoplasmic interactions and bidirectional transmbrane signalling. Current Opinion in Cell Biology, 8, 657– 669 Domloge-Hultsch, N., Anhalt, G.J., Gammon, W.R., Lazarova, Z., Briggaman, R., Welch, M., Jabs, D.A., Huff, C. and Yancey, K.B. (1994) Antiepiligrin cicatricial pemphigoid. A subepithelial bullous disorder. Arch. Dermatol. USA, 130, 1521–1529. Dowling, J., Yu, Q.-C. and Fuchs, E. (1996) β4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J. Cell Biol., 134, 599–572. Eady, R.A.J. and Dunhill, M.G. (1994) Epidermolysis bullosa: hereditary skin fragility diseases as paragdigms in cell biology. Arch Dermatol. Res., 287, 2–9. Ellison, J.E. and Garrod, D.R. (1984) Anchoring filaments of the amphibian epidermal-dermal junction transverse the basal lamina entirely from the plasma membrane of hemidesmosomes to the dermis. J. Cell Sci., 72, 163–172. Errante, L.D., Wiche, G. and Shaw, G. (1994) Distribution of plectin, an intermediate filament-associated protein, in the adult rat central nervous system. J. Neurosei. Res., 37, 515–528. Fine, J.-D., Stenn, J., Johnson, L., Wright, T., Bock, H.-G.O. and Horiguchi, Y. (1989) Autosomal recessive epidermolysis bullosa simplex: generalized phenotypic features suggestive of junctional or dystrophic epidermolysis bullosa, and association with neuromuscular disease. Arch Dermatol., 125, 931–938. Foisner, Q., Leichtfried, F.E., Herman, H., Small, J.V., Lawson, D. and Wiche, G. (1988) Cytoskeleton-associated plectin: in situ localization, in vitro reconstitution, and binding to immobilized intermediate filament proteins. J. Cell Biol., 106, 723–733. Foisner, R. and Wiche, G. (1987) Structure and hydrodynamic properties of plectin molecules. J. Mol. Biol., 198, 515–531. Gache, Y., Chavanas, S., Lacour, J.P., Wiche, G., Owaribe, K., Meneguzzi, G. and Ortonne, J.P. (1996) Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy. J. Clin. Invest., 91, 2289–2298. Garrod, D.R. (1993) Desmosomes and hemidesmosomes. Current Opinion in Cell Biology, 5, 30– 40. Georges-Labouesse, E., Messaddeq, N., Yehia, G., Cadalbert, L., Dierch, A. and Le Meur, M. (1996) Absence of the alpha-6 integrin leads to epidermolysis bullosa and neonatal death in mice. Nat. Genet., 13, 370–373. Gerecke, D.R., Wagman, D.W., Champliaud, M.-F. and Burgeson, R.E. (1994) The complete primary structure for a novel laminin chain, the laminin B1K chain. J. Biol. Chem., 269, 11073–11080. Giancotti, F.G., Stepp, M.A., Suzuki. S., Engvall, E. and Ruoslahti, E. (1992) Proteolytic processing of endogenous and recombinant β4 integrin subunit. J. Cell Biol., 118, 951–959.


29

Giudice, G.J., Emery, D.J. and Diaz, L.A. (1992) Cloning and primary structural analysis of the bullous pemphigoid autoantigen BP180. J. Invest. Dermatol., 99, 243–250. Giudice, G.J., Emery, D.J., Zelickson, B.D., Anhalt, G.L., Liu, Z. and Diaz, L.A. (1993) Bullous pemphigoid and herpes gestationis autoantibodies recognize a common noncollagenous site on the BP180 ectodomain. J. Immunol., 151, 5742–5750. Green, K.L., Virata, M.L.A., Elgart, G.W., Stanley, J.R. and Parry, D.A. (1992) Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: Members of a new gene family involved in organisation of intermediate filaments. Int.J. Biol. Macromol., 14, 145–153. Gou, L., Degenstein, L., Dowling, J., Yu, Q.C., Wollmann, R., Perman, B. and Fuchs, E. (1995) Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration. Cell, 81, 233–243. Green, K.J. and Jones, J.C.R. (1996) Desmosomes and hemidesmosomes: structure and function of the molecular components. FASEB J., 10, 871–881. Heagerty, A.H.M., Kennedy, A.R., Eady, R.A.J., Hsi, B.-L., Verrando, P., Yeh, C.-J. and Ortonne, J.P. (1986) GB3 monoclonal antibody for diagnosis of junctional epidermolysis bullosa [Letter]. Lancet, 1, 860. Hieda, Y., Nishizawa, Y., Uematsu, J. and Owaribe, K. (1992) Identification of a new major hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes. J. Cell Biol., 116, 1497–1506. Hirako, Y., Usukura, J., Nishizawa, Y. and Orawibe, K. (1996) Demonstration of the molecular shape of BPI80, a 180kDa bullous pemphigoid antigen and its potential for trimer formation. J. Biol. Chem., 271, 13739–13745. Hogervorst, F., Kuikman, L, von Dem Borne, A.E.G.Kr. and Sonnenberg, A. (1990) Cloning and sequence analysis of β-4 cDNA: an integrin subunit that contains a unique 118kD cytopolasmic domain. EMBO J., 9, 765–770. Hogervorst, F., Admiraal, L.G., Niessen, C., Kuikman, I., Janssen, H., Daams, H. and Sonnenberg, A. (1993) Biochemical characterisation and tissue distribution of the A and B variants of the integrin α6 subunit. J. Cell Biol., 121, 179–191. Hopkinson, S.B., Baker, S.E. and Jones, J.R.C. (1995) Molecular genetic studies of a human epidermal autoantigen (the 180-kD bullous pemphigoid antigen/BP180): identification of functionally important sequences within the BP180 molecule and α6 integrin. J. Cell Biol., 130, 117–125. Hopkinson, S.B. and Jones, J.C.R. (1996) Type XVII collagen and collagen-like molecules: related by more that a common motif. Seminars in Cell and Dev. Biol., 7, 659–666. Hopkinson, S.B., Ridelle, K.S. and Jones, J.C.R. (1992) Cytoplasmic domain of the 180-kD bullous pemphigoid antigen, a hemidesmosomal component: molecular and cell biological characterization. J. Invest. Dermatol., 99, 262–270. Humphries, M.J. (1996) Integrin activation: the link between ligand binding and signal transduction. Current Opinion in Cell Biology, 8, 632–640. Jones, J.C.R., Asmuth, J., Baker. S.E., Langhofer, M., Roth, S.L. and Hopkinson, S.B. (1994) Hemidesmosomes: extracellular matrix/intermediate filament connectors. Exp. Cell Res., 213, 1–11. Jonkman, M.F., De Jong, M.C.J.M., Heeres, K., Pas, H.H., Van der Meer, J.B, Owaribe, K., Martinez de Velasco, A.M., Niessen, C.M. and Sonnenberg, A. (1995) 180-kD bullous pemphigoid antigen (BP180) is deficient in generalized atrophic benign epidermolysis bullosa. J. Clin. Invest., 95, 1345–1352. Jonkman, M.F., De Jong, M.C.J.M., Heeres, K., Steijlen, P.M., Orawibe, K., Küster, W., Menser, M., Gedde-Dahl, T. and Sonnenberg, A. (1996) Generalised atrophic benign epidermolysis bullosa. Either 180-kD bullous pemphigoid or laminin-5 deficiency. Arch. Dermatol., 132, 145–150. Kelly, D.E. (1966) Fine structure of desmosomes, hemidesmosomes and an adepidermal globular layer in developing newt epidermis. J. Cell. Biol., 26, 51–72. Kallunki, P., Sainio, K., Eddy, R., Byers, M., Kallunki, T., Sariola, H., Beck, K., Hirvoneu, H., Shaws, T.B. and Tryggvason, K. (1992) A truncated laminin chain homologous to the B2 chain: structure-, spatial expression, and chromosomal assignment. J. Cell Biol., 119, 679–693. Kitajima, Y., Owada, M.K., Fujisawa, Y., Seishima, M., Yaioita, H., Hirako, Y. and Orawibe, K. (1995) A hemidesmosomal transmembrane collagenous molecule, the 180-kDa bullous pemphigoid antigen (BPAII),

30


phosphyalated with 12–0-tetradecanoylphorbol-13-acetate be in a human squamous carcinoma cell line (DEM-1). Epithelial Cell Biol., 4, 70–75. Kivirikko, S., McGrath, J.A., Baudoin, C., Aberdam, D., Ciatti, S., Dunnill, M.G., McMillan, J.R., Eady, R.A., Ortonne, J.P. and Meneguzzi, G. et al. (1995) A homozygous nonesense mutation in the α3 chain gene of laminin 5 (LAMA3) in lethal (Herlitz) junctional epidermolysis bullosa. Hum. Mol. Genet., 4, 959–962. Klatte, D.H., Kurplakus, M.A., Greling, K.A. and Jones, J.C.R. (1989) Immunochemical characterization of three components of the hemidesmosome and their expression in cultured epithelial cells. J. Cell Biol., 109, 3377–3390. Kletter, G., Evans, O.B., Lee, J.A., Melvin, B., Yates, A.B., and Bock, H.- G.O. (1989) Congenital muscular dystrophy and epidermolysis bullosa simplex. J. Pediatr., 114, 104– 107. Kurpakus, M.A., Stock, E.L. and Jones, J.C.R. (1990) Analysis of wound healing in an in vitro model: early appearance of laminin and a 125 X 103 Mr polypeptide during adhesion complex formation. J. Cell Sci., 96, 651–660. Langhofer, M., Baker, S.E., Hopkinson, S.B. and Jones, J.C.R. (1994) Hemidesmosomes: Form, function and assembly. In “Molecular Biology of Desmosomes and Hemidesmosomes” (ed. J.E.Collins and D.R.Garrod, Austin: R.G.Landes Co. pp. 69–86. Legan, P.V., Collins, J.E. and Garrod, D.R. (1992) The molecular biology of desmosomes and hemidesmosomes: ‘What’s in a name?’. BioEssays, 14, 385– 393. Li, K., Tamai, K., Tan, E.M.L. and Uitto, J. (1993) Cloning of type XVII collagen: complimentary and genonic DNA sequences of mouse 180-kilodalton bullous pemphigoid antigen (BP180) predict an interrupted collagen domain, a transmembrane segment, and unusual features in the 5’ end of the gene and the 3’—untranslated region of the mRNA. J. Biol. Chem., 268, 8825–8834. Liu, C-G., Maercker, C., Cartoum, M.J., Hauptmann, R. and Wiche, G. (1996) Human plectin — organisation of the gene, sequence analysis and chromosome location (8q 24). Proc. Natl. Acad. Sci. USA, 93, 4278–4283. Liu, Z., Diaz, L.A., Troy, J.L., Taylor, J.F., Emery, D.J., Fairley, J.A. and Guidice, G.J. (1993) A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180. J. Clin Invest., 92, 2480–2488. Liu, Z., Giudice, G.L., Swartz, S.J., Fairly, J.A., Till, G.O., Troy, J.L. and Diaz, L.A. (1995) The role of complement in experimental bullous pemphigoid. J. Clin Invest., 95, 1539– 1544. Mainiero, F., Pepe, A., Yeon, M., Ren, Y. and Giancotti, F.G. (1996)The intracellular functions of α6β4 integrin are regulated by EGF. J. Cell Biol., 134, 241–253. Mainiero, F., Pepe, A., Wary, K.K., Spinardi, L., Mohammadi, M., Schlessinger, J. and Giancotti, F.G. (1995) Signal transduction by the α6β4 integrin: distinct β4 subunit sites mediate recruitment of Shc/Grb2 and association with the cytoskeleton of hemidesmosomes. EMBO J., 14, 4470– 4481. Marinkovich, M.P., Lunstrum, G.P., Keene, D.R. and Burgeson, R.E. (1992) The dermalepidermal junction of human skin contains a novel laminin variant. Biol. Chem., 119, 695– 703. Marinkovich, M.P., Taylor, T.B., Keen, B.R., Burgeson, R.E. and Zone, J.J. (1996) LAD-1, the linear IgA bullous dermatosis autoantigen, is a novel 120-kD anchoring filament protein synthesized by epidermal cells. J. Invest. Dermatol., 106, 734–738. Mayer, U., Pöschl E, Gerecke, D.R., Wagman, D.W., Burgeson, R.E. and Timpi, R. (1995) Low nidogen affinity of laminin-5 can be attributed to two serine residues in EDF-like motif γ21114. FEES Lett., 365, 129–132. McGrath, J.A., Darling, T., Gatalica, B., Poula-Gubo, G., Hintner, H., Christiano, A.M., Yamsey, K. and Uitto, J. (1996) A homozygous deletion mutation in the gene encoding the 180-kDa bullous pemphigoid antigen (BPAG2) in a family with generalized atrophic benign epidermolysis bullosa. J. Invest. Dermatol., 106, 771–774. McGrath, J.A., Gatalica, B., Christiano, A.M., Li, K., Owaribe, K., McMillan, J.R., Eady, R.A.J. and Uitto, J. (1995) Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa. Nat. Genet., 11, 83–86. Mclean, W.H.I., Pulkkinen, L., Smith, F.J.D., Rugg, E.L., Lane, E.B., Bullrich, F., Burgeson, R.E., Amano, S., Hudson, D.L. and Owaribe, K. et al. (199) Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organisation. Genes Dev., 10, 1724–1735.


31

Mutasim, D.F., Takahashi, Y., Labib, R.S., Anhalt., G.J., Patel, H.P. and Diaz, L.A. (1985) A pool of bullous pemphigoid antigen(s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex. J. Invest. Dermatol., 84, 47–53. Niemi, K.-M., Sommer, H., Kero, M., Kanerva, L. and Haltia, M. (1988) Epidermolysis bullosa simple associated with muscular dystrophy with recessive inheritance. Arch. Dermatol., 124, 551–554. Niessen, C.M., Cremona, O., Daams, H., Ferraresi, S., Sonnenberg, A. and Marchisio, P.C. (1994) Expression of the integrin α6β4 in peripheral nerves: localization in Schwann and perineural cells and different variants of the β4 subunit. J. Cell Sci., 107, 543–552. Niessen, C.M., Hogervost, F., Jaspars, L.H., De Melker, A.A., Delwel, G.O., Hulsman, E.H.M., Kuikman, I. and Sonnenberg, A. (1994) The α6β4 integrin is a receptor for both laminin and kalinin. Exp. Cell Res., 211, 360–367. Niessen, C.M., Van der Raaij-Heimer L.M.H., Hulsman, E.H.M., Van der Neut, R., Jonkman, M.F. and Sonnenberg, A. (1996) Deficiency of the integrin β4 subunit in junctional epidermolysis bullosa with pyloric atresia: consequences for hemidesmosome formation and adhesion properties. J. Cell Set., 109, 1695–1706. Owaribe, K., Nishizawa, Y. and Franke, W.W. (1991) Isolation and characterisation of hemidesmosomes from bovine corneal epithelium. Exp. Cell Res., 192, 622–630. Pulkkinen, L., Christiano, A.M., Airenne, T., Haakana, H., Tryggvason, K. and Uitto, J. (1994) Mutations in the γ2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa. Nat. Genet., 6, 293–289. Pulkkinen, L., Smith, F.J.D., Shimizu, H., Murata, S., Yaoita, H., Hachisuka, H., Nishikawa, T., Mclean, W.H.I, and Uitto, J. (1996) Homozygous deletion mutations in the plectin gene (PLEC1) in patients with epidermolysis bullosa simplex associated with late-onset muscular dystrophy. Human molecular Genetics, 5, 1539–1546. Ridelle, K.S., Green, K.J. and Jones, J.C.R. (1991) Formation of hemidesmosomes in vitro by a transformed rat bladder cell line. J. Cell Biol., 112, 159–168. Rouselle, P., Lunstrum, G.P., Keene, D.R. and Burgeson, R.E. (1991) Kalinin: an epithelium-specific basement membrane adhesion molecule that is a components of anchoring filaments. J. Cell Biol., 114, 567–576. Ryan, M.C., Tizard, R., VanDevanter, D.R. and Carter, W.G. (1994) Cloning of the lam A3 gene encoding the α3 chain of the adhesive ligand epiligin. J. Biol. Chem., 269, 22779– 22787. Sawamura, D., Ki, K., Nomura, K., Sugita, Y., Christiano, A.M. and Uitto, J. (1991) Bullous pemphigoid antigen: cDNA cloning, cellular expression and evidence for polymorphism of the human gene. J. Invest. Dermatol., 96, 908–915. Schwartz, M.A., Orawibe, K., Kartenbeck, J. and Franke, W.W. (1990) Desmosomes ans hemidesmosomes: constitutive molecular components. Annu. Rev. Cell Biol., 6, 461–491 Seiffert, G.J., Lawson. and Wiche, G. (1992) Immunolocalisation of the intermediate filament associated protein plectin at focal contact and actin stress fibres. Eur. J. Cell Biol. ,59, 138–147. Skalli, O., Jones, J.C.R. and Gagescu, R.D. (1994) IFAP 300 is common to desmosomes and hemidesmosomes and is a possible linker of immediate filaments to these junctions. J. Cell Biol., 125, 159–170. Smith, F.J.D., Eady, R.A.J., Leigh, I.M., McMillan, J.R., Rugg, E.L., Kelsell, S.P., Bryant, S.P., Spurr, N.K., Geddes, J.F., Kirtschig, G., Milana, G., de Bono, A.G., Owaribe, K., Wiche, G., Pulkkinen, L., Uitto, J., McLean, W.H.I, and Lane, E.B. (1996) Plectin deficiency results in muscular dystrophy with epidermolysis bullosa. Nat. Genet., 13, 450– 457. Sonnenberg, A. and Linders, C.T.J. (1990) The α6β4 protein complexes: tissue distribution and biochemical properties. J. Cell Sci., 96, 207–217. Sonnenberg, A., Calafat, J., Janssen, H., Daams, H., van der Raan-Helmer, L.M.H., Baker, J., Falcioni, R., Kennel, S.J., Aplin, J.D., Loizidou, M. and Garrod, D. (1991) Integrin α6β4 complex is located in hemidesmosomes suggesting major role in epidermal cellbasement membrane adhesion. J. Cell Biol., 113, 907–917. Sonnenberg, A., De Melker, A.A., Martines de Valesco, A.M., Janssen, H., Calafat, J. and Niessen, C.M. (1991) Formation of hemidesmosomes in cells of a transformed murine mammary tumour cell line and mechanisms involved in adherance of these cells to laminin and kalinin. J. Cell Sci., 106, 1083–1102.

32


Sorokin, L., Sonnenberg, A., Aumailey, M., Timpl, R. and Ekblom, P. (1990) Recognition of the laminin E8 cellbinding site by an integrin possessing the α6 subunit is essential for epithelial polarization in the developing kidney tubules. J. Cell Biol., 111, 1265–1273. Spinardi, L., Ren, Y.L, Saunders, R. and Giancotti, F.G. (1993) The β4 subunit cytoplasmic domain mediates the interaction of α6β4 integrin with the cytoskeleton of hemidesmosomes. Mol. Biol. Cell, 4, 871–884. Spinardi, L., Einheber, S., Cullen, T., Milner, T.A. and Giancotti, F.G. (1995) A recombinant tail-less integrin β4 subunit disrupts hemidesmodomes, but does not supress α6β4-mediated cell adhesion to laminins. J. Cell Biol., 129, 473–487. Stanley, J.R. (1995) Autoantibodies against adhesion molecules and structures in blistering skin diseases. J. Exp. Med., 181, 1–4. Stappenback, T.S. and Green, K.J. (1992) The desmoplakin carboxyl terminus co-aligns with and specifically disrupts intermediate filament networks when expressed in cultured cells. J. Cell Biol., 116, 1197–1209. Stepp, M.A., Spurr-Michaud, S., Tisdale, A., Elwell, J. and Gipson, I.K. (1990) α6β4 integrin heterodimer is a component of hemidesmosomes. Proc. Natl Acad. Sci. USA, 87, 8970– 8974. Sutherland, A.E., Calerco, P.G. and Damsky, C.H. (1993) Developmental regulation of integrin expression at the time of implantation in the mouse embryo. Development, 119, 1175– 1186. Suzuki, S. and Naitoh, Y. (1990) Amino acid sequence of a normal integrin β4 subunit and primary expression of the mRNA in epithelial cells. EMBO J., 9, 757–763. Svitkina, T.M., Verkhovsky, A.B. and Borisy, G.G. (1996) Plectin sidearms mediate interaction of immediate filaments with microtubules and other components of the cytoskeleton. J. Cell Biol., 135, 991–1007. Tamura, R.N., Rozzo, L., Starr, L., Chambers, J., Reichardt, L.F., Cooper, H.M. and Quaranta, V. (1990) Epithelial integrin α6β4: Complete primary structure of α6 and variant forms of β4. Cell Biol., 111, 1593–1604. Tanaka, T., Korman, N.J., Shimizu, H., Eady, R.A.J., Klaus-Kovtun, V., Cehrs, K. and Stanley, J. (1990) Production of rabbit antibodies against carboxy-terminal epitopes encoded by bullous pemphigoid cDNA. J. Invest. Dermatol., 94, 617–623. Tanaka, T., Parry, D.A.D., Klaus-Kovtun, V., Steinert, P.M. and Stanley, J.R. (1991) Comparisons of molecularly cloned bullous pemphigoid antigen to desmoplakin I confirms that they define a new family of cell adhesion junction plaque proteins. J. Biol Chem., 266, 12555–12559. Tang, H.-Y., Chaffotte, A.-F. and Thacher, S.M. (1996) Structural analysis of the predicted coiled—coil rod domain of the cytoplasmic bullous pemphigoid antigen (BPAG1). J. Biol. Chem., 271, 9716–9722. Tidman, M.J. and Eady, R.J. (1986) Hemidesmosome heterogenity in junctional epidermolysis bullosa revealed by morphometric analysis. J. Invest. Dermatol., 86, 51–56. Uematsu, J., Nishizawa, Y., Sonnenberg, A. and Owaribe, K. (1994) Demonstration of type II hemidesmosomes in a mammary gland epithelial cell line, BMGE-H. J. Biochem., 115, 469–476. Vailly, J., Pulkkinen, L. Christiano, A.M., Tryggvason, K., Uitto, J., Ortonne, J.P. and Meneguzzi, G. (1995) Identification of a homozygous exon-skipping mutation in the LAMC2 gene in a patient with Herlitz’s junctional epidermolysis bullosa. J. Invest. Dermatol., 104, 434–437. Van der Neut, R., Krimpenfort, P., Calafat, J., Niessen, C.M. and Sonnenberg, A. (1996) Epithelial detachment due to absence of hemidesmosomes in integrin β4 null mice. Nat. Genet., 13, 366–369. Vidal, F., Aberdam, D, Miguel, C., Christiano, A.M., Pulkkinien, L., Uitto, J., Ortonne, J.P. and Meneguzzi, G. (1995) Integrin β4 mutations associated with junctional epidermolysis bullosa with pyloric atresia. Nat. Genet., 10, 229–324. Weiss, P. and Ferris, W. (1954) Electronmicrographs of larval amphibian epidermis. Exp. Cell Res., 6, 546–549. Wiche, G., Beckor, B., Luber, K., Weitzor, G., Castannon, M.J., Haudtman, R., Stravowac and Stewart, M. (1991) Cloning and sequencing of rat plectin indicates a 466-kDa polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil. J. Cell Biol., 114, 83–89. Wiche, G., Krepler, R., Artlieb, U., Pytela, R. and Denk, H. (1983) Occurrence and localization of plectin in tissues. J. Cell Biol., 97, 887–901.


33

Wiche, G., Krepler, R., Artlieb, U., Artlieb, U., Pytela, R. and Aberer, W. (1984) Identification of plectin in different human cell types and immunolocalization at epithelial cell surface membrane. J. Cell Biol., 155, 43–49. Xia, Y., Gil, S.G. and Carter, W.G. (1995) Anchorage mediated by integrin α6β4 to laminin 5 (epillgrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein. J. Cell Biol., 132, 727–740. Yang, Y., Dowling, J., Yu, Q.C., Kouklis, P., Cleveland, D.W. and Fuchs, E. (1996) An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell, 86, 655–665.

NOTES ADDED IN PROOF Since this review was submitted, a number of important developments relating to our understanding of molecular interactions in hemidesmosomes gave taken place. Beginning with the anchoring fibrils and progressing through the structure to the keratin filaments, these are as follows. The noncollagenous NC1 domain of type VII collagen, the major component of anchoring fibrils, has been produced in recombinant form and shown to possess binding affinity for type IV collagen, fibronectin, type I collagen and laminin 5/ 6 complex (Chen et al., 1997). Such multiple interactions are consistent with the role of type VII collagen in dermal-epidermal adhesion. Monomeric laminin 5 (LN5) has been shown to react with NC1 domain of type VII collagen via the NH2 terminal region of its β3 and/or γ2 chains (Rouselle et al., 1997). This work suggests that anchoring filaments contain monomeric laminin 5 and that LN5 complexes with LN6 and 7 are located in inter-hemidesmosomal regions of the basement membrane. It is also consistent with location of the NH2-domain of LN5 in the lamina densa and the COOH-domain adjacent to the plasma membrane. A recently identified protein, fibulin-2, has been shown to bind to a specific nine amino acid sequence in the LN5 γ2 chain and also to bind LN1 (Utani et al., 1997). This is important for linking LN5 to other components of the basement membrane. Immunogold labelling with bullous pemphigoid and cicatricial pemphigoid sera shown that BP 180 epitopes for the former lie within the lamina lucida and for the latter within the lamina densa (Bedane et al., 1997). This suggests that BP180 has an extended configuration with its COOH-terminus in the laminia densa. The study also supports the hypothesis that BP180 contributes to anchoring filaments (see also Masanuga et al., 1997). Borradori et al. (1997) showed that localization of BP180 to hemidesmosomes is mediated by a segment of the cytoplasmic domain that spans 265 amino acids at the NH2 terminus, and that this localization is dependent on sequences in the cytoplasmic domain of β4 integrin. Subsequently, Borradori et al. (1998) showed that 36 amino acid deletion from the BP180 NH2 terminal region prevented localization to hemidesmosomes, and further showed that the subcellular localization of BP230 appears to be determined by BP180. Both Gagnoux-Palacios et al. (1997) and Aho and Uitto (1998) have shown that the second Type III fibronection repeat region of the β4 integrin cytoplasmic domain appears to be involved in interaction with BP180. A number of recent papers have demonstrated interaction between the cytplasmic domain of β4 integrin and HD1/ plectin (Sanchez-Aparicio et al., 1991; Niessen et al., 1997a,b; Gagnoux-Palacios et al., 1997; Resniczek et al., 1998). Niessen et al. (1997a) show association between recombinant β4 and HD1, and that β4 appears to be involved in localizing HD1 to hemidesmosomes. Niessen et al. (1997b) conclude that a region of the β4 tail that includes the second fibronection III repeat and a 27 amino acid portion of the connecting segment is involved in targeting α6β4 to hemidesmosomes and in localizing HD1/plectin. Plectin (—/—) mice die shortly postnatally with severe skin blistering and show significantly reduced numbers of hemidesmosomes, resembling the skin of patients with EBS-MD (Andra et al., 1997). McMillan et al. (1998) show that association of keratin filaments with hemidesmosomes is significantly reduced. In addition to the above, numerous disease associated genetic defects of the hemidesmosome complex have been reported. Unfortunately these cannot be detailed in this short note. Reviews of the

34


hemidesmosome-basement membrane region have also appeared (Burgeson and Christiano, 1997; Jones et al., 1998; Pulkkinen and Uitto, 1998). REFERENCES Aho, S. and Uitto, J. (1998) Direct interaction between the intracellular domains of bullous pemphigoid antigen 2 (BP180) and beta 4 integrin, hemidesmosomal components of basal keratinocytes. Biochem. Biophys. Res. Commun., 243, 694–699 Andra, K., Lassmann, H.P., Bittner, R., Shorny, S., Fassler, R., Prost, F. and Wiche, G. (1997). Targeted inactivation of lectin reveals essential function in maintaining the integrity of skin, muscle and heart cytoarchitecture. Genes. Dev., 11, 3143–3156. Bedane, C., McMillan, J.R., Balding, S.D., Bernard, P., Prost, C., Bonnetblanc, J.M., Diaz, L.A., Eady, R.A. and Giudice, G.J. (1997) Bullous pemphigoid and cicatricial pemphigoid autoantibodies react with ultrastructurally separable epitopes on the BP180 ectodomain: evidence that BP180 spans the lamina lucida. J. Invest. Dermatol., 108, 901–907. Borradori, L., Koch, P.J., Niessen, C.M., Erkeland, S., van Leusden, M.R. and Sonnenberg, A. (1997) The localization of bullous pemphigoid antigen 180 (BP180) in hemidesmosomes is mediated by its cytoplasmic domain and seems to be regulated by the beta4 integrin subunit. J. Cell Biol., 136, 1333–1347. Borradori, L., Chavanas, D., Schaapveld, R.Q., Gagnoux- Palacios, L., Calafat, J., Meneguzzi, G. and Sonneberg, A. (1998) Role of the bullous pemphigoid antigen 180 (BP180) in the assembly of hemidesmosomes and cell adhesion —reexpression of BP180 in generalized atrophic benign epidermolysis bullosa keratinocytes. Exp. Cell. Res., 239, 473–476. Burgeson, R E. and Christiano, A.M. (1997) The dermal-epidermal junction. Curr. Opin. Cell. Biol., 9, 651–658. Chen, M., Marinkovich, M., Veis, A., Cai, X., Rao, C.N., O’Toole, E.A. and Woodley, D.T (1997) Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components. A potential role in epidermal-dermal adherence in human skin. J. Biol. Chem., 272, 14516–14522. Gagnoux-Palacios, L., Gache, Y., Ortonne, J.P. and Meneguzzi, G. (1997) Hemidesmosome assembly assessed by expression of a wild-type integrin beta 4 cDNA in junctional epidermolysis bullosa keratinocytes. Lab. Invest., 77, 459–468. Jones, J.C.R., Hopkinson, S.B. and Goldfinger, L.E. (1998) Structure and assembly of hemidesmosomes. BioEssays, 20, 488–494. Masunaga, T., Shimizu, H., Yee, C., Borradori, L., Lazarova, Z., Nishikawa, T. and Yancey, K.B. (1997) The extracellular domain of BPAG2 localizes to anchoring filaments and its carboxyl terminus extends to the lamina densa of normal human epidermal basement membrane. J. Invest. Dermatol., 109, 200–206. McMillan, J.R., McGrath, J.A., Tidman, M.J. and Eady, R.A. (1998) Hemidesmosomes show abnormal association with the keratin filament network in junctional forms of epidermolysis bullosa. J. Invest. Dermatol., 110, 132–137. Niessen, C.M., Hulsman, E.H., Oomen, L.C., Kuikman, I. and Sonnenberg, A. (1997b) A minimal region on the integrin beta4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells. J. Cell Set., 110, 1705–1716. Niessen, C.M., Hulsman, E.H., Rots, E.S., Sanchez-Aparicio, P. and Sonnenburg, A. (1997) Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells. Mol. Biol. Cell, 8, 555–566. Pulkkinen, L. and Uitto, J. (1998) Hemidesmosomal variants of epidermolysis bullosa. Mutations in the alpha6beta2 integrin and the 180-kD bullous pemphigoid antigen/type XVII collagen genes. Exp. Dermatol., 7, 46–64. Rezniczek, G.A., de-Pereda, J.M., Reipert, S. and Wiche, G. (1998)Linking integrin alpha6beta4based cell adhesion to the intermediate filament cytoskeleton: direct interaction between the beta4 subunit and plectin at multiple molecular sites. J. Cell. Biol., 141, 209–225.


35

Rousselle, P., Keene, D.R., Ruggiero, F., Champliaud, M.F., Rest, M. and Burgeson, R.E. (1997) Laminin 5 binds the NC-1 domain of type VII collagen. J. Cell Biol., 138, 719– 728. Sanchez-Aparicio, P., Martinez de Velasco, A.M., Niessen, C.M., Borradori, L., Kuikman, I., Hulsman, E.H., Fassler, R., Owaribe, K. and Sonnenberg, A. (1997) The subcellular distribution of the high molecular mass protein, HD1, is determined by the cytoplasmic domain of the integrin beta 4 subunit. J. Cell Set., 110, 169–78. Utani, A., Nomizu, M. and Yamada, Y. (1997) Fibulin-2 binds to the short arms of laminin5 and laminin-1 via conserved amino acid sequences. J. Biol. Chem., 272, 2814–2820.

3. EPITHELIAL POLARITY AND SORTING PETER SCHEIFFELE and KAI SIMONS Cell Biology Programme, European Molecular Biology Laboratory, Postfach 102209, 69112 Heidelberg, Germany Fax: x-6221–387512; Tel: x-6221–387334

INTRODUCTION Epithelia form boundaries between different compartments in the body and allow vectorial flow of molecules and ions from one to the other side (Simons and Fuller, 1985). To serve the transport functions and to protect the cells from external environment, proteins and lipids have to be distributed polarized over the apical and basolateral plasma membrane domains. Generation and maintenance of surface polarity requires sorting and polarized transport of newly synthesized surface molecules (Rodriguez-Boulan and Powell, 1992; Simons, 1995). In recent studies determinants for intracellular sorting into the apical and basolateral pathways have been identified. The two routes appear to use fundamentally different mechanisms. Apical and basolateral determinants are not restricted to proteins in epithelial cells, but appear to function in all cell types to generate polarity. Therefore, epithelial cells provide a good tool to study general mechanisms of membrane trafficking which cannot be easily analysed in cells lacking obvious surface polarity. The aim of this review is to analyse the sorting mechanisms responsible for the polarized distribution of plasma membrane proteins in epithelia. MODEL SYSTEMS Sorting has been studied in different cell lines developing polarized monolayers in vitro when grown on a permeable filter support: Madin-Darby canine kidney (MDCK) cells, Caco-2 (human intestinal), Fischer rat thyroid (FRT) cells, LLCPK1(pig kidney), retinal pigment epithelial cells (RPE) and hepatocytes. One important outcome from these studies was that the different epithelial cells use different delivery routes for biosynthetic transport (Rodriguez-Boulan and Powell, 1992). While apical and basolateral proteins in MDCK and FRT cells are sorted mainly in the TGN, hepatocytes route plasma membrane proteins first to their basolateral surface and then transcytose the apical components. Caco-2 cells have an intermediate phenotype as apical proteins can use both, a direct apical route and an indirect route via the basolateral membrane (Rodriguez-Boulan and Powell, 1992). Therefore, the same protein can be differently targeted in different cell lines. Analysis of these variations might help to understand the importance and function of individual components of the sorting machinery. Results obtained in culture systems do not always, reflect the distribution of the protein in epithelial tissues, because surface distribution can vary for instance in case of the LDL-receptor or the transferrin-receptor. Proteins can also change polarity under different physiological conditions, a process which has been termed plasticity of epithelial polarity (Schwartz et al., 1985). As most published studies on protein sorting in epithelial cells have been carried out in the MDCK


37

cell line this review will in the following first describe signals and mechanisms identified there and then relate them to observations made in other systems. SORTING SIGNALS Secretory Proteins A large number of exogenous secretory proteins including lysozyme, rat growth hormone (rGH), and alpha-2-microglobulin which were assumed not to contain infomation for polarized sorting, has been expressed in the MDCK cell line (Table 1 ). Many of these proteins were secreted in a nonpolarized fashion into the apical and basolateral media, usually somewhat more to the basolateral side, probably following the bulk fluid flow (Gottlieb et al., 1986; Kondor-Koch et al., 1985). This demonstrated that polarized secretion in MDCK cells requires luminal signals. Polarized apical secretion has been detected for two endogenous glycoproteins of unknown function, gp80 and gp20 (Urban et al., 1987; Ullrich et al. ,1991), whereas the extracellular matrix components laminin and heparan-sulfate proteoglycan were secreted from the basolateral membrane (Caplan and Jamieson, 1987). While signals for basolateral secretion are not known, N-glycosylation has been shown to be involved in apical secretion (Scheiffele et al., 1995). The initial observation was that the endogenous glycoprotein gp80 was secreted in a nonpolarized fashion when N-glycosylation was inhibited with the drug tunicamycin (Urban et al., 1987). Rat growth hormone (rGH) is unglycosylated and only to 35% apically secreted. When one or two N-glycosylation sites were introduced, the glycosylated forms of the protein were targeted with increasing efficiency to the apical membrane (65 or 92% apical, respectively; Figure 1) (Scheiffele et al., 1995). Therefore, N-glycans function as a transplantable apical determinant. Analysis of previously performed studies revealed that N-glycans might be generally employed for apical secretion in epithelial cells. As observed for the singly and doubly glycosylated rGH the sorting efficiency will depend on the number of glycans present and possibly also on their accessibility. However, proteins such as gp20 which are not N- but O-glycosylated and the hepatitis B surface antigen which is not secreted as a conventional soluble protein but as a lipoprotein particle (Gonzalez et al., 1993) must carry other signals (Table 1). Treatment of cells with inhibitors of terminal glycosylation (deoxymannojirimycin or swainsonine) does not influence polarity of the apical protein gp80, therefore no complex type glycans are required, but rather the GlcNAc-Man-core (Parczyk and Koch-Brandt, 1991; Wagner et al., 1995). So far the role of N-glycans in the secretory pathway was thought to be restricted to folding and quality control in the ER (Helenius, 1994), the mannose-6-phosphate signal for lysosomal transport being the only exception. Now it appears that glycosylation is also used as a general determinant for apical transport (Fiedler and Simons, 1995). An open Table 1 Polarized secretion from MDCK cells. Apical secretion gp80 erythropoietin hCBG gp20

N-glycosyl.

Reference

required required required –

(Urban et al., 1987) (Kitagawa et al., 1994) (Musto, 1993) (Ullrich et al., 1991)

38


N-glycosyl.

Reference

HBsAg not required (Gonzalez et al., 1993) endoglucanase E + (Soole et al., 1992) NEP (sec) + (Corbeil et al., 1992) APN (sec) + (Vogel et al., 1992) plgAR (sec) + (Mostov et al., 1987) Thy-1 (sec) + (Powell et al, 1991) PLAP (sec) + (Brown et al., 1989) DAF (sec) + (Lisanti et al., 1989) NA (sec) + (Kundu and Nayak, 1994) Basolateral secretion Laminin + (Caplan and Jamieson, 1987) HSPG + (Caplan and Jamieson, 1987) gD-1 sec + (Lisanti et al., 1989) Nonpolar secretion growth hormone, lysozyme, α-2– (Gottlieb et al., 1986) microglobulin, Ig K-chain, prochymosin lysozyme – (Kondor-Koch et al., 1985) uteroglobin – (Vogel et al., 1993) cystatin C – (Vogel et al., 1992) HA (sec), VSV G (sec) + (Gonzalez et al., 1987) uPA – (Ragno et al., 1992) hGH – (Lisanti et al., 1989) ‘Sec’ indicates anchor-minus forms of transmembrane proteins. hCBP, human corticosteroid binding globulin; HBsAg, hepatitis B surface antigen; NEP, neutral endopeptidase; APN, aminopeptidase N; plgAR, polymeric immunoglobulin A-receptor; PLAP, placenta! alkaline phosphatase; DAF, decay accelerating factor; NA influenza virus neuraminidase; HSPG, heparansulfate proteoglycan; gD-1, herpes simplex virus glycoprotein D; HA, influenza virus hemagglutinin; VSV G, vesicular stomatitis virus glycoprotein; uPA, mouse urokinase-type plasminogen activator; hGH, human growth hormone.

question is how the glycosylation signal functions. Glycans might be used to change physical properties of proteins for sorting. Another possibility is that the carbohydrate moiety is directly recognized by a lectinlike sorting receptor which concentrates the protein in apical carrier vesicles (see below). Since also basolaterally secreted proteins are N-glycosylated they must carry dominant sorting determinants which would allow their segregation from the apical glycoproteins, either earlier in the secretory pathway or by binding to basolateral receptors with higher affinity than the putative apical lectin. The ectodomains of many membrane proteins have been expressed as anchor-less secreted forms to analyse their targeting information. For viral proteins the


39

Figure 1 Polarized secretion of singly and doubly glycosylated forms of rat growth hormone (GH). Secretion from stably transfected MDCK cells into the apical and basolateral (BL) media was analysed by western blotting and quantified by densitometric scanning (Scheiffele et al., 1995).

results were often inconsistent and even contradictory. However, for cellular proteins the ectodomain was often found to be secreted apically, independently of the polarity of the membrane protein (Table 1). The fact that all the analysed proteins were N- glycosylated could explain the presence of a apical sorting signal in their ectodomain (Simons, 1995). Basolateral Membrane Proteins Sorting signals of basolateral transmembrane proteins are located to their cytoplasmic tails (Hunziker et al., 1991). In many cases truncation of the cytoplasmic tail or mutation of crucial cytoplasmic amino acids results in apical sorting, probably facilitated by the carbohydrate signal in the ectodomain (Table 2, Figure 2). On the other hand the basolateral signal when transplanted onto an apical protein will always dominate over the apical signal present and result in basolateral sorting. Sorting signals therefore function hierarchically, the basolateral information being dominant (Matter and Mellman, 1994; Simons, 1995). Basolateral signals have been extensively characterised by mutagenesis. Two classes of signals have been defined, one related to clathrin-coated pit localisation motifs and another unrelated motif (Matter and Mellman, 1994). Many of the clathrin-coated pit related signals depend on tyrosine residues, but the requirements for amino acids surrounding the critical tyrosine are different for basolateral sorting Table 2 Sorting of tail-minus forms of basolateral transmembrane proteins. Reference Apical pIgAR

(Casanova et al., 1991)

40


Reference LDL-receptor (Hunziker et al., 1991) LAP (Prill et al., 1993) Fc-receptor (Hunziker et al., 1991) Igp 120 (Hunziker et al., 1991) EGF-receptor (Hobert and Carlin, 1995) Nonpolarized transferrin-receptor (Kundu and Nayak, 1994) VSV G (Thomas et al., 1993) ASGP-receptor (Geffen et al., 1993) N-cadherin* (Koller and Ranscht, 1996) *tail-minus N-cadeherin is basolaterally stabilized. plgAR, polymeric immunoglobulin A-receptor; LDL, low-densitylipoprotein; LAP, lysosomal acid phosphatase; EGF, epidermal growth factor; ASGP, asialoglycoprotein.

and endocytosis. Interestingly, replacement of the tyrosine in the cytoplasmic tail of lysosomal acid phosphatase with phenylalanine blocks endocytosis while basolateral sorting is not significantly affected (Prill et al., 1993). In other proteins signals for endocytosis and basolateral sorting are physically separated. The cytoplasmic tail of low density lipoprotein (LDL)-receptor contains two targeting determinants. The proximal one overlaps with the clathrin-coated pit signal and can be inactivated by replacement of the critical tyrosine by alanine (mutant A18; Figure 3). The resulting receptor is not endocytosed efficiently through clathrin-coated pits, but

Figure 2 Sorting of truncated basolateral transmembrane proteins (BL) in the trans-Golgi-network (TGN). As in the case of LDL-receptor and other proteins (table 2) deletion of the cytoplasmic signal results in apical (AP) sorting and removal of the membrane anchor results in apical secretion of the ectodomain.

the distal determinant is still sufficient for basolateral targeting. On the other hand truncation of the cytoplasmic tail leaving only 22 cytoplasmic residues (mutant CT22; Figure 3) results in an apical receptor which can be efficiently endocytosed via clathrin coated pits (Matter et al., 1992; Matter et al., 1994). The fact that basolateral sorting information can overlap with endocytic signals suggests that the machinery mediating sorting and clathrin-dependent endocytosis might be functionally similar. Secondary structure


41

Figure 3 Signals in the cytoplasmic tail of LDL-receptor and plgA-receptor. Crucial ami no acid residues are shown in bold. (A) The wild-type (WT) LDL-recepotr contains two basolateral determinants. In the mutant A18 the tyrosine residue of the clathrin-coated pit internalisation signal (underlined) is mutated to alanine. CT22 is a truncation mutant containing only the membrane proximal 22 amino acids of the cytoplasmic tail (Matter and Mellman, 1994). (B) Minimal cytoplasmic signal (truncation mutant stop670) and secondary structure of the polymeric Immunoglobulin A-receptor (plgA-R). The indicated mutation of the serine residue to aspartate (mimicking phosphorylation) results in apical sorting of the receptor (Aroeti et al., 1993).

analysis of some basolateral signals reveals additional similarities as they adopt a p-turn conformation which can also be found for endocytosis signals (Figure 3) (Aroeti et al., 1993; Vaux, 1992). Surface plasmon resonance studies have shown that the in vitro binding of the cytoplasmic adaptor complexes AP-1 and AP-2 to sorting signals correlates with sorting efficiency and endocytosis (Heilker et al., 1996). A more detailed analysis of signals carying information for sorting but not for endocytosis might reveal differences in binding of the Golgi adaptors versus the plasma membrane adaptor AP-2. Also the lipid environment will influence adaptor binding to cytoplasmic signals (Rapoport et al., 1997). However, so far basolateral markers have not been detected in exocytic clathrin-coated vesicles, and the coat components employed remain to be discovered. Apical Membrane Proteins GPI-anchored proteins The glycosylphosphatidylinositol (GPI)-anchor was the first apical signal to be identified. Addition of a GPI-anchor is sufficient to target proteins to the apical plasma membrane (Brown et al., 1989; Lisanti et al., 1989). When the cytoplasmic tail and transmembrane domain of basolateral proteins like vesicular

42


stomatitis virus glycoprotein (VSV G) or herpes simplex virus glycoprotein D was replaced by a GPIanchor, the chimeric proteins were transported apically. The apical polarity is not simply due to deletion of the cytoplasmic basolateral signal because also a nonpolarized secreted protein, human growth hormone, is directed apically when fused to a GPI-moiety (Lisanti et al., 1989). As the human growth hormone is an unglycosylated protein this also demonstrates that GPI-anchored proteins do not need N-glycosylation for apical polarity. Therefore, the GPI-anchor is sufficient for apical targeting and must somehow be recognized by the sorting machinery. GPI-anchored proteins enter a detergent-insoluble glycolipid-rich complex (DIG) during transport to the apical cell surface which contains a specific subset of lipids and transmembrane proteins (Brown and Rose, 1992; Melkonian et al., 1995). The inclusion into these complexes could reflect the recognition of the GPIanchor by the sorting machinery and will be discussed later. Apical transmembrane proteins Two different signals seem to mediate apical transport of transmembrane proteins. One, most likely involving glycosylation, is located in the ectodomain (Matter and Mellman, 1994; Simons, 1995). As mentioned above this could explain why secretory forms of apical and basolateral transmembrane proteins are secreted apically and deletion of the basolateral signal in a transmembrane protein mostly results in apical delivery (Figure 2). A second signal was proposed to be located in the transmembrane domain (TMD) of the type II transmembrane protein influenza virus neuraminidase (Kundu et al., 1996). A number of chimeric proteins assembled from the basolateral transferrin receptor and the apical neuraminidase were analysed. Truncation of the cytoplasmic signal in transferrin receptor yielded a nonpolarized transported molecule. When the TMD of the neuraminidase was introduced into this truncated receptor the protein was transported apically. It is not clear why the cytoplasmic deletion mutant of the transferrin receptor is not targeted apically as observed for many other basolateral transmembrane proteins (Table 2). This could suggest that N-glycosylation is not sufficient for apical targeting of some transmembrane proteins although it can mediate apical secretion of their ectodomains. Apical targeting of transmembrane proteins could generally need TMD determinants. According to this, proteins like the LDL-receptor would contain such a determinant which must be recessive versus the cytoplasmic basolateral signal. Another explanation would be that the carbohydrate moieties in the transferrin receptor are not sufficiently exposed to be recognized by the apical sorting machinery. Only analysis of unglycosylated transmembrane proteins will clarify this question and the involvment of other proteinaceus signals for apical sorting cannot be excluded. Like GPI-anchored proteins also several apical transmembrane proteins are found in DIGs, whereas basolateral transmembrane proteins are excluded from these complexes (Kurzchalia et al., 1992; Danielsen, 1995). In the following we will discuss the nature of these microdomains and then propose a model for sorting into the apical pathway of MDCK cells. SPHINGOLIPID-CHOLESTEROL RAFTS AND APICAL TRANSPORT The raft-hypothesis is based on the observation that not only apical proteins but also glycosphingolipids are sorted to the apical membrane (van Meer et al., 1987) (see below). Transport of the lipids and proteins was proposed to be coupled because GPI-anchored proteins and some apical transmembrane proteins could be recovered together with glycosphingolipids and cholesterol in a detergent-insoluble glycolipid-enriched complex (DIG) after extraction with Triton X-100 at 4°C. This was further supported by the fact that also


43

Figure 4 Model for the assembly of raft lipids. Sphingomyelin and glycosphingolipids are enriched in the exoplasmic leaflet and glycerolipids in the cytoplasmic leaflet. Cholesterol fills space under the large headgroups of sphingolipids or extends interdigitating fatty acyl chains in the opposite leaflet (Simons and Ikonen, 1997).

apical transport vesicles contain a detergent-insoluble subcomplex (Brown and Rose, 1992; Kurzchalia et al., 1992; Skibbens et al., 1989). Evidence and a model for the structure of sphingolipid-cholesterol rafts has been recently summarized (Simons and Ikonen, 1997). Briefly, glycosphingolipids and cholesterol would form microdomains and thereby create a lateral organisation of cellular membranes. Inclusion of lipids into the microdomain would be favoured by long saturated fatty acid chains (Schroeder et al., 1994). As glycosphingolipids have large headgroups, their lateral association would tend to create voids in the exoplasmic leaflet of the membrane which could be filled with cholesterol molecules (Simons and Ikonen, 1997) (Figure 4). These sphingolipidcholesterol assemblies would be small and dynamic, and float like rafts in the surrounding membrane consisting of glycerophospholipids and (“non-raft”) cholesterol (Harder and Simons, 1997; Simons and Ikonen, 1997). GPI-anchored proteins like placental alkaline phosphatase (PLAP) can directly associate with the rafts by their two mostly saturated fatty acid chains in the phosphatidylinositol (C16:0 and C18:0) (Schroeder et al., 1994; Redman et al., 1994). For the apical transmembrane protein influenza virus hemagglutinin (HA) bulky hydrophobic amino acids in the transmembrane domain (TMD) were shown to be crucial for association with the rafts. In both cases cellular proteins were not required for binding to raft lipids, therefore this interaction is completely encoded in the protein (Schroeder et al., 1994; Scheiffele et al., 1997). Whether the TMD determinant of HA also functions in apical sorting remains to be shown. However, the apical TMD signal in the influenza virus neuraminidase (NA) also seems to mediate raft association (Kundu et al., 1996). Therefore, TMD determinants might be generally used for apical sorting by intercalating proteins directly into the lipid environment which recruits the apical sorting machinery.

44


The proposed model suggests a crucial role for cholesterol in rafts. Recent experiments demonstrated that cholesterol is required for association of HA and GPI-anchored proteins to rafts (Scheiffele et al., 1991; Hanada et al., 1995). Cholesterol depletion by a steroid binding cyclodextrin inhibits apical transport of HA and causes missorting of HA to the basolateral membrane domain whereas basolateral transport of VSV G is not significantly affected (Keller and Simons, 1998). This establishes the importance of sphingolipidcholesterol rafts for the apical pathway in epithelial cells. One problem is that not all apical proteins are found in DIGs (Danielsen, 1995; Arreaza and Brown, 1995). Therefore, direct and tight raft association by a TMD determinant cannot be the only mechanism for apical sorting. It seems most likely that similarly as for apical secretion also the N-glycosylation in the ectodomain contributes to apical targeting (Scheiffele et al., 1995). Some proteins might exhibit a low or no affinity to rafts, but they would associate to the apical sorting machinery by their carbohydrates. Other proteins (glycosylated or unglycosylated) could bind with high affinity to rafts by their TMDs. Therefore both signals would not be strictly required but could act cooperatively. However, another possibility is that also a raft independent pathway to the apical surface exists. VIP36, a leguminous lectin homologue, has been identified in TGN-derived vesicle preparations (Fiedler et al., 1994). The protein belongs to a new family of intracellular lectins including ERGIC53 (human), Emp47p and YK24 (yeast) which share about 20% sequence identity with each other and with members of the leguminous lectin family (Fiedler and Simons, 1994) (Schröder et al., 1995). While ERGIC53 is localized to the ER-Golgi-intermediate compartment (Schweizer et al., 1988), VIP36 resides at steady state in the Golgi-complex and might recycle between the Golgi-complex and the plasma membrane (Fiedler et al., 1994). VIP36 can also be recovered in detergent-insoluble complexes, but in contrast to other raft associated proteins its interaction is strongly pH-dependent as it is weak at neutral pH and much stronger at pH 6.2 (Scheiffele and Simons, unpublished)—approximately the pH of the TGN (Anderson and Orci, 1988). We propose that VIP36 functions as a clustering factor which binds to carbohydrate residues of glycoproteins in the TGN, promoted by the high local concentration and the chemical conditions. The binding would increase the affinity of VIP36 and the cargo to the sphingolipid-cholesterol rafts. VIP36 is an oligomer and could form larger assemblies of cargo molecules in rafts which could then bud off as apical vesicles. As the raft association of VIP36 is strongly pH dependent, the whole complex will disassemble after arrival at the plasma membrane to release cargo proteins and allow recycling of VIP36 and other components of the apical targeting machinery back to the Golgi-complex. The hypothesised function of VIP36 would be to cluster glycoproteins in the TGN. Interestingly, antibody clustering of raft proteins on the cell surface has been demonstrated to concentrate them in a raft lipid environment and also to increase their detergent insolubility (Harder et al., 1998). Similarly, VIP36 clustered on the cell surface by antibodies is partially recruited to caveolae which can be conceived as large immobilized raft domains (Fiedler et al., 1994). This antibody clustering effect might mimic the recruitment of VIP36 to caveolin-1-containing patches of the TGN which will form the apical transport vesicles. The observations made for clustered proteins also suggest that oligomerisation and multivalency are important parameters for raft association in vivo (Harder and Simons, 1997). TRANSCYTOSIS Besides the direct route from the TGN to the apical surface also an indirect transcytotic route exists which has been well characterised for the polymeric IgA receptor (plgAR) (Mostov et al., 1992). The protein is first targeted from the TGN to the basolateral plasma membrane by its cytoplasmic sorting signal. The receptor then is endocytosed and delivered to basolateral endosomes where a serine residue in the cytoplasmic tail of


45

the receptor is phosphorylated (Casanova et al., 1990). Subsequently, the protein is directed to the apical recycling compartment, a tubular structure underlying the apical plasma membrane, and finally reaches the apical surface (Apodaca et al., 1994; Barroso and Sztul, 1994). The function of the phosphorylation event is most likely to inactivate the basolateral signal which would allow the recessive apical signal in the ectodomain to come into play and target the protein apically. This is supported by the fact that a serine to aspartate mutant (with the aspartate mimicking the phosphorylation, see Figure 3) is already in the TGN directed to the apical membrane (Aroeti and Mostov, 1994). Therefore, transcytosis does not appear to require specific signals, but rather the inactivation of the basolateral signal in endosomes to reveal the recessive apical sorting information in the ectodomain. This scenario implies that the sorting signals functioning in the TGN can be also decoded in endosomes (see below). Regulation of membrane trafficking has been analysed using different inhibitors and activators of protein kinases and trimeric G proteins. For example activation of Gs by aluminium tetrafluoride causes stimulation of apical but inhibition of basolateral transport (Pimplikar and Simons, 1993). Also stimulation of the expected downstream molecules adenylate cyclase and protein kinase A increased apical TGN-surface transport and at the same time stimulated apical transcytosis (Hansen and Casanova, 1994; Pimplikar and Simons, 1994; Mostov and Cardone, 1995). As both pathways respond in the same way to pharmacological treatments they might be similarly regulated. One the other hand apical transport does not appear to pass through the apical recycling compartment, but is thought to occur direct. Also the machinery for fusion of transcytotic vesicles with the apical plasma membrane seems to be different than that of TGN-derived vesicles and does not appear to involve raft formation (see below). Future work will have to establish the similarities and differences between the two (direct and indirect) apical pathways. LIPID SORTING The apical and basolateral plasma membrane domains have different lipid compositions which are achieved by vectorial transport of lipids. Intermixing of the lipids in the outer leaflet of the lipid bilayer is prevented by the tight junctions (van Meer et al., 1986). Analysis of lipid sorting has received considerable attention, also because according to the raft hypothesis it should be prerequisite for protein sorting. Initially, the lipid contents of viruses budding in a polarized fashion from MDCK cells was determined. When the phospholipid compositions of the basolateral vesicular stomatitis viruses (VSV) and the apical fowl plague viruses (FPV) produced in polarized MDCK cell monolayers were compared, VSV showed an enrichment in sphingomyelin, phosphatidylinositol and phosphatidylcholine whereas FPV was enriched in phosphatidylserine and phosphatidylethanolamine. As the phospholipid compositions of the viruses produced in unpolarized MDCK cells were very similar to each other, the detected differences do reflect the composition of the apical and basolateral plasma membrane domain rather than preferences of lipid incorporation by the different viruses (van Meer and Simons, 1982). Sorting of sphingomyelin and glycosphingolipids in the exocytic pathway has been analysed with the fluorescently labelled short-chain ceramide analog N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocapryl sphingosine (C6-NBD-ceramide) (Lipsky and Pagano, 1983). C6-NBD-ceramide can be introduced into cells at low temperature and is concentrated in the Golgi complex. Here it will be converted to C6-NBDsphingomyelin and C6-NBD-glucosylceramide which then move to the plasma membrane (Lipsky and Pagano, 1983). Recently, it was demonstrated that a multidrug resistance protein localized to the apical surface of polarized epithelial cells can translocate these short-chain lipids from the cytosol through the plasma membrane (van Helvoort et al., 1996). Therefore, part of the apical NBD-signal measured, can be independent of vesicular transport. This makes the interpretation of previous studies employing C6-NBD-

46


ceramide difficult. However, also in permeabilized cells C6-NBD-glycosylceramide was preferentially transported to the apical membrane when cytosolic short-chain lipids were captured with BSA (Kobayashi et al., 1992). The other problem with C6-NBD-ceramide is that it is lacking the long and saturated fatty acid chains, but instead carries the NBD-group after a C6-chain. Thereby, most of the raft character of the molecule is removed and C6-NBD-sphingomyelin indeed rather behaves like a bulk-membrane marker than like a raft lipid (Simons and Ikonen, 1997). C6-NBD-glycosylceramide might retain some raft lipid properties by its bulky headgroup, capable of interacting with other glycolipid headgroups. However, lipid polarity and sorting of raft lipids to the apical membrane of MDCK II cells is confirmed by the fact that also endogenous glycolipids such as the Forssman antigen are enriched in the apical plasma membrane (van Genderen et al., 1991). SORTING IN OTHER MODEL SYSTEMS Variant and Mutant MDCK Strains Two lectin-resistant cell lines derived from the parental MDCK cells have been obtained which retained the characteristic structural and functional epithelial polarity (Meiss et al., 1982). A ricin-resistant line (MDCKRCAR) carries a defect in an UDP-galactose transporter in the Golgi complex and therefore is unable to incorporate galactose and sialic acid into glycoproteins. Another concanavalin A-resistant line (MDCKConAR), with a so far undefined defect, transfers unusual core oligosaccharides on glycoproteins. Despite the defects in N-glycosylation both cell lines target gp80 apically, underlining that the apical N-glycan signal apparently does not require specific terminal sugar structures (see above) (Parczyk and Koch-Brandt, 1991; Wagner et al., 1995). Basolateral missorting in MDCK-RCAR cells was observed for the apical glycoprotein gp114. However, treatment with glycosylation inhibitors revealed no requirement for terminal sugar modifications in sorting (Le Bivic et al., 1993). All other apical proteins analysed so far are correctly sorted in MDCK-RCAR cells and in contrast to wild-type MDCK cells gp114 in MDCK-RCAR cells is efficiently endocytosed. This suggested that the observed missorting might be due to a mutation in gp114 creating a dominant basolateral signal rather than being a consequence of the aberrant N-glycosylation (Le Bivic et al., 1993). MDCK-ConAR cells transport some GPI-anchored proteins in an unpolarized fashion, but sort four out of five detectable endogenous GPI-anchored proteins, gp80, influenza virus hemagglutinin, and C6-NBDglycosylceramide normally to the apical membrane (Lisanti et al., 1990). Also the partially missorted GPIanchored proteins can associate with rafts (Zurzolo et al., 1994). It is not known whether GPI-anchored proteins, transmembrane proteins and secretory proteins generally use the same vesicles for surface delivery. One explanation for missorting of GPI-anchored proteins could be that their carrier vesicles are partially misstargeted or that some of the molecules can be accomodated in basolateral vesicles. Unpolarized delivery of a GPI-anchored protein is also observed in a special clone of MDCK cells, termed MDCK/J (Mays et al., 1995). In MDCK/J cells also C6-NBD-glycosylceramide and the Na+,K+ATPase are equally transported to the apical and basolateral membrane, but at steady state Na+,K+-ATPase is found in the basolateral membrane where it is stabilised by interaction with the membrane skeleton (Hammerton et al., 1991). This is in contrast to another clone termed MDCK/G which sorts GPI-anchored proteins and C6-NBD-glycosylceramide apically and Na+,K+-ATPase exclusively to the basolateral membrane. Treatment of MDCK/ G cells with Fumonisin B1, an inhibitor of sphingolipid synthesis, converts them to the same sorting phenotype as MDCK/J cells (Mays et al., 1995). This clearly demonstrates a requirement for lipids in polarized sorting. Na+,K+-ATPase appears to be excluded from


47

sphingolipid-cholesterol rafts which transport GPI-anchored proteins and C6-NBD-glycosylceramide. This example also illustrates the complexity of the sorting processes as in MDCK/J cells transport of GPIanchored proteins and C6-NBD-glycosylceramide is coupled, whereas some GPI-anchored proteins appear idependently sorted in MDCK-ConAR cells. However, as mentioned above, C6-NBD-glycosylceramide might not always reflect sorting of endogenous glycosphingolipids. Influenza virus hemagglutinin and gp80 are directly transported to the apical membrane, also in the clone MDCK/J cells (Mays et al., 1995). As suggested above this could mean that parallel pathways exist to the apical surface. FRT Cells The rat thyroid cell line FRT shows a very interesting sorting phenotype. In fully polarized cells proteins mainly use the direct route from the TGN to the cell surface as in MDCK cells (Zurzolo et al., 1992). One striking difference is, however, that FRT cells sort some GPI-anchored proteins preferentially to the basolateral plasma membrane domain (Zurzolo et al., 1993). Also C6-NBD-glycosylceramide is sorted basolaterally (Zurzolo et al., 1994). According to their detergent-solubility the GPI-anchored protein gD1DAF is not found associated with raft lipids, in contrast to MDCK and MDCK-ConAR cells where even basolaterally missorted GPI-anchored proteins are detergent-insoluble (Zurzolo et al., 1994). On the other hand other apical proteins as influenza virus HA, dipeptidyl peptidase IV and gp80 are transported with the same polarity in FRT as in MDCK cells (Zurzolo et al., 1992; Graichen et al., 1996). How could the sorting phenotype found in FRT cells be explained ? Sorting of the GPI-anchored proteins seems to follow the transport of NBD-GlcCer, but is diverged from the apical pathway which is assumed to use raft association and carbohydrates for sorting. Raft lipids in detergent-insoluble assemblies exist in FRT cells, but obviously the analysed GPI-anchored proteins cannot interact with rafts (Zurzolo et al., 1994). One explanation for this would be that the GPI-anchor added has a different structure; for example it could contain unsaturated fatty acids and therefore not partition into rafts. Indeed the GPI structure can differ in different cell lines (McConville and Ferguson, 1993) but has not yet been analysed in FRT cells. Another explanation would be that a protein factor is required to insert GPI-anchored proteins into rafts during biosynthetic traffic. Transfer of GPI-anchored PLAP in raft lipid containing liposomes results in detergent insolubility of the molecule (Schroeder et al., 1994). However, when isolated GPI-anchored proteins are transplanted into living cells incubation for prolonged times is required before they acquire detergent insolubility (van den Berg et al., 1995). This demonstrates that accomodating a GPI-anchored protein in the raft lipid environment is not a trivial process but might indeed need chaperone like cellular protein factors. One exciting observation was that FRT cells lack caveolin-1 (Zurzolo et al., 1994), a cholesterol binding protein implicated in vesicular transport and structural component of cell surface caveolae (Murata et al., 1995; Fra et al., 1995; Scheiffele et al., 1998). However, expression of caveolin-1 in FRT cells did not restore apical sorting or raft association of GPI-anchored proteins (Lipardi et al., 1998). Therefore, other or additional components could be lacking. While this problem still remains to be resolved it emphasizes again that apical components can be diverged into different routes. Caco-2 Cells One system where apical signals seem to be more stringently decoded than in MDCK cells is the human intestinal cell line Caco-2. Secretory proteins without signals are almost exclusively released in the basolateral medium which demonstrates that the capacity of the apical pathway is small (Rindler and Traber, 1988). In MDCK cells the same proteins are secreted in a nonpolarized fashion (Gottlieb et al., 1986).

48


However, a direct apical pathway for secretory proteins exists as gp80, the apical marker protein in MDCK cells, is secreted from the apical surface of Caco-2 cells (Appel and Koch-Brandt, 1994). The distribution of most membrane proteins at steady state in Caco-2 cells is the same as in MDCK cells. However, not all apical membrane proteins are transported via a direct route from the TGN, but they are first delivered to the basolateral membrane and then subsequently transcytosed (Matter et al., 1990; Le Bivic et al., 1990). GPIanchored proteins which have an optimal membrane anchor for raft association reach the apical membrane by the direct pathway and they have been shown to enter DIGs during transport (Garcia et al., 1993). Also sucrose-isomaltase which is partially insoluble can be transported by the direct route (Garcia et al., 1993). We speculate that proteins which can associate avidly with rafts will be transported via the direct route, whereas proteins which only loosly associate with rafts travel via the indirect route as they do not have sufficient affinity to the apical sorting machinery in the TGN. These proteins might be included without signals and tolerated as “stow-aways” in the basolateral vesicles (see also below). As the capacity of the basolateral route is much higher than that of the direct apical route this passive forward transport could be sufficient for effective surface delivery. An other explanation would be that apical and basolateral proteins are efficiently sorted in the TGN but that some apical vesicles dock and fuse with the basolateral membrane. However, as some proteins mainly travel along the direct route whereas others only use the indirect route this is not very likely. Hepatocytes Hepatocytes appear not to use a direct apical targeting route. Instead all proteins are first delivered to the basolateral membrane and apical components then transcytosed (Hubbard et al., 1989). So far no apical secretion from hepatocytes has been detected and also raft associated proteins like the GPI-anchored 5'nucleotidase are delivered to the apical membrane via an indirect route (Schell et al., 1992). As endocytosis of raft proteins from the basolateral membrane occurs only slowly, this creates considerable delay in apical surface delivery. Two scenarios can be imagined for this sorting phenotype: either all proteins are transported in the same carriers from the TGN to the basolateral plasma membrane or apical and basolateral proteins are packaged into different vesicles as in MDCK cells, but they are all targeted to the basolateral plasma membrane. This could be achieved by placing the docking and fusion machinery for both routes to the basolateral membrane. The involvement of different vesicle populations in basolateral transport has been suggested recently (Nickel et al., 1994), however, it is not yet understood if they separate apically or basolaterally directed cargo. RPE The retinal pigment epithelium (RPE) is derived from the neuroectoderm. The particularity of this cell type is that its apical surface is not free: microvilli and lamellipodia interact with photoreceptor outer segments directly and through the interphotoreceptor matrix (Zinn and Marmor, 1979). A stable cell line, termed RPEJ, has been created which forms polarized monolayers and resembles primary cultures in vitro (Nabi et al., 1993). Budding of influenza and vesicular stomatitis virus occurs with the same polarity as in MDCK cells, suggesting that the envelope proteins are sorted with the same polarity (Nabi et al., 1993). The absence of the interphotoreceptor matrix in cultured cells accounts for several differences in the steady state localisation of membrane proteins in vitro vs. in vivo (Gundersen et al., 1993) (see below).


49

DEVELOPMENT OF POLARITY During the establishment of cell surface polarity epithelial cells undergo dramatic changes. At areas of cellcell contacts adherens junctions, tight junctions, and desmosomes are formed which are crucial to build up a tight monolayer. Two factors appear to determine initial polarity: interactions with the substratum and cellcell contacts (Eaton and Simons, 1995). Single cells attached to a substratum exhibit polarity as they promote polarized apical and basolateral budding of influenza or vesicular stomatitis virus, respectively (Rodriguez-Boulan et al., 1983). Such polarized release of viral particles is also observed in absence of a substratum when cells are grown as small aggregates in suspension. Influenza viruses bud from the free surface whereas VSV preferentially buds from cell-cell contact sites (Rodriguez-Boulan et al., 1983). This demonstrates that cells are able to distinguish apical and basolateral components already in the initial stages of polarization. MDCK cells secrete extracellular matrix components from their basolateral side where also integrins for substratum adhesion are expressed (Caplan and Jamieson, 1987; Ojakian and Schwimmer, 1994). This allows cells in suspension culture to form cysts with the apical membrane outside and the basolateral inside, binding to the selfproduced matrix (Wang et al., 1990). Cell-cell contacts are mediated by cadherins (Kemler, 1992). In MDCK cells the basolateral E-cadherin forms Ca2+-dependent contacts with the neighbouring cells and thereby fixes the cytoskeleton (Gumbiner et al., 1988). After adsorption to the substratum MDCK cells in culture form E-cadherin mediated contacts. Already two hours later the apical protein gp135 is transported polarized to the apical pole (Wollner et al., 1992). Instead exclusive basolateral delivery of proteins occurs first 48 hours after the initial cell-cell contacts (Wollner et al., 1992). However, a polarized distribution of the basolateral proteins is achieved earlier by selective stabilization in the basolateral plasma membrane. It appears that interactions with the cytoskeleton facilitate early establishment of basolateral polarity, whereas first in fully polarized cells exocytosis occurs with high fidelity to the right target domain. When cells during polarization are cultured in medium with low Ca2+ concentrations (1–5 μM) apical polarization is maintained whereas no polarity of basolateral proteins can be achieved (Vega-Salas et al., 1987). This highlights the importance of the assembly of the cytoskeleton for the basolateral pathway. The organization of the cytoskeleton changes during polarization. In unpolarized MDCK cells most microtubules are emerging from the centrioles in the perinuclear region and extend their plus-ends to the cell periphery. After cell-cell contacts are established the two centrioles separate and move to the apical pole of the cells (Buendia et al., 1989). This process depends on the cell-cell contacts as disruption of the intercellular junctions with low Ca2+ medium causes the centrioles to move back to the center of the cell (Buendia et al., 1989). In fully polarized cells microtubules span the apical-basal axis with their minus ends underneath the apical membranes. These polar microtubular tracks are used for exocytosis and different motor proteins are involved in apical and basolateral transport (Lafont et al., 1994). The microtubules have an increased stability in the fully polarized cells and do not only emerge from centrioles. Consistently, two pools of γ-tubulin have been detected, one non-centriolar and another associated to centrioles (Meads and Schroer, 1995). E-cadherin expressing cells localize the ankyrin-spectrin membrane cytoskeleton to the basolateral membrane. Anchoring of Na+, K+-ATPase to spectrin is used to stabilize the protein selectively in the basolateral plasma membrane (Hammerton et al., 1991), and ectopic expression of E-cadherin in nonpolarized fibroblasts is sufficient to localize Na+, K+-ATPase to cell-cell contact sites (McNeill et al., 1990) (see also below). Similarly, the apical cytoskeleton can be used to localize an epithelial Na+-channel via a SH3 -domain interaction to the apical membrane (Rotin et al., 1994).

50


PLASTICITY OF EPITHELIAL POLARITY The polarity of proteins in vivo can change during differentiation or depending on growth conditions. A reversal of polarity can be induced by external cues like the electrical potential across the membrane and extracellular matrix components. For example the polarity of band 3, a Cl/HCO3 exchanger in intercalated epithelial cells of the renal collecting tubule, reverses when animals are fed an acid diet (Schwartz et al., 1985). This change in polarity can be mimicked in cultured cells depending on the density they are plated. It turned out that a 230 kDa extracellular matrix protein was capable of inducing the change of polarity (van Adelsberg et al., 1994). If this is achieved by domain specific stabilization or by including band 3 into different transport vesicles is not known. Also targeting of the LDL-receptor varies in different tissues. Using transgenic mice it has been demonstrated that basolateral delivery in hepatocytes depends on the same cytoplasmic signal as identified in cultured cells (Yokode et al., 1992). However, in kidney the protein was apically expressed (Pathak et al., 1990). The presence of the described hierarchically acting signals for polarized sorting might allow to modulate polarity. Basolateral signals could be inactivated by cell-specific post-translational modifications, thereby activating the recessive apical signal (Casanova et al., 1990; Simons, 1995). Such post-translational modifications could be induced by cell-cell signalling or extracellular cues as mentioned above. Cell-cell contacts and adhesion can give crucial cues for epithelial polarization. The cell adhesion molecule N-CAM is apically polarized in the RPE but found on the basolateral membrane in cultured cells. The contact with the neural retina which is lacking in vitro was suggested to be required for the apical localisation (Gundersen et al., 1993). Also Na+, K+-ATPase is restricted to the apical membrane of the RPE but is distributed in a nonpolarized fashion in RPE-J cells in vitro. Expression of E-cadherin results in basolateral accumulation of the ankyrin-spectrin cytoskeleton and thereby the Na+, K+-ATPase is also moved basolaterally. Concomitantly, expression of an ankyrin isoform is induced (Marrs et al., 1995). Therefore, cell adhesion molecules can be employed to determine epithelial polarity. As described before apical or basolateral pathways can be selectively influenced by regulatory components as trimeric G proteins and protein kinases (Mostov and Cardone, 1995) and therefore developmental regulation of polarity could also be achieved by stimulation of one pathway increasing its capacity. SORTING IN ENDOSOMES Sorting signals are not only decoded in the TGN. As obvious in the case of Caco2 cells, the basolateral endosomes are capable of distinguishing apical and basolateral proteins because apical proteins on the indirect route are efficiently transcytosed (Matter et al., 1990). Dissection of the signals present in the LDLreceptor demonstrated that the same signals mediating sorting in the TGN of MDCK cells also function in endosomes (Matter et al., 1993). When the distal signal in the cytoplasmic tail of the receptor was inactivated the weaker proximal signal mediated basolateral sorting and subsequently endocytosis (see Figure 3). From the basolateral endosomes the receptor was then efficiently transcytosed to the apical recycling endosome and plasma membrane. The endocytic pathways originating from the basolateral and apical surface meet in the apical recycling compartment (Odorizzi et al., 1996). From here transferrin receptors can be sorted back to the basolateral surface and apically recycling proteins are redirected to the apical plasma membrane. Also morphological studies by electron microscopy support the idea that endosomes are capable of sorting apical and basolateral components because morphologically different types of vesicles can bud from the compartment (Odorizzi et al., 1996). However, the main sorting event in the exocytic pathway of MDCK cells occurs in the TGN from where two distinct apical and basolateral vesicle


51

populations are released (Wandinger-Ness et al., 1990). Those vesicles have been postulated to be directly delivered to the respective plasma membrane domain, but recently evidence was provided that the basolateral transport occurs via endosomes. The transferrin receptor could be recovered before surface arrival from an endosomal fraction labeled with the fluid-phase marker horseradish peroxidase, whereas the (apical) GPI-anchored protein PLAP was excluded from this fraction (Futter et al., 1995). Secondly, after release from the TGN the basolateral asialoglycoprotein receptor H1 was transiently detected in an immunoisolated endosomal fraction using the mannose 6-phosphate receptor as a marker (Leitinger et al., 1995). The passage of the basolateral route through an acidic compartment has also been suggested when it was observed that weak bases like NH4Cl block basolateral secretion (Caplan and Jamieson, 1987). Under these conditions also nonpolarized secreted proteins like rat growth hormone and lysozyme are directed to the apical membrane (Scheiffele and Simons, unpublished), possibly because of a general block of vesicle budding from the basolateral endosomes (Aniento et al., 1996). The similarity of basolateral sorting signals and endocytosis signals further suggests that the clathrin-mediated endocytic pathway and the basolateral route could converge in endosomes. MACHINERY FOR DOCKING AND FUSION Not only the nature of the sorting signals differs in the two TGN-surface routes of MDCK cells. Dissection of the transport machinery has revealed further dramatic differences. Basolateral transport depends on the same machinery for docking and fusion as so far characterised for synaptic vesicles and ER-Golgi transport (Ikonen et al., 1995; Rothman, 1994). The involvement of NEM-sensitive factor (NSF), the soluble NSF attachment proteins (SNAPs) and the SNAP receptors (SNAREs) was demonstrated by sensitivity of basolateral delivery to NEM, to NSF antibodies and to neurotoxins which cleave and thereby inactivate SNAREs. Removal of rab proteins from cells, which might regulate the assembly of the SNARE complexes, also caused an inhibition of basolateral transport. On the other hand apical transport was insensitive to these treatments revealing different requirements for docking and fusion (Ikonen et al., 1995). The following considerations might explain the need for a different machinery in apical transport: The lipid composition assumed to reside in the apical vesicles is very different than that found in COP coated vesicles which use the NSF/SNAP/SNARE system (Brügger et al., 1997). Also apical proteins do not use cytoplasmic signals which in the NSF/SNAP/SNARE system are required to recruit coat proteins (Fiedler et al., 1996). Finally, the existence of two different principles for docking and fusion would strictly prevent delivery of the content of mistargeted vesicles with the wrong plasma membrane. But how is specific docking and fusion of apical vesicles achieved ? This process is so far not understood. However, a member of the annexin protein family, annexin 13b, has been isolated from MDCK cells which localizes to apical vesicles and the apical plasma membrane (Lafont et al., 1998). Annexins have been postulated to perform multiple functions, one of them being membrane fusion (Moss, 1992). As annexins are calcium-dependent phospholipid binding proteins and able to form oligomers (Luecke et al., 1995), one working model is that an annexin complex on the transport vesicle would bind a second complex attached to the plasma membrane. After this docking step fusion could occur, also mediated by the coupled annexins. While this model is still speculative it has been demonstrated that annexin 13b can associate to sphingolipid-cholesterol rafts independently of calcium (Lafont et al., 1998). Antibodies to annexin 13b and recombinant annexin 13b specifically block apical transport of HA whereas basolateral transport of VSV G is not affected (Lafont et al., 1998). Therefore, annexin 13b could indeed function in the apical pathway.

52


ANALOGY OF NEURONAL AND EPITHELIAL POLARITY Neurons are a striking example of polarized cells. Comparison of neuronal and epithelial polarity suggested that the same sorting signals and mechanism for transport could be used, with the axonal pathway representing an apical route and the dendritical a basolateral one (Rodriguez-Boulan and Powell, 1992; De Hoop and Dotti, 1993). In agreement with this hypothesis influenza and Semliki Forest viruses bud from the axons or the dendrites, respectively (Dotti and Simons, 1990), and GPI-anchored proteins are targeted preferentially to the axons (Dotti et al., 1991). Rab8, the GTPase regulating basolateral transport in MDCK cells, is involved in traffic to the dendrites (Huber et al., 1993) suggesting that also the machinery for transport is similar. Like the apical pathway, also axonal transport employs sphingolipid-cholesterol rafts. Thy-1, a GPI-anchored protein, and influenza virus hemagglutinin are found in DIGs during axonal transport (Ledesma et al., 1998). As hippocampal neurons in culture pass through different stages of polarity, sorting can be analysed and compared between premature and mature cells. After establishment of an axon stage three neurons sort dendritical proteins efficiently but deliver HA and Thy-1 unpolarized and fail to associate them with DIGs (Dotti and Simons, 1990; Ledesma et al., 1998). In fully mature stage 5 neurons HA and Thy-1 are sorted and found in DIGs. It is possible that the raft pathway is not or only in a premature form present in stage 3 neurons and therefore Thy1 and HA are delivered unsorted. Interestingly, the lack of DIG association in the stage 3 cells correlates with the presence of only low amounts of the raft lipid sphingomyelin which seems to be much more abundant in stage 5 cells (Ledesma et al., 1998). Sorting in neurons will not be identical to sorting in MDCK cells. As already indicated by the heterogeneity among different epithelial cell types, variations will occur. However, we expect that neurons, like epithelial cells, make use of two post-Golgi routes which are functionally similar to the apical and basolateral pathways (Simons et al., 1993). APICAL AND BASOLATERAL COGNATE ROUTES The two targeting pathways of polarized cells could be generated in two different ways. A new epithelial route could be induced upon differentiation to the polar phenotype or two previously existing routes could be diverged and focussed to different domains by reorganization of the cytoskeleton and establishment of the tight junctions (Rodriguez-Boulan and Powell, 1992). When signals for transport and the mechanisms for docking and fusion had been identified in the polarized cell system this provided the tools to distinguish the two pathways experimentally in nonpolarized cells. It turned out that surface transport of a basolateral protein was also signal mediated in nonpolarized fibroblasts as peptides encoding the basolateral signal of VSV G could specifically block its delivery (Müsch et al., 1996). Furthermore, docking and fusion of the basolateral marker in nonpolarized cells required the NSF/SNAP/SNARE machinery as in MDCK cells. Instead surface transport of the apical marker HA in nonpolarized cells was independent of these factors, but the protein was found associated with sphingolipid-cholesterol rafts (Yoshimori et al., 1996). Therefore, apical and basolateral cognate routes do exist in nonpolarized cells and function similarly as in epithelial cells. Why should cells with a continuous plasma membrane have two different routes to the surface ? The signals identified in polarized cells will allow directed surface delivery. It has been observed that some proteins are preferentially targeted to the leading edge of fibroblasts where they might be used for locomotion (Singer and Kupfer, 1986; Bretscher, 1996; Bretscher, 1992a). This polarized exocytosis on the biosynthetic pathway and from endosomes could provide components required for extension of the cell: new membrane and receptors for attachment to the extracellular matrix. It appears that the basolateral cognate route is used for this targeting step (Figure 5A, B) as so far all molecules identified are basolateral proteins:


53

the transferrin receptor (Hopkins et al., 1994), VSV G (Bergmann et al., 1983) and some integrins (Bretscher, 1992b). Interestingly, not all integrins are recycling efficiently. While α5β1 and α6β4 are circulating, α3β1 and α4β1 do not follow the same route (Bretscher, 1992b). This could be explained by the fact that α3β1 and α4β1 are found in big complexes with the detergent insoluble proteins CD9 and CD81 which should allow segregation from the basolateral cognate endocytic route (Berditchevski et al., 1996; Mannion et al., 1996; Shevchenko et al., 1997). When rab8, the GTPase regulating basolateral transport in epithelial cells, is expressed as constitutively active mutant in fibroblasts, the cells rearrange their cytoskeleton and form long extensions (Peränen et al., 1996). The basolateral cognate vesicles are targeted into these processes and accumulate their cargo in the tip. Like in the leading edge new membrane and the basolateral integrins are required for outgrowth and will now be directed to the place of their function. Also molecules on the biosynthetic route are transported in a polarized fashion. While VSV G is concentrated in the extensions, influenza virus HA is transported without preference (Peränen et al., 1996). Similar polarized exocytosis is observed in the budding yeast Saccharomyces cerevisiae. Polarity is developed by assembly of an asymmetric cytoskeleton which directs the secretory pathway to the side of bud formation (Drubin and Nelson, 1996). Secretion and addition of new membrane occurs preferentially at the bud. This is probably achieved by transporting the vesicles along the cytoskeleton (Drubin et al., 1993) and by placing the docking machinery to the growth site (TerBush and Novick, 1995). In analogy to the apical and basolateral cognate routes in flbroblasts this route would represent a basolateral cognate pathway (Figure 5D). However, it is still very speculative if in yeast a raft pathway with similar principles as in mammalian cells exists. Parallel biosynthetic pathways to the plasma membrane have been described. From the Golgi complex two populations of exocytic vesicles are produced, one carrying cell-surface components and another transporting the periplasmic enzymes invertase and acid phosphatase (Harsay and Bretscher, 1995). However, in late secretory mutants (sec1, sec4 and sec6) delivery of both vesicle populations is blocked, suggesting that they are transported by functionally similar pathways and not apical and basolateral cognate routes (Harsay and Bretscher, 1995). However, rafts might also exist in yeast. DIGs can be isolated as in mam malian cells (Kübler et al., 1996), but their composition has not yet been carefully analysed. Interestingly, the rate of ER to Golgi transport of a GPI-anchored protein was shown to be enhanced by ceramide synthesis whereas transport of soluble and transmembrane proteins was not changed (Horvath et al., 1994). Therefore, GPI-anchored proteins in yeast might be transported in raft-like lipid microdomains. Another example of polarization of nonepithelial cells is observed for osteoclasts (Salo et al., 1996). Resorbing osteoclasts secrete protons and enzymes from their ruffled membrane to degrade bone matrix components. Analysis of the targeting of the viral marker proteins VSV G and influenza virus HA demonstrated that the continuous basal membrane facing the bone marrow and extracellular fluid is divided into two different subdomains, recieving apical and basolateral cognate traffic (Figure 5C) (Salo et al., 1996). Bone degradation products are specifically transcytosed from the ruffled membrane to the apical cognate domain (Salo et al., 1997; Nesbitt and Horton, 1997). Whether sphingolipid-cholesterol rafts operate in the biosynthetic or transcytotic routes to this domain is not known. TWO CIRCUITS? Analysis of the post-Golgi membrane traffic in polarized and nonpolarized cells revealed two functionally distinct routes. It has been proposed that these two exocytic routes represent parts of two general circuits which operate in intracellular transport (Simons and Ikonen, 1997). One would be the basolateral circuit

54


Figure 5 Apical (grey) and basolateral (black) exocytic (cognate) routes in epithelial cells (A), migrating flbroblasts (B), osteoclasts (C), and (postulated) yeast (D).

employing cytoplasmic signals and the other the apical circuit using raft association and carbohydrates. The exocytic routes are balanced by apical and basolateral endocytosis processes. The clathrin-pathway, like the basolateral endocytosis, would make use of cytoplasmic signals and adaptors whereas the apical route would be raft-mediated, depending on clustering of the internalized proteins, cholesterol and actin (Simons and Ikonen, 1997; Rothberg et al., 1990; Deckert et al., 1996). Although named “apical” and “basolateral” the circuits are not anymore restricted to apical and basolateral surfaces in epithelial cells, where they were defined as the preferred mechanism used for the respective exocytosis route. Instead the circuits meet in most compartments of the cell as clathrin-coated pits and clathrin-mediated endocytosis also occur on the apical plasma membrane and raft associated proteins can be found on the basolateral surface (Melkonian et al., 1995; Simons and Ikonen, 1997). Proteins transported on one route would be excluded from the other, as raft markers like GPI-anchored proteins or influenza virus HA do not interact with clathrin-coated pits (Lazarovits and Roth, 1988; Bretscher et al., 1980). When raft association is weakend by mutation of the HA TMD determinant the


55

mutant protein can be endocytosed through clathrin-coated pits, although it does not contain a cytoplasmic endocytosis signal (Lazarovits et al., 1996). Probably, the mutant HA can be tolerated rather than being actively included. Also the GPI-anchored prion protein can switch to the basolateral circuit. It contains a proteinaceus signal which is required for its clathrin-mediated endocytosis (Shyng et al., 1995), probably by attaching it to a receptor with a cytoplasmic internalization signal. The plgAR changes for transcytosis from the basolateral to the apical route, controlled by inactivation of its cytoplasmic signal (Aroeti and Mostov, 1994). SUMMARY Identification of signals and transport mechanisms in epithelial cells demonstrated that the apical and basolateral routes use different principles. While basolateral sorting depends on cytoplasmic proteinaceus signals, apical targeting is based on carbohydrate-protein and lipid-protein interactions. The observations made in non-epithelial cells demonstrate that none of the pathways is a unique and specialized mechanism of epithelial cells. It rather appears that the principles of apical and basolateral transport are used to build up two independent circuits in the cell which are used to create surface polarity. As the signals directing proteins into the different circuits function hierarchically, proteins can be switched from one to the other pathway. This allows plasticity at the level of intracellular sorting which can be employed to serve the needs of different cell types. REFERENCES Anderson, R.G.W. and Orci, L. (1988) A view of acidic intracellular compartments. J. Cell Biol., 106, 539–543. Aniento, F., Gu, F., Parton, R.G. and Gruenberg, J. (1996) An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J. Cell Biol., 133, 29–41. Apodaca, G., Katz, L.A. and Mostov, K.E. (1994) Receptor-Mediated Transcytosis of IgA in MDCK Cells Is via Apical Recycling Endosomes. J. Cell Biol., 125, 67–86. Appel, D. and Koch-Brandt, C. (1994) Sorting of a secretory protein (gp80) to the apical surface of Caco-2 cells. J. Cell Sci., 107, 553–559. Aroeti, B., Kosen, P.A., Kuntz, I.D., Cohen, F.E. and Mostov, K.E. (1993) Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor. J. Cell Biol., 123, 1149–60. Aroeti, B. and Mostov, K.E. (1994) Polarized sorting of the polymeric immunoglobulin receptor in the exocytotic and endocytotic pathways is controlled by the same amino acids. EMBO J., 13, 2297–304. Arreaza, G. and Brown, D.A. (1995) Sorting and intracellular trafficking of a glycosylphosphatidylinositol-anchored protein and two hybrid transmembrane proteins with the same ectodomain in Madin-Darby canine kidney epithelial cells. J. Biol. Chem., 270, 23641– 23647. Barroso, M. and Sztul, E.S. (1994) Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome. J. Cell Biol., 124, 83–100. Berditchevski, F., Zutter, M.M. and Hemler, M.E. (1996) Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins). Mol. Biol. Cell, 1, 193–207. Bergmann, J.E., Kupfer, A. and Singer, S.J. (1983) Membrane insertion at the leading edge of motile fibroblasts. Proc. Natl. Acad. Sci. USA, 80, 1367–71. Bretscher, M.S. (1992a) Cells can use their transferrin receptors for locomotion. Embo J., 11, 383–9. Bretscher, M.S. (1992b) Circulating integrins: α5β1, α6β4 and Mac-1, but not α3β1, α4β1 or LFA-1. EMBO J., 11, 405–10. Bretscher, M.S. (1996) Moving membrane up to the front of migrating cells. Cell, 85, 465–7.

56


Bretscher, M.S., Thomson, J.N. and Pearse, B.M.F. (1980) Coated pits act as molecular filters. Proc. Natl. Acad. Set. USA, 77, 4156–4159. Brown, D.A., Crise, B. and Rose, J.K. (1989) Mechanism of membrane anchoring affects polarized expression of two proteins in MDCK cells. Science, 245, 1499–1501. Brown, D.A. and Rose, J.K. (1992) Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell, 68, 533–544. Brügger, B., Helms, J.B., Lehmann, W.-D. and Wieland, F.T. (1996) Lipid sorting during the budding of COPI coated vesicles from CHO Golgi membranes. Mol. Biol. Cell, 7(Supplement), 600a. Buendia, B., Bré, M.-H., Griffiths, G. and Karsenti, E. (1989) Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells. J. Cell. Biol., 110, 1123–1135. Caplan, M.J., Stow, J.L., Newman, A.P., Madri, J., Anderson, H.C., Farguhar, M.G., Palade, G.E. and Jamieson, J.D. (1987) Dependence on pH of polarized sorting of secreted proteins. Nature, 329, 632–635. Casanova, J.E., Apodaca, G. and Mostov, K.E. (1991) An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor. Cell, 66, 65–75. Casanova, J.E., Breitfeld, P.P., Ross, S.A. and Mostov, K.E. (1990) Phosphorylation of the polymeric Immunoglobulin Receptor Required for Its Efficient Transcytosis. Science, 248, 742–745. Corbeil, D., Boileau, G., Lemay, G. and Crine, P. (1992) Expression and Polarized Apical Secretion in Madin-Darby Canine Kidney Cells of a Recombinant Soluble Form of Neutral Endopeptidase Lacking the Cytosolic and Transmembrane Domains. J. Biol. Chem., 267, 2798–2801. Danielsen, E.M. (1995) Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes. Biochemistry, 34, 1596–1605. De Hoop, M.J. and Dotti, C.G. (1993) Membrane traffic in polarized neurons in culture. J. Cell Sci. Suppl., 17, 85–92. Deckert, M., Ticchioni, M. and Bernard, A. (1996) Endocytosis of GPI-anchored proteins in human lymphocytes: role of glycolipid-based domains, actin cytosceleton, and protein kinases. J. Cell Biol., 133, 791–799. Dotti, C., Parton, R.G. and Simons, K. (1991) Polarized sorting of glypiated proteins in hippocampal neurons. Nature, 349, 158–161. Dotti, C. and Simons, K. (1990) Polarized sorting of viral glycoproteins to the axon and dendrites of hippocampal neurons in culture. Cell, 62, 63–72. Drubin, D.G., Jones, H.D. and Wertman, K.F. (1993) Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Moll. Biol. Cell, 4, 1277–1294. Drubin, D.G. and Nelson, W.J. (1996) Origins of cell polarity. Cell, 84, 335–44. Eaton, S. and Simons, K. (1995) Apical, basal, and lateral cues for epithelial polarization. Cell, 82, 5–8. Fiedler, K., Parton, R.G., Kellner, R., Etzold, T. and Simons, K. (1994) VIP36, a novel component of glycolipid rafts and exocytic carrier vesicles in epithelial cells. EMBO J., 13, 1729–1740. Fiedler, K. and Simons, K. (1994) A putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins. Cell, 77, 625–626. Fiedler, K. and Simons, K. (1995) The role of N-glycans in the secretory pathway. Cell, 81, 1–20. Fiedler, K., Veit, M., Stamnes, M.A. and Rothman, J.E. (1996) Bimodal interaction of coatomer with the p24 family of putative cargo receptors. Science., 273, 1396–9. Fra, A.M., Williamson, E., Simons, K. and Parton, R.G. (1995) De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin. Proc. Natl. Acad. Sci. USA, 92, 8655–9. Futter, C.E., Connolly, C.N., Cutler, D.F. and Hopkins, C.R. (1995) Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell surface. J. Biol. Chem., 270, 10999–1003. Garcia, M., Mirre, C., Quaroni, A., Reggio, H. and Le Bivic, A.L. (1993) GPI-anchored proteins associate to form microdomains during their intracellular transport in Caco-2 cells. J. Cell Sci., 104, 1281–1290. Geffen, I., Fuhrer, C., Leitinger, B., Weiss, M., Huggel, K., Griffiths, G. and Spiess, M. (1993) Related signals for endocytosis and basolateral sorting of the asialoglycoprotein receptor. J. Biol. Chem., 268, 20772–20777.


57

Gonzalez, A., Nicovani, S. and Juica, F. (1993) Apical secretion of Hepatites B surface antigen from transfected MadinDarby Canine Kidney Cells. J. Biol. Chem., 268, 6662–6667. Gonzalez, A., Rizzolo, L., Rindler, M., Adesnik, M., Sabatini, D.D. and Gottlieb, T. (1987) Nonpolarized secretion of truncated forms of the influenza hemagglutinin and the vesicular stomatitis virus G protein from MDCK cells. Proc. Natl. Acad. Sci. USA, 84, 3738– 3742. Gottlieb, T.A., Beaudry, G., Rizzolo, L., Colman, A., Rindler, M., Adesnik, M. and Sabatini, D.D. (1986) Secretion of endogenous and exogenous proteins from polarized MDCK cell monolayers. Proc. Natl. Acad. Sci. USA, 83, 2100–2104. Graichen, R., Losch, A., Appel, D. and Koch, B.C. (1996) Glycolipid-independent sorting of a secretory glycoprotein to the apical surface of polarized epithelial cells. J. Biol. Chem., 271, 15854–7. Gumbiner, B., Stevenson, B. and Grimaldi, A. (1988) The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex. J. Cell Biol., 107, 1575–1587. Gundersen, D., Powell, S.K. and Rodriguez, B.E. (1993) Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina. J. Cell Biol., 121, 335–43. Hammerton, R.W., Krzeminski, K.A., Mays, R.W, Ryan, T.A., Wollner, D.A. and Nelson, W.J. (1991) Mechanism for regulating cell surface distribution of Na+,K+ATPase in polarized epithelial cells. Science, 847–850. Hanada, K., Nishijima, M., Akamatsu, Y. and Pagano, R.E. (1995) Both sphingolipids and cholesterol participate in the detergent insolubility of alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein, in mammalian membranes. J. Biol. Chem., 270, 6254–6260. Hansen, S.H. and Casanova, J.E. (1994) Gs alpha stimulates transcytosis and apical secretion in MDCK cells through cAMP and protein kinase A.J. Cell Biol., 126, 677–687. Harder, T. and Simons, K. (1997) Caveolae, DIGs, and the dynamics of sphingolipd-cholesterol microdomains. Curr. Op. Cell Biol., 9, 534–542. Harder, T., Scheiffele, P., Verleade, P. and Simons, K. (1998) Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol., 140, 929– 942. Harsay, E. and Bretscher, A. (1995) Parallel secretory pathways to the cell surface in yeast. J. Cell Biol., 131, 297–310. Heilker, R., Manning, K.U., Zuber, J.F. and Spiess, M. (1996) In vitro binding of ciathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting. Embo J., 15, 2893–9. Helenius, A. (1994) How N-Linked Oligosaccharides Affect Glycoprotein Folding in the Endoplasmic Reticulum. Mol. Biol. Cell., 5, 253–265. Hobert, M. and Carlin, C. (1995) Cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells. J. Cell Physiol., 162,434– 446. Hopkins, C.R., Gibson, A., Shipman, M., Strickland, D.K. and Trowbridge, I.S. (1994) In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella. J. Cell Biol., 125, 1265–74. Horvath, A., Sutterlin, C., Manning, K.U., Movva, N.R. and Riezman, H. (1994) Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast. Embo J., 13, 3687–95. Hubbard, A.L., Stieger, B. and Bartles, J.R. (1989) Biogenesis of Endogenous Plasma Membrane Proteins in Epithelial Cells. Annu. Rev. Physiol., 51, 735–770. Huber, L.A., de Hoope, M.J., Dupree, P., Zerial, M., Simons, K. and Dotti, C. (1993) Protein transport to the dendritic plasma membrane of cultured neurons is regulated by rab8p. J. Cell Biol., 123, 47–55. Hunziker, W., Harter, C., Matter, K. and Mellman, I. (1991) Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell, 66, 1–20. Ikonen, E., Tagaya, M., Ullrich, O., Montecucco, C. and Simons, K. (1995) Different requirements for NSF, SNAP, and Rab proteins in apical and basolateral transport in MDCK cells. Cell, 81, 571–580. Keller, P. and Simons, K. (1998) Cholesterol is required for surface transport of influenza virus hemagglutinin. J. Cell Biol., 140, 1357–1367. Kemler, R. (1992) Classical cadherins. Semin. Cell Biol., 3, 149–55.

58


Kitagawa, Y., Sano, Y., Ueda, M., Higashio, K., Narita, H., Okano, M., Matsumoto, S.I. and Sasaki, R. (1994) Nglycosylation of erythropoietin is critical for apical secretion by Madin-Darby canine kidney cells. Exp. Cell Res., 213, 449–457. Kobayashi, T., Pimplikar, S.W., Parton, R.G., Bhakdi, S. and Simons, K. (1992) Sphingolipid transport from the transGolgi network to the apical surface in permeabilized MDCK cells. FEBS, 300, 227–231. Koller, E. and Ranscht, B. (1996) Differential targeting of T- and N-cadherin in polarized epithelial cells. J. Biol. Chem., 271, 30061–30067. Kondor-Koch, C.B., Bravo, R., Fuller, S.D., Cutler, D. and Garoff, H. (1985) Exocytic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK. Cell, 43, 297–306. Kübler, E., Dohlman, H.G. and Lisanti, M.P. (1996) Identification of Triton X-100 insoluble membrane domains in the yeast saccharomyces cerivisiae. J. Biol. Chem., 271, 32975–32980. Kundu, A., Avalos, R.T., Sanderson, C.M. and Nayak, D.P. (1996) Transmembrane domain of influenza virus neuraminidase, a type II protein, possesses an apical sorting signal in polarized MDCK cells. J. Virol., 70, 6508–6515. Kundu, A. and Nayak, D.P. (1994) Analysis of the signals for polarized transport of influenza virus (A/WSN/33) neuraminidase and human transferrin receptor, type II transmembrane proteins. J. Virol., 68, 1812–1818. Kurzchalia, T.V., Dupree, P., Parton, R.G., Kellner, R., Virta, H., Lehnert, M. and Simons, K. (1992) VIP21, a 21-kD membrane protein is an integral component of trans-Golgi-network-derived transport vesicles. J. Cell Biol., 118, 1003–1014. Lafont, F., Burkhardt, J.K. and Simons, K. (1994) Involvement of microtubule motors in basolateral and apical transport in kidney cells. Nature, 372, 801–803. Lafont, F., Lecat, S., Verkade, P. and Simons, K. (1998) Annexin 13b associates with lipid microdomains to function in apical delivery. J. Cell Biol., 142, 1413–1427. Lazarovits, J., Nairn, H.Y., Rodriguez, A.C., Wang, R.-H., Fire, E., Bird, C., Henis, Y.I. and Roth, M.G. (1996) Endocytosis of chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits. J. Cell Biol., 134, 339–348. Lazarovits, J. and Roth, M. (1988) A single amino acid change in the cytoplasmic domain allows the influenza virus haemagglutinin to be endocytosed through coated pits. Cell, 53, 743–752. Le Bivic, A., Garcia, M. and Rodriguez, B.E. (1993) Ricin-resistant Madin-Darby canine kidney cells missort a major endogenous apical sialoglycoprotein. J. Biol Chem., 268, 6909–16. Le Bivic, A., Quaroni, A., Nichols, B. and Rodriguez-Boulan, E. (1990) Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line. J. Cell Biol., 111, 1351–1361. Ledesma, M.D., Simons, K. and Dotti, C. (1998) Neuronal polarity: essential role for protein-lipid complexes in axonal sorting. Proc. Natl. Acad. Sci. USA, 95, 3966–3971. Leitinger, B., Hille-Rehfeld, A. and Spiess, M. (1995) Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes. Proc. Natl. Acad. Sci. USA, 92, 10109–10113. Lipardi, C., Mora, R., Colomer, V., Paladino, S., Nitsch, L., Rodriguez-Boulan, E. and Zurzolo, C. (1998) Caveolin transfection results in caveolae formation but not apical sorting of glycosylphosphatidylinositol (GPI) — anchored proteins in epithelial cells. J. Cell Biol., 140, 617–626. Lipsky, N.G. and Pagano, R.E. (1983) Sphingolipid metabolism in cultured fibroblasts: Microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc. Natl. Acad. Sci. USA, 80, 2608–2612. Lisanti, M.P., Caras, I.W., Davitz, M.A. and Rodriguez-Boulan, E. (1989) A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells. J. Cell Biol., 109, 2145–2156. Lisanti, M.P., Le Bivic, A., Saltiel, A.R. and Rodriguez-Boulan, E. (1990) Preferred Apical Distribution of GlycosylPhosphatidyl (GPI) Anchored Proteins: A Highly Conserved Feature of the Polarized Epithelial Cell Phenotype. J. Membrane Biol., 113, 155–167. Luecke, H., Chang, B.T., Mailliard, W.S., Schlaepfer, D.D. and Haigler, H.T. (1995) Crystal structure of the annexin XII hexamer and implications for bilayer insertion. Nature, 378, 512–5.


59

Mannion, B.A., Berditchevski, F., Kraeft, S.K., Chen, L.B. and Hemler, M.E. (1996) Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/ CD29). J. Immunol., 157, 2039–47. Marrs, J.A., Andersson, F.C., Jeong, M.C., Cohen, G.L., Zurzolo, C., Nabi, I.R., Rodriguez, B.E. and Nelson, W.J. (1995) Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules. J. Cell Biol., 129, 507–19. Matter, K., Brauchbar, M., Bucher, K. and Hauri, H.-P. (1990) Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells. Cell., 60, 429–37. Matter, K., Hunziker, W. and Mellman, I. (1992) Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell., 71, 741–753. Matter, K. and Mellman, I. (1994) Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr. Opin. Cell Biol., 6, 545–554. Matter, K., Whitney, J.A., Yamamoto, E.M. and Mellman, I. (1993) Common Signals Control Low Density Lipoprotein Receptor Sorting in Endosomes and the Golgi Complex of MDCK Cells. Cell, 14, 1053–1064. Matter, K., Yamamoto, E.M. and Mellman, I. (1994) Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells. J. Cell Biol., 126, 991–1004. Mays, R.W., Siemers, K.A., Fritz, B.A., Lowe, A.W., van Meer, G. and Nelson, W.J. (1995) Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells. J. Cell Biol., 130, 1105–1115. McConville, M.J. and Ferguson, M.A. (1993) The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem. J. McNeiil, H., Ozawa, M., Kemler, R. and Nelson, W.J. (1990) Novel function of the cell adhesion molecule uvomorulin as inducer of cell surface polarity. Cell, 62, 309–316. Meads, T. and Schroer, T.A. (1995) Polarity and nucleation of microtubules in polarized epithelial cells. Cell Motil. Cytoskeleton, 32, 273–88. Meiss, H.K., Green, R.F. and Rodriguez-Boulan, E. (1982) Lectin-resistant mutants of polarized epithelial cells. Mol. Cell Biol., 2, 1287–1294. Melkonian, K.A., Chu, T., Tortorella, L.B. and Brown, D.A. (1995) Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells. Biochemistry, 34, 16161–70. Moss, S.E. (1992). The Annexins. In The Annexins (ed. S.E. Moss), vol. pp. 1–9. London: Portland Press. Mostov, K., Apodaca, G., Aroeti, B. and Okamoto, C. (1992) Plasma membrane protein sorting in polarized epithelial cells. J. Cell Biol., 116, 577–583. Mostov, K.E. and Cardone, M.H. (1995) Regulation of protein traffic in polarized epithelial cells. Bioessays, 17, 129–138. Mostov, K.E., Harris, J.M. and Breitfeld, P.P. (1987) An anchor-minus form of the polymeric immunoglobulin receptor is secreted predominantly apically in Madin-Darby Canine Kidney cells. J. Cell Biol., 105, 2031–2036. Murata, M., Peranen, J., Schreiner, R., Wieland, F., Kurzchalia, T.V. and Simons, K. (1995) VIP21/caveolin is a cholesterol-binding protein. Proc. Natl. Acad. Sci. USA, 92, 10339– 43. Müsch, A., Xu, H., Shields, D. and Rodriguez-Boulan, E. (1996) Transport of vesicular stomatitis virus to the cell surface is signal mediated in polarized and nonpolarized cells. J. Cell Biol., 133, 543–558. Musto, N.A. (1993) Polarized secretion of Human Corticoid Steroid Binding Globulin by MDCK and BeWo Cells. Exp. Cell Res., 209, 271–276. Nabi, I.R., Mathews, A.P., Cohen, G.L., Gundersen, D. and Rodriguez, B.E. (1993) Immortalization of polarized rat retinal pigment epithelium. J. Cell Sci. Nesbitt, S.A. and Horton, M.A. (1997) Trafficking of matrix collagens through bone resorbing osteoclasts. Science, 276, 266–269. Nickel, W., Huber, L.A., Kahn, R.A., Kipper, N., Barthel, A., Fasshauer, D. and Soling, H.D. (1994) ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes. J. Cell Biol., 125, 721–32.

60


Odorizzi, G., Pearse, A., Domingo, D., Trowbridge, I.S. and Hopkins, C.R. (1996) Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism. J. Cell Biol., 135, 139–52. Ojakian, G.K. and Schwimmer, R. (1994) Regulation of epithelial cell surface polarity reversal by beta 1 integrins. J. Cell Sci. Parczyk, K. and Koch-Brandt, C. (1991) The role of carbohydrates in vectorial exocytosis. FEBS, 278, 267–270. Pathak, R.K., Yokode, M., Hammer, R.E., Hofmann, S.L., Brown, M.S., Goldstein, J.L. and Anderson, R.G. (1990) Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice. J. Cell Biol., 111, 347–59. Peränen, J., Auvinen, P., Virta, H., Wepf, R. and Simons, K. (1996) Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts. J. Cell Biol., 135, 153–67. Pimplikar, S. and Simons, K. (1993) Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature, 362, 456–458. Pimplikar, S.W. and Simons, K. (1994) Activators of protein kinase A stimulate apical but not basolateral transport in epithelial Madin-Darby canine kidney cells. J. Biol. Chem., 269, 19054–19059. Powell, S.K., Lisanti, M.P. and Rodriguez-Boulan, E. (1991) Thy-1 expresses two signals for apical localization in epithelial cells. Am. J. Physiol., 260, C715–720. Prill, V., Lehmann, L., von, F.K. and Peters, C. (1993) The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells. Embo J., 12, 2181–93. Ragno, P., Estreicher, A., Gos, A., Wohlwend, A., Belin, D. and Vassalli, J.-D. (1992) Polarized secretion of urokinasetype plasminogen activator by epithelial cells. Exp. Cell Res., 203, 236–243. Rapoport, I., Miyazaki, M., Boll, W., Duckworth, B., Cantley, L.C., Shoelson, S. and Kirchhausen, T. (1997) Regulatory interactions in the recognition of endocytic sorting signals by AP-2 complexes. EMBO J., 16, 2240–2250. Redman, C.A., Thomas-Oates, J.E., Ogata, S., Ikehara, Y. and Ferguson, M.A.J. (1994) Structure of the glycosylphosphatidylinositol membrane anchor of human placental alkaline phosphatase. Biochem. J., 302, 861–865. Rindler, M J. and Traber, M.G. (1988) A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells. J. Cell Biol., 107, 471–9. Rodriguez-Boulan, E., Paskiet, K.T. and Sabatini, D.D. (1983) Assembly of enveloped viruses in Madin-Darby canine kidney cells: polarized budding from single attached cells and from clusters of cells in suspension. J. Cell Biol., 96, 866–874. Rodriguez-Boulan, E. and Powell, S.K. (1992) Polarity of epithelial and neuronal cells. Annu. Rev. Cell Biol., 8, 395–427. Rothberg, K.G., Ying, Y.S., Kamen, B.A. and Anderson, R.G. (1990) Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5– methyltetrahydrofolate. J. Cell Biol. Rothman, J.E. (1994) Mechanisms of intracellular protein transport. Nature, 372, 55–63. Rotin, D., Barsagi, D., Obrodovich, H., Merilainen, J., Lehto, V.P., Canessa, C.M., Rossier, B.C. and Downey, G.P. (1994) An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane. EMBO J., 13, 4440– 4450. Salo, J., Lehenkari, P., Mulari, M., Metsikkö, K. and Väänänen, H.K. (1997) Removal of osteoclast bone resorption products by transcytosis. Science., 276, 270–273. Salo, J., Metsikkö, K., Palokangas, H., Lehenkari, P. and Väänänen, H.K. (1996) Bone-resorbing osteoclasts reveal a dynamic division of basal plasma membrane into two different domains. J. Cell Sci., 109, 301–307. Scheiffele, P., Peränen, J. and Simons, K. (1995) N-glycans as apical sorting signals in epithelial cells. Nature, 378, 96–98. Scheiffele, P., Roth, M.G. and Simons, K. (1997) Interaction of influenza hemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain. EMBO J., 16, 5501–5508.


61

Scheiffele, P., Verkade, P., Fra, A.M., Virta, H., Simons, K. and Ikonen, E. (1998) Caveolin-1 and -2 in the exocytic pathway of MDCK cells. J. Cell Biol., 140, 795–806. Schell, M.J., Maurice, M., Stieger, B. and Hubbard, A.L. (1992) 5´nucleotidase is sorted to the apical domain of hepatocytes via an indirect route [published erratum appears in J. Cell Biol., 1993 Nov; 123(3): following 767]. J. Cell Biol., 119, 1173–82. Schröder, S., Schimmoller, F., Singer-Kruger, B. and Riezman, H. (1995) The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the retl-1 mutation in alpha-COP. J. Cell Biol., 131, 895–912. Schroeder, R., London, E. and Brown, D. (1994) Interactions between saturated acyl chains confer detergent resistance to lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc. Natl. Acad. Set. USA., 91, 12130–12134. Schwartz, G.J., Barasch, J. and Al, A.Q. (1985) Plasticity of functional epithelial polarity. Nature, 318, 368–71. Schweizer, A., Fransen, J.A.M., Bachi, T., Ginsel, L. and Hauri, H.P. (1988) Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubular vesicular compartment at the cis-side of the Golgi apparatus. J. Cell Biol., 107, 1643–1653. Shevchenko, A., Keller, P., Scheiffele, P., Mann, M. and Simons, K. (1997) Identification of components of trans-Golgi network-derived transport vesicles and detergent-insoluble complexes by nanoelectrospray tandem mass spectrometry. Electrophoresis, 18, 2591–2600. Shyng, S.L., Moulder, K.L., Lesko, A. and Harris, D.A. (1995) The N-terminal domain of a glycolipid-anchored prion protein is essential for its endocytosis via clathrin-coated pits. J. Biol Chem., 270, 14793–800. Simons, K. (1995) Biogenesis of epithelial cell surface polarity. Harvey Lect., 89, 125–146. Simons, K., Dupree, P., Fiedler, K., Huber, L.A., Kobayashi, T., Kurzchalia, T., Olkkonen, V., Pimplikar, S., Parton, R. and Dotti, C. (1993). Biogenesis of cell-surface polarity in epithelial cells and neurons.In Cold Spring Harbor Symposia for Quantitative Biology 57 (ed. pp.Cold Spring Harbor: Simons, K. and Fuller, S.D. (1985) Cell surface polarity in epithelia. Annu. Rev. Cell Biol., 1, 243–288. Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes. Nature, 387, 569–572. Singer, S.J. and Kupfer, A. (1986) The directed migration of eukaryotic cells. Annu Rev. Cell Biol., 2, 337–65. Skibbens, J.E., Roth, M.G. and Matlin, K.S. (1989) Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts. J. Cell Biol., 108, 821–832. Soole, K.L., Hall, J., Jepson, M.A., Hazlewood, G.P., Gilbert, H.J. and Hirst, B.H. (1992) Constitutive secretion of a bacterial enzyme by polarized cells. J. Cell Sci., 102, 495–504. TerBush, D.R. and Novick, P. (1995) Sec6, Sec8, and Sec15 5 are components of a multisubunit complex which localizes to small bud tips in saccharomyces cerevisiae. J. Cell Biol., 130, Thomas, D.C., Brewer, C.B. and Roth, M.G. (1993) Vesicular stomatitis virus glycoprotein contains a dominant cytoplasmic basolateral sorting signal critically dependent upon a tyrosine. J. Biol. Chem., 268, 3313–3320. Ullrich, O., Mann, K., Haase, W. and Koch-Brandt, C. (1991) Biosynthesis and secretion of an Osteopontin-related 20 kDa polypeptide in the Madin-Darby Canine Kidney Cell line. J. Biol. Chem., 266, 3518–3525. Urban, J., Parczyk, K., Leutz, A., Kayne, M. and Kondor-Koch, C. (1987) Constitutive apical secretion of an 80-kD sulfated glycoprotein complex in the polarized epithelial MadinDarly canine kidney cell line. J. Cell Biol., 105, 2735–2743. van Adelsberg, J., Edwards, J.C., Takito, J., Kiss, B. and Al-Awqati, Q. (1994) An induced extracellular matrix protein reverses the polarity of band 3 in intercalated epithelial cells. Cell, 76, 1053–61. van den Berg, C., Cinek, T., Hallett, M.B., Horejsi, V. and Morgan, B.P. (1995) Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca (2+)-signaling competent. J. Cell Biol., 131, 669–77. van Genderen, I.L., van Meer, G., Slot, J.W., Geuze, H.J. and Voorhout, W. (1991) Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immunoelectron microscopy after freeze-substitution. J. Cell Biol., 115, 1009–1019.

62


van Helvoort, A., Smith, A.J., Sprong, H., Fritzsche, I., Schinkel, A.H., Borst, P. and van Meer, G. (1996) MDR1 Pglycoprotein is a lipid translocase of broad specefkity, while MDR3 P-glycoprotein specifically translocates phosphatidylcholine. Cell, 87, 507–517. van Meer, G., Gumbiner, B. and Simons, K. (1986) The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next. Nature, 322, 639–641. van Meer, G. and Simons, K. (1982) Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions. EMBO J., 1, 847–852. van Meer, G., Stelzer, E.H.K., Wijnaendts-van-Resandt, R.W. and Simons, K. (1987) Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J. Cell Biol., 105, 1623– 1635. Vaux, D. (1992) The structure of an endocytosis signal. Trends Cell Biol., 2, 189–192. Vega-Salas, D.E., Salas, P.J.I., Gundersen, D. and Rodriguez-Boulan, E. (1987) Formation of the apical pole of epithelial (Madin-Darby Canine Kidney) cells: Polarity of an apical protein is independent of tight junctions while segregation of a basolateral marker requires cell-cell interactions. J. Cell. Biol., 104, 905–916. Vogel, L.K., Spiess, M., Sjöström, H. and Norén, O. (1992) Evidence for an apical sorting signal on the ectodomain of human aminopeptidase N. J. Biol. Chem., 267, 2794–2797. Vogel, L.K., Suske, G., Beato, M., Norén, O. and Sjöström, H. (1993) Uteroglobin, an apically secreted protein of the uterine epithelium, is secreted non-polarized from MDCK cells and mainly basolateral from Caco-2 cells. FEBS, 330, 293–296. Wagner, M., Morgans, C. and Koch-Brandt, C. (1995) The oligosaccharides have an essential but indirect role in sorting gp80 (clusterin, TRPM-2) to the apical surface of MDCK cells. EJCB, 67, 84–88. Wandinger-Ness, A., Bennet, M.K., Antony, C. and Simons, K. (1990) Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J. Cell Biol., 111, 987–1000. Wang, A.Z., Ojakian, G.K. and Nelson, W.J. (1990) Steps in the morphogenesis of a polarized epithelium. I. Uncoupling the roles of cell-cell and cell-substratum contact in establishing plasma membrane polarity in multicellular epithelial (MDCK) cysts. J. Cell Sci. Wollner, D.A., Krzeminski, K.A. and Nelson, W.J. (1992) Remodeling the cell surface distribution of membrane proteins during the development of epithelial cell polarity. J. Cell Biol., 116, 889–99. Yokode, M., Pathak, R.K., Hammer, R.E., Brown, M.S., Goldstein, J.L. and Anderson, R.G. (1992) Cytoplasmic sequence required for basolateral targeting of LDL receptor in livers of transgenic mice. J. Cell Biol., 117, 39–46. Yoshimori, T., Keller, P., Roth, M.G. and Simons, K. (1996) Different Biosynthetic Transport Routes to the Plasma Membrane in BHK and CHO Cells. J. Cell Biol., 133, 247–256. Zinn, K.M. and Marmor, M.F. (1979). The retinal pigment epithelium. In (ed. pp. Cambridge, MA: Harvard University Press. Zurzolo, C., Le Bivic, A., Quaroni, A., Nitsch, L. and Rodriguez-Boulan, E. (1992) Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line. EMBO J., 11, 2337–2344. Zurzolo, C., Lisanti, M.P., Caras, I.W., Nitsch, L. and Rodriguez-Boulan, E. (1993) Glycosylphosphatidylinositolanchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells. J. Cell Biol., 121, 1031–1039. Zurzolo, C., Polistina, C., Saini, M., Gentile, R., Aloj, L., Migliaccio, G., Bonatti, S. and Nitsch, L. (1992) Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues. J. Cell Biol., 117, 551–64. Zurzolo, C., van’t Hof, W, van Meer, G. and Rodriguez-Boulan, E. (1994) VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells. EMBO J., 13, 42–53.

4. EPITHELIAL GENE REGULATION ROBIN M. WATTS and HANS-ULRICH BERNARD Institute of Molecular and Cell Biology, National University of Singapore, Singapore 117609, Tel.: 65–778–8823; Fax: 65–779–1117; E-mail: [email protected]

INTRODUCTION While many proteins with functions related to metabolism, structure or gene expression are present in the majority of the cells of a differentiated organism, the unique functions of specific cell types depend on the exclusive expression of particular genes. Immunoglobulins are expressed by B lymphocytes, myosin preferentially by muscle cells, α-crystallin by cells of the ocular lens. Many different mechanisms can be conceived that might account for the establishment of such cell type specific protein expression during embryogenesis and development, and its maintainance in the differentiated cell. Genes may become altered during lineage determination or differentiation, for example by methylation or alteration of the chromatin structure, affecting accessibility of the DNA by transcription factors. Regulatory chain reactions, instigated by environmental cues, could lead to synthesis of activators or repressers that may ultimately modulate transcription and translation. These mechanisms require cis-reponsive elements, which are positioned in the coding, intronic, or flanking sequences of the genes, and are often targets of trans-acting factors. The limiting complexity of the genome requires the postulation of "combinatorial" gene regulation, whereby the interplay of different widely expressed regulatory proteins creates spatial and temporal specificity (Ernst and Smale, 1995). There is now a wealth of information about these regulatory phenomena, and the technology is in place to expand this knowledge: analyses of chromatin structure and transcript boundaries help to localize cis-responsive elements, transgenes reveal their inclusion or loss in the cloning process, and DNAse protections and gelshifts identify and define trans-acting factors. However, it is probably fair to state that we presently lack a complete understanding of the exact regulation of most genes. The epithelial tissues of the body perform very diverse functions and differ in their capabilities between anatomical locations. Epithelia may be absorptive, secretory, ciliated, or keratinised. Morphologically the cells may be cuboidal, columnar or squamous. Some epithelia stratify into multiple layers of cells, while simple epithelia retain a monolayer structure. A number of proteins are found in common between epithelia developmentally derived from the endoderm, ectoderm, or interconverted from mesenchyme, such as Ecadherin (Hennig et al., 1996) and transglutaminase 1 (Lee et al., 1996). Many epithelia express keratin genes, and some maintain the capacity to switch between epithelial expression programs, such as the induction of squamous differentiation markers upon injury of tracheobronchial epithelium (Saunders et al., 1993). Morphologically different epithelia, and even similar epithelia from different anatomical sites, often show divergence in protein composition. The cytokeratins K1 and K10 are expressed in differentiating epidermal keratinocytes, while K4 and K13 are found in mucosal stratified epithelia, and K8 and K18 in many simple epithelia. Stratified epithelia express different proteins in different layers, for example, K5 and K14 in basal cells, of cutaneous epithelia K1 and K10 in suprabasal layers, and filaggrin and loricrin in

64


terminally differentiated cells. It would therefore seem there is not a single regulatory mechanism resulting in the various epithelial phenotypes, but rather a multitude of mechanisms. The first line of experiments must be separate enquiries into the regulation of each of these genes. The hope is, of course, to find generalizations, for example mechanisms shared by genes such as those encoding copolymerizing pairs of cytokeratins, or possibly even general mechanisms shared by all epithelial lineages, which are further modulated in each differentiation type. PARADIGMS OF SPECIFIC TRANSCRIPTIONAL MODULATION For a number of tissues regulatory mechanisms influencing tissue or stage specificity of gene expression are known. It may be useful to reconsider some paradigms before inquiring into mechanisms of epithelial specific expression. (1) MyoD may serve as a paradigm of cell-type specificity induced by a single transcription factor or a family of factors targetting unique cis-responsive elements. MyoD is one of four helix-loop-helix proteins functioning as “master regulatory” muscle transcription factors. Expression of myoD, myogenin, myf-5 or myf-6 in a variety of non-muscle cell types results in the instigation of a muscle specific transcription program and the production of a muscle phenotype. The myoD family proteins form heterodimers with ubiquitous helix-loop-helix E proteins and bind E-box DNA motifs, cis-acting sequences of most muscle specific promoters and enhancers (reviewed in Olson and Klein, 1994). Developmentally, myf5 and myoD expression preceed and modulate myogenesis, and are themselves restricted in expression by transcriptional activators, DNA methylation status, and mRNA stability (see Weintraub et al., 1991 for review). (2) OCA-B is a coactivator responsible for cell-type specificity. For B-cell restricted transcription of immunoglobulin genes, a conserved eight base pair (“octamer”) element is critical. Two proteins that bind these motifs, Oct-1 and Oct-2, are present in B-cells, and as Oct-2 expression is largely limited to B lymphocytes, it was regarded to be responsible for specific activation. However, it seems Oct-2 alone does not suffice to activate B-cell transcription, and recent work has characterised a B-cell specific coactivator, termed OCA-B (Luo and Roeder, 1995). As a coactivator, OCA-B does not itself bind DNA, but directly contacts bound Oct-1 or Oct-2 and interacts with the general transcription machinery. When added to HeLa extracts, OCA-B is capable of activating B-cell specific promoters. Specific transcription is thus dependent (i) on the presence of appropriatey positioned octamer motifs in the regulatory regions of target genes, (ii) the correct trans-acting element (Oct-1 or Oct-2), and (iii) the bridging coactivator OCA-B. (3) Liver-specific transcription is an example of cell-specific mechanisms brought about by (i) multiple unrelated transcription factors, which (ii) are not strictly tissue-specific. Several important DNA binding proteins have been repeatedly encountered in control regions of liver-specific genes, namely hepatocyte nuclear factor 1 (HNF-1), -3, -4, and CCAAT/enhancer binding protein (C/EBP), while binding sites for widely distributed factors such as AP-1 and NFI complement enhancer specificity. The crucial difference between hepatic and non-hepatic tissues is the co-occurrence and abundance of HNF-1, -3, -4, and C/EBP (Xanthopolous et al., 1991). Somatic cell hybrid experiments indicate that the loss of HNF-4 and HNF-1, but not of other factors, correlates with loss of hepatic functions (Griffo et al., 1993). There may be a regulatory hierarchy between HNF-1 and -4, as HNF-4 appears to be essential to regulate HNF-1 expression (Kuo et al., 1992). Positive and negative interactions between liver enriched factors contribute to specificity, but vary in effect depending on the placement of binding


65

sites in different genes (Rouet et al., 1995). This expression of hepatocyte specific proteins in the liver parenchyme under the control of hepatocyte-enriched factors is apparently possible against a background of epithelial-specific transcription as the same cells also express the primitive cytokeratins, K8 and K18. K14 is also expressed in the liver in epithelial cells of the bilary ducts, which are derived from the same precursor cells as hepatocytes, an example of the plasticity of epithelial specific transcriptional regulation in differentiation (Blouin et al., 1992) and neoplastic disease (Quinlan et al., 1985; Smedts et al., 1993). (4) Developmental globin gene switching is the best studied example of regulation on the level of chromatin structure. The erythroid specific and temporally controlled transcription of the human β-globin cluster is determined by the locus control region (LCR) (reviewed by Crossley and Orkin, 1993), a regulatory element located 5' of the gene cluster and comprised of four DNase I hypersensitive subdomains. These subdomains bind erythroid enriched (GATA-1, NF-E2) and ubiquitous (Sp1) factors. A single enhancer is required for the separate control of each of the five globin genes in the cluster, which are either expressed in an embryonic (ε), fetal (Gγ, Aγ) or adult (δ, β) specific manner. The model held to account for this globin gene switching is the competition between promoters for the shared enhancer, which assists in the formation and maintenance of an open chromatin structure at the active locus. At each stage of development the precise array of transcription factors bound at the individual promoters, or the distance of the promoter from the LCR can dictate exactly which set of sequences the LCR interacts with. An adult stage-specific protein, NF-E4, may switch the balance in favour of expression of the p gene over the ε gene, while other proteins may bend the DNA in a manner that decreases likelihood of enhancer interaction. EPITHELIAL GENE REGULATION The purpose of this review is to address epithelial gene regulation. Specifically, we address the transcriptional control of a subset of broadly expressed cellular genes as well as that of an epithelial specific virus. It would not be feasible to include the literature about subjects as diverse as transcriptional regulation in epithelia of the liver, the kidney, or the intestines (to name but a few), or developmental processes during embryogenesis. As different epithelia mediate different processes, it is not surprising to note transcription factors which predominate in each tissue type. Examples include murine homologues (Cdx-1, Cdx-2) of the Drosophila homeobox gene, caudal, which contribute to the specific activation of a number of intestine specific genes (Beck et al., 1995; Suh et al., 1994), the epithelially enriched transcription factor, wingedhelix nude, important in regulating keratinocyte and thymic epithelial differentiation (Brissette et al., 1996; Nehls et al., 1996), and thyroid transcription factor 1, which participates in regulating genes specific for the respiratory epithelium (Ikeda et al., 1996). Most of these genes possess binding sites for “ubiquitous” transcription factors in addition to the specific elements. Epithelia can be considered to be the most basic cell type, as the 8-cell compacted morula is comprised of epitheloid cells, which develop characteristics of adult epithelia after the transition to the blastocyst. This trophoectodermal epithelium is destined to form the placenta, however, by implantation, primitive embryonic endodermal and ectodermal cells are determined (Fleming and Johnson, 1988). From these early epithelial cells differentiates the mesenchyme, which in some cases converts back into epithelium. A model of the epithelial cell as the “default phenotype” has been proposed (Frisch, 1994), whereby in the absence of cell type specific factors, ubiquitous factors activate transcription programs that result in an epithelial phenotype. Such a model would seem plausible considering the fluidity of the epithelial state. It would inherently require the down-regulation of epithelial genes in non-epithelial cells, which is in fact observed

66


for keratin genes in keratinocyte/ fibroblast hybrids (Peehl and Stanbridge, 1981) and for cytokeratin K8 repression in embryonic and carcinoma cells (Cremisi and Duprey, 1987). K18 and K8 Cytokeratin 18 (K18) is expressed in simple epithelia such as the linings of the urinary bladder, the endocervix, lung alveoli, the gall bladder, the small intestine and the colon (Quinlan et al., 1985). K18 copolymerizes with K8 to form the first intermediate filaments expressed during development (Neznanov and Oshima, 1993). The temporal and spatial coincidence of these proteins invites one to hypothesize that similar mechanisms may govern the transcription of their genes. K8/ K18 are occasionally expressed concomitantly with K7 and K19, which also preferentially occur in simple epithelia, suggesting different regulatory mechanisms for the two keratin pairs. The K18 protein is encoded in 7 exons that extend over a genomic region of 4 kb. Transfection of a human K18 genomic clone comprising all 7 exons and 2 kb 5' and 4 kb 3' flanking region led to promiscuous expression in fibroblasts (Kulesh and Oshima, 1988), while in transgenic mice expression was appropriate with regard to both tissue specificity and efficiency (Abe and Oshima, 1990). In search of potential cisresponsive elements responsible for K18 expression, 7 DNase I hypersensitive sites have been mapped in permissive cells of these transgenic mice. Two sites were in an Alu repeat 5' of the K18 promoter, one site identified the promoter itself, three sites were in the first intron, and one site in the sixth exon (Neznanov and Oshima, 1993). The Alu repeats and their RNA polymerase III promoter insulates the K18 transcriptional unit from surrounding sequences, permitting copy number-dependent expression of the transgene (Thorey et al., 1993). Activation of the K18 promoter depends on at least one of four potential Sp1 binding sites, and up-regulation of Sp1 activity may be one of several mechanisms that contribute to cell type specificity of K18 expression and its alteration during cancer progression (Gunther et al., 1995; Apt et al., 1996). The hypersensitive sites in the first intron identify an enhancer with an Ets and an AP-1 site, the latter mediating K18 induction by members of the jun and fos families (Oshima et al., 1990). These sites also mediate K18 induction by oncogenes that stimulate the ras signalling pathway, a possible explanation for K18 expression in a variety of tumors (Pankov et al., 1994a). K18 induction during retinoic acid induced differentiation of embryonic carcinoma and embryonic stem cells to extraembryonic endoderm involves not only the expression of favorable Ets and AP-1 factors (Ets-2, c-jun and JunB respectively), but also the relief of three intronic silencing elements (Pankov et al., 1994b). The chromatin conformation of the promoter and 5' regions of K18 also seem to provide a level of control between expressing and nonexpressing tissues (Neznanov and Oshima, 1993). The hypersensitive site in the sixth exon identifies a strong enhancer, which has not yet been characterized in detail, but contains putative AP-1 and NFI-binding sites. The mouse K8 homologue (Endo A) is influenced by a 3' enhancer, whose function depends on multiple ets-binding sites (Fujimura et al., 1994), the more crucial of which bind an Ets protein related to Ets-2 (Fujimura et al., 1994). AP-1 binding sites also feature in the first intron and 3' downstream region of murine K8 (Tamai et al., 1991). It is not known whether this similarity between the K8 and K18 genes is central to the molecular mechanism of their co-expression. Studies of the human K8 gene in transgenic mice have revealed intragenic sequences essential for transgene expression. The 12 kb DNA fragment used contained the gene, 1.1 kb of upstream and 3.2 kb of downstream sequence, however the transgene expression efficiency was compromised with respect to the endogenous gene, indicating more distal regulatory elements may be required (Casanova et al., 1995). The 5' region and first intron of human K8


67

contain consensus Sp1 elements (one and three motifs, respectively) and the intron additionally contains an AP-1 site and an Alu element (Krauss and Franke, 1990). K5 and K14 Cytokeratin 5 (K5) and K14 are expressed in mitotic basal cells of the majority of stratified epithelia, but are transcriptionally down-regulated in suprabasal cells, where their place is taken by K1 and K10 (in the epidermis) or K4 and K13 (in mucosal epithelium). The human K5 promoter has a complex structure of adjacent or overlapping binding sites for AP-2, Sp1 and other yet unidentified factors (Byrne and Fuchs, 1993; Ohtsuki et al., 1993). In transgenic mice, transgenes with K5 promoter sequences are expressed in a cell type specific manner, but lose the restriction for basal cells. This observation indicates additional regulatory elements reside in a fragment encompassing 6 kb of the 5'-flanking region, which determine differentiation specificity (Byrne and Fuchs, 1993). Genomic sequences extending 5.3 kb upstream from the 5' end of the bovine K5 transcript suffice to give epithelial and basal cell specific expression in transgenic mice, proximal sequences extending up to –1.3 kb were inactive (Ramirez et al., 1994). This is in contrast to the findings with the human K5 gene, where the promoter alone demonstrated specific expression, as there is significant similarity between the human and bovine K5 promoter sequences and complete conservation of an AP-2, “site A” and an Sp1 binding motif. Strangely, in in vitro transfection experiments with bovine K5 constructs, enhancer elements between –1009 to –762 were sufficient for tissue specific expression. The only cis-responsive element that was detected in this fragment was an AP-1 site. Unexpectedly, this AP-1 site was protected by nuclear extracts from fibroblasts as well as epithelial cells, while activity mediated by this site was specific for epithelial cells, suggesting the involvement of sequences surrounding the core AP-1 site or differences in the composition of AP-1 in different cell types (Casatorres et al., 1994). The promoter of the human K14 gene demonstrates some sequence similarity with the K5 promoter and possibly similar cis-reponsive elements. These elements include AP-2 and Sp1 binding sites (Leask et al., 1990; Leask et al., 1991; Ohtsuki et al., 1993). Both K5 and K14 are transcriptionally down-regulated by retinoic acid treatment, an effect mediated by 5' regulatory sequences (Stellmach et al., 1991). A retinoic acid and thyroid hormone receptor responsive element has been identified in the K14 promoter, and sequence similarities with the K5 promoter support gene regulation by these hormones (Tomic-Canic et al., 1992). K1 Cytokeratin 1 (K1) is expressed in epidermal keratinocytes, its expression up-regulated in cells of the spinous layer and then down-regulated as cells mature and progress into the granular layer of the epidermis. A number of factors that modulate in vitro epidermal differentiation also modulate K1 transcription, such as calcium and vitamin D3, which induce differentiation and K1, and retinoic acid treatment, which suppresses different ion and K1 (for references see Lu et al., 1994). An enhancer 1.7 kb downstream from the coding regions of K1 mediates calcium inducible and epithelial specific transcription. Cell specificity and calcium inducibility are controlled by separate elements, as removal of the most 3' 207 bp generated a calcium responsive enhancer active in both fibroblasts and epithelia (Huff et al., 1993). The element responsible for cell specific repression was not identified. Footprinting of the calcium responsive element identified closely spaced AP-1 and steroid hormone binding sites, the former mediating regulation by calcium (Lu et al., 1994). It is conceivable that the calcium response originates from transcriptional modulation of the c-fos gene,

68


which encodes a subunit of AP-1 (Collart et al., 1991), but it is unclear why this response has not been reported for other epithelial enhancers with AP-1 sites. Vitamin D3 suppresses the calcium response, an effect antagonized by the coaddition of retinoic acid, possibly due to competition of the two receptors for their common coactivator, RXR (Lu et al., 1993). K6, K16, and K17 The cytokeratins 6 (K6) and K16 are expressed in the root sheaths of hair follicles, in suprabasal cells of some internal stratified epithelia, and in the epidermis during hyperproliferative situations such as tumorigenesis and wound healing. The bovine K6 promoter has been studied in transgenic mice and in vitro (Ramirez et al., 1995; Navarro et al., 1995), and the human K6 promoter in vitro only (Bernerd et al., 1993; Jiang et al., 1993). The K6 promoters of both species are similar and may contain identical cis-responsive elements. While promoter fragments from both species including up to 650 bp 5'-flanking sequence were active in transfections, the bovine K6 promoter did not stimulate reporter gene expression in transgenic mice. The sequences contained in the 9 kb upstream of the promoter segment necessary for expression in vivo were not identified (Ramirez et al., 1995). Seven factor binding sites have been identified in footprint and gelshift experiments of the bovine and human K6 promoter segments, namely AP-1, AP-2, and retinoic acid response elements and binding sites for unknown factors. One of these elements mediated an epidermal growth factor response, and this element has similarity to a promoter element of the K16 gene with the same function (Jiang et al., 1993). K17 is not expressed in healthy skin, but only under various pathological conditions. A 450 promoter fragment of the human K17 gene contains binding sites for NFI, AP-2, and Sp1, an interferon-gammaresponse element, and three binding sites for unidentified factors. Mutagenesis confirmed that these binding sites are functional as cis-responsive elements (Milisavljevic et al., 1996). Loricrin Loricrin is a major component of the cell envelope of terminally differentiating statifled squamous keratinocytes, and its expression is regulated during differentiation. Transcripts appear in upper spinous layers and accumulate in the granular layer of the epidermis (Mehrel et al., 1990). The loricrin expression pattern is similar to that of filaggrin, but differs from the spinous layer specific transcription of K1. Transcription of all three genes correlates with the endogenous transepidermal calcium gradient and can be modulated in cell culture by the concentration of calcium ions. To localize elements governing the transcription of mouse loricrin, the single coding exon of a 6.5 kb genomic clone was replaced with reporter genes. The constructs retained 1.5 kb of 5'-flanking region, a short non-coding exon, a 1.1 kb intron in the 5'-region and 2.2 kb of the 3'-flanking sequences (DiSepio et al., 1995). High levels of reporter product were observed after transfection into murine keratinocytes cultured under high or low calcium, while there was no expression in dermal fibroblasts. Transgenic mice harboring the promoter-reporter construct showed strong expression of the transgene not only in the differentiated layers of the epidermis but also in the undifferentiated basal layer, indicating that elements within the non-coding 6.5 kb of DNA are sufficient for tissue specific regulation, but insufficient for modulation during differentiation. Surprisingly, deletions up to 60 bp 5' of the transcription start site retained high levels of promoter activity, with footprint and gelshift experiments pinpointed an AP-1 site as responsible for this activity (DiSepio et al., 1995). This AP-1 site is conserved between the mouse and human loricrin promoters, and its mutation abolished promoter activity. It is not known why the loricrin


69

AP-1 site does not suffice to couple transcription to calcium induced differentiation, in the manner in which the AP-1 site of K1 appears to. Differentation responsive regulatory elements may reside outside the 6.5 kb, as a human loricrin transgene with 9 kb 5'-flanking and 9 kb 3'-flanking DNA responded to differentiation in transgenic mice (Yoneda and Steinert, 1993). Involucrin Involucrin is enzymatically cross-linked with other proteins of the cornified envelope produced in the upper layer of the epidermis. Keratinocyte differentiation as well as involucrin expression is induced upon TPA (12-O-tetradecanoyl phorbol13-acetate) treatment of cells. Analysis of the 5' control region of the gene revealed several TPA response elements, which are bound by AP-1 (Welter et al., 1995). Although footprinting studies to map binding sites for other factors have not been performed, mutation of two AP-1 sites diminished epithelial specific promoter activity of a 2.5 kb subclone of the 5' region by 80%. Bandshifts with extracts from TPA treated keratinocytes showed enhanced binding of AP-1 to the involucrin promoter, and these complexes were comprised of the subunits Fra1, JunB and JunD (Welter et al., 1995). POU-domain factors also regulate a 2.5 kb involucrin promoter construct, with Oct-1, Oct-2, Brn4, Oct-6/SCIP and Skn1i/a inhibiting basal and TPA induced promoter activity (Welter et al., 1996). Interestingly this down-regulation is not dependent on octamer binding sites, a phenomenon observed in a number of other promoters (Monuki et al., 1993). E-cadherin E-cadherin, also referred to as uvomorulin, is an epithelial specific transmembrane protein involved in the formation of adherens junctions via the interaction between extracellular domains of E-cadherin molecules of neighboring cells. Intracellularly, E-cadherin complexes with catenins and actin and therefore mediates between the extracellular matrix and the cytoskeleton, with possible consequences for intracellular signalling. E-cadherin is widely expressed in epithelial cells of all differentiation types, including the morula stage of embryogenesis, but is lacking in other cell types. E-cadherin is encoded by 16 exons that extend over a genomic segment of at least 40 kb (Ringwald et al., 1991). Some part of the regulation of E-cadherin expression may occur on the level of chromatin condensation, as five DNase hypersensitive sites found in E-cadherin expressing cells are inaccessible in non-expressing lines (Hennig et al., 1995). Three of these sites map to a 400 bp segment upstream of the transcription start, while the other two are located 700 bp and 1.2 kb downstream, in the first and the second intron, respectively. Part of the epithelial specificity of E-cadherin transcription resides in the promoter, as genomic segments including 178 bp upstream of the start of the human gene (Behrens et al., 1991) or 1.5 kb around the start of the mouse gene (Ringwald et al., 1991) suffice for cell type specificity in transfection assays. While the E-cadherin promoter lacks a TATA box, three defined footprints in this promoter fragment protect sequences that are conserved between the mouse and the human E-cadherin promoters, and center on positions –79, –61, and –39, 5' of the transcription start. They are referred to as the E-palindrome, CCAAT-box and GC region. It is not known which factor is bound by the E-palindrome, although the sequence may suggest a member of the helix-loop-helix factor family. The factor occurs in epithelial cells as well as in fibroblasts, but down-regulates transcription only in the latter cells (Hennig et al., 1996). The CCAAT-box is homologous to a half-palindromic NFI-binding site (Gloss et al., 1989), but affinity to the factor CP1 cannot be excluded (Chodosh et al., 1988). The 33 bp GC region binds AP-2 (Hennig et al., 1996) but is also modulated by Sp1 and Sp3, as expected from the presence of a GGGCGG Spl-binding site

70


(Apt et al., 1996). Specificity of E-cadherin transcription is further stimulated by an intronic enhancer with three binding sites. Two of these sites are differentially protected by nuclear extracts from E-cadherin expressing and non-expressing cells, respectively. The protected sequences are suggestive of AP-2 and H4TF-1 binding sites, but binding of these factors has yet to be confirmed. Transglutaminase and Profilaggrin Transglutaminase 1 is expressed in all tissues of epithelial origin, while transglutaminase 3 is specific for the terminal differentiation of the epidermis. Although not functionally examined, AP-1, Sp1, NFI, and AP-2 motifs feature in the 5' regulatory region of the transglutaminase 1 gene (Yamanishi et al., 1992; Saunders et al., 1993). The transglutaminase 3 gene has an epithelial specific promoter which relies upon cooperativity between an Sp1 and an Ets-like motif, although elements required for differentiation-specific transcription lie outside the promoter proximal 3 kb (Lee et al., 1996). Profilaggrin is expressed in the granular layer of the skin. The precursor protein is proteolytically cleaved and dephosphorylated to give the active filaggrin, which assists in the aggregation of keratin filaments in the cornified layer. Sequences indicative of overlapping AP-1 and retinoic acid/thyroid hormone response elements were identified in the first intron of the profilaggrin gene, and although only a short stretch of 5' sequences were available for analysis, the absence of AP-2 motifs is notable (Presland et al., 1992). Human papillomaviruses Human papillomaviruses (HPVs) strictly require epithelial host cells. HPVs infect the basal cells of stratified mucosal or cutaneous epithelia, and express viral E6 and E7 proteins which induce continuing mitoses in the normally mitotically inactive suprabasal layers by annihilating p53 and pRb cell cycle control (for a review see Howley, 1996). More than 70 HPV types are known, and each HPV type infects preferentially (but not exclusively) either skin, mucosal stratified epithelia, or the simple columnar epithelium of the endocervix. Infections of other simple epithelia accessible to the infectious agent, such as those of the colon or lung have not been observed. Most research has centered on HPV-11, which causes genital and laryngeal warts, and HPV-16 and HPV-18, which cause cervical carcinomas. The specificity of HPV for epithelia does not arise from preferential infection, but from selective activation of HPV transcription, with about 500 bp of the 8 kb viral genome functioning as an epithelial specific enhancer-promoter (Cripe et al., 1987; Gloss et al., 1987). The enhancer-promoter of HPV-16 is regulated by at least nine different transcription factors, AP-1, NFI, Oct-1, the progesterone receptor, Sp1, TEF1, TEF2, YY1, and the HPV transcription factor E2 (for reviews see Bernard and Apt, 1994; O’Connor et al., 1995). Investigators of HPV transcription have a unique research tool at their disposal, namely the sequence comparison of the genomes of many different HPV types. As the enhancer-promoter regions of different HPV types are more than 80% dissimilar, it is likely that conserved regions are maintained because they are required for aspects of HPV biology, such as its epithelial specificity. Surprisingly, most or possibly all of the 9 different types of transcription factor binding sites are conserved between the 35 HPV types that infect mucosal epithelia. It is also of interest that activation of HPV transcription occurs equally well in cell lines that represent the natural targets of HPVs, such as those derived from cervical carcinomas, as in cells that are normally not infected by mucosal HPVs, e.g. cells derived from the epidermis or mammary carcinomas (Chong et al., 1991). The search for epithelially restricted factors involved in HPV transcription has not yielded tangible data. A suspected keratinocyte specific element, the CK 8-mer, binds the ubiquitous factor NFI (Gloss et al.,


71

1989), a presumed keratinocyte specific factor, KRF1, is probably widespread (Kyo et al., 1995), and a binding site in HPV-18 for Skn-1, a tissue restricted POU-domain factor, lies outside the enhancer-promoter segment with epithelial specific function (Yukawa et al., 1996). A highly conserved octamer-NFI hybrid motif is not targeted by Skn-1 (O’Connor and Bernard, 1995). There is support for the hypothesis that epithelial specificity is induced by cooperativity between various ubiquitous transcription factors, and that some of these factors have epithelial specific functions, although they bind ubiquitously. Epithelial cells possess an excess of the activator Sp1 over the represser Sp3, such that binding sites in epithelia are largely occupied by the activator, whereas the reverse is true in fibroblasts (Apt et al., 1996). NFI binding factors are comprised of the activator NFI-CTF, rather the repressor NFI-X1, in epithelia, and the opposite is found in fibroblasts (Apt et al., 1994). Jun-B is a major Jun family AP-1 subunit expressed in epithelia, whereas cJun is expressed in fibroblasts, and is unable to transactivate an HPV promoter (Thierry et al., 1992), while a coactivator for TEF1 is missing in many cell types that do not support HPV transcription (Ishiji et al., 1992). The potential epithelial regulation of the repressor YY1 has not yet been examined (Bauknecht et al., 1992; O’Connor et al., 1996). ARE THERE EPITHELIAL SPECIFIC TRANSCRIPTION FACTORS? Research on epithelial gene regulation has been inspired by the hope to detect epithelial specific transcription factors. Assumptions about such hypothetical factors include that they would (i) bind to unique nucleotide sequences and (ii) be present only in epithelial cells, and absent elsewhere. If such factors were to be found, the genes encoding these proteins may be regulated themselves by some epithelial specific mechanism, which may point to the regulatory cascades responsible for epithelial determination. A number of factors have been proposed as epithelial specific regulators, but none have so far satisfied all the criteria. One of the first possible epithelial specific cis-responsive elements arose from the observation that the promoters of several cytokeratin genes share little similarity besides the TATA box and, in a 5'-position, the sequence AAPuCCAAA, a similarity deemed to be too unlikely to be explained by chance alone (Blessing et al., 1987). The authors did not study the functions of this potential element, and later found that enhancer sequences of the bovine cytokeratin IV* gene excluded this element and yet directed epithelial expression (Blessing et al., 1989). In spite of these empirical limitations, the sequence AAPuCCAAA became widely cited as a “CK element” and generated excitement, particularly as similar sequences were found in the enhancers of HPVs (Cripe et al., 1990; Fuchs, 1993). While no epithelial restricted factor appeared to bind this sequence, it became clear that the CK element is one out of many similar motifs that represent halfpalindromic NFI-binding sites (Gloss et al., 1989). In the light of more recent observations that the NFI factor family is capable of compositional variation, it must be concluded that these sequences do cooperate in epithelial specific transcription, although the term “CK element” represents a misnomer. A different sequence, GCCTGCAGGC, located 5' from the TATA box of the human K14 gene, was found to control transcription of K14 in conjunction with another distal element (Leask et al., 1990). The factor was given the provisional name, KER-1, but was later found to be identical to AP-2 (Fuchs, 1993). Similarly, an activator of keratin transcription during Xenopus embryogenesis found to be enriched in skin, was termed KTF-1, while it later became clear that it is closely related or identical to AP-2 (Snape et al., 1991). Although AP-2 has been found to be significantly more abundant in epidermal keratinocytes than in other unrelated tissues, it does not seem to be sufficient for keratinocyte specific gene expression (Leask et al., 1991; Mitchell et al., 1991). Anderson and coworkers (1993) have decribed the factor Skn-1, a member of the POU-domain containing transcription factor family. POU-domain factors play important regulatory roles in development

72


and differentiation, including cell specification. Not surprisingly, expression of the majority of these factors is enriched in a subset of tissues (Wegner et al., 1993). Skn-1 i is a transcriptional represser related to Oct-2, which is found in greatest abundance in the epidermis. Studies with the related and most probably identical factors, Epoc-1 and Oct-11 indicate expression also occurs in the thymus, stomach and testis (Yukawa at al., 1993; Goldsborough et al., 1993). An alternative transcription activating spliced form, Skn-1 a, increases the complexity of Skn-1 factors. Skn-1a probably interferes with the inhibitory action of Skn-1 i in regions of the epidermis in which both factors are expressed (Anderson et al., 1993). A natural cis-responsive element targeted by Skn-1 is not yet known, although a positive influence on the K10 promoter could be measured. Oct-6 (SCIP, Tst-1) also appears to be involved in epidermal gene regulation, although the factor is also expressed in a number of other stratified epithelia (Faus et al., 1994). Basonuclin is a nuclear protein specific to keratinocytes, and occurs in hair follicles and in the epidermis, where it is confined to cells of the basal layer. As basonuclin possesses zinc fingers and other sequence motifs suggestive of transcription factors, it is presumed to bind DNA and regulate epithelially expressed genes, although neither has yet been demonstrated (Tseng and Green, 1992). SPECIFICITY THROUGH UBIQUITOUS TRANSCRIPTION FACTORS From what has been said it will be clear that there is presently no strong evidence for the transcriptional activation of any gene by strictly epithelial specific transcription factors. Factors such as Skn-1 and basonuclin, that may be epithelial specific, may not be directly involved in the regulation of any of the genes addressed here. Other factors initially surmised to be epithelial specific may be only epithelially enriched, such as AP-2, or, like CK/NFI, mistaken in their identity. Against this background one has to conclude that epithelial specific mechanisms have to be searched for elsewhere, for example in the properties of factors that, at first glance, were considered to occur ubiquitously. This hypothesis is complemented by the intriguing observation that epithelial genes are not regulated by any random combination of the hundreds of transcription factors known to date, but that a very small set of factors are shared by many (although possibly not all) epithelial specific enhancers and promoters. Among these, regulation by AP-1, AP-2, NFI, Sp1 and the retinoic acid receptor has been observed most frequently. AP-1: AP-1 sites have occurred frequently as important epithelial cis-responsive elements. They have been discussed in connection with epithelial specificity (Chong et al., 1990; Thierry et al., 1992; DiSepio et al., 1995), activation in embryonal stem cells (Kulesh and Oshima, 1988), and calcium responsiveness (Lu et al., 1994). There is no problem in associating pleiotropic functions with the same or similar cisresponsive elements, as AP-1 can have very diverse properties: AP-1 factors are formed as homo- or heterodimers of the products of the jun and the fos-gene family. The differentially regulated genes of these families give rise to proteins with diverse functions, based both on the properties of the subunits and the combined properties of the dimers, furthermore the subunits can undergo post-translational modifications in response to intracellular signals (for reviews see Angel and Herrlich, 1995). The differential subunit composition of AP-1 between epithelial and non-epithelial cells, combined with an increased binding affinity, has been proposed to explain the factor’s contribution to specificity. The Jun family members junB and jun D, and fos family proteins Fra-1 and Fra-2 have been identified in AP-1 binding complexes in the epidermis (Thierry et al., 1992; Welter at al., 1995; Rutberg et al., 1996). Transfected c-jun is unable to activate the epithelial specific HPV-18 enhancer, which is preferentially bound by JunB in keratinocyte extracts (Thierry et al., 1992), while protein kinase C is required for functional changes during epidermal differentiation (Rutberg et al., 1996). Quite a number of studies of the expression of AP-1 subunits in situ have been performed, but post translational modifications, interactions with AP-1 coactivators (Claret et al.,


73

1996), or interference by a cytosolic inhibitor of binding (Auwerx and Sassoni-Corsi, 1991), have not yet been detected. AP-2: In recent years the term AP-2 has been expanded to include a number of related proteins. Three distinct genes encode AP-2, AP-2b and AP2.2, all of which are transcriptional activators, and AP-2B, a negative regulator of transcription, is derived by alternative splicing of AP-2 transcripts (Buettner et al., 1993). Multiple forms of AP-2 are expressed during early embryogenesis, principally in the neural crest. As cell lineages diverge, diffential AP-2 expression becomes established. AP-2, AP2.2 and AP-2b are found in the surface ectoderm and facial mesenchyme , however AP2.2 is enriched in nasal and oral epithelia, while AP-2 and AP-2b are differentially expressed in various sectors of the midbrain (Moser et al., 1995; Chazaud et al., 1996; Mitchel et al., 1991). AP-2 expression preceeds that of K5 in the developing surface ectoderm, and functionally interacts with the promoters of K5, K14, K1 (Leask et al., 1990; Leask et al., 1991) and Ecadherin (Hennig et al., 1996). The multiplicity of AP-2 proteins may allow a different set of genes to be expressed in each tissue type. The proteins may also be modulated in activity by signalling molecules such as protein kinase C and cAMP (Lüscher et al., 1989). It is also of interest that the AP-2 promoter is itself bound by ubiquitous factors, namely AP-2, NFI and Oct factors (Greaser et al., 1996). NFI: Nuclear factor I was originally believed to be derived from a single gene whose differentially spliced products could bind DNA as homo- or heterodimers of the spliced forms. Later, it became clear that there were four NFI genes, NFI-A, B, C, and X, whose products could be differentially expressed, spliced and could readily heterodimerize (for references see Apt et al., 1993 and 1994). Among the four genes, the NFI-X gene is poorly expressed in epithelial cells, but strongly in mesenchymal cells, and its major splice product, NFI-X 1, is a potent antagonist of activation by NFI-C (Apt et al., 1994). In other words, NFI binding sites function as epithelial specific cis-responsive elements, with activating NFI-C homodimers binding in epithelia, and repressing NFI-X homo- or heterodimers binding in mesenchymal cells. Sp1: Sp1 sites are found in epithelial as well as in ubiquitously active promoters. Sp-factors are not the products of a single gene, but of four different genes, two of which (Sp1 and Sp3) are expressed in epithelial as well as mesenchymal cells. Sp1 has transcriptional activation functions, however Sp3 is a represser (Hagen et al., 1994). While in case of NFI, the represser NFI-X is up-regulated to give nonepithelial repression, there is little modulation of the represser Sp3. In contrast, the activator Sp1 is upregulated in epithelial cells such as HeLa, and weakly expressed in fibroblasts. This leads to an epithelial preferential activation of promoters with Sp-factor binding sites, independent whether the linked gene is naturally epithelial specific or ubiquitously transcribed (Apt et al., 1996). RAR/RXR: Retinoids have long been known to play a role in development and morphogenesis, and act via members of the DNA binding nuclear hormone receptor families RAR and RXR. The retinoic acid receptors (RARα, β, γ) are activated by both all-trans and 9-cis retinoic acids, whereas the retinoid X receptors (RXRα, β, γ) bind only the 9-cis isomer. RXRα is expressed late in fetal development in epithelial tissues such as the linings of the nasal and oral cavities and the stomach, and is enriched in the epidermis, RXRβ is more ubiquitously expressed, and RXRγ in myogenic lineages and otic epithelium (Dollé et al., 1994). RARγ is the major epithelial retinoic acid receptor, and is found in the skin, oral cavity, oesophagus and stomach (Ruberte et al., 1990). There are differences in retinoic acid receptors between different epithelia such as the simple columnar and stratified squamous epithelia of the cervix (Darwiche et al., 1994). Retinoids are generally regarded to inhibit epidermal differentiation, although a minimal level is required for maintenance of normal cervical epithelial differentiation (Darwiche et al., 1994). Epidermally targeted expression of a dominant negative RAR results in an abnormal skin phenotype in transgenic mice, disrupting the commitment to differentiation (Saitou et al., 1995). Similarly, RARs are essential for suprabasal differentiation processes and thus required for skin maturation (Imakado et al., 1995). It is

74


important to note that the broad effects of retinoic acid on epithelial genes may not necessarily be mediated by response elements in the control regions of the genes themselves, but may be due to effects on the regulators of transcription, such as the binding interference of RARs and RXRs on AP-1. TRANSCRIPTIONAL CONTROL TRIGGERED BY THE EXTRACELLULAR MATRIX AND BY GROWTH FACTORS A basement membrane comprised of extracellular matrix (ECM) containing a basal lamina and often a sublaminal layer of collagen fibrils separates epithelial sheets from their underlying supporting tissues. The interaction of epithelial cells and the ECM has significant effects on differentiation and transcription, depending on the composition of the matrix (reviewed in Adams and Watt, 1993). An example is the culture of mouse mammary epithelial cells on reconstituted ECM in the presence of lactogenic hormones, which results in the formation of alveolar-like structures, induction of milk protein expression and secretion of milk fat globules dependent on the ECM component laminin (Aggeler et al., 1991; Streuli et al., 1995). Cells posses fibronectin or integrin receptors that help to transduce external information, such as the compostion of the ECM, to the cytosol. Integrin receptors are heterodimers of many different α and β family subunits, and the heterodimer composition determines the receptor specificity for ECM components. The cytosolic signalling cascade stimulated by the integrin receptor/ligand interaction (reviewed in Clark and Brugge, 1995) and the determination of ECM responsive DNA elements (Schmidhauser et al., 1992) are currently under study. It is of interest to note that epithelial cell-matrix interactions are essential to cell survival (Frisch and Francis, 1994), as removal of matrix or neutralisation of β1 intergrins with antibodies induces interleukin-1β converting enzyme, and apoptosis ensues (Boudreau et al., 1995). The normal loss of surface β1 integrins from differentiating epidermal cells is dependent on functional E-cadherin complexes, which raises questions about overlap in signalling targets between the two (Hodivala and Watt, 1994). Epithelial cells are subject to a number of other signal stimuli. Proliferation inducing signals are provided by EGF, TGF-α, KGF and some cytokines, whereas members of the TGF-β family appear to be involved in withdrawal from the cell cycle (Fuchs, 1993). A number of epithelially enriched receptor tyrosine kinases (c-met, c-ros, c-neu, KGF receptor) are involved in transducing signals from the mesenchyme, influencing cell motiiity, differentiation and branching morphogenesis during development and the wound response (Sachs et al., 1996). Signal transduction through the E-cadherin/β-catenin complex results in the nuclear trans location and DNA interaction of a lymphoid enhancer-binding factor (LEF)/β-catenin complex (Behrens et al., 1996). It is interesting to note that LEF-1 binding motifs are present in hair keratin promoters (Zhou et al., 1995). An epithelial protein kinase C isoform is also involved in the TPA induction of tranglutaminase 1 (Ueda et al., 1996). CONCLUSIONS Studies of the transcriptional control of epithelial specific genes have identified epithelial regulatory elements, which on further examination have been found to bind apparently ubiquitous factors. An hypothesis is gaining credence, that such specificity is a result of the interplay of these ubiquitous factors, in the absence of other cell type determining proteins. Further research has indicated that some of these ubiquitously binding factors vary functionally between cell types. Expression of different members of a gene family, or differently spliced forms of a single gene can have important functional ramifications. Although the possibility of epithelial specific coactivators has not been thoroughly examined, it is clear that AP-1, AP-2, NFI-C, and Sp1 can all stimulate epithelial specific as well as differentiation specific transcription, but none


75

of them is sufficient in isolation to instigate these programs. In spite of this progress in the identification of some components of the epithelial specific transcription program, detailed studies of the mechanistic intricacies, and of different regulatory systems such as those that act on the level of chromatin conformation, remain challenges for the future. REFERENCES Abe, M. and Oshima, R.G. (1990) A single human keratin 18 gene is expressed in diverse epithelial cells of transgenic mice. J. Cell Biol., 111, 1197–1207. Adams, J.C. and Watt, F.M. (1993) Regulation of development and differentiation by the extracellular matrix. Development, 117, 1183–1198. Aggeler, J., Ward, J., Blackie, L.M., Barcellos-Hoff, M.H., Streuli, C.H. and Bissell, M.J. (1991) Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo. J. Cell Science, 99, 407–417. Andersen, B., Schonemann, M.D., Flynn, S.E., Pearse, R.P. 2nd., Singh, H. and Rosenfeld, M.G. (1993) Skn1a and Skn-1i: two functionally distinct Oct-2 related factors expressed in the epidermis. Science, 260, 78–82. Angel, P.E. and Herrlich, P.A. (eds.) (1995) The Fos and Jun families of transcription factors. CRC Press, Boca Raton. Apt, D., Chong, T., Liu, Y. and Bernard, H.U. (1993) Nuclear factor I and epithelial cell-specific transcription of human papillomavirus type 16. J. Virol., 67, 4455–4463. Apt, D., Liu Y., Bernard, H.U. (1994) Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell type specific manner. Nucleic Acids Res., 22, 3825–3833. Apt, D., Watts, R.M., Suske, G. and Bernard, H.U. (1996) High Sp1/Sp3 rations in epithelial cells during epithelial differentiation and cellular transformation correlate with the activation of the HPV-16 promoter. Virology, 224, 281–291. Auwerx, J. and Sassoni-Corsi, P. (1991) IP-1: a dominant inhibitor of fos/jun whose activity is modulated by phosphorylation. Cell, 64, 983–993. Bauknecht, T. Angel, P., Royer, H.D. and zur Hausen, H. (1992) Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interactin with the transcriptional represser YY1. EMBO J., 11, 4607–4617. Beck, R, Erler, T., Russell, A. and James, R. (1995) Expression of Cdx-2 in the mouse embryo and placenta: possible role in patterning of the extra-embryonic membranes. Dev. Dyn., 204, 219–227. Behrens, J., Löwrick, O., Klein-Hitpass, L. and Birchmeier, W. (1991) The E-cadherin promoter: Functional analysis of a G-C-rich region and an epithelial cell-specific palindromic regulatory element. Proc. Natl. Acad. Sci. USA, 88, 11495–11499. Behrens, J., von Kries, J.P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Birchmeier, W. (1996) Functional interaction of beta-catenin with the transcription factor LEF-1. Nature, 382, 638–642. Bernard, H.U. and Apt, D. (1994) Transcriptional control and cell type specificity of HPV gene expression. Arch. Dermatology, 130, 210–215. Bernerd, F., Magnaldo, T., Freedberg, I.M. and Blumenberg, M. (1993) Expression of the carcinoma-associated keratin K6 and the role of AP-1 proto-oncoproteins. Gene Expr., 3, 187–199. Blessing, M., Zentgraf, H. and Jorcano, J.L. (1987) Differentially expressed bovine cytokeratin genes. Analysis of gene linkage and evolutionary conservation of 5'-upstream sequences. EMBO J., 6, 567–575. Blessing, M., Jorcano, J.L. and Franke, W.W. (1989) Enhancer elements directing cell-type-specific expression of cytokeratin genes and changes of the epithelial cytoskeleton by transfections of hybrid cytokeratin genes. EMBO J., 8, 117–126. Blouin, R., Blouin, M.J., Royal, L, Grenier, A., Roop, D.R., Loranger, A. and Marceau, N. (1992) Cytokeratin 14 expression in rat liver cells in culture and localization in vim. Differentiation, 52, 45–54.

76


Boudreau, N., Sympson, C.J., Werb, Z. and Bissell, M.J. (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science, 261, 891–893. Brissette, J.L., Kamimura, J., Lee, D. and Dotto, G.P. (1996) The product of the mouse nude locus, Whn, regulates the balance between epithelial cell growth and differentiation. Gems and Dev., 10, 2212–2221. Buettner, R., Kannan, P., Imhof, A., Bauer, R., Yim, S.O., Glockshuber, R., Van Dyke, M.W. and Tainsky, M.A. (1993) An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by Ap-2. Mol. Cell Biol., 13,4174–4185. Byrne, C. and Fuchs, E. (1993) Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice. Mol. Cell. Biol., 13, 3176–3190. Casanova, L., Bravo, A., Were, F., Ramirez, A., Jorcano, J.J. and Vidal, M. (1995) Tissuespecific and efficient expression of the human simple epithelial keratin 8 gene in transgenic mice. J. Cell Sci., 108, 811–820. Casatorres, J., Navarro, J.M., Blessing, M. and Jorcano, J.L. (1994) Analysis of the control of expression and tissue specificity of the keratin 5 gene, characteristic of basal keratinocytes. J. Biol. Chem., 269, 20489–20496. Chazaud, C., Oulad-Abdelghani, M., Bouillet, P., Decimo, D., Chambon, P. and Dolle P. (1996) AP-2.2, a novel gene related to AP-2, is expressed in the forebrain, limbs and face during mouse embryogenesis. Mech. Dev., 54, 83–94. Chodosh, L.A., Baldwin, A.S., Carthew, R.W. and Sharp, P.A. (1988) Human CCAAT-binding proteins have heterologous subunits. Cell, 53, 11–24. Chong, T., Chan, W.K. and Bernard, H.U. (1990) Cooperative activation of the enhancer of human papillomavirus 16 by nuclear factor I, AP1, steroid receptors and a possibly novel transcription factor, PVF: A model for the composition of genital papillomavirus enhancers. Nucleic Acids Res., 18, 465–470. Chong, T., Apt, D., Gloss, B., Isa, M. and Bernard, H.U. (1991) The enhancer of human papillomavirus-16: Binding sites for the ubiquitous transcription factors oct-1, NFA, TEF-2, NFI and AP1 participate in the epithelial specific transcription. J. Virol., 65, 5933–5943. Claret, F.-X., Hibi, M., Dhut, S., Toda, T. and Karin, M. (1996) A new group of conserved coactivators that increase the specificity of AP-1 transcription factors. Nature, 383, 453– 457. Clark, E.A. and Brugge, J.S. (1995) Integrins and signal transduction pathways: the road taken. Science, 233–239. Collart, M.A., Tourkine, N., Belin, D., Vassalli, P., Jeanteur, P. and Blanchard, J.M. (1991) c-fos gene transcription in murine macrophages is modulated by a calcium-dependent block to elongation in intron 1. Mol. Cell Biol., 11, 2826–2831. Creaser, P.C., D-Argenio, D.A. and Williams, T. (1996) Comparative and functional analysis of the AP-2 promoter indicates that conserved octamer and initiator elements are critical for activity. Nucleic Acids Res., 24, 2597–2605. Cremisi, C. and Duprey, P. (1987) A labile inhibitor blocks endo A gene transcription in murine undifferentiated embryonal carcinoma cells. Nucleic Acids Res., 15, 6105–6116. Cripe, T.C., Haugen, T.H., Turk, J.P., Tabatabai, F, Schmid, P.G., Dürst, M., Gissmann, L., Roman, A. and Turek, L. (1987) Transcriptional regulation of the human papillomavirus 16 E6–E7 promoter by a keratinocyte-dependent enhancer and by viral E2 trans-activator and repressor gene products: Implications for cervical carcinogenesis. EMBO J., 6, 3745– 3753. Crossley, M. and Orkin, S.H. (1993) Regulation of the β-globin locus. Curr. Opin. Genet. Dev., 3, 232–237. Darwiche, N., Celli, G. and De Luca, L.M. (1994) Specificity of retinoid receptor gene expression in mouse cervical epithelia. Endocrinology, 134, 2018–2025. DiSepio, D., Jones, A., Longley, M.A., Bundman, D., Rothnagel, J.A. and Roop, D.R. (1995) The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiationspecific expression. J. Biol. Chem., 270, 10792–10799. Dolle, P., Fraulob, V., Kastner, P. and Chambon, P. (1994) Developmental expression of murine retinoid X receptor (RXR) genes. Mech. Dev., 45, 91–104. Earnst, P. and Smale, S.T. (1995) Combinatorial regulation of transcription II: the immunoglobulin m heavy chain gene. Immunity, 2, 427–438. Faus, I., Hsu, H-J. and Fuchs, E. (1994) Oct-6: a regulator of keratinocyte gene expression in stratified squamous epithelia. Mol. Cell. Biol., 14, 3263–3275.


77

Fleming, T.P. and Johnson, M.H. (1988) From the egg to epithelium. Ann. Rev. Cell Biol., 4, 459–485. Frisch, S.M. (1994) Ela induces the expression of epithelial characteristics. J. Cell Biol., 127, 1085–1096. Frisch, S.M. and Francis, H. (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol., 124, 619–626. Fuchs, E. (1993) Epidermal differentiation and keratin gene expression. J. Cell Set., 17, 197– 208. Fujimura, Y., Yamamoto, H., Hamazato, F. and Nozaki, M. (1994) One of two ets-binding sites in the cytokeratin endoA enhancer is essential for enhancer activity and binds to ets-2 related proteins. Nucleic Acids Res., 22, 613–618. Gloss, B., Bernard, H.U., Seedorf, K. and Klock, G. (1987) The upstream regulatory region of the human papillomavirus-16 contains an E2 protein independent enhancer which is specific for cervical carcinoma cells and regulated by glucocorticoid hormones. EMBO J., 6, 3735–3743. Gloss, B., Yeo-Gloss, M., Meisterernst, M., Rogge, L., Winnacker, E.L. and Bernard, H.U. (1989) Clusters of nuclear factor I binding sites identify enhancers of several papillomaviruses but alone are not sufficient for enhancer function. Nucleic Acids Res., 17, 3519–3533. Goldsborough, A.S., Healy, L.E., Copeland, N.G., Gilbert, D.J.Jenkins, N.A., Willison, K.S. and Ashworth, A. (1993) Cloning, chromosomal localization and expression pattern of the POU domain gene Oct-11. Nucleic Acids Res., 21, 127–134. Griffo, G., Hamon-Benais, C., Angrand, P.O., Fox, M., West, L., Lecoq, O., Povey, S., Cassio, D. and Weiss, M. (1993) HNF4 and HNF1 as well as a panel of hepatic functions are extinguished and reexpressed in parallel in chromosomally reduced rat hepatoma-human fibroblast hybrids. J. Cell Biol., 121, 887–898. Gunther, M., Frebourg, T., Laithier, M., Fossar, N., Bouziane-Ouartini, M., Lavialle, C. and Brison, O. (1995) An Sp1 binding site and the minimal promoter contribute to overexpression of the cytokeratin 18 gene in tumorigenic clones relative to that in nontumorigenic clones of a human carcinoma cell line. Mol. Cell Biol., 15, 2490–2499. Hagen, G., Müller, S., Beato, M. and Suske, G. (1994) Sp1-mediated transcriptional activation is repressed by Sp3. EMBO J., 13, 3843–3851. Hennig, G., Behrens, J., Truss, M., Frisch, S., Reichmann, E. and Birchmeier, W. (1995) Progression of carcinoma cells is associated with alterations in chromatin structure and factor binding at the E-cadherin promoter in vivo. Oncogene, 11, 475–484. Hennig, G., Löwrick, O., Birchmeier, W. and Behrens, J. (1996) Mechanisms identified in the transcriptional control of epithelial gene expression. J. Biol. Chem., 271, 595–602. Hodivala, K.J. and Watt, F.M. (1994) Evidence that cadherins play a role in the downregulation of integrin expression that occurs during keratinocyte terminal differentiation. J. Cell Biol., 124, 589–600. Howley, P.M. (1996) Papillomavirinae: The viruses and their replication. In: Fields, B.N., Knipe, D.M. and Howley, P.M. (eds.) Fields Virology. Lippincott-Raven Publ. Philadelphia. Huff, C.A., Yuspa, S.H. and Rosenthal, D. (1993) Identification of a control elements 3' to the human keratin 1 gene that regulate cell type and differentiation-specific expression. J. Biol. Chem., 268, 377–384. Ikeda, K., Shaw-White, J.R., Wert, S.E. and Whitsett, J.A. (1996) Hepatocyte nuclear factor 3 activates transcription of thyroid transcription factor 1 in respiratory epithelial cells. Molec. Cell Bio., 16, 3626–3636. Imakado, S., Bickenbach, J.R., Bundman, D.S., Rothnagel, J.A., Attar, P.S., Wang, X.J., Walczak, V.R., Wisniewski, S., Pote, J. and Gorden, J.S. (1995) Targeting expression of a dominant-negative retinoic acid receptor mutant in the epidermis of transgenic mice results in loss of barrier function. Genes Dev., 9, 317–328. Ishiji, T., Lace, M.J., Parkkinen, S., Anderson, R.D., Haugen, T.H., Cripe, T.P., Xiao, J.H., Davidson, I., Chambon, P. and Turek, L.P. (1992) Transcriptional enhancer factor (TEF)-1 and its cell-specific co-activator activate human papillomavirus-16 E6 and E7 oncogene transcription in keratinocytes and cervical carcinoma cells. EMBO J., 11, 2271–2281. Jiang, C.K., Magnaldo, T., Ohtsuki, M., Freedberg, I.M., Bernerd, F and Blumenberg, M. (1993) Epidermal growth factor and transforming growth factor a specifically induce the activation- and hyperproliferation-associated keratins 6 and 16. Proc. Natl. Acad. Sci. USA, 90, 6786–6790.

78


Krauss, S. and Franke, W.W. (1990) Organisation and sequence of the human gene encoding cytokeratin 8. Gene, 86, 241–249. Kulesh, D.A. and Oshima, R.G. (1988) Cloning of the human keratin 18 gene and its expression in nonepithelial mouse cells. Mol. Cell Biol., 8, 1540–1550. Kuo, C.J., Conley, P.B., Chen, L., Sladek, F.M. Darnell, J.E. and Crabtree, G.R. (1992), Transcriptional hierarchy involved in mammalian cell-type specification. Nature, 355, 457–461. Isaacs, H.V., Tannahill, D. and Slack, J.M. (1992) Expression of a novel FGF in the Xenopus embryo.A new candidate inducing factor for mesoderm formation and anteroposterior specification. Development, 114, 711–20. Isaacs, H.V., Pownall, M.E. and Slack, J.M. (1994) eFGF regulates Xbra expression during Xenopus gastrulation. Embo J., 13, 4469–81. Ishikawa, T., Yoshioka, H., Ohuchi, H., Noji, S. and Nohno, T. (1995) Truncated type II receptor for BMP-4 induces secondary axial structures in Xenopus embryos. Biochem. Biophys. Res. Commun., 216, 26–33. Jones, C.M., Lyons, K.M., Lapan, P.M., Wright, C.V. and Hogan, B.L. (1992) DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing factor in Xenopus mesoderm induction. Development, 115, 639–47. Jones, C.M., Kuehn, M.R., Hogan, B.L., Smith, J.C. and Wright, C.V. (1995) Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation. Development, 121, 3651–62. Jones, C.M. (1996) Signalling by TGF-β family members: short-range effects of Xnr-2 and BMP-4 contrast with the long-range effects of activin. Current Biology, 6, 1468–75. Jones, C.M., Dale, L., Hogan, B.L., Wright, C.V. and Smith, J.C. (1996) Bone morphogenetic protein-4 (BMP-4) acts during gastrula stages to cause ventralization of Xenopus embryos. Development, 122, 1545–54. Kageura, H. and Yamana, K. (1983) Pattern regulation in isolated halves and blastomeres of early Xenopus laevis. J. Embryol. Exp. Morphol., 74, 221–34. Karnovsky, A. and Klymkowsky, M.W. (1995) Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin. Proc. Natl. Acad. Sci. USA, 92, 4522–26. Kaufmann, E., Paul, H., Friedle, H., Metz, A., Scheucher, M., Clement, J.H. and Knöchel, W. (1996) Antagonistic actions of activin A and BMP-2/4 control dorsal lip-specific activation of the early response gene XFD-1' in Xenopus laevis embryos. EMBO J., 15, 6739–9. Keller, R. (1991) Early embryonic development of Xenopus laevis. Methods Cell Biol., 36, 61– 113. Keller, R. and Winklbauer, R. (1992) Cellular basis of amphibian gastrulation. Curr. Top. Dev. Biol., 27, 39–89. Kelley, C., Yee, K., Harland, R. and Zon, L.I. (1994) Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. Dev. Biol., 165, 193–205. Kessler, D.S. and Melton, D.A. (1994) Vertebrate embryonic induction: mesodermal and neural patterning. Science, 266, 596–604. Kessler, D.S. and Melton, D.A. (1995) Induction of dorsal mesoderm by soluble, mature Vg1 protein. Development, 121, 2155–64. Kimelman, D. and Maas, A. (1992) Induction of dorsal and ventral mesoderm by ectopically expressed Xenopus basic fibroblast growth factor. Development, 114, 261–9. Kinoshita, K., Bessho, T. and Asashima, M. (1993) Competence prepattern in the animal hemisphere of the 8-cell-stage Xenopus embryo. Dev. Biol., 160, 276–84. Kinoshita, N., Minshull, J. and Kirschner, M.W. (1995) The identification of two novel ligands of the FGF receptor by a yeast screening method and their activity in Xenopus development. Cell, 83, 621–30. Klein, P. and Melton, D.A. (1996) A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA, 93, 8455–59. Kloc, M., Spohr, G. and Etkin, L.D. (1993) Translocation of repetitive RNA sequences with the germ plasm in Xenopus oocytes. Science, 262, 1712–4. Olson, E.N. and Klein, W.H. (1994) bHLH factors in muscle development: dead lines and commitments, what to leave in and what to leave out. Genes Dev., 8, 1–8. Oshima, R.G., Abrams, L. and Kulesh, D. (1990) Activation of an intron enhancer within the keratin 18 gene by expression of c-fos and c-jun in undifferentiated F9 embryonal carcinoma cells. Genes Dev., 4, 835–838.


79

Pankov, R., Neznanov, N., Umezawa, A. and Oshima, R.G. (1994a) AP-1, ETS and transcriptional silencers regulate retinoic acid-dependent induction of keratin 18 in embryonic cells. Mol. Cell Biol., 14, 7744–7757. Pankov, R., Umezawa, A., Maki, R., Der, C.J., Hauser, C.A. and Oshima, R.G. (1994b) Oncogene activation of human keratin 18 transcription via the Ras signal transduction pathway. Proc. Natl. Acad. Sci., 91, 873–877. Peehl, D.M. and Stanbridge, E.J. (1981) Characterisation of human keratinocyte x HeLa somatic cell hybrids. Int. J. Cancer, 27, 625–635. Presland, R.B., Haydock, P.V., Fleckman, P., Nirunsuksiri, W. and Dale, B.A. (1992) Characterisation of the human epidermal profilaggrin gene. J. Biol. Chem., 261, 23772–23781. Quinlan, R.A., Schiller, D.L., Hatzfeld, M., Achtstatter, T., Moll, R., Jorcano, J.L., Magin, T.M. and Franke, W.W. (1985) Patterns of expression and organization of cytokeratin intermediate filaments.In: Wang, E., Fischman, D., Liem, R.K.H. and Sun, T.T. (eds.) Intermediate Filaments. Annals New York Acad. Sci., 455, 282–306. Ramirez, A., Bravo, A., Jorcano, J.L. and Vidal, M. (1994) Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development. Differentiation, 58, 53–64. Ramirez, A., Vidal, M., Bravo, A., Larcher, F. and Jorcano, J.L. (1995) A 5'-upstream region of a bovine keratin 6 gene confers tissue-specific expression and hyperproliferaton-related induction in transgenic mice. Proc. Natl. Acad. Sci. USA, 92, 4783–4787. Ringwald, M., Baribault, H., Schmidt, C. and Kemler, R. (1991) The structure of the gene coding for the mouse cell adhesion molecule uvomorulin. Nucleic Acids Res., 23, 6533– 6539. Rouet, P., Raguenez, G., Tronche, F, Mfou’ou, V. and Salier, J.-P. (1995) Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF-1, -3 and -4 in the liver specific enhancer for the human a-1-microglobulin/ bikunin precursor. Nucleic Acids Res., 23, 395–404. Ruberte, E., Dolle, P., Krust, A., Zelent, A., Morriss-Kay, G. and Chambon, P. (1990) Specific spatial and temporal distribution of retinoic acid receptor gamma transcripts during mouse embrygenesis. Development, 108, 213–222. Rutberg, S.E., Saez, E., Glick, A., Dlugosz, A.A., Spiegeleman, B.M. and Yuspa, S.H. (1996) Differentiation, of mouse keratinocytes is accompanied by PKC-dependent changes in AP-1 proteins. Oncogene, 13, 167–176. Sachs, M., Weidner, K.M., Brinkman, V., Walther, I., Obermeier, A., Ullrich, A. and Birchmeier, W. (1996) Motogenic and morphogenic activity of epithelial receptor tyrosine kinases. J. Cell Biol., 133, 1095–1107. Saitou, M., Sugai, S., Tanaka, T., Shimouchi, K., Fuchs, E., Narumiya, S. and Kakizuka, A. (1995) Inhibition of skin development by targeted expression of a dominant-negative retinoic acid receptor. Nature, 374, 159–162. Saunders, N.A., Bernacki, S.H., Vollberg, T.M. and Jetten, A.T. (1993) Regulation of transglutaminase type I expression in squamous differentiating rabbit tracheal epithelial cells and human epidemal keratinocytes: effects of retinoic acid and phorbol esters. Mol. Endocrinol., 7, 387–398. Schmidhauser, C., Casperson, G.F., Myers, C.A., Sanzo, K.T., Bolten, S. and Bissell, M.J. (1992) A novel transcription enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of β-casein gene expression. Mol. Biol. Cell, 3, 699–709. Smedts, F., Ramaekers, F.C.S. and Vooijs, P.G. (1993) The dynamics of keratin expression in malignant transformation of cervical epithelium: A review. Obstetrics Gynecology, 82, 465– 474. Snape, A.M., Winning, R.S. and Sargent, T.D. (1991) Transcription factor AP-2 is tissue-specific in Xenopus and is closely related or identical to keratin transcription factor 1 (KTF-1) Development, 113, 283–293. Stellmach, V., Leask, A. and Fuchs, E. (1991) Retinoid-mediated transcriptional regulation of keratin genes in human epidermal and squamous cell carcinoma cells. Proc. Natl. Acad. Set. USA, 88, 4582–4586. Streuli, C.H., Schmidhauser, C., Bailey, N., Yurchenco, P., Skubitz, A.P.N., Roskelly, C. and Bissell, M. (1995) Laminin mediates tissue-specific gene expression in mammary epithelia. J. Cell Biol., 129, 591–603. Suh, E., Chen, L., Taylor, J. and Traber, P.G. (1994) A homeodomain protein related to caudal regulates intestinespecific gene transcription. Mol. Cell Biol., 14, 7340–7351. Tamai, Y., Takemoto, Y., Matsumoto, M., Morita, T., Matsushiro, A. and Nozaki, M. (1991) Sequence of the Endo A gene encoding mouse cytokeratin and its methylation state in the CpG-rich region. Gene, 104, 169–176. Thierry, F., Spyrou, G., Yaniv, M. and Howley, P.M. (1992) Two AP1 sites binding jun B are essential for human papillomavirus type 18 transcription in keratinocytes. J. Virol., 66, 3740–3748.

80


Thorey, I.S., Cecena, G., Reynolds, W. and Oshima, R.. (1993) Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice. Mol. Cell Biol., 13, 6742–6751. Tomic-Canic, M., Sunjevaric, I, Freedberg, I.M. and Blumenberg, M. (1992) Identification of the retinoic acid and thyroid hormone receptor responsive element in the human K14 keratin gene. J. Invest. Dermatol., 99, 842–847. Tseng, H. and Green, H. (1992) Basonuclin: a keratinocyte protein with multiple paired zinc fingers. Proc. Natl. Acad. Sci. USA, 89, 10311–10315. Ueda, E., Ohno, S., Kuroki, T., Livneh, E., Yamada, K., Yamanishi, K. and Yasuno, H. (1996) The eta isoform of protein kinase C mediates transcriptional activation of the human transglutaminase 1 gene. J. Biol. Chem., 271, 9790–9794. Wegner, M., Drolet, D.W. and Rosenfeld, M.G. (1993) POU-domain proteins: structure and function of developmental regulators. Curr. Opin. Cell Biol., 5, 488–498. Weintraub, H., Davis, R., Tapscott, S., Thayer, M., Krause, M., Benezra, R., Blackwell, K., Turner, D., Rupp, R., Hollenberg, S., Zhuang, Y. and Lasser, A., (1991) The myoD gene family: nodal point during specification of the muscle cell lineage. Science, 251, 761–766. Welter, J.F., Crish, J.F., Agarwal, C. and Eckert, R.L. (1995) Fos-related antigen (Fra-1), junB and junD activate human involucrin promoter transcription by binding to proximal and distal AP1 sites to mediate phorbal ester effects on proximal activity. J. Biol. Chem., 270, 12614–12622. Welter, J.F., Gali, H., Crish, J.F. and Eckert, R.L. (1996) Regulation of human involucrin promoter activity by POU domain proteins. J. Biol. Cbem., 271, 14727–14733. Xanthopolous, K.G., Prezioso, V.R., Chen, W.S., Sladek, F.M., Cortese, R. and Darnell, J.E. (1991) The different tissue transcription patterns of genes for HNF-1, C/EBP, HNF-3 and HNF-4, protein factors that govern liver-specific transcription. Proc. Natl. Acad. Sci. USA, 88, 3807–3811. Yamanishi, K., Inazawa, J., Liew, F.-M., Nonomura, K., Ariyama, T., Yasuno, H., Abe, T., Doi, H., Hirano, J. and Fukushima, S. (1992) Structure of the gene for human transglutaminase 1. J Biol. Chem., 267, 17858–17863. Yoneda, K. and Steinert, P. (1993) Overexpression of human loricrin in transgenic mice produces a normal phenotype. Proc. Natl. Acad. Sci. USA, 90, 10754–10758. Yukawa, K., Butz, K., Yasui, T., Kikutani, H. and Hoppe-Seyler, F. (1996) Regulation of human papillomavims transcription by the differentiation-dependent epithelial factor Epoc-1/skn-1a. J. Virol., 70, 10–16. Yukawa, K., Yasui, T., Yamamoto, A., Shiku, H., Kishimoto, T. and Kikutani, H. (1993) Epoc-1: a POU-domain gene expressed in murine epidermal basal cells and thymic stromal cells. Gene, 133, 163–169. Zhou, P., Byrne, C., Jacobs, J. and Fuchs, E. (1995) Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate. Genes Dev., 9, 570–583.

5. CROSS-TALK BETWEEN APICAL AND BASOLATERAL DOMAINS OF EPITHELIAL CELLS REGULATES MICROVILLUS ASSEMBLY MONIQUE ARPIN, TIZIANA CREPALDI† and DANIEL LOUVARD Laboratoire de Morphogenèse et Signalisation Cellulaires, 26 rue d’Ulm, UMR 144 CNRS-lnstitut Curie. 75231 Paris Cedex 05, France Epithelial cells have a surface that is well adapted to their function as a selective barrier between two compartments. As with all cells, the surface is composed of a network of actin filaments closely associated with the plasma membrane. This network has several different functions. It is required for the development and maintenance of cell polarity, it regulates the functions of membrane proteins, and it participates in transducing signals from the exterior of cells to intracellular compartments. The actin network can be subdivided into actin-based multiprotein complexes such as those found at sites of cell to cell or cell-to-substratum contacts or at the apical domain of epithelial cells. These complexes are composed of numerous actin binding proteins and signalling molecules. The precise composition of each complex reflects an activation state of the cell and a particular role in cell polarity. One of the challenges of cell biology is to understand how assembly of these dynamic structures is regulated. Moreover, as the actin network appears to have an essential role in many signalling pathways that control cell processes, it will be important to understand the relationship between actin cytoskeleton and signalling proteins that function at the membrane. In this review, we describe recent advances in the understanding of the actin cytoskeleton and cell signalling. We focus on the apical domain of epithelial cells. This domain is assembled during terminal cellular differentiation, and presents an ideal model in which to study signals that trigger assembly of actin complexes. A—APICAL EPITHELIAL CELL SURFACE ORGANIZATION The Apical Domain of Epithelial Cells is Adapted to Specific Functions In most epithelial cell monolayers, the apical surface is in contact with the lumen cavity. Depending on the tissue origin of the epithelial cells, this surface may have specialized functions. These functions depend on modifications of the membrane components and of the underlying actin cytoskeleton to form specialized structu res.Whereas in the past it was believed that the actin cytoskeleton had only a structural role in forming these structures it is clear now that its role is paramount. Although most epithelial cells have microvilli on their apical domain, in only few cell types are these microvilli highly organized. The microvilli present at the surface of absorptive cells have been the most extensively analysed. Absorptive cells of the intestine or kidney have provided a unique model to analyse

†Institute for Cancer Research (I.R.C.C.) and Department of Biomedical Sciences, University of Torino Medical School, Strada provincial 142, km 3.5. 10060 Torino, Italy.

82


the actin cytoskeleton at the plasma membrane of epithelial cells. The hallmark of these terminally differentiated cells is their apical surface which has thousands of microvilli having a uniform length and width that, together with the terminal web, form an organelle named the brush border. Experimentally, this organelle became paradigm for studying membrane cytoskeleton interactions, because it could be isolated biochemically. The stereocilia of auditory cells are another example of remarkable structures formed by the actin cytoskeleton. Unlike intestinal cells, hair cells have a polarized staircase of stereocilia. Depending upon the location in the cochlea, each hair cell displays a specific number of stereocilia of a particular length (Tilney and Tilney, 1992). Stereocilia are well adapted for their function in the conversion of mechanical stimulus into an electrical signal. Another type of cell surface specialization is seen in placenta. In this tissue, microvilli participate in endocytosis, hence as the microvilli are relatively more dynamic they tend to be less well structured and less uniform than intestinal microvilli. These few examples indicate how specialised cell surfaces have structures that are adapted to their function. One of the first steps in understanding the relationship between the structure and the functions of specialised surfaces was the biochemical identification of their components. When purified proteins were available, reconstitution experiments were performed in vitro to analyze the contribution of each component in the elaboration of these membrane specializations. Microvilli assembly has also been analysed during development by immunohistochemical approaches. The role of specific proteins in the assembly of cytoskeletal complexes has been assessed by production of cloned proteins by transfection or by introduction by microinjection in model cells in culture. Gene targetting and reverse genetics have recently provided other means to assess the function of individual components. The biochemical properties of the major actin binding proteins that organise the actin filament bundles have been described in several reviews (Bretscher, 1991; Heintzelman and Mooseker, 1992). For this reason, we will only briefly summarise our current understanding of this structure. Rather, we will focus upon these topics: the role of the actin binding proteins in microvillus assembly, the signals that control assembly of the brush border, and the role of the microfilaments in various membrane protein functions. The Microvillar Actin Cytoskeleton of Epithelial Cells The brush border of absorptive epithelial cells is composed of the microvilli and the terminal web. The microvillus core contains 20 bundled actin filaments aligned with the same polarity (Mooseker and Tilney, 1975). These filament bundles are anchored at the tip of the microvilli in a dense structure that is not well characterized. The rootlets of the bundles are connected to the terminal web, by another actin complex which also contains myosin II and nonerythroid spectrin proteins (Heintzelman and Mooseker, 1992). Because our knowledge of the terminal web is less advanced we will only discuss the structure of the microvilli. Intestinal microvilli, that retain their structural integrity can be prepared from isolated brush border (Bretscher and Weber, 1978; Matsudaira and Burgess, 1979). This technique has facilitated identification of the four major actin binding proteins associated with the core bundle (Bretscher and Weber, 1979). Two of these proteins, villin and I-fimbrin, bind directly to actin filaments. The third protein, brush border myosin I complexed with the calmodulin, forms the arms that laterally attach the core bundles to the membrane. Finally, a multifunctional protein, ezrin, is localized underneath the plasma membrane of the brush border. In addition to these major proteins, other actin interacting proteins have been recently identified.


83

The microvillus core bundles are assembled by a specific set of proteins The core bundles contain actin binding proteins different from those found associated with other microfilament arrays present in non-muscle cells. Each microvillar protein belong to a family of protein with related structure. Properties of microvillar cytoskeletal proteins of intestinal cells In Vitro Activities

Ligands

Homologous Proteins

VILLIN 92,5 kD

Capping Nucleating Severing Bundling

F/G Actin Ca2+ PiP2

I-FIMBRIN

Bundling

F-Actin Ca2+, Mg2+

BRUSH-BORDER MYOSIN I 110 kD

Mechanoenzyme

F-Actin Mg2+ ATP Phospholipids Calmodulin F-Actin PiP2 CD44/Rho-GDI CD43 I-CAMI PKA EBP50 RhoGDI

Advillin Supervillin Protovillin Quail gene product Dematin Gelsolin Severin Fragmin L/T-Fimbrin/Plastin Dystrophin Spectrin/Fodrin α-actinin Filamin ABP 120 Myosins Iα

EZRIN 67 kD

Radixin Moesin Merlin/Schwannomin EM 10 PTPH1 PTPase MEG Band 4.1 Talin

Villin Villin is a 92, 5 kDa protein (Bretscher and Weber, 1979) whose expression is restricted to the epithelial cells of the uro-genital and digestive tracts (Maunoury et al., 1988; 1992). High levels of villin correlate with cells that present a well organised brush border. Villin has structural features that are also found in gelsolin, fragmin and severin. These proteins can sever, nucleate and cap actin filaments in a calcium dependent manner (Friederich et al., 1990). In addition to these activities, villin can bundle actin filaments, an activity that is due to a carboxy-terminal domain, the headpiece, that is not present in either gelsolin, fragmin or severin (Glenney et al., 1981a; Bazari et al., 1988; Arpin et al., 1988; Hartwig and Kwiatkowski, 1991). Recently, a number of proteins homologous to villin have been identified which all contain a villin-like headpiece. Supervillin displays a carboxy-terminal half homologous to villin particularly in F-actin binding sites and an amino-terminal half which contains four potential nuclear targeting signals (Pestonjamasp et al., 1997). Advillin is expressed in various tissues and shares primary

84


structure homology with villin along its entire length (Marks et al., 1998). Protovillin in Dictyostelium amoebae, also contains six domain repeats fused to a villin-like headpiece (Hofmann et al., 1993). However, unlike villin, protovillin shows no F-actin severing, or cross-linking activities. In Drosophila, the product of quail gene is homologous to villin. It bundles actin filaments during oogenesis (Mahajan-Miklos and Cooley, 1994). A domain homologous to the villin headpiece has also been identified at the carboxyterminus end of dematin (Rana et al., 1993; Azim et al., 1995). This protein has an actin bundling activity which is regulated by phosphorylation (Husain-Chishti et al., 1988). Among the above homologues of villin, advillin displays the highest homology with villin carboxy-terminal amino-acids which are essential for actin binding (Friederich et al., 1992; Doering and Matsudaira, 1996). But advillin displays a broader pattern of expression than villin. Fimbrin/Plastin Since the identification of fimbrin in microvilli, it has been subsequently shown by anti-fimbrin antibodies that fimbrin is associated with actin bundles in a wide variety of cells (Bretscher and Weber, 1980). More recently, highly related proteins named plastins, have been identified in many tissues and in many eucaryotes including yeast (Lin et al.,1988, 1994; de Arruda et al., 1990; Adams et al.,1991). Plastin isoforms have a tissue specific distribution. L-plastin/fimbrin is produced normally in hematopoietic cells but it has been identified in solid tumour cells from different tissus. T-plastin/fimbrin is produced in epithelial and mesenchymal cells. Another fimbrin isoform, I-plastin, is produced only in the brush border of intestinal and kidney cells (Lin et al., 1994). In vitro biochemical assays show that fimbrin/ plastin are monomeric proteins with F-actin bundling activity (Glenney et al., 1981b). This activity on filament organisation is differently regulated by ionic concentration or phosphorylation (Namba et al., 1992; Messier et al., 1993; Lin et al., 1994). Analysis of amino-acid sequences reveal that these proteins have common domain with the actin cross-linking family proteins (Matsudaira, 1991). Interestingly, despite highly related amino-acid sequence, members of this family show distinct affinities for different actin filament arrays. Unconventional myosins Myosins are motor proteins that convert chemical energy into mechanical force. Isolation of many myosin proteins from different tissue types has revealed the diversity of this superfamily (Cheney and Mooseker, 1995; Hasson and Mooseker, 1996). A non conventional myosin I, brush border myosin I (BBMI), is one of the major proteins of the brush border of absorptive cells. (Matsudaira and Burgess, 1979). The biochemical activity of BBMI is well characterized, it forms a complex with calmodulin and tethers the actin bundle cores to the plasma membrane (for review see Mooseker et al., 1991). It is not yet known precisely how BBMI binds to the plasma membrane, although it may require an interaction with acidic phospholipids (Hayden et al., 1990) and binding to a novel membrane zipper protein (Bikle et al., 1996). In addition to BBMI, members of the classes V and VI non conventional myosins have been identified in the intestinal brush border cytoskeleton (Heintzelman et al., 1994). Myosin-VI localizes to both the terminal web and the microvilli, whereas myosin-V is localised in the terminal web and at the distal ends of the microvilli (Heintzelman et al., 1994). Two myosins, myosin-IA and myosin-IB, have been identified in the Drosophila brush border microvilli (Morgan et al., 1994, 1995). The two proteins have different intracellular locations. In the adult gut, myosinIB is primarily located in the microvilli whereas myosin-IA is found in both the terminal web and the


85

microvilli (Morgan et al., 1995). Yet more informations are necessary in order to get a full biochemical characterization of these myosins and a better understanding of their functions which may be very diverse. Ezrin Ezrin belongs to the band 4.1 protein superfamily (Arpin et al., 1994) whose members display an homologous domain, the FERM domain, which mediates their attachment to the plasma membrane (Chishti et al., 1998). Ezrin was first isolated from intestinal epithelial cells then from placenta microvilli and human choriocarcinoma cells (Bretscher, 1983; 1989; Pakkanen et al., 1987). It displays high percentage aminoacid sequence similarity with radixin and moesin proteins (Tsukita et al., 1997) and constitutes the ERM subfamily. Whereas ezrin is restricted to the actin-rich structures of epithelial cells, moesin is found in a restricted number of epithelial cells and in endothelial cells (Berryman et al., 1993). In cultured cells, however, this almost exclusive distribution of these two proteins is lost (Franck et al., 1993). Unlike villin, brush border myosin I and I-fimbrin, ezrin distribution is not restricted to cells with well organized brush border. It is not known if microvillus specific isoform of ezrin exists. Based on its subcellular localization and its homology with other band 4.1 proteins, it was proposed that ezrin may play a role in linking the microfilaments to the plasma membrane. This hypothesis was supported by transfection studies, in which the NH2-terminal domain of ezrin was located to the membrane whereas the carboxy-terminal domain localized with actin filaments such as stress fibers (Algrain et al., 1993). The identification of domains with distinct binding properties suggested that the membrane and actin binding sites are masked in ezrin. Indeed, it was shown that masking is caused by the ability of ezrin to form homoor heterodimers with other ERM proteins (Gary and Bretscher, 1993; Andreoli et al., 1994; Gary and Bretscher, 1995). Several membrane binding partners of ezrin have been identified. Ezrin coimmunoprecipitate with CD44 and this interaction, in vitro, requires PIP2 (Tsukita et al., 1994; Hirao et al., 1996). However, the expression pattern of CD44 is not restricted to epithelial cells. Moreover, CD44 has been shown to localize at the basolateral surface in some epithelial cell types, suggesting that either interaction occurs via cytoplasmic ezrin, or that a CD44 isoform is present on the microvillus membrane. An interaction of ezrin with the intercellular adhesion molecules CD43, ICAM-2, ICAM-3 has also been reported (Helander et al., 1996; Serrador et al., 1997, Yonemura et al., 1998; Legg and Isacke, 1998). The amino-terminal domain of ezrin associates with EBP50, a phosphoprotein containing PDZ domains (Reczek et al., 1997; Reczek and Bretscher, 1998). Interestingly, EBP50 appears to link ezrin to integral membrane proteins. Indeed, EBP50 interacts with the cystic fibrosis transmembrane conductance regulator (CFTR) (Short et al., 1998). Moreover, the rabbit homologue of EBP50, NHE-RF, is a cofactor involved in the protein kinase A regulation of the renal Na+/H+ exchanger (Weinman et al., 1995). Yun et al. (1997) recently showed that E3KARP, another cofactor of the Na+/H+ ion exchanger which shares homology with the amino-terminal domain of NHE-RF binds to the NHE3 Na+/H+ exchanger and subjects it to PKA regulation. One role of ezrin would be to localize the kinase to its functional sites, i.e. to the microvilli. This hypothesis is supported by the observation that, in gastric parietal cells, ezrin anchors the c-AMP dependent protein kinase (type II-A-kinase) to the microvjllar plasma membrane (Dransfield et al., 1997). In this latter case ezrin is thought to play a major role in membrane reorganization during parietal cell stimulation localizing membrane proteins in subregion of epithelial cells. The nature of the binding of ezrin to actin and the role of ezrin in actin organization remain poorly understood. The carboxy-terminal domain which is masked in the purified protein (Gary and Bretscher, 1995) contains the F-actin binding site (Turunen et al., 1994; Pestonjamasp et al., 1995). Using a solid

86


phase assay, Roy et al. have found an F-actin binding site in the amino-terminal domain and a G-actin binding site was mapped to residues 288–310 (Roy et al., 1997). Although these actin binding sites were identified with muscle α-actin, it was reported that ezrin binds only to the p-actin isoform (Shuster and Herman, 1995; Yao et al., 1996) and in one case this binding was found to be calcium dependent (Shuster and Herman, 1995). The microvillus cytoskeletal protein composition correlates with the specific functions of this organelle The core bundle proteins of intestinal microvilli are similar to those of proximal tubule cells of kidney, another cell type specialized in absorption (Coudrier et al., 1988) although the microvilli of the proximal tubule cells contain moesin preferentially expressed in endothelial cells, in addition to ezrin (Berryman et al., 1993). The actin binding proteins associated with actin filament bundles of stereocilia differ from those found in the intestinal or kidney microvilli (Tilney et al., 1989; Shepherd et al., 1989; Drenckhahn et al., 1991). Stereocilia do not contain villin but do produce fimbrin. Several unconventional myosins have been characterized in hair cells (Hasson et al., 1997) and have been shown to be essential for hearing and balance. Myosin Ib restricted to the tip of the hair bundles (Gillespie et al., 1993) has been postulated to be an actinbased adaptation motor (Gillespie, 1996). Myosin VI is enriched in the cutilar plate of hair cells. Mutants in its gene, Snell’s waltzer, leads to a profound deafness (Avraham et al., 1995). In hair cells, myosin VIIa is distributed along the entire length of Stereocilia and in the cutilar plate, whereas in the rat retina, this myosin is enriched at the apical plasma membrane of the retinal pigmented epithelia (Hasson et al., 1995). Mutation in myosin VIIa gene, responsible for Usher syndrome type 1B, is a frequent cause of deafness and blindness in humans (Weil et al., 1995). Because many events occurring at the placental microvilli are regulated by calcium, placental microvillar proteins have been isolated on the basis of their calcium sensitivity. For example, a-actinin and ezrin are released from microvillar cytoskeleton by micromolar levels of free calcium. In contrast, other polypeptides of various molecular weight are associated with the cytoskeleton at millimolar levels of free calcium (Edwards and Booth, 1987). Altogether, these observations indicate that it exists a close relationship between the molecular composition of the actin filaments and the function of epithelial cells to which these microvilli are associated. Assembly of the Intestinal Microvilli How are these different components assembled to form a microvillus? Experiments with purified components —actin, villin and BBMI—showed that the filament bundles obtained in vitro are similar to microvillus core of intestine with the brush border myosin I-calmodulin complex forming the lateral arms. However, two differences are found between the reconstituted bundles and those of in vivo. Microvilli bundles are of uniform length and of fixed numbers, whereas bundles made in vitro are of undeterminate length and have fewer number of filaments. This suggests that cells can control these parameters (Coluccio and Bretscher, 1989). Analysis of the pattern of expression of the genes coding for these proteins and analysis of proteins during embryogenesis and in the adult during terminal differentiation along the crypt-villus axis has revealed that this structure does not auto-assemble. Assembly of the microvillus core is strictly regulated


87

both spatially and temporally (Shibayama et al., 1987; Maunoury et al., 1988, 1992; Ezzel et al., 1989; Heintzelman et al., 1990a,b;. Chafel et al., 1995). All the proteins are present before being localized to the apical surface of the cells. For example, ezrin is already produced in oocyte and is present throughout preimplantation of the embryo, long before a rudimentary gut is formed. Before compaction ezrin is located at the cell cortex. It becomes restricted to the apical microvilli of the outer cells and remains there until after the 8 to 16 cell stage (Louvet et al., 1996). Thus, the localization of ezrin to the microvilli correlates with the polarization of the cells. During mouse embryogenesis, the maturation of the intestinal epithelium is characterized by a transition from a thick pseudostratified structure to simple columnar cells (from day 11 to day 15 embryo, depending on the intestinal segments). Ezrin polarization has already occurred in the pseudo-stratified epithelium at day 10 (J.J.Fontaine, personnal communication). The apical localization of ezrin precedes that of villin. Villin localizes to the apex of cells when rudimentary microvilli appear at day 12.5 on the surface (Ezzel et al., 1989; Maunoury et al., 1992; Shibayama et al., 1987; Heintzelman and Mooseker, 1990a,b). Immunofluorescence analysis of fimbrin production in intestinal cells in embryo from days 10.5 to 16.5 revealed that genes coding for the fimbrin isoforms are sequentially switched on and off during development (Chafel et al., 1995). L- and T-fimbrin, the two non intestinal epithelial cell isoforms, are first produced at day 10.5. Whereas T-fimbrin is predominantly located at the apex of the cells, L-fimbrin is found at the basal surface of the epithelial cells. I-fimbrin is synthesized at day 14.5 and has an apical localization by day 16.5 which coincides with the appearance of well organized microvilli and with the disappearance of L- and T-fimbrin isoforms. As a consequence, rudimentary microvilli observed at the early stage of development are organized by villin and T-fimbrin (Chafel et al., 1995). From immunological studies, myosin I is the last of the major proteins to localize in the microvilli and this corresponds to the final maturation step of this structure (Shibayama et al., 1987). The timing of the appearance of major microvillus proteins was important. However, these descriptive studies formed the basis of other questions: i) what is the mechanism for initiation of microvillus assembly? ii) what are the signals that control initiation of microvillus assembly?. To answer these questions several different approaches have been used in an attempt to integrate information in a general scheme. Contribution of cytoskeletal proteins to intestinal microvillus assembly Villin The function of villin in vivo was tested by producing high level of the protein by transient transfection of villin cDNA into fibroblasts that normally do not produce the protein (Friederich et al., 1989) and later by microinjecting the protein into cultured cells (Franck et al., 1990). In both cases, villin induced the growth of microvilli on the dorsal surface of the cells and reorganized stress fibers. Villin core protein (lacking the head piece domain), did not induce these morphological modifications (Friederich et al., 1989). Moreover, when villin synthesis was impaired by ablation of mRNA by antisense, the human adenocarcinoma cell line CaCO2 which displays a well organized brush border, did not assemble this structure (Costa de Beauregard et al., 1995). The morphogenic activity of villin in vivo, was attributed to a cluster of charged amino-acids (KKEK) in the carboxy-terminus of the protein. Experiments in vitro, using a 22 amino acid synthetic peptide comprising this cluster of amino acids confirmed that this motif is part of an actin binding site (Friederich et al., 1992). Additional residues in the villin headpiece which define the F-actin binding site have been identified by cysteine scanning mutagenesis (Doering and Matsudaira, 1996).

88


The transfection experiments, however, did not address the question of whether these microvilli are formed de novo or from preexisting nucleation sites. Two lines of evidence suggest that nucleation is not the mechanism by which villin induces microvillus assembly. When cells were transfected with villin cDNA and treated with low level of cytochalasin D, the growth of microvilli was inhibited suggesting that the microvillus actin filaments are not capped by villin (Friederich et a!., 1993). Second, a villin variant unable to nucleate actin monomers in vitro, still induced microvillus formation in transfected cells (Friederich et al., personal communication). Altogether, these observations suggest that villin does not play a role in the initiation of actin polymerisation. Although these experiments, performed in cultured cells, emphasize the role of villin in microvillus formation a different result was obtained in villin null mice. Intestinal cells of these villin null mice are viable and display a brush border (Robine et al., 1996; Pinson et al., 1998). The differences in responses of cells in culture and cells of villin null mice, may be explained by one of several proposals, i) In villin null mice, microvillus morphogenesis is performed by a villin-related protein. For example, advillin displays a strong similarity with villin and is present in intestinal cells, ii) Another possibility is that fimbrin can compensate villin function. Indeed, fimbrin like villin can cross-link actin filaments, iii) Villin may have a function which is unrelated to its activities in vitro. In this case, the knock out of villin, would induce subtle changes associated with modification of the physiology of intestinal mucosa, which remain to be identified. Fimbrin/Plastin Formation of rudimentary microvilli, during development, is associated with expression of fimbrin T whereas well organized microvilli are present after synthesis of I-fimbrin (Chafel et al., 1995). To test the roles of these proteins, LLC-PK1 cells which are derived from proximal tubule of kidney and which display a brush border were transfected with cDNAs coding for T- or L-fimbrin. Overproduction of L-fimbrin had no effect on the shape of the microvilli. Overproduction of T-fimbrin induced shape changes in microvilli: they were longer and more densely packed. However, they were shorter and less organized than microvilli induced by overproduction of villin in LLC-PK1 cells (Arpin et al., 1994). The morphology of the villininduced microvilli in these transfected cells was reminiscent of kidney brush border microvilli (Coudrier et al., 1988). These results support the observations on T-fimbrin and microvilli during development and indicate that T-fimbrin does not have the ability to organize microvillar microfilaments to form a highly organized brush border. Ezrin Insights into the function of ezrin in brush border assembly have been obtained by several different techniques. Takeuchi et al. showed that ezrin and other ERM proteins play a role in cell-cell adhesion and microvillus formation by using antisense oligonucleotides. The antisense oligonucleotides to the coding sequence of the three proteins are required to perturb cell-substrate, cell-cell adhesion and microvilli formation (Takeuchi et al., 1994). These experiments suggest that ERM play a role at an early stage of cell adhesion because preformed microvilli appear more resistant to antisense ablation than adhesion structures (Takeuchi et al., 1994). We have used a different approach to show that ezrin is an essential component-of brush border microvilli. We have produced the globular amino-terminal domain of ezrin in stably transfected LLC-PK1 cells. These transfected cells have a more flbroblastic phenotype: they are elongated and display thick actin cables. Upon production of the amino-terminal domain of ezrin, the apical microvilli disappear and a


89

concomittant increase in the number and size of stress fibers occurs. Immunofluorescence studies indicated that in these cells, both the amino-terminal domain and the endogenous ezrin were mainly concentrated at the periphery of the cells (Crepaldi et al., 1997). A role for ezrin in brush border microvilli can be proposed to explain this dominant-negative effect, i) The amino-terminal domain may bind to a regulatory component, ii) It may inhibit the activity of the carboxy-terminal domain which displays a morphogenic effect when overproduced in insect cells (Martin et al., 1995). iii) The amino-terminal domain may prevent oligomerization of endogenous ezrin (Berryman et al., 1995). The oligomerization properties of ezrin deserves special attention. In placental microvilli, ezrin exists as dimeric and higher oligomeric forms (Berryman et al., 1995). By mechanisms which remain to be elucidated, cell activation leads to oligomer formation, unmasking of the F-actin binding site and possibly a membrane binding site. Phosphorylation of ezrin on one or more serine, threonine or tyrosine residues may represent one activation step. For example, treatment of A431 cells with EGF, induces ezrin phosphorylation, oligomerization, and appearance of microvilli at the surface of the cells (Bretscher, 1989; Berryman et al., 1995). In this case, oligomerization of ezrin persists after a reduction in the level of phosphorylation. Recently it has been shown that ezrin threonine residue 567 is phosphorylated by the Rhoassociated kinase (Rho-kinase). It has been proposed that this phosphorylation suppresses the head to tail interaction of this protein hence keeping it in an open, active conformation (Matsui et al., 1998). In favor of this proposal, Chen et al. have reported that dephosphorylation of ezrin, on serine/ threonine residues, induced by anoxia leads to dissociation of the brush border membrane from the cytoskeleton and breakdown of renal microvilli (Chen et al., 1995). At an early stage of apoptosis induced in L cells Kondo et al. have observed the breakdown of the microvill and the translocation of ERM from the microvilli to the cytoplasm with the concomitant dephosphorylation of these proteins (Kondo et al., 1997). B—SIGNALS AFFECTING MICROVILLUS ASSEMBLY AND FUNCTIONS Cell shape determination is essential for proper functioning of epithelial cells. Lack of apical microvilli leads to severe defects as observed, for example, in the familial microvillous atrophy (Phillips and Schmitz, 1992). Several genetic and biochemical evidence suggest that signals, originating from the cell surface, trigger epithelial differentiation and morphogenesis. There are few data on how signals at the cell surface are integrated and transduced to microvillar cytoskeletal and membrane components. Some signals origi nate from cell-cell or cell-matrix contacts, since it has been shown that cell adhesion plays a role in establishment of cell polarity. Tyrosine kinase receptors specific to epithelial cells, have been shown to have a role in cell movement and cell morphogenesis in epithelial cells in culture. Ion exchangers or ion channels present in the plasma membrane of microvilli can be regulated by signals originating from receptors present at the baso-lateral surface of the cells. The actin cytoskeleton is believed to play a role in these signal pathways as well. Understanding how the cytoskeleton participates in signal transduction represents a new challenge. Role of Cell Adhesion in the Generation of an Apical Domain of Epithelial Cells Treatment of cells in culture by calcium depletion has shown that apical and basolateral domains are generated in response to independent signals. Under these conditions, functional E-cadherin is lost, tight and adherens junctions do not form. Proteins that are normally restricted to basolateral domains are dispersed at the surface of the cells. Nevertheless, apical microvilli form (Vega-Salas et al., 1987). In other experiments it has been shown that integrin mediated-interactions can elicit the development of the apical membrane.

90


MDCK cell cysts grown in suspension reverse their surface polarity when incubated within collagen gels (Ojakian and Schwimmer, 1994). This effect is inhibited by treatment with antibodies to β1-integrin. In kidney organo-cultures the development of tubular epithelium is inhibited by treatment with antibodies to α6-subunit of integrin, the receptor for laminin A (Klein et al., 1988). Altogether, these experiments suggest that the apical and baso-lateral domains of epithelial cells can be organized separately. The formation of the apical domain would depend on integrin-mediated interaction with the substratum. Genetic analysis of Drosophila mutants for crumbs genes indicates that the crumbs gene encodes a transmembrane protein with EGF-like and laminin A-like repeats. Crumbs protein has a crucial role in defining the apical membrane domain. Analysis of a crumbs mutant with truncated cytoplasmic domain revealed that cells lost polarity. Overproduction of the crumbs protein or overproduction of the membrane bound cytoplasmic domain resulted in expansion of the apical surface, reduction of the basolateral surface and extensive reorganization of the cytoskeleton. How the crumbs protein confers apical character to a membrane domain is not understood, but it likely involves interaction with cytoskeletal proteins resulting in changes in cell shape (Wodarz et al., 1995; Grawe et al., 1996). Regulatory Signals in Microvillus Assembly Microvillus assembly may be controlled by many signalling pathways that converge upon cytoskeletal proteins targets. Among the factors able to induce epithelial cell morphogenesis the role of the hepatocyte growth factor and its receptor c-met have been extensively investigated, c-met receptor tyrosine kinase is able to induce morphogenesis of epithelial cells in three-dimensional matrices (Sachs et al., 1996). The proteins that interact with the cytoplasmic domain of the c-met receptor and the different signal transduction pathways activated upon hepatocyte growth factor (HGF) treatment have been extensively studied by different groups. The down-stream events involved in cell motility or morphogenesis, pertaining to the actin cytoskeleton are very poorly understood. Changes in actin organization is a prerequisite for cell scattering and cell motility as shown by treatment of epithelial cell sheets with the hepatocyte growth factor (Stoker et al., 1987; Gherardi et al., 1989) for invasiveness (Weidner et al., 1990) or for morphogenesis (Montesano et al., 1991). Ezrin has long been recognized as a substrate for tyrosine, threonine and serine kinases (Gould et al., 1986; Bretscher, 1989). Depending on the stimuli, different residues can be phosphorylated. Epidermal growth factor (EGF) phosphorylates ezrin on two residues (Y145 and 353) in human epidermoid carcinoma A431 cells (Krieg and Hunter, 1992) with a concomitant redistribution of ezrin in membrane ruffles (Bretscher, 1989). In parietal cells, EGF stimulates phosphorylation of ezrin on serine residues different from those phosphorylated by histamine stimulation (Urushidani et al., 1989; Whitney et al., 1990). We have shown that ezrin is a downstream substratre of the tyrosine kinase activity of the HGF-receptor. Ezrin becomes phosphorylated on tyrosine residues when LLC-PK1 cells are treated with HGF. In MDCK cells a complex of ezrin, c-met receptor and the c-yes kinase has been isolated by co-immunoprecipitation. In addition, overproduction of ezrin in epithelial cells enhanced the motility or the morphogenesis of epithelial cells cultured in collagen matrix, induced by HGF. The motility and the tubulogenesis of kidney cells are inhibited by overproduction of the amino-terminal domain of ezrin. These experiments provide evidence that ezrin is able to convey the signals elicited by the HGF receptor to the actin cytoskeleton machinery (Crepaldi et al., 1997). Recently, Gautreau et al. have shown that ezrin is necessary for signaling survival dependent on cellsubstratum adhesion. A kidney-derived epithelial cell line over-producing a mutant in which tyrosine 353 was changed to phenylalanine (Y353F) undergoes apoptosis, when cultured in a collagen type I matrix


91

(Gautreau et al., unpublished results). The mechanism by which ezrin signals survival involves activation of the PI3-K/Akt pathway. The multiple roles of ezrin such as cell scattering, cell morphogenesis or cell survival may be dependent upon its subcellular localization. Changes in its conformation may result in its association with different cytoplasmic or membrane proteins which in turn may regulate its different activities. Signals resulting of the activation of membrane receptors can be transduced through the inositol phospholipids which generate second messengers by the action of phospholipases C or phosphoinositide-3OH kinase (PI3-K). Activation of phospholipases C leads to an increase in the intracellular calcium concentration and to the stimulation of the protein kinase C. Calcium regulates the activities of microvillus actin binding proteins. In vitro, villin activities are regulated in a calcium-dependent manner (For review see Bretscher, 1991). I-Fimbrin also binds calcium but the dissociation constant is higher than that of villin (10 μM) and millimolar Mg2+ saturate this site. Cross-linking activity of I-fimbrin is inhibited in presence of calcium (Glenney et al., 1981b; Lin et al., 1994). The binding of the myosin-I to calmodulin and the mechanochemical activity of the protein are also regulated by calcium (Collins et al., 1990; Wolenski et al., 1993). In vivo, the microvillar proteins are likely regulated by calcium since the intestinal epithelium is the place of calcium transport. PiP2 also regulates the actin cytoskeleton by binding numerous actin interacting proteins as shown in vitro (Janmey, 1994). For example, the F-actin severing activity of villin is inhibited by PiP2 (Janmey and Matsudaira, 1988; Janmey et al., 1992). Ezrin binds PiP2 in vitro (Niggli et al., 1995). Recently, insights into the mechanisms whereby membrane-cytoskeleton association of ezrin is regulated by phosphatidylinositol turnover and Rho-dependent signaling pathways have been obtained. It was first shown that association of ezrin with the transmembrane protein CD44 is enhanced by 4, 5-PiP2 in vitro (Hirao et al., 1996). In addition, this association in vivo is regulated by Rho, a small GTPase involved in actin cytoskeleton organization in responses to extracellular signals. A cryptic binding site for Rho-GDI in the amino-terminal domain of ERM activates Rho by releasing Rho-GDI inhibition (Takahashi et al., 1997). Since the small GTPase Rho is an upstream regulator of 4, 5-PiP2 level in plasma membrane (Chong et al., 1994; Ren and Schwartz, 1998) this suggested that PIP2 may regulate the actin-membrane linking activity of ezrin through the Rho pathway (Kotani et al., 1997; Shaw et al., 1998). Whether ezrin plays a role in the actin filament microvillus assembly is not known. However, Rho-dependent formation of actin stress fibers requires ERM molecules (Mackay et al., 1997). Moreover, phosphorylation of a conserved threonine residue in the carboxy-terminal part of ERM, under the double control of Rhokinase and myosin light chain phosphatase, may change ERM conformation (Matsui et al., 1998; Fukata et al., 1998), and therefore account for ERM activation down-stream of Rho. Role of Actin Cytoskeleton in Membrane Protein Regulation Several observations suggest that signals originating from receptors located at the baso-lateral surface of cells can act on microvillar membrane proteins through cytoskeletal proteins. In ileal absorptive cells, the cholinergic agonist carbachol inhibits Na+ absorption and the brush-border Na+/H+ exchanger upon binding to its baso-lateral membrane receptors. Activation of these receptors leads to an increase in the calcium and diacylglycerol levels with the translocation of active protein kinase C and PLC-γ1 onto the brush border (Donowitz et al., 1989; Cohen et al., 1991; Khurana et al., 1996a). This process activates a tyrosine kinase. Khurana et al., have shown that stimulation of the G-protein-linked receptor by carbachol induces tyrosine phosphorylation of villin. The tyrosine-phosphorylated form of villin was complexed with the PLC-γl in the detergent-soluble fraction (S.Khurana et al., personnal communication). Interestingly, the villin insoluble

92


fraction was not phosphorylated. This is the first report of villin being the substrate of tyrosine kinase, and it raises very interesting questions for further work. These observations suggest a new function for villin. It could regulate the function of membrane proteins such as the Na+/H+ exchanger through changes in actin filament organization. Conversely, stimulation of Na+ absorption and of the Na+/H+ exchanger is induced by EGF and its receptor also located on the baso-lateral surface (Donowitz et al., 1994). This stimulation involves an increase in both the amount and activity of the apical membrane PI3-kinase (Khurana et al., 1996b). It has been shown that the apically located, renal amiloride sensitive Na+ channels interacts directly with the actin cytoskeleton (for review see Cantiello, 1995). Cantiello and coworkers suggest that actin filaments are involved in the regulation of amphibian renal epithelial Na+ channels. In patch-clamp experiments they showed that actin filaments stimulate Na+ channels present on the apical surface of a cell line derived from the distal amphibian nephron. They also showed that the protein kinase A mediates actin phosphorylation and that only phosphorylated actin filaments can activate the channels (Prat et al., 1993; Cantiello et al., 1991; Berdiev et al., 1996). The renal epithelial protein (Apx) which modulates the activity of this channel is also regulated by the actin cytoskeleton (Prat et al., 1996). One proposal is that this protein directly interacts with actin via two putative actin binding sites similar to those found in villin and human p-spectrin (Staub et al., 1992). Moreover, the putative pore forming subunit of the rat amiloride-sensitive Na+ channel (arENaC) binds α-spectrin in vivo via the SH3 domain of spectrin. This interaction might serve as the apical signal of this channel (Rotin et al., 1994). Cytoskeletal proteins have also been shown to play a role in sensory signal transduction. Genetic identification of unconventional myosins as responsible of deafness has provided some clues on the role of the hair cell actin cytoskeleton in the conversion of mechanical forces into electrical signals. Several observation suggest that myosin-lp is coupled to the tip link associated cation channels. Opening of these channels occurs in response to mechanical stimulation of the hair cell bundles. The mechanical properties of this motor is regulated by Ca++ (Zhu et al., 1996). CONCLUSIONS AND PERSPECTIVES New concepts related to the relationship between cytoskeletal—membrane proteins have emerged from recent studies. Microvillar cytoskeletal proteins can integrate various signals transduced from receptors and from adhesion molecules through different pathways. Changes in actin organization can modulate the activity of membrane proteins, such as channels, exchangers. In turn, these membrane proteins may modulate actin filament organization resulting in sensitive feedback loops. The most exciting work appears to be the analysis of membrane-cytoskeleton interactions through the analysis of networks of protein-protein interaction. The clarification of signal transduction pathways that origin from the cell surface receptors to the F-actin will result in the identification of the components involved in signalling. These studies will have to take also in consideration the post-translational modification such as phosphorylation, lipid binding, that can change the conformation of the protein and therefore its function. Development of genetic approaches will undoubtly provide some clues on how these multimolecular complexes are built and function. Screening for genetic defects will be particularly useful when biochemical approaches are difficult. Knock out of genes will constitute another means to assess the function of the cytoskeletal proteins.


93

Scheme of signal transduction pathways between basolateral and apical domains of epithelial cells. Agonists can activate G-protein linked receptors and tyrosine kinase receptors located on the basolateral surface of the epithelial cells. Signal transduction leads to changes in microvillar cytoskeleton organization and changes in the functions of proteins at the plasma membrane. V villin; E ezrin; Y c-yes; P phosphate; EGF epidermal growth factor; HGF hepatocyte growth factor; Agonists such as carbachol, vasopressin.

ACKNOWLEDGEMENTS We thank our colleagues for discussions and advices, specially E.Coudrier, E. Friederich, R.Golsteyn and P.Poullet for their help and the critical reading of the manuscript. The authors wish to thank S.Khurana (Johns Hopkins University, Baltimore), E.Friederich, J.J.Fontaine for communicating their results before publication. The author’s work was supported by grants from the Association pour la recherche sur le cancer (ARC-N ° 1312), the Ligue Nationale Franchise contre le cancer, the Biomed Program (BMH4-CT95–0090). REFERENCES Adams, A.E.M., Botstein, D. and Drubin, D.G. (1991) Requirement of yeast fimbrin for actin organization and morphogenesis in vitro. Nature, 354, 404–408. Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993) Ezrin contains cytoskeleton and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J. Cell Biol., 120, 129–139. Andreoli, C., Martin, M., Leborgne, R., Reggio, H. and Mangeat, P. (1994) Ezrin has properties to self-associate at the plasma membrane. J. Cell Sci., 107, 2509–2521. Arpin, M., Pringault, E., Finidori, J., Garcia, A., Jeltsch, J.-M., Vandekerckhove, J. and Louvard, D. (1988) Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity. J. Cell Biol., 107, 1759–1766.

94


Arpin, M., Algrain, M. and Louvard, D. (1994) Membrane-actin filament connections: an increasing diversity of players related to band 4.1. Curr. Opin. Cell Biol., 6, 136–141. Arpin, M., Friederich, E., Algrain, M., Vernel, F. and Louvard, D. (1994) Functional differences between L- and TPlastin isoforms. J. Cell Biol., 127, 1995–2008. Avraham, K.B., Hasson, T., Steel, K.P., Kingsley, D.M., Russell, L.B., Mooseker, M.S., Copeland, N.G. and Jenkins, N.A. (1995) The mouse Snell’s waltzer deafness gene encodes an unconventional myosin required for structural integrity of inner hair cells. Nature Genet., 11, 369–375. Azim, A.C., Knoll, J.H.M., Beggs, A.H. and Chishti, A.H. (1995) Isoform cloning, actin binding, and chromosomal localization of human erythroid dematin, a member of the villin superfamily. J. Biol. Chem., 270, 17407–17413. Bazari, W.L., Matsudaira, P.T., Wallek, M., Smeal, J., Jakes, R. and Ahmed, Y. (1988) Villin sequence and peptide map identify six homologous domains. Proc. Natl. Acad. Set. USA, 85, 4986–4990. Berdiev, B.K., Prat, A.G., Cantiello, H.F., Ausiello, D.A., Fuller, C.M., Jovov, B., Benos, D.J. and Ismailov, I.I. (1996) Regulation of epithelial sodium channels by short actin filaments. J. Biol. Chem., 271, 17704–17710. Berryman, M., Franck, Z. and Bretscher, A. (1993) Ezrin is concentrated in the apical microvilli of a wide variety of epithelial cells whereas moesin is found primarily in endothelial cells. J. Cell Sci, 105, 1025–1043. Berryman, M., Gary, R. and Bretscher, A. (1995) Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J. Cell Biol., 131, 1231–1242. Bikle, D.D., Munson, S. and Komuves, L. (1996) Zipper protein, a B-G protein with the ability to regulate actin/myosin I interactions in the intestinal brush border. J. Biol. Chem., 271, 9075–9083. Bretscher, A. and Weber, K. (1978) Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy. J. Cell Biol., 79, 839–845. Bretscher, A. and Weber, K. (1979) Villin: the major microfilament-associated protein of the intestinal microvillus. Proc. Natl. Acad. Sci. USA, 76, 2321–2325. Bretscher, A. and Weber, K. (1980) Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures. J. Cell Biol., 86, 335–340. Bretscher, A. (1983) Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells. J. Cell Biol., 97, 425–432. Bretscher, A. (1989) Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J. Cell Biol., 108, 921–30. Bretscher, A. (1991) Microfilament structure and function in the cortical cytoskeleton. Annu. Rev. Cell Biol., 7, 337–74. Cantiello, H.F. (1995) Role of the actin cytoskeleton on epithelial Na+ channel regulation. Kidney Int., 48, 970–984. Cantiello, H.F., Stow, J., Prat, A.G. and Ausiello, D.A. (1991) Actin filaments regulate epithelial Na+ channel activity. Am. J. Pbysiol., 261, C882–C888. Chafel, M.M., Shen, W. and Matsudaira, P. (1995) Sequential expression and differential localization of I-, L-, and Tfimbrin during differentiation of the mouse intestine and yolk sac. Developmental Dynamics, 203, 141–151. Chen, J., Cohn, J.A. and Mandel, L.J. (1995) Dephosphorylation of ezrin as an early event in renal microvillar breakdown and anoxic injury. Proc. Natl. Acad. Set. USA, 92, 7495– 7499. Cheney, R.E. and Mooseker, M.S. (1995) Unconventional myosins. Annu. Rev. Cell Dev. Biol., 11, 633–675. Chong, L.D., Traynor-Kaplan, A., Bokoch, G.M. and Schwartz, M.A. (1994) The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell, 79, 507–513. Cohen, M.E., Wesolek, J., McCullen, J., Rys-Sikora, K., Pandol, S., Rood, R.P., Sharp, G.W.G. and Donowitz, M. (1991) Carbachol- and elevated Ca2+-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells. J. Clin. Invest., 88, 855–863. Collins, K., Sellers, J.R. and Matsudaira, P. (1990) Calmodulin dissociation regulates brush border myosin I (110-kDcalmodulin) mechanochemical activity in vitro. J. Cell Biol., 110, 1137–1147. Coluccio and Bretscher (1989) Reassociation of microvillar core proteins : making a microvillar core in vitro. J. Cell Biol., 108, 495. Costa de Beauregard, M.-A., Pringault, E., Robine, S. and Louvard, D. (1995) Suppression of villin expression by antisense RNA impairs brush border assembly in polarized epithelial intestinal cells. EMBO J., 14, 409-421.


95

Coudrier, E., Kerjaschki, D. and Louvard, D. (1988) Cytoskeleton organization and submembranous interactions in intestinal and renal brush borders. Kidney Int., 34, 309–320. de Arruda, M.V., Watson, S., Lin, C.-S., Leavitt, J. and Matsudaira, P. (1990) Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins. J. Cell Biol., 111, 1069–1079. Donowitz, M., Cohen, M.E., Gould, M. and Sharp, G.W.G. (1989) Elevated intracellular Ca2+acts through protein kinase C to regulate rabbit ileal NaCl absorption. J. Clin. Invest., 83, 1953–1962. Donowitz, M., Montgomery, J.L., Walker, M.S. and Cohen, M.E. (1994) Brush-border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na (+)-H+ exchange. Am. J. Physiol., 266, G647–656. Dransfield, D.T., Bradford, A.J., Smith, J., Martin, M., Roy, C., Mangeat, P.H. and Goldenring, J.R. (1997) Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J., 16, 35–43. Drenckhahn, D., Engel, K., Höfer, D., Merte, C., Tilney, L. and Tilney, M. (1991) Three different actin filament assemblies occur in every hair cell: each contains a specific actin cross-linking protein. J. Cell Biol., 112, 641–651. Edwards, H.C. and Booth, A.G. (1987) Calcium-sensitive, lipid-binding cytoskeletal proteins of the human placental microvillar region. J. Cell Biol., 105, 303–311. Ezzell, R.M., Chafel, M.M. and Matsudaira, P.T. (1989) Differential localization of villin and fimbrin during development of the mouse viseral endoderm and intestinal epithelium. Development, 106, 407–419. Franck, Z., Footer, M. and Bretscher, A. (1990) Microinjection of villin into cultured cells induces rapid and longlasting changes in cell morphology but does not inhibit cytoki-nesis, cell motility, or membrane ruffling. J. Cell Biol., 111, 2475–2485. Franck, Z., Gary, R. and Bretscher, A. (1993) Moesin, like ezrin, colocalizes with actin in the cortical cytoskeleton in cultured cells, but its expression is more variable. J. Cell Sci., 105, 219–231. Friederich, E., Huet, C., Arpin, M. and Louvard, D. (1989) Villin induces microvilli growth and actin redistribution in transfected fibroblasts. Cell, 59, 461–475. Friederich, E., Pringault, E., Arpin, M. and Louvard, D. (1990) From the structure to the function of villin, an actin-binding protein of the brush border. Bioessays, 12, 403–408. Friederich, E., Vancompernolle, K., Huet, C., Goethals, M., Finidori, J., Vandekerckhove, J. and Louvard, D. (1992) An actin-binding site containing a conserved motif of charged amino acid residues is essential for the morphogenic effect of villin. Cell, 70, 81–92. Friederich, E., Kreis, T.E. and Louvard, D. (1993) Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D. J. Cell Sci., 105, 765–775. Gary, R. and Bretscher, A. (1993) Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins. Proc. Nat. Acad. Sci. USA, 90, 10846–50. Gary, R. and Bretscher, A. (1995) Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site. Mol. Biol. Cell, 6, 1061–1075. Gherardi, E., Gray, J., Stoker, M., Ferryman, M. and Furlong, R. (1989) Purification of scatter factor, a fibroblastderived basic protein that modulates epithelial interactions and movement. Proc. Natl. Acad. Sci. USA, 86, 5844–5848. Gillespie, P.G., Wagner, M.C. and Hudspeth, A.J. (1993) Identification of a 120-kD hair-bundle myosin I located near stereociliary tips. Neuron, 11, 581–594. Gillespie, P.G. (1996) Deaf and dizzy mice with mutated myosin motors. Nature Med., 2, 21– 29. Glenney, J.R.J., Geisler, N., Kaulfus, P. and Weber, K. (1981a) Demonstration of at least two different actin-binding sites in villin, a calcium-regulated modulator of F-actin organization. J. Biol. Chm., 256, 8156–8161. Glenney, J.R.J., Kaulfus, P., Matsudaira, P. and Weber, K. (1981b) F-actin binding and bundling properties of fimbrin, a major cytoskeletal protein of microvillus core filaments. J. Biol. Chem., 256, 9283–9288. Gould, K., Cooper, J., Bretscher, A. and Hunter, T. (1986) The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein. J. Cell Biol., 102, 660–669. Grawe, F., Wodarz, A., Lee, B., Knust, E. and Skaer, H. (1996) The Drosophila genes crumbs and Stardust are involved in the biogenesis of adherens junctions. Development, 122, 951– 959.

96


Hartwig, J.H. and Kwiatkowski, D.J. (1991) Actin-binding proteins. Curr. Opin. Cell Biol., 3, 87–97. Hasson, T., Heintzelman, M.B., Santos-Sacchi, J., Corey, D.P. and Mooseker, M.S. (1995) Expression in cochlea and retina of myosin VIIa, the gene product defective in Usher syndrome type IB. Proc. Natl. Acad. Sci. USA, 92, 9815–9819, Hasson, T. and Mooseker, M.S. (1996) Vertebrate unconventional myosins. J. Biol. Chem., 271, 16431–16434. Hayden, S.M., Wolenski, J.S. and Mooseker, M.S. (1990) Binding of brush border myosin I to phospholipid vesicles. J. Cell Biol., 111, 443–451. Heintzelman, M.B. and Mooseker, M.S. (1990a) Assembly of the brush border cytoskeleton: changes in the distribution of microvillar core proteins during enterocyte differentiation in adult chicken intestine. Cell Motil. Cytoskel., 15, 12–22. Heintzelman, M.B. and Mooseker, M.S. (1990b) Structural and compositional analysis of early stages in microvillus assembly in the enterocyte of the chick embryo. Differentiation, 43, 175–182. Heintzelman, M.B. and Mooseker, M.S. (1992) Assembly of the intestinal brush border cytoskeleton. Current Topics in Developmental Biology, 26, 93–122. Heintzelman, M.B., Hasson, T. and Mooseker, M.S. (1994) Multiple unconventional myosin domains of the intestinal brush border cytoskeleton. J. Cell Set., 107, 3535–3543. Hirao, M., Sato, N., Kondo, T., Yonemura, S., Monden, M., Sasaki, T., Takai, Y., Tsukita, S. and Tsukita, S. (1996) Regulation mechanism of ERM (ezrin/radixin/moesin) protein/ plasma membrane association: possible involvement of phosphatidylinositol turnover and rho-dependent signaling pathway. J. Cell Biol., 135, 37–51. Hofmann, A., Noegel, A.A., Bomblies, L, Lottspeich, F. and Schleicher, M. (1993) The 100 kDa F-actin capping protein of Dictyostelium amoebae is a villin prototype (protovillin) FEES Lett., 328, 71–76. Husain-Chishti, A., Levin, A. and Branton, D. (1988) Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9. Nature, 324, 718–721. Janmey, P.A. and Matsudaira, P.T. (1988) Functional comparison of villin and gelsolin. Effects of Ca2+, KC1, and polyphosphoinositides. J. Biol.Chem., 263, 16738–16743. Janmey, P.A., Lamb, J., Allen, P.G. and Matsudaira, P.T. (1992) Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin. J. Biol. Chem., 267, 11818– 11823. Janmey, P.A. (1994) Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. Annu. Rev. Physiol., 56, 169–191. Klein, G., Langegger, M., Timpl, R. and Ekblom, P. (1988) Role of laminin A chain in the development of epithelial cell polarity. Cell, 55, 331–341. Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W.A., Rhee, S.G., Donowitz, M. and Cohen, M.E. (1996a) Asymmetric signal transduction in polarized ileal Na+-absorbing cells: carbachol activates brush-border but not basolateral-membrane PIP2—PLC and translocates PLC-γ1 only to the brush border. Biochem. J., 313, 509–518. Khurana, S., Nath, S.K., Levine, S.A., Bowser, J.M., Tse, C.-M., Cohen, M.E. and Donowitz, M. (1996b) Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na+/ H+ exchange. J. Biol. Chem., 271, 9919–9927. Krieg, J. and Hunter, T. (1992) Identification of the two major epidermal growth factor-induced tyrosine phosphorylation sites in the microvillar core protein ezrin. J. Biol. Chem., 267, 19258–65. Lin, C.-S., Aebersold, R.H., Kent, S.B., Varma, M. and Leavitt, J. (1988) Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts. Mol. Cell Biol., 8, 4659–4668. Lin, C.-S., Shen, W., Chen, Z.P., Tu, Y.-H. and Matsudaira, P. (1994) Identification of I-plastin, a human fimbrin isoform expressed in intestine and kidney. Mol. Cell. Biol., 14, 2457– 2467. Louvet, S., Aghion, J., Santa-Maria, A., Mangeat, P. and Maro, B. (1996) Ezrin becomes restricted to outer cells following asymmetrical division in the preimplantation mouse embryo. Dev. Biol., 177, 568–579. Mahajan-Miklos, S. and Cooley, L. (1994) The villin-like protein encoded by the drosophila quail gene is required for actin bundle assembly during oogenesis. Cell, 78, 291–301. Martin, M., Andréoli, C., Sahuquet, A., Montcourrier, P., Algrain, M. and Mangeat, P. (1995) Ezrin NH2-terminal domain inhibits the cell extension activity of the COOH-terminal domain. J. Cell Biol., 128, 1081–1093.


97

Matsudaira, P. (1991) Modular organization of actin crosslinking proteins. TIBS, 16, 87–92. Matsudaira, P.T. and Burgess, D.R. (1979) Identification and organization of the components in the isolated microvillus cytoskeleton. J. Cell Biol., 83, 667–673. Maunoury, R., Robine, S., Pringault, E., Huet, C., Guénet, J.L., Gaillard, J.A. and Louvard, D. (1988) Villin expression in the visceral endoderm and in the gut anlage during early mouse embryogenesis. EMBO J., 7, 3321–3329. Maunoury, R., Robine, S., Pringault, E., Leonard, N., Gaillard, J.A. and Louvard, D. (1992) Developmental regulation of villin gene expression in the epithelial cell lineages of mouse digestive and urogenital tracts. Development, 115, 717–28. Messier, J.M., Shaw, L.M., Chafel, M., Matsudaira, P. and Mercuric, A.M. (1993) Fimbrin localized to an insoluble cytoskeletal fraction is constitutively phosphorylated on its headpiece domain in adherent macrophages. Cell Motil. Cytoskel., 25, 223–233. Montesano, R., Matsumoto, K., Nakamura, T. and Orci, L. (1991) Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell, 67, 901–908. Mooseker, M.S. and Tilney, L.G. (1975) Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J. Cell Biol., 67, 725–743. Mooseker, M.S., Wolenski, J.S., Coleman, T.R., Hayden, S.M., Cheney, R.E., Espreafico, E., Heintzelman, M.B. and Peterson, M.D. (1991) in Ordering the membrane-cytoskeleton trilayer, (Mooseker, M.S. and Morrow, J. eds), pp. 31–55, Academic Press, Orlando, FL. Morgan, N.S., Skovronsky, D.M., Artavanis-Tsakonas, S. and Mooseker, M.S. (1994) The molecular cloning and characterization of Drosophila melanogaster myosin-IA and myosin-IB. J. Mol. Biol., 239, 347–356. Morgan, N.S., Heintzelman, M.B. and Mooseker, M.S. (1995) Characterization of myosin-IA and myosin-IB, two unconventional myosins associated with the Drosophila brush border cytoskeleton. Dev. Biol., 172, 51–71. Namba, Y., Ito, M., Zu, Y., Shigesada, K. and Maruyama, K. (1992) Human T cell L-plastin bundles actin filaments in a calcium dependent manner. J. Biochem., 112, 503–507. Niggli, V., Andréoli, C., Roy, C. and Mangeat, P. (1995) Identification of a phosphatidylinositol-4.5-bisphosphatebinding domain in the N-terminal region of ezrin. FEBS Letters, 376, 172–176. Ojakian, G.K. and Schwimmer, R. (1994) Regulation of epithelial cell surface polarity reversal by bl integrins. J. Cell Sci., 107, 561–576. Pakkanen, R., Hedman, K., Turunen, O., Wahlstrom, T. and Vaheri, A. (1987) Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma ceils. J. Histocbem. Cytochem., 35, 809–816. Pestonjamasp, K., Amieva, M., Strassel, C., Nauseef, M., Furthmayr, H. and Luna, E. (1995) Moesin, ezrin and p205 are actin-binding proteins associated with neutrophil plasma membranes. Molecular Biology of the Cell, 6, 247–259. Phillips, A.D. and Schmitz, J. (1992) Familial microvillous atrophy: a clinicopathological survey of 23 cases. J. Pediatr. Gastro. Nutri., 14, 380–396. Prat, A.G., Bertorello, A.M., Ausiello, D.A. and Cantiello, H.F. (1993) Activation of epithelial Na+ channels by protein kinase A requires actin filaments. Am. J. Physiol., 265, C224–C233. Prat, A.G., Holtzman, E.J., Brown, D., Cunningham, C.C., Reisin, I.L., Kleyman, T.R., McLaughlin, M., Jackson, J.G.R., Lydon, J. and Cantiello, H.F. (1996) Renal epithelial protein (Apx) is an actin cytoskeleton-regulated Na+ channel. J. Biol. Chem., 271, 18045–18053. Rana, A.P., Ruff, P., Maalouf, G J., Speicher, D.W. and Chishti, A.H. (1993) Cloning of human erythroid dematin reveals another member of the villin family. Proc. Natl. Acad. Sci. USA, 90, 6651–6655. Robine, S., Jaisser, F., Cohen-Tanoudji, M., Antony, C., Pehau, G., Ferrary, E., Ouelbani, L., El Marjou, F., Costa de Beauregard, M.A., Babinet, C. and Louvard, D. Mol. Biol. Cell, 7, Abstr. 3156. Rotin, D., Bar-Sagi, D., O’Brodovich, H., Merilainen, J., Lehto, V.P., Canessa, C.M., Rossier, B.C. and Downey, G.P. (1994) An SH3 binding region in the epithelial Na+ channel (arENaC) mediates its localization at the apical membrane. EMBO J., 13, 4440–4450. Sachs, M., Weidner, K.M., Brinkmann, V., Walther, I., Obermeier, A., Ullrich, A. and Birchmeier, W. (1996) Motogenic and morphogenic activity of epithelial receptor tyrosine kinases. J. Cell Biol., 133, 1095–1107.

98


Shepherd, G.M.G., Barres, B.A. and Corey, D.P. (1989) “Bundle blot" purification and initial protein characterization of hair cell stereocilia. Proc. Natl. Acad. Sci. USA, 86,4973–977. Shibayama, T., Carboni, J. and Mooseker, M. (1987) Assembly of intestinal brush border: appearance and redistribution of microvillar core proteins in developing chick enterocytes. J. Cell Biol., 105, 335–344. Shuster, C. and Herman, I. (1995) Indirect association of ezrin with F-actin: isoform specificity and calcium sensitivity. J. Cell Biol., 128, 837–848. Staub, O., Verrey, F., Kleyman, T.R., Benos, D.J., Rossier, B.C. and Kraehenbuhl, J.-P. (1992) Primary structure of an apical protein from Xenopus laevis that participates in amiloride-sensitive sodium channel activity. J. Cell Biol., 119, 1497–1506. Stoker, M., Gherardi, E., Perryman, M. and Gray, J. (1987) Scatter factor is a fibroblast-derived modulator of epithelial cell motility. Nature, 327, 239–242. Takeuchi, K., Sato, N., Kasahara, H., Funayama, N., Nagafuchi, A., Yonemura, S. and Tsukita, S. (1994) Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J. Cell Biol., 125, 1371–1384. Tilney, M., Tilney, L., Stephens, R., Merte, C.D.D., Cotanche, D. and Bretscher, A. (1989) Preliminary biochemical characterization of the stereocila and cuticular plate of hair cells of the chick cochlea. J. Cell Biol., 109, 1711–1723. Tilney, L.G. and Tilney, M.S. (1992) Actin filaments, stereocilia, and hair cells: how cells count and measure. Annu. Rev. Cell Biol. 8, 257–274. Tsukita, S., Oishi, K., Sato, N., Sagara, J., Kawai, A. and Tsukita, S. (1994) ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J. Cell Biol., 126, 391–401. Tsukita, S., Yonemura, S. and Tsukita, S. (1997) ERM (ezrin/radixin/moesin) family: from cytoskeleton to signal transduction. Curr. Opin. Cell Biol., 9, 70–75. Turunen, O., Wahlstrom, T. and Vaheri, A. (1994) Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family. J. Cell Biol., 126, 1445–1453. Urushidani, T., Hanzel, D. and Forte, J. (1989) Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation. Am. J. Physiol., 256, 1070–1081. Vega-Salas, D.E., Salas, P.J.I., Gundersen, D. and Rodriguez-Boulan, E. (1987) Formation of the apical pole of epithelial (Madin-Darby Canine Kidney) cells: polarity of an apical protein is independent of tight junctions while segregation of a basolateral marker requires cell-cell interactions. J. Cell Biol., 104, 905–916. Weidner, K.M., Behrens, J., Vandekerckhove, J. and Birchmeier, W. (1990) Scatter factor: Molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell Biol., 111, 2097–2108. Weil, D., Blanchard, S., Kaplan, J., Guilford, P., Gibson, F., Walsh, J., Mburu, P., Varela, A., Levilliers, J., Weston, M.D., Kelley, P.M., Kimberling, W.J., Wagenaar, M., Levi-Acobas, F., Larget-Piet, D., Munnich, A., Steel, K.P., Brown, S.D.M. and Petit, C. (1995) Defective myosin VIIa gene responsible for Usher syndrome type 1B. Nature, 374, 60– 61. Whitney, A.B., Okamoto, C.T., Hanzel, D.K. and Forte, J.G. (1990) Epidermal growth factor (EGF) stimulates phosphorylation of an 80 kDa protein at the apical surface of parietal cells. FASEB J., 4, A488. Wodarz, A., Hinz, U., Engelbert, M. and Knust, E. (1995) Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of drosophila. Cell, 82, 67–76. Wolenski, J.S., Hayden, S.M., Forscher, P. and Mooseker, M.S. (1993) Calcium-calmodulin and regulation of brush border myosin-I MgATPase and mechanochemistry. J. Cell Biol., 122, 613–621. Yao, X., Cheng, L. and Forte, J.G. (1996) Biochemical characterization of ezrin-actin interaction. J. Biol. Chem., 271, 7224–7229. Zhu, T., Sata, M. and Ikebe, M. (1996) Functional expression of mammalian myosin I beta: analysis of its motor activity. Biochemistry, 35, 513–522.

6. BASEMENT MEMBRANES NEIL R.SMYTH and MATS PAULSSON Institute for Biochemistry, Medical Faculty, University of Cologne, Joseph-StelzmannStrafie. 52, D-50931 Cologne, Germany

INTRODUCTION The basement membrane is one of the characteristic structures of the differentiated epithelium and is important for the development of epithelial polarity. The most useful definition of the basement membrane is probably still a morphological one, i.e. that of the basal lamina as observed in the electron microscope. It is a thin sheetlike extracellular matrix network, found at all borders between epithelia or endothelia and the underlying stroma. This extracellular matrix layer is present also in other types of tissues e.g. surrounding muscle and fat cells and ensheathing peripheral nerves (Vracko, 1974). It appears that the basement membranes are more heterogeneous than has been assumed. As they are present at such different locations, they also differ in their morphology, in their molecular composition, and in their functional properties (Figure 1). Basement membranes may also be defined on the basis of their molecular composition. All basement membranes appear to contain a network of collagen type IV in which the meshes are filled with a laminin gel. Also the nidogen (alternatively called entactin) molecule which connects laminin to collagen IV (Aumailley et al., 1989) and the proteoglycan perlecan appear to be ubiquitous. The same molecules may, however, also be found as diffusible components, at least in embryonal connective tissue (Kücherer-Ehret et al., 1990; Thomas and Dziadek, 1993; Ekblom et al., 1994), or organized into networks that do not have the other characteristics of a basement membrane, e.g. the pericelluar matrix of fibroblasts (Schittny et al., 1988). Basement membranes have a number of essential functions. They serve as a mechanical support for the attachment of cells and thereby they allow an orderly formation of cell layers. Simultaneously they provide a barrier for the diffusion of macromolecules and for the migration of cells so dividing the organism into compartments. Because of the roles in creating and maintaining a tissue architecture basement membranes are ubiquitous and probably necessary in multicellular organisms. They not only contain spatial information but also regulate the phenotype of neighbouring cells through receptor mediated transmembrane contacts with the cytoskeleton and with intracellular signalling systems. Basement membranes contain a depot of tightly bound growth factors and may modulate the activity of these substances by the way they present them to the cells. We can identify sites in basement membrane molecules that bind to other extracellular proteins and carbohydrates and mediate the formation of a supramolecular structure (Timpl, 1996) as well as other domains that bind to cellular receptors (Aumailley et al., 1996). This functional diversity within the molecules has been achieved by evolution of modular proteins made up of many independently folded protein domains and undergoing a complex set of posttranslational modifications.

100


Figure 1 The many locations and functions of basement membranes as illustrated by an embryonic day 4.5 mouse embryo implanting into the uterine wall. Laminin was detected by immunofluorescence microscopy using antibodies raised against murine laminin-1. Basement membranes containing laminin are seen in the embryo under the parietal endodermal cells and overlying the trophectoderm (a). This basement membrane will later become Reichert’s membrane. Maternal basement membranes are seen under the uterine epithelium (b) and surrounding blood vessels and glands in the endometrium (c). Cells will migrate along the basement membrane formed over the trophectoderm. Later Reichert’s membrane helps delimit the embryo from the maternal tissue and is required to form a intact yolk sac (Williamson et al., 1997). The basement membranes in the vessel walls will serve as a substrate for endothelial cells and contribute to the permeability barrier between blood and interstitial fluid.

Basement membranes in different tissues are not uniform in molecular composition and supramolecular structure (Paulsson, 1993) and undergo marked changes during development (Ekblom et al., 1996; Miner et al., 1997). This diversity requires a strictly regulated expression of basement membrane genes; a selfassembly which might be aided by the cellular elements; and an controlled apparatus for their degradation. The spatio-temporal control of basement membrane characteristics is still poorly understood, but of great importance if we assume that these characteristics in turn can act as cues for cellular differentiation. Another component in the cellular perception of basement membranes is the differential expression of a repertoire of specific cell surface receptors for the constituent proteins. At present a number of binding sites for receptors in basement membrane proteins have been defined. The interplay of these sites and the receptors and the integration of the intracellular signals transduced have not been characterised. The emphasis has so far been on the integrin class of transmembrane receptors but increasing evidence shows that also other membrane proteins, such as α-dystroglycan, play a role.


101

This chapter should serve to give an overall view of the structure and molecular composition of basement membranes. In addition, we will highlight the diversity of basement membrane proteins and their receptors. Examples of the role of basement membranes and their constituent proteins in epithelial-mesenchymal interactions will be given in later chapters. MORPHOLOGY OF BASEMENT MEMBRANES Basement membranes are typically between 20 and 300 nm thick. Using conventional preparation methods for electron microscopy a lamina lucida or lamina rara, adjacent to the epithelial cells, may be distinguished from a further removed lamina densa. Apposed to some basement membranes, a pars fibroreticularis forms the transition to the underlying connective tissue stroma. It could well be that the differences in electron densities that have given rise to the nomenclature are preparation artefacts as they can not reproduced with cryofixation methods for electron microscopy (Chan and Inoue, 1994). Even so they are useful in helping us to distinguish the common single basement membrane from the double one found in the glomerulus and the rare multilayered ones, as in the parietal yolk sac of rodents. Basement membranes underlying stratified epithelia show another type of specialisation. They are connected with the hemidesmosomes by anchoring filaments and with the stroma through anchoring fibrils. The former contain laminins 5, 6, and 7 (see below) while the latter which contribute to the pars fibroreticularis are made up from collagen type VII (Burgeson, 1993). Specialised areas of distinct molecular composition may occur within basement membranes. At the motor endplate, the composition of the skeletal muscle basement membrane differs clearly from the surrounding areas. Here an abundant expression of laminins containing the β2 chain (Hunter et al., 1989) as well as of certain agrin variants (Reist et al., 1987) are detected. These proteins appear essential for the formation of the neuromuscular junction and agrin is able to guide a regenerating nerve back to its original synaptic site (McMahan, 1990). THE MOLECULAR STRUCTURE OF BASEMENT MEMBRANES All basement membranes contain four groups of components, namely representitives of the collagen IV family, members of the laminin family, nidogens and a special set of proteoglycans. In addition to these ubiquitous molecules there are other basement membrane associated proteins which have a more restricted presence (Lindblom and Paulsson, 1996). As an increasing number of collagen IV and laminin family members have been traced it has become evident from expression data, disease linkage studies, and gene inactivation experiments that heterogeneity with regard to those molecules is important in defining the physiological role of the particular tissue. Collagen IV Collagen IV is the only collagen present in all basement membranes and forms the scaffold of the basal lamina. The collagen IV polypeptide family to date consists of six members α1(IV) to α6(IV) giving the potential for great variability in the trimeric molecule (for refs. see Lindblom and Paulsson, 1996). The collagen IV α-chains have a common structure with three distinct domains (Figure 2). The central and major part of the molecule is a 360 nm long stretch containing Gly Xaa Yaa tripeptide repeats which forms the triple helix together with two other α(IV) chains. This stretch contains numerous imperfections in the tripeptide pattern which may serve to increase the flexibility of the molecules. At the C-terminus there is a

102


noncollagenous, globular domain (NC1) while at the N-terminus a non-triple helical hinge domain divides the major triple helix from the 30 nm long triple helical 7S domain (Timpl et al., 1981). The genes for the six α-chains have been shown to be ordered in three pairs upon the genome, with α1– α2, α3–α4, and α5–α6 in a head-to-head orientation (Pöschl et al., 1988; Soininen et al., 1988; Zhou et al., 1993; Sugimoto et al., 1994). Analysis of the amino acid sequences has shown that by homology the family can be divided into α1, α3, α5 and α2, α4, α6 subgroups. A given chain is well conserved through evolution with the NC1 and 7S domains being especially maintained (Leinonen et al., 1994) probably due to their importance in the formation of dimers and tetramers in the collagen IV network (Figure 2). Despite the great potential for formation of different trimers, there is data on the in vivo occurence only for two heterotrimers. These are (α1)2α2 (Trüeb et al., 1982), which is by far the most common form of collagen IV and the rarer (α3)2α4 present in the glomerular basement membrane (Butkowski et al., 1989; Johansson et al., 1992). The trimeric molecules containing α5 and α6 have not yet been described. However, the α5 chain is more widespread, being more highly expressed in the kidney (Butkowski et al., 1989), than the α6 which is most abundant in the lung and oesophagus (Hostikka et al., 1990; Zhou et al., 1993). While the α5 chain colocalises with the α6 chain in the skin and other tissues by immunohistochemistry, triple staining also shows it occurring with α3 and α4 chains in the glomerular basement membrane (Ninomiya et al., 1995). In most aspects the biosynthesis of collagen type IV proceeds in the same manner as well studied for the fibrillar collagens (Prockop et al., 1979a,b). Important steps are the hydroxylation of proline and lysine residues, folding of the triple helix, and glycosylation. The three chains then interact by the NC1 domains and the formation of a triple helix follows in a C- to N-terminal direction (Dölz et al., 1988). The molecule is stabilised by disulphide bonds present in the NC1, triple-helical and 7S domains (Siebold et al., 1987, 1988). Unlike in the fibrillar collagens, the specialised domains at the N- and C-termini are retained. After secretion, the trimers self-associate to give the supramolecular network (Oberbäumer et al., 1982; Blumberg et al., 1986). Several forms of intermolecular associations have been described (Figure 2). Two NC1 domains may interact in a head-to-head manner producing a dimer and a tetramer may be formed by the lateral interaction of four 7S domains (Timpl et al., 1981). Both of these structures are stabilised by the rearrangement of intramolecular disulphide bonds and in the case of the 7S domain interaction also by intermolecular lysine-derived crosslinking (Siebold et al., 1987, 1988). The end-to-end interactions alone would produce a model network with a pore size of 800 nm (distance between two identical crosslinking sites, Timpl et al., 1981) which is substantially larger than that seen in electron microscopy studies of the collagen IV network as it occurs in the tissue (Yurchenco and Ruben, 1987, 1988). This discrepancy is explained by the formation of other less defined interactions where the triple helices from more than one trimer wind about each other giving supercoiled structures (Figure 2). The possibility for specific supramolecular interactions between different trimers allows great ordered heterogeneity throughout a basement membrane as well as the potential for localised variation in the collagen IV suprastructure. The importance of collagen IV in basement membrane function and hence in development is highlighted by the fact that Gly to Glu mutations in a single Gly Xaa Yaa repeat in the α1 (IV) lead to lethality in late embryogenesis for C. elegans (Guo etal., 1991). The less widespread α3 and α5 chains have been implicated in a number of human disease syndromes (Hudson et al., 1993). In Goodpasture’s syndrome, characterised by glomerular nephritis and pulmonary haemorrhage, the clinical picture is related to auto-antibody production against the NC1 domain of the α3(IV) chain (the Goodpasture antigen; Saus et al., 1988; Kalluri et al., 1991; Neilson et al., 1993). Mutations in the gene coding for α5 (IV) have been shown in Alport’s syndrome, a dominant X-linked progressive nephritis with ultrastructural disruption of the glomerular basement membrane often accompanied by deafness and lenticular and retinal changes (Barker et al., 1990; Zhou et al., 1991).


103

Figure 2 A schematic view of the structure of collagen IV and the various interactions leading to the formation of a collagen IV network.

Laminins and Nidogen (Entactin) The laminins form the second network within the basement membrane. They are the major non-collagenous protein components, with the original molecule having been isolated from embryonic carcinoma cells (Chung et al., 1979) and EHS tumour cells (Timpl et al., 1979). This has now been shown to be the prototype (laminin-1) for a wide family and there is evidence for 11 members (nomenclature, see Burgeson et al., 1994; Miner et al., 1997). Laminin is a multidomain heterotrimer formed by the combination of one each of an α, a β and a γ chain. Laminin-1 is a 800 kDa glycoprotein dissociating into the 400 kDa α1 chain and β1 and γl chains each of 200 kDa. It has a cross structure (Engel et al., 1981) with one long and three short arms (Figure 3). The short arms are formed from the three free N-terminal ends of the a, β and γ chains. These parts of the β and γ chains contain two globular domains while there are three in the short arm contributed by the α chain (Figure 4). The globules are interspersed by multiple epidermal-growth-factorlike (EGF) domains forming rods. The laminin EGF domains differ from that originally described in having 8 cysteine residues as compared to the 6 cysteine motif seen in the epidermal growth factors and so have been re-designated as LE (laminin EGF) domains (Maurer and Engel, 1996). The long arm is a triple coiledcoil α-helix maintaining the trimer which is further stabilized by the presence of interchain disulphide bonds at both ends of the coiled-coil (Barlow et al., 1984; Paulsson et al., 1985). While the α-helical region continues to the C-terminal end of the β and γ chains the a chain has a large C-terminal extension, the G domain, which contains five independently folding globular domains (Figure 4; Sasaki et al., 1988). The β chains have a break in the α-helical region which is well conserved through this subfamily.

104


Figure 3 Rotary shadowing electron microsopy of (a, b) laminin-nidogen complexes and of (c) dissociated nidogen molecules, (a) shows an overview, while (b) and (c) display selected particles. Also note the single molecule of contaminating collagen IV in the lower right part of panel a. Bar=100 nm. (From Paulsson et al., 1987a, with permission)

The α1, β1 and γ1 subunits have been shown to be representatives of three distinct but closely related gene families of which there are now five, three, and two members respectively (Figure 4). Certain chains have been described as maintaining the domain structure of those in the original laminin-1, e.g. α2 and β2 (Maurer and Engel, 1996), while others have N terminal truncations presumably acquired in the process of evolutionary duplication e.g. α4, and γ2 (Maurer and Engel, 1996). The trimeric molecules formed by the combination of these chains may appear to have no short arms e.g. laminin 5 (α3, β3, γ2; Rousselle et al., 1991) or have a Y shape on electron microscopy e.g. laminin 7 (α3, β2, γ1 Champliaud et al., 1996). The


105

individual chains are each the product of their own distinct genes, designated in the fashion LAMA1 for the α1 chain, LAMC2 for the γ2 chain etc. (Burgeson et al., 1994). Recently there have been description of tissue specific RNA splicing of the α3 (Galliano et al., 1995; Miner et al., 1997) and γ2 chains (Airenne et al., 1996). Work on the mechanisms of laminin biosynthesis has been largely limited to laminin-1. All the fundamental features of the biosynthesis appear to be similar to those found for other secreted glycoproteins and the central issue is therefore the assembly of the laminin trimer. All steps in trimer formation are intracellular. Although there is still some controversy, most in vivo studies show that a βγ dimer is formed as the first intermediate. This dimer can be detected early in a disulphide linked form while the α chain is seen as a separate entity in SDS-PAGE gels run under nonreducing conditions. As determined from the nature and extent of glycosylation of biosynthesis intermediates, the subsequent formation of a disulphide bonded αβγ trimer is essential for transport from the endoplasmic reticulum to the Golgi apparatus (Morita et al., 1985; Peters et al., 1985). The absence of one of the chains dramatically reduces the secretion of the other chains (Yurchenco et al., 1997; Smyth and Edgar, unpublished) and it appears that the insertion of the a chain is a physiological prerequisite for secretion. All laminins so far isolated have turned out to be trimers, though the a chain can be secreted in a monomeric form by cultured cells (Yurchenco et al., 1997). The laminin trimer is formed by assembly of the coiled-coil α-helical regions of the three chains which results in the formation of the long arm structure (Paulsson et al., 1985). The amino acid sequence in these regions must contain additional information beyond the basic heptad repeat with hydrophobic residues in positions 1 and 4, as many aspects of the assembly process may be reproduced with purified proteins in vitro. The most striking feature showing this specificity is the fact that all laminins studied have turned out to be heterotrimers. Several in vitro studies employing proteolytic fragments (Hunter et al., 1990, 1992) or recombinant peptides (Utani et al., 1994; Kammerer et al., 1994, 1995; Antonsson et al., 1995) from the coiled-coil domains have shown that other assembly products are much less stable than the heterotrimers. Attempts have been made to identify shorter peptide sequences that direct the assembly, but these studies have not yet been conclusive. Ionic bridges between the different chains probably play a role in increasing the stability beyond that created by interactions between the hydrophobic residues in the heptad repeat (Beck et al., 1992), but experimental results point to additional mechanisms being important. These considerations are significant for understanding the formation of different trimeric molecules. With the presence of 10 genetically distinct polypeptides there is the possibility of 30 separate forms of the complete heterotrimer. As with collagen IV this potential is not met, presumably due to the spatio-temporal regulation of laminin gene expression and to the variable stability of the differently composed coiled-coils. A single cell population can produce more than one trimeric laminin molecule and may be induced to switch from producing one trimer to another (Tokida et al., 1990). The formation of a particular trimer is in part due to the amino acid sequence of the chains as certain pairwise combinations interact more strongly than others (Beck et al., 1992). Molecular chaperones may however also play a role in directing the assembly of a certain trimer. Upon secretion the laminin molecules aggregate to form a meshwork. This has been shown in vitro to occur by self-assembly and to be dependent upon the concentration of divalent cations, especially Ca2+ (Yurchenco et al., 1985; Paulsson, 1988). Indeed the presence of chelating agents will prevent assembly (Yurchenco et al., 1985) and has been used to extract laminin from the basement membranes in tissues (Paulsson et al., 1987a; Paulsson and Saladin, 1989). At high concentra tions and in the presence of Ca2+ the laminin molecules align to produce an array connected by the short arms with the terminal globular domains interacting to stabilise the structure (Yurchenco et al., 1992). Calcium ions are believed to produce a confbrmational change in the N-terminal domains allowing aggregation (Paulsson, 1988; Paulsson et al.,

106


Figure 4 Domain structure of vertebrate laminin chains. The large circles and rectangles towards the N-terminus (top) designate the globular domains in the short arms, while the smaller circles indicate EGF-like domains. The coiled-coil regions are shown as a long rod, and at the C-termini of the α chains the five globules of the G domain are shown as circles of intermediate size. The circles are filled in different ways to indicate further sequence homologies. (Modified from Maurer and Engel, 1996)

1988). There is also data suggesting that Ca2+ may stabilise the short arms from proteolytic cleavage


107

(Paulsson et al., 1988). The ion binding sites, essential for these phenomena, have not yet been elucidated. The removal of the α1 chain by antisense methods (De Arcangelis et al., 1996) or the yl chain by homologous recombination (Smyth and Edgar, unpublished) prevented the formation of a laminin network by cells normally producing these chains. Further, these manipulations stopped the laying down of a basement membrane, suggesting that laminin is needed for the aggregation of collagen IV in cell culture systems. The loss of laminin receptors such as β1-containing integrins also prevents the production of the laminin network suggesting a receptor mediated assembly of the basement membrane (Timpl, 1996; DiPersio et al., 1997). The functions of laminin are related to its multidomain structure, which allows many interactions both with other proteins of the basement membrane and with cell surface receptors. Laminin binds particularly strongly to nidogen (also called entactin; Carlin et al., 1981) which links laminin to collagen IV and so interconnecting the two networks (Aumailley et al., 1989). Nidogen was in early studies seen as a 150 kDa protein that cofractionated with laminin in immunoprecipitation from tissue culture medium (Hogan et al., 1980). It was purified from the culture medium of teratocarcinoma cells (Carlin et al., 1981) and from the EHS tumour matrix (Timpl et al., 1983; Paulsson et al., 1986, 1987a). Structural studies of the EHS protein (Paulsson et al., 1986) and recombinantly prepared material (Fox et al., 1991) revealed an elongated structure with three globular domains, G1 to G3, connected by a short polypeptide link between G1 and G2 and a longer rod between G2 and G3. The rod is made up of EGF-like motifs which, in contrast to the ones in laminin, have the classical 6 cysteine pattern (Durkin et al., 1988; Mann et al., 1989). By electron microscopy of EDTA-extracted laminin-nidogen complexes it was possible to show that a terminal globular domain of nidogen binds to one of the short arms of laminin (Figure 3; Paulsson et al., 1987a). This interaction has in the meantime been studied in great detail and we now know that the C terminal nidogen domain G3 (Fox et al., 1991) binds with a very high affinity to EGF repeat 4 in domain III of the γ1 chain (Mayer et al., 1993a; Pöschl et al., 1994). Demonstration of high affinity binding of domain G2 in nidogen to collagen IV (Aumailley et al., 1989; Fox et al., 1991) showed that nidogen links the laminin and collagen IV networks. Interestingly, some of the residues essential for nidogen binding have not been conserved in domain III of the γ2 chain and accordingly laminin-5 (α3β3γ2) does not show affinity for nidogen (Mayer et al., 1995). This may be an evolutionary adaptation to the fact that laminin-5 is to form anchoring filaments and not basement membranes proper. Nidogen may also link laminin to other proteins in the basement membrane as it binds to the core protein of the basement membrane proteoglycan perlecan (Battaglia et al., 1992; Reinhardt et al., 1993) and to fibulin-1 and -2 (Pan et al., 1993; Sasaki et al., 1995). Through this central role in basement membrane assembly nidogen may direct or modulate the whole process. At least in some locations in the developing embryo nidogen is synthesized by the mesenchyme (Thomas and Dziadek, 1993; Ekblom et al., 1994). Perhaps basement membrane formation is induced by its diffusing to meet its future interaction partners which are contributed by the epithelial cells. Nidogen may also be a main target for degradation in turnover and remodelling of basement membrane. It was early noticed that it is particularly easily degraded by proteases (Dziadek et al., 1985) and more recently it has been shown to contain distinct cleavage sites for members of the family of matrix metalloproteinases (Mayer et al., 1993b; Alexander et al., 1996). Recent work has shown that also nidogen is only one member of a family of proteins and this fact may also contribute to basement membrane heterogeneity (Ohno, 1996). Basement Membrane Proteoglycans Basement membranes have a net negative charge, which is of importance for their filtration properties (Comper et al., 1996). In classical work the molecular component mainly contributing the anionic sites to

108


the glomerular basement membrane was identified as heparan sulphate (Kanwar and Farquhar, 1979a,b). Somewhat later chondroitin sulphate was also shown to be present in a variety of basement membranes (Couchman et al., 1984). The bulk of the heparan sulphate is found in the proteoglycan perlecan (Noonan et al., 1991), though agrin has also been shown to sometimes carry this glycosaminoglycan (Tsen et al., 1995; Denzer et al., 1995). Most of the chondroitin sulphate appears to be bound to the proteoglycan bamacan (Wu and Couchman, 1997) but also perlecan may carry chondroitin sulphate (Danielson et al., 1992). Perlecan has a core protein of about 400 kDa which is substituted with three heparan sulphate chains of 35–40 kDa each, when the proteoglycan is isolated from the mouse EHS tumour (Paulsson et al., 1987b). There is evidence for heterogeneity in its glycosylation when isolated from different sources (Pejler et al., 1987). The name perlecan is due to the pronounced domain structure which gives it the appearance of a string of pearls as observed in the electron microscope. The complete coding sequence for perlecan has been determined from mouse (Noonan et al., 1991) and man (Kallunki and Tryggvason, 1992; Murdoch et al., 1992). The N-terminal domain I contains all three heparan sulphate attachment sites and is followed by domain II which is made up from cysteine-rich repeats with homology to those in the LDL receptor. Domain III contains four subdomains consisting of laminin-like EGF repeats, while domain IV contains numerous immunoglobulin-like repeats with a highest homology to those in N-CAM. Domain V is made up from three globular regions separated by two EGF-like repeats. The globular regions are homologous to the G domains at the C-terminus of the laminin α chains. The anchorage of perlecan into the basement membrane network is not fully understood though there is evidence for self-interactions between core proteins (Yurchenco et al., 1987), interactions between the core protein and nidogen and between the heparan sulphate and laminin (Battaglia et al., 1992). These affinities can be demonstrated in vitro but it is not yet clear if they also play a role in assembly in vivo. Perlecan is tightly bound to the rest of the basement membrane as denaturing solvents are needed for its solubilization from tissue. It may contribute to the interactions of the basement membrane with cells as several integrins have been shown to bind to the core protein (Hayashi et al., 1992; Battaglia et al., 1993; Chakravarti et al., 1995). Heparan sulphate in basement membranes binds growth factors, in particular basic FGF (Baird and Ling, 1987; Jeanny et al., 1987; Folkman et al., 1988), and perlecan modulates the activity of basic FGF in tissues (Aviezer et al., 1994). The presence of structures within the heparan sulphate chains with high affinity for antithrombin III (Pejler et al., 1987) indicate the potential for interactions with the coagulation system. Further, expression of perlecan has been implicated as a factor in the formation of amyloid deposits (Ailles et al., 1993; Snow et al., 1994). Bamacan (basement membrane-associated chondroitin sulphate proteoglycan) is the second basement membrane proteoglycan that has been studied in some detail. It is found in almost all basement membranes, with the exception of the glomerular basement membrane (McCarthy et al., 1989; McCarthy and Couchman, 1990; McCarthy et al., 1993). Its core protein has a molecular mass of 138 kDa and is made up by three presumably globular domains separated by sequence stretches that are predicted to form coiled-coil αhelices (Wu and Couchman, 1997). The chondroitin sulphate attachment sites are most likely to be located in the peripheral portions of the core protein sequence. The sequence does not have any clear homology to other extracellular proteins and bamacan appears to represent a gene that has developed independently. Bamacan has not yet been studied extensively in functional assays but the hypothesis has been put forward that it serves to stabilize basement membrane structure (Wu and Couchman, 1997).


109

Agrin Among the many proteins that occur in minor amounts or only in some basement membranes, agrin has proven to be of such biological importance that it deserves a special mention. While collagen IV, laminin, nidogen and heparan sulphate proteoglycans could be detected through being abundant in extracts of basement membrane containing tissue or in the culture media of basement membrane producing cell lines, it needed sophisticated biological assays to identify agrin. After denervation of muscle a regeneration occurs leading to the formation of a new synapse in exactly the same position on the muscle fiber as the previous one. There must therefore be molecules present on the surface of the muscle, e.g. in the basement membrane, that constitue a biological memory and direct the formation of the synapse. Such a molecule, with the property of inducing the patching of acetylcholine receptors and acetylcholine esterase, may of course also be expected to be of great importance in the formation of the peripheral nervous system during embryonal development. In a very systematic and elegant set of biological experiments McMahan and colleagues defined the properties of this protein (reviewed in McMahan and Wallace, 1989; McMahan, 1990; Hall and Sanes, 1993) and provided the information required for the subsequent isolation of agrin from the electrical organ of Torpedo (Nitkin et al., 1987), a tissue particularly rich in synapses. The amino acid sequence of agrin revealed it to be a multidomain protein with a molecular mass of about 225 kDa (Rupp et al., 1991; Tsim et al., 1992; Smith et al., 1992). There is some variability in structure between species and in addition the potential for alternative splicing (see below). Agrin may occur as a heparan sulphate proteoglycan and then has a total molecular mass of 400–600 kDa (Tsen et al., 1995; Denzer et al., 1995). The rat and chick proteins at their N-termini carry 9 repeats with 6 cysteines each, homologous to the pancreatic serine trypsin inhibitor type I (PSTI). Interspaced between PSTI-repeat 8 and 9, a sequence with homology to domain III of laminin chains is found. PSTI 9 is followed by two serineand threonine-rich regions which may be subject to O-linked glycosylation. Towards the C-terminus four EGFlike repeats with six cysteines are interspaced by sequences with homology to the laminin α chain. Alternative splicing occurs both at the Nand C-terminal ends and this splicing may drastically influence the functional properties of the protein. Inserts at or close to the C-terminus yield a high activity in assays that measure the aggregation of the acetylcholine receptor (for refs. see Gesemann et al., 1996), while an insert at the N-terminus yields a form with stronger binding to the extracellular matrix (Denzer et al., 1995). At least one agrin splice variant is found in extracellular matrix outside the nervous system (Godfrey et al., 1988; Godfrey, 1991). This form does not induce aggregation of the acetycholine receptor (Ruegg et al., 1992). Isoforms that do not play a role in synapse formation might serve other purposes in basement membranes. The agrin present at the neuromuscular junction is synthesized in the ventral horn of the spinal cord, transported to the nerve terminal, released and inserted in to the synaptic basement membrane (for refs see Kröger and Mann, 1996). The interactions involved in the anchorage in the basement membrane are not known, but must be rather strong as agrin remains associated with the basement membrane for weeks after denervation (Reist et al., 1987). Cellular receptors, such as α-dystroglycan may be involved in its anchorage (see below). Other forms of agrin are presumably produced and secreted locally as they have also been found far from nerve endings (Godfrey et al., 1988; Godfrey, 1991). BIOLOGICAL EFFECTS OF THE BASEMENT MEMBRANE The basement membrane and some of its major protein components have been shown to play major roles in the modulation of many aspects of cellular activity. These include altering cellular adhesion, spreading and

110


migration as well as roles in differentiation, acting as survival or anti-apoptosis factors. Most studies suffer from the limitation that they were performed in cell culture with pure proteins coated to plastic. While results obtained with the isokted proteins or protein domains are certainly significant, the three-dimensional structure of the basement membrane will undoubtedly also be important in defining the overall biological effects. Experiments have been performed with gels prepared from basement membrane extracts, but these also contain high concentrations of various growth factors that may or may not occur in basement membranes in vivo. Results which have been gained by inhibition with antibodies to basement membrane molecules themselves or their cellular receptors in organ cultures and in whole embryos are probably more representative of the in vivo situation (see Chapter 12). The present concepts will be challenged in the next years through the increasing use of gene targeting, resulting in animals where single basement membrane protein or receptor genes have been inactivated. It appears that laminin and collagen IV are major mediators of contacts between the basement membrane and cells. Binding sites in laminin for cellular receptors have been identified by use of proteolytic fragments of laminin-1 and recombinant domains of this and some other laminins. Many studies have also employed synthetic peptides derived from the laminin sequence that appear to mimic some of the properties of the trimeric laminin molecule (Yamada, 1991; Yamada and Kleinman, 1992). The latter studies remain controversial as they do not take into account the need for proper folding to create a high affinity binding site and as the effects obtained with the peptides were often not conclusively shown to be correlates of those of the intact protein (see e.g. Brandenberger et al., 1996). Many of the results obtained with synthetic peptides could be due to de novo activities not found in the same sequences when present in native and intact laminin. The most prominent binding sites in laminin have been localised to the distal portion of the long arm (Deutzmann et al., 1990). α6β1 integrin binds to sites at the transition between the coiled-coil α-helix and the globular G domain. The activity of of the corresponding proteolytic fragments or recombinant domains critically depend on the presence of all three laminin chains in a proper assembly. α-dystroglycan binds to a fragment consisting of two most C-terminal subdomains of G domain and is contributed solely by the a chain. In collagen IV binding sites for α1β1 and α2β1 integrins have been localised to the major triple helix and appear to consist of an RGD like arrangement of amino acid residues which are in different chains and are sterically brought together through the assembly of the triple helix (Eble et al., 1993). Basement Membrane-binding Integrins The most studied receptors for basement membrane proteins are members of the integrin family (Figure 5). These are transmembrane proteins which act either as cell-cell or cell-matrix adhesion molecules (Hynes, 1992). In addition to linking the extracellular matrix with the intracellular structural proteins, they are true receptors acting as signal transducers carrying information to the cell from its extracellular environment and effecting a wide spectrum of cellular activities (outside-in signalling). Some evidence suggest modulation of integrin function by modification of their intracellular domains (inside-out signalling). Integrins are noncovalently linked heterodimers formed of a member each from the α and β subfamily, both units being required to allow ligand binding which is divalent cation dependent (Hynes, 1992). Seven of the 15 α members and two of the eight β members have been shown to have basement membrane binding properties, giving eight active dimers (Figure 5). Further diversity in integrin activity is rendered by the presence of three isoforms of the β1 integrin as well as time- or tissue-specific alternative splicing of the α3, α6 and α7 chains which appears to alter the biological roles of these molecules. It is believed that the intracellular


111

Figure 5 The family of basement membrane-binding integrins. The individual chains are given in circles and the connecting lines show the dimer combinations that have been demonstrated (Modified from Aumailley et al., 1996).

domains of integrins are in direct contact with cytoskeleton-associated proteins, but also that they are coupled to intracellular signalling pathways (Kim and Yamada, 1997). Most basement membrane-binding integrins are of the β1 family with α6β1 binding all and α3β1 many laminin isoforms. α7β1 is particularly highly expressed in muscle where it functions by binding to the abundant laminins 2 and 4 in the muscle basement membrane (von der Mark et al., 1991). Of the β4 integrin family only α6β4 has been shown to be a receptor for laminins. It binds laminin-1 and -5 and is of particular interest because of its distribution, being restricted mainly to the basal surface of epithelial cells, suggesting a role in maintaining the epithelial integrity. α1β1 and α2β1 interact with collagen IV (see above). Other Receptors for Basement Membrane Proteins A number of other cell surface molecules have been described as binding partners and hence receptors for the basement membrane proteins. These include the so called 67 kDa laminin binding protein, the enzyme β1,4-galactosyltransferase and in particular α-dystroglycan. α-dystroglycan is a part of the dystrophin complex first described on the sarcolemma (IbraghimovBeskrovnaya et al., 1992, Henry and Campbell, 1996). It is the product of a gene encoding for a precursor protein containing both the α-and β-dystroglycan subunits(Ibraghimov-Beskrovnaya et al., 1993). α-

112


dystroglycan is attached at the cell surface to β-dystroglycan and adhalin. These are two of the five transmembrane proteins making up the membrane-bound part of the dystrophin complex, β-dystroglycan binds to dystrophin which in turn interacts with the cytoskeleton. The interaction between laminin-2 and αdystroglycan is of high affinity (Gee et al., 1993). Binding has been shown to occur at the C-terminal end of the laminin a chain and in the case of muscle cells it is the α2 chain which is believed to be the ligand for αdystroglycan. Through this attachment the basement membrane is linked to the cytoskeleton and it is believed that this connection is required to reduce stress placed upon the sarcolemma in normal muscle contraction and its loss results in muscular dystrophy. Mutations in the LAMA2 gene have been shown to be present in human congenital muscular dystrophy (CMD) and in the dy dystrophic mouse strain (Sunada et al., 1994; Tome et al., 1994; Henry and Campbell, 1996; Xu et al., 1994). As further work has shown that αdystroglycan interacts with the C-terminal part of also the α1 laminin chain (Gee et al., 1993; Yamada et al., 1994) and as α-dystroglycan is expressed in a wide variety of cell types it is believed that this interaction may play wider roles in development than merely that shown for the skeletal muscle (Durbeej et al., 1995, 1997). Indeed, the knock-out of α-dystroglycan produces a lethality at embryonic day 6, due to abnormal attachment to Reichert’s membrane or imperfect formation of this structure (Williamson et al., 1997). Thus a lethality occurs far earlier than the start of myogenesis. CONCLUDING REMARK The basement membrane has been shown to be essential for epithelial integrity and development. As little as a decade ago the basement membrane was considered as having an unvarying structure with merely one form of laminin, one of collagen IV and one of nidogen. However, over the intervening time an increasing diversity in these molecules has been uncovered. It is the importance of this diversity and its role in structural, developmental and physiological biology which is now just beginning to be understood. ACKNOWLEDGEMENTS The authors are grateful to Christian Frie, Jörg Karolat, Patrik Maurer and Jan Modregger for their help in preparing figures. REFERENCES Allies, L., Kisilevsky, R. and Young, I.D. (1993) Induction of perlecan gene expression precedes amyloid formation during experimental murine AA amyloidogenesis. Lab. Invest., 69, 443–448. Airenne, T., Haakana, H., Sainio, K., Kallunki, P., Sariola, H. and Tryggvason, K. (1996) Structure of the human laminin α2 gene (LAMC2): alternative splicing with different tissue distribution of two transcripts. G monies, 32, 54–64. Alexander, C.M., Howard, E.W., Bissell, M.J. and Werb, Z. (1996) Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J. Cell Biol., 135, 1669–1677. Antonsson, P., Kammerer, R., Schulthess, T., Hänisch, G. and Engel, J. (1995) Stabilization of the α-helical coiled-coil domain in laminin by C-terminal disulfide bonds. J. Mol. Biol., 250, 74–79. Aumailley, M., Wiedemann, H., Mann, K. and Timpl, R. (1989) Binding of nidogen and the laminin-nidogen complex to basement membrane collagen type IV. Eur. J. Biochem., 184, 241–248. Aumailley, M., Gimond, C. and Rousselle, P. (1996) Integrin-mediated cellular ineractions with laminin. In: The laminins (Ekblom, P. and Timpl, R., eds) pp. 127–158, Harwood Academic Publishers, Amsterdam.


113

Aviezer, D., Hecht, D., Safran, M., Eisinger, M., David, G. and Yayon, A. (1994) Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis and angiogenesis. Cell, 79, 1005–1013. Baird, A. and Ling, N. (1987) Fibroblast growth factors are present in the extracellular matrix produced by endothelial cells in vitro: Implications for the role of heparinase-like enzymes in the neovascular response. Biochem. Biophys. Res. Commun., 142, 428–435. Barker, D.F., Hostikka, S.L., Zhou, J., Chow, L.T., Oliphant, A.R., Gerken, S.C., Gregory, M.C., Skolnick, M.H., Atkin, C.L. and Tryggvason, K. (1990) Identification of mutations in the COL4A5 collagen gene in Alport syndrome. Science, 248, 1224–1227. Barlow, D.P., Green, N.M., Kurkinen, M. and Hogan, B.L.M. (1984) Sequencing of laminin B chain cDNAs reveals Cterminal regions of coiled-coil alpha helix. EMBO J., 3, 2355– 2362. Battaglia, C., Mayer, U., Aumailley, M. and Timpl, R. (1992) Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core. Eur. J. Biochem., 208, 359–366. Battaglia, C., Aumailley, M., Mann, K., Mayer, U. and Timpl, R. (1993) Structural basis of β1 integrin-mediated cell adhesion to a large heparan sulfate proteoglycan from basement membranes. Eur. J. Cell Biol., 61, 92–99 Beck, K., Dixon, T.W., Engel, J. and Parry, D.A.D. (1992) Ionic interactions in the coiled-coil domain of laminin determine the specificity of chain assembly. J. Mol. Biol., 231, 311–323. Blumberg, B., Fessler, L.I., Kurkinen, M. and Fessler, J.H. (1986) Biosynthesis and supramolecular assembly of procollagen IV in neonatal lung. J. Cell Biol., 103, 1711–1719. Brandenberger, R., Kammerer, R.A., Engel, J. and Chiquet, M. (1996) Native chick laminin-4 containing the β2 chain (s-laminin) promotes motor axon growth. J. Cell Biol., 135, 1583–1592. Burgeson, R.E. (1993) Dermal-epidermal adhesion in skin. In: Molecular and cellular aspects of basement membranes (Rohrbach, D.H. and Timpl, R., ed.) pp. 49–66, Academic Press, Orlando. Burgeson, R.E., Chiquet, M., Deutzmann, R., Ekblom, P., Engel, J., Kleinman, H., Martin, G.R., Meneguzzi, G., Paulsson, M., Sanes, J., Tirnpl, R., Tryggvason, K., Yamada, Y. and Yurchenco, P.D. (1994) A new nomenclature for the laminins. Matrix Biol., 14, 209–211. Butkowski, R.J., Wieslander, J., Kleppel, M., Michael, A.F. and Fish, A.J. (1989) Basement membrane collagen in the kidney: Regional localization of novel chains related to collagen IV Kidney Int., 35, 1195–1202. Carlin, B., Jaffe, R., Bender, B. and Chung, A.E. (1981) Entactin, a novel basal lamina-associated sulfated glycoprotein. J. Biol. Chem., 256, 5209–5214. Chakravarti, S., Horchar, T., Jefferson, B., Laurie, G.W. and Hassell, J.R. (1995) Recombinant domain III of perlecan promotes cell attachment through its RGDS sequence. J. Biol. Chem., 270, 404–409. Champliaud, M.F., Lunstrum, G.P., Rousselle, P., Nishiyama, T., Keene, D.R. and Burgeson, R.E. (1996) Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attchment. J. Cell Biol., 132, 1189–1198. Chan, F.L. and Inoue, S. (1994) Lamina lucida of basement membrane: An artefact. Microsc. Res. Techn., 28, 48–59. Chung, A.E., Jaffe, R., Freeman, I.L., Vergnes, J.-P., Braginski, J.E. and Carlin, B. (1979) Properties of a basement membrane-related glycoprotein synthesized in culture by a mouse embryonal carcinoma-derived cell line. Cell, 16, 277–287. Comper, W.D., Glasgow, E.F. and Singh, A.K. (1996) The glomerulus.In: Extracellular matrices, Vol. I: Tissue function (Comper, W.D., ed) pp. 410–442, Gordon and Breach, Camberwell. Couchman, J.R., Caterson, B., Christner, J.E. and Baker, J.R. (1984) Mapping by monoclonal antibody detection of glycosaminoglycans in connective tissues. Nature, 307, 650–652. Danielson, K.G., Martinez-Hernandez, A., Hassell, J.R. and lozzo, R. (1992) Establishment of a cell line from the EHS tumor: Biosynthesis of basement membrane constituents and characterization of a hybrid proteoglycan containing heparan and chondroitin sulfate chains. Matrix, 11, 22–35. De Arcangelis, A., Neuville, P., Boukamel, R., Lefebvre, O., Kedinger, M. and Simon-Assmann, P. (1996) Inhibition of laminin a 1-chain expression leads to alteration of basement membrane assembly and cell differentiation. J. Cell Biol., 133, 417–430.

114


Denzer, A.J., Gesemann, M., Schumacher, B. and Ruegg, M. (1995) An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix. J. Cell Biol., 131, 1547–1560. Deutzmann, R., Aumailley, M., Wiedemann, H., Pysny, W., Timpl, R. and Edgar, D. (1990) Cell adhesion, spreading and neurite stimulation by laminin fragment E8 depends on maintenance of secondary and tertiary structure in its rod and globular domain. Eur. J. Biocbem., 191, 513–522. DiPersio, C.M., Hodivala-Dilke, K.M., Jaenisch, R., Kreidberg, J.A. and Hynes, R.O. (1997) α3β1 integrin is required for normal development of the epidermal basement membrane. J. Cell Biol., 137, 729–742. Dölz, R., Engel, J. and Kühn, K. (1988) Folding of collagen IV. Eur. J. Biochem., 178, 357– 366. Durbeej, M., Larsson, E., Ibraghimov-Beskrovnaya, O., Roberds, S.L., Campbell, K.P. and Ekblom, P. (1995) Nonmuscle α-dystroglycan is involved in epithelial development. J. Cell Biol., 130, 79–91. Durbeej, M., Jung, D., Hjalt, T., Campbell, K.P. and Ekblom, P. (1997) Transient expression of Dp140, a product of the Duchenne muscular dystrophy locus, during kidney tubulogenesis. Dev. Biol., 181, 156–167. Durkin, M.E., Chakravarti, S., Bartos, B.B., Liu, S.-H., Friedman, R.L. and Chung, A.E. (1988) Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor. J. Cell Biol., 107, 2749–2756. Dziadek, M., Paulsson, M. and Timpl, R. (1985) Identification and interaction repertoire of large forms of the basement membrane protein nidogen. EMBO J., 4, 2513–2518. Eble, J.A., Golbik, R., Mann, K. and Kühn, K. (1993) The α1β1 integrin recognition site of the basement membrane collagen molecule {α1(IV)]2α2(VI). EMBO J., 12, 4795– 4802. Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., Chu, M.-L., Mayer, U. and Timpl, R. (1994) Role of mesenchymal nidogen for epithelial morphogenesis in vitro. Development, 120, 2003–2014. Ekblom, P., Durbeej, M. and Ekblom, M. (1996) Laminin isoforms in development. In: The laminins (Ekblom, P. and Timpl, R., eds) pp. 185–216, Harwood Academic Publishers, Amsterdam. Engel, J., Odermatt, E., Engel, A., Madri, J.A., Furthmayr, H., Rohde, H. and Timpl, R. (1981) Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. J. Mol. Biol., 150, 97–120. Folkman, J., Klagsbrun, M., Sasse, J., Wadzinski, M., Ingber, D. and Vlodavsky, I. (1988) A heparin-binding angiogenic protein—basic fibroblast growth factor—is stored within basement membrane. Am. J. Pathol., 130, 393–400. Fox, J.W., Mayer, U., Nischt, R., Aumailley, M., Reinhardt, D., Wiedemann, H., Mann, K., Timpl, R., Krieg, T., Engel, J. and Chu, M.-L. (1991) Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen IV. EMBO J., 10, 3137–3146. Galliano, M.F., Aberdam, D., Aguzzi, A., Ortonne, J.P. and Meneguzzi, G. (1995) Cloning and complete primary structure of the mouse laminin α3 chain. Distinct expression pattern of the laminin α3A and α3B chain isoforms. J. Biol. Chem., 270, 21820–21826. Gee, S.H., Blacher, R.W., Douville, P.J., Provost, P.R., Yurchenco, P.D. and Carbonetto, S. (1993) Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan and binds with high affinity to the major heparin binding domain of laminin. J. Biol. Chem., 268, 14972–14980. Gesemann, M., Cavalli, V., Denzer, A.J., Brancaccio, A., Schumacher, B. and Ruegg, M. (1996) Alternative splicing of agrin alters its binding to heparin, dystroglycan and the putative agrin receptor. Neuron, 16, 755–767. Godfrey, E.W., Dietz, M.E., Morstad, A.L., Wallskog, P.A. and Yorde, D.E. (1988) Acetyl-choline receptoraggregating proteins are associated with the extracellular matrix of many tissues in torpedo. J. Cell Biol., 106, 1263–1272. Godfrey, E.W. (1991) Comparison of agrin-like proteins from the extracellular matrix of chicken kidney and muscle with neural agrin, a synapse organizing protein. Exp. Cell Res., 195, 99–109. Guo, X., Johnson, J.J. and Kramer, J.M. (1991) Embryonic lethality caused by mutations in basement membrane collagen of C. elegam. Nature, 349, 707–709. Hall, Z.W. and Sanes, J.R. (1993) Synaptic structure and development: The neuromuscular junction. Cell, 72/Neuron, 10, (Suppl.) 99–121.


115

Hayashi, K., Madri, J.A. and Yurchenco, P.D. (1992) Endothelial cells interact with the core protein of basement membrane perlecan through β1 and β3 integrins: An adhesion modulated by glycosaminoglycan. J. Cell Biol., 119, 945–959. Henry, M.D. and Campbell, K.P (1996) Dystroglycan: an extracellular matrix receptor linked to the cytoskeleton. Curr. Opin. Cell Biol., 5, 625–631. Hogan, B.L.M., Cooper, A.R. and Kurkinen, M. (1980) Incorporation into Reichert’s membrane of laminin-like extracellular proteins synthesized by parietal endoderm cells of the mouse embryo. Dev. Biol., 80, 289–300. Hostikka, S.L., Eddy, R.L., Byers, M.G., Hoyhtya, M., Shows, T.B. and Tryggvason, K. (1990) Identification of a distinct type IV collagen a chain with restricted kidney distribution and assignment of its gene to the locus of X chromosome-linked Alport syndrome. Proc. Natl. Acad. Sci. USA, 87, 1606–1610. Hudson, B.G., Reeders, S.T. and Tryggvason, K. (1993) Type IV collagen: structure, gene organization and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis. J. Biol. Chem., 268, 26033–26036. Hunter, D.D., Shah, V., Merlie, J.P. and Sanes, J.R. (1989) A laminin-like adhesive protein concentrated in the synaptic cleft of the neuromuscular junction. Nature, 338, 229–234. Hunter, I., Schulthess, T., Bruch, M., Beck, K. and Engel, J. (1990) Evidence for a specific mechanism of laminin assembly. Eur. J. Biochem., 188, 205–211. Hunter, I., Schulthess, T. and Engel, J. (1992) Laminin chain assembly by triple and double stranded coiled-coil structures. J. Biol. Chem., 267, 6006–6011. Hynes, R.O. (1992) Integrins: versatility, modulation and signaling in cell adhesion. Cell, 69, 11–25. Ibraghimov-Beskrovnaya, O., Ervasti, J.M., Leveille, C.J., Slaughter, S.A., Sernett, S.W. and Campbell, K.P. (1992) Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature, 355, 696–702. Ibraghimov-Beskrovnaya, O., Milatovich, A., Ozcelik, T., Yang, B., Koepnick, K., Francke, U. and Campbell, K.P. (1993) Human dystroglycan: skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosmal localization. Hum. Mol. Genet., 2, 1651–1657. Jeanny, J.C., Fayein, N., Moenner, M., Chevallier, B., Barritault, D. and Courtois, Y. (1987) Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes. Exp. Cell Res., 171, 63–75. Johansson, C., Butkowski, R. and Wieslander, J. (1992) The structural organization of type IV collagen. Identification of three NC1 populations in the glomerular basement membrane. J. Biol. Chem., 267, 24533–24537. Kallunki, P. and Tryggvason, K. (1992) Human basement membrane heparan sulfate proteoglycan core protein: A 467kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules and epidermal growth factor. J. Cell Biol., 116; 559–571. Kalluri, R., Gunwar, S., Reeders, S.T., Morrison, K.C., Mariyama, M., Ebner, K.E., Noelken, M.E. and Hudson, B.G. (1991) Goodpasture syndrome. Localization of the epitope for the autoantibodies to the carboxyl-terminal region of the α3(IV) chain of basement membrane collagen. J. Biol. Chem., 266, 24018–24024. Kammerer, R., Antonsson, P., Schulthess, T. and Engel, J. (1994) Sequential stabilization of the α-helical coiled-coil involved in chain assembly of laminin as revealed by recombinant fragments. Matrix Biol., 14, 417–418. Kammerer, R., Antonsson, P., Schulthess, T., Fauser, C. and Engel, J. (1995) Chain recognition in the C-terminal αhelical coiled-coil region of laminin. J. Mol. Biol., 250, 64–73. Kanwar, Y.S. and Farquhar, M.G. (1979a) Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes. J. Cell Biol., 81, 137–153. Kanwar, Y.S. and Farquhar, M.G. (1979b) Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membrane. Proc. Natl. Acad. Sci. USA, 76, 4493–497. Kim, L.T. and Yamada, K.M. (1997) The regulation of expression of integrin receptors. Proc. Soc. Exp. Biol. Med., 214, 123–131. Kröger, S. and Mann, S. (1996) Biochemical and functional characterization of basal laminabound agrin in the chick central nervous system. Eur. J. Neurosci., 8, 500–509.

116


Kücherer-Ehret, A., Pottgiesser, J., Kreutzberg, G.W., Thoenen, H. and Edgar, D. (1990) Developmental loss of laminin from the interstitial extracellular matrix correlates with decreased laminin gene expression. Development, 110, 1285–1293. Leinonen, A., Mariyama, M., Mochizuki, T., Tryggvason, K. and Reeders, S.T. (1994) Complete primary structure of the human type IV collagen α4(IV) chain. Comparison with structure and expression of the other α(IV) chains. J. Biol. Chem., 269, 26172–26177. Lindblom, A. and Paulsson, M. (1996) Basement membranes. In: Extracellular matrices, Vol. I: Tissue function (Comper, W.D., ed) pp. 133–174, Gordon and Breach, Camberwell. Mann, K., Deutzmann, R., Aumailley, M., Timpl, R., Raimondi, L., Yamada, Y., Pan, T.-c., Conway, D. and Chu, M.-L. (1989) Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells. (1989) EMBO J., 8, 65–72. Maurer, P. and Engel, J. (1996) Structure of laminins and their chain assembly. In: The laminins (Ekblom, P. and Timpl, R., eds) pp. 27–50, Harwood Academic Publishers, Amsterdam. Mayer, U., Nischt, R., Pöschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y. and Timpl, R. (1993a) A single EGFlike motif of laminin is responsible for high affinity nidogen binding. EMBO J., 12, 1879–1885. Mayer, U., Mann, K., Timpl, R. and Murphy, G. (1993b) Sites of nidogen cleavage by proteases involved in tissue homeostasis and remodelling. Eur. J. Biochem., 217, 877–884. Mayer, U., Pöschl, E., Gerecke, D.R., Wagman. D.W., Burgeson, R.E. and Timpl, R. (1995) Low nidogen affinity of laminin-5 can be attributed to two serine residues in EGF-like motif γ2III4. FEBS Lett., 365, 129–132. McCarthy, K.J., Accavitti, M.A. and Couchman, J.R. (1989) Immunological characterization of a basement membranespecific chondroitin sulfate proteoglycan. J. Cell Biol., 109, 3187–3198. McCarthy, K.J. and Couchman, J.R. (1990) Basement membrane chondroitin sulfate proteoglycans: Localization in adult rat tissues. J. Histochem. Cytochem., 38, 1479–1486. McCarthy, K.J., Bynum, K., St. John, P.L., Abrahamson, D.R. and Couchman, J.R. (1993) Basement membrane proteoglycans in glomerular morhogenesis: Chondroitin sulfate proteoglycan is temporally and spatially restricted during development. J. Histochem. Cytochem., 41, 401–414. McMahan, U.J. and Wallace, B.G. (1989) Molecules in the basal lamina that direct the formation of synaptic specializations at neuromuscular junctions. Dev. Neurosci., 11, 227– 247. McMahan, U.J. (1990) The agrin hypothesis. Cold Spring Harbor Symp., 55, 407–418. Miner, J.H., Patton, B.L., Lentz, S.I., Gilbert, D.J., Snider, W.D., Jenkins, N.A., Copeland, N.G. and Sanes, J.R. (1997) The laminin α chains: Expression, developmental transitions and chromosomal locations of α1–5, identification of heterotrimeric laminins 8–11 and cloning of a novel α3 isoform. J. Cell Biol., 137, 685–701. Morita, A., Sugimoto, E. and Kitagawa, Y. (1985) Post-translational assembly and glycosylation of laminin subunits in parietal endoderm-like F9 cells. Biochem. J., 229, 259–264. Murdoch, A.D., Dodge, G.R., Cohen, I., Tuan, R.S. and lozzo, R.V. (1992) Primary sequence of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules and epidermal growth factor. J. Biol. Chem., 267, 8544–8557. Neilson, E.G., Kalluri, R., Sun, M.J., Gunwar, S., Danoff, T., Mariyama, M., Myers, J.C., Reeders, S.T. and Hudson, B.G. (1993) Specificity of Goodpasture autoantibodies for the recombinant noncollagenous domains of human type IV collagen. J. Biol. Chem., 268, 8402–8405. Ninomiya, Y., Kagawa, M., lyama, K., Naito, I., Kishiro, Y., Seyer, J.M., Sugimoto, M., Oohashi, T. and Sado, Y. (1995) Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies. J. Cell Biol., 130, 1219–1229. Nitkin, R.M., Smith, M.A., Magill, C., Fallon, J.R., Yao, Y.-M.M., Wallace, B.G. and McMahan, U.J. (1987) Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J. Cell Biol., 105, 2471–2478. Noonan, D.M., Fulle, A., Valente, P., Cai, S., Horigan, E., Sasaki, M., Yamada, Y. and Hassell, J.R. (1991) The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity


117

with laminin A-chain, low density lipoprotein-receptor and the neural cell adhesion molecule. J. Biol. Chem., 266, 22939–22947. Oberbäumer, I., Wiedemarin, H., Timpl, R. and Kühn, K. (1982) Shape and assembly of type IV procollagen obtained from cell culture. EMBO J., 1, 805–810. Ohno, I. Human osteoblast mRNA for osteonidogen, complete cds. DDBJ/EMBL/GenBank, Accession D86425. Pan, T.-C, Kluge, M., Zhang, R.-Z., Mayer, U., Timpl, R. and Chu, M.-L. (1993) Sequence of extracellular mouse protein BM-90/fibulin and its calcium-dependent binding to other basement-membrane ligands. Eur. J. Biochem., 215, 733–740. Paulsson, M., Deutzmann, R., Timpl, R., Dalzoppo, D., Odermatt, E. and Engel, J. (1985) Evidence for coiled-coil αhelical regions in the long arm of laminin. EMBO J., 4, 309– 316. Paulsson, M., Deutzmann, R., Dziadek, M., Nowack, H., Timpl, R., Weber, S. and Engel, J. (1986) Purification and structural characterization of intact and fragmented nidogen obtained from a basement membrane tumor. Eur. J. Biochem., 156, 467–478. Paulsson, M., Aumailley, M., Deutzmann, R., Timpl, R., Beck, K. and Engel, J. (1987a) Laminin-nidogen complex. Extraction with chelating agents and structural characterization. Eur. J. Biochem., 166, 11–19 Paulsson, M., Yurchenco, P.D., Ruben, G.C., Engel, J. and Timpl, R. (1987b) Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane. J. Mol. Biol., 197, 297–313. Paulsson, M. (1988) The role of Ca2+ binding in the self-aggregation of laminin-nidogen complexes, J. Biol. Chem., 263, 5425–5430. Paulsson, M., Saladin, K. and Landwehr, R. (1988) Binding of Ca2+ influences susceptibility of laminin to proteolytic digestion and interactions between domain-specific laminin fragments. Eur. J. Biochem., 177, 477–481. Paulsson, M. and Saladin, K. (1989) Mouse heart laminin. Purification of the native protein and structural comparison with Engelbreth-Holm-Swarm tumor laminin.J. Biol. Chem., 264, 18726–18732. Paulsson, M. (1993) Laminin and collagen IV variants and heterogeneity in basement membrane composition. In: Molecular and cellular aspects of basement membranes (Rohrbach, D.H. and Timpl, R., eds.), pp. 177–187, Academic Press, Orlando. Pejler, G., Bäckström, G., Lindahl, U., Paulsson, M., Dziadek, M., Fujiwara, S. and Timpl, R. (1987) Structure and affinity for antithrombin of heparan sulfate chains derived from basement membrane proteoglycans. J. Biol. Chem., 262, 5036–5043. Peters, B.P., Hartle, R.J., Krzesicki, R.F., Kroll, T.G., Perini, F., Balun, J.E., Goldstein, I.J. and Ruddon, R.W. (1985) The biosynthesis, processing and secretion of laminin by human choriocarcinoma cells. J. Biol. Chem., 260, 14732–14742. Pöschl, E., Pollner, R. and Kühn, K. (1988) The genes for the α1(IV) and α2(IV) chains of human basement membrane collagen type IV are arranged head-to-head and separated by a bidirectional promoter of unique structure. EMBO J., 7, 2687–2695. Pöschl, E., Fox, J.W., Block, D., Mayer, U. and Timpl, R. (1994) Two non-contiguous regions contribute to nidogen binding to a single EGF-like domain of the laminin β1 chain. EMBO J., 13, 3741–3747. Prockop, D.J., Kivirikko, K.I., Tuderman, L. and Guzman, N.A. (1979a) The biosynthesis of collagen and its disorders. New Engl. J. Med., 301, 13–23. Prockop, D.J., Kivirikko, K.I., Tuderman, L. and Guzman, N.A. (1979b) The biosynthesis of collagen and its disorders. New Engl. J. Med., 301, 77–85. Reinhardt, D., Mann, K., Nischt, R., Fox, J.W., Chu, M.-L., Krieg, T. and Timpl, R. (1993) Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan and zinc. J. Biol. Chem., 268, 10881–10887. Reist, N.E., Magill, C. and McMahan, U.J. (1987) Agrin-like molecules at synaptic sites in normal, denervated and damaged skeletal muscles. J. Cell Biol., 105, 2457–2469. Rousselle, P., Lunstrum, G.P., Keene, D.R. and Burgeson, R.E. (1991) Kalinin: An epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments. J. Cell Biol., 114, 567–576. Ruegg, M.A., Tsim, K.W., Horton, S.E., Kroger, S., Escher, G., Gensch, E.M. and McMahan, U.J. (1992) The agrin gene codes for a family of basal lamina proteins that differ in function and distribution. Neuron, 8, 691–699.

118


Rupp, F., Payan, D.G., Magill-Solc, C., Cowan, D.M. and Scheller, R.H. (1991) Structure and expression of rat agrin. Neuron, 6, 811–823. Sasaki, M., Kleinman, H.K., Huber, H., Deutzmann, R. and Yamada, Y. (1988) Laminin, a multidomain protein. The A chain has a unique globular domain and homology with the basement membrane proteoglycan and the laminin B chains. J. Biol. Chem., 263, 16536–16544. Sasaki, T., Kostka, G., Göhring, W., Wiedemann, H., Mann, K., Chu, M.-L. and Timpl, R. (1995) Structural characterization of two variants of fibulin-1 that differ in nidogen affinity. J. Mol. Biol., 245, 241–250. Saus, J., Wieslander, J., Langeveld, J.P.M., Quinones, S. and Hudson, B.G. (1988) Identification of the Goodpasture antigen as the α3(IV) chain of collagen IV. J. Biol. Chem., 263, 13374–13380. Schittny, J.C., Timpl, R. and Engel, J. (1988) High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen and heparan sulphate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations. J. Cell Biol., 107, 1599–1610. Siebold, B., Qian, R.-Q., Glanville, R.W., Hofmann, H., Deutzmann, R. and Kühn, K. (1987) Construction of a model for the aggregation and cross-linking region (7S domain) of type IV collagen based upon an evaluation of the primary structure of the α1 and α2 chains in this region. Eur. J. Biochem., 168, 569–575. Siebold, B., Deutzmann, R. and Kühn, K. (1988) The arrangement of intra- and intermolecular disulfide bonds in the carboxyterminal, non-collagenous aggregation and cross-linking domain of basement-membrane type IV collagen. Eur. J. Biochem., 176, 617–624. Smith, M.A., Magill-Solc, C., Rupp, E, Yao, Y.-M.M., Schilling, J.W., Snow, P. and McMahan, U.J. (1992) Isolation and characterization of a cDNA that encodes an agrin homolog in the marine ray. Mol. Cell. Neurosci., 3, 406–417. Snow, A.D., Sekiguchi, R., Nochlin, D., Eraser, P., Kimata, K., Mizutani, A., Arai, M., Shreier, W.A. and Morgan, D.G. (1994) An important role of heparan sulfate proteoglycan (perlecan) in a model system for the deposition of fibrillar Ab-amyloid in rat brain. Neuron, 12, 219–234. Soininen, R., Huotari, M., Hostikka, S.L., Prockop, D.J. and Tryggvason, K. (1988) The structural genes for α1 and α2 chains of human type IV collagen are divergently encoded on opposite DNA strands and have an overlapping promoter region. J. Biol. Chem., 263, 17217–17220. Sugimoto, M., Oohashi, T. and Ninomiya, Y. (1994) The genes COL4A6 and COL4A6, coding for basement membrane collagen chains α5(IV) and α6(IV), are located head-to-head in close proximity on human chromosome Xq22 and COL4A6 is transcribed from two alternative promoters. Proc. Natl. Acad. Sci. USA, 91, 11679–11683. Sunada, Y., Bernier, S.M., Kozak, C.A., Yamada, Y. and Campbell, K.P. (1994) Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin M chain to dy locus. J. Biol. Chem., 269, 13729–13732. Thomas, T. and Dziadek, M. (1993) Genes coding for basement membrane glycoproteins laminin, nidogen and collagen IV are differentially expressed in the nervous system and by epithelial, endothelial and mesenchymal cells of the mouse embryo. Exp. Cell Res., 208, 54–67. Timpl, R., Rohde, H., Gehron Robey, P., Rennard, S.L, Foidart, J.-M. and Martin, G.R. (1979) Laminin—A glycoprotein from basement membranes. J. Biol. Chem,, 254, 9933–9937. Timpl, R., Wiedemann, H., van Delden, V., Furthmayr, H. and Kühn, K. (1981) A network model for the organization of type IV collagen molecules in basement membranes. Eur. J. Biochem., 120, 203–211. Timpl, R., Dziadek, M., Fujiwara, S., Nowack, H. and Wick, G. (1983) Nidogen: a new, self-aggregating basement membrane protein. Eur. J. Biochem., 137, 455–465. Timpl, R. (1996) Binding of laminin to extracellular matrix components. In: The laminins (Ekblom, P. and Timpl, R., eds) pp. 97–126, Harwood Academic Publishers, Amsterdam. Tokida, Y., Aratani, Y., Morita, A. and Kitagawa, Y. (1990) Production of two variant laminin forms by endothelial cells and shift of their relative levels by angiostatic steroids. J. Biol. Chem., 265, 18123–18129. Tome, F.M., Evangelista, T., Leclerc, A., Sunada, Y., Manole, E., Estournet, B., Barois, A., Campbell, K.P. and Fardeau, M. (1994) Congenital muscle dystrophy with merosin deficiency. C.R. Acad. Sci. III, 317, 351–357. Trüeb, B., Gröbli, B., Spiess, M., Odermatt, B.F. and Winterhalter, K.H. (1982) Basement membrane (type IV) collagen is a heteropolymer. J. Biol. Chem., 257, 5239–5245.


119

Tsen, G.W., Halfter, W., Kröger, S. and Cole, G.J. (1995) Agrin is a heparan sulfate proteoglycan. J. Biol. Chem., 270, 3392–3399. Tsim, K.W.K, Ruegg, M.A., Escher, G., Kröger, S. and McMahan, U.J. (1992) cDNA that encodes active agrin. Neuron, 8, 677–689. Utani, A., Nomizu, M., Timpl, R., Roller, P.P. and Yamada, Y. (1994) Laminin chain assembly. Specific sequences at the C-terminus of the long arm are required for the formation of specific double- and triple-stranded coiled-coil structures. J. Biol. Chem., 269, 19167– 19175. von der Mark, H., Dürr, J., Sonnenberg, A., von der Mark, K., Deutzmann, R. and Goodman, S.L. (1991) Skeletal myoblasts utilize a novel β1-series integrin and not α6β1 for binding to the E8 and T8 fragments of laminin. J. Biol. Chem., 266, 23593–23601. Vracko, R. (1974) Basal lamina scaffold-Anatomy and significance for maintenance of orderly tissue structure. Am. J. Pathol., 77, 313–346. Williamson, R.A., Henry, M.D., Daniels, K.J., Hrstka, R.F., Lee, J.C., Sunada, Y., Ibraghimov-Beskrovnaya, O. and Campbell, K.P. (1997) Dystroglycan is essential for early embryonic development: disruption of Reichert’s membrane in Dag 1-null mice. Hum. Mol. Genet., 6, 831–841. Wu, R.R. and Couchman, J.R. (1997) cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs. J. Cell Biol., 136, 433–444. Xu, H., Christmas, P., Wu, X.R., Wewer, U.M. and Engvall, E. (1994) Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc. Natl. Acad. Sci. USA, 91, 5572–5576. Yamada, H., Shimizu, T., Tanaka, T., Campbell, K.P. and Matsumura, K. (1994) Dystroglycan is a binding protein of laminin and merosin in peripheral nerve. FEBS Lett., 352, 49– 53. Yamada, K.M. (1991) Adhesive recognition sequences. J. Biol. Chem., 266, 12809–12812. Yamada, Y. and Kleinman, H.K. (1992) Functional domains of cell adhesion molecules. Curr. Opin. Cell Biol., 4, 819–823. Yurchenco, P.D., Tsilibary, E.C., Charonis, A.S. and Furthmayr, H. (1985) Laminin polymerization in vitro. Evidence for a two-step assembly with domain specificity. J. Biol. Chem., 260, 7636–7644. Yurchenco, P.D. and Ruben, G.C. (1987) Basement membrane structure in situ: Evidence for lateral associations in the type IV collagen network. J. Cell Biol., 105, 2559–2568. Yurchenco, P.D., Cheng, Y.-S. and Ruben, G.C. (1987) Self-assembly of a high molecular weight basement membrane proteoglycan into dimers and oligomers. J. Biol. Chem., 262, 17668–17676. Yurchenco, P.D. and Ruben, G.C. (1988) Type IV collagen lateral associations in the EHS tumor matrix. Comparison with amniotic and in vitro networks. Am. J. Pathol., 132, 278– 291. Yurchenco, P.D., Cheng, Y.-S. and Colgnato, H. (1992) Laminin forms an independent network in basement membranes. J. Cell Biol., 117, 1119–1133. Yurchenco, P.D. and O’Rear, J (1993) Supramolecular organization of basement membranes. In: Molecular and cellular aspects of basement membranes (Rohrbach, D.H. and Timpl, R., eds.) pp. 20–47, Academic Press, Orlando. Yurchenco, P.D., Quan, Y., Colognato, H., Mathus, T., Harrison, D., Yamada, Y. and O’Rear, J.J. (1997) The α-chain of laminin-1 is independently secreted and drives secretion of its β- and a-chain partners. Proc. Natl. Acad. Sci. USA, 94, 10189–10194. Zhou, J., Barker, D.F., Hostikka, S.L., Gregory, M.C., Atkin, C.L. and Tryggvason, K. (1991) Single base mutation in α5 collagen chain gene converting a conserved cysteine to serine in Alport syndrome. Genomics, 9, 10–18. Zhou, J., Mochizuki, T., Smeets, H., Antignac, C., Laurila, P., de Paepe, A., Tryggvason, K. and Reeders, S.T. (1993) Deletion of the paired α5(IV) and α6(IV) collagen genes in inherited smooth muscle tumors. Science, 261, 1167–1169.

EPITHELIA IN DEVELOPMENT

7. DEVELOPMENT OF EPITHELIAL CELL POLARITY IN DROSOPHILA ELISABETH KNUST Institut fur Genetik, Heinrich-Heine Universität Düsseldorf, U niversitdtsstr. 1, 40225 Düsseldorf, Germany Tel.: (211)–811–3504; Fax: (211)–811–2279; E-mail: [email protected] SUMMARY The cleavage divisions in the early Drosophila embryo take place in the absence of cytokinesis, thus forming a large multinuclear cell. The transition from this syncycial stage to the first epithelium, the blastoderm, is achieved by the gradual ingrowth of the egg’s plasma membrane. Later, those parts of this epithelium, which contribute to the ectodermal epithelia, undergo extensive morphogenetic movements necessary for the elaboration of the final body plan. This chapter will summarise our current knowledge on the mechanisms and molecules controlling the establishment and maintenance of epithelial cell polarity. Surprisingly, despite the enormous differences in the generation of the blastoderm stage between vertebrates and Drosophila, some of the mechanisms required for the establishment and maintenance of epithelial cell polarity appear to be conserved and make use of the same molecules during the development of the respective epithelia. This chapter summarises aspects of Drosophila epithelial development and points out at novel mechanisms, for which no counterparts have been identified in vertebrates so far. The molecular and functional analysis of these mechanisms in Drosophila will contribute to our understanding of the genetic and molecular basis of the development of epithelial cell polarity, and will certainly have important implications for the development of epithelia in general. INTRODUCTION One of the basic questions with respect to epithelial development is concerned with the establishment and maintenance of epithelial cell polarity. This polarity is manifested by an asymmetric distribution of organelles and molecules within the cell and the separation of the plasma membrane into two distinct domains: the apical side faces a lumen or the outside of the organism, while the baso-lateral side contacts the neighboring cells and, in many cases, a basal membrane, which underlies the epithelium. The lateral surfaces often develop various types of junctional structures, some of which ensure close contacts between adjacent cells and/or act as diffusion barriers between apical and basolateral membrane domains, while others provide channels to allow intercellular communication (see Garrod and Collins, 1992, for review). Epithelia are found in many different phyla of the animal kingdom, from the most primitive metazoa, such as the Coelenterata, to vertebrates. In all these organisms, epithelia perform several important functions: they provide the basis for extensive morphogenetic movements during development, they form diffusion barriers between different compartments of the body and they control the targeted transport of ions and molecules. Despite these more or less common functions, two structural differences can be observed

122


between epithelia of vertebrates and invertebrates: first, vertebrate epithelia form tight junctions apical to the adherens junctions, which serve two functions: i) they provide a selective paracellular diffusion barrier to the passage of ions and block the passage of macromolecules between the apical and the luminal side of epithelia. ii) Tight junctions provide a barrier to restrict the free diffusion of proteins and lipids within the outer leaflet of the plasma membrane, thus contributing to cell polarity (see Garrod and Collins, 1992, for review). Tight junctions have not been identified in epithelia of invertebrates so far, but only in a few other tissues (e.g. the blood-brain barrier of some insects; Lane and Skaer, 1980). The second difference between epithelia of vertebrates and invertebrates consists in the presence of septate junctions basal to the adherens junctions in epithelia of invertebrates (Noirot-Timothée and Noirot, 1980), which may serve a similar function as tight junctions in vertebrates (Wood, 1990). In order to understand the development of epithelia, and in particular epithelial cell polarity, it is important to study both vertebrates and invertebrates. This will allow us to detect common themes, but may also lead us to uncover novel strategies. In this respect, Drosophila offers the unique possibility to study the development of epithelial cell polarity in vivo, by inducing and characterising mutant phenotypes. This will not only tell us important aspects concerning the function of a particular gene and its product, but will also allow to study genetic interactions and relationships, which will eventually unravel complete genetic pathways and networks. In this chapter, I will start by summarising what is known about the formation of the first epithelium in the Drosophila embryo and the establishment of cell polarity in this tissue. Subsequently, I will give a summary of our current knowledge on the genes and their products required to maintain epithelial cell polarity. Some of these genes are already known from vertebrates, while others are novel genes, which thus may uncover additional aspects required to maintain cell polarity. Finally, the chapter will close by giving a brief summary of data providing some insight into the function and composition of septate junctions, and their possible involvement in controlling epithelial cell polarity. FORMATION OF THE FIRST EPITHELIUM, THE CELLULAR BLASTODERM The development of the Drosophila embryo is initiated by 13 synchronous nuclear divisions, which are not associated with cytokinesis. During nuclear division cycle 10, most of the nuclei have reached the periphery of the egg, where most of the egg s cytoplasm is concentrated, and align just beneath the egg membrane. The next three divisions take place at the periphery and give rise to the syncycial blastoderm about two hours after fertilisation. Finally, with the onset of the 14th mitotic cycle, cellularisation is achieved by the gradual ingrowth of the egg’s plasma membrane between the nuclei, resulting in the formation of the cellular blastoderm about three hours after fertilisation. The growing of the membranes can be subdivided into two phases (see Foe et al. 1993, and Warn and Robert-Nicoud, 1992, for reviews). In the first—slow— phase, the plasma membrane protrudes between the individual nuclei. According to Sanders (1975) the material required for membrane formation is provided by so called lamellar bodies, structures visible in the electronmicroscope, which gradually disappear as the membranes grow (Sanders, 1975). So far, however, the nature of this material is not known. The transition from slow phase to fast phase of membrane growth occurs as the leading tip of the membrane furrows extend as far inwards as the base of the nuclei. During the fast phase of membrane growth the microvilli, which are abundant on the surface of the embryo prior to this stage, disappear, suggesting that they provide the material for the growing membrane during the fast phase of cellularisation (Mahowald, 1963", Fullilove and Jacobson, 1971; Foe and Alberts, 1983). So within a surprisingly short period of only about 80 minutes, the unicellular egg is transformed into the cellular blastoderm, a polarised, single layered epithelial sheet of about 6000 cells surrounding the yolk.


123

The next question is: what are the mechanisms that lead to the separation into an apical and a basal membrane domain at the blastoderm stage? ESTABLISHMENT OF CELL SURFACE POLARITY IN THE DROSOPHILA BLASTODERM The development of cell surface polarity, i.e. the separation of the physically continuous plasma membrane into an apical and a baso-lateral domain, is a gradual process, which is initiated during the extension of the plasma membranes, but completed only after formation of the cells. Based on observations obtained by light microscopy and transmission electronmicroscopy, it seems that the apical membrane of the blastodermal cells is derived from the egg’s plasma membrane, while the components of the baso-lateral membrane are delivered from two different sources: during the first, slow, phase, membrane material is provided by stacked leaflets of bilipid membrane, the lamellar bodies, which are deposited into the egg by the mother and which gradually disappear as the membranes grow. The second component of the lateral membrane is provided by the highly pleated egg’s plasma membrane itself, which is unfolded during the fast phase, allowing the rapid growth of the lateral membranes. Therefore, the egg’s plasma membrane contributes to the prospective apical and lateral domains, implying that the separation of membrane domains is achieved only after cellularisation is nearly complete. This leads us to the question at what point and how the two membrane surfaces separate. The cells themselves have already an inherent polarity during their formation, visible, for example, by the apical localisation of the centrosomes or the apical-basal orientation of the microtubules. Nevertheless, cell surface polarity develops only gradually, as demonstrated by using various marker proteins (Müller and Wieschaus, 1996). The transmembrane protein Neurotactin (Hortsch et al., 1990) is initially detectable in the peripheral cytoplasm at the beginning of cellularisation. During the fast phase of membrane growth, Neurotactin becomes localised to the lateral plasma membrane and broadly overlaps with the localisation of ARMADILLO. ARMADILLO, a cytoplasmic protein of the adherens junction homologous to the vertebrate β-catenin (Peifer and Wieschaus, 1990), is redistributed at mid cellularisation towards the apical part of the lateral membrane and is subsequently found concentrated at the sites of the prospective zonulae adherentes (ZA), belt like structures encircling the apical side of the cell. In parallel, NEUROTACTIN is still found apically and basally to the prospective ZA. Once germ band extension is initiated, NEUROTACTIN is exlusively found basally to ARMADILLO and the ZA in the lateral membrane domain. This suggests that only at this stage a complete separation into three distinct membrane domains has been achieved: a basolateral domain, expressing NEUROTACTIN, a junctional domain expressing components of the ZA, such as ARMADILLO, and an apical domain expressing neither of them (Müller and Wieschaus, 1996). So far, no zygotically expressed protein has been described that is expressed in the apical membrane of the blastoderm epithelium. Besides SHARK, a cytoplasmic tyrosine kinase containing a Src homology-2 (SH2) domain and ankyrin repeats, for which no function has been described until now (Ferrante et al., 1995) the transmembrane protein CRUMBS is one of the earliest proteins detected on the apical membrane (see also below). Although crumbs RNA and CRUMBS protein are already present in the blastoderm, the protein is initially not associated with the plasma membrane, but is diffusely distributed in the cell below the apical membrane (Wodarz et al., 1993). At the onset of gastrulation, as soon as the first morphogenetic movements occur, CRUMBS protein can be detected exclusively in the apical plasma membrane, indicating the presence or maturation of the apical membrane domain.

124


Further support for the establishment of an apical domain only at this stage comes from the analysis of heterologous marker proteins in Drosophila. Human placental alkaline phosphatase (PLAP) is a glycosylphosphatidyl inositol (GPI)-linked protein, which is targeted to the apical plasma membrane when transfected into Madin-Darbin canine kidney (MDCK) cells. However, upon fusion of alkaline phosphatase to the transmembrane and cytoplasmic domain of vesicular stomatitis virus G protein (PLAPG), the fusion protein is localised at the baso-lateral membrane domain in MDCK cells (Brown et al., 1989). When these proteins are expressed in the Drosophila embryo under the control of an inducible heat-shock promoter, PLAPG can be found basolaterally throughout development in all epithelia analysed (Shiel and Caplan, 1995). Surprisingly, the GPI-linked PLAP was localised also basolaterally in the cellular blastoderm, and persisted in this membrane domain in all surface epithelia. However, in those epithelia that invaginate and contribute to the internal structures of the embryo, the GPI-linked PLAP becomes redistributed to the apical membrane as soon as invagination occurs. This suggests either the presence of a switch in the sorting machinery of GPI-linked proteins, or the induction of such a machinery only at this developmental stage. The change in the subcellular distribution of both CRUMBS and the GPI-linked PLAP at a similar developmental stage gives rise to several open questions: What are the sorting signals in these proteins? What are the cellular components that recognise these signals and why are they not acting at the cellular blastoderm? What is the difference between surface epithelia, which express PLAP basolaterally, and internal epithelia, which express PLAP apically? Why do these two kinds of epithelia do not make any difference with respect to the sorting of CRUMBS, which is localised apically in both kinds of epithelia? MAINTENANCE OF EPITHELIAL CELL POLARITY IN THE DROSOPHILA EMBRYO All ectodermal epithelia in Drosophila are derived from the first epithelium, the blastoderm, which undergoes various morphogenetic movements. During these processes, the polarised phenotype of the blastodermal cells of the Drosophila embryo has to be maintained in order to perform the various functions, such as invaginations or directional transport of ions or molecules during absorptive or secretory processes. Vertebrate epitheiia use two main mechanisms in order to maintain epithelial cell polarity: i) Tight junctions, located at the extreme end of the lateral plasma membrane, provide a barrier that prevents the free diffusion of proteins and lipids within the two membrane domains, ii) The basal lamina, a special form of the extracellular matrix (ECM) serves as substrate for epithelia to adhere, but has also important functions for their polarised organisation in some instances, e.g. in the mammary epithelia (see Talhouk et al., 1993, for review). Strikingly, there are no tight junctions in the epithelia of the Drosophila embryo and the deposition of a basal lamina only occurs at later stages of embryogenesis. Instead, blastodermal cells are directly contacting the yolk. The question is therefore, which alternative strategies for maintenance of epithelial cell polarity can be discovered in the Drosophila embryo. In the next paragraphs I will first discuss data suggesting that the cytoskeleton and cell-cell adhesion may play important functions also in the fly embryo, and will then summarise data on two genes of Drosophila, crumbs and Stardust, which are required for the maintenance of epithelial cell polarity in the embryo, and for which no vertebrate counterparts have been described so far.


125

The Cytoskeleton and Epithelial Cell Polarity of the Blastoderm Once epithelial polarity is established, the next question to be addressed concerns the mechanism(s) that maintain the various proteins precisely at a particular region within the membrane, thus excluding them from other regions during and after cellularisation. Any separation of proteins occurring prior to and during the formation of the lateral membrane by unfolding of the apical membrane (fast phase of membrane growth) is likely to be controlled by mechanisms that involve interactions with cytoplasmic components rather than by a barrier with a function comparable to that of tight junctions. It is well established that the membrane cytoskeleton, which is closely juxtaposed to the inner surface of the membrane, not only functions to maintain the integrity of the membrane, but also facilitates adhesion, signal transduction and cell polarity (see Luna and Hitt, 1992, for review). Using various antibodies directed against components of the cytoskeleton, it be comes obvious that the cortical cytoskeleton is already compartmentalised prior to the onset of cellularisation and that some of its components are localised to specific membrane domains during the formation of the cells. For example, actin and some actin-binding proteins are concentrated in caps over the interphase nuclei of the syncycial blastoderm, and become reorganised to the so called transient membrane furrows, which anticipate the later membrane furrows (Miller et al., 1989). The actin binding protein βH-spectrin is associated with the transient mitotic furrows in the syncytial blastoderm. During cellularisation βH-spectrin can be found both in a circumferential band localised apicolaterally, but also associated with the leading edge of the ingrowing membrane; the latter staining disappears once cellularisation is complete (Thomas and Kiehart, 1994). In other systems spectrin has already been demonstrated to mediate interactions with integral membrane proteins, e.g. with the rat epithelial amiloridesensitive Na+-channel, which is localised in the apical membrane via interactions of a proline-rich sequence in its cytoplasmic tail with the SH3-domain of α-spectrin (Rotin et al., 1994). Thus it is tempting to speculate that also in the case of blastoderm formation in the Drosophila embryo spectrin (but also others) may serve similar functions. Development of Adherens Junctions in the Drosophila Embryo Adherens junctions are multiprotein complexes mediating close cell-cell contacts (see Garrod and Collins, 1992; Gumbiner, 1996, for reviews). One of the central components is the Ca2+-dependent, homophilic adhesion molecule E-cadherin. Its cytoplasmic tail associates with cytosolic proteins, called catenins, some of which attach to microfilaments (see Kemler, 1993; Marrs and Nelson, 1996, for reviews). In this way modulation of cell shape can be transmitted directly from cell to cell. In addition, recent evidence points to adherens junctions as the sites at which signals can be transferred in both directions, i.e. from inside out and vice versa (reviewed by Rantsch, 1994; Peifer, 1995). In Drosophila, the following homologs of vertebrate adherens junction proteins (reviewed in Marrs and Nelson, 1996) have been isolated: Drosophila E-cadherin is encoded by the shotgun locus (Oda et al., 1994; Tepaβ et al., 1996; Uemura et al., 1996), β-catenin is encoded by the armadillo gene (Peifer and Wieschaus, 1990) and α-catenin also has its homolog (Oda et al., 1993) (see below). The formation of adherens junctions in the Drosophila embryo is a gradual process and has been described in great detail at the electronmicroscopic level (Eichenberger-Glinz, 1979; Tepaβ and Hartenstein, 1994a). Additional molecular data were provided from the analysis of the developmental expression of those proteins, which constitute the adherens junctions, namely α- and β-catenin/ARMADILLO and DE-cadherin/SHOTGUN. Another helpful marker to follow the biogenesis of this type of junction is an antibody directed against phosphotyrosine, an epitope that is highly enriched at the sites of the adherens junctions (Woods and Bryant, 1993; Müller and Wieschaus, 1996). The analysis shows that

126


localisation of these proteins anticipates the formation of the adherens junctions. Shortly after the formation of the cellular blastoderm and long before the zonulae adherentes can be detected at the ultrastructural level, α-catenin, β-catenin/ARMADILLO and DE-cadherin/SHOTGUN are enriched in a band encircling the most apical region of the lateral membrane, while lower amounts of these proteins can also be detected along the lateral membranes. After the onset of gastrulation, the proteins mentioned are highly concentrated in the most apico-lateral region of the cells, thus clearly outlining the cell surfaces. This subcellular localisation is maintained until the end of embryogenesis. At the ultrastructural level the first adherens type junctions detectable are the spot adherens junctions (SAJ) in the cellular blastoderm distributed over the lateral borders of the cell membranes (Tepaβ and Hartenstein, 1994a; Grawe et al., 1996). In the next step, the SAJs begin to cluster at the apical most region of the lateral membrane and the zonulae adherentes start to develop. These adherens junctions are maintained throughout development in all ectodermal epithelia except the Malpighian tubules, where they disappear at later stages (Tepaβ and Hartenstein, 1994). What is the function of β-catenin/ARMADILLO and DE-cadherin/SHOTGUN during the development of epithelia in Drosophila, and what happens in their absence? Initially armadillo has been identified by its mutant phenotype as a segment polarity gene. This phenotype is due to the second role of armadillo, which is in the transduction of the WINGLESS signal required for cell fate determination (Peifer et al., 1991). The analysis of the development of cell adhesion in embryos lacking a functional armadillo gene has been hampered by the fact that a considerable amount of its gene product is provided maternally and deposited into the egg. Removing just the zygotic component still leaves sufficient protein to confer proper adhesiveness, and only interferes with its function in transmitting the WINGLESS signal. However, complete removal of the maternal contribution of gene expression blocks oogenesis, since the protein is required in the germ line for proper development of the oocyte. An allele (armxp33), which encodes a truncated version of the protein (Orsulic and Peifer, 1996), was analysed in order to understand the function of armadillo in the formation of embryonic epithelia. In homozygous mutant germ cells this defective protein provides sufficient function to allow the development of a few egg chambers, such that females with a mosaic germ line produce some eggs, which can be fertilised and initiate embryonic development (Cox et al., 1996). If these eggs are fertilised by a sperm carrying a wild-type armadillo allele, embryos develop almost normally. If the sperm does not contribute a functional copy of the gene, development is severely impeded. Cellularisation proceeds normally, but as soon as gastrulation starts, the epithelial structure disintegrates and the cells adopt a multilayered, mesenchym-like appearance. DE-cadherin/SHOTGUN and phosphotyrosine can be found on all cell membranes, instead of being concentrated apically. The cells round up and lose their columnar appearance as well as the contact to their neighbours. Consequently, morphogenetic movements, such as the formation of the ventral furrow or germ band extension, do not proceed normally (Cox et al., 1996; Müller and Wieschaus, 1996). The gene shotgun, which encodes DE-cadherin (Oda et al., 1994; Tepaβ et al., 1996; Uemura et al., 1996), also has a considerable maternal component. Embryos without a functional copy of shotgun lack most of the head and the ventral epidermis. Strikingly, although the gene is expressed in nearly all epithelia (Oda et al., 1994, Tepaβ et al., 1996; Uemura et al., 1996), some of them appear morphological and in their expression of marker proteins completely normal in shotgun mutants, while others are heavily affected. The severity of the defects is dependent upon the degree of morphogenetic activity. Morphogenetic activities include processes such as imaginations or delaminations of individual cells, which seem to require specialised adhesive mechanisms to ensure the stability and integrity of the epithelia. Epithelia most affected by the loss of the zygotic shotgun function are those that show vigorous morphogenetic activity, such as the neuroectoderm, where about a third of the cells delaminate as neuroblasts. Also affected are the Malpighian tubules, which normally achieve a two to eightfold extension of their length by cell


127

rearrangement (Tepaβ et al., 1996). Removal of maternal E-cadherin results in defects in both active and inactive epithelia, suggesting that the structural integrity of the different epithelia depends on different amounts of DE-cadherin. Nevertheless, the distribution of α- and β-catenin is severely affected in all epithelial cells without zygotic shotgun function, suggesting that maternal cadherin allows the initial assembly of junctions, but is not sufficient for the maintenance of ARMADILLO and α-catenin localisation at the junctions. Such weakened junctions, while sufficient to maintain the integrity of static epithelia, do not provide sufficient strength for active epithelia to sustain strong morphogenetic movements. Maintenance of Epithelial Cell Polarity in the Drosophila Embryo by the Genes crumbs and Stardust Crumbs and Stardust mutants exhibit a very similar phenotype As mentioned above, there is no ECM underlying the early epithelia in the Dro-sophila embryo, which could provide basal signals to control polarity, as described for example in the trophectoderm of the early mouse embryo (see Collins and Fleming, 1995, for review). However, genetic analyses have uncovered Drosophila genes, which seem to act from the apical, rather than the basal side to sustain preestablished epithelial polarity. Embryos mutant for either crumbs or Stardust fail to maintain cell polarity in those epithelia that are derived from the blastodermal ectoderm, namely the amnioserosa, the epidermis, the foreand hindgut, the tracheae, the Malpighian tubules and the salivary glands. Depending on the epithelium considered, the defects range from slight disturbances in epithelial organisation, disruption of epithelial integrity to widespread cell death, e.g. in the epidermis (Tepaβ and Knust, 1990, 1991) (Figure 1). A detailed analysis of these two mutants revealed that both, the biogenesis of adherens junctions and maintenance of cell surface polarity are disrupted. In embryos mutant for crumbs, spot adherens junctions (SAJs) develop initially normal. However, at the time when ZAs develop in the epidermis of wild-type embryos, crumbs mutant embryos fail to assemble these structures at the apical end of the lateral borders (Grawe et al., 1996; Tepaβ, 1996). Later on, cell death occurs in many ectodermally derived epithelia (Tepaβ and Knust, 1990). Although the terminal phenotype of Stardust mutant embryos is nearly identical to that of crumbs mutants (Tepaβ and Knust, 1990), an earlier defect with respect to adherens junction formation is observed. In embryos mutant for Stardust, the number of SAJs in the lateral cell membranes are dramatically reduced from the earliest appearances of SAJs. As in crumbs mutant embryos, ZA’s fail to develop and only remnants of spot adherens junctions can be seen on the lateral borders of the epidermal primordium (Grawe et al., 1996). The failure to build a proper ZA is preceded by an abnormal distribution of DE-cadherin and ARMADILLO (Grawe et al., 1996; Müller and Wieschaus, 1996; Tepaβ, 1996). Initially their expression is normal at the blastoderm stage and the onset of gastrulation, however, shortly after β-catenin/ARMADILLO and DE-cadherin/SHOTGUN are localised in a punctated pattern in the cytoplasm. At the end of germ band extension, the amount of both proteins detectable at the apicolateral membrane is strongly reduced. In general, the defects in the distribution of DE-cadherin and ARMADILLO seem to be more severe in Stardust than in crumbs mutant embryos of similar age. Based on their nearly identical phenotypes, it was proposed that crumbs and Stardust are elements in the same pathway. This was confirmed by the observation that the severity of the phenotype of mutants homozygous for null mutations in crumbs is dramatically attenuated by an additional copy of the wild-type Stardust gene: the integrity of the epidermis was almost completely restored and only minor defects were detected. No rescuing effect of a duplication of crumbs on Stardust embryos was observed. This suggests

128


Figure 1 Sagittal sections of embryos during germ band extension. Shown are part of the germ band from a wild-type (A) and a homozygous mutant crumbs11A22 (B) embryo. While the wild-type embryo exhibits a well organized, palisadelike arrangement of the epithelial cell, cells in the mutant embryo have lost their polarity, indicated by their round shape and their irregular arrangement; occacionally, they are lying on top of each other. Dead cells (black pycnotic bodies; arrowheads), which are pushed into the interior in wild-type embryos, fail to be internalized in the mutant.

that the latter acts downstream of crumbs, a relationship that should be understood in a formal genetic sense, allowing no predictions as to the molecular nature of these interactions (Tepaβ and Knust, 1993). Molecular characterisation of the CRUMBS protein So far, only the crumbs gene has been characterised molecularly (Tepaβ et al., 1990). Its derived amino acid sequence predicts a transmembrane protein of 2146 amino acids. The small cytoplasmic domain of 37 amino acids shows no significant sequence similarity to other sequences in the databases. The large extracellular portion includes 30 epidermal growth factor (EGF)-like repeats and four laminin A G-domainlike repeats (Figure 2A). Both motifs are assumed to mediate protein-protein interactions, but so far no partner(s) of the CRUMBS protein have been identified. Antibodies raised against the extracellular portion of the CRUMBS protein show that the tissues that express CRUMBS are the same as those affected in CRUMBS mutants, i.e. the epidermis, the fore- and hindgut, the tracheae, the salivary glands and the Malpighian tubules. As revealed by immuno-electronmicroscopy, the crumbs protein is associated exclusively with the apical membrane, being particularly enriched at the sites of membrane contact apical to the ZA (Tepaβ, 1996). This suggests that CRUMBS is not a constituent of the ZA, but rather supports the


129

Figure 2 Characteristics of the CRUMBS protein in wild-type and crumbs8F105 mutant. (A) Schematic representation of CRUMBS. SP: signal peptide, TM: transmembrane domain. The open boxes represent EGF-like repeats, the black regions correspond to the laminin A G domain-like repeats. (B) Amino acid sequence of the cytoplasmic domain of the wild-type and the crumbs8F05 mutant, respectively. A single nucleotide exchange in the mutant protein introduces a premature stop codon, thus removing the 23 carboxy-terminal amino acids.

view that this protein may be involved in delimiting the site at which the components of the zonula adherens assemble. The molecular analysis of crumbs8F105, an EMS-induced null allele, has yielded important insights into the function of the wild-type protein. The mutation is caused by a single base exchange, which leads to the introduction of a premature stop codon, thus eliminating the C-terminal 23 amino acids of the short cytoplasmic domain (Figure 2B). The mutant embryos produce normal amounts of CRUMBS protein, which, in contrast to the wild-type, is not restricted to the apical plasma membrane, but rather can be found in the cytoplasm and on all membranes. This behaviour in crumbs mutants is similar to that of other apical proteins, e.g. YELLOW (Wodarz et al., 1993). Further results support the view that crumbs plays a key role in specifying the apical plasma membrane domain of ectodermal epithelial cells in Drosophila. Overexpression of CRUMBS by means of the GAL4 system results in an considerable expansion of the apical plasma membrane and concomitant reduction of the baso-lateral domain (Wodarz et al., 1995). This phenotype is associated with the misdistribution of βHeavy spectrin, a component of the membrane cytoskeleton, and the ectopic deposition of cuticle into more basal regions of the plasma membrane. Strikingly, overexpression of the membrane bound cytoplasmic portion of CRUMBS alone is sufficient to produce this dominant phenotype, which strengthens the importance of the cytoplasmic tail for the function of the CRUMBS protein (Wodarz et al., 1995). In contrast and most surprisingly the extracellular portion of CRUMBS, either membrane bound or secreted, has no effect on embryonic or adult development when overexpressed (Wodarz et al., 1995; M.Engelbert and E.Knust, unpublished). SEPTATE JUNCTIONS, DHR-/PDZ-PROTEINS AND EPITHELIAL CELL POLARITY Septate junctions (SJ) are prominent structures in the epithelia of invertebrates, which are not found in vertebrates (Lane and Skaer, 1980). They are characterised by a region in which the plasma membranes of neighboring cells come to lie into close apposition with each other. The space between the membranes is filled with electron-dense material, which, depending on its structure, allows to discriminate two types of SJs: smooth SJs, found in the gut and the Malpighian tubules, and pleated SJs, characterised by a ladder-like

130


structure spanning the cleft between adjacent membranes, and common to all ectodermally derived epithelia with the exception of the Malpighian tubules (Lane and Skaer, 1980; Tepaβ and Hartenstein, 1994). In addition, pleated SJs can be found in the glial sheaths. SJs are assumed to perform similar functions as tight junctions in vertebrate cells, namely to restrict the diffusion of proteins and lipids between the apical and the basolateral membrane domains. However, only in the case of Hydra this function could be demonstrated experimentally (Wood, 1990). The question arises which are the constituents required for the biogenesis of SJs and whether they are involved in the establishment and/or maintenance of cell polarity. Since SJ develop rather late, their function could be required only at later stages of embryogenesis (Tepaβ and Hartenstein, 1994). To date, only three proteins have been described to be associated with septate junctions in Drosophila: the proteins encoded by the genes discs-large, coracle and Neurexin IV. Neurexins are transmembrane proteins originally identified as synapse-specific receptors for the venom of the black widow spider, latrotoxin (Petrenko et al., 1991). The only Drosophila neurexin identified do date, Neurexin IV, is associated with septate junctions of epithelial and glial cells (Baumgartner et al., 1996). Loss of Neurexin IV function results in the mislocalisation of CORACLE, the Drosophila homolog of protein 4.1, which is normally also localised at septate junctions (Fehon et al., 1994). Furthermore, the septa typical for pleated septate junctions are lost. As a consequence, the brain-blood barrier is completely abolished in mutants lacking a functional neurexin gene, pointing to an important function of septate junctions as intercellular barriers (Baumgartner et al., 1996). Embryos homozygous mutant for neurexin loss-of-function mutations show defects in dorsal closure similar as coracle mutants, but no phenotype with respect to epithelial cell polarity has been described so far. The third protein associated with septate junctions in Drosophila is DISCS-LARGE, a member of the so called MAGUK (membrane associated guanylate kinase) family, which is characterised by three protein motifs: an SH3-(Src-homolgy region-3) domain, a guanylate kinase (GUK) domain and one or more repeats of 80–90 amino acids, called DHR-(Discs large homology region)/PDZ-(PSD-95, Dlg, ZO-1) domain (Woods and Bryant, 1991; see Anderson, 1996, for review). Many members of this family are localised at the plasma membrane, some of them in close association with junctional structures, such as the septate junctions (DISCS-LARGE), the tight junction (ZO-1) or synapses (PSD-95 and others). SH3- and PDZdomains are supposed to mediate protein-protein interactions. In the case of the PDZ-domain, for example, a direct interaction between the cytoplasmic tail of the NMDA receptor and one of the three PDZ-domains of PSD-95 could be demonstrated (Kornau et al., 1995; see also Anderson, 1996, for review). Mutants in discs-large lead to neoplastic overgrowth of larval imaginal discs, single layered epithelial cell sheets, which give rise to most of the external parts of the adult fly (Woods and Bryant, 1991; Woods et al., 1996). This phenotype led to its classification as a tumor suppressor gene, and suggested its participation in cell-cell communication (see Bryant et al., 1993, for review). In addition, discs-large is required for the organisation of the cytoskeleton and the control of apicobasal polarity of epithelial cells, including the formation of the septate junctions and the proper localisation of other proteins, e.g. CORACLE, to the septate junctions and EXPANDED to the apical domain (Boedigheimer and Laughon, 1993). Adherens junctions are still present, but often mislocalised in discs-large mutant imaginal discs (Woods et al., 1996). The various functions of the DISCS-LARGE protein could be attributed to different domains: mutations in the SH3 and GUK domain cause loss of normal proliferation, but did not affect the other functions.


131

PERSPECTIVES The data available so far suggest that the establishment and maintenance of epithelial cell polarity in the Drosophila embryo relies on various components, such as proper cell-cell adhesion, formation of apical and baso-lateral membrane domains and functional septate junctions. In addition, we may expect that mechanisms similar to those known in vertebrates, required for the transport and targeting of proteins to the respective membrane domains (see Rodriguez-Boulan and Nelson, 1989; Rodriguez-Boulan and Powell, 1992, for reviews), as well as various components of the cytoskeleton are also involved in this process. Therefore, Drosophila will continue to serve as a model organism to study the cellular and molecular basis of epithelial cell polarity, which is crucial for all multicellular organisms. ACKNOWLEDGEMENT I would like to thank José A. Campos-Ortega, Kevin Johnson, Arno Müller and Andreas Wodarz for constructive criticisms on the manuscript. Work in the author’s lab is supported by the Deutsche Forschungsgemeinschaft. REFERENCES Anderson, J.M. (1996) Cell signalling: MAGUK magic. Curr. Biol., 6, 382–384. Baumgartner, S.A., Littleton, J.T., Broadie, K., Bhat, M.A., Harbecke, R., Lengyel, J.A., Chiquet-Ehrismann, R., Prokop, A. and Bellen, H.J. (1996) A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function. Cell, 87, 1059–1068. Boedigheimer, M. and Laughon, A. (1993) Expanded: a gene involved in the control of cell proliferation in imaginal discs. Development, 118, 1291–1301. Brown, D.A. , Crise, B. and Rose, J.K. (1989) Mechanism of membrane anchoring affects polarised expression of two proteins in MDCK cells. Science, 245, 1499–1501. Bryant, P.J., Watson, K.L., Justice, R.W. and Woods, D.F. (1993) Tumor suppressor genes encoding proteins required for cell interactions and signal transduction in Drosophila. Development Suppl., 239–249. Collins, J.E. and Fleming, T.P. (1995) Epithelial differentiation in the mouse preimplantation embryo: making adhesive cell contacts for the first time. Trends Biochem. Sci., 20, 307– 312. Cox, R.T., Kirkpatrick, C. and Peifer, M. (1996) Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis. J. Cell Biol., 134, 133–148. Eichenberger-Glinz, S. (1979) Intercellular junctions during development and in tissue cultures of Drosophila melanogaster. Roux’s Arch. Dev. Biol., 186, 333–349. Fehon, R.G.A., Dawson, I. and Artavanis-Tsakonas, S. (1994) Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development, 120, 545–557. Ferrante, A.W., Reinke, R. and Stanley, E.R. (1995) Shark, a Src homology 2, ankyrin repeat, tyrosine kinase, is expressed on the apical surfaces of all ectodermal epithelia. Proc. Natl. Acad. Sci. USA, 92, 1911–1915. Foe, v.E. and Alberts, B.M. (1983) Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. J. Cell Sci., 61, 31–70. Foe, V.E., Odell, G.M. and Edgar, B.A. (1993) Mitosis and morphogenesis in the Drosophila embryo. In The development of Drosophila melanogaster. eds. M. Bate and A. Martinez-Arias. (Cold Spring Harbor Laboratory Press, pp. 149–300. Fullilove, S.L. and Jacobson, A.G. (1971) Nuclear elongation and cytokinesis in Drosophila montana. Dev. Biol., 26, 560–577.

132


Garrod, D.R. and Collins, J.E. (1992) Intercellular junctions and cell adhesion in epithelial cells. In Epithelial organization and development (ed. T.P. Fleming), pp. 1–52. Chapman and Hall. London. Glasgow. New York. Tokyo. Melbourne. Madras. Grawe, F., Wodarz, A., Lee, B., Knust, E. and Skaer, H. (1996) The Drosophila genes crumbs and Stardust are involved in the biogenesis of adherens junctions. Development, 122, 951– 959. Gumbiner, B.M. (1996) Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell, 84, 345–357. Hortsch, M., Patel, N.H., Bieber, A.J., Traquina, Z.R. and Goodman, C.S. (1990) Drosophila neurotactin, a surface glycoprotein with homology to serine esterases, is dynamically expressed during embryogenesis. Development, 110, 1327–1340. Kemler, R. (1993) From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet., 9, 317–321. Kornau, H.-C, Schenker, L.T., Kennedy, M.B. and Seeburg, P.H. (1995) Domain interactions between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science, 269, 1737–1740. Lane, N.J. and Skaer, H.B. (1980) Intercellular junctions in insect tissues. In: Advances in Insect Physiology. Eds. M.J. Berridge, J.E. Trehern and V.B. Wigglesworth (London: Academic Press) pp. 35–213. Luna, E.J. and Hitt, A.L. (1992) Cytoskeleton-plasma membrane interactions. Science, 258, 955–964. Mahowald, A.P. (1963) Ultrastructural differentiations during formation of the blastoderm in the Drosophila melanogaster embryo. Dev. Biol., 8, 186–204. Marrs, J.A. and Nelson, W.J. (1996) Cadherin cell adhesion molecules in differentiation and embryogenesis. Int. Rev. Cytol., 165, 159–205. Miller, K.G., Field, C.M. and Alberts, B.M. (1989) Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography. J. Cell Biol., 109, 2963–2975. Müller, H.-A.J. and Wieschaus, E. (1996) armadillo, bazooka and Stardust are critical for formation of the zonula adherens and maintenance of the polarised blastoderm epithelium in Drosophila. J. Cell Biol., 134, 149–163. Noirot-Timothée, C. and Noirot, C. (1980) Septate and scalariform junctions in arthropods. Int. Rev. Cytol., 63, 97–140. Oda, H., Uemura, Shiomi, K., Nafafuchi, A., Tsukita, S. and Takeichi, M. (1993) Identification of a Drosophila homologue of α-catenin and its association with the armadillo protein. J. Cell Biol., 121, 1133–1140. Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M. (1994) A Drosophila homolog of cadherin associated with Armadillo and essential for embryonic cell-cell adhesion. Dev. Biol., 165, 716–726. Orsulic, S. and Peifer, M. (1996) An in vivo structure-function relationship of Armadillo, the β-catenin homologue, reveals both separate and overlapping regions of the protein required for cell adhesion and for wingless signaling. J. Cell Biol., 134, 1283–1300. Peifer, M. (1995) Cell adhesion and signal transduction: the Armadillo connection. Trends Cell Biol., 5, 224–229. Peifer, M., Rauskolb, C., Williams, M., Riggleman, B. and Wieschaus, E. (1991) The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation. Development, 111, 1029–1043. Peifer, M. and Wieschaus, E. (1990) The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin. Cell, 63, 1167– 1178. Petrenko, A.G., Perin, M.S., Davletov, B.A., Ushkaryov, Y.A., Geppert, M. and Sudhof, T.C. (1991) Binding of synaptotagmin to the α receptor implicates both in synaptic vesicle exocytosis. Nature, 353, 65–68. Rantsch, B. (1994) Cadherins and catenins: interactions and functions in embryonic development. Curr. Op. Cell Biol., 6, 740–746. Rodriguez-Boulan, E. and Nelson, W.J. (1989) Morphogenesis of the polarized epithelial cell phenotype. Science, 245, 718–725. Rodriguez-Boulan, E. and Powell, S.K. (1992) Polarity of epithelial and neuronal cells. Ann. Rev. Cell Biol. 8, 395–427. Rotin, D., Bar-Sagi, D., O’Brodovich, H.O., Merilainen, J., Lehto, V.P., Canessa, C.M., Rossier, B.C. and Downey, G.P. (1994) An SH3 binding region in the epithelial Na+ channel αrENaC mediates its localization at the apical membrane. EMBO J., 13, 4440–4450.


133

Sanders, E.J. (1975) Aspects of furrow membrane formation in the cleaving Drosophila embryo. Cell Tiss. Res., 156, 463–474. Shiel, M.J. and Kaplan, M.J. (1995) Developmental regulation of membrane protein sorting in Drosophila embryos. Am. J. Physiol., 269, 207–216. Talhouk, R.S., Werb, L. and Bissell, M.J. (1992) Functional interplay between extracellular matrix and extracellularmatrix-degrading proteinases in the mammary gland: a coordinate system for regulating mammary epithelial function. In Epithelial organization and development (Fleming, T.P., ed.), pp. 329–351, Chapman & Hall. TepaB, U. (1996) Crumbs, a component of the apical membrane, is required for zonula adherens formation in primary epithelia of Drosophila. Dev. Biol., 177, 217–225. TepaB, U. and Hartenstein, V. (1994) The development of cellular junctions in the Drosophila embryo. Dev. Biol., 161, 563–596. TepaB, U. and Knust, E. (1990) Phenotypic and developmental analysis of mutations at the crumbs locus, a gene required for the development of epithelia in Drosophila melanogaster. Roux’s Arch. Dev. Biol., 199, 189–206. TepaB, U. and Knust, E. (1993) crumbs and Stardust act in a genetic pathway that controls the organization of epithelia in Drosophila melanogaster. Dev. Biol., 159, 311–326. TepaB, U., Gruszynski-DeFeo, E., Haag, T.A., Omatyar, L., Török, T. and Hartenstein, V. (1996) shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neuroectoderm and other morphogenetically active epithelia. Genes Dev., 10, 672–685. TepaB, U., Theres, C. and Knust, E. (1990) crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell, 61, 787–799. Thomas, G.H. and Kiehart, D.P. (1994) beta-heavy-spectrin has a restricted tissue and subcellular distribution during Drosophila embryogenesis. Development, 120, 2039–2050. Uemura, T., Oda, H., Kraut, R., Hayashi, S. Kataoka, Y. and Takeichi, M. (1996) Zygotic DE-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Dev., 10, 659–671. Warn, R.M. and Robert-Nicoud, M. (1992) The formation and fate of the blastoderm epithelium of the Drosophila embryo. In Epithelial organization and development (ed. T.P. Fleming), pp. 137–161. Chapman & Hall. London. Glasgow. New York. Tokyo. Melbourne. Madras. Wood, R.L. (1990) The septate junctions limits the mobility of lipophilic markers in plasma membranes of Hydra vulgaris (attenuata) Cell Tissue Res., 259, 61–66. Woods, D.F. and Bryant, P.J. (1989) Molecular cloning of the lethal (I) discs large-1 oncogene of Drosophila. Dev. Biol., 134, 222–235. Woods, D.F. and Bryant, P.J. (1991) The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell, 66, 451–464. Woods, D.F., Bryant, P.J. (1993) ZO-1, DlgA and PSD-95/SAP90: Homologous proteins in tight, septate and synaptic cell junctions. Mech. Dev., 44, 85–89. Woods, D.F., Hough, C, Peel, D., Callaini, G. and Bryant, P.J. (1996) Dlg protein is required for junction structure, cell polarity, and proliferation control in Drosophila epithelia. J. Cell Biol, 134, 1469–1482.

8. MOLECULAR ASPECTS OF EARLY MESODERM DEVELOPMENT IN AMPHIBIAN EMBRYOS CHRISTOF NIEHRS and BEATRIZ FERREIRO Division of Molecular Embryology, Deutsches Krebsforschungszemtrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

SUMMARY The earliest inductive event during amphibian embryogenesis is the formation of mesoderm, a key determinant for the vertebrate body plan. While it is established that growth factors inducing mesoderm belong to the TGF-β and FGF families, the identity of the individual genes functioning in vivo is still unclear, though the number of candidates is increasing. The wnt- and Bmp—signaling pathways are involved in dorsal and ventral mesoderm formation, respectively. Further patterning of lateral mesoderm is mediated by the Bmp-4 morphogen, whose function is antagonized by noggin and chordin, both acting in the organizer. Homeobox genes rate among the most prominent groups of transcription factors involved in mesoderm formation. They are regulated by growth factors and themselves induce additional signalling factors, thus leading to a hierarchical cascade of inductive events. INTRODUCTION Axis formation in the vertebrate embryo is critically dependent on mesoderm. During gastrulation, the early dorsal mesoderm, or Spemann organizer, has a central inductive role. The Spemann organizer transplanted to the ventral side of a host embryo induces a secondary axis, that mainly consists of host tissue. The Spemann organizer dorsalizes surrounding mesoderm and induces and patterns the neural plate (Spemann and Mangold, 1924; reviewed by Hamburger, 1988). In later embryonic stages, the inductive properties of the mesoderm are also important where epithelio-mesenchymal interactions are neccesary for organogenesis (Birchmeier and Birchmeier, 1993). Finally, skeleton, muscle, connective tissue, heart, endocrine- and exocrine organs, as well as blood and the immune system develop from this germ layer. In this chapter, we will give a brief introduction into premolecular studies and then review signaling factors and intracellular transducers thought to play a role in mesoderm induction and patterning. For related developmental aspects such as later axial development, as well as neural induction and patterning we refer to other reviews (Slack and Tannahill, 1992; Ferreiro and Harris, 1994; Kessler and Melton, 1994; Krumlauf, 1994; Papalopupu and Kintner, 1994; Reilly and Melton, 1996). In the amphibian tadpole, all features characteristic of the vertebrate body plan are present, and like in all other vertebrates, five main tissues of the mesoderm can be distinguished dorso-ventrally: notochord, somites, pronephros, lateral plate and blood anlage (Figure 1). In the early amphibian embryo, mesoderm originates from the so-called marginal zone, a ring of cells present in the equatorial region of the early gastrula. Based on self-differentiation capacity, five different areas of the marginal zone can be distinguished: the dorsal domain differentiates into notochord, the dorsolateral domain into muscle, the


135

Figure 1 Development of Xenopus mesoderm. Mesoderm develops from an equatorial belt of cells located between the animal and vegetal pole of the gastrula embryo, the marginal zone. The marginal zone is already specified dorsoventrally and will give rise to a characteristic sequence of mesodermal tissues shown for the tadpole embryo on the right.

lateral and ventrolateral domain into pronephros, blood, and mesenchyme, and the ventral domain into blood and mesenchyme (Figure 1) (Dale and Slack, 1987a). Thus, the dorso-ventral pattern of these tissues is already specified in the gastrula marginal zone. Hence, the elucidation of the molecular mechanisms underlying pattern formation in the marginal zone is of prime importance for understanding mesodermal development. Many embryological data support the notion that dorsal and ventral mesoderm arise as the consequence of an inductive process. Prospective mesodermal blastomers isolated from the animal half of the 8-cell stage embryo develop into ectoderm, whereas they differentiate into mesoderm when explanted at the blastula stage (Nakamura et al., 1970; Kageura and Yamana, 1983; Gurdon et al., 1985). This suggests that mesodermal differentiation requires an instructive signal. Nieuwkoop showed that animal cells in contact with vegetal cells form mesoderm, and that the inductive properties of ventral and dorsal vegetal cells are different (Nieuwkoop, 1969; Nieuwkoop, 1973). Ventral vegetal cells induce blood and mesenchyme, i.e. ventral mesoderm, whereas dorsal vegetal blastomers induce notochord and somites, i.e. dorsal mesoderm. From these findings it was concluded that formation of dorsal mesoderm, and therefore of the organizer with all its inductive capacities, is itself the consequence of an inductive process with the signal originating from dorsal vegetal cells, also called the Nieuwkoop center. However, there are also data that argue against an inductive mechanism, and rather for the involvement of cytoplasmic determinants in mesodermal formation. Explantation experiments analogous to those of Nakamura et al. (1970) and of Kageura and Yamana (1983) showed that animal cells of the 16-cell (Gallagher et al., 1991) and even of the 8-cell stage (Grunz, 1994) could autonomously differentiate into dorsal mesoderm. Moreover, the dorsal and ventral marker genes goosecoid (gsc) and Xwnt-8 are expressed in dorsal and ventral blastomers, respectively, even when early blastomeres are dissociated, i.e. in the absence of an external signal. In contrast, the pan-mesodermal marker gene Xbra requires cell-cell interactions for its expression (Lemaire and Gurdon, 1994). Apparently, mesoderm formation can to a

136


certain extent also take place in the absence of cell-cell interactions, as a consequence of the action of cytoplasmic determinants. These determinants, however, could very well be identical to the mesodermal inducers, if they are capable of autoinduction. Possibly only mesoderm that forms in the animal half of the marginal zone needs an external signal, whereas the more vegetal part of the marginal zone contains mesodermal inducers enabling it to differentiate autonomously. Cell-cell interactions are not only necessary for initial mesoderm induction, but also during gastrulation. Thus, dissociated mesodermal cells do not terminally differentiate (Symes et al., 1988; C.N. unpublished observations); the dorsal mesoderm can still be ventralized until the late gastrula stage by treatment with suramin, which appears to inhibit certain cell-cell interactions (Grunz, 1992; Oschwald et al., 1993); and during gastrulation a critical mass of prospective muscle cells is necessary for cell differentiation, a phenomenon known as community effect (Gurdon et al., 1992; Gurdon et al., 1993). The factors involved in these late cell-cell interactions and in the community effect are still unknown, but are probably not identical to the primary mesodermal inducers (Symes et al., 1988; Gurdon et al., 1993). Mesodermal cells are not determined until early blastula, since the development of prospective mesodermal cells can be changed when transplanted (Heasman et al., 1984), which is consistent with the observation that the dorsal signal starts to pass between vegetal masses and animal caps only after the early blastula stage (Wylie et al., 1996). The temporal competence of the ectoderm for mesodermal induction is limited to the early gastrula, and this may serve to regulate the width of the mesodermal marginal zone (Reilly and Melton, 1996). In addition, dorsal ectoderm shows a greater competence than ventral ectoderm to be induced to dorsal mesoderm (Sokol and Melton, 1991; Godsave and Shiurba, 1992; Kinoshita et al., 1993), pointing to the possible influence of a dorsal determinant on the ectoderm (Sokol, 1993; see below). The search for mesoderm-inducing factors benefited greatly from the technically simple animal cap assay (reviewed in Smith and Howard, 1992). Induction of dorsal mesoderm is accompanied by elongation of animal explants, mimicking gastrulation movements, while induction of ventral mesoderm leads to the formation of characteristic fluid-filled vesicles, and thus both types can be easily scored. This assay led to the discovery of many growth factors that, even at high dilution, are still capable to induce mesoderm in animal caps. Most of these growth factors belong to two families, the transforming growth factor β (TGF-β) family, and the fibroblast frowth factor (FGF) family. In addition, a number of other factors, known as mesoderm modifiers, do not induce mesoderm de novo, but do have an effect on the differentiation pattern, either dorsalizing or ventralizing existing mesoderm. PATTERN FORMATION IN THE MESODERM: SIGNALS AND DETERMINANTS Dorsal Mesoderm Cortical rotation and dorsal determinants The unfertilized Xenopus egg shows animal-vegetal asymmetry in pigmentation and yolk distribution. The animal half shows a stronger pigmentation and contains fewer and smaller yolk platelets. Multiple mRNAs are asymmetrically localized in the egg, and may play a role in the formation of the primary animal-vegetal axis. At the animal side are localized an-1, 2 encoding ubiquitin-related proteins (Linnen et al., 1993) and an-3 encoding an ATP-dependent DNA helicase (Gururajan et al, 1991), whereas at the vegetal side are localized mRNAs encoding the growth factors Vg1 (Rebagliati et al., 1985) and Xwnt-11 (Ku and Melton,


137

1993), Xcat-2 which shows homology to the Drosophila gene nanos (Mosquera et al., 1993), and the repetitive RNAs Xlsirte (Kloc et al, 1993). Fertilzation gives rise to a new axis, the axis of bilateral symmetry. The site where the sperm enters determines the axis of dorso-ventral asymmetry such that the entry point becomes the ventral side of the embryo. By some unknown mechanism the microtubular aster, which forms from the sperm centriole, initiates a directional cytoplasmic reaction during the first cell division. In this process, the cortex, a layer of cytoplasm just below the cell membrane, rotates with respect to the more internal cytoplasm toward the prospective dorsal side (see Elinson and Holowacz, 1995, for review). The sperm entry point dictates the direction of rotation. The cortical reaction is accompanied by shifting of the central cytoplasm, the significance of which for dorso-ventral axis formation is at present not clear (Danilchick et al., 1991; Brown et al., 1993; Denegre and Danilchik, 1993). The cortical rotation is tightly linked to the shift of a dorsal cytoplasmic determinant to the prospective dorsal side. This maternal determinant is at first localized in the vegetal cortex of the egg and can be transplanted to the ventral side of a host embryo, leading to the induction of secondary embryonic axes (Curtis, I960; Yuge et al., 1990; Fujisue et al., 1993; Holowacz and Elinson, 1993; Holowacz and Elinson, 1995; Sakai, 1996). mRNA from dorsal animal blastomers also seems to have an inductive effect (Hainski and Moody, 1992). The identity of the dorsal determinant is still unclear, although it is thought to be a component of the wingless-signal transduction pathway (see below). The cortical rotation and the shifting of the dorsal determinant is brought about by a half-sphere of parallel microtubules, which bind to the vegetal pole during the first cell division (see Elinson and Holowacz, 1995, for review). The majority of these microtubules points with their plus-ends towards the dorsal side. Movement of the dorsal cytoplasmic determinant takes place through a kinesin-mediated transport along microtubules (Houliston and Elinson, 1991). It is unclear whether the microtubules themselves glide along each other, or whether they serve as a scaffold for the transport of material (Houliston, 1994; Houliston et al., 1994; Larabell et al., 1996). UV-irradiation of early embryos inhibits the formation of the parallel microtubules, and leads to ventralized embryos with mesoderm consisting only of blood and mesenchyme. By contrast, Li-treatment during early cell division stages leads to a complete dorsalization of the embryos, whose mesoderm consists solely of notochord (Elinson and Holowacz, 1995). Wingless (wnt) growth factor family Whereas products of genes of the wnt-1 class typically do not induce mesoderm, they are able to dorsalize ventral mesoderm (Christian et al., 1992). The wnt-signaling pathway which is locally activated in dprsal tissues by cortical rotation is assumed to be the main signal of the Nieuwkoop center inducing the organizer (for reviews, see Gumbiner, 1995; Miller and Moon, 1996). This view is supported by observations that wnt gene products and downstream components of the wnt signal transduction pathway are able to ectopically induce a second organizer in a non-cell-autonomous fashion, and that dominant-negative mutants of components of this transduction pathway inhibit the induction of the organizer (Smith and Harland, 1991; Sokol et al., 1991; Heasman et al., 1994; Dominguez et al., 1995; Guger and Gumbiner, 1995; He et al., 1995; Karnovsky and Klymkowsky, 1995; Pierce and Kimelman, 1995; Sokol et al., 1995; Behrens et al., 1996; Huber et al., 1996; Wylie et al., 1996). Also the well-known dorsalizing action of Li seems to be due to its direct activation of the wnt-pathway (Klein and Melton, 1996). Activation of the wnt signal transduction pathway is followed by the accumulation in the nucleus of p-catenin, complexed with the transcription factor LEF-1/XTcf-3 (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996). The

138


earliest timepoint in which nuclear localized β-catenin is found more in the dorsal than in the ventral blastomers is the 16-cell stage (Miller and Moon, 1996), and this dorsal accumulation continues until the end of the blastula stage (Schneider et al., 1996). This contrasts with the observation that the dorsal signal may pass only after the early blastula stage (Wylie et al., 1996) and suggests that the signal induced by LEF-1/β-catenin requires zygotic transcription, starting only at MBT. However, at present, there are no genes of the wnt-1 class known that are selectively expressed in the Nieuwkoop center. Xwnt-8b is a maternally expressed wnt-gene which can induce secondary embryonic axes, but it is an open question whether it is selectively active at the dorsal side (Cui et al., 1995). Since a dominant-negative wnt does not interfere with organizer formation (Hoppler et al., 1996) it appears likely that activation of the wnt signal transduction pathway by cortical rotation does not take place through a wntligand, but through activation of an intracellular component of the pathway. Transforming growth factor (TGF)-β family Activin was the first chemically defined rnesoderm inducer, and is the best characterized. Activin and related growth factors of the TGF-β family are, with an effective concentration of ca. 0.1ng/ml, the most potent mesoderm inducers in the animal cap assay. Dose-response experiments with activin have provided strong evidence for the role of a TGF-β morphogen in dorso-ventral mesoderm development. Activin is able, in increasing concentrations, to induce a ventral-dorsal sequence of mesodermal tissues in animal cap cells (see for reviews Sive, 1993; Smith, 1995; Tiedemann et al., 1995). These inductions involve thresholds, and not continuous transitions (Green and Smith, 1990; Green et al., 1992). Gurdon and collaborators have developed an assay system to study dorso-ventral pattern formation in the mesoderm by activin in animal caps in situ. In this system, an animal cap is combined in a sandwich with an activin source. The inducer is either another animal cap injected with activin mRNA, or a chromatographic bead soaked with activin. By means of in situ hybridization, the expression of mesodermal marker genes can then be detected as concentric rings in the surrounding tissue. As one would expect when activin indeed functions as a dorso-ventral morphogen, expression of the dorsal marker gene gsc was observed close to the activin source, while the pan-mesodermal marker gene Xbra was expressed further away (Gurdon et al., 1994). Cells respond to activin in a ratchet-like fashion, “remembering” the highest concentration exposed to (Gurdon et al., 1995). However, activin is probably not an authentic mesoderm inducer, since (1) it is present at extremely low concentrations in the embryo (Dohrmann et al., 1993; Rebagliati and Dawid, 1993) (2) The stepwise inductions (thresholds) cannot be demonstrated with activin when the analysis of marker gene expression is done at the gastrula instead of the neurula stage. Rather, a gradual transition in the induction of mesodermal tissues is observed with increasing doses of activin (Green et al., 1994; Wilson and Melton, 1994). These results point towards secondary interactions after activin-treatment of animal cap cells. (3) Overexpression of the activin-specific inhibitor Follistatin, in concentrations sufficient to inhibit mesoderm induction by activin in vitro, has no effect on the physiological mesoderm induction (Schulte-Merker et al., 1994) or dorso-anteriorizes embryos (Kessler and Melton, 1995; Sasai et al., 1995), and (4) mouse embryos, in which the activin A and B genes have been inactivated by homologous recombination, show normal mesoderm formation (Matzuk et al., 1995). Taken together, the above experiments with activin primarily show how responding cells can behave rather than revealing a physiological role of this factor in the embryo. At present it is assumed that Vg1 is a physiological mesoderm inducer since it is expressed in vegetal cells of the Xenopus blastula (Rebagliati et al., 1985; Melton, 1987). Microinjected Vg1 mRNA induces a


139

ventral-dorsal sequence of mesoderm, like activin, in a dose-dependent manner (Kessler and Melton, 1995). However, it is only active when its preprodomain is replaced with that of another TGF-β growth factor (Dale et al., 1993; Thomsen and Melton, 1993; Schulte-Merker et al., 1994; Kessler and Melton, 1995). It is assumed that specific proteases locally activate Vg1 in the blastula. TGF-β receptors are heteromers of type I and type II Ser-Thr protein kinases. Type II receptors bind the ligand and form a signaling complex with a type I receptor which becomes in turn phosphorylated and activated. Type I kinase subsequently phosphorylates an intracellular transcription factor, of the so-called ‘mothers against dpp’ (Smad) class, which regulates the expression of target genes (see section Intracellular regulators: ventral mesoderm; Massague, 1996; Niehrs, 1996). Experiments with the receptors confirm the notion that TGF-β signals are necessary for the mesoderm formation. Overexpression of type I and type II receptors in Xenopus ventral mesoderm leads to the induction of dorsal mesoderm (Kondo et al., 1991; Hemmati-Brivanlou et al., 1992; Mathews et al., 1992; Mahony and Gurdon, 1995). Dominant-negative TGF-β receptors of which the cytoplasmic domain has been artificially deleted play an important role in studying mesoderm formation. These receptors are still able to form complexes with wild type receptors, but cannot transduce the signal, thus inhibiting endogenous signal transduction. Such dominant-negative TGF-β receptors have been characterized for the activin type II receptor. Its expression inhibits the induction of both ventral and dorsal mesoderm. The specificity of the effect is unclear, however, since the activin type II receptor also interacts with type I receptors for other ligands (Massague, 1996). Ventral Mesoderm For a long time it was assumed that ground-state ventral mesoderm plays no active role in patterning, since in transplantation experiments the organizer is dominant over ventral mesoderm (Slack, 1994). However, experiments with dominant-negative receptors demonstrated that signals from the fibroblast growth factorand the bone morphogenetic protein families are required for the differentiation of the ventral mesoderm and that ventral signals can be dominant over dorsal. Fibroblast growth factor (FGF) family Members of the FGF family are potent inducers of ventral and lateral mesoderm. Basic FGF and FGF-2 are maternally expressed but not, as would be expected of a mesodermal inducer, in vegetal, but in animal cells. From the midblastula transition (MBT) onwards, when zygotic transcription starts, eFGF and FGF-3 genes are expressed in the mesoderm, and they may serve a mesoderm-maintenance function (Slack, 1994). FRL1 and FRL2, although not related to FGF growth factors, can activate FGF-receptors and are expressed in gastrula and neurula embryos (Kinoshita et al., 1995). Experiments with a dominant-negative FGF receptor demonstrated that FGF signals are required for the induction of ventral and dorsal mesoderm except head mesoderm (Amaya et al., 1991; Amaya et al., 1993; Northrop et al., 1995). FGF signals cooperate with activin-like signals in the induction of mesoderm (Cornell and Kimelman, 1994; LaBonne and Whitman, 1994; Cornell et al., 1995). This could explain why mesoderm formation is restricted to the equatorial marginal zone. Since maternal FGF-2 is expressed in the ectodermal animal caps and may have a limited range of action, the superimposition of a vegetal activin-like signal with an animal FGF signal in the equatorial area would lead to a functional overlap of both signals, and thus to the formation of mesoderm in the marginal zone (Cornell et al., 1995). Data obtained in

140


Ambystoma showing that explants of the marginal zone with ectoderm form more mesoderm than explants without it, also support an active role of the ectoderm in mesoderm induction (Tiedemann, 1993). Bone morphogenetic proteins (Bmp) family Bmp-2, –4, and 7 belong to the Bmp subclass of the TGF-β family, and are expressed in early Xenopus embryos (Köster et al., 1991; Dale et al., 1992; Jones et al., 1992; Nishimatsu et al., 1992; Nishimatsu et al., 1993; Clement et al., 1995; Hemmati-Brivanlou and Thomsen, 1995). Bmps selectively induce ventral mesoderm in animal caps and their overexpression even leads to ventralization of dorsal mesoderm (Dale et al., 1992; Jones et al., 1992), distinguishing them from other inducers such as FGF. A dominance of ventral over dorsal mesoderm had not been observed in tissue combination experiments, possibly because the amount of secreted growth factor is too low or the range of action too short for being dominant. Bmp-2 is mainly expressed maternally, and is an excellent candidate for a ventral vegetal signal for mesoderm induction (Köster et al., 1991). Bmp-4 and Bmp-7 are mainly expressed after MBT, i.e. after mesoderm induction has taken place. These factors are therefore probably not of importance for mesoderm induction, but could relay the specification of the ventral mesoderm from Bmp-2 in the gastrula (Dale et al., 1992; Jones et al., 1992; Fainsod et al., 1994; Schmidt et al., 1995; Jones et al., 1996). Experiments with dominant-negative Bmp-2/4 type I receptors have shown that Bmp signals are required for the induction of ventral, but not dorsal mesoderm. In contrast to the dominant-negative FGF receptor (Amaya et al., 1993), the expression of the dominant-negative Bmp-2/4 receptor in ventral mesoderm leads to the formation of dorsal mesoderm (Graff et al., 1994; Maeno et al., 1994; Suzuki et al., 1994; Ishikawa et al., 1995). It was further shown for the dominant-negative Bmp-2/4 receptor that it does not interfere with activin signaling, supporting the specificity of the effects observed (Graff et al., 1994). These are important differences between FGF- and Bmp signals, which point towards FGFs being primarily active in mesoderm induction and Bmps in dorso-ventral patterning. Specifically, Bmps unlike FGF are able to induce globin expression, the most ventral mesodermal marker (Graff et al., 1994; Clement et al., 1995; Zhang and Evans, 1996). Interestingly, Bmp signals are not only required for ventral mesoderm differentiation within the mesoderm, but also in the surrounding ectoderm. This is based on the observation that animal caps expressing a dominant-negative Bmp-2/4 receptor dorsalize sandwiched ventral marginal zone explants (Maeno et al., 1994). This demonstrates again an active, signaling function of the ectoderm in mesoderm formation. Wingless (wnt) family The biological activity of Xwnt-8 has occasionally led to confusion. Microinjection of Xwnt-8 mRNA into ventral blastomers leads to formation of complete secondary embryonic axes (Smith and Harland, 1991; Sokol et al., 1991). The induction is non-cell-autonomous, because microinjected cells are not necessarily part of the embryonic axis but can be endodermal. These properties correspond to those of the Nieuwkoop centre signal. However, Xwnt-8 is not expressed in the Nieuwkoop centre but in ventral mesoderm, and after MBT. Indeed, ectopic dorsal expression of Xwnt-8 after MBT, by microinjecting a plasmid in which Xwnt-8 expression is under the control of the cytoskeletal actin promotor, leads to ventralization of embryos, and notochordal mesoderm differentiates into somites (Christian and Moon, 1993). The same phenotype is observed after Li-treatment of gastrulae, consistent with its direct activation of the wnt-pathway. Xwnt-8 probably plays a role in the differentiation of the lateral mesoderm (Hoppler et al., 1996), and the induction


141

Figure 2 Functional screen for the identification of dorsalizing genes. In this functional screen a plasmid library is plated at desired pool size. Embryos are microinjected with mRNA transcribed from the pools and scored for rescue of UV-ventralization (Smith and Harland, 1991) or secondary axis formation (Lemaire et al., 1995).

of secondary axes is only the reflection of an activity related to the wnt-signaling pathway of the Nieuwkoop centre. Signals of the wnt-1 class, to which Xwnt-8 belongs, are transduced through the classical wnt pathway. The wnt-1 class is antagonized by the wnt-5A class, the overexpression of which decreases cell adhesion and blocks the ectopic induction of secondary embryonic axes by Xwnt-8 (Du et al., 1995; Torres et al., 1996). The ventralizing effect of plasmid-driven Xwnt-8 can be also inhibited by wnt-5A (Torres et al., 1996). Xwnt-11, a member of the wnt-5A class, is expressed in the dorsal marginal zone of the gastrula (Ku and Melton, 1993) raising the possibility that Xwnt-11 and Xwnt-8 are physiological antagonists. Other factors It was recently demonstrated that the product of lunatic fringe (lFng), a Xenopus homologue of the drosophila gene fringe, can induce ventral mesoderm. Fringe is a novel, secreted protein and plays a role in dorso-ventral pattern formation in the Drosophila wing. A receptor or signaling pathway for fringe is as yet unknown. IFng is maternal and ubiquitously expressed in the blastula and gastrula stages (Wu et al., 1996). Lateral Mesoderm Dorsalization In his organizer experiments, Spemann already demonstrated that ventral mesoderm of the lateral plate type differentiates into muscle under the influence of an implanted organizer, i.e. a more dorsal mesodermal tissue (“assimilative induction"). This pointed to an inductive function of the organizer in muscle formation. In addition, comparison between the maps of cell fate and cell specification strengthens the view that muscle differentiation indeed requires an inductive signal from the organizer (Dale and Slack, 1987a,b). It was accordingly demonstrated that isolated ventral mesoderm in contact with dorsal mesoderm (organizer) differentiates into somites rather than lateral plate. Similarly, contact of ventral with dorsolateral mesoderm leads to differentiation of pronephros rather than lateral plate or blood (Yamada, 1950; Smith arid Slack, 1983).

142


These results indicate that dorsal and dorsolateral mesoderm emitt a dorsalizing signal, which leads to pattern formation in the ventral mesoderm. Based on these findings, the three-signal model for mesoderm development was formulated (Smith and Slack, 1983). Signals emitted from the Spemann organizer Candidates for the dorsalizing signal emitted from the organizer are the secreted factors noggin (Smith and Harland, 1992; Smith et al., 1993) chordin (Sasai et al., 1994; Sasai et al., 1995), follistatin (HemmatiBrivanlou et al., 1994) and nodal related (Xnr) 1–3 (Jones et al., 1995; Smith et al., 1995). All are expressed in the organizer and, when overexpressed in ventral mesoderm, lead to dorsalization. Both noggin and Xnr3 were cloned by a powerful functional screen pioneered by Smith and Harland (1991, 1992), involving injection of pools of mRNA, scoring for phenotypic effects and sib selection of active clones (Figure 2). Noggin and chordin do not seem to transmit their signals through their own receptors. Instead, these factors directly bind to Bmp-2 and Bmp-4, thereby inactivating them (Piccolo et al., 1996; Zimmerman et al., 1996). Follistatin probably functions through a similar mechanism (A. Fainsod, D. Kessler, personal communication). Thus, one mechanism for dorsalization is to antagonize ventralizing Bmp signals in the marginal zone. Chordin is homologous to the Drosophila gene short gastrulation (sog), which plays an important role in the dorso-ventral development of the fly-blastoderm. In Drosophila, sog antagonizes the Bmp-4 homolog decapentaplegic (dpp) (Francois et al., 1994), and overexpression of Drosophila sog in Xenopus has effects similar to overexpression of chordin (Holley et al., 1995; Schmidt et al., 1995). Dpp functions as a morphogen, and regulates in a dose-dependent fashion the development of ectodermal structures in the fly embryo (Ferguson and Anderson, 1992; Wharton et al., 1993). The discovery of the homology between the antagonizing sytems chordin/sog and Bmp-4/dpp in Xenopus and Drosophila strongly argues in favour of the thesis of Geoffrey St. Hilaire, that vertebrates and arthropodes have homolo gous body plans, and that during evolution an inversion of the dorso-ventral axes must have taken place (Arendt and Nübler-Jung, 1994; De Robertis and Sasai, 1996). Similar to dpp regulation of dorso-ventral pattern formation in both, Drosophila ectoderm (Ferguson and Anderson, 1992; Wharton et al., 1993) and mesoderm (Staehling-Hampton et al., 1994; Frasch, 1995), it seems that the Bmp-4/chordin system regulates dorso-ventral pattern formation of all three germ layers in the frog. In ectoderm, chordin induces—and Bmp-4 inhibits neural tissue formation and instead induces epidermis (Sasai et al., 1995; Wilson and Hemmati-Brivanlou, 1995), whereas in endoderm Bmp-4 represses and chordin induces dorsal endoderm (Sasai et al., 1996). It is an intriguing possibility that the signal dorsalizing mesoderm is identical to the signal inducing neural tissue, since noggin (Lamb et al., 1993), follistatin (Hemmati-Brivanlou et al., 1994), chordin (Sasai et al., 1995) and Xnr3 (Glinka et al., 1996) all are able to directly neuralize animal caps without inducing mesoderm. Nodal was first discovered in the mouse, where mutation of the gene leads to defects in gastrulation (Zhou et al., 1993). Xnr1 and 2 are expressed in the subepithelial cells and in the epithelium of the organizer and are able to induce also dorsal mesoderm in animal caps (Jones et al., 1995). In contrast to activin, however, the products of both genes also dorsalize ventral mesoderm. Xnr3, on the other hand, is expressed only in the epithelial layer of the organizer (Ecochard et al., 1995; Smith et al., 1995; Glinka et al., 1996). Xnr3 has a dorsalizing effect only at relatively high concentrations compared to other members of the TGF-β family (Smith et al., 1995; Glinka et al., 1996). Nevertheless, the epithelial layer of the organizer has a strong dorsalizing effect after transplantation (Shih and Keller, 1992), even though chordin


143

and noggin are expresed at higher levels in the deep cells than in the epithelial layer of the organizer (Smith and Harland, 1992; Sasai et al., 1994; Glinka et al., 1996). The potent dorsalizing activity of the organizer epithelium seems to be mediated by the synergistic actions of Xwnt-11 and Xnr3, which are coexpressed in the epithelial layer, and cooperate in the induction of secondary embryonic axes by a factor of 50–100 fold. The mechanism of this synergy does not involve the classical wnt signaling pathway, but a transduction pathway that can also be induced by activin (Glinka et al., 1996). Xnr1 and noggin synergize in the induction of secondary embryonic axes with complete heads (Lustig et al., 1996). It is therefore possible that Xnr1 is an inducer of head mesoderm, which is in turn responsible for the induction of anterior structures. The Bmp-4 morphogen and dorsal-ventral antagonism The homology between dorso-ventral axes formation in Drosophila and Xenopus raises the question if Bmp-4 functions also as a morphogen in Xenopus mesoderm. In organizer explants, Bmp-4 induces somites, pronephros, and blood in a dose-dependent manner, and a dominant-negative Bmp-2/4 receptor dosedependently induces pronephros, somites, ajid notochord in explants from ventral mesoderm. This is accompanied by a dose-dependent regulation of expression of marginal zone marker genes (Dosch et al., 1997). This is best illustrated by the behavior of the dorsolateral marker Xmyf-5 (Figure 3). Bmp-4 is expressed outside of the organizer in the early gastrula, but mRNA expression does not appear to be graded, as would be expected of a morphogen (Fainsod et al., 1994; Schmidt et al., 1995). Since noggin is able to dorsalize ventral mesoderm in a dose-dependent manner (Dosch et al., 1997), and since noggin and chordin can inactivate Bmp-4 by directly binding to it (Piccolo et al., 1996; Zimmerman et al., 1996), it is an obvious assumption that not the concentration of the Bmp-4 protein, but rather its activity is graded by the action of chordin and noggin. When these proteins diffuse from the dorsal towards the ventral side, they may increasingly inactivate Bmp-4 in the vicinity of the organizer. Although the Bmp-4 activity gradient remains to be detected by some direct means, both noggin and Bmp-4 can signal long-range in the marginal zone as would be expected in this model (Dosch et al., 1997). Interestingly, in the animal cap Bmp-4 appears to have short-range action (Jones, 1996). This suggests regional differences in diffusibility of Bmp-4. The shape of the Bmp-4 activity gradient may be therefore influenced by these differences. Relay effects of Bmp-4 are unlikely because, even though Bmp-4 has a long-range action, its intracellular effector XSmad-1 does not (Dosch et al., 1997). In contrast, activin, Vg1, TGFβ1 (Reilly and Melton, 1996), as well as Xnr-2 proteins (Jones et al., 1996) are hardly able to diffuse from expressing embryonic cells, although the data are conflicting for activin (Jones et al., 1996). Because of this it is suspected that their effects are mediated via additional inducers by a relay-like mechanism (Reilly and Melton, 1996). How are the activin dose-response data, mentioned earlier, to be interpreted in the light of these findings? An important observation is that a mixture of cells, induced with high and low concentrations of activin, does not differentiate into a mixture of ventral and dorsal cells, as one would expect if activin were a morphogen. Instead, these cells differentiate into muscle (Wilson and Melton, 1994), i.e. a ‘consensus’ is formed. Activin can induce both noggin and ventral mesoderm, and therefore probably also Bmp-4 expression (Green et al., 1992; Green et al., 1994; Wilson and Melton, 1994). If a population of nogginexpressing cells were mixed with Bmp-4-expressing cells, the Bmp-4 morphogen model would predict a differentiation of lateral mesoderm, as is observed experimentally. Therefore, the effects of activin may be by signal-relay via Bmp-4 and noggin, ckordin, and follistatin. In summary, a primary function of the organizer is to antagonize the effect of Bmp-4, and to grade its activity. Like dpp in Drosophila, Bmp-4, as an instructive morphogen, regulates dorso-ventral pattern

144


Figure 3 Dose-dependent regulation of Xmyf-5 by Bmp-4 signaling. Xmyf-5 expression occurs in two dorsolateral stripes in the marginal zone of the Xenopus gastrula, shown in vegetal view, dorsal side up. Ectopic expression of Bmp-4 or dominant-negative (dn) Bmp-4 receptor regulates the pattern of Xmyf-5 expression at different doses (low, high), indicating that stripe formation occurs within a window of the Bmp-4 morphogen gradient. (From Dosch et al., 1997).

formation in the marginal zone. What appears macroscopically as induction is microscopically modulation of a ventral morphogen read-out. Since Bmp-4 and its antagonists noggin, chordin, and follistatin are also expressed in many other regions of the early embryo, a function as a morphogen in other morphogenetic processes is not unlikely. A model for the dorsoventral regulation of marker genes by Bmp-4 and noggin is given in Figure 4. It is surprising that noggin and Bmp-4 are stably expressed side by side, even though they negatively affect each other (Reem-Kalma et al., 1995). Model calculations for a mutually inhibiting system lead to the expectation that such a system is labile and sensitive to changes in the expression of one of its components,


145

Figure 4 Model of mesodermal patterning at the gastrula stage by a Bmp-4 morphogen gradient. Mesoderm specification in the gastrula marginal zone, shown schematically in lateral view. Top, Bmp-4 is uniformly expressed in the marginal zone except in the dorsal domain (thin line). Noggin is expressed in the dorsal domain (thick line). Their protein products overlap dorsolaterally leading to attenuated Bmp-4 activity (dashed line), resulting in an activity gradient of Bmp-4. Distinct thresholds within this gradient specify dorsal (future notochord), dorsolateral (future muscle) and lateroventral (future blood, lateral plate, pronephros) positional values. Chordin is inhibited by Bmp-4 outside the organizer domain. Xmyf-5 has a requirement for low- and is inhibited by high Bmp-4 dose. Xvent-2 has a requirement for low Bmp-4 dose. Xvent-1 has a high dose requirement for Bmp-4 (Dosch et al., 1997).

readily tipping the balance into one (dorsal) or the other (ventral) direction. The mutually inhibiting anterior and posterior segments of the Drosophila blastoderm could for example not coexist, if the domains would not mutually stabilize each other. This long range help’ is mediated by the secreted factors wingless and hedgehog (Meinhardt, 1994). An analogous Xenopus factor which is ventralizing even though it is expressed in the dorsal domain, is the product of the Bmp gene Admp (Moos et al., 1995), which could have a ‘long range help’ function for Bmp-4 expression. It is unlikely, that all mesoderm is regulated by the Bmp-4 system. For example, the secondary embryonic axes induced by dominant negative Bmp-2/4 receptors lack complete heads (Graff et al., 1994; Suzuki et al., 1994). A prerequisite for head induction is the presence of head mesoderm, and its formation requires apparenly not only the absence of Bmp-4 signals, but also the presence of special inducers which may be released from the Nieuwkoop center. A candidate for a head inducer is cerberus, which is expressed in the head mesoderm and which induces heads when overexpressed in ventral vegetal cells. Cerberus suppresses the formation of trunk structures and the expression of the trunk organizer marker gene gsc (Bouwmeester et al., 1996).

146


In Table 1 are listed signaling factors inducing or patterning mesoderm. Table 1 Signaling factors involved in Xenopus mesoderm development. Genes encoding secreted factors and expressed in Xenopus embryos are listed. Dorsal (d), lateral (1), or ventral (v) mesoderm inducing potential in animal cap assays is indicated as is information on mesoderm patterning potential, typically tested by expression in the marginal zone and scoring for dorsalization or ventralization of mesodermal differentiation. A further distinction is the effect observed of expression before (pre) or after (post) midblastula transition (MBT). Some wnt-genes do not appear to affect dorsoventral differentiation but inhibit dorsal gastrulation movements (inhib. movements). MesodermPatterning Factor

Mesoderm

pre

Induction

MBT

TGF-β family Activin dose-dep. d/v

d

Vg1

dose-dep. d/v

d

Bmp-2

v

v

Bmp-4

v

dose-dep. d/v

Bmp-7

v

v

ADMP Xnr1 Xnr2 Xnr3

v, 1 dose-dep. d/v no

v d d d

post References no

v

d

FGF family FGF-2 v, 1 FGF-3 eFGF FRL1 FRL2 Wnt family Xwnt-8 Xwnt-3A Xwnt-5A

Kimelman and Maas, 1992; Thompson and Slack, 1992. Tannahill et al., 1992. Isaacs et al., 1992. Kinoshita et al., 1995. Kinoshita et al., 1995.

v, 1 v, 1 v, 1 v, 1 v no

Green and Smith, 1990; Sokol et al., 1991; Green et al., 1992; Smith et al., 1993. Dale et al., 1993; Thomsen and Melton, 1993; Kessler and Melton, 1995. Clement et al., 1995; Hemmati-Brivanlou and Thomsen, 1995. Köster et al., 1991; Dale et al., 1992; Jones et al., 1992; Jones et al., 1996; Zhang and Evans, 1996; Dosch et al., 1997. Hawley et al., 1995; Yamashita et al., 1995. Moos et al., 1995. Jones et al., 1995. Jones et al., 1995. Ecochard et al., 1995; Smith et al., 1995; Glinka et al., 1996.

d d no, inhib. movements

v

Christian and Moon, 1993. Moon et al., 1993b; Wolda et al., 1993. Moon et al., 1993a; Torres et al., 1996.


147

INTRACELLULAR REGULATORS Pan-mesoderm An important molecular marker and regulator of the mesoderm anlage is encoded by brachyury (Xbra), which is expressed in the marginal zone from the onset of zygotic gene expression. Xbra encodes a transcription factor, which is necessary and sufficient in Xenopus for differentiation of mesoderm, except head mesoderm (Smith et al., 1991; Cuniiffe and Smith, 1992; Conlon et al., 1996). Xbra and FGF signals are coupled in an autoregulatory loop. This interaction may function as a molecular switch. Once a cell has received a FGF-signal, it will produce more FGF via Xbra, and a stable state of cell differentiation is reached (Isaacs et al., 1994; Schulte-Merker and Smith, 1995). Other pan-mesodermal genes expressed at gastrula stage are the homeobox genes Xcad3 (Northrop and Kimelman, 1994), Mox2 (Candia and Wright, 1995), and the zinc finger gene Xpo (Sato and Sargent, 1991). The expression of Xcad3 is positively regulated by both the FGF- and Bmp signaling pathways, and one signal can replace the other. However, unlike ventrolaterally BMP signals are not required in the dorsal domain, activation there takes place only through the FGF pathway (Northrop et al., 1995). The expression of pan-mesodermal genes apparently involves distinct dorsal and ventral regulatory modules. Dorsal Mesoderm In the dorsal marginal zone a number of transcription factors are expressed, which play a role in the regulation of dorsal specification. On the basis of the activity of the genes encoding these factors in gainand loss-of-function experiments, their function can be assigned to either the head- or the trunk organizer, terms that Spemann and Mangold used to distinguish between the different inducing capacities of early and late organizer tissue. Molecular domains of the organizer As already suggested by the different inducing properties of head and trunk organizer, the Spemann organizer is not a homogeneous tissue, but consists of distinct cell types (Vodicka and Gerhart, 1995; Bouwmeester et al., 1996). The expression of different genes defines at least four cell types in the organizer: (1) epithelial cells (Xnr3); (2) subepithelial cells (Xbra); (3) deep cells (gsc chordiri); and (4) endoderm (cerberus) (see Figure 5). These different cell types have different cell fates and inducing capacities (Table 2). How these different domains are established and maintained is unclear at present. Head organizer Ortbodenticle-2 (otx-2) is, like gsc and Xanf-1, expressed in the deep cells and induces anterior structures when overexpressed (Blitz and Cho, 1995; Pannese et al., 1995). Table 2 Properties of organizer domains organizer domain epithelium

subepithelial cells

cell Fate

Chorda-mesoderm Prechordal plate headmesenchyme pharynx Xnot, Xbra chordin, gsc

marker Gene

archenteron roof (Xenopus only) Xnr3

deep cells

leading edge foregut liver cerberus

148


Figure 5 Molecular domains in the organizer. A Xenopus gastrula at stage 11 is shown schematically in dorso-vegetal view. The domains of expression of cerberus, gsc/chordin/Xlim-1, Xbra and Xnr3 define at least four different domains within the organizer. The properties of these domains are summarized in Table 2. (after Bouwmeester et al., 1996). organizer domain epithelium

subepithelial cells

deep cells

leading edge

induction of

somites neural tube

convergent extension

forebrain, eyes split of eye-anlage branchial arches heart cement gland “spreading migration”

cement gland

autonomous gastrulation movements

midbrain hindbrain neural tube floor plate somites convergent extension

Since otx-2 mouse null mutants show strong head defects, the gene plays a central role in the head organizer (Acampora et al., 1995; Matsuo et al., 1995; Ang et al., 1996). A similar phenotype is observed when the mouse homolog of Xlim-1 is inactivated (Shawlot and Behringer, 1995). In Xenopus, overexpression of Xlim-1 has hardly any effects (Taira et al., 1992). However, Xlim-1 can be activated by mutagenesis of the Lim-domains, leading to secondary axes formation when injected at the ventral side, revealing its dorsalizing action (Taira et al., 1994). The product of the homeobox gene siamois (sia) is a downstream regulator of the dorsalizing wnt signaling pathway, sia is selectively expressed in the dorsal mesendoderm and is able to induce an organizer and secondary embryos with a complete head in a non-cell-autonomous


149

fashion (Lemaire et al., 1995). The expression of sia is not regulated by mesoderm inducers and it is probably a direct target gene of the Nieuwkoop center signal (Carnac et al., 1996). The relationship between these genes and the head-inducing cerberus is at present unknown. Trunk organizer The homeobox genes goosecoid (Cho et al., 1991) and Xanf-1 (Zaraisky et al., 1992; Zaraisky et al., 1995) are expressed in the deep cells and later in head mesoderm. When expressed ectopically in the ventral side of the embryo, gsc and Xanf-1 are able to cell-autonomously induce a second axes which however lacks a head. The products of both genes cause anterior cell movements in a cell-autonomous fashion (Niehrs et al., 1993; Zaraisky et al., 1995). While mouse mutants with an inactivated gsc gene do not show any early embryonic defects (Rivera et al., 1995; Yamada et al., 1995), a possibly reduntant, second gsc gene was recently discovered in chicken (M. Kessel, personal communication). The function for gsc in the trunk organizer is also supported by the observations that embryos are ventralized by the injection of antisense gsc mRNA, and ectopically express Xwnt-8. The phenotype of these embryos consists of a variable loss of head- and trunk structures (Steinbeisser et al., 1995). The winged-helix transcription factors XFKH-1/ XFD-1/pintallavis (Dirksen and Jamrich, 1992; Knöchel et al., 1992; Ruiz i Altaba and Jessell, 1992) are expressed in the gastrula organizer and when inactivated in the mouse lead to trunk defects (Ang and Rossant, 1994; Weinstein et al., 1994). Pintallavis, when coinjected with Xbra in Xenopus, induces notochord and somites (Cunliffe and Smith, 1994) consistent with a role in the trunk organizer. The product of the homeobox gene Xnot is a regulator of notochord development. It is expressed in the dorsal involuting marginal zone, and also in the notochord arising from these cells (Gont et al., 1993; von Dassow et al., 1993). Overexpression of Xnot mRNA leads to an enlarged notochord in Xenopus (Gont et al., 1996), and zebrafish mutants in which the Xnot homologue floating head is mutated do not form notochord (Talbot et al., 1995). Both gsc and Xnot directly or indirectly induce chordin expression (Sasai et al., 1994). This fact probably explains the dose-dependent dorsalization of ventral mesoderm by gsc. Expression of gsc mRNA in increasing concentrations in ventral marginal zone explants leads to the differentiation of pronephros, muscle, and notochord in vitro (Niehrs et al., 1994). It is likely that gsc induces increasing amounts of chordin that will antagonize Bmp-4 proportionally in the explants. In vivo, gsc mRNA is expressed in a dorso-ventral gradient in the organizer and may also induce chordin dose-dependently. Therefore, the gsc gradient could be of physiological significance to pattern mesoderm. Since gsc and Bmp-4 negatively regulate each other (Fainsod et al., 1994; Steinbeisser et al., 1995), graded gsc expression may arise through this feedback loop. Lateral Mesoderm Dorsolateral mesoderm mainly develops into muscle, and lateral mesoderm into pronephros (Dale and Slack, 1987a). The myogenic gene Xmyf-5 is a marker for dorsolateral mesoderm already in early gastrula (Dosch et al., 1997). Consistent with the role as a muscle regulatory gene in the gastrula are the observations that microinjection of Xmyf-5 mRNA induces the expression of muscle genes (Hopwood et al., 1991), and that mouse mutants in which both myf-5 and myoD are inactivated show severe muscular defects (Rudnicki et al., 1993). The dorsolateral domain in the gastrula is also defined by the expression of the genes Xvent-1 and Xvent-2. Both genes are lateroventrally expressed, but the dorsal boundary of Xvent-2 expression is closer to the organizer than that of Xvent-1. The difference in expression of both genes

150


coincides with the dorsolateral, Xmyf-5 expressing domain (Dosch et al., 1997). At present, there is no marker gene for the lateral domain in the gastrula which gives rise to the pronephros. In neurula-embryos, Xlim-1 is expressed in the pronephros-anlage (Taira et al., 1994), and the homologous mouse gene is important for kidney differentiation (Shawlot and Behringer, 1995). Ventral Mesoderm The mesoderm-inducing FGF signals are, in Xenopus, intracellularly mediated by the well-documented ras signaling pathway (Whitman and Melton, 1992; MacNicol et al., 1993; Hartley et al., 1994; Gotoh et al., 1995; LaBonne et al., 1995; MacNicol et al., 1995; Tang et al., 1995; Umbhauer et al., 1995; Dong et al., 1996). As described, mesoderm induction by activin (Cornell and Kimelman, 1994; LaBonne and Whitman, 1994; Cornell et al., 1995) and Vg1 (Schulte-Merker et al., 1994) depends on the simultaneous presence of FGF signals. This probably explains why activin and Bmp-4 signaling (Xu et al., 1996) are also influenced by the ras signaling pathway. Since dominant-negative raf (MacNicol et al., 1993; Graves et al., 1994) and AP1/Jun (Dong et al., 1996) inhibit the FGF-, but not activin-mediated mesoderm induction, the nodal point of the FGF-activin synergism is apparently upstream of these components. In contrast to FGF-signals, relatively little is known about how TGF-β signals are transduced. The transcription factors XSmad-1 and XSmad-2 (Xmad-1 and Xmad-2) can selectively mediate the induction of Bmp-4 and activin target genes, respectively (Graff et al., 1996; Thomsen, 1996). Both factors synergize with XSmad-4 (Lagna et al., 1996). TJie activation of the phosphatidylinositol cycle (PIC) may also be involved in the Bmp-4 signaling pathway. Li+ is an inhibitor of the PIC and dorsalizes ventral mesoderm, which can be prevented by coinjection of inositol (Busa and Gimlich, 1989). On the other hand, activation of the PIC by microinjection of PIC-coupled 5-hydroxytryptamine receptor (and subsequent incubation with 5hydroxytryptamine) leads to ventralization of dorsal mesoderm (Ault et al., 1996). However, the mechanism of action of Li+ probably also involves activation of the wnt-signaling pathway, because Li+ ions inhibit glycogen-synthase-kinase-3 (GSK-3), a represser of wnt-signaling (Klein and Melton, 1996). From this it could be suggested that GSK-3 may activate the PIC. The Bmp-4 signal appears to be mediated by a novel class of homeobox genes, which we call vent-genes. They were found through a large scale screen for mesodermal marker genes using whole-mount in situ hybridization. Xvent-1 (Gawantka et al., 1995) and Xvent-2 (Onichtchouk et al., 1996) show close similarity within but not outside the homeodomain. Both genes were independently described by others, who named them PV.1 (Tidman-Ault et al., 1996) (Xvent-1) and Vox (Schmidt et al., 1996), Xbr1 (Papalopulu and Kintner, 1996), and Xom (Ladher et al., 1996) (Xvent-2). In the gastrula, both genes are expressed in the animal cap and in the marginal zone outside of the organizer. Bmp-4 signaling is both necessary and sufficient for vent gene expression (Gawantka et al., 1995; Ladher et al., 1996; Onichtchouk et al., 1996; Scmidt et al., 1996; Tidman-Ault et al., 1996) and Xvent-2/Xom expression is an immediate-early response to Bmp-4 signaling (Ladher et al., 1996). The expression of both vent genes overlaps with the Bmp-4 expression pattern in gastrula and neurula. Overexpression on the dorsal side of both vent genes leads to ventralization of embryos. Xvent-2 can ventralize more strongly than Xvent-1 and, like Bmp-4, leads dose-dependently to the formation of so-called belly-piece or “Bauchstück” embryos which consist only of blood and mesenchyme. The RNA dose required for this is quite high, however, so that Xvent-2 may require cofactors in vivo for functioning efficiently. Indeed, coexpression of Xvent-1 and Xvent-2 has an additive ventralizing effect (unpublished results). PV1/Xvent-1 and Xvent-2 expression revert the dorsalization by the dominant-negative Bmp2/4 receptor, and therefore appear to function downstream of Bmp-4- (Tidman-Ault et al., 1996; Onichtchouk et


151

al., 1996). Taken together, these data indicate that Xvent gene products mediate the Bmp-4 signal (Gawantka et al., 1995; Ladher et al., 1996; Onichtchouk et al., 1996; Schmidt et al., 1996; Tidman-Ault et al., 1996). Because of the different expression of the vent genes and their different potency for ventralization, the question arises whether this reflects different subfunctions in the transduction of the Bmp-4 signal. Loss-of-function experiments are important to address this point. Xhox-3 is a ventralizing Xenopus homologue of Drosophila even-skipped, a gene belonging to the pairrule class. Xbox-3 is expressed in the ventroposterior mesoderm later than the vent-genes (Ruiz i Altaba and Melton, 1989a), and is induced by them (Gawantka et al., 1995; Ladher et al., 1996; Onichtchouk et al., 1996; Schmidt et al., 1996; Tidman-Ault et al., 1996). Xhox-3 seems to be necessary for posterior development (Ruiz i Altaba and Melton, 1989b,c). Mix-1 is expressed in mesoderm and endoderm already in late blastula (Rosa, 1989), and has a strong ventralizing effect when overexpressed. It is expressed both dorsally and ventrally, but its ventralizing function seems to be neutralized dorsally through interaction with the product of the homeobox gene siamois, with which it forms heteromeric complexes. Dominant-negative Mix-1 can revert ventralization caused by Bmp-4 overexpression (Mead et al., 1996). The zinc-finger genes GATA-1 and -2 are like the vent-genes expressed ventrally in the gastrula, and later in the blood-anlage (Kelley et al., 1994; Walmsley et al., 1994; Bertwistle et al., 1996), where they play an important role at least in the mouse, as shown by targeted mutations (Pevny et al., 1991; Tsai et al., 1994). The hierarchical relationship of these ventralizing genes, and whether they regulate distinct subfunctions of the Bmp-4 signal pathway and if so, which, will need to be addressed in the future. To this end, dominantnegative constructs will be usefull. It seems possible to generate such constructs by point mutations in the homeodomain (Mead et al., 1996), or by fusion with transcriptional repressor domains (Conlon et al., 1996). PROMOTOR ANALYSIS OF TARGET GENES Another way to elucidate the mechanism of mesoderm induction is to investigate promoters of genes that are induced by mesoderm inducers in the absence of protein synthesis, i.e. genes that are expressed as an immediate early response to mesoderm induction. Such analyses have been done for the activin-inducible genes gsc (Watabe et al., 1995), Mix2 (Chen et al., 1996), HNF1-α (Weber et al., 1996), and XFD-1 (Kaufmann et al., 1996). It is noteworthy that the identified activin-responsive elements appear different in all cases, which points towards multiple branchings of the activin-like signaling pathway. The activin element in Mix2 is regulated by a complex of FAST-1, a maternal winged-helix transcription factor, and XSmad-2 (Chen et al., 1996). For the specific dorsal expression of gsc, factors binding to an activin- and a wnt-responsive element synergize in its promoter. The activin responsive element is active everywhere in the vegetal hemisphere, whereas the wnt-responsive element is active only in the dorsovegetal region (Watabe et al., 1995). However, by in situ hybridization gsc expression can only be demonstrated in the organizer. This points towards the existence of an additional, as yet unknown, repressing and probably Bmp-4 responsive element in the gsc promoter such as is found in the XFD-1 promoter (Kaufmann et al., 1996). It should be kept in mind, however, that an endogenous activin-like activity is ubiquitously present in the entire vegetal half of the embryo. This is consistent with the view that the general induction of mesoderm is mediated by an activin-like signal, such as Vg1 (Rebagliati et al., 1985; Dale et al., 1993; Thomsen and Melton, 1993; Kessler and Melton, 1995) but that induction of the dorsal side requires an additional wnt-like signal. As a consequence, there is no need for separate dorsal and ventral mesoderm inducers in the embryo, since wntsignals modulate its dorso-ventral character. Mesoderm induction and dorso-ventral patterning would thus

152


be mechanistically uncoupled. The identification and characterization of Bmp-responsive elements will help to clarify this issue. GASTRULATION MOVEMENTS Gastrulation is a central morphogenetic process during embryogenesis. In this topological transformation, the three germ layers (ecto-, meso-, and endoderm) adopt their final position through migration of cells. The gastrulation movements originate in the organizer where they are also most prominent. Two different types of cell movement can be distinguished in the organizer: the involution of the deep cells (prospective head endomesodermal cells), and the convergent extension of the subepithelial cells (prospective chordamesoderm) (Keller and Winklbauer, 1992). Gsc is expressed mainly in deep (Cho et al., 1991), and Xnot in subepithelial cells (Gont et al., 1993; von Dassow et al., 1993). The driving force of the gastrulation movements in Xenopus are the subepithelial cells since they, unlike the deep cells, can autonomously perform gastrulation movements accompanied by the typical tissue stretching in vitro. On the other hand, in vitro, unlike subepithelial cells, deep cells move on a fibronectin matrix as single cells, and extend filopodia (“spread cells” or “migrating cells"). Subepithelial cells can show both, a “spread cell” phenotype, and extend lamellipodia, which rotate around the cells (“circus movements”) (Winklbauer et al., 1992). Convergent extension and spreading do not seem to be causally coupled, however, since also ventral mesodermal cells, which do not go through convergent extension, show spreading (Howard and Smith, 1993; Niehrs et al., 1993). It therefore seems that spreading on fibronectin is a general property of mesodermal cells. Technically, gastrulation movements in Xenopus can be investigated in vitro with explants and dissociated single cells, especially using time-lapse video (Keller, 1991)- Gastrulation movements can be easily followed after fixation using lineage-tracers, among which colloidal gold is particularly useful because of the high contrast labeling it provides (Niehrs and De Robertis, 1991). Little is known about the cell-biological mechanism and the regulators of these regionally different cell movements. In vitro, several growth factors are able to regulate the adhesion- and movement properties of animal cap cells. Activin can dose-dependently induce “circus movements” and, at high doses, “spread cells” (Howard and Smith, 1993; Symes et al., 1994). FGF induces, like low doses of activin, to a small extent the extension of fllo- and lamellipodia and “circus movements” (Howard and Smith, 1993; Minoura et al., 1995). Platelet derived growth factor (PDGF) in combination with activin, can induce spreading which is mediated by phosphatidylinositol 3-kinase (Symes and Mercola, 1996). It seems that in vivo PDGF-signals are also able to regulate gastrulation movements. PDGF-A is expressed in the gastrula ectoderm and PDGF-receptor-a (PDGFR) is expressed in mesoderm. Overexpression of a dominant-negative PDGFR selectively inhibits proper involution and the closing of the blastopore, but not convergent extension, without having a significant effect on the expression of the mesodermal marker genes gsc and Xbra (Ataliotis et al., 1995). However, many of the injected cells die, so that an unspecific mechanism cannot be excluded. In addition, the fact that the PDGFR and its ligand are not expressed in a dorso-ventral discriminating fashion is not in agreement with the assumed role of PDGF in the regulation of dorsal gastrulation movements. Possibly, dorso-ventral located cofactors play a role in this process. Finally, since closing of the blastopore is generally ascribed to convergent extension (Keller and Winklbauer, 1992), its inhibition by a dominant-negative PDGFR is also inconsistent with a selective inhibition of involution, suggesting that only a subcomponent of convergent extension is impaired, such as adhesion to the blastocoel roof.


153

Similarly, genes of the wnt-5A class interfere with elongation movements induced by activin without interfering with dorsal mesoderm differentiation (Moon et al., 1993a). Taken together, the regulation of regional gastrulation movements cannot yet be explained in a straightforward manner by the function of individual growth factors. A problem encountered with in vitro analysis is that the processes of cell-cell interactions, in groups of mesodermal cells, cannot be derived from the behaviour of single cells on a substrate. More complex in vitro assays would be useful to address this issue. Since products of homeobox genes can regulate regional cell movements in other systems, such as e.g. mab-5 in C. elegans (Costa et al., 1988), a similar role in Xenopus is not unlikely. Microinjection of gsc mRNA confers to ventral cells the movement-properties of the dorsal deep cells. Microinjected ventral cells move into the anterior direction in secondary axes. Surprisingly, even prospective chordamesodermal cells, which already express gsc to a certain extent, change their movement properties with overexpression and move further into the anterior direction than during normogenesis, and differentiate into head mesoderm. The anterior movement induced by gsc can also be demonstrated in vitro, and the difference between injected and non-injected cells becomes apparent only when they move as a cohort, and not as single cells on fibronectin (Niehrs et al., 1993). This suggests that the gsc-induced cell movements are based on cellcell interactions. Similar data were recently obtained for the gjf-related homeobox gene Xanf-1 (Zaraisky et al., 1995). The Hox gene XlHbox6 is expressed in Xenopus mesoderm in the lateral plate, i.e. a tissue that does not show any prominent convergent extension or involution during gastrulation (Wright et al., 1990). On the other hand, Microinjection of XlHbox6 mRNA into dorsal cells leads to inhibition of convergent extension, and microinjected cells do not differentiate anymore into chordamesoderm (Niehrs and De Robertis, 1991). In the above examples with transcription factors changed cell movements are linked to changes in cell fate. Whether the effects on cell differentiation occur primarily through inhibition of cell movements, or through changes in the specification of gastrula cells, cannot be distinguished. This is a fundamental question in gastrulation. Can morphogenetic movements be uncoupled from dorsal cell differentiation, or not? The experiments with PDGF and Xwnt-5A suggest that movements may not be required for dorsal mesoderm differentiation, but more experiments under conditions that exclude neurulation movements are required to settle this question. It is intruiging that the cadherin/β-catenin system is involved in both, celladhesion as well as cell-signaling by the wnt pathway (reviewed by Miller and Moon, 1996). Until now, no experimental conditions are known, which exclusively induce cell movements but not differentiation. This may reflect the lack of sufficiently specific methods to separate these intertwined processes. To obtain a better understanding about the regulation of cell movements, it will therefore be important to isolate the target genes regulated by the homeobox-transcription factors, and to investigate their function. MESODERM INDUCTION: SUMMARY AND PERSPECTIVE Experience has shown, that certain aspects of the over-all picture of the genesis of the mesoderm will always need to be revised; new molecules with new properties are found, and the complexity of known molecular interactions continues to increase. The following model (Figure 6), which contains essential components of the three-signal model of Smith and Slack (1983) can be derived from the available molecular and embryological data. The strategy employed by the embryo rests upon the interaction between neighbouring groups of cells, at the border of which a new group of cells splits off through cell-cell interactions. In this way, first mesoderm is formed at

154


Figure 6 Model of early mesodermal patterning during Xenopus embryogenesis. Mesoderm arises in the marginal zone of the blastula by the superimposition of an animal FGF and a vegetal activin-like signal (e.g.Vg1). Both signals induce mesoderm of ventral type, expressing Bmp-4 (Mesoderm induction). Dorsally, cortical rotation leads to the displacement of a dorsal determinant, activating the w«J-pathway and inducing dorsal- or organizer mesoderm (O) (Nieuwkoop signalling). The organizer generates via the dorsal inducers noggin and chordin a gradient of the ventral morphogen BMP-4 by attenuating its activity locally (BMP-4 gradient formation). Distinct threshold concentrations of BMP-4 specify dorsolateral mesoderm as notochord (No), muscle (Mu) pronephros (Pro) and blood (Mesoderm specification).

the interphase between endoderm and ectoderm through the process of mesoderm induction. Subsequently, lateral mesoderm is formed by the interaction of organizer and non-organizer mesoderm. This picture agrees with the view of early authors like Holtfreter, who pictured embryonic development as a hierarchical series of inductive events. At the same time the old ideas about gradients in axis development seem to find justification, since the mechanism of dorsalization appears to rely on a Bmp-4 gradient. Although during the past five years great strides forward were made in the molecular analysis of the Spemann organizer and mesoderm formation, obviously more questions have been raised than answered. A number of unsolved questions (which we find) of great importance should therefore conclude this chapter. Mesoderm Induction and Dorsal Determinant Which are the actual mesoderm inducers? If Vg1 is one of them, how is it activated? Is the ground state of mesoderm ventral? How is the width of the marginal zone regulated? It seems to be firmly established, that the dorsal determinant which induces the organizer makes use of the wnt-signaling pathway. It is completely unclear, however, how cortical rotation is linked to the wnt-pathway, and what is the Nieuwkoop centre signal which ultimately induces the organizer.


155

Induction by the Organizer Substantial progress has been made in the analysis of the trunk and head organizer and in the identification of inducers. But how are the various organizer domains induced? Which role do they play in the induction and patterning of the neural plate and gastrulation movements? How are the inducers in the organizer linked to the Hox gene expression and to the anterior posterior pattern formation? Antagonizing the Organizer The Bmp-4 signaling pathway plays a central role in antagonizing the organizer, and its components are at present being characterized. However, what is its relationship to the lateralizing Xwnt-8 signal? Equally complex is the relationship between the ventralizing homeobox genes. How are they hierarchically linked, do they perform partial functions in ventral and lateral mesoderm, and if so, which? Dorso-ventral Pattern Formation by the Organizer How is the Bmp-4 activity gradient translated into the discrete differentiation capacities of the ventral and lateral mesoderm? Is there already in the gastrula a pronephros-specific molecular regionalization? Does a “long-range help” mechanism, analogous to the Drosophila blastoderm, exist between organizer- and nonorganizer mesoderm for their stable coexistence? What is the actual shape of the gradient and how is the shape regulated? Gastrulation Movements Are morphogenetic movements coupled to cell differentiation? If so, what is the molecular mechanism? If not, what are the regulators of cell movement? Which factors regulate the regional specificity of cell movements (convergent extension, spreading migration)? ACKNOWLEDGEMENTS We thank Dr. D.Kessler and R.Dosch for critically reading and Dr. R.Hartong for help with writing the manuscript. This work was supported by grant Ni 286/ 4–1 from the Deutsche Forschungsgemeinshaft to C.N. and a fellowship of the European Community to B.E REFERENCES Acampora, D., Mazan, S., Lallemand, Y., Avantaggiato, V., Maury, M., Simeone, A. and Brulet, P. (1995) Forebrain and midbrain regions are deleted in Otx2—/— mutants due to a defective anterior neuroectoderm specification during gastrulation. Development, 121,3279– 90. Amaya, E., Musci, T.J. and Kirschner, M.W. (1991) Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell, 66, 257– 70. Amaya, E., Stein, P.A., Musci, T.J. and Kirschner, M.W. (1993) FGF signalling in the early specification of mesoderm in Xenopus. Development, 118, 477–87. Ang, S.L. and Rossant, J. (1994) HNF-3 beta is essential for node and notochord formation in mouse development. Cell, 78, 561–74.

156


Ang, S.L., Jin, O., Rhinn, M., Daigle, N., Stevenson, L. and Rossant, J. (1996) A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development, 122, 243–52. Arendt, D. and Nübler-Jung, K. (1994) Inversion of dorsoventral axis? Nature, 371, 26. Ataliotis, P., Symes, K., Chou, M.M., Ho, L. and Mercola, M. (1995) PDGF signalling is required for gastrulation of Xenopus laevis. Development, 121, 3099–110. Auk, K.T., Durmowicz, G., Galione, A., Harger, P.L. and Busa, W.B. (1996) Modulation of Xenopus embryo mesoderm-speciflc gene expression and dorsoanterior patterning by receptors that activate the phosphatidylinositol cycle signal transduction pathway. Development, 122, 2033–41. Behrens, J., J.P, K., Kühl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Birchmeier, W. (1996) Functional interaction of beta-catenin with the transcription factor LEF-1. Nature, 382, 638–42. Bertwistle, D., Walmsley, M.E., Read, E.M., Pizzey, J.A. and Patient, R.K. (1996) GATA factors and the origins of adult and embryonic blood in Xenopus: responses to retinoic acid. Mechanisms of Development, 57, 199–214. Birchmeier, C. and Birchmeier, W. (1993) Molecular aspects of mesenchymal-epithelial interactions. Annu. Rev. Cell Biol. ,9,511–40. Blitz, I.L. and Cho, K.W.Y. (1995) Anterior neuroectoderm is progressively induced during gastrulation: the role of the Xenopus homeobox gene orthodenticle. Development, 121, 993–1004. Bouwmeester, T., Kim, S.-H., Sasai, Y., Lu, B. and De Robertis, E.M. (1996) Cerberus is a head-inducing secreted factor expressed in the anterior endoderm of Spemann’s organizer. Nature, 382, 595–601. Brown, E.E., Denegre, J.M. and Danilchik, M.V. (1993) Deep cytoplasmic rearrangements in ventralized Xenopus embryos. Dev. Biol., 160, 148–56. Busa, W.B. and Gimlich, R.L. (1989) Lithium-induced teratogenesis in frog embryos prevented by a polyphosphoinositide cycle intermediate or a diacylglycerol analog. Dev. Biol., 132, 315–24. Candia, A.F. and Wright, C.V. (1995) The expression pattern of Xenopus Mox-2 implies a role in initial mesodermal differentiation. Mech. Dev., 52, 27–36. Carnac, G., Kodjachbachian, L., Gurdon, J.B. and Lemaire, P. (1996) The homeobox gene siamois is a target of the wnt dorsalization pathway and triggers organiser activity in the absence of mesoderm. Development, 122, 3055–65. Chakrabarti, A., Matthews, G., Colman, A. and Dale, L. (1992) Secretory and inductive properties of Drosophila wingless protein in Xenopus oocytes and embryos. Development, 115, 355–69. Chen, X., Rubock, M.J. and Whitman, M. (1996) A transcriptional partner for MAD proteins in TGF-beta signalling. Nature, 383, 691–96. Cho, K.W., Blumberg, B., Steinbeisser, H. and De Robertis, E.M. (1991) Molecular nature of Spemann’s organizer: the role of the Xenopus homeobox gene goosecoid. Cell, 67,1111– 20. Christian, J.L., McMahon, J.A., McMahon, A.P. and Moon, R.T. (1991) Xwnt-8, a Xenopus Wnt-l/int-1 — related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis. Development, 111, 1045– 55. Christian, J.L., Olson, D.J. and Moon, R.T. (1992) Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm. Embo J., 11, 33–41. Christian, J.L. and Moon, R.T. (1993) Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes Dev., 7, 13–28. Clement, J.H., Fettes, P., Knochel, S., Lef, J. and Knöchel, W. (1995) Bone morphogenetic protein 2 in the early development of Xenopus laevis. Mechanisms Development, 52, 357– 70. Conlon, F., Sedgwick, S.G., Weston, K.M. and Smith, J.C. (1996) Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm. Development, 122, 2427–35. Cornell, R.A. and Kimelman, D. (1994) Activin-mediated mesoderm induction requires FGF. Development, 120, 453–62. Cornell, R.A., Musci, T.J. and Kimelman, D. (1995) FGF is a prospective competence factor for early activin-type signals in Xenopus mesoderm induction. Development, 121, 2429– 37.


157

Costa, M., Weir, M., Coulson, A., Sulston, J. and Kenyon, C. (1988) Posterior pattern formation in C. elegans involves position-specific expression of a gene containing a homeobox. Cell, 55, 747–56. Cui, Y., Brown, J.D., Moon, R.T. and Christian, J.L. (1995) Xwnt-8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis. Development, 121, 2177–86. Cunliffe, V. and Smith, J.C. (1992) Ectopic mesoderm formation in Xenopus embryos caused by widespread expression of a Brachyury homologue. Nature, 358, 427–30. Cunliffe, V. and Smith, J.C. (1994) Specification of mesodermal pattern in Xenopus laevis by interactions between Brachyury, noggin and Xwnt-8. Embo J., 13, 349–59. Curtis, A.S.G. (1960) J.Embryol. exp. Morphol., 8, 163–73. Dale, L. and Slack, J.M. (1987a) Regional specification within the mesoderm of early embryos of Xenopus laevis. Development, 100, 279–95. Dale, L. and Slack, J.M. (1987b) Fate map for the 32 — cell stage of Xenopus laevis. Development, 99, 527–51. Dale, L., Howes, G., Price, B.M. and Smith, J.C. (1992) Bone morphogenetic protein 4: a ventralizing factor in early Xenopus development. Development, 115, 573–85. Dale, L., Matthews, G. and Colman, A. (1993) Secretion and mesoderm-inducing activity of the TGF-β-related domain of Xenopus Vg1. Embo J., 12, 4471–80. Danilchick, M., Peng, H.B. and Kay, B.K. (1991) Xenopus laevis: Practical uses in cell and molecular biology. Pictorial collage of embryonic stages. Methods Cell Biol., 36, 679–81. Denegre, J.M. and Danilchik, M.V. (1993) Deep cytoplasmic rearrangements in axis-respecified Xenopus embryos. Dev. Biol, 160, 157–64. De Robertis, E.M. and Sasai, Y. (1996) A common plan for dorsoventral patterning in Bilateria. Nature, 380, 37–40. Dirksen, M.L. and Jamrich, M. (1992) A novel, activin-inducible, blastopore lip-specific gene of Xenopus laevis contains a fork-head DNA-binding domain. Genes Dev., 6, 599–608. Dohrmann, C.E., Hemmati-Brivanlou,.A., Thomsen, G.H., Fields, A., Woolf, T.M. and Melton, D.A. (1993) Expression of activin mRNA during early development in Xenopus laevis. Dev. Biol., 157, 474–83. Dominguez, I., Itoh, K. and Sokol, S.Y. (1995) Role of glycogen synthase kinase 3 beta as a negative regulator of dorsoventral axis formation in Xenopus embryos. Proc. Natl. I. Acad. Sci. USA, 92, 8498–502. Dong, Z., Xu, R.H., Kim, J., Zhan, S.N., Ma, W.Y., Colburn, N.H. and Kung, H. (1996) AP-1/jun is required for early Xenopus development and mediates mesoderm induction by fibroblast growth factor but not by activin. J. Biol. Chem., 271, 9942–6. Dosch, R., Gawantka, V., Delius, H., Blumenstock, C. and Niehrs, C. (1997) Bmp-4 acts as a morphogen in dorsoventral mesoderm patterning in Xenopus. Development, 124, 2325– 34. Du, S.J., Purcell, S.M., Christian, J.L., McGrew, L.L. and Moon, R.T. (1995) Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos. Mol. Cell Biol., 15, 2625–34. Ecochard, V., Cayrol, C., Foulquier, F., Zaraisky, A. and Duprat, A.M. (1995) A novel TGF-β like gene, fugacin, specifically expressed in the Spemann organizer of Xenopus. Developmental Biol., 172, 699–703. Elinson, R.P. and Holowacz, T. (1995) Specifying the dorsoanterior axis in frogs: 70 years since Spemann and Mangold. Curr. Top. Dev. Biol., 30, 253–85. Fainsod, A., Steinbeisser, H. and De Robertis, E.M. (1994) On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo. Embo J., 13, 5015–25. Ferguson, E.L. and Anderson, K.V. (1992) Decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo. Cell, 71, 451–61. Ferreiro, B. and Harris, W.A. (1994) Neurogenesis in Xenopus: a molecular genetic perspective. In: Advances in Genetics, 31, 29–78. J.C. Hall and J.C. Dunlap (eds). Academic Press. Francois, V., Solloway, M., O’Neill, J.W., Emery, J. and Bier, E. (1994) Dorsal-ventral patterning of the Drosophila embryo depends on a putative negative growth factor encoded by the short gastrulation gene. Genes Dev., 8, 2602–16.

158


Frasch, M. (1995) Induction of visceral and cardiac mesoderm by ectodermal Dpp in the early Drosophila embryo. Nature, 374, 464–67. Fujisue, M., Kobayakawa, Y. and Yamana, K. (1993) Occurence of dorsal axis-inducing activity around the vegetal pole of an uncleaved Xenopus egg and displacement to the equatorial region by cortical rotation. Development, 118, 163–70. Gallagher, B.C., Hainski, A.M. and Moody, S.A. (1991) Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus. Development, 112, 1103–14. Gawantka, V., Delius, H., Hirschfeld, K., Blumenstock, C. and Niehrs, C. (1995) Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1. Embo J., 14, 6268– 79. Glinka, A., Delius, H., Blumenstock, C. and Niehrs, C. (1996) Combinatorial signalling by Xwnt-11 and Xnr3 in the organizer epithelium. Mech. Dev., 60, 1–11. Godsave, S.F. and Shiurba, R.A. (1992) Xenopus blastulae show regional differences in competence for mesoderm induction: correlation with endogenous basic fibroblast growth factor levels. Dev. Biol., 151, 506–15. Gont, L.K., Steinbeisser, H., Blumberg, B. and De Robertis, E.M. (1993) Tail formation as a continuation of gastrulation: the multiple cell populations of the Xenopus tailbud derive from the late blastopore lip. Development, 119, 991–1004. Gont, L.K., Fainsod, A., Kim, S.H. and De Robertis, E.M. (1996) Overexpression of the homeobox gene Xnot-2 leads to notochord formation in Xenopus. Dev. Biol., 174, 174– 8. Gotoh, Y., Masuyama, N., Suzuki, A., Ueno, N. and Nishida, E. (1995) Involvement of the MAP kinase cascade in Xenopus mesoderm induction. Embo J., 14, 2491–8. Graff, J.M., Thies, R.S., Song, J.J., Celeste, A.J. and Melton, D.A. (1994) Studies with a Xenopus BMP receptor suggest that ventral mesoderm-inducing signals override dorsal signals in vivo. Cell, 79, 169–79. Graff, J.M., Bansal, A. and Melton, D.A. (1996) Xenopus Mad proteins transduce distinct subsets of signals for the TGFb superfamily. Cell, 85, 479–87. Graves, L.M., Northrop, J.L., Potts, B.C., Krebs, E.G. and Kimelman, D. (1994) Fibroblast growth factor, but not activin, is a potent activator of mitogen-activated protein kinase in Xenopus explants. Proc. Natl. I. Acad. Sci. USA, 91, 1662–6. Green, J.B. and Smith, J.C. (1990) Graded changes in dose of a Xenopus activin A homologue elicit stepwise transitions in embryonic cell fate. Nature, 347, 391–4. Green, J.B.A., New, H.V. and Smith, J.C. (1992) Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm. Cell, 71, 731–39. Green, J.B.A., Smith, J.C. and Gerhard, J.C. (1994) Slow emergence of a multithreshold response to activin requires cell-contact-dependent sharpeneing but not prepattern. Development, 120, 2271–78. Grunz, H. (1992) Suramin changes the fate of Spemann’s organizer and prevents neural induction in Xenopus laevis. Mech. Dev., 38, 133–41. Grunz, H. (1994) The four animal blastomeres of the eight-cell stage of Xenopus laevis are intrinsically capable of differentiating into dorsal mesodermal derivatives. Int.J. Dev. Biol., 38, 69–76. Guger, K.A. and Gumbiner, B.M. (1995) beta-Catenin has Wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning. Dev. Biol., 172, 115– 25. Gumbiner, B.M. (1995) Signal transduction of beta-catenin. Curr. Opin. Cell. Biol., 7, 634– 40. Gurdon, J.B., Mohun, T.J., Fairman, S. and Brennan, S. (1985) All components required for the eventual activation of muscle-specific actin genes are localized in the subequatorial region of an uncleaved amphibian egg. Proc. Natl. Acad. Sci. USA, 82, 139–43. Gurdon, J.B., Kao, K., Kato, K. and Hopwood, N.D. (1992) Muscle gene activation in Xenopus requires intercellular communication during gastrula as well as blastula stages. Dev. Suppl., 1992, 137–42. Gurdon, J.B., Kato, K. and Lemaire, P. (1993) The community effect, dorsalization and mesoderm induction. Current Opinion Genet Develop., 3, 662–7. Gurdon, J.B., Harger, P., Mitchell, A. and Lemaire, P. (1994) Activin signalling and response to a morphogen gradient. Nature, 371, 487–92.


159

Gurdon, J.B., Mitchell, A., and Mahony, D. (1995) Direct and continuous assessment by cells of their position in a morphogen gradient. Nature, 376, 520–1. Gururajan, R., Perry, O.K., Melton, D.A. and Weeks, D.L. (1991) The Xenopus localized messenger RNA An3 may encode an ATP-dependent RNA helicase. Nature, 349, 717–9. Hainski, A.M. and Moody, S.A. (1992) Xenopus maternal RNAs from a dorsal animal blastomere induce a secondary axis in host embryos. Development, 116, 347–55. Hamburger, V. (1988) “The heritage of experimental embryology: Hans Spemann and the organizer.” Oxford Univ. Press, New York. Hartley, R.S., Lewellyn, A.L. and Mailer, J.L. (1994) MAP kinase is activated during mesoderm induction in Xenopus laevis. Dev. Biol., 163, 521–4. Hawley, S.H., Wunnenberg, S.K., Hashimoto, C., Laurent, M.N., Watabe, T., Blumberg, B.W. and Cho, K.W. (1995) Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction. Genes Dev., 9, 2923–35. He, X., Saint, J.J., Woodgett, J.R., Varmus, H.E. and Dawid, I.B. (1995) Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature, 374, 617–22. Heasman, J., Wylie, C.C., Hausen, P. and Smith, J.C. (1984) Fates and states of determination of single vegetal pole blastomeres of X. laevis. Cell, 37, 185–94. Heasman, J., Crawford, A., Goldstone, K., Garner, H.P., Gumbiner, B., McCrea, P., Kintner, C., Noro, C.Y. and Wylie, C. (1994) Overexpression of cadherins and underexpression of beta-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell, 79, 791–803. Hemmati-Brivanlou, A., Wright, D.A. and Melton, D.A. (1992) Embryonic expression and functional analysis of a Xenopus activin receptor. Dev. Dyn., 194, 1–11. Hemmati-Brivanlou, A., Kelly, O.G. and Melton, D.A. (1994) Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell, 77, 283–95. Hemmati-Brivanlou, A. and Thomsen, G.H. (1995) Ventral mesodermal patterning in Xenopus embryos: expression patterns and activities of BMP-2 and BMP-4. Dev. Genet., 17, 78–89. Holley, S.A., Jackson, P.D., Sasai, Y., Lu, B., De Robertis, E.M., Hoffmann, M. and Ferguson, E.L. (1995) A conserved system for dorso-ventral patterning in insects and vertebrates involving sog and chordin. Nature, 376, 249–53. Holowacz, T. and Elinson, R.P. (1993) Cortical cytoplasm, which induces dorsal axis formation in Xenopus, is inactivated by UV irradiation of the oocyte. Development, 119, 277–85. Holowacz, T. and Elinson, R.P. (1995) Properties of the dorsal activity found in the vegetal cortical cytoplasm of Xenopus eggs. Development, 121, 2789–98. Hoppler, S., Brown, J.D. and Moon, R.T. (1996) Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos. Genes and Development, 10, 2805–17. Hopwood, N.D., Pluck, A. and Gurdon, J.B. (1991) Xenopus Myf-5 marks early muscle cells and can activate muscle genes ectopically in early embryos. Development, 111, 551–60. Houliston, E. and Elinson, R.P. (1991) Patterns of microtubule polymerization relating to cortical rotation in Xenopus laevis eggs. Development, 112, 107–17. Houliston, E. (1994) Microtubule translocation and polymerisation during cortical rotation in Xenopus eggs. Development, 120, 1213–20. Houliston, E., Le, G.R., Kress, M., Philippe, M. and Le, G.K. (1994) The kinesin-related protein Eg5 associates with both interphase and spindle microtubules during Xenopus early development. Dev. Biol., 164, 147–59. Howard, J.E. and Smith, J.C. (1993) Analysis of gastrulation: different types of gastrulation movement are induced by different mesoderm-inducing factors in Xenopus laevis. Mech. Dev., 43, 37–48. Huber, O., Korn, R., Mclaughlin, J., Ohsugi, M., Herrmann, B.G. and Kemler, R. (1996) Nuclear localization of p-catenin by interaction with transcription factor LEF-1. Mech. Dev., 59, 3–10. Isaacs, H.V., Tannahill, D. and Slack, J.M. (1992) Expression of a novel FGF in the Xenopus embryo. A new candidate inducing factor for mesoderm formation and anteroposterior specification. Development, 114, 711–20.

160


Isaacs, H.V., Pownall, M.E. and Slack, J.M. (1994) eFGF regulates Xbra expression during Xenopus gastrulation. Embo J., 13, 4469–81. Ishikawa, T., Yoshioka, H., Ohuchi, H., Noji, S. and Nohno, T. (1995) Truncated type II receptor for BMP-4 induces secondary axial structures in Xenopus embryos. Biochem. Biophys. Res. Commun., 216, 26–33. Jones, C.M., Lyons, K.M., Lapan, P.M., Wright, C.V. and Hogan, B.L. (1992) DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing factor in Xenopus mesoderm induction. Development, 115, 639–47. Jones, C.M., Kuehn, M.R., Hogan, B.L., Smith, J.C. and Wright, C.V. (1995) Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation. Development, 121, 3651–62. Jones, C.M. (1996) Signalling by TGF-β family members: short-range effects of Xnr-2 and BMP-4 contrast with the long-range effects of activin. Current Biology, 6, 1468–75. Jones, C.M., Dale, L., Hogan, B.L., Wright, C.V. and Smith, J.C. (1996) Bone morphogenetic protein-4 (BMP-4) acts during gastrula stages to cause ventralization of Xenopus embryos. Development, 122, 1545–54. Kageura, H. and Yamana, K. (1983) Pattern regulation in isolated halves and blastomeres ofearly Xenopus laevis. J. Embryol. Exp. Morphol., 74, 221–34. Karnovsky, A. and Klymkowsky, M.W. (1995) Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin. Proc. Natl. Acad. Sci. USA, 92, 4522–26. Kaufmann, E., Paul, H., Friedle, H., Metz, A., Scheucher, M., Clement, J.H. and Knöchel, W. (1996) Antagonistic actions of activin A and BMP-2/4 control dorsal lip-specific activation of the early response gene XFD-1' in Xenopus laevis embryos. EMBO J., 15, 6739–9 Keller, R. (1991) Early embryonic development of Xenopus laevis. Methods Cell Biol., 36, 61– 113. Keller, R. and Winklbauer, R. (1992) Cellular basis of amphibian gastrulation. Curr. Top. Dev. Biol., 27, 39–89. Kelley, C., Yee, K., Harland, R. and Zon, L.I. (1994) Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. Dev. Biol., 165, 193–205. Kessler, D.S. and Melton, D.A. (1994) Vertebrate embryonic induction: mesodermal and neural patterning. Science, 266, 596–604. Kessler, D.S. and Melton, D.A. (1995) Induction of dorsal mesoderm by soluble, mature Vg1 protein. Development, 121, 2155–64. Kimelman, D. and Maas, A. (1992) Induction of dorsal and ventral mesoderm by ectopically expressed Xenopus basic fibroblast growth factor. Development, 114, 261–9. Kinoshita, K., Bessho, T. and Asashima, M. (1993) Competence prepattern in the animal hemisphere of the 8-cell-stage Xenopus embryo. Dev. Biol., 160, 276–84. Kinoshita, N., Minshull, J. and Kirschner, M.W. (1995) The identification of two novel ligands of the FGF receptor by a yeast screening method and their activity in Xenopus development. Cell, 83, 621–30. Klein, P. and Melton, D.A. (1996) A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA, 93, 8455–59. Kloc, M., Spohr, G. and Etkin, L.D. (1993) Translocation of repetitive RNA sequences with the germ plasm in Xenopus oocytes. Science, 262, 1712–4. Knöchel, S., Lef, J., Clement, J., Klocke, B., Hille, S., Koster, M. and Knöchel, W. (1992) Activin A induced expression of a fork head related gene in posterior chordamesoderm (notochord) of Xenopus laevis embryos. Mech. Dev., 38, 157–65. Kondo, M., Tashiro, K., Fujii, G., Asano, M., Miyoshi, R., Yamada, R., Muramatsu, M. and Shiokawa, K. (1991) Activin receptor mRNA is expressed early in Xenopus embryogenesis and the level of the expression affects the body axis formation. Biochem. Biophys. Res. Commun., 181, 684–90. Koster, M., Plessow, S., Clement, J.H., Lorenz, A., Tiedemann, H. and Knöchel, W. (1991) Bone morphogenetic protein 4 (BMP-4), a member of the TGF-beta family, in early embryos of Xenopus laevis: analysis of mesoderm inducing activity. Mech. Dev., 33, 191–9. Krumlauf, R. (1994) Hox genes in vertebrate development. Cell, 78, 191–201. Ku, M. and Melton, D.A. (1993) Xwnt-11: a maternally expressed Xenopus wnt gene. Development, 119, 1161–73.


161

LaBonne, C. and Whitman, M. (1994) Mesoderm induction by activin requires FGF-mediated intracellular signals. Development, 120, 463–72. LaBonne, C., Burke, B. and Whitman, M. (1995) Role of MAP kinase in mesoderm induction and axial patterning during Xenopus development. Development, 121, 1475–86. Ladher, R., Mohun, T.J., Smith, J.C. and Snape, A.M. (1996) Xom: a Xenopus homeobox gene that mediates the early effects of BMP-4. Development, 122, 2385–94. Lagna, G., Hata, A., Hemmati-Brivanlou, A., Massague, J. (1996) Partnership between DPC4 and SMAD proteins in TGF-β signalling pathways. Nature, 383, 832–6 Lamb, T.M., Knecht, A.K., Smith, W.C., Stachel, S.E., Economides, A.N., Stahl, N., Yancopolous, G.D. and Harland, R.M. (1993) Neural induction by the secreted polypeptide noggin. Science, 262, 713–8. Larabell, C.A., Rowning, B.A., Wells, J., Wu, M. and Gerhard, J.C. (1996) Confocal microscopy analysis of living Xenopus eggs and the mechanism of cortical rotation. Development, 122, 1281–89. Lemaire, P. and Gurdon, J.B. (1994) A role for cytoplasmic determinants in mesoderm patterning: cell-autonomous activation of the goosecoid and Xwnt-8 genes along the dorsoventral axis of early Xenopus embryos. Development, 120, 1191–9. Lemaire, P., Garrett, N. and Gurdon, J.B. (1995) Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell, 81, 85–94. Linnen, J.M., Bailey, C.P. and Weeks, D.L. (1993) Two related localized mRNAs from Xenopus laevis encode ubiquitin-like fusion proteins. Gene, 128, 181–8. Lustig, K.D., Kroll, K., Sun, E., Ramos, R., Elmendorf, H. and Kirschner, M.W. (1996) A xenopus nodal-related gene that acts in synergy with noggin to induce complete secondary axis and notochord formation. Development, 122, 3275–82. MacNicol, A.M., Muslin, A.J. and Williams, L.T. (1993) Raf-1 kinase is essential for early Xenopus development and mediates the induction of mesoderm by FGF. Cell, 73, 571– 83. MacNicol, A.M., Muslin, A.J., Howard, E.L., Kikuchi, A., MacNicol, M.C. and Williams, L.T. (1995) Regulation of Raf-1-dependent signaling during early Xenopus development. Mol. Cell Biol., 15, 6686–93. Maeno, M., Ong, R.C., Suzuki, A., Ueno, N. and Kung, H.F. (1994) A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: roles of animal pole tissue in the development of ventral mesoderm. Proc, Natl. Acad. Set. USA, 91, 10260–4. Mahony, D. and Gurdon, J.B. (1995) A type 1 serine/threonine kinase receptor that can dorsalize mesoderm in Xenopus. Proc. Natl. Acad. Sci. USA, 92, 6474–8. Massague, J. (1996) TGF-beta signalling: Receptors, Transducers, and Mad proteins. Cell, 85, 947–50. Mathews, L.S., Vale, W.W. and Kintner, C.R. (1992) Cloning of a second type of activin receptor and functional characterization in Xenopus embryos. Science, 255, 1702–5. Matsuo, I., Kuratani, S., Kimura, C., Takeda, N. and Aizawa, S. (1995) Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev., 9, 2646–58. Matzuk, M.M., Kumar, T.R., Vassalli, A., Bickenbach, J.R., Roop, D.R., Jaenisch, R. and Bradley, A. (1995) Functional analysis of activins during mammalian development. Nature, 374, 354–56. Mead, P.E., Brivanlou, I.H., Kelley, C.M. and Zon, L.I. (1996) BMP-4-responsive regulation of dorsal-ventral patterning by the homeobox protein Mix.1. Nature, 382, 357–60. Meinhardt, H. (1994) Biological pattern formation: new observations provide support for theoretical predictions. Bioessays, 16, 627–32. Melton, D.A. (1987) Translocation of a localized maternal mRNA to the vegetal pole of Xenopus oocytes. Nature, 328, 80–2. Miller, J. and Moon, R.T. (1996) Signal transduction through beta-catenin and specification of cell fate during embryogenesis. Genes Develop., 10, 2527–39. Minoura, I., Nakamura, H., Tashiro, K. and Shiokawa, K. (1995) Stimulation of circus movement by activin, βFGF and TGF-β 2 in isolated animal cap cells of Xenopus laevis. Mech. Dev., 49, 65–9.

162


Molenaar, M., Wetering, M.V.D., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destree, O. and Clevers, H. (1996) Xtcf-3 transcription factor mediates b-catenin-induced axis formation in xenopus embryos. Cell, 86, 391–399. Moon, R.T., Campbell, R.M., Christian, J.L., McGrew, L.L., Shih, J. and Eraser, S. (1993a) Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development, 119, 97–111. Moon, R.T., DeMarais, A. and Olson, D.J. (1993b) Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement. J. Cell Sci. Suppl., 17, 183–8. Moos, M.J., Wang, S. and Krinks, M. (1995) Anti-dorsalizing morphogenetic protein is a novel TGF-beta homolog expressed in the Spemann organizer. Development, 121, 4293–301. Mosquera, L., Forristall, C., Zhou, Y. and King, M.L. (1993) A mRNA localized to the vegetal cortex of Xenopus oocytes encodes a protein with a nanos-like zinc finger domain. Development, 117, 377–86. Nakamura, O., Takasaki, H. and Mizohata, T. (1970) Differentiation during cleavage in Xenopus laevis. I. Aquisition of self-differentiation capacity of the dorsal marginal zone. Proc. Jpn. Acad., 46, 694–99 Niehrs, C. and De Robertis, E.M. (1991) Ectopic expression of a homeobox gene changes cell fate in Xenopus embryos in a position-specific manner. Embo J., 10, 3621–9. Niehrs, C., Keller, R., Cho, K.W. and De Robertis, E.M. (1993) The homeobox gene goosecoid controls cell migration in Xenopus embryos. Cell, 72, 491–503. Niehrs, C., Steinbeisser, H. and De Robertis, E.M. (1994) Mesodermal patterning by a gradient of the vertebrate homeobox gene goosecoid. Science, 263, 817–20. Niehrs, C. (1996) Mad connection to the nucleus. Nature, 381, 561–62. Nieuwkoop, P.D. (1969) The formation of mesoderm in Urodelean Amphibians. I Induction by the Endoderm. Roux. Arch., 162, 341–73. Nieuwkoop, P.D. (1973) The organisation centre of the amphibian embryo, its origin, spatial organisation and morphogenetic action. Adv. Morph., 10, 1–39. Nishimatsu, S., Suzuki, A., Shoda, A., Murakami, K. and Ueno, N. (1992) Genes for bone morphogenetic proteins are differentially transcribed in early amphibian embryos. Biochem. Biophys. Res. Commun., 186, 1487–95. Nishimatsu, S., Takebayashi, K., Suzuki, A., Murakami, K. and Ueno, N. (1993) Immunodetection of Xenopus bone morphogenetic protein-4 in early embryos. Growth Factors, 8, 173–6. Northrop, J.L. and Kimelman, D. (1994) Dorsal-ventral differences in Xcad-3 expression in response to FGF-mediated induction in Xenopus. Dev. Biol., 161, 490–503. Northrop, J., Woods, A., Seger, R., Suzuki, A., Ueno, N., Krebs, E. and Kimelman, D. (1995) BMP-4 regulates the dorsal-ventral differences in FGF/MAPKK-mediated mesoderm induction in Xenopus. Dev. Biol., 172, 242–52. Onichtchouk, D., Gawantka, V., Dosch, R., Delius, H., Hirschfeld, K., Blumenstock, C. and Niehrs, C. (1996) The Xvent-2 homeobox gene is part of the BMP-4 signaling pathway controling dorsoventral patterning of Xenopus mesoderm. Development, 122, 3045–53. Oschwald, R., Clement, J.H., Knöchel, W. and Grunz, H. (1993) Suramin prevents transcription of dorsal marker genes in Xenopus laevis embryos, isolated dorsal blastopore lips and activin A induced animal caps. Mech. Dev., 43, 121–33. Pannese, M., Polo, C., Andreazzoli, M., Vignali, R., Kablar, B., Barsacchi, G. and Boncinelli, E. (1995) The Xenopus homologue of Otx2 is a maternal homeobox gene that demarcates and specifies anterior body regions. Development, 121, 707–20. Papalopupu, N. and Kintner, C. (1994) Molecular genetics of neurulation. Ciba Found. Symp., 181, 90–99. Papalopulu, N. and Kintner, C. (1996) A Xenopus gene, Xbr-1, defines a novel class of homeobox genes and is expressed in the dorsal ciliary margin of the eye. Developmental Biol., 174, 104–14. Pevny, L., Simon, M.C., Robertson, E., Klein, W.H., Tsai, S.F., D’Agati, V., Orkin, S.H. and Costantini, F. (1991) Erythroid differentiation in chimaeric mice blocked by a targeted mutation in the gene for transcription factor GATA-1. Nature, 349, 257–60.


163

Piccolo, S., Sasai, Y., Lu, B. and De Robertis, E.M. (1996) Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4. Cell, 86, 589–98. Pierce, S.B. and Kimelman, D. (1995) Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3. Development, 121, 755–65. Rebagliati, M.R., Weeks, D.L., Harvey, R.P. and Melton, D.A. (1985) Identification and cloning of localized maternal RNAs from Xenopus eggs. Cell, 42, 769–71. Rebagliati, M.R. and Dawid, I.B. (1993) Expression of activin transcripts in follicle cells and oocytes of Xenopus laevis. Dev. Biol., 159, 574–80. Reem-Kalma, Y., Lamb, T. and Frank, D. (1995) Competition between noggin and bone morphogenetic protein 4 activities may regulate dorsalization during Xenopus development. Proc. Natl. Acad. Sci. USA, 92, 12141–5. Reilly, K.M. and Melton, D.A. (1996) Short-range signaling by candidate morphogens of the TGF-β family and evidence for a relay mechanism of induction. Cell, 86, 743–54. Rivera, P.J., Mallo, M., Gendron, M.M., Gridley, T. and Behringer, R.R. (1995) Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development. Development, 121, 3005–12. Rosa, F.M. (1989) Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos. Cell, 57, 965–74. Rudnicki, M.A., Schnegelsberg, P.N., Stead, R.H., Braun, T., Arnold, H.H. and Jaenisch, R. (1993) MyoD or Myf-5 is required for the formation of skeletal muscle. Cell, 75, 1351–9. Ruiz i Altaba, A. and Melton, D.A. (1989a) Bimodal and graded expression of the Xenopus homeobox gene Xhox3 during embryonic development. Development, 106, 173–83. Ruiz i Altaba, A. and Melton, D.A. (1989b) Involvement of the Xenopus homeobox gene Xhox-3 in pattern formation along the anterior-posterior axis. Cell, 57, 317–26 Ruiz i Altaba, A. and Melton, D.A. (1989c) Interaction between peptide growth factors and homeobox genes in the establishment of anterior-posterior polarity in frog embryos. Nature, 341, 33–8 Ruiz i.Altaba, A. and Jessell, T.M. (1992) Pintallavis, a gene expressed in the organizer and midline cells of frog embryos: involvement in the development of the neural axis. Development, 116, 81–93. Sakai, M. (1996) The vegetal determinants required for the Spemann organizer move equatorially during the first cell cycle. Development, 122, 2207–14. Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L.K. and De Robertis, E.M. (1994) Xenopus chordin: a novel dorsalizing factor activated by organizer-specific homeobox genes. Cell, 79, 779–90. Sasai, Y., Lu, B., Steinbeisser, H. and De Robertis, E.M. (1995) Regulation of neural induction by the Chd and Bmp-4 antagonistic patterning signals in Xenopus. Nature, 376, 333–6. Sasai, Y., Lu, B., Piccolo, S. and De Robertis, E.M. (1996) Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps. EMBO J., 15, 4547–55. Sato, S.M. and Sargent, T.D. (1991) Localized and inducible expression of Xenopus-posterior (Xpo), a novel gene active in early frog embryos, encoding a protein with a ‘CCHC’ finger domain. Development, 112, 747–53. Schmidt, J., Francois, V., Bier, E. and Kimelman, D. (1995) Drosophila short gastrulation induces an ectopic axis in Xenopus: evidence for conserved mechanisms of dorsal-ventral patterning. Development, 121, 4319–28. Schmidt, J.E., Suzuki, A., Ueno, N. and Kimelman, D. (1995) Localized BMP-4 mediates dorsal/ventral patterning in the early Xenopus embryo. Dev. Biol., 169, 37–50. Schmidt, J.E., vonDassow, G. and Kimelman, D. (1996) Regulation of dorsal-ventral patterning: the ventralizing effects of the novel Xenopus homeobox gene Vox. Development, 122,1711– 21. Schneider, S., Steinbeisser, H., Warga, R.M. and Hausen, P. (1996) beta catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mechanisms Develop., 57, 191–8. Schulte-Merker, S., Smith, J.C. and Dale, L. (1994) Effects of truncated activin and FGF receptors and of follistatin on the inducing activities of BVg1 and activin: does activin play a role in mesoderm induction? EMBO J., 15, 3533–41.

164


Schulte-Merker, S. and Smith, J.C. (1995) Mesoderm formation in response to Brachyury requires FGF signalling. Curr. Biol., 5, 62–7. Shawlot, W. and Behringer, R.R. (1995) Requirement for Lim1 in head-organizer function. Nature, 374, 425–30. Shih, J. and Keller, R. (1992) The epithelium of the dorsal marginal zone of Xenopus has organizer properties. Development, 116, 887–99. Sive, H.L. (1993) The frog prince-ss: a molecular formula for dorsoventral patterning in Xenopus. Genes Dev., 7, 1–12. Slack, J.M. and Tannahill, D. (1992) Mechanism of anterioposterior axis specification in vertebrates. Development, 114, 285–302. Slack, J.M. (1994) Inducing factors in Xenopus early embryos. Curr. Biol., 4, 116–26. Smith, J.C. and Slack, J.M. (1983) Dorsalization and neural induction: properties of the organizer in Xenopus laevis. J. Embryol. Exp. Morphol., 78, 299–317. Smith, J.C., Price, B.M., Green, J.B., Weigel, D. and Herrmann, B.G. (1991) Expression of a Xenopus homolog of Brachyury (T) is an immediate-early response to mesoderm induction. Cell, 67, 79–87. Smith, J.C. and Howard, J.E. (1992) Mesoderm-inducing factors and the control of gastmlation. Dev. Suppl., 1992, 127–36. Smith, J.C., Cunliffe, V., Green, J.B. and New, H.V. (1993) Intercellular signalling in mesoderm formation during amphibian development. Pkilos. Trans. R. Soc. Lond. B Biol. Sci., 340, 287–96. Smith, J.C. (1995) Mesoderm-inducing factors and mesodermal patterning. Curr. Opin. Cell Biol., 7, 856–61. Smith, W.C. and Harland, R.M. (1991) Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center. Cell, 67, 753–65. Smith, W.C. and Harland, R.M. (1992) Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos. Cell, 70, 829–40. Smith, W.C., Knecht, A.K., Wu, M. and Harland, R.M. (1993) Secreted noggin protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm. Nature, 361, 547–9. Smith, W.C., McKendry, R., Ribisi, S.J. and Harland, R.M. (1995) A nodal-related gene defines a physical and functional domain within the Spemann organizer. Cell, 82, 37–46. Sokol, S., Christian, J.L., Moon, R.T. and Melton, D.A. (1991) Injected Wnt RNA induces a complete body axis in Xenopus embryos. Cell, 67, 741–52. Sokol, S. and Melton, D.A. (1991) Pre-existent pattern in Xenopus animal pole cells revealed by induction with activin. Nature, 351, 409–11. Sokol, S.Y. (1993) Mesoderm formation in Xenopus ectodermal explants overexpressing Xwnt8: evidence for a cooperating signal reaching the animal pole by gastmlation. Development, 118, 1335–42. Sokol, S.Y., Klingensmith, J., Perrimon, N. and Itoh, K. (1995) Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled. Development, 121, 1637–47. Spemann, H. and Mangold, H. (1924) Über Induktion von Embryonalanlagen durch Implantation artfremder Organisatoren. Arch. Mikrosk. Anat. Entweicklungsmechan., 100, 599– 638. Staehling-Hampton, K., Hoffmann, F.M., Baylies, M.K., Rushton, E. and Bate, M. (1994) dpp induces mesodermal gene expression in Drosophila. Nature, 372, 783–6. Steinbeisser, H., Fainsod, A., Niehrs, C., Sasai, Y. and De Robertis, E.M. (1995) The role of gsc and BMP-4 in dorsalventral patterning of the marginal zone in Xenopus: a loss-of-function study using antisense RNA. Embo J., 14, 5230–43. Suzuki, A., Thies, R.S., Yamaji, N., Song, J.J., Wozney, J.M., Murakami, K. and Ueno, N. (1994) A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo. Proc. Natl. Acad. Sci. USA, 91, 10255–9. Symes, K., Yaqoob, M. and Smith, J.C. (1988) Mesoderm induction in Xenopus laevis: responding cells must be in contact for mesoderm formation but suppression of epidermal differentiation can occur in single cells. Development, 104, 609–18. Symes, K., Yordan, C. and Mercola, M. (1994) Morphological differences in Xenopus embryonic mesodermal cells are specified as an early response to distinct threshold concentrations of activin. Development, 120, 2339–46.


165

Symes, K. and Mercola, M. (1996) Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA, 93, 9641–4. Taira, M., Jamrich, M., Good, P.J. and Dawid, I.B. (1992) The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos. Genes Dev., 6, 356–66. Taira, M., Otani, H., Jamrich, M. and Dawid, I.B. (1994) Expression of the LIM class homeobox gene Xlim-1 in pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic acid and exogastrulation. Development, 120, 1525–36. Taira, M., Otani, H., Saint, J.J. and Dawid, I.B. (1994) Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus. Nature, 372, 677–9. Talbot, W.S., Trevarrow, B., Halpern, M.E., Melby, A.E., Farr, G., Postlethwait, J.H., Jowett, T., Kimmel, C.B. and Kimelman, D. (1995) A homeobox gene essential for zebrafish notochord development. Nature, 378, 150–7. Tang, T.L., Freeman, R.J., O’Reilly, A.M., Neel, B.C. and Sokol, S.Y. (1995) The SH2– containing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development. Cell, 80, 473–83. Tannahill, D., Isaacs, H.V., Close, M.J., Peters, G. and Slack, J.M. (1992) Developmental expression of the Xenopus int-2 (FGF-3) gene: activation by mesodermal and neural induction. Development, 115, 695–702. Thompson, J. and Slack, J.M. (1992) Over-expression of fibroblast growth factors in Xenopus embryos. Mech. Dev., 38, 175–82. Thomsen, G.H. and Melton, D.A. (1993) Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell, 74, 433–41. Thomsen, G.H. (1996) Xenopus mothers against decapentaplegic is an embryonic ventralizing agent that acts downstream of the BMP-2/4 receptor. Development, 122, 2359–2366. Tidman-Ault, C., Dirksen, M.L. and Jamrich, M. (1996) A novel homeobox gene PV.1 mediates induction of ventral mesoderm in Xenopus embryos. Proc. Natl. Acad. Sci. USA, 93, 6415–20. Tiedemann, H. (1993) Mesoderm differentiation in early amphibian embryos depends on the animal cap. Roux. Arch. Develop. Biol., 203, 28–33. Tiedemann, H., Tiedemann, H., Grunz, H. and Knöchel, W. (1995) Molecular Mechanisms of tissue determination and pattern formation in amphibian embryos. Naturwissenschaften, 82, 123–34. Torres, M.A., Yang, S.J., Purcell, S.M., DeMarais, A.A., McGrew, L.L. and Moon, R.T. (1996) Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5 A class and by a dominant negative cadherin in early Xenopus development. J. Cell Biol., 133, 1123–37. Tsai, F.Y., Keller, G., Kuo, F.C., Weiss, M., Chen, J., Rosenblatt, M., Alt, F.W. and Orkin, S.H. (1994) An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature, 371, 221–6. Umbhauer, M., Marshall, C.J., Mason, C.S., Old, R.W. and Smith, J.C. (1995) Mesoderm induction in Xenopus caused by activation of MAP kinase. Nature, 376, 58–62. Vodicka, M.A. and Gerhart, J.C. (1995) Blastomere derivation and domains of gene expression in the Spemann Organizer of Xenopus laevis. Development, 121, 3205–3518. von Dassow, G., Schmidt, J.E. and Kimelman, D. (1993) Induction of the Xenopus organizer: expression and regulation of Xnot, a novel FGF and activin-regulated homeo box gene. Genes Dev., 7, 355–66. Walmsley, M.E., Guille, M.J., Bertwistle, D., Smith, J.C., Pizzey, J.A. and Patient, R.K. (1994) Negative control of Xenopus GATA-2 by activin and noggin with eventual expression in precursors of the ventral blood islands. Development, 120, 2519–29. Watabe, T., Kim, S., Candia, A., Rothbacher, U., Hashimoto, C., Inoue, K. and Cho, K.W. (1995) Molecular mechanisms of Spemann’s s organizer formation: conserved growth factor synergy between Xenopus and mouse. Genes Dev., 9, 3038–50. Weber, H., Holewa, B., Jones, E.A. and Ryffel, G.U. (1996) Mesoderm and endoderm differentiation in animal cap explants: identification of the HNF4-binding site as an activin A responsive element in the Xenopus HNF1 alpha promoter. Development, 122, 1975–84.

166


Weinstein, D.C., Ruiz i Altaba, A., Chen, W.S., Hoodless, P., Prezioso, V.R., Jessell, T.M. and Darnell, J.J. (1994) The winged-helix transcription factor HNF-3 beta is required for notochord development in the mouse embryo. Cell, 78, 575–88. Wharton, K.A., Ray, R.P. and Gelbart, W.M. (1993) An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo. Development, 117, 807–22. Whitman, M. and Melton, D.A. (1992) Involvement of p21ras in Xenopus mesoderm induction. Nature, 357, 252–4. Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M, Joseph, E. and Heasman, J. (1996) Maternal β-catenin establishes a dorsal signal in early Xenopus embryos. Development, 122, 2987–2996. Wilson, P.A. and Melton, D.A. (1994) Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Curr. Biol., 4, 676–86. Wilson, P.A. and Hemmati-Brivanlou,.A. (1995) Induction of epidermis and inhibition of neural fate by Bmp-4. Nature, 376, 331–3. Winklbauer, R., Selchow, A., Nagel, M. and Angres, B. (1992) Cell interaction and its role in mesoderm cell migration during Xenopus gastrulation. Dev. Dyn., 195, 290–302. Wolda, S.L., Moody, C.J. and Moon, R.T. (1993) Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos. Dev. Biol., 155, 46–57. Wright, C.V., Morita, E.A., Wilkin, D.J. and De Robertis, E.M. (1990) The Xenopus XIHbox 6 homeo protein, a marker of posterior neural induction, is expressed in proliferating neurons. Development, 109, 225–34. Wu, J.Y., Wen, L., Zhang, W.J. and Rao, Y. (1996) The secreted product of Xenopus gene lunatic Fringe, a vertebrate signaling molecule. Science, 273, 355–8. Xu, R.H., Dong, Z., Maeno, M., Kim, J., Suzuki, A., Ueno, N., Sredni, D., Colburn, N.H. and Kung, H.F. (1996) Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development. Proc. Natl. Acad. Sci. USA, 93, 834–8. Yamada, T. (1950) Dorsalization of the ventral marginal zone of the Triturus gastrula. I. Biol. Bull., 98, 98–121. Yamada, G., Mansouri, A., Torres, M., Stuart, E.T., Blum, M., Schultz, M., De Robertis, E.M. and Gruss, P. (1995) Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death. Development, 121, 2917–22. Yamashita, H., Ten Dijke, P., Huylebroeck, D., Sampath, T.K., Andries, M., Smith, J.C., Heldin, C.H. and Miyazono, K. (1995) Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J. Cell Biol., 130, 217–26. Yuge, M., Kobayakawa, Y., Fujisue, M. and Yamana, K. (1990) A cytoplasmic determinant for dorsal axis formation in an early embryo of Xenopus laevis. Development, 110, 1051– 6. Zaraisky, A.G., Lukyanov, S.A., Vasiliev, O.L., Smirnov, Y.V., Belyavsky, A.V. and Kazanskaya, O.V. (1992) A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo. Dev. Biol., 152, 373–82. Zaraisky, A.G., Ecochard, V., Kazanskaya, O.V., Lukyanov, S.A., Fesenko, I.V. and Duprat, A.M. (1995) The homeobox-containing gene XANF-1 may control development of the Spemann organizer. Development, 121, 3839–47. Zhang, C. and Evans, T. (1996) BMP-like signals are required after the midblastula transition for blood cell development. Dev. Genet., 18, 267–78. Zhou, X., Sasaki, H., Lowe, L., Hogan, B.L.M. and Kuehn, M.R. (1993) Nodal is a novel TGF-b-like gene expressed in the mouse node during gastrulation. Nature, 361, 543–7. Zimmerman, L.B., De Jesús-Escobar, J.-E. and Harland, R.M. (1996) The Spemann Organizer Signal Noggin Binds and Inactivates Bone Morphogenetic Protein-4. Cell, 86, 599–606.

9. CELL ADHESION AND SIGNALLING IN EARLY XENOPUS DEVELOPMENT DORIS WEDLICH and MICHAEL KÜHL Abteilung Biochemie, Universität Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany

The Xenopus embryo is the organism par excellence in demonstrating how signalling of diffusible inducing factors is coupled to cell-cell adhesion in the process of pattern formation. This becomes most obvious in the double function of β-catenin as stabilizer of the cadherin/catenin adhesion complex and as transducer of the Xwnt signal into the nucleus. Despite this example of a direct molecular link between signalling and cell adhesion more indirect evidence is given considering the activity of mesoderm inducers like activin, TGFβ, bFGF or BMP. It seems, that the two subjects cell-sorting and tissue induction initially studied in amphibians by Townes and Holtfreter (1955) and Spemann and Mangold (1924) are coming together after at least 40 years. Beyond the current most exciting cadherin/Xwnt signalling connection we also try to include in this article important observations that give us an idea how cell-cell adhesion goes along with mesoderm induction in general. As outlined in Figure 1 we will touch the themes (1) competition between cell adhesion and signal pathways, (2) modulation of cell adhesion by signal molecules and (3) cell adhesion as prerequisite for cell response to inducing factors. CADHERINS AND Wnt SIGNALLING β-Catenin, the homologue to armadillo in Drosophila, was initially identified as cytosolic protein anchoring cadherins via α-catenin to the cytoskeleton (Kemler, 1993; Takeichi, 1995). Recently, a novel function of pcatenin as nuclear protein was deciphered: its capacity to induce a secondary axis (Funayama et al., 1995). The requirement of β-catenin in the cadherin/catenin adhesion complex was proven by expression of cadherin mutants lacking the β-catenin binding site in murine fibroblasts. Cadherin mediated cell adhesion was abolished (McNeill et al., 1990; Nagafuchi and Takeichi, 1988). In the Xenopus embryo, comparable experiments were performed injecting in vitro transcribed mRNAs coding for E-, XB/ U-, or EP/C-cadherin mutants deficient for β-catenin binding. Dominant negative expression of all these mutants results in localized adhesion defects depending on the specific spatial expression pattern of the analyzed cadherin (Kühl and Wedlich, 1996). The phenotypes of these embryos appear quite normal despite a shortening of the anterior-posterior axis, reduced head structures and enlarged proctodeums (Figure 2A, B). Mesodermal and neural differentiation was almost unaffected. These phenotypes arise whenever convergent extension movements (Keller, 1986) during gastrulation are impaired. In contrast to cytoplasmic cadherin deletion mutants, overexpression of extracellular truncated forms at the dorsal side of Xenopus embryos launch the molecular linkage between cadherin mediated cell adhesion and dorsoventral axis formation. In these embryos adhesion defects are set out because the cadherin mutant is unable to bind the homophilic partner molecule while its cytoplasmic domain competes with the wildtype form for β-catenin binding. Furthermore, the supply of additional β-catenin binding sites depletes the

168


Figure 1 Schematic summary of the topics handled in this review. These include in particular the signal transduction pathway initiated by class I Xwnt molecules and its interferences with the cadherin cell-cell adhesion system.

cytoplasm and the nucleus from β-catenin that is normally required for axis induction. This leads to embryos lacking axial structures in addition to posteriorized phenotypes caused by adhesion defects (Figure 2C, Figure 3 and Holt et al., 1994; Fagotto et al., 1996). A similar phenotype was described when βcatenin mRNA was destroyed by an oligonucleotide knock-out approach (Heasman et al., 1994). Both phenotypes, posteriorized embryos as well as loss of axial structures caused by reduced cell adhesion and/or depletion of cytosolic β-catenin, can be correlated to different classes of the Xwnt family (Moon, 1993). In Xenopus, we discern two functional classes of Wnt proteins: members of class I (Xwnt 8, Xwnt 8b, Xwnt 3A, Xwnt 1) are able to induce a secondary axis when expressed on the ventral side of the embryo while those of class II (Xwnt 5 A, Xwnt 4, Xwnt 11) seem to modulate cell-adhesion (Du et al., 1995; Torres et al., 1996). THE FUNCTION OF β-CATENIN IN THE CLASS I Xwnt SIGNAL PATHWAY Although the function of β-catenin in the cadherin adhesion complex was intensively studied during the last couple of years its localization in the nuclei after overexpression in ventral blastomeres and the formation of a secondary axis in Xenopus embryos (Funayama et al., 1995) set a new landmark in our view of mesoderm induction. Remarkably, the nuclear localization of β-catenin has also been reported for the endogenous molecule (Schneider et al., 1996). During blastula stage, β-catenin is transiently found in the nuclei of dorsal marginal cells in the Xenopus and the zebraflsh embryo. Genetic analyses in Drosophila facilitates to discover the cytosolic/nuclear function of β-catenin. The identification of armadillo as member of the wingless (wg) signalling cascade (Peifer et al., 1991; Nordermeer et al., 1994) and its striking homology to β-catenin (McCrea et al., 1991; Butz et al., 1992)


169

Figure 2 Effect of overexpressing cadherin mutants on the dorsal side of the Xenopus embryo (A,C) or β-catenin on the ventral side of the embryo (D). (A) A posteriorized phenoptype is obtained after injection of either an extracellular or an intracellular deleted mutant of XB/ U-cadherin into the two dorsal blastomeres of a 4 cell stage Xenopus embryo. The anterior part of the embryo is reduced or even missing. (C) The embryos shown here are completely ventralized and lack dorsal structures. This phenotype is only observed after injection of the extracellular deletion mutant of XB/Ucadherin. (D) Injection of human β-catenin into the ventral cells of a 4 cell stage embryo leads to the formation of secondary body axis. Both, (C) and (D) emphazise the importance of β-catenin for dorsoventral patterning. An uninjected control embryo is shown in (B).

resulted in an outburst of experiments confirming the evolutionary conservation of this signal pathway from worms to higher vertebrates. Drosophila wg but also the vertebrate homologues, the Wnt gene-family, encode glycosilated secreted proteins with conserved cysteine residues. In vertebrates, this family consists, at the moment, of 15 different molecules and this might not be the end of the line. The direct molecular link between β-catenin and Wnt signalling with respect to class I members was exemplary demonstrated in Xenopus using Xwnt 8 probes. Moreover, all so far known members of the wingless cascade in Drosophila have been proved to function similarily in the Xwnt 8 signal pathway (Figure 4 and Table 1). Most recently, the missing links at both ends of this rainbow have been discovered using the advantages of both experimental systems. The wg receptor frizzled (Dfz2) was identified in Drosophila (Bhanot et al., 1996). The functional

170


Figure 3 β-catenin is involved in both, cell adhesion and Wnt signalling. A known down-stream partner of β-catenin in Wnt signalling is given by LEF-1. Thus, LEF-1 and cadherins compete for β-catenin binding. Table 1 Summary of Xenopus phenotypes arisen by injection of molecules involved in Wnt signalling. Gene

ventral injection

dorsal injection

References

Class I Xwnt

–axis duplication –dominant negative abolishes double axis formation by Xwnt 8 –axis duplication

–dominant negative has no effect

Moon, 1993; Hoppler et al., 1996

Xdsh GSK-3β

–dominant negative results in axis duplication

β-catenin

–axis duplication

XTCF-3

–dominant negative abolishes double axis formation by βcatenin –wild-type has no effect –axis duplication –synergy with β-catenin –axis duplication

LEF-1 siamois

–dominant negative has no effect

Rothbächer et al., 1995; Sokol, 1996 –wild-type ventralizes the embryo He et al., 1995; Pierce and Kimelman, 1995; Dominguez et al., 1995; Yost et al., 1996 –oligo-knock out ventralizes the Heasman et al., 1994; embryo Guger and Gumbiner, 1995; –β-catenin depletion by cadherins Holt et al., 1994 ventralizes the embryo Funayama et al., 1995; Fagotto et al., 1996 –dominant negative ventralizes Molenaar et al., 1996 the embryo

–dominant negative ventralizes the embryo –not done

Behrens et al., 1996 Huber et al., 1996 Lemaire et al., 1995


171

Figure 4 Detailed scheme of the Xwnt signalling pathway. (For details see text). Gene

ventral injection

dorsal injection

References Carnac et al., 1996

interaction of β-catenin with the transcription factor LEF-l/Tcf-3 in axis formation was figured out in Xenopus (Behrens et al., 1996; Molenaar et al., 1996). Combining the data of both systems our present understanding of the β-catenin/Wnt signalling connection can be summarized as follows (Figure 4): Frizzled (Dfz2) binds the Wnt/wg protein. Most probably this interaction is conserved also in vertebrates, since Mfz4, Hfz5, Mfz7 and Mfz8 can bind Drosophila wg (Bhanot et al., 1996) and Rfz1 is able to interact with Xwnt 8 (Yang-Snyder et al., 1996). Since several vertebrate frizzled homologues have been described (Wang et al., 1996) one might think that for each Wnt gene exist, although with some overlapping specificities, a corresponding receptor. It is speculated that the interaction of Wnt/wg with the receptor is transfered into the cell through a conformational change of the latter as it has also been suggested for Gprotein coupled receptors. The intracellular C-terminus of fz proteins contains a S/T-X-V motif which has been shown to interact with PDZ (also called DHR) domains (Saras and Heldin, 1996). Indeed, the next known downstream component of the wg pathway, dishevelled (Dsh) contains such a PDZ domain (Klingenschmith et al., 1994). In this context it is remarkably that overexpression of Rfz1 (Yang-Snyder et al., 1996) in the animal cap of Xenopus embryos results in a shift of Xdsh from the cytosol to the membrane. A similar effect has been reported in Drosophila cl-8 cells (Yanagawa et al., 1995). Dsh is hyperphosphorylated and shifted to the membrane in response to a wg signal. Both effects might depend on the presence of Dfz2 since Dsh is not phosphorylated and membrane located in Schneider S2 cells. This cell line does not express the wg receptor.

172


Dishevelled inactivates shaggy/zeste white 3 (Siegfried et al., 1994). This serin/ threonine kinase is the invertebrate homologue of the vertebrate gene glykogen synthase kinase 3 (gsk-3). In Xenopus, either injection of Xdsh mRNA or mRNA encoding for a kinase-dead mutant of Xgsk-3β results in axis duplication (Rothbächer et al., 1995; Sokol et al., 1995; He et al., 1995; Pierce and Kimelman, 1995; Dominguez et al., 1995). Recently, it has been shown (Yost et al., 1996) that the N-terminal region of pcatenin is a direct target for Xgsk-3β. If one delets or mutates this part of the β-catenin, its stability and inducing capacity is significantly increased compared to the wild-type form. Overexpression of the kinase-dead mutant of Xgsk-3β also results in an accumulation of cytosolic p-catenin. In other words: A Wnt signal leading to an inhibition of Xgsk-3β stabilizes and accumulates cytosolic β-catenin. Karnovsky and Klymkowsky (1995) presented Xenopus data that plakoglobin is able to replace β-catenin in axis induction. However, it remains to be shown whether endogenous plakoglobin is also involved in the Xwnt signalling pathway. Most probably, a Xenopus homologue to the tumor suppressor gene APC (adenomatous polyposis coli) acts as counterplayer in destabilizing cytosolic β-catenin although at present no data about this molecule are available. Analyzing different mammalian cell lines Rubinfeld et al. (1996) and Papkoff et al. (1996) have demonstrated that the APC gene product binds gsk-3β and β-catenin thereby fixing enzyme and substrate in juxtaposition and optimizing the catalytic process. Beyond this operator task the APC protein may also function as proteolytic enzyme degrading β-catenin. A domain separated from the p-catenin binding epitope that destabilizes β-catenin has been identified in the internal region of APC (Munemitsu et al., 1995). This accumulation of β-catenin seems to be a prerequisite for its nuclear uptake. The observation of nuclear β-catenin is definetively coupled with Wnt signalling. The transport of β-catenin into the nucleus in Xenopus embryos can be stimulated by injection of Xwnt 8 mRNA, by a kinase-dead mutant of Xgsk-3β and by lithium chloride (Schneider et al., 1996; Yost et al., 1996). The latter is an inhibitor of gsk-3β (Klein and Melton, 1996; Stambolic et al., 1996) and is often used to dorsalize Xenopus embryos. Conversely, ventralization of Xenopus embryos by UV treatment prevents the nuclear localization of β-catenin. β-catenin does not contain a known nuclear localization signal or a known DNA binding motif. The two most challenging questions: (1) how is β-catenin translocated into the nucleus and (2) if there, how does β-catenin alter the genetic program, were answered by the identification of one protein, the transcription factor LEF-1/Tcf3. Using the yeast two hybrid screen two groups independently identified the HMG box transcription factor LEF-1, respectively its Xenopus homologue Tcf-3, as direct binding partner of β-catenin (Behrens et al., 1996; Molenaar et al., 1996). The interaction of both proteins, surprisingly, is sufficient to transport pcatenin into the nucleus (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996). The function of LEF-1, however, goes beyond that of a simple transporter. Like β-catenin, LEF-1 possess axis inducing capacity when injected into the ventral half of the Xenopus embryo (Behrens et al., 1996; Huber et al., 1996). This capacity depends on its interaction with β-catenin. Moreover, the properties of the transcription factor LEF-1 are modified by β-catenin. As an architectural transcription LEF-1 bends DNA containing the LEF-1 target site (Giese et al., 1992, Grosschedl et al., 1994). This bending is altered by the interaction of β-catenin with LEF-1 which might influence the expression of target genes. Detailed promoter analyses of known target genes of the Wnt/wg signalling cascade will reveal how this bending effect alters the activity of reporter genes. Target genes in Xenopus are the homeobox gene siamois (Lemaire et al., 1995; Carnac et al., 1996; Brannon and Kimelman, 1996) and the TGF-β homologue nodal


173

related 3 (ndr3) (Smith et al., 1995) while engrailed is a target gene in Drosophila (for a review of Drosophila wg signalling: Klingensmith and Nusse, 1994). THE CONTRIBUTION OF ENDOGENOUS CLASS I Xwnt MOLECULES IN MESODERM FORMATION REMAINS ELUSIVE The aforementioned data concerning Wnt signalling during Xenopus dorsoventral axis formation were all raised in an artifical system: the ectopic expression of Xwnt molecules on the ventral side of the embryo which results in axis duplication. These experiments fail to explain the role of endogenous Xwnt signals in axis formation as long as loss of function phenotypes are unknown. Here, the overexpression of cadherins or their extracellular mutants (Figure 1C) on the dorsal side of the embryo can give us important hints. Overexpression of the complete protein is sufficient to tether β-catenin to the plasma membrane while the cytoplasmic pool is depleted. This has been shown detailed in cadherin transfected XTC cells (Finnemann et al., 1995). Consistent with these observations, overexpression of EP/Ccadherin, N-cadherin or XB/U-cadherin in the dorsal blastomeres results in loss of dorsal structures (Holt et al., 1994; Fagotto et al., 1996, own unpublished data). These data strongly suggest a function of β-catenin in endogenous axis formation which has originally been implicated by β-catenin oligo nucleotide knock-out studies by Heasman et al. (1994). However, this raises the question whether the other members up- and downstream of β-catenin in the Wnt pathway are also involved in dorsoventral patterning, thus questioning the integration of endogenous Xwnt within the formation of the dorsal axis. Surprisingly, the most recent findings of Hoppler et al. (1996) show that it is not. A dominant negative Wnt construct which is able to suppress the axis inducing capacity of Xwnt 8 on the ventral side of the embryo, does not interfere with endogenous axis specification. The same result was obtained with a dominant negative Xdsh mutant (Sokol, 1996). In addition, none of the temporal and spatial Xwnt expression patterns, so far known (Figure 5), correlates with one of the dorsalizing centers, the Nieuwcoop Center or the Spemann organiser. At the moment, β-catenin is the first molecule of the Wnt signalling cascade for which an endogenous function in dorsoventral patterning has been demonstrated undoubtedly. Experimental results by Wylie et al. (1996) now support the idea of β-catenin functioning as a competence modifier. In this sense β-catenin is not involved in mesoderm induction but rather in establishing a dorsal fate. Moreover, detailed explanation and heterochronous recombination experiments give evidence Table 2 Molecules that are involved in dorsoventral patterning of mesoderm during early Xenopus development except for class I Wnt members. Gene

ventral injection

dorsal injection

BMP-4/2

–ventralization –injection of truncated BMP-4 receptor leads to axis duplication

Activin

–partial secondary axis –injection of type II receptor may lead to axis duplication –Injection of BMP4-Vg1 leads to axis duplication

–strong ventralization of the embryo Suzuki et al., 1994 Maeno et al., 1994 Schmidt et al., 1995 Clement et al., 1995 –type I receptor can partially rescue Thomsen et al., 1990 ventralized embryos Mathews et al., 1992 Mahony and Gurdon, 1995 –rescue of UV-ventralized embryos Dale et al., 1993 Thomsen and Melton, 1993 Kessler and Melton, 1995 –not done Sasai et al., 1994

Vg1

chordin

–axis duplication

References

174


Gene

ventral injection

dorsal injection

References

noggin

–in cooperation with Xnr1 axis duplication

–rescue of UV-ventralized embryos

Smith et al., 1993 Lustig et al., 1996

that the dorsal signal of maternal β-catenin acts at or shortly after mid blastula transition (MBT) (Wylie et al., 1996). This is consistent with the transient nuclear localization of endogenous p-catenin at the dorsal side during blastula stage (Schneider et al., 1996). Therefore, it is assumed that the accumulation of cytosolic βcatenin at the dorsal side is directly linked to the cortical rotation of the Xenopus egg following fertilization and that its accumulation is not induced by an endogenous Xwnt signal (Miller and Moon, 1996). CROSSTALKS BETWEEN SIGNAL PATHWAYS INFLUENCING β-CATENIN Despite different Wnt signals, several growth factors are active in induction and patterning of mesoderm during Xenopus embryogenesis. How all these signals cooperate is rarely understood. At the moment, there are only a few hints concerning a crosstalk of signalling pathways, e.g. between activin and bFGF (LaBonne and Whitman, 1994; Cornell and Kimelman, 1994). Most recently, Cui et al. (1996) reported that both, signalling of Xwnt 8b/P~catenin and Vg1, a member of the TGF-β family, are required to restore complete axial mesoderm in animal caps of UV-treated embryos. Up to date, a direct molecular coupling of these signal pathways has not been described. However, an indirect influence of class I Xwnt molecules (Olson et al., 1991) activin (Olson and Moon, 1992) and β-catenin (Guger and Gumbiner, 1995) is suggested since overexpression of these molecules increases gap junction permeability which might enhance the diffusion of signal molecules. In mammalian cells a direct molecular interaction between the EGF-receptor and β-catenin was reported (Hoschuetzky et al., 1994) and is discussed between the bFGF receptor and N-cadherin (Williams et al. 1994; Mason, 1994). These observations nourish speculations that β-catenin could function as signal transducer in other pathways during Xenopus embryogensis as well. For two other members of the Wnt signalling cascade, Dsh and gsk, which act upstream of p-catenin, it has already been shown that they also participate in other signal cascades. In Drosophila, dsh does not only contribute to wg signalling but also inhibits Notch (Ruel et al., 1993; Axelrod et al., 1996) by direct binding to its cytoplasmic domain. Notch is a membrane integrated receptor that regulates the signal response of neighbouring cells. The Xenopus homologue of Notch, Xotch, is ubiquitously expressed throughout embryogenesis, but with slightly higher degree in dorsal mesoderm (Coffman et al., 1990). Overexpression of an extracellular deletion mutant results in neural and mesodermal hypertrophy. The competence of animal cap cells to respond to neural or mesodermal inducers is temporally extended (Coffman et al., 1993). Remarkably, the embryos injected with the Xotch mutant develop dorsal axial structures although this construct should also function as a dominant negative mutant in endogenous Xwnt signalling. This result again argues against a function of a endogenous Xwnt in axis formation as mentioned before. Later in embryogenesis several Xwnt genes as well as Xotch are expressed in a variety of neural tissues. Therefore, it is most likely that neural and neuronal development is controlled by a reciprocal interaction of Wnt/wg and Notch signals. A relais-like function seems to inhere the gsk-3β since various incoming signals use this molecule to be transmitted into different responses (Plyte et al., 1992). For example, gsk-3 has recently been demonstrated to be regulated by wg via protein kinase C (Cook et al., 1996). At the moment it is not evident whether this


175

Figure 5 Early expression of Xwnts during Xenopus embryogenesis. (A) The spatial expression of Xwnt 8, 8b and 11 at blastula stage is illustrated. Xwnt 8 is expressed in the equatorial (marginal) zone on the ventral and ventro-lateral side (Smith and Harland, 1991). Xwnt 11 is expressed on the dorsal side of the embryo. The expression domain extends the Spemann organiser (Ku and Melton, 1993). In contrast to Xwnt 8 and Xwnt 11, no whole mount in situ hybridizations are available for Xwnt 8b. RT-PCR analyses revealed that Xwnt 8b is expressed throughout the whole embryo in an animal-vegetal gradient (Cui et al., 1995). (B) Temporal expression of early Xwnt genes. Data are from: Xwnt 5 A (Moon et al., 1993), Xwnt 8 (Smith and Harland, 1991), Xwnt 8b(Cui et al., 1995) and Xwnt 11 (Ku and Melton, 1993).

176


observation reflects the involvement of PKC in the Wnt signalling cascade in general or whether this is due to a special physiological background in mouse 10T1/ 2 fibroblasts. In addition to β-catenin other proteins have been reported as substrates of gsk-3β. These include several transcription factors like c-Myc, c-Myb and c-Jun (Plyte et al., 1992). It will be of high interest to elucidate whether these substrates are also used in Wnt signalling and how does this influence Xenopus development. Xwnt MOLECULES AS MODIFIERS OF ADHESION STRENGTH Transfection of mammalian cell lines with Wnt-1 cDNA, a class I member, results in stabilization of the cadherin/β-catenin and the APC/β-catenin complex, increase in β-catenin and plakoglobin synthesis and in stabilization of β-catenin and plakoglobin monomeres (Hinck et al., 1994; Papkoff et al., 1996). This correlates with a strengthening of the calcium-dependent cell-cell adhesion (Bradley et al., 1993; Hinck et al., 1994). Comparable experiments considering the stability of cadherin/catenin complexes or monomeres fail to be done in Xenopus embryo injected with Xwnt RNA. However, Torres et al. (1996) reported that overexpression of Xwnt 5 A, a class II member, leads to a decrease in Ca2+-dependent cell-cell adhesion and inhibition of convergent extension movements. In contrast, mRNA injection of the class I member Xwnt 8 neither results in loss of cell-adhesion nor in distortions of morphogenetic movements. The observed posteriorized Xwnt 5A phenotypes were identical to those derived by overexpression of cadherin deletion mutants (Torres et al., 1996; Lee and Gumbiner, 1995; Kühl et al., 1996). Loss of cell adhesion due to Xwnt 5A overexpression was rescued by co-injection of N-cadherin RNA (Torres et al., 1996). In addition to Xwnt 5A, all so far known members of the class II Xwnt proteins (Xwnt 4 and Xwnt 11) inhibit convergent extension movements or lead to posteriorized phenotypes (Du et al., 1995). Convergent extension cell movement appears as useful indicator of altered cell adhesion. The identity in phenotypes and the loss of calcium dependent cell adhesion in Xenopus embryos overexpressing Xwnt 5 A, Xwnt 4, Xwnt 11 or cadherin deletion mutants addresses the question whether class II Xwnt proteins regulate cadherin meditated cell adhesion and if so how they drive it. With reservations to the small number of data up to now, it is an attractive hypothesis that Xwnt 5 A, Xwnt 4 and Xwnt 11 function as regional modifiers of cell adhesion strength in early Xenopus development. THE COMPETENCE OF CELLS TO RESPOND TO INDUCING SIGNALS DEPENDS ON CELL ADHESION Considering cell adhesion and signalling in Xenopus embryogenesis one may not disregard the mutual interaction between both processes. Brieher and Gumbiner (1994), for example observered a reduced EP/Ccadherin mediated cell adhesion following activin treatment of animal caps. The mode of its molecular regulation is unclear. Conversely, reduction in cell adhesion by dominant negative expression of nonfunctional N-Cadherin mutants led to an incompetence of muscle progenitor cells to differentiate into muscle cells (Holt et al., 1994). Earlier studies by Sargent et al. (1986) who analyzed the expression of mesodermal marker genes in embryos dissociated by depletion of bivalent cations from the culture medium came to comparable results: the activation of the muscle specific α-actin gene was blocked whereas the synthesis of markers for ectoderm and endoderm was not altered. Beyond the requirement of cell-cell contact for muscle differentiation the correct specification of the mesoderm in general seems to depend on cell adhesion. When animal cap cells dispersed by depletion of divalent cations from the culture medium were treated with different concentrations of activin they expressed a strikingly different pattern of mesodermal marker genes than those which were allowed to reaggregate directly after activin treatment


177

(Wilson and Melton, 1994). For example, no signal for cardiac act in and Xwnt 8 was observed, gsc expression was increased while Xbra expression was reduced. Dissociation of marginal zone explants at mid to late blastula stages and cultivation of these dispersed cells until gastrula stage also results in the lack of mesodermal markers (Xnwt8, Xbra, 1A11, Xlim1) as reported by Sokol (1994). However, in contrast to Wilson and Melton (1994) he also found an inhibition of gcs expression. The latter might be due to the fact that different combinations and concentrations of inducing factors act in the marginal zone compared to activin treated animal caps. Nevertheless, the most exciting observation is the requirement of cell contacts for the induction of Xwnt 8 which was found in both experimental approaches. This leads back to the challenging question whether cadherins but also class II Xwnt molecules contribute to mesoderm specification by regulating the expression and activity of class I Xwnt members. The tight coupling of both processes cell adhesion and class I Xwnt mesoderm patterning in Xenopus makes it difficult to distinguish between both functions in the cadherin or the β-catenin molecule as it was puzzled out in Drosophila (Sanson et al., 1996, Orsulic and Peifer, 1996). CONCLUSION Considering inductive signalling pathways in Xenopus development in context with cell adhesion processes, a regulatory network becomes obvious including cell adhesion as an important separate factor. A mutual interaction between signalling and cell adhesion is given: signal molecules like activin and Wnt molecules can modify adhesive strength. Conversely, cell adhesion itself contributes to correct signal response committing cells to differentiate into a certain cell type. The bifunctional character of β-catenin, as adhesion mediator and as signal transducer, represents an elegant molecular example how such mutual interaction can be established. Finally, the balance between the two functions of β-catenin also suggests that the tissue integrity of the embryo and its mesodermal patterning are closely linked. REFERENCES Axelrod, J., Matsuno, K., Atavanis-Tsakonas, S. and Perrimon, N. (1996) Interaction between wingless and notch signaling pathways mediated by dishevelled. Science, 271,1826–1832. Behrens, J., von Kries, J.P., Kühl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Birchmeier, W. (1996) Functional interaction of β-catenin with the transcription factor LEF-1, Nature, 382, 638–642. Bhanot, P., Brink, M., Samos, C.H., Hsieh, J.-C, Wang, Y., Macke, J.P., Nathans, D. and Nusse, R. (1996) A new member of the frizzled family from Drosophila functions as a wingless receptor. Nature, 382, 225–230. Bradley, R.S., Cowin, P. and Brown, A.M.C. (1993) Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion. J. Cell Biol., 123, 1857–1865. Brannon, M. and Kimelman, D. (1996) Acivation of siamois by the wnt pathway. Dev. Biol.,180, 344–347. Brieher, W.M. and Gumbiner, B.M. (1994) Regulation of C-cadherin function during activin induced morphogenesis of Xenopus animal caps. J. Cell Biol., 126, 519–527. Butz, S., Stappert, J., Weissing, H. and Kemler, R. (1992) Plakoglobin and β-catenin: distinct but closely related. Science, 257, 1142–1143. Carnac, G., Kodjabachian, L., Gurdon, J. and Lemaire, P. (1996) The homeobox gene siamois is a target of the wnt dorsalisation pathway and triggers organiser activity in the absence of mesoderm. Development, 122, 3055–3065. Clement, J.H., Fettes, P., Knöchel, S., Lef, J. and Knöchel, W. (1995) Bone morphogenetic protein 2 in the early development of Xenopus laevis. Mech. Dev., 52, 357–370.

178


Cornell, R.A. and Kimelman, D. (1994) Activin-mediated mesoderm induction requires FGF. Development, 120, 453–462. Cui, Y., Brown, J.D., Moon, R.T. and Christian, J.L. (1995) Xwnt-8b: a maternally expressed Xenopus Wnt gene with a potential role in establishing the dorsoventral axis. Development, 121, 2177–2186. Cui, Y., Tian, Q, and Chriatian, J. (1996) Synergistic effects of Vg1 and Wnt signals in the specification of dorsal mesoderm and endoderm. Dev. Biol., 180, 22–34. Coffmann, C., Harris, W. and Kintner, C. (1990) Xotch, the Xenopus homolog of Drosodhila Notch. Science, 249, 1438–1441. Coffmann, C., Skoglund, P., Harris, W. and Kintner, C. (1993) Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell, 73, 659–671. Cook, D., Fry, M.J., Hughes, K., Sumathipala, R., Woodgett, J. and Dale, T.C. (1996) Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C. EMBO J., 15, 4526–4536. Dale, L., Matthews, G. and Colman, A. (1993) Secretion and mesoderm-inducing activity of the TGF-b-related domain of Xenopus Vg1. EMBO J., 12, 4471–4480. Dominguez, I., Itoh, K. and Sokol, S. (1995) Role of glycogen synthase kinase 3β as a negative regulator of dorsoventral axis formation in Xenopus embryos. Proc. Nat. Acad. Set. USA, 92, 8498–8502. Du, S., Purcell, S., Christian, J., McGrew, L.L. and Moon, R.T. (1995) Identification of different classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos. Mol. Cell. Biol., 15, 2625–2634. Fagotto, F, Funayama, N., Glück, U. and Gumbiner, B.M. (1996) Binding to cadherins antagonizes the signaling activity of β-catenin during axis formation in Xenopus. J. Cell Biol., 132, 1105–1114. Finnemann, S., Kühl, M., Otto, G. and Wedlich, D. (1995) Cadherin transfection of Xenopus XTC cells downregulates expression of substrate adhesion molecules. Mol. Cell. Biol., 15, 5082–5091. Funayama, N., Fagotto, F., McCrea, P. and Gumbiner, B. (1995) Embryonic axis induction by the armadillo repeat domain of β-catenin: evidence for intracellular signalling. J. Cell Biol., 128, 959–968. Giese, K., Cox, J. and Grosschedl, R. (1992) The HMG box domain of lymphoid enhancer factor 1 bends DNA and facilitates assembly of functional nucleoprotein structure.Cell, 69, 185–195. Grosschedl, R., Giese, K. and Pagel, J. (1994) HMG domain proteins: architectural elements in the assembly of nucleoprotein structures. TIG, 10, 94–100. Guger, K. and Gumbiner, B.M. (1995) β-catenin has wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning. Dev. Biol., 172, 115– 125. He, X., Saint-Jeannet, J.P., Woodgett, J.R., Vermus, H. and Dawid, I. (1995) Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos. Nature, 374, 617– 622. Heasman, J., Crawford, A., Goldstone, K., Garner-Hamrick, P., Gumbiner, B., McCrea, P., Kintner, C., Yoshida Noro, C. and Wylie, C. (1994) Overexpression of cadherins and underexpression of β-catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell, 79, 791–803. Hinck, L., Nelson, W.J. and Papkoff, J. (1994) Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing βcatenin binding to the cell adhesion protein cadherin. J. Cell Biol., 124, 729–741. Holt, C., Lemaire, P. and Gurdon, J. (1994) Cadherin-mediated cell interactions are necessary for the activation of MyoD in Xenopus mesoderm. Proc. Nat. Acad. Sci USA, 91, 10844– 10848. Hoppler, S., Brown, J.D. and Moon, R.T. (1996) Expression of a dominant negative Wnt blocks induction of MyoD in Xenopus embryos. Gen. Dev., 10, 2805–2817. Hoschuetzky, H., Aberle, H. and Kemler, R. (1994) β-catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J. Cell Biol., 127, 1375– 1380. Huber, O., Korn, R., McLaughlin, J., Ohsugi, M., Herrmann, B.G. and Kemler, R. (1996) Nuclear localization of βcatenin by interaction with transcription factor LEF-1. Mech. Dev., 59, 3–10. Karnovsky, A. and Klymkovsky, M.W. (1995) Anterior axis duplication in Xenopus induced by the over-expression of the cadherin-binding protein plakoglobin. Proc. Nat. Acad. Sci. USA, 92, 4522–526.


179

Keller, R. (1986). The cellular basis of amphibian gastrulation. In: Developmental biology, vol. 2 (ed. L.W. Browder), pp. 241–323, New York, Plenum Press. Kemler, R. (1993) From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. TIG, 9, 317–321. Kessler, D. and Melton, D. (1995) Induction of dorsal mesoderm by soluble, mature Vg1 protein. Development, 121, 2155–2164. Klein, P.S. and Melton, D. (1996) A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA, 93, 8455–8459. Klingensmith, J. and Nusse, R. (1994) Signaling by wingless in Drosophila. Dev. Biol., 166, 396–414. Klingensmith, J., Nusse, R. and Perrimon, N. (1994) The Drosophila segment polarity gene dishevelled encodes a novel protein required for response to the wingless signal. Gen. Dev., 8, 118–130. Kühl, M. and Wedlich, D. (1996) Xenopus cadherins: Sorting out types and functions in embryogenesis. Dev. Dynam., 207, 121–134. Kühl, M., Finnemann, S., Binder, O. and Wedlich, D. (1996) Dominant negative expression of a cytoplasmically deleted mutant of XB/U-cadherin disturbs mesoderm migration during gastrulation in Xenopus laevis. Mech. Dev., 54, 71–82. Ku, M. and Melton, D.A. (1993) Xwnt-11: a maternally expressed Xenopus wnt gene. Development, 119, 1161–1173. LaBonne, C. and Whitman, M. (1994) Mesoderm induction by activin requires FGF-mediated intracellular signals. Development, 120, 463–472. Lee, C.H. and Gumbiner, B. (1995) Disruption of gastrulation movements in Xenopus by a dominant negative mutant for C-cadherin. Dev. Biol., 171, 363–373. Lemaire, P., Garrett, N. and Gurdon, J.B. (1995) Expression cloning of siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell, 81, 85–94. Lustig, K.D., Kroll, K., Sun, E., Ramos, R., Elmendorf, H. and Kirschner, W. (1996) A Xenopus nodal-related gene that acts in synergy with noggin to induce complete secondary axis and notochord formation. Development, 122, 3275–3282. Maeno, M., Ong, R.S., Suzuki, A., Ueno, N. and Kunk, H. (1994) A truncated bone morphogenetic protein 4 receptor alters the fate of ventral mesoderm to dorsal mesoderm: Roles of animal pole tissue in the development of ventral mesoderm. Proc. Natl. Acad. Set. USA, 91, 10260–10264. Mahony, D. and Gurdon, J.B. (1995) A type 1 serine/thereonine kinase recptor that can doralize mesoderm in Xenopus. Proc. Natl. Acad. Set. USA, 92, 6474–6478. Mason, I. (1994) Do adhesion molecules signal via FGF receptors? Curr. Biol., 4, 1158–1161. Mathews, L., Vale, W.W. and Kintner, C. (1992) Cloning of a second type of activin receptor and functional characterization in Xenopus embryos. Science, 255, 1702–1705. McCrea, P., Turck, C.W. and Gumbiner, B. (1991) A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. Science, 254, 1359–1361. McNeill, H., Ozawa, M., Kemler, R. and Nelson, W.J. (1990) Novel function of the cell adhesion molecule uvomorulin as an inducer of cell surface polarity. Cell, 62, 309–316. Miller, J. and Moon, R.T. (1996) Signal transduction through β-catenin and specification of cell fate during embryogenesis. Gen. Dev., 10, 2527–2539. Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destrée, O. and Clevers, H. (1996) Xtcf-3 transcription factor mediates β-catenin-induced axis formation in Xenopus embryos. Cell, 86, 391–399. Moon, R.T. (1993) In pursuit of the functions of the Wnt family of developmental regulators: insights from Xenopus laevis. BioEssays, 15, 91–97. Moon, R.T., Campbell, R.M., Christian, J.L., McGrew, L.L., Shih, J. and Eraser, S. (1993) Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development, 119, 97–111.

180


Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B. and Polakis, P. (1995) Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumor suppressor protein. Proc. Nat. Acad. Sci. USA, 92, 3046–3050. Nagafuchi, A. and Takeichi, M. (1988) Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO J., 7, 3679–3684. Nordermeer, J., Klingensmith, J., Perrimon, N. and Nusse, R. (1994) Dishevelled and armadillo act in the wingless signalling pathway in Drosophila. Nature, 367, 80–83. Olson, D.J. and Moon, R.T. (1992) Distinct effects of ectopic expression of Wnt-1, activin B and bFGF on gap junction permeability in 32–cell Xenopus embryos. Dev. Biol., 151, 204–212. Olson, D.J., Christian,J. and Moon, R.T. (1991) Effect of Wnt-1 and related proteins on gap junctional communication in Xenopus embryos. Science, 252, 1173–1176. Orsulic, S. and Peifer, M. (1996) An in vivo structure function study of armadillo, the β-catenin homologue, reveals both seperate and overlapping regions of the protein required for cell adhesion and for wingless signaling. J. Cell Biol., 134, 1283–1300. Papkoff, J., Rubinfeld, R., Schryver, B. and Polakis, P. (1996) Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. Mol. Cell. Biol., 16, 2128–2134. Peifer, M., Rauskolb, C., Williams, M., Riggleman, B. and Wieschaus, E. (1991) The segment polarity gene armadillo interacts with the wingless signaling pathway in both embryonic and adult pattern formation. Development, 111, 1029–1043. Pierce, S. and Kimelman, D. (1995) Regulation of Spemann organizer formation by the intracellular kinase Xgsk-3. Development, 121, 755–765. Plyte, S.E., Hughes, K., Nikolakaki, E., Pulverer, B. and Woodgett, J.R. (1992) Glycogen synthase kinase-3: functions in oncogenesis and development. Biochim. Biophys. Acta, 1114, 147–162. Rothbächer, U., Laurent, M.N., Blitz, I.L., Watabe, T., Marsh, J.L. and Cho, K.W.Y. (1995) Functional conservation of the wnt signalling pathway revealed by ectopic expression of Drosophila dishevelled in Xenopus. Dev. Biol., 170, 717–721. Rubinfeld, B., Albert, I., Porifiri, E., Fiol, C., Munemitsu, S. and Polakis, P. (1996) Binding of GSK3β to the APC-βcatenin complex and regulation of complex assembly. Science, 272, 1023–1026. Ruel, L., Bourouis, M., Heitzler, P., Pantesco, V. and Simpson, P. (1993) Drosophila shaggy kinase and rat glycogen synthase kinase-3 have conserved activities and act downstream of notch. Nature, 362, 557–559. Sanson, B., White, P. and Vincent, J.P. (1996) Uncoupling cadherin-based adhesion from wingless signalling in Drosophila. Nature, 383, 627–630. Saras, J. and Heldin, C.H. (1996) PDZ domains bind carboxy-terminal sequences of target proteins. Trend. Biochem. Sci., 21, 455–458. Sargent, T.D., Jamrich. M. and Dawid, I. (1986) Cell interactions and the control of gene activity during early development of Xenopus laevis. Dev. Biol., 114, 238–246. Sasai, Y., Lu, B., Steinbeisser, H., Geissert, D., Gont, L. and DeRobertis, E. (1994) Xenopus Chordin: A novel dorsalizing factor activated by organizer-specific homeobox genes. Cell, 79, 779–790. Schmidt, J., Suzuki, A., Ueno, N. and Kimelman, D. (1995) Localized BMP-4 mediates dorsal/ ventral patterning in the early Xenopus embryo. Dev. Biol., 169, 37–50. Schneider, S., Steinbeisser, H., Warga, R. and Hausen, P. (1996) β-catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech. Dev., 57, 191– 198. Siegfried, E., Wilder, E.L. and Perrimon, N. (1994) Components of wingless signalling in Drosophila. Nature, 367, 76–79. Smith, W.C. and Harland, R.M. (1991) Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center. Cell, 67, 753–765. Smith, W.C., Knecht, A., Wu, M. and Harland, R. (1993) Secreted noggin protein mimics the Speman organizer in dorsalizing Xenopus mesoderm. Nature, 361, 547–549. Smith, W.C., McKendry, R., Ribisi, S. and Harland, R. (1995) A nodal-related gene defines a physical and functional domain within the Speman organizer. Cell, 82, 37–46.


181

Sokol, S. (1994) The pregastrula establishment of gene expression pattern in Xenopus embryos: Requirements for local cell interactions and for protein synthesis. Dev. Biol., 166, 782– 788. Sokol, S. (1996) Analysis of dishevelled signalling pathways during Xenopus development. Curr. Biol., 6, 1456–1467. Sokol, S.Y., Klingensmith, J., Perrimon, N. and Itoh, K. (1995) Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled. Development, 121, 1637–1647. Spemann, H. and Mangold, H. (1924) Über Induktion von Embryonalanlagen durch Implantation artfremder Organisatoren. Arch. f. mikr. Anat. u. Entw. Mech., 100, 599–638. Stambolic, V., Ruel, L. and Woodgett, J.R. (1996) Lithium inhibts glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells. Curr. Biol., 6, 1664–1668. Suzuki, A., Thies, R.S., Yamaji, N., Song, J., Wozney, J., Murakami, K. and Ueno, N. (1994) A truncated bone morphogenetic protein receptor affects dorsal-ventral patterning in the early Xenopus embryo. Proc. Natl. Acad. Set. USA, 91, 10255–10259. Takeichi, M. (1995) Morphogenetic roles of classical cadherins. Curr. Op. Cell Biol., 7, 619– 627. Thomsen, G. and Melton, D. (1993) Processed Vg1 protein is an axial mesoderm inducer in Xenopus. Cell, 74, 433–441. Thomsen, G., Woolf, T., Whitman, M., Sokol, S., Vaughan, J., Vale, W. and Melton, D. (1990) Activins are expressed early in Xenopus embryogenesis and can induce axial mesoderm and anterior structures. Cell, 63, 485–493. Torres, M., Yang-Snyder, J.A., Purcell, S.M., DeMarais, A., McGrew, L.L. and Moon, R.T. (1996) Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A Class and by a dominant negative cadherin in early Xenopus development. J. Cell Biol., 133, 1123–1137. Townes, P. and Holtfreter, J. (1955) Directed movements and selective adhesion of embryonic amphibian cells. J. Exp. Zool., 128, 53–120. Wang, Y., Macke, J.P., Abella, B.S., Andreasson, K., Worley, P., Gilbert, D., Copeiand, N.G., Jenkins, N.A. and Nathans, J. (1996) A large family of putative transmembrane receptors homologous to the product of the Drosophila tissue polarity gene frizzled. J. Biol. Chem., 271, 4468–4476. Williams, E.J., Furness, J., Walsh, F.S. and Doherty, P. (1994) Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM and N-cadherin. Neuron, 13, 583– 594. Wilson, P. and Melton, D. (1994) Mesodermal patterning by an inducer gradient depends on secondary cell-cell communication. Curr. Biol., 4, 676–686. Wylie, C., Kofron, M., Payne, C., Anderson, R., Hosobuchi, M., Joseph, E. and Heasman, J. (1996) Maternal β-catenin establishes a ‘dorsal signal’ in early Xenopus embryos. Development, 122, 2987–2996. Yanagawa, S., van Leeuwen, F., Wodarz, A., Klingensmith, J. and Nusse, R. (1995) The dishevelled protein is modified by wingless signaling in Drosophila. Gen. Dev., 9, 1087– 1097. Yang-Snyder, J., Miller, J., Brown,.J., Lai, C. and Moon, R.T. (1996) A frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr. Biol., 6, 1302–1306. Yost, C., Torres, M., Miller, J.R., Huang, E., Kimelman, D. and Moon, R.T. (1996) The axis-inducing activity, stability and subcellular distribution of β-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Gen. Dev., 10, 1443–1454.

10. TYROSINE KINASE RECEPTORS AS DRIVING FORCES FOR GROWTH, MOTTLTTY AND DIFFERENTIATION OF EPITHELIAL CELLS WALTER BIRCHMEIER1, CARMEN BIRCHMEIER2, VOLKER BRINKMANN1 AND CATHERIN NIEMANN1 1 Department of Growth and Differentiation, 2Department of Genetics and Structural Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D.13125 Berlin, Germany

SUMMARY During development, distinct tissues arise by differentiation and morphogenesis from initially uniform cells. We will here concentrate on the discussion of the morphogenesis of epithelia, which form an astounding array of different structures, e.g. hollow cysts, single or multi-layered plates or branched tubules. Signals for differentiation and morphogenesis of epithelia are frequently given by surrounding mesenchymal tissuses which grow and differentiate concomitantly. Tyrosine kinase receptors and their ligands can constitute paracrine signaling systems which provide a molecular basis for signal exchange between different cellular compartments like mesenchyme and epithelia. Scatter factor/hepatocyte growth factor and the c-met receptor, neuregulins and the family of c-erbB receptors, FGFs and their receptors as well as GDNF and cret constitute such paracrine signaling systems, which control growth, morphogenesis and differentiation of epithelia. Recent experiments with cultured epithelial cells have shown that different tyrosine kinase receptors can elicit different morphogenic responses. The important roles of tyrosine kinase receptors and their ligands in the development of epithelia were substantiated by the analysis of mice that carry targeted mutations or transdominant receptors. Together, these results demonstrate an essential role of such paracrine acting signaling systems in the control of the development of epithelial cells types. Epithelia form continuous mono- and multilayered sheets of tightly adhesive cells. Characteristic for epithelia are specialized organelle-like structures, like tight junctions, adherens junctions and desmosomes, that are responsible for intercellular contacts. Hemidesmosomes form contacts to the acellular basement membranes. An additional characteristic of many epithelial cells is their polarization, that is the distinct morphological appearance of basal, lateral and apical surfaces. A consequence of the laterally located tight junctions is the inhibition of free diffusion in the cellular membrane, and therefore adhesion molecules or tyrosine kinase receptors can be exclusively or predominantely expressed on the basolateral side of epithelial cells (Rodriguez-Boulan and Nelson, 1989; Schwarz et al., 1990; Birchmeier and Birchmeier, 1993; see also articles by Garrod, Knust, Tsukita et al., and Scheiffele and Simons in this book). In most organs mesenchymal cells are found below the epithelia. The mesenchymal cells are morphologically distinct, usually non-polarized, loosely associated and express a characteristic set of genes (Hay, 1990; Ekblom, 1992). In general, a basement membrane separates the epithelial from the mesenchymal cell compartment. The basement membrane is synthesized by both, epithelial and mesenchymal Correspondence: Walter Birchmeier: Tel: +49–30–9406 3737 or 3800; Fax: +49–30–9406 2656.


183

cells, and contains extracellular matrix molecules like laminin, collagen IV, nidogen/ entactin and basement membrane proteoglycans (Timpl, 1989; see also article by Paulsson in this book). Tyrosine kinase receptors and their ligands can form both autocrine or paracrine signaling systems. Recent evidence demonstrates that a paracrine signaling mode is typical for several receptors-ligand pairs which affect epithelia. For example, scatter factor (SF), also known as hepatocyte growth factor (HGF), was initially described as fibroblast-derived factor that scatters epithelial cells (Stoker et al., 1987; Miyazawa et al., 1989; Nakamura et al., 1989; Weidner et al., 1991). The specific receptor of SF/HGF was found to be cmet (Bottaro etal., 1991; Naldini et al., 1991) which is expressed by epithelia, cells of the myogenic lineage and by neuronal cells. In situ hybridization analysis suggested a paracrine signaling mode for SF/HGF and c-met in the developing mouse; SF/HGF is expressed by various types of mesenchymal cells located in the vicinity of epithelial or muscle cells that express c-met (Sonnenberg et al., 1993). Neuregulin-1, also known as NDF, heregulin, GGF or ARIA, is a member of the EGF family of growth factors (Peles and Yarden, 1993). The high affinity receptors of neuregulin-1 are the erbB3 and erbB4 receptor tyrosine kinases; the erbB2 receptor functions as essential co-receptor (Carraway & Cantley, 1994). Neuregulin-1 is produced predominantly by mesenchymal and neuronal cells, whereas the receptors are expressed in epithelia, muscle and Schwann cells as well as in other cells of the nervous system (Marchionni et al., 1993; OrrUrtreger et al., 1993; Meyer and Birchmeier, 1994; Meyer et al., 1997). FGFs were initially identified as potent growth factors for fibroblasts and other cells types, as oncogenes activated by mouse mammary tumor virus (MMTV) or isolated in transformation assays using NIH 3T3 cells (Burgess and Maciag, 1989). Four receptors for FGFs exist in mammals that have overlapping specificities for the many FGFs (Johnson and Williams, 1993). FGFs and their receptor form frequently paracrine signaling system in development, which are known to regulate various developmental events (cf. article by Werner in this book). The c-ret receptor was identified because of its oncogenic potential when mutated (Takahashi et al., 1985). The ligand of c-ret was recently found to correspond to GDNF (glial-derived neurotrophic factor) (Durbec et al., 1996; Treanor et al., 1996; Vega et al., 1996). c-Ret is expressed in epithelia of the kidney and in cells of the peripheral nervous system, whereas GDNF is produced by kidney mesenchyme and by mesenchyme located at the sites of development of the sympathetic and enteric nervous system (Pachnis et al., 1993; Choi and Bohn, 1995; Trupp et al., 1995; cf. also article by Schuchardt et al., in this book). Other epithelial receptor tyrosine kinases exist, to which no ligand was assigned yet, such as c-ros (Sonnenberg et al., 1991). Indirect evidence suggest a paracrine signalling modus for c-ros. THE ROLE OF SF/HGF AND NEUREGULIN IN THE DEVELOPMENT OF BREAST EPITHELIA SF/HGF induces various cellular responses on epithelial and other cells in culture: (i) it promotes growth of primary hepatocyes and other epithelial cells, (ii) it can induce increased motility (scattering) of epithelial and endothelial cells, (iii) it has an unusual activity, the ability to induce branching morphogenesis of MDCK (kidney) and other epithelial cells in three-dimensional matrices, and (iv) it promotes outgrowth of axons from developing motoneurons (Stoker, et al., 1987; Miyazawa et al., 1989; Nakamura et al., 1989; Weidner et al., 1991; Montesano et al., 1991a,b; Bussolino et al., 1992; Grant et al., 1993; Brinkmann et al., 1995; Bladt et al., 1996; Ebens et al., 1996). Neuregulin also induces morphological changes in breast carcinoma cells, which are accompanied by terminal differentiation, i.e. accumulation of fat droplets or βcasein secretion (Peles et al., 1992). Other carcinoma cells respond to neuregulin by growth (Holmes et al., 1992). In addition, skeletal muscle and cell of neuronal origin, i.e. glial cells, oligodentrocytes and retinal neurons have been reported to respond to neuregulin in cell culture (Marchionni et al., 1993; Falls et al.,

184


1993; Shah et al., 1994; Jo et al., 1995; Dong et al., 1995; Morrissey et al., 1995; Bermingham et al., 1996; Canoll et al., 1996; Trachtenberg and Thompson, 1996). We recently studied the role of SF/HGF and neuregulin in development of the mammary epithelium, using organ culture of mouse mammary glands (Yang et al., 1995). In vivo, the epithelia of the mammary glands undergo first a period of growth and branching during puberty; later during pregnancy, alveolar differentiation and milk production occurs. Both processes are under central hormonal control. Branching and alveolar morphogenesis can be recapitulated in organ culture. Addition of exogenous SF/HGF to mammary glands in organ culture strongly promotes epithelial growth and branching, whereas exogenous neuregulin induces alveolar maturation followed by terminal differentiation characterized by milk production. Corresponding antisense oligonucleotides prevent branching morphogenesis or alveolar differentiation, whereas the addition of exogenous factor overcomes the inhibitory effect of the antisense oligonucleotides. Interestingly, both factors are specifically expressed in the mesenchyme of the mammary gland during development: SF/HGF during puberty and neuregulin during pregnancy (Yang et al., 1995). Neuregulin is not at all expressed during puberty and involution (when the lactating glands are remodeled and return to a ‘pre-pregnancy’ state) but is specifically turned on late in pregnancy. It is then expressed at high levels. In contrast, the receptors of both these factors (i.e. the c-met receptor and the members of the cerbB family) do not appear to be developmentally regulated in the mammary gland. This indicates that the central hormonal control acts by regulating the production of local, mesenchymal factors during two phases of mammary gland development: branching and alveolar maturation. The mesenchymal factors then promote growth and differentiation of the epithelium in the mammary gland (Yang, et al., 1995). The two phases of epithelial morphogenesis in the mammary gland, branching and alveolar morphogenesis, could recently be recapitulated in cell culture. Upon addition of SF/HGF, EpH4 mammary gland epithelial cells form branched tubules in a matrigel matrix. Subsequent exposure to neuregulin induces alveolar-like bulbs along the tubules (Niemann et al., 1989). Branching morphogenesis was also induced by the recently identified substrate of c-Met (Weidner et al., 1996, see also below). Alveolar development was promoted by NGF in cells expressing a trk-erbB2 hybrid (cf. Sachs et al., 1995). Thus, among the neuregulin receptors present in epithelial cells (erbB2, erbB3 and erbB4), the erbB2 mediated signal alone is sufficient to induce alveolar differentiation. We conclude form these experiments that mammary gland epithelial cells respond to signals given by different factors (and mediated by different receptors) in a specific manner, that affects their morphogenic response. Thus, signalling specificity of these tyrosine kinase receptors exist. OTHER MORPHOGENIC RESPONSES INDUCED BY SF/HGF AND C-MET IN EPITHELIAL CELLS Montesano et al. (1991a,b) have first observed that HGF/SF induces branching tubular structures of kidney (MDCK) epithelial cells in three-dimensional collagen gels. Similar branching tubules can be seen when breast or prostate epithelial cells are exposed to the factor (Soreano et al., 1994; Brinkmann et al., 1995). However, tubules are not the only structures induced: Colon carcinoma cells can form crypt-like organoids that build a well developed brush border towards the lumen. Moreover, HGF/SF stimulates formation of alveolar-like structures in lung epithelial cells and induces formation of hollow spheres in pancreas epithelial cells. HGF/SF induces therfore complex morphogenic responses in epithelial cells derived form diverse organs; the epithelial structures formed in vitro are similar to the ones observed in vivo. Apparently, an intrinsic program which is determined by the organ of origin governs the detailed structures formed by the cells (Brinkmann et al., 1995).


185

THE CELLULAR RESPONSES TOWARDS HGF/SF AR GENERATED BY A SPECIFIC SUBSTRATE OF C-MET, GAB1 The experiments in cell culture and developmental studies (cf. below) have indicated that receptor tyrosine kinases take over a variety of functions in epithelia. For instance, many receptors such as c-erbB2, c-ros, the KGF receptor, or trk A can scatter kidney epithelial cells but are unable to induce the formation of branching tubules (cf. Sachs et al., 1996). However, a novel chimeric receptor with the ability to form tubules could be created by fusing the COOH-terminal region of c-met (which contains the major substrate binding sites; cf. Ponzetto et al., 1994; Fixman et al., 1995; Weidner et al., 1995) to the COOH-terminus of the kinase domain of trk A (Sachs et al., 1996). Thus the C-terminal part of c-met mediates the signal required for the formation of branched tubules in kidney epithelial cells. We hypothesised that this site (named bidentate docking site, it contains two tyrosine residues Y1347 and Y1354) may bind specific substrates which should be different from the more common substrates shared by other receptors. We used the yeast 2-hybrid system to detect such specific substrates of the c-met receptor and identified Gab1 (Weidner et al., 1996). Gab1 had been previously isolated as a protein able to bind Grb2 (HolgadoMadruga et al., 1996). Gab1 binds strongly to c-met but not to a dozen other tyrosine kinases from different families (Weidner et al., 1996). Moreover, Gab1 did not interact with mutant c-met protein that lacks kinase activity or the C-terminal tail. Binding was impaired when tyrosine residue Y1347 and Y1353 were mutated, and mutations of both tyrosine residues (the bidentate docking site) prevented Gab1 binding completely (Weidner et al., 1996). Gab1 is a member of a family of multiadapter proteins that includes DOS (daughter of sevenless) and IRS (insulin receptor substrates I and II). This family is characterized by a pleckstrin homology domain at the NH2-terminus and by multiple binding sites for proteins containing SH2 and SH3 domains. We have identified a new phosphotyrosine interaction domain of Gab1 (MBD, c-met binding domain, from amino acids 450–532) which binds with an as yet unique specificity to a c-met tyrosyl phosphopeptide (Weidner et al., 1996). Importantly, expression of Gab1 in kidney epithelial cells induces the formation of branching tubular structures in the absence of HGF/SF. Our data therefore support the concept that branching morphogenesis of c-met is a specific cellular response to this receptor that requires unique substrates such as Gab1. THE ROLE OF HGF/SF AND C-MET IN METASTASIS Several experimental and clinical studies indicate that HGF/SF and its receptor c-met play a role in tumour progression and in particular, in invasion and metastasis (cf. Weidner et al., 1990; Rong et al., 1993, 1995; Bellusci et al., 1994; Yamashita et al., 1994; Rosen et al., 1994; Ferracini et al., 1995; Joseph et al., 1995; Meiners et al., 1997). In order to examine the role of HGF/SF in metastasis, we have chosen an animal model system which comprises several steps of the metastatic cascade and reflects aspects of metastasis of human breast carcinomas. HGF/SF-transfected human MDA MB435 breast carcinoma cells were transplanted into the orthotopic site, the mammary fat pad of nude mice, which results in the formation of lung metastasis. We observed high metastasis rates in HGF/SF-transfected cells (and also of neuregulintransfected cells) in comparison to non-transfected cells. These data indicate that HGF/SF is in fact a strong inducer of metastasis of carcinoma cells (Meiners et al., 1997). The question arises how one factor, HGF/SF, can induce formation of complex epithelial structures (branched tubules) and increase metastasis, i.e. induce two seemingly unrelated and even opposing cellular responses. It should be noted that formation and remodelling of complex epithelial structures also requires cell movement, i.e. breaking and reforming of cell-cell contacts. Furthermore, the metastatic MDA MB435

186


cells lack expression of the cell adhesion molecule E-cadherin (Frixen et al., 1991); these cells therefore do not adhere well to each other. Re-expression of E-cadherin in MDA MB435 cells reconstitutes adhesion and polarity, i.e. characteristics of epithelial cells, and accordingly reduces invasion and metastasis (Meiners et al., 1997). Moreover, E-cadherin expression in part reconstitutes the ability of MDA MD435 cells to form tubular structures upon HGF/ SF stimulation (Meiners et al., 1997). Our data indicate that epithelial (carcinoma) cells which maintain their epithelial differentiation program respond toward HGF/ SF by morphogenesis. In contrast, carcinoma cells which have lost epithelial characteristics, i.e. have undergone epithelial-mesenchymal transitions, respond toward HGF/SF by invasion and metastasis (cf. also article of Grassi et al. in this book). Thus, the cellular responses to HGF/SF appear to depend on the differentiation state of the cells: Morphogenesis requires epithelial differentiation and cell polarity, whereas metastatic responses are observed when epithelial characteristics are lost. SF/HGF AND THE C-MET RECEPTOR CONTROL GROWTH AND MOTILITY OF CELLS IN VIVO Targeted mutagenesis in embryonal stem (ES) cells by homologous recombination was employed to ablate the SF/HGF and the c-met genes (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995). The mutant ES cells were then used to generate mice that carry the mutation. Both mutations show the same phenotype when present in a homozygous state. The identical phenotypes is genetic evidence for an absence of redundancy in the SF/HGF and c-met signaling system: SF/HGF appears to be the only functional ligand for c-met, and c-met the only receptor for SF/HGF. The targeted mutations of c-met and SF/HGF affect development of the embryonal liver, the placenta and distinct skeletal muscles (Bladt et al., 1995). The liver is reduced in size, and histological damage of variable severity is observed in the parenchyme. That indeed the hepatocellular compartment, and not the embryonal hematopoietic system (which is also located in the liver) is primarily affected by the mutation, was verified by the analysis of chimeric animals: c-met—/—cells cannot contribute to the adult liver, but participate normally in generation of blood and various hematopoietic cell types (Bladt et al., 1995). The placenta, particularly the labyrinth layer, is also affected by a lack of SF/HGF or c-met (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995). In addition, a complete absence of particular muscles was observed in the mutant embryos (Bladt, et al., 1995): Skeletal muscles of the limb, the diaphragm and the internal lingual muscles are lacking, whereas all other skeletal muscles can form. The primary cause of this defect is an absence of migration of muscle precursors cells from the dermomyotomes of the somite to the anlage of the limb, diaphragm or tongue. As consequence, muscles at these sites cannot form (Bladt et al., 1995). The muscle precursor cells located in the ventro-lateral dermomyotome express the c-met receptor. The signal that induces a mesenchymal-epithelial conversion of the somite cells and migration to the limb bud is known to be given by limb mesenchyme (Hayashi et al., 1995). Indeed, the medial mesenchyme of the limb anlage expresses SF/HGF. Moreover, we recently demonstrated in chick embryos that ectopic application of SF/HGF induces epithelial-mesenchymal conversion and emigration of dermomyotomal cells (Brand-Saberi et al., 1996; cf. also Heymann et al., 1996). Thus, SF/HGF acts in vivo as a paracrine and as a locally acting factor during development of various organs. All phenotypes in the mutant mice reflect disturbances in the interactions between different cell types. Different cellular functions are affected: growth and morphogenesis of epithelial cells in the liver and placenta, and migration of myogenic precursor cells that originate from the somites.


187

NEUREGULIN AND THE C-ERBB RECEPTOR TAKE OVER MULTIPLE FUNCTIONS IN DEVELOPMENT The functions of neuregulin and its receptors, c-erbB2, erbB3 and c-erbB4 receptors has also been analyzed genetically in mice (Gassmann et al., 1995; Lee et al., 1995; Meyer and Birchmeier, 1995, Riethmacher et al., 1997). Multiple, independent defects in development of the heart and peripheral nervous system were observed in such mutant animals. Mice homozygous for mutations in neuregulin, erbB2 and erbB4 die early in development, i.e. on day 10 of gestation. The embryonal death is caused by a defect in development of the heart. During development, the endothelial lining of the heart (the endocardium) produces neuregulin, which is an essential signal for trabeculation of the heart muscle (myocardium) (Meyer and Birchmeier, 1995). In the heart, the neuregulin signal is received by the erbB4 and erbB2 receptor (Gassmann, et al., 1995; Lee et al., 1995; Meyer and Birchmeier, 1995). Since only erbB4, but not erbB2 can bind neuregulin with high affinity, erbB2 functions in vivo as essential co-receptor for erbB4. Moreover, severe defects in development of the peripheral nervous system are detected in animals that lack a functional neuregulin, erbB2 or erbB3 gene (Meyer and Birchmeier, 1995; Lee et al., 1995; Riethmacher et al., 1997). These include defects in the development of cranial ganglia and Schwann cells. ErbB4 is not required for neuregulin signaling in development of the peripheral nervous system. Instead, the erbB3 receptor, again together with erbB2 as co-receptor, mediates the essential signals of this growth factor in the peripheral nervous system. From the neuregulin gene, various isoforms are produced which are expressed in distinct and dynamic patterns during development and in the adult (Chen et al., 1994; Corfas et al., 1995; Meyer et al., 1997). This indicated that the neuregulin isoforms take over additional functions in embryogenesis and in the adult. Experiments in cell culture demonstrate that neuregulin affects Schwann cell precursors, immature and mature Schwann cells, and influences cell fate decisions, growth, survival and maturation of cells in this lineage (Marchionni et al., 1993; Dong et al., 1995). Direct axonal contact has been long known to induce DNA synthesis in Schwann cell precursors. Antibodies against erbB2 inhibit this axon-driven DNA proliferation, indicating that the neuronal signal is given by an axon-bound isoform of neuregulin (Morrissey et al., 1995). Mice that lack neuregulin, erbB2 or erbB3 display a reduction in early Schwann cell precursors at early developmental stages; erbB3 mutant mice can survive to brith and show then a absence of Schwann cells that accompagny axons of sensory and motorneurons (Riethmacher et al., 1997). Moreover, experimental evidence indicate that neuregulin provides an important signal for maturation of the neuromuscular junction (Falls et al., 1993; Jo et al., 1995) and for alveolar differentiation of the mammary gland epithelium (Peles et al., 1992; Yang et al., 1995; see also above). The strong expression of neuregulin observed in the central nervous system of the perinatal animal implies additional functions in the development, maintenance or function of the brain or spinal cord. The lethality of embryos that carry mutations in the neuregulin or SF/HGF signaling systems has prevented us from investigating other additional roles, particularly the expected function in epithelial development of the mammary gland. Isoform specific mutations (Peles and Yarden, 1993; Meyer et al., 1997), as well as tissue specific or inducible ablation (Gu et al., 1994; Kühn et al., 1995) of the genes encoding factors or receptors will allow to address these question in the future. FGF’S AND THEIR RECEPTORS FUNCTION IN DEVELOPMENT OF EPITHELIA The FGF family of growth factors and receptor take over many important functions including development of limbs and skeleton (Yamaguchi and Rossant, 1995). We will discuss here in detail only the functional roles in development of epithelia. A transdominant FGF2 receptor was used to analyze the functional role of

188


this receptor which binds KGF, but also other members of the FGF family of growth factors. This receptor mutant lacks the kinase domain, forms inactive heterodimers and thus prevents cross-phosphorylation and signaling through the intact receptor. When expressed under a lung-specific promoter (surfactant promoter) in the mouse, this receptor interferes with branching of the lung epithelium and formation of alveoli. Instead, two undifferentiated tubes are formed that extend from the bifurcation of the tracheae (Peters et al., 1994). The transdominant receptor also interferes with development of the skin when expressed in keratinocytes in vivo, and prevents normal wound healing (Werner et al., 1993; Werner et al., 1994). Expressed under the control of the keratin 6 promoter which directs the transdominant receptor to the suprabasal layer of the skin, epidermal thickening and disrupted organization of epidermal keratinocytes was observed. Expressed under the control of the keratin 14 promoter which directs the transdominant receptor to undifferentiated keratinocytes in the basal level of the skin, epidermal atrophy and a severe delay and reduced capacity of wound healing is observed (cf. also article by Werner in this book). The function of FGFs and their receptors in branching morphogenesis of the lung has well conserved in evolution (Sutherland et al., 1996). The Drosophila gene branchless it is an essential gene for determining the tracheal branching pattern and encodes a homologue of mammalian FGFs. It is expressed dynamically in clusters of cells surrounding the developing tracheal system at each position where a new branch will form. Localized misexpression of branchless can direct branch formation and outgrowth to new positions (Sutherland et al., 1996). The work in Drosophila represents a clear example of the important role of a growth factor in determining branching morphogenesis and the branching patterns. THE C-RET RECEPTOR The c-ret receptor transmits signals given by GDNF (glial-derived neurotrophic factor), and requires a coreceptor for its function. Ret was identified originally as a gene able to induce malignant transformation when mutated (Takahashi et al., 1985). Mutation in c-ret have been identified in thyroid cancers and in inherited multiple endocrine neoplasia (MEN 2a and b; Grieco et al., 1990; Mulligam et al., 1993); the germline mutations found in the ret gene of MEN2A and MEN2B patients were recently demonstrated to upregulate the tyrosine kinase activity of the receptor, rendering it independent of the presence of the putative ligand (Santoro et al., 1995). The c-ret receptor is expressed in few distinct cells only: Epithelia of the meso- and metanephros (i.e. Wolffian duct and ureter) and neural crest cells and their derivatives (Pachnis et al., 1993). A targeted mutation in the c-ret gene of the mouse has demonstrated essential roles of c-ret in development of the kidney and the enteric nervous system (Schuchardt et al., 1994). In the kidney, c-ret is produced initially in the entire ureter and consequently in the very tips of the ureter bud, where branching morphogenesis occurs. The ligand, GDNF, is produced in the mesenchyme of the kidney anlage. Mice that carry homozygous mutations in c-ret or the GDNF genes have renal agenesis or severe dysgenesis, caused by lack of growth and branching of the ureter (Moore et al., 1996; Pichel et al., 1996; Sánchez et al., 1996). In addition, enteric ganglia that derive from the neural crest do not form, and as a consequence, intestinal movements are absent and the intestine does not function. Heterozygous animals are healthy (cf. also article by Schuchardt et al. in this book). Interestingly, in humans an inherited disorder of enteric ganglia, Hirschsprung disease, is caused by mutations in the c-ret gene (Romeo et al., 1994; Edery et al., 1994). Here, one copy of an intact allele is apparently not sufficient for c-ret function, and consequently Hirschsprung disease is dominantly inherited.


189

CONCLUSIONS AND FUTURE PERSPECTIVES We have discussed here the recent identifications of the physiological functions of a collection of tyrosine kinase receptors in epithelia. These receptors were originally identified as oncogenes; many of the receptors were implicated in the development of human epithelial tumors (carcinomas). The recent evidence now demonstrates that these tyrosine kinase receptors have different roles in growth or survival of cells, in morphogenesis of epithelia, but also function in other cells types like muscle precursor or neural crest cells. Particular revealing is the genetic findings of the importance of tyrosine kinase receptors, which mediate cellular interactions, for instance between mesenchyme and epithelia. The list of receptors discussed here is by no means exhaustive, and many more of such signaling systems are currently studied. In all cases discussed here, the targeted mutation have lead to embryonal or perinatal death, and thus have yet not revealed physiological roles of the signaling systems in the adult. In the future, conditional genetic alterations (Gu et al., 1994) will allow the analysis of additional functions in other development stages. REFERENCES Bellusci, S., G.Moens, G.Gaudino, P.Comoglio, T.Nakamura, J.P.Thiery, and J.Jouanneau (1994) Creation of an hepatocyte growth factor/scatter factor autocrine loop in carcinoma cells induces invasive properties associated with increased tumorigenicity. Oncogene, 9, 1091–1099. Bermingham McDonogh, O., K.L.McCabe and T.A.Reh (1996) Effects of GGF/neuregulins on neuronal survival and neurite outgrowth correlate with erbB2/neu expression in developing rat retina. Development, 122, 1427–1438. Birchmeier, C. and W.Birchmeier (1993) Molecular aspects of mesenchymal-epithelial interactions. Annu. Rev. Cell Biol., 9, 511–540. Bladt, F., D.Riethmacher, S.Isenmann, A.Aguzzi and C.Birchmeier (1995) Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud. Nature, 376, 768–671. Bottaro, D.P., J.S.Rubin, D.L.Faletto, A.M.Chan, T.E.Kmiecik, G.F.Vande Woude and S.A.Aaronson (1991) Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science, 251, 802–804. Brand-Saberi, B., T.S.Muller, J.Wilting, B.Christ and C.Birchmeier (1996) Scatter factor/ hepatocyte growth factor (SF/ HGF) induces emigration of myogenic cells at interlimb level in vivo. Dev. Biol., 179, 303–308. Brinkmann, V., H.Foroutan, M.Sachs, K.M.Weidner and W.Birchmeier (1995) Hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells. J. Cell Biol., 131, 1573–1586. Burgess, W.H. and T.Maciag (1989. The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem., 58, 575–606. Bussolino, F., M.F.Di Renzo, M.Ziche, E.Bocchietto, M.Olivero, L.Naldini, G.Gaudino, L.Tamagnone, A.Coffer and P.M.Comoglio (1992) Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J. Cell Biol., 119, 629–641. Canoll, P.D., J.M.Musacchio, R.Hardy, R.Reynolds, M.A.Marchionni and J.L.Salzer (1996) GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors. Neuron, 17, 229–243. Carraway III, K.L. and L.C.Cantley (1994) A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling. Cell, 78, 5–8. Chen, M.S., O.Bermingham McDonogh, F.T.Danehy, Jr., C.Nolan, S.S.Scherer, J.Lucas, D. Gwynne and M.A. Marchionni (1994) Expression of multiple neuregulin transcripts in postnatal rat brains. J. Comp. Neurol., 349, 389–400. Choi Lundberg, D.L. and M.C.Bohn (1995) Ontogeny and distribution of glial cell line-derived neurotrophic factor (GDNF) mRNA in rat. Brain Res. Dev. Brain Res., 85, 80– 88.

190


Corfas, G., K.M.Rosen, H.Aratake, R.Krauss and G.D.Fischbach (1995) Differential expression of ARIA isoforms in the rat brain. Neuron, 14, 103–115. Dong, Z., A.Brennan, N.Liu, Y.Yarden, G.Lefkowitz, R Mirsky and K.R.Jessen (1995) Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation and maturation of rat Schwann cell precursors. Neuron, 15, 585–596. Durbec, P., C.V.Marcos Gutierrez, C.Kilkenny, M.Grigoriou, K.Wartiowaara, P.Suvanto, D.Smith, B.Ponder, F.Costantini and V.Pachnis (1996) GDNF signalling through the Ret receptor tyrosine kinase. Nature, 381, 789–793. Ebens, A., K.Brose, E.D.Leonardo, M.G.Hanson, Jr., F.Bladt, C.Birchmeier, B.A.Barres and M.Tessier Lavigne (1996) Hepatocyte growth factor/scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons. Neuron, 17, 1157– 1172. Edery, P., S.Lyonnet, L.M.Mulligan, A.Pelet, E.Dow, L.Abel, S.Holder, C.Nihoul Fekete, B.A.Ponder and A.Munnich (1994) Mutations of the RET proto-oncogene in Hirschsprung’s disease. Nature, 367, 378–380. Ekblom, P. (1992) Renal Development. In The Kidney: Physiology and Pathophysiology. D.W. Seldin and G.Giebisch, editors. Raven Press, Ltd. New York. 475–501. Falls, D.L., K.M.Rosen, G.Corfas, W.S.Lane and G.D.Fischbach (1993) ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family. Cell, 72, 801–815. Ferracini, R., M.F.Di Renzo, K.Scotlandi, N.Baldini, M.Olivero, P.Lollini, O.Cremona, M.Campanacci and P.M.Comoglio (1995) The Met/HGF receptor is over-expressed in human osteosarcomas and is activated by either a paracrine or an autocrine circuit. Oncogene, 10, 739–749. Fixman, E.D., M.A.Naujokas, G.A.Rodrigues, M.F.Moran and M.Park (1995) Efficient cell transformation by the TprMet oncoprotein is dependent upon tyrosine 489 in the carboxy-terminus. Oncogene, 10, 237–249. Frixen, U.H., J.Behrens, M.Sachs, G.Eberle, B.Voss, A.Warda, D.Lochner and W. Birchmeier (1991) E-cadherinmediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J. Cell Biol., 113, 173–185. Gassmann, M., F.Casagranda, D.Orioli, H.Simon, C.Lai, R.Klein and G.Lemke (1995) Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature, 378, 390–394. Grant, D.S., H.K.Kleinman, I.D.Goldberg, M.M.Bhargava, B.J.Nickoloff, J.L.Kinsella, P.Polverini and E.M.Rosen (1993) Scatter factor induces blood vessel formation in vivo. Proc. Natl. Acad. Sci. USA, 90, 1937–1941. Grieco, M., M.Santoro, M.T.Berlingieri, R.M.Melillo, R.Donghi, I.Bongarzone, M.A. Pierotti, G.Delia Porta, A.Fusco and G.Vecchio (1990) PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell, 60, 557–563. Gu, H., J.D.Marth, P.C.Orban, H.Mossmann and K.Rajewsky (1994) Deletion of a DNA polymerase beta gene segment in T cells using cell type-specific gene targeting. Science, 265, 103–106. Hay, E.D. (1990) Role of cell-matrix contacts in cell migration and epithelial-mesenchymal transformation. Cell Differ. Dev., 32, 367–375. Hayashi, K. and E.Ozawa (1995) Myogenic cell migration from somites is induced by tissue contact with medial region of the presumptive limb mesoderm in chick embryos. Development, 121, 661–669. Heymann, S., M.Koudrova, H.Arnold, M.Koster and T.Braun (1996) Regulation and function of SF/HGF during migration of limb muscle precursor cells in chicken. Dev. Biol., 180, 566–578. Holgado-Madruga, M., D.R.Emlet, D.K.Moscatello, A.K.Godwin and A.J.Wong (1996) A Grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature, 379, 560–564. Holmes, W.E., M.X.Sliwkowski, R.W.Akita, W.J.Henzel, J.Lee, J.W.Park, D.Yansura, N. Abadi, H. Raab, G.D. Lewis et al. (1992) Identification of heregulin, a specific activator of p185erbB2. Science, 256, 1205–1210. Jo, S.A., X.Zhu, M.A.Marchionni and S.J.Burden (1995) Neuregulins are concentrated at nerve-muscle synapses and activate ACh-receptor gene expression. Nature, 373, 158–161. Johnson, D.E. and L.T.Williams (1993) Structural and functional diversity in the FGF receptor multigene family. Adv. Cancer Res., 60, 1–41. Joseph, A., G.H.Weiss, L.Jin, A.Fuchs, S.Chowdhury, P.O’Shaugnessy, I.D.Goldberg and E.M.Rosen (1995) Expression of scatter factor in human bladder carcinoma. J. Natl. Cancer Inst., 87, 372–377.


191

Kühn, R., F.Schwenk, M.Aguet and K.Rajewsky (1995) Inducibie gene targeting in mice. Science, 269, 1427–1429. Lee, K.F., H.Simon, H.Chen, B.Bates, M.C.Hung and C.Hauser (1995) Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature, 378; 394–398. Marchionni, M.A., A.D.Goodearl, M.S.Chen, O.Bermingham McDonogh, C.Kirk, M. Hendricks, F.Danehy, D.Misumi, J.Sudhalter, K.Kobayashi et al (1993) Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system. Nature, 362, 312–318. Meiners, S., V.Brinkmann, H.Naundorf and W.Birchmeier (1998) Role of morphogenetic factors in metastasis of mammary carcinoma cells. Oncogene, 16, 9–20. Meyer, D. and C.Birchmeier (1994) Distinct isoforms of neuregulin are expressed in mesenchymal and neuronal cells during mouse development. Proc. Natl. tl. Acad. Sci. USA, 91, 1064–1068. Meyer, D. and C.Birchmeier (1995) Multiple essential functions of neuregulin in development. Nature, 378, 386–390. Meyer, D., T.Yamaai, A.Garratt, E.RiethmacherSonnenberg, D.Kane, L.E.Theill and C. Birchmeier (1997) Isoformspecific expression and function of neuregulin. Development, 124, 3575–3586. Miyazawa, K., H.Tsubouchi, D.Naka, K.Takahashi, M.Okigaki, N.Arakaki, H.Nakayama, S.Hirono, O.Sakiyama et al. (1989) Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem. Biophys. Res. Commun., 163, 967–973. Montesano, R., K.Matsumoto, T.Nakamura and L.Orci (199 la) Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell, 67, 901–908. Montesano, R., G.Schaller and L.Orci (1991b) Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell, 66, 697–711. Morrissey, T.K., A.D.Levi, A.Nuijens, M.X.Sliwkowski and R.P.Bunge (1995) Axon-induced mitogenesis of human Schwann cells involves heregulin and p185erbB2. Proc. Natl. Acad. Sci. USA, 92, 1431–1435. Mulligan, L.M., J.B.Kwok, C.S.Healey, M.J.Elsdon, C.Eng, E.Gardner, D.R.Love, S.E. Mole, J.K.Moore, L.Papi et al. (1993) Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature, 363, 458–460. Nakamura, T., T.Nishizawa, M.Hagiya, T.Seki, M.Shimonishi, A.Sugimura, K.Tashiro and S.Shimizu (1989) Molecular cloning and expression of human hepatocyte growth factor. Nature, 342, 440–443. Naldini, L., K.M.Weidner, E.Vigna, G.Gaudino, A.Bardelli, C.Ponzetto, R.P.Narsimhan, G.Hartmann, R.Zarnegar, G.K.Michalopoulos, W.Birchmeier and P.M.Comoglio (1991) Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J., 10, 2867–2878. Niemann, C., V.Brinkmann, E.Spitzer, G.Hartmann, M.Sachs, H.Naundorf and W. Birchmeier (1998) Reconstruction of mammary gland development in vitro: Requirement of c-met and c-erbB2 signalling for branching and alveolar morphogenesis. J. Cell Biol., in press. Orr-Urtreger, A., L.Trakhtenbrot, R.Ben Levy, D.Wen, G.Rechavi, P.Lonai and Y.Yarden (1993) Neural expression and chromosomal mapping of Neu differentiation factor to 8p12–p21. Proc. Natl. Acad. Sci. U.S. A., 90, 1867–1871. Pachnis, V., B.Mankoo and F.Costantini (1993) Expression of the c-ret proto-oncogene during mouse embryogenesis. Development, 119, 1005–1017. Peles, E., S.S.Bacus, R.A.Koski, H.S.Lu, D.Wen, S.G.Ogden, R.B.Levy and Y.Yarden (1992) Isolation of the neu/ HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells. Cell, 69, 205–216. Peles, E. and Y.Yarden (1993) Neu and its ligands: from an oncogene to neural factors. Bioessays, 15, 815–824. Peters, K., S.Werner, X.Liao, S.Wert, J.Whitsett and L.Williams (1994) Targeted expression of a dominant negative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. EMBO J., 13, 3296–3301. Pichel,J.G., L.Shen, H.Z.Sheng, A.C.Granholm, J.Drago, A.Grinberg, E.J.Lee, S.P.Huang, M.Saarma, B.J.Hoffer, H.Sariola and H.Westphal (1996) Defects in enteric innervation and kidney development in mice lacking GDNF. Nature, 382, 73–76.

192


Ponzetto, C., A.Bardelli, Z.Zhen, F.Maina, P.dalla Zonca, S.Giordano, A.Graziani, G. Panayotou and P.M.Comoglio (1994) A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/ scatter factor receptor family. Cell, 77, 261–271. Riethmacher, D., E.Sonnenberg-Riethmacher, V.Brinkmann, T.Yamaai, G.R.Lewin and C. Birchmeier (1997) Severe neuropathies in mice with targeted mutations in the ErbB3 receptor. Nature, 389, 725–730. Rodriguez Boulan, E. and W.J.Nelson (1989) Morphogenesis of the polarized epithelial cell phenotype. Science, 245, 718–725. Romeo, G., P.Ronchetto, Y.Luo, V.Barone, M.Seri, I.Ceccherini, B.Pasini, R.Bocciardi, M.Lerone, H.Kääriainen and G.Martucciello (1994) Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung’s s disease. Nature, 367, 377–378. Rong, S., L.A.Donehower, M.F.Hansen, L.Strong, M.Tainsky, M.Jeffers, J.H.Resau, E. Hudson, I.Tsarfaty and G.F.Vande Woude (1995) Met proto-oncogene product is overexpressed in tumors of p53—deficient mice and tumors of Li-Fraumeni patients. Cancer Res, 55, 1963–1970. Rong, S., M.Oskarsson, D.Faletto, I.Tsarfaty, J.H.Resau, T.Nakamura, E.Rosen, R.F. Hopkins,3d and G.F.Vande Woude (1993) Tumorigenesis induced by coexpression of human hepatocyte growth factor and the human met protooncogene leads to high levels of expression of the ligand and receptor. Cell Growth Differ., 4, 563–569. Rosen, E.M., J.Knesel, I.D.Goldberg, L.Jin, M.Bhargava, A.Joseph, R.Zitnik, J.Wines, M.Kelley and S.Rockwell (1994) Scatter factor modulates the metastatic phenotype of the EMT6 mouse mammary tumor. Int. J. Cancer, 57, 706–714. Sachs, M., K.M.Weidner, V.Brinkmann, I.Walther, A.Obermeier, A.Ullrich and W. Birchmeier (1996) Motogenic and morphogenic activity of epithelial receptor tyrosine kinases. J. Cell Biol., 133, 1095–1107. Sanchez, M.P., I.Silos Santiago, J.Frisen, B.He, S.A.Lira and M.Barbacid (1996) Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature, 382, 70–73. Santoro, M., F.Carlomagno, A.Romano, D.P.Bottaro, N.A.Dathan, M.Grieco, A.Fusco, G.Vecchio, B.Matoskova, M.H.Kraus and et al. (1995) Activation of RET as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science, 267, 381– 383. Schmidt, C., F.Bladt, S.Goedecke, V.Brinkmann, W.Zschiesche, M.Sharpe, E.Gherardi and C.Birchmeier (1995) Scatter factor/hepatocyte growth factor is essential for liver development. Nature, 373, 699–702. Schuchardt, A., V.D’Agati, L.Larsson Blomberg, F.Costantini and V.Pachnis (1994) Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature, 367, 380–383. Schwarz, M.A., K.Owaribe, J.Kartenbeck and W.W Franke (1990) Desmosomes and hemidesmosomes: constitutive molecular components. Annu. Rev. Cell Biol., 6, 461–491. Shah, N.M., M.A.Marchionni, I.Isaacs, P.Stroobant and D.J.Anderson (1994) Glial growth factor restricts mammalian neural crest stem cells to a glial fate. Cell, 77, 349–360. Sonnenberg, E., A.Godecke, B.Walter, F.Bladt and C.Birchmeier (1991) Transient and locally restricted expression of the rosl protooncogene during mouse development. EMBO J., 10, 3693–3702. Sonnenberg, E., D.Meyer, K.M.Weidner and C.Birchmeier (1993) Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development. J. Cell Biol., 123, 223– 335. Soriano, J.V., M.S.Pepper, T.Nakamura, L.Orci and R.Montesano (1995) Hepatocyte growth factor stimulates extensive development of branching duct-like structures by cloned mammary gland epithelial cells. J. Cell Set., 108, 413–420. Stoker, M., E.Gherardi, M.Ferryman and J.Gray (1987) Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature, 327, 239–242. Sutherland, D., C.Samakovlis and M.A Krasnow (1996) branchless encodes a Drosophila FGF homolog that controls tracheal cell migration and the pattern of branching. Cell, 87, 1091–1101. Takahashi, M., J.Ritz and G.M.Cooper (1985) Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell, 42, 581–588. Timpl, R. (1989) Structure and biological activity of basement membrane proteins. Eur. J. Biochem., 180, 487–502.


193

Trachtenberg, J.T. and W.J.Thompson (1996) Schwann cell apoptosis at developing neuromuscular junctions is regulated by glial growth factor. Nature, 379, 174–177. Treanor, J.J., L.Goodman, F.de Sauvage, D.M.Stone, K.T.Poulsen, C.D.Beck, C.Gray, M.P. Armanini, R.A.Pollock, F.Hefti, H.S.Phillips, A.Goddard, M.W.Moore, A.Buj Bello, A.M.Davies, N.Asai, M.Takahashi, R.Vandlen, C.E.Henderson and A.Rosenthal (1996) Characterization of a multicomponent receptor for GDNF. Nature, 382, 80–83. Trupp, M., M.Ryden, H.Jornvall, H.Funakoshi, T.Timmusk, E.Arenas and C.F.Ibanez (1995) Peripheral expression and biological activities of GDNF, a new neurotrophic factor for avian and mammalian peripheral neurons. J. Cell Biol., 130, 137–148. Uehara, Y., O.Minowa, C.Mori, K.Shiota, J.Kuno, T.Noda and N.Kitamura (1995) Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor. Nature, 373, 702–705. Vega, Q.C., C.A.Worby, M.S.Lechner, J.E.Dixon and G.R.Dressler (1996) Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis. Proc. Natl. Acad. Set. USA, 93, 10657–10661. Weidner, K.M., J.Behrens, J.Vandekerckhove and W.Birchmeier (1990) Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell Biol., 111,2097–2108. Weidner, K.M., N.Arakaki, G.Hartmann, J.Vandekerckhove, S.Weingart, H.Rieder, C. Fonatsch, H.Tsubouchi, T.Hishida, Y.Daikuhara and W.Birchmeier (1991) Evidence for the identity of human scatter factor and human hepatocyte growth factor. Proc. Natl. Acad. Sci. USA, 88, 7001–7005. Weidner, K.M., M.Sachs, D.Riethmacher and W.Birchmeier (1995) Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells. Proc. Natl. Acad. Sci. U.S. A., 92, 2597–2601. Weidner, K.M., S.Di Cesare, M.Sachs, V.Brinkmann, J.Behrens and W.Birchmeier (1996) Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature, 384, 173–176. Werner, S., W.Weinberg, X.Liao, K.G.Peters, M.Blessing, S.H.Yuspa, R.L.Weiner and L.T.Williams (1993) Targeted expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J., 12, 2635–2643. Werner, S., H.Smola, X.Liao, M.T.Longaker, T.Krieg, P.H.Hofschneider and L.T.Williams (1994) The function of KGF in morphogenesis of epithelium and reepithelialization of wounds. Science, 266, 819–822. Yamaguchi, T.P. and J.Rossant (1995) Fibroblast growth factors in mammalian development. Curr. Opin. Genet. Dev., 5, 485–491. Yamashita, J., M.Ogawa, S.Yamashita, K.Nomura, M.Kuramoto, T.Saishoji and S.Shin (1994) Immunoreactive hepatocyte growth factor is a strong and independent predictor of recurrence and survival in human breast cancer. Cancer Res., 54, 1630–1633. Yang, Y., E.Spitzer, D.Meyer, M.Sachs, C.Niemann, G.Hartmann, K.M.Weidner, C. Birchmeier and W.Birchmeier (1995) Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland. J. Cell Biol., 131, 215–226.

11. THE FUNCTION OF FIBROBIAST GROWTH FACTORS IN SKIN MORPHOGENESIS AND WOUND REPAIR SABINE WERNER Max-Planck-Institut fur Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Tel.: 089–8578–2269; Fax: 089–8578–2814

FIBROBIAST GROWTH FACTORS AND THEIR RECEPTORS Fibroblast growth factors (FGFs) comprise a growing group of structurally similar polypeptide mitogens which currently includes 18 different members. Besides the initially discovered acidic FGF (aFGF, FGF-1) (Jaye et al., 1986) and basic FGF (bFGF, FGF-2) (Abraham et al., 1986), the family also includes the products of the proto-oncogenes int-2 (FGF-3), (Moore et al., 1986), hst (FGF-4) (Delli-Bovi et al., 1987), FGF-5 (Zhan et al., 1988), and FGF-6 (Maries et al., 1989), as well keratinocyte growth factor (KGF, FGF-7) (Rubin et al., 1989), androgen-induced growth factor (AIGF, FGF-8) (Tanaka et al., 1992), gliaactivating factor (GAF, FGF-9) (Miyamoto et al., 1993), four fibroblast growth factor homologous factors (FHFs, FGFs 11–14) (Smallwood et al., 1996), FGF-15 (McWhirter et al., 1997), FGF-16 (Miyake et al., 1998), FGF-17 (Hoshikawa et al., 1998), and FGF-18 (Ohbayashi et al., 1998). All members of the FGF family are between 155 and 268 amino acids in length and share a conserved central region of approximately 140 amino acids. They all bind heparin, heparan sulfate proteoglycans and glycosaminoglycans and strongly concentrate in the extracellular matrix (for review see Basilico and Moscatelli, 1992). Most of the FGFs have a classical signal sequence which is located at the aminoterminus or—in the case of FGF-9—in the middle of the protein. These FGFs are therefore efficiently secreted via the endoplasmic reticulum-Golgi secretory pathway. By contrast, aFGF and bFGF, as well as the recently identified fibroblast growth factor homologous factors lack a classical signal sequence and, therefore, are inefficiently secreted. Most members of the FGF family have a very broad mitogenic spectrum (reviewed by Basilico and Moscatelli, 1992). Thus they stimulate proliferation of a variety of cells of mesodermal, ectodermal and also endodermal origin. Furthermore, they can act as neurotrophic and angiogenic factors in vivo (Folkman and Klagsbrun, 1987; Morrison et al., 1986; Walicke et al., 1986). The only known exception is KGF which is a highly specific mitogen for different types of epithelial cells (Rubin et al., 1989; Finch et al., 1989). In contrast to many other growth factors, FGFs are not only mitogenic, but also have the capacity to modify differentiation processes in vitro and in vivo (for review see Basilico and Moscatelli, 1992). Finally, bFGF can protect various types of neurons in vivo (Anderson et al., 1988; Peterson et al., 1996), and keratinocyte growth factor has been shown to exert a protective effect on epithelial cells (Panos et al., 1995), presumably by induction of enzymes which can detoxify reactive oxygen species (Panos et al., 1995; Frank et al., 1997). The biologic effects of flbroblast growth factors are mediated via three different types of binding partners: heparan sulphate oligosaccharides (reviewed by Basilico and Moscatelli, 1992), a low-affinity, cysteine-rich transmembrane FGF-binding protein (Burrus et al., 1992), and four high-affinity transmembrane FGF


195

receptors of the tyrosine kinase family (reviewed by Johnson and Williams, 1993). The FGF receptors are transmembrane protein tyrosine kinases with either two or three immunoglobulin-like domains and a highly acidic region in the extracellular part of the receptor. Binding to these receptors results in receptor oligomerization and initiation of the intracellular signal transduction cascade (reviewed by Johnson and Williams, 1993). A series of studies from different laboratories has shed light on the complexity of the FGF/FGF receptor binding pattern. It has been demonstrated that different members of the FGF family bind to the four known FGF receptors (FGFR1-FGFR4) with different affinities. Whereas KGF, for example, only binds to a splice variant of FGFR2, aFGF has been shown to bind to all known FGF receptors with high affinity (summarized by Ornitz et al., 1996). Additional complexity in the FGF/ FGFR binding pattern is generated by alternative splicing in the extracellular domain. It has been shown that the use of alternative exons for the carboxyterminal half of the third immunoglobulin-like domain of FGF receptors 1, 2, and 3 generates receptor variants with different ligand-binding specificities (Werner et al., 1992a; Miki et al., 1992; Chellaiah et al., 1994). These alternative exons were designated IIIa, Illb and IIIc. The Illb receptor variant of FGFR2 is of particular importance, since it is the only type of FGF receptor which binds KGF (Miki et al., 1992). Whereas aFGF and bFGF are widely expressed in many embryonic and adult tissues and organs, the other FGFs have a much more limited tissue distribution, especially in the adult organism. Thus expression of FGF-3 and FGF-4 has not been detected in normal adult mice, FGF-5 is only found in the brain and in hair follicles (Haub et al., 1990; Hebert et al., 1994), and FGF-6 is specifically expressed in the adult muscle (deLapeyriere et al., 1990), By contrast, all known FGFs are expressed at multiple sites during embryonic development. Most importantly, a highly tissue-specific expression during organogenesis has been found for all members of the FGF family, suggesting that they could act as differentiation signals at specific steps in development (Wilkinson et al., 1988, 1989; Gonzalez et al., 1990; Hebert et al., 1991; Haub and Goldfarb, 1991; Niswander and Martin, 1992; deLapeyriere et al., 1993; Han and Martin, 1993; Mason et al., 1994; Crossley and Martin, 1995; MacArthur et al., 1995; Yamasaki et al., 1996; Smallwood et al., 1996). The broad expression pattern of the FGFs during mouse organogenesis correlates with the expression pattern of the FGF receptors, and expression of the four FGF receptors has been found in many embryonic tissues (Orr-Urtreger et al., 1991; Stark et al., 1991; Peters et al., 1992; Peters et al., 1993; Orr-Urtreger et al., 1993). Each of the FGFR genes has a unique pattern of expression, indicating that each receptor has specific functions during organogenesis. EXPRESSION OF FIBROBLAST GROWTH FACTORS AND THEIR RECEPTORS IN THE DEVELOPING AND ADULT SKIN To date, a series of data are available demonstrating expression of FGFs and FGF receptors at specific sites in the developing and adult skin (summarized in Table 1). Most of these studies were carried out with mouse tissue, but the few results which were obtained with rat, human and ovine tissue most often correlate with the available mouse data. As shown in Table 1, all four FGF receptors as well as aFGF, bFGF, FGF-5, KGF and FGF-10 have been detected in the developing and adult skin, whereby FGF-5 and FGFR4 expression has only been found in hair follicles. In interfollicular skin, aFGF mRNA could be detected in the dermis and epidermis of mouse tail skin (Werner et al., 1993). In addition, aFGF protein was found in the epidermis of human and ovine skin (du Cros et al., 1993; Hughes and Hall, 1993). A series of immunohistochemical studies demonstrated the presence of bFGF protein in the basement membrane in

196


various species but also in different layers of the epidermis (Gonzalez et al., 1990; duCros et al., 1993; Hughes and Hall, 1993). Finally, KGF and FGF-10 mRNAs were found in the dermis of embryonic (Figure 1) and adult skin (Werner et al., 1992b; 1993; Beer et al., 1997). In addition to the ligands, FGFR1, FGFR2 and also FGFR3 have been detected in the dermis and epidermis of various species. Whereas FGFR1 is predominantly expressed in the developing and adult dermis, FGFR2 transcripts are most abundant in the epidermis (Wanaka et al., 1991; Orr-Urtreger et al., 1991, 1993; Peters et al., 1992; Werner et al., 1992b). Interestingly, the two different splice variants of FGFR2 are differentially expressed in the developing skin. Whereas FGFR2-IIIc expression is restricted to the dermis and the loose mesenchyme, the KGF receptor variant FGFR2-IIIb is exclusively expressed in keratinocytes (Orr-Urtreger et al., 1993). This differential expression is demonstrated in Figure 1. In addition to interfollicular skin, FGFs and their receptors are expressed at various places in the hair follicle, whereby each gene has a unique expression pattern (Orr-Urtreger et al., 1991, 1993; Peters et al., 1992; duCros et al., 1993; Hughes and Hall, 1993; Hebert et al., 1994 (see Figure 4b), Danilenko et al., 1995a; Rosenquist and Martin, 1996). Interestingly, expression of FGFs and FGF receptors is strongly dependent on the stage of the hair growth cycle, suggesting multiple roles of FGFs in the regulation of hair growth (Rosenquist and Martin, 1996). EXPRESSION OF FIBROBLAST GROWTH FACTORS AND THEIR RECEPTORS DURING WOUND REPAIR Given the potent mitogenic and chemotactic activity of FGFs for many skin-derived cells (see below), researchers have long speculated on a possible role of FGFs in wound repair. This hypothesis was supported by a series of studies which demonstrated a significant stimulation of the healing process by locally applied FGFs, particularly in animals with wound healing defects (reviewed by Abraham and Klagsbrun, 1996). Therefore, we speculated on a possible role of endogenous FGFs in wound repair. To address this question, we determined the FGF and FGFR mRNA expression levels in normal skin and in wound tissue of fullthickness Table 1 Expression of FGFs and FGF receptors in developing and adult skin aFGF: • Dermal and epidermal compartments of adult mouse tail skin (RNase protection assay: Werner et al., 1993) • Basal keratinocytes, follicle cell aggregations of the developing (76d) ovine skin (Immunofluorescence: du Cros et al., 1993) • Epidermis, proximal regions of the pilary canal, suprabulbar cells and keratinocytes of the keratogenous zone of the wool follicle of adult ovine skin (Immunofluorescence: du Cros et al., 1993) • Suprabasal layers of the epidermis, media of arterioles and venules of adult human skin (Immunohistochemistry: Hughes and Hall, 1993) bFGF: • Stratum granulosum of the epidermis, cell-dense areas of the dermis, basement membranes of embryonic (18d) rat skin (Immunohistochemistry: Gonzalez et al., 1990) • Dermal compartment of adult mouse tail skin (RNase protection assay: Werner et al., 1993) • Intermediate layer and periderm of the epidermis, dermal-epidermal junction and basement-membrane zone of the follicles of the developing (76d) ovine skin (Immunofluorescence: du Cros et al., 1993) • Suprabasal cells of the epidermis, basement membrane zone adjacent to the outer root sheath and the bulb matrix of adult ovine skin (Immunofluorescence: du Cros et al., 1993)


197

•

Basal layer of the epidermis, sweat and sebaceous glands, hair follicles, and blood vessels of adult human skin (Immunohistochemistry: Schulze-Osthoff et al., 1990; Hughes and Hall, 1993) FGF-3 (int-2), FGF-4 (hst): not detected FGF-5: • Dermal compartment of mouse tail skin (RNase protection assay: Werner et al., 1993) • Outer root sheath of anagen hair follicles of mouse back skin (In situ hybridization: Hebert et al., 1994) FGF-6: not detected FGF-7 (KGF): • Dermal compartment of adult mouse back skin and tail skin (In situ hybridization: Werner et al., 1992; RNase protection assay: Werner et al., 1993) • Dermis of adult human skin (RNase protection assay: S. Werner, unpublished data) • Dermis of embryonic (d14.5 and 15.5) mouse skin (In situ, hybridization: Mason et al., 1994) • Dermal papilla of the hair follicles of embryonic (d.17.5 and 20.5) and newborn rat back skin (In situ hybridization: Danilenko et al., 1995a) • Dermal papilla of the hair follicles of adult mouse back skin (In situ hybridization: Rosenquist and Martin, 1996) FGF-8, FGF-9: not detected FGFR1: • Compact mesenchyme of the developing (17d) rat skin (In situ hybridization: Wanaka et al., 1991) • Mesenchyme of the developing mouse skin (9.5–12.5d) (In situ hybridization: Orr-Urtreger et al., 1991; Peters et al., 1992) • Dermal mesenchymal cells just below the epidermis and dermal papilla cells of the hair follicles of the 14.5 and 16.5d mouse embryo (In situ hybridization: Orr-Urtreger et al., 1991; Peters et al., 1992) • Dermal compartment and (at lower levels) epidermal compartment of mouse tail skin (RNase protection assay: Werner et al., 1993) • Microvasculature of adult human skin (Immunohistochemistry: Hughes and Hall, 1993) • Dermal papilla of the anagen hair follicles of adult mouse skin (In situ hybridization: Rosenquist and Martin, 1996) FGF-10: • Dermal compartment of adult mouse tail skin (RNase protection assay: Beer et al., 1997) FGFR2: • Surface ectoderm of the developing mouse skin (d9.5–12.5) (In situ hybridization: Orr-Urtreger et al., 1991; Peters et al., 1992) • Basal and lower suprabasal layers of the epidermis, hair bulb and epithelial root sheath of the hair follicles of 14.5 and 16.5d mouse skin (In situ hybridization: Orr-Urtreger et al., 1991; Peters et al., 1992) • Epidermis of the adult mouse back and tail skin (KGF receptor variant=FGFR2-IIIb) (In situ hybridization: Werner et al., 1992; RNase protection assay: Werner et al., 1993, Immunohistochemistry: S. Werner, unpublished data) • Surface ectoderm of the mouse embryo (d10 and d12) and periderm of the embryonic mouse epidermis (d14.5) (KGF receptor variant) (In situ hybridization: Orr-Urtreger et al., 1993) • Dermis and loose mesenchyme of embryonic (d14.5) mouse skin (FGFR2-IIIc) (In situ hybridization: OrrUrtreger et al., 1993) • Suprabasal cells of the epidermis of adult human skin (KGF receptor variant) (Immunofluorescence: LaRochelle et al., 1995; Marchese et al., 1995)

198


•

Basal keratinocytes and hair follicle keratinocytes of embryonic (d17.5 and 20.5) and newborn rat skin (KGF receptor variant=FGFR2-IIIb) (In situ hybridization: Danilenko et al., 1995a) • Hair matrix cells from the base of the follicle to the top of the hair bulb, adjacent to the dermal papilla of adult mouse back skin (In situ hybridization: Rosenquist and Martin, 1996) FGFR3: • Dermal (particularly FGFR3-IIIc) and epidermal (particularly FGFR3-IIIb) compartments of adult mouse tail skin (RNase protection assay: Werner et al., 1993) • Hair bulb of the follicles of adult mouse back skin (In situ hybridization: Rosenquist and Martin, 1996) FGFR4: • Inner and outer root sheath of the hair follicle neck and hair matrix in adult mouse back skin (In situ hybridization: Rosenquist and Martin, 1996) • Not expressed in skin of 17–18 week human fetus (Partanen et al., 1991)

excisional mouse wounds at various time points after injury. We thereby found expression of aFGF, bFGF, FGF-5, KGF, FGF-10, FGFR1, FGFR2, FGFR3, and FGFR4 in normal skin, whereas transcripts encoding FGF-3, FGF-4, FGF-8 and FGF-9 could not be detected (Werner et al., 1992b; S. Werner, unpublished data; Beer et al., 1997). After injury, no significant regulation of the FGF receptors was observed. However, aFGF, bFGF, FGF-5 and particularly KGF were expressed at significantly higher levels in the wounded tissue compared to non-wounded skin (summarized in Table 2). The most striking induction was obtained for KGF. Thus at day 1 after injury KGF mRNA levels were more than 150-fold higher compared to the basal levels (Werner et al., 1992b). In situ hybridization and immunohistochemical staining of the wound tissue demonstrated the presence of KGF mRNA in dermal fibroblasts below the wound and at the wound edge, whereas KGF receptor transcripts and also the corresponding protein were exclusively detected in keratinocytes of the epidermis and the hair follicles (Werner et al., 1992b; S. Werner, unpublished data). These cells migrate into the wound and subsequently proliferate, which finally leads to complete wound reepithelialization (reviewed by Clark, 1991). This expression pattern of KGF and its receptor suggested that Table 2 mRNA Expression of FGFs and FGF receptors in mouse skin and in full-thickness excisional wounds (Werner et al., 1992b; S. Werner, unpublished data; Beer et al., 1997) FGF-1 FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF- 10 FGFR1 FGFR2 FGFR3

skin

1d wound

5d wound

(+) (+) – – (+) – + – – + ++ ++ +

++ + – – + – +++ – – + ++ ++ +

+ ++ – – (+) – +++ – – + ++ ++ ++


skin

1d wound

199

5d wound

FGFR4 (+) (+) (+) Expression levels: -: not detectable; (+): low; +: medium; + + : strong; + + + : very strong

Figure 1 Comparison of FGF-7 (KGF), FGFR-IHc and FGFR2-IIIb expression in skin, bone and muscle of the developing mouse embryo by in situ hybridization. A and D: bright field illumination, B, C, E, F and G: dark field illumination. A-C: tongue and mandibula, 15.5 days p.c., B: hybridization with FGFR2-IIIb (KGFR); C: hybridization with FGF-7 (KGF). D-G: Cross-section at the level of the forelimbs, 15.5 days p.c., E: Hybridization with FGFR2-IIIb (KGFR), F: hybridization with FGF-7 (KGF), G: hybridization with FGFR2-IIIc. Note the specific expression of FGFR2-IIIb in the epithelium and the specific expression of FGFR2-IIIc in the mesenchyme. Abbreviations: am, abdominal muscles; bi, biceps humeri; ha, heart; hu, humerus, lu, lung; mo, molar tooth; sb, sternebrae; sd, salivary duct; se, surface ectoderm; to, tongue. Size bars: A, 300 mm; D, 1mm. This figure shows work carried out in Dr. Peter Lonai’s laboratory, Weizmann Institute, Israel, and was kindly provided by Dr. Peter Lonai.

200


Figure 2 Expression of KGF in normal and wounded human skin. RNA was isolated from human non-wounded skin and from excisional wounds. 10 µg total cellular RNA were analyzed by RNase protection assay for the presence of KGF mRNA. The time after injury is indicated above each lane. 30 µg tRNA were used as a negative control. 1000 cpm of the radiolabeled antisense riboprobe were loaded in the lane labeled “probe” and used as a size marker.

dermally derived KGF could stimulate wound reepithelialization in a paracrine manner. Increased KGF expression was subsequently also observed in human excisional wounds (Marchese et al., 1995) and incisional wounds (Figure 2; S.Werner, unpublished data). Marchese et al. (1995) also studied the distribution of functional KGF receptors in the wound using a KGF-immunoglobulin G fusion protein. Interestingly, a striking decrease in receptor protein expression was found during the intermediate period of reepithelialization, although KGF receptor transcripts were abundant in the keratinocytes at the wound edge. These results suggested functional receptor downregulation in the presence of increased KGF. Taken together, these wound healing studies suggested an important role of KGF in wound reepithelialization. This hypothesis was supported by two follow-up studies from our laboratory which demonstrated reduced KGF expression during wound repair in healing-impaired animals (Werner et al., 1994a; Brauchle et al., 1995). TRANSGENIC MOUSE STUDIES TO ASSESS FGF FUNCTION IN THE SKIN Given the broad expression pattern of FGFs and FGF receptors in the developing and adult skin, researchers from many different fields have become interested in the function of FGFs in this tissue. Until recently, only in vitro data were available which demonstrated the mitogenic and in some cases also chemotactic effects of various FGFs on skin-derived cells, including fibroblasts, keratinocytes, melanocytes and microvascular endothelial cells (reviewed by Pittelkow et al., 1991; Basilico and Moscatelli, 1992). Although these tissue culture studies suggested an important role of FGFs in the skin, the in vivo function of FGFs in this tissue has long been unclear. During the past few years, transgenic mouse studies have revealed exciting and unexpected roles of FGFs in skin development and wound repair. These studies include gain of function experiments (overexpression of FGFs in the skin) but also loss of function experiments such as gene knock-


201

outs by homologous recombination in embryonic stem cells and targeted overexpression of dominantnegative FGF receptor mutants. The results of these experiments demonstrate an important function of FGF-5 and KGF in hair follicle morphogenesis, a novel role of FGF in keratinocyte organization and differentiation, and a role of keratinocyte growth factor receptor ligands in keratinocyte proliferation and wound reepithelialization (summarized in Table 3). Expression of a Dominant Negative FGF Receptor in Suprabasal Layers of the Epidermis of Transgenic Mice As summarized above, aFGF, bFGF as well as several FGF receptors are present in the epidermis. Furthermore, several members of the FGF family have been shown to be mitogenic for keratinocytes (Shipley et al., 1989; Rubin et al., 1989). Therefore, we speculated about a possible role of FGFs in keratinocyte proliferation, differentiation and/or organization. To test this possibility, we intended to block FGFR function in the epidermis of transgenic mice. However, a knockout experiment was obviously not suitable for this purpose, since FGF receptors have a broad tissue distribution and targeted disruption of these genes is likely to cause early Table 3 FGF/FGF receptor transgenic mice with skin phenotypes 1.

2.

3.

4. 5. 6.

Overexpression of KGF in basal keratinocytes of the epidermis (Guo et al., 1993). Phenotype: Epidermal hyperthickening, increased proliferation of basal keratinocytes, defects in keratinocyte differentiation, suppression of hair follicle morphogenesis and adipogenesis, gross transformations in the epidermis with age. Expression of a dominant-negative FGFR1 in suprabasal keratinocytes of the epidermis (Werner et al., 1993) Phenotype: Epidermal hyperthickening, hyperproliferation of basal keratinocytes, defects in keratinocyte organization and differentiation. Expression of a dominant-negative KGF receptor in basal keratinocytes of the epidermis (Werner et al., 1994b) Phenotype: Epidermal atrophy, pycnotic nuclei of the basal cells, reduced number of hair follicles, defects in hair follicle morphology, severely reduced wound reepithelialization. FGF-5 knockout (Hebert et al., 1994) Phenotype: Abnormally long hair. Allelic with go (angora) mutation. FGF-7 (KGF) knockout (Guo et al., 1996) Phenotype: Rough hair coat, similar to rough mutant mice. Overexpression of KGF in the liver (Nguyen et al., 1996) Phenotype (skin): Cutaneous papillomatous acanthosis and hyperkeratosis, reduced number of hair follicles.

embryonic lethality, as demonstrated by the FGFR1 knockout (Deng et al., 1994; Yamaguchi et al., 1994). Thus the importance of FGFs in the skin would be obscured. For the selective blockade of FGFR function in the skin, we developed a new strategy which is based on targeted expression of dominant-negative FGF receptors. Recent studies in Xenopus oocytes have shown that signal transduction through tyrosine kinase receptors can be blocked by Overexpression of dominant-negative receptor mutants in the same cell (Amaya et al., 1991; Ueno et al., 1991, 1992, 1993). These mutant receptors lack a functional tyrosine kinase domain (Honegger et al., 1990; Ueno et al., 1991). Upon ligand binding, they form non-functional heterodimers with full-length wild-type receptors, thereby blocking signal transduction (Figure 1) (Honegger et al., 1990; Kashles et al., 1991; Ueno et al., 1991). Most importantly, the dominant-negative action is specific for each growth factor receptor; for example, a truncated FGF receptor only blocks signal transduction through FGF receptors but not through the platelet-derived growth factor receptor (Ueno et al., 1992). The only prerequisite for the dominant-negative effect of the truncated receptor is that both the mutant and the wild-type receptor bind to the same ligand (Ueno et al., 1993). Therefore, a truncated FGFR1 will not only block signaling through wild-type FGFR1, but also through wild-type FGFR2 or

202


FGFR3, provided that the truncated and the wild-type receptor bind to the same type of FGF (Ueno et al., 1992; Ueno et al., 1993). Thus a truncated FGF receptor should inhibit the response of all FGF receptors which bind the same FGF. In order to act as a dominant-negative mutant, the truncated receptor has to be expressed at significantly higher levels than the endogenous wild-type receptor. It was therefore essential to use a very strong promoter for our transgenic mouse studies. Furthermore, it was important to specifically target the transgene to the skin in order to avoid side effects in other tissues and organs. We therefore decided to use a keratin promoter, since keratins are (i) highly expressed in epithelial cells and (ii) regulated in a tissue- and differentiation-specific manner (Moll et al., 1982). Thus basal, non-differentiated keratinocytes of the epidermis and outer root sheath cells of the hair follicles express keratins 5 and 14, whereas differentiated keratinocytes in the suprabasal layers of the epidermis and in the hair follicles express another pair of keratins (keratins 1 and 10). The promoters of these keratins have been cloned and their tissue-specificity has been analyzed in transgenic mice (Vassar et al., 1989; Bailleul et al., 1990; Greenhalgh et al., 1993; Ramirez et al., 1994). They can therefore be used to target transgenes to specific layers of the epidermis. In a first step, we used a suprabasal keratin 10 promoter (Rieger et al., 1985) to target a dominantnegative FGFR1-IIIc mutant to suprabasal cells of the epidermis. This form of receptor was chosen, since it binds aFGF and bFGF with high affinity but not KGF. It should therefore block the action of aFGF and bFGF in the suprabasal cells but not that of dermally-derived KGF. The transgenic mice were analyzed for expression of the truncated receptor in different parts of the skin and in other organs. Using RNase protection assays and in situ hybridization we demonstrated high levels of transgene mRNA in suprabasal keratinocytes of ear and tail epidermis and lower levels in the epidermis of trunk skin. These differences in transgene expression levels correlate with the expression levels of endogenous K10 which are highest in the epidermis of the tail and the ear and significantly lower in the epidermis of the trunk. No transgene expression was detected in other organs, demonstrating that we had indeed expressed the transgene in a tissue-specific manner. Although the transgenic mice looked macroscopically normal, a histological analysis of the skin revealed a significant difference in the epidermis of the tails and ears of transgenic mice, whereas the trunk skin appeared normal. Characteristic features of transgenic mouse tail epidermis included hyperthickening, the presence of several layers of nucleated cells with basal cell-like morphology in the suprabasal layers and a high degree of disorganization in the basal and lower suprabasal layers (Figure 3). These abnormalities were only observed in mice which had at least a 20-fold overexpression of the truncated receptor compared to the endogenous receptor. This is consistent with in vitro data which demonstrated that a 10–50-fold excess of the mutated receptor is necessary to block signal transduction (Ueno et al., 1992). This finding also explains the lack of phenotypic abnormalities in the skin of the trunk, since the transgene is expressed at much lower levels in trunk skin compared to tail skin. Further characterization of the tail skin revealed a significant hyperproliferation of the basal keratinocytes, although these cells did not express the transgene. This finding suggests that the basal cell compartment is stimulated as a consequence of the action of the transgene in the suprabasal compartment, demonstrating a significant influence of the differentiated suprabasal cells on the proliferation rate of the underlying basal cells. In addition to the severe disorganization of the epidermis, we also found significant defects in the differentiation pattern of the keratinocytes as demonstrated by immunostaining with antibodies directed against differentiation-specific keratins. As mentioned above, basal keratinocytes exclusively express keratins 5 and 14 (Fuchs and Green, 1980; Nelson and Sun, 1983; Roop et al., 1983). In suprabasal keratinocytes, K1 and K10 mRNAs and proteins are expressed at high levels, whereas K5 and K14


203

Figure 3 Phenotypic abnormalites in the tail skin of transgenic mice expressing a dominant-negative FGFR1 in suprabasal keratinocytes. Tail skin from a control mouse (A), a heterozygous transgenic mouse (B) and a homozygous transgenic mouse (C) from the same litter was fixed in 4% paraformaldehyde and paraffin-embedded. 6 µm sections were stained with hematoxylin and eosin. Note the presence of several layers of nucleated cells above the basal layer in transgenic mice and their high degree of disorganization. (Adapted from Werner et al., 1993, with permission).

expression is markedly downregulated (Fuchs and Green, 1980). Whereas expression of the suprabasal

204


keratins K1 and K10 appeared normal in transgenic mouse epidermis, the number of layers expressing high levels of the basal keratins K5 and K14 was significantly increased. Furthermore, we found an abnormal expression pattern of keratin 6. In normal skin, K6 expression is restricted to the hair follicles and is only transiently expressed in the proliferating epidermis of wounds. However, it is aberrantly expressed in the suprabasal keratinocytes in hyperplastic, neoplastic and psoriatic skin (Moll et al., 1982; Weiss et al., 1984; Stoler et al., 1988). A similar interfollicular expression of K6 was found in transgenic mouse epidermis. These results demonstrate that inhibition of FGF function in the differentiating cells of the epidermis changed the regular expression pattern of these proteins and therefore disrupted the normal program of keratinocyte differentiation. Taken together, this study provided the first evidence for an important role of FGF in keratinocyte organization and differentiation. As discussed above, the abnormalities seen in the transgenic mice are likely to be the result of the inhibition of aFGF or bFGF action, or of an as yet unknown FGFR1-IIIc ligand. However, the biological action of KGF should not be inhibited by the transgene, since it does not bind this ligand. To prove this hypothesis, we subsequently expressed a dominant-negative KGFR (FGFR2-IIIb) mutant under the control of the same promoter in the epidermis. Due to the binding specificities of this receptor, it should not only inhibit the function of aFGF and bFGF, but also that of KGF. Thus if KGF had an effect on suprabasal keratinocytes, one would expect a different phenotype compared to the abnormalities obtained with the dominant-negative FGFR1 which only blocks aFGF and bFGF function. However, the phenotypic abnormalities observed upon expression of the two different forms of truncated receptor were identical (S.W., unpublished data), indicating that KGF is indeed not involved in the regulation of suprabasal keratinocyte organization and differentiation. A possible ligand for the FGF receptors in the suprabasal cells is aFGF, since the FGF receptors which are expressed in the suprabasal cells of the epidermis (FGFR2-IIIb and FGFR3IIIb) bind this type of FGF with high affinity (Miki et al., 1992; Chellaiah et al., 1994). Furthermore, aFGF mRNA was shown to be expressed in these cells (Werner et al., 1993) and aFGF protein was detected in the epidermis (du Cros et al., 1993). Although aFGF is normally not secreted, it might become available upon the naturally occurring death of these cells. Alternatively, an as yet unknown FGF could activate the FGF receptors in the suprabasal keratinocytes. FGF-5 Knockout (Hebert et al., 1994) To date, functional gene knock-outs have been published for FGF-3, FGF-4, FGF-5, FGF-7, FGF-8, FGFR1, FGFR2 and FGFR3. Whereas the deletion of the FGF-4, FGF-8, FGFR1 and FGFR2 genes resulted in early embryonic lethality (Feldman et al., 1995; Deng et al., 1994; Yamaguchi et al., 1994; Arman et al., 1998; Meyers et al., 1998), the other knock-out mice were born alive and revealed specific defects in various tissues and organs (Mansour et al., 1993; Hebert et al., 1994; Colvin et al., 1996; Deng et al., 1996; Guo et al., 1996). Interestingly, a skin phenotype was observed for FGF-5 and FGF-7 null mice. During mouse development, FGF-5 is expressed just prior to the start of gastrulation, whereby it seems to be restricted to the embryonic ectoderm (Haub and Goldfarb, 1991; Hebert et al., 1991). At later stages in development, FGF-5 mRNA was found in the somitic myotome and the precursors of specific skeletal muscles (Haub and Goldfarb, 1991), and in the adult animal FGF-5 is expressed in the spinal cord and hippocampus (Haub et al., 1990). It was therefore expected that defects in these organs would be found in mice which lack a functional FGF-5 gene. Surprisingly, mice homozygous for a predicted null allele of the FGF-5 gene (Hebert et al., 1994) were found healthy and fertile and appeared phenotypically normal. However, within 3 weeks after birth, their hair grew noticeably longer than that of their heterozygous and wild-type littermates (Fig.4A). This result provided the first evidence for a role of FGF-5 in the limitation of hair elongation.


205

Interestingly, the phenotype of FGF-5 knockout mice appeared identical to that of mice homozygous for the spontaneous mutation angora (go). The angora gene had been defined by two recessive mutations that cause the production of abnormally long hair (Dickie, 1963; Konyukhov and Berdaliev, 1990). Indeed, a genomic complementation test between FGF-5 neo and go showed that the two alleles fail to complement one another, demonstrating that go is a mutant allele of the FGF-5 gene. A subsequent molecular study demonstrated the lack of RNA containing exons 2 and 3 of FGF-5 in tissue from go/go mice, suggesting that go is a null allele of FGF-5 (Hebert et al., 1994). The long hair phenotype of FGF-5 knockout mice was surprising, since FGF-5 expression in the hair follicle had not been previously described. However, a detailed in situ hybridization study revealed the presence of FGF-5 mRNA in the outer root sheath surrounding the transient portion of the hair follicle and in a small population of cells at the base of the follicle (Figure 4B). Interestingly, expression of this gene was dependent on the stage of the hair cycle. The latter is divided into three phases: anagen, the stage during which the follicle is regenerated and a new hair is produced; catagen, the stage during which hair elongation ceases and the hair follicle regresses; and telogen, the stage during which the follicle is at rest. FGF-5 mRNA was exclusively detected during the late anagen phase (anagen VI), suggesting that it normally acts at a specific stage of the hair cycle. In go mice, the only observed abnormality is that anagen VI, the phase of active hair growth, is extended (Pennycuik and Raphael, 1984). These observations, together with the FGF-5 expression and knockout data indicate that FGF-5 normally induces the transition from anagen VI to catagen. The mechanism of action of FGF-5 in the cycling hair is presently unknown. However, studies from the same laboratory demonstrated expression of a high-affinity receptor for FGF-5 (FGFR1-IIIc) in the dermal papilla (Rosenquist and Martin, 1996). Furthermore, dermal papilla cells have been shown to respond to FGF-5 (Rosenquist and Martin, 1996). These data suggest that dermal papilla cells are stimulated by FGF-5 in a paracrine manner during the late phase of anagen, resulting in the initiation of catagen. One response of the dermal papilla cells to FGF-5 might be the down-regulation of KGF expression. KGF is found at high levels in the dermal papilla during anagen, but not during catagen or telogen (Rosenquist and Martin, 1996). Since KGF is a potent growth factor for keratinocytes (Rubin et al., 1989) and since KGF receptors are abundant in the hair matrix cells nearest the dermal papilla (Orr-Urtreger et al., 1993; Danilenko et al., 1995a; Rosenquist and Martin, 1996), it might normally stimulate hair matrix cell proliferation and thus hair growth. This hypothesis is supported by the hair growth abnormalities in mice which express a dominant-negative KGF receptor in the skin (Werner et al., 1994b, see below) and by the potent stimulatory effect of exogenous KGF on hair growth in two distinct murine models of alopecia (Danilenko et al., 1995a). Thus a down-regulation of KGF expression by dermal papilla cells as a consequence of FGF-5 action might be a mechanism by which FGF-5 could indirectly induce apoptosis in the nearby hair matrix. However, a direct effect of FGF-5 on epithelial cells might also be possible. Taken together, these data demonstrated a novel role of FGF-5 and possibly of other FGFs in the regulation of the hair growth cycle. Overexpression of KGF in the Epidermis of Transgenic Mice (Guo et al., 1993) KGF is a potent and highly specific growth factor for several types of epithelial cells, including keratinocytes of the skin (Rubin et al., 1989; Finch et al., 1989). Furthermore, it has been shown to stimulate migration of keratinocytes (Tsuboi et al., 1993), to inhibit the terminal differentiation of these cells in vitro (Hines and Allen-Hoffmann, 1996), and to exert a protective effect on its target cells (Panos et al., 1995; Frank et al., 1997). To determine the in vivo function of KGF in the skin, several transgenic mouse models have been established. In a first approach, Guo et al. (1993) expressed KGF in the skin of

206


Figure 4 The role of FGF-5 in hair growth. (A) Phenotype of FGF-5 mutant homozygous mice. The animal on the right (front) is a mutant homozygous mouse. The animal on the left is a normal control mouse. Note the exceptionally long hair of the mutant mouse. (B)-(E): Longitudinal sections of dorsal skin taken from Swiss-Webster (albino) mice at various times after birth: P6 (B), P12 (C and E), and P18 (D). Staining for alkaline phosphatase activity identifies cells expressing FGF-5 mRNA. Early in anagen VI (P6), no FGF-5 mRNA is detected above background. Late in anagen VI (P12), staining is detected in the lower third of the outer root sheath of all follicles. In catagen (P18), no FGF-5 mRNA is detected. Abbreviations: dp, dermal papilla, hmc, hair matrix cells, ors, outer root sheath. Magnification for (B)-(D), 160x, for (E), 80x. (Adapted from Hebert et al., 1994 with permission). This figure shows work carried out in Dr. Gail Martin’s laboratory, University of California San Francisco, and was kindly provided by Dr. Gail Martin.

transgenic mice under the control of the basal keratin 14 promoter. This promoter targets transgenes to stratified squamous epithelia (Vassar et al., 1989). In the skin, the K14 promoter is active in basal


207

keratinocytes as well as in keratinocytes of the outer root sheath of the epidermis (Vassar et al., 1989). Since these cells express functional KGF receptors (Werner et al., 1992b; Orr-Urtreger et al., 1993; Marchese et al., 1995), the expression of KGF in these cells resulted in an autocrine action of this growth factor. This is in contrast to the normal in vivo situation, where KGF acts in a paracrine manner. Surprisingly, most of the transgenic mice were extremely frail, and died within a few hours after birth. They were characterized by wrinkled skin, pronounced eyelids, and sparser whiskers. The few transgenic animals which survived to adulthood lagged in weight gain and produced excessive saliva. Furthermore, they were characterized by an age-independent sparsity of hair. A detailed histopathological analysis of the skin of the transgenic mice revealed severe abnormalities, including epidermal hyperthickening due to the presence of an increased number of spinous and granular layers and to a lesser extent also stratum corneum layers, an abnormally high density of cells within the epidermis, and immature appearance of the keratinocytes with an increased nucleus to cytoplasm ratio. Furthermore, these mice had a dramatically reduced number of hair follicles, although the morphology of the remaining follicles was normal. The reason for the reduced number of hair follicles is presently unknown. However, it is unlikely to be the result of transgene expression in the follicle, since the latter occurs only after birth, when hair morphogenesis is complete (Kopan and Fuchs, 1989). By contrast, the excess of KGF produced in the developing epidermis might have interfered with hair follicle morphogenesis, and the authors suggested that the unregulated, increased growth response might have blocked the mesenchymal-epithelial signalling which is essential for hair follicle morphogenesis. Despite the specific action of KGF on epithelial cells, the skin of transgenic mice was also characterized by an increased number of immature fibroblasts beneath the hyperthickened epidermis and a severe reduction in fat, presumably as a result of an as yet unknown indirect mechanism. A detailed ultrastructural and immunohistochemical characterization of the transgenic mouse epidermis revealed severe defects in the process of terminal differentiation. Thus there was a high number of nucleated cells which reached the skin surface, a phenomenon which is characteristic for hyperproliferative skin. Furthermore, keratin 14 staining was not restricted to the basal layer but extended through all suprabasal layers, and keratin 6 was aberrantly expressed in the suprabasal layers of the epidermis. The hyperproliferative phenotype was confirmed by bromodeoxyuridine incorporation studies, whereby a twofold stimulation of basal cell proliferation was detected. Whereas in newborn KGF-expressing transgenic animals no signs of disorganization or transformation were detected, early signs of transformation appeared in certain regions of hyperthickened skin of older KGF-expressing transgenic animals. These included epidermal invaginations into the dermis, irregularities in epidermal thickening, and aberrations in epidermal organization. However, no visible signs of skin papillomas were detected. These results suggested a role of KGF in transformation processes of the epidermis, although additional signals are obviously required for the induction of skin tumorigenesis. EXPRESSION OF KERATINOCYTE GROWTH FACTOR IN EMBRYONIC LIVER OF TRANSGENIC MICE (NGUYEN ET AL., 1996) The potent role of KGF for keratinocyte proliferation in vivo was confirmed in a study by Nguyen et al. (1996), in which KGF was overexpressed in the embryonic liver of transgenic mice using a human apolipoprotein E promoter. These mice had severe abnormalities in several organs, since the recombinant KGF was efficiently secreted into the bloodstream. In the skin, KGF caused marked cutaneous papillomatous acanthosis and hyperkeratosis. Furthermore, the number of developing hair follicles compared to non-transgenic littermates was significantly reduced. Thus the presence of increased levels of KGF in the developing skin does obviously interfere with hair follicle morphogenesis. By contrast, systemic

208


administration of KGF to adult nude mice caused follicular hypertrophy (Danilenko et al., 1995), suggesting an important role of KGF for hair follicle growth in the adult animal. The latter hypothesis was supported by results obtained by targeted expression of a dominant-negative KGF receptor in the epidermis of transgenic mice (see below). EXPRESSION OF A DOMINANT-NEGATIVE KGF RECEPTOR IN BASAL KERATINOCYTES OF THE EPIDERMIS OF TRANSGENIC MICE (WERNER ET AL., 1994B) To further determine the function of KGF and its receptor in skin morphogenesis and wound repair, we expressed a dominant-negative KGF receptor (FGFR2-IIIb) mutant in the epidermis of transgenic mice using a keratin 14 promoter. A truncated FGFR2-IIIb was used as a transgene because this splice variant of FGFR2 is expressed at high levels in the epidermis (see above) and because it is the only known highaffinity receptor for KGF and should therefore mediate the action of this mitogen in the skin. However, since this receptor variant also binds aFGF, FGF-3 and to a lesser extent also bFGF, the truncated receptor should block the function of all these ligands. Since KGF is the most abundant FGF in normal and particularly in wounded skin, it seems likely that the effect of this factor is particularly affected by the truncated receptor. We obtained three transgenic mouse lines which expressed high levels of the truncated receptor in basal keratinocytes and in the hair follicles. Expression of transgene mRNA was detected exclusively in the skin and was found at similar levels in the skin of the ear, the tail, the back and the belly. All transgenic mice looked macroscopically normal, but they revealed significant histological abnormalities in the skin. Characteristic features of transgenic mouse skin were atrophy and disorganization of the epidermis (Figure 5B) and the presence of pycnotic nuclei in the basal cells (Figure 5B). By contrast, overexpression of KGF in the epidermis had caused epidermal hypertrophy and increased size of the nuclei (Guo et al., 1993), suggesting that the level of KGF and its activity do normally regulate epidermal thickness and the nucleus to cytoplasm ratio. In addition to the abnormalities in the epidermis, the transgenic mice revealed an abnormal morphology of the hair follicles and a 60–80% reduction in the number of hair follicles (Figure 5D). Interestingly, these defects were not detectable in newborn animals and appeared only 3 weeks after birth. Thus the first hair cycle appeared normal, whereas subsequent hair follicle morphogenesis was distorted. The histological defects seen in the hair follicles correlated with a severe delay in hair re-growth when the hairs were pulled out after the first hair cycle. These findings suggest that the onset of transgene expression in the adult hair follicle and the subsequent blockade of KGF receptor signalling in the outer root sheath cells had inhibited the normal progression through the hair cycle. This hypothesis is consistent with results of other studies which suggested an important role of KGF in hair growth and differentiation (Danilenko et al., 1995a; Rosenquist and Martin, 1996). Taken together, the abnormalities seen in the transgenic mice suggest a particularly important role of KGF receptor signalling in keratinocyte proliferation and organization as well as in hair follicle morphogenesis in the adult animal. By contrast, KGF and its receptor are obviously less important for skin development. Surprisingly, we also detected phenotypic abnormalities in the dermis of transgenic mice (Figure 5D). This part of the skin was characterized by severe thickening with a gradual replacement of adipose tissue by connective tissue. Since the transgene is not expressed in interfollicular dermis, indirect mechanisms must be responsible for this phenomenon. Thus inhibition of FGF receptor function in basal keratinocytes not only had direct effects on the target cells but also long-term indirect effects which might result from abnormal expression of other factors as a consequence of the KGF receptor blockade in keratinocytes.


209

Figure 5 Phenotypic abnormalities in the skin of transgenic mice expressing a dominant-negative KGF receptor mutant in the basal cells of the epidermis. A dominant-negative KGF receptor (FGFR2-IIIb) mutant was expressed in the basal cells of the epidermis of transgenic mice using a human keratin 14 promoter and regulatory elements of the human growth hormone gene. Back skin from a control mouse (A,C) and a heterozygous transgenic litter mate (B,D) was fixed overnight in 4% paraformaldehyde and paraffin-embedded. Sections (6 µm) were stained by hematoxylin/eosin. E, epidermis; D, dermis; H, hair follicle. Note the severe atrophy in transgenic mouse epidermis (compare A and B), the reduced number of hair follicles and the replacement of fatty tissue by connective tissue (compare C and D). E and F: A single full-thickness excisional wound was made on the back of a transgenic mouse (F) and a control littermate (E). Mice were killed at day 5 after injury. Wounds were isolated, bisected, fixed in 4% paraformaldehyde, and paraffinembedded. 6 µm sections from the middle of the wound were stained with hematoxylin-eosin. H, hair follicle; HE, hyperproliferative epithelium, Es, eschar. Note the severely reduced reepithelialization in transgenic animals.

Due to the high expression of KGF during wound repair, we speculated about a role of KGF and its receptor in re-epithelialization. To test this possibility, we generated full-thickness excisional wounds on the backs of transgenic mice and control litter mates. Histological analysis of the wounds revealed a severe delay in wound re-epithelialization in the transgenic animals. At day 5 after injury, a highly hyperthickened epithelium had formed at the wound edge of control mice and the wounds were almost completely reepithelialized (Figure 5E). By contrast, hyperthickening of the epithelium was hardly detectable in transgenic mice and only a small percentage of the wound was covered by migrating and proliferating keratinocytes (Figure 5F). Quantitation of the cell proliferation rate by incorporation of bromodeoxyuridine

210


demonstrated a more than 90% reduced proliferation rate of the keratinocytes at the wound edge in transgenic mice. Thus a blockade of FGF receptor signalling had inhibited wound re-epithelialization. These results demonstrated an important role of KGF receptor signalling in wound healing, although the type of KGF receptor ligand which is responsible for this defect was not defined by this study. However, the strong induction of KGF expression after skin injury in normal mice (Werner et al., 1992b), and the reduced induction of this factor in wounds of healing-impaired mice (Werner et al., 1994a; Brauchle et al., 1995) suggested KGF as the most important FGF involved in these processes. This hypothesis was supported by a series of wound healing studies where exogenous KGF was applied to different types of wounds. In most cases, KGF caused a significant stimulation of the wound healing process, whereby re-epithelialization was particularly improved (Staiano-Coico et al., 1993; Pierce et al., 1994; Danilenko et al., 1995b; Wu et al., 1996). KGF Knockout (Guo et al., 1996) To further determine the role of KGF in the development of the skin and of other tissues, Guo et al. (1996) used embryonic stem cell technology to generate mice lacking KGF. The obtained knockout mice appeared normal with the exception of the fur which appeared mat and greasy, especially in male animals. The rough and unkempt appearance resembled the phenotype seen in the recessive mouse mutant rough (ro) (Falconer and Snell, 1953), whose mutation maps at or near the KGF locus. Despite these obvious hair abnormalities, the morphology of hair follicles in KGF knockout mice was indistinguishable from control follicles. Although KGF is widely expressed during development and in the adult animal, no histological defects could be detected in the KGF knockout mice. Most surprisingly, even the healing process of incisional wounds appeared normal, demonstrating that wounds can heal in the absence of KGF. It will, however, be interesting to study the healing process of excisional wounds in these animals, since the extent of reepithelialization is much higher in excisional than in incisional wounds. The lack of obvious phenotypic abnormalities in the KGF knockout mice is in contrast to the results obtained with dominant-negative KGF receptors. Thus targeted expression of such a receptor mutant in the developing lung inhibited lung branching morphogenesis (Peters et al., 1994), a blockade of KGF receptor signaling in the mammary gland impaired the pregnancy-dependent growth of this organ (Jackson et al., 1997); and targeted expression of the same receptor mutant in the skin caused severe abnormalities in normal skin and inhibited wound re-epithelialization (Werner et al., 1994b, see above). The reason for these discrepancies is presently unclear. Although it might be possible that KGF is indeed not involved in these processes, this seems very unlikely, since the pattern of KGF expression correlates very well with its postulated functions in the lung, the mammary gland and in normal and wounded skin. The most likely explanation for the discrepancies between the knockout and the dominant-negative receptor results is a redundancy in ligand signalling. Although KGF might normally be the most important KGF receptor ligand in these tissues, the lack of this gene in KGF knockout mice might be compensated by other known KGF receptor ligands such as aFGF, bFGF and FGF-3 or by novel FGFs such as the recently identified FGF-10 (Yamasaki et al., 1996; Beer et al., 1997; Igarashi et al., 1998). Such a compensatory effect might be particularly efficient during embryonic development. By contrast, suppression of KGF expression in the adult animal could be more harmful, and this hypothesis is supported by the severe wound healing defect seen in glucocorticoid-treated mice in which KGF expression is significantly reduced (Brauchle et al., 1995). Studies using neutralizing KGF antibodies during wound repair should help to further clarify this issue. Furthermore, the tissue-specific knockout of the FGFR2-IIIb splice variant as well as double knockouts


211

of different ligands of this receptor will shed light on the role of the KGF receptor and the various types of FGF in many tissues, including normal and wounded skin. CONCLUSIONS To date, a series of expression studies as well as some recent transgenic mouse studies have provided insight into the function of FGFs in normal and wounded skin. These experiments have revealed an important role of KGF receptor signalling in skin morphogenesis and wound re-epithelialization, although the ligand(s) which is (are) responsible for these effects has (have) yet to be identified. Furthermore, FGF-5 and KGF have been identified as potent modulators of hair follicle morphogenesis and hair growth. Finally, a novel role of FGFs in keratinocyte organization and differentiation has been discovered. Future FGF and FGF receptor knockout studies as well as the use of different dominant-negative receptor mutants under the control of various skin-specific promoters will undoubtedly reveal additional important functions of FGFs in this tissue. ACKNOWLEDGEMENTS I am particularly grateful to Dr. Peter Lonai, the Weizmann Institute, Rehovot, Israel, and to Dr. Gail Martin, University of California San Francisco, for kindly providing Figure 1 and Figure 4, respectively. I also thank my co-workers for providing unpublished data, Dr. L.T.Williams, Chiron Corporation, Emeryville, for his invaluable contributions to the dominant-negative receptor studies, and T. Coutts for help with the manuscript. Work in my laboratory is supported by the Max-Planck-Gesellschaft, the Deutsche Forschungsgemeinschaft, the German Ministry for Education and Research (BMBF), a Human Frontier Science Grant the Stiftung VERUM and a Hermann-and-Lilly Schilling award. REFERENCES Abraham, J.A., Whang, J.L., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz, D. and Fiddes, J.C. (1986) Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J., 5, 2523–2528. Abraham, L.A. and Klagsbrun, M. (1996) Modulation of wound repair by members of the fibroblast growth factor family. In R.A.F. Clark, (ed.) The Molecular and Cellular Biology of Wound Repair (2nd ed.), Plenum Press, New York, pp. 195–248. Amaya, E., Musci, T.J. and Kirschner, M.W. (1991) Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell, 66, 257– 270. Anderson, K.J., Dam, D., Lee, S. and Cotman, C.W. (1988) Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo. Nature, 332, 360–361. Arman, E., Haffnerkrausz, R., Chen, Y., Heath, J.K. and Lonai, P. (1998) Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc. Natl. Acad. Sci. USA, 95, 5082–5087. Bailleul, B., Surani, M.A., White, S., Barton, S.C., Brown, K., Blessing, M., Jorcano, J. and Balmain, A. (1990) Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter. Cell, 62, 697–708. Basilico, C. and Moscatelli, D. (1992). The FGF family of growth factors and oncogenes. Adv. Cancer Res., 59, 115–165. Beer, H.-D., Florence, C., Dammeier, J., McGuire, L., Werner, S. and Duan, D.R. (1997) Mouse fibroblast growth factor 10: cDNA cloning, protein characterization, and regulation of mRNA expression. Oncogene, 15, 2211–2218.

212


Brauchle, M., Fässler, R. and Werner, S. (1995). Suppression of keratinocyte growth factor expression by glucocorticoids in vitro and during wound healing. J. Invest. Dermatol., 105, 579–584. Burrus, L.W., Zuber, M.E., Lueddecke, B.A. and Olwin, B.B. (1992) Identification of a cysteine-rich receptor for fibroblast growth factors. Mol. Cell Biol., 12, 5600–5609. Chellaiah, A.T., McEwen, D.G., Werner, S., Xu, J. and Ornitz, D.M. (1994) Fibroblast growth factor receptor 3: Alternative splicing in immunoglobulin-like domain III creates a receptor highly specific for acidic FGF/FGF-1. J. Biol. Chem., 269, 11620–11627. Clark, R.A.F. (1991) Cutaneous wound repair. In: L.A. Goldsmith (ed.), Physiology, Biochemistry and Molecular Biology of the skin, Oxford University Press, New York, pp. 576–601. Colvin, J.S., Bohne, B.A., Harding, G.W, McEwen, D.G. and Ornitz, D.M. (1996) Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nature Gen., 12, 390–397. Crossley, P.H. and Martin, G.R. (1995) The mouse FGF8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development, 121, 439–451. Danilenko, D.M., Ring, B.D., Yanagihara, D., Benson, W., Wiemann, B., Starnes, C.O. and Pierce, G.F. (1995a) Keratinocyte growth factor is an important endogenous mediator of hair follicle growth, development and differentiation. Am. J. Pathol., 147, 145–154. Danilenko, D.M., Ring, B.D., Tarpley, J.E., Morris, B., Van, G.Y, Morawiecki, A., Callahan, W., Goldenberg, M., Hershenson, S. and Pierce, G.F. (1995b) Growth factors in porcine full and partial thickness burn repair—differing targets and effects of keratinocyte growth factor, platelet-derived growth factor-BB, epidermal growth factor and neu differentiation factor. Am. J. Pathol., 147, 1261–1277. deLapeyriere, O., Rosnet, O., Benharroch, D., Raybaud, F., Marchetto, S., Planche, J., Galland, F., Mattei, M.-G., Copeland, N.G., Jenkins, N.A., Coulier, F. and Birnbaum, D. (1990) Structure, chromosome mapping and expression of the murine FGF-6 gene. Oncogene, 5, 823–831. deLapeyriere, O., Ollendorf, V., Planche, J., Ott, M.O., Pizette, S., Coulier, F. and Birnbaum, D. (1993) Expression of the FGF-6 gene is restricted to developing skeletal muscle in the mouse embry. Development, 118, 601–611. Delli-Bovi, P., Curatola, A.M., Kern, F.G., Greco, A., Ittmann, M. and Basilico, C. (1987) An oncogene isolated by transfection of Kaposi’s sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell, 50, 729–737. Deng, C.-X., Wynshaw-Boris, A., Shen, M.M., Daugherty, C., Ornitz, D.M. and Leder, P. (1994) Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes and Dev., 8, 3045–3057. Deng, C.-X., Wynshaw-Boris, A., Zhou, F., Kuo, A. and Leder, P. (1996) Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell, 84, 911–921. Dickie, M.N. (1963) New mutations. I. Angora. Mouse News Lett., 29, 39. du Cros, D.L., Isaacs, K. and Moore, P.M. (1993) Distribution of acidic and basic fibroblast growth factors in ovine skin during follicle morphogenesis. J. Cell Sci., 105, 667–674. Falconer, D.S. and Snell, G.D. (1953). Two new hair mutants: rough and frizzy. J. Hered., 43, 53–57. Feldman, B., Poueymirou, W., Papaioannou, V.E., Dechiara, T.M. and Goldfarb, M. (1995) Requirement of FGF-4 for postimplantation mouse development. Science, 261, 246–249. Finch, P.W., Rubin, J.S., Miki, T., Ron, D. and Aaronson, S.A. (1989) Human KGF is FGF-related with properties of a paracrine effector of epithelial cells. Science, 145, 752–755. Folkman, J. and Klagsbrun, M. (1987). Angiogenic factors. Science, 235, 442–447. Frank, S., Munz, B. and Werner, S. (1997) The human homologue of a bovine non-selenium glutathione peroxidase is a novel keratinocyte growth factor-regulated gene. Oncogene, 14, 915–921. Fuchs, E. and Green, H. (1980) Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell, 19, 1033–1042. Greenhalgh, D.A., Rothnagel, J.A., Quintanilla, M.I., Orengo, C.C., Gagne, T.A., Bundman, D.S., Longley, M.A. and Roop, D.R. (1993) Induction of epidermal hyperplasia, hyperkeratosis and papillomas in transgenic mice by a targeted v-Ha-ras oncogene. Mol. Carcinog., 7, 99–110.


213

Gonzalez, A.M., Buscaglia, M., Ong, M. and Baird, A. (1990) Distribution of basic fibroblast growth factor in the 18day rat fetus: Localization in the basement membranes of diverese tissues. J. Cell. Biol., 110, 753–765. Guo, L., Yu, Q.-C. and Fuchs, E. (1996) Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice. EMBO J., 12, 973–986. Guo, L., Degenstein, L. and Fuchs, E. (1996) Keratinocyte growth factor is required for hair development but not for wound healing. Genes and Dev., 10, 165–175. Han, J.-K. and Martin, G.R. (1993) Embryonic expression of FGF-6 is restricted to the skeletal muscle lineage. Dev. Biol., 158, 549–554. Haub, O., Drucker, B. and Goldfarb, M. (1990) Expression of the murine fibroblast growth factor 5 gene in the adult central nervous system. Proc. Natl. Acad. Sci. USA, 87, 8022– 8026. Haub, O. and Goldfarb, M. (1991) Expression of the fibroblast growth factor-5 gene in the mouse embryo. Development, 112, 397–406. Hebert, J.M., Boyle, M. and Martin, G.R. (1991) mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation. Development, 112, 407–415. Hebert, J.M., Rosenquist, T., Götz, J. and Martin, G.R. (1994) FGF-5 as a regulator of the hair growth cycle: Evidence from targeted and spontaneous mutations. Cell, 78, 1017– 1025. Hines, M.D. and Allen-Hoffmann, L. (1996) Keratinocyte growth factor inhibits cross-linked envelope formation and nucleosomal fragmentation in cultured human keratinocytes. J. Biol. Chem., 271, 6245–6251. Honegger, A.M., Schmidt, A., Ullrich, A. and Schlessinger, J. (1990) Evidence for Epidermal Growth Factor (EGF)induced intermolecular autophosphorylation of the EGF receptors in living cells. Mol. Cell Biol., 10, 4035–4044. Hoshikawa, M., Ohbayashi, N., Yonamine, A., Konishi, M., Ozaki, K., Fukui, S. and Itoh, N. (1998) Structure and expression of a novel fibroblast growth factor, FGF-17, preferentially expressed in the embryonic brain. Biochem. Biophys. Res. Commun., 244, 187–191. Hughes, S.E. and Hall, P.A. (1993) Immunolocalization of fibroblast growth factor receptor 1 and its ligands in human tissues. Lab. Invest., 69, 173–182. Igarashi, M., Finch, P.W. and Aaronson, S.A. (1998) Characterization of recombinant human fibroblast growth factor (FGF)-IO reveals functional similarities with keratinocyte growth factor (FGF-7). J. Biol. Chem., 273, 13230–13235. Jackson, D., Bresnick, J., Rosewell, I., Crafton, T., Poulsom, R., Stamp, G. and Dickson, C. (1997) Fibroblast growth factor receptor signalling has a role in lobuloalveolar development of the mammary gland. J. Cell Set., 110, 1261–1268. Jaye, M., Howk, R., Burgess, W., Ricca, G.A., Chiu, I.M., Ravera, M.W., O’Brien, S.J., Modi, W.S., Maciag, T. and Drohan, W.N. (1986) Human endothelial cell growth factor: cloning, nucleotide sequence and chromosome localization. Science, 233, 541–545. Johnson, D.E. and Williams, L.T. (1993) Structural and functional diversity in the FGF receptor multigene family. Adv. Cancer Res., 60, 1–41. Kashles, O., Yarden, Y., Fischer, R., Ullrich, A. and Schlessinger, J. (1991) A dominant negative mutation suppresses the function of normal Epidermal Growth Factor receptors by heterodimerization. Mol. Cell. Biol., 11, 1454–1463. Konyukhov, B.V. and Berdaliev, A.S. (1990) Analysis of the angora-Y gene expression. Mouse Genome, 87, 94–95. Kopan, R. and Fuchs, E. (1989) A new look into an old problem: keratins as tools to investigate determination, morphogenesis and differentiation in skin. Genes and Dev., 3, 1–15. LaRochelle, W.J., Dirsch, O.R., Finch, P.W., Cheon, H.-G., May, M., Marchese, C., Pierce, J.H. and Aaronson, S.A. (1995) Specific receptor detection by a functional keratinocyte growth factor-immunoglobulin chimera. J. Cell Biol., 129, 357–366. MacArthur, C.A., Lawshe, A., Xu, Y., Santos-Ortega, S., Heikinheimo, M., Chellaiah, A.T. and Ornitz, D.M. (1995) FGF-8 isoforms activate receptor splice forms that are expressed in mesenchymal regions of mouse development. Development, 121, 3603–3613. Mansour, S.L., Goddard, J.M. and Capecchi, M.R. (1993) Mice homozygous for a targeted disruption of the protooncogene int-2 have developmental defects in the tail and inner ear. Development, 117, 13–28.

214


Maries, I., Adelaide, J., Raybaud, F., Mattei, M.G., Coulier, F., Planche, J., deLapeyriere, O. and Birnbaum, D. (1989) Characterization of the HST-related FGF-6 gene, a new member of the fibroblast growth factor gene family. Oncogene, 4, 335–340. Mason, Y.J., Fuller-Pace, F., Smith, R. and Dickson, C. (1994) FGF-7 (Keratinocyte growth factor) expression during mouse development suggests roles in myogenesis, forebrain regionalisation and epithelial-mesenchymal interactions. Mech. Dev., 45, 15–30. Marchese, C., Chedid, M., Dirsch, O.R., Csaky, K.G., Santanelli, F., Latini, C., LaRochelle, W.J., Torrisi, M.R. and Aaronson, S.A. (1995) Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin. J. Exp. Med., 182, 1369–1376. Mcwhirter, J.R., Goulding, M., Weiner, J.A., Chun, J., Murre, C. (1997) A novel fibroblast growth factor gene expressed in the developing nervous system is a downstream target of the chimeric homeodomain oncoprotein E2A-PBX1. Development,124, 3221–3232. Meyers, E.N., Lewandoski, M. and Martin, G.R. (1998) An FGF-8 mutant allelic series generated by ere- and flpmediated recombination. Nature Gen., 18, 138–141. Miki, T., Bottaro, D.P., Fleming, T.P., Smith, C.L., Burgess, W.H., Chan, A.M.-L. and Aaronson, S.A. (1992) Determination of ligand-binding specificity by alternative splicing: Two distinct growth factor receptors encoded by a single gene. Proc. Natl. Acad. Sci. USA, 89, 246–250. Miyake, A., Konishi, M., Martin, F.H., Hernday, N.A., Ozaki, K., Yamamoto, S., Mikami, T., Arakawa, T. and Itoh, N. (1998) Structure and expression of a novel member, FGF-16, of the fibroblast growth factor family. Biochem. Biophys. Res. Commun., 243, 148–152. Miyamoto, M., Naruo, K.-L, Seko, C., Matsumoto, S., Kondo, T. and Kurokawa, T. (1993) Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property. Mol. Cell Biol., 13, 4251– 4259. Moll, R., Franke, W.W., Schiller, D.L., Geiger, B. and Krepler, R. (1982) The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors and cultured cells. Cell, 31, 11–24. Moore, R., Casey, G., Brookes, S., Dixon, M., Peters, G. and Dickson, D. (1986) Sequence, topography and protein coding potential of mouse int-2: A putative oncogene activated by mouse mammary tumour virus. EMBO J., 5, 919–926. Morrison, R.S., Sharma, A., deVellis, J. and Bradshaw, R.A. (1986) Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture. Proc. Natl. Acad. Sci. USA, 83, 7537–7541. Nelson, W. and Sun, T.-T. (1983) The 50- and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. J. Cell Biol., 97, 244–251. Nguyen, H.Q., Danilenko, D.M., Bucay, N., Derose, M.L., Van, G.Y., Thomason, A. and Simonet, W.S. (1996) Expression of keratinocyte growth factor in embryonic liver of transgenic mice causes changes in epithelial growth and differentiation resulting in poly cystic kidneys and other organ malformations. Oncogene, 12, 2109–2119. Niswander, L. and Martin, G.R. (1992) FGF-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development, 114, 755–768. Ohbayashi, N., Hoshikawa, M., Kimura, S., Yamasaki, M., Fukui, S. and Itoh, N. (1998) Structure and expression of the mRNA encoding a novel fibroblast growth factor, FGF-18. J. Biol. Chem., 273, 18161–18164. Ornitz, D.M., Xu, J.S., Colvin, J.S., McEwen, D.G., MacArthur, C.A., Coulier, F., Gao, G., Goldfarb, M. (1996) Receptor specificity of the fibroblast growth factor family. J. Biol. Chem., 271, 15292–15297. Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y. and Lonai, P. (1991) Developmental expression of two murine fibroblast growth factor receptors, fig and bek. Development, 113, 1419–1434. Orr-Urtreger, A., Bedford, M.T., Burakova, T., Arman, E., Zimmer, Y., Yayon, A., Givol, D. and Lonai, P. (1993) Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev. Biol, 158, 475–486. Panos, R.J., Bak, P.M., Simonet, W.S., Rubin, J.S. and Smith, L.J. (1995) Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats. J. Clin. Invest., 96, 2026–2033.


215

Partanen, J., Mäkelä, T.P., Eerola, E., Korhonen, J., Hirvonen, H., Claesson-Welsh, L. and Alitalo, K. (1991) FGFR-4, a novel acidic fibroblast growth factor receptor with a distinct expression pattern. EMBO J., 10, 1347–1354. Pennycuik, P.R. and Raphael, K.A. (1984) The angora locus (go) in the mouse: hair morphology, duration of the growth cycle and site of action. Genet. Res., 44, 283–291. Peters, K.G., Werner, S., Chen, G. and Williams, L.T. (1992) Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development, 114, 233–243. Peters, K.G., Ornitz, D., Werner, S. and Williams, L.T. (1993) Unique expression pattern of the mouse FGF receptor 3 gene during organogenesis. Dev. Biol., 155, 423–430. Peters, K.G., Werner, S., Liao, X., Wert, S., Whitsett, J. and Williams, L.T. (1994) Targeted expression of a dominantnegative FGF receptor blocks branching morphogenesis and epithelial differentiation of the mouse lung. EMBO J., 13, 3296–3301. Peterson, D.A., Lucidi-Phillipi, C.A., Murphy, D.P., Ray, J. and Gage, F.H. (1996) Fibroblast growth factor-2 protects entorhinal layer II glutamatergic neurons from axotomy-induced death. J. Neurosci., 16, 886–898. Pierce, G.F., Yanagihara, D., Klopchin, K., Danilenko, D.M., Hsu, E., Kenney, W.C. and Morris, C.F. (1994) Stimulation of all epithelial elements during skin regeneration by keratinocyte growth factor. J. Exp. Med., 179, 831–840. Pittelkow, M.R., Coffey R.J. Jr. and Moses, H.L. (1991) Transforming growth factor-p and other growth factors. In L.A. Goldsmith (ed.), Physiology, Biochemistry and Molecular Biology of the Skin, Oxford University Press, New York, pp. 351–384. Ramirez, A., Bravo, A., Jorcano, J.L. and Vidal, M. (1994) Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type specific expression of a LacZ gene in the adult and during development. Differentiation, 58, 53–64. Rieger, M., Jorcano, J.L. and Franke, W.W. (1985) Complete sequence of a bovine type I cytokeratin gene: conserved and variable intron positions in genes of polypeptides of the same cytokeratin subfamily. EMBO J., 4, 2261–2267. Roop, D.R., Hawley-Nelson, P., Cheng, C.K. and Yuspa, S.H. (1983) Keratin gene expression in mouse epidermis and cultured epidermal cells. Proc. Natl. Acad. Sci. USA, 80, 716– 720. Rosenquist, T.A. and Martin, G.R. (1996) Fibroblast growth factor signalling in the hair growth cycle: Expression of the fibroblast growth factor receptor and ligand genes in the murine hair follicle. Dev. Dyn., 205, 379–386. Rubin, J.S., Osada, H., Finch, P.W., Taylor, W.G., Rudikoff, S., Aaronson, S.A. (1989) Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc. Natl. Acad. Sci. USA, 86, 802–806. Schulze-Osthoff, K., Risau, W., Vollmer, E. and Sorg, C. (1990) In situ detection of basic fibroblast growth factor by highly specific antibodies. Am. J. Pathol., 137, 85–92. Shipley, G.D., Keeble, W.W., Hendrickson, J.E., Coffey, R.J. Jr. and Pittelkow, M.R. (1989) Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor. J. Cell. Physiol., 138, 511–518. Smallwood, P.M., Munoz-Sanjuan, I., Tong, P., Macke, J.P., Hendry, S.H.C., Gilbert, D.J., Copeland, N.G., Jenkins, N.A. and Nathans, J. (1996) Fibroblast growth factor (FGF) homologous factors: New members of the FGF family implicated in nervous system development. Proc. Natl. Acad. Sci. USA, 93, 9850–9857 Staiano-Coico, L., Krueger, J.G., Rubin, J.S., D’limi, S., Vallat, V.P., Valentino, L., Fahey III, T., Hawes, A., Kingston, G., Madden, M.R., Mathwich, M., Gottlieb, A.B. and Aaronson, S.A. (1993) Human keratinocyte growth factor effects in a porcine model of epidermal wound healing. J. Exp. Med., 178, 865–878. Stark, K.L., McMahon, J.A. and McMahon, A.P. (1991) FGFR-4, a new member of the fibroblast growth factor receptor family, expressed in the definitive endoderm and skeletal muscle lineages of the mouse. Development, 113, 641–651. Stoler, A., Kopan, R., Duvic, M. and Fuchs, E. (1988) Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation. J. Cell. Biol., 107, 427–446.

216


Tanaka, A., Miyamota, K., Minamino, N., Takeda, M., Sato, B., Matsuo, H. and Matsumoto, K. (1992) Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells. Proc. Natl. Acad. Set. USA, 89, 8928–8932. Tsuboi, R., Sato, C., Kurita, Y., Ron, D., Rubin, J.S. and Ogawa, H. (1993) Keratinocyte growth factor (FGF-7) stimulates migration and plasminogen activator activity of normal human keratinocytes. J. Invest. Dermatol., 101, 49–53. Ueno, H., Colbert, H., Escobedo, J.A., Williams, L.T. (1991) Inhibition of PDGF p-receptor by co-expression of a truncated receptor. Science, 252, 844–848. Ueno, H., Gunn, M., Dell, K., Tseng, A. and Williams, L.T. (1992) A truncated form of fibroblast growth factor receptor 1 inhibits signal transduction by multiple types of fibroblast growth factor receptor. J. Biol. Chem., 267, 1470–1476. Ueno, H., Escobedo, J.A. and Williams, L.T. (1993) Dominant-negative mutations of platelet-derived growth factor (PDGF) receptors. Inhibition of receptor function by ligand-dependent formation of heterodimers between PDGF alpha- and beta-receptors. J. Biol. Chem., 268, 22814–22819. Vassar, R., Rosenberg, M., Ross, S., Tyner, A. and Fuchs, E. (1989) Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. Proc. Natl. Acad. Sci. USA, 86, 1563–1567. Walicke, P., Cowan, W.M., Ueno, N., Baird, A. and Guillemin R. (1986) Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. USA, 83, 3012–3016. Wanaka, A., Milbrandt, J. and Johnson, E.M. Jr. (1991) Expression of FGF receptor gene in rat development. Development, 111, 455–468. Weiss, R.A., Eichner, R. and Sun, T.-T. (1984) Monoclonal antibody analysis of keratin expression in epidermal diseases: A 48- and 58-kdalton keratin as molecular markers for hyperproliferative keratinocytes. J. Cell Biol., 98, 1397–1406. Werner, S., Duan, D.-S. R., deVries, C., Peters, K.G. Johnson, D.E. and Williams, L.T (1992a) Differential splicing in the extracellular region of FGF receptor 1 generates receptor variants with different ligand binding specificities. Mol. Cell Biol., 12, 82–88. Werner, S., Peters, K.G., Longaker, M.T., Fuller-Pace, F, Banda, M. and Williams, L.T. (1992b) Large induction of keratinocyte growth factor expression in the dermis during wound healing. Proc. Natl. Acad. Sci. USA, 89, 6896–6900. Werner, S., Weinberg, W., Liao, X., Peters, K.G., Blessing, M., Yuspa, SH., Weiner, R. and Williams, L.T. (1993) Expression of a dominant-negative FGF receptor mutant in the epidermis of transgenic mice reveals a role of FGF in keratinocyte organization and differentiation. EMBO J., 12, 2635–3643. Werner, S., Breeden, M., Hübner, G., Greenhalgh, D.G. and Longaker, M.T. (1994a) Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse. J. Invest. Dermatol., 103, 469–473. Werner, S., Smola, H., Liao, X., Longaker, M.T, Krieg, T., Hofschneider, P.H. and Williams, L.T. (1994b) The function of KGF in epithelial morphogenesis and wound reepithelialization. Science, 266, 819–822. Wilkinson, D.G., Peters, G., Dickson, C. and McMahon, A.P. (1988) Expression of the FGF related proto-oncogene int-2 during gastrulation and neurulation in the mouse. EMBO J., 7, 691–695. Wilkinson, D.G, Bhatt, S. and McMahon, A.P. (1989) Expression of the FGF-related protooncogene int-2 suggests multiple roles in fetal development. Development, 105, 131–136. Wu, L.C., Pierce, G.F., Galiano, R.D. and Mustoe, T.A. (1996) Keratinocyte growth factor induces granulation tissue in ischemic dermal wounds—importance of epithelial — mesenchymal cell interactions. Arch. Surg., 131, 660–666. Yamaguchi, T.P., Harpal, K., Henkemeyer, M. and Rossant, J. (1994) FGFR-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes and Dev., 8, 3032–3044. Yamasaki, M., Miyake, A., Tagashira, S. and Itoh, N. (1996) Structure and expression of the rat mRNA encoding a novel member of the fibroblast growth factor family. J. Biol. Chem., 271, 15918–15921. Zhan, X., Bates, B., Hu, X. and Goldfarb, M. (1988) The human FGF-5 oncogene encodes a novel protein related to flbroblast growth factors. Mol. Cell Biol., 8, 3487–3495.

217

12. LAMININ ISOFORMS AND EPITHELIAL DEVELOPMENT PETER EKBLOM, ERIK FORSBERG, DONALD GULLBERG AND MARJA EKBLOM Department of Animal Physiology, Uppsala University, Biomedical Center BOX 596, SE-751 24 Uppsala, Sweden Tel. 46–18–4714211; Fax: 46–18508095; E-mail: [email protected] INTRODUCTION Epithelial morphogenesis during embryonic development is driven by interactions between epithelium and the adjacent mesenchyme (Grobstein 1967). In the past 10 years major advances have been made in the clarification of the molecular basis of epithelial-mesenchymal interactions. They are controlled by paracrine interactions between growth factors and their receptors, direct cell-cell interactions, and by interactions between extracellular matrix and its receptors. For all these three interactions, several crucial molecules have been identified. Here we will describe the expression and possible role of some of the major extracellular matrix proteins involved in epithelial-mesenchymal interactions. BASEMENT MEMBRANE COMPONENTS Epithelial cells are firmly attached to a thin sheet of extracellular matrix, the basement membrane or basal lamina. This sheet can barely be seen by light microscopy, but electron microscopy reveals a thin continuous membrane of approximately 100 nm. As described in Chapter 1, all basement membranes contain various isoforms of collagen IV, proteoglycans, and glycoproteins such as the laminins and nidogen. Basement membranes form very early during development of epithelial sheets, suggesting that they are important for the epithelial morphogenesis. Many different approaches have suggested that basement membrane components influence the phenotype and functions of many cell types, in addition to being structural components (Kleinman et al., 1986; Grant et al., 1990; Hay 1993; Yurchenco and O’Rear, 1994; Ekblom, 1996; Timpl and Brown, 1996). We recently reviewed some of the current knowledge about the expression and role of laminins during epithelial morphogenesis (Ekblom et al., 1998). Several recent novel findings justify this update and a more comprehensive coverage. As described in detail in Chapter 6 by Smyth and Paulsson all well characterized laminins have been found to be heterotrimers of α, β and γ chains. Five α chains, three β chains and two γ chains are known for humans and mice (Timpl and Brown, 1996) but it is likely that several other chains will soon be described. The number of laminin heterotrimers cannot be estimated at present, but will certainly be far more than 11. Some of the laminin chains are rather ubiquitously expressed and are found in most laminins. The γ1 chain is present in all but one of the well described laminins. By binding to each other, the laminins form huge networks within the basement membranes. The type IV collagens form another network, but there seems to be no direct binding of this network to the laminin network. Instead, the two networks are held together by smaller connecting molecules such as nidogen, a 150 kDa glycoprotein found in all basement membranes. In addition, epithelial basement membranes contain the proteoglycans perlecan, bamacan and agrin


219

(Couchman et al., 1996; Ekblom, 1996; Timpl and Brown, 1996; Burgeson and Christiano, 1997). Agrin binds with high affinity to laminins (Denzer et al., 1997, 1998). Differential expression of isoforms of proteoglycans, collagen IV and laminin lead to region-specific basement membranes. Variations in the molecular composition of them are also achieved by different amounts of associated proteins. For example, fibulins and flbronectin are extracellular matrix components found prominently in mesenchymal tissue compartments, but during discrete stags of development, they may also be present in basement membranes. Changes in the molecular composition of basement membranes probably influence the biological properties of the basement membranes, but this issue has not been studied in detail. THE DISTRIBUTION OF LAMININ CHAINS IN DEVELOPING EPITHELIA Both during preimplantation development and onset of organogenesis, laminins are expressed already at onset of epithelial morphogenesis in the mouse embryo. This became evident as soon as antibodies against the major basement membrane components became available almost 20 years ago. In the first immunofluorescence studies, polyclonal antibodies which detect α1, β1 and γ1 chains were used (Ekblom et al., 1980; Leivo et al., 1980), so the chain composition of the detected laminins did not become clear. The γ1 chain and to a large extent also the β1 chain are found in most basement membranes. Hence, when antibodies against these chains are used in immunohistology, all basement membranes will be stained. It was therefore initially not clear that basement membranes are molecularly heterogeneous, and that profound changes in the expression pattern of different laminin chains occur during embryogenesis. Distribution of Laminin α1 Chain in the Body So far most in vitro studies about the biological role of laminins are based on studies with laminin-1. A major reason for this is that it can be obtained from a mouse transplantable tumor. Many cells types can attach to laminin-1, and many of these cells are stimulated to differentiate by the molecule. An important issue is whether this reflects the in vivo situation. Some clues about this issue can be obtained by studying its tissue distribution, which can be determined to a large extent by studying the distribution of the α1 chain. Curiously, the issue about al expression has been much debated. We have always argued that this chain in non-neural embryonic and newborn mouse tissues is confined to epithelial basement membranes, and with few exceptions is not expressed in endothelium or muscle. Furthermore, we found that in epithelia the α1 chain was confined to a subset of basement membranes (Klein et al., 1988; Ekblom et al., 1990; Klein et al., 1990). Our view of a restricted expression in epithelial basement membrane was compatible with a few mRNA studies of human embryonic and adult tissues or cells (Kleinman et al., 1987; Nissinen et al., 1991; Van den Heuvel and Abrahamson, 1993; Vuolteenaho et al., 1994). However, two influencial articles (Engvall et al., 1990; Sanes et al., 1990) suggested a much broader distribution of the al chain, and several subsequent studies likewise argued strongly for a broad distribution. Laminin al chain was thus claimed to be prominent in basement membranes of muscle, endothelial and practically all epithelial sheets (Engvall et al., 1990; Sanes et al., 1990; Mundegar et al., 1995; Virtanen et al., 1995) in several species (human, rat, hamster and guinea pig). The reports of a broad distribution were based on analyses of adult tissues, whereas we mainly studied mouse tissues and embryonic stages (Ekblom et al., 1990; Klein et al., 1990; Sorokin et al., 1992). It is important to note that different reagents were used. The reports suggesting a broad distribution used a monoclonal antibody 4C7 (Engvall et al., 1990), whereas we used well characterized polyclonal (Klein et al., 1988, 1990) and monoclonal antibodies (Sorokin et al., 1992) against the E3 fragment of mouse laminin-1. Since antibody 4C7 and other similar monoclonals generated and

220


characterized in a similar manner (Engvall et al., 1990) are commercially available under different names, they have been extensively used as reagents to study α1 chain. In order to resolve the controversy about laminin α1 expression, we have recently performed three detailed studies in mouse, rat and human tissues. New antibodies reacting with recombinant mouse or human α1 chain epitopes were raised. In a first approach, two different antisera against the carboxyterminal globular domains (the E3 fragment) were tested in rat tissue sections (Durbeej et al., 1996). Both a new polyclonal antibody made against mouse recombinant E3 and previously described α1 chain-specific antibodies selectively stained certain epithelial basement membranes in rat, and showed no reactivity with endothelium or muscle in rat kidneys or other tissues. No evidence for a “broad” distribution of α1 chain was thus found. The distribution of α1 and β2 appeared to be mutually exclusive, raising the possibility that laminin-3 (α1β2γ1) is rare. Thus, according to our studies, laminin-3 is not found in endothelium or in the glomerular basement membrane (Durbeej et al., 1996), as previously thought (Engvall et al., 1990). In a second approach, new monoclonal antibodies against mouse laminin α1 chain were produced (Sorokin et al., 1997a). The previously produced antibodies were against the E3 fragment, the most carboxyterminal end of the α1 chain (Sorokin et al., 1992). It could be argued that E3 is selectively lost by proteolysis in vivo. Another alternative is that it is masked in immunofluorescence, for instance by regionspecific proteoglycans, and not accessible to antibodies in tissue sections. The new monoclonal antibodies made against the α1 chain did not react with E3 in ELISA but reacted with the adjacent fragment E8. In contrast, the previously made antibodies were specific for the E3 fragment. Both the new and previous monoclonals reacted with the recombinantly produced globular regions G1–G5, derived from the α1 chain. The conclusion is that the new monoclonals are specific for the α1-chain sequences within the E8 fragment. In immunofluorescence, the new monoclonals selectively stained the epithelium in the kidney and showed no reactivity against blood vessel or muscle basement membranes. The selective staining of α1 chain was seen both in embryonic and adult mouse kidneys (Sorokin et al., 1997a). One example is the glomerular basement membrane, which in newborn mice does not stain with antibodies against the α1 chain (Figure 1), but instead stains brightly with antibodies against laminin α5 chain (Sorokin et al., 1997b). In a third approach, the specificity of 4C7 antibody (Engvall et al., 1990) was clarified (Tiger et al., 1997). To this end, polyclonal antibodies against human α1 and α5 chain were first produced. The antibody against α1 chain were raised against recombinant human E3 produced in human cells. In culture medium from cells producing both α1 and α5 mRNA, antibodies against al chain detected a 400 kDa in immunoprecipitation, whereas the polyclonal α5 antibody detected a smaller 380 kDa protein. It was thus significant that antibody 4C7 also precipitated a 380 kDa protein rather than a 400 kDa protein (Figure 2). Furthermore, in immunoblots polyclonal antibodies against α5 chain but not the polyclonal antibodies against α1 chain reacted with the 4C7-precipitate. Finally, it was demonstrated that the antibody against human α1 chain in immunofluorescence selectively stained basement membranes of proximal tubules, but did not stain basement membranes of blood vessels or glomeruli (Tiger et al., 1997). This restricted distribution was in complete agreement with our previous results in mouse (Ekblom et al., 1990; Sorokin et al., 1997a) and rat (Durbeej et al., 1996). In contrast, the 4C7 antibody shows a broad reactivity in immunofluorescence, similar to the pattern seen by monoclonal antibodies against mouse α5 chain (Sorokin et al., 1997a,b). In conclusion, recent studies in mouse (Sorokin et al., 1997a), rat (Durbeej et al., 1996) and human (Tiger et al., 1997) tissues have clarified the controversy about the distribution of laminin al chain. The laminin α1 chain is selectively expressed in a subset of epithelial basement membranes in mouse, rat and human tissues, and it does not have a broad distribution. This was described for embryonic and newborn mice already a long time ago (Klein et al., 1990; Ekblom et al., 1990). Instead, it is the laminin α5 chain which


221

Figure 1 Laminin α1 and α5 in kidneys of newborn mice. Immunofluorescence demonstrate selective expression of laminin α1 chain in basement membranes of proximal kidney tubules (A), whereas laminin α5 chain is strongly expressed in the glomerular basement membrane (B). From Sorokin et al., 1997a.

has a broad distribution in the adult. Since most studied about the distribution in adult tissues have been performed with antibody 4C7 or similarly generated antibodies with yet unclarified specificity, we still do not know very well where laminin α1 in the adult is expressed. Only adult kidney has been studied in more detail so far in mouse (Sorokin et al., 1997a,b) and human samples (Tiger et al., 1997). The identification of the 4C7 antigen as laminin α5 chain has many other implications. Several functional studies have been performed with commercial laminin preparations isolated with the 4C7 antibody, in the assumption that human laminin-1 was used. These studies and the immunohistological studies with 4C7 have to be reinterpreted.

222


Figure 2 Monoclonal antibody 4C7 detects laminin α5 chain. Immunoprecipitation was performed from material of a cell line that produced both laminin α1 and α5 chains. All antibodies precipitated 200 kDa β/γ chains. In addition, polyclonal antibodies against γ/β chains precipitated both a 400 kDa α1 chain and a 380 kDa α5 chain. Polyclonal antibodies against α1 chain precipitated a 400 kDa α1 chain but not a 380 kDa α5 chain. In contrast, polyclonal antibodies against α5 chain precipitated a 380 Kda α5 chain but not a 400 kDa α1 chain. Significantly, the 4C7 antibody precipitated a 380 kDa α5 chain but not a 400 kDa α1 chain. From Tiger et al. (1997).

Expression of the α1 mRNA Northern blot and in situ hybridization studies of embryonic tissues studies support the view of a selective expression of laminin α1 chain in epithelium in non-neural embryonic tissues. In embryonic kidneys the α1 chain mRNA is strictly produced by epithelial cells, as shown several years ago (Ekblom et al., 1990). These studies have subsequently been confirmed and extended. The close association of al mRNA expression was noted in other embryonic organs as well, but in some embryonic tissues a thin sheet of mesenchyme adjacent to the epithelium produces the α1 mRNA (Thomas and Dziadek, 1993). For example, mesenchymal cells close to the stalk of the epithelia are the main producers of laminin α1 during branching epithelial morphogenesis of lung and salivary gland, although the protein is deposited in the epithelial basement membranes. Interestingly, the α1 mRNA were produced by the mesenchyme only in the stalk area during branching, whereas an epithelial origin was noted in the tip of the branching lung and salivary gland epithelia (Thomas and Dziadek, 1994; Kadoya et al., 1995). In conclusion, the expression of the α1 chain mRNA is during embryogenesis associated with epithelial-mesenchymal interactions, and the protein is deposited into the epithelial basement membranes. The other cell types which are attached to basement membranes, such as muscle, fat, and endothelium produce very little if any laminin α1 chain. Two exceptions have been noted for muscle; strong expression was seen in the atrium of the embryonic heart (Klein et al., 1990) and some expression in the embryonic myotendinous junction (Patton et al., 1997). This does not exclude the possibility that some endothelial and fat cells at some developmental stages produce some α1 chain. Another potentially important expression site is the nervous system (Vuolteenaho et al., 1994) but it will not be covered here.


223

Expression of the Laminin α2–5 Chains in the Embryo Cells which produce basement membranes but lack the laminin α1 chain should produce some other α chain. This argument is based on the fact that laminins are found in all basement membranes, and on the assumption that all laminins are heterotrimers. Several reports have demonstrated that laminin α2 and α4 mRNAs are made by embryonic mesenchymal cells and muscle. Some endothelial cells also produce α4 chain but many endothelial cells produce none of the described a chain (Frieser et al., 1997). Their expression patterns are thus somewhat complex (Frieser et al., 1997; livanainen et al., 1997) but will not be described here since they are mainly “mesenchymal” laminins. Although most epithelial basement membranes during the earliest stages of epithelial development contain the al chain (and thus probably laminin-1), several epithelial sheets lose the α1 chain due to a drastic decrease in the expression of the mRNA upon further differentiation. The α1 chain is frequently then replaced by the α5 chain. Particularly clear examples of the switch from α1 to α5 chain are the collecting ducts and distal tubules of the kidney. No expression of α5 chain was noted in embryonic mesenchyme, and in the embryonic stage only a few endothelial cell types produced it (Durbeej et al., 1996). These findings were subsequently confirmed and extended for several other tissues, by more detailed in situ hybridization studies and by immunofluorescence using well characterized monoclonal antibodies against the α5 chain. As tissues mature, the α5 chain becomes very prominent and is well expressed and much more broadly distributed than α1 (Durbeej et al., 1996; Mineretal., 1997; Sorokineta/., 1997a, b; Salmivirta et al., 1997). Another α chain made by several epithelial cells is the α3 chain. Recently a novel splice variant of laminin α3 chain, encoding a ‘full-size’ laminin chain, was identified in the mouse and named α3B (Miner et al., 1997). The previously described ‘truncated’ laminin α3 chain, named α3A is present in laminins –5, – 6 and –7. Northern blot data indicate the presence of mRNA for the a3B splice variant in adult brain, lung and kidney (Miner et al., 1997). Studies of mRNA encoding the corresponding human ‘full-sized’ laminin α3 chain variant, named laminin α3B, indicate a more widespread tissue expression than the truncated human α3A. In studies of human tissues the highest RNA levels for laminin α3B were found in uterus, mammary gland, lung and brain chain (Doliana et al., 1997). Interestingly only the α3A variant was detected in adult human kidney. Protein data on the α3B variant is still lacking. With available antibodies specific for the two variants it will be interesting to determine the distribution of the two splice forms. It will also be important to determine which beta and gamma chains the α3B form associates with. The nomenclature for laminin isoforms might need revision to account for the existence of a full length as well as a truncated a chains. Laminin α3A (previously called α3) is part of a common laminin heterotrimer, laminin-5 located in epithelial basement membranes such as skin, rnucosa, lung and intestine, and developing tooth (Carter et al., 1991; Kallunki et al., 1992; Aberdam et al., 1994; Stahl et al., 1995; Orian-Rosseau et al., 1996; Salmivirta et al., 1997). Laminin-5, also known as nicein/kalinin/epiligrin, is composed of the subunits α3, β3 and γ2 (Matsui et al., 1995; Burgeson and Christiano, 1997). Laminin-5 has been suggested to be involved in branching epithelial morphogenesis (Stahl et al., 1995). Its importance for epidermal cell attachment is clear, since mutations of laminin-5 cause skin blistering leading to death in early childhood (Uitto et al., 1994). In line with these findings keratinocytes have been found to bind avidly to laminin-5 in vitro and can promote the formation of hemidesmosomes in vitro (Hormia et al., 1995). Recently it was proposed that proteolytic cleavage of the 00 chain in laminin-5 enhances hemidesmosome formation. The laminin α3 chain is frequently proteolytically cleaved from a 190 kDa to a 160 kDa protein (Goldfinger et al., 1998). Laminin-5 induced hemidesmosome formation requires binding to cell surface receptors. Both α3 β1 and α6β4 integrins bind laminin-5 and they are produced by many epithelial cells, including keratinocytes (Carter et al., 1991; Niessen et al., 1994). Laminin-5 binding to these receptors could be important also for

224


reepitelialization during wound healing (Kainulainen et al., 1998). Laminin-5 remains well expressed in many carcinomas and in their metastases, and it has been proposed that some tumor cells of epithelial origin use laminin-5 to migrate and invade into tissues (Pyke et al., 1995; Tani et al., 1996). There are Many Epithelial Laminins In conclusion, it is apparent that several different laminin chains can be found in epithelial basement membranes. The type made depends on the differentiation stage, and type of epithelia. It is likely that novel laminin chains still will be found, but at present we can distinguish three classes of epithelial laminins, those containing either α1, α3 or α5 chains. Major epithelial heterotrimers are laminin-1 (α1β1γ1), laminin-10 (α5β1γ1), and laminin-5 (α3β3γ2). Laminin-5 (α3β3γ2) has for some time by many considered to be “the epithelial laminin”, but as we have argued here laminin-α1 is also an epithelial laminin, since it largely is restricted to basement membranes of embryonic epithelium. In addition, α5 containing laminins probably associated with β1 and γ1 are expressed in many embryonic epithelia lacking laminin α1. Whereas the α1 and α3A chain are more strictly epithelial, the α5 has a broader distribution; it is found in developing muscle basement membranes and a few endothelial basement membranes. After birth, α5 chain gradually becomes prominent in most endothelial basement membranes. The α5 chain mRNA levels remain high in several adult tissues (Durbeej et al., 1995; Miner et al., 1997). It is thus clear that the α5 chain in the adult stage is the prominent a chain as far as expression is concerned. This can be contrasted with the α1 chain, which is hard to detect by Northern blots from adult tissues, and is restricted to a few epithelial basement membranes in the adult stage. Turnover rates may of course vary, so it would be important to study the distribution of the different α chains proteins in the adult stage. Although the different α chains to some extent are found in different basement membranes, there are many overlaps. Epidermal basement membranes provide extreme examples of basement membranes containing several laminins. The isoforms in adult skin have been covered in more detail recently (Burgeson and Christiano, 1997). Another type of complexity is the rapid switch in isoform expression which occurs during embryonic development. Such transitions have been described for several developing epithelia, including gastrointestinal, lung and kidney epithelia. For all these tissue it has been suggested that laminin α1 chain could be important for epithelial development. The possible role of laminins for lung development has recently been reviewed (Dunsmore and Rannels, 1996; Schuger, 1997). We will therefore here mainly describe laminin chains during development of kidney tubules, and selected aspects about laminin isoforms in some parts of the developing gastrointestinal tract. LAMININ ISOFORMS AND RECEPTORS IN KIDNEY TUBULES In the developing kidney three major cell lineages can be distinguished at early stages, the epithelial ureter bud, the metanephrogenic mesenchyme, and blood vessels. Each cell lineage expresses different laminin isoforms at this stage, and as development proceeds, changes in the isoform expression occur for each cell lineage. The ureter bud develops into collecting ducts, whereas the mesenchyme will form both different types of stroma and a new epithelium. We will here mainly describe the laminin isoforms during mesenchyme conversion to epithelia. This conversion can be induced also in vitro. In an in vitro culture, the first morphological sign of epithelial development is a condensation of the mesenchyme (36h). At about 48h the cells form distinct tubules with an underlying basement membrane, and by 72h the cells are polarized with specialized basal, lateral and apical surfaces. The 48– 72h stages correspond roughly to the S-shaped stage of in vivo development (Grobstein, 1967; Saxén et al., 1968).


225

Of the five known α chains, three chains (α4, α1 and α5) are prominently expressed during kidney tubulogenesis but each at different developmental stages. The two other chains (α2, α3) are found in embryonic kidneys but they are not made by developing tubule cells. The α2 chain is made mainly by those mesenchymal which do not convert into epithelium, whereas the α3 polypeptide has been detected in basement membranes of the embryonic collecting ducts and ureter (Abderdam et al., 1994). None of the known α chains seem to be expressed in undifferentiated mesenchyme, suggesting the presence of novel a chains. Formation of mesenchymal condensates correlates with strong induction of α4 chain expression (livanainen et al., 1997; Miner et al., 1997; Sorokin et al., 1997a), followed by a slightly later induction of the α1 chain during overt tubulogenesis. The α4 expression declines fast and is no longer detected in Sshaped tubules which instead express α1. The α1 chain remains expressed in proximal tubules but gradually disappears from distal tubules and glomerular podocytes. As development proceeds and the S-shaped tubules elongate to form distinct distal and proximal tubules and glomerular epithelial sheets, the α5 chain gradually appears in all these epithelial cells (Miner et al., 1997; Sorokin et al., 1997b). It is likely that each of the three α-chains produced during kidney tubulogenesis are associated with β1 and γ1 chains since both chains have been demonstrated during tubulogenesis in condensates, S-shaped tubules and during tubule elongation (Ekblom et al., 1990) whereas β2 chain is not present in any of these stages (Durbeej et al., 1996). However, the presence of not yet characterized β or γ chains cannot be excluded. Antibody perturbation suggests a role for laminin-α1 in kidney morphogenesis (Figure 3). Its expression also correlates well with onset of basement membrane biogenesis of kidney tubules. In a kidney organ culture system, antibodies against E8 and E3 fragment of laminin-1 perturbed development of kidney tubules (Klein et al., 1988; Ekblom et al., 1990; Sorokin et al., 1992). The E8 fragment contains parts from three chains (α1β1γ1). Since β1 and γ1 are present in many laminins, the interpretation of the results with anti-E8 antibodies is not straightforward. In contrast, the E3 fragment is formed exclusively by one chain, α1. Our hypothesis is thus that this chain could be important for initiation of basement membrane formation and epithelial morphogenesis. The results in the kidney organ culture system (Klein et al., 1988) led us to search for epithelial receptors for the E3 and E8 fragments of laminin-1. Many different laminin receptors may exist, but the so far best characterized laminin receptors are certain integrins and dystroglycan. In vitro organ culture data suggest that some of these could be involved in kidney tubulogenesis. Dystroglycan—A Major Laminin Receptor of Kidney Tubules Dystroglycan is a high-affinity receptor for laminin-2 in muscle, but it also binds the E3 fragment of laminin-1 (Gee et al., 1993; Ibraghimov-Beskrovnaya et al., 1993; Henry and Campbell, 1996). Dystroglycan is identical to cranin, a laminin-binding protein first found from nervous tissue (Smalheiser and Schartz, 1987; Smalheiser and Kim, 1995). Northern blotting of various non-muscle tissues showed that dystroglycan is found in several tissues (Ibraghimov-Beskrovnaya et al., 1993). This could be due to production by smooth muscle, but in situ hybridization of embryonic tissues revealed that epithelial cells were main sources of dystroglycan mRNA in non-muscle tissues in addition to smooth muscle (Durbeej et al., 1995). In the embryo and in adult, dystroglycan is present in the basement membrane region of epithelial cells (Durbeej et al., 1995, 1998). Antibodies which block dystroglycan-laminin binding perturbed development of kidney epithelial cells in vitro (Durbeej et al., 1995). Further evidence for a role of dystroglycan in non-muscle tissues was provided by gene targeting in mice. Dystroglycan null mice show the first signs of defects on day 5–6 of embryonic development, and do not initiate gastrulation. Reichert’s membrane was found to be disrupted in these mutant mice, suggesting that dystroglycan is needed for the interaction between the endodermal cells and Reichert’s membrane (Williamson et al., 1997). It means that

226


Figure 3 Perturbation of kidney tubulogenesis in vitro by monoclonal antibody 200 against E3 fragment of laminin-1. Organ cultures of kidney mesenchyme was cultured for three days in the presence of antibodies and then analyzed by histology. Immunofluoresence shows that control antibodies which do not inhibit development penetrates into the tissues (A). Well developed tubules were noted in cultures treated with non-blocking control antibodies against α1 chain (B) but in the presence of monoclonal antibody 200, only few tubules formed and they lacked a proper lumen (C). From Sorokin et al. (1992).

the dystroglycan —/— embryos die before myogenesis begins. Taken together, all currently available studies suggest a role for dystroglycan in epithelial morphogenesis. It is important to note that dystroglycan in epithelial cells not only binds laminin-1 but also other basement membrane components. Agrin is found in many epithelial basement membranes, and binds dystroglycan with high affinity (Gesemann et al., 1998). Epithelial cells can produce many laminins which might bind dystroglycan and we do not know the exact composition of the laminins in Reichert’s membrane. Integrins of Developing Kidney Tubules Many integrins (including α1β1, α2β1, α3β1, α6β1, α6β4, α7β1, α9β1 act as laminin receptors in vitro (Sonnenberg et al., 1990; Aumailley et al., 1996), but some of these bind with much higher affinity to collagen (Kühn, 1996). Several studies have demonstrated the expression of various integrin subunits in embryonic kidneys and in cell lines from adult kidneys. The β1 subunit is present in virtually all cell types of the developing kidneys but no β4 was detected in embryonic kidneys by in situ hybridization (Falk et al., 1996). Antibodies against β1 integrin subunit were found to block kidney development in vitro, whereas control antibodies against E-cadherin failed to do so (Falk et al., 1996). These studies suggest a role for β1 containing integrins in kidney development. Epithelial cells derived from adult kidney can reverse their polarity when cultured in the presence of antibodies against β1 integrin (Ojakian and Schwimmer, 1994;


227

Zuk and Matlin, 1996). Hence, it seems that β1 containing integrin are important both for the development and maintenance of epithelial cell phenotype. It is difficult to study this by genetic means, because mice lacking the β1 subunit die very early during embryogenesis, on day 4–5 (Fässler and Meyer, 1995; Stephens et al., 1995). Recently, an in vivo approach to study the role β1 integrins for basement membrane assembly has been reported (Sasaki et al., 1998). Integrin β1 deficient embryonic stem cells were injected into mice and were found to produce teratomas. In the tumors, and also in embryoid bodies derived from the integrin β1 deficient cells (Bloch et al., 1997) the ultrastructural organization of the basement membranes was extensively disorganized. In addition, a selective and rather unexpected 40–60% downregulation 3 of expression of laminin α1, β1 and γ1 chain mRNAs was seen. No similar downregulation was seen for mRNAs of several other extracellular matrix proteins such as nidogen, fibulins, and perlecan. The selective downregulation of laminin-1 chains could be verified at the protein level, which also showed that nidogen protein levels were reduced despite normal mRNA levels. These studies emphasize the importance of β1 integrins for proper basement membrane assembly, but the interpretation of the results is not straightforward. The abnormalities in the basement membranes could have been caused directly by the lack of the pi integrins, or by the drastic reduction of the amount of laminin-1 (Sasaki et al., 1998). It will be interesting to learn whether β1 integrins also in other experimental model systems affect the biosynthesis of laminin chains. Several integrin a chains can associate with the β1 integrin subunit, and many of these are produced by some of the cell types of either developing or adult kidney tubules. Several laminin-binding integrins are expressed in the developing kidney. The α6β1 is a well known and specific laminin-binding integrin (Aumailley et al., 1996). At onset of kidney tubulogenesis, the α6 subunit is absent in mesenchyme but it appears during tubulogenesis when basement membrane formation begins, and is then expressed in Sshaped tubules in vivo. Detailed mRNA studies showed that the α6B variant is the predominant form, and that the α6 mRNA increases about 10-fold during onset of basement membrane formation, concomitantly with a similar increase in expression of laminin α1 chain (Ekblom et al., 1990; Falk et al., 1996). Antibodies against α6 chain have been shown to perturb kidney development in vitro (Falk et al., 1996), but mice lacking the α6 integrin undergo normal embryogenesis and consequently also have normal kidneys at birth. These mice die only after birth, apparently because of skin blisters as the epidermis cannot attach to its basement membrane (Georges-Labousesse et al., 1996). The morphologically normal embryogenesis suggests compensation by other subunits in the α6 —/— knock out mice, but it cannot be excluded that that the effects of antibodies in the organ culture studies (Sorokin et al., 1990; Falk et al., 1996) are artifacts. Antibody application may lead to improper signaling by the integrins, and antibody preparations may contain some impurities. Evidence suggesting toxic impurities in commercial anti-integrin α6 antibody preparations has been presented (Chen et al., 1997). These possibilities should be studied in the embryonic kidney organ culture system. It is at present unclear how important the α6 integrin subunit is for kidney development in vivo, or for branching epithelial morphogenesis in general. Several other α-chains which heterodimerize with the β1 integrin subunit are produced by some of the kidney cells. Some of the kidney tubules express either integrin α2 or α3 chains in addition to the more ubiquitous α6. Thus, distal tubules contain much integrin α2, whereas the podocytes of the glomeruli contain integrin α3 (Korhonen et al., 1990). It is thus interesting that mice lacking 00 integrin have abnormal glomeruli (Kreidberg et al., 1996). The integrin α3β1 binds laminin-5 (Carter et al., 1991) but possibly also other laminins (Gehlsen et al., 1989). Laminin-1 does not bind well to integrin α3β1 (Aumailley et al., 1996) so one possibility is that the other laminin isoform which in one study bound integrin α3β1 (Gehlsen et al., 1989) was laminin-10 (α5β1γ1) or -11 (α5β2γ1) rather than laminin-1 (α1β1γ1). The phenotype of the integrin α3 null mice with selective defects in the glomeruli

228


fits well with this proposal, since it now is clear that α5 laminin chain is strongly expressed in glomeruli (Durbeej et al., 1996; Miner et al., 1997; Sorokin et al, 1997a). It is likely that developing kidney tubules contain yet additional integrin a subunits, and some of these may form laminin-binding complexes. Some evidence for novel integrins come from studies of epithelial cell lines from adult kidney. In the established cell line MDCK large amounts of integrin α2β1 and α3β1 were found (Schoenenberger et al., 1994). These mainly bind collagen IV but weakly also to laminins (Kühn, 1996). In addition, Schoenenberger et al. (1994) found an unidentified integrin subunit, the vitronectin receptor αvβ3, and some α6β4 which is also a laminin-1 receptor (Aumailley et al., 1996). The strong expression of α2β1 suggests that the MDCK cell lines is derived from distal tubules. A fairly similar integrin expression pattern was found in primary cell cultures enriched for epithelial cells of kidney cortex (Simon et al., 1994). In the primary cell cultures, the initial attachment to laminin-1 was found to be dependent on integrin α6β1 and α3β1. Interestingly, the attachment was in some preparations due to an unidentified β1 integrin. After immunodepletion of membrane extracts with α1, α2, α3, α5, α6, and αv antibodies, the β1 integrin antibody still precipitated an integrin. Its electrophoretic mobility differed from the laminin binding α7β1 integrin (Simon et al., 1994). One newly described subunit is integrin α8, and genetic studies suggest that it is important for the earliest stages of kidney tubulogenesis (Müller et al., 1997) but there is so far no evidence that it bind laminins. In conclusion, kidney tubules express many integrins, and probably even several laminin-binding integrins. The receptor repertoire is different in the different segments of the tubules, and complex developmental transitions occur. The binding affinities of the different tubular integrins to different laminins is not well known, and even less is at present known about the biological significance of these interactions. The binding of α6β1 to laminin-1 has been well studied in cell attachment assays using established cell lines (Aumailley et al., 1996) and for some time it has been thought that interaction between laminin-1 and integrin α6β1 is of major importance for kidney tubulogenesis (Sorokin et al., 1990). However, the physiological role of this interaction during kidney development is at present unclear, because a crucial role was not demonstrated in genetic studies (Georges-Labousesse et al., 1996). Further studies at many different levels are required to clarify the biological role of the many integrins during kidney tubulogenesis. LAMININ ISOFORMS IN THE DEVELOPING GASTROINTESTINAL TRACT The embryonic salivary gland has been much used to study the importance of basement membrane components for development (Bernfield et al., 1984). During early stages of salivary glands, the epithelial basement membrane components contain small amounts of laminin al chain, but a soon as the epithelium begins to branch, expression Is lost and instead α5 chain is expressed (Kadoya et al., 1995; Durbeej et al., 1996). Antibodies against the E3 fragment of laminin, al chain can perturb branching morphogenesis (Figure 4) apparently by blocking some early event of basement membrane assembly. In organ cultures treated with monoclonal anti-E3 antibodies, other basement membrane components such as nidogen and laminin-1 (Figure 4) could not be detected in the tip of the epithelial branches Antibodies against a integrin α6 did not have such an effect (Kadoya et al., 1995). These finding raise the possibility that E3 binding to dystroglycan initiates basement membrane assembly. Laminin α-chains in the developing mouse intestinal epithelial basement membrane include laminin α1, α2, α3 and α5 chains. As in many other comparisons of laminin α1 chain distribution in mouse and human tissues, a discrepancy was noted between the distribution in mouse and human because the monoclonal antibody 4C7 was used as a reagent for the human laminin α1 chain. For mouse tissues, the monoclonal


229

Figure 4 Monoclonal antibody 200 disrupts basement membrane assembly in organ cultures of submandibular gland. Submandibular glands were cultured for two days in the presence of antibodies against E3 fragment of laminin-1 (A—C) or antibodies against integrin α6 (D) and then stained with antibodies against E-cadherin (A), nidogen (B), or laminin-1 (C, D). In the tips of the branching gland, no nidogen or laminin-1 was seen in explants treated with the anti-E3 antibodies. From Kadoya et al. (1995).

antibodies of Sorokin et al. (1992) were used. They revealed that the α1 chain is located only in the crypt region of the intestine (Simon-Assmann et al., 1995), as opposed to the widespread distribution of the 4C7 antigen in human intestine (Simon-Assmann et al., 1994; Bealieu, 1997). With the clarification of the 4C7 antigen as laminin α5 (Tiger et al., 1997), the reason for the discrepancy is now clear. There is some evidence for a role of laminins in intestinal epithelial development. Culture of rat intestinal epithelium together with mouse basement membrane components stimulate the differentiation of the cells (Hahn et al., 1990). Laminins may be the key components in the basement membranes. A heterotrimeric human laminin preparation was found to stimulate human enterocytes, but the nature of the heterotrimer was not quite clear (Vachon and Beaulieu, 1995). The distribution of α1 chain the crypts suggest a role for it in early epithelial intestine development, or in proliferation. In an attempt to study the role of laminin α1 chain for epithelial polarity and basement membrane assembly, an antisense approach to suppress laminin α1 chain mRNA was used by DeArcangelis et al., 1996. Sequences of the α1 chain in the antisense direction were transfected in the human colon adenocarcinoma cell line Caco-2 which has the capacity to differentiate in vitro. Anti-sense transfected cells lost epithelial polarity, differentiated poorly and were unable to assemble a basement membrane. As a tool to monitor the synthesis of laminin-1, the 4C7 antibody was used. Interestingly, the 4C7 antigen expression was found to be drastically diminished in the transfected cells (DeArcangellis et al., 1996). The interpretation of the findings is thus not straightforward. Complicated

230


Figure 5 Perturbation of kidney development with antibodies against the EGF-like domains of laminin γ1 chain which harbor the nidogen-binding site. Kidneys are much smaller and epithelial sheets do not branch well when cultured in the presence of antibodies against the nidogen-binding site (A) as compared with kidney cultured in the presence of nonimmune IgG (B). From Ekblom et al. (1994).

indirect effects of the treatment cannot be excluded, and therefore it is still an open question whether laminin α1 chain is important for intestinal epithelial cell development. THE ASSEMBLY OF EPITHELIAL BASEMENT MEMBRANES EXTRACELLULARLY In the extracellular space laminins bind to each other, and to other basement membrane components. Three extracellular interactions will be covered here, laminin self-assembly, and binding of laminins to nidogen and to agrin. Laminin-self assembly and the high affinity binding of laminins to nidogen (also called entactin) have been extensively studied. In vitro studies with organ cultures and blocking antibodies suggest that some of these assembly processes could be important for morphogenesis. Recently, it has been shown that laminin-1 binds with high affinity to agrin, which is found in many epithelial basement membranes. Whether agrin binding to laminin-1 is of developmental importance is unclear. The laminin-laminin interactions leads to the formation of large laminin-networks located close to the cell surface. Laminin self-assembly occurs via short arms of the β1 and γ1 chains, where the most aminoterminal ends form the binding sites (Yurchenco and O’Rear, 1994; Timpl and Brown, 1996). Antibodies against these parts perturb epithelial morphogenesis in organ and cell cultures of embryonic lung epithelia. It has therefore been suggested that the extracellular assembly of a laminin network is required for epithelial morphogenesis cells (Schuger et al., 1995; 1998). The β1 and γ1 chains are found in many laminins, including laminins-1 and −10, which both are found in many epithelia, including the developing lung epithelium (Durbeej et al., 1996) which were used to study the role of laminin self-assembly for development (Schuger et al., 1998). There is good evidence that the nidogen-laminin interactions are important for epithelial-mesenchymal interactions. The high affinity binding of nidogen to laminin γ1 chain (Kd 0.5 nM) has been mapped to a few amino acids in one LE repeat (III4). Since γ1 chain is part of many laminin, this repeat is found in the short arm of many laminins, including laminins-1 and -10 (Mayer et al., 1993; Timpl and Brown 1996). Antibodies which block this interaction inhibit branching epithelial morphogenesis of kidney (Figure 5), lung and salivary gland in organ cultures in vitro (Ekblom et al., 1994; Kadoya et al., 1997). During


231

normal development, the nidogen polypeptide is found ubiquitously in basement membranes in these organs, already during onset of organogenesis. By immunofluorescence, only very little nidogen can be detected in the mesenchyme. Nevertheless, nidogen mRNA has been detected exclusively in the mesenchyme at sites of epithelial-mesenchymal interactions (Thomas and Dziadek, 1994; Ekblom et al., 1994; Fleischmajer et al., 1995; Senior et al., 1996; Kadoya et al., 1997). Certain collagen IV isoforms and perlecan are also produced by mesenchyme during embryogenesis (Dziadek, 1995; Simon-Assmann et al., 1995). The strict mesenchymal production of nidogen is quite interesting since nidogen could be a key organizer of basement membrane assembly. The two major networks in basement membranes, collagen IV and laminin, are bound to each other by nidogen (Mayer et al., 1993; Timpl and Brown, 1996). Classical organ culture studies performed a long time ago have demonstrated that mesenchymal cells are important for morphogenesis of epithelium (Grobstein, 1967), and it has been a major goal to identify the mesenchymal morphogens. Several growth factors have been shown to be important, and apparently induce the pattern of epithelial branching. Yet, several additional mesenchymal factors are required for proper morphogenesis. These factors include mesenchymal extracellular matrix components. Tenascin-C and epimorphin are two mesenchymal extracellular matrix components suggested to be involved in the regulation of epithelial morphogenesis (Chiquet-Ehrismann et al., 1986; Hirai et al., 1992, 1998). Our studies suggest that nidogen could be such a mesenchymal factor (Ekblom et al., 1994). How nidogen-laminin interactions influence epithelial-mesenchymal interactions is not yet quite clear. They could affect both the epithelial cells by influencing basement membrane assembly, but could also directly stimulate mesenchymal/ stromal differentiation. Nidogen is easily cleaved by common proteases unless it is bound to laminin-1. This could explain why it is detected only in epithelial basement membranes and not in mesenchyme, although its mRNA is produced by the mesenchyme. Recently, we found a way to increase the expression of the nidogen polypeptide in the mesenchyme. Epidermal growth factor (EGF) profoundly increased nidogen mRNA and protein expression both in the mesenchyme and the basement membrane. Interestingly, antibodies against the nidogen binding site of laminin γ1 chain no longer inhibited morphogenesis in organ cultures treated with EGF. It suggests that the nidogen which accumulates in response to EGF competes with the antibody (Kadoya et al., 1997). Taken together, all current results suggest an important role for the nidogenbinding site of laminin γ1 in epithelial-mesenchymal interactions, particularly in the kidney but also in some other organs (Figure 6). The hypothesis of a crucial role of nidogen-laminin interactions is presently being tested by several genetic means. Attempts to generate mice lacking nidogen are underway in several laboratories. Genetically altered laminin γ1 chains lacking only the nidogen-binding site are currently also constructed (R. Timpl, U. Mayer, personal communication). Compensation by overexpression of other molecules may occur in some of these animals.. There is now evidence for additional nidogens, and they may have similar roles as nidogen-1. Recent studies suggest that agrin is a major laminin-binding protein in epithelial basement membranes. Agrin is a proteoglycan of basement membranes initially identified from the neuromuscular junction (McMahan, 1990; Ruegg et al., 1992; Rupp et al., 1992). But agrin in neuromuscular junction is a particular splice variant with capacity to induce synaptic specilizations in the neuromuscular junction. Other variants found in non-neuronal and non-muscle tissues, in epithelial basement membranes (Reist et al., 1987; Godfrey, 1991; Gesemann et al., 1998) lack this capacity, but may have different functions. Agrin binds dystroglycan (Sugiyama et al., 1994; Gesemann et al., 1996), and agrin variants from lung and kidney have been shown to bind dystroglycan with very high affinity (Kd 2 nM, Gesemann et al., 1998). Agrin also binds to laminins, and binding of agrin to laminin-1 is also of high affinity (Kd 5 nM, Denzer et al., 1997). The agrin-binding site of laminin-1 was recently mapped to central region of the three-stranded coiled-coil domain in the long arm (Denzer et al., 1998). Both the expression pattern and the dual high affinity binding

232


Figure 6 Scheme of interactions between laminin-1 and nidogen derived from mesenchyme and the interactions of laminin-1 with two receptor complexes on the basal cell surface of an epithelial cell. The interactions between the E3 fragment and dystroglycan may initiate basement membrane assembly.

to both laminin-1 and its receptor suggest that agrin could be of importance for the integrity of epithelial sheets. It is also a major constituent of the glomerular basement membrane (Groffen et al., 1998) where it might interact with dystroglycan (Durbeej et al., 1998). Agrin-deficient mice seem to have no gross abnormalities in epithelia (Gautam et al., 1996), but in these mice only certain agrin exons were removed. Total agrin expression seemed to be drastically reduced in these mice (Gautam et al., 1996), but agrin binding to dystroglycan and laminin-1 is of high affinity. Low-level expression of some agrin variants may therefore be sufficient for functional interactions. Animals completely lacking agrin may be needed to reveal the importance of agrin in epithelia (Gesemann et al., 1998). Embryonic organ cultures studies with blocking antibodies might also be valuable.


233

CONCLUDING REMARKS Several recent observations have clarified how laminins might affect epithelial morphogenesis. Figure 6 shows a current model of the interactions between laminin1 with nidogen-1, and with cell surface components on the basal side of epithelial cells. Many major epithelial laminins and their receptors have been identified, and we are beginning to understand extracellular protein-protein interactions which drive basement membrane assembly. Some epithelial cell types express several laminin α-chains, so it will be relevant to test how the different laminins affect epithelial cells. Important recent discoveries are the identification of dystroglycan as a laminin and agrin receptor in muscle and epithelia, and nidogen as a highaffinity laminin-binding protein important for epithelial-mesenchymal interactions. Many genetic approaches about the nidogens and dystroglycan are currently underway in different laboratories. Several other exciting future issues can be envisioned. Suggestions about the role of laminins, based on cell and organ culture techniques, will soon be tested by genetic means in various model organisms. It will also be interesting to test how the different laminins affect gene expression in vitro. The few studies available clearly suggest that laminins stimulate gene expression (Streuli et al., 1996), but different isoforms presumably influence cells in different ways. In a recent study it was found that the intracellular filamentous structures become different if cells are plated on either laminin-5 or laminin-1 (Dogic et al., 1998). Laminin-10 or other epithelial laminins may lead to other types of rearrangements of the filaments. In order to understand such processes, it will be important to identify the receptors binding to newly discovered laminins. Another interesting future issue to study is the polar secretion of the laminins. Epithelial cell polarity has been much studied, but a slightly neglected issue is the relationship between epithelial cell polarity and basement membrane assembly. Laminins and other basement membrane constituents are deposited only basally, but the mechanisms controlling this polar secretion are totally unknown. An understanding of the directed secretion may tell us much about the polarity of the cells also. In most in vitro cell culture assays designed to study cell polarity, only the “apical” and “basolateral” pathways are assayed. In the future, it might be of relevance to develop techniques to distinguish between the basal and lateral path-way. For such issues, detailed studies about the processing of laminins and their receptors may be rewarding. REFERENCES Abderdam, D., Aguzzi, A., Baudoin, C., Galliano, M.-F., Ortonne, J.-P. and Meneguzzi, G. (1994) Developmental expression of nicein adhesion protein (laminin-5) subunits suggests multiple morphogenetic roles. Cell Adhesion Commun., 2, 115–129. Aumailley, M., Gimond, C. and Rouselle, P. (1996) Integrin-mediated cellular interactions with laminins. In The laminins, eds. P. Ekblom, R. Timpl. pp. 127–158. Amsterdam: Harwood Academic Press. Beaulieu, J.-F. (1997) Extracellular matrix components and integrins in relationship to human intestinal epithelial cell differentiation. Progr. Histochem. Cytochem., 31, 1–76 Bernfield, M., Banerjee, S.D., Koda, J.E. and Rapraeger, A.C. (1984) Remodelling of the basement membrane: morphogenesis and maturation. Ciba Found. Symp., 108, 179–196 Bloch, W., Forsberg, E., Lentini, S., Brakebusch, C., Martin, K., Krell, H.W., Weidle, U.H., Addicks, K. and Fässler, R. (1997) β1 integrin is essential for teratoma growth and angiogenesis. J. Cell Biol., 139, 265–278. Burgeson, R. and Christiano, A.M. (1997) The dermal-epidermal junction. Ctfrr. Op. Cell Biol., 7, 651–658. Carter, W.G., Ryan, M.C. and Gahr, P.J. (1991). Epiligrin, a new cell adhesion ligand for integrin α3β1 in epithelial basement membranes. Cell, 65, 599–610.

234


Chen, L., Shick, V., Matter, M.L., Laurie, S.M., Ogle, R.C. and Laurie, G.M.W. (1997) Laminin E8 alveolarization site; heparin sensitivity, cell surface receptors and role in cell spreading. Am.J. Physiol., 272, L494-L503. Chiquet-Ehrismann, R., Mackie, E.J., Pearson, J. and Sakakura, T. (1986) Tenascin, an extracellular protein involved in tissue interactions during fetal development and oncogenesis. Cell, 47, 131–139. Couchman, J.R., Kapoor, R., Sthanam, M. and Wu, R.R. (1996) Perlecan and basement membrane-chondroitin sulfate proteoglycan (bamacan) are two basement membrane chondroitin/dermatan sulfate proteoglycans in the Engelbreth-Holm-Swarm tumor matrix. J. Biol. Chem., 271, 9595–9602. De Arcangelis, A., Neuville, P., Boukamel, R., Lefebre, O., Kedinger, M., and Simon-Assmann, P. (1996) Inhibition of laminin a 1-chain expression leads to alteration of basement membrane assembly and cell differentiation. J. Cell Biol., 133, 47–430. Denser, A.J., Brandenberger, R., Gesemann, M., Chiquet, M. and Ruegg, M.A. (1997) Agrin binds to the nerve-muscle basal lamina via laminin. J. Cell Biol., 137, 671–683. Denzer, A.J., Gesemann, M., Schumacher, B. and Ruegg, M.A. (1995) An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix. J. Cell Biol., 131, 1547–1560. Denzer, A.J., Schulthess, T., Fauser, C., Schumacher, B., Kammerer, R.A., Engel, J. and Ruegg, M.A. (1998) Electron microscopic structure of agrin and mapping of its binding site in laminin-1. EMBO J., 17, 335–343. Dogic, D., Rouselle, P. and Aumailley, M. (1998). Cell adhesion to laminin-1 and 5 induces isoform-specific clustering of integrins and other focal adhesion components. (1998) J. Cell Sci., 111, 793–802. Doliana, R., Bellina, I., Bucciotti, F., Mongiat, M., Perris, R. and Colombatti, A. (1997) The human α3B is a ‘full-sized’ laminin chain variant with a more widespread tissue expression than the truncated α3A. FEBS Lett., 417, 65–70. Durbeej, M., Fecker, L., Salmivirta, K., Hjalt, T., Klein, G., Sorokin, L., Timpl, R., Ebendal, T., Ekblom, P. and Ekblom, M. (1996) Expression of laminin α1, α5 and β2 chain during embryogenesis of the kidney and vasculature. Matrix Biol., 15, 397–413. Durbeej, M., Henry, M., Ferletta, M., Campbell, K.P. and Ekblom, P. (1998) Distribution of dystroglycan in normal adult mouse tissues. J. Histochem. Cytochem., 46, 449–457. Durbeej, M., Larsson, E., Ibraghimov-Beskrovnaya, O., Roberds, S.L., Campbell, K.P. and Ekblom, P. (1995) Nonmuscle α-dystroglycan is involved in epithelial development. J. Cell Biol., 130, 79–91. Dunsmore, S.E. and Rannels, D.E. (1996) Extracellular matrix biology of the lung. Am. J. Physiol., 270, L3–L27. Dziadek, M. (1995) Role of laminin-nidogen complexes in basement membrane formation during embryonic development. Experientia, 51, 901–903. Ekblom, P. (1996) Receptors for laminins during epithelial morphogenesis. Curr. Op. Cell Biol., 8, 700–706. Ekblom, P., Alitalo, K., Vaheri, A., Timpl, R. and Saxén, L. (1980) Induction of a basement membrane glycoprotein in embryonic kidney, possible role of laminin in morphogenesis. Proc. Natl. Acad. Sci. USA, 77, 485–489. Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., Chu, M.-L, Mayer, U. and Timpl, R. (1994) Role of mesenchymal nidogen for epithelial morphogenesis in vitro. Development, 120, 2003–2014. Ekblom, M., Klein, G., Mugrauer, G., Fecker, L., Deutzmann, R., Timpl, R. and Ekblom, P. (1990) Transient and locally restricted expression of laminin α chain mRNA by developing epithelial cells during kidney organogenesis. Cell, 60, 337–346. Ekblom, M., Falk, M., Salmivirta, K., Durbeej, M. and Ekblom, P. (1998) Laminin isoforms and epithelial development. Ann. N.Y. Acad. (in press). Engvall, E., Earwicker, D., Haaparanta, T., Ruoslahti, E. and Sanes, J.R. (1990) Distribution and isolation of four laminin variants; restricted tissue distribution and heterotrimers assembled from five different subunits. Cell Regul., 1, 731–740. Falk, M., Salmivirta, K., Durbeej, M., Larsson, E., Ekblom, M., Vestweber, D. and Ekblom, P. (1996) Integrin α6Bβ1 is involved in kidney tubulogenesis in vitro. J. Cell Sci., 109, 2801–2810. Fleischmajer, R., Schechter, A., Bruns, M., Perlish, J., MacDonald, E., Pan, T., Timpl, R. and Chu, M.-L. (1995) Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro. J. Invest. Dermatol., 105, 597–601.


235

Frieser, M., Nöckel, H., Pausch, F., Röder, C., Hahn, A., Deutzmann, R. and Sorokin, L.M. (1997) Cloning of the mouse laminin α4 gene, expression in a subset of endothelium. Eur. J. Biochem., 247, 727–735. Fässler, R. and Meyer, M. (1995) Consequences of lack of β1 integrin gene expression in mice. Genes & Dev., 9, 1896–1908. Gautam, M., Noakes, P.G., Moscoso, L., Rupp, F, Scheller, R.H., Merlie, J.P. and Sanes, J.R. (1996) Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell, 85, 525–535. Gee, S.H., Blacker, R.W., Douville, P.J., Provost, P.R., Yurchenco, P.D. and Carbonetto, S. (1993) Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan and binds with high affinity to the major heparin binding domain of laminin. J. Biol. Chem., 268, 14972–14980. Gehlsen, K.R., Dickerson, K., Argraves, W.S. and Ruoslahti, E. (1989) Subunit structure of a laminin-binding integrin and localization of its binding site on laminin. J. Biol. Chem., 264, 19034–19038. Georges-Labousesse, E., Messadeq, N., Yehia, G., Cadalbert, L., Dierich, A. and Le Meur, M. (1996) Absence of integrin α6 leads to epidermolysis bullosa and neonatal death in mice. Nature Genet., 13, 370–373. Gesemann, M., Brancaccio, A., Schumacher, B. and Ruegg, M.A. (1998) Agrin is a high-affinity binding protein of dystroglycan in non-muscle tissue. J. Biol. Chem., 273, 600– 605. Godfrey, E.W. (1991). Comparison of agrin-like proteins from the extracellular matrix of chicken kidney and muscle with neural agrin, a synapse organizing protein. Exp. Cell Res., 195, 99–109. Goldfmger, L.E., Stack, M.S. and Jones, J.C.R. (1998) Processing of laminin-5 and its functional consequences, role of plasmin and tissue-type plasminogen activator. J. Cell Biol., 141, 255–265. Grant, D.S., Kleinman, H.K. and Martin, G.R. (1990) The role of basement membranes in vascular development. Ann. N.Y. Acad. Set., 588, 61–72. Grobstein, C. (1967) Mechanism of organogenetic tissue interaction. Natl. Cancer Inst. Monogr., 26, 279–299. Groffen, A.J., Ruegg, M.A., Dijkman, H., van de Velden, T.J., Buskens, C.A., van den Born, J., Assmann, K.J., Monnens, L.A., Veerkamp, J.H. and van den Heuvel, L.P. (1998) Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane. J. Histochem. Cytochem. 46, 19–27. Hahn, U., Stallmach, A., Hahn, E.G. and Riecken, E.O. (1990) Basement membrane components are potent promoters of rat intestinal epithelial cell differentiation in vitro. Gastroenterology., 98, 322–335. Hay, E.D. (1993) Extracellular matrix alters epithelial differentiation. Curr.-Op. Cell Biol., 5, 1029–1035. Henry, M. and Campbell, K.P (1996) Dystroglycan, an extracellular matrix recepor linked to the cytoskeleton. Curr. Op. Cell Biol., 8, 625–631. Hirai, Y., Takabe, M., Takashima, M., Kobayashi, S. and Takeichi, M. (1992) Epimorphin, a mesenchymal protein essential for epithelial morphogenesis. Cell, 69, 471–481. Hirai, Y., Lochter, A., Galosy, S., Koshida, S., Niwa, S. and Bissell, M. (1998) Epimorphin functions as a key morphoregulator for mammary epithelial cells. J. Cell Biol. 140, 159– 169. Hormia, M., Falk-Marzillier, J., Plopper, G., Tamura, R.N., Jones, J.C., and Qauranta, V. (1995) Rapid spreading and mature hemidesmosome formation in HaCat keratinocytes induced by incubation with soluble laminin-5. J. Invest Derm., 105, 557–561. Ibraghimov-Beskrovnaya, O., Milatovich, A., Ozcelik, T., Yang, B., Koepnick, K., Francke, U. and Campbell, K.P. (1993) Human dystroglycan, skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosomal localization. Hum. Mol. Genet., 2, 1651–1657. livanainen, A., Kortesmaa, J., Sahlberg, C., Morita, T., Bergmann, U., Thesleff, I. and Tryggvason, K. (1997) Primary structure, developmental expression and immunolocalization of the murine laminin α4 chain. J. Biol. Chem., 272, 27862–27868. Kadoya, Y., Kadoya, K., Durbeej, M., Holmvall, K., Sorokin, L. and Ekblom, P. (1995) Antibodies against domain E3 of laminin-1 and integrin α6 perturb branching epithelial morphogenesis of submandibular gland, but by different modes. J. Cell Biol., 129, 521– 534. Kadoya, Y., Salmivirta, K., Talts, J., Kadoya, Y., Mayer, U., Timpl, R., and Ekblom, P. (1997) Importance of nidogen binding to laminin γ1 chain for branching epithelial morphogenesis of the submandibular gland. Development, 124, 683–691.

236


Kainulainen, T., Häkkinen, L., Hamidi, S., Larjava, K., Kallioinen, M., Peltonen, J., Salo, T., Larjava, H. and Oikarinen, A. (1998) Laminin-5 expression is independent of the injury and the microenvironment during reepithelializaiton of wounds. J. Histochem. Cytochem., 46, 353–360. Kallunki, P., Sainio, K., Eddy, R., Byers, M., Sariola, H., Beck, K., Hirvonen, H., Shows, T.B. and Tryggvason, K. (1992) A trunctated laminin chain homologous to the B2 chain: Structure, spatial expression and chromosomal assignment. J. Cell Biol., 119, 679–693. Klein, G., Ekblom, M., Fecker, L., Timpl, R. and Ekblom, P. (1990) Differential expression of laminin A and B chains during development of embryonic mouse organs. Development, 110, 823–837. Klein G., Langegger, M., Timpl, R. and Ekblom, P. (1988) Role of laminin a chain for the development of epithelial cell polarity. Cell, 55, 331–341. Kleinman, H.K., McGarvey, M.L., Hassell, J.R., Cannon, V.L., Laurie, G.W. and Martin, G.R. (1986) Basement membrane complexes with biological activity. Biochemistry, 25, 312–318. Kleinman, H.K., Ebihara, I., Killen, P.D., Sasaki, M., Cannon, F., Yamda, Y. and Martin, G.R. (1987) Genes for basement membrane components are coordinately expressed in differntiating F9 cells but not in normal adult murine tissues. Dev. Biol., 122, 373–378. Korhonen, M., Ylänne, J., Laitinen, L. and Virtanen, I. (1990) The α1–α6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. J. Cell Biol., 111, 1245–1254. Kreidberg, J.A., Donovan, M.J., Goldstein, S.L., Rennke, H., Shepherd, K., Jones, R.C. and Jaenisch, R. (1996) α3β1 integrin has a crucial role in kidney and lung organogenesis. Development, 122, 3537–3547. Kühn, K. (1996) Extracellular matrix constituents as integrin ligands. In Integrin-ligand interaction, eds. J.A.Eble and K.Kühn. pp. 41–84. R.G. Landes Company, Austin, Texas. Leivo, I., Vaheri, A., Timpl, R. and Wartiovaara, J. (1980) Appearance and distribution of collagens and laminin in the early mouse embryo. Dev. Biol., 76, 100–114. Matsui, C., Wang, C.K, Nelson, C.F. Bauer, E.A. and Hoeffner, W.K. (1995) The assembly of laminin-5 subunits. J. Biol. Chem., 270, 23496–23503. Mayer, U., Nischt, R., Pöschl, E., Mann, K., Fukuda, K., Gerl, M., Yamada, Y. and Timpl, R. (1993) A single EGF-like motif of laminin is responsible for high affinity nidogen binding. EMBO J., 12, 1879–1885. McMahan, U.J. (1990) The agrin hypothesis. Cold Spring Harbor Symp. Quant. Biol., 55, 407– 418. Miner, J.H., Patton, B.L., Lentz, S.I., Gilbert, D.J., Snider, W.D., Jenkins, N.A., Copeland, N.G. and Sanes, J.R. (1997) The laminin a chains: Expression, developmental transitions and chromosomal locations of α1–α5, identification of heterotrimeric laminins 8–11, and cloning of a novel α3 isoform. J. Cell Biol., 137, 685–701. Mundegar, R.R., von Oertzen, J. and Zierz, S. (1995) Increased laminin A expression in regenerating myofibers in neuromuscular disorders. Muscle Nerve, 18, 992–999. Müller, U., Wang, D., Denda, S., Meneses, J.J., Pedersen, R.A. and Reichardt, L.F. (1997) Integrin α8β1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell, 88, 603–613. Niessen, C.M., Hogervorst, F., Jaspar, L.H., de Melker, A., Delwel, G.O., Hulman, E.H., Kuikman, I. and Sonnenberg, A. (1994) The α6β4 integrin is a receptor for both laminin and kalinin. Exp. Cell Res., 211, 360–367. Nissinen, M., Vuolteenaho, R., Boot-Handford, R., Kallunki, P. and Tryggvason, K. (1991) Primary structure of the human laminin A chain. Limited expression in human tissues. Biochem. J., 276, 369–379. Ojakian, G.K. and Schwimmer, R. (1994) Regulation of epithelial cell surface polarity by β1 integrins. J. Cell Sci., 107, 561–576. Orian-Rousseau, V., Aberdam, D., Fontao, L., Chevalier, L., Meneguzzi, G., Kedinger, M. and Simon-Assmann, P. (1996). Developmental expression of laminin-5 and HD1 in the intestine, epithelial to mesenchymal shift for the laminin γ2 chain subunit deposition. Dev. Dyn., 206, 12–23. Patton, B.L., Miner, J.H., Chiu, A.Y. and Sanes, J.R. (1997) Distribution and function of laminins in the neuromuscular system of developing, adult and mutant mice. J. Cell Biol., 139, 1507–1521. Pyke, C., Salo, S., Ralfkioer, E., Romer, J., Dano, K. and Tryggvason, K. (1995) Laminin-5 is a marker of invading cancer in some human carcinomas and is coexpressed with the receptor for urokinase plasminogen activator in budding cancer cells in colon adenocarcinomas. Cancer Res., 55, 4132–4139.


237

Reist, N.E., Magill, C. and McMahan, U.J. (1987). Agrin-like molecules at synaptic sites in normal, denervated and damaged skeletal muscles. J. Cell Biol., 105, 2457–2469. Ruegg, M.A., Tsim, K.W.K., Horton, S.E., Kröger, S., Escher, G., Gensch, E.M. and McMahan, U.J. (1992) The agrin gene codes for a family of basal lamina proteins that differ in function and distribution. Neuron, 8, 691–699. Rupp, F., Özçelik, T., Linial, M., Peterson, K., Francke, U. and Scheller, R. (1992) Structure and chromosomal localization of the mammalian agrin gene. J. Neurosci., 12, 3535–3544. Salmivirta, K., Sorokin, L.M. and Ekblom, P. (1997) Differential expression of laminin a chains during murine tooth development. Dev. Dyn., 210, 206–215. Sanes J.R., Engvall, E., Butkowsky, R. and Hunter, D.D. (1990) Molecular heterogeneity of basement membranes, isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J. Cell Biol., 111, 1685–1699. Sasaki, T., Forsberg, E., Bloch, W., Addicks, K., Fässler, R. and Timpl, R. (1998) Deficiency of β1 integrins in teratoma interferes with basement membrane assembly and laminin1 expression. Exp. Cell Res., 238, 70–81. Saxén, L., Koskimies, O., Lahti, A., Miettinen, H., Rapola, J. and Wartiovaara, J. (1968) Differentiation of kidney mesenchyme in an experimental model system. Adv. Morphog., 7, 251–293. Schoenenberger, C, Zuk, A., Zinkl, G.M., Kendall, D. and Matlin, K.S. (1994) Integrin expression and localization in normal MDCK cells and transformed MDCK cells lacking apical polarity. J. Cell Set., 107, 527–541. Schuger, L. (1997). Laminins in lung development. Exp. Lung Res., 23, 1119–1129. Schuger, L., Skubitz, A.P.M., Morenas, A. and Gilbridge, K. (1995) Two separate domains of laminin promote lung organogenesis by different mechanisms of action. Dev. Biol. 169, 520–532. Schuger, L., Yurchenco, P., Relan, N.K. and Yang, Y. (1998). Laminin fragment E4 inhibition studies, basement membrane assembly and embryonic lung epithelial cell polarization requires laminin polymerization. Int. J. Dev. Biol., 42, 217–220. Senior, R., Griffin, G.L., Mudd, S., Moxley, M.A., Longmore, W.J. and Pierce, R.A. (1996) Entactin expression by rat lung and rat alveolar epithelial cells. Am. J. Resp. Cell Mol. Biol., 14, 239–247. Simon, E.E., Liu, C.H., Das, M., Nigam, S., Broekelman, T.J. and McDonald, J.A. (1994) Characterization of integrins in cultured human renal cortical tubule epithelial cells. Am. J. Physiol., 267, F612–623. Simon-Assmann, P., Duclos, B., Orian-Rousseau, V., Arnold, C., Mathelin, C., Engvall, E. and Kedinger, M. (1994). Differential expression of laminin isoforms and α6β4 integrin subunits in the developing human and mouse intestine. Dev. Dyn., 201, 71–85. Simon-Assmann, P., Kedinger, M., Dearcangelis, A., Rousseau, V. and Simo, P. (1995) Extracellular matrix components in intestinal development. Experientia, 51, 883–900. Smalheiser, N.R. and Schwartz, N.B. (1987) Cranin a laminin-binding protein of cell membranes. Proc. Natl. Acad. Sci. USA, 84, 6457–6461. Smalheiser, N.R. and Kim, E. (1995) Purfication of cranin, a laminin binding membrane protein identity with dystroglycan and reassessment of its carbohydrate moieties. J. Biol. Chem., 270, 15425–15433. Sonnenberg, A., Linder, C., Daams, J.H. and Kennel, S.J. (1990) The α6β1 (VLA-6) and α6β4 protein complexes. Tissue distribution and biochemical complexes. J. Cell Sci., 96, 207– 217. Sorokin, L.M., Conzelmann, S., Ekblom, P., Battaglia, C., Aumailley, M. and Timpl, R. (1992) Monoclonal antibodies against laminin A chain fragment E3 and their effects on binding to cells and proteoglycan and on kidney development. Exp. Cell Res., 201, 137–144. Sorokin, L.M., Pausch, F., Durbeej, M. and Ekblom, P. (1997a) Differential expression of five laminin a chains in developing and adult mouse kidney. Dev. Dyn., 210, 446–462. Sorokin, L.M., Pausch, F., Frieser, M., Kröger, S., Ohage, E. and Deutzmann, R. (1997b) Developmental regulation of the laminin α5 chain suggests a role in epithelial and endothelial cell maturation. Dev. Biol., 189, 285–300. Stahl, S., Weitzmann, S. and Jones, J.C.R. (1995) The role of laminin-5 and its receptors in mammary epithelial cell branching morphogenesis. J. Cell Sci., 110, 55–63. Stephens, L.E., Sutherland, A.E., Klimanskaya, I.V., Andrieux, A., Meneses, J., Pederesen, R.A. and Damsky, C.M. (1995) Deletion of β1 integrin in mice results in inner cell mass failure and peri- implantation lethality. Genes & Dev., 9, 1883–1895.

238


Streuli, C. (1996) Basement membrane as a differentiation and survival factor. In The laminins. Ekblom, P. and Timpl, R. (Eds.). Harwood Academic Publishers. Amsterdam, pp. 217– 234. Sugiyama, J., Bowen, D.C. and Hall, Z.W. (1994) Dystroglycan binds nerve and muscle agrin. Neuron. 13, 103–115. Tani, T., Lumme, A., Kivilaakso, E., Kiviluoto, T., Burgeson, R.E., Kangas, L., Leivo, I. and Virtanen, I. (1997) Pancreatic carcinomas deposit laminin-5, preferably adhere to laminin-5 and migrate on the newly deposited basement membrane. Am. J. Pathol., 151, 1289– 1302. Thomas, T. and Dziadek, M. (1993) Genes coding for basement membrane glycoproteins laminin, nidogen and collagen IV are differentially expressed in the nervous system and by epithelial, endothelial and mesenchymal cells of the mouse embryo. Exp. Cell Res., 208, 54–67. Thomas, T. and Dziadek, M. (1994) Expression of collagen α1 (IV), laminin and nidogen genes in the embryonic lung, implications for branching morphogenesis. Mech. Devi., 45, 193– 201. Tiger, C.-F., Champliaud., M., Pedrosa-Domellof, F., Thornell, L., Ekblom, P. and Gullberg, D. (1997) Presence of laminin α5 chain and lack of laminin α1 chain during human muscle development and in muscular dystrophies. J. Biol. Chem., 272, 28590–28595. Timpl, R. and Brown, J. (1996) Supramolecular assembly of basement membranes. Bioessays, 18, 123–132. Uitto, J. Pulkkinen, L. and Christiano, A.M. (1994). Molecular basis of the dystrophic and junctional forms of of edidermolysis bullosa, mutations in the type VII collagen and kalinin (laminin 5) genes. J. Invest. Derm., 103, 39–46 Vachon, P.H. and Bealieu, J.F. (1995) Extracellular heterotrimeric laminin promotes differentiation in human enterocytes. Am. J. Physiol., 268, G857–G867. Van den Heuvel, G.B. and Abrahamson, D.R. (1993) Quantitation and localization of laminin A, B1 and B2 chain RNA transcripts in developing kidney. Am. J. Physiol., 265, F293– F299. Virtanen, I., Laitinen, L. and Korhonen, M. (1995) Differential expression of laminin polypeptides in developing and adult human kidney. J. Histochem. Cytochem., 43, 621–628. Vuolteenaho, R., Nissinen, M., Sainio, K., Byers, M., Eddy, P., Hirvonen, H., Shows, T., Sariola, H., Engvall, E. and Tryggvason, K. (1994) Human laminin M chain (merosin): complete primary structure, chromosomal assignment and expression of the M and A chain in human fetal tissues. J. Cell Biol., 124, 381–394. Williamson, R.A., Henry, M.D., Daniels, K.J., Hrstka, R.F., Lee, J.C., Sunada, Y., Ibraghimov-Beskrovnaya, O. and Campbell, K.P. (1997) Dystroglycan is essential for early embryonic development: disruption of Reichert’s membrane in Dag 1-null mice. Hum. Mol. Genet., 6, 831–841. Yurchenco, P. and O’Rear, J.J. (1994) Basal lamina assembly. Curr. Op. Cell Biol., 6, 674–681. Zuk, A. and Matlin, K.S. (1996) Apical β1 integrin in polarized MDCK cells mediate tubulocyst formation in response to type I collagen overlay. J. Cell Set., 109, 1875–1889.

13. THE ROLE OF RET RECEPTOR TYROSINE KINASE AND GLIAL DERIVED NEUROTROPHlC FACTOR IN DEVELOPMENT OF THE EXCRETORY AND PERIPHERAL NERVOUS SYSTEMS ANITA SCHUCHARDT1, PASCALS DURBEC2, CAMELIA V. MARCOSGUTIERREZ2, VASSILIS PACHNIS2 and FRANK COSTANTINI1 1 Department of Genetics and Development, College of Physicians and Surgeons, Columbia

University, New York, NY 10032, USA and 2Division of Developmental Neurobiology, National Institute for Medical Research, London NW7 1AA, UK OVERVIEW The receptor tyrosine kinase RET has recently been shown to serve as a functional receptor for the glialderived neurotrophic factor (GDNF). RET binds to GDNF in conjunction with an accessory receptor chain, GDNFR-α. These three proteins are expressed in a number of sites in the developing peripheral and central nervous systems, as well as in the developing excretory system. Studies of mice with targeted mutations in the genes encoding RET and GDNF have established that both genes are required for the normal development of the metanephric (adult) kidney, and for the enteric nervous system and parts of the sympathetic nervous system. The development of the metanephric kidney is unique among glandular organs because the epithelial components derive from mesenchyme as well as epithelium. The epithelial component, the ureteric bud, induces the metanephric mesenchyme to condense into epithelial vesicles and differentiate into the nephrons of the mature kidney. Simultaneously, the metanephric mesenchyme induces growth and arborization of the ureteric bud which eventually forms the collecting system of the kidney. RET is expressed in the ureteric bud of the developing kidney, while GDNF expression is localized to the metanephric mesenchyme, and together they mediate the inductive effect of the metanephric mesenchyme on the ureteric bud. The peripheral nervous system is formed by the migration and differentiation of neural crest cells, many of which express RET, while GDNF is expressed in a complementary pattern in the surrounding mesenchyme. GDNF/RET signaling is of particular importance for the survival and proliferation of a specific subpopulation of neural crest, the sympathoenteric lineage, which form the parts of the enteric and sympathetic nervous systems. INTRODUCTION RET is a Member of the Receptor Tyrosine Kinase Superfamily The predicted structure of the RET protein places it in the receptor tyrosine kinase (RTK) superfamily (Takahashi et al., 1988; Takahashi and Cooper, 1987; Hanks, 1991). This group of proteins is defined by the presence of an extracellular domain which binds ligand, a transmembrane domain, and an intracellular domain which contains a regulated catalytic kinase activity (reviewed in White, 1991). Binding of ligand to the extracellular domain activates the intracellular kinase activity, triggering a signaling cascade which can Corresponding Author: F. Costantini. Tel: (212) 305–6814; Fax: (212) 923–2090; E-mail: [email protected]

240


affect gene transcription, cell metabolism, cell proliferation and cytoskeletal architecture. The precise mechanism by which this activation occurs is not known, but it appears to involve the formation of receptor dimers and structural changes within the receptor. Upon activation of the tyrosine kinase activity, intermolecular trans-phosphorylation of the receptors is observed. The phosphorylated tyrosines regulate interactions between the receptor and its substrates, some of which become activated themselves either through phosphorylation or structural changes. The subset of signaling proteins which interact with the phosphorylated receptor is determined partly by the amino acids surrounding the phosphorylated tyrosines within the receptor. Thus, different receptor tyrosine kinases can produce different responses within the same cell. However, specificity is also determined in part by the expression profile of the signaling proteins within a cell. Therefore, one receptor may produce very different responses when expressed in various cell types. Because one of the effects of activated RTKs can be cell proliferation, deregulation of the catalytic kinase activity often results in uncontrolled growth, or oncogenesis. The RET gene was initially isolated in a rearranged constitutively active form as an oncogene (Takahashi et al., 1985; Takahashi et al., 1988; Takahashi and Cooper, 1987). When expressed in transgenic mice, this rearranged form can cause various types of tumors depending on the promoter used and the resulting sites of expression (Iwamoto et al., 1990, 1991a, 1991b). Gain-of-function mutations of RET are associated with the familial cancer syndromes Multiple Endocrine Neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma (FMTC), which are characterized by an increased tendency to develop endocrine neoplasias and phaeochromocytomas (Mulligan et al., 1993; Donis-Keller et al., 1993; Hofstra et al., 1994; Santoro et al., 1995). GDNF is a Ligand for RET GDNF (glial cell-line derived neurotrophic factor) has recently been identified as a ligand for RET. GDNF is classified as a distantly related (20% similarity) member of the TGF-β family, based on the presence of a TGF-β-like precursor structure and dimeric subunit composition and the presence of a cysteine knot motif which is typical of family members (Lin et al., 1993). As its name implies, GDNF was initially isolated from a glial cell line as a neurotrophic factor which increases the survival of midbrain dopaminergic neurons (Lin et al., 1993). GDNF has since been shown to have a neurotrophic effect on several other types of neurons from the central and peripheral nervous systems, including spinal motor neurons, and adrenergic neurons in the CNS and autonomic, enteric and subsets of sensory neuroblasts and neurons in the PNS (Arenas et al., 1995; Buj-Bello et al., 1995; Henderson, C.E., 1994). Several lines of evidence suggested that GDNF could be a ligand for RET (Massague, 1996): All GDNF responsive cells expressed RET (Abantaggiato et al., 1994; Pachnis et al., 1993; Tsuzuki et al., 1995), during development, GDNF expression is localized to regions where expression of a RET ligand would be et al., 1996); the phenotypes of mice which lacked a functional GDNF gene were expected (Durbec et al., 1996a; Hellmich et al., 1996; Sánchez et al., 1996; Suvanto very similar to the phenotypes of mice lacking a functional RET gene (Moore et al., 1996; Pichel et al., 1996; Sánchez et al., 1996; Schuchardt et al., 1994); and the addition of GDNF induces neuronal outgrowth in explants isolated from normal but not RET-deficient embryos (Durbec et al., 1996a). Studies showing that GDNF could bind to RET and stimulate RET kinase activity confirmed that GDNF is indeed a RET ligand (Durbec et al., 1996a; Jing et al., 1996; Treanor et al., 1996; Trupp et al., 1996; Vega et al., 1996; Worby et al., 1996).


241

Figure 1 A model for the interactions between RET, GDNFR-α and GDNF (Jing et al., 1996; Treanor et al., 1996; Massagué, 1996). GDNF (a) first binds to the GPI-linked cell surface protein GDNFR-α (b), and this dimer then binds to extracellular domain of the RET receptor tyrosine kinase (c), allowing dimerization and autophosphorylaton of RET.

GDNFR-α Mediates the Interaction between RET and GDNF Interestingly, GDNF does not appear to directly bind to RET. Instead the interaction appears to be mediated by a GPI-linked extracellular protein called GDNFR-α. This protein was discovered during attempts to isolate a receptor for GDNF via expression cloning (Jing et al., 1996; Treanor et al., 1996). GDNFR-α lacks a cytoplasmic domain, but is absolutely required for GDNF to associate with RET and to stimulate RET kinase activity. When cells were treated with PI-PLC to disrupt the GPI linkage, GDNF failed to bind RET or induce RET phosphorylation. Both activities could be restored by adding soluble GDNFR-α with GDNF. Overexpression of GDNFR-α in cells which expressed RET augmented the binding of GDNF to RET. Cross-linking and immunoprecipitation studies showed that the three proteins could be immunoprecipitated in the same complex, and also showed that GDNFR-α could bind GDNF either as a monomer or a dimer in the absence of RET (Jing et al., 1996; Treanor et al., 1996). A model for the interaction between GDNF, GDNFR-α, and RE, based on the available data, is illustrated in Figure 1. According to this model, GDNFRα first binds GDNF and this complex is then recognized by RET, resulting in dimerization, activation of the kinase domain and receptor trans-phosphorylation. Evidence for the importance of dimerization in RET signaling comes from an MEN2A mutation which results in steady-state dimerization of RET and constitutive activation of the RET kinase (Asai et al., 1995; Santoro et al., 1995). The interaction between GDNF, GDNFR-α and RET is unique in several ways. GDNF is the first TGF-β family member to have a receptor tyrosine kinase instead of a receptor serine/threonine kinase as a signaling partner. In addition, although other receptors bind ligands in association with other molecules (e.g., FGF binds to RTKs in association with heparan sulfate glycosaminoglycans, and TGF-β interacts with serine/ threonine kinase receptors in conjunction with the membrane protein, betaglycan; Schlessinger et al., 1995), the dependence of the GDNF/RET interaction on GDNFR-α is unusually strict (Massagué, 1996). The ability of soluble GDNFR-α to bind to GDNF and mediate its interaction with RET suggests that the function of GDNFR-α is not merely to limit diffusion of RET. Because the requirement for GDNFR-α to

242


mediate GDNF binding to RET is absolute, GDNFR-α may be altering the structure of GDNF, and perhaps the ligand for RET should be thought of as GDNF bound to GDNFR-α (Massagué, 1996). RET AND GDNF ARE REQUIRED FOR NORMAL DEVELOPMENT OF THE EXCRETORY AND NERVOUS SYSTEMS Expression of RET, GDNF and GDNFR-α During Development The ability of receptor tyrosine kinases to convert an extracellular signal into an intracellular one suggests that they would play a vital role during development when intercellular communication is crucial. Indeed many RTKs have been shown to be essential for the normal development of C.elegans, Drosophila and mouse. During murine development, the RET proto-oncogene and GDNF are expressed primarily in the nervous and excretory systems (Abantaggiato et al., 1994; Hellmich et al., 1996; Pachnis et al., 1993; Suvanto et al., 1996; Tsuzuki et al., 1995). In the central nervous system, RET expression is first detected at E8.5 in the ventral neural tube. Motor neurons later develop at this site and RET expression persists in these cells. By E13.5, RET mRNA can be seen in the neural retina of the developing eye and by postnatal day 7, it has become restricted to the layers of the retina containing the ganglion, amacrine and horizontal cells. RET message is also detected in the ventral part of the midbrain beginning at E12.0 and continuing through birth. Within the peripheral nervous system, RET mRNA is initially found in all migrating truncal neural crest cells and subsequently is identified in most of their neuronal derivatives, including neurons of the sympathetic ganglia and enteric nervous system, and in subsets of neurons in the dorsal root ganglia. Figure 2 (B, D, E) illustrates the expression of RET during the development of the enteric nervous system. In the cranial region, only the migrating neural crest cells at the level of rhombomere IV express RET message and later in development, message is initially detected only in the cranial ganglion derived from this neural crest, the facialacoustic ganglion. At later developmental stages, all cranial ganglia express RET Within the developing excretory system, RET mRNA is restricted to the nephric (Wolffian) duct and its derivative, the ureteric bud (UB) (Pachnis et al., 1993). RET message is first detected at E8.5–E10 in the nephric duct of the pronephric and mesonephric system. A gradient of RET mRNA is observed with the highest concentration at the caudal end of the nephric duct, where the ureteric bud will later develop (Figure 3B). When the UB evaginates, grows into the nearby metanephric mesenchyme, and branches for the first time (E11.0–E11.5), RET is expressed throughout the UB (Figure 3C). At later stages, when the UB is elongating and branching repeatedly within the outer nephrogenic zone of the developing kidney, RET expression is restricted to the growing and branching tips of the UB (Figure 3F). As might be expected for a ligand of RET, the expression of GDNF during development is similar and complementary to that of RET (Durbec et al., 1996a; Hellmich et al., 1996; Sánchez et al., 1996). For example, as early as embryonic day 12.5 (E12.5), GDNF expression is observed in skeletal muscle, a potential target for spinal motor neurons. During embryogenesis, GDNF mRNA is prevalent in the mesenchymal tissues of the digestive tract (Figure 2), which are colonized by the RET expressing neural crest cells that later develop into the enteric nervous system. In the developing excretory system, GDNF is first expressed in the mesenchyme adjacent to the mesonephric duct, before ureteric bud outgrowth. At E11. 5, GDNF mRNA is localized to the metanephric mesenchymal cells surrounding the ureteric bud (Figure 3C). Although the expression of GDNF in the metanephric blastema before ureteric bud outgrowth has not been reported, evidence from RET and GDNF knockout mice (see below) implies that GDNF is expressed in the blastema at this stage. At later stages, GDNF mRNA is restricted to the mesenchymal cells


243

in the outer nephrogenic zone, surrounding the RET expressing UB tips (Figure 3D, E). GDNF expression is not observed in any differentiated metanephric mesenchymal derivatives. The expression of GDNFR-α has been studied in less detail. However, GDNFR-α transcripts are found in the central nervous system of the E15.5 rat in spinal motor neurons and in the dopaminergic neurons of the ventral midbrain. At this stage, GDNFR-α mRNA is also detected in the peripheral nervous system in subsets of neurons within the dorsal root ganglia. In the excretory system of E15.5 rat, GDNFR-α is confined to the tips of the ureteric bud at the periphery of the kidney. While these sites of GDNFR-α expression co-localize with RET expression, there are exceptions to this pattern. Within the gastrointestinal tract, GDNFR-α expression co-localizes with GDNF expression in the smooth and striated muscle which surrounds the enteric nervous system (Treanor et al., 1996). Mice Lacking RET or GDNF Display Defects in the Nervous System and Excretory Systems The importance of the RET/GDNF signaling system during development has been demonstrated by the generation of mice which lack functional RET or GDNF. Mice homozygous for a mutant allele (ret-k−) designed to abolish RET kinase activity die within twenty-four hours of birth, and display severe renal defects, in addition to an absence of the neurons and glia which form the superior cervical ganglia and the enteric nervous system (Schuchardt et al., 1994). The excretory system defects in the ret-k−/ ret-k− mice range from complete absence of the kidneys on one or both sides (unilateral or bilateral renal agenesis), to formation of a blind-ending ureter with no detectable renal tissue, to the presence of small, severely dysplastic kidney rudiments. The phenotypes of mice lacking GDNF are almost identical, with slight variations in severity (Moore et al., 1996; Pichel et al., 1996; Sánchez et al., 1996). In these mice, the enteric nervous system is absent, and the excretory system displays a range of defects, although bilateral renal agenesis is more frequent than in ret-k−/ ret-k− mice. One difference between RET and GDNF homozygous mutant mice is that in the latter, the superior cervical ganglia are present and display only a 20% reduction in the number of neurons. In addition, GDNF mutant mice display reductions in the number of neurons (20–40%) in the trigeminal motor nucleus, the spinal motor column, petrosal-nodose ganglia, and dorsal root ganglia. These neuronal populations have not been examined in enough detail to determine if they are affected in the ret-k−/ret-k− mice. THE ROLE OF RET/GDNF SIGNALING IN THE DEVELOPMENT OF THE PERIPHERAL NERVOUS SYSTEM RET is essential for the normal development of the enteric nervous system. Individuals heterozygous for loss-of-function mutations in RET develop Hirschprung’s disease, congenital megacolon caused by absence of enteric ganglia from the terminal colon (Edery et al., 1994; Romeo et al., 1994), and mice homozygous for loss of RET kinase activity display absence of most enteric ganglia (Durbec et al., 1996b; Schuchardt et al., 1994). The neurons and glia of the enteric nervous system develop from vagal neural crest cells (originating at the level of somites 1–7) and sacral neural crest cells (originating posterior to somite 28), which migrate into and colonize the muscle layers of the gastrointestinal tract (Serbedzija et al., 199?; LeDouarin and Teillet, 1973). RET mRNA is expressed within the migrating neural crest cells, both before and after they enter the gastrointestinal tract, and in the differentiated neurons (Pachnis et al., 1993). In ret-k−/ret-k− newborn mice, neurons and glia are absent from the distal stomach, duodenum, and small and large intestine (Figure 4a, b). However, a significant (although reduced compared to wild-type) number

244


Figure 2 Comparative analysis of GDNF and RET expression in the developing ENS. A and B, Adjacent sagittal sections through the gastrointestinal duct of a E10.5 mouse embryo stained with a GDNF (A) or RET (B) probe. GDNF mRNA is observed throughout the entire length of the gut, with the highest expression levels detected in the stomach (s). Similarly, c-Ret-expressing cells are detected in the developing esophagus (es) and intestine (i) although a larger number of positive cells is seen in the stomach, region. C and D, Adjacent sagittal sections through the intestinal loops of a E11.5 mouse embryo hybridized with a GDNF (C) or RET (D) probe. GDNF expression is detected throughout the gut mesenchyme where scattered RET-positive cells are observed. The levels of GDNF mRNA expression are directly proportional to the number of RET-positive cells. Dotted lines in D represent the boundaries between the gut endoderm and mesenchyme. E, Sagittal section of the intestinal region of a E12.5 mouse embryo hybridized with a GDNF probe (green precipitate) and a RET probe (black precipitate). GDNF expression is observed in the gut mesenchyme, while transcripts for RET are synthesized by the enteric neuron precursors. F and G, Transverse sections through the gut of a E11.5 and a E13.5 mouse embryo hybridized with a GDNF probe. At E11.5 transcripts for GDNF are detected throughout the gut mesenchyme while by E13.5 they become restricted to the outer muscle layer of the gut where enteric neuroblasts lie.

of cells expressing neuronal and glial markers could be detected in the esophagus and stomach of homozygous mutant embryos and newborn mice (Durbec et al., 1996b). Markers for neuronal precursor


245

Figure 3 Comparative analysis of GDNF and RET expression in the excretory system of the mouse embryo. Sagittal sections from E10.5 (A, B), E11.5 (C), E13.5 (D) and E15.5 (E,F) mouse embryos stained with GDNF (A, C, D, E) and RET (B, C, F) probes by in situ hybridization. A and B, Adjacent sagittal sections illustrating the expression of GDNF (A) and RET (B) transcripts in the developing excretory system of a E 10.5 mouse embryo. Transcripts for GDNF are observed in the uncondensed mesenchyme (m) adjacent to the RET-expressing caudal end of the mesonephric duct (md). C, Sagittal section of the metanephric region of an E11.5 mouse embryo hybridized with a GDNF (black precipitate) and a RET (blue precipitate) probes. High levels of RET mRNA are observed in the ureteric bud and its branches (ub), while GDNF expression is detected in the surrounding condensing metanephric mesenchyme (m). D, Sagittal section through the kidney of an E13.5 mouse embryo hybridized with a GDNF probe. Signal is observed in the outer zone of the kidney, surrounding the ureteric bud branches. E and F, Adjacent sagittal sections of the kidney of an E15.5 mouse embryo hybridized with GDNF (E) and a RET (F) probes. Transcripts for both genes are restricted to the outer zone of the kidney. Here, RET mRNA is detected at the tips of the urteric bud branches while GDNF mRNA is observed in the surrounding tissue, mu, mullerian duct.

cells and neural crest revealed the presence of these cells in the esophagus, stomach and proximal loop of the intestine of E10.5 wild-type embryos, but only in the esophagus and proximal stomach of homozygous

246


mutant embryos. Therefore, RET-deflcient neural crest cells appear to be able to migrate into the foregut of mutant embryos and differentiate into neurons and glia (although in smaller than normal numbers), but are unable to successfully colonize the wall of the mid- and hind-gut (Durbec et al., 1996b). The other neural crest derivative that is severely affected in the homozygous ret-k− mice is the superior cervical ganglion (SCG) of the sympathetic chain (the other sympathetic ganglia appear unaffected) (Durbec et al., 1996b). The use of sympathoblast markers revealed that at E10–10.5, the sympathetic chain in homozygous mutant embryos (including the most anterior portion which presumably gives rise to the SCG) is indistinguishable from wild-type. However, by E12.5 the SCG had disappeared in ret-k−/ret-k− embryos (Figure 4c, d), although all other ganglia appeared to be forming normally. Therefore, in parallel with what is observed in the ENS, RET-deficient neural crest cells appear able to migrate into the region of the SCG and begin to differentiate, but are unable to complete later stages of gangliogenesis. Interestingly, in situ hybridization studies reveal that the precursors to the ENS and the SCG appear to share a common origin from the vagal crest and transiently form a common pool of RET-positive cells in the mesenchyme between the dorsal aorta and the foregut (Durbec et al., 1996b). Lineage studies confirmed the common origin of the SCG and the ENS in the vagal crest and restricted it further to the anterior vagal neural crest which arises in the postotic hindbrain (somites 1–5) (Durbec et al., 1996b). Consistent with previous reports (LeDouarin and Teillet, 1973; LeDouarin, 1986) these lineage studies demonstrated that the rest of the sympathetic chain is derived from more posterior truncal neural crest, but more interestingly, the lineage studies also revealed that a subpopulation of the ENS precursors that colonize the foregut derives from vagal neural crest which is located in the trunk (somites 6–7). Therefore, the ENS of the midand hindgut and the SCG, which are more sensitive to loss of RET function, appear to derive from a common origin, the anterior vagal neural crest, and appear to represent a distinct “sympathoenteric” lineage (Durbec et al., 1996b). RET signaling appears to play a major role in the survival and proliferation of the progenitors of this lineage. Although in the ret-k−/ret-k− mice, migration and early differentiation of the SCG and ENS precursors appear normal, an affect on these early steps can not be ruled out. Mice lacking GDNF also display defects in the ENS and SCG (Moore et al., 1996; Pichel et al., 1996; Sánchez et al., 1996). The phenotype in the enteric nervous system of GDNF-deficient mice appears very similar to that of RET-deficient mice. One group (Sánchez et al., 1996) reported a complete absence of enteric ganglia in the stomach and esophagus of mutant newborns, while another (Moore et al., 1996) observed a few ganglia present in the stomach. Enteric ganglia are missing from the small and large intestine of all the mutant newborns. The small and large intestine of E12.5 embryos which lack GDNF don’t contain peripherin-positive and p75-positive neuroblasts, but p75-positive neuroblasts are observed in the esophagus and stomach at this stage (Moore et al., 1996; Sánchez et al., 1996). These results confirm the findings in the RET-deficient mice, and suggest that the migration and early differentiation steps are largely normal in the GDNF-deficient mice, with the mutation primarily affecting later stages of survival and proliferation. In contrast, although absence of GDNF also affects the SCG, the phenotype is much less severe than that observed in the absence of functional RET. Only 20–35% of the SCG neurons are missing in mice homozygous for mutations in GDNF (Moore et al., 1996; Sánchez et al., 1996), whereas the entire ganglia are absent in the ret-k−/ ret-k− animals (Durbec et al., 1996b). This discrepancy may suggest the presence of another ligand which can interact with RET and partially compensate for the absence of GDNF in the SCG.


247

Figure 4 Defects in the enteric and sympathetic nervous system of homozygous ret-k− mice, a and b, absence of neurons from the gut of mutant newborn animals (reproduced from Schuchardt et al., 1994). The two immunofluorescence micrographs show sections of large intestine from heterozygous (a) and homozygous (b) animals, stained with antibodies against peripherin, an intermediate filament protein expressed in enteric neurons. Neurons of the myenteric plexus (arrow) and submucosal plexus are present in the gut wall of the heterozygote (a) but absent in the homozygous mutant animal (b). c and d, absence of the superior cervical ganglion (SCG) from homozygous mutant E12.5 embryos (reproduced from Durbec et al., 1996b). Transverse sections of heterozygous (a) or homozygous(d) embryos were stained with antibodies against the neuronal marker tyrosine hydroxylase (TH). Strongly TH-positive SCG precursors were present around the dorsal aorta (A) of the heterozygous embryos, while they were absent from the homozygous embryos. NG, nodose ganglion.

THE ROLE OF RET/GDNF SIGNALING IN RENAL DEVELOPMENT Overview of Kidney Development The development of the excretory system in mammals is characterized by a progression through three

248


successive overlapping systems: the pronephros, the mesonephros and the metanephros (Saxen, 1987). The pronephros and mesonephros are transient systems comprised of the nephric duct and a series of tubules and glomeruli located in the adjacent nephric cord. Development of the metanephros, or mature kidney, begins when the caudal tip of the nephric duct (also called the Wolffian duct at this stage) evaginates, forming the ureteric bud which grows caudally into a distinct region of adjacent mesenchyme, the metanephric blastema. Formation of the metanephric kidney occurs through a series of reciprocal inductive interactions between the ureteric bud and the surrounding metanephric mesenchyme (reviewed by Bard, 1992; Ekblom, 1992; Hardman et al., 1994; Saxen, 1987). The metanephric mesenchyme induces the ureteric bud to grow and branch eventually resulting in the formation of the collecting system (Erickson, 1968; Grobstein, 1953a, 1955). The precise pattern of growth and branching of the ureteric bud determines the histoarchitecture of the mature kidney (Saxen, 1987). The ureteric bud induces the metanephric mesenchyme to condense into epithelial vesicles which eventually differentiate into the mature parts of the nephron: the proximal and distal collecting tubules, glomeruli, and Henle’s loops (Grobstein, 1955, 1956; Saxen, 1970). RET and GDNF are Required for Normal Metanephric Kidney Development Until recently, the receptors and ligands involved in the inductive interactions which produce the mature kidney were not known. Expression studies suggested that RET and GDNF were good candidates for mediating one of these inductions and these suspicions were confirmed by the inability of mice which lack functional RET or GDNF to develop normal kidneys. Mice lacking RET or GDNF display a range of excretory system defects Newborn mice homozygous for the ret-k– allele display a variety of excretory system defects ranging from renal agenesis to the formation of small dysplastic kidneys (Figure 5) (Schuchardt et al., 1994). In mice homozygous for GDNF mutations, a higher frequency of renal agenesis was observed, with the occasional presence of a microscopic kidney (Moore et al., 1996; Pichel et al., 1996; Sánchez et al., 1996). This difference in phenotypic severity is probably due to the different genetic backgrounds of the strains carrying the two mutations, because we have observed that when the ret-k− mutation is carried on an inbred background, the proportion of mutant mice displaying renal agenesis increases from 33% to 68% (unpublished data), which is comparable to that observed in the GDNF-deficient mice. Thirty to forty percent of mice heterozygous for null mutations in GDNF also display defects in the excretory system, which range from unilateral and bilateral renal agenesis to hypoplasia or dysplasia. In contrast, the excretory systems of mice heterozygous for the ret-k− allele appear indistinguishable from wild type, regardless of their genetic background. The presence of excretory system defects in the GDNF heterozygotes, but not the ret-k − heterozygotes suggests that the amount of GDNF is a limiting factor in the development of the metanephros. The absence of RET inhibits ureteric bud growth and branching At E10.5 in the ret-k− homozygotes, the development of the excretory system appears normal. The Wolffian duct has extended and contacted the cloaca, and the mesonephros is grossly normal in both the RETdeficient and GDNF-deficient embryos, suggesting that RET is not required for extension of the Wolffian duct or development of the mesonephros (Schuchardt et al., 1996). The effect of the ret-k− mutation on the


249

development of the excretory system is first apparent at E1 1.0 when 50% of the homozygous embryos fail to develop a ureteric bud on one or both sides (Figure 6D) (Schuchardt et al., 1996). Failure of the ureteric bud to form at the appropriate time is also observed in embryos lacking GDNF (Moore et al., 1996; Pichel et al., 1996; Sánchez et al., 1996), suggesting that renal agenesis is caused by failure of the ureteric bud to evaginate from the Wolffian duct. In RET-deficient embryos, when a ureteric bud was observed, its growth and branching were delayed (Figure 6B) (Schuchardt et al., 1996). In some cases, the ureteric buds had failed to grow into the mesenchyme (Figure 6C), and these presumably develop into the blind-ending ureters observed in some newborn mutant mice. In cases where the ureteric bud had contacted the metanephric mesenchyme, its branching was delayed and often appeared abnormal. The first branching event is always symmetrical in wild-type embryos (Figure 6E), but in ret-k− homozygous embryos, one branch was often longer than the other (Figure 6F), or the ureteric bud appeared curved but not branched (perhaps because only a single branch had grown). The decreased and delayed branching of the ureteric bud in the metanephric mesenchyme is consistent with the small size and limited development of the collecting system of the hypodysplastic kidneys which develop in the RET-deficient mice (Figure 5f, g). Ureteric buds from ret-k−/ ret-k− embryos which were separated from the surrounding mesenchyme and cultured with isolated wildtype mesenchymes showed a greatly decreased tendency to survive and branch compared to wild-type ureteric buds. On the other hand, mutant metanephric mesenchyme was fully competent to induce growth and branching when recombined with wild type ureteric buds (Schuchardt et al., 1996). These results, combined with the expression of RET within the ureteric bud (Pachnis et al., 1993), suggests that RET is required within the ureteric bud for normal growth and branching. This hypothesis is further supported by the observation that high concentrations of exogenous GDNF can increase the number of ureteric bud branches in cultured kidney rudiments (Vega et al., 1996). Defects in the metanephric mesenchyme of RET-deficient mice are secondary to those in the ureteric bud The small dysplastic kidneys seen in some ret-k-/ret-k- newborn mice have fewer nephrons than normal kidneys and contain an abundance of loose mesenchyme which is not normally observed in the newborn kidney (Schuchardt et al., 1994). Histological analyses of mutant embryos revealed that the development of the metanephric mesenchyme was affected in the ret-k−/ret-k− embryos (Schuchardt et al., 1996). When the metanephric blastemas of age-matched wild-type and mutant embryos were compared, condensation of the metanephric mesenchyme of the mutant embryos appeared delayed (Figure 6B). The retardation in condensation could have been caused by delayed entrance of the ureteric bud into the metanephric mesenchyme or the reduced size of the bud. However, it was also observed when comparing wild-type and mutant metanephric blastemas in which the development of the ureteric buds had progressed to the same stage. Ex vivo experiments in which mutant metanephric mesenchymes were combined with wild-type spinal cord, a potent inducer of tubulogenesis, revealed no difference in the extent or timing of tubulogenesis in the mutant and wild-type mesenchymes (Schuchardt et al., 1996). Therefore, the decreased amount of tubulogenesis observed in kidney rudiments from RET-deficient embryos is probably a secondary effect of defects in the growth and branching of the ureteric bud.

250


Figure 5 Renal defects in RET mutant newborn mice, a-d, dissected urogenital systems from one wild type (a) and three homozygous ret-k− mutant newborn mice (b-d). The mutant mice displayed small kidney rudiments (both sides of the specimen shown in b and the left side of the specimen in c), blind-ending ureters with no renal tissue (d), or the absence of both ureter and kidney (right side of specimen in c). a, adrenal; k, kidney, u, ureter; v, vas deferens; e, epididymis; t, testes; b, bladder; d, dorsal aorta; o, ovary; ut, uterine horn, e-f, histological sections of a wild type newborn kidney (e) and a mutant kidney rudiment (f and g), stained with H&E. All three sections are shown at the same magnification. The normal kidney shows a distinct nephrogenic zone (top), cortex and medulla (lower right), while the mutant kidney rudiment displays a disorganized parenchyma, reduced branching of the ureter (arrowhead in f), dilated tubules and Bowman’s capsules, and abundant primitive mesenchyme. Reproduced from Schuchardt et al. (1994).

RET/GDNF signaling is required for evagination of the ureteric bud and its subsequent growth and branching The accumulated data suggests a model for the role of the RET/GDNF signaling system which is illustrated in Figure 7. The failure of ureteric buds to develop in a large proportion of RET-deficient or GDNFdeficient mice suggests that GDNF stimulates the evagination of the ureteric bud from the Wolffian duct. This evagination always occurs at the tip of the Wolffian duct and RET expression is greatest in this location, suggesting that the location of ureteric bud outgrowth is controlled by a threshold mechanism,


251

whereby a certain level of RET activation must be achieved to stimulate evagination. Normally, this level of activation can only be achieved at the tip of the Wolffian duct. In support of this idea, addition of GDNFcontaining beads to cultures containing the Wolffian duct and the surrounding mesenchyme sometimes causes supernumerary ureteric buds to develop (Pichel et al., 1996). Detailed histological analyses of metanephros development in ret-k−/ret-k− embryos suggests that if a ureteric bud has not formed from the Wolffian duct by E1 1.0 it will not form (Schuchardt et al., 1996). Restrictions on the time at which ureteric bud evagination can occur could be caused by changes in the levels of RET or secondary proteins within the Wolffian duct. Alternatively, the distance between the metanephric mesenchyme and Wolffian duct may simply have increased to such an extent that insufficient GDNF can reach the RET receptors on the Wolffian duct. Once the ureteric bud has formed, its subsequent growth and branching continues to be controlled by the RET/GDNF signaling system. In contrast to other glandular organs, such as the salivary gland, where the branches of the epithelial component appear to be formed when the tips are cleaved by a ‘tourniquet’ of extracellular matrix (Nakanishi and Ishii, 1989), the branches of the ureteric bud appear to form as bumps evaginating from a smooth tube (Davies et al., 1995). Therefore, the arborization of the ureteric bud within the metanephric mesenchyme may occur via the same mechanism as its initial evagination from the Wolffian duct and be controlled in a similar manner by RET and GDNF. The delayed and asymmetrical branching of the mutant ureteric bud may be viewed as a stochastic process whereby the formation of each branch relies on overcoming a threshold level of stimulation. Because the ureteric bud can form, branch and grow, albeit at lower frequency and rate, in mice lacking RET or GDNF, another receptor/ligand system(s) must be able to stimulate evagination of the ureteric bud and its subsequent growth and arborization. CONCLUSIONS AND FUTURE QUESTIONS The studies summarized here have established that RET and GDNFR-α comprise a functional receptor for GDNF, and that GDNF, GDNFR-α and RET mediate critical inductive interactions during the development of the metanephric kidney and parts of the peripheral nervous system. However, many interesting questions concerning the developmental roles of this signaling system remain to be addressed. How are the specific patterns of expression of RET, GDNF and GDNFR-α regulated during development? In the kidney, do the limited sites of expression of RET, GDNF and/or GDNFR-α determine the specific patterns of ureteric bud branching morphogenesis? In the nervous system, is the RET/GDNF signaling system control for migration patterns of neural crest cell subsets, or is it required for their proliferation or survival? What are the components of the signaling pathways down-stream from RET in different cell types ? What genes are turned on or off by RET/ GDNF signaling? Some of these questions are likely to be answered in the next few years. REFERENCES Abantaggiato, V., Dathan, N.A., Grieco, M., Fabien, N., Lazzaro, D., Fusco, A., Simeone, A. and Santoro, M., (1994) Developmental expression of the RET protooncogene. Cell Growth Diff., 5, 305–311. Arenas, E., Trupp, M., Akerud, P. and Ibáñez, C.F. (1995) GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo. Neuron, 15, 1465–1473. Asai, N., Iwashita, T., Matsuyama, M. and Takahashi, M. (1995). Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations. Mol.Cell Biol., 15, 1613–9.

252


Figure 6 Failure of ureteric bud and metanephric mesenchyme to develop normally in RET mutant embryos. A-D, sagittal sections through metanephric rudiments from a wild type embryo (A) and homozygous ret-k− embryos (B, C and D) at E11.5. In the normal embryo (A), the ureteric bud (u) has grown from the Wolffian duct (w) and branched once within the metanephric mesenchyme (m), and mesenchymal condensation (arrow) is seen around the ureteric bud tips. In the specimen in B, ureteric bud branching is retarded and no mesenchymal condensation is observed. C exemplifies a metanephros in which the ureteric bud has formed, but has not entered the mesenchyme, and D shows a metanephros in which no ureteric bud has formed, although the Wolffian duct and metanephric mesenchyme are present. Scale bar in A=100 m.m. E and F, wild type (E) and mutant (F) ureteric buds (arrows) after enzymatic separation from the metanephric mesenchyme (arrowheads). The wild type ureteric bud is symmetrically branched, while the mutant bud has branched only in one direction. Scale bars in E and F=200 μm. Reproduced from Schuchardt et al. (1996). Bard, J.B. (1992) The development of the mouse kidney—mouse embyrogenesis writ small. Curr. Opin. Genet. Dev., 2, 580–595.


253

Figure 7 A model of the role of RET and GDNF in kidney development. A, RET receptors (Y) are expressed in the posterior Wolffian duct, and GDNF produced by the metanephric blastema stimulates nearby Wolffian duct cells to proliferate, causing an evagination, the ureteric bud. B, the ureteric bud grows toward the metanephric blastema, possibly along a gradient of GDNF. C and D, after the ureteric bud has grown into the metanephric blastema, GDNF stimulates the formation and growth of the two symmetrical branches. E, at later stages, RET expression is restricted to cells at the tips of the ureteric bud, and GDNF produced by the surrounding mesenchyme continues to stimulate growth and branching at the bud tips. The co-receptor GDNFR-α, which is required for binding of GDNF to RET, has not included in the model, as its sites of expression during kidney development have not been thoroughly characterized. Reproduced from Schuchardt et al. (1996). Buj-Bello, A., Buchman, V.L., Horton, A., Rosenthal, A. and Davies, A.M. (1995) GDNF is an age-specific survival factor for sensory and autonomic motor neurons. Neuron, 15, 821–828. Davies, J., Lyon, M., Gallagher, J. and Garrod, D. (1995) Sulphated proteoglycan is required for collecting duct growth and branching but not nephron formation during kidney development. Development, 113, 193–198. Donis-Keller, H., Dou, S., Chi, D., Carlson, K.M., Toshima, K., Lairmore, T.C., Howe, J.R., Moley, J.F., Goodfellow, P. and Wells, Jr., S.A. (1993) Mutations in the RET protooncogene are associated with MEN 2A and FMTC. Hum. Mol. Genet., 2, 851–856. Durbec, P., Marcos-Gutierrez, C.V., Kilkenny, C., Grigoriou, M., Wartiowaara, K., Suranto, P., Smith, D., Ponder, B., Costantini, F., Saarma, M., Sariola, H. and Pachnis, V. (1996a) GDNF signalling through the RET receptor tyrosine kinase. Nature, 381, 789–792. Durbec, P., Larsson-Blomberg, L.B., Schuchardt, A., Costantini, F. and Pachnis, V. (1996b) Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. Development, 122, 349–358. Edery, P., Lyonnet, S., Mulligan, L.M., Pelet, A., Dow, E., Abel, L, Holder, S., Nihoul Fekete, C., Ponder, B.A. and Munnich, A. (1994) Mutations of the RET proto-oncogene in Hirschprung’s disease. Nature, 367, 378–380. Ekblom, P. (1992) Renal Development. In D.W.Seldin and G.Giebisch (eds.), The Kidney: Physiology and Pathophysiology, Raven Press, New York, pp. 475–501. Erickson, R.A. (1968) Inductive interactions in the development of the mouse metanephros. J. Exp. Zool., 169, 33–42.

254


Grobstein, C. (1953) Inductive epithelio-mesenchymal interaction in cultured organ rudiments of the mouse. Science, 118, 52–55. Grobstein, C. (1955) Inductive interaction in the development of the mouse metanephros. J. Exp. Zool., 130, 319–340. Grobstein, C. (1956) Trans-filter induction of tubules in mouse metanephrogenic mesenchyme. Exp. Cell Res., 10, 424–440. Hanks, S.K. (1991) Eukaryotic protein kinases. Curr. Opin. Struct. Biol., 1, 369–383. Hardman, P., Kolatsi, M., Winyar, P.J., Towers, P.R. and Woolf, A.S. (1994) Branching out with the ureteric bud. Exp. Nephrol., 2, 211–219. Hellmich, H.L., Kos, L., Cho, E.S., Mahon, K.A. and Zimmer, A. (1996) Embryonic expression of glial cell-line derived neurotrophic factor (GDNF) suggests multiple developmental roles in neural differentiation and epithelialmesenchymal interactions. Mech. Dev., 54, 95–105. Henderson, C.E., Phillips, H.S., Pollock, R.A., Davies, A.M., Lemeulle, C., Armanini, M., Simpson, L.C., Moffet, B., Vandlen, R.A., Koliatsos, V.E. and Rosenthal, A. (1994) GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science, 266, 1062–1064. Hofstra, R., Landsvater, R., Ceccherini, I., Stulp, R., Stelwagen, T., Luo, Y., Pasini, B., Hoppener, J., van Amstel, H., Romeo, G., Lips, C. and Buys, C. (1994) A mutation in the ret proto-oncogene associated with multiple endocrine neoplasia type 2B and sporadic medullary thryoid carcinoma. Nature, 367, 375–376. Iwamoto, T., Pu, M., Ito, M., Takahashi, M., Isobe, K., Nagase, F., Kawashima, K., Ichihara, M. and Nakashima, I. (1991a) Preferential development of pre-B lymphomas with drastically down-regulated N-myc in the Eµ-ret transgenic mice. Eur. J. Immunol., 21, 1809– 1814. Iwamoto, T., Takahashi, M., Ito, M., Hamaguchi, M., Isobe, K., Misawa, N., Asai, J., Yoshida, T. and Nakashima, I. (1990) Oncongenicity of the ret transforming gen in MMTV/ret transgenic mice. Oncogene, 5, 535–542. Iwamoto, T., Takahashi, M., Ito, M., Hamatani, K., Ohbayashi, M., Wajjwalku, W., Isobe, K. and Nakashima, I. (1991b) Aberrant melanogenesis and melanocytic tumour development in transgenic mice that carry a metallothionein/rc?/ fusion gene. EMBO J., 10, 3167–3175. Jing, S., Wen, D., Yu, Y., Hoist, P.L., Luo, Y., Fang, M., Tamir, R., Antonio, L., Hu, Z., cupples, R., Louis, J.-C, Hu, S., Altrock, B.W. and Fox, G.M. (1996) GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNFR-α, a novel receptor for GDNF. Cell, 85, 1113–1124. LeDouarin, N. and Teillet, M.A. (1973) The migration o neural crest cells to the wall of the digestive tract in avian embryos. J. Embryol. Exp. Morphol., 30, 31–48. Lin, L.-F.H., Doherty, D.H., Lile, J.D., Bektesh, S. and Collins, F. (1993) GDNF: a glial cell line-derived neurotrophic factor for mid-brain domapinergic neurons. Science, 260, 1130– 1132. Massagué, J. (1996) Crossing receptor boundaries. Nature, 382, 29–30. Moore, M.W., Klein, R.D., Farinas, I., Sauer, H., Armanini, M., Phillips, H., Reichardt, L.F., Ryan, A.M., CarverMoore, K. and Rosenthal, A. (1996) Renal and neuronal abnormalities in mice lacking GDNF. Nature, 382, 76–79. Mulligan, L., Kwok, J., Healey, C., Elsdon, M., Eng, C., Gardner, E., Love, D., Mole, S., Moore, J., Papi, L., Ponder, M., Telenius, H., Tunnacliffe, A. and Ponder, A. (1993) Germ-line mutations of the ret proto-oncogene in multiple endocrine neoplasia type 2 A. Nature, 363, 458–460. Nakanishi, Y. and Ishii, T. (1989) Epithelial shape change in mouse embryonic submandibular gland: modulation by extracellular matrix components. Bioessays, 11, 163–167. Pachnis, V., Mankoo, B.S. and Costantini, F. (1993) Expression of the c-ret proto-oncogene during mouse embryogenesis. Development, 119, 1005–1117. Pichel, J.G., Shen, L., Sheng, H.Z., Granholm, A.-C., Drago, J., Grinberg, A., Lee, E.J., Puang, S.P., Saarma, M., Hoffer, B.J., Sariola, H. and Westphal, H. (1996) Defects in enteric innervation and kidney development in mice lacking GDNF. Nature, 382, 73–76. Romeo, G., Ronchetto, P., Luo, Y., Barone, V., Seri, M., Ceccherini, I., Pasini, B., Bocciardi, R., Lerone, M., Kaariainen, H. and Maartucciello, G. (1994) Point mutations affecting the tyrosine kinase domain of the ret protooncogene in Hirschsprung’s disease. Nature, 367, 377–387.


255

Sánchez, M.P., Silos-Santiago, I., Frisén, J., He, B., Lira, S.A. and Barbacid, M. (1996) Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature, 382, 70–73. Santoro, M., Carlomagno, F., Romeo, A., bottaro, D., Dathan, N., Grieco, M., Fusco, A., Vecchio, G., Matoskova, B., Kraus, M. and Di Fiore, P. (1995) Activation of ret as a dominant transforming gene by germline mutations of MEN2A and MEN2B. Science, 261, 381–383. Saxen, L. (1970) Failure to demonstrate tubule induction in a heterologous mesenchyme. Dev. Biol., 23, 511–523. Saxen, L. (1987) Organogenesis of the Kidney. Cambridge University Press, Cambridge. Schlessinger, J., Lax, I. and Lemmon, M. (1995). Regulation of growth factor activation by proteoglycans: what is the role of the low affinity receptors? Cell, 83, 357–60. Schuchardt, A., D’Agati, V., Larsson-Blomberg, L., Costantini, F. and Pachnis, V. (1994) Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature, 367, 380–383. Schuchardt, A., D’Agati, V., Pachnis, V. and Costantini, F. (1996) Renal agenesis and hypodysplasia in ret-k- mutant mice result from defects in ureteric bud development. Development, 122, 1919–1929. Serbedzija, G.N., Burgan, S., Fraser, S.E. and Bronner-Fraser, M. (1991) Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos. Development, 111, 857–866. Suvanto, P., Hiltunen, J.O., Arumae, U., Moshnyakov, M., Sariola, H., Sainio, K. and Saarma, M. (1996) Localization of glial cell line-derived neurotrophic factor (GDNF) mRNA in embryonic rat by in situ hybridization. Eur. J. Neurosci., 8, 816–822. Takahashi, M., Buma, Y., Iwamoto, T., Inaguma, Y., Ikeda, H. and Hiai, H. (1988) Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene, 3, 571–578. Takahashi, M. and Cooper, G.M. (1987) ret transforming gene encodes a fusion protein homologous to tyrosine kinases. Mol. Cell. Biol., 7, 1378–1385. Takahashi, M., Ritz, J. and Cooper, G.M. (1985) Activation of a novel human transforming gene, ret, by DNA rearrangement. Cell, 42, 581–588. Treanor, J.J.S., Goodman, L., de Sauvage, F., Stone, D.M., Poulsen, K.T., Beck, C.D., Gray, C., Armanini, M.P., Pollock, R.A., Hefti, F., Phillips, H.S., Goddard, A., Moore, M.W., Buj-Bello, A., Davies, A.M., Asai, N., Takahashi, M., Vandlen, R., Henderson, C.E. and Rosenthal, A. (1996) Characterization of a multicomponent receptor for GDNF. Nature, 382, 80–83. Trupp, M., Arenas, E., Fainzilber, M., Nilsson, A.-S., Sieber, B.-A., Grigoriou, M., Kilkenny, C., Salazar-Grueso, E., Pachnis, V., Arumäe, U., Sariola, H., Saarma, M. and Ibáñez, C.F. (1996) Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature, 381, 785–789. Tsuzuki, T., Takahashi, M., Asai, N., Iwashita, T., Matsuyuma, M. and Asai, J. (1995) Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues. Oncogene, 10, 191–198. Vega, Q.C., Worby, C.A., Lechner, M.S., Dixon, J.E. and Dressler, G.R. (1996). Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis. Proc. Natl. Acad. Sci. USA, 93, 10657–10661. White, M.F. (1991) Structure and function of tyrosine kinase receptors. J. Bioenerg. Biomemb., 23, 63–82. Worby, C.A., Vega, Q.C., Zhao, Y., Chao, H.H.J., Seasholtz, A.F. and Dixon, J.E. (1996). Glial cell line-derived neurotrophic factor signals through the RET receptor and activates mitogen-activated protein kinase. J. Biol. Chem., 271, 23619–23622.

14. EPITHELIAL-MESENCHYMAL SIGNALING IN THE REGULATION OF EPITHELIAL MORPHOGENESIS AND APOPTOSIS DURING TOOTH DEVELOPMENT ANNE VAAHTOKARI and IRMA THESLEFF Developmental Biology Programme, Institute of Biotechnology, PO Box 56, University of Helsinki, FIN-00014 Helsinki, Finland Most organs form from epithelial and mesenchymal components, and reciprocal interactions between the two tissues constitute a central regulatory mechanism of epithelial morphogenesis. The growth of the epithelium as well as the folding and branching of epithelial sheets determine the shape of the organs. During recent years, it has become evident that the molecular mechanisms of developmental regulation have been conserved to a great extent during evolution and that similar signaling pathways regulate the morphogenesis of different organs. In this paper we review recent data demonstrating the roles of different signals, such as bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs) and Sonic hedgehog, in the regulation of tooth morphogenesis. We present our results demonstrating the presence of a signaling center in the tooth germ, the enamel knot, which is a transient epithelial structure expressing Bmps, Fgfs and Sonic hedgehog. Finally, we discuss our results showing that apoptosis is developmentally regulated in the epithelial cells, including the enamel knot, during tooth morphogenesis, and that apoptosis can be inhibited by epidermal growth factor (EGF) and FGF. A general role for programmed cell death in silencing embryonic signaling centers is suggested. INTRODUCTION Epithelial-mesenchymal Interactions In an embryo, the developing organs must grow, achieve their final forms and be prepared to start their specialized functions. These goals are reached by cell proliferation, cell and tissue morphogenesis as well as cell differentiation, which are intricately linked together. Most organs are derived from epithelial and mesenchymal tissues, which must interact reciprocally for development to proceed normally (Gurdon, 1992). The epithelial-mesenchymal interactions can be mediated by direct cell-cell contacts, cell-matrix interactions and soluble factors (Grobstein, 1955; Saxén et al., 1976). During kidney development, cell-cell interactions are required (Lehtonen et al., 1975). The basement membrane, lying between the epithelium and the mesenchyme, is able to trigger intracellular signal transduction, which leads e.g. to cell differentiation and tissue patterning in the developing kidney and other organs (Gumbiner, 1996). Soluble mediators of tissue interactions are mostly proteins, often belonging to growth factor families. The responding tissue must be able to receive the inductive signals, i.e. be competent. For example, competent cells may express suitable receptors on their surfaces to bind matrix molecules or soluble ligands (Jessell and Melton, 1992). Usually the tissue, after receiving an inductive signal from an adjacent tissue, sends a new message to the inducer tissue. Thus, the epithelial-mesenchymal signaling is reciprocal and continuous during organ development. However, molecular mechanisms that mediate information between tissues have, until recently, been largely unknown. There is even more limited information about genes


257

effecting the shape of various organs. On the other hand, cell proliferation and differentiation have been relatively well characterized at the molecular level. Epithelial Morphogenesis The developing teeth, hairs and exocrine glands all have an epithelial component derived from ectoderm, and their early stages resemble each other considerably (Thesleff et al., 1995b). These organs start their development from an epithelial thickening, which subsequently invaginates into the underlying mesenchyme and forms an epithelial bud. The bud stage as well as the subsequent branching and folding of epithelial sheets are also characteristic for many other developing organs. It is conceivable that these structural resemblances in the early organ rudiments reflect similar molecular mechanisms underlying these developmental processes, and there is increasing evidence that different organs use identical molecular pathways for their initial construction. For example, members of the epidermal growth factor (EGF) family seem to be involved in the regulation of epithelial morphogenesis in several developing organs (Partanen and Thesleff, 1987; Miettinen et al., 1995). EGF can partially rescue Tabby mice, which have defects in teeth, hair and exocrine glands (Blecher et al., 1990). The defective gene in Tabby mice is homologous to the gene causing the human syndrome, anhidrotic ectodermal dysplasia (EDA). These genes have been recently cloned and shown to encode a novel transmembrane protein with so far unknown functions (Kere et al., 1996; Srivastava et al., 1997). This review focuses on molecules which appear to be involved in epithelial-mesenchymal interactions and epithelial morphogenesis during organogenesis. The developing mouse tooth is a good model system for such studies, as it can be studied in vitro, and the cultured dental tissues can be experimentally manipulated. Incisors and molars also have characteristic shapes allowing the analysis of the regulation of pattern and morphogenesis. In addition, terminal cell differentiation occurs only during the later stages of morphogenesis. Thus, different developmental processes can be studied separately. TOOTH DEVELOPMENT Initiation, bud and cap stages: In rodents, the dentition lacks canines and premolars, and there is only one set of teeth, consisting of one incisor and three molars in each half of the maxilla and mandible. The jaws are derived from the first branchial arch and the frontonasal process. In mice, initiation of molar tooth development is morphologically evident after midgestation, at embryonic day 10–11 (E10–E11), when the dentally committed oral epithelium locally thickens (Figure 1). At the bud stage (E12–E13), the dental epithelium invades the underlying neural crest-derived jaw mesenchyme, which condenses around the epithelial bud. These condensed mesenchymal cells become committed to dental papilla mesenchyme and dental follicle cells. During the cap stage (E14–15), the dental epithelium changes its shape into a cap, enclosing the dental papilla mesenchyme and starting the development of the first cusp. The central cells of the dental epithelium become clustered into a distinct, transient morphological structure, the primary enamel knot. Moreover, the dental follicle is formed around the developing tooth (Figure 1). Early bell stage: At the following early bell stage (E16–E18), other cusps of the molars start to develop, and transient epithelial secondary enamel knots become visible at the tips of the developing cusps. The dental mesenchymal cells adjacent to the basement membrane separating the dental epithelium and mesenchyme start to differentiate into odontoblasts. In the dental epithelium, the cells of the inner enamel epithelium begin their differentiation into ameloblasts. Both preodontoblasts and preameloblasts first stop proliferating, polarize and then start to secrete their cell-specific matrices, dentin and enamel, respectively.

258


The differentiation of odontoblasts and ameloblasts starts at the tips of the cusps and gradually proceeds along the slopes of the cusps. The dental mesenchyme forms the pulp of the tooth, which becomes innervated around birth (Mohamed and Atkinson, 1983). Late bell stage: At the late bell (crown) stage (postnatal days 0–7, P0–P7), both the odontoblasts and ameloblasts become terminally differentiated, the basement membrane between these cells degrades (Sahlberg et al., 1992), and the morphogenesis of the tooth crown is completed. Subsequently, dentin becomes mineralized, the roots start to form, and the supporting tissues of the tooth differentiate. The epithelial part of the tooth is reduced and is finally completely degraded as the tooth erupts about three weeks after birth (Figure 1). During cytodifferentiation, reciprocal epithelial-mesenchymal interactions also play crucial roles (Thesleff, 1995; Thesleff and Åberg, 1997) but they are not discussed in this review. EPITHELIAL-MESENCHYMAL INTERACTIONS DURING TOOTH DEVELOPMENT Shift of Odontogenic Potential Prior to the bud stage of tooth development, the potential for tooth formation resides in the dental epithelium. This has been shown by using experimental tissue recombinants: when the early (E9–E11) mouse presumptive dental epithelium was combined with second branchial arch mesenchyme, which will not normally participate in tooth development, and the recombined tissues were cultured in the anterior chamber of the eye, a tooth-like structure was formed (Mina and Kollar, 1987; Lumsden, 1988). In contrast, when the E9–E11 presumptive dental mesen chyme was cultured with a non-dentally committed second branchial arch epithelium, no tooth germ was generated. However, when the same experiments were made by using older tissues (E12–E13), the results were reversed: now the dental mesenchyme could instruct a non-dental epithelium to partake in tooth formation, whereas the dental epithelium was unable to construct a tooth germ with non-dental mesenchyme (Mina and Kollar, 1987). Thus, between E11 and E12 there seems to be a shift of odontogenic potential from the dental epithelium to the dental mesenchyme. It is still unclear if the oral epithelial cells are the very first cells possessing capacity for tooth formation or if there is an earlier shift of odontogenic potential from the neural crest-derived mesenchymal cells to the presumptive dental pithe lium; by Dil labeling it has been verified that migrating midbrain neural crest cells contribute to molar development in rats (Imai et al., 1996). The neural crest cells that colonize other branchial arches than the first one, retain their characteristic patterns of Hox gene expression after leaving the neural tube. For neural crest cells migrating to the first branchial arch, it may be that non-clustered homeobox-containing genes such as Dlx and Msx genes have a similar patterning role (MacKenzie et al., 1991, 1992; Dollé et al., 1992; Robinson and Mahon, 1994). Indeed, Dlx and Msx genes have been speculated to determine the capacity of the neural crest cells to participate in the development of the dentitions and to regulate the patterning of teeth (Weiss et al., 1994; Sharpe, 1995). Molecules Associated with Odontogenic Potential During the initiation of tooth development, intense and localized expression of a number of different molecules has been observed in the dental epithelium. These include the extracellular matrix (ECM) receptor β5 integrin as well as transcription factors Msx-2, Lef-1, Otlx-2 and tlx-1 (Oosterwegel et al., 1993; Jowett et al., 1993; Raju et al., 1993; Yamada et al., 1994; Mucchielli et al., 1997). The putative transmembrane receptors Notch-1, -2 and -3 on the other hand, are downregulated in the dental epithelium


259

Figure 1 Development of a mouse molar tooth. Schematic drawings of (A-D, F) frontal or (E) sagittal sections. (A) The first visible sign of tooth development is the local thickening of the oral epithelium. (B) At the bud stage, the dental epithelium invaginates into the underlying neural crest-derived mesenchyme. (C) During the cap stage, the transient primary enamel knot in the dental epithelium is visible, and the dental follicle is formed. The developing cervical loop is demarcated. (D) At the bell stage, morphogenesis of the cusps and cell differentiation proceed. The bordered region indicates a developing cusp. (E) At the late bell stage, cuspal morphogenesis is completed, and terminal differentiation of epithelial ameloblasts and mesenchyme-derived odontoblasts takes place. (F) The erupted tooth. The epithelial tissue has degraded and the roots have developed. The dental pulp is formed by the mesenchymal cells, ab, ameloblasts; dc, developing cusp; de, dental epithelium; df, dental follicle; dl, dental lamina; dm, dental mesenchyme; jm, jaw mesenchyme; ob, odontoblasts; oe, oral epithelium; pek, primary enamel knot; sek, secondary enamel knot; si, stratum intermedium; sr, stellate reticulum.

but continue to be expressed elsewhere in the oral epithelium (Mitsiadis et al., 1995). These genes may be involved in the determination of the dental epithelium. Signal molecules in the BMP and FGF families are also expressed in the epithelium and may have roles in mediating epithelial-mesenchymal interactions at this stage (Figure 2, Vainio et al., 1993; Thesleff et al., 1995a, Kettunen et al., 1997). Of the epithelial factors mentioned above, the expression of pj integrin, Msx-2, Lef-1, tlx-1 and Bmp-4 shift to the dental mesenchyme concurrently with the odontogenic potential, whereas the expression of Fgf-8 and Fgf-9 is downregulated (Kettunen and Thesleff, 1998). Otlx-2 remains expressed specifically in the dental epithelium throughout tooth morphogenesis (Mucchielli et al., 1997). Gene expression patterns during tooth development can be viewed in a graphical www database (Gene expression in tooth, 1998). At the bud stage, several molecules are upregulated in the condensing dental mesenchyme. These include the ECM molecule tenascin, the growth factors Fgf-3 and activin βA subunit, as well as transcription factors AP 2.2, Egr-1, Msx-1, N-myc, Sna and Tcf-1 (Thesleff et al., 1987; Wilkinson et al., 1989; Smith et al., 1992; Yamada et al., 1992; Karavanova et al., 1992; Jowett et al., 1993; Oosterwegel et al., 1993; Roberts and Earth, 1994; Feijen et al., 1994; Chazaud et al., 1996). As hepatocyte growth factor (HGF) becomes

260


expressed in the dental mesenchyme during cap stage and as its receptor is expressed in the dental epithelium, it is conceivably a mesenchymal signal participating in the regulation of epithelial development (Sonnenberg et al., 1993). Molecules Necessary for Tooth Development If mice lack Msx-1, Lef-1 or platelet-derived growth factor (PDGF) receptor alpha subunit, their tooth development is arrested around the bud stage (Morrison-Graham et al., 1992; van Genderen et al., 1994; Satokata and Maas, 1994). Moreover, in p53-deficient mice, there are defects in upper incisor development (Armstrong et al., 1995), and in mice lacking activin βA subunit, follistatin or type II activin receptor, the incisors are also missing (Matzuk et al., 1995a,b,c). In bud-staged tooth germs, antisense oligonucleotides specific for precursor of substance P, which is a tachykinin neurotransmitter expressed in the dental mesenchyme, block morphogenesis in vitro (Weil et al., 1995). Similarly, antisense inhibition of HGF expression also perturbs tooth morphogenesis (Tabata et al., 1996). It is noteworthy that all the molecules that have so far been shown to be necessary for tooth morphogenesis are associated with epithelialmesenchymal interactions thus pointing out the central roles of epithelial-mesenchymal interactions for tooth morphogenesis. As in these functional studies also other organs were affected, they support the notion that the molecular mechanisms of epithelial-mesenchymal interactions are conserved. SIGNALING MOLECULES REGULATING TOOTH MORPHOGENESIS Bone Morphogenetic Proteins BMPs belong to the TGF-β superfamily. BMP-2 and BMP-4 share about 90% sequence similarity and 75% similarity with the Drosophila decapentaplegic (dpp) (Kingsley, 1994; Reddi, 1994). BMPs-5–8 are about 90% identical to one another, and 70% identical to the Drosophila 60A product (Reddi, 1994). BMPs initiate cartilage and bone formation, and regulate chemotaxis, proliferation and differentiation (Reddi, 1994). BMP type I and II serine/threonine kinase receptors are homologous to TGF-β receptors. In contrast to TGF-β receptors, the ligands bind to type I and II BMP receptors separately (Rosenzweig et al., 1995). However, the maximal ligand binding and optimal signal transduction seem to be achieved only when type I and II BMP receptors cooperate (Liu et al., 1995; Nohno et al., 1995). Bmp-4 is expressed in mouse embryos in developing limbs, heart, facial processes, neuroepithelium of the developing pituitary gland, and condensed mesenchyme of developing whisker follicles (Jones et al., 1991). In transgenic mice, ectopic expression of Bmp-4 during hair and whisker follicle development results in a decrease in cell proliferation and changes in cytokeratin expression (Blessing et al., 1993). Mouse embryos lacking BMP-4 die before gastrulation and show little or no mesoderm differentiation (Winnier et al., 1995). During chick development, BMP-4 may also act as a lateralizing signal in the somite patterning, and in rhombomeres, BMP-4 mediates apoptosis (Graham et al., 1994; Pourquié et al., 1996). Misexpression of Bmp-4 in embryonic lung epithelium results in aberrant lung development (Bellusci et al., 1996). BMP-7 protein has been localized in chondrocytes, osteoblasts and in basement membranes of developing organs (Vukicevic et al., 1994). In mice deficient of BMP-7, kidney development is impaired causing death shortly after birth. In addition, the mutant mice have eye defects because of abnormal lens induction and skeletal abnormalities in the ribs, skull and digits (Luo et al., 1995; Dudley et al., 1995).


261

During early tooth development, Bmp-2 transcripts have been detected in the dental epithelium from the bud stage to the early bell stage, and subsequently in the central cells of the dental mesenchyme in mouse embryos (Lyons et al., 1990; Vainio et al., 1993; Thesleff, 1995). Bmp-4 has been localized in the dental epithelium during the initiation of tooth development, and at the bud stage, the expression shifts gradually to the dental mesenchyme, which correlates with the shift of odontogenic potential (Mina and Kollar, 1987; Vainio et al., 1993). Bmp-7 transcripts have been detected in the inner dental epithelium and dental pulpal cells during embryonic tooth development (Helder et al., 1995). Transcripts of the Alk-2B receptor, which may bind BMP-7, are expressed in the dental lamina at the bud stage (Verschueren et al., 1995). Bmp-2, Bmp-4 and Bmp-7 are also expressed in the enamel knot of the dental epithelium (see below; Figure 2) (Vaahtokari et al., 1996a; Åberg et al., 1997). Application of BMP-2 and BMP-4 on E11 dental mesenchyme in vitro induces transcription factors Msx-1, Msx-2 and Egr-1 (Vainio et al., 1993). BMP-4 also activates the HMG box gene Lef-1 as well as its own expression in the dental mesenchyme, and Msx-1 is required for this autoinduction (Kratochwil et al., 1996; Chen et al., 1996). The data strongly suggest that BMP-mediated signaling is involved in the transition of odontogenic potential from the dental epithelium to the dental mesenchyme, and that Msx-1 acts both upstream and downstream of BMP-4. Fibroblast Growth Factors Currently, the fibroblast growth factor (FGF) family has nine structurally related mammalian members: FGF-1 (acidic FGF, aFGF), FGF-2 (basic FGF, bFGF), FGF-3 (int-2), FGF-4 (Hst-1, kFGF), FGF-5, FGF-6 (hst-2), FGF-7 (keratinocyte growth factor, KGF), FGF-8, and FGF-9 (glia-activating factor or GAP) (Muenke and Schell, 1995). Use of alternative initiation codons and alternative splicing generate several isoforms of FGFs (Lappi, 1995). Additionally, posttranslational modifications of FGFs occur (Mason, 1994). At the moment, four mammalian FGF tyrosine kinase receptors (FGFR) are known (FGFR1-FGFR4) (Muenke and Schell, 1995). Several isoforms of the FGFRs are generated by alternative splicing, and some of them are secreted proteins (Johnson et al., 1991). All FGFs bind heparin, and heparan sulfate-containing cell surface proteoglycans function as low-affinity FGF binding proteins, which are needed for the binding of FGFs to their high-affinity receptors (Baird, 1994; Salmivirta and Jalkanen, 1995). FGFs are associated with several cellular functions including regulation of cell proliferation, migration, differentiation and survival, angiogenesis, responses to wounding, extension of neurites, and malignant transformation (Mason, 1994; Muenke and Schell, 1995; Slavin, 1995). FGFs are important regulators of embryonic development, and e.g. in mesoderm induction, FGFs act mainly as ventrolateral inducers (Slack, 1994). However, each FGF has a specific expression pattern and distinct target cells (Mason, 1994). Mutants in the extracellular region of the human FGFRs result in disorders of bone development (Muenke and Schell, 1995). Homozygous FGFR1 and FGFR2 deficient mice die during early embryogenesis, whereas disruption of mouse Fgfr-3 gene results in defects in bone growth (Yamaguchi et al., 1994; Deng et al., 1994, 1996). Of the members of the FGF family, FGF-4 and FGF-8 are the best candidates for mediators of the epithelial-mesenchymal interactions during tooth development. Fgf-4 is expressed during gastrulation, myogenesis, limb, mammary gland and tooth development in mice (Niswander and Martin, 1992; ColemanKrnacik and Rosen, 1994). Homozygous mouse embryos lacking FGF-4 gene do not develop after implantation (Feldman et al., 1995). FGF-4 can replace the apical ectodermal ridge (AER) during limb development and induce ectopic limb formation (Niswander et al., 1993; Mima et al., 1995; Cohn et al.,

262


1995). However, as Fgf-8 is expressed earlier than Fgf-4 in the developing limb, and as both proteins use same receptors, FGF-4 may induce the signaling pathway used by FGF-8 during normal development (Crossley and Martin, 1995). Thus, FGF-8 produced by the AER may initiate limb formation, and FGF-4 may function during later stages of limb development (Crossley et al., 1996b). In addition, FGF-8 can induce midbrain development (Crossley et al., 1996a). Before initiation of tooth development, Fgf-4 is expressed in E8.5–9 branchial arch ectoderm (Drucker and Goldfarb, 1993). In the tooth germ, Fgf-4 is transiently expressed only in the enamel knots at the cap and bell stages (Figures 2 and 3), and FGF-4 stimulates cell proliferation both in the dental epithelium and mesenchyme (Jernvall et al., 1994). In addition, FGF-1, FGF-2 and FGF-4 induce syndecan1 expression in the dental mesenchyme in a manner that requires Msx-1 (Chen et al., 1996). Syndecan-1 is a transmembrane proteoglycan, which functions as a co-receptor for FGF-1, FGF-2 and FGF-4 (Salmivirta and Jalkanen, 1995), and whose expression in the dental mesenchyme is induced by the dental epithelium (Vainio et al., 1989). Fgf-8 is present in the surface ectoderm of the first branchial arch before and during the initiation of tooth development (Figure 2) (Heikinheimo et al., 1994; Kettunen and Thesleff, 1998). Sonic Hedgehog In Drosophila, the hedgehog (hh) gene has a central role in patterning the tissues of the developing fly (Nüsslein-Volhard and Wieschaus, 1980; Ingham, 1994). As a segment polarity gene, hedgehog acts in a concentration-dependent manner by specifying cell types within each embryonic segment (Heemskerk and Dinardo, 1994). The mouse homologs of hedgehog are Sonic hedgehog (Shh), Desert hedgehog (Dhh) and Indian hedgehog (Ihh). Sonic hedgehog undergoes autoproteolytic cleavage to an amino-terminal fragment and a carboxyterminal fragment (Bumcrot et al., 1995; Porter et al., 1995). All signaling activity of Sonic hedgehog resides within its amino-terminal domain, although it is not clear how its long-range activity is mediated (Fan et al., 1995; Martí et al., 1995a). In Drosophila hedgehog, cholesterol is covalently attached to the amino-terminal signaling domain during autoprocessing and the carboxylterminal domain acts as an intramolecular cholesterol transferase (Porter et al., 1996). There is recent data suggesting that abnormal cholesterol metabolism may affect the function of Sonic hedgehog (Kelley et al., 1996). The tumor suppressor gene patched is the receptor for Sonic hedgehog (Marigo et al., 1996; Stone et al., 1996). Cubitus interruptus (ci), which belongs to the Gli family of zinc finger proteins, forms a negative feedback loop with patched and thus regulates expression of hedgehog target genes (Alexandre et al., 1996; Hepker et al., 1997). Sonic hedgehog is expressed in several regions that have organizing activity, including the node, notochord, floorplate of the neural tube and the zone of polarizing activity (ZPA) in the developing limbs (Smith, 1994). In addition, Sonic hedgehog is detected in epithelial tissues in developing organs during epithelial-mesenchymal interactions (Chang et al., 1994; Bitgood and McMahon, 1995; Iseki et al., 1996). In addition to the tooth, these tissues include hair, whisker, rugae, gut, bladder, urethra, vas deferens and lung. During tooth morphogenesis, Sonic hedgehog is expressed at the tip of the budding epithelium, which subsequently forms the enamel knot, and at bell stage in the inner enamel epithelium (Bitgood and McMahon, 1995; Iseki et al., 1996; our unpublished results). Moreover, Sonic hedgehog appears to induce Bmp-4 and Hoxd genes in the adjacent mesoderm during chick hindgut formation (Roberts et al., 1995). Targeted disruption of Sonic hedgehog in mice results in defects in patterning of brain, spinal cord, the axial skeleton, limbs and other tissues (Chiang et al., 1996).


263

Figure 2 Histological appearance of tooth morphogenesis from initiation to cap stage and expression of some signal molecules in the dental epithelium. (A) Frontal section through the head of an E11 mouse embryo. Thickenings of the oral epithelium are evident at the sites of upper and lower molar tooth germs, and they express intensely Fgf-8 (B) Higher magnification of the epithelial thickenings in A. (C) Bud stage molar tooth germ in the mandible of an E13 embryo. Bmp-2 expression is evident in the enamel knot area of the dental epithelium. (C) Cap stage molar tooth germ from an E14 mouse embryo. Fgf-4 is expressed intensely in the enamel knot.

ENAMEL KNOT AS AN EPITHELIAL SIGNALING CENTER At the cap stage of tooth development, the cells of the central dental epithelium form a transient structure called the enamel knot, which expresses Fgf-4 and is mitotically inactive (Niswander and Martin, 1992; Jernvall et al., 1994). As FGF-4 enhances proliferation in dental tissues, we have suggested that the enamel knot directs the morphogenesis of tooth cusps by stimulating neighboring tissues to grow, while the enamel knot itself remains non-proliferating (Jernvall et al., 1994). Our subsequent analysis of expression of signaling molecules other than Fgf-4 in the enamel knot cells, by using in situ hybridization, BrdU incorporation, and three-dimensional computer reconstructions of serial sections, suggests that the enamel knot in fact is a signaling center (Vaahtokari et al., 1996a).

264


Localization of Sonic Hedgehog, Bmp-2, Bmp-4, Bmp-7, Fgf-4 and Lack of Cell Proliferation in the Enamel Knot At the bud stage, the cells at the tip of the dental epithelium do not yet form a distinct cell cluster, but the formation of the enamel knot is evident as lack of BrdU incorporation at the tip. At the cap stage, the enamel knot region is clearly a discrete morphological structure, and its cells remain non-proliferative. The enamel knot cells express Sonic hedgehog, Bmp-2, and Bmp-7 at the bud and cap stages, but the expression of Bmp-4 and Fgf-4 is only seen at the cap stage (Vaahtokari et al., 1996a). Bmp-3, Bmp-5 and Bmp-6 transcripts are not detected in the dental epithelium at the bud and cap stages (Åberg et al., 1997). Three-dimensional computer reconstructions of serial sections reveal correlations between the distribution domains of Sonic hedgehog, Bmp-2 and non-dividing cells at the bud and cap stages (Figure 3). However, Bmp-7 expression covers a larger area than do Sonic hedgehog and Bmp-2 transcripts. In comparison to Bmp-2 and Bmp-7, expression of Bmp-4 starts later in the enamel knot and is only detected in the distal half of the enamel knot. The distribution domain of Fgf-4 RNA is a few cell layers smaller than the area of non-proliferating cells (Jernvall et al., 1994). It has been demonstrated by using experimental tissue recombinations that tooth development is directed by the dental epithelium before the early bud stage (E12), after which the dental mesenchyme possesses the capability to conduct the development of an epithelium towards a dental phenotype (Kollar and Baird, 1970). Thus, the formation, and perhaps maintenance, of the enamel knot may be guided by the dental mesenchyme. In tissue recombinants, in which a dental mesenchyme with an odontogenic potential instructs a non-dental epithelium to participate in tooth formation, the induction of an enamel knot in the epithelium may be needed for further development. At the moment, there is no data about ectopic enamel knot formation in tooth recombinants. Recent data from out laboratory indicate that BMP-4 may be a mesenchymal signal regulating the development of the enamel knot. BMP-4 releasing beads induce the expression of p21, a cyclin dependent kinase inhibitor, which marks the enamel knot from its beginning (Jernvall et al., 1998). The enamel knot may be important in the transition of the tooth germ from the bud stage to the cap stage. In transgenic mice lacking Msx-1 or Lef-1, tooth development is arrested at the bud stage (van Genderen et al., 1994; Satokata and Maas, 1994). Thus, it is possible that these genes are involved either in the regulation or function of the enamel knot, or that the signaling molecules produced by the enamel knot exert their effects in the dental mesenchyme through these genes. The Enamel Knot Resembles Other Embryonic Signaling Centers Signaling centers of the limb bud: The ZPA region of the posterior limb mesoderm is required to provide the anterior-posterior polarity of the limb and to maintain and polarize the AER, which defines the proximal-distal axis of the limb (Perrimon, 1995). It has been demonstrated that Sonic hedgehog expression colocalizes with ZPA activity, and that it may mediate the polarizing activity of the ZPA (Riddle et al., 1993). Like the ZPA, Sonic hedgehog expression regulates nested expression of the Hoxd genes. Thus, Sonic hedgehog patterns the anteroposterior limb axis (Riddle et al., 1993). Once induced in the ZPA, Sonic hedgehog expression is dependent on signals from the posterior AER (Laufer et al., 1994). The replacement of the AER with FGF-4-soaked beads results in advancing chick limb development and the maintenance of Sonic hedgehog expression (Laufer et al., 1994; Niswander et al., 1993, 1994). On the other hand, Sonic hedgehog induces expression of FGF-4 in the posterior AER (Riddle et al., 1993; Niswander et al., 1994; Laufer et al., 1994). Thus, there is a positive feedback loop between Sonic hedgehog and FGF-4.


265

Figure 3 Cell division and gene expression in the putative signaling center, the enamel knot. Three-dimensional computer reconstructions of the bud (E13, upper row) and cap (E14, lower row) stages of tooth development showing the distribution patterns of cell proliferation (BrdU incorporation), and the expression of signaling molecules (in situ hybridization). The gray epithelial-mesenchymal interface is shown from the oral side, and the anterior ends of the tooth germs are towards the observer. Lack of mitotic activity and expression of Sonic hedgehog, Bmp-2 and Bmp-7 are detected in the enamel knot as early as the bud stage, whereas the expression of Bmp-4 and Fgf-4 is seen in the enamel knot only at the cap stage. The distribution of Bmp-7 covers the largest area at both the bud and cap stages, while Bmp-4 expression is restricted to the distal half of the enamel knot.

Bmp-2, Bmp-4 and Bmp-7 are expressed in both signaling centers, but Bmp-4 and Bmp-7 are also found elsewhere in the developing limb tissues (Francis et al., 1994; Lyons et al., 1995). Cooperation between the Sonic hedgehog and FGF-4 signals is required for maintenance of Bmp-2 expression in the limb bud, and Bmp-2 seems to be part of the response pathway to the polarizing signal (Laufer et al., 1994; Francis et al., 1994). On the other hand, BMP-2 is known to inhibit the mitogenic effects of FGF-4 in limb tissues (Niswander and Martin, 1993). As all these signaling molecules are expressed in the enamel knot, it is plausible that the enamel knot may have the functions of both AER and ZPA, in other words, to be capable of regulating both growth and patterning. Other embryonic signaling centers: In the notochord, which regulates dorsoventral patterning of the neural tube, Sonic hedgehog, Bmp-2 and Bmp-7 are colocalized (Lyons et al., 1995). Dorsal ectoderm expresses Bmp-4 which appears to be involved in dorsalization of the neural tube (Liem et al., 1995). During chick feather development, the apical regions of the feather placode and barb ridges express Sonic hedgehog, and the diffuse expression of Bmp-2 and Fgf-4 overlap these areas (Nohno et al., 1995). During chick branchial arch development, expression patterns of Sonic hedgehog, Bmp-7 and Fgf-8 overlap in the posterior ectodermal margin of the second branchial arch (Wall and Hogan, 1995). In the developing lung, Sonic hedgehog, Bmp-4 and Bmp-7 are expressed in the distal tips of the bronchial tubes during branching morphogenesis, and overexpression of Sonic hedgehog suggests that it regulates lung mesenchymal cell proliferation (Bitgood and McMahon, 1995; Urase et al., 1996; Bellusci et al., 1996, 1997). Thus, the same

266


molecules seem to be expressed in specific regions in developing organs, perhaps forming signaling centers which may direct the patterning of neighboring tissues during embryogenesis. Possible Roles for the Signaling Molecules of the Enamel Knot BMPs: In the developing limb, BMP-2 counteracts the mitogenic effects of FGF-4 (Niswander and Martin, 1993). In an analogous way, BMP-2 produced by enamel knot cells may regulate the proliferative effects of FGF-4 secreted by the same cells. BMP-4 regulates apoptosis in odd-numbered rhombomeres as well as in the interdigital mesenchyme (Graham et al., 1993; Ganan et al., 1996; Zou and Niswander, 1996). The enamel knot cells undergo apoptosis starting from the distal aspect of the knot, and BMP-4 expression is intimately associated with apoptosis (Jernvall et al., 1998). It is possible that BMP-4 mediates cell death in the enamel knot and thus terminates its signaling. Fgf-4: FGF-4 acts as a mitogen in developing dental tissues in vitro, and thus, it may stimulate growth of the cusps (Jernvall et al., 1994). It has recently been shown that FGF-4 strongly downregulates the expression of Fgfr-2 but not Fgfr-1 (Ali et al., 1995). Both Fgf receptors have been observed during tooth development, but a more detailed analysis of their expression patterns is needed to evaluate the possible significance of FGF receptor downregulation by FGF-4 in dental tissues (Peters et al., 1992; Orr-Urtreger et al., 1993). Sonic hedgehog: In Drosophila, hedgehog regulates the expression of dpp, which is homologous to vertebrate Bmp-2 and Bmp-4, during patterning of the imaginal discs. In vertebrates, Sonic hedgehog induces the expression of Bmp-2 in the limb bud (Laufer et al., 1994), and Bmp-4, in the developing hindgut (Roberts et al., 1995). Therefore, in the enamel knot, Sonic hedgehog may regulate the expression of Bmps. Patched, the hedgehog receptor, is expressed in dental tissues which do not express Sonic hedgehog (our unpublished results). Enamel Knot May Direct Cuspal Morphogenesis The enamel knot may have a role in the formation of secondary enamel knots, possibly by regulating growth and gene expression in both the epithelium and mesenchyme. In the epithelium, the expression of Sonic hedgehog and Bmp-2 spreads laterally along the inner enamel epithelium at the bell stage (Vainio et al., 1993; our unpublished results). This expansion of expression may regulate the formation of secondary enamel knots at the tips of the developing cusps. The secondary enamel knots express Fgf-4 (Jernvall et al., 1994), and they may control the growth of the cusps at the bell stage, when the primary enamel knot has disappeared. Heterochrony means temporal changes in the appearance and rate of developmental processes which may be achieved by different timing in the expression of certain genes, whereas dissimilar growth rates of different parts of the organisms are termed allometry (Gilbert, 1994). Both heterochrony and allometry are important developmental mechanisms generating structural evolutionary changes (Gilbert, 1994). The characteristic tooth shapes in different species may result from heterochrony in the formation of enamel knots, which in turn may cause allometric changes in tooth development (Jernvall, 1995). In other words, in species with divergent tooth shapes, the enamel knots may appear at different times during tooth development, which leads to disparity in cuspal sizes, and these quantitative changes may gradually evolve into qualitative changes in the number of cusps. Thus, the enamel knots may have important evolutionary roles. In conclusion, the data presented here suggest that the enamel knot is an embryonic signaling center which may provide positional information for the development of the tooth crown.


267

APOPTOSIS IN DENTAL EPITHELIAL MORPHOGENESIS Non-pathological cell death is referred to as programmed cell death (PCD), because the cell is thought to activate an intrinsic death machinery and kill itself (Steller, 1995). On the other hand, necrosis is a pathological form of cell death which takes place when the plasma membrane of the cell is damaged. The characteristic features of apoptosis, which is the most common form of programmed cell death, are shrinkage of the cell, cleavage of the genomic DNA by activation of endogenous proteases, nuclear and cytoplasmic condensation, and fragmentation of cytoplasm and nucleus into membrane-bound vesicles (apoptotic bodies). Apoptotic bodies are rapidly, and specifically, engulfed by macrophages or neighboring cells (Rotello, 1994; Hopkinson-Woolley et al., 1994). Extracellular signals regulating apoptosis: In many types of mammalian cells, cell survival is dependent on a constant supply of survival signals provided by neighboring cells and the ECM (Thompson, 1995; Coucouvanis and Martin, 1995). For example, deprivation of growth factors leads to the induction of apoptosis (Collins et al., 1994), and basement membrane ECM, which suppresses apoptosis of mammary epithelial cells, has been shown to regulate interleukin-1 p-converting enzyme (ICE) expression negatively (Boudreau et al., 1995). Apoptosis can also be induced by positive modulators, such as glucocorticoids in thymocyte death and Müllerian inhibiting substance (MIS) in regression of the Müllerian duct (Thompson, 1995). In addition, the cells fated to undergo programmed cell death must be competent to respond to the appearance or loss of external signals leading to cell death (Schwartz and Osborne, 1993). Functions of apoptosis: During embryonic development, apoptosis has been suggested to regulate the sizes of cell populations, and in this way to match e.g. innervation to the size of the muscle (Raff et al., 1993). Moreover, apoptosis has been proposed to eliminate cells which have fulfilled their transitory purposes and are no longer useful for the embryo, to remove cells that have migrated to a wrong place, to select the fittest cells for survival and to sculpture tissues to a correct shape (Raff, 1992; Raff et al., 1993). As evidence for the last function, apoptosis has been found to be necessary for the separation of the digits in the developing limb (van der Hoeven, 1994; Zou and Niswander 1996). Until recent novel techniques were available, apoptosis was studied mainly in developing nervous and immune systems, but its role in other embryonic processes, including organogenesis, was largely ignored (Raff et al., 1993). For example, the kidney rudiment is one of the most well-studied developing organs, but the first report on apoptosis during renal development was not published until some years ago (Koseki et al., 1992; Coles et al., 1993). Additionally, apoptosis takes place during primary and secondary palatal fusion as well as in lens development (Mori et al., 1994; Pellier and Astic, 1994; Morgenbesser et al., 1994). We have localized apoptosis during tooth morphogenesis and analyzed its regulation (Vaahtokari et al., 1996b). Methodological Aspects Difficulties in detecting apoptosis: It has been difficult to notice even large-scale cell death by using traditional histological methods (Coles et al., 1993). The process of apoptosis takes place within one to two hours and apoptotic cells are rapidly recognized and eliminated by macrophages and other phagocytic cells (Coles et al., 1993; Martin et al., 1994). For example, in the rat retina, where about 100 000 (50%) of the retinal ganglion cells die over a period of several days, the percentage of dying cells (using morphological criteria) is about 0.5% in the tissue sections studied (Perry et al., 1983). Thus, serial sections are needed to detect apoptosis reliably in tissues, otherwise even massive cell death can take place unnoticed. The fairly recently developed Tunel staining, which labels cells and in which chromosomal DNA is fragmented, has made it considerably easier and more reliable to identify apoptotic cells (Gavrieli et al., 1992).

268


Figure 4 Distribution of apoptotic cells during molar tooth development. Tunel staining analysis of serial sections. (A) No apoptosis is detected during the initiation of tooth development. (B) At the early bud stage, apoptosis is detected in the dental epithelium adjacent to the oral epithelium. (C) At the late bud stage, apoptosis extends from the oral epithelium to the tip of the dental epithelium. (D) At the cap stage, apoptosis is abundant in the dental lamina and primary enamel knot. (E) At early bell stage, apoptotic cells are located in the dental follicle, outer enamel epithelium, dental lamina, and secondary enamel knot cells as well as in the stratum intermedium cells next to the enamel knots and in the presumptive secondary tooth bud. (F) At the late bell stage, apoptosis is seen in all cell types of the epithelial enamel organ. See Figure 1 for structural details.

Tunel and Nile Blue stainings: We localized apoptosis by Tunel staining and used several sets of serial sections for each developmental stage studied (Vaahtokari et al., 1996b). In addition, whole mount Nile Blue stainings, which detect apoptotic but not necrotic cells, were used for examining developing jaws and limbs. For cultured explants, both Nile Blue and Tunel stainings were used. No staining was observed in the negative controls for Tunel staining, and the positive controls, which had been treated with deoxyribonuclease I before DNA end labeling, were entirely stained. Both Tunel and Nile Blue stainings detected apoptosis in the AER of E10– 12.5 limb buds and, at E14, between the separating digits, as described earlier (Hinchliffe, 1982; Lee et al., 1993). Developmentally Regulated Apoptosis in Dental Epithelium Apoptosis has been suggested to be involved in the regulation of tissue size, which implies that cells can die at random in the tissue; in this case, there is no need for strict temporal and spatial regulation of apoptosis. However, the distribution pattern of apoptotic cells detected suggests that programmed cell death may be carefully controlled during tooth development and possibly has roles in dental morphogenesis, in particular in the invagination process, silencing of enamel knot signaling as well as in the removal of epithelial cells during advanced tooth development (Figure 4). Apoptosis in the Invagination of Epithelium At the bud stage of molar development, a streak of apoptotic cells was detected stretching from the oral epithelium to the tip of the epithelial dental bud. As the epithelial bud intruded deeper into the mandibular arch mesenchyme, apoptotic epithelial cells were detected closer to the tip of the bud. This suggests that apoptosis may have a role in the invagination process of the dental epithelium. Bud formation is a common stage for most organs, and apoptosis may have a general role in the shaping of the budding epithelium. Apoptosis was also detected in the toothless diastema region in the upper, but not the lower, E13 jaw. This correlates with the earlier observation that rudimentary tooth buds develop in the maxilla but degrade later, whereas in the mandible, no invaginating dental buds have been reported (Peterková et al., 1993). Thus, apoptosis seen in the maxillary diastema may be involved in the invagination of diastema dental rudiments.


269

Apoptosis in the Removal of Embryonic Signaling Centers Enamel knots are transient structures and putative signaling centers for tooth development (see above). Apoptosis was observed both in the primary enamel knot at the cap stage (E14–15) and in the secondary enamel knots at the bell stage (E18), suggesting that cell death may be the mechanism for their disappearance. Thus, apoptosis is possibly the means to terminate the signaling functions of the enamel knot cells. We detected apoptosis in the AER of E11–E13 limb buds, which represent another embryonic signaling center (see above). Cell death has been localized earlier in the AER but it has not been connected to signaling but rather to the separation of digits which, however, takes place later than the cell death detected (Lee et al., 1993; Coelho et al., 1993). We propose that apoptosis in both the enamel knots and AER may be a way to stop the expression of signaling molecules after they have served their purpose. In other embryonic signaling and organizing centers, the transient inductive actions may also be silenced by cell death. Temporal differences (heterochrony) in apoptotic cell death in enamel knots may lead to shorter or longer periods of signaling, which in turn may result in differences in the size and shape of the cusps. In an analogous way, heterochrony in apoptosis of the AER may be associated with evolutionary changes in limb morphology. Thus, apoptosis may be a thus far neglected evolutionary mechanism to realize heterochrony. Apoptosis in the Disruption of Dental Epithelium The epithelial component of the developing tooth, the enamel organ, reduces in size in postnatal mice during enamel formation, and finally disappears completely as the tooth erupts. At bell stage, cell death was located in all dental epithelial cell types: ameloblasts, which face the dental mesenchyme and secrete enamel; stratum intermedium cells, which are intimately associated with the ameloblast layer; dental lamina cells, which connect the tooth germ to the oral epithelium; stellate reticulum cells, which form the central part of the enamel organ (Figure 4). Based on our results, it is conceivable that a large number of epithelial cells undergo apoptosis, which agrees with the earlier estimation from kinetic studies that approximately half of the ameloblasts die during maturation stage (Smith and Warshawsky, 1977). Furthermore, apoptosis has recently been detected in ameloblasts during the eruption of mouse molars (Shibata et al., 1995). The closeness of dying stratum intermedium cells and ameloblasts suggests that apoptosis in the two cell types may be linked. Stratum intermedium cells are connected to each other and to ameloblasts by desmosomes and gap junctions, although their exact function is unclear (Ten Cate, 1994). It is possible that this coupling is also involved in the regulation of apoptosis. Cell death in epidermal cells may be a specialized form of differentiation, as many cellular and molecular events are shared by apoptosis and keratinocyte terminal differentiation (Polakowska et al., 1994). The same may be true of the enamel organ cells. Association of apoptosis with gene expression: The expression of several genes has been reported to coincide with cell death in various tissues, but the associations appear not to be general (Osborne and Schwartz, 1994; McConkey and Orrenius, 1994). Of such genes, the expression patterns of Egr-1, N-myc, cfos, Msx-2 and Bcl-2 have been analysed during tooth development, but our results indicate no codistribution with apoptosis, except that Msx-2 shows a partial colocalization with apoptosis in the enamel knot (Hirning et al., 1991; Yamada et al., 1992; Karavanova et al., 1992; MacKenzie et al., 1992; Smeyne et al., 1993; Slootweg and de Weger, 1994). The association of Msx-2 and apoptosis has also been found in odd-numbered rhombomeres, in which expression of Msx-2 precedes apoptosis (Graham et al., 1993).

270


Prevention of Apoptosis in Cultured Dental Tissues by Epithelial-mesenchymal Interactions and by EGF and FGF-4 The possible effects of epithelial-mesenchymal interactions on apoptosis were studied in tissue cultures in which epithelial and mesenchymal components of tooth germs at the bud stage were cultured either in isolation or together for 16–24 hours. After culture, apoptosis was detected by using Tunel and Nile Blue stainings. When isolated epithelia and mesenchymes from E13–E13.5 tooth germs were cultured together overnight, apoptosis was prevented at the epithelial-mesenchymal interphase in both the mesenchyme and epithelium. However, when either tissue was cultured in isolation, apoptosis was detected in the whole explant. The epithelial-mesenchymal interactions regulate many aspects of tooth development and they may also mediate survival signals between the interacting tissues. Egf and Fgf-4 are expressed during tooth development, and these proteins act as survival factors in other developmental systems (Cobo et al., 1992; Niswander and Martin, 1992; Heikinheimo et al., 1993; MacCabe, 1993). The effects of EGF and FGF-4 on cell death were determined by culturing isolated dental mesenchymes from E13–E13.5 tooth germs in the presence of EGF or FGF-4 soaked agarose or heparin acrylic beads. Apoptosis was prevented by EGF and FGF-4 in a concentration-dependent manner (Figure 5). EGF inhibited apoptosis only when beads were soaked in 125 ng EGF/100 beads, lower (25 and 75 ng per 100 beads) or higher amounts (250, 600 and 1000 ng per 100 beads) of EGF did not have any effect on cell death. On the other hand, 25–250 ng of FGF-4 prevented apoptosis in the dental mesenchyme (Vaahtokari et al., 1996b). It has been shown that at least certain cells undergo apoptosis if deprived of EGF or FGF (Collins et al., 1994). These growth factors may function as survival factors in other developing organs as well, and it has indeed been shown that in the developing kidney, EGF can inhibit apoptosis (Koseki et al., 1992; Coles et al., 1993), and in the developing limb, the AER-derived FGF-2 rescues mesodermal cells from apoptosis (MacCabe, 1993). In addition, lens fibre cells are dependent on FGF for their survival (Chow et al., 1995). Exogenous EGF has been shown to prevent degradation of dental follicle in tooth germs in vitro (Partanen et al., 1985). Furthermore, Egf-specific antisense oligodeoxynucleotides in tooth germ cultures impair tooth morphogenesis (Kronmiller et al., 1991; Hu et al., 1992). The authors have suggested that the effects are due to the mitogenic effects of EGF (Partanen et al., 1985; Hu et al., 1992), but they may also be interpreted so that EGF acts as a survival factor. FGFs are expressed in dental epithelium during several stages of tooth morphogenesis, and hence it is possible that they function as survival factors for dental mesenchyme. The only cells in the developing tooth expressing Fgf-4 are enamel knot cells, which do not proliferate, although FGF-4 acts as a mitogen for dental tissues in vitro (Jernvall et al., 1994). This unresponsiveness of enamel knot cells to FGF-4 may be due to the lack of expression of proper FGF receptors or there may be some other differences between the enamel knot cells and their neighbors. In transgenic mice expressing a dominant-negative form of the murine FGFR-1 in the lens, the mice display diminished differentiation and apoptosis of fibre cells (Chow et al., 1995). Inability to respond to FGF-4 may lead to apoptosis, which may terminate the signaling from the enamel knot. CONCLUDING REMARKS Tooth development, like that of other organs, is a complex system consisting of an interplay of several simultaneous processes. However, the developmental mechanisms are organized in a hierarchical way: at the molecular level, there are signaling molecules which bind to their receptors, and the signal transduction pathways which lead to changes in gene expression. At the cellular level, a single cell can divide, migrate,


271

differentiate or die, whereas at the tissue level, pattern formation takes place. Naturally, these kinds of definitions are oversimplifications, but they give, nevertheless, conceptual tools with which the difficulties in understanding embryonic development can be tackled. In this review, roles in tooth development are suggested for signaling molecules triggering separate signal transduction pathways (BMPs, FGFs and Sonic Hedgehog), for a particular signaling center which may direct local growth and patterning (enamel knot), and for a general developmental mechanism which may regulate growth and morphogenesis (apoptosis). Until recently, there have been only sparse glimpses of the connections between the various levels of the developmental processes despite of the ever-increasing amount of data on genes involved in embryogenesis. At the moment, the intricate molecular networks regulating organogenesis are gradually being explored, which reveals relationships between various molecular pathways. The rapidly advancing techniques of molecular biology and genetics will hopefully give tools in the near future for turning the data presented in this review into a coherent picture of the regulation of tooth development, which will give insight into organ development in general. REFERENCES Åberg, T., Wozney, J. and Thesleff, I. (1997) Expression patterns of BMPs in the developing mouse tooth suggests roles in morphogenesis and cell differentiation. Dev. Dyn., 210,407– 416. Alexandre, C., Jacinto, A. and Ingham, P.W. (1996) Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the Cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes Dev., 10, 2003– 2013. Ali, J., Mansukhani, A. and Basilico, C. (1995) Fibroblast growth factor receptors 1 and 2 are differentially regulated in murine embryonal carcinoma cells and in response to fibroblast growth factor-4. J. Cell. Phys., 165, 438–448. Armstrong, J.F., Kaufman, M.H., Harrison, D.J. and Clarke, A.R. (1995) High-frequency developmental abnormalities in p53-deficient mice. Curr. Biol., 5, 931–936. Baird, A. (1994) Fibroblast growth factors: activities and significance of non-neurotrophin growth factors. Curr. Opin. Neurobiol., 4, 78–86. Bellusci, S., Henderson, R., Winnier, G., Oikawa, T. and Hogan, B.L. (1996) Evidence from normal expression and targeted misexpression that bone morphogenetic protein (Bmp-4) plays a role in mouse embryonic lung morphogenesis. Development, 122, 1693–702. Bellusci, S., Furuta, Y., Rush, M.G., Henderson, R., Winnier, G. and Hogan, B.L.M. (1997) Involvement of Sonic hedgehog (Shh) in mouse embryonic lung growth and morphogenesis. Development, 124, 53–63. Bitgood, M.J. and McMahon, A.R (1995) Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Dev. Biol., 172, 126–158. Blecher, S.R., Kapalanga, J. and Lalonde, D. (1990) Induction of sweat glands by epidermal growth factor in murine Xlinked anhidrotic ectodermal dysplasia. Nature, 354, 542–544. Blessing, M., Nanney, L.B., King, L.E., Jones, C.M. and Hogan, B.L. (1993) Transgenic mice as a model to study the role of TGF-beta-related molecules in hair follicles. Genes Dev., 7, 204–215. Boudreau, N., Sympson, C.J., Werb, Z. and Bissell, M.J. (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science, 267, 891–893. Bumcrot, D.A., Takada, R. and McMahon, A.R (1995) Proteolytic processing yields two secreted forms of Sonic hedgehog. Mol. Cell Biol., 15, 2294–2303. Chang, D.T., López, A., von Kessler, D.R, Chiang, C., Simandl, B.K., Zhao, R., Seldin, M.F., Fallon, J.F. and Beachy, P.A. (1994) Products, genetic linkage and limb patterning activity of a murine hedgehog gene. Development, 120, 3339–3353.

272


Figure 5 Effects of epithelial-mesenchymal interactions and FGF-4 on apoptosis in dental mesecnhyme. Whole mount (A,B) TUNEL and (D,F) Nile Blue stainings of E13 dental mesenchymes (dm) cultured together for 16–24 h with (A) E13 dental epithelium (de), (B) BSA releasing agarose bead, (C,D) FGF-4 releasing heparin acrylic bead (E,F) FGF-4 releasing agarose bead. (A) Apoptosis is absent in the epithelium and mesenchyme at the tissue interface although the other areas of the explant undergo apoptosis. (B) Apoptosis is seen throughout the explant, also around the hole in the tissue which indicates the location of the BSA releasing bead during culture. (C) A translucent zone is detected around the FGF-4 releasing heparin acrylic bead before the staining. (D) Apoptosis is prevented around the FGF-4 bead in an area approximately corresponding to the translucent zone. (E) A translucent zone is also located around the FGF-4 releasing agarose bead. (F) As in (D), apoptosis is suppressed around the FGF-4 releasing bead. Chazaud, C., Oulad-Abdelghani, M., Bouillet, P., Décimo, D,, Chambon, P. and Dollé, P. (1996) AP-2.2, a novel gene related to AP-2, is expressed in the forebrain, limbs and face during mouse embryogenesis. Mech. Dev., 54, 83–94.


273

Chen, Y.P., Bei, M., Woo, I., Satokata, I. and Maas, R. (1996) Msx1 controls inductive signaling in mammalian tooth morphogenesis. Development, 122, 3035–3044. Chiang, C., Litingtung, Y., Lee, E., Young, K.E., Corden, J.L., Westphal, H. and Beachy, P.A. (1996) Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature, 383, 407–413. Chow, R.L., Roux, G.D., Roghani, M., Palmer, M.A., Rifkin, D.B., Moscatelli, D.A. and Lang, R.A. (1995) FGF suppresses apoptosis and induces differentiation of fibre cells in the mouse lens. Development, 121, 4383–4393. Cobo, J., Hernández, L.C., del Valle, M.E., Viljande, M. and Vega, J.A. (1992) Immunohis-tochemical localization of epidermal growth factor and its receptor during odontogenesis in the rat. Eur. J. Orthod., 14, 333–338. Coelho, C.N., Upholt, W.B. and Kosher, R.A. (1993) The expression pattern of the chicken homeobox-containing gene GHox-7 in developing polydactylous limb buds suggests its involvement in apical ectodermal ridge-directed outgrowth of limb mesoderm and in programmed cell death. Differentiation, 52, 129–137. Cohn, M.J., Izpisúa-Belmonte, J.C., Abud, H., Heath, J.K. and Tickle, C. (1995) Fibroblast growth factors induce additional limb development from the flank of chick embryos. Cell, 80, 739–746. Coleman-Krnacik, S. and Rosen, J.M. (1994) Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development. Mol. Endocrinol., 8, 218–229 Coles, H.S.R., Burne, J.F. and Raff, M.C. (1993) Large-scale normal cell death in the developing rat kidney and its reduction by epidermal growth factor. Development, 118, 777– 784. Collins, M.K.L., Perkins, G.R., Rodriguez-Tarduchy, G., Nieto, M.A. and López-Rivas, A. (1994) Growth factors as survival factors: regulation of apoptosis. BioEssays, 16, 133–138. Coucouvanis, E. and Martin, G.R. (1995) Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell, 83, 279–287. Crossley, P.H. and Martin, G.R. (1995) The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development, 121, 439–51. Crossley, P.H., Martinez, S. and Martin, G.R. (1996a) Midbrain development induced by FGF8 in the chick embryo. Nature, 380, 66–68. Crossley, P.H., Minowada, G., MacArthur, G.R. and Martin, G.R. (1996b) Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell, 84, 127– 136. Deng, C.-X., Wynshaw-Boris, A., Shen, M.M., Daugherty, C., Ornitz, D.M. and Leder, P. (1994) Murine FGFR-1 is required for early postimplantation growth and axial organization. Genes Dev., 8, 3045–3057. Deng, C.-X., Wynshaw-Boris, A., Zhou, F, Kuo, A. and Leder, P. (1996) Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell, 84, 911–921. Dollé, P., Price, M. and Duboule, D. (1992) Expression of the murine Dlx-1 homeobox gene during facial, ocular and limb development. Differentiation, 49, 93–99. Drucker, B J. and Goldfarb, M. (1993) Murine FGF-4 gene expression is spatially restricted within embryonic skeletal muscle and other tissues. Mech. Dev., 40, 155–163. Dudley, A.T., Lyons, K.M. and Robertson, E.J. (1995) A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev., 9, 2795– 2807. Fan, C.-M., Porter, J.A., Chiang, C, Chang, D.T., Beachy, P. and Tessier-Lavigne, M. (1995) Long-range sclerotome induction by Sonic hedgehog: Direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signalling pathway. Cell, 81, 457– 465. Feijen, A., Goumans, M.J. and van den Eijnden-van Raaij, A.J.M. (1994) Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins. Development, 120, 3621–3637. Feldman, B., Poueymirou, W., Papaioannou, V.E., DeChiara, T.M. and Goldfarb, M. (1995) Requirement of FGF-4 for postimplantation mouse development. Science, 261, 246–249. Francis, P.H., Richardson, M.K., Brickell, P.M. and Tickle, C. (1994) Bone morphogenetic proteins and a signalling pathway that controls patterning in the developing chick limb. Development, 120, 209–218.

274


Ganan, Y., Macias, D., Duterque-Coquillaud, M. Ros, M.A. and Hurle, J.M. (1996) Role of TGF betas and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod. Development, 122, 2349– 57. Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119, 493–501. Gene expression in tooth (1998) http://honeybee.helsinki.fi/toothexp Gilbert, S.F. (1994) Developmental Biology, Sinauer Associates, Sunderland. Graham, A., Heyman, I. and Lumsden, A. (1993). Even-numbered rhombomeres control the apoptotic elimination of neural crest cells from odd-numbered rhombomeres in the chick hindbrain. Development, 119, 233–245. Graham, A., Francis-West, P., Brickell, P. and Lumsden, A. (1994) The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature, 372, 684–686. Grobstein, C. (1955) Inductive interactions in the development of the mouse metanephros. J. Exp. Zool., 130, 319–340. Gumbiner, B.M. (1996) Cell adhesion: The molecular basis of tissue architecture and morphogenesis. Cell, 84, 345–357. Gurdon, J.B. (1992) The generation of diversity and pattern in animal development. Cell, 68, 185–199. Heemskerk, J. and Dinardo, S. (1994) Drosophila hedgehog acts as a morphogen in cellular patterning. Cell, 76, 449–460. Heikinheimo, K., Voutilainen, R., Happonen, R.-P. and Miettinen, P.J. (1993) EGF receptor and its ligands, EGF and TGF-α, in developing and neoplastic odontogenic tissues. Int. J. Dev. Biol., 37, 387–396. Heikinheimo, M., Lawshe, A., Shckleford, G.M., Wilson, D.B. and MacArthur, C.A. (1994) Fgf-8 expression in the postgastrulation mouse suggests roles in the development of the face, limb and central nervous system. Mech. Dev., 48, 129–138. Helder, M.N., Ozkaynak, E., Sampath, K.T., Luyten, F.P., Latin, V., Oppermann, H. and Vukicevic, S. (1995) Expression pattern of osteogenic protein-1 (bone morphogenetic protein-7) in human and mouse development. J. Histochem. Cytochem., 43, 1035–1044. Hepker, J., Wang, Q.T., Motzny, C.K., Holmgren, R. and Orenic, T.V. (1997) Drosophila Cubitus interruptus forms a negative feedback loop with patched and regulates expression of hedgehog target genes. Development, 124, 549–558. Hinchliffe, J. (1982) Cell death in vertebrate limb morphogenesis. In J. Harrison and V. Navaratnam, (eds.), Progress in Anatomy,Cambridge University Press, Cambridge, pp. 1– 19. Hirning, U., Schmid, P., Schulz, W.A., Rettenberg, G. and Hameister, H. (1991) A comparative analysis of N-myc and c-myc expression and cellular proliferation in mouse organogenesis. Mech. Dev., 33, 119–126. Hopkinson-Woolley, J., Hughes, D., Gordon, S. and Martin, P. (1994) Macrophage recruitment during limb development and wound healing in the embryonic and foetal mouse. J. Cell Set., 107, 1159–1167. Hu, C.-C, Sakakura, Y., Sasano, Y., Shum, L., Bringas, Jr. P., Werb, Z. and Slavkin, H.C. (1992) Endogenous epidermal growth factor regulates the timing and pattern of embryonic mouse molar tooth morphogenesis. Int. J. Dev. Biol., 36, 505–516. Imai, H., Osumi-Yamashita, N., Nimomiya, Y. and Eto, K. (1996) Contribution of early-emigrating midbrain crest cells to the dental mesenchyme of mandibular molar teeth in rat embryos. Dev. Biol., 176, 151–165. Ingham, P.W. (1994) Hedgehog points the way. Curr. Biol., 4, 347–350. Iseki, S., Araga, A., Ohuchi, H., Nohno, T., Yoshika, H, Hayashi, F. and Noji, S. (1996) Sonic hedgehog is expressed in epithelial cells during development of whisker, hair, and tooth. Biochem. Biophys. Res. Commun., 218, 688–693. Jernvall, J. (1995) Mammalian molar cusp patterns: Developmental mechanisms of diversity. Acta Zool. Fennica, 198, 1–61. Jernvall, J., Kettunen P., Karavanova, L, Martin, L.B. and Thesleff, I. (1994) Evidence for the role of the enamel knot as a control center in mammalian tooth cusp formation: non-dividing cells express growth stimulating Fgf-4 gene. Int. J. Dev. Biol., 38, 463–469.


275

Jernvall, J., Åberg, T., Kettunen, P., Keränen, S. and Thesleff, I. (1997) The life history of an embryonic signaling center: BMP-4 induces p21 and is associated with apoptosis in the mouse tooth enamel knot. Development, 125, 161–169. Jessell, T.M. and Melton, D.A. (1992). Diffusible factors in vertebrate embryonic induction. Cell, 68, 257–270. Johnson, D.E., Lu, J., Chen, H., Werner, S. and Williams, L.T (1991) The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain. Mol. Cell. Biol., 11, 4627–4634. Jones, C.M., Lyons, K.M. and Hogan, B.L.M. (1991) Involvement of Bone Morphogenetic Protein-4 (BMP-4) and Vgr-1 in morphogenesis and neurogenesis in the mouse. Development, 111, 531–542. Jowett, A.K., Vainio, S., Ferguson, M.W.J., Sharpe, P.T. and Thesleff, I. (1993) Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth. Development, 117, 461–470. Karavanova, I., Vainio, S. and Thesleff, I. (1992) Transient and recurrent expression of the Egr-1 gene in epithelial and mesenchymal cells during tooth morphogenesis suggests in-volvement in tissue interactions and in determination of cell fate. Mech. Dev., 39, 41–50. Kelley, R.I., Roessler, E., Hennekam, R.C.M., Feldman, G.I., Kosaki, K.Jones, M.C., Palumbos, J.C. and Muenke, M. (1996) Holoprosencephaly in RSH/Smith-Lemli-Opitz syndrome — does abnormal cholesterol metabolism affect the function of Sonic hedgehog. Am. J. Med. Gen., 66, 478–484. Kere, J., Srivastava, A.K., Montonen, O., Zonana, J., Thomas, N., Ferguson, B., Munoz, F., Morgan, D., Clarke, A., Baybayan, P. et al. (1996) X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein. Nat. Genet., 13, 409–416. Kettunen, P. and Thesleff, I. (1998) Expression and function of FGFs-4, -8 and -9 suggest functional redundancy and repetitive use as epithelial signals during tooth morphogenesis. Dev. Dyn., 211, 256–268. Kingsley, D.M. (1994) The TGF-β superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev., 8, 133–146. Kollar, E.J. and Baird, G.R. (1970) Tissue interactions in embryonic mouse tooth germs: II. The inductive role of the dental papilla. J. Embryol. Exp. Morph., 24, 173–186. Koseki, C., Herziinger, D. and Al-Awqati, Q. (1992) Apoptosis in metanephric development. J. Cell Biol., 119, 1327–1333. Kratochwil, K., Dull, M., Farinas, I., Galceran, J. and Grosschedl, R. (1996) Lef1 expression is activated by BMP-4 and regulates inductive tissue interactions in tooth and hair development. Genes Dev., 10, 1382–1394. Kronmiller, J.E., Upholt, W.B. and Kollar, E.J. (1991) EGF antisense oligodeoxynucleotides block murine odontogenesis in vitro. Dev. Biol., 147, 485–488. Lappi, D.A. (1995) Tumor targeting through flbroblast growth factor receptors. Semin. Cancer Biol., 6, 279–288. Laufer, E., Nelson, C.E., Johnson, R.L., Morgan, B.A. and Tabin, C. (1994) Sonic hedgehog and FGF-4 act through a signaling cascade and feedback loop to integrate growth and patterning on the developing limb bud. Cell, 79, 993–1003. Lee, K.K., Chan, W.Y. and Sze, L.Y. (1993) Histogenetic potential of rat hind-limb interdigital tissues prior to and during the onset of programmed cell death. Anat. Rec., 236, 568– 572. Lehtonen, E., Wartiovaara, J., Nordling, S. and Saxén, L. (1975) Demonstration of cytoplasmic processes in Millipore filters permitting kidney tubule induction. J. Embryol. Exp. Morphol., 33, 187–203. Liem, Jr., K.F., Tremml, G., Roelink, H. and Jessell, T.M. (1995) Dorsal differentiation of neural plate cells induced by BMP-mediated signals from epidermal ectoderm. Cell, 82, 969–979. Liu, P., Ventura, F., Doody, J. and Massagué, J. (1995) Human type II receptor for bone morphogenic proteins (BMPs): Extension of the two-kinase receptor model to the BMPs. Mol. Cell Biol., 15, 3479–3486. Lumsden, A.G.S. (1988) Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ. Development, 103, 155–169. Luo, G., Hofmann, C., Bronckers, A.L.J.J., Sohocki, M., Bradley, A. and Karsenty, G. (1995) BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev., 9, 2808–2820.

276


Lyons, K.M., Pelton, R.W. and Hogan, B.L. (1990) Organogenesis and pattern formation in the mouse: RNA distribution patterns suggest a role for Bone Morphogenetic Protein-2A (BMP-2A). Development, 109, 833–844. Lyons, K.M., Hogan, B.L.M. and Robertson, E.J. (1995) Colocalization of BMP 7 and BMP 2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development. Mech. Dev., 50, 71–83. MacCabe, J.A. (1993) Many chick embryo limb mesoderm cells require b-FGF for survival. Prog. Clin. Biol. Res., 383, 611–623. MacKenzie, A., Leeming, G.L., Jowett, A.K., Ferguson, M.W.J. and Sharpe, P.T. (1991) The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth develoment in vivo and in vitro. Development, 111, 269–285. MacKenzie, A., Ferguson, M.W.J. and Sharpe, P.T. (1992) Expression patterns of the homeobox gene, Hox-8, in the mouse embryo suggest a role in specifying tooth initiation and shape. Development, 115, 403–420. Marigo, V., Davey, R.A., Zuo, Y., Cunningham, J.M. and Tabin, C.J. (1996) Biochemical evidence that Patched is the hedgehog receptor. Nature, 384, 176–179. Martí, E., Bumcrot, D.A., Takada, R. and McMahon, A.P. (1995a) Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants. Nature, 375, 322–325. Martin, S.J., Green, D.R. and Cotter, T.G. (1994) Dicing with death: dissecting the components of the apoptosis machinery. Trends Biochem. Sci., 19, 26–30. Mason, I.J. (1994). The ins and outs of fibroblast growth factors. Cell, 78, 547–552. Matzuk, M.M., Kumar, T.R. and Bradley, A. (1995a) Different phenotypes for mice deficient in either activins or activin receptor type II. Nature, 374, 356–360. Matzuk, M.M., Kumar, T.R., Vassalli, A., Bickenbach, J.R., Roop, D.R., Jaenisch, R. and Bradley, A. (1995b) Functional analysis of activins during mammalian development. Nature, 374, 354–356. Matzuk, M.M., Lu, N., Vogel, H., Sellheyer, K., Roop, D.R. and Bradley, A. (1995c) Multiple defects and perinatal death in mice deficient in follistatin. Nature, 374, 360–363. McConkey, D.J. and Orrenius, S. (1994) Signal transduction pathways to apoptosis. Trends Cell Biol., 4, 370–375. Miettinen, P.J., Berger, J.E., Meneses, J., Phung, Y., Pedersen, R.A., Werb, Z. and Derynck, R. (1995) Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature, 376, 337–341. Mima, T., Ohuchi, H., Noji, S. and Mikawa, T. (1995) FGF can induce outgrowth of somatic mesoderm both inside and outside of limb-forming regions. Dev. Biol., 167, 617–620. Mina, M. and Kollar, E.J. (1987) The induction of odontogenesis in non-dental mesenchyme combined with early murine mandibular arch epithelium. Archs. Oral Biol., 32, 123–127. Mitsiadis, T., Lardelli, M., Lendahl, U. and Thesleff, I. (1995) Expression of Notch 1, 2, and 3 is regulated by epithelialmesenchymal interactions and retinoic acid in the developing mouse tooth and associated with determination of ameloblast cell fate. J. Cell Biol., 130, 407–418. Mohamed, S.S. and Atkinson, M.E. (1983) A histological study of the innervation of developing mouse teeth. J. Anat., 136, 735–749. Morgenbesser, S.D., Williams, B.O., Jacks, T. and DePinho, R.A. (1994) p53-dependent apoptosis produced by Rbdeficiency in the developing mouse lens. Nature, 371, 72–74. Mori, C., Nakamura, N., Okamoto, Y., Osawa, M. and Shiota, K. (1994) Cytochemical identification of programmed cell death in the fusing fetal mouse palate by specific labelling of DNA fragmentation. Anat. Embryol., 190, 21–28. Morrison-Graham, K., Schatteman, G.C., Bork, T., Bowen-Pope, D.F. and Weston, J.A. (1992) A PDGF receptor mutation in the mouse (Patch) perturbs the development of a non-neuronal subset of neural crest-derived cells. Development, 115, 133–142. Mucchielli, M.-L., Mitsiadis, T.A., Raffo, S., Brunet, J.-F., Proust, J.-P. and Goridis, C. (1997) Mouse Otlx2/RIEG expression in the odontogenic epithelium precedes tooth initiation and requires mesenchyme-derived signals for its maintenance. Dev. Biol., 189, 275–284. Muenke, M. and Schell, U. (1995) Fibroblast-growth-factor receptor mutations in human skeletal disorders. Trends Genet., 11, 308–313.


277

Niswander, L. and Martin, G.R. (1992) Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development, 114, 755–768. Niswander, L. and Martin, G.R. (1993) FGF-4 and BMP-2 have opposite effects on limb growth. Nature, 361, 68–71. Niswander, L., Ickle, C., Vogel, A., Booth, I. and Martin, G.R. (1993) FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell, 75, 579– 587. Niswander, L., Jeffrey, S., Martin, G.R. and Tickle, C. (1994) A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature, 371, 609–612. Nohno, T., Ishikawa, T., Saito, T., Hosokawa, K., Noji, S., Wolsing, D.H. and Rosenbaum, J.S. (1995) Identification of a human type II receptor for bone morphogenetic protein-4 that forms differential heteromeric complexes with bone morphogenetic protein type I receptors. J. Biol. Chem., 270, 22522–22526. Nüsslein-Volhard, C. and Wieschaus, E. (1980) Mutations affecting segment number and polarity on Drosophila. Nature, 287, 795–801. Oosterwegel, M., van de Watering, M., Timmermann, J., Kruisbeek, A., Oestree, O., Meijlink, F. and Clevers, H. (1993) Differential expression of the HMG box factors TCF-1 and LEF-1. Development, 118, 439–448. Orr-Urtreger, A., Bedford, M.T., Burakova, T., Arman, E., Zimmer, Y., Yayon, A., Givol, D. and Lonai, P. (1993) Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev. Biol., 158, 475–486. Osborne, B.A. and Schwartz, L.M. (1994) Essential genes that regulate apoptosis. Trends Cell Biol., 4, 394–399. Partanen, A.-M. and Thesleff, I. (1987) Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages. Dev. Biol., 120, 186–197. Partanen, A.-M., Ekblom, P. and Thesleff, I. (1985) Epidermal growth factor inhibits morphogenesis and cell differentiation in cultured mouse embryonic teeth. Dev. Biol., 111, 84–94. Pellier, V. and Astic, L. (1994) Detection of apoptosis by electron microscopy and in situ labelling in the rat olfactory pit. Neuro. Report, 5, 1429–1432. Perrimon, N. (1995) Hedgehog and beyond. Cell, 80, 517–520. Perry, V., Henderson, Z. and Linden, R. (1983) Postnatal changes in retinal ganglion cell and optic axon populations in the pigmented rat. J. Comp. Neurol., 219, 356–368. Peterková, R., Peterka, M. and Ruch, J.V. (1993) Morphometric analysis of potential maxillary diastemal dental anlagen in three strains of mice. J. Cran. Genet. Dev. Biol., 13, 213–222. Peters, K.G., Werner, S., Chen, G. and Williams, L.T. (1992) Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development, 114, 233–243. Polakowska, R.R., Piacentini, M., Bartlett, R., Goldsmith, L.A. and Haake, A.R. (1994) Apoptosis in human skin development: morphogenesis, periderm, and stem cells. Dev. Dyn., 199, 176–188. Porter, J.A., von Kessler, D.P., Ekker, S.C., Young, K.E., Lee, J.J., Moses, K. and Beachy, P.A. (1995) The product of hedgehog autoproteolytic cleavage active in local and long-range signalling. Nature, 374, 363–366. Porter, J.A., Young, K.E. and Beachy, P.A. (1996) Cholesterol modification of Hedgehog signaling proteins in animal development. Science, 274, 255–259. Pourquié, O., Fan, C.-M., Coltey, M., Hirsinger, E., Watanabe, Y., Bréant, C., Francis-West, P., Brickell, P., TessierLavigne, M. and Le Douarin, N.M. (1996) Lateral and axial signals involved in avian somite patterning: a role for BMP4. Cell, 84, 461–471. Raff, M.C. (1992) Social controls on cell survival and cell death. Nature, 356, 397–400. Raff, M.C., Barres, B.A., Burne, J.F., Coles, H.S., Ishizaki, Y and Jacobson, M.D. (1993) Programmed cell death and the control of cell survival: lessons from the nervous system. Science, 262, 695–700. Raju, K., Tang, S., Dubé, I.D., Kamel-Reid, S., Bryce, D.M and Breitman, M.L. (1993) Characterization and developmental expression of Tlx-1, the murine homolog of HOX11. Meek. Dev., 44, 51–64. Reddi, A.H. (1994) Bone and cartilage differentiation. Curr. Opin. Genet. Dev., 4, 737–744. Riddle, R.D., Johnson, R.L., Laufer, E. and Tabin, C. (1993) Sonic hedgehog mediates polarizing activity of the ZPA. Cell, 75, 1401–1416.

278


Roberts, D.J., Johnson, R.L., Burke, A.C., Nelson, C.E., Morgan, B.A. and Tabin, C. (1995) Sonic hedgehog is an endodermal signal inducing BMP-4 and Hox genes during induction and regionalization of the chick hindgut. Development, 121, 3163–3174. Roberts, V.J. and Barth, S.L. (1994) Expression of messenger ribonucleic acids encoding the inhibin/activin system during mid- and late-gestation rat embryogenesis. Endocrinology, 134, 914–923. Robinson, G.W. and Mahon, K.A. (1994) Differential and overlapping expression domains of Dlx-2 and Dlx-3 suggest distinct roles for Distal-less homeobox genes in craniofacial development. Mech. Dev., 48, 199–215. Rosenzweig, B.L., Imamura, T., Okadome, T., Cox, G.N., Yamashita, H., ten Dijke, P., Heldin, C.H. and Miyazono, K. (1995) Cloning and characterization of a human type II receptor for bone morphogenetic proteins. Proc. Natl. Acad. Set., 92, 7632–7636. Rotello, R.J., Fernandez, P.-A. and Yuan, J. (1994) Anti-apogens and anti-engulfens: monoclonal antibodies reveal specific antigens on apoptotic and engulfment cells during chicken embryonic development. Development, 120, 1421–1431. Sahlberg, C., Reponen, P., Tryggvason, K. and Thesleff, I. (1992) Association between the expression of murine 72 kDa type IV collagenase by odontoblasts and basement membrane degradation during mouse tooth development. Arch. Oral Biol., 37, 1021–30. Salmivirta, M. and Jalkanen, M. (1995) Syndecan family of cell surface proteoglycans: developmentally regulated receptors for extracellular effector molecules. Experientia, 51, 863–872. Satokata, I. and Maas R. (1994) Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet., 6, 348–356. Saxén, L., Lehtonen, E., Karkinen-Jääskeläinen, M., Nordling, S. and Wartiovaara, J. (1976) Are morphogenetic tissue interactions mediated by transmissible signal substances or through cell contacts? Nature, 259, 662–663. Schwartz, L.M. and Osborne, B.A. (1993) Programmed cell death, apoptosis and killer genes. Immunol. Today, 14, 582–590. Sharpe, P.T. (1995) Homeobox genes and orofacial development. Connect. Tissue Res., 32, 17– 25. Shibata, S., Suzuki, S., Tengan, T. and Yamashita, Y. (1995) A histochemical study of apoptosis in the reduced ameloblasts of erupting mouse molars. Arch. Oral Biol., 40, 677–680. Slack, J.M.W. (1994) Inducing factor in Xenopus early embryos. Curr. Biol., 4, 116–126. Slavin, J. (1995) Fibroblast growth factors—at the heart of angiogenesis. Cell Biol. Internat., 19, 431–444. Slootweg, P.J. and de Weger, R.A. (1994) Immunohistochemical demonstration of bcl-2 protein in human tooth germs. Arch. Oral Biol., 39, 545–550. Smeyne, R.J., Vendrell, M., Hayward, M., Baker, S.J., Miao, G.G., Schilling, K., Robertson, L.M., Curran, T. and Morgan, J.I. (1993) Continuous c-fos expression precedes programmed cell death in vivo. Nature, 363, 166–169. Smith, C.E. and Warshawsky, H. (1977) Quantitative analysis of cell turnover in the enamel organ of the rat incisor. Evidence for ameloblast death immediately after enamel matrix secretion. Anat. Rec., 187, 63–98. Smith, D.E., Franco del Amo, F. and Gridley, T. (1992) Isolation of Sna, a mouse gene homologous to the Drosophila genes snail and escargot: its expression pattern suggests multiple roles during postimplantation development. Development, 116, 1033–1039. Smith, J.C. (1994) Hedgehog, the floor plate, and the zone of polarizing activity. Cell, 76, 193–196. Sonnenberg, E., Meyer, D., Weidner, K.M. and Birchmeier, C. (1993) Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelial during mouse development. J. Cell Biol., 123, 223– 235. Srivastava, A.K., Pispa, J., Hartung, A.J., Du, Y., Ezer, S., Jenks, T., Shimada, T., Pekkanen, M., Ko, M.S.H., Thesleff, I., Kere, J. and Schlessinger, D. (1997) The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene, which reveals novel mouse and human exons and encodes a protein (ectodysplasin) with collagenous domains. Proc. Natl. Acad. Sci. (USA), 94, 13069–13074. Steller, H. (1995) Mechanisms and genes of cellular suicide. Science, 267, 1445–1449.


279

Stone, D.M., Hynes, M., Armanini, M., Swanson, T.A., Gu, Q.M., Johnson, R.L., Scott,M. P., Pennica, D., Goddard, A., Phillips, H., Noll, M., Hooper, J.E., Desauvage, F. and Rosenthal, A. (1996) The tumour-suppressor gene Patched encodes a candidate receptor for Sonic hedgehog. Nature, 384, 129–134. Tabata, M.J., Kim, K., Liu, J.-G, Yamashita, K., Matsumura, T., Kato, J., Iwamoto, M., Wakisaka, S., Matsumoto, K., Nakamura, T., Kumegawa, M. and Kurisu, K. (1996) Hepatocyte growth factor is involved in the morphogenesis of tooth germ in murine molars. Development, 122, 1243–1251. Ten Cate, A.R. (1994) Oral Histology, Mosby-Year Book, St. Louis. Thesleff, I. and Åberg, T. (1997) Tooth morphogenesis and the differentiation of ameloblasts. In Ciba Foundation Symposium no 205: Dental Enamel, John Wiley & Sons Ltd, Sussex, pp. 3–17. Thesleff, I. (1995) Differentiation of ameloblasts and its regulation by epithelial-mesenchymal interactions. In C. Robinson, J. Kirkham and R. Shore, (eds.), Dental enamel: formation to destruction, CRC Press, Boca Raton. Thesleff, I., Mackie, E., Vainio, S. and Chiquet-Ehrismann, R. (1987) Changes in the distribution of tenascin during tooth development. Development, 101, 289–296. Thesleff, I., Vaahtokari, A., Kettunen, P. and Åberg, T. (1995a) Epithelial-mesenchymal signaling during tooth development. Connect. Tissue Res., 32, 1–4. Thesleff, I., Vaahtokari, A. and Partanen, A.-M. (1995b) Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int. J. Dev. Biol., 39, 35–50. Thompson, C.B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science, 261, 1456–1462. Urase, K., Mukasa, T., Igarashi, H., Ishii, Y., Yasugi, S., Momoi, M.Y and Momoi, T. (1996) Spatial expression of Sonic hedgehog in the lung epithelium during branching morphogenesis. Biochem. Biophys. Res. Comm., 225, 161–166. Vaahtokari, A., Åberg, T., Jernvall, J., Keränen, S. and Thesleff, I. (1996a) The enamel knot as a signaling center in the developing mouse tooth. Mech. Dev., 54, 39–43. Vaahtokari, A., Åberg, T. and Thesleff, I. (1996b) Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development, 122, 121–126. Vainio, S., Jalkanen, M. and Thesleff, I. (1989) Syndecan and tenascin expression is induced by epithelialmesenchymal interactions in embryonic tooth mesenchyme. J. Cell Biol., 108, 1945–1954. Vainio, S., Karavanova, I., Jowett, A.K. and Thesleff, I. (1993) Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell, 75, 45–58. van der Hoeven, F., Schimmang, T., Volkman, A., Mattei, M.-G., Kyewski, B. and Rüther, U. (1994) Programmed cell death is affected in the novel mouse mutant Fused toes (Ft). Development, 120, 2601–2607. van Genderen, C., Okamura, R.M., Fariñas, I., Quo, R.-G., Parslow, T.G., Bruhn, L. and Grosschedl, R. (1994) Developoment of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1 — deficient mice. Genes. Dev., 8, 2691– 2703. Verschueren, K., Dewulf, N., Goumans, M.-J., Lonnoy, O., Feijen, A., Grimsby, S., Vande Spiegle, K., ten Dijke, P., Morén, A., Vanscheeuwijck, P., Heldin, C.-H., Miyazono, K., Mummery, C., Van Den Eijnden-Van Raaij, J. and Huylebroeck, D. (1995) Expression of type I and type IB receptors for activin in midgestation mouse embryos suggests distinct functions in organogenesis. Mech. Dev., 52, 109–123. Vukicevic, S., Latin, V., Chen, P., Batorsky, R., Reddi, A.H. and Sampath, T.K. (1994) Localization of osteogenic protein-1 (bone morphogenetic protein-7) during human embryonic development: high affinity binding to basement membranes. Biochem. Biophys. Res. Comm., 198, 693–700. Wall, N.A. and Hogan, B.L.M. (1995) Expression of bone morphogenetic protein-4 (Bmp-4), bone morphogenetic protein-7 (Bmp-7), fibroblast growth factor-8 (Fgf-8) and Sonic hedgehog (Shh) during branchial arch development in the chick. Mech. Dev., 53, 383– 392. Weil, M., Itin, A. and Ja Keshet, E. (1995) A role for mesenchyme-derived tachykinins in tooth and mammary gland morphogenesis. Development; 121, 2419–2428. Weiss, K.M., Bollekens, J., Ruddle, F.H. and Takashita, K. (1994) Distal-less and other homeobox genes in the development of the dentition. J. Exp. Zool., 270, 273–84.

280


Wilkinson, D.G., Bhatt, S. and McMahon, A.P. (1989) Expression pattern of the FGF-related proto-oncogene int-2 suggests multiple roles in fetal development. Development, 105,131– 136. Winnier, G., Blessing, M., Labosky, P.A. and Hogan, B.L. (1995) Bone morphogenetic protein-4 is required for mesoderm formation and patterning in the mouse. Genes Dev., 9, 2105– 2116. Yamada, S., Ikeda, M. and Eto, K. (1992) Differential expression of c-myc and N-myc during oral organogenesis of the mouse embryo. Dev. Growth Differ., 34, 239–251. Yamada, S., Yamada, K.M. and Brown, K.E. (1994) Integrin regulatory switching in development: oscillation of β5 integrin mRNA expression during epithelial-mesenchymal interactions in tooth development. Int. J. Dev. Biol., 38, 553–556. Yamaguchi, T.P., Harpal, K., Henkemeyer, M. and Rossant, J. (1994) fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Gems Dev., 8, 3032–3044. Zou, H. and Niswander, L. (1996) Requirement for BMP signaling in interdigital apoptosis and scale formation. Science, 272, 738–41.

15. BIOLOGICAL FUNCTIONS OF GAP JUNCTION CHANNELS REVEALED BY TARGETED CONNEXIN DEFICIENT MICE KLAUS WILLECKE and THOMAS OTT Abteilung Molekulargenettk, Institut fur Genetik, Römerstrafie 164, 53117 Bonn, Germany INTRODUCTION Gap junctions have been first described by electron microscopy and electrophysiology as aggregates of electrically conductive cell-to-cell channels that were permeable for ions and metabolites. During the last 15 years the subunit proteins and corresponding genes of gap junctions have been characterized, but only indirect evidence for the functional relevance of gap junction channels in mammalian cells could be obtained. The discovery of the first inherited human mutation that affected a gap junctional gene (Bergoffen et al., 1993) and, especially, the generation and characterization of mice that lack gap junctional proteins made it possible to understand the consequences of gap junctional functions in whole organisms. Thus, we are at the beginning of an exciting period when long standing hypotheses regarding gap junctional functions can be directly tested in targeted mouse mutants. Here we have attempted to summarize the present insights into the biological function of gap junctions, based mainly on investigations of connexin defective mice and humans. The family of gap junctional proteins, the connexins, consists in rodents of at least 13 members (for recent reviews see Bruzzone et al., 1996; Goodenough et al., 1996; Kumar and Gilula, 1996) which are closely related in sequence and topology. The different connexins are abbreviated as Cx followed by a number which represents the round molecular mass (in kDa) of the corresponding protein. All known connexins are transmembrane proteins that cross the plasma membrane four times. The carboxy and the amino terminal ends of the proteins are located on the cytoplasmic side of the membrane with the C-terminal and the central cytoplasmic loop to be the most divergent regions of the connexins. The different cytoplasmic domains are probably major targets for phosphorylation by protein kinases triggered by various signal transduction pathways. The two extracellular loops of connexin proteins are closely related between different members of the connexin family, and, especially, the strong conservation of two cysteine motifs in each of these loops seems to be important for proper protein folding and oligomerization, as well as for docking of two connexin hemichannels in contacting plasma membranes. All connexin genes cloned from rodent or human genomes show a similar genomic organization. The coding sequence is located on the second exon, whereas the first exon contains the 5' untranslated leader sequence. The intron between these two exons is highly variable in length between different connexin genes. At least for the rat and mouse connexin32 (Cx32) gene, alternative tissue-specific promoters and exonl sequences exist which add to the diversity of connexin gene expression (Neuhaus et al., 1995; Söhl et al., 1996). A functional gap junctional channel is formed by docking of two hemichannels (connexons) located in contacting plasma membranes of neighbouring cells. Each connexon is usually composed of six identical connexin subunits. Two hemichannels, each one built up of a different type of connexin, can interact to form

282


heterotypic gap junction channels. Recently, it has been shown that different connexin proteins can contribute to the same heteromeric hemichannel (Stauffer, 1995; Jiang and Goodenough, 1996), but the biological relevance of heterotypic and heteromeric gap junction channels remains to be demonstrated (cf. Kumar and Gilula, 1996). The docking (“coupling”) of two connexons results in formation of a pore between the two contacting cells. The permeability of these pores depends on the type of connexin but, in general, small molecules and metabolites up to a molecular mass of 1.000 Da can diffuse through gap junction channels in mammals. Molecules that can diffuse through gap junction channels include ions as well as metabolites such as second messenger molecules like cAMP, cGMP, inositol 1, 4, 5 trisphosphate and calcium ions (Saez et al., 1989, and references therein). Thus, metabolic signals could be transmitted through gap junctions to generate coordinated and synchronized activity in tissues. Expression of at least one connexin protein has been found in every cell type analyzed, except skeletal muscle, late stages of hematopoietic cells and certain adult neurons. Expression of connexins can overlap in various tissues and cell types. It is not yet clear, whether only one type of connexin is sufficient for functional gap junctions. Recent results, summarized below, suggest that different types of connexin channels fulfill different physiological functions. Table 1 lists the known murine connexin genes and their main pattern of expression in tissues and cell types. As far as it has been investigated, all types of murine connexin genes are also present in the human genome, although their cell type specific expression pattern can differ, possibly an adaptation to unknown specific physiological needs of different organisms (cf. Gros and Jongsma, 1996). Several levels of regulation of connexin expression and gap junctional channel activity have become obvious during recent years and can differ between different channels, i.e. initiation of cell type specific transcription, stability of connexin mRNAs, posttranslational modification of connexin proteins (phosphorylation, acylation, ubiquination), oligomerization of connexin subunits, and compatibility of connexin hemichannels (reviewed by Bruzzone et al., 1996). Several examples of incompatibility of connexins have been described, most prominently Cx40 and Cx43 (Bruzzone et al., 1993; Haubrich et al., 1996). The incompatibility of these connexins contributes to the propagation of action potentials through Cx40 containing electrical synapses (i.e. gap junctions) in conductive myocardium which is surrounded by Cx43 expressing working myocardium. In certain established liver derived cell lines, formation of functional gap junctions is dependent on expression of the adhesion protein E-cadherin (Jongen et al., 1991). Thus, it is likely that the expression of certain cell adhesion proteins is a precondition or, at least, contributes to the functional assembly of gap junction channels. It has been suggested in several reports (Steinberg et al., 1994; Elfgang et al., 1995; Veenstra et al., 1996) that the permeability of connexin channels to different tracer molecules, including fluorescent dyes, is different. Thus, connexin channels appear to be selective towards ions and small molecular weight compounds. However, the selectivity of connexin channels towards anions and cations is several orders of magnitude lower than for anion and cation channels in plasma membranes. The selectivity of connexin channels for metabolites and second messenger molecules is likely to be relevant for their distinct biological functions. Again, this prediction has to be experimentally proven. The almost ubiquitous existence of gap junctions between mammalian cells has triggered many hypotheses and speculations regarding possible functions during development, differentiation, cellular growth control, physiology and synchronization. In the past it was impossible to prove these hypotheses since no highly specific and selective inhibitors of gap junctional functions were known. For electrophysiological studies, halothane and octanol have frequently been used to block gap junctional coupling, although these compounds also affect the membrane potential and other membrane properties, at least at higher concentrations. More recently, glycyrrhetinic acid (cf. Guan et al., 1996) and anandamide (Venance et al., 1995) have been characterized as inhibitors of gap junctional function. Since these compounds are likely to


283

alter general membrane properties, it still needs to be shown that their inhibitory effect on functional gap junction channels is due to direct interaction with connexins rather than an indirect consequence of altered membrane parameters. Unfortunately, no naturally occurring mouse mutant has been identified that lacks connexin protein. On the other hand, several tumor cell lines have been established that express very little or none of the connexin proteins tested (reviewed by Yamasaki and Naus, 1996). In these cases, however, it is not known whether lack of connexin expression is the cause or the consequence of aberrant control of cell proliferation. So far, only two human connexin mutations have been described. The first one was a myelination disorder affecting peripheral nerves that is associated with a defect in the Cx32 gene (Bergoffen et al., 1993). The second mutation was found in the human Cx43 gene of certain patients exhibiting visceral atrial heterotaxia (Britz-Cunningham et al., 1995). These results still need to be confirmed, since a recent report could not provide more evidence for involvement of Cx43 defects in the hearts of further patients suffering from visceral atrial heterotaxia (Gebbia et al., 1996). It is hoped that further human inherited diseases can be correlated with connexin defects, based on comparisons of chromosomal locations of connexin genes and the inherited altered human phenotype. Due to progress in the transgenic mouse technology it is now possible to modulate, change or modify expression of the gene of interest. In addition, targeted deletion or replacement of mouse genes in specific cell types by homologous recombination in embryonic stem cells can be expected to deepen our understanding of gene functions and interactions in the mouse (Bradley and Liu, 1996). DELETION OF THE MOUSE CONNEXIN43 GENE The first connexin gene that was genetically targeted by homologous recombination in mouse embryonic stem cells was coding for Cx43 (Reaume et al., 1995). This gene is first expressed with the onset of zygotic transcription at the 8-cell stage of the early mouse embryo, suggesting a function in communication between cells of the early embryo. Expression of Cx43 is maintained in the embryo after implantation and also found in adult mice in various cell types such as working cardiomyocytes, astrocytes, fibroblasts, smooth muscle cells and endothelial cells. Gene targeting of Cx43 was performed in embryonic stem cells with a replacement strategy using a promoterless neomycin resistance gene to increase the targeting frequency. Reintroduction of the modified gene into the germline and inbreeding of heterozygous mice revealed an early postnatal lethality of homozygous Cx43 deficient mice (Reaume et al., 1995). The young mice die immediately after birth and show a cyanotic appearance suggesting a disturbance in pulmonary gas exchange. Surprisingly, despite the broad and very early developmental expression of Cx43, there was no obvious gross morphological abnormality in Cx43 deficient mice. Histological analysis revealed an enlargement of the conus region, located near the right ventricle of the heart, and an abnormal morphology of the right ventricular outflow tract, leading to pulmonary stenosis. A functional assay using a resin, soaked with methyl methacrylate, confirmed closure of the vessel. Thus, the reason for postnatal death is the failure to pump blood from the right ventricle into the lung, a failure that can be compensated until term by connection of the blood circuits of mother and fetus. So far, the results do not answer why the phenotypic abnormality of Cx43 null mutant mice is only visible in such distinct structures as the outflow tract of the heart and is not seen in other structures that express high levels of Cx43, like the endothelium and glial astrocytes of the brain. One possibility is that other connexins can compensate for critical functions of Cx43 in non-myocardial cells during development, so that the Cx43 deficient embryo and fetus can develop to term. Alternatively, it is possible that the early postnatal lethality prevents other subtle malfunctions to develop. In order to study whether or not Cx43 has an essential function in postnatal development, one

284


would like to turn off Cx43 expression experimentally at distinct time points. This could be done with a dominant negative Cx43 gene that is expressed in later stages of development or by use of Cre/loxP mediated tissue specific homologous recombination. In the last experimental approach, the Cre recombinase gene of the P1 phage is expressed in transgenic mice under control of a cell type specific promoter. The Cre recombinase can delete gene sequences which are flanked (“floxed”) by inserted recognition motifs (loxP sequences) (Gu et al., 1994). What is the function of Cx43 during heart development? Beside Cx43, at least three other connexins are expressed in the developing and adult heart (cf. Gros and Jongsma, 1996). Cx45 was found together with Cx43 in intercalated discs of the myocardium. Cx37 expression seems to be restricted to endothelial cells together with expression of Cx40. Furthermore, murine Cx40 is widely expressed in fetal heart two weeks after conception and becomes restricted to conductive myocardium at late pregnancy and thereafter (Delorme et al., 1995), whereas Cx43 is mainly expressed in working myocardium. Cells that form the right outflow tract of myocardium are developmentally derived from a subpopulation of neural crest cells that migrate to the developing heart and differentiate there. Possibly the exact timing of Cx43 expression in the subpopulation of neural crest cells is critical for the cells to migrate into the heart (cf. Lo, 1996). DELETION OF THE MOUSE CONNEXIN32 GENE Two connexin proteins, Cx32 and Cx26, are expressed in mouse hepatocytes at a ratio of about 2:1. We have previously shown that expression of these connexin proteins correlated with expression of other hepatocyte specific marker proteins (Stutenkemper et al., 1992). Both proteins have been localized in the same gap junctional plaque of hepatocytes (Nicholson et al., 1987). It is not clear whether differentiation of liver cells depends on gap junctional communication through connexin32 and −26 channels. In order to study the function of the connexin32 gene, a promoterless neo gene was inserted into the reading frame of the gene followed by a polyoma enhancer element to increase expression of the neo gene. Unexpectedly, Cx32 deficient mice showed no obvious phenotypic alteration, compared to wild type mice. The Cx32 deficient mice were viable and fertile but weighed on the average 17 to 20% less than wild type animals. More detailed analysis revealed a 60 to 70% decrease in the amount of Cx26 protein in liver and a thousandfold decreased size of liver gap junctional plaques (Nelles et al., 1996). Nevertheless, Cx32 deficient hepatocytes, when cultured for three days, still showed 30 to 40% cell to cell transfer of microinjected Lucifer yellow, supporting the hypothesis that the remaining Cx26 gap junctional channels may be sufficient to maintain homeostasis in liver (Temme et al., 1997). Then, we discovered that the Cx32 deficiency influenced glucose mobilization after electrical stimulation of sympathetic liver nerves. It was found that the glucose release after electrical stimulation was decreased by about 78%, demonstrating that the remaining Cx26 gap junctional channels could not propagate the nerve derived signals through the liver acinus (Nelles et al., 1996). Stimulation of sympathetic nerves in wild type and mutated mice leads to release of the neurotransmitter noradrenaline which binds to receptors on hepatocytes near the nerve endings. In mouse liver, innervation is much more scarce than in human liver. It is therefore assumed that inositol 1, 4, 5 trisphosphate (IP3) is released inside hepatocytes in response to noradrenaline mediated signal transduction (Gardemann et al., 1992). In wild type hepatocytes, IP3 is supposed to diffuse through gap junction channels into adjacent hepatocytes, thereby also triggering release of glucose from glycogen. In Cx32 deficient liver, glucose release is strongly decreased although gap junctional coupling through Cx26 channels is still maintained. This result suggests that the efficiency of the remaining Cx26 channels is not sufficient for equilibration of IP3 between coupled hepatocytes. It has been reported that Cx32 channels show a preference for anions over cations (cf. Veenstra et al., 1996). Furthermore, murine Cx32 channels


285

expressed in Xenopus oocytes or human HeLa cells showed an estimated five to sixfold greater permeability to Lucifer yellow than Cx26 channels (Cao et al., 1998). If this is also valid for permeability of IP3, it would explain the strong decrease of IP3 mediated glucose release in Cx32 deficient liver. The second messenger IP3 has a life time inside cells in the order of one second (cf. Kasai and Petersen, 1994). Thus, we hypothesize that a five- to sixfold difference in IP3 permeability between Cx32 and Cx26 channels could explain the decrease of glucose mobilization in Cx32 deficient hepatocytes that still maintain gap junctional communication through Cx26 channels. The Cx32 protein is also expressed in Schwann cells of peripheral nerves where Cx32 containing “reflexive gap junctions” in the nodes of Ranvier and Schmidt-Lanterman incisures connect myelin sheeths around the axon (Bergoffen et al., 1993). Cx32 mutations in humans were found to cause the neurodegenerative Charcot-Marie-Tooth disease (X-type) (Bergoffen et al., 1993), which is usually recognized only in the second decade of life in human patients, and which is characterized by demyelination and degeneration of peripheral nerves. Our analysis of Cx32 mice at the age of three months revealed no abnormal myelination or nerve conduction (Nelles et al., 1996). However, analysis of Cx32 deficient mice at older age yielded different results. At an age of four to eight months, these mice showed mild but statistically significant changes in conduction properties of peripheral nerves. After distal sciatic nerve stimulation, an increase in the latency and duration of the muscle response was noticed. After proximal stimulation, a reduction of the amplitude of the muscle response (M-amplitude) was detected. In one year old mice, a slight but significant decrease of the M-amplitude after facial nerve stimulation was also seen. The ultrastructural analysis of femoral nerves showed structures indicative of myelin degeneration (“onion bulbs"), abnormally thick periaxonal collars and non-compacted regions of myelin (Anzini et al., 1997). Thus, older Cx32 deficient mice showed similar phenotypic symptoms as human patients with a mild form of X-linked Charcot-Marie-Tooth disease (CMTX). These mice can function as a mouse model for understanding the onset and progression of the human CMTX disease. When we compared the number of proliferating hepatocytes in the liver of Cx32 deficient mice and control mice, we found that female Cx32 deficient mice showed 14-fold, and male mice four-fold higher incorporation of bromodeoxyuridine than wildtype mice. Furthermore, twelve months old male and female Cx32 deficient mice showed 25-fold and eight-fold more spontaneous liver tumors, respectively, compared to wild type animals. Intraperitoneal injection of the carcinogen diethylnitrosamine in Cx32 deficient mice two weeks after birth, led after one year to more liver tumors and accelerated growth of hepatocarcinomas than in control animals (Temme et al., 1997). Since ploidy and liver weight were not significantly altered, although incorporation of bromodeoxyuridine was strongly increased in the liver of Cx32 deficient mice, turnover of Cx32 deficient hepatocytes was probably higher in mutant mice. We concluded that spontaneously or chemically initiated hepatocytes, deficient in Cx32 mediated gap junctional communication, had a higher probability to escape from homeostatic growth control than corresponding wild type hepatocytes. The differences between male and female mice had been observed before (Poole and Drinkwater, 1996), and can be correlated to sex hormone levels in the animals. The finding that Cx32 deficient mice develop more and earlier liver tumors than wild type controls demonstrated that Cx32 containing gap junctions lower the risk of carcinogenesis in mouse liver. Since the total amount of Cx26 protein is decreased by about two thirds in the liver of Cx32 deficient mice, the effects which we observed in these animals could be influenced by the decrease of the Cx26 protein. As mentioned before, however, we found significant cell to cell transfer of microinjected Lucifer yellow in cultured Cx32 deficient hepatocytes, which was probably due to homotypic Cx26 gap junction channels. Thus, one would have to assume that the remaining Cx26 channels were insufficient for normal growth control among Cx32 deficient hepatocytes. We think it more likely that the effects were primarily caused by the absence of Cx32

286


channels. Since both connexins have been shown to interact in heterotypic and, possibly, in heteromeric channels, however, it is likely that intercellular transduction of signals through gap junctions between normal hepatocytes is mediated by interacting Cx32 and Cx26 proteins. In contrast to the high frequency of hepatic tumors in Cx32 deficient mice, there is no report in the literature that human CMTX patients suffer from an increased frequency of liver tumors. Since the frequency of CMTX patients in the total human population is rather rare, it is possible that a statistically significant increase in the incidence of liver tumors in these patients has not been noticed, so far. In addition, the spontaneous occurrence of hepatocellular carcinomas among humans in the Western world is below one in fifty thousand individuals. This is more than three orders of magnitude lower than the spontaneous occurrence of liver tumors in wild type mice. Thus, even if the frequency of spontaneous hepatocarcinomas is increased in CMTX patients relative to wild type Caucasians, similarly as in the Cx32 deficient mice, we think it is unlikely to be recognized because of the rareness of CMTX individuals. Cx32 deficient mice should be an excellent system to study the release of gap junction mediated tumor suppression in the liver. DELETION OF THE MOUSE CONNEXIN26 GENE We deleted the mouse Cx26 gene in embryonic stem cells, using a replacement vector with a neo cassette driven by the phosphoglycerate kinase promoter, and replacing most of the coding exon2 sequence of the Cx26 gene. After introduction of this gene defect into the germ line and inbreeding of heterozygous defective mice, we found that homozygous Cx26 defective mice died around 10.5 dpc, in utero. At this time, the mutant embryos were only about three quarters as big as wild type littermates, but otherwise showed no sign of morphological abnormality. At 9 dpc, both homozygous mutant and wild type embryos exhibited about the same size (Gabriel et al., 1998). At this time of development, Cx26 is expressed in the skin of the embryo and, with increasing intensity, between the syncytiotrophoblast layer I and II in the labyrinth region of the placenta that separates maternal blood from the embryonic blood vessels. It had been suggested before that glucose is taken up from maternal blood into the syncytiotrophoblast layer I via glucose transporter protein I, diffuses through Cx26 containing gap junctions into syncytiotrophoblast layer II, from where it can be released into the fetal blood stream, again, via glucose transporter protein I (Shin et al., 1996). If this hypothesis is correct, transport of glucose from maternal to fetal blood should be impaired in homozygous Cx26 deficient mouse embryos. We found that uptake of l4C-3-Omethylglucose, a non-metabolizable glucose derivative, from maternal blood into homozygous Cx26 deficient mice was only half as efficient as in heterozygous and wild type littermates. The remaining uptake of glucose in homozygous Cx26 deficient embryos probably occurred via the yolk sac, an extraembryonic structure that has supporting and placenta-like functions during early embryonic development. After 9 dpc of mouse pregnancy, the placenta is formed and is beginning to substitute for yolk sac functions. Thus, the reason for lethality of homozygous Cx26 deficient embryos seems to be insufficient nutrition through the labyrinth region of the placenta. Apparently, the demand for nutrients in the growing embryo is so high at this stage of development that it cannot be fulfilled through uptake via the yolk sac. It is interesting to note that the Cx26 channels are used for uptake of essential nutrients through the syncytiotrophoblast layers of the mouse placenta. It is unlikely that short lived second messenger molecules are transported to a large extent from maternal to fetal blood through Cx26 gap junction channels. Thus, we speculate that Cx26 channels may be less suitable for the intercellular diffusion of second messenger molecules than Cx32 channels but could be more appropriate for diffusion of more stable nutrients. The embryonic lethality of Cx26 deficient mice precludes studies of functional relevance of these channels in adult liver. This question can be tackled in the future, however, by liver specific deletion of the Cx26 gene,


287

using the Cre/loxP technique (Gu et al., 1994) or by inducible induction of the Cre/loxP system, for example through the use of tetracycline dependent promoters (Kistner et al., 1996). DELETION OF THE MOUSE CONNEXIN37 GENE Very recently, Cx37 deficient mice have been described (Simon et al., 1997). Although both sexes of homozygous Cx37 defective mice were viable, female mice turned out to be infertile. This infertility is marked by a lack of mature Graafian follicles, failure to ovulate, and inappropriate formation of corpora lutea. This phenotype is likely due to defective gap junction mediated communication between the oocyte and surrounding granulosa cells. No transfer of neurobiotin tracer between the microinjected oocyte and surrounding granulosa cells was seen in Cx37 deficient mice, whereas granulosa cells were strongly coupled via Cx43 containing gap junctions. Expression of Cx37 has been demonstrated in the oocyte but not in granulosa cells. Thus, at present, it is not clear whether homotypic Cx37 channels or, possibly, heterotypic Cx37/Cx43 channels mediate gap junctional coupling between the oocyte and surrounding granulosa cells. The observed phenotypic alteration suggests that oocytes send a signal to surrounding granulosa cells, in order to inhibit formation of corpora lutea. This is in contrast to the previous notion that granulosa cells produce cAMP that triggers protein kinase A to arrest oocytes in prophase I of meiosis. In this model, impaired gap junctional coupling would lead to inappropriate maturation of the oocyte. Future analyses will have to show, whether Cx37 deficient mice could be a model organism for the study of female infertility in humans. DELETION OF THE MOUSE CONNEXIN46 GENE Gong et al. (1996) have recently reported, that Cx46 deficient mice (called α3 deficient mice, cf. Table 1) were viable and fertile but developed early onset cataracts in their eye lenses that increased in intensity with age. Homozygous Cx46 deficient Table 1 Murine connexin genes Cx Gene

Group

Mr

Tissue or Cell Type

Phenotype of Cx Gene Deletion in the Mouse

Cx26

β2

26.5

embryonic lethality at 10.5 dpc, deficient uptake of glucose through the placenta of the placenta

Cx30 Cx30.3 Cx31 Cx31.1 Cx32

β6 β5 β3 β4 β1

30.4 30.3 31 31.1 32

hepatocytes, pinealocytes, leptomeningealocy tes, pancreas, endometrium, syncytiotrophoblasts brain, skin skin keratinocytes, placenta Skin hepatocytes, certain neurons, kidney, oligodendrocytes, pancreas, Schwann cells

Cx33 Cx36 Cx37

α7 α? α4

32.9 36.1 37.6

testes retina, brain lung, endothelium, oocyte

decreased release of glucose upon electrical stimulation of hepatic nerves, high incidence of hepatocarcinogenesis, myelin defects

female infertility

288


Cx Gene

Group

Mr

Cx40

α5

40.4

Tissue or Cell Type

Phenotype of Cx Gene Deletion in the Mouse

lung, endothelium, smooth muscle cells, conductive myocardium Cx43 α1 43.1 working myocardium, lethality shortly after birth, closed myometrium, endothelium, outflow tract from right astrocytes, fibroblasts myocardial ventricle Cx45 α6 45 kidney, skin, heart Cx46 α3 46 lens fiber cells age-related formation of cataracts Cx50 α8 50 lens fiber cells All connexin (Cx) genes listed were mentioned with references in recent reviews (Goodenough et al., 1996; Bruzzone et al., 1996), except the mouse connexin gene 30, which was described by Dahl et al. (1996). Connexin 36 has been reported by Condorelli et al. (1998) and Söhl et al. (1998). Mr, relative molecular weight. The nomenclature using Greek letters was described by Kumar and Gilula (1992). Note that the list of tissue or cell type specific expression pattern is incomplete.

mice exhibited several-fold decreased amounts of Cx50 (connexin α8) in their lenses and much less gap junctional plaques. This is reminiscent of the decreased expression of Cx26 in Cx32 deficient livers (cf. Nelles et al., 1996). Cx46 and Cx50 have previously been colocalized in lens fiber cells (Jiang and Goodenough, 1995). Heterozygous Cx46 deficient mice were indistinguishable from their wild type littermates and did not develop cataracts. It is not yet known whether human cataract formation could also be triggered by defects in the Cx46 gene. CONCLUSIONS AND OUTLOOK The deletion of connexin genes in mice has already yielded many new insights into the biological function of connexin channels. For the first time, it has become possible to study whole animals that lack certain connexin proteins completely. The phenotypes of the six connexin gene deletions described so far were all different, confirming the notion that the function of connexin channels is different in various cell types and tissues. If the hypothesis turns out to be correct that connexin channels are adapted to intercellular transfer of second messenger molecules and/or other metabolites such as glucose, different intercellular signal transduction mechanisms may be mediated by different connexin channels. This would provide yet another reason for the surprising diversity of connexin channels. Gap junctions among excitable and non-excitable cells appear to function as a network for signal equilibration between the same or different cell types. This seems to coordinate and optimize cellular activity, and to guarantee homeostatic growth control in tissues. The detailed purpose of this network is only beginning to be explored. When more mouse mutants of connexin channels will be characterized, the functional importance of the gap junctional network for long term homeostasis in the mammalian organism will become more apparent. It can already be predicted that characterization of targeted connexin mutants in mice will greatly stimulate our knowledge in molecular physiology. The different connexin mutants are also likely to provide answers to the question whether connexin channels show selective permeability to certain metabolites. Even in those cases where the physiological consequences of inactivating connexin channels differ between mouse and humans, one can expect to gain further functional insights from characterization of the mouse mutants. The ability to delete mouse genes cell type specifically (Tsien et al., 1996) and to replace genes by one another in mice (cf.


289

Bradley and Liu, 1996) will also yield more information about the molecular and cellular biology of gap junctions. NOTE ADDED IN PROOF This manuscript was written in March 1997. Some references, submitted for publication, were updated in September 1998. Recently, connexin40 deficient mice have been generated and characterized by two groups (Simon et al., 1998; Kirchhoff et al., 1998). ACKNOWLEDGEMENT Research in our laboratory has been supported by grants of the Deutsche Forschungsgemeinschart (SFB 284, C1), the Deutsche Krebshilfe, and the Fonds der Chemischen Industrie to K.W. REFERENCES Anzini, P., Neuberg, D.H.H., Schachner, M., Nelles, E., Willecke, K., Zielasek, J., Toyka, K., Suter, U. and Martini, R. (1997) Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin32. J. Neurosci., 17, 4545–4551. Bergoffen, J., Scherer, S.S., Wang, S., Oronzi, S.M., Bone, L.J., Paul, D.L., Chen, K., Lensch, M.W., Chance, P.F. and Fischbeck, K.H. (1993) Connexin mutants in X-linked Charcot-Marie-Tooth disease. Science, 262, 2039–2042. Bradley, A. and Liu, P. (1996) Target practice in transgenics. Nature Genetics, 14, 121–123. Britz-Cunningham, S.H., Shah, M.M., Zuppan, C.W. and Fletcher, W.H. (1995) Mutations in the connexin43 gap junction gene in patients with heart malfunctions and defects of laterality. New Engl. J. Med., 332, 1323–1329. Bruzzone, R., Haefliger, J.A., Gimlich, R.L. and Paul, D.L. (1993) Connexin40, a component of gap junctions in vascular endothelium, is restricted in its ability to interact with other connexins. Mol. Biol. Cell, 4, 7–20. Bruzzone, R., White, T.W and Paul, D.L. (1996) Connections with connexins: the molecular basis of direct intercellular signaling. Europ. J. Biochem., 238, 1–27. Cao, F., Eckert, R., Elfgang, C., Nitsche, J.M., Snyder, S.A., Hülser, D.F., Willecke, K. and Nicholson, B.J. (1998) A quantitative analysis of connexin-specific differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J. Cell Science, 111, 31– 43. Condorelli, D.F., Parenti, R., Spindler, F., Salinato, A.T., Bellnardo, N., Cardile, V. and Cicirata, F. (1998) Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons. Europ. J. Neuroscience, 10, 1202–1208. Dahl, E. (1996) Molecular cloning and functional expression of mouse connexin30, a gap junction gene highly expressed in adult brain and skin. J. Biol. Chem., 271, 17903–17910. Delorme, B., Dahl, E., Jany-Guichard, T., Maries, I., Briand, J.P., Willecke, K., Gros, D. and Theveniau-Ruissy, M. (1995) Developmental regulation of Cx40. Developmental Dynamics, 294, 358–371. Elfgang, C., Eckert, R., Lichtenberg-Fraté, H., Butterweck, A., Traub, O., Klein R.A., Hülser, D.F. and Willecke, K. (1995) Specific permeability and selective formation of gap junction channels in connexin transfected HeLa cells. J. Cell Biol., 129, 805–817. Gabriel, H.D., Jung, D., Bützler, C., Temme, A., Traub, O., Winterhager, E. and Willecke, K. (1998) Transplacental uptake of glucose is decreased in embryonic lethal connexin26 deficient mice. J. Cell Biol, 140, 1453–1461. Gardemann, A., Puschel, G.P. and Jungermann, K. (1992) Nervous control of liver metabolism and hemodynamics. Eur. J. Biochem., 207, 399–411. Gebbia, M., Jeffrey, A., Towbin, M.D. and Casey, B. (1996) Failure to detect connexin43 mutations in 38 cases of sporadic and familial heterotaxy. Circulation, 94, 1909–1912.

290


Gong, X., Li, E., Klier, G., Huang, Q., Wu, Y., Lei, H., Kumar, N.M., Horwitz, J. and Gilula, N.B. (1997) Disruption of α3 Connexin gene leads to proteolysis and cataractogenesis in mice. Cell, 91, 833–844. Goodenough, D.A., Golliger, J.A. and Paul D.L. (1996) Connexins, connexons and intercellular communication. Annual Rev. Biochem., 65, 475–502. Gros, D.B. and Jongsma, H.J. (1996) Connexins in mammalian heart function. Bio Essays, 8, 719–730. Gu, H., Marth, J.D., Orban, P.C., Mossmann, H. and Rajewsky, K. (1994) Deletion of a DNA Polymerase β gene segment in T cells using cell type-specific gene targeting. Science, 265, 103–106 Guan, X., Wilson, S., Schlender, K.K. and Ruch, R.R. (1996) Gap junction disassembly and connexin43 dephosphorylation induced by 18β-glycyrrhetinic acid Mol. Carcinogenesis, 16, 157–164. Haubrich, S., Schwarz, H.J., Bukauskas, F., Lichtenberg-Fraté, H., Traub, O., Weingart, R. and Willecke, K. (1996) Incompatibility of connexin40 and 43 hemichannels in gap junctions between mammalian cells is determined by intracellular domains. Mol. Biol. Cell, 7, 1195–2006. Jiang, J.X., White, T.W. and Goodenough, D.A. (1995) Changes in connexin expression and distribution during chick lens development. Devel. Biol., 168, 649–661. Jiang, J.X. and Goodenough, D.A. (1996) Heteromeric connexins in lens gap junction channels. Proc. Natl. Acad. Set. USA, 93, 1287–1291. Jongen, W.M.F., Fitzgerald, D.J., Asamoto, M., Piccoli, C., Slaga, T.J., Gros, D., Takeichi, M. and Yamasaki, H. (1991) Regulation of connexin43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin. J. Cell Biol., 114, 545–555. Kasai, H. and Petersen, O.H. (1994) Spatial dynamics of second messengers: IP3 and cAMP as long-range and associative messengers. Trends Neurosc., 17, 95–101. Kirchhoff, S., Nelles, E., Hagendorff, A., Krüger, O., Traub, O. and Willecke, K. (1998) Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40 deficient mice. Current Biol., 8, 299–302. Kistner, A., Gossen, M., Zimmermann, F., Jerecic, J., Ullmer, C., Lubbert, H. and Bujard, H. (1996) Doxycyclinemediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl. Acad. Sci. USA, 93, 10933–10938. Kumar, N.M. and Gilula, N.B. (1992) Molecular biology and genetics of gap junction channels. Sem. Cell Biol., 3, 3–16. Kumar, N.M. and Gilula, N.B. (1996) The gap junction communication channel. Cell, 84, 381–388. Lo, C.W. (1996) The role of gap junction membrane channels in development. J. Bioenerg. Biomembranes, 28, 379–385. Nelles, E., Bützler, C., Jung, D., Temme, A., Gabriel, H.-D., Dahl, U., Traub, O., Stümpel, F., Jungermann, K., Zielasek, J., Toyka, K.V., Dermietzel, R. and Willecke, K. (1996) Defective propagation of signals generated by sympathetic nerve stimulation in the liver of connexin32-deficient mice. Proc. Natl. Acad. Sci. USA, 93, 9565–9570. Neuhaus, I.M., Dahl, G. and Werner, R. (1995) Use of alternative promoters for tissue specific expression of the gene coding for connexin32. Gene, 158, 257–262. Nicholson, B.J., Dermietzel, R., Teplow, D., Traub, O., Willecke, K. and Revel, J.-P. (1987) Two homologous components of hepatic gap junctions. Nature, 329, 732–734. Poole, T.M. and Drinkwater, N.R. (1996) Strain dependent effects of sex hormones on hepatocarcinogenesis in mice. Carcinogenesis, 17, 191–196. Reaume, A.C., de Sousa, P.A., Kulkarni, S., Langille, B.L., Zhu, D., Davies, T.C., Jujena, S.C., Kidder, G.M. and Rossant, J. (1995) Cardiac malformations in neonatal mice lacking connexin43. Science, 267, 1831–1834. Saez, J.C., Conner, J.A., Spray, D.C. and Bennett, M.V.L. (1989) Hepatocyte gap junctions are permeable to the second messenger inositol 1,4,5 trisphosphate and to calcium ions. Proc. Natl. Acad. Sci. USA, 86, 2708–2712. Shin, D.C., Suzuki, T., Matsuzaki, T., Tanaka, S., Kuraoka, A., Shibita, Y. and Takata, K. (1996) Immunolocation of GLUT1 and connexin26 in the rat placenta. Cell Tiss. Res., 285, 83– 89. Simon, A.M., Goodenough, D.A. and Paul, D.L. (1998) Mice lacking connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block. Current Biol., 8, 295–298.


291

Simon, A.M., Goodenough, D.A., Li, E. and Paul, D.L. (1997) Female infertility in mice lacking connexin37. Nature, 385, 525–529. Söhl, G., Degen, J., Teubner, B. and Willecke, K. (1998) The murine gap junction gene connexin36 is highly expressed in mouse retina and regulated during brain development. FEES Letters, 428, 27–31. Söhl, G., Gillen, G., Bosse, F., Gleichmann, M., Müller, H.W. and Willecke, K. (1996) A second alternative transcript of the gap junction gene connexin32 is expressed in murine Schwann cells and modulated in injured sciatic nerve. Europ. J. Cell Biol., 69, 267–275. Stauffer, K.A. (1995) The gap junction proteins β1 connexin (connexin32) and β2 connexin (connexin26) can form heteromeric hemichannels. J. Biol. Chem., 270, 6788–6772. Steinberg, T.H., Civitelli, R., Gerst, S.T., Robertson, A.J., Hick, E., Veenstra, R.D., Wang, H.Z., Warlow, P.M., Westphale, E.M., Laing, J. and Beyer, E.G. (1994) Connexin43 and connexin45 form gap junctions with different molecular permeabilities in osteoblastic cells. EMBO J., 13, 744–750. Stutenkemper, R., Geisse, S., Schwarz, J.H., Look, J., Traub, O., Nicholson, B.J. and Willecke, K. (1992) The hepatocyte-specific phenotype of murine liver cells correlates with high expression of connexin32 and connexin26 but very low expression of connexin43. Exp. Cell Res., 201, 43–54. Temme, A., Buchmann, A., Gabriel, H.D., Nelles, E., Schwarz, M. and Willecke, K. (1997) High evidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32. Current Biol., 7, 713–716. Tsien, J.Z., Chen, D.F., Gerber, D., Tom, C., Mercer, E.H., Anderson, D.J., Mayford, M., Kandel, E.R. and Tonegawa, S. (1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell, 87, 1317–1326. Veenstra, R.D. (1996) Size and selectivity of gap junction channels formed from different connexins. J. Bioeng. Biomembr., 28, 327–337. Venance, L., Piomelli, D., Glowinski, J. and Giaume, C. (1995) Inhibition by anandamide and intercellular calcium signalling in striatal astrocytes. Nature, 376, 590–594. Yamasaki, H. and Naus, C.C.G. (1996) Role of connexin genes in growth control. Carcinogenesis, 17, 1199–1213.

EPITHELIA IN DISEASE

16. BEAUTY IS SKIN DEEP: BIOLOGY AND GENETICS OF THE EPIDERMAL KERATINS ELAINE FUCHS Department of Molecular Genetics and Cell Biology, The Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637 7, USA Tel: 773–702–1347; Fax: 773–702–0141; Federal Express: 1293–9242–8 When Dr. Howard Green and coworkers devised methods to culture human epidermal keratinocytes under conditions where they could be propagated for several hundred generations without undergoing senescence (Rheinwald and Green, 1975; 1977), it opened the door for biochemical expeditions into the intricate molecular pathways that orchestrate development and differentiation in the epidermis. While there are still many mysteries underlying the mechanisms of epidermal differentiation, we know much more about this complex program than ever before. Much of what we know about the epidermis of the skin has been derived from a foundation of knowledge built from studying epidermal keratinocytes in tissue culture. Below, I review what is known about the program of epidermal differentiation, pointing out differences between culture and in vivo studies where they are relevant. THE PROGRAM OF EPIDERMAL GROWTH AND DIFFERENTIATION The epidermis is a stratified squamous epithelium which covers the surface of our body. As such, it serves an important protective function, which it manifests by building an extensive cytoskeletal architecture composed of 10 nm keratin filaments. The mitotically active cells of the epidermis are tucked safely away in the innermost, i.e. basal layer, attached to an underlying basement membrane composed of extracellular matrix. Within this layer of cells are a population of stem cells, which periodically give rise to “transit amplifying” basal cells (Barrandon and Green, 1987). Putative stem cells in vitro express a higher level of β1 integrin on their surface than the transit amplifying basal cells that have a more limited lifespan (Jones et al., 1995; Hotchin et al., 1995). α3β1 integrin, which is activated by the ligand laminin V, is thought to be essential for the proper survival and functioning of the mitotically active keratinocyte (for review, see Watt et al., 1993). α6β4 integrin, which also uses laminin V as ligand, forms the core of the hemidesmosomes, which are specialized membrane plaque structures lining the base of basal epidermal cells in vivo. α6β4 differs from most integrins in that it connects to keratin filaments on its cytoplasmic surface. While the importance of α3β1 integrin has been underscored from cell culture studies, where few hemidesmosomes are formed, it is clear from gene targeting studies that in vivo, α6β4 play a key role in cellsubstratum attachment, a necessary feature for epidermal survival (Dowling et al., 1996; van der Neut et al., 1996; Georges-Labouesse et al., 1996). In vitro (Frisch and Francis, 1994) and in vivo (Dowling et al., 1996), when basal epidermal cells detach from their underlying basement membrane, they trigger the apoptosis program of cell death. Once a transit amplifying cell has exhausted its limited supply of cell divisions, it withdraws from the cell cycle, commits to differentiate terminally, and moves outward towards the skin surface (Figure 1). In transit, the cell undergoes a variety of morphological and biochemical changes that culminate in the

294


Figure 1 The Epidermis and Keratin Expression. Epidermis. On the left is a cross section of human skin stained with hematoxylin and eosin. The four major steps in epidermal differentiation are: a) an innermost, basal layer of mitotically active cells; b) 3–6 layers of spinous cells that are still transcriptionally active but are no longer dividing; these cells devote most of their translational machinery to expressing keratins; c) 1–3 layers of granular cells that are transcriptionally active and synthesize the final components necessary for barrier function. Basal epidermal cells express keratins 5 and 14. As basal cells commit to terminally differentiate, they switch off the expression of K5 and K14, and induce the expression of K1 and K10. As epidermal cells move up through the spinous layers, they express K2e, which can pair with K10. Some keratins are expressed in the epidermis under special circumstances: during wound-healing, spinous cells induce the exrpession of keratins K6 and K16; K9 is unique to the suprabasal layers of palmar and plantar skin. Squames sloughed from the skin surface are merely dead sacs, chock full of keratin macrofibrils (Reprinted by permission of The New England Journal of Medicine, Amy S. Paller, M.D., Andrew J.Snyder, B.A., YiuMo Chan, B.S., Qian-Chun Yu, Ph.D., Elizabeth Hutton, B.S., Gianluca Tadini, M.D., and Elaine Fuchs, Ph.D., 331, 1410, 1994. Copyright (1994). Massachmettes Medical Society. All rights reserved, see also Fuchs, 1997).

production of dead, flattened squames that are then shed from the skin surface, continually replaced by inner layer cells moving outward. Every two to four weeks, there is a fresh epidermis to confront new traumas, and to keep microorganisms out and essential bodily fluids in. Despite what is likely to be permanent withdrawal from the cell cycle, a terminally differentiating keratinocyte is still active transcriptionally. In suprabasal as well as basal cells, keratinocytes produce an extensive cytoskeletal architecture of keratin filaments (for reviews, see Fuchs and Byrne, 1994; Fuchs, 1995). Keratins constitute ~10–20% of the total protein of dividing keratinocytes and up to 85% of the total protein of fully differentiated cells. While keratins are most abundant in the epidermis and its appendages, there are approximately 30 different keratin genes that are differentially expressed in all epithelial tissues at various stages of differentiation and development (Moll et al., 1982). Keratins are often expressed as specific protein pairs, forming obligatory heteropolymers composed of stable heterodimers (Fuchs et al.,


295

1981; Moll et al., 1982; Hanukoglu and Fuchs, 1983; Coulombe et al., 1990; Hatzfeld and Weber, 1990b; Steinert, 1990). There are four major keratin genes expressed in the epidermis. In vivo and in vitro, mitotically active epidermal cells express keratin K5 and its partner K14 (Fuchs and Green, 1980; Moll et al., 1982; Nelson and Sun, 1983). As keratinocytes withdraw from the cell cycle and commit to differentiate terminally, they switch off K5 and K14 and induce the expression of K1 and K10 (Fuchs and Green, 1980). In cultured mouse keratinocytes, this switch occurs quite efficiently upon calcium induction (Hennings et al., 1980; Roop et al., 1987). Cultured human epidermal cells are less sensitive to calcium, but the switch can be induced moderately by either (a) withdrawal of retinoids from the culture medium (Fuchs and Green, 1981) or (b) culturing of epidermal cells at the air-liquid interface on a floating raft of collagen embedded with dermal fibroblasts (Asselineau et al., 1985; Kopan et al., 1987). In vivo, two additional partners for K10 are expressed in differentiating epidermal cells: K2e is expressed late in differentiation, appearing in the upper spinous layers of normal epidermis (Collin et al., 1992); K9 is unique to the skin of the palms, the base of the foot and calluses (Fuchs and Green, 1980; Langbein et al., 1994). In tissue culture in vitro, and during wound-healing in vivo, a new set of keratins, K6 and K16, are induced in spinous cells; this keratin pair is also expressed in the outer root sheath of the hair follicle (Sun et al., 1984; Mansbridge and Knapp, 1987; Paladini et al., 1996). The cortex of the developing hair shaft differs completely from the epidermis in that a new set of keratins, the Ha and Hb keratins, are expressed (Moll et al., 1982; Lynch et al., 1986; Stark et al., 1987; Coulombe et al., 1989). In all, there are more than 30 different keratin genes in the human genome. These genes are differentially expressed not only in the epidermis and its appendages, but in virtually every epithelial tissue of the body (Moll et al., 1982). What is the functional significance of the complexity of keratins in the skin? While we don’t yet know the underlying molecular explanation, it seems likely that the multiplicity of sequences enables the keratinocytes to meet different structural needs. For instance, mitotically active keratinocytes have keratin filaments that are relatively disperse in the cytoplasm, whereas K1 and K10 filaments of terminally differentiating cells form much thicker bundles of 10 nm filaments. At the ultrastructural level, the dense bundles of keratin filaments in the suprabasal layers exhibit a spiny-like appearance which gives the spinous layer its name. These features appear to be imparted by the keratin sequences themselves, since in vitro, K5 and K14 assemble into discrete 10 nm filaments, while K1 and K10 assemble into filaments that tend to aggregate (Eichner et al., 1986). While different keratins assemble into 10 nm filaments with their own particular physical and chemical properties, these filaments also associate with different proteins and structures within the epidermis. Thus, an epidermal cell is not simply a bag of keratin filaments, but rather these filaments have architecture. The architecture of keratin filaments appears in part to be determined by their specialized head and tail domains (see below), which are likely to protrude along the filament surface and impart to the filaments the ability not only to associate with themselves, but also to make contacts with other proteins in the cell. Thus for example, keratin IFs are likely to attach to hemidesmosomes through specific sequences that are not conserved among all keratins. A hemidesmosomal protein involved in the attachment is BPAG1e, or the epidermal form of bullous pemphigoid antigen 1 (Yang et al., 1996). This 230 kDa protein, first identified by a monospecific antisera isolated from patients with the autoimmune disease bullous pemphigoid, is localized to the inner plate of the hemidesmosome, where the α6β4 containing membrane plaque interfaces with keratin filaments (Stanley et al., 1993; Guo et al., 1995). Gene targeting of the BPAG1 gene results in a severing of the connection between keratin filaments and hemidesmosomes, with a concomitant generation of a narrow zone of mechanical fragility just above the sheet of hemidesmosomes within a basal epidermal cell (Guo et al., 1995). In striking contrast to the loss of α6β4 integrins (Dowling et al., 1996; van

296


der Neut, et al., 1996; Georges-Labouesse et al., 1996), hemidesmosomes still form normally, and the attachment to the underlying basement membrane appears unperturbed (Guo et al., 1995). However, in the absence of BPAG1e, an epidermal cell cannot respond to wound-healing as rapidly as it can when BPAG1e is present (Guo et al., 1995). The underlying explanation for this remains unknown, but it could be reflective of a perturbation in molecular signalling between α3β1 and α6β4 junctions. Clearly for cell migration to take place, both junctions must be made and broken in a periodic fashion. Keratin filaments also attach to desmosomes, which are membranous plaques involved in cell-cell interactions within the epidermis (Stappenbeck and Green, 1992, 1993; Kouklis et al., 1994). While ultrastructurally, desmosomes resemble hemidesmosomes, they are composed of different proteins. At the core of the desmosome is a set of desmosomal cadherins, referred to as desmogleins and desmocollins (for review, see Garrod, 1993). Plakoglobin and plakophilin are β-catenin-like molecules that bind to the desmosomal cadherins. Unlike β-catenin, however, which associates indirectly with the actin cytoskeleton, these molecules mediate the association of desmosomes with keratin filaments. At least one mediator in this event is desmoplakin, which shares sequence similarities with BPAG1e (Green, et al., 1992). Envoplakin, another member of this family, may play a similar role at later stages of epidermal differentiation (Ruhrberg et al., 1996). In vitro studies indicate that desmoplakin’s carboxy terminal tail domain associates directly with the amino terminal head domain of K5 and K1 (Kouklis et al., 1994). This interaction appears to be regulated in vivo by virtue of phosphorylation by an as yet unidentified serine/threonine kinase (Stappenbeck et al., 1994). Gene targeting studies on desmoglein and desmocollin genes have only recently been conducted (Koch et al., 1997). However, recently, a dominant negative desmoglein 3 was expressed in the epidermis using transgenic mouse technology (Allen et al., 1996). While the levels of transgenic Dsg3 seemed to be low relative to endogenous desmogleins, marked abnormalities occurred in the skin of these mice. Most notable was a thickening and hyperkeratosis of the footpad skin and a prominent thinning of the hair coat (Allen et al., 1996). Signs of keratinocyte degeneration were also found in some regions of the skin. Collectively, these features resemble to some extent those seen in the balding mutant mouse (Sundberg, 1994), which maps chromosomally to the locus where the desmosomal cadherins reside and which was recently shown to be a Dsg 3 mutant mouse (Koch et al., 1997). Interestingly, the hyperkeratosis seen in the Dsg3 mutant mice resembled that seen in affected family members with non-epidermolytic hyperkeratosis, a human disorder which in one family mapped to the desmoglein/desmocollin locus on chromosome 18 (Hennies et al., 1995; Kelsell et al., 1995). The extent to which these similarities to human diseases are real awaits further studies. However, the results thus far are suggestive that defects in desmosomes can influence not only cell-cell adhesion, but also features of epidermal and hair follicle differentiation as well. Precisely how cells make and break these contacts as they progress through their differentiation pathway to the skin surface has remained a mystery, and will be of significant interest to study in the future. As keratinocytes differentiate, they encounter newly synthesized IF- associated proteins (IFAPs) such as filaggrin, a granular layer protein which promotes the bundling of keratin filaments into large macrofibrils (Dale et al., 1978). Recently, it was shown that these macrofibrils associate with the cornified envelope through specific contacts that are unique to K1 and K10 (Kimonis et al., 1994; Steinert and Marekov, 1995). Envoplakin also becomes cross-linked into the cornified envelope (Ruhrberg et al., 1996). The cornified envelope forms late in the terminal differentiation process. It consists of many different proteins, of which involucrin and loricrin are major components (Simon and Green, 1985; Steinert and Marekov, 1995), deposited beneath the plasma membrane and interconnected by y-glutamyl lysine crossbridges (Rice et al., 1979). Defects in loricrin have recently been shown to give rise to a form of Vohlwinkel’s Syndrome,


297

typified by perturbations in the cornified envelope (Maestrini et al., 1996). Activated by epidermal transglutaminases, the process culminates in the production of a sac in which to hold the macrofibrils of keratin filaments. Once the cornified envelope is completed and filaggrin has cemented the keratin fibers together, the job of creating an elaborate protective cytoskeleton is finished. The transcriptional and translational machineries of the terminally differentiated keratinocyte are destroyed, and the organelles, including nucleus, are degraded. Cells that are sloughed from the skin surface are metabolically inert, dead sacs chock full of keratin fibers. KERATIN FILAMENTS: ASSEMBLY INTO 10 NM FILAMENTS Keratins are remarkable in their ability to self-assemble into 10 nm intermediate filaments (IFs; Steinert et al., 1976; for review, see Fuchs and Weber, 1994). In vitro, assembly occurs in the absence of any auxiliary proteins or factors, leaving one to wonder how this process is controlled in vivo. In the electron microscope, keratin IFs appear as ropes of three or four strands, called protofibrils (Aebi et al., 1983; Steven et al., 1983). Two protofilaments comprise each protofibril; the width of each protofllament is equivalent to two antiparallel, linear chains of IF protein dimers (Parry, et al., 1977; Aebi et al., 1983, 1986; Conway and Parry, 1988; Parry and Squire, 1998). The two polypeptides in each dimer are aligned in parallel (Parry et al., 1977), and dimer subunits are linked head to tail (Aebi et al., 1986). Subunits in adjacent chains are thought to be arranged in a half-staggered and antiparallel position relative to each other (Parry et al., 1977; Conway and Parry, 1988; Geisler et al., 1992; Steinert and Parry, 1993; Steinert et al., 1993). Figure 2 illustrates the general features of 10 nm keratin filament structure. Keratins are typified by their central, 310 amino acid residue “rod,” flanked by nonhelical amino “head” and carboxy “tail” domains (Hanukoglu and Fuchs, 1982, 1983; Steinert et al., 1983). The rod is largely αhelical and contains heptad repeats of hydrophobic residues, resulting in a hydrophobic coil circumventing the helical surface (Pauling and Corey, 1953; Crick, 1953). Two keratin polypeptides coil together by virtue of these hydrophobic interactions. While conceptually convenient, the rod domain is not a continuous αhelix, but rather it is interspersed with three short linker segments predicted to perturb the α-helix (Figure 2; Hanukoglu and Fuchs, 1983; Conway and Parry, 1988). How these localized perturbations contribute to filament structure is unknown. However, their evolutionary conservation underscores their importance. Keratin heterodimers consist of a member of each of two distinct sequence classes, which share only ~25– 35% sequence identity within the rod segments (Fuchs et al., 1981; Hanukoglu and Fuchs, 1983). The sequences at the beginning and end of the rods are highly conserved even across subtypes. These rod ends are the most critical for the assembly of 10 nm keratin filaments in vivo and in vitro (Albers and Fuchs, 1987, 1989; Lu and Lane, 1990; Hatzfeld and Weber, 1990a; Coulombe et al., 1990; Wilson et al., 1992; Letai et al., 1992), and even subtle point mutations in these segments can disrupt filament formation (Letai et al., 1992). In contrast, proline mutations more centrally in the rod are often not as deleterious to the assembly process (Letai et al., 1992). In contrast to the α-helical rod domain, the nonhelical head and tail segments of keratins are highly variable in size and in sequence and are less important for 10 nm filament structure. Of the heads and tails of a keratin heteropolymer, only the type II keratin head domain is critically involved in 10 nm filament assembly (Wilson et al., 1992; Steinert and Parry, 1993). Intriguingly, the head and tail sequences within a keratin filament are more accessible to enzymatic reagents than the rod domain (Steinert et al., 1983). These data suggest that the end domains may play a specialized role in cytoarchitecture by participating in interfilament associations and in the interaction of keratin filaments with other proteins in the cell.

298


Figure 2 Model of keratin filament assembly. (A). Stick figure depicts the structure of a keratin heterodimer (Hanukoglu and Fuchs, 1982, 1983; Conway and Parry, 1988). Boxes denote the α-helical segments of the coiled-coil rod; arrow indicates direction of polypeptides, from base (N-terminus) to tip (C-terminus). Large boxes encompass the α-helical rod domain, interrupted by short non-helical linker segments. Hatched boxes denote highly conserved ends of the rod. Thinner bars denote non-helical head and tail domains. Small black bar in each arrow denotes the sequence in the L1–2 linker region, which is relevant in W-C EBS mutations. (B). Putative arrangement of dimer subunits in the 10 nm keratin filament. Model is adapted from that previously described (Fuchs, 1994; permission from the Journal of Cell Biology).

GENETIC DISORDERS OF THE EPIDERMAL KERATINS: EPIDERMOLYSIS BULLOSA SIMPLEX Given that keratins are the major structural proteins of the epidermis and its appendages, it seemed natural to expect that there might be genetic disorders of keratins and their associated proteins. To begin to explore this possibility, researchers in the field took an unconventional approach for the times, and focused on first identifying the critical domains of the epidermal keratin polypeptides and then creating transgenic mice expressing keratin gene mutations. The first molecular mutagenesis studies on keratins were conducted on K14, the first keratin cDNA and gene that had been cloned and characterized (Albers and Fuchs, 1987,1989; Coulombe et al., 1990). Deletion mutations in K14 behaved in a dominant negative fashion when expressed by transient transfection in cultured human epidermal keratinocytes (Albers and Fuchs, 1987,1989). Removing the head and tail domains of K14 had little or no effect on keratin network formation, but even small deletions at the highly conserved rod ends resulted in a complete collapse of the endogenous keratin network, leaving punctate aggregates of protein at the cell periphery. In vitro filament assembly studies produced similar results, demonstrating that as little as 1% of some severely disrupting keratin 14 mutants could perturb filament assembly when in the presence of wild-type K5 and 99% wild-type K14 (Coulombe et al., 1990).


299

Subsequent studies with keratin 5 mutations yielded similar findings, except in this case, the K5 head domain seemed to be more important for filament assembly than the K14 head domain (Wilson et al., 1992; Letai et al., 1992). This was the first indication of several that the two keratin polypeptides may have subtly different roles in overall 10 nm filament structure. Similar investigations by other groups studying additional keratins or other IF proteins strengthened these conclusions, namely that the amino and carboxy ends of the α-helical rod domain are particularly critical for the assembly process (for review, see Fuchs and Weber, 1994). Based on these findings, the majority of human keratin disorders would be expected to display an autosomal dominant pattern of inheritance, and mutations or deletions in the conserved rod end domains should account for the most severe forms of the disorders. To provide clues as to which human genetic diseases were likely to be keratin disorders, transgenic mouse technology was used to target the expression of the mutant, epitope-tagged human K14 genes to the skin (Vassar et al., 1991; Coulombe et al., 1991a). To drive expression, the human K14 promoter was used, since it had previously been shown that this promoter is faithfully active in the basal layer of the epidermis of transgenic mice (Vassar et al., 1989). In the first set of experiments, transgenic mice expressing a carboxy truncated, severely disrupting mutant human K14 gene displayed the clinical and pathological features of the Dowling-Meara subtype of Epidermolysis Bullosa Simplex (EBS) (Vassar et al., 1991; Table 1). Of the three major subtypes of EBS, D-M EBS is the most severe and the rarest, affecting ~1:100, 000 in the population. Clinical features are present at birth and include mechanical stress-induced skin blistering due to cytolysis within the basal layer of the epidermis. By electron microscopy, clumps or aggregates of keratin are present in the basal, but not the suprabasal layers of D-M EBS epidermis (Figure 3A; Anton-Lamprecht, 1994 and references therein). These features were uniformly present in the K14 mutant-expressing mice (Vassar et al., 1991). Additionally, the basal clumps of keratin protein labeled with antibodies against the transgene and endogenous K14 and K5 proteins (Vassar et al., 1991). In contrast, mice expressing more mildly disrupting K14 mutants exhibited features more typical of WeberCockayne EBS (Coulombe et al., 1991a). These mice displayed blistering that was largely confined to palmoplantar skin; although an ultrastructural study revealed marked signs of basal cell cytolysis, there were few if any discernible aberrations in K5/K14 keratin filament networks (Coulombe et al., 1991a). Given the striking parallels in the clinical features of the transgenic mice and human EBS patients, EBS was predicted to be a disorder of keratins 14 and 5 (Vassar et al., 1991). Moreover, the three major subtypes of EBS were predicted to be dependent upon where the mutations resided in the keratins, and the degree to which those mutations perturb the overall filament assembly process (Coulombe et al., 1991a). These hypotheses contradicted those derived from biochemical and genetic mapping studies of the 1980’s. However, they were consistent with early electron microscopy reports suggesting that perturbations in keratin filament networks are early events in the blistering process (Anton-Lamprecht and Schnyder, 1982). Similarities between cultured D-M EBS keratinocytes and keratinocytes transfected with mutant keratin genes had also been noted (Kitajima et al., 1989), lending further support to this hypothesis. Table 1 Characteristics of Epidermolysis Bullosa Simplex Feature

Dowling-Meara

Koebner

Weber-Cockayne

Autosomal dominant Skin blistering Basal cell cytolysis discernable abnormalities in basal keratin network

+ entire body + +

+ body + +

+ hands/feet + ±

300


Feature

Dowling-Meara

Koebner

Weber-Cockayne

keratin clumping in basal layer

+

—

—

One of the great advantages in analyzing the genetic basis of a skin disorder is that it is possible to culture most of the cell types found in the skin, including epidermal keratinocytes. From a simple 4mm skin biopsy, sufficient keratinocytes can be generated for keratin protein isolation and filament assembly, immunofluorescence, and mRNA/DNA isolation and sequence analysis. Interestingly, despite the prevalence of basal cell cytolysis in the skin of Dowling Meara patients, basal cell cultures from these patients were surprisingly viable (Kitajima et al., 1989; Coulombe et al., 1991b). In the first sequence analysis of keratin cDNAs cloned from cultured Dowling-Meara EBS keratinocytes, point mutations were found in one of two K14 alleles in a highly conserved arginine residue at position 125 (R125) (Coulombe et al., 1991b). Located at the amino end of the α-helical rod domain of K14, this residue was mutated to a histidine in one patient and a cysteine in an unrelated second patient (Coulombe et al., 1991b). This arginine codon (CGC) appears to be a hotspot for mutagenesis by methylation and subsequent deamination (Youssoufian et al., 1988). It is also a residue conserved even in the distantly related IF protein of the snail (Dodemont et al., 1990), and is a residue that is essential for filament assembly (Coulombe et al., 1991b). This residue is mutated in >40% of all the Dowling-Meara EBS patients analyzed thus far (Coulombe et al., 1991b; Lane et al., 1992; Hovnanian et al., 1993; Stephens et al., 1993; Chen et al., 1995; Chan et al., 1996; Umeki et al, 1996). Mutations in K5 also give rise to Dowling-Meara EBS (Lane et al., 1992; Hovnanian et al., 1993; Nomura et al., 1996; Irvine et al., 1997a), as expected based upon the strong propensity of K5 to heterodimerize with K14 (Coulombe et al., 1990). Table 2 summarizes the locations of these mutations and their clinical severity. In contrast to Dowling-Meara EBS, patients with Koebner EBS tend to have mutations, often proline substitutions, more centrally within the α-helical rod domain (Bonifas et al., 1991; Dong et al., 1993; Humphries et al., 1993, 1996; Yamanishi et al., 1994). Interestingly, proline mutations more centrally in the K14 or K5 rod domain are often less severely disrupting in a filament assembly assay than are more subtle mutations at the rod ends (Letai et al., 1992, 1993). In the first Koebner study conducted, it was shown that the defect mapped to a location on chromosome 17q12–21 (Bonifas et al., 1991), where the functional K14 gene was known to reside (Rosenberg et al., 1988). Weber-Cockayne EBS is also a disorder of K5 and K14. This was first suggested by genetic mapping analysis (Bonifas et al., 1991), localizing a defect in a Weber-Cockayne family to chromosome 12q11–12, i.e. the locus where the K5 gene was known to reside (Rosenberg et al., 1991). It was later demonstrated by identifying specific K5 mutations in affected family members (Chan et al., 1993, 1994a; Rugg et al., 1993;Ehrlich et al., 1995; Matsuki et al., 1995; Nomura et al., 1997a). In contrast to D-M and K EBS, most of the W-C mutations are located in the non-helical segments of the keratin polypeptide, and many of the mutations are in K5 rather than K14. Many of the W-C EBS mutations identified thus far reside in the nonhelical linker segment (L1–2) that separates helix 1B from helix 2A (Rugg et al., 1993; Chan et al., 1994a; Table 2). However, there must be other as yet unidentified locations for K5 or K14 mutations, as judged by the fact that sequence analysis of a number of DNAs from W-C EBS patients has often failed to reveal mutations within the L1–2 linker segment or the non-helical K5 head domain (K.Barrios and E.Fuchs, unpublished data). When the EBS mutations were engineered in the context of their wild-type keratin and assayed for their ability to affect filament assembly, it was discovered that most of the Dowling-Meara mutations perturb filament elongation and give rise to short 10 nm filaments (Coulombe et al., 1991b; Letai et al., 1993; Chan


301

Figure 3 Ultrastructural Characteristics of Clinically Severe EBS and EH. (A). Electron microscopy was performed on glutaraldehyde fixed, ultrathin sections of a skin biopsy from a patient with Dowling-Meara EBS (for method, see Cheng et al., 1992). Note the presence of large clumps of amorphous keratin in the innermost, basal cells of the epidermis. These clumps sometimes persist in the suprabasal layers, particularly in very severe cases such as the one shown here. Clumping of keratin often precedes overt signs of cytolysis and/or degeneration (asterisk). (B). Electron microscopy of ultrathin sections of a skin biopsy from a patient with severe EH. Note absence of keratin clumps in the basal layer, but presence of clumps and signs of cytolysis in the suprabasal layers. Abbreviations: kc, keratin clumps; Nu, nucleus; asterisk, cytolysis/degeneration. Bar represents µm in (A) and 4 µm in (B). (Reproduced from Molecular Biology of the Cell (1997), with permission of the American Society for Cell Biology) [Electron microscopy by Dr. Q.-C. Yu].

et al., 1994a). In contrast, when combined with their wild-type partner keratin, the L1–2 linker mutations produce filaments which are often unraveled, suggesting that the L1–2 residues may be involved in the lateral positioning or the association of the linear arrays of dimers that exist within a 10 nm filament (Chan et al., 1994a). Taken together, it appears that the end domains of the rod are especially critical for filament elongation, while the central linker segment plays a role in lateral packing of subunits. Additionally, these findings imply that filament length is more critical to mechanical strength than lateral associations between protofibrils and/ or protofilaments. Finally, while there are certainly exceptions to this rule, there is a clear correlation between the severity of the disease, the location of the mutation and the degree to which a K5/ K14 mutation affects 10 nm filament assembly. What does our understanding of keratin filament assembly and EBS tell us about the fiinction(s) of keratin filaments in the skin? It is intriguing that defects in K5 or K14 lead to basal cell cytolysis in response to mechanical stress. A priori, this might be reflective of a key function for keratin filaments in maintaining mechanical integrity within a basal epidermal cell. Alternatively, however, the cytolysis may

302


not be dependent upon keratin per se, but rather the accumulation of large clumps or aggregates of protein in the cytoplasm. This could compromise the physiology of the keratinocyte, and lead to release of proteases and cell destruction. Gene targeting technology in mice (Lloyd et al., 1995), coupled with analysis of several rare autosomal recessive cases of EBS (Chan et al., 1994b; Rugg et al., 1994; Jonkman et al., 1996; Corden et al., 1998), unequivocally addressed keratin function. In the absence of K14, K5 is unstable and turns over rapidly. There is a residual keratin network composed of K15 and K5 filaments (Lloyd et al., 1995), but this wispy filamentous network is apparently not sufficient to sustain mechanical trauma: mice and humans alike undergo blistering as a consequence of basal cell cytolysis. From these studies it is clear that an important function of the basal epidermal keratin network is to impart mechanical integrity to the keratinocyte, without which the cell becomes fragile and prone to rupture upon physical trauma. Additional insights to keratin function have surfaced in exploring patients with EBS and mottled pigmentation. Patients with this disorder appear to have perturbations in the distribution of melanosomes within their epidermal basal cells. It was surprising to find that affected members of two apparently unrelated families with this disorder have the exact same point substitution, a proline to a leucine Table 2 Summary of Mutations Found in Patients with Keratin Disorders Gene

Disease

Mutation

Domain

K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K14 K5 K5 K5 K5 K5 K5 K5 K5

W-C, K EBS D-M EBS K EBS D-M EBS D-M EBS D-M EBS D-M EBS EBS-rec EBS-rec W-C EBS K EBS D-M, K EBS EBS-rec K EBS K EBS K EBS W-C EBS EBS MP D-M EBS W-C EBS K-EBS-hom D-M EBS W-C EBS W-C EBS W-C EBS

M1 191 Q120R L122F R125H (9) R125C (7) R125S Y129D E144A 3' splice V270M M272R A274D (3) W305X ∆E375 I377N L384P (2) R388C P24L (3) ∆30 aa I161S (8) K173N L174F N193K M327T N329K

1A 1A 1A 1A 1A 1A 1A 1A 1B L12 L12 L12 2B 2B 2B 2B 2B N-term H1/1A H1 1A 1A 1A L12 L12


Gene

Disease

Mutation

Domain

K5 K5 K5 K5 K5 K5 K5 K10 K10 K10 K10 K10 K10 K10 K10 K10 K10 K10 K10 K1 K1 K1 K1 K1 K1

W-C EBS W-C EBS K EBS W-C EBS D-M EBS D-M EBS D-M EBS EH, EN EH EH, EN EH, EN EH EH EH EH EH EH EH-mild EH PPK-mild EH-mild EH-mild EH-mild EH EH

D328V R331C L463P (2) L463P I467T E475G E477K M150R N154H R156H (6) R156C (3) R156P R156S A158P Y160D Y160N L161S K439E L442Q K73I R178P V154G L160P S185P N187S (2)

L12 L12 2B 2B 2B 2B 2B 1A 1A 1A 1A 1A 1A 1A 1A 1A 1A 2B 2B V1 H1 H1 H1 1A 1A

Table 2 Summary of Mutations Found in Patients with Keratin Disorders Gene

Disease

Mutation

Domain

K1 K1 K1 K1 K9 K9 K9 K9 K9 K9 K9 K9

EH EH EH EH EPPK EPPK EPPK EPPK EPPK EPPK EPPK EPPK

N187H S192P Y481C E489Q M1 56V L159V N160Y N160K N160S R162Q R162W (5) L16TS

1A 1A 2B 2B 1A 1A 1A 1A 1A 1A 1A 1A

303

304

Gene


Disease

Mutation

K9 EPPK Q171P K2e EH Q187P K2e EH T485P K2e EH E493K (8) K2e EH E494K K6a PC ∆N170 K16 PC L130P K17 PC N92S (4) K17 PC N92D K17 PC N92H K17 PC R94H (2) K17 PC Y98D Hb6 Monilethrix E410K (3) Hb6 Monilethrix E410D Hb1 Monilethrix E403K K13 WSN L:P K4 WSN ∆N K12 MCD R135G K12 MCD R135I K12 MCD R135T K12 MCD L140R K12 MCD V143L K12 MCD Y428D K3 MCD E509K EBS, Epidermolysis Bullosa Simplex WC, Weber-Cockayne DM, Dowling Meara K, Koebner MP, Mottled Pigmentation EH, Epidermolytic Hyperkeratosis EN, Epidermal nevi of the epidermolytic hyperkeratosis type IBS, Ichthyosis bullosa of Siemens WSN, White sponge nevus MCD, Meesmann’s corneal dystrophy PC; pachyonychia congenita

Domain 1A 1A 2B 2B 2B 1A 1A 1A 1A 1A 1A 1A 2B 2B 2B 1A 1A 1A 1A 1A 1A 1A 2B 2B

at residue 24, in the non-helical head domain of K5 (Uttam et al., 1996; Irvine et al., 1997b). Given that EBS can be caused by so many different mutations, this makes it unlikely that a second mutation is responsible for the mottled pigmentation phenotype. The P24L substitution is intriguing in that a) it is within a region likely to protrude along the filament surface (Steinert et al., 1983), b) it is predicted to cause a significant conformational change in the K5 head domain, and c) the K5 head domain associates with desmoplakin, a desmosomal protein (Stappenbeck and Green, 1992; Kouklis et al., 1994). Whether this change might alter the transfer of melanin granules from the melanocyte to the keratinocyte, or whether it


305

might perturb the normal positioning of the protective melanin granules over the nucleus of the mitotically active basal keratinocyte remains to be explored. However, it is interesting that while long regarded as strictly a microtubule-driven process, melanosome distribution has recently been shown to be aberrant in the myosin V null mouse (Mercer et al., 1991), and now a keratin mutation may affect the process as well. One possible explanation is that connections exist between different cytoskeletal components, and that by perturbing one network, an effect on another filament system occurs. Another complex disorder involving EBS is that of EBS with Muscular Dystrophy. A priori, it is possible that patients who suffer from these two disorders have two different genetic lesions: one, in K5 or K14, that gives rise to EBS and the other in one of the dystrophin-associated proteins, that gives rise to Muscular Dystrophy. Interestingly, it was recently shown that patients with this complex disorder have a single genetic lesion. Autosomal recessive or compound heterozygous mutations in plectin (HD1) seem to be responsible for the defects in skin and in muscle, where this protein is known to be expressed (McLean et al., 1996; Gache et al., 1996). Plectin is a protein associated with the hemidesmosome, and related to a neuronal splice form of BPAG1 (BPAG1n), recently shown to associate with both the actin and the intermediate filament cytoskeleton (Yang et al., 1996). Ablation of the BPAG1 gene in mice gives rise to EBS and sensory neuron disease (Guo et al., 1995), reflective of the expression pattern of this gene (Yang et al., 1996; Brown et al., 1995). Ablation of plectin in mice gives rise to EBS and muscle defects (Andra et al., 1997). Thus, it is now clear that EBS can be generated in several different ways that go beyond simple mutations in the K5 or K14 gene. EPIDERMOLYTIC HYPERKERATOSIS AND OTHER SUPRABASAL DISORDERS OF KERATIN Epidermolytic Hyperkeratosis has a number of striking, mirror-image parallels with EBS (for review, see Anton-Lamprecht, 1994). Patients with EH have normal basal cells, but display clumps of keratin protein in their suprabasal keratinocytes and exhibit extensive cytolysis in their suprabasal layers (Figure 3B). Since epidermal cells undergo a switch from K5/K14 to K1/K10 as they exit the basal layer (Fuchs and Green, 1980), it took little intuition to extrapolate from the K14 transgenic mouse studies and predict that EH would be a disorder of K1/K10 (Vassar et al., 1991). Indeed, transgenic mice expressing a mutant K10 gene were soon found to display characteristic features of EH (Fuchs et al., 1992), and genetic mapping data (Compton et al., 1992) and sequence analyses (Rothnagel et al., 1992; Cheng et al., 1992; Chipev et al., 1992) revealed that affected members of EH families have point mutations in their K1 or K10 genes. In fact, the parallels between EBS and EH are even greater than initially imagined: many of the most severe cases of EH display mutations in the highly conserved ends of the rod domain, and in many cases, the exact same residue mutated in K14 or K5 to give rise to EBS is mutated in K10 or K1 to give rise to EH (Cheng et al., 1992; Rothnagel et al., 1992; 1993; Chipev et al., 1992; 1994; Syder et al., 1994; Huber et al., 1994; McLean et al., 1994; Yang et al., 1994; 1997a). Mutations in the K2e gene also occur, and consistent with the late onset of K2e expression (Collin et al., 1992), these patients exhibit only mild clinical features even when the mutated residue is in the highly conserved rod end (Rothnagel et al., 1994; McLean et al., 1994; Kremer et al., 1994; Yang et al., 1996b; Jones et al., 1997). An additional mild case, often referred to as the Siemens subtype, has also been found to harbor a K10 mutation, but in this case, the mutation falls outside the highly conserved end domains of the rod segment (Syder et al., 1994).

306


Epidermolytic Palmoplantar Keratoderma (EPPK) is similar to EH except that the disorder is restricted to palmoplantar skin. Given that K9 is a keratin which had previously been shown to be expressed specifically only in the suprabasal layers of palmo and plantar skin (Fuchs and Green, 1980; Langbein et al., 1986), EPPK was a candidate for K9 gene defects. Indeed, K9 mutations have been discovered in the rod end segments of K9 in a number of different EPPK patients (Torchard et al., 1994; Reis et al., 1994; Hennies et al., 1994; Bonifas et al., 1994; Rothnagel et al., 1995; Navsaria et al., 1995; Kobayashi et al., 1996; Endo et al., 1997; Nomura et al., 1997b). Based on parallels between W-C EBS and EPPK, some patients might also be expected to have mild defects in their K1 or K10 genes. Intriguingly, there are noncytolytic forms of PPK, and one of these was recently shown to have a point mutation in a lysine residue located in the head domain of K1, at a considerable distance from the rod segment (Kimonis et al., 1994). In the 10 nm filament, this portion of the head domain is thought to protrude along the filament surface (Steinert et al., 1983). This residue is also quite highly conserved, and in K5, this residue is in a domain that participates in the direct association between desmoplakin and keratin (Kouklis et al., 1994). This raises the possibility that some forms of PPK might involve subtle perturbations in desmosomes and/ or their IF connections, as suggested from Dsg3 mice studies (Allen et al., 1996; Koch et al., 1997). An interesting situation arises in a rare form of Epidermal Nevus (EN), where patients with this disorder display mosaic patterns of what resembles EH along their body surface (Fitzpatrick et al., 1993). These patients often produce offspring with full blown EH. When patients with this disorder were analyzed genetically, it was discovered that affected body regions are heterozygous for a mutation in K1 or K10, while unaffected body regions have the wild-type sequence (Pallet et al., 1994). These data suggest that these EN patients acquire a post-zygotic mutation in their K10 or K1 gene, and display clinically mosaic patterns of EH due to clonal expansion of the mutant cells (Paller et al., 1994). If a mutant cell is transmitted through the germline, the genetic mosaicsm is lost and full blown EH results. No clinically mosaic disorder of EBS has ever been described, and most likely, it does not exist. Why not? If a post-zygotic mutation occurs in the K5 or K14 gene, the basal layer of the epidermis would be expected to contain a mixed population of mutant and wild-type keratinocytes. The wild-type keratinocytes would be able to withstand physical trauma and would continue to divide laterally to populate the basal layer. The mutant keratinocytes on the other hand would undergo cytolysis in time, and their popultation would dwindle relative to that of the wild-type cells. Eventually, wild-type cells would prevail and account for the bulk of the cells in the basal layer. The result would be a clinically normal appearance, despite the genetic mosaicism. In contrast, a genetically mosaic defect in K1 or K10 would not manifest itself clinically until the cell had already left the basal layer: at this point, the cell is locked into upward movement and cannot move laterally. Thus, what begins as genetically mosaic persists as clinically mosaic for any biochemical marker that is expressed after a cell has undergone a commitment to terminally differentiate. PACHYONYCHYA CONGENITA: A DISORDER OF THE WOUND-HEALING/ OUTER ROOT SHEATH KERATINS Pachyonychya Congenita (PC) is an autosomal dominant, degenerative disorder characterized by a thickening of the skin that surrounds the hair follicles and the hands and feet. It is accompanied by defects in the nails and oral tissues (Fitzpatrick et al., 1993). Based on the additional ultrastructural abnormalities in keratin filaments, it has traditionally been classified clinically as a keratinization disorder, and often discussed in the context of EBS, EH and PPK. Given the keratin filament abnormalities and the location of the keratinocytes that exhibit signs of degeneration in PC, PC fit the paradigm to be a disorder of K6, K16


307

and K17. It is now known that PC genetically maps to the type I and type II keratin gene clusters on chromosome 17 and 12, respectively (Munro et al., 1994), and also harbors mutations in each of these three genes in different PC families (McLean et al., 1995; Bowden et al., 1995; Smith et al., 1997; 1998; Fujimoto et al., 1998). BEYOND THE SKIN: DELVING DEEPER BENEATH THE BODY SURFACE There are more than 30 different keratin genes and a total of nearly 60 different intermediate filament genes in the human genome. Thus, it was just a matter of time before additional disorders of IF proteins were discovered that extended beyond the skin. The first of these is White Sponge Nevus (WSN), a rare autosomal dominant disorder typified by white, thick plaques in the oral (primarily buccal) mucosa, occasionally accompanied by esophageal, genitalial and/or rectal involvement. Thickening of the epithelium, degeneration of the suprabasal cells, and clumps or aggregates of keratin filaments signify this disorder as a candidate for a keratin defect. Based on the distribution of clinical abnormalities, and the particular cells involved, defects in K4 and K13 are expected (Moll et al., 1982). In the past year, K4 and K13 mutations in WSN patients were identified by several laboratories (Richard et al., 1995; Rugg et al., 1995). Monilethrix and Meesman’s Corneal Dystrophy are other recent additions to the growing list of IF disorders (Irvine et al., 1996c; Winter et al., 1997a; 1997b; 1998). Disorders of the neurofilament genes also seem likely, given their prominence in large myelinated axons of motor neurons. There are several neurological disorders that fit the paradigm for a genetic disorder of IFs. Amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease), infantile spinal muscular atrophy, and hereditary sensory motor neuropathy are all clinically characterized by progressive muscle denervation and atrophy arising from motor neuron degeneration. Most importantly, aggregates of neurofilament proteins and aberrant accumulation of NFs in motor neuron cell bodies are associated with these disorders. Recently, it was shown that mice engineered to express a mutant NF transgene exhibit pathological features of ALS, including axonal and perikaryal swellings, slowed axonal transport, and selective degeneration of spinal motor neurons, resulting in neurogenic atrophy of the skeletal muscle (Xu et al., 1993; Cote et al., 1993; for review, see Lee and Cleveland, 1994). This said, it remains unclear as to whether the NF-H mutations found in individuals with sporadic ALS are causative (Figlewicz et al., 1994), and in many ALS cases, mutations in NF genes have not been found, despite considerable searching (Williamson et al., 1998). Another candidate disorder of IFs includes those forms of generalized myopathies that are typified by not only cell degeneration, but also aberrations in their desmin IF networks. Perturbations in the desmin IF network have been observed in a number of distal myopathies (Helliwell et al., 1994; Horowitz and Schmalbruch, 1994). Although no linkage has yet been found between any form of myopathies and the desmin locus on chromosome 2, several incidences have recently been reported uncovering desmin point substitutions in severe myopathies (Goldfarb et al., 1998; Munoz-Marmol et al., 1998). Functional studies suggest a correlation between these substitutions and desmin filament assembly. In summary, our knowledge of epidermal keratins has brought us to the basis of genetic disorders of the skin specifically and to genetic disorders of the cytoskeleton more generally. By understanding the functional significance of keratin filaments, it has been possible to extrapolate to the general importance of IFs in higher eukaryotic organisms. By elucidating the molecular nature of cytoarchitecture within the epidermis and by understanding how epidermal cells divide and differentiate, it should be possible in the future to continue to make further forages into the broader significance of human biology.

308


ACKNOWLEDGMENTS I would like to extend a very special thank you to past members of my laboratory, who over the years have contributed to many of the different findings regarding the epidermal keratins and their relation to human disease. Some of these former students and postdoctorates now have their own laboratories: Dr. Pierre Coulombe (Johns Hopkins University), Dr. Kathryn Albers (University of Kentucky), Dr. Douglas Marchuk (Duke University), Dr. Angela Tyner (University of Illinois, Chicago), Dr. Rafi Kopan (Washington University, St. Louis), Dr. Robert Vassar (Amgen Corporation, CA), Dr. Mary Beth McCormick (University of Wisconsin, Madison), and Dr. Qian-Chun Yu (Wistar Institute, Philadelphia). Others are graduate students, postdoctoral fellows, interns or residents at major academic institutions in the country: Dr. Anthony Letai (Harvard Medical School), Dr. Yiumo Chan (Harvard Medical School), Dr. Jian Cheng and Andrew S. Syder (Washington University, St. Louis). Others are still with my group: Ms. Linda Degenstein, Ms. Geraldine Strasser, Dr. James Dowling and Dr. Yanmin Yang. I’d like to thank the many dermatologists who have collaborated with us over the years: first and foremost, Dr. Amy S. Paller (Children’s Hospital, Northwestern University), and also Dr. Jo-David Fine (University of North Carolina), Dr. Jouni Uitto (Thomas Jefferson University), Dr. Tobias Gedde-Dahl (University of Oslo, Norway), Dr. Ingrun Anton-Lamprecht (University of Heidelberg, Germany) and Dr. Leonard Milstone (Yale University). Finally, I’d like to thank my colleagues in the field who have contributed so heavily to the understanding of keratin filament architecture and gene expression. REFERENCES Aebi, U., Cohn, J.B. and Gerace, L.L. (1986) The nuclear lamina is a meshwork of intermediate-type filaments. Nature, 323, 560–564. Aebi, U., Fowler, W.E., Rew, P., Sun, T.-T (1983) The fibrillar substructure of keratin filaments unraveled. J. Cell Biol., 97, 1131–1143. Albers, K. and Fuchs, E. (1987) The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines. J. Cell Biol., 105, 791–806. Albers, K. and Fuchs, E. (1989) Expression of mutant keratin cDNAs in epithelial cells reveals possible mechanisms for initiation and assembly of intermediate filaments. J. Cell Biol., 108, 1477–1493. Allen, E., Yu, Q.-C. and Fuchs, E. (1996) Abnormalities in desmosomes, proliferation and differentiation in the epidermis of mice expressing a mutant desmosomal cadherin. J. Cell Biol., 133, 1367–1382. Andra, K., Lassmann, H., Bittner, R., Shorny, S., Fassler, R., Propst, F. and Wiche, G. (1997) Targeted inactivation of plectin reveals essential function in maintaining the integrity of skin, muscle, and heart cytoarchitecture. Genes Dev, 11, 3143–3156. Anton-Lamprecht, I. (1994) Ultrastructural identification of basic abnormalities as clues to genetic disorders of the epidermis. J. Invest. Dermatol., 103, 65–125. Anton-Lamprecht, I. and Schnyder, V.W. (1982) Epidermolysis bullosa herpetiformis Dowling-Meara: report of a case and pathogenesis. Dermatolog., 164, 221–235. Asselineau, D., Bernhard, B., Bailly, C. and Darmon, M. (1985) Epidermal morphogenesis and induction of the 67kd keratin polypeptide by culture of human keratinocytes at the liquid-air interface. Exp. Cell Res., 159, 536–539. Barrandon, Y. and Green, H. (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl. Acad. Sci. USA, 84, 2302–2306. Bonifas, J.M., Matsumura, K., Chen, M.A., Berth-Jones, J., Hutchison, P.E., Zloczower, M., Fritsch, P.O. and Epstein, E.H. Jr. (1994) Mutations of keratin 9 in two families with palmoplantar epidermolytic hyperkeratosis. J. Invest. Dermatol., 103, 474–477.


309

Bonifas, J.M., Rothman, A.L., Epstein, E.H. (1991) Epidermolysis bullosa simplex: Evidence in two families for keratin gene abnormalities. Science, 254, 1202–1205. Bowden, P.E., Haley, J.L., Kansky, A., Rothnagel, J.A., Jones, D.O. and Turner, R.J. (1995) Mutation of a type II keratin gene (K6a) in pachyonychia congenita. Nat. Genet., 10, 363– 365. Brown, A., Bernier, G., Mathieu, M., Rossant, J. and Kothary, R. (1995) The mouse dystonia musculorum gene is a neural isoform of bullous pemphigoid antigen 1. Nat. Genet., 10, 301–306. Chan, Y.-M., Cheng, J., Gedde-Dahl, T., Niemi, K.-M. and Fuchs, E. (1996) Genetic analysis of a severe case of dowling-meara epidermolysis bullosa simplex. J. Invest. Dermatol., 3106, 327–334. Chan, Y.-M., Yu, Q.-C, Fine, J.-D., Fuchs, E. (1993) The genetic basis of Weber-cockayne epidermolysis bullosa simplex. Proc. Natl. Acad. Sci. USA, 90, 7414–7418. Chan, Y.-M., Yu, Q.C., LeBlanc-Straceski, J., Christiano, A., Pulkkinen, L., Kucherlapati, R.S., Uitto, J. and Fuchs, E. (1994a) Mutations in the non-helical linker segment L1–2 of keratin 5 in patients with Weber-Cockayne epidermolysis bullosa simplex. J. Cell Sci., 107, 765–774. Chan, Y.-M., Anton-Lamprecht, I., Yu, Q.-C., Jackel, A., Zabel, B., Ernst, J.-P. and Fuchs, E. (1994b) A human keratin 14 “knockout”: the absence of K14 leads to severe epidermolysis builosa simplex and a function for an intermediate filament protein. Genes and Development, 8, 2574–2587. Chen, H., Bonifas, J.M., Matsumura, K., Ikeda, S. and Leyden, W.A. (1995) Keratin 14 gene mutations in patients with epidermolysis bullosa simplex. J. Invest. Dermatol., 105, 629– 632. Cheng, J., Syder, A.J., Yu, Q.C., Letai, A., Paller, A.S. and Fuchs, E. (1992) The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes. Cell, 70, 811–819. Chipev, C.C., Korge, B.P., Markova, N., Bale, S.J., DiGiovanna, J.J., Compton, J.G. and Steinert, P.M. (1992) A leucine —»proline mutation in the H1 subdomain of keratin 1 causes epidermolytic hyperkeratosis. Cell, 70, 821–828. Chipev, C.C., Yang, J.M., DiGiovanna, J.J., Steinert, P.M., Markov, L., Compton, J.G. and Bale, S.J. (1994) Preferential sites in keratin 10 that are mutated in epidermolytic hyperkeratosis. Am. J. Hum. Genet., 54, 179–190. Collin, C., Moll, R., Kubicka, S., Ouhayoun, J.P. and Franke, W.W. (1992) Characterization of human cytokeratin 2, an epidermal cytoskeletal protein synthesized late during differentiation. Exp. Cell Res., 202, 132–141. Compton, J.G., DiGiovanna, J.J., Santucci, S.K., Kearns, K.S., Amos, C.I., Abangan, D.L., Korge, B.P., McBride, O.W., Steinert, P.M. and Bale, S.J. (1992) Linkage of epidermolytic hyperkeratosis to the type II keratin gene cluster on chromosome 12q. Nat. Genet., 1, 301–305. Conti, C.J. (1994) The Mouse Skin as a Model for Chemical Carcinogenesis. in J.P. Sunderberg, (ed), Handbook of Mouse Mutations with Skin and Hair Abnormalities, CRC Press, Inc., Boca Raton, pp. 39–46. Conway, J.F. and Parry, D.A.D. (1988) Intermediate filament structure: 3. Analysis of sequence homologies. Int. J. Biol. Macromol., 10, 79–98. Cooper, D.N. and Youssoufian, H. (1988) The CpG dinucleotide and human genetic disease. Hum. Genet., 78, 151–155. Corden, L.D., Mellerio, J.E., Gratian, M.J., Eady, R.A., Harper, J.I., Lacour, M., Magee, G., Lane, E.B., McGrath, J.A. and McLean, W.H. (1998) Homozygous nonsense mutation in helix 2 of K14 causes severe recessive epidermolysis bullosa simplex. Hum Mutat, 11, 279–285. Cote, F., Collard, J-F. and Julien, J-P. (1993) Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis. Cell, 73, 35–46. Coulombe, P., Chan, Y.-M., Albers, K. and Fuchs, E. (1990) Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro. J. Cell Biol., 111, 3049–3064. Coulombe, P.A., Hutton, M.E., Letai, A., Hebert, A., Paller, A.S. and Fuchs, E. (1991b) Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell, 66, 1301–1311. Coulombe, P.A., Hutton, M.E., Vassar, R. and Fuchs, E. (1991a) A function for keratins and a common thread among different types of epidermolysis bullosa simplex diseases. J. Cell Biol., 115, 1661–1674. Coulombe, P.A., Kopan, R. and Fuchs, E. (1989) Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation. J. Cell Biol., 109, 2295–2312. Crick, F.H.C. (1953) The packing of alpha-helices: simple coiled-coils. Acta Cry St., 6, 689– 697.

310


Dale, B.A., Holbrook, K.A. and Steinert, P.M. (1978) Assembly of stratum corneum basic protein and keratin filaments in macrofibrils. Nature, 276, 729–731. Dodemont, H., Riemer, D. and Weber, K. (1990) Structure of an invertebrate gene encoding cytoplasmic intermediate filament (IF) proteins: implications from the origin and the diversification of IF proteins. Embo J., 9, 4083–4094. Dong, W., Ryynanen, M. and Uitto, J. (1993) Identification of a leucine-to-proline mutation in the keratin 5 gene in a family with the generalized kobner type of epidermolysis bullosa simplex (EBS). Hum. Mutat., 2, 94–102. Dowling, J., Yu, Q.-C. and Fuchs, E. (1996) Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival. J. Cell Biol., 134, 559– 572. Ehrlich, P., Sybert, V.P., Spencer, A. and Stephens, K. (1995) A common keratin 5 gene mutation in epidermolysis bullosa simplex—Weber-Cockayne. J. Invest. Dermatol., 104, 877–879. Eichner, R., Sun, T.-T. and Aebi, U. (1986) The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments. J. Cell Biol., 102, 1767– 1777. Endo, H., Hatamochi, A. and Shinkkai, H. (1997) A novel mutation of a leucine residue in coil 1A of keratin 9 in epidermolytic palmoplantar keratoderma. J Invest Dermatol, 109, 113–115. Figlewicz, D.A., Krizus, A., Martinoli, M.G., Meininger, V., Dib, M., Rouleau, G.A. and Julien, J.-P (1994) Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral schlerosis. Hum. Mol. Genet, 3, 1757– 1761. Fitzpatrick, T.B., Eisen, A.Z., Wolff, K. Freedberg, I.M. and Austen, K.F. (eds.), (1993) Dermatology in General Medicine, Fourth Ed. Vol. I and II. McGraw-Hill, Inc., New York, pp. 1–2961. Frisch, S.M. and Francis, H. (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J. Cell Biol., 124, 619–626. Fuchs, E. (1995) Keratins and the Skin [Review]. Annual Review of Cell and Developmental Biology, 11, 123–153. Fuchs, E. and Byrne, C. (1994) The Epidermis: rising to the surface. Curr. Opin. Genet. Develop, 4, 725–736. Fuchs, E., Coppock, S., Green, H. and Cleveland, D. (1981) Two distinct classes of keratin genes and their evolutionary significance. Cell, 27, 75–84. Fuchs, E., Esteves, R.A. and Coulombe, P.A. (1992) Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for Epidermolytic Hyperkeratosis. Proc. Natl. Acad. Set. USA, 89, 6906–6910. Fuchs, E. and Green, H. (1980) Changes in keratin gene expression during terminal differentiation of the keratinocyte. Cell, 19, 1033–1042. Fuchs, E. and Green, H. (1981) Regulation of terminal differentiation of cultured human keratinocytes by vitamin A. Cell, 25, 617–625. Fuchs, E. and Weber, K. (1994) Intermediate filaments: structure, dynamics, function, and disease. Ann. Rev. Biochem., 63, 345–382. Fujimoto, W., Nakanishi, G. Hirakawa, S., Nakanishi, T., Shimo, T., Takigawa, M. and Arata, J. (1998) Pachyonychia congenita type 2, keratin 17 mutation in a Japanese case. J. Am. Acad. Dermatol., 38, 1007–1009. Gache, Y., Chavanas, S., Lacour, J.P., Wiche, G., Owaribe, K., Meneguzzi, G. and Ortonne, J.P. (1996) Defective espression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystophy. Journal of Clinical Investigation, 97(10, May 15), 2289–2298. Garrod, D.R. (1993) Desmosomes and hemidesmosomes. Curr. Opin. Cell Biol., 5, 30–40. Geisler, N., Schunemann, J. and Weber, K. (1992) Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofllaments. Eur. J. Biochem., 206, 841–852. Georges-Labouesse, E., Messaddeq, N., Cadalbert, L., Dierich, A. and Le Meur, M. (1996) Absence of integrin alpha 6 leads to epidermolysis bullosa and neonatal death in mice. Nat. Genet., 13, 370–373. Goldfarb, L.G., Park, K.Y., Cervenakova, L., Gorokhova, S., Lee, H.S., Vasconcelos, O., Nagle, J.W., Semino-Mora, C., Sivakumar, K. and Dalakas, M.C. (1998) Missense mutations in desmin associated with familial cardiac and skeletal myopathy. Nature Genetics, 19, 402– 403. Green, K.J., Stappenbeck, T.S., Parry, D.A. and Virata, M.L. (1992) Structure of desmoplakin and its association with intermediate filaments. J. of Dermatol., 19, 765–769.


311

Green, K.J., Virata, M.L.A., Elgart, G.W., Stanley, J.R., Parry, D.A.D. (1992) Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of new gene family involved in organization of intermediate filaments. Int.J. Biol. Macromol., 14, 145–153. Guo, L., Degenstein, L., Dowling, J., Yu, Q.-C., Wollmann, R., Perman, B. and Fuchs, E. (1995) Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified squamous epithelia and severe neurologic degeneration. Cell, 81, 233– 243. Hanukoglu, I. and Fuchs, E. (1982) The cDNA sequence of a human epidermal keratin: divergence of sequence but conservation of structure among intermediate filament proteins. Cell, 31, 243–252. Hanukoglu, I. and Fuchs, E. (1983) The cDNA sequence of a type II cytoskeletal keratin reveals constant and variable structural domains among keratins. Cell, 33, 915–924. Hatzfeld, M. and Weber, K. (1990b) The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: Use of site-specific mutagenesis and recombinant protein expression. J. Cell Biol., 110, 1199–1210. Hatzfeld, M. and Weber, K. (1990a) Modulation of keratin intermediate filament assembly by single amino acid exchanges in the consensus sequence at the C-terminal end of the rod domain. J. Cell Sci., 99, 351–362. Helliwell, T.R., Green, A.R., Green, A., Edwards, R.H. (1994) Hereditary distal myopathy with granule-filamentous cytoplasmic inclusions containing desmin, dystrophin and vimentin. J. Neuro. Sci., 124, 174–187. Hennies, H.C., Kuster, W., Mischke, D. and Reis, A. (1995) Localization of a locus for the striated form of palmoplantar keratoderma to chromosome 18q near the desmosomal cadherin gene cluster. Hum. Mol. Genet, 4, 1015–1020. Hennies, H.C., Zehender, D., Kunze, J., Kuster, W. and Reis, A. (1994) Keratin 9 gene mutational heterogeneity in patients with epidermolytic palmoplantar keratoderma. Human Genetics 93, 649–654. Hennings, H.D., Michael, D., Cheng, M.C., Steinert, P., Holbrook, K. and Yuspa, S.H. (1980) Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell, 19, 245–254. Horowitz, S.H. and Schmalbruch, H. (1994) Autosomal dominant distal myopathy with desmin storage: a clinicopathologic and electrophysiologic study of a large kinship. Muscle and Nerve, 17, 151–160. Hotchin, N.A., Gandarillas, A. and Watt, F.M. (1995) Regulation of cell surface beta 1 integrin levels during keratinocyte terminal differentiation. J. Cell Biol., 128, 1209–1219. Hovnanian, A., Pollack, E., Hilal, L., Rochat, A., Prost, C., Barrandon, Y. and Goosens, M.A. (1993) A missense mutation in the rod domain of keratin 14 associated with recessive epidermolysis bullosa simplex. Nat. Genet., 3, 327–332. Huber, M., Scaletta, C., Benathan, M., Frenk, E., Greenhalgh, D.A., Rothnagel, J.A., Roop, D.R. and Hohl, D. (1994) Abnormal keratin 1 and 10 cytoskeleton in cultured keratinocytes from epidermolytic hyperkeratosis caused by keratin 10 mutations. J. Invest. Dermatol., 102, 691–694. Humphries, M.M., Mansergh, F.C., Kiang, A.S., Jordan, S.A., Sheils, D.M., Martin, M.J., Farrar, G.J., Kenna, P.F., Young, M.M. and Humphries, P. (1996) Three keratin gene mutations account for the majority of dominant simplex epidermolysis bullosa cases within the population of Ireland. Hum Mutat, 8, 57–63. Humphries, M.M., Sheils, D.M., Farrar, G.J., Kumar-Singh, R., Kenna, P.F., Mansergh, F.C., Jordan, S.A., Young, M., Humphries, P. (1993) A mutation (Met→Arg) in the type I keratin (K14) gene responsible for autosomal dominant epidermolysis bullosa simplex. Hum. Mutat., 2, 37–42. Irvine, A.D., McKenna, K.E., Bingham, A., Nevin, N.C. and Hughes, A.E. (1997a) A novel mutation in the helix termination peptide of keratin 5 causing epidermolysis bullosa simplex Dowling-Meara. J. Invest. Dermatol., 109, 816–816. Irvine, A.D., McKenna, K.E., Jenkinson, H. and Hughes, A.E. (1997b) A mutation in the V1 domain of keratin 5 causes epidermolysis bullosa simplex with mottled pigmentation. J. Invest. Dermatol., 108, 809–810. Irvine, A.D., Corden, L.D., Swensson, O., Swensson, B., Moore, J.E., Frazer, D.G., Smith, F.J.D., Knowlton, R.G., Christophers, E. and Rochels, R. (1997c) Mutations in corneaspecific keratin K3 or K12 genes cause Meesmann’s corneal dystrophy. Nat. Genet., 16, 184–187.

312


Jones, D.O., Watts, C., Mills, C., Sharpe, G., Marks, R. and Bowden, P.E. (1997) A new keratin 2e mutation in ichthyosis bullosa of Siemens. J. Invest. Dermatol., 108, 354–356. Jones, P.H., Harper, S. and Watt, F.M. (1995) Stem cell patterning and fate in human epidermis. Cell, 80, 83–93. Jonkman, M.F., Heeres, K., Pas, H.H., van Luyn, M.J.A., Elema, J.D., Corden, L.D., Smith, F.J.D., McLean, W.H.I., Ramaekers, F.C.S., Burton, M. and Scheffer, H. (1996) Effects of Keratin 14 Ablation on the Clinical and Cellular Phenotype in a Kindred with Recessive Epidermolysis Bullosa Simplex. Journal of Investigative Dermatology, 107, 764–769. Kelsell, D.P., Stevens, H.P., Ratnavel, R., Bryant, S.P., Bishop, D.T., Leigh, I.M. and Spurr, N.K. (1995) Genetic linkage studies in non-epidermolytic palmoplantar keratoderma: evidence for heterogeneity. Hum. Mol. Genet, 4, 1021–1025. Kimonis, V., DiGiovanna J.J., Yang, J.M., Doyle, S.Z., Bale, S.J. and Compton, J.G. (1994). A mutation in the V1 end domain of keratin 1 in non-epidermolytic palmar-plantar keratoderma. J. Invest. Dermatol. 103, 764–769. Kitajima, Y., Inoue, S. and Yaoita, H. (1989) Abnormal organization of keratin intermediate filaments in cultured keratinocytes of epidermolysis bullosa simplex. Arch. Dermatol. Res., 281, 5–10. Kobayashi, S., Tanaka, T., Matsuyoshi, N. and Imamura, S., (1996). Keratin 9 point mutation in the pedigree of epidermolytic hereditary palmoplantar keratoderma perturbs keratin intermediate filament network formation. FEES Letters, 386, 149–155. Koch, P.J., Mahoney, M.G., Ishikawa, H., Pulkkinen, L., Uitto, J., Shultz, L., Murphy, G.F., Whitaker-Menezes, D. and Stanley, J.R. (1997) Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris. J. Cell Biol., 137, 1091–1102. Kopan, R., Traska, G. and Fuchs, E. (1987) Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization. J. Cell Biol., 105, 427–440. Kouklis, P., Hutton, E. and Fuchs, E. (1994) Making the connection: keratin intermediate filaments and desmosomes proteins. J. Cell Biol., 127, 1049–1060. Kremer, H., Zeeuwen, P., McLean, W.H., Mariman, E.C., Lane, E.B., van de Kerkhof, C.M., Ropers, H.H. and Steijlen, P.M. (1994) Ichthyosis bullosa of Siemens is caused by mutations in the keratin 2e gene. J. Invest. Dermatol., 103, 286–289. Lane, E.B., Rugg, E.L., Navsaria, H., Leigh, I.M., Heagerty, A.H.M., Ishida-Yamamoto, A. and Eady, R.A.J. (1992) A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature, 356, 244–246. Langbein, L., Heid, H.W., Moll, I. and Franke, W.W. (1993) Molecular characterization of the body site-specific human epidermal cytokeratin 9-' cDNA cloning, amino acid sequence, and tissue specificity of gene expression. Differentiation, 55, 57–71. Lee, M.K. and Cleveland, D.W. (1994) Neurofilament function and dysfunction: involvement in axonal growth and neuronal disease. [Review]. Curr. Opin. Cell Biol., 6, 34–40. Letai, A., Coulombe, P. and Fuchs, E. (1992) Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations. J. Cell Biol., 116, 1181–1195. Letai, A., Coulombe, P.A., McCormick, M.B., Yu, Q.-C, Hutton, E. and Fuchs, E. (1993) Disease severity correlates with position of keratin point mutations in patients with epidermolysis bullosa simplex. Proc. Natl. Acad. Sci. USA, 90, 3197–3201. Lloyd, C., Yu, Q.-C., Cheng, J., Turksen, K., Degenstein, L, Hutton, E. and Fuchs, E. (1995) The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14. J. Cell Biol., 129, 1329–1344. Lu, X. and Lane, E.B. (1990) Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation. Cell, 62, 681–696. Lynch, M.H., O’Guin, W.M., Hardy, C., Mak, L. and Sun, T.-T. (1986) Acidic and basic hair/ nail (“hard”) keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to “soft” keratins. J. Cell Biol, 103, 2593–2606.


313

Maestrini, E., Monaco, A.P., McGrath, J.A., Ishida-Yamamoto, A., Camisa, C., Hovnanian, A., Weeks, D.E., Lathrop, M., Uitto, J. and Christiano, A.M. (1996) A molecular defect in loricrin, the major component of the cornified cell envelope, underlies Vohwinkel’s syndrome. Nat. Genet., 13, 70–77. Mansbridge, J.N. and Knapp, A.M. (1987) Changes in keratinocyte maturation during wound healing. J. Invest. Derm., 89, 253–262. Matsuki, M., Hashimoto, K., Yoshikawa, K., Yasuno, H. and Yamanishi, K. (1995) Epidermolysis bullosa simplex (Weber-Cockayne) associated with a novel missense mutation of Asp328 to Val in Linker 12 domain of keratin 5. Human Molecular Genetics, 4, 1999– 2000. McLean, W.H., Eady, R.A., Dopping-Hepenstal, P.J., McMillan, J.R., Leigh, I.M., Navsaria, H.A., Higgins, C, Harper, J.I., Paige, D.G. and Morley, S.M. (1994) Mutations in the rod 1A domain of keratins 1 and 10 in bullous congenital ichtyosiform erythroderma (BCIE). J. Invest. Dermatol., 102, 24–30. McLean, W.H., Pulkkinen, L., Smith, F.J., Rugg, E.L., Lane, E.B., Bullrich, F., Burgeson, R.E., Amano, S., Hudson, D.L., Owaribe, K., McGrath, J.A., McMillan, J.R., Eady, R.A., Leigh, I.M., Christiano, A.M. and Uitto, J. (1996) Loss of plectin causes epidermolysis bullosa with muscular dystrophy: cDNA cloning and genomic organization. Genes & Development, 10(14, Jul 15), 1724–1735. McLean, W.H., Rugg, E.L., Lunny, D.P., Morley, S.M., Lane, E.B., Swensson, O., Dopping-Hepenstal, P.J., Griffiths, W.A., Eady, R.A. and Higgins, C. (1995) Keratin 16 and keratin 17 mutations cause pachyonychia congenita. Nat. Genet., 9, 273–278. Mercer, J.A., Seperack, P.K., Strobel, M.C., Copeland, N.G. and Jenkins, N.A. (1991) Novel myosin heavy chain encoded by murine dilute coat colour locus [published erratum appears in Nature 1991 Aug 8, 352 (6335):547]. Nature, 349, 709–713. Moll, R., Franke, W.W., Schiller, D.L., Geiger, B. and Krepler, R. (1982) The catalog of human cytokeratins: Patterns of expression in normal epithelia, tumors, and cultured cells. Cell, 31, 11–24. Munoz-Marmol, A.M., Strasser, G., Isamat, M., Coulombe, P., Yang, Y., Roca, X., Vela, E., Mate, J.L., Coll, J., Fernandez-Figueras, M.T., Navas-Palacios, J.J., Ariza, A. and Fuchs, E. (1998) A dysfunctional desmin mutation in a patient with severe generalized myopathy. PNAS USA. Munro, C.S., Carter, S., Bryce, S., Hall, M., Rees, J.L., Kunkeler, L., Stephenson, A. and Strachan, T. (1994) A gene for pachyonychia congenita is closely linked to the keratin gene cluster on 17ql2-q21. J. Med. Genet., 31, 675–678. Navsaria, H.A., Swensson, O., Ratnavel, R.C., Shamsher, M., McLean W.H., Lane E.B., Griffiths, D., Eady R.A. and Leigh, I.M. (1995) Ultrastructural changes resulting from keratin 9 gene mutations in two families with epidermolytic palmoplantar keratoderma. J. Invest. Dermatol. 104, 425–429. Nelson, W. and Sun, T.-T. (1983) The 50– and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. J. Cell Biol., 97, 244–251. Nomura, K., Umeki, K., Meng, X., Tamai, K., Sawamura, D., Hosokawa, M., Miyazawa, T., Funayama, M. and Hashimoto, I. (1997a) A keratin K5 mutation (Leu 63-Pro) in a family with the Weber-Cockayne type of epidermolysis bullosa simplex. Arch Dermatol Res, 289, 493–495. Nomura, K., Meng, X., Umeki, K., Tamai, K., Sawamura, D., Hashimoto, I. and Kikuchi, T. (1997b) A keratin K10 gene mutation in a Japenese patient with epidermolytic hyperkeratosis. Jpn J. Hum Genet., 42, 217–223. Nomura, K., Shimizu, H., Meng, X., Umeki, K., Tamai, K., Sawamura, D., Nagao, K., Kawakami, T., Nishikawa, T. and Hashimoto, I. (1996) A novel keratin K5 gene mutation in Dowling-Meara epidermolysis bullosa simplex. J. Invest. Dermatol., 107, 253–254. Paladini, R.D., Takahashi, K., Bravo, N.S. and Coulombe, P.A. (1996) Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16. J. Cell Biol., 132, 381–397. Paller, A.S., Syder, A.J., Chan, Y.M., Yu, Q.C., Hutton, E., Tadini, G. and Fuchs, E. (1994) A direct link between clinical and genetic mosaicism: the genetic basis of a form of epidermal nevus. N. Engl. J. Med., 331, 1408–1415. Parry, D.A.D. and North, A.C.T. (1998) Hard alpha-Keratin intermediate filament chains: substructure of the N- and Cterminal domains and the predicted structure and function of the C-Terminal domains of Type I and Type II chains. J. Struct. Biol., 122, 67–75.

314


Parry, D.A.D., Crewther, W.G., Fraser, R.D. and MacRae, T.P. (1977) Structure of -keratin: structural implication of the amino acid sequence of the type I and type II chain segments. J. Mol. Biol., 113, 449–454. Pauling, L. and Corey, R.B. (1953) Compound helical configurations of polypeptide chains: structure of proteins of the α-keratin type. Nature, 171, 59–61. Reis, A., Hennies, H.C., Langbein, L., Digweed, M., Mischke, D., Drechsler, M., Schrock, E., Royer-Pokora, B., Franke, W., Sperling, K. and Kuster, W. (1994) Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK). Nat. Genet., 6, 174–179. Rheinwald, J.G. and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell, 6, 331–343. Rheinwald, J.G. and Green, H. (1977) Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes. Nature, 265, 421–424. Rice, R.H. and Green, H. (1979) Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: activation of the cross-linking by calcium ions. Cell, 18, 681–694. Richard, G., DeLaurenzi, V., Didona, B., Bale, S.J. and Compton, J.G. (1995) Keratin 13 point mutation underlies the hereditary mucosal epithelia disorder white sponge nevus. Nat. Genet., 11, 453–455. Roop, D.R., Huitfeldt, H., Kilkenny, A. and Yuspa, S.H. (1987) Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins. Differentiation, 35, 143–150. Rosenberg, M., Fuchs, E., Le Beau, M.M., Eddy, R. and Shows, T.B. (1991) Three epidermal and one epithelial keratin gene map to human chromosome 12. Cell Cytogen., 57, 33– 38. Rosenberg, M., RayChaudhury, A., Shows, T.B., LeBeau, M.M. and Fuchs, E. (1988) A group of type I keratin genes on human chromosome 17: characterization and expression. Mol. Cell. Biol., 8, 722–736. Rothnagel, J.A., Dominey, A.M., Dempsey, L.D., Longley, M.A., Greenhalgh, D.A,, Gagne, T.A., Huber, M., Frenk, E., Hohl, D. and Roop, D.R. (1992) Mutations in the rod domains of keratins 1 and 10 in epidermolytic hyperkeratosis. Science, 257, 1128–1130. Rothnagel, J.A., Fisher, M.R, Axtell, S.M., Pittelkow, M.R., Anton-Lamprecht, I., Huber, M., Hohl, D. and Roop, D.R. (1993) A mutational hot spot in keratin 10 (KRT 10) in patients with epidermolytic hyperkeratosis. Hum. Mol. Genet, 2, 2147–2150. Rothnagel, J.A., Traupe, H., Wojcik, S., Huber, M., Hohl, D., Pittelkow, M.R., Saeki, H., Ishibashi, Y. and Roop, D.R. (1994) Mutations in the rod domain of keratin 2e in patients with ichthyosis bullosa of Siemens. Nat. Genet., 7, 485–490. Rothnagel, J.A., Wojcik, S., Liefer, K.M., Dominey, A.M., Huber, M., Hohl, D. and Roop, D.R. (1995) Mutations in the 1A domain of keratin 9 in patients with epidermolytic palmoplantar keratoderma. J. Invest. Dermatol., 104, 430–433. Rugg, E.L., McLean, W.H.I., Allison, WE., Lunny, D.R, Macleod, R.I., Felix, D.H., Lane, E.B. and Munro, C.S. (1995) A mutation in the mucosal keratin K4 is associated with oral white sponge nevus. Nat. Genet., 11, 450–452. Rugg, E.L., McLean, W.H.I., Lane, E.B., Pitera, R., McMillan, J.R., Dopping-Hepenstal, P.J.C., Navsaria, H.A., Leigh, I.M., Eady, R.A.J. (1994) A functional “knockout” of human keratin 14. Genes and Development, 8, 2563–2573. Rugg, E.L., Morley, S.M., Smith, F.J.D., Boxer, M., Tidman, M.J., Navsaria, H., Leigh, I.M. and Lane, E.B. (1993) Missing links: Weber-cockayne keratin mutations implicate the L12 linker domain in effective cytoskeleton function. Nat. Genet., 5, 294–300. Ruhrberg, C, Hajibagheri, M.A., Simon, M., Dooley, T.P. and Watt, P.M. (1996) Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin. J. Cell Biol., 134, 715–729. Simon, M. and Green, H. (1985) Enzymatic cross-linking of involucrin and other proteins by keratinocyte participates in vitro. Cell, 40, 677–683. Smith, F.J.D., Jonkman, M.F., van Goor, H., Coleman, C.M., Covello, S.P., Uitto, J. and McLean, W.H.I. (1998) A mutation in human keratin K6b produces a phenocopy of the K17 disorder pachyonychia congenita type 2. Hum. Mol. Genet., 7, 1143–1148.


315

Smith, F.J., Corden, L.D., Rugg, E.L., Ratnavel, R., Leigh, I.M., Moss, C., Tidman, M.J., Hohl, D., Huber, M., Kunkeler, L., Munro, C.S., Lane, E.B. and McLean, W.B. (1997) Missense mutations in keratin 17 cause either pachyonychia congenita type 2 or a phenotype resembling steatocystoma multiplex. J. Invest. Dermatol., 108, 220–223. Stanley, J.R. (1993) Cell adhesion molecules as targets of autoantibodies in pemphigus and pemphigoid, bullous diseases due to defective epidermal cell adhesion. Adv. Immunol., 53, 291–325. Stappenbeck, T.S., Bornslaeger, E.A., Corcoran, C.M., Luu, H.H., Virata, M.L. and Green, K.J. (1993) Functional analysis of desmoplakin domains: specification of the interaction with keratin versus vimentin intermediate filament networks. J. Cell Biol., 123, 691–705. Stappenbeck, T.S. and Green, K.J. (1992) The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells. J. Cell Biol., 116, 1197–1209. Stappenbeck, T.S., Lamb, J.A., Corcoran, C.M. and Green, K.J. (1994) Phosphorylation of the desmoplakin COOH terminus negatively regulates its interation with keratin intermediate filament networks. J. Biol. Chem., 269, 29351–29354. Stark, H.-J., Breitkreutz, D., Limat, A., Bowden, P. and Fusenig, N.E. (1987) Keratins of the human hair follicle: “hyperproliferative” keratins consistently expressed in outer root sheath cells in vivo and in vitro. Differentiation, 35, 236–248. Steinert, P.M. (1990) The two-chain coiled-coil molecular of native epidermal keratin intermediate filaments is a type Itype II heterodimer. J. Biol. Chem., 265, 8766–8774. Steinert, P.M., Idler, W.W. and Zimmerman, S.B. (1976) Self-assembly of bovine epidermal keratin filaments in vitro. J. Mol. Biol., 108, 547–567. Steinert, P.M. and Marekov, L.N. (1995) The proteins elafin, filaggrin, keratin intermediate filaments, loricrin, and small proline-rich proteins 1 and 2 are isodipeptide cross-linked components of the human epidermal cornified cell envelope. J. Biol. Chem., 270, 17702– 17711. Steinert, P.M., Marekov, L.N., Fraser, R.D.B. and Parry, D.A.D. (1993) Keratin intermediate filament structure: crosslinking studies yield quantitative information on molecular dimensions and mechanisms of assembly. J. Mol. Biol., 230, 436– 452. Steinert, P.M. and Parry, D.A.D. (1993) The conserved H1 domain of the type II keratin 1 chain plays an essential role in the alignment of nearest neighbor molecules in mouse and human keratin 1/keratin 10 intermediate filaments at the two-to four-molecule level of structure. J. Biol. Chem., 268, 2878–2887. Steinert, P.M., Rice, R.H., Roop, D.R., Trus, B.L. and Steven, A.C. (1983) Complete amino acid sequence of a mouse epidermal keratin subunit and implications for the structure of intermediate filaments. Nature, 302, 794–800. Steinert, P.M., Steven, A.C. and Roop, D.R. (1983) Structural features of epidermal keratin filaments reassembled in vitro. J. Invest. Dermatol., 81, 86s-90s. Stephens, K., Sybert, V.P., Wijsman, E.M., Ehrlich, P. and Spencer, A. (1993) A keratin 14 mutational hot spot for epidermolysis bullosa simplex, dowling-meara: implications for diagnosis. J. Invest. Dermatol., 101, 240–243. Steven, A., Hainfeld, J., Trus, B., Wall, J. and Steinert, P. (1983) Epidermal keratin filaments assembled in vitro have masses-per-unit length that scale according to average subunit mass: structural basis for homologous packing of subunits in intermediate filaments. J. Cell Biol., 97, 1939–1944. Sun, T.-T., Eichner, R., Schermer, A., Cooper, D., Nelson, W.G. and Weiss, R.A. (1984) The Transformed Phenotype. In Levine, A., Topp,W, Woude, G. van de and Watson, J.D. (ed.), The Cancer Cell, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 169–176. Syder, A.J., Yu, Q.-C, Paller, A.S., Giudice, G., Pearson, R. and Fuchs, E. (1994) Genetic mutations in the K1 and K10 genes of patients with epidermolytic hyperkeratosis: Correlation between location and disease severity. J. Clin. Invest., 93, 1533–1542. Torchard, D., Blanchet-Bardon, C, Serova, O., Langbein, L., Narod, S., Janin, N., Goguel, A.F., Bernheim, A., Franke, W.W., Lenoir, G.M. and Feunteun, J. (1994) Epidermolytic palmoplantar keratoderma cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer. Nat. Genet., 6, 106–109.

316


Umeki, K., Nomura, K., Harada, K. and Hashimoto, I. (1996) A keratin K14 gene mutation in a Japanese patient with the Dowling-Meara type of epidermolysis bullosa simplex. J. Dermatol. Sci., 11, 64–69. Uttam, J., Hutton, E., Coulombe, P., Anton-Lamprecht, I., Yu, Q.-C., Gedde-Dahl, T., Fine, J.-D. and Fuchs, E. (1996) The genetic basis of epidermolysis bullosa simplex with mottled pigmentation. Proc. Natl. Acad. Set. USA, 93, 9079–9084. van der Neut, R., Krimpenfort, P., Calafat, J., Niessen, C.M. and Sonnenberg, A. (1996) Epithelial detachment due to absence of hemidesmosomes in integrin beta 4 null mice. Nat. Genet., 13, 366–369. Vassar, R., Coulombe, P.A., Degenstein, L., Albers, K. and Fuchs, E. (1991) Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell, 64, 365–380. Vassar, R., Rosenberg, M., Ross, S., Tyner, A. and Fuchs, E. (1989) Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice. Proc. Natl. Acad. Sci. USA, 86, 1563–1567. Watt, F.M., Kubler, D., Hotchin, N.A., Nicholson, L.J. and Adams, J.C. (1993) Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions. J. Cell Sci., 106, 175–182. Williamson, T.L., Bruijin, L.I., Zhu, Q., Anderson, K.L., Anderson, S.D., Julien, J.P. and Cleveland, D.W. (1998) Absence of neurofilaments reduces the selective vulnerability of motor neurons and slows disease caused by a familial amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutant. Proc. Natl. Acad. Sci. USA., 95, 9631–9636. Wilson, A.K., Coulombe, P.A. and Fuchs, E. (1992) The roles of K5 and K14 head, tail and R/KLLEGE domains in keratin filament assembly in vitro. J. Cell Biol., 119, 401– 414. Winter, H., Rogers, M.A., Langbein, L., Stevens, H.P., Leigh, I.M., Labreze, C., Roul, S., Taieb, A., Krieg, T. and Schweizer, J. (1997a) Mutations in the hair cortex keratin hHb6 cause the inherited hair disease monilethrix. Nat. Genet., 16, 372–374. Winter, H., Rogers, M.A., Gebhardt, M., Wollina, U., Boxall, L., Chitayat, D., Babul-Hirji, R., Stevens, H.P., Zlotogorski, A. and Schweizer, J. (1997b) A new mutation in the type II hair cortex keratin hHb1 involved in the inherited hair disorder monilethrix. Hum. Genet., 101, 165–169. Winter, H., Labreze, C. Chapalain, V., Surleve-Bazeille, J.E., Mercier, M., Rogers, M.A., Taieb, A. and Schweizer, J. (1998) A variable monilethrix phenotype associated with a novel mutation, Glu402Lys, in the helix termination motif of the type II hair keratin hHb1. J. Invest. Dermatol., 111, 169–172. Xu, Z., Cork, L.C., Griffin, J.W. and Cleveland, D.W. (1993) Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease. Cell, 73, 23–33. Yamanishi, K., Matsuki, M., Konishi, K. and Yasuno, H. (1994) A novel mutation of Ieu122 to Phe at a highly conserved hydrophobic residue in the helix initiation motif of keratin 14 in epidermolysis bullosa simplex. Hum. Mol. Genet, 3, 1171–1172. Yang, J.M., Yoneda, K., Morita, E., Imamura, S., Nam, K., Lee, E.S. and Steinert, P.M. (1997a) An alanine to proline mutation in the 1A rod domain of the keratin 10 chain in epidermolytic hyperkeratosis. J. Invest. Dermatol., 109, 692–694. Yang, J.M., Chipev, C.C., DiGiovanna, J.J., Bale, S.J., Marekov, L.N., Steinert, P.M. and Compton, J.G. (1994) Mutations in the H1 and 1A domains in the keratin 1 gene in epidermolytic hyperkeratosis. J. Invest. Dermatol., 102, 17–23. Yang, Y., Dowling, J., Yu, Q.-C, Kouklis, P., Cleveland, D.W. and Fuchs, E. (1996) An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments. Cell. 86, 655–665.

17. CULTURED KERATINOCYTE GRAFTS AS PROTOTYPES FOR EPITHELIAL MORPHOGENESIS DURING WOUND HEALING CAROLYN C.COMPTON Gastrointestinal Pathology Department, Harvard Medical School Massachusetts General Hospital, Boston, Massachusetts, USA Fax: 617–726–6875; 617–720–0215 HUMAN KERATINOCYTE GRAFTS: BACKGROUND AND DEVELOPMENT In 1975, Rheinwald and Green reported a cell culture method that made rapid in vitro propagation of human keratinocytes possible for the first time. Human keratinocytes had previously proven difficult merely to maintain in tissue culture, and they had never before been serially subcultivated (Andreassi, 1992). Thus, the method represented a significant breakthrough in cell cultivation technology. The culture technique was based on the use of sublethally irradiated 3T3 fibroblast feeder layers and of culture medium supplemented with ingredients such as fetal calf serum, glucocorticoids, cholera toxin (Green, 1978), and epidermal growth factor (Rheinwald and Green, 1977). The system so efficiently supported keratinocyte growth that large quantities of cultured epithelium could be produced within a relatively short period from a small sample of normal skin. In fact, the surface area of a donor skin biopsy could be expanded 5,000- to 10,000fold within two to three weeks by this technique, making it possible to produce sheets of keratinocytes with an aggregate surface area large enough to cover the entire human body from a skin biopsy the size of a postage stamp (Green et al., 1979). In 1979, Green, Kehinde and Thomas reported that a confluent culture of keratinocytes grown by this method could be released intact from the culture vessel by the neutral proteinase Dispase. This enzyme had the ability to selectively disrupt the strong bond between the basal keratinocytes and the culture vessel surface without disrupting intercellular junctions within the cell sheet. The result was a potentially graftable epithelial unit. However, it remained to be demonstrated that these delicate keratinocyte sheets were capable of surviving transplantation to the relatively hostile environment of a living wound. In order to test the performance of human keratinocyte sheets as grafts, short-term pilot studies were performed in athymic mice (Banks-Schlegel and Green, 1980). In these experiments, the cell sheets not only survived transplantation, they appeared to attach spontaneously the surface of a fresh cutaneous wound and to rapidly undergo both stratification and terminal differentiation. The ability to produce graftable sheets of human epidermal cells on demand suggested exciting new possibilities for the treatment of clinical disorders in which skin is in high demand and short supply. In particular, massive burn injury seemed an obvious target for therapeutic use of this readily available source of epithelium. Experimental clinical studies were initiated in burn patients, and the results indicated that cultured epithelial autografts (CEA) produced by the method of Rheinwald and Green could be transplanted successfully to escharectomized fullthickness wounds (O’Connor et al., 1981; O’Connor et al., 1984). Despite the demonstration that clinical use of CEA was feasible, general pessimism about the performance of cultured epithelial grafts or, indeed, any skin graft lacking a dermis remained widespread in the medical arena. Based on long clinical experience with conventional skin grafts, it was widely held that dermis was an absolute requirement for skin graft survival and that postgrafting outcome, both cosmetic and functional

318


was directly proportional to the amount of dermis in the graft. Therefore, on a fundamental level, pure epithelial grafts were considered unacceptable and even untransplantable by the vast majority of surgeons. A turning point was reached, when Gallico et al. (1984) reported the success of the first large-scale therapeutic use of CEA in two massively burned patients. The unprecedented survival of these patients who had sustained deep burns over 98% and 99% of their body surfaces, respectively, demonstrated unequivocally that CEA could be life-saving. Thereafter, CEA produced by the method of Rheinwald and Green began to be used worldwide for burn wound treatment (Eldad et al., 1987; Bettex-Galland et al., 1988; Herzog et al., 1988; Kumagai et al., 1988; DeLuca et al., 1989; Munster et al., 1990; Teepe et al., 1990), and the successful use of CEA for other clinical disorders such as giant congenital nevi (Gallico et al., 1989) began to be reported. In addition to the expanding use of autologous keratinocyte grafts, clinical investigators began to experiment with the use of cultured epithelial grafts derived from allogeneic sources. Considerable clinical and scientific controversy soon arose concerning the antigenicity of cultured keratinocyte grafts in general and the ability of cultured allogeneic grafts, in specific, to survive permanently following transplantation. From the outset, it was universally anticipated that autologous keratinocyte grafts would be immunologically accepted following transplantation and that permanent survival would, at least, be feasible on this basis. However, several investigators reported what appeared to be prolonged or permanent engraftment of allogeneic keratinocyte grafts in patients (Hefton et al., 1983; Thivolet et al., 1986; Faure et al., 1987), and it was hypothesized that alterations in antigen expression, especially class II histocompatibility antigens, occurring in vitro might allow cultured allografts to escape immunologic rejection. It had been demonstrated that human keratinocytes cease to express HLA-DR in culture (Morhenn et al., 1982; Hefton et al., 1984) and that Langerhans cells, which constitutively express HLA-DR, do not survive in vitro cultivation (Stingl et al., 1978). Certainly, the possibility of indefinitely long or permanent survival of cultured allografts was attractive. If the hypothesis were correct, both the labor intensiveness and the high cost of CEA therapy would be greatly reduced, given that many patients could be treated with grafts produced from a single healthy donor. Furthermore, it would be possible to produce allogeneic grafts prospectively so that they could be made immediately available on clinical demand. However, in a carefully controlled clinical study in which the depth of wounds to which cultured allografts were transplanted was documented histologically, it was demonstrated that re-epithelialization occurred only in partial-thickness wounds and never in full-thickness wounds (Mad den et al., 1986). The results suggested that what had previously been interpreted as permanent engraftment of cultured allografts was more likely to have been spontaneous re-epithelialization of wounds containing residual appendigeal structures that had been clinically estimated to be full-thickness in depth. Indeed, it appeared that cultured allografts might accelerate perifollicular outgrowth in partial-thickness wounds, leading to rapid wound resurfacing that might easily be misinterpreted as engraftment of the keratinocyte sheet. Definitive proof that cultured allografts do not survive permanently finally came from studies in which molecular markers were used to document the origin of the epidermis of allografttreated wounds. In all cases, whether analyzed by ABO blood group antigens (Kaawach et al., 1991; Zhao et al., 1992), class I histocompatibility antigens (van Gielen et al., 1987), y-chromosomal material (Brain et al., 1989; Burt et al., 1989; Phillips et al., 1990; Kaawach etal., 1991; Zhao et al., 1992) or DNA “fingerprints” (DeLuca et al., 1989; Phillips et al., 1990; van de Merwe et al., 1990), the epidermis of wounds resurfaced following cultured allograft application was ultimately found to have the molecular profile of the patient rather than that of the graft. However, it was noted that cultured allograft survival was prolonged compared to conventional allogeneic skin grafts (Thivolet et al., 1986).


319

With clarification of the fundamental clinical behavior of CEA, scientific focus on CEA as a unique system for the study of human skin biology increased. Biopsy studies in patients with wounds grafted with CEA and in animal models of CEA grafting began to focus on specific postgrafting biologic events occurring during wound healing and culminating in skin regeneration. In the CEA system, a relatively uniform, preformed confluent sheet of undifferentiated keratinocytes simultaneously contact living stroma upon transplantation and synchronously initiate both basement membrane zone reconstruction and epidermal differentiation. Thus, from a morphogenetic perspective, CEA represented an unprecedented opportunity to define the sequential events in these processes because they could be studied in isolation from epithelial migration for the first time. Epidermal dendritic cell behavior could be studied from new perspectives as well in the CEA system. Initially being devoid of Langerhans cells and Merkel cells as well as being unpigmented, CEA provided a vehicle for the study of both epidermal repopulation by dendritic cells and initiation of melanocyte function. Finally, they constituted an ideal system for studying epithelialconnective tissue interactions during human wound healing. THE CULTURED EPITHELIAL GRAFT Donor Cell Adhesion and Proliferation In Vitro In the first step of CEA production, the donor skin is separated at the dermoepidermal junction, the epidermis minced and trypsinized, and the resultant single cell suspension plated at relatively high density onto sublethally irradiated 3T3 fibroblast feeder layers. Since the initial cell suspension contains keratinocytes from all layers of the epidermis, most of which are either terminally differentiated or irrevocably committed to terminal differentiation and have withdrawn from the cell cycle, only 1%–10% (most commonly, 3%–5%) of the cells plated in the primary cultures have the capacity to proliferate and generate colonies. These clonogenic cells must first attach to the culture flask surface before beginning division, surface adhesion being an absolute requirement for growth common to all normal epithelia. Extracellular matrix proteins including basal lamina glycoproteins and other adhesion molecules laid down on the culture vessel surface by 3T3 fibroblast feeder layer cells may be important in facilitating this process. It has been shown that 3T3 cells deposit a pericellular matrix rich in type IV collagen, laminin, interstitial procollagens, flbronectin and connective tissue glycoproteins (Alitalo, et al., 1982). The major class of receptors by which keratinocytes adhere to extracellular matrix proteins are the β1 integrins, and in both normal epidermis and stratified keratinocyte cultures, β1 integrin expression is largely confined to the basal proliferative layer (Carter et al., 1990; Adams and Watt, 1990; Adams and Watt, 1991; Hertle et al., 1991; Hertle et al., 1992). In normal skin, in fact, down-regulation of integrins may be one of the mechanisms by which epidermal basal cell progeny that have withdrawn from the cell cycle and committed to terminal differentiation are selectively released from the basal layer in the initial phase of their outward movement towards the skin surface. Even among proliferative basal cells, however, constitutive expression of integrins is dependent upon contact with extracellular matrix. With disruption of such contact, loss of integrins from the cell surface occurs over a period of hours. Thus, during CEA production, plating of disaggregated keratinocytes in suspension must be carried out promptly to minimize the loss of surface integrins that might compromise surface adhesion by the colony-forming cells. Once plated under appropriate conditions, however, colony-forming cells attach to the matrix-coated surface and immediately polarize, with integrins responsible for cell-substrate adhesion segregating to the basal surface within 6–12 hours (DeLuca and Cancedda, 1992).

320


Cell division, once initiated in culture, is rapid. When the colonies are small, growth is exponential, and the doubling time of the cells is only about 17 hours. Although the feeder fibroblasts are viable and actively support the growth of the keratinocyte, they do not divide by virtue of their prior radiation exposure. Their exact role in the growth support of keratinocytes is unknown, but it hypothesized that secretion of stimulatory cytokines and growth factors or uptake of growth inhibitory substances produced by the keratinocytes or both of these actions may be involved. As essential as these function might be, however, they are short-lived by default because the feeder cells themselves are quickly undermined by the rapidly growing and ultimately eliminated from the system during periodic exchanges of culture medium (effete medium is drained off and replaced by fresh medium every few days). Although keratinocyte division is initially exponential and remains so while colonies are small, sustained rapid growth is dependent upon stimulation of outward migration of the cells at the colony periphery. Promotion of centrifugal migration is mediated by epidermal growth factor, an additive to the keratinocyte culture medium that also stimulates keratinocyte mitosis (Barrandon and Green, 1987a). Under these ideal conditions, the growing colonies expand radially up to 2 mm per day (De Luca and Cancedda, 1992). Within a few days, when the culture approaches confluence, the cells may then be disaggregated and replated into secondary cultures for further expansion. On serial subcultivation, the cells may be plated at much lower density because the plating efficiency (i.e., the proportion of cells plated that will form colonies) of this preselected population of proliferative cells is far greater than that of cells derived from normal epidermis in steady state. In secondary or tertiary cultures, from 30–70% of the cells will form colonies. Clonal Heterogeneity Among Donor Cells Not all colony-forming cells display equal proliferative potential in culture. Even under optimally defined conditions, some display finite growth capacity and others have very high proliferative potential (Barrandon and Green, 1987b). The heterogeneity among clonogenic cells correlates with current concepts of proliferating populations in the normal epidermis. Although proliferation in normal epidermis is restricted to the basal layer, only a fraction of basal cells have the capacity for continuing self-renewal. These cells give rise to all other cells and are known as stem cells. The daughter cells generated from a stem cell division differ from stem cells in having a limited capacity for division before becoming committed to terminal differentiation. They are known as transit amplifying cells, and their ability to divide is believed to be determined by external influences increasing the demand for epidermal growth (e.g., epidermal injury). With their restricted division potential, they transiently amplify the epidermal cell number, but after a relatively small number of divisions, all the progeny (termed “postmitotic cells”) go on to terminal differentiation and death. In culture, the cells with limited growth potential (no more than 15 cell generations) that form small abortive colonies when isolated and cultivated as clones, termed paraclones (Barrandon and Green, 1987b), are thought to correspond to transit amplifying cells. The clonogenic cells with high proliferative capacity that do not display the presenescent behavior of paraclones have been termed holoclones. These cells form large, round colonies when isolated as clones and can give rise to both additional holoclones and to paraclones, indicating that they are at the origin of the cell lineage. For this reason and because they exhibit a high capacity for self-renewal, holoclones are thought to represent epidermal stem cells. Colony-forming cells with proliferative capacity intermediate between paraand holoclones are known as meroclones. Initial studies have indicated that the holoclone population of normal skin decreases with biologic age (Barrandon and Green, 1987b). This observation would suggest that, overall, the proliferative capacity of CEA produced from young individuals might be superior to that of CEA produced from aged patients by


321

virtue of having a higher proportion of holoclones, but this possibility has yet to be tested. Even if true, however, there are no clinical data to suggest that graft performance is improved as a result. For CEA, the lability of holoclones in culture is a far more important issue than the initial proportion of holoclones in the donor skin epidermis. Holoclones are known to degenerate into mero- and paraclones under suboptimal culture conditions, and widespread degradation of holoclones might be expected to adversely affect graft quality in a critical fashion. The in vitro lability of holoclones mandates that strictly defined culture conditions, ones known to preserve holoclones, be maintained during CEA production. CEA at Transplantation: Morphologic and Biologic Profile The cultures that are to be used as grafts are allowed to grow to confluence or post-confluence over about 7 to 10 days. Just prior to grafting, they are Dispase-released from the flask and washed with serum-free medium to eliminate any (possibly-antigenic) bovine protein and to remove any residual 3T3 cells. Dispase release has two major effects on cell structure. The first is a dramatic change in cell shape throughout the graft. Prior to enzymatic release, the cells in all layers of the graft are markedly attenuated and have flat contours. With detachment from culture vessel surface, the cells spontaneously round up into a cuboidal shape (Figure 1). The overall result is a 50%–60% reduction in the surface area of the graft. The polarity of the cell sheet is maintained, however, and the basal layer is readily discernible by the more uniform polygonal shape and greater basophilia of the cells (Figure 1). The second major effect of Dispase release is the effacement of all basal cell attachment specializations along the plasma membrane. In attached cultures, deposits of extracellular material resembling basal lamina are seen on the plastic surface of the culture flask subjacent to the basal cells, and collections of electron-dense material resembling the attachment plaques of immature hemidesmosomes appear along the cytoplasmic surface of the plasma membrane of basal cells (Compton et al., 1989). Basal cell attachment plaques are associated with slender bands of extracellular electron-dense material located approximately 10 nm from the outer leaflet of the plasma membrane that correspond to hemidesmosomal sub-basilar dense plaques (Figure 2). These attachment specializations have been termed “stable anchoring contacts” and have been shown to contain both α6β4 integrin and bullous pemphigoid antigen (Watt and Hertle, 1994), demonstrating their analogy to hemidesmosomes. With Dispase detachment, these primitive hemidesmosomal junctions are no longer visible at the cell surface on ultrastructural examination (Compton et al. 1989). However, it is possible that they are merely internalized within the cell as they are in intact epidermis upon Dispase-detachment from its basement membrane (Poumay et al., 1994). Strong staining for both α6β4 integrin and bullous pemphigoid antigen, structural components of hemidesmosomes (Stepp et al., 1990), is seen circumferentially along the plasma membranes of basal cells of released grafts by immunohistochemistry (Figure 3). In addition, many suprabasal cells also express α6β4 integrin (Figure 3). In contrast, there is no ultrastructural evidence of anchoring fibril formation in vitro, and immunohistochemical stains for collagen VII are typically negative. Histologically, released cell sheets vary from about 3–9 cells layers in thickness (Figure 1). The confluent cell sheet is not uniformly stratified because the processes of differentiation and stratification begin first in the centers of expanding colonies and occur later at the colony peripheries. Although the basal layer is well-defined and well-oriented, the suprabasal layers are less well organized and typically show little or no terminal differentiation (Compton et al., 1989). Granular and cornified cell layers are typically lacking, but occasional rare superficial cells may show keratohyaline granule formation and exhibit filaggrin expression by immunohistochemistry. Immunohistochemical stains of CEA for epidermal differentiation markers reveal aberrant patterns of expression. For example, in normal skin, expression of involucrin, one of the protein elements of the cross-

322


Figure 1 Dispase-released cultured keratinocyte graft. The cell sheet is only three to four cell layers thick in this area and shows no granular or cornified layer differentiation. After release, the cells assume a cuboidal shape, and the cells basal layer is discernible by its ventral polarization, greater basophilia and smaller average cell size.

linked envelope of cornified cells, is restricted to superficial cells (i.e., those of the upper stratum spinosum and stratum granulosum). In CEA, however, involucrin expression is premature and is seem in all cells immediately above the basal layer. Similarly, normal expression of transglutaminase, the enzyme that crosslinks the proteins of the cornified envelope, is also limited to cells in the superficial epidermis (granular cells), but in CEA, cells in every layer of the stratified sheet express this protein (Figure 4). Cytokeratin expression is also altered in CEA. Cytokeratins 5 and 14, the major cytoskeletal structural proteins of the basal layer, are abruptly down-regulated in the suprabasal cells in normal skin. In CEA, expression of this keratin pair is appropriately strong in the basal layer, but it persists in suprabasal cells throughout all layers of the graft. In contrast, cytokeratins 1 and 10, the major keratins in the suprabasal layers of the normal epidermis, are expressed erratically, if at all, in CEA. By immunohistochemical staining, sparse numbers of single cells in random distribution within the suprabasal layers of the graft may be see to strongly express keratin 10, but expression of keratin 1 is rarely seen at all. Instead, keratins 6 and 16 (“hyperproliferative” keratins) are expressed in the suprabasal keratinocytes of the graft, but expression is patchy rather than uniform in distribution. Even in the presence of normal connective tissue matrix or cellular elements, cultured keratinocytes show aberrant expression of differentiation markers and cytokeratins. Panepithelial expression of keratin 14 and premature expression of involucrin also occur in organotypic cultures of keratinocytes in which they are either cocultured with dermal cells (fibroblasts or endothelial cells) or placed atop de-epidermized dermis (Fusenig, 1994). Although it has been suggested that keratin 1 expression is greater when cultured keratinocytes are grown on connective tissue substrates (i.e., dermis or collagen), the expression of keratins 1 and 10 is delayed and dyssynchronous even in this setting. Many of these same alterations in keratinocyte differentiation are also seen in vivo in hyperplastic skin diseases and in normal epidermis during wound healing (Fusenig, 1994). Thus, it is appears that the dysregulation of keratinocyte differentiation that occurs


323

Figure 2 Ultrastructure of the attachment face of a keratinocyte graft in culture. The attachment face (ventral surface) of the basal cell shows attachment specializations that resemble primitive hemidesmosomes, but no anchoring fibrils are seen.

in vitro is not significantly altered by connective tissue interactions may be an intrinsic property of keratinocytes in hyperproliferative states. CEA produce an array of cytokines (Table 1) that are known to play key roles in the regulation of autocrine and paracrine cellular responses (McKay and Leigh, 1991; Stoof et al., 1994). They also express a variety of other substances, such as enzymes and adhesion molecules, that mediate specific biologic processes known to be important in wound healing (Table 2). For the cytokines, however, immunohistochemical staining patterns are variable, both regionally within a single graft and among different CEA. Both qualitative and quantitative variations in expression of some cytokines, like TGFα and TGFβ1, can be seen among CEA generated from skin of different body-site origins. This observation is confirmed by quantitative Northern blots of the corresponding mRNAs for these cytokines, suggesting intrinsic body-site specific differences in cytokine expression potentials. In contrast, the biologic age of the keratinocytes does not appear to substantively affect cytokine expression. For example, comparable amounts of TGFα mRNA are expressed in CEA generated from the same sun-protected body site of males aged 7 months to 82 years (Compton et al., 1995). Expression of TGFβ1 mRNA Table 1 Cytokines Expressed by Cultured Human Keratinocytes Amphiregulin (AR) Basic fibroblast growth factor (bFGF) Granulocyte-Colony stimulating factor (G-CSF) Granulocyte/macrophage-Colony stimulating factor (GM-CSF) Human growth factor (huGRO) Interferon-γ-induced protein (γ-IP-10) Interleukin-1α (IL1α)

324


Figure 3 Distribution of α6β4 integrin in Dispase-released cultured keratinocyte graft. By immunohistochemistry, circumferential plasma membrane staining for α6β4 integrin can be seen on basal cells and some suprabasal cells. Interleukin-1β (IL1β) Interleukin-6 (IL1) Interleukin-8 (IL1) Interleukin-10 (IL10) Macrophage chemotactic and activating factor (MCAF) Macrophage-Colony stimulating factor (M-CSF) Nerve growth factor (NGF) Platelet-derived growth factor AA (PDGF-AA) Platelet-derived growth factor BB (PDGF-BB) Transforming growth factor α (TGFα) Transforming growth factor β1 (TGFβ1) Transforming growth factor β2 (TGFβ2) Transforming growth factor β3 (TGFβ3) Tumor necrosis factor α (TNFα) Vascular endothelial growth factor (VEGF) Table 2 Other Products of Human Keratinocytes in Culture with Potential for Modulating Wound Healing Collagen IV Collagen VII Collagenase Fibronectin Glycosaminoglycan


325

Laminin Matrix metalloprotease-1 (MMP-1) Matrix metalloprotease-3 (MMP-3) Plasminogen activating factor (PA) Tissue inhibitor of metalloproteases (TIMP) Thrombomodulin Thrombospondin

Figure 4 Premature expression of transglutaminase in cultured keratinocyte graft. By immunohistochemistry, inappropriate expression of transglutaminase, a marker of terminal differentiation in normal epidermis, is seen throughout all layers of the cell sheet.

in CEA is similarly unaffected by the age of the donor keratinocytes (Compton et al., 1994). CEA that have been generated from non-acral skin contain variable numbers of melanocytes, that appear unevenly distributed amongst the basal keratinocytes when visualized by tyrosine-DOPA staining. Melanocytes not only survive but proliferate in coculture with keratinocytes, and keratinocytes play an active role in inducing melanocyte growth. However, frozen storage of epidermal cell suspensions between passages has a detrimental affect on the accompanying melanocyte population. Although cryopreservation has little impact on keratinocyte viability or function (e.g., plating efficiency) and is a technique commonly used to coordinate CEA production with clinical grafting schedules, it decreases both melanocyte number and function in the progeny CEA. Melanosome transfer between melanocytes and keratinocytes will begin in vitro in fresh cultures (DeLuca et al., 1988), but in cultures generated from frozen cells, melanosome transfer is delayed, often for many weeks after graft transplantation (Compton et al., 1989).

326


POST-TRANSPLANTATION EVENTS IN SKIN REGENERATION Epidermal Differentiation CEA differentiate rapidly after transplantation. Within 3–5 days postgrafting, CEA begin to resemble normal epidermis on clinical inspection, the result of opacity to light acquired as a result of the dramatic increase in graft thickness and the differentiation of both granular and cornified cell layers (Figure 5). Rapid differentiation is important clinically because the fluid-loss and bacterial-invasion barrier functions of epidermis are provided primarily by the terminally differentiated cell layers. Although fully differentiated, the grafts are moderately hypertrophic at this stage, about twice normal thickness. The basal contour of the new epidermis varies from jagged to flat, but true rete ridges are lacking. Jagged contours at the interface with the wound bed typically even out within a week or two (Figure 6) and remain flat until rete ridges are regenerated months later (Compton et al., 1989; Compton 1992; Compton, 1996). Commonly, parakeratosis and/or hyperkeratosis, corresponding to mild aberrations in epidermal differentiation, may occur during the first week or two after grafting, but typically resolve spontaneously unless the wound site is irritated or inflamed (Compton et al., 1989). Increased rates of cell division and aberrations in biochemical markers of keratinocyte differentiation characterize the early postgrafting period for CEA. After transplantation of CEA, premature expression of involucrin in the immediate suprabasal layer persists for several weeks to months (Figure 7) , but normalization of filaggrin expression occurs rapidly after grafting and coincides with the appearance of a granular layer in the differentiating graft. Keratinocyte hyperproliferation is reflected in the large number of mitotic figures within the basal layer (Figure 5) and the increased proportion of basal cells expressing Ki67, a nuclear antigen expressed only by actively cycling cells. Only 5–10% of basal cells in normal steady-state skin express this proliferation marker. In contrast, approximately one third to one half of all basal cells in CEA express Ki67 in the early postgrafting period (Figure 8). The increased proliferative activity characteristic of epidermis newly regenerated from CEA is relatively short-lived, however, and by 3 to 4 months postgrafting, the proportion of basal cells actively engaged in the cell cycle is normal. In contrast to actual hyperproliferation in CEA, however, expression of “hyperproliferative” keratins (i.e., cytokeratins 6 and 16) is extremely protracted in transplanted CEA. Immunostaining for cytokeratins 6 and 16 is strong and diffuse throughout the newly formed epidermis from the earliest postgrafting time points (Figure 9) and may persist for as long as one year or more postgrafting (Compton, 1996). In some cases, hyperproliferative keratin expression is seen up to two years after grafting and, in burn patients, may correspond to continuing irritation at the graft site from compression garments or other therapeutic maneuvers. Normalization of the profile of expression of the major epidermal cytokeratins within the newly regenerated epidermis is correspondingly slow. During the first month postgrafting, immunohistochemical staining shows strong expression of basal cell cytokeratins 5 and 14 throughout the epidermis (Figure 10). The generation of suprabasal cytokeratins 1 and 10 is typically slow and discordant (Figure 11). Expression of keratins 5 and 14 recedes to the basal layer by about 6 to 12 months, but normal suprabasal patterns of expression of keratins 1 and 10 may not be seen for more than a year postgrafting, coincident with regressing expression of keratins 6 and 16. Attachment to the Wound Bed and Basement Membrane Zone Formation Like keratinocyte adhesion to the culture flask after plating, initial adhesion of CEA to the wound bed stroma postgrafting is mediated by integrins displayed on the basal cells. Therefore, if transplantation is carried out


327

Figure 5 Cultured epithelial autograft at 6 days postgrafting. A hyperplastic, fully stratified epidermis has regenerated from the cultured graft. The epidermis shows mitotic figures in the basal layer, consistent with hyperproliferation.

rapidly following the release of the grafts from the culture flask (before β1 integrins would be lost from the contact face of the basal cells), immediate adhesion is observed. In fact, this is observed in nude mice grafting models in which the time from graft release to surgical application is controlled and striping of the graft from wound shortly after application can be carried out without clinical consequence. In this setting, grafts become very difficult to dislodge from the wound without within 30 to 60 minutes following transplantation (Compton, unpublished results). Permanent attachment to the wound bed, however, requires the reformation of all the structural elements of the basement membrane zone as well as the generation of a normal complement of hemidesmosomes. As judged electron microscopically by the structural maturity of the nascent elements, the process is largely complete within a few weeks postgrafting (Figure 12) (Compton et al., 1996). However, morphometric studies have indicated that some aspects of anchoring fibril regeneration take years to fully complete (see below). At the time of grafting and for 24 to 48 hours thereafter, there is no definite evidence of hemidesmosomal structure along the attachment face of the graft by ultrastructural examination. Beginning at about day 3 postgrafting, however, condensations of electron material resembling dense plaques appear focally along the plasma membrane and by one week postgrafting, small, but fully formed hemidesmosomes can be definitively identified (Regauer et al., 1990). Similarly, collections of electron dense material consistent with basal lamina are seen extracellularly at the attachment face of the graft beginning at about day 3 postgrafting and some show perpendicular insertion of slender anchoring fibrils (see below). The newly forming hemidesmosomes, basal lamina and anchoring fibrils all appear together in these discontinuous foci along the attachment face of the graft. The finding suggests that their formation is somehow linked and that their generation is initiated simultaneously at these “nucleation foci”. By 3–4 weeks postgrafting, the basal lamina reaches confluency, and hemidesmosomes appear fully mature and show cytoplasmic tonofilament insertion (Figure 12) (Compton et al., 1989).

328


Figure 6 Cultured epithelial autograft at 6 days postgrafting. The subepithelial connective tissue consists of collagen-poor, glycosaminoglycan-rich granulation growing up from the underlying muscle fascia. The epidermis has a slightly irregular basal contour but no rete ridges.

At 1 week postgrafting, immunohistochemical localization of type VII collagen, the major structural component of anchoring fibrils, shows nearly continuous staining along the CEA attachment face. However, by electron microscopy, the newly formed anchoring fibrils are extremely thin and are recognizable only by their characteristic periodicity. Despite their structural immaturity, the nascent anchoring fibrils demonstrated a normal pattern of ultrastructural immunolabeling for type VII collagen at insertion points into the lamina densa and subjacent anchoring plaques (Figure 12) (Regauer et al., 1990; Compton et al., 1996). Morphometrical analysis has shown that, as early as 2 weeks postgrafting, anchoring fibril population densities are equal to those of normal site-matched skin and remain normal thereafter through 6 years of follow-up. However, until 3 years postgrafting, anchoring fibril diameters are narrow compared to those of normal skin. The depth of extension of anchoring fibril-anchoring plaque lattices into the subjacent stroma is normal by 3 weeks but, after 1 month, is typically 1.5- to 2-fold greater than normal (Compton et al., 1996). Overall, studies of anchoring fibril regeneration by CEA have indicated that: (1) an entire population of anchoring fibrils is formed immediately, in what appears to be a preprogrammed fashion, by basal keratinocytes following contact with living stromal tissue; (2) once reformed, anchoring fibril populations remain normal in density over time; (3) nascent anchoring fibrils are thin and require several years to reach full maturity; and (4) abnormally thick anchoring fibril lattices may form over time in the skin regenerated from CEA, at least when grafted to full-thickness wounds. Donor Site-Specific Differentiation of Transplanted CEA Reexpression of site-specific epidermal differentiation after transplantation has been demonstrated in CEA derived from sole skin grafted to various diverse body sites. Sole and palm skin both have a highly distinctive phenotype and are unique in their expression of cytokeratin 9. These unusual site-specific differentiation features can be identified easily by objective criteria. As judged by the reappearance of these


329

Figure 7 Involucrin expression in a cultured epithelial autograft at 2.5 months postgrafting. By immunohistochemistry, an abnormal pattern of involucrin expression in all suprabasal layers can be seen.

features, sole-derived CEA have been shown to reexpress their native epithelial phenotype within a few weeks postgrafting and maintain that phenotype long-term through 6 years of follow-up. Although evident by histological criteria alone, reexpression of sole-specific phenotype has been confirmed by immunohistochemistry and immunoblotting using a monospecific antibody to cytokeratin 9 (Compton, 1992; Compton, 1993; Compton et al., 1998). These studies indicate that site-specificity may be an intrinsic property of epithelium and that, in a permissive environment, epidermal cells have the capacity to express a site-specific phenotype in the absence of dermis. Therefore, for the best cosmetic and functional outcome when used clinically, CEA should be generated from donor skin with the same phenotype desired in the graft-derived skin. Dendritic Cell Repopulation (1) Langerhans Cells Cutaneous Langerhans cells are bone marrow derived dendritic cells that participate in cutaneous immune responses as antigen-presenting cells. In normal skin, the density of intraepidermal Langerhans cells ranges from about 6 to 20 (mean 11) Langerhans cells/mm of epidermis. (Horton, Allen and MacDonald, 1984; Compton, Kupper and Nadire, 1996) In organ cultures of normal skin, Langerhans cells may persist in diminished numbers, but in cell culture, they do not survive at all. Thus, CEA contain no Langerhans cells at the time of grafting. However, immunohistochemical studies using antibody to CD1a, a T cell ligand strongly expressed by Langerhans cells, demonstrate rapid repopulation of CEA by Langerhans cells after transplantation, the result of transmigration from underlying connective tissues, lymphatics, or blood vessels. Langerhans cells appear in small numbers in the CEA-derived epidermis at 5 days postgrafting, the earliest biopsy time point in studies of this phenomenon. However, complete restoration of normal

330


Figure 8 Proportion of cycling cells within a cultured epithelial autograft at 6 months postgrafting. Immunolocalization of Ki67 (nuclear staining) reveals that approximately every third cell within the basal layer is engaged in the cell cycle, a dramatic increase over the normal proportion of cycling basal cells (about one per 10–20 basal cells).

population densities takes months (Figure 13). In healthy patients (e.g., those with giant congenital nevi) normal densities are reached within 2 to 6 months. In debilitated patients (e.g., those with massive burns), the time required for complete epidermal repopulation by Langerhans cells is prolonged and may take more than a year. Once repopulation is complete, the population density of Langerhans cells remains normal thereafter, through a follow-up period of 11 years (Compton, Kupper and Nadire, 1996). (2) Melanocytes The number and distribution of melanocytes present in CEA at the time of grafting is variable, due in large part to freezing of cells between passages during graft production (see above). Scarcity of melanocy tes and uneven distribution is typical in grafts generated from cells that have been subjected to freezing. At the time of grafting, CEA rarely show evidence of active melanin transfer to keratinocytes, and even following transplantation, functional epidermal-melanin units may be slow to be established (Figure 14). Thus, epidermal pigmentation may take several weeks to months to develop in transplanted CEA (Compton et al., 1989). However, when fully restored, the melanin retention pattern in the epidermis is usually normal and racially appropriate, and the normal pattern, once attained, remains normal long-term (Figure 15). However, areas of hyper- or hypopigmentation may occur in some cases. Hyperpigmentation, with abnormal persistence of melanin within the superficial epidermal layers is usually transient unless the epidermis is chronically exposed to ultraviolet irradiation. In contrast, regional hypopigmentation is usually permanent, and on biopsy, the uninvolved skin area is typically devoid of melanocytes (Compton, Warland and Kratz, 1998). The findings suggest that melanocytes have a limited capacity for lateral movement in epidermis and will not freely redistribute themselves either in CEA or in CEA-derived epidermis. This behavior is not


331

Figure 9 Expression of the hyperproliferative keratins 6 and 16 in a cultured epithelial autograft at 1 month postgrafting. By immunohistochemistry, strong diffuse suprabasal expression keratins 6 and 16 can be seen within the newly regenerated epidermis.

restricted to melanocytes in CEA. Clinical examples of the failure of melanocytes to migrate laterally into areas of depigmentation caused by cutaneous injury are numerous. (3) Merkel Cells Merkel cells are epidermal neuroendocrine cells. In normal skin, their distribution is variable, and their density is body site-dependent, being greatest in skin specialized for sensory perception such as the palms, fingertips, and soles. Merkel cells are innervated by sensory nerve fibers, forming Merkel cell-neurite complexes that are believed to act as slowly adapting mechanoreceptors. They contain cytoplasmic densecore secretory granules, suggesting a neuroendocrine function, but the express functional role of human Merkel cells is poorly defined. Merkel cells are absent from CEA prior to transplantation but can be seen in epidermis regenerated from CEA as early as 3 weeks postgrafting using immunohistochemical staining (for cytokeratin 18 or neuron-specific enolase) or by electron microscopy (Compton et al., 1990). The observed appearance of Merkel cells in CEA represents the first documented example of de novo Merkel cell differentiation in post-natal life. It constitutes direct evidence that Merkel cells derive from neuroendocrine differentiation of epithelial cells and disproves the hypothesis that Merkel cells are of neural crest origin, as has been previously thought. The nascent Merkel cells are characteristically located in the basal layer or between the basal layer and the basement membrane, usually occurring singly but sometimes in pairs or clusters (Compton et al., 1990). In contrast to the dendritic appearance of Merkel cells in age- and body-site-matched normal skin, Merkel cells in epidermis regenerated from CEA are round to ovoid and lack dendrites. Overall, the average density of regenerated Merkel cell populations in CEA is lower than that of normal sole skin, but the ranges overlap broadly. Intriguingly, Merkel cell differentiation has been observed to date only in CEA derived from sole skin (Compton et al., 1990). Despite extensive examination by immunostaining, Merkel cells have not been

332


Figure 10 Inappropriate suprabasal expression of keratins 5 and 14 in a cultured epithelial autograft at 2.5 months postgrafting. By immunohistochemistry, the CEA shows strong suprabasal expression of the keratin pair that is confined largely to the basal layer in normal epidermis.

found in the epidermis regenerated from CEA derived from axilla, thorax, thigh, back, buttock, or groin skin over a 6-year follow-up period. This suggests that the capacity for neuroendocrine differentiation is a sitespecific property of sole-derived stem cells and may account for the comparatively marked Merkel cell density in soles (and palms) compared to other body sites. Although reasonable to suspect that palm-derived CEA might also manifest de novo Merkel cell differentiation, they are rarely produced for clinical use and have never been studied. Another unexpected aspect of Merkel cell differentiation in CEA is its seemingly complete independence from neural influences (Compton et al., 1990). The intimate structural association of sensory neurites and Merkel cells in normal skin would suggest a critical interactive function or, possibly, an essential interdependency. However, nerves do not regenerate normally in the connective tissue “neodermis” subjacent to CEA and never re-innervate the regenerated epidermis (see below). Thus, Merkel cells arise and survive in CEA in the complete absence of innervating neurites and, in most cases, in the absence of any neighboring neural structures. The findings demonstrate that neither the initiation nor the maintenance of Merkel cell differentiation in skin depends on innervation. However, it is possible that the development of dendritic morphology may be innervation dependent. Rete Ridge Formation in CEA and Regeneration of Derails from Wound Bed Granulation Tissue A dynamic relationship clearly exists between CEA and the connective tissue stroma to which it is transplanted. CEA have a profound modulatory impact on the stroma, and the stroma is, in turn, capable of markedly altering CEA function. The effect of CEA on stroma has been most clearly illustrated by the


333

Figure 11 Weak, irregular suprabasal expression of keratin 1 in a cultured epithelial autograft at 1 month postgrafting. Keratin 1, the major structural protein of the suprabasal strata of normal epidermis, is slow to be expressed in CEA postgrafting.

changes induced over time in the granulation tissue that forms the base of the full-thickness wounds to which CEA have been transplanted (Figure 6). Over the first 6–8 weeks postgrafting, this immature, undifferentiated connective tissue matures into normal scar (Figure 16). At about 6 months to a year postgrafting, CEA develop rete ridges and the subjacent scar tissue begins to remodel to produce a bilayered distribution pattern of collagen resembling that of the papillary and reticular dermis. In the superficial neodermis, capillary arcades develop that interdigitate with the nascent rete ridges of the CEA-derived epidermis, reproducing the vascular architecture of normal papillary dermis (Compton et al., 1989). Full maturation of the neodermis is a slow process, however. For several years postgrafting, the CEA neodermis lacks elastic tissue and is, therefore, structurally incomplete. Not until about 5 years postgrafting, does elastin begin to appear in the CEA neodermis, but when it does regenerate, its distribution is comparable to that of normal dermis (i.e., relatively scant in the superficial dermis but abundant in the deep dermis). Controlled prospective studies have shown that regeneration of structurally normal dermis from granulation tissue is a phenomenon uniquely associated with CEA and does not occur with standard meshed split-thickness skin grafts (Compton, 1992; Compton, 1996). The ability of CEA to induce dermal regeneration may be linked to their ability to regulate tissue growth, differentiation, and inflammatory reactions through the production of cytokines and other biologically active molecules (see above). Theoretically, aspects of this function that could alter outcome in wound healing include the tempo of production and release of modulatory molecules by CEA after contacting the wound, the absolute amounts of each product elaborated by the graft, and the relative proportions of molecules with antagonistic or synergistic activities that are released simultaneously by the graft. In studies of CEA transplanted to subcutaneous wounds in athymic mice, harvested at short intervals (i.e., 30 min, 1, 3, 6, 12, and 24 hr, and 2, 4, 7, and 15 days), and lysed to measure the intracellular content of TGFα, TGFβ1, and bFGF, it has been shown that patterns of cytokine expression by cultured keratinocytes after transplantation to a wound are highly orchestrated. Initially, the intracellular content of TGFα is high but falls steadily up to

334


Figure 12 Ultrastructure of the attachment face of a cultured keratinocyte autograft at 4 weeks postgrafting. A complete basal lamina has reformed, hemidesmosomes are fully mature, and slender anchoring fibrils are seen. The nonhelical poles of the anchoring fibrils and the anchoring plaques into which they insert are labeled by an immunogold technique using an antibody to human type VII collagen.

2 days postgrafting when it again rises and remains steady at about one third of the in vitro value. This pattern indicates rapid release of TGFα in the immediate postgrafting period. Intracellular content bFGF varies little over the same time frame, indicating either little output or balanced production and release. In contrast, TGFβ1 levels rise 7-fold by day 4 but decrease to 4-fold greater than in vitro levels by day 14, indicating rapid production but slow release of this cytokine (Compton, unpublished results). Exactly how these patterns of cytokine expression might alter the wound healing process is unknown at present, but it is reasonable to hypothesize that they may contribute to the wound healing promotional impact of cultured grafts, both autologous and allogeneic, that has been observed clinically. Just as CEA modulate connective tissue biology, the stroma to which CEA are transplanted has the reciprocal ability to alter graft differentiation and maturation. This dynamic interaction has been demonstrated by both clinical studies and animal models in which CEA have been grafted to different types of substrates. In patients, normal human dermal matrix (derived from cadaver allodermis that is allowed to engraft in the wound bed) markedly accelerates both the normalization of cytokeratin programs and the regeneration of rete ridges in CEA after grafting to full-thickness wounds (Compton et al., 1993). The overall “take” rate of CEA is also improved by allodermis (Odessey, 1992; Hickerson et al., 1994). In porcine animal models, similar improvements in clinical outcome and graft maturation are also obtained with wound preparation using allodermis (Kangesu et al., 1993; Navsaria et al., 1994). Synthetic dermal substrates constructed of collagen and glycosaminoglycans and engineered to have biomechanical properties similar to those of dermis have also been shown to increase CEA engraftment rates and to accelerate cultured graft maturation (Orgill et al., 1998).


335

Figure 13 Langerhans cells in cultured epithelial autograft at 1 year postgrafting. A normal population density of Langerhans cells is seen within the epidermis regenerated from cultured epithelial autografts in a burn patient.

Graft Reinnervation Immunohistochemical studies of nerve regeneration using antibodies to neurofilament (NF) subunits (NF200, NF160, NF68), nerve growth factor receptor, and tyrosine hydroxylase (sympathetic autonomic nerves) have shown that a normal cutaneous innervation pattern is not reestablished in the skin regenerated from CEA grafted to full-thickness wounds. The NF-positive, tyrosine hydroxylase-negative nerves considered to be sensory fibers appear in a perivascular location in the superficial wound bed beneath CEA within 4–8 weeks postgrafting and persist for years. However, free superficial dermal and intraepithelial nerve endings consistent with sensory nerves, as seen in normal age- and site-matched skin, do not regenerate. Neurophysiological tests of sensory nerve function in skin regenerated from CEA have confirmed that the structural abnormality is also associated with dysfunction. Thick fiber sensory function (vibratory sensation) is found to be fully reconstituted within 3 years postgrafting, but fine fiber sensory function (temperature sensation) remains subnormal within this time frame (Compton, 1996). Sympathetic autonomic nerves, identified by immunohistochemical labeling using antibody to tyrosine hydroxylase, are observed only in the deep regions of the wound bed or neodermis but never in superficial subepithelial regions. However, tests of micro-circulatory function have shown no abnormalities in skin regenerated from CEA. Stability of CEA-Derived Skin on Long-term Follow-up Long-term follow-up studies spanning 11 years have demonstrated that the skin which regenerates from CEA is structurally stable. The CEA neodermis maintains its histologically normal appearance long-term (Figure 17), and in serial biopsies of a single CEA graft-site, the elastin content of the extracellular matrix either remains stable or increases slightly over time (Figure 18). The epidermis also maintains its fully mature histologic appearance but is devoid of appendages (i.e., hair follicles and eccrine glands). Although epidermal appendages never regenerate from CEA, they also fail to regenerate in healed interstices of

336


Figure 14 Melanocytes in cultured epithelial autograft at 1 week postgrafting. By argentaffin staining, melanocytes can be seen to contain melanin (black granules) but no melanin transfer to surrounding keratinocytes is observed.

standard meshed split-thickness skin grafts that re-epithelialize via epithelial migration. In fact, regeneration of epidermal appendages from undifferentiated post-natal human keratinocytes has never been demonstrated in any experimental or clinical setting. It is possible that post-natal keratinocyte stem cells are no longer fully pluripotential and, unlike their fetal counterparts, lack the ability to form appendage buds. Alternatively, it is possible that the cells are capable of giving rise to appendages, but the necessary inductive environment and/or the signals for appendage bud formation are lacking. Apart from the absence of appendages, the epidermis of CEA is histologically indistinguishable from that of body site-matched (i.e., derived from the same body site as the CEA) normal controls. The epidermal is normal in thickness and the epidermal strata are normally proportioned. Normal differentiation is evidenced by the restriction of involucrin expression to the upper epidermal strata (Figure 19) and by a normal pattern of cytokeratin expression, with keratins 5 and 14 confined to the basal layer (Figure 20) and keratins 1 and 10 diffusely present throughout the suprabasal layers (Figure 21). A normal proportion of cycling cells is seen in the basal layer (Figure 22), and no indications of premature aging (atrophy), such as flattening of rete ridges or epidermal thinning, are present. Even in sun-exposed areas, no cytologic atypia is seen in the CEA-derived epidermis. To date, there have been no reports of either dysplasia or neoplastic transformation in skin regenerated from CEA, suggesting that CEA are relatively genetically stable, both in vitro and posttransplantation.

CONCLUSIONS Cultured keratinocyte grafts have not only revolutionized the treatment of acute life-threatening wounds, they have provided an opportunity to study the biology of epithelial (and dermal) morphogenesis during wound healing in human beings from a completely unique perspective. Studies of these grafts have provided


337

Figure 15 Repigmented cultured epithelial autograft at 11 years postgrafting. Argentaffin stains reveal a normal Caucasian pattern of epidermal pigmentation in this CEA from a white patient at long-term follow-up.

new insights into the biological behavior of keratinocytes and have focused attention on the dynamic role of epithelium in the wound healing process. Prior to the advent of cultured keratinocyte grafting, both clinical and experimental wound healing studies were primarily focused on connective tissue. Frequently, the epithelium was either viewed as a passive bystander in the process or was ignored altogether. The epithelium is now seen as having primary, direct regulatory functions in a host of regenerative and developmental processes. Its potential for modulating wound healing has lead to its use as a topical agent for the treatment of problematic wounds. Its ready availability and its accessibility post-transplantation have made it attractive as a possible vehicle for gene therapy. The keratinocyte graft continues to be a biologically fascinating and clinically exciting entity. REFERENCES Adams, J.C. and Watt, F.M. (1990) Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes α5β1 integrin loss from the cell surface. Cell, 63, 425–435. Adams, J.C. and Watt, F.M. (1991) Expression of β1, β3, β4 and β5 integrins by human epidermal keratinocytes and nondifferentiating keratinocytes. J. Cell Biol., 115, 829–841. Alitalo, K., Kuismanen, E., Myllala, R., Kiistala, U., Asko-Seljavaara, S. and Vaheri, A. (1982) Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells. J. Cell Biol., 94, 497–505. Andreassi, L. (1992) History of keratinocyte cultivation. Burns, 18 (Suppl.), S2–S4. Banks-Schlegel, S. and Green, H. (1980). Formation of epidermis by serially cultivated human epidermal cells transplanted as an epithelium to athymic mice. Transplantation, 29, 308– 313. Barrandon, Y. and Green, H. (1987a) Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-α and epidermal growth factor. Cell, 50, 1131–1137.

338


Figure 16 Neodermis of CEA at 6 months postgrafting. An elastic tissue-trichrome stain shows a dense type I collagen (blue-stained) in lamellar array, a random distribution of blood vessels and a lack of black-staining elastin. The features are histologically characteristic of normal scar. Barrandon, Y. and Green, H. (1987b) Three clonal types of keratinocytes with different capacities for multiplication. Proc. Natl. Acad. Set,, USA, 84, 2303–2306. Bettex-Galland, M., Slongo, T., Hunziker, T., Wiesmann, U. and Bettex, M. (1988) Use of cultured keratinocytes in the treatment of severe burns. Z. Kinderchir., 43, 224–228. Brain, A., Purkis, P., Coates, P., Hackett, M., Navsaria, H. and Leigh, I.M. (1989) Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed associated with probe specific for Y chromosome. Br. Med. J., 298, 917–919. Burt, A.M., Pallet, C.D., Sloane, J.P., O’Hare, M.J., Schafler, K.F., Yardeni, P., Eldad, A., Clarke, J.A. and Gusterson, B.A. (1989) Survival of cultured allografts in patients with burns assessed with probe specific for Y-chromosome. Br. Med. J., 298, 915–917. Carter, W.G., Wayner, E.A., Bouchard, T.S. and Kaur, P. (1990) The role of integrins α2β1 and CX^pj in cell-cell and cell-substrate adhesion of human epidermal cells. J. Cell Biol., 110, 1387–1404. Compton, C.C. (1996) Cultured keratinocyte grafts for burn wound resurfacing: review of observations from an 11-year biopsy study. Wounds, 8, 125–133. Compton, C.C. (1992) The biology of cultured epithelial autografts: an eight year study in pediatric burn patients. Eur. J. Pediatr. Surg., 2, 216–222. Compton, C.C. (1993) Wound healing potential of cultured epithelium. Wounds, 5, 97–110. Compton, C.C., Gill, J.M., Bradford, D.A., Regauer, S., Gallico, G.G. and O’Connor, N.E. (1989) Skin regenerated from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. Lab. Invest., 60, 600–612. Compton, C.C., Hickerson. W., Nadire, K.B. and Press, W. (1993) Acceleration of skin regeneration from cultured epithelial autografts by transplantation to homograft dermis. J. Bum Care Rehabil., 14, 653–662. Compton, C.C., Kupper, T.S. and Nadire, K.B. (1996) HIV-infected Langerhans cells constitute a significant proportion of the epidermal Langerhans cell population throughout the course of HIV disease. J. Invest. Dermatol., 107, 822–826.


339

Figure 17 Cultured epithelial autograft at 11 years postgrafting. The epidermis lacks appendages but is normal in thickness, has a normal rete ridge pattern and shows a remodeled neodermis. Compton, C.C., Nadire, K.B., Regaur, S., Warland, G., Landry, D.B., O’Connor, N.E. and Gallico, G.G., III. (1998) Cultured human sole-derived keratinocyte grafts re-express site-specific differentiation after transplantation. Differentiation, 64, 45–53. Compton, C.C., Press, W., Gill, J.M., Bantick, G., Nadire, K.B., Warland, G., Fallon, J.T., III and Vamvakas, E.C. (1996) The generation of anchoring fibrils by epidermal keratinocytes: a quantitative long-term study. Epith. Cell Biol., 4, 93–103. Compton, C.C., Regauer, S., Seiler, G.R. and Landry, D.B. (1990) Human Merkel cells in skin derived from cultured keratinocyte grafts. Lab. Invest., 2, 233–241. Compton, C.C., Tong, Y., Trookman. N., Zhao. H. and Roy, D. (1995) TGFα gene expression in cultured human keratinocytes is unaffected by cellular aging. Arch. Dermatol., 131, 683–690. Compton, C.C., Tong, Y., Trookman, N., Zhao, H. and Roy, D. (1994) TGFβ1 gene expression in cultured human keratinocytes does not decrease with biological age. J. Invest. Dermatol., 103, 127–133. Compton, C.C., Warland, G. and Kratz, G. (1998) Melanocytes in cultured epithelial grafts are depleted with serial subcultivation and cryopreservation. J. Burn Care Rehabil., 19, 330–336. De Luca, M., Albanese, E., Bondanza, S., Megna, M., Ugozzoli, L, Molina, F., Cancedda, R., Sant, P.L., Bormioli, M., Stella, M. and Magliacani, G. (1989) Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium, fresh or preserved in a frozen state. Burns, 15, 303–309. De Luca, M. and Cancedda, R. (1992) Culture of human epithelium. Burns (Suppl.), 18, S5– S10. De Luca, M., Franzi, A.T., D’Anna, F., Zicca, A., Albanese, E., Bondanza, S. and Cancedda, R. (1988) Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium. Eur. J. Cell Biol., 46, 176–180. Eldad, A., Burt, A., Clarke, J.A. (1987) Cultured epithelium as a skin substitute. Burns, 13, 173–180. Faure, M., Mauduit, G., Schmitt, D., Kanitakis, J., Demidem, A. and Thivolet, R. (1987) Growth and differentiation of human epidermal cultures used as auto and allografts in humans. Br. J. Dermatol., 116, 161–170.

340


Figure 18 Neodermis of cultured epithelial autograft at 11 years postgrafting. An elastic tissue-trichrome stain shows that the neodermis has a bilayered distribution of collagen (blue-green stain) and an abundant elastin network (black-staining fibrils) in the regenerated reticular dermis. Fusenig, N.E. (1994) Epithelial-mesenchymal interactions regulate keratinocyte growth and differentiation in vitro. In I.M.Leigh, E.B.Lane and F.M.Watt, (eds.), The Keratinocyte Handbook, Cambridge University Press, Great Britain, pp. 71–94. Gallico, G.G., O’Connor, N.E., Compton, C.C., Kehinde, O. and Green, H. (1984) Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med., 311, 448–451. Gallico, G.G., O’Connor, N.E., Compton, C.C., Remensnyder, J.P., Kehinde, O. and Green, H. (1989) Cultured epithelial autografts for giant congenital nevi. J. Plast. Reconstr. Surg., 84, 1–9. Green, H. (1978) Cyclic AMP in relation to proliferation of the epidermal cell—a new view. Cell, 15, 801–815. Green, H., Kehinde, O. and Thomas, J. (1979) Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc. Natl. tl. Acad. Set., 76, 5665–5668. Hefton, J.M., Amberson, J.B., Biozes, D.G. and Weksler, M.E. (1984) Loss of HLA-DR expression by human epidermal cells after growth in culture. J. Invest. Dermatol., 83, 48– 50. Hefton, J.M., Madden, M.R., Finkelstein, J.L. and Shires, G.T. (1983) Grafting of burn patients with allografts of cultured epidermal cells. Lancet, 2, 428–430. Hertle, M.D., Adams, J.C. and Watt, F.M. (1991) Integrin expression during human epidermal development in vivo and in vitro. Development, 112, 193–206. Hertle, M.D., Kubler, M.-D., Leigh, I.M. and Watt, F.M. (1992) Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis. J. Clin. Invest., 89, 1892– 1901. Herzog, S.R., Meyer, A., Woodley, D. and Peterson, H.D. (1988) Wound coverage with cultured autologous keratinocytes: use after burn wound excision, including biopsy followup. Trauma, 28, 195–198. Hickerson, W., Compton, C.C., Fletchall, S. and Smith, L.R. (1994) Cultured epidermal autografts and allodermis combination for permanent burn wound coverage. Burns, 20 (Suppl. 1), S52–S55.


341

Figure 19 Involucrin expression in cultured epithelial autograft at 11 years postgrafting. A normal pattern of involucrin expression limited to the upper third of the epidermis is seen.

Figure 20 Keratin 14 expression in cultured epithelial autograft at 11 years postgrafting A normal pattern of basal expression of keratin 14, with down-regulation in suprabasal layers is seen. Morton, J J., Allen, M.H. and MacDonald, D.M. (1984) An assessment of Langerhans cell quantification in tissue sections. J. Am. Acad. Dermatol., 11, 591–593. Kaawach, W.F., Oliver, A.M., Weiler-Mithoff, D.R., Abramovich, D.R. and Rayner, C.R. (1991) Survival assessment of cultured epidermal allografts applied over partial thickness wounds. Br. J. Plast. Surg., 44, 321–324. Kangesu, T., Navsaria, H.A., Manek, S., Fryer, P.R., Leigh, I.M. and Green, C.J. (1993) Keratodermal grafts: the importance of dermis for the in vivo growth of cultured keratinocytes. Br. J. Plast. Surg., 46, 401–409

342


Figure 21 Keratin 10 expression in cultured epithelial autograft at 11 years postgrafting A normal pattern of strong diffuse suprabasal expression of keratin 10 is seen. Kumagai, N., Nishina, H., Tanabe, H., Hosaka, T., Ishida, H. and Ogino, Y. (1988) Clinical application of autologous cultured epithelia for the treatment of burn wounds and burn scars. Plast. Reconstr. Surg., 82, 99–108. Madden, M.R., Finkelstein, J.L., Staiano-Coco, L., Goodwin, C.W., Shires, G.T., Nolan, E.E. and Hefton, J.M. (1986) Grafting of cultured allogeneic epidermis on second and third degree burn wounds on 26 patients. J. Trauma, 26, 955–960. McKay. I.A. and Leigh, I.M. (1991) Epidermal cytokines and their roles in cutaneous wound healing. Br. J. Dermatol., 124, 513–518. Morhenn, V.B., Benike, C.J., Cox, A.J., Charron, D.J. and Engelman, E.G. (1982) Cultured human epidermal cells do not synthesize HLA-DR. J. Invest. Dermatol., 78, 32–37. Munster, A.M., Weiner, S.H. and Spence, R.J. (1990) Cultured epidermis for the coverage of massive burn wounds. Ann. Surg., 211, 676–680. Navsaria, H.A., Kangesu, T., Manek, S., Green, C.J. and Leigh, I.M. (1994) An animal model to study the significance of dermis for grafting cultured keratinocytes. Burns, 20 (Suppl. 1), S57–S60. O’Connor, N., Mulliken, J.B., Banks-Schlegel, S., Kehinde, O. and Green, H. (1981) Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet, 1, 75–78. O’Connor, N., Gallico, G., Compton, C.C., Kehinde, O. and Green, H. (1984) Grafting of burns with cultured epithelium prepared from autologous epidermal cells. II. Intermediate term results on three pediatric patients. In T.K. Hunt, K.B.Heppenstall, E.Pines and D.Ropee, (eds.), Soft and Hard Tissue Repair, Biological and Clinical Aspects. Vol II, Surgical Seminar Series, Prager Scientific, New York, NY, pp. 283–292. Odessey, R. (1992) Multicenter experience with cultured epidermal autograft for treatment of burns. J. Burn Care Rehabil., 13, 174–180. Orgill, D.P., Butler, C., Regan, J.F., Barlow, S.M., Yannas, I.V. and Compton, C.C. (1998) Vascularized collagengycosaminoglycan matrix provides a dermal substrate and improves take of cultured epithelial autografts. Plast. Reconstr. Surg., 102, 423–429. Phillips, T.J., Bhawan, J., Leigh, I.M., Baum, H.J. and Gilchrest, B.A. (1990) Cultured epidermal autografts and allografts: a study of differentiation and allograft survival. J. Amer. Acad. Dermatol., 23, 189–198.


343

Figure 22 Proportion of cycling cells within a cultured epithelial autograft at 6 months postgrafting by immunolocalization of Ki67 antigen. The proportion of cycling cells is seen to be normal (i.e., about one per 10–20 basal cells). Poumay, Y., Roland, I.H., Leclercq-Smekens, M. and Leloup, R. (1994) Basal detachment of the epidermis using Dispase: tissue spacial organization and fate of integrin 0X5^4 and hemidesmosomes. J. Invest. Dermatol., 102, 111–117. Regauer, S., Seiler, G.R., Barrandon, Y., Easley, K.W. and Compton, C.C. (1990) Epithelial origin of cutaneous anchoring fibrils. J. Cell Biol., 111, 2109–2115. Rheinwald, J.G. and Green, H. (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell, 6, 331–344. Rheinwald, J.G. and Green, H. (1977) Epidermal growth factor and multiplication of cultured human epidermal keratinocytes. Nature, 265, 421–424. Stepp, M.A., Spurr-Michaud, S., Tisdale, A., Elwell, J. and Gipson, I.K. (1990) α6β4 integrin heterodimer is a component of hemidesmosomes. Proc. Natl. Acad. Set. USA, 87, 8970– 8974. Stingl, G., Katz, S.I., Clement, L., Green, I. and Shevack, E.M. (1978) Immunologic function of la-bearing epidermal Langerhans cells. J. Immunology, 121, 2005–2010. Stoof, T.J., Boorsma, R.M. and Nickoloff, B.J. (1994) Keratinocytes and immunological cytokines. In I.M.Leigh, E.B.Lane and F.M.Watt, (eds.), The Keratinocyte Handbook, Cambridge University Press, Great Britain, pp. 365–399. Teepe, R.G.C., Kreis, R.W., Koebrugge, E.J., Kempenaar, J.A., Vloemans, A.F.P.M., Hermans, R.P., Boxma, H., Dokter, J., Hermans, J., Ponec, M. and Vermeer, B.J. (1990) The use of cultured autologous epidermis in the treatment of extensive burn wounds. J. Trauma, 30, 269–275. Thivolet, J., Faure, M., Demidem, A. and Mauduit, G. (1986) Long term survival and immunological tolerance of human epidermal allografts produced in culture. Transplantation, 42, 274–280. van der Merwe, A.E., Mattheyse, F.J., Bedford, M., van Welden, P.D. and Roseeuw, D. (1990) Allografted keratinocytes used to accelerate the treatment of burn wounds are replaced by recipient cells. Burns, 16, 193–197. van Gielen, V., Faure, M., Mauduit, G. and Thivolet, J. (1987) Progressive replacement of human cultured epithelial allografts by recipient cells as evidenced by HLA class 1 antigen expression. Dermatologica, 175, 166–170.

344


Watt, F.M and Hertle, M.D. (1994) Keratinocyte integrins. In I.M. Leigh, E.B.Lane and F.M. Watt, (eds.), The Keratinocyte Handbook, Cambridge University Press, Great Britain, pp. 3153–164. Zhao, Y.B., Zhao, X.F., Lu, S.Z., Wang, X., Huang, S.Z. and Zhuo, X.T. (1992) Clinical observations and methods for identifying the existence of cultured epidermal allografts. Burns. 18. 4–8.

18. MODULATION OF EPITHELIAL TRAITS DURING THE PROGRESSION OF CARCINOMAS M.GRASSI, J.P.THIERY and J.JOUANNEAU Laboratoire de morphogenese cellulaire et de progression tumorale, UMR 144, CNRSlnstitut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France Tel: 01–42–34–63–50; Fax: 01–42–34–63–49; E-mail: [email protected] INTRODUCTION Normal epithelial cells are characterized by the tight association of individual cells through various junctional structures. In the past few years, information has been gathered on possible molecular mechanisms underlying reduced cell-cell adhesion in carcinomas. Tumors of epithelial origin are of major medical importance, since more than 90% of all fatal malignancies are carcinomas. In human carcinomas, the loss of epithelial differentiation is a prognostic marker and correlates with a poor outcome of the disease (Gabbert et al., 1985). The transition from in situ tumor growth to invasive disease is defined by the ability of the tumor cells at the primary site to invade local tissues (cf. Figure 1). This process is marked by loss of epithelial differentiation and development of a more fibroblastic or mesenchymal appearance (Birchmeier et al., 1993). This is characterized by reduced intercellular adhesion and by greater motility and invasiveness of the tumor cells. The ability to form metastases is critically dependent on a permanent or transient loss of epithelial characteristics (Birchmeier and Behrens, 1994). Here we will discuss various mechanisms that regulate the transition between epithelia and mesenchyme during tumor progression. ALTERATIONS OF CELL ADHESION INDUCE MODULATION OF EPITHELIAL TRAITS Desmosomes and adherens junctions constitute the main adhesive junctions between epithelial cells. Adherens junctions are specialized regions of the plasma membrane where the transmembrane cell adhesion molecule E-cadherin recognizes and binds the E-cadherin present on neighboring cells in a Ca2+-dependent manner. E-cadherin is a 120 kDa transmembrane glycoprotein, from which an extracellular 80 kDa tryptic fragment can be released in the presence of Ca2+. The cDNA of E-cadherin encodes a signal peptide at the ammo-terminus, a large extracellular domain with five repeated domains important in Ca2+-binding, a single transmembrane sequence, and a 15 kDa cytoplasmic domain (Takeichi, 1991). The cytoplasmic portion of E-cadherin is associated with a group of proteins, named catenins, which mediate interactions with the microfilament network. This interaction is essential for the integrity of adherens junctions. E-cadherin binds directly to β-catenin, which binds to α-catenin, and α-catenin connects the complex to the cytoskeleton. γcatenin (plakoglobin) is related to β-catenin, and interacts in a similar manner with E-cadherin and α-catenin (Huelsken et al., 1994; Naethke et al., 1994). Any significant change in the expression and/or the structure of one of the essential components of the adherens junctions can lead to junctional disassembly and, consequently, to invasive carcinoma cells. Various types of human carcinomas were examined for the defects in one of the constituents of intercellular

346


Figure 1 Transition from in situ carcinoma to invasive disease. This process is characterized by loss of epithelial differentiation and development of a more fibroblastic appearance. The tumor cell loses its attachment to the primary tumor mass, creates a pathway through the surrounding stroma or basement membrane, and moves through the pathway it has created. It reaches the circulation, migrates through the barrier into the secondary sites, and colonizes and proliferates in distant organs.

junctions. A correlation between the dysfunction of E-cadherin-mediated cellular adhesion, by downregulation of E-cadherin expression or by mutation in the E-cadherin gene, and invasive potential of many types of carcinomas has emerged from these studies. In head and neck cancers for example, downregulation of E-cadherin expression correlates with loss of epithelial differentiation of the tumors and with the formation of lymph node metastases (Schipper et al., 1991). In vitro, the invasiveness of carcinoma cells with a fibroblastoid phenotype can be prevented by transfection with E-cadherin cDNA and can be induced again by treatment of the transfected cells with anti-E-cadherin monoclonal antibodies. These findings indicate that the loss of E-cadherin is a critical step in the dedifferentiation of epithelial cells and the acquisition of invasive properties and, furthermore, that E-cadherin acts as a tumor (invasion) suppressor (Frixen et al., 1991). Nevertheless, in a few reported cases, alterations in cell-cell adhesion between carcinoma cells result from loss of α- or β-catenin, due to gene mutations (Shimoyama et al., 1992; Kawanishi et al., 1995). In some puzzling cases, no down-regulation of the expression of any protein of the cadherin adhesive complex was observed, suggesting that posttranslational modifications might account for the reduced cell-cell adhesion observed. β- and γ-catenins are phosphorylated during EGF and HGF/SF- in vitro stimulation of carcinoma cell scattering (Hoschuetzky, 1994). MDCK cells transformed with a temperature-sensitive mutant of v-src exhibit, at the permissive temperature, an increase in tyrosine phosphorylation of E-cadherin and β-catenin, concomitant with the inactivation of the cadherin adhesion system. It has therefore been hypothesized that tyrosine phosphorylation of catenins by c-src might result in the disturbance of the adhesion complexes and


347

lead to a non-adhesive state of epithelial cells (Behrens et al., 1993). In epithelial tumor cells overexpressing the EGF receptor, EGF modified the functional association between E-cadherin and actin filaments through tyrosine phosphorylation of the cadherin/catenin complex, and thereby made the adhesion molecule incompetent (Fujii et al., 1996). However, the effects of catenin phosphorylation on the function of the adhesion complex remain to be clarified. Like E-cadherin, APC, another tumor suppressor gene product, can bind to β-catenin and γ-catenin. Mutations of APC appear to initiate sporadic and inherited forms of human colorectal cancer (Rubinfeld et al., 1993; Su et al., 1993). As β-catenin modulates E-cadherin function in cell junctions, APC binding to pcatenin might affect cell adhesion. Binding of β-catenin to APC is dependent on phosphorylation of the central region of APC by glycogen synthase kinase-3β (GSK-3β). When β-catenin is present in excess, APC might bind to GSK-3β, undergo phosphorylation, and down-regulate intracellular β-catenin (Rubinfeld et al., 1996). The dysfunction of APC in tumors may be related to its inability to decrease cytoplasmic βcatenin levels. In addition to mutation and posttranslational modifications, adherens junction integrity can also be affected by changes in the regulation of gene expression of particular junctional constituents. A significant variation in the battery of function-ally important transcription factors which regulate the expression of the adherens junction proteins might lead to changes in the invasiveness of carcinoma cells. Two regulatory mechanisms could control the epithelial specificity of E-cadherin expression: suppression of promoter activity in mesenchymal cells by the palindromic element E-Pal and an enhancement of activity in epithelial cells by both E-Pal and the epithelium-specific enhancer (Hennig et al., 1996). CpG methylation of the promoter may also be a mechanism of E-cadherin inactivation in human carcinomas. The E-cadherin promoter is hypermethylated in E-cadherin-negative cell lines, whereas it is not in E-cadherin-positive ones. Furthermore, treatment of E-cadherin-negative cells with a demethylating agent caused gene expression reversion, leading to epithelial morphogenesis with acquisition of the homophilic cell-cell adhesive property (Yoshiura et al., 1995). It has recently become clear, however, that loss of epithelial differentiation in carcinoma cells in vitro and in vivo can also occur without the loss of E-cadherin. For instance, treatment of epithelial cells with scatter factor/hepatocyte growth factor (SF/HGF), which is a growth and motility factor for various cell types, leads to the dissociation of the cells, induces cell motility following a change from an epithelial to a fibroblastoid shape, and leads to invasiveness into collagen gels, but does not induce a loss of E-cadherin (Weidner et al., 1990; Stoker et al., 1987). Similar results were observed after epithelio/mesenchymal transition of rat bladder carcinoma cells with acidic fibroblast growth factor (Boyer et al., 1992). Furthermore, in a number of ‘scattered’ gastric carcinomas, E-cadherin was found to be present (Shimoyama et al., 1991). CELL SCATTERING AND MOTILITY FACTORS INDUCE MODULATION OF EPITHELIAL TRAITS There is increasing evidence that the cell adhesion machinery is subject to regulation by soluble factors that lead to cell dispersion and enhanced cell motility and therefore might also promote invasion of tumor cells. The most potent and general epithelial cell dissociating cytokine is HGF/SF (Hepatocyte Growth Factor/ Scatter Factor). HGF/SF is a 92 kDa heparin binding glycoprotein which is converted into disulfide-linked 62 kDa and 34/32 kDa subunits by proteolytic cleavage (Weidner et al., 1990). HGF/SF binds to and activates a tyrosine kinase receptor, the product of the c-met proto-oncogene (Bottaro et al., 1991; Naldini et al., 1991). This

348


factor, which is predominantly secreted by mesenchymal cells, is capable of dispersing a variety of normal and tumor cells (Rosen et al., 1994). It has recently been shown that HGF/SF is expressed in breast carcinomas, and that the level of expression correlates with the state of tumor progression (Yamashita et al., 1994). HGF/SF may function in the prostate gland as a paracrine growth factor, synthesized by stromal cells and targeting epithelial cells. The expression of c-Met is up-regulated by androgen deprivation and c-Met appears to be preferentially expressed on androgen-insensitive metastatic cells. These data suggest a possible linkage of c-Met expression with prostatic carcinoma progression (Humphrey et al., 1995). Furthermore, expression of the c-Met receptor may add a selective growth advantage to a narrow subset of differentiated ovarian cancers in premenopausal patients (Di Renzo et al., 1994). Finally, HGF/SF is overproduced by stromal cells within a variety of tumors as a consequence of malignant tumor cells secretion of SF-inducing factors (Rosen et al., 1994; Rosen and Goldberg, 1995). The contribution of the stroma to tumor development and progression has been recognized for some time. Stromal cells actively interact with tumor epithelia and support malignant growth. Fetal fibroblasts secrete a “migration stimulating factor” (MSF), a 192 kDa protein. Fibroblasts obtained from a majority of breast tumors resemble fetal cells in terms of their apparently persistent production of a MSF, which is not made by their adult counterparts. MSF stimulates the synthesis of high molecular weight hyaluronic acid (HA). Recent data suggest that the observed effect of MSF on fibroblast migration is actually a consequence of its effect on HA synthesis. It may perturb epithelial-stromal interactions in a manner which promotes the invasive behavior of the emerging neoplastic cell population. Whether this involves permissive or inductive mechanisms remains to be elucidated. Soluble factors produced by the emerging population of preneoplastic cells may promote the proliferative expansion of pre-existing MSF-producing cells, and/or induce MSF synthesis in facultative flbroblasts (Schor et al., 1993; Schor, 1995). A variety of other growth factors (e.g. EGF, PDGF, TGFβ, FGF-I), cytokines, and motility factors, and some of the above-mentioned factors, being autocrine, could accumulate within the tumor microenvironment, and contribute to tumor growth, invasion, and metastasis. Tumor cell autocrine motility factor (AMF), a 55 kDa protein, induces directed and random migration of AMF-producing tumor cells (Liotta et al., 1986). AMF induces cellular responses by binding to its receptor, a 78 kDa cell surface glycoprotein (gp78) and activating a transduction cascade (Silletti et al., 1991). AMF and its receptor have been postulated to play a role in tumor cell metastasis. It has been demonstrated that expression of the AMF receptor correlates with malignant potential in human bladder cell lines, as well as disease stage and recurrence in human bladder specimens. Upregulation of gp78 expression is concomitant with disease progression in bladder cancer and with the downregulation of E-cadherin, consistent with a shift from a sedentary to a motile cellular phenotype (Otto et al., 1994; Silletti et al., 1995). AMF is the previously cloned cytokine and enzyme designated as neuroleukin and phosphohexose isomerase (PHI). PHI catalyzes isomerization of glucose 6-phosphate to fructose 6-phosphate. Specific PHI inhibitors block both enzymatic activity and AMF-induced cell motility (Watanabe et al., 1996). Thus, the role of the AMF enzymatic activity has yet to be elucidated. It could modify the gp78 receptor, and as a result induce a transduction cascade. AMF is not the only motility factor with an enzymatic activity however, and a new kind of transduction pathway might be involved. Autotaxin (ATX) is a 125-kDa glycoprotein, previously isolated and purified from the conditioned medium of human melanoma cells, which elicits chemotactic and chemokinetic responses at picomolar to nanomolar concentrations (Stracke et al., 1992). ATX has been cloned and its deduced amino acid sequence is homologous to the phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha) (Murata et al., 1994). The phosphodiesterase enzymatic site is linked to motility stimulation, suggesting that this exo/ecto-enzyme


349

may participate in an extracellular cascade of cellular activation involving the metabolism of extracellular nucleotides (Stracke et al., 1996). It appears that there is a wide range of motility and scattering factors, all of them triggering cell surface receptors and leading to similar changes in epithelial cell morphology; cell ruffling, cell scattering, formation of lamellipodia or filopodia, and motility (Sachs et al., 1996). SIGNALING PATHWAYS IMPLICATED IN CELL SCATTERING The signaling pathways generated by different scattering factors might vary according to the type of inducers. Tyrosine kinase activities are generally implicated in cytoskeletal modifications leading to cell motility. The interaction of HGF/SF with its receptor, the tyrosine-kinase c-Met, triggers phosphorylation of two carboxyl-terminal tyrosines essential for signal transduction (Y1349VHVNATY1356VNV). These phosphotyrosines are binding sites for the SH2 domains of p85, pp60src, PL-Cγ, and She (Ponzetto et al., 1994; Pelicci et al., 1995), and for the proline-rich domain of Gab1 (Weidner etal, 1996). Y1356VNV also has the ability of binding STAT, a factor required for signal transduction and activation of transcription, which is induced by HGF/SF (Boccaccio et al., 1998), and Grb2, the adaptator for the Ras guanylnucleotide exchanger SOS. Ras can be activated either directly via Grb2/SOS recruitment to the activated HGF/SF receptor, or indirectly through She phosphorylation (Pelicci et al., 1995). GTP-Ras activates the mitogen-activated-protein kinase (MAPkinase) cascade, but although MAPkinase activation and c-fos induction play an essential role in the signaling pathway leading to cell growth, they are not required for the motility response induced by HGF/ SF (Nagamine et al., 1996). In MDCK cells, Ras ‘is necessary together with Rac, another GTP-binding protein, to induce ‘spreading’ and actin reorganization in the first few hours following HGF/SF treatment. Progression from spreading into full scattering requires a third signal, which is likely to be phosphatidylinositol 3-kinase (PI3-K) (Ridley et al., 1995; Nobes et al., 1995). PI3-K consists of two subunits, p85 and p110. p85 binds to tyrosine kinase receptors at phosphotyrosine residues, and p110 is the catalytic subunit. It stimulates the phosphorylation of phosphoinositides to produce PI(3)P, PI(3, 4)P2 and PI(3, 4, 5)P3 (Panayotou et al., 1992). A localized concentration of PIP3 around activated receptors might bind actin filament-capping proteins such as gelsolin, thereby exposing the barbed ends of actin filaments and enabling them to act as F-actin nucleation sites. As yet however, it has not been shown that PIP3 can bind to any capping proteins. PIP3 has also been reported to activate PKCζ, and it is possible that this in turn phosphorylates proteins involved in membrane ruffling (Ridley, 1994). The p85 subunit of PI3-K has a Src homology 3 (SH3) domain which could be important for receptor function , since it binds to dynamin, a protein implicated in regulating clathrin coatmediated endocytosis (Gout et al., 1993; Van der Bliek et al., 1993). In addition p85 has homology to GTPase activating proteins (GAPs) specific for Rho family proteins. This domain may interact with Rac leading to Rac activation and membrane ruffling, (cf. Figure 2) Activated Rac may also activate Rho, another GTP-binding protein. GTP-Rho stimulates focal adhesion and stress fibre assembly (Ridley and Hall, 1992). Focal adhesions are formed where stress fibres terminate at the plasma membrane, and where integrins and many other proteins are clustered forming a link between the extracellular matrix and the actin cytoskeleton. Rho may stimulate the activation of a tyrosine kinase that leads to the accumulation of phosphotyrosine-containing proteins in focal adhesions, in particular pp125FAK, which then phosphorylates tensin and paxillin, two components of focal adhesions, leading to the formation of stress fibres (Ridley and Hall, 1994).

350


Figure 2 A model for Rac activation leading to membrane ruffling. In resting cells, Rac is normally complexed in the cytosol with a GDI (Guanine nucleotide Dissociation Inhibitor), preventing nucleotide exchange. Upon activation of cells by an extracellular factor, this complex dissociates and Rac is able to bind to the membrane. In the case of tyrosine kinase receptors, a Grb2—like protein brings a Rac exchange factor (Rac EF) to the membrane by binding via a SH2 domain to an activated receptor. This leads to the activation of Rac and stimulation of downstream events leading to membrane ruffling. Alterations in phospholipid metabolism by PI3-kinase (PI3-K) may also be required for membrane ruffling, but whether it acts upstream or downstream of Rac is not known; may be by stimulating the GTPase activity of Rac. (Ridley, 1994)

The loss of epithelial traits could be associated with the activation of a mesenchymal genetic program and/or with the repression of an epithelial genetic program. The putative master genes necessary for such a cell shape modification are being actively sought. A CARCINOMA MODEL To investigate the role of epithelial to mesenchymal cell transition in the invasive and progressive carcinoma process, we have used a model system consisting of the NBT-II cell line, which derives from a chemically-induced rat bladder carcinoma (Toyoshima et al., 1971). Unusually, these epithelial cells possess the ability to disassemble their junctions and dissociate upon exogenous addition of growth factors. The changes involved in this transition are summarized in Table 1. The activity of growth factors involves triggering tyrosine kinase type receptors, such as the FGF-1, EGF, TGFα and HGF/SF receptors (Valles et al., 1990; Gavrilovic et al., 1990; Bellusci et al., 1994a; Rodier et al., 1995). NBT-II cells constitutively producing one such scattering factor after stable transfection acquire the dissociated mesenchymal phenotype. Moreover, these cells acquired invasive properties in vitro (Jouanneau et al., 1991; Tucker et al., 1991; Bellusci et al., 1994a) and grow as solid carcinomas in nude mice with reduced lag time for appearance of the tumor when compared to untransfected NBT-II cells (Jouanneau et al., 1991; Bellusci et al., 1994a). These effects have been demonstrated to be due to an autocrine loop in the


351

Table 1 Some traits of epithelial and mesenchymal morphologies of NBT-II cells. (1) Boyer et al., 1989; (2) Boyer et al., 1992; (3) Savagner et al., 1994; (4) Gavrilovic et al., 1990, Jouanneau et al., 1991; (5) Valles et al., 1996; (6) Savagner et al., 1997 (submitted article). Epithelial Morphology

Mesenchymal Morphology

desmosomes(1)

Surface Keratins (1) Restricted distribution of E-cadherin(2) FGFR2b(3) Restricted distribution of α2β1 and α3β1 integrins(5)

Internalized desmosomes(1) Vimentin (1) Diffuse distribution of E-cadherin(2) FGFR2c(3) collagenase production(4) Diffuse distribution of α2β1 and α3β1 integrins(5) Upregulation of α2β1 integrin(5) Transient expression of the Zinc finger protein Slug(6)

case of HGF/SF (Bellusci et al., 1994a) and could be either autocrine and/or intracrine in the case of FGF-1 producing cells, due to the lack of obvious secretion of the growth factor. These results suggest that carcinoma cells producing a scattering factor acquire aggressive properties in vivo. Furthermore, we have demonstrated that FGF-1 producing cells are not dominant in vivo for proliferation among the NBT-II parental cells. In a mixed tumor cell population, a few cells producing the growth/ scatter factor mediate a community effect resulting in a collective behavior of the cells (Jouanneau et al., 1994). This mechanism may contribute to the progression of in situ carcinoma towards invasive and metastatic tumors. The nature of the interaction between the different subsets of tumor cells remains to be elucidated. Several hypotheses have to be considered, and among them the direct paracrine hypothesis. Alternatively, cell communication within a tumor cell collective could be mediated by gap junctions; the tumor cells could be coupled biochemically by passage through the gap junctions of small molecules such as second messengers. These hypotheses are currently under investigation. We have investigated whether an endogenous growth/scatter factor could be naturally produced by a subset of cells during tumor progression, we have selected in vivo a monoclonal metastatic variant of the rat bladder carcinoma NBT-II cells, named M-NBT-II. In vivo, these cells are more tumorigenic than the parental ones, and in culture, these cells have a mesenchymal phenotype. Interestingly, they produce and secrete a scattering activity that dissociates not only the parental epithelial NBT-II cells but also epithelial MDCK cells in culture (Bellusci et al., 1994b). This autocrine activity, termed Scatter Factor Like (SFL), exerts biological effects similar to those of HGF/SF (cf. Figure 3). Nevertheless, we have demonstrated that SFL is neither HGF/SF nor a variant of this growth factor (Bellusci et al., 1994b). Interestingly, SFLproducing cells are able to mediate a community effect in experimental carcinoma (Bellusci et al., 1994c). CONCLUSION In addition to adhesion molecules and invasogenic cytokines, other factors may contribute to the invasive tumor phenotype, among them the metalloproteases (MMPs) and their inhibitors (Liotta, 1986b). Type IV procollagenases appear to be produced by stromal fibroblasts in response to factors secreted by tumor cells at the leading edge of an invading tumor. Moreover, these enzymes may be activated by proteins located on the tumor cell membrane (Shi and Liu, 1995). Studies of tumor-derived MMP-inducers and tumor membrane-associated MMP activators may provide promising diagnostic and therapeutic tools in cancer

352


Figure 3 HGF/SF and SFL are both able to dissociate MDCK cells. MDCK ceils were cultured in 6-well plates and incubated with either standard medium (control), or human recombinant HGF/SF (Genentech) at 10 ng/mL (rhSF), or MNBT-II conditioned medium concentrated with a Centricon 30 and used at 20 U/mL (SFL) (1 U of activity is definied as the lowest amount of factor per mL that clearly dissociates MDCK cells (Bellusci et al., 1994a)). Cells were examined by contrast phase microscopy after 24 hours.

metastasis; in addition, the process of tumor neoangiogenesis itself requires somewhat similar proteolytic events and plays a major role not only in tumor growth, but also in the development of metastases. Permanent and/or transient loss of control over growth and scatter factors, their receptors, cell to cell adhesion molecules and interactions with the tumor microenvironment could lead to the loss of epithelial features, and therefore contribute to the progression of carcinoma towards an undifferentiated, invasive, and more metastatic tumor state. REFERENCES Behrens, J., L.Vakaet, R.Friis, E.Winterhager, F.Van Roy, M.M.Mareel and W.Birchmeier (1993) Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature-sensitive v-SRC gene. J.Cell Biol., 120, 757–66. Bellusci, S., G.Moens, G.Gaudino, P.Comoglio, T.Nakamura, J. P.Thiery and J.Jouanneau (1994a) Creation of an hepatocyte growth factor/scatter factor autocrine loop in carcinoma cells induces invasive properties associated with increased tumorigenicity. Oncogene, 9, 1091-9. Bellusci, S., G.Moens, J.P.Thiery and J.Jouanneau (1994b) A scatter factor-like factor is produced by a metastatic variant of a rat bladder carcinoma cell line. J. Cell Set., 107, 1277–1287. Bellusci, S., G.Moens, A.Delouvee, J.P.Thiery and J.Jouanneau (1994c) SFL production by carcinoma cells induces the aggressive properties of nonproducing cells in vivo via a community effect. Invasion Metastasis, 14, 319–328. Birchmeier, W. and Behrens, J. (1994) Cadherin expression in carcinomas: role in the formation of cell junctions and the prevention of invasiveness. Bioch. Biophys. Acta, 1198, 11–26. Birchmeier, W., Weidner, K.M. and Behrens, J. (1993) Molecular mechanisms leading to loss of differentiation and gain of invasiveness in epithelial cells. J. Cell Set. Suppl., 17, 159– 64.


353

Boccaccio, C., Ando, M., Tamagnone, L., Bardelli, A., Michieli, P., Baltistini, C. and Comoglio, P.M. (1998) Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature, 391, 285–8. Bottaro, D.P., J.S.Rubin, D.L.Faletto, A.M.Chan, T.E.Kmiecik, G.F.Vande Woude and S.A.Aaronson (1991) Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science, 251, 802-4. Boyer, B., G.C.Tucker, A.M.Valles, W.W.Franke and J.P.Thiery (1989) Rearrangements of desmosomal and cytoskeletal proteins during the transition from epithelial to fibroblastoid organization in cultured rat bladder carcinoma cells. J. Cell Biol., 109, 1495–509. Boyer, B., S. Dufour and J.P. Thiery (1992) E-cadherin expression during the acidic FGF-induced dispersion of a rat bladder carcinoma cell line. Exp. Cell Res., 201, 347–57. Di Renzo, M.F., M.Olivero, D.Katsaros, T.Crepaldi, P.Gaglia, P.Zola, P.Sismondi and P.M.Comoglio (1994) Overexpression of the Met/HGF receptor in ovarian cancer. Int. J. Cancer, 58, 658–62. Frixen, U.H., J.Behrens, M.Sachs, G.Eberle, B.Voss, A.Warda, D.Lochner and W. Birchmeier (1991) E-cadherinmediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J. Cell Biol., 113, 173–85. Fujii, K., F.Furukawa and N.Matsuyoshi (1996) Ligand activation of overexpressed epidermal growth factor receptor results in colony dissociation and disturbed E-cadherin function in HSC-1 human cutaneous squamous carcinoma cells. Exp. Cell Res., 223, 50–62. Gabbert, H., Wagner, R., Moll, R. and Gerharz, C.D. (1985) Tumor dedifferentiation: An important step in tumor invasion. Clin. Exp. Metastasis, 3, 257–79. Gavrilovic, J., G.Moens, J.P.Thiery and J.Jouanneau (1990) Expression of transfected transforming growth factor alpha induces a motile fibroblast-like phenotype with extracellular matrix-degrading potential in a rat bladder carcinoma cell line. Cell Regul. , 1, 1003–14. Gout, I., R.Dhand, I.D.Hiles, M.J.Fry, G. Panayotou, P.Das, O.Truong, N.F.Totty, J. Hsuan, G.W.Booker et al. (1993) The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 75, 25–36. Hennig, G., O.Lowrick, W.Birchmeier and J.Behrens (1996) Mechanisms identified in the transcriptional control of epithelial gene expression. J. Biol. Chem., 271, 595–602. Hoschuetzky, H., H.Aberle and R.Kemler (1994) Beta-catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J. Cell Biol., 127, 1375–1380. Hulsken, J., W.Birchmeier and J.Behrens (1994) E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton. J. Cell Biol., 127, 2061–2069. Humphrey, P.A., X.Zhu, R.Zarnegar, P.E. Swanson, T.L.Ratliff, R.T.Vollmer and M.L.Day (1995) Hepatocyte growth factor and its receptor (c-MET) in prostatic carcinoma. Am. J. Pathol., 147, 386–96. Jouanneau, J., G.Moens, Y.Bourgeois, M.F.Poupon and J.P.Thiery (1994) A minority of carcinoma cells producing acidic fibroblast growth factor induces a community effect for tumor progression. Proc. Natl Acad. Sci. USA, 91, 286–90. Jouanneau, J., J.Gavrilovic, D.Caruelle, M. Jaye, G. Moens, J.P.Caruelle and J.P.Thiery (1991) Secreted or nonsecreted forms of acidic fibroblast growth factor produced by transfected epithelial cells influence cell morphology, motility, and invasive potential. Proc. Natl Acad Sci. USA, 88, 2893–7. Kawanishi, J., J.Kato, K.Sasaki, S.Fujii, N.Watanabe and Y.Niitsu (1995) Loss of E-cadherin-dependent cell-cell adhesion due to mutation of the beta-catenin gene in a human cancer cell line, HSC-39. Mol. Cell Biol., 15, 1175–81. Liotta, L.A., R.Mandler, G.Murano, D.A.Katz, R.K.Gordon, P.K.Chiang and E.Schiffmann (1986a) Tumor cell autocrine motility factor. Proc. Natl Acad. Sci. USA, 83, 3302–6. Liotta, L.A. (1986b) Tumor invasion and metastases—role of the extracellular matrix: Rhoads Memorial Award lecture. Cancer Res., 46, 1–7. Murata, J., H.Y.Lee, T.Clair, H.C.Krutzsch, A.A.Arestad, M.E.Sobel, L.A.Liotta andM. L.Stracke (1994) cDNA cloning of the human tumor motility-stimulating protein, autotaxin, reveals a homology with phosphodiesterases. J. Biol. Chem., 269, 30479–84. Naethke, I.S., L.Hinck, J.R.Swedlow, J.Papkoff and W.J.Nelson (1994) Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells. J. Cell Biol., 125, 1341–52.

354


Nagamine, K., S.Shibamoto, K.Takeuchi, K.Miyazawa, N.Kitamura, Y.Chatani, M.Kohno and F. Ito (1996) Dissociation of c-fos induction and mitogen-activated-protein kinase activation from the hepatocyte-growth-factorinduced motility response in human gastric carcinoma cells. Eur. J. Biochem., 236, 476–81 Naldini, L., K.M.Weidner, E.Vigna, G.Gaudino, A.Bardelli, C.Ponzetto, R.P.Narsimhan, G.Hartmann, R.Zarnegar, G.K.Michalopoulos et al. (1991) Scatter factor and hepatocyte growth factor are indistinguishable ligands for the MET receptor. EMBO J., 10, 2867– 78. Nobes, C.D., P.Hawkins, L.Stephens and A.Hall (1995) Activation of the small GTP-binding proteins rho and rac by growth factor receptors. J. Cell Sci., 108, 225–33. Otto, T., W.Birchmeier, U.Schmidt, A.Hinke, J.Schipper, H.Rubben and A.Raz (1994) Inverse relation of E-cadherin and autocrine motility factor receptor expression as a prognostic factor in patients with bladder carcinomas. Cancer Res., 54, 3120–3. Panayotou, G. and M.D.Waterfield (1992) Phosphatidylinositol 3-kinase: a key enzyme in diverse signalling processes. Trends Cell Biol., 2, 358–60. Pelicci, G., S.Giordano, Z.Zhen, A.E.Salcini, L.Lanfrancone, A.Bardelli, G.Panayotou, M.D.Waterfield, C. Ponzett, P.G.Pelicci et al. (1995) The motogenic and mitogenic responses to HGF are amplified by the She adaptor protein. Oncogene, 10, 1631–8. Ponzetto, C., A.Barcjelli, Z.Zhen, F.Maina, P.dalla Zonca, S.Giordano, A.Graziani, G. Panayotou and P.M.Comoglio (1994) A multifunctional docking site mediates signaling and transformation by the hepatocyte growth factor/ scatter factor receptor family. Cell, 77, 261–71. Ridley, A.J. and Hall, A. (1992) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell, 70, 389–99. Ridley, A.J., P.M.Comoglio and A.Hall (1995) Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol. Cell. Biol., 15, 1110–22. Ridley, A.J. and Hall, A. (1994) Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J., 13, 2600–10. Ridley, A.J. (1994) Membrane ruffling and signal transduction. Bioessays, 16, 321–7. Rodier, J.M., A.M.Valles, M.Denoyelle, J.P.Thiery and B.Boyer (1995) pp60c-src is a positive regulator of growth factor-induced cell scattering in a rat bladder carcinoma cell line. J. Cell Biol., 131, 761–773. Rosen, E.M. and Goldberg, I.D. (1995) Regulation of scatter factor (hepatocyte growth factor) production by tumorstromal interaction, in Epithelial-Mesenchymal Interactions in Cancer, ed. by Goldberg and Rosen, pp. 17–32. Rosen, E.M., A.Joseph, L.Jin, S.Rockwell, J.A.Elias, J.Knesel, J.Wines, J.McClellan, M.J. Kluger, I.D. Goldberg et al. (1994) Regulation of scatter factor production via a soluble inducing factor. J. Cell Biol., 127, 1783–7. Rubinfeld, B., B.Souza, I.Albert, O.Muller, S.H.Chamberlain, F.R.Masiarz, S.Munemitsu and P. Polakis (1993) Association of the APC gene product with beta-catenin. Science, 262, 1731–4. Rubinfeld, B., I.Albert, E.Porfiri, C.Fiol, S.Munemitsu and P.Polakis (1996) Binding of GSK3beta to the APC-betacatenin complex and regulation of complex assembly. Science, 272, 1023–6. Sachs, M., K.M.Weidner, V.Brinkmann, I.Walther, A.Obermeier, A.Ullrich and W. Birchmeier (1996) Motogenic and morphogenic activity of epithelial receptor tyrosine kinases. J.Cell Biol., 133, 1095–107. Savagner, P., A.M.Valles, J.Jouanneau, K.M.Yamada and J.P.Thiery (1994) Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells. Mol. Biol. Cell, 5, 851–862. Schimoyama, Y. and Hirohashi, S. (1991) Expression of E- and P-cadherin in gastric carcinomas. Cancer Res., 51, 2185–92. Schipper, J.H., Frixen, U.H., Behrens, J., Unger, A., Jahnke, K. and Birchmeier, W. (1991) E-cadherin expression in squamous cell carcinomas of head and neck: inverse correlation with tumor dedifferentiation and lymph node metastasis. Cancer Res., 51, 6328–37. Schor, S.L., A.M.Grey, I.Ellis, A.M.Schor, B.Coles and R.Murphy (1993) Migration stimulating factor (MSF): its structure, mode of action and possible function in health and disease. Symp. Soc. Exp. Biol., 47, 235–51.


355

Schor, S.L. (1995) Fibroblast subpopulations as accelerators of tumor progression: The role of migrating stimulating factor, in Epithelial-Mesenchymal Interactions in Cancer, ed. by Goldberg and Rosen, 273–96. Shi, Y.E. and Y.Liu (1995) Stromal-epithelial interaction in type IV collagenase expression and activation: The role in cancer metastasis. Epithelial-Mesenchymal Interactions in Cancer, ed. by Goldberg and Rosen, pp. 215–34. Shimoyama, Y., A.Nagafuchi, S.Fujita, M.Gotoh, M.Takeichi, S.Tsukita and S.Hirohashi (1992) Cadherin dysfunction in a human cancer cell line: possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesiveness. Cancer Res., 52, 5770–4. Silletti, S., H.Watanabe, V.Hogan, I.R.Nabi and A.Raz (1991) Purification of B16-F1 melanoma autocrine motility factor and its receptor. Cancer Res., 51, 3507–11. Silletti, S., J.P.Yao, K.J.Pienta and A.Raz (1995) Loss of cell-contact regulation and altered responses to autocrine motility factor correlate with increased malignancy in prostate cancer cells. Int. J. Cancer, 63, 100–105. Stoker, M., E.Gherardi, M.Ferryman and J.Gray (1987) Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature, 327, 239–42. Stracke, M.L., H.C.Krutzsch, E.J.Unsworth, A.Arestad, V.Cioce, E.Schiffmann and L.A. Liotta (1992) Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J. Biol. Chem., 267, 2524–9. Stracke, M.L., T.Clair, S.Aznavoorian, P.Mulvaney and L.A.Liotta (1996) The exophosphodiesterase catalytic site of autotaxin is necessary for motility stimulation. Clinical and Experimental Metastasis, 14, 18. Su, L.K., B.Vogelstein and K.W.Kinzler (1993) Association of the APC tumor suppressor protein with catenins. Science, 262, 1734–7. Takeichi, M. (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science, 251, 1451–5. Toyoshima, K., N.Ito, Y.Hiasa, Y.Kamamoto and S.Makiura (1971) Tissue culture of urinary bladder tumor induced in a rat by N-butyl-N-(-4-hydroxybutyl)nitrosamine: establishment of cell line, Nara Bladder Tumor II. J. Natl Cancer Inst., 47, 979–85. Tucker, G.C., A.Delouvee, J.Jouanneau, J.Gavrilovic, G.Moens, A.M.Valles and J.P.Thiery (1991) Amplification of invasiveness in organotypic cultures after NBT-II rat bladder carcinoma stimulation with in vitro scattering factors. Invasion Metastasis, 11, 297–309. Valles, A.M., B.Boyer, J.Badet, G.C.Tucker, D.Barritault and J.P.Thiery (1990) Acidic fibroblast growth factor is a modulator of epithelial plasticity in a rat bladder carcinoma cell line. Proc. Natl. Acad. Sci. USA, 87, 1124–8. Valles, A.M., B.Boyer, G.Tarone and J.P.Thiery (1996) α2β1 integrin is required for the collagen and FGF-1 induced cell dispersion in a rat bladder carcinoma cell line. Cell Adh. Comm., 4, 187–99. Van der Bliek, A.M., T.E.Redelmeier, H.Damke, E.J.Tisdale, E.M.Meyerowitz and S.L. Schmid (1993) Mutations in human dynamin block an intermediate stage in coated vesicle formation. J. Cell Biol., 122, 553–63. Watanabe, H., K.Takehana, M.Date, T.Shinozaki and A.Raz (1996) Tumor cell autocrine motility factor is the neuroleukin/phosphohexose isomerase polypeptide. Cancer Res., 56, 2960–3. Weidner, K.M., J.Behrens, J.Vandekerckhove and W.Birchmeier (1990) Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells. J. Cell Biol., 111, 2097–108. Weidner, K.M., S.Di Cesare, M.Sachs, V.Brinkmann, J.Behrens and W.Birchmeier (1996) Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature, 384, 173–6. Yamashita, J., M.Ogawa, S.Yamashita, K.Nomura, M.Kuramoto, T.Saishoji and S.Shin (1994) Immunoreactive hepatocyte growth factor is a strong and independent predictor of recurrence and survival in human breast cancer. Cancer Res., 54, 1630–3. Yoshiura, K., Y.Kanai, A.Ochiai, Y.Shimoyama, T.Sugimura and S.Hirohashi (1995) Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc. Natl. Acad. Sci. USA, 92, 7416–7419.

19. MULTIPLE MECHANISMS FOR INACTIVATION OF ECADHERIN CELL ADHESION SYSTEM IN CANCER ATSUSHI OCHIAI, YAE KANAI and SETSUO HIROHASHI Pathology Division, National Cancer Center Research Institute, 5–1–1 Tsukiji, Chuo-ku, Tokyo 104, Japan The E-cadherin cell adhesion system controls epithelial cell polarity and tissue morphology. Cell polarity disturbance and destruction of normal tissue morphology are histological features of human cancers. Cancer invasion is initiated by cell dissociation from primary cancer nests, due to reduced intercellular adhesion, and the E-cadherin cell adhesion system is known to act as an “invasion-suppressor” in human cancers. The system is inactivated by various mechanisms, including reduced expression, genetic alteration and/or tyrosine phosphorylation of E-cadherin and its undercoat proteins, termed catenins. The system interacts directly with products of oncogenes, e.g. c-erbB-2 protein, epidermal growth factor receptor, and of tumor suppressor gene APC protein, through β-catenin and may participate in regulation of cell growth and differentiation. Transfection of truncated P~catenin, which dominant-negatively inhibits the E-cadherin and c-erbB-2 protein interaction, into human cancer cell lines showing strong tyrosine phosphorylation of β-catenin reduced peritoneal dissemination and induced well differentiated histological features of the tumors that developed in vivo, in comparison with control cells. Regulation of the system by tyrosine phosphorylation of β-catenin may be of importance in signal transduction pathways contributing to the determination of biological properties of human cancers. BASIC PROPERTIES OF THE CADHERIN SYSTEM IN CANCER Cell-cell adhesion determines cell polarity and participates in histogenesis. As early as the 1940s, the mutual adhesiveness of cancer cells was known to be significantly weaker than that of the corresponding normal cells (Coman, 1944; McCutcheon et al., 1948). Reduced intercellular adhesiveness enables cancer cells to disobey the social order, resulting in destruction of the histological structure, which is the morphological hallmark of malignant tumors. Reduced intercellular adhesiveness is also considered to be indispensable for the critical steps of multistage carcinogenesis, invasion and metastasis. Most cadherin molecules are integral membrane glycoproteins that show homophilic binding specificity and act as Ca2+-dependent cell-cell adhesion molecules (reviewed by Takeichi, 1991). In epithelial cells, E (epithelial)-cadherin (120 kDa) molecules accumulate to form typical, belt-like, cell-cell adherens junctions (Geiger and Ginsberg, 1991). Through its cytoplasmic domain, E-cadherin associates with catenins, which anchor it to the actin cytoskeleton, and molecular interactions among these proteins are considered indispensable for the E-cadherin cell adhesion system (Nagafuchi and Takeichi, 1988; Ozawa et al., 1989, 1990).

Corresponding author: Setsuo Hirohashi, Pathology Division, National Cancer Center, Research Institute, 5–1–1 Tsukiji, Chuo-ku, Tokyo 104, Japan. Tel: 81–3–3542–2511; Fax: 81–3–3248–2737; E-mail: [email protected]


357

Substantial evidence indicating the E-cadherin cell adhesion system is involved in cancer invasion and metastasis has accumulated. Suppression of E-cadherin activity may trigger the release of cancer cells from primary cancer nests and confer invasive properties on tumors. In fact, in vitro experiments using cell lines have revealed that E-cadherin has invasion-suppressive properties: non-invasive epithelial cells acquired the ability to invade collagen gels when anti-E-cadherin antibodies (Behrens et al., 1989) or plasmids encoding E-cadherin-specific antisense RNA (Vleminckx et al., 1991) were added to their cultures. Examination of Ecadherin protein expression in various cell lines (Vleminckx et al., 1991; Mareel et al., 1991; Sommers et al., 1991; Frixen et al., 1991) revealed that non-invasive cells with epithelioid phenotypes expressed Ecadherin, whereas invasive cells with fibroblastoid phenotypes did not. Moreover, the invasive potential of the latter was suppressed when the cell lines were transfected with E-cadherin cDNA (Frixen et al., 1991). These in vitro findings suggest E-cadherin plays a key role in the maintenance of regulated epithelial morphology and that breakdown of the system confers invasive potential on cancer cells. In this review, we discuss the mechanisms responsible for dysfunction of this system in human cancers. REDUCED EXPRESSION OF E-CADHERIN In an attempt to elucidate the significance of the invasion-suppressive properties of E-cadherin in human cancerous tissue in vivo, we performed immunohistochemical examinations of gastric carcinoma specimens from 54 patients (Shimoyama and Hirohashi, 1991) using a monoclonal antibody, HECD-1, which recognizes human E-cadherin (Shimoyama et al., 1989). We found that E-cadherin was expressed uniformly in most of the differentiated and adherent-type undifferentiated gastric adenocarcinomas, whereas it was not detected in four surgical specimens of scattered-type undifferentiated gastric adenocarcinomas lacking tight intercellular adhesion (Shimoyama and Hirohashi, 1991). These findings indicated that loss of E-cadherin expression freed cells from tight cell-cell association and conferred invasive properties on them. Several other studies on E-cadherin protein expression in various human primary, e.g. stomach (Oka et al., 1993; Mayer et al., 1993), head and neck (Schipper et al., 1993), esophageal (Shiozaki et al., 1993), prostate (Umbas et al., 1993) and breast (Rasbridge et al., 1993), cancers have been reported (reviewed by Birchmeier and Behrens, 1994). Generally, E-cadherin expression was strong in well differentiated cancers, which maintained their intercellular adhesiveness and had less invasive properties, but was weaker in undifferentiated cancers, which had lost their cell-cell adhesiveness and showed strong invasive tendencies. Therefore, the E-cadherin-mediated invasion suppressor system was considered to be inactivated when Ecadherin expression was reduced in vivo. Moreover, significant correlations between abnormalities of Ecadherin expression and the clinical outcomes of patients with cancers derived from the bladder (Bringuier et al., 1993) and head and neck (Mattussen et al., 1993) have been reported. In order to clarify the mechanism responsible for regulation of E-cadherin expression in cancers, we cloned and sequenced the promoter region of the human E-cadherin gene. The chloramphenicol acetyltransferase (CAT) assay revealed epithelial cell-specific promoter activity in the –191 to +94 human E-cadherin promoter/ CAT construct (Yoshiura et al., 1995). This active promoter region contains CpG islands and subsequently, we demonstrated CpG methylation around the promoter region of the E-cadherin gene and induction of E-cadherin expression following treatment with the DNA methyltransferase inhibitor 5-azacytidine in human cancer cell lines lacking E-cadherin expression, suggesting that E-cadherin expression in human cancer cells is regulated by CpG methylation around the promoter region (Yoshiura et al., 1995). The inaccessibility of the promoter region of the E-cadherin gene to transcription factors in human cancer cell lines has also been proposed to be due to CpG methylation (Henning et al., 1995).

358


In vivo, CpG methylation around the promoter region of the E-cadherin gene was detected in 67% of primary hepatocellular carcinomas and correlated significantly with immunohistochemically detected reduction of E-cadherin expression (Figure 1, Kanai et al., 1997). CpG methylation around the promoter region, which increases during the progression from the precancerous conditions, chronic hepatitis and cirrhosis, to hepatocellular carcinomas, may participate in hepatocarcinogenesis through reduction of Ecadherin expression, resulting in loss of intercellular adhesiveness, loss of contact inhibition and destruction of tissue morphology (Kanai et al., 1997). However, we also found that cancer cells of quite a few undifferentiated gastric adenocarcinomas with scattered cell growth patterns showed definite E-cadherin expression (Shimoyama and Hirohashi, 1991), which raised the question why were these cancer cells unable to form tight cell-cell associations? Although E-cadherin was expressed strongly in some cancers showing scattered-type invasion, the distribution of immunoreactive E-cadherin was abnormal, indicating that E-cadherin dysfunction may be mediated, at least in part, by structural abnormalities of normally expressed non-functional molecules. Dysfunction of cellcell adhesion in tumors showing strong E-cadherin expression may also be due to abnormalities of associated proteins. In order to investigate these possibilities, we examined human carcinoma cell lines lacking tight cell-cell adhesion. GENETIC ALTERATION OF E-CADHERIN We focused on structural abnormalities of E-cadherin in cancer cells. Twelve human cancer cell lines showing loose cell-cell adhesion were investigated for E-cadherin gene mutations, and genetic alterations of E-cadherin were detected in two of them, MKN45 and KATO-III. The MKN 45 cell line was established from a poorly differentiated human gastric adenocarcinoma (Motoyama and Watanabe, 1983) and these cells expressed E-cadherin mRNA strongly. Northern blot analysis showed the size of this mRNA was normal and the protein expression level, detected by Western blot analysis, was similar to that in control cell lines. RNA sequencing revealed a 12-bp deletion in the mRNA, which resulted in a 4-amino acid deletion between the Ca2+-binding motifs of the 1st cadherin domain (Oda et al., 1994). At the DNA level, an 18-bp deletion around the exon 6-intron 6 boundary resulted in the loss of the entire “donor consensus sequence (Padgett et al., 1986)” for RNA splicing. Instead of this “true” donor signal, the cryptic GpT dinucleotides positioned 12-bp upstream and surrounding the consensus sequence were used for RNA splicing (Oda et al., 1994). It is conceivable that the deletion in MKN 45 cells altered the conformation around the Ca2+-binding motif of normally expressed E-cadherin and reduced the cell-cell adhesion potential. The KATO-III cell line was established from a clinical specimen of a signet ring cell carcinoma of the stomach taken from a patient (Motoyama and Watanabe, 1983). The amount of E-cadherin mRNA KATOIII cells expressed, evaluated by Northern blot analysis, was reduced appreciably in comparison with other cell lines possessing tight cell-cell adhesion and Western blot analysis failed to detect E-cadherin in the former. Although Southern blot analysis detected no gross abnormalities in the E-cadherin gene, direct sequencing of all the shifted bands revealed by the RT-PCR-SSCP method demonstrated the presence of four different mRNA transcripts containing insertions of 7, 25, 42 and approximately 150 bp (Oda et al., 1994) (Figure 2). The DNA point mutation in KATO-III cells, a G to A base substitution of the last 3' nucleotide of exon 7, also affected the donor consensus sequence (Oda et al., 1994). The reported association between mutations and degradation of mRNA is also noteworthy (Maquat et al., 1994; Daar and Maquat, 1994): structural abnormalities of the E-cadherin gene may result in rapid post-transcriptional degradation of mRNA in KATO III cells.


359

Figure 1 (A) CpG methylation around the promoter region of the E-cadherin gene in normal liver (N) and hepatocellular carcinoma (T) tissues. High-molecular-weight DNA was digested with Eco RI and Bam HI (EB); Eco RI, Bam HI and Msp I (EBM) or Eco RI, Bam HI and Hpa II (EBH), subjected to electrophoresis, transferred to nitrocellulose membranes and hybridized with a DNA probe including of the E-cadherin promoter. Neither EBM nor EBH digestion of N yielded definite bands, whereas EB digestion yielded 2.6-kb DNA bands, indicating that the CpG sites around the promoter region were not methylated. Both EB and EBH digestion of T yielded 2.6-kb DNA bands, whereas EBM digestion yielded no definite bands, indicating that the CpG sites around the promoter region were methylated. (B) Immunohistochemical examination using a monoclonal antibody against E-cadherin, HECD-1. Hepatocellular carcinoma cells had lost E-cadherin immunoreactivity (T), whereas the surrounding non-cancerous hepatocytes showed strong Ecadherin immunoreactivity at the cell-cell borders (N).

In both the above cell lines, loss of heterozygosity at the E-cadherin locus was confirmed by PCR-SSCP analysis, using intronic primers, of exons 6 and 7 of genomic E-cadherin (Oda et al., 1994). E-cadherin dysfunction in these cell lines may be attributable to a combination of the loss of one allele and a mutation in the other. It is noteworthy that loss of heterozygosity on the long arm of chromosome 16, to which the E-

360


cadherin gene has been assigned (Mansouri et al., 1988), was detected frequently in human metastasizing cancers (Tsuda et al., 1990). In fact, in vivo, we detected E-cadherin gene mutations and loss of heterozygosity on the long arm of chromosome 16 in invasive lobular carcinomas of the breast (Kanai et al., 1994), whereas ductal carcinomas of the breast did not show such mutations and a high incidence of exon skipping-type mutations in diffuse-type stomach adenocarcinomas was reported (Becker et al., 1994). Furthermore, we detected exon skipping-type mutations accompanied by loss of heterozygosity at the E-cadherin locus even in the intramucosal lesions of signet ring cell carcinomas of the stomach (Muta et al., 1996). Taken together, these findings suggest strongly that E-cadherin gene inactivation by a unique mechanism, which follows the twohit theory of classical tumor-suppressor genes, is involved in the developmental stages of diffuse-type cancers. GENETIC ALTERATIONS AND REDUCED EXPRESSION OF α-CATENIN We have also focused on abnormalities of catenins. Immunofluorescence cytochemical analysis of PC9 cells revealed E-cadherin all along the cell membranes and Western blot analysis showed that the molecular sizes of the E-cadherin expressed on PC9 and control cells were the same (Shimoyama et al., 1992). However, PC9 cells showed a low rate of E-cadherin-mediated aggregation in the presence of Ca2+. The immunoprecipitate obtained from PC9 cells using an anti-E-cadherin monoclonal antibody, HECD-1, contained a protein considered to be β-catenin, but no band at the position corresponding to α-catenin was detected and Western blotting using an anti-α-catemn monoclonal antibody, α-18, detected no α-catenin in PC9 cells (Shimoyama et al., 1992). In order to study a-catenin abnormalities more precisely, we isolated clones of human α-catenin cDNA using a mouse cDNA (Nagafuchi et al., 1991) as a probe and determined their nucleotide sequences (Oda et al., 1993). Northern blot analysis using the newly obtained human α-catenin cDNA probe revealed faint 3. 0- and 2.7-kb bands in PC9 cells and cloning sequence analysis revealed that these cells possessed two distinct clones, one with a 957–and the other with a 76l-bp deletion (Oda et al., 1993). The former resulted in a 319-amino acid deletion and the latter caused a frameshift resulting in a stop codon four codons after the breakpoint. The breakpoints of these two clones, the 5' margins of the deleted regions, were both GpT and their 3' margins were at identical positions, suggesting that incorrect donor-like sites were employed in RNA splicing. Southern blot analysis demonstrated that part of the α-catenin gene, which corresponded to the deleted region in the mRNA, was deleted homologously in PC9 cells (Oda et al., 1993). Thus, mRNA instability may result in markedly reduced α-catenin expression in PC9 cells, as observed with E-cadherin in KATO-III cells. Hirano et al. (1992) reported that PC9 cells regain their cell-cell adhesion potential when they were transfected with mouse α-catenin cDNA which provided the first evidence in vitro that a-catenin abnormalities caused dysfunction of the E-cadherin-mediated intercellular adhesion system in cancer cells. In fact, we found that α-catenin expression was often reduced in adenocarci-nomas of the stomach and breast, in which cancer cells are characteristically detached from each other and infiltrate the stroma in a scattered manner (Ochiai et al., 1994a), indicating an association between reduced α-catenin expression and a morphologically invasive tendency in vivo.


361

Figure 2 (A) Schematic representation of the molecular mechanisms responsible for producing four different mRNA clones through splicing errors in KATO-III cells. A single base substitution, G to A (indicated by bold italic letters), reduces the value of the donor site signal GpT (open circles) of the exon 7-intron 7 boundary. Use of “incorrect” GpTs (solid circles) alters the RNA splicing process, producing 7–, 25–, 42– and approximately 150– bp insertions in the intron sequences. (B) PCR-SSCP analysis, using intronic primers, of exon 7 of the E-cadherin gene in KATO-III cells. Only shifted bands of exons 7, representing the mutated allele, were detected, indicating that the normal allele was absent.

GENETIC ALTERATIONS AND TYROSINE PHOSPHORYLATION OF β-CATENIN We also analyzed two human stomach cancer cell lines, HSC-39 and HSC-40A, showing loose cell-cell adhesion and an identical 321-bp in-frame β-catenin mRNA deletion was identified in both (Oyama et al., 1994). The truncated β-catenin lacked the region through which β-catenin is believed to interact with acatenin. In an immunoprecipitation experiment using an anti-E-cadherin monoclonal antibody, HECD-1, the truncated β-catenin co-precipitated with E-cadherin, whereas α-catenin did not, indicating that the interaction between α-catenin and E-cadherin is not direct, but mediated by β-catenin (Oyama et al., 1994).

362


This study provided the first evidence of a genetic abnormality of β-catenin that disrupts the interaction between E-cadherin and α-catenin in and may participate in the loosely adhesive growth pattern of human cancer cell lines. Moreover, both cell lines originated from the ascites of one patient, but were established independently using different methods, suggesting that the mutation had already occurred and might have played a role in the invasive growth pattern of the tumor in vivo. Southern blot analysis of these cell lines disclosed homozygous deletion of part of the β-catenin gene in both and this could be a mechanism that inactivates the E-cadherin system. Recently, mutations at the N-terminal site of β-catenin, which interacts with glycogen synthase kinase-3β (GSK-3β), a mammalian homolog of ZW3 kinase of Dorsophila, in primary colon cancers lacking APC tumor suppressor gene mutations were reported (Morin et al., 1997). The interaction between the GSK-3β phosphorylation site of β-catenin and APC protein is considered essential for the tumor suppressive effect of APC and mutations of the APC gene, as well as those of b-catenin inhibit this effect. We found that the deleted β-catenin in the HSC-39 and 40A cell lines also included this functionally significant phosphorylation site. Aberrant tyrosine phosphorylation of β-catenin is another mechanism that has been found to contribute to the disruption of cadherin function: as Matsuyoshi et al. (1992) reported that tyrosine phosphorylation levels of β-catenin correlated with cell adhesion dysfunction in jfr-transformed cells. We carried out Western blotting analysis using an anti-phosphotyrosine monoclonal antibody followed by immunoprecipitation with HECD-1 and demonstrated strong tyrosine phosphorylation of β-catenin and Ecadherin in loosely adherent cancer cell lines, which showed neither mutations nor reduced expression of Ecadherin or α- and β-catenins. SIGNAL TRANSDUCTION BETWEEN GROWTH FACTORS AND THE ECADHERIN CELL ADHESION SYSTEM Undifferentiated adenocarcinomas, which often possess structurally altered cadherins or catenins, as discussed in this chapter, invade as a single cells without recovering their glandular structures or cancer cell nests in entire cancer tissues, including metastatic sites (Ochiai et al., 1994). On the other hand, the cancer cells of differentiated adenocarcinomas form glands and tumor cell nests, displaying tight cell-cell connections in most of the tumor tissue. It is well known that such differentiated adenocarcinomas frequently lose their glandular structures at the invading fronts and then recover cell-cell adhesiveness and form glands in metastatic sites (Jass et al., 1986; Hase et al., 1993; Ono et al., 1996). Such reversibility of the E-cadherin-mediated cell adhesion system indicates the presence of another cell dissociation mechanisms that contributes to reversible cadherin dysfunction in cancer cells. One such putative mechanism is aberrant protein tyrosine phosphorylation of β-catenin (Matsuyoshi et al., 1992; Shibamoto et al., 1994). We demonstrated physical associations between the c-erbB-2 gene product and β-catenin in human adenocarcinoma cell lines (Ochiai et al., 1994b; Kanai et al., 1994b) and an association between β-catenin and the epidermal growth factor (EGF) receptor, which is highly homologous with c-erbB-2, in a squamous cell carcinoma cell line has been reported (Hoschuetzky et al., 1994). Amplification of the c-erbB-2 gene was reported to correlate with a poor prognosis in patients with breast and gastric adenocarcinomas (Slamon et al., 1989; Wright et al., 1989; Uchino et al., 1993). In order to investigate signal transduction between the c-erbB-2 gene product and β-catenin, we examined the total phosphotyrosine levels, phosphotyrosine levels of c-erbB-2 and β-catenin and morphological changes in human adenocarcinoma cells after stimulation or inhibition of their tyrosine kinase activities. Tyrosine phosphorylation level alterations in human cancer cells are accompanied by morphological changes. Treatment of a human poorly differentiated adenocarcinoma cell line, TMK-1, with transforming


363

Figure 3 Alterations in the morphology of (A, B) and localization of phosphorylated tyrosine residues, detected by immunocytochemical staining (C, D), in TMK-1 cells after treatment with 10nM TGF-α (B and D) or no treatment (A and C). TMK-1 cells showed loose cell-cell connections, a scattered distribution and markedly increased tyrosine phosphorylation after TGF-α treatment.

growth factor α (TGF-α) caused rapid and dramatic morphological changes (Figure 3A, B). Within a few minutes, some TMK-1 cells started to become round or spindle-shaped, cell movement was promoted markedly and within an hour, most of the cells were scattered and had lost their cell-cell connections. Tyrosine phosphorylation in both the cytoplasm and cell membranes of TMK-1 cells had increased markedly after stimulation with TGF-α for 15 min, when maximal phosphorylation of both the c-erbR-2 gene product and β-catenin was also observed, and declined gradually thereafter (Figure 4). TGF-α induced phosphorylation of c-erbB-2 and β-catenin was confirmed by the in vitro kinase assay in a cell-free system. TGF-α treatment inhibited cadherin-dependent TMK-1 cell aggregation significantly (p

Epithelial Morphogenesis in Development and Disease (Cell Adhesion and Communication)

Cell Junctions: Adhesion, Development, and Disease

Cell Adhesion and Migration in Skin Disease (Cell Adhesion and Communication)

Tissue Remodeling and Epithelial Morphogenesis

Cell Migration in Development and Disease

Epithelial Cell Culture Protocols

Adhesion Molecules in Health and Disease

Molecular Basis of Epithelial Appendage Morphogenesis

Epithelial Cell Culture Protocols

Advances in Molecular and Cell Biology Volume 16: Cell Adhesion

Epithelial Cell Culture Protocols

Fungal Morphogenesis (Developmental and Cell Biology Series)

Mouse Development: Patterning, Morphogenesis, and Organogenesis

Intercellular Signaling in Development and Disease: Cell Signaling Collection

Cell adhesion: fundamentals and biotechnological applications

Centrosomes in Development and Disease

Receptors of Cell Adhesion and Cellular Recognition

Sickle Cell Disease (Genes and Disease)

Stem Cell Biology in Health and Disease

Pancreatic Beta Cell in Health and Disease

Insect Development: Morphogenesis, Molting and Metamorphosis

Cell Communication in Nervous and Immune Systems

Cell Communication in Nervous and Immune System

Cell Communication in Nervous and Immune System

Cress Cell Adhesion and Cytoskeletal Molecules in Metastasis

Plant Cytoskeleton in Cell Differentiation and Development

The Cell Cycle and Development

The Cell Cycle and Development

Cell Cycle in Development

Cell Fusion in Health and Disease: II: Cell Fusion in Disease

Molecular Basis Of Epithelial Appendage Morphogenesis (Molecular Biology Intelligence Unit)

Epithelial Morphogenesis in Development and Disease (Cell Adhesion and Communication)

Cell Junctions: Adhesion, Development, and Disease

Cell Adhesion and Migration in Skin Disease (Cell Adhesion and Communication)

Tissue Remodeling and Epithelial Morphogenesis

Cell Migration in Development and Disease

Epithelial Cell Culture Protocols

Adhesion Molecules in Health and Disease

Molecular Basis of Epithelial Appendage Morphogenesis

Epithelial Cell Culture Protocols

Advances in Molecular and Cell Biology Volume 16: Cell Adhesion

Epithelial Cell Culture Protocols

Fungal Morphogenesis (Developmental and Cell Biology Series)

Mouse Development: Patterning, Morphogenesis, and Organogenesis

Intercellular Signaling in Development and Disease: Cell Signaling Collection

Cell adhesion: fundamentals and biotechnological applications

Centrosomes in Development and Disease

Receptors of Cell Adhesion and Cellular Recognition

Sickle Cell Disease (Genes and Disease)

Stem Cell Biology in Health and Disease

Pancreatic Beta Cell in Health and Disease

Insect Development: Morphogenesis, Molting and Metamorphosis

Cell Communication in Nervous and Immune Systems

Cell Communication in Nervous and Immune System

Cell Communication in Nervous and Immune System

Cress Cell Adhesion and Cytoskeletal Molecules in Metastasis

Plant Cytoskeleton in Cell Differentiation and Development

The Cell Cycle and Development

The Cell Cycle and Development

Cell Cycle in Development

Cell Fusion in Health and Disease: II: Cell Fusion in Disease

Molecular Basis Of Epithelial Appendage Morphogenesis (Molecular Biology Intelligence Unit)

Recommend Documents