International Review of Cytology: A Survey of Cell Biology, Volume 180

VOLUME 180 SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik 1949...

Author: Kwang W. Jeon

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VOLUME 180

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

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

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

M. Melkonian Keith E. Mostov Andreas Oksche Vladimir R. Pantic L. Evans Roth Jozef St. Schell Manfred Schliwa Wilfred 0. Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

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

VOLUME 180

ACADEMIC PRESS San Diego London Boston New York

Sydney Tokyo Toronto

Fronr cover photogrph: The cytoplasm of mouse basophils shows large numbers of perigranular vesicles. (For more details, see Chapter 3, Figure 6a.)

This book is printed o n acid-free paper.

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Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy ee is the same as for current chapters. 0074-7696/98 $25.00

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525 B Street, Suite 1900, San Diego, v92101-4495, USA http://www.apnet.com Academic Press Limited 24-28 Oval Road, London NWI 7DX, UK http://www.hbuk.co.uk/ap/ International Standard Book Number: 0-12-364584-0 PRINTED IN THE UNTIED STATES OF AMEIUCA 98 99 0 0 0 1 02 0 3 E B 9 8 7 6

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3 2 1

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

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Gene Expression during Amphibian Limb Regeneration Jacqueline Geraudie and Patrizia Ferretti I. II. 111. IV. V.

.................................. Introduction .......... Molecules Regulated duri .............................. Growth Control in the Blastema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genes Controlling Morphogenesis in the Regenerating Limb ..................... Concluding Remarks ..................................... References ....................................

1 4

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23

Biochemistry of the Extracellular Matrix of Volvox Manfred Sumper and Armin Hallmann I. 11. 111. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ultrastructure of the Volvox ECM . . . . . . . . . Biochemical Characterization of ECM Components ECM Biogenesis and Remodeling , ..................................... ................ Relationship to Higher Plant ECMs . . . . . . . .

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

51 54 56 62 74

75 79

Cell Biology of the Basophil Ann M. Dvorak I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Basophil Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

87 90

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CONTENTS

Ill. IV. V. VI. VII. VIII. IX. X. XI. XII.

Basophils in Disease . . . . . . . . . . . ................................... Basophils as Secretory Cells . . . . ......... ............... Development of Tools for Advanced Cell-Biological Studies of Basophils , , , , , , , , , , , Vesicles as Prominent Transport Organelles in the Cytoplasm of Basophils . . . . . . . . . Proof of a Degranulation Model Identified in Human Basophils in 1975 . . . . . . Charcot-Leyden Crystal Protein Distributionin Actively Degranulating Human Basophils Histamine Distribution in FMLP4timulated Human Basophil Granules . . . . . . . . . . . . . .............. Recovery of Basophils from Secretion . . Morphometric Analysis of Basophil Degra Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

97 98 110 122

157 192

212 214

Membrane Receptors for Endocytosis in the Renal Proximal Tubule Erik Its0 Christensen, Henrik Birn, Pierre Verroust, and S ~ r e nK. Moestrup I. 11. 111. IV. V. VI. VII.

Introduction , . , . . , . . , , , , , , , , . , . , , . , . , , , , , , . , , , , . , , , , . , , , , . , , . , . , , . , . , , . Ultrastructure of the Endocytic Apparatus in the Proximal Tubule . . . . . . . . . . . . . . . . . Megalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gp28Ollntrinsic Factor Receptor (IFR) . . . . . . . . . . . . . . . . IGF-IIIMan-6-P Receptor., , , . Folate Receptor. . . . . . . . . . . . . . . . . . ............. Concluding Remarks . . . . . . . . ....................... References . . . . . . . . . . . . . . . . . . . ...............................

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

237 239 243

272 272

285

CONTRIBUTORS

Number in parentheses indicate the pages on which the authors' contributions begin.

Henrik Birn (237), Department of Cell BioIogy, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark Erik Its0 Christensen(237), Department of CeII Biology, Institute ofAnatomy, University of Aarhus, DK-8000 Aarhus C, Denmark Ann M. Dvorak (87), Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 022 15 Patrizia Ferretti (1), Developmental Biology Unit, Institute of Child Health, London WClN IEH, United Kingdom Jacqueline Geraudie (1), Laboratoire de Biologie du Developpement, Universite Paris 7, Denis Diderot Case 7077, 75251 Paris Cedex 05, France Arrnin Hallman (51), Lehrstuhl Biochemie I, Universitat Regensberg, 0-93053 Regensberg, Germany Ssren K. Moestrup (237), Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark Manfred Sumper (51), Lehrstuhl Biochemie I, Universitat Regensberg, 0-93053 Regensberg, Germany Pierre Verroust (237), INSERM U64, Hljpital Tenon4, F-75970 Paris Cedex20, France

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Gene Expression during Amphibian Limb Regeneration‘ Jacqueline Geraudie* and Patrizia Ferrettit *Laboratoirede Biologie du DCveloppement, UniversitC Pans 7, Case 7077 75251 Paris Cedex 05, France; and tDevelopmenta1 Biology Unit, Institute of Child Health, UCL, London WClN IEH, United Kingdom

Limb regeneration in adult urodeles is an important phenomenon that poses fundamental questions both in biology and in medicine. In this review, we focus on recent advances in the characterization of the regeneration blastema at cellular and molecular levels and on the current understanding of the molecular basis of limb regeneration and its relationship to development. In particular, we discuss (i) the spatiotemporal distribution of genes and gene products in the mesenchyme and wound epidermis of the regenerating limb, (ii) how growth is controlled in the regeneration blastema, and (iii) molecules that are likely to be involved in patterning the regenerating limb such as homeobox genes and retinoids. KEY WORDS: Blastema, Regeneration, Limb, Urodeles, Extracellular matrix, Growth factors, Cytoskeleton, Homeobox, Retinoids, Wound epidermis.

1. Introduction Limb regeneration involves a series of hierarchical events leading to the replacement of the missing part and can be described as a developmental event induced within the context of an adult animal. It was Spallanzani who, in 1768, brought to the attention of the scientific community his striking observations on the regenerative ability of the legs of aquatic salamanders and asked the fundamental question, “how comes it to pass that other land animals are not endowed with the same power?” Despite the many approaches that have been taken to examine and manipulate the system since Spallanzani’sinitial observations, the regenerative process has

’ This work is dedicated to the memory of Michele Gtraudie. Infernorional Review of Cvrology, Vol. 180

0074-76%/98$25.00

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Copyright 8 1998 by Academic Press. All rights of reproduction in any form reserved.

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JACQUELINE GERAUDIE AND PATRlZlA FERRETTI

retained much of its mysteries. It is hoped that these will be unveiled in a not-too-distant future thanks to the multidisciplinary approach and new methodologies in cell and molecular biology that are now applied to the study of this remarkable phenomenon. The stereotyped sequence of morphological and cytological events that give rise to the newly formed limb has been widely reviewed (Singer, 1952; Thornton, 1968; Iten and Bryant, 1973; Wallace, 1981; Sicard, 1985; Tsonis, 1991,1996; Stocum, 1995) and will be briefly reiterated here. In this review, we will be mainly concerned with recent advances in the characterization of the regeneration blastema at the molecular level and the current understanding of the molecular basis of limb regeneration. The molecular characterization of regeneration blastemas started about 14 years ago when the then novel monoclonal antibody (mAb) technology was used to produce a number of valuable reagents directed against developmentally regulated antigens in the mesenchymal progenitor cells of regenerating limbs (blastemal cells) and in the specialized overlying wound epidermis (Kintner and Brockes, 1984; Tassava et al., 1986). Subsequently, construction and screening of several limb blastema libraries has allowed identification and analysis of the spatiotemporal pattern of expression of several genes. Novel approaches to elucidate the functional significance of some of these genes (Schilthuis et al., 1993; Pecorino et al., 1994, 1996), together with the attempt to identify useful mutants (Del Rio-Tsonis et al., 1992) and produce transgenic urodeles (Y. Le Parco, personal communication), have been recently developed. Analysis of the expression and regulation of genes and gene products associated with limb regeneration, and its comparison with limb bud development, will help to answer a question central to the phenomenon of limb regeneration: To what extent are the molecular signals governing limb development and morphogenesis reiterated during limb regeneration and to what extent does regeneration set its own developmental program? Regeneration of an adult limb is mainly studied using aquatic urodeles (Duellman and Trueb, 1986), such as newts (Notophthalmus, Pleurodeles, and Cynops) and axolotls (Ambystoma),because they display the highest regenerative capability. Amputation or severe tissue damage are the injuries that trigger regeneration and initiate regrowth and morphogenesis, leading to replacement of the lost part (Singer, 1952). The basic events that occur following amputation are epithelialization of the cut surface and formation of a specialized wound epidermis, accumulation of blastemal cells by dedifferentiation of stump tissue cells, proliferation, redifferentiation, and morphogenesis. Closure of the wound is the first process that occurs following limb amputation. Initially, wound healing of the cut surface of the stump is accomplished by migration of stump keratinocytes from the edges of the

GENE EXPRESSION DURING AMPHIBIAN LIMB REGENERATION

3

wound toward its center (Hay and Fishman, 1961; Repesh and Oberpriller, 1980), as observed following skin injury of other body parts. However, wound healing in the stump skin, besides occurring without any scarring, is not simply another example of skin repair. The wound epidermis of regenerating limbs does not rapidly form a basement membrane, like in injured skin in other vertebrates, but rather maintains a direct contact with the underlying mesenchyme (Salpeter and Singer, 1960). This is believed to be important for the extensive remodeling, growth, and patterning of the underlying mesenchymal tissues observed during regeneration. Identification and characterization of the molecular mechanisms controlling formation and maintenance of the limb wound epidermis is important in order to understand how the wound epidermis affects, and is affected by, the underlying mesenchyme. It may also help to gain insights into why skin injury induces scarring in certain parts of the urodele body but not in the regenerating limb. Following reepitheliahation of the wound, a limited histolysis of the stump tissues is observed. This is carried out through activation of several enzymes and remodeling of the extracellular matrix architecture, which modifies the local organization of the tissues (Schmidt, 1968). White blood cells (granulocytes, lymphocytes, and macrophages) invade the injured region and are involved in clearing cell debris and, as a consequence, edema formation can occur. Blastemal cells start to be released from the stump at this stage. The origin of the progenitor cells has been widely studied, and it has been shown that, with the exception of the epidermis, all stump tissues, including dermis, muscle, bone cartilage and nerve, are a source of blastemal cells (Wallace, 1981; Liversage, 1991; Ferretti and Brockes, 1991). Accumulation of blastemal cells is believed to occur through a process of "dedifferentiation" whereby mature differentiated cells lose their differentiated phenotype and reenter the cell cycle. Alternatively, reserve cells present in the stump might be recruited to form a blastema following amputation. However, there is no clear evidence for the existence of such a cell population in stump tissues. A possible exception is the muscle, in which cells with myogenic potential located outside the muscle basement membrane, the postsatellite cells, have been described (Cameron et al., 1986). However, the issue of the origin of progenitor cells in the adult limb, and of how they are activated and released to form the blastema, has not been fully elucidated, although the use of modern methodology has allowed progress toward this goal in the past few years (Brockes, 1994, 1998). Blastemal cells start to proliferate about 3 or 4 days after amputation and their division is under the control of growth factors originating from the peripheral nervous system, the wound epidermis, and possibly the blastemal cells themselves. The blastema develops steadily through stages defined as

4


early, medium, and late bud (Singer, 1952). Cell differentiation begins after the late bud stage and proceeds in a proximal to distal direction through palette (or paddle), notch, and digit stage (Fig. 1). Complete morphological and functional reconstruction of the missing part is achieved between 6 and 10 weeks postamputation, depending on the species, the age of the animal, and the temperature at which the animal is kept. For clarity of presentation, we will discuss changes in gene expression in the wound epidermis (WE) and mesenchyme separately. However, it is important to remember that the functions of WE and blastemal cells are closely integrated (Fig. 2).

II. Molecules Regulated during Regeneration

A. Wound Epidermis The WE of the regenerating adult limb, like that of the developing limb in urodeles, does not form a prominent ridge. In this respect, it is different from the apical ectodermal ridge of the chick limb bud. However, in common with the apical ectodermal ridge is the fact that both epithelia do not have a basal membrane, and both are in direct contact with the underlying mesenchyme. One day after amputation, cells in all layers of the WE contain [3H]thymidine (Riddiford, 1960; Hay and Fishman, 1961), a feature not found in normal epidermis in which stem cells are located in the basal (germinative) layer (Barrandon, 1993). Thymidine incorporation, however,

FIG. 1 Successive stages of regeneration of forelimbs of Ambystoma maculatum larvae amputated at the mid-humerus level and analyzed in longitudinal tissue sections (A-F) and whole mount skeletal preparations stained for cartilage with methylene blue (G and H). The rate of regeneration depends on the temperature, on the urodele species studied and is faster in larvae than in adults. The sequence of events during limb regeneration, however, is essentially the same in all urodeles. (A) Within 5 days after amputation, a few blastemal cells have accumulated beneath the WE at the tip of the damaged stump tissues. (B) Early bud blastema stage. ( C ) Medium bud blastema stage. (D) Late bud stage. (E) Early redifferentiation stage: Note that cartilage is differentiating at the tip of the humerus and cartilage condensation of radius and ulna is beginning. (F) Notch stage (two digits). The carpals and the first two fingers have started to differentiate. A basement membrane has become visible between the epidermis covering the regenerate and the underlying tissues. (G) Three-finger stage. X, unsegmented carpal mesenchyme; d l and d2, carpals articulating with digits 1 and 2. (H) Four-finger stage. All carpals have differentiated. r, carpal radiale; u, carpal ulnare; cl, carpal centrale; d3 and d4, carpals articulating with digits 3 and 4. The first three digits are already fully shaped (photographs provided by D. Stocum).

-_

,/a....

1-3 days preblastema

WE4 (actin-binding protein) WE6 (cytoskeletal protein) 9G 1 collagen XI1 fibronectin tenascin

__ -

metalloproteinases GAGS hyaluronate 22/18 (cytoskeletal protein) 22/31 (vimentin) Hox sonic hedgehog -bandedhedgehog hyaluronidase collagen laminin ST1 -CRABP I same expression as at 1-3d WE3 (actin-binding protein) NvKlI (type I1cytokeratin) KGFR 1 17C-I neuropepbdes

-

4-7 days early blastema

same expression as at 1-3d NvK8 (cytokeratin) NvK18 (cytokeratin) NCAM tenascin fibronectin type I and XI1 collagen FGFRl

-

8-20 days blastemdcone FIG. 2 Drawing summarizing changes in gene and protein expression in the WE and blastemal cells of regenerating urodele limbs at different times after amputation. The day range indicated at each stage reflects the differences in rate of regeneration between species and serves only as a rough guide.


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is very seldom observed at later stages of regeneration in the thickened WE of the limb. The WE has phagocytic activity and also has a role in the cytolysis of the stump tissues, which may favor the release of blastema cells (Singer and Salpeter, 1961; Thornton, 1968; Stocum, 1985). It is well established that inhibition of the regenerative process can be produced by removal of the WE, by covering the cut surface with a skin flap of normal epidermis, or by sawing the skin edges at the amputation site (Stocum, 1985). This is similar to the effect induced by stripping the apical ectodermal ridge of chick and frog limb buds in that it impairs limb growth and produces truncated limbs (Saunders, 1948; Tschumi, 1957). However, whereas the apical ectodermal ridge does not regenerate, the WE regrows very rapidly following its removal. The presence of this specialized epidermis and an adequate nerve supply provide the conditions necessary for initiating the process of recruitment of progenitor cells from the mature tissue of the stump and their proliferation. Changes in the morphology of the WE are reflected by significant changes in the molecular composition of the cells in its different layers. Early cytochemical studies by Schmidt (1968) have demonstrated that the WE synthetizes a variety of enzymes such as alkaline and acid phosphatases, nucleoside phosphatases, lactate dehydrogenase, and esterases. A significant increase in our knowledge of the molecular differences between normal epidermis, epidermis of the developing limb buds, and WE, however, was obtained only when specific antibodies and cDNA/RNA probes were developed. The molecular differences between the WE and the normal epidermis are summarized in Table I, and their possible role in the regenerate outgrowth is discussed below. Homeobox-containing genes expressed in the epidermis, such as members of the dlx family, and some of the molecules that are upregulated both in the WE and in blastemal cells, such as NCAM, tenascin, RARS1, will be discussed later (Fig. 2). The antigen identified by mAb WE3 is expressed in a few small round cells in the epidermis, in cells of the integumentary glands, and in some internal tissues of the adult Nofophfhalmusviridescens (Tassava et aZ., 1986; Goldhamer et aZ., 1989). WE3 is first detected in the basal layers of the WE during the second week after amputation (Tassava etal., 1986). Reactivity of mAb WE3 is not species specific because a positive signal is also observed in the WE of regenerating Pleurodeles walfl limbs (Tassava et al., 1993). Upregulation and maintenance of expression of WE3 are correlated with the increase in mitotic activity of the underlying progenitor cells: Its expression is high during the period of blastemal cells accumulation and growth and is downregulated when the regenerate reaches the digit stage (Fig. 1). WE3 reactivity, which is initially granular in appearance, becomes filamentous and associated with the cytoskeleton during blastemal growth (Goldhamer et aZ., 1989). It has been shown that this mAb recognizes a

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JACQUELINE GERAUDIE AND PATRIZIA FERRETTI

TABLE I Molecules Expressed in the Limb Wound Epidermis (WE) in the Newt, Nofophthalmus viridescens (Unless Otherwise Specified)

Molecule

Detection

Normal limb epidermis

Reference

-

mAb WE3 Actin-binding glycoprotein WE3 (putative carbonic anhydrase)

Some round cells Goldhamer et al. (1989), Tassava et al. (1993), (particularly mitochondriaTassava and Acton (1989) rich cells) Negative

Castilla and Tassava, (1992), Tassava et a/. (1993)

mAb WE6 Cytoskeletonassociated protein WE6 9G1, intracellular mAb 9G1 (axolotl) component (not yet characterized)

Some cells

Estrada et al. (1992)

Not reported

Onda and Tassava (1991)

NvKII type I1 epidermal keratin

Negative

Ferretti et al. (1989, 1993), Ferretti and Ghosh (1997)

mAb 117C1 117C intracellular component (not yet characterized)

Very low levels

Koshiba et al. (1994)

KGFR (variant of FGFR2)

KGFR riboprobe

Very low levels

Poulin et a/. (1993), Poulin and Chiu (1995)

Substance P

Polyclonal antiserum Not reported

Globus and Alles (1990)

Neurotensin


Globus and Alles (1990)

P-Endorphins


Vethamany-Globus (1987)

N-CAM Type XI1 collagen

Polyclonal antiserum Absent mAb MT2 Absent IS-2 riboprobe NvTN.l riboprobe Present (low mAb MT1 levels in basal layer) mAb 55C12 (axolotl)

Maier and Miller (1992) Klatt et al. (1992), Wei et al. (1995)

Fibronectin

Polyclonal antiserum Absent

Repesh et al. (1982). Gulati etal. (1983) Nace and Tassava (1995)

RARSl

mAb MT4 Polyclonal antisera (FW6 and RP8)

Actin-binding protein WE4

Tenascin

mAb WE4

mAb LPlK NvKII riboprobes

Many nuclei

Onda et al. (1990) Onda et al. (1991) Koshiba et al. (1994)

Hill et al. (1993)


9

43- or 44-kDa doublet (Castilla and Tassava, 1992) identified as an actinbinding protein(s) (Tassava et af., 1993). WE3 is coexpressed with the enzyme carbonic anhydrase and it has been proposed that both molecules could be involved in controlling the osmotic regulation and the ionic composition of the blastema (Goldhamer et af., 1989). For example, they could regulate the pH of the intercellular fluid,whose variation might then activate the metabolism of progenitor cells, and possibly their reentry into the cell cycle. Further studies will be needed to clarify whether WE3 itself is a carbonic anhydrase, a possibility supported by its expression in cells of the unamputated limb and in cells of the gastrointestinal tract with secretory/ ion transport function. In addition, this antigen is upregulated by retinoic acid (RA), which has, among many other effects that will be discussed later, the well-known property of inducing secretory differentiation (Tassava, 1992). This strengthens the possibility that WE3 may have a role in secretory/transport function in the adult epidermis. Finally, WE3 is absent in the ectoderm of the limb bud (Tassava and Acton, 1989), in which either a different or no secretory/transport activity is required. The WE3 doublet eluted from a denaturating gel was used as an immunogen for the production of monoclonal antibodies (Castilla and Tassava, 1992). One of the mAbs that was generated, WE4, presented a pattern of reactivity partly overlapping with that of mAb WE3. The two antigens are probably physiologically related because they are coexpressed in many cell types and are both actin-binding proteins (Tassava et af., 1993). However, there are also some significant differences in their expression pattern. Unlike WE3, WE4 is not present in normal limb epidermis of N. viridescens. In addition, WE4, unlike WE3, is expressed in the WE as early as 2 days after amputation and also in the germinative layer of the stump epidermis. Therefore, WE4 expression appears to be induced in response to amputation and is maintained up to the digit stage, longer than WE3 expression. WE4 pattern of expression in P. waltl is very similar to that described in N. viridescens, except for the normal limb epidermis that is WECpositive in P. waltf (Tassava et af., 1993). In order to identify molecules regulated by RA in the WE, Estrada et a2. (1993) immunized mice with the WE of newts that had been treated with RA. One of the mAbs they isolated, WE6, recognizes a cytoskeleton protein that is detected as early as 1 day after amputation in all layers of the WE and does not undergo a transition from a granular to a filamentous form as does WE3. In contrast to WE3 however, WE6 is not regulated by RA and is found in epidermal cells of the stump adjacent to the lesion site but not in the epidermis of the proximal region of the stump tissues. Another antigenic determinant expressed in all cells of the WE has been identified by using the 9G1 mAb (Onda and Tassava, 1991). 9G1 mAb recognizes an intracellular antigen(s), and its reactivity is diffuse and granu-

10


lar in appearance. In young axolotl larvae, but not in adult newts, 9G1 is expressed at most stages of limb regeneration but is absent in normal skin epidermis. However, 9G1 reactivity is not restricted to the WE; it is also found in dedifferentiating chondrocytes and blastemal cells, in which a filamentous network is observed. 9G1 is also induced in the basal layer of WE in the flank and in the underlying mesenchymal cells. Therefore, its expression is associated with wound healing rather than specifically with limb regeneration. Their presence could be related both to specific functions of these molecules during healing and to the age of the experimental animals. Because 9G1 expression was studied in premetamorphosis axolotls, it cannot be ruled out that regulation of this molecule is under the control of thyroid hormones (Onda and Tassava, 1991). In denervated early blastemas, when proliferation of blastemal cells is nerve dependent, 9G1 expression is abolished in the WE but not in blastemal cells (Onda and Tassava, 1991). This suggests that the nerve controls, either directly or indirectly, the phenotype of the cells in the WE. NvKII is a WE-associated keratin that was originally identified as one of the two proteins recognized by the mAb LPlK in the newt, N . viridescens (Ferretti et al., 1989,1991,1993; Ferretti and Ghosh, 1997). To date, NvKII is the only WE marker that has been cloned. NvKII shares a high percentage of amino acid homology with human keratin 5 (K5) and 6 (K6), which are markers of different states of epidermal differentiation. Interestingly, K6 is induced in response to skin injury and in highly proliferative epithelia. Notwithstanding the sequence homology between NvKII and K6 there are some important differences in the expression and regulation of these keratins in newts and humans. NvKII expression is not induced simply in response to injury, as is K6, because it is not detected following injury of the newt flank skin. In addition, it is not expressed in regenerating upper and lower jaws, but it is expressed in cells of the WE of regenerating limbs, in which it is first detected around the time when proliferation of blastemal cells begins (4 or 5 days after amputation). Staining with mAb LPlK does not show any reactivity in the ectoderm of the developing newt limb (Ferretti et al., 1989). Both K6 and NvKII respond to RA treatment, but whereas K6 is upregulated by RA, NvKII is dramatically downregulated. Its transcript levels are higher in distal than in proximal blastemas, but the apparent difference observed in its expression in proximal and distal normal limbs appears to be due to the blastema-like quality of the fingertips, which express many regeneration-associated antigens (Ferretti and Ghosh, 1997). The need for such a specific expression of this cytokeratin in the limb WE is not understood, but the facts that NvKII is higher in distal than in proximal blastemas and is regulated by RA suggest that it may be expressed in cells that play a role in specifying positional information.


11

Strong reactivity with the 117C1 mAb is observed in the cytoplasm of WE and blastema cells, but only faint staining is detectable in epidermis, dermis, muscle, perichondrium, and cartilage of normal limbs (Koshiba et al., 1994). The molecules recognized by the 117C1 mAb have molecular weights of approximately 130,200, and 240 kDa, as established by Western blotting, but their exact nature is not known. The WE has been shown to contain neuropeptides (Vethamany-Globus, 1987; Globus and Alles, 1990). Among these, there is substance P (SP), an undecapeptide that functions as a neurotransmitter in the central and peripheral nervous system and appears to have a mitogenic effect on the blastemal cells in newt limbs and in planaria (Globus et al., 1983; Salo and Baguna, 1986). Reactivity with an antibody to SP is observed in the suprabasal layers of the WE, but it has not been shown whether similar reactivity is present in normal skin. By radioimmunoassay high levels of SP are also detected in the blastema mesenchyme, and this discrepancy has not yet been convincingly explained (Globus and Alles, 1990). It will be important to establish whether, as suggested, SP is present in different forms in the mesenchyme (soluble form) and in the WE (bound to membrane receptors). Immunoreactivity for endogenous opioid peptides (P-EP, P-endorphin-like molecule), which are known to induce analgesia in man, has also been observed in the suprabasal layers of the WE cells of the blastema (Vethamany-Globus, 1987) and may be important for reducing the local pain stimuli induced by amputation. High plasma P-EP levels are maintained during limb regeneration, possibly to reduce pain perception in the amputated animal. In contrast to SP and P-EP, neurotensin-like immunoreactivity is detected only in the basal layer of the WE, and low levels of expression of other peptides belonging to the tachykinin family have been reported in limb blastemas (Globus and Alles, 1990). The precise role of the neuropeptides found in the WE is unknown, and it is also unknown whether their expression reflects a general response to injury or is specific to limb regeneration. The information so far available about the molecular composition of the WE (Fig. 2) reveals that whereas some of the dramatic changes induced by amputation are equivalent to those induced by skin wounding in other body parts, others are intimately linked to the process of limb regeneration. Furthermore, the fact that expression of some of these antigens recapitulates their developmental regulation in the limb bud supports the view that the ectoderm of the developing bud and the WE may play similar roles in the ontogenesis of embryonic and adult limbs. In conclusion, the WE is a very complex structure that encompasses features of both embryonic ectoderm and injured adult skin (9G1) and also appears to have some traits of its own (WE3 and NvKII). It is interesting that both WE-specific molecules (WE3 and NvKII), a cytoskeleton-associated molecule, and a cytoskeletal

12


protein, respectively, are regulated by RA. Because RA affects growth and patterning of the regenerating limb, this suggests that the cytoskeleton may play an important role in controlling the physiological state of the cells of the WE and possibly epithelial-mesenchymal interactions. Analysis of potential molecular mechanisms underlying the permissive role of the WE on growth and patterning of blastemal cells is in progress, and recent works from Bryant’s, Tassava’s, and Boilly’s laboratories suggest that fibroblast growth factor (FGF) is present in the WE and may play an equivalent role in limb development and regeneration (see Section 111).

B. Blastemal Mesenchyme 1. Preblastema We use the term “preblastema” to refer to the regenerative phase between the time of amputation and the onset of cell division, which includes closing of the wound, and the initial phase of formation blastemal cells and their accumulation beneath the wound epidermis. Significant changes in the connective tissues at the level of amputation are apparent within 24-48 h after surgery. As mentioned in the Introduction, the first event is degradation of certain components of the extracellular matrix (ECM), upregulation of others, and partial histolysis of the distal stump tissues to release the progenitor cells that will form the blastema. One of the molecules that is rapidly degraded following amputation is collagen, which cannot be detected by biochemical assays at the early stages of regeneration (Grillo et al., 1968; Dresden and Gross, 1970). Collagen belongs to a large multigene family consisting of at least 15 different species (types I-XV collagen). Only some of the newt collagen types have been identified and studied in the regenerating limb. Type I collagen distribution has been studied by transmission electron microscopy (TEM), given its characteristic periodic cross-striation (GCraudie and Singer, 1981). The nucleotide sequence of a1 chain of type XI1 collagen was isolated by using the MT2 mAb, and its pattern of expression was examined by both immunocytochemistry and in situ hybridization (Klatt et al., 1992; Wei et al., 1995). In normal limbs, type XI1 collagen is expressed in tendons and perichondrium but is not expressed in the mesenchyme at the site of amputation. However, it is upregulated in the cells of the basal layer of the WE in 3day regenerates (Wei et al., 1995). It has been shown that collagenolytic enzymes are activated in the stump upon amputation, at the time of histolysis and dedifferentiation (Grillo et al., 1968;Dresden and Gross, 1970). Recently, several enzymes of the matrix metalloproteinases (MMPs) family, which include collagenases, gelatinases,


13

and stromelysins, have been identified in newt and axolotl limbs (Yang and Bryant, 1994; Miyazaki et al., 1996). The MMP clones isolated in the newt encode for a collagenase, a gelatinase also isolated in axolotl, and a stromelysin. None of these enzymes are detected in normal limbs, but they are all upregulated as early as 2 days after amputation, although their later time course of expression differs slightly. Another ECM molecule that disappears following amputation is laminin, a complex glycoprotein that is present in basement membranes, provides a site of attachment for epithelial cells, and plays a role in the maintenance of tissue integrity (Timpl, 1989). This is of great functional importance because, as discussed previously, epithelial-mesenchymal interactions are indispensable for the progression of regeneration and are impaired by the presence of a basement membrane. Fibronectin is an ECM glycoprotein that in normal limbs colocalizes with laminin and type IV collagen in basement membranes. Unlike laminin, fibronectin is detected in the WE 24 h after amputation, during the initial phase of histolysis of stump tissues (Repesh et aL, 1982; Gulati et al., 1983; Nace and Tassava, 1995), whereas the fibronectin transcript is localized in the basal cells of the WE, the protein is detected in the acellular space beneath it. The presence of fibronectin in the WE is interesting because normally this protein is produced by mesenchymal cells and is not expressed in normal epidermis (Nace and Tassava, 1995). It cannot be ruled out that some of the fibronectin observed at this stage is due to deposition of blood fibronectin. Fibronectin may play a role in the migration of dedifferentiating cells, and later of blastemal cells, because receptors for its RGDS cell binding motif are present on epidermal cells (Donaldson and Mahan, 1987). An ECM component that appears to be different from any of those already characterized in the newt and is downregulated following amputation is identified by the mAb Stump 1(ST1) (Yang et aL, 1992). This reagent was obtained by immunizing mice with newt homogenates of regenerating retinas and lenses. ST1 is expressed in the epimysium, perimysium, perineurium, tendons, blood vessels, and around the epidermal glands and skeletal tissues of normal limbs, but it is downregulated in the distal stump region and is not detectable in the early blastema. Hyaluronate and chondroitin sulfate are the major large hydrophilic polyanionic glycosaminoglycans of the ECM associated with blastema formation and cell differentiation in the regenerating limb (Toole and Gross, 1971; Mescher and Munaim, 1986; Mescher and Cox, 1988). Hyaluronate is first synthesized at the time of dedifferentiation, when concomitantly hyaluronidase activity disappears (Smith et aL, 1975; Mescher and Munaim, 1986). It has been suggested that this polysaccharide, which induces tissue hydration, provides an environment suitable for cell migration (Toole et al., 1984) and could facilitate the migration of progenitor cells from the

14


stump by promoting expansion of the distal portion of the stump (Mescher and Cox, 1988). Together, these changes in the extracellular environment are thought to be fundamental for the initiation of blastema formation, but their precise role in the cascade of events leading to the release of blastemal cells from the stump has yet to be elucidated. Cell migration of stump dermal fibroblasts, and of cells that are released from the cut stump myofibers, cartilage, and myelin, occurs early after amputation (Hay and Fishman, 1961; Gardiner et al., 1986; Kintner and Brockes, 1985; Muneoka et al., 1986). The mesenchymal progenitor cells that can be observed within 24 h after amputation at the stump tip express vimentin and another cytoskeletal protein, 22/18, which is not detectable in cryostat sections of normal limbs (Kintner and Brockes, 1984, 1985; Gordon and Brockes, 1988). 22/18 appears to identify a conformational change in an intermediate filament component, demonstrating that a significant reorganization of the cytoskeleton occurs during formation of blastemal cells (Ferretti and Brockes, 1990), possibly related to modifications in cell-extracellular matrix interactions. Interestingly, 22/18 is expressed in blastemal cells whose division depends on the presence of the nerve, but mesenchymal cells of the limb bud, whose development is nerve independent, do not express it (Fekete and Brockes, 1987). However, induction of 22/18 expression in the regenerate is not under nerve control, confirming that initial formation of blastemal cells and their release from the stump tissues do not require innervation (Tassava and Olsen, 1985). In the distal portion of the stump, 22/18 reactivity is also observed in some mononucleated cells at the cut surface of the muscle and of the nerve, which appear to be Schwann cells being released from the myelin sheath because they also express the Schwann cell marker Leu-7 (Gordon and Brockes, 1988). The existence of Leu-7-22/18-positive cells migrating from the stump supports previous morphological observations that Schwann cells contribute to blastema formation (Maden, 1977). Expression of the myelin protein PO is also briefly observed in the Schwann cells moving away from the nerve stump but is rapidly downregulated (Kintner and Brockes, 1985). Other genes that are upregulated within 24 h after amputation and are important for patterning, such as Hox genes, will be discussed in Section IV. How blastemal cells originate from the stump tissues, and the molecular mechanism governing this process, is still unclear. Two possible origins of blastemal cells are dedifferentiation of mature tissues of the stump, which would regress to an undifferentiated state and reenter the mitotic cycle, and activation of populations of normally quiescent stem cells in the different stump tissues. The two possibilities are not mutually exclusive, and different tissues may contribute cells to the blastema through different mechanisms. In either case, changes in the phenotype of cells at the cut


15

surface of the stump occur, as reflected by the appearance of 22/18 reactivity, and they may be triggered by the changes in their extracellular environment discussed previously. To date, the only tissue in which convincing evidence of dedifferentiation has been presented is the muscle. However, this is also the only tissue in which the presence of muscle progenitor cells (postsatellite cells), which may be similar to the satellite cells of the mammalian muscle, has been shown in in v i m studies (Popiela, 1976; Cameron et al., 1986; Carlson, 1988; Lyons and Buckingham, 1992). It is conceivable that there are two different modes of rebuilding muscle depending on the type of damage. Also, whereas postsatellite cells may be used in muscle repair, the process of dedifferentiation may be required only for muscle regeneration after limb amputation (Carlson, 1979; Griffin et al., 1987; Ferretti and Corcoran, manuscript in preparation). The occurrence of muscle dedifferentiation was first observed by Hay (Hay, 1959; Hay and Fishman, 1961) in TEM sections of stump muscle, in which single nuclei of the severed myofiber appeared to pinch off from the degenerating fiber and migrate away as mononucleated cells. This interpretation has been supported by double-labeling experiments in which some blastemal cells have been shown to coexpress 22/18 and the muscle marker 12/101 (Kintner and Brockes, 1984) and recently by elegant tracing experiments (Lo et af., 1993). Ten days after implantation in the blastema of myotubes prelabeled with [3H]thymidine and rhodamine-dextran in culture, mononucleated cells containing the labels are detected in viva These cells seem to have the ability to fuse because some labeled myotubes are present in the blastemas. These results indicate that the implanted myotubes have indeed undergone a process of dedifferentiation, which results in the production of mononucleated myogenic progenitor cells. Interestingly, a few of these cells appear to be able to differentiate into chondrocytes, supporting the view that at least some transdifferentiation can occur during limb regeneration, as also suggested by the use of tissue-specific hypomethylation sites as lineage markers (Casimir et al., 1988; Ferretti and Brockes, 1991). Other tissues contributing cells to the blastema are skeletal tissue and fibroblasts. The contribution of periosteal cells to blastema formation has been demonstrated by histological analysis and grafting experiments reviewed elsewhere (Wallace, 1981). However, skeletal tissues seem to contribute only 2% cells, whereas dermal fibroblasts contribute approximately 48% (Namenwirth, 1974; Muneoka et al., 1986). 2. Blastema

The blastemal cells that accumulate at the distal tip of the stump begin to proliferate between 3 and 5 days after amputation while release of blastemal

16

JACQUELINE G6RAUDIE AND PATRlZlA FERRET1

cells from the stump is still occurring. At this stage expression of other two cytoskeletal proteins, the keratins 8 and 18 now cloned in the newt (Ferretti et al., 1993; Corcoran and Ferretti, 1997), is upregulated in blastemal cells. Activation of these genes in the mesenchyme is rather surprising because they are normally restricted to simple epithelia. Their expression might be simply due to the need for a more rigid skeleton in the growing blastema than in limb bud development, where NvK8 and NvK18 are not observed. However, recent experiments have shown that antisense oligonucleotides to K8 and K18 inhibit cell division in cultured blastemal cells, suggesting a causal relationship between keratin expression and blastema growth (Corcoran and Ferretti, 1997). Different regulation by RA of these keratins in myogenic cells cultures originating either from normal limb or regeneration blastema suggests two possible origins of myogenic cells; one from satellite cells, which would be activated for muscle repair, and one from dedifferentiation of mature muscle, which would occur in the process of epimorphic regeneration (Carlson, 1979; Ferretti and Corcoran, manuscript in preparation). Complex functional links between the organization of cytoskeletal elements, cell surface molecules, and extracellular matrix components appear to control changes in cell shape, movement, and fate (Daniels and Solursh, 1991).The identification of numerous changes in the cytoskeleton of blastema1 cells suggeststhat such changes may be causally related to dedifferentiation, growth, and redifferentiation of blastemal cells. Therefore, it will be important to further investigate the role of cytoskeletal proteins during regeneration. It has been shown that myosin larval forms are temporarily expressed in early blastemas, and that they are replaced by adult isoforms at the onset of differentiation (Saadi et al., 1993). The changes in myosin isoforms do not follow the pattern observed during limb bud development, possibly because this is controlled by thyroid hormone in the embryonic limb, but not in the adult regenerate. Recent analysis of the expression of some of the helix-loop-helix transcription factors that control myogenesis during development, such as Myf5 and MRF-4, has revealed that Myf-5, which is the first of the myogenic genes expressed during embryogensis, is detected by Northern blotting throughout the regenerative process (Simon et al., 1995). However, it is unknown whether Myf-5 is expressed in a discrete population of blastemal cells or in all of them. This information would be very valuable because it would allow one to gain some insight in cell lineages in the blastema. In contrast, the MRF-4 transcript is not detected in the blastema but is abundant in adult myofibers (Simon et al., 1995). It has been proposed that there is a causal relationship between MRF-4 downregulation in the blastema and myofiber dedifferentiation in the stump. However, it is also possible


17

that its downregulation, like that of the 12/101 protein, is the consequence and not the cause of dedifferentiation. MRF-4 reexpression follows the pattern of expression of the muscle-specific myosin gene observed in the late regenerates, at the time of muscle cell differentiation. The extracellular environment of the blastema is also highly dynamic. In early blastemas, type XI1 collagen is expressed not only in the WE but also in the mesenchyme (Klatt et al., 1992; Wie et al., 1995). The spatiotemporal distribution of this molecule suggests that it could be secreted by the W E into the mesenchymal compartment before mesenchymal cells begin to synthesize it themselves. The level of expression of type XI1 collagen decreases in the WE of late blastemas, whereas at this stage it is still high in the mesenchyme. With the progression of differentiation, however, collagen XI1 is also downregulated in the mesenchyme, with the exception of the perichondrium. Another type of collagen detected in the mesenchyme of early blastemas is type I collagen, and its expression is maintained throughout regeneration (GCraudie and Singer, 1981). Increased activity of prolyl hydroxylase, an enzyme involved in hydroxylation of the prolyl residues present in collagen molecules (Colquhoun and Dresden, 1983), and massive collagen biosynthesis are detected at the onset of digit regeneration (Mailman and Dresden, 1976). Fibronectin, like type XI1 collagen, begins to be synthesized by mesenchyma1 cells at the early bud stage and is released in the extracellular space (Nace and Tassava, 1995), as during development of the chick limb bud (Tomasek et al, 1982). During muscle differentiation, blastemal cells with myogenic potential are surrounded by fibronectin, align along the longitudinal axis of the regenerate, and fuse; after cell fusion, fibronectin immunoreactivity decreases. Finally, fibronectin is also transiently present in differentiating chondroblasts and hypertrophic chondrocytes. Like fibronectin, laminin is detected during redifferentiation in regenerating myotubes and around the chondrocyte lacunae, where laminin is also normally present (Gulati ef al., 1983). However, neither laminin nor ST1 are detected in the undifferentiated blastema, but both are reexpressed at the time of cell differentiation. The sequence of expression of glycosaminoglycans in the course of limb regeneration parallels that described in the avian developing limb bud (Toole, 1973). Whereas high levels of hyaluronate are detected at all blastema stages, hyaluronidase levels are extremely low until the onset of differentiation. Increase in hyaluronidase activity, and consequent decrease in hyaluronate levels, might favor the onset of vascularization of the blastema observed in late buds (Peadon and Singer, 1966; West er al., 1985). Chondroitin sulfate, which is initially very low and slowly increases during blastema growth, becomes the primary GAG present at the onset of chon-

18


drification of the regenerating skeleton (Toole and Gross, 1971; Smith et al., 1975). Although it is known that heparan sulfate is expressed both in the blastema mesenchyme and in the W E (Boilly et al., 1995), detailed analysis of the time course of its expression during regeneration is still missing. Immunocytochemical studies have shown that the ECM glycoprotein tenascin is first detected at the WE/mesenchyme borders and in a few mesenchymal cells beneath it 5 days after amputation, although tenascin mRNA is already transcribed in the W E at 2 days (Onda et a/., 1990, 1991; Koshiba et al., 1994). Transcription in the distal portion of the WE, particularly in the basal cells, is maintained throughout the regenerative process, and it is therefore likely that the tenascin detected at the epithelialmesenchymal interface is mainly synthetized by the WE. Intracellular tenascin-positive reactivity is detected in some cells in the wound epidermis. In the blastema mesenchyme tenascin is uniformly distributed and is lost with the progression of differentiation in a proximal to distal fashion. Work by Koshiba et al. (1994) has suggested that tenascin reactivity is initially reduced in retinoid-treated blastemas and is later found in areas where active proliferation takes place. Although numerous studies have investigated changes in the ECM during regeneration, little is known about the expression and role of cell-cell adhesion molecules. N-CAM, which functions as a homophilic ligand in cell-cell interactions and serves as an adhesive substrate for guidance of growth cones, has been detected in the WE and mesenchyme of the blastema and in cultured blastemal cells, but it is not known when it is first expressed (Maier et al., 1986; Ferretti and Brockes, 1990). Interestingly, infusion of anti-N-CAM Fab fragments in the blastema in vivo retards its growth, as observed after partial denervation (Maier et al., 1986). The fact that growth inhibition is not total may be due to technical reasons, such as the infusion protocol, the stability of the antibody, and its distribution in the blastema. Why anti-N-CAM infusion induces growth retardation must still be clarified, but it may be the consequence of either inhibition of blastemal cell interactions or inhibition of axonal growth in the blastema. Whereas N-CAM is widely expressed in the blastema, the distribution of other adhesion molecules that contain the L1 and L2 carbohydrate epitopes and are known to be involved in neural cell-cell interactions is more restricted. In fact, an anti-Ll antibody and the monoclonal antibodies HNK-1/Leu-7 (anti-L2) label only a subpopulation of blastemal cells that are believed to originate from dedifferentiation of the Schwann cells of the nerve stump (Gordon and Brockes, 1988; Maier and Miller, 1992). The distribution of other molecules involved in adhesion mechanisms, such as integrins, cadherins, or selectins, during limb regeneration has not been thoroughly investigated. Results from Tsonis (1996) and our laboratories (J. GCraudie and P. Ferretti, unpublished results) have indicated that


19

a3, a6, av, and 0 3 are upregulated in the blastema, whereas a l , a4, and

01 integrin subunits are downregulated. Whereas a 4 was detected in muscle, chondrocytes, and skin but not in limb blastemal cells in axolotl, the newt tail blastema appeared to express this integrin subunit. It is not clear whether this reflects a difference in reactivity across species or differences between regenerating limbs and tails. It is apparent from the studies discussed here and summarized in Table I1 and Fig. 2 that many ECM and membrane proteins with a wide range of adhesion properties are expressed in a coordinated spatiotemporal fashion during regeneration. This is not surprising because changes in cell interactions and cell migration are certainly key factors in blastema formation. Attempting to understand the role played by different molecules in cell-cell and cell-matrix interactions during limb regeneration is going to be an exciting but formidable task.

111. Growth Control in the Blastema As mentioned in the Introduction, early regeneration blastemas require the presence of both the WE and an adequate level of nervous supply for their outgrowth (Todd, 1823; Singer, 1974; Tassava and Mescher, 1975; Carlone and Mescher, 1985; Sicard, 1985; Brockes, 1987; Fekete et al., 1987; Singer and GCraudie, 1991). In addition, the “hormonal milieu” appears to be important for blastema growth, but it is difficult to define the specific role of individual hormones (Tassava, 1969, 1983; Sicard, 1985). Because of the dramatic growth arrest induced by denervating the limb, which can be reversed by treatment with neural extracts, much experimental work on growth control in the blastema in vivo has focused on the comparison between normal and denervated blastemas. When a limb is denervated prior to amputation, wound healing and accumulation of blastemal cells occur normally, but the blastemal cells do not proliferate and regeneration is inhibited (Singer, 1952,1974; Thornton, 1970). On the contrary, if the limb is denervated after a blastema has formed, regeneration proceeds, but the regenerated limb is smaller in size. Therefore, regeneration depends on the presence of the nerve only during the phase of rapid proliferation of blastemal cells (Singer and Craven, 1948). The nervous system is believed to control cell growth predominantly during the G1 phase of the cell cycle (Goldhamer and Tassava, 1987), and it has been postulated that this action is exercised either directly or indirectly by neurotrophic factors secreted by the nerves (Singer, 1974; Singer et al., 1976; Jabaily and Singer, 1977; Brockes, 1987; Ferretti and Brockes, 1991; Dinsmore and Mescher, 1998).

TABLE I1 Gene Expression in the Limb Blastema Mesenchyme

Molecule ECM molecules Metalloproteinases Type XI1 collagen Type I collagen Laminin Fibronectin

Tenascin GAGS

Surface molecules N-CAM L2 epitope (HNKl)* L1 PO" Cytoskeleton 22/18 intermediate filament component Keratin 8

Detection Riboprobes Biochemical IS-2 riboprobe mAb MTZ TEM Polyclonal antiserum

Yang and Bryant (1994), Miyazaki et al. (1996) Klatt et al. (1992) Wei et al. (1995) Gtraudie and Singer (1981) Gulati et al. (1983), Maier and Miller (1992) Repesh et al. (1982), Gulati er Polyclonal antiserum mAb MT4 al. (1983), Maier and Miller (1992), Nace and Tassava (1995) NvTN.l riboprobe mAb MTl Onda et al. (1990, 1991). mAb 55C12 (axolotl) Koshiba et al. (1994) Biochemical Toole and Gross (1971), Mescher and Munaim (1986), Mescher and Cox (1988), Boilly et al. (1995) Polyclonal antiserum mAb Leu-7 Polyclonal antiserum mAbs 3013, 30114, 30/26 mAb 22/18

Vimentin

NvK8 riboprobe mAbs LPlWLE41 NvKlS riboprobe mAbs RGE53, CK18.2 mAb 22131

Keratin 14 GFAP Myosin

mAb Polyclonal antiserum Riboprobe

Keratin 18

Cell membrane receptors Riboprobes FGFRl bek (variant of FGFR2) Riboprobes Insulin receptor Others Sarcoplasmic reticulum protein" CRABP

Reference

Maier et al. (1986) Fekete and Brockes (1987), Gordon and Brockes (1988) Maier and Miller (1992) Kintner and Brockes (1985) Kintner and Brockes (1985), Ferretti and Brockes (1990) Ferretti et al. (1989, 1993) Corcoran and Ferretti (1997) Ferretti et al. (1989, 1993) Corcoran and Ferretti (1997) Kintner and Brockes (1985), Fekete and Brockes (1987), Tsonis et al. (1992) Maier and Miller (1992) Casimir et al. (1985)

Polyclonal antiserum

Poulin et al. (1993) Poulin et al. (1993), Poulin and Chiu (1995) Foty and Liversage (1993)

mAb 12/101

Kintner and Brockes (1985)

Biochemical

Keeble and Maden (1986), McCormick et al. (1988) . ,

"Detected in a few cells at early stages of regeneration; 12/101 is reexpressed in a few blastemal cells at the bud stage.


21

In the early blastema axotomy induces a transient outburst in DNA, RNA, and protein synthesis; this is followed by a 40-50% decrease in their levels within 48 h and growth arrest of the blastema (Dresden, 1969; Lebowitz and Singer, 1970; Singer and Caston, 1972; Smith et al., 1975; GCraudie and Singer, 1978; Mescher and Cox, 1989). Recently, expression of some molecules involved in the control of the cell cycle, such as the protooncogenes c-myc (Geraudie et ul., 1990), Ki-ras (see AndCol et ul., 1990; Y. AndCol and J. Geraudie, unpublished results), rus (Tsonis, 1991), ski (Ludolph et al., 1995), and the marker of S phase proliferating cell nuclear antigen (PCNA), has been examined in regenerating limbs. It has been shown that both c-myc (GCraudie et al., 1990) and p53 (see Tchang et al., 1993; F. Tchang and J. GCraudie, unpublished results) are upregulated in regenerating limbs of Xenopus froglets compared to controls, whereas PCNA levels do not change significantly (Lemaitre et al., 1992). Expression of ski has been reported in different stages of axolotl limb blastemas by Northern analysis (Ludolph et ul., 1995). However, because this protooncogene appears to be expressed at high levels in differentiated muscle, cellular localization will be necessary to rule out the possibility that its expression is due to contamination with muscle tissue at the early stages of regeneration and to muscle redifferentiation at the later ones. Denervation results in significant changes in the expression of many of these molecules in the regenerating limbs. As demonstrated by Northern blot, Xenopus c-myc (Gtraudie et al., 1990) and Ki-rm (Y. And601 and J. Geraudie, unpublished results) transcripts are downregulated in denervated limbs. However, a transient accumulation of the c-myc protein in the regenerate is observed. Furthermore, PCNA is significantly upregulated for at least 4 days after denervation (Lemaitre et al., 1992); the significance of this accumulation is unclear, and it will be important to examine changes in PCNA expression in normal and denervated blastemas in species with a higher regenerative capability than Xenopus. Altogether, the upregulation of the c-myc and PCNA proteins has led to the proposal that nervous system normally exerts a negative control on their expression (Lemaitre et al., 1992). This suggests that the nerve might control proliferation of blastema1 cells through both positive and negative activities. Identification of the neurotrophic molecule(s) postulated by Singer (1974) has proved difficult, but during the past few years it has become apparent that both mitogenic and trophic factors are required for blastema growth. Molecules that seem to satisfy at least some of the properties expected of a “neurotrophic factor” (Brockes, 1984) are members of the FGF fibroblast growth factor and neuregulin/glial growth factor (GGF) families and the iron transport protein transferrin. Also, insulin appears to be important in limb regeneration (Foty and Liversage, 1993), and factors such as EGF and substance P, but not PDGF and NGF, have been shown

22


to be mitogenic for blastemal cells in vitro (Mescher and Loh, 1981; Smith and Globus, 1989; Smith et al., 1995). Whereas relatively little information is available on the GGF-like activity demonstrated in regeneration blastemas (Brockes and Kintner, 1986), more is known about FGFs and transferrin in regenerating limbs, and it is possible that these are indeed the key mitogenic and trophic factors required for nerve-dependent blastemal cell proliferation. The mitogenic effect of FGF was initially suggested by the fact that infusion of crude FGF preparations in denervated limb blastema could reverse growth arrest (Mescher and Gospodarowicz, 1979; Gospodarowicz and Mescher, 1980). The presence of both acidic (FGF-1) and basic FGF (FGF-2) in the limb blastema was later shown by binding assays, Western blot analysis, and immunocytochemistry (Albert et al., 1987; Boilly and Albert, 1990; Boilly et al., 1991; Mullen et al., 1996; Zenjary et al., 1996). Strong FGF-1 and FGF-2 immunoreactivity is detected in the wound epidermis, but only FGF-1 reactivity seems to be present in the blastema mesenchyme (Mullen et af., 1996; Zenjary et al., 1996). As mentioned earlier, lack of a wound epidermis, as well as removal of the apical ectodermal ridge in developing limb buds, inhibits limb outgrowth. In the limb bud this effect can be reverted by FGF application (Niswander et al., 1993; Fallon et al., 1994), and FGF can also induce “regeneration” of the embryonic limb (Taylor et al., 1994; Kostakopoulou et al., 1996). It will be important to establish whether, in a similar fashion, FGF can compensate for the lack of a WE in regenerating limbs; however, given the fast regrowth of the WE after its removal, it may not be a trivial task to address this issue. Recent work has shown that FGF-1 is also transcribed in the cell bodies of the neurones innervating the regenerating limb (Dinsmore and Mescher, 1998), and that the FGF-2 protein is present in the nerves in which its levels, like those in the wound epidermis, are significantly decreased following denervation (Mullen et a/.,1996). Furthermore, implantation of a FGF-2 bead rescues, at least in part, the growth inhibition induced by severing the nerve. These results point to a causal link between FGF-2 mitogenic activity and nerve-dependent growth in limb blastemas. Also, FGF-1 appears to control blastemal cell proliferation, as suggested by in vivo experiments using neutralizing antibodies and molecules that inhibit growth factor binding (Zenjary et al., 1996), but whether FGF-1 activity and nerve dependency are linked has yet to be elucidated. The presence of other members of the family, such as FGF-4 and FGF-8, that appear to be involved in limb patterning during development (Niswander et al., 1993; Crossley et al., 1996) has not yet been reported in the regenerating newt limb. FGF signaling is mediated by a family of tyrosine kinase transmembrane receptors, the FGF receptors (FGFRs). Distinct spatiotemporal expression of FGFR-1 and two variants of FGFR-2 (bek and KGFR) has been observed


23

by in situ hybridization in newt blastemas (Poulin et al., 1993, Poulin and Chiu, 1995). FGFR-1 transcription is observed in blastemal cells during the early stages of regeneration, whereas FGFR-2 message is detected in the basal layer of the WE and in periosteal cells of the bone stump, and its expression is maintained in regenerating cartilage. In conclusion, although other mitogenic factors may be involved in blastemal cell growth, the pattern of expression of FGFs and FGFRs together with the functional experiments carried out so far strongly suggest that these molecules play an important role in limb outgrowth during regeneration. It is unclear, however, whether they also play a role in patterning of the regenerating limb, as they do during limb development (Izpis6a-Belmonte et al., 1992; Laufer et al., 1994; Niswander et al., 1994; Ferretti and Tickle, 1997). The intracellular events involved in the transduction of mitogenic signals have not been thoroughly investigated. However, the fact that FGFRs can activate phospholipase C in other species, and that nerve extracts and substance P induce formation of inositol phosphates (Smith et al., 1995), indicates that the inositol phospholipid signaling pathways is involved in mitogenic signal transduction in the blastema. A list of urodele genes encoding for soluble signaling molecules that have been detected in limb blastemas is given in Table 111. As mentioned previously, another factor that appears to play a key role in keeping the blastemal cells cycling is transferrin. In fact, both chelation of ferric ions and the use of an antitransferrin antibody prevents the mitogenic effect of neural extracts (Munaim and Mescher, 1986; Albert and Boilly, 1988; Dinsmore and Mescher, 1998). Transferrin is abundant in Schwann cells and in peripheral nerves, where it is transported both anterogradely and retrogradely. A significant decrease in transferrin levels is observed in

TABLE Ill

Urcdele Genes Encoding for Soluble Signaling Molecules Gene

Urodele amphibian

Reference Takabatake et al. (1996), Imokawa and Yoshizato (1997)

sonic hedgehog

Cynops

banded hedgehog

Notophthalmus viridescens Stark et al. (manuscript in preparation)

FGF

Cynops

Imokawa ef al. (manuscript in preparation)

Wnt-?

Cynops

Imokawa et al. (manuscript in preparation)

Wnt-5"

Axolotl

Busse and Seguin (1993) Caubit et al. (1996)

Wnt-SA," -5B,a -7A" Pleurodeles wait1 a

Genes whose expression in limb regeneration blastemas has not been studied.

24

JACQUELINE GERAUDIE AND PATRlZlA FERRET1

the growth-arrested denervated blastemas (Mescher and Munaim, 1984; Kiffmeyer et al., 1991; Mescher and Kiffmeyer, 1992), and, at least a short time after denervation, application of transferrin significantly increases [3H]thymidine incorporation in blastemal cells in vivo (Mescher and Kiffmeyer, 1992). It will be important to establish in long-term experiments whether application of FGF and transferrin can fully substitute for the nerve and results into normal regeneration of denervated blastemas. The recent advancements in the identification of mitogenic and trophic factors required for blastemal growth discussed previously, the increasing understanding of the molecular mechanisms controlling reentry into the cell cycle following amputation (Tanaka et ul., 1997), and the analysis of the “immortal” nature of blastemal cells in culture (Ferretti and Brockes, 1988; Powell et a1.,1997) are currently very exciting and promising areas of research in the regeneration field. In fact, the regenerating limb provides one of the very few opportunities to study the issues of reversal of the differentiated state, reentry into the cell cycle, and control of proliferation in an adult organism in a nononcogenic context.

IV. Genes Controlling Morphogenesis in the Regenerating Limb

In previous sections we discussed the changes in the phenotype of blastemal and WE cells and some of the differences in gene expression in adult limb blastemas and in developing limb buds. These molecular differences, together with the fact that cell division is under nerve control during regeneration but not during development, are likely to reflect the different origin of limb progenitor cells in embryos and adults. Therefore, at least in regard to limb progenitors, regeneration does not simply recapitulate development. In contrast, patterning of the regenerating limb is likely to be governed by the same set of genes that have been shown to play an important role during development. This section will review the information currently available on such genes in the regenerating limb. Among the molecules that appear to play a key role in patterning of the limb are retinoids and their receptors, segment polarity genes (sonic hedgehog, wnt, and engruiled), FGFs and their receptors (FGFRs), bone morphogenetic proteins (bmp), and homeobox-containing genes (e.g., hoxa and hoxd clusters, msx-1 and -2, dlx). The homologs of many of these genes have now been cloned in urodeles (Table IV), although much information on their expression in regenerating limbs, especially at the cellular level, is still missing. The emerging picture, as outlined in more detail below, is that expression of all the genes believed to play a role in patterning of the limb bud studied so far is either maintained in the adult urodele limb or

25


TABLE IV Homeobox-Containing Genes and Retinoid Acid Receptors Gene

Urodele amphibian

Reference

HOXa-4, -S, -7, -9, -10, -11, -13

Axolotl

Gardiner et al. (1995)

Hoxa-4, -9 Hox-~II

Pleurodeles waltl

Nicolas et al. (1995)

Notophthalmus viridescens

Beauchemin et al. (1994) Beauchemin and Savard (1993) Gardiner et al. (1995) Nicolas et al. (1995)

Hox-~~ H o x - ~ -66 ~,


Hoxb-3, -9

Axolotl Pleurodeles waltl

HOXC-6,-I0

Notophthalrnus viridescens

HOXC-12

Pleurodeles waltl

HOXC-13 Hoxd-10, -I1

Axolotl Notophthalmus viridescens

H o x ~ - 8 ,-10, -I1

Axolotl

Hoxd-I3

Pleurodeles waltl

MSX-1


Msx-I and Msx-2

Axolotl

dlx-related genes


Dlx-3 DLU-3'

Axolotl Pleurodeles waltl Notophthalmus viridescens

T-box (TBX) RARa, RARP, RARS ~~


Savard et al. (1988), Tabin (1989), Simon and Tabin (1993), Savard and Tremblay (1995) Nicolas et 01. (1995) Gardiner et al. (1995) Brown and Brockes (1991), Simon and Tabin (1993) Gardiner et al. (1995) Nicolas et al. (1995) Crews et al. (1995), Simon et al. (1995) Gardiner et al. (1995) Beauchemin and Savard (1992, 1993), Gardiner et al. (1993) Mullen et al. (1996) Nicolas et al. (1996) Simon et al. (1997) Giguere et al. (1989), Ragsdale et at. (1989, 1992a,b)

~

Expression in regeneration blastemas has not been studied.

reinduced following amputation. However, it appears that the pattern of expression of at least some of these genes is somehow different from that observed in the developing bud.

A. Retinoids and Their Receptors Retinoids (vitamin A and its derivatives) appear to be required for normal limb development and when administered exogenously can severely affect

26

JACQUELINE GERAUDIE AND PATRlZlA FERREnl

patterning of developing and regenerating limbs. Therefore, effects of retinoids on developing and regenerating limbs have been the focus of many studies and reviews (Brockes, 1990; Stocum and Maden, 1990; Stocum, 1991; Bryant and Gardiner, 1992; Mendelsohn et al., 1992; Lohnes et al., 1994; Tickle and Eichele, 1994). In developing chick limb buds, beads soaked with all-trans-RA can mimic the effect of the zone of polarizing activity (ZPA) when implanted in the anterior margin of the bud and induce anteroposterior mirror-image duplications (Tickle et al., 1982;Tickle, 1991). A similar effect can also be induced by R A in developing anurans limbs (Migliorini Bruschelli and Rosi, 1971; Niazi and Saxena, 1978), but there is no evidence that such an effect can be induced in developing urodeles. During regeneration exogenous retinoids can affect patterning of all the three limb axes (Stocum, 1991), besides temporarily delaying blastema growth and inducing apoptosis in the mesenchymal cells of the blastema (Geraudie and Ferretti, 1997) and the appearance of ciliated and secretory cells in the WE (Maden, 1983; Scadding, 1989). However, the most striking effect of retinoids on limb regeneration in urodeles is the dose-dependent induction along the proximodistal axis of supernumerary structures, which are more proximal in identity than the amputated ones (Maden, 1982; Thomas and Stocum, 1984;Stocum, 1991). Because normally only structures distal to the amputation plane regenerate, the effect induced by retinoids along the proximodistal axis of the limb is often described as a “proximalization” of the positional identity of the blastema (Fig. 3). It has been reported that implantation of RA-soaked silastin blocks in regenerating axolotl limbs induces formation of supernumerary limbs (Maden et al., 1985). From this study, though, it is not clear whether this is due to localized toxicity, which would induce destruction of the tissue surrounding the RA-soaked implant and trigger formation of an accessory blastema, or to a specific effect of R A on the anteroposterior axis. This is a possibility because using surgically made half limbs it has been demonstrated that retinoic acid can affect the anteroposterior and dorsoventral axes of regenerating limbs (Kim and Stocum, 1986; Ludolph et al., 1990; Monkemeyer et al., 1992). In axolotl, equal concentrations of exogenously administered retinoids have apparently different effects on developing and regenerating limbs because the developing limb does not show duplications along the proximodistal or anteroposterior axis but is hypomorphic (Scadding and Maden, 1986). This may simply be due to differences in sensitivity to the toxic effects of exogenously administered retinoids on progenitor cells of developing and regenerating limbs. Such a possibility is consistent with the differences in cell phenotype and control of cell division in developing limb buds and regeneration blastemas, which were discussed previously. However, because RA-induced duplications along the proximodistal axis of developing limbs have not been observed in other species, it is also possible that such


27

FIG. 3 RA effects on proximodistal duplication in the regenerating limb are dose dependent. Axolotl forelimbs were amputated at the wrist level as indicated by the line, and the skeletal pattern was assessed in whole mount limb preparations stained with methylene blue. (A) Control regenerate (injected intraperitoneally 4 days postamputation with the RA vehicle, DMSO); note that the eight carpals and four digits that had been removed have regenerated. (B) Low dose of RA; note the duplication of a third of the radius and ulna. (C) Intermediate dose of RA; note the duplication of complete radius and ulna. (D) Dose of RA inducing maximal duplication (100-150 pg RA/g body wt); note the complete duplication of humerus, radius and ulna and the partial shoulder girdle (arrow). u, ulna; r, radius (photographs provided by D. Stocum; from Stocum and Crawford (1987).

a response cannot be evoked in the developing urodele limb. In contrast, the lack of duplications along the anteroposterior axis may indeed be explained by the fact that the treatment by immersion used by Scadding and Maden (1986) does not allow a sufficient increase in R A concentration specifically in the anterior margin of the limb bud when used at low doses. Treatment with RA at higher concentrations, however, could have a generalized toxic effect resulting in hypomorphic limbs. Although the exact relationship between RA-induced effects during development and regeneration has not yet been fully elucidated, it is evident that exogenous retinoids can dramatically affect patterning of regenerating limbs. This raises the question of whether endogenous retinoids play a role in limb regeneration as suggested in limb development. In order to address this issue, various groups have taken complementary approaches and started to assess the presence of retinoids, of their cytoplasmic binding proteins, and of nuclear receptors in the limb blastema. The levels of various retinoids in the limb regeneration blastema of axolotl have been measured by using high-performance liquid chromatography (Scadding and Maden, 1994). This study has demonstrated that en-

28


dogenous all-trans-RA is indeed present in a gradient in regenerating limb blastemas and that it is three- to fivefold more concentrated posteriorly than anteriorly. In contrast, no significant anteroposterior gradient of either retinol, although its levels are slightly higher posteriorly, or 3,4dehydroretinol have been observed. The levels of another naturally occurring retinoid, 3,4-didehydroretinoic acid, which is a powerful morphogen in the chick wing bud (Thaller and Eichele, 1990; Eager e f al., 1991), have not been measured in this study. Although currently there are no data available on the concentrations of RA, retinol, and 3,4-dehydroretinol in axolotl limb buds, the RA gradient measured in the regeneration blastema parallels that observed in the chick wing bud (Eichele and Thaller, 1987), and therefore it has been suggested that endogenous RA may play the same role in developing and regenerating limbs at least on patterning of the anteroposterior axis (Scadding and Maden, 1994). Scadding and Maden (1994) have also compared retinoid levels in blastema1 cells and wound epidermis and found that RA and retinol levels in the mesenchyme and WE are very similar, whereas 3,4-dehydroretinol concentration is much higher in the mesenchyme than in the epidermis. It is not yet known, however, whether this vitamin A metabolite may also play a role in limb patterning. In contrast, another naturally occurring isomer of RA, 9-cis-retinoic acid, which is about 25 time more potent than RA in inducing anteroposterior duplications in the chick wing bud (Thaller et al., 1993), is synthesized and secreted by the WE of the newt limb blastema (Vivian0 et at., 1995).Its levels in the WE appear to be significantly higher than those of RA, and 9-cis-retinoic acid synthesis and release by the WE not only may be important for the maintenance of the WE but also may affect the behavior of the underlying mesenchymal cells. In fact, it has been shown that 9-cis-retinoic acid is more potent than RA in proximalizing regenerating axolotl limbs (Tsonis et al., 1994). Retinoid-binding proteins are cytoplasmic proteins that may not only translocate retinoids to the nucleus but also play an important role in controlling the amount of free retinoids present in the cell and, as a consequence, the level of activation of their nuclear receptors. Two retinoic acid-binding proteins, CRABP-I and CRABP-11, and two retinol-binding proteins, CRBP-I and CRBP-11,have been identified in birds and mammals, and their distribution during development has been thoroughly studied (Maden, 1991; Donovan et al., 1995). It has been shown that a CRABP is present in limb regeneration blastemas (Keeble and Maden, 1986; McCormick et al., 198S), and that its concentration is about fourfold higher in regenerating than in normal limbs of axolotl, by binding assays using [11,123H]all-truns-RA (Keeble and Maden, 1986). Ludolph et al. (1993) isolated a partial DNA sequence encoding for the axolotl CRABP-I, and Northern blot analysis indicates that CRABP-I is not expressed at detectable levels


29

in the regeneration blastema, but it is present in unamputated limbs. Therefore, it is likely that the CRBP, which has been shown to be upregulated in the blastema by Keeble and Maden (1986), is the axolotl homolog of CRABP-11. It is not known whether the CRABP expressed in the regenerating axolotl limb is upregulated by RA as is CRABP-I1 in other species (Astrom et al., 1992), and whether it is asymmetrically distributed in the blastema. In chick limb buds CRABP is more concentrated anteriorly than posteriorly (Maden et al., 1988),whereas RA levels are higher posteriorly (Eichele and Thaller, 1987). It has therefore been suggested that CRABP might be involved in steepening the gradient of RA along the anteroposterior axis by sequestering it and decreasing its availability to the receptors (Maden et al., 1988). However, much of the recent work has undermined the RA gradient hypothesis and supported an involvement of RA in the establishment and maintenance of the zone of polarizing activity (Brockes, 1990; Noji et al., 1991; Wanek et al., 1991). Furthermore, the fact that significant differences in the level of different retinoids and of CRABPs (Scott et al., 1994) have been found in the developing limb of different species, together with the variable phenotype in CRABP transgenic mice (Wei and Chen, 1991), makes it very difficult to build a clear and solid picture of the developmental role of these molecules. Currently, one should neither assume that their role in limb development in different species is absolutely equivalent nor too easily extrapolate from limb development to regeneration. Much more information on urodele CRABPs and CRBPs, their regulation, and distribution will be needed in order to assess their possible role in modulating retinoid-induced effects in regenerating limbs. Whereas only one of the retinoid-binding proteins has been cloned in an urodele species and the function of this protein family has yet to be studied thoroughly in the limb regeneration blastema, more information concerning retinoid nuclear receptors and how they may mediate the multiple effects of retinoids in different systems has been accumulated in the past few years (Means and Gudas, 1995). The retinoid receptors (RARs and RXRs) belong to the steroidkhyroid hormone receptor superfamily and activate transcription by binding either as homodimers or as heterodimers to specific response elements of target genes. Three RARs, a,l3, and y, and three RXRs, a,p and y, with multiple spliced variants have been identified in mammals (Leid et al., 1992; Mangelsdorf et al., 1993). RA has been shown to have a high affinity for the RARs but low affinity for RXRs. The retinoid 3,4-didehydroretinoic acid, which can induce digit duplications in chick limb buds, also displays low affinity for RXR (Eager et al., 1991). In contrast, 9-cis-retinoic acid has been shown to bind with an affinity 40-fold higher than RA to the RXRs (Mangelsdorf et al., 1992). The fact that RARs and RXRs have different activities and distinct patterns of expression

30


in the developing mammalian limb has raised the question of their role in the patterning of limb regenerates. Five RARs have so far been identified in the newt, N. viridescens, and three of them are clearly the newt homologs of mammalian RARal, RARa2, and RARP (Giguere et al., 1989; Ragsdale et al., 1989, 1992a,b). In contrast, no urodele RXR has yet been cloned. Full sequences of the newt R A R d and RARa2 are available (Ragsdale et aL, 1989, 1992a), but only the partial sequence of RARP has been published (Giguere etal., 1989).The other two newt RARs, although related to mammalian RARy, have been named RAR61 and RARE? because of significant differences in the amino acid sequence of regions A (N terminus) and F (C terminus), which in the case of the other RARs are highly conserved across species (Ragsdale et al., 1989, 1992b). RARSl contains two methionine initiators, and analysis with a panel of polyclonal antibodies specific for different regions of the protein suggests that they are both used in the limb regeneration blastema and produce 61a and 61b receptors (Hill et al., 1993). RARal, RARp,and RARS;! have also been detected in the blastema, but the levels of these transcripts appear to be lower than those of RAR61 (Giguere et al., 1989; Ragsdale et al., 1989, 1992b). The high levels of RARSl in limb and limb blastemas and its unique sequence characteristics in the A region, compared to all the other vertebrate RARs, have raised the question of whether expression of this receptor might be of particular significance within the context of regeneration and prompted its thorough characterization. Reactivity on blastema sections with anti-61 antibodies has shown that RARG is expressed in about 50% of the nuclei in the epidermis and mesenchyme of both normal and regenerating limbs. No differences were observed between proximal and distal blastemas, but a higher percentage of blastema cells expressing RAR61 (70-80%) was observed just beneath the WE (Hill e f al., 1993). Furthermore, no significant change in RARS1 expression was induced following injection of a proximalizing dose of RA. The lack of a gradient of RAR61 and of RA-induced changes in its levels and distribution observed by immunocytochemistry is consistent with the analysis of its expression carried out by RNAase protection (Ragsdale et al., 1992b). Because of the wide tissue distribution of RARG1 and its significant upregulation in the blastema compared to the normal limb, Hill et al. (1993) suggested that its expression may be causally linked to the ability of RARSpositive cells to be recruited into the blastema following amputation. If this were the case one would expect that the majority of cells throughout the blastema, and not just a small population of cells beneath the wound epidermis, would express this gene. The possible significance of this pattern of RAR6 expression has yet to be clarified.


31

No information about the cellular distribution of RARa in normal and regenerating limbs is available, and the RARP transcript appears to be expressed throughout the blastema, without any apparent anteroposterior gradient or proximodistal gradient (Giguere et aZ., 1989). Analysis of RARG1 expression by immunocytochemistry, in conjunction with analysis of the expression of RARa and RARM by RNAase protection in cultured blastemal cells, has demonstrated that different types of receptors are coexpressed with RARG1 at least in subpopulations of these cells (Ragsdale et aZ., 1992a,b). In order to tackle the complex issue of the role of retinoids in limb regeneration and of which RARs may mediate the different responses to RA in this system, Brockes and colleagues have constructed a series of chimeric receptors and introduced them either into cultured cells (COS cells and limb blastema cells) or in the regenerating limb in v i v a Cultured blastemal cells were transfected either by microinjecting the plasmids into the nucleus or by using a biolistic particle delivery system (Brockes, 1994). In some experiments the transfected cells were reimplanted into the regenerating limb (Brockes, 1992). In vivo transfections were carried out by using the biolistic particle delivery system (Brockes, 1994; Pecorino et a[., 1994,1996). This experimental approach of combining the use of chimeric receptors and transfection in vitro and in vivo is proving very valuable for elucidating the role of retinoids and their receptors in the regenerating limb. Cultured newt limb cells transfected with a CAT reporter gene carrying the RA-responsive element of RARP respond to RA in a dose-dependent fashion and are diagnostic of environmental differences in proximal and distal blastemas (Brockes, 1992). In fact, when they are implanted at different axial levels in the regenerating limb of a newt injected with a proximalizing dose of RA, the level of normalized CAT activity is higher proximally than distally, suggesting the existence of a proximodistal gradient of RA. However, the nature of the environmental differences revealed by these experiments is not yet known. Because RA inhibits blastema growth for about a week after a single injection in vivo and dramatically decreases [3H]thymidine incorporation in cultured blastemal cells, RARa1-thyroid hormone T3 receptor a (TRa) and RAR61-TRa chimeras have been used to establish which of these RARs mediates RA-induced growth inhibition (Schilthuis et aZ., 1993). These studies have shown that stimulation of chimeric receptors with T3 can induce growth inhibition equivalent to that induced by RA in cultured blastemal cells transfected with the RARal-TRa receptor but not in cells transfected with RARG1-TRa. When WE cells are transfected in vivo with RARG1-TRa, T3 treatment can induce expression of the antigen WE3, a marker of secretory differentiation specific for the WE that is upregulated by RA (Tassava, 1992; see Section Il), in the transfected cells. In contrast,

32


no WE3 expression has been detected in WE cells transfected with the chimeric receptor RARal-TRa (Pecorino et al., 1994). Therefore, RARa1 is involved in growth inhibition but not in the regulation of WE3, and the opposite is true in the case of RARG1. However, RARG1 is unable to change the proximodistal identity of blastemal cells in the limb, whereas RAR62, although expressed at lower levels in limb blastemas, can mediate the proximalizing effect of RA (Pecorino et al., 1994, 1996). In conclusion, the ability of RA to affect limb patterning, its presence in regenerating limbs (Scadding and Maden, 1994; Vivian0 et al., 1995), and the demonstration that different retinoic acid receptors are expressed in the regenerate and mediate different functions (Schilthuis et al., 1993; Pecorino et al., 1994, 1996) support the view that endogenous retinoids play important and varied roles in limb regeneration. With the powerful tools now available and judiciously designed experiments, such as those discussed previously, it should be possible to definitely clarify this issue in the near future.

8. Homeobox-Containing Genes Developing limb buds of birds and mammals express numerous genes of the hox complex, which consists of four clusters, hoxu, hoxb, hoxc, and hoxd. A similar organization of the hox genes clusters has been recently demonstrated in urodeles (Belleville et ul., 1992). In higher vertebrates, genes of the hoxa and hoxd cluster are expressed in a coordinated fashion in specific overlapping domains along the proximodistal and anteroposterior axis, respectively, during limb development. This pattern of expression has led to the idea that they may encode position in the limb bud, and for this reason these hox gene clusters have been more thoroughly studied in developing limbs. Their possible role in setting the proximodistal and anteroposterior axes has been supported by a large bulk of experiments in chick and mouse in which overexpression and knockout of these genes have resulted in patterning abnormalities (IzpisuaBelmonte and Duboule, 1992; Morgan et al., 1992; Doll6 et al., 1993; Small and Potter, 1993; Davis and Capecchi, 1994; Davis et al., 1995; Favier et al., 1995; Yokouchi et al., 1995). Genes of the hoxb and hoxc clusters are also expressed during limb development, but no coordinated pattern of expression of the genes in developing limbs has so far become apparent. Nonetheless, interesting expression patterns in limbs of wild-type mice, and the limb phenotypes in mice transgenic for members of these clusters, suggest that hoxb and hoxc may also be pivotal to correct limb patterning. Hoxb genes may be important in inducing limb outgrowth at the appropriate level along the body axis, and it has been recently suggested that hoxc


33

genes may be important in specifying the proximal region of the limb (humerus and femur) (Davis et al., 1995). It is therefore becoming apparent that different combinations of hox genes have a role in determining different regions of the limb during development (Ferretti and Tickle, 1997). Furthermore, some hox genes contain RAREs and are regulated by RA both in vitro and in vivo (Boncinelli et al., 1991; Marshall et al., 1994; Ogura and Evans, 1995). Because of their important role in limb development and because of their responsiveness to retinoids, a major effort in identifying urodele homologs of homeoboxcontaining genes, and in assessing their potential role in limb regeneration, has been undertaken. A list of homeobox-containing genes and of other genes believed to play a significant role in limb patterning that have been cloned in urodeles is given in Table IV. Nvhoxcd (former hvHbox I ) was the first hox gene to be isolated and characterized in urodeles (Savard et al., 1988). In species that cannot regenerate their limbs in adulthood, including Xenopus, hoxcd is expressed in developing but not in adult limbs. In contrast, in adult newts this gene is expressed in both normal and regenerating forelimbs and hindlimbs in a proximodistal gradient (Savard et al., 1988; Tabin, 1989; Savard and Tremblay, 1995). It has therefore been proposed that its expression may be causally related to the regenerative ability of the adult urodele limb. Another gene of the same cluster identified in N. viridescens is Nvhoxc10 (former hox-3.6) (Simon and Tabin, 1993). Nvhoxc-I0 mRNA is expressed in the normal limb and is upregulated during regeneration, reaching its highest level in the undifferentiated mid-bud blastema. No significant difference between proximal and distal blastemas is observed, and expression is restricted to the mesenchyme. With the progression of regeneration, Nvhoxc-10 expression is gradually downregulated to the levels detected in the normal limb. Nvhoxc-10, like a spliced from of Nvhoxcd (Savard et al., 1988; Savard and Tremblay, 1995), is also expressed in normal and regenerating tails but, unlike Nvhoxc-6, is not detected in either normal or regenerating forelimbs (Simon and Tabin, 1993). This parallels the pattern of expression of hoxc genes in developing mouse limbs. In fact, hoxc-ll is expressed in hindlimb but not in forelimb mesenchyme (Peterson et al., 1994). In contrast, hoxc-8 (former hox-3.1) appears to be expressed in both developing hindlimbs and forelimbs, as revealed by analysis of a reporter gene carried out in hoxc-8 null mutations obtained through homologous recombination (Le Mouellic et al., 1992). Because expression of both newt hoxc genes studied so far is maintained in the adult limb, it will be interesting to establish whether this property extends to other members of this cluster and could indeed be causally related to the regenerative capability of the urodele limb.

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Numerous urodele genes belonging to the hoxa and hoxd clusters have been cloned (Table IV), but full DNA sequences are currently available only for hoxa-9 and hoxa-13, which have been isolated from an axolotl limb blastema cDNA library (Gardiner et al., 1995), and hoxa-11, hoxd-10, and hoxd-11, which have been cloned in the newt, N. viridescens (Brown and Brockes, 1991; Simon and Tabin, 1993; Beauchemin et al., 1994). The level of expression of the hoxd genes, like that of Nvhoxc-6, is higher in proximal than distal blastemas. Like the hoxc genes, hoxa and hoxd genes are also expressed in the undifferentiated mesenchyme but not in the WE of the regenerating limb. However, in contrast to Nvhoxc-6 and Nvhoxc10, most of these genes are not expressed in normal adult limbs but are induced following amputation. Only the newt hoxa-11 seems to depart from this behavior, and it is detected in the normal limb, primarily in muscle and bone. It will be interesting to establish whether the axolotl hoxa11 behaves like its newt counterpart because this would confirm that its regulation in the adult limb is different from that of other members of the same cluster. The fact that hoxa genes in the adult limb may not be expressed colinearily as during limb development is also supported by the work of Gardiner and colleagues (1995). Analysis of hoxa-9 and hoxa-11 transcripts in the axolotl regeneration blastemas by whole mount in situ hybridization has shown that, in most limbs, both genes are expressed beneath the WE 24 hours after amputation,independently from the level at which the limb was cut. Nonetheless, a detailed time course of the expression of hoxa genes between 0 and 48 h after amputation will have to be carried out to clearly establish that their activation in regenerating limbs does not follow the colinearity rule. The cellular localization of the hoxd genes and their spatiotemporal pattern of expression early after limb amputation are not yet known. However, analysis of hoxd-11 and hoxd-12 in regenerating zebrafish fins suggests that although expression of genes of this cluster is reinduced following amputation, their spatial pattern of expression in the regenerate is different (J. GCraudie et al., unpublished results) from that observed during development (Sordino et al., 1995). In addition, they both are detected at 24 h after amputation. Therefore, it appears that, although the same set of genes expressed during development is used to rebuild the missing part of a lower vertebrate appendage, the role they play in the process may be, at least at the early stages of regeneration, somehow different. Partial sequences of genes of the hoxb cluster have been isolated from regenerating axolotl limbs, but no information on their spatiotemporal pattern of expression during regeneration, or on whether they are expressed in normal limbs, is available (Gardiner et al., 1995). In the mouse, hoxb-8 (former hox-3.1) is expressed initially in the lateral plate mesoderm posterior to somite 9 and then in the posterior part of the mouse limb bud in a

GENE EXPRESSION DURING AMPHl6lAN LIMB REGENERATION

35

region corresponding to the ZPA (CharitC et al., 1994). On the basis of this pattern of expression and of the anteroposterior limb duplication observed in transgenic embryos ectopically expressing hoxb-8, it has been suggested that hoxb-8 may have a role in the establishment of the ZPA (Charitt el al., 1994). It will therefore be important to look into the expression of this neglected gene cluster in the limb blastema because it may prove very informative regarding the establishment of limbness and early patterning mechanisms in development and regeneration. Because of the patterning effects induced by R A on regenerating limbs, and the fact that R A has been shown to regulate expression of certain hox genes (Boncinelli et al., 1991; Marshall et al., 1994; Ogura and Evans 1995), much work has been aimed at assessing its effects on hox genes in regenerating limbs. Most of these studies have been carried out by RNAase protection because, until recently, there have been technical problems with the detection of hox transcripts by in situ hybridization in urodeles. Analysis of hox transcript by RNAase protection in the newt has been carried out, at the earliest, 5 days after R A injections. Because the initial effect induced by R A is inhibition of blastema growth, it is difficult to collect a sufficient amount of material that is not contaminated by stump tissues for RNA analysis at earlier stages. Under the experimental conditions used, hoxd10 expression appears to be upregulated by R A (Simon and Tabin, 1993), whereas the more 5' hoxd-11 is not (Brown and Brockes, 1991). In contrast, in axolotl limb blastemas analyzed by in situ hybridization, the more 5' hoxa-13, but not hoxa-9, appears to be downregulated by R A (Gardiner et al., 1995). In order to confirm that there is indeed a difference in the regulation of hox genes along the complex it will be important to examine the response to R A of other members of these two clusters, preferably in the same species. Besides the hox gene family, other families of homeobox-containing genes, such as the msx family (related to Drosophila msh) and the dlx family (related to Drosophila distal-less), appear to play important roles in the development of limbs and face, in which epithelial-mesenchymal interactions are pivotal to the developmental process. Genes belonging to the msx family are expressed both in the ridge and in the mesenchyme of developing appendages (limbs and fins) and are believed to be important in maintaining cells in an undifferentiated state in the progress zone (Hill et al., 1989; Robert et al., 1989, 1991; Davidson et al., 1991; Song et al., 1992; Vogel et al., 1995; Akimenko et al., 1994a). Interestingly, in higher vertebrates high levels of msx-1 in the fingertip of developing digits appear to correlate with significant regenerative capability (Reginelli et al., 1995). Only the newt msx-1 has been studied in detail during limb regeneration (Crews et al., 1995; Simon et al., 1995), but a partial sequence of axolotl

36


msx-2 has been isolated (see Table IV). Parallel to the pattern of expression observed in other vertebrates during limb development, the newt msx-1 is found both in the blastema mesenchyme and in the WE (Crews et aL, 1995). These authors, unlike Simon et al., (1995), detect comparable levels of msx2 in unamputated limbs by both Northern blot and RNAase protection analysis, and surprisingly the exposure times required to detect msx-l in the two studies are significantly different (14 days and 2 or 3 days, respectively). Whether these discrepancies could be partly due to the different probes used to detect the transcript 5' coding region in the case of Crews et al. and 3' untranslated region in the case Simon et al. and/or to the presence/ absence in the normal limb samples of the fingertips, which appear to be blastema-like (Ferretti and Ghosh, 1997), is currently unclear. Finally, functional studies have indicated that in urodeles, unlike in mammals, msxI does not seem to affect either proliferation or differentiation of newt myogenic cells in culture (Crews et al., 1995). More work will be required to clarify the different expression results obtained and to establish which role msx-I might play in limb regeneration. On the basis of much work carried out in Drosophila and expression in the apical ectodermal ridge of developing limbs and fins (Akimenko et al., 1994b), genes of the dlx family are thought to play a role in limb outgrowth and in the establishment of proximodistal pattern (Cohen et al., 1989; Doll6 et al., 1992; Bulfone et al., 1993; Simeone el aL, 1994; Diaz-Benjumena et al,, 1994; Morass0 et al., 1995). Two newt dlx genes have been identified, NvHBox-4 and NvHBox-5 (Beauchemin and Savard, 1992); the homeodomain of NvBox-4 is identical at the amino acid level of that of mouse, axolotl, and Pleurodeles dlx-3 (Mullen et al., 1996; Nicolas et al., 1996). Analysis by Northern blot of newt dlxs has shown that both genes are ubiquitously expressed in the adult skin, and that their levels in normal and regenerating limbs, at different axial levels, are comparable. In contrast, the axolotl dlx-3 has recently been shown to be upregulated in the WE of regenerating limbs by both Northern blot and in situ hybridization (Mullen et al., 1996). Furthermore, its levels are higher in the WE of distal blastemas than in that of proximal blastemas. In denervated blastemas both regeneration and expression of dlx-3 are inhibited, but both effects can be reversed by FGF-2-soaked beads. Dlx-3 is also downregulated by R A treatment, but by the time this compound has cleared from the limb and growth resumes, dlx-3 has returned to normal levels. Together, these results suggest that expression of dlx-3 and limb outgrowth may be linked. If NvHBox-4 and axolotl dlx-3 are truly homologous, it is difficult to understand why their patterns of expression and regulation appear to be rather different in two related species with comparable regenerative capabilities.


37

Recently, by using a differential display technique, a newt gene belonging to the T-box family of transcription factors, which includes brachyury, has been isolated (Simon et al., 1997). This gene appears to be upregulated in forelimb but not in hindlimb blastemas. Given the proposed important role of T-box genes in the evolution of fin and limb morphogenesis (GibsonBrown et al., 1996),the newt T-box gene identified might play an important role in controlling forelimb “identity” in the regenerate. C. Segment Polarity Genes

Although some of the urodele segment polarity genes have been cloned (Table 111), information about their distribution and possible role in the regenerating limbs is still minimal. At the last International Conference on Limb Development and Regeneration (1996), it was shown that two hedgehog genes sonic hedgehog (shh) and banded hedgehog (bhh), are expressed in developing and regenerating limbs. Whereas bhh expression is clearly detected throughout developing and regenerating limb buds (D. Stark, P. Gates, J. Brockes, and P. Ferretti, unpublished results), it has been suggested that shh has a posterior domain of expression in the regenerating blastema as well as in the developing limb bud (Imokawa and Yoshizato, 1997; Endo et al., 1997). If this is indeed the case, shh distribution in newt limb blastemas would be different from that observed in another regenerating appendage, the zebrafish fin. In fact, although shh is expressed in posterior mesenchyme of early fin buds (Akimenko and Ekker, 1995), no posterior localization is observed when its expression is reinduced following fin amputation (Laforest et al., manuscript in preparation). Because indiun hedgehog, the mammalian homolog of bhh, is not expressed in early limb buds, it is conceivable that expression of bhh in the developing and regenerating newt limbs may be related to the regenerative capability in this species. Bhh may either play a role in dedifferentiation, or be implicated in the establishment of positional identity since it is significantly upregulated by RA. It is not clear how expression of hedgehog genes in the blastema is regulated. It will be important to establish whether in the regenerating limb there is a loop including hedgehog genes, FGF, and RA that links outgrowth with patterning as suggested in the chick limb bud (Izpisua-Belmonte et al., 1992; Laufer er al., 1994; Niswander er al., 1994; Stratford et aL, 1996). If this is the case, application of both FGF and RA to digit stumps in mammals may constitute a first step toward inducing regeneration in higher vertebrates. In summary, the bulk of data currently available on patterning of the regenerating limb support the view that the same key molecules are used to build both embryonic and adult limbs. However, there are indications

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that at the early bud stages they may be used in a slightly different fashion, and this might be due to differences in the origin of the progenitor cells during development and regeneration and to the presence of positional cues in the stump of the adult newt limb.

V. Concluding Remarks Much information on the molecular basis of regeneration has been gained since Spallanzani’s (1768) first report of this extraordinary phenomenon. In the past decade in particular, the application of the most advanced experimental tools has given great impetus to the molecular analysis of epimorphic regeneration. In this article we have attempted to give a comprehensive overview of the expression patterns and possible roles of molecules, a few specific to regeneration and many common to development, that have been studied within the context of limb regeneration in urodeles. Although much work has yet to be done if we are to induce significant regeneration in mammals, the rapidly growing understanding of this phenomenon in amphibians is shifting this prospect from the realm of science fiction to that of possibility. Acknowledgments The authors thank all the colleagues for providing data prior to their publication and Dr. D. Stocum for supplying Figs. 1 and 2. JG was partly supported by Laboratoire de Biologie du DCveloppement des Poissons (Professor J. M. Vernier), Universite Paris-Sud XI and UA CNRS 1134. PF was supported by The Wellcome Trust and the MRC.

References Akimenko, M. A,, and Ekker, M. (1995). Anterior duplication of the Sonic hedgehog expression pattern in the pectoral fin buds of zebrafish treated with retinoic acid. Dev. Biol. 170,243-247. Akimenko, M. A., Johnson, S. L., Westerfield, M., and Ekker, M. (1994a). Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish. Development 121,347-357. Akimenko, M. A., Ekker, M., Wegner, J., Lin, W., and Westerfield, M. (1994b). Combinatorial expression of three zebrafish genes related to distal-less: Part of a homeobox gene code for the head. .I. Meurosci. 14, 3475-3486. Albert, P., and Boilly, B. (1988). Effect of transferrin on amphibian limb regeneration: A blastema cell culture study. Roux’s Arch. Dev. Biol. 197, 193-196. Albert, P., Boilly, B., Courty, J., and Barritault, D. (1987). Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or I1 (basic or acidic FGF). Cell Differ. 21, 63-68. AndCol, Y., Gusse, M., and MCchali, M. (1990). Characterization and expression of a Xenopus ras during oogenesis and development. Dev. Biol. 139,24-34.


39

Astrom, A., Pettersson, U., and Voorhees, J. J. (1992). Structure of the human cellular retinoic acid-binding protein I1 gene. Early transcriptional regulation by retinoic acid. J. Biol. Chem. 267,25251-25255. Barrandon, Y . (1993). The epidermal stem cell: An overview. In “Epithelial Stem Cells. Part 1. Keratinocytes. Seminars in Developmental Biology” (C. Potten, Ed.). Academic Press, New York. Beauchemin, M., and Savard, P. (1992). Two distal-less related homeobox-containing genes expressed in regeneration blastemas of the newt. Dev. B i d . 154,55-65. Beauchemin, M., Noiseux, N., Tremblay, M., and Savard, P. (1994). Expression of Hox A l l in the limb and the regeneration blastema of adult newt. Int. J. Dev. Bid. 38,641-649. Belleville, S., Beauchemin, M., Tremblay, M., Noiseux, N., and Savard, P. (1992). Homeoboxcontaining genes in the newt are organized in clusters similar to other vertebrates. Gene 114, 179-186. Boilly, B., and Albert, P. (1988). Blastema cell proliferation in vitro: Effects of limb amputation on the mitogenic activity of spinal cord extracts. Biol. Cell 62, 183-187. Boilly, B., and Albert, P. (1990). in vitro control of blastema cell proliferation by extracts from epidermal cap and mesenchyme of regenerating limbs of axolotls. Roux’s Arch. Dev. Biol. 198, 443-441. Boilly, B., Cavanaugh, K. P., Thomas, D., Hondermarck, H., Bryant, S. V., and Bradshaw, R. A. (1991). Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotls and binds specifically to blastema tissue. Dev. B i d . 145, 302-310. Boilly, B., Oudghir, M., Deudon, E., Boilly-Marer, Y., and Hondermarck, H. (1995). Nerve dependent sulphated glycosaminoglycan synthesis in limb regeneration of the newt Pleurodeles wall. Roux’s Arch. Dev. Biol. 204, 509-512. Boncinelli, E., Simeone, A., Acampora, D., and Mavilio, F. (1991). Hox gene activation by retinoic acid. Trends Genet. 7,329-334. Brockes, J. P. (1984). Mitogenic growth factors and nerve dependence of limb regeneration. Science 225, 1280-1287. Brockes, J. P. (1987). The nerve dependence of amphibian limb regeneration. J. Exp. B i d . 132,79-91. Brockes, J . P. (1989). Retinoids, homeobox genes, and limb morphogenesis. Neuron 2,12851294. Brockes, J . P. (1990). Retinoic acid and limb regeneration. J. Cell Sci. Suppl. 13, 191-198. Brockes, J. P. (1992). Introduction of a retinoid reporter gene into the urodele limb blastema. Proc. Natl. Acad. Sci. USA 89, 11386-11390. Brockes, J. P. (1994). New approaches to amphibian limb regeneration. Trends Genet. 10, 169-173. Brockes, J. P. (1998). Progenitor cells for regeneration: Origin by reversal of the differentiated state. In “Cell and Molecular Biology of Regeneration” (P. Ferretti and J. GCraudie, Eds.), pp. 63-77. Wiley, Chichester, UK. Brockes, J. P., and Kintner, C . R. (1986). Glial growth factor and nerve-dependent proliferation in the regeneration blastema of urodele amphibians. Cell 45,301-306. Brown, R., and Brockes, J. P. (1991). Identification and expression of a regeneration-specific homeobox gene in the newt limb blastema. Development 111,489-496. Bryant, S. V., and Gardiner, D. M. (1992). Retinoic acid, local cell-cell interactions, and pattern formation in vertebrate limbs. Dew. Biol. 152, 1-25. Bulfone, A,, Kim, H. J., Puelles, L., Porteus, M. H., Grippo, J. F., and Rubenstein, J. L. (1993). The mouse Dlx-2 (Tes-I) gene is expressed in spatially restricted domains of the forebrain, face and limbs in midgestation mouse embryos. Mech. Dev. 40,129-140. Busse, U., and Seguin, C. (1993). Isolation of cDNAs for two closely related members of the axolotl Wnr family, Awnt-SA and Awnt-SB, and analysis of their expression during development. Mech. Dev. 40,63-72.

40


Cameron, J. A., Hilgers, A. R., and Hintenberger, T. J. (1986). Evidence that reserve cells are a source of a regenerated adult newt muscle in vitro. Nature (London) 321, 607-610. Carlone, R. L., and Mescher, A. L. (1985). Trophic factors from nerves. In “Regulation of Vertebrate Limb Regeneration” (R. E. Sicard, Ed.), pp. 93-105. Oxford Univ. Press, London. Carlson, B. M. (1979). The relationship between the tissue and epimorphic regeneration of muscle. In “Muscle Regeneration” (A. Mauro, Ed.), pp. 57-71. Raven Press, New York. Carlson, B. M. (1988). Some aspects of regeneration in skeletal and cardiac muscle. In “Recent Trends in Regeneration Reseach” (V. Kiortsis, S. Koussoulakos, and H. Wallace, Eds.), NATO AS1 Series, Life Sciences, Vol. 12, pp. 147-157. Plenum. Casimir, C. M., Gates, P. B., Patient, R. K., and Brockes, J. P. (1988).Evidence for dedifferentiation and metaplasia in amphibian limb regeneration from inheritance of DNA methylation. Development 104,657-668. Castilla, M., and Tassava, R. A. (1992). Extraction of the WE3 antigen and comparison of reactivities of mAb WE3 and WE4 in adult newt regenerate epithelium and body tissues. In “Monographs in Developmental Biology” (H. W. Sauer, Ed.), Vol. 23, pp. 116-130. Karger, Basel. Caubit, X., Nicolas, S., Shi, D. L., and Le Parco, Y. (1997). Reactivation and graded expression pattern of Wnt-ZOagene during early regeneration stages of adult tail in Amphibian urodele Pleurodeles waltl. Dev. Dyn.208, 139-148. CharitC, J., de Graaff, W., and Deschamps, J. (1994). Ectopic expression of Hoxb-8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures. Cell 78, 589-601. Choo, A. F., Logan, D. M., and Rathbone, M. (1978). Nerve trophic effects: An in vitro assay for factors involved in regulation of protein synthesis in regenerating amphibians limbs. J. EXP.2001.206,347-354. Cohen, S. M., Bronner, G.,Kuttner, F., Jurgens, G., and Jackle, H. (1989). Distal-less encodes a homeodomain protein required for limb development in Drosophila. Nature 338,432-434. Colquhoun, J. E., and Dresden, M. H. (1983). Prolyl hydroxylase activity during newt forelimb regeneration. Dev. Biol. 95, 193-199. Corcoran, J. P. T., and Ferretti, P. (1997). Keratin 8 and 18 expression in mesenchymal progenitor cells of regenerating limbs is associated with cell proliferation and differentiation. Dev. Dyn. 210,355-370. Crews, L., Gates, P. B., Brown, R., Joliot, A., Foley, C., Brockes, J. P., and Gann, A. A. (1995). Expression and activity of the newt Msx-I gene in relation to limb regeneration. Proc. R. SOC. London B. Biol. Sci. 259,161-171. Crossley, P. H., Minowada, G. Macarthur, C. A., and Martin, G. R. (1996). Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell 84, 127-136. Daniels, K., and Solursh, M. (1991). Modulation of chondrogenesis by the cytoskeletal and extracellular matrix. J. Cell Sci. 100,249-254. Davidson, D. R., Crawley, A., Hill, R. E., and Tickle, C. (1991). Position-dependentexpression of two related homeobox genes in developing vertebrate limbs. Nature (London) 352, 429-431. Davis, A. P., and Capecchi, M. R. (1994). Axial homeosis and appendicular skeleton defects in mice with a targeted disruption of hoxd-21. Development 1243,2187-2198, Davis, A. P., Witte, D. P., Hsieh-Li, H. M., Potter, S. S., and Capecchi, M. (1995). Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11. Nature (London) 375,791-795. Del Rio-Tsonis, K., Washabaugh, C. H., and Tsonis, P. A. (1992). The mutant axolotl short toes exhibits impaired limb regeneration and abnormal basement membrane formation. Proc. Natl. Acad. Sci. USA 89,5502-5506. Diaz-Benjumena,F. J., Cohen, B., and Cohen, S. M. (1994). Cell interaction between compartments establishes the proximal-distal axis of Drosophila legs. Nature 372, 175-179.


41

Dinsmore, C. E., and Mescher, A. L. (1998). The role of the nervous system in regeneration. In “Cell and Molecular Biology of Regeneration” (P. Ferretti and J. GCraudie, Eds.), pp. 79-108. Wiley, Chichester, UK. DollC, P., Price, M., and Duboule, D. (1992). Expression of the murine DLx-I homeobox gene during facial, ocular and limb development. Differentiation 49, 93-99. DollC, P., Dierich, A., LeMeur, M., Schimmang,T., Schuhbaur,B., Chambon, P., and Duboule, D. (1993). Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice with neotenic limbs. Cell 75,431-441. Donaldson, D. J., and Mahan, J. T. (1987). Newt epidermal cell migration in vitro and in vivo appears to involve Arg-Gly-Asp-Ser receptors. J. Cell Sci. 87,525-534. Donovan, M., Olofsson, B., Gustafson, A. L., Dencker, L., and Eriksson, U. (1995). The cellular retinoic acid binding proteins. J. Steroid Biochem. Mol. Biol. 53, 459-465. Dresden, M. H. (1969). Denervation effects on newt limb regeneration: DNA, RNA and protein synthesis. Dev. Biol. 19,311-320. Dresden, M. H., and Gross, J. (1970). The collagenolytic enzyme of the regenerating limb of the newt Triturus viridescens. Dev. Biol. 22, 129-137. Duellman, W. E., and Trueb, L. (1986). “Biology of amphibians.” McGraw-Hill, New York. Eager, N. S., Rowe, A., and Brickell, P. M. (1991). A member of the chicken RXR family of nuclear receptors activates transcription in response to retinoic acid. FEBS Lett. 292, 103-106. Eichele, G., and Thaller, C. (1987). Characterization and concentration gradients of a morphogenetically active retinoid in the chick limb bud. J. Cell Biol. 105, 1917-1923. Endo, T., Yokoyama, H., Tamura, K., and Ide, H. (1997). Shh expression in developing and regenerating limb buds of Xenopus laevis. Dev. Dyn. 209,227-232. Estrada, C. M., Park, C. D., Castilla, M., and Tassava, R. A. (1993). Monoclonal antibody WE6 identifies an antigen that is up-regulated in the wound epithelium of newts and frogs. In “Limb Development and Regeneration, Part A” (J. F. Fallon, P. F. Goetinck, R. 0. Kelley, and D. L. Stocum, Eds.), pp. 271-282. Wiley, New York. Fallon, J. F., Lopez, A., Ros, M., Savage, M. P., Olwin, B. B., and Simandl, B. K. (1994). FGF2: Apical ectodermal ridge growth signal for chick limb development. Science 264,104-107. Favier, B., LeMeur, M., Chambon, P., and Doll& P. (1995). Axial skeleton homeosis and forelimb malformations in Hoxd-11 mutant mice. Proc. Natl. Acad. Sci. USA 92,310-314. Fekete, D., and Brockes, J. P. (1987). A monoclonal antibody detects a differencein the cellular composition of developing and regenerating limbs of newts. Development 99, 589-602. Fekete, D., Ferretti, P., Gordon, H., and Brockes, J. P. (1987). Ontogeny of nerve dependence of amphibian limb regeneration. In “From Message to Mind Directions in Developmental Neurobiology” (S. S. Easter, K. F. Barrald, and B. M. Carlson, Eds.), pp. 29-42. Sinauer, Sunderland, MA. Ferretti, P., and Brockes, J. P. (1988). Culture of newt cells from different tissues and their expression of a regeneration associated antigen. J. Exp. Zool. 247, 77-91. Ferretti, P., and Brockes, J. P. (1990). The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt, Notophthalmur viridescens, during limb regeneration. Cell Tissue Res. 259, 483-493. Ferretti, P., and Brockes, J. P. (1991). Cell origin and identity in limb regeneration and development. Glia 4,214-224. Ferretti, P., and Ghosh, S. (1997). Expression of regeneration-associatedcytoskeletalproteins reveals differences and similarities between regenerating organs. Dev. Dyn. 210,288-304. Ferretti, P., and Tickle, C. (1997). The limbs. In “Embryos, Genes and Birth Defects” (P. Thorogood, Ed.), pp. 101-132. Wiley, Chichester, UK. Ferretti, P., Fekete, D. M., Patterson, M., and Lane, E. B. (1989). Transient expression of simple epithelial keratins by mesenchymal cells of regenerating newt limb. Dev. Biol. 133,415-424.

42


Ferretti, P., Brockes, J. P., and Brown, R. (1991). A new type I1 keratin restricted to normal and regenerating limbs and tails is responsive to retinoic acid. Development 111,497-507. Ferretti, P., Corcoran, J. P. T., and Ghosh, S. (1993). Expression and regulation of keratins in the wound epithelium and mesenchyme of the regenerating newt limb. Prog. Clin. Biol. Res. 383A, 261-269. Foty, R. A., and Liversage, R. A. (1993). Detection of insulin receptors in newt liver and forelimb regenerates and the effects of local insulin deprivation on epimorphic regeneration. J. EXP. ZOO^. 266,299-311. Gardiner, D. M., Muneoka, K., and Bryant, S. V. (1986). The migration of dermal cells during blastema formation in axolotls. Dev. Biol. 118,488-493. Gardiner, D. M., Blumberg, B., and Bryant, S. V. (1993). Expression of homeobox genes in limb regeneration. Prog. Clin. Biol. Res. 383A, 31-40. Gardiner, D. M., Blumberg, B., Komine, Y., and Bryant, S. V. (1995). Regulation of HoxA expression in developing and regenerating axolotl limbs. Development U1, 1731-1741. Geraudie, J., and Ferretti, P. (1997). Correlation between RA-induced apoptosis and patterning defects in regenerating fins and limbs. In/. J. Dev. Biol. 41, 529-532. Gkraudie, J., and Singer, M. (1978). Nerve-dependent macromolecular synthesis in the epidermis and blastema of the adult newt limb regenerate. J. Exp. Zool. 203,455-460. Geraudie, J., and Singer, M. (1981). Scanning electron microscopy of the normal and denervated limb regenerate in the newt, Notophthalmus, including observations on embryonic amphibian limb-bud mesenchyme and blastema of the fish-fin regenerate. Am. J. Anat. 162,7347. GCraudie, J., Hourdry, J., Vriz, S., Singer, M., and Mechali, M. (1990). Enhanced c-myc gene expression during forelimb regeneration outgrowth in the young Xenopus luevis. Proc. Nutl. Acad. Sci. USA 87,3797-3801. Gibson-Brown, J. J., Agulnik, S. I., Chapman, D. L., Alexiou, M., Garvey, N., Silver, L. M., and Papaioannou, V. E. (1996). Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech. Dev. 56, 93-101. Giguere, V., Ong, E. S.,Evans, R. M., and Tabin, C. J. (1989). Spatial and temporal expression of the retinoic acid receptor in the regenerating amphibian limb. Nature (London) 337, 566-569. Globus, M., and Alles, P. (1990). A search for immunoreactive substance P and other neural peptides in the limb regenerate of the newt, Notophthalmus viridescens. J. Exp. Zool. 254, 165-176. Globus, M., Vethamany-Globus, S., Kesik, A., and Milton, G. (1983). Roles of neural peptide substance P and calcium in the newt Notophthalmus viridescens. In “Limb Development and Regeneration” (J. F. Fallon and A. I. Caplan, Eds.), pp. 513-523. A. R. Liss, New York. Goldhamer, D. J., and Tassava, R. A. (1987). An analysis of proliferative activity in innervated and denervated forelimb regenerates of the newt, Notophthalmus viridescens. Development 100,619-628. Goldhamer, D. J., Tomlinson, B. L., and Tassava, R. A. (1989). A developmentally regulated wound epithelial antigen of the newt limb regenerate is also present in a variety of secretory/ transport cell types. Dev. B i d . 135, 392-404. Gordon, H., and Brockes, J. P. (1988). Appearance and regulation of an antigen associated with limb regeneration in Nofophthulmus viridescens. J . Exp. Zool. 247, 232-243. Gospodarowicz, D., and Mescher, A. L. (1980). Fibroblast growth factor and the control of vertebrate regeneration and repair. Ann. N. Y. Acad. Sci. 339, 151-174. Griffin, K. J. P., Fekete, D. M., and Carlson, B. M. (1987). A monoclonal antibody stains myogenic cells in regenerating newt muscle. Development 101, 267-277. Grillo, H. L., Lapikre, C. M., Dresden, M. H., and Gross, J. (1968). Collagenolytic activity in regenerating forelimbs of the adult newt (Triturus viridescens). Dev. Biol. 17, 571-583. Gulati, A. K., Zalewski, A. A., and Reddi, A. (1983). An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt. Dev. Biol. 46,355-365.

GENE EXPRESSION DURING AMPHIBIAN

LIMB REGENERATION

43

Hay, E. D. (1959). Microscopic observations of muscle dedifferentiation in regenerating Ambiystoma limbs. Dev. Biol. 1, 555-585. Hay, E. D., and Fishman, D. A. (1961). Origin of the blastema in regenerating limbs of the newt Triturus viridescens. Dev. Biol. 3,26-59. Hill, D. S., Ragsdale, C. W., Jr., and Brockes, J. P. (1993). Isoform-specific immunological detection of newt retinoic acid receptor d l in normal and regenerating limbs. Development 117, 937-945. Hill, R. E., Jones, P. F., Rees, A. R., Sime, C. M., Justice, M. J., Copeland, N. G., Jenkins, N. A., Graham, E., and Davidson, D. R. (1989). A new family of mouse homeobox containing genes: Molecular structure, chromosomal location and developmental expression of Hox7.1. Genes Dev. 3, 26-37. Hinds, P. W., and Weinberg, R. A. (1994). Tumor suppressor genes. Curr. Opin. Gene Dev. 4,135-141. Imokawa, Y., and Yoshizato, K. (1997). Expression of Sonic hedgehog gene in regenerating newt limb blastemas recapitulates that in developing limb buds. Proc. Nutl. Acud. Sci. USA 94, 9159-9164. Izpisua-Belmonte. J. C., and Duboule, D. (1992). Homeobox and pattern formation in the vertebrate limb. Dev. Biol. 152,26-36. Izpisua-Belmonte, J. C., Brown, J. M., Duboule, D., and Tickle, C. (1992). Expression of hox-4 genes in the chick wing links pattern-formation to the epithelial mesenchymal interactions that mediate growth. EMBO J. 11, 1451-1457. Iten, L. E., and Bryant, S. V. (1973). Forelimb regeneration from different levels of amputation in the newt, Notophthalmus viridescens: Length, rate and stages. Rou’s Arch. Dev. Biol. 173,263-282. Jabaily, J., and Singer, M. (1977). Neurotrophic stimulation of DNA synthesis in the regenerating forelimb of the newt, Triturus. J. Exp. Zool. 199,251-256. Keeble, S., and Maden, M. (1986). Retinoic acid-binding protein in the axolotl: Distribution in mature tissue and time of appearance during limb regeneration. Dev. Biol. 117,435-441. Kiffmeyer, W. R., Tomusk, E. V., and Mescher, A. L. (1991). Axonal transport and release of transferrin in nerves of regenerating amphibian limbs. Dev. Biol. 147,392-402. Kim, W. S., and Stocum, D. L. (1986). Retinoic acid modifies positional memory in the anteroposterior axis of regenerating axolotl limbs. Dev. Biol. 114, 170-179. Kintner, C., and Brockes, J. P. (1984). Monoclonal antibodies identify blastemal cells derived from dedifferentiating muscle in newt limb regeneration. Nature (London) 308,67-69. Kintner, C., and Brockes, J. P. (1985). Monoclonal antibodies to the cells of a regenerating limb. J. Embryol. Exp. Morphol. 89, 37-51. Klatt, K. P., Yang, E. V., and Tassava, R. A. (1992). Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is colocalized with tenascin during adult newt limb regeneration. Differentiation 50, 133-140. Koshiba, K., Tamura, K., and Ide, H. (1994). Expression of regeneration-associated antigens in normal and retinoid-treated regenerating limbs of Ambystoma mexicanum. Dev. Growth Differ. 36, 357-364. Kostakopoulou, K., Vogel, A,, Brickell, P., and Tickle, C. (1996). “Regeneration” of wing bud stumps of chick embryos and reactivation of Msx-1 and Shh expression in response to FGF-4 and ridge signals. Mech. Dev. 55, 119-131. 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 of the developing limb bud. Cell 79, 993-1003. Lebowitz, P., and Singer, M. (1970). Neurotrophic control of protein synthesis in the regenerating limb of the newt, Triturus. Nature (London) 225, 824-827. Leid, M., Kastner, P., and Chambon, P. (1992). Multiplicity generates diversity in the retinoic acid signalling pathways. Trends Biochem. Sci. 17,427-433.

44

JACQUELINE GERAUDIE AND PATRIZIA FERRE?TI

Lemaitre, J. M., Mkchali, M., and GCraudie, J. (1992). Nerve-dependent expression of c-myc protein during forelimb regeneration of Xenopus laevis froglets. Inf.J. Dev. Biol. 36,483-489. Le Mouellic, H., Lallemand, Y . ,and BrOlet, P. (1992). Homeosis in the mouse induced by a null mutation in the Hox 3.1 gene. Cell 69, 251-264. Liversage, R. A. (1991). Origin of the blastema cells in epimorphic regeneration of urodele appendages: A history of ideas. In “A History of Regeneration Research” (C. E. Dinsmore, Ed.), pp. 179-199. Cambridge Univ. Press, Cambridge, UK. Lo, D. C., Allen, F., and Brockes, J. P. (1993). Reversal of muscle differentiation during urodele limb regeneration. Proc. Natl. Acad. Sci. USA 90,7230-7234. Lohnes, D., Mark, M., Mendelsohn, C., DollC, P., Dierich, A,, Gorry, P., Gansmuller, A,, and Chambon, P. (1994). Function of the retinoic acid receptors (RARs) during development. (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development 120, 2723-2748. Ludolph,D. C.,Cameron, J. A.,andStocum,D. L. (1990).Theeffect ofretinoicacidonpositional memory in the dorsoventral axis of regenerating axolotl limbs. Dev. Biol. 140,41-52. Ludolph, D. C., Cameron, J. A,, Neff, A. W., and Stocum, D. L. (1993). Cloning and tissuespecific expression of the axolotl cellular retinoic acid-binding protein. Dev. Growth Differ. 35,341-347. Ludolph, D. C., Neff, A. W., Parker, M. A., Mescher, A. L., Smith, R. C. and Malacinski, G. M. (1995). Cloning and expression of the axolotl proto-oncogene ski. Biochem. Biophys. Acta 1260, 102-104. Lyons, G. E., and Buckingham, M. (1992). Developmental regulation of myogenesis in the mouse. Sem. Dev. Biol. 3,243-253. Maden, M. (1977). The role of Schwann cells in paradoxical regeneration in the axolotl. J. Embryol. Exp. Morphol. 41, 1-13. Maden, M. (1982). Vitamin A and pattern formation in the regenerating limb. Nature (London) 295,672-675. Maden, M. (1983). The effect of vitamin A on the regenerating axolotl limb. J. Embryol. Exp. Morphol. 77,273-295. Maden, M. (1991). Retinoid-binding proteins in the embryo. Sem. Dev. Biol. 2, 161-170. Maden, M., Keeble, S., and Cox, R. A. (1985). The characteristics of local application of retinoic acid to the regenerating axolotl limb. R o u ’ s Arch. Dev. Biol. 194,228-235. Maden, M., Ong, D. E., Summerbell, D., and Chytil, F. (1988). Spatial distribution of cellular protein-binding to retinoic acid in the chick limb bud. Nature (London) 335, 733-735. Maier, C. E., and Miller, R. H. (1992). In vifro and in vivo characterization of blastemal cells from regenerating newt limbs. J. Exp. Zool. 262, 180-192. Maier, C. E., Watanabe, M., Singer, M., McQuarrie, I. G., Sunshine, J., and Rutishauser, U. (1986). Expression and function of neural cell adhesion molecule during limb regeneration. Proc. Natl. Acad. Sci. USA 86, 8395-8399. Mailman, M. L., and Dresden,M. H. (1976).Collagen metabolism in the regenerating forelimb of Nofophthalmus viridescens: Synthesis, accumulation and maturation. Dev. Biol. 50,378-394. Mangelsdorf, D. J., Borgmeyer, U., Heyman, R. A., Yang Zhou, J., Ong, E. S., Oro, A. E., Kakizuka, A,, and Evans, R. M. (1992). Characterization of three RXR genes that mediate the action of 9-cir retinoic acid. Genes Dev. 6,329-344. Mangelsdorf, D. J., Kliewer, S. A., Kakizuka, A., Umesono, K., and Evans, R. M. (1993). Retinoid receptors. Recent Prog. Hormone Res. 48,99-121. Marshall, H., Studer, M., Popperl, H., Aparicio, S., Kuroiwa, A., Brenner, S., and Krumlauf, R. (1994). A conserved retinoic acid response element required for early expression of the homeobox gene Hoxb-I. Nature (London) 370,567-571. McCormick, A. M., Shubeita, H. E., and Stocum, D. L. (1988). Cellular retinoic acid binding protein: Detection and quantitation in regenerating axolotl limbs. J. Exp. Zool. 245,270-276. Means, A. L., and Gudas, L. J. (1995). The role of retinoids in vertebrate development. Annu. Rev. Biochem. 64.201-233.


45

Mendelsohn, C., Ruberte, E., and Chambon, P. (1992). Retinoid receptors in vertebrate limb development. Dev. Biol. 152, 50-61. Mescher, A. L. (1993). Development and regeneration of limbs in the Short toes axolotl mutant. In “Limb Development and Regeneration, Part A ’ (J. F. Fallon, P. F. Goetinck, R. 0. Kelley, and D. L. Stocum, Eds.), pp. 181-191. Wiley, New York. Mescher, A. L., and Cox, C. A. (1988). Hyaluronate accumulation and nerve-dependent growth during regeneration of larval Ambystoma limbs. Differentiation 38, 161-168. Mescher, A. L., and Gospodarowicz, D. (1979). Mitogenic effect of a growth factor derived from myelin on denervated regenerates of newt forelimbs. J. Exp. 2001.207,497-503. Mescher, A. L., and Kiffmeyer, W. R. (1992). Axonal release of transfemn in peripheral nerves of axolotl during regeneration. Monogr. Dev.B i d . 23, 100-109. Mescher, A. L., and Loh, J.-J. (1981). Newt forelimb regeneration blastemas in vitro: Cellular response to explantation and effects of various growth factors. J. Exp. 2001.216,235-245. Mescher, A. L., and Munaim, S. I. (1984). “Trophic” effect of transferrin on amphibian limb regeneration blastemas. J. Exp. 2001. 230,485-490. Mescher, A. L., and Munaim, S. I. (1986). Changes in the extracellular matrix and glycosaminoglycan synthesis during the initiation of regeneration in adult newt forelimbs. Anat. Rec. 214,424-431. Migliorini Bruschelli, G., and Rosi, G. (1971). Polymelia induced by vitamin A in embryos of Bufo vulgaris. Rev. Biol. 64, 277-283. Miller, S. D., Farmer, G., and Prives, C. (1995). p53 inhibits DNA replication in vitro in a DNA binding dependent manner. Mol. Cell. B i d . 15, 6554-6560. Miyazaki, K., Uchiyama, K., Imokawa, Y., and Yoshizato,K. (1996). Cloning and characterization of cDNAs for matrix metalloproteinase of regenerating newt limbs. Proc. Natl. Acad. Sci. USA 93,6819-6824. Monkemeyer, J., Ludolph, D. C., Cameron, J. A., and Stocum, D. L. (1992). Retinoic acidinduced change in anteroposterior positional identity in regenerating axolotl limbs is dosedependent. Dev. Dyn. 193,286-294. Morasso, M. I., Mahon, K. A., and Sargent, T. D. (1995). A Xenopus distal-less gene in transgenic mice. Conserved regulation in distal limb epidermis and other sites of epithelialmesenchymal interaction. Proc. Natl. Acad. Sci. USA 92, 3968-3972. Morgan, B. A., Izpisua-Belmonte, J. C., Duboule, D., and Tabin, C. J. (1992). Targeted misexpression of hox-4.6 in the avian limb bud causes apparent homeotic transformations. Nature (London) 358,236-239. Mullen, L. M., Bryant, S. V., Torok, M. A., Blumberg, B., and Gardiner, D. M. (1996). Nerve dependency of regeneration: The role of Distal-less and FGF signaling in amphibian limb regeneration. Development 122,3487-3497. Munaim, S. I., and Mescher, A. L. (1986). Transferrin and the trophic effect of neural tissue on amphibian limb regeneration blastemas. Dev. Biol. 116, 138-142. Muneoka, K., Fox, W., and Bryant, S. V. (1986). Cellular contribution from dermis and cartilage to the regenerating limb blastema in axolotl. Dev. Biol. 116,256-260. Nace, J. D., and Tassava, R. A. (1995). Examination of fibronectin distribution and its sources in the regenerating newt limb by immunocytochemistry and in situ hybridization. Dev. Dyn. 202,153-164. Namenwirth, M. (1974). The inheritance of cell differentiation during limb regeneration in the axolotl. Dev. Biol. 41, 42-56. Niazi, I. A., and Saxena, S. (1978). Abnormal hindlimb regeneration in tadpole of the toad, Bufo andersonii, exposed to excess vitamin A. Folia Biol. (Krakow) 26,3-11. Nicolas, S., Massacrier, A., Caubit, X., Cau, P., and Le Parco, Y. (1996). A distal-less-like gene is induced in the regenerating central nervous system of the urodele Pleurodeles waltl. Mech. Dev. 56,209-220. Niswander, L., Tickle, C., Vogel, A., Booth, I., and Martin, G. L. (1993). FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell 75,579-587.

46


Niswander, L., Jeffrey, S., Martin, G. R., and Tickle, C. (1994). A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature (London) 371,609-612. Noji, S.,Nohno, T., Koyama, E., Muto, K., Ohyama, K., Aoki, Y., Tamura, K., Ohsugi, K., Ide, H., and Taniguchi, S. (1991). Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud. Nature (London) 350,83-86. Ogura, T., and Evans, R.M. (1995). Evidence for two distinct retinoic acid response pathways for HOXBl gene regulation. Proc. Natl. Acad. Sci. USA 92, 392-396. Onda, H.,and Tassava, R. A. (1991). Expression of the 9G1 antigen in the apical cap of axolotl regenerates requires nerves and mesenchyme. J. Exp. 2001.257, 336-349. Onda, H., Goldhamer, D. J., and Tassava, R. A. (1990). An extracellular matrix molecule of newt and axolotl regenerating limb blastemas and embryonic limb buds: Immunological relationship of MT1 antigen with tenascin. Development 108, 657-668. Onda, H., Poulin, M. L., Tassava, R. A., and Chiu, I. M. (1991). Characterization of a newt tenascin cDNA and localization of tenascin mRNA during newt limb regeneration by in situ hybridization. Dev. B i d . 148, 219-232. Peadon, A. M., and Singer, M. (1966). The blood vessels of the regenerating limbs of the adult newt, Triturus. J. Morphol. 118, 79-90. Pecorino, L.T., and Lo, D. C. (1992). Having a blast with gene transfer. Curr. Biol. 2,31-32. Pecorino, L.T., Lo, C. D., and Brockes, J. P. (1994). Isoform-specific induction of a retinoidresponsive antigen after biolistic transfection of chimaeric retinoic acidlthyroid hormone receptors into a regenerating limb. Development 120,325-333. Pecorino, L.T.,Entwistle, A,, and Brockes, J. P. (1996). Activation of a single retinoic acid receptor isoform mediates proximodistal respecification. Curr. Biol. 6, 563-569. Peterson, R. L., Papenbrock, T., Davda, M. M., and Awgulewitsch, A. (1994). The murine Hoxc cluster contains five neighboring AbdB-related Hox genes that show unique spatially coordinated expression in posterior embryonic subregions. Mech. Dev.47,253-260. Popiela, H.(1976). Muscle satellite cells in urodele amphibians: Facilitated identification of satellite cells using ruthenium red staining. 1. Exp. Zool. 198,57-64. Poulin, M. L.,and Chiu, I. M. (1995). Re-programming of expression of the KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens). Dev. Dyn. 202,378-387. Poulin, M. L.,Patrie, K. M., Botelho, M. J.,Tassava, R. A., and Chiu, I. M. (1993). Heterogeneity in the expression of fibroblast growth factor receptors during limb regeneration in newts (Notophthalmus viridescens). Development 119,353-361. Powell, A. J., Gates, P. B., Wylie, D., Velloso, C. P., Brockes, J. P., and Jat, P. S. (1997). Immortalization of rodent embryo fibroblasts by a 3’ untranslated region from the newt limb blastema. Submitted for publication. Ragsdale, C. W., Jr., Petkovich, M., Gates, P. B., Chambon, P., and Brockes, J. P. (1989). Identification of a novel retinoic acid receptor in regenerative tissues of the newt. Nature (London) 341, 654-657. Ragsdale,C. W.,Jr.,Gates,P. B.,andBrockes,J.P.(1992a).Identification andexpressionpattern of a second isoform of the newt alpha retinoic acid receptor. Nucleic Acids Res. 20,5851. Ragsdale, C. W., Jr., Gates, P. B., Hill, D. S., and Brockes, J. P. (1992b). Delta retinoic acid receptor isoform 6 is distinguished by its exceptional N-terminal sequence and abundance in the limb regeneration blastema. Mech. Dev. 40,99-112. Reginelli, A.D., Wang, Y. Q., Sassoon, D., and Muneoka, K. (1995). Digit tip regeneration correlates with regions of Msxl (Hox-7)expression in fetal and newborn mice. Development 121, 1065-1076. Repesh, L. A., and Oberpriller, J. C. (1980). Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Norophthalmus viridescens. Am. 1.Anat. 159, 187-208. Repesh, L. A,, Fitzgerald, T. J., and Furtch, L. T. (1982). Changes in the distribution of fibronectin during limb regeneration in newts using immunocytochemistry. Differentiation 22,229-240.


47

Riddiford, L. M. (1960). Autoradiographic studies of tritiated thymidine infused into the blastema of the early regenerate in the adult newt, Triturus. J. Exp. 2001.144, 25-32. Robert, B., Sassoon, D., Jacq, B., Gehring, W., and Buckingham, M. (1989). Hox-7, a mouse homeobox gene with a novel pattern of expression during embryogenesis. EMBO J. 8,91-100. Robert, B., Lyons, G., Simandl, B. K., Kuroiwa, A., and Buckingham, M. (1991). The apical ectodermal ridge regulates Hox-7 and Hox-8 gene expression in developing chick limb buds. Genes Dev. 5,2363-2374. Saadi, A., Gallien, C. L., Guyot-Lenfant, M., and Chanoine, C. (1993). A new approach of urodele amphibian limb regeneration: Study of myosin isoforms and their control by thyroid hormone. Mech. Dev. 43,49-56. Salo, E., and Baguna, J. (1986). Stimulation of cellular proliferation and differentiation in the intact and regenerating planarian Dugesia ( G ) figrina by the neuropeptide substance P. J. EXP.2001.237,129-135. Salpeter, M., and Singer, M. (1960). The fine structure of mesenchymatous cells in the regenerating forelimb of the adult newt Triturus. Dev. B i d . 2,516-534. Saunders, J. W., Jr. (1948). The proximo-distal sequence of origin of the parts of chick wing and the role of the ectoderm. J . Exp. 2001.108,363-403. Savard, P., and Tremblay, M. (1995). Differential regulation of Hox C6 in the appendages of adult urodeles and anurans. J. Mol. Biol. 249,879-889. Savard, P., Gates, P. B., and Brockes, J. P. (1988). Position dependent expression of a homeobox gene transcript in relation to amphibian limb regeneration. EMBO J. 7,4275-4282. Scadding, S. R. (1989). Histological effects vitamin A on limb regeneration in the larval axolotl, Ambystoma mexicanum. Can. J. 2001.68, 159-167. Scadding, S . R., and Maden, M. (1986). Comparison of the effects of vitamin A on limb development and regeneration in the axolotl, Ambystoma mexicanum. J. Embryol. Exp. Morphol. 91,19-34. Scadding, S . R., and Maden, M. (1994). Retinoic acid gradients during limb regeneration. Dev. Biol. 162,608-617. Schilthuis, J. G., Gann, A. A,, and Brockes, J. P. (1993). Chimeric retinoic acidlthyroid hormone receptors implicate RAR-alpha 1 as mediating growth inhibition by retinoic acid. EMBO J. 12,3459-3466. Schmidt, A. J. (1968). “Cellular Biology of Vertebrate Regeneration and Repair,” pp. 420. Univ. of Chicago Press, Chicago. Scott, W. J., Jr., Walter, R., Tzimas, G., Sass, J. O., Nau, H., and Collins, M. D. (1994). Endogenous status of retinoids and their cytosolic binding proteins in limb buds of chick vs mouse embryos. Dev. Biol. 165,397-409. Sicard, R. E. (1985). “Regulation of Vertebrate Limb Regeneration,” pp. 185. Oxford Univ. Press, Oxford, UK. Simeone, A., Acampora, D., Pannese, M., D’Esposito, M., Stornaiuolo, A., Gulisano, M., Mallamaci, A., Kastury, K., Druck, T., and Huebner, K. (1994). Cloning and characterization of two members of the vertebrate Dlx gene family. Proc. Nafl.Acad. Sci. USA 91,2250-2254. Simon, H. G., Kittappa, R., Khan, P. A,, Tsilfidis, C., Liversage, R. A., and Oppenheimer, S. (1997). A novel family of T-box genes in urodele amphibian limb development and regeneration: Candidate genes involved in vertebrate forelimb/hindlimb patterning. Development W, 1355-1366. Simon, H. G., and Tabin, C. J. (1993). Analysis of Hox-4.5 and Hox-3.6 expression during newt limb regeneration: Differential regulation of paralogous Hox genes suggest different roles for members of different Hox clusters. Development 111, 1397-1407. Simon, H.-G.,Nelson, C., Goff, D., Laufer, E., Morgan, B., and Tabin, C. J. (1995). Differential expression of myogenic regulator genes and msx-1 during dedifferentiation of regenerating amphibian limbs. Dev. Dyn. 202, 1-12. Singer, M. (1952). The influence of the nerve in regeneration of the amphibian extremity. Q. Rev. Biol. 27, 169-200.

48

JACQUELINE GERAUDIE AND PATRlZlA FERRE3lI

Singer, M. (1974). Neurotrophic control of limb regeneration in the newt. Ann. N. Y. Acud. Sci. 228,308-322. Singer, M., and Caston, J. D. (1972). Neurotrophic dependence of macromolecular synthesis in the early limb regenerate of the newt, Triturus. J. Embryol. Exp. Morphol. 28,l-11. Singer,M., and Craven, L. (1948).The growth and morphogenesis of the regenerating forelimb of adult Triturus following denervation at various stages of development. 1. Exp. Zool. 108,279-308. Singer, M., and GCraudie, J. (1991). The neurotrophic phenomenon: Its history during limb regeneration in the newt. In “A History of Regeneration Research. Milestones in the Evolution of a Science” (C. E. Dinsmore, Ed.), pp. 101-112. Cambridge Univ. Press, Cambridge, UK. Singer, M., and Salpeter M. M. (1961). Regeneration in vertebrates: The role of the wound epithelium. In “Growth of the Living Systems” (M. X. Zarrow, Ed.), pp. 277-311. Basic Books, New York. Singer, M., Maier, C. E., and McNutt, W. S. (1976). Neurotrophic activities of brain extracts in forelimb regeneration of the urodele, Trimrus. J. Exp. 2001.196,131-150. Small, K. M., and Potter, S. S. (1993). Homeotic transformations and limb defects in Hox A l l mutant mice. Genes Dev. 7,2318-2328. Smith, G. N., Toole, B. P., and Gross, J. (1975). Hyaluronidase activity and glycosaminoglycan synthesis in the amputated newt limb: Comparison of denervated, nonregenerating limbs with regenerates. Dev. B i d . 43,221-232. Smith, M. J., and Globus, M. (1989). Multiple interactions in juxtaposed monolayers of amphibian neuronal epidermal and mesodermal limb blastema cells. In Virro Cell Dev. Biol. 25, 849-856. Smith, M. J., Globus, M., and Vethamany-Globus, S. (1995). Nerve extracts and substance P activate the phosphatidylinositol signaling pathway and mitogenesis in newt forelimb regenerates. Dev. Biol. 167,239-251. Song, K., Wang, Y., and Sassoon, D. (1992). Expression of Hox-7.1 in myoblasts inhibits terminal differentiation and induces cell transformation. Nature 360,477-481. Sordino, P., Vanderhoeven, F., and Duboule, D. (1995). Hox gene expression in teleost fins and the origin of vertebrate digits. Nature (London) 375,678-681. Spallanzani, L. (1768). Prodromo di un opera da imprimersi sopra la riproduzioni animal. Giovanni Montanari, Modena. “An essay on Animal Reproduction” (Translated in 1769 by M. Maty). T. Becket and DeHondt, London. Stocum, D. L. (1985). The role of the skin in urodele limb regeneration. In “Regulation of Vertebrate Limb Regeneration” (R. E. Sicard, Ed.), pp. 32-53. Oxford Univ. Press, Oxford, UK. Stocum, D. L. (1991). Retinoic acid and limb regeneration. Sem. Dev. Biol. 2, 199-210. Stocum, D. L. (1995). “Wound Repair, Regeneration and Artificial Tissues.” R. G . Landes, Austin, TX. Stocum, D. L., and Maden, M. (1990). Regenerating limbs. Methods Enzymol. 190 (Part B), 189-200. Stratford, T., Horton, C., and Maden, M. (1996). Retinoic acid is required for the initiation of outgrowth in the chick limb bud. Curr. B i d . 6, 1124-1133. Tabin, C. J. (1989). Isolation of potential vertebrate limb-identity genes. Development 105, 813-820. Tanaka, E. M., Gann, A. A. F., Gates, P. B., and Brockes, J. P. (1997). Newt myotubes re-enter the cell cycle by phosphorylation of the retinoblastoma protein. J. Cell Biol. 136, 155-165. Tassava, R. A. (1969). Hormonal and nutritional requirements for limb regeneration and survival of adult newts. J. Exp. Zool. 170,33-54. Tassava, R. A. (1983). Limb regeneration to digit stages occurs in well-fed adult newts after hypophysectomy. J. Exp. 2001.225,433-441.


49

Tassava, R. A. (1992). Retinoic acid enhances monoclonal antibody WE3 reactivity in the regenerate epithelium of the adult newt. 1. Morphol. 213, 159-169. Tassava, R. A., and Acton, R. D. (1989). Distribution of a wound epithelium antigen in embryonic tissues of newts and salamanders. Ohio J. Sci. 89, 12-15. Tassava, R. A., and Mescher, A. L. (1975). The role of injury, nerves, and wound epidermis during the initiation of amphibian limb regeneration. Differentiation 4, 23-24. Tassava, R. A., and Olsen, C. L. (1985). Neurotrophic influences on cellular proliferation in urodele limb regeneration: In vivo experiments. In “Regulation of Vertebrate Limb Regeneration” (R. E. Sicard, Ed.), pp. 81-91. Oxford Univ. Press, Oxford, UK. Tassava, R. A., Johnson-Wint, B., and Gross, J. (1986). Regenerate epithelium and skin glands of the adult newt react to the same monoclonal antibody. J. Exp. 2001.239,229-240. Tassava, R. A., Castilla, M., Arsanto, J. P., and Thouveny, Y. (1993). The wound epithelium of the regenerating limbs of Pleurodeles waltl and Notophthalmus viridescens: Studies with mAbs WE3 and WE4, phalloidin, and DNase 1. J. Exp. Zool. 267, 180-187. Taylor, G. P., Anderson, R., Reginelli, A. D., and Muneoka, K. (1994). FGF-2 induces regeneration of the chick limb bud. Dev. Biol. 163, 282-284. Tchang, F., Gusse, M., Soussi, T., and MBchali, M. (1993). Stabilization and expression of high levels of p53 during early development in Xenopus laevis. Dev. Biol. 159,163-172. Thaller, C., and Eichele, G. (1990). Isolation of 3,4-didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud. Nature (London) 345, 815-819. Thaller, C., Hofmann, C., and Eichele, G. (1993). 9-cis-retinoic acid, a potent inducer of digit pattern duplications in the chick wing bud. Development 118,957-965. Thoms, S . D., and Stocum, D. L. (1984). Retinoic acid-induced pattern duplication in regenerating urodele limbs. Dev. Biol. 103, 319-328. Thornton, C. S. (1968). Amphibian limb regeneration. In “Advances in Morphogenesis” (M. Abercrombie, J. Brachet, and T. J. King, Eds.), Vol. 7, pp. 205-249. Academic Press, New York. Thornton, C. S. (1970). Amphibian limb regeneration and its relation to nerves. Am. Zool. 10,113-118. Tickle, C. (1991). Retinoic acid and chick limb development. Development (Suppl. l), 113-121. Tickle, C., and Eichele, G. (1994). Vertebrate limb development. Annu. Rev. Cell. Biol. 10, 121-152. Tickle, C., Alberts, B. M., Wolpert, L., and Lee, J. (1982). Local application of retinoic acid to the limb bud mimics the action of the polarizing region. Nafure (London) 2%, 564-565. Timpl, R. (1989). Structure and activity of basement membrane proteins. Eur. J. Biochem. 180,487-502. Todd, T. J . (1823). On the process of reproduction of the members of the aquatic salamander. Q. J. Sci. 16, 84-96. Tomasek, J. J., Mazurkiewicz, J. E., and Newman, S. A. (1982). Non-uniform distribution of fibronectin during avian limb development. Dev. Biol. 90, 118-126. Toole, B. P. (1973). Hyaluronate and hyaluronidase in morphogenesis and differentiation. Am. 2001.W, 1061-1065. Toole, B. P., and Gross, J. (1971). The extracellular matrix of the regenerating newt limb: Synthesis and removal of hyaluronate prior to differentiation. Dev. Biol. 25, 57-77. Toole, B. P., Goldberg, R. L., Chi-Rosso, G., Underhill, C. B., and Orkin, R. W. (1984). Hyaluronate cell-interactions. In “The Role of Extracellular Matrix in Development” (R. L. Trelstad, Ed.), A. R. Liss, New York. Tschumi, P. A. (1957). The growth of the hind limb bud of Xenopus Iaevb and its dependence upon the epidermis. J. Anat. 91,149-173. Tsonis, P. A. (1991). Amphibian limb regeneration. In vivo 5, 541-550. Tsonis, P. A. (1996). “Amphibian Limb Regeneration.” Cambridge Univ. Press, Cambridge, UK.

50

JACQUELINE GERAUDIE AND PATRlZlA FERREllI

Tsonis, P. A,, Washabaugh, C. H., and del Rio-Tsonis, K. (1994). Morphogenetic effects of 9-cis-retinoic acid on the regenerating limbs of the axolotl. Roux’ Arch. Dev. Biol. 203, 230-234. Vethamany-Globus, S . (1987). Hormone action in newt limb regeneration: Insulin and endorphins. Biochem. Cell Biol. 65, 730-738. Viviano, C. M., Horton, C. E., Maden, M., and Brockes, J. P. (1995). Synthesis and release of 9-cis retinoic acid by the urodele wound epidermis. Development 121,3753-3762. Vogel, A., Roberts-Clarke, D., and Niswander, L. (1995). Effect of FGF on gene expression in chick limb bud cells in vivo and in vim. Dev. Biol. 171, 507-520. Wallace, H. (1981). “Vertebrate Limb Regeneration.” Wiley, Chichester, UK. Wanek, N., Gardiner, D. M., Muneoka, K., and Bryant, S . V. (1991). Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud. Nature (London) 380, 81-83. Wei, L. N., and Chen, G. J. (1991). Production and analyses of transgenic mice with ectopic expression of cellular retinoic acid-binding protein. Biochem. Biophys. Res. Commun. 179,210-216. Wei, Y., Yang, E. V., Klatt, K. P., and Tassava, R. A. (1995). Monoclonal antibody MT2 identifies the urodele a1 chain of Type XI1 collagen, a developmentally regulated extracellular matrix protein in regenerating newt limbs. Dev. Biol. 168, 503-513. West, D. C., Hampson, I. N., Arnold, F., and Kumar, S . (1985). Angiogenesis induced by degradation products of hyaluronic acid. Science 288, 1324-1326. Yang, E. V., Shima, D. T., and Tassava, R. A. (1992). Monoclonal antibody ST1 identifies an antigen that is abundant in the axolotl and newt limb stump but is absent from the undifferentiated regenerate. J. Exp. Zool. 264, 337-350. Yang, E. V., and Bryant, S . V. (1994). Developmental regulation of a matrix metalloproteinase during regeneration of axolotl appendages. Dev. B i d . 166,696-703. Yokouchi, Y., Nakazato, S., Yamamoto, M., Goto, Y., Kameda, T., Iba, H., and Kuroiwa, A . (1995). Misexpression of Hoxa-I3 induces cartilage homeotic transformation and changes cell adhesiveness in chick limb buds. Genes Dev. 9, 2509-2522. Zenjari, C., Boilly-Marer, Y., Desbiens, X., Oudghir, M., Hondermarck, H., and BoiIIy, B. (1996). Experimental evidence for FGF-1 control of blastema cell proliferation during limb . Biol. 40, 965-971. regeneration of the amphibian Pleurodeles waltl. Int. .IDev.

Biochemistry of the Extracellular Matrix of Volvox Manfred Sumper and Armin Hallmann Lehrstuhl Biochemie I, Universitat Regensburg, D-93053 Regensburg, Germany

The volvocine algae range in complexity from unicellular Cblamydomonas to multicellular organisms in the genus Volvox. The transition from unicellularity to multicellularity in the Volvocales is a recent event in evolution. Thus, these organisms provide a unique opportunity for exploring the development of a complex extracellular matrix (ECM) from the cell wall of a unicellular ancestor. The ECM of Volvox is divided into four main zones: The flagellar, boundary, cellular, and deep zones. Each zone is defined by ultrastructure and by characteristic ECM glycoproteins. Voivox ECM is modified under developmental control or in response to external stimuli, like the sexinducing pheromone or stress factors. The structures of more than 10 ECM glycoproteins from a single species of Volvox are now known in molecular detail and are compared to other algal and plant cell walllECM glycoproteins. Although usually classified as hydroxyproline-rich glycoproteins, the striking feature of all algal ECM glycoproteins is a modular composition. Rod-shaped hydroxyproline-rich modules are combined with hydroxyproline-free domains that meet the multiple functional requirements of a complex ECM. The algal ECM provides another example of the combinatorial advantage of shuffling modules that is so evident in the evolution of the metazoan ECMs. KEY WORDS: Extracellular matrix, Green algae, Volvocales, Volvox, Cell wall, Glycoproteins, Plant development.

1. Introduction The extracellular matrix (ECM) of a multicellular organism is a complex organelle that serves structural as well as nonstructural functions. It provides a scaffolding to create and to stabilize the physical structure of tissues. In addition, the ECM mediates many developmental responses of cells includInfemnfronal Review of Cyrology, Val. 180 0074-76%/98 $25.00

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Copyright D 1998 by Academic Press. All rights of reproduction in any form reserved.

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ing regulation of growth and differentiation, wound repair, and pathogen defense. It is now recognized that common principles operate in the design of ECMs in plants and animals: extended and usually crosslinked glycoproteins with repeating sequence motives provide tensile strength, and polypeptides organized in a modular fashion have been evolved to allow both specific targeting and controlled enzymatic actions within the ECM network. The development of a complex ECM from a simple cell wall was one of the prerequisites to promote the evolutionary transition from unicellularity to multicellularity. A family of organisms collectively referred to as the Volvocaceae provides the unique opportunity for exploring molecular genetic pathways that led from unicellularity to multicellularity. The volvocine algae range in complexity from unicellular Chlamydomonus through colonial genera (such as Gonium, Pandorim, and Eudorina) to multicellular organisms, with differentiated cells and complete division of labor, in the genus Volvox. The multicellular members in the order contain 2" Chlamydornonas-like cells (the maximum value of n being a species character) held together in a predictable pattern by a hydroxyproline-rich ECM. Volvox is among the simplest multicellular organisms and yet it shares many features that characterize the life cycles and developmental histories of much more complex organisms. The rRNA sequence data indicate that the transition from unicellularity to multicellularity within the volvocine algae probably happened within the past 50-75 million years and is therefore a very recent event in evolution (Rausch et al., 1989; Larson et al., 1992). The changes within genomes required to achieve this fundamental event may not yet be obscured by genetic drift and, therefore, it should be possible to analyze the evolutionary sequence from an ancestor resembling Chlamydomonas to the multicellular members of the Volvocales. Recent progress in the development of nuclear transformation (Kindle, 1990; Schiedlmeier et al., 1994), the introduction of selectable markers (Kindle et al., 1989; Debuchy et al., 1989; Stevens et af., 1996; Schiedlmeier et al., 1994; Hallmann and Sumper, 1996) and reporter genes (Davies et al., 1992; Hallmann and Sumper, 1994b), and gene replacement by homologous recombination in Chlamydomonas (Sodeinde and Kindle, 1993; Gumpel et al., 1994; Nelson and Lefebvre, 1995) as well as in Volvox carteri (Hallmann et al., 1997) has made it possible to apply the powerful strategies of molecular genetics. Cell walls and the ECM of the volvocine algae (Miller et al., 1974) are entirely assembled from glycoproteins with a high content of hydroxyproline (hydroxyproline-rich glycoproteins, HRGPs). The walls lack cellulose, hemicelluloses, pectins, and lignin (Adair et al., 1987). HRGPs also represent a main constituent of higher plant ECMs and much work has been done to analyze the structures of these proteins (Cooper et al., 1984; ShoWalter and Varner, 1989; Varner and Lin, 1989; Kieliszewski and Lamport,

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THE Vo/vox ECM

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1994). However, there are few examples in the literature where multiple HRGPs have been examined in molecular detail from a single species or from closely related species. This would allow a more integrated approach to elucidate the structure, assembly, and function of ECM proteins. In recent years, this type of analysis has been initiated with volvocine algae and the emerging picture will be summarized in this review. V. carteri f. nagariensis has become the standard subject of Volvox research, owing to its genetic accessibility. v.carteri is composed of only two cell types: 2000-4000 biflagellate Chlurnydornonas-like somatic cells are arranged in a monolayer at the surface of a hollow sphere and 16 much larger reproductive cells (“gonidia”) lie just below the somatic cell sheet (Starr, 1969) (Fig. 1). Eleven or twelve rapid and synchronous cleavage divisions of a gonidium generate all the cells of an adult organism. An asymmetric division of 16 cells at the stage of the 32-cell embryo delineates 16 new reproductive cells from the somatic cell initials which continue cleavage. At the termination of cell divisions, the embryo enters the process of inversion, thereby turning the embryo inside-out. After inversion, the somatic cells begin to secrete ECM material, causing each cell to move apart from its neighbors. The organism now grows in size but not cell

FIG. 1 Asexual spheroid of Vdvox carferi containing 16 large gonidia (asexual reproductive cells), which just have initiated embryogenesis. The small dots represent 2000-4000 terminally differentiated somatic cells. Magnification, 80X.

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number. When the daughter spheroids are about a quarter of their final size they are released from the parent organism through holes formed by local enzymatic degradation of the ECM. A number of previous reviews describe main topics of Volvox biology in detail: a perfect introduction of the organism (Starr, 1970), genetic and biochemical aspects (Kirk and Harper, 1986), ontogenetic and phylogenetic aspects (Kirk, 1988; Schmitt et al., 1992), as well as morphological and physiological aspects (Desnitski, 1995; Nozaki, 1996).

II. Ultrastructure of the Volvox ECM The ECM of representatives of the genus Volvox consists of a large number of anatomically distinct structures arranged in a defined spatial pattern. Based on light- and electron-microscopic observations, David Kirk and co1986) proposed a highly useful system of nomenclature workers (Kirk et d., that greatly facilitates discussion of comparative morphology and phylogeny of the ECM. In this nomenclature the ECM is divided into four main zones: the flagellar zone (FZ), the boundary zone (BZ), the cellular zone (CZ), and the deep zone (DZ). In the stylized drawings of Figs. 2 and 3 these

cz

DZ

FIG. 2 Highly stylized cross section of a Volvox carteri spheroid emphasizing the major compartments of the ECM. CZ, cellular zone; DZ, deep zone; G, gonidium; S, somatic cell. For details, see Fig. 3.

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BZ

CZ

FIG. 3 Stylized drawing of a portion of a Volvox carteri spheroid illustrating ECM zones and subzones according to Kirk er al. (1986). FZ, flagellar zone; BZ, boundary zone; CZ, cellular zone. Subzones are as described in the text.

zones are defined in a cross section of V. carteri, the species from which most structural data are available. Each of these major zones is subdivided in a hierarchical fashion to define observable substructures. This terminology is entirely based on morphological criteria and therefore does not imply any similarities or differences with respect to the biochemical properties among the various compartments. The definitions of zones and subzones given by Kirk et al. (1986) are as follows.

A. The Flagellar Zone The FZ (Fig. 3) includes all ECM specializations seen only on or in the immediate vicinity of the flagellum. The FZ is further subdivided into three compartments: FZ1 contains all coatings and appendages of the flagellar

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membrane. FZ2 includes all components that create the flagellar collar. FZ3 describes modifications of the boundary and, or, cellular zones in the region traversed by the flagellum.

B. The Boundary Zone The BZ (Fig. 3) includes those components of the ECM that, except in periflagellar regions, appear to be continuous over the surface of the organism but are not structurally continuous with deeper layers. Again, three subzones are distinguishable. Because BZ2 (tripartite layer) is highly conserved in all Volvocales examined to date, it was used as a reference point in numbering the components of the boundary zone. The tripartite layer corresponds to layers W2-W6 in the ECM terminology of Roberts (1974) and contains the crystalline layer. Therefore, the subzones BZ1 and BZ3 are those components external (BZ1) and internal (BZ3) to BZ2. C. The Cellular Zone

The CZ (Figs. 2 and 3) includes components lying internal to the boundary zone and exhibiting specializations around individual cells (in unicellular Volvocales the boundary zone and cellular zone are synonymous). The coherent meshwork of ECM filaments attached to the plasmalemma of each cell body is denoted as CZ1. CZ2 describes the relatively amorphous components filling all portions of the CZ not occupied by more highly structured components. The distinct fibrous material that creates chambers around individual cells is defined as CZ3.

D. The Deep Zone The DZ (Fig. 2) contains all ECM components internal to the cellular zone. More specifically, DZ1 is a fibrous layer enclosing DZ2. DZ2 appears as a relatively amorphous component filling the deepest regions of the spheroid and is by far the largest region of the spheroid in most Volvox species.

111. Biochemical Characterization of ECM Components

To some extent, the four ECM zones defined by morphological characteristics also behave as structural units during biochemical fractionation. For


57

instance, treatment of Volvox spheroids with chaotropic agents selectively extracts main parts of the BZ. On the other hand, a very mild mechanical treatment that introduces slits into Volvox spheroids causes selective liberation of most of the highly viscous material of the DZ. Treatment of Volvox spheroids with hot SDS removes all soluble molecules, but it leaves the ECM appearing largely intact. Colorless Volvox “ghosts” are produced which mainly consist of covalently crosslinked components of the ECM. These “ghosts” exhibit typical morphological features of CZ. Finally, a number of procedures are known that selectively remove all the flagella from a Volvox spheroid (Snell, 1983).

A. Boundary and Flagellar Zone All members of the algal order Volvocales have cell walls containing a crystalline layer overlying an amorphous inner layer (Goodenough and Heuser, 1985; Roberts et af., 1985). From studies of a large number of Volvocales species, Roberts and co-workers concluded that the cell walls of these algae fall into four major structural classes (Roberts, 1974; Roberts et al., 1982). Volvox and some other colonial Volvocales have a wall structure shared by Chlamydomonas reinhardtii (Roberts et al,, 1982; Roberts et al., 1985). In C. reinhardtii, the crystalline layer is composed of a number of hydroxyproline-rich glycoproteins that may be disassembled by chaotropic agents and recrystallized in vitro (Hills et al., 1975; Catt et al., 1976, 1978; Roberts, 1974, 1979; Roberts et al., 1980; Goodenough et af., 1986). As pointed out by Kirk et al. (1986), it is the boundary zone (tripartite layer, BZ2) corresponding to layers W2-W6 of Chlamydomonas that is closely related in both these organisms, whereas the inner zones of the cell walls/ECMs diverge and define species specificity. Previous work on the species-specific sensitivity of the inner wall structures to lytic enzymes provided an experimental basis for this generalization (Claes, 1971; Schlosser, 1976,1984;Matsuda etal., 1987).Proof for the evolutionary relationship between C. reinhardtii and V. carteri boundary zones was obtained by performing interspecific reconstitutions of cell walls. Adair et al. (1987) solubilized the crystalline layers of V. carteri and demonstrated that the insoluble layers of C. reinhardtii allowed nucleated assembly of the solubilized BZ2 components to yield hybrid walls. Vice versa, V . carteri can nucleate the assembly of C. reinhardtii crystalline layer, but not that from Chlamydomonas eugametos. Therefore, it is plausible to expect homologies among the glycoproteins of the boundary zones. Morphological and some biochemical data are available for the corresponding components of the crystalline layer of C. reinhardtii. Four glycoproteins are extractable; three are HRGPs (GP1, GP2, and GP3), and one is glycine-rich (GP 1.5) (Good-

58


enough et al., 1986; Goodenough and Heuser, 1988). Partial sequences deduced from cDNA clones have been published for GP1 and GP2 (Adair and Apt, 1990; data library accession nos. M58496 and M58597). Strangely, the reading frame used for prediction of the amino acid sequence of GP2 contains two TAG stop codons and therefore requires reexamination. Peptide-mapping studies, immunological cross-reactivity, and electronmicroscopic data support the presence of GP2 homologues in V. curteri (Adair and Appel, 1989; Goodenough and Heuser, 1988). As yet, only a single gene encoding a boundary zone glycoprotein from Volvox (ISG, see Section IV.A.2) has been cloned and characterized.

6. Cellular Zone As mentioned above, extraction of asexually growing Volvox spheroids with 1%SDS containing 0.5 M NaCl at 95°C solubilizes all cytoplasmic proteins and produces colorless Volvox “ghosts” that still exhibit features characteristic of the ECM morphology. In particular, the honeycomb-like cellular compartments (CZ3) remain intact, indicating the existence of covalent crosslinks in the coherent network constituting this ECM subzone. Such an insoluble polymer poses particular problems for a biochemical characterization of the building blocks involved. Therefore, it was important to realize that insoluble “ghosts” are nearly quantitatively converted to soluble components if treated with anhydrous hydrogen fluoride (HF). This procedure is known to cleave 0-glycosidic linkages without affecting peptide bonds (Mort and Lamport, 1977). Thus, HF cleavage enables the characterization of all the polypeptides present in the insoluble fraction of the Volvox ECM. SDS-PAGE analysis of HF-solubilized “ghosts” exhibits two main polypeptides with molecular masses around 60 and 70 kDa that were identified as being derived from the (deglycosylated) glycoproteins SSG 185 and pherophorin I (Fig. 4). 1. The ECM Glycoprotein SSG 185

The sulfated glycoprotein SSG 185has been characterized as the monomeric precursor of the CZ3 substructure using immunological techniques (Wenzl el al., 1984; Ertl et al., 1989).The primary structure of the SSG 185 polypeptide chain has been derived from cDNA and genomic DNA. A central domain of the protein, 80 amino acid residues long, consists almost exclusively of hydroxyproline residues. Most of these hydroxyproline residues are glycosylated with 1,Zlinked di- and tri-arabinosides. Proteolysis of SSG 185 results in a large, completely resistant 145-kDa fragment with a high content of hydroxyproline that represents this central domain of SSG 185.

59


Coomassie stain

,SSG 185 antibody

FIG.4 Main components of the SDS-insoluble Volvox ECM. Volvox “ghosts,” representing the SDS-insoluble ECM, were solubilized by treatment with anhydrous hydrogen fluoride at o”C, loaded onto a SDS-polyacrylamide gel, and transferred by Western blotting onto a membrane. Proteins (deglycosylated) were stained with Coomassie blue (“Coomassie stuin”). Immune detection was performed with a monoclonal antibody directed against pherophorin I (“Pherophorin 1 antibody”) and a polyclonal antibody directed agains SSG 185 (“SSG 185 antibody”). Both times the second antibody was an anti-IgG alkaline phosphatase conjugate.

Most probably, the secondary structure of such a domain is the polyproline I1 helix conformation. This is the most extended helix formed by polypeptides with three residues per turn and a pitch of 0.94 nm. As revealed by EM, the central domain of SSG 185 indeed shows a rod-shaped morphology (Ertl et aZ., 1989).Attached to this central rod is a 21-nm-longpolysaccharide with highly acidic properties. This polysaccharide consists of a 1,3-linked polymannan backbone and each mannose unit carries at position 6 a 1,2linked di-arabinoside side chain (Fig. 5). The striking feature of this saccharide is its extreme degree of sulfation. Each arabinose carries two sulfate groups and the mannoses of the backbone are sulfated at position 4. Remarkably, the degree of sulfation in this SSG 185 polysaccharide is found to change under developmental control (see Section IV.B.l). The high density of negative charges should exert a strong influence on the overall physicochemical properties of the C Z . Cations and positively charged ECM proteins should bind to this strong cation exchanger. Incorporation studies with radioactive phosphate led to the detection of another unique structural element within the central rod-shaped domain of SSG 185. A phosphodiester bridging two arabinose residues via their 5positions was isolated from hydrolysates of polymeric SSG 185 (Holst et aZ., 1989).The possible function of this phosphodiester as a crosslink within the ECM is discussed in Section 1V.D.

60


1

I-

O$O-

5

FIG. 5 Proposed chemical structure of the sulfated saccharide covalently linked to SSG 185.

The N-terminal (about 230 amino acid residues) as well as the C-terminal (about 165 amino acid residues) extensions of the central hydroxyprolinerich domain exhibit no unusual amino acid preference except for cysteine. A sequence comparison of the N- and C-terminal domains of SSG 185 remarkably reveals a significant degree of homology (24% identity and 54% similarity over 206 residues). SSG 185 oligomers obtained by limited proteolytic degradation of polymeric SSG 185 were studied by electron microscopy. From the corresponding EM pictures, an overlapping staggered assembly of the monomeric units has been deduced. Monoclonal antibodies raised against the protease-resistant 145-kDa glycopeptide were shown to prevent in vivo expansion of young Volvox spheroids, presumably by inhibiting the insertion and polymerization of SSG 185 monomers into the CZ3 zone (Ertl et al., 1989). The in viva kinetics of SSG 185 polymerization could be followed by pulse-chase labeling experiments with radioactive sulfate (Wenzl et al., 1984). 2. Pherophorin I

The sex-inducing pheromone of Volvox is a glycoprotein. The pherophorins, a newly discovered ECM protein family, are multidomain glycoproteins with homology to this sex-inducing pheromone in their C-terminal domain (Sumper et al., 1993). Recent evidence suggests that pherophorins represent a major family of ECM glycoproteins with amazingly different properties with respect of localization within the ECM. Pherophorins I and I l l are constitutively expressed in asexually growing Volvox spheroids (God1 et al., 1995). Besides SSG 185, pherophorin I is a main constituent of the C Z


61

(Fig. 4). In contrast to SSG 185, pherophorins I and I11 can be selectively extracted from Volvox “ghosts” by treatment with EDTA, indicating a noncovalent fixation of pherophorins within the CZ. Mg2+or Ca2+bridges may be involved in this interaction. The primary structure of pherophorin I was deduced from cDNA sequence analysis. A proline-rich (most probably hydroxyproline-rich) stretch of about 23 amino acid residues around position 170 indicates the existence of two well-separated domains in this polypeptide. Both these domains exhibit no unusual amino acid preference. Surprisingly, the N-terminal domain shares 21% identity and 56% similarity over 220 residues with the corresponding N-terminal domain of SSG 185. The biochemistry and ECM localizations of other members of the pherophorin family that are expressed under the control of the sex-inducing pheromone are described below (Section IV.B.2).

C. Deep Zone Although the constituents of the deep zone can easily be obtained, until recently very little was known about the biochemistry of this compartment. An early paper (Gilles et al., 1983) described an extracellular glycoprotein (290 kDa) of the deep zone that was reported to be phosphorylated on serine residues (“pp290”). Upon application of sex-inducing pheromone, pp290 was found to be replaced by two other phosphorylated proteins (“pp240” and “pp120”). A recently initiated biochemical analysis could not confirm the originally published data. These glycoproteins are not phosphoproteins, rather they bear their phosphate residues on saccharide chains. Again, the phosphodiester arabinose-5-phospho-5’-arabinose originally characterized in SSG 185 could be identified as a main structural element of pp290 and pp240 (Wenzl and Sumper, unpublished results). Therefore, we replace the term pp290 with HRGP-290 for the following reasons: The deglycosylated polypeptide turned out to be nearly resistant to proteases like pronase or subtilisin. Amino acid sequence analysis by automated Edman degradation yielded only hydroxyproline signals over 40 cycles. Amino acid analysis exhibited nearly exclusively hydroxyproline (Gorlach, 1994). Unfortunately, this strange composition excludes the cloning of the corresponding gene on the basis of amino acid sequence data. HRGP-290 contains the neutral sugars arabinose and galactose in a 1: 1 ratio, and permethylation analysis exhibited a terminally bound furanosidic galactose and 1,2,5-substituted arabinose as main products (Wenzl, Riegel, and Sumper, unpublished results). Figure 6 shows the composition of a deep zone extract after pulse labeling with [14C]hydrogencarbonate and [33P]phosphate,respectively. Relatively few labeled components are seen

62

MANFRED SUMPER AND ARMlN HALLMANN 14c 33p

I

I

kDa 290 b

HRGP-290

240 b

120 w

FIG. 6 Composition of a deep zone (DZ) extract after pulse labeling with [‘“Clhydrogen carbonate (‘“‘C”) and [33P]phosphate (“33P’).Fluorograrn of an SDS-polyacrylarnide gel. HRGP-290 represents a main constituent of the DZ.

and HRGP-290 represents a main constituent of the deep zone. Upon a chase, HRGP-290 turns out to be the monomeric precursor of an insoluble polymer. HRGP-290 is a likely precursor for the fibrous polymer found in the deep zone and defined as subzone DZ1. An early report by Mitchell (1980) described the existence of a polyhydroxyproline in Volvox. Possibly, these early data were derived from HRGP-290. Wounding of Volvox spheroids (see Section IV.C.2) induces biosynthesis of a multidomain protease and a lysozymekhitinase and both these enzymes were identified as components of the deep zone. Another newly identified constituent of the deep zone is pherophorin-S (see Section IV.B.2.b).

IV. ECM Biogenesis and Remodeling Scanning electron microscopy of cleaving embryos gave no evidence of any extracellular matrix in the vicinity of embryonic cells (Green and Kirk, 1981). Therefore, it was concluded that embryogenesis is the only stage of the life cycle during which Volvox cells appear to be totally devoid of any extracellular matrix (Kirk and Harper, 1986). At the end of embryogenesis, shortly after inversion, the close contacts between blastomeres are broken, permitting cells to draw away from one another as the young spheroid


63

rapidly expands by deposition of ECM material. It is this stage of development at which massive production of ECM molecules and their assembly are initiated. Uncleaved gonidia as well as early embryos can easily be removed from the mother spheroid without affecting their developmental program (Starr, 1969,1970). This fact is an ideal prerequisite to resolve by pulse-chase labeling experiments the stage dependence of production and kinetics of assembly for individual ECM proteins.

A. Embryogenesis

1. Algal Cell Adhesion Molecule (Algal CAM) Proof that plants possess homologues of animal adhesion proteins has been lacking for a long time. The blastomeres of a developing Volvox embryo establish close contacts with their neighbors in a precisely predictable pattern. But even for Volvox embryos, it was concluded from electronmicroscopic investigations that embryonic cells of Volvox are exclusively linked by cytoplasmic bridges (Green and Kirk, 1981; Kirk and Harper, 1986) and do not possess cell-cell contacts mediated by cell adhesion molecules or an ECM. Considering the known importance of animal cell adhesion molecules in developmental processes, the nature of cell-cell contacts of Volvox embryos was reinvestigated using a biochemical approach (Huber and Sumper, 1994). Monoclonal antibodies were raised against a crude membrane fraction from Volvox embryos. The resulting monoclonals were then screened for their capability to interfere in vivo with cell-cell contact formation during embryogenesis. Several monoclonals were found that were able to disrupt cell-cell contacts of the 4-cell embryo. Disruption of these cell-cell contacts results in a big hole in the center of a 4-cell embryo. Confocal laser scanning immunofluorescence microscopy localized the corresponding antigen at the cell-cell contacts of the developing embryo, as expected for a potential CAM. Therefore, this antigen was denoted Algal CAM. Algal CAM was purified, and amino acid sequence data allowed the cloning of its cDNA and genomic DNA. The deduced amino acid sequence reveals a multidomain structure of Algal CAM with unexpected homologies to known plant as well as animal protein families. N-terminally located is a proline-rich domain with Ser-Pro-Pro-Pro-Pro repeats diagnostic of the extensins from higher plant cell walls. This plant-specific domain is followed by two repeats with homology to fasciclin I, a cell adhesion molecule involved in the neuronal development of Drosophilu. The fasciclin I protein in Drosophilu is a homophilic cell adhesion molecule (Elkins et ul., 1990), expressed on the surface of a subset of fasciculating axons, and seems to be involved in growth cone extension and/or guidance (Zinn el

64


al., 1988). Fasciclin I is composed of four homologous repeats of about 150 amino acids each (Zinn et al., 1988). Algal CAM contains two copies of related repeats and, therefore, represents the first plant homologue of animal cell adhesion molecules. Different variants of Algal CAM are produced under developmental control by alternative splicing. The C-terminal part of Algal CAM can be replaced by a different amino acid sequence that is encoded by an additional exon. This alternative sequence may represent a glycosyl-phosphatidylinositol (GPI) anchor addition signal (reviewed by Englund, 1993). The potential GPI-anchored variant of Algal CAM is absent in early embryos and becomes detectable at or immediately after the differentiating cleavage (i.e., the transition from the 32- to the 64-cell embryo). Four-cell embryos treated with Algal CAM antibody undergo a lower number of cleavages and produce only a few reproductive cells. These observations suggest an important function of Algal CAM variants during Volvox embryogenesis and support an early model (Sumper, 1979) using properties of cell adhesion molecules to explain the main features of Volvox embryogenesis: in a slightly modified form, this model is able to offer a mechanism by which the embryo could realize arrival at the 32-cell stage. At this stage, the differentiating cell cleavage takes place in exactly 16 blastomeres. Algal CAM antibody given at late stages of embryogenesis does no longer disrupt cell-cell contacts, but causes a quite different phenotype: the antibody selectively inhibits the process of embryonic inversion. 2. The ECM Glycoprotein ISG An ECM protein (denoted as ISG) with remarkable properties was discovered by pulse-labeling studies with radioactive sulfate over the period of embryonic inversion (Wenzl and Sumper, 1982; Schlipfenbacher et al., 1986). ISG is a sulfated, extracellular glycoprotein synthesized for less than 10 min in inverting Volvox embryos as well as in inverting sperm cell packets. Subsequently, ISG has been characterized by studies of protein chemistry and electron microscopy. The primary structure of ISG has been derived from genomic DNA and cDNA (Ertl et al., 1992). ISG is composed of a globular and a rod-shaped domain. The rod-shaped domain is again related to the extensin family with numerous repeats of Ser-(Hyp),-6 motifs. Virtually all of the hydroxy amino acids in this region appear to be glycosylated, with the dominant sugars being arabinose and galactose. Deglycosylation reduces the apparent molecular mass of ISG from about 200 kDa to about 60 kDa. The N-terminally located globular domain exhibits no unusual amino acid composition. The length of the rod-shaped domain was determined by electron microscopy to be 57 nm. Purified ISG oligomerizes to star-like particles with a central knob and a variable number of the 57-nm arms.

BIOCHEMISTRY OF THE Vo/vox ECM

65

Although ISG is synthesized only during an extremely short period in the 48-h life cycle of Volvox, the mature glycoprotein remains stable for at least 24 h, as revealed by pulse-chase experiments with radioactive sulfate. Immunofluorescencemicroscopy, using polyclonal antibodies raised against the globular domain of ISG produced in Escherichia coli by recombinant DNA technology, localizes ISG in the flagellar tunnel region and in the boundary zone of the ECM (Ertl et al., 1992). A distinct feature at the Cterminal end of ISG is the occurrence of a cluster of five positively charged amino acid residues. A synthetic decapeptide matching this sequence disturbed the early stages of ECM biogenesis and assembly at concentrations M without affecting the viability of Volvox cells. This as low as 5 X observation suggests that ISG may participate in early processes that organize ECM assembly. Based on these results and on the remarkable phenotype exhibited by a temperature-sensitive flagellaless mutant (flgCII) studied by Huskey (1979), an interesting hypothesis for the function of ISG was articulated by David Kirk (personal communication). When the mutant f?gCll was held at the restrictive temperature during embryonic inversion and early postembryonic development and then shifted to the permissive temperature, spheroids developed flagella that appeared to beat normally, but the spheroids were incapable of any coordinated swimming behavior. It turned out that the cells of the spheroid were randomly oriented with respect to the anterior-posterior axis of the spheroid and thus flagella of different cells were working antagonistically rather than in a coordinated manner. Therefore, it is concluded that flagella must be present at the time when the ECM is first laid down in order for the cells to be locked into the correct position within the spheroid. A very important early stage in ECM construction, prior to the breakdown of embryonic cell-cell interactions (cytoplasmic bridges and/or cell-cell contacts), should therefore involve deposition of boundary-zone matrix material around the bases of the flagella, and also out over the rest of the surface of the inverted embryo. This would permanently trap all flagella and hence the cells, thereby fixing the cellular orientations that had been established during embryonic cleavages. ISG, the first ECM component of the postinversion embryo to be synthesized, is a likely candidate to serve this function. If so, ISG would also assist in nucleation and assembly of other ECM components. This proposed scenario would explain the observation that a peptide analogue of ISG applied at the early stages of ECM biogenesis has such a drastic effect on the organization of all parts of the ECM. The striking developmental control of ISG synthesis operates at the level of transcription. ISG mRNA is virtually absent at the very beginning of the inversion process. A high level is observed during inversion and, toward

66


the end of inversion, the level of ISG mRNA again significantly decreases (Ertl et a[., 1992). The characterization of Algal CAM and ISG proves the existence of ECM glycoproteins during both the early and the late phase of embryogenesis. Possibly, these “embryonic” ECM molecules are not yet organized in supramolecular networks and may therefore escape from microscopic detection during embryogenesis. A putative Chlamydomonas cell wall glycoprotein identified by its cDNA clone is related to the globular domain of ISG (Woessner et al., 1994). Remarkably, the rod-shaped domain with the typical Ser-(Pro)4-6repeats in ISG is replaced by a different rod-shaped element with (Ser-Pro), repeats in the Chlamydomonas homologue. B. Modifications under Developmental Control

1. Asexual Life Cycle: SSG 185 Pulse-labeling studies with radioactive sulfate at different developmental stages revealed the existence of variants of the ECM glycoprotein SSG 185 that are defined by different electrophoretic mobilities in SDS-PAGE. A given variant is reproducibly synthesized at a defined stage of development. For instance, at the time of early embryogenesis, the somatic cells of the mother spheroid synthesize a SSG 185 with a significantly larger apparent molecular mass compared to the variant produced at later stages of embryogenesis (Wenzl et al., 1984). The molecular basis for this difference resides in the sulfated polysaccharide attached to SSG 185. In particular, SSG 185 variants with a slower electrophoretic mobility lack the sulfate group at the C 4-positions in the polymannan backbone (Mengele, 1988). These modifications of the SSG 185 monomer remain conserved in the polymer, indicating structural modifications of the ECM at defined developmental stages. At present, the biological reason for this ECM modulation is unknown. An exciting recent discovery in plant ECM biology has been the demonstration that ECM molecules may influence cell fate in plants (Brownlee and Berger, 1995). It appears that information may be stored in the structure of ECM. Somatic cells of Volvox exhibit programmed cell death (Pommerville and Kochert, 1982) and this event is influenced by the development program: In sexual (egg-containing) organisms, which do not enter embryonic cleavages, programmed cell death is delayed by 96 h. It is tempting to speculate that ECM modifications might be involved in this cross-talk between asexual embryos (or eggs) and somatic cells. 2. Sexual Development

Although V. carteri reproduces asexually much of the time, in nature it reproduces sexually at least once each year. V. carteri lives in temporary


67

ponds that dry out in the heat of late summer. Asexual Volvox algae would die in minutes once the pond dried out, but V. carteri is able to escape this catastrophe by switching to the sexual life cycle shortly before the pond dries up, producing dormant zygotes that survive the drought of late summer and the cold of winter. When rain fills the ponds in spring, the zygotes hatch out to establish a new generation of asexually reproducing individuals. The stimulus for switching from the asexual to the sexual mode of reproduction in V. carteri is known to be a sex-inducing pheromone, a 32-kDa glycoprotein (Starr, 1970; Tschochner et al., 1987; Mages et al., 1988). This pheromone is one of the most potent biological effector molecules known. It triggers sexual development of gonidia at concentrations as low as M and initiates a modified developmental program that results in the production of eggs or sperm packets, depending on the genetic sex of the individual. What is the source of this sex-inducing pheromone and how do these simple organisms predict the coming of unfavorable conditions to produce a sexual generation just in time? Kirk and Kirk (1986) found a simple explanation: the sexual cycle is initiated by a heat shock that causes the somatic cells of the asexual Volvox spheroid to produce the sex-inducing pheromone. The level of pheromone is then further amplified by the ability of sperm cells to produce more sex-inducingpheromone (Starr, 1970;Gilles et al., 1984). By this strategy, all members of the population within a pond or lake become synchronously converted to the sexual pathway. A particularly fascinating problem is the molecular mechanism which enables the sex-inducing pheromone to act at a concentration as low as l O - I 7 M. A final answer is not yet available despite the efforts of several groups. However, it is established that the first biochemical responses to the sex-inducing pheromone are structural modifications within the ECM (Wenzl and Sumper, 1982, 1986; Gilles et al., 1983; Sumper et al., 1993). In particular, the earliest response, detectable a few minutes after the application of the pheromone, is the synthesis of a sulfated ECM glycoprotein, pherophorin 11, with an apparent molecular mass of 70 kDa. a. Pherophorin ZZ After application of the sex-inducing pheromone, the somatic cells (which are not the ultimate target of the pheromone’s message!) of an asexually growing Volvox spheroid respond with a strong induction of pherophorin I1 synthesis. As in the case of constitutively expressed pherophorin I, pherophorin I1 is again found to be localized in the insoluble CZ of the ECM. The primary structure of pherophorin I1 was deduced from cDNA sequence analysis (Sumper et al., 1993). A short proline-rich (most probably hydroxyproline-rich) stretch of about 12 amino acid residues around position 170 separates two domains that share sequence identities with the corresponding domains of pherophorin I (Fig. 7). The C-terminal domain exhibits 28% identity and 68% similarity with the sex-inducing pheromone. About 10 copies of pherophorin 11-related


69

genes are found to exist, at least 3 of them in tandem arrangement, in the Volvox genome (Godl et al., 1995). This high gene dose may explain the massive production of pherophorin I1 in response to pheromone application. Even after its deposition within the ECM, pherophorin I1 (in contrast to pherophorin I ) turned out to be an unstable protein being proteolytically processed in a highly specific manner. With a half-life of about 6 h, pherophorin I1 is cleaved into a 42-kDa and a 30-kDa fragment. The cleavage site is exactly adjacent to the polyproline spacer. Interestingly, pherophorin I exhibits an insertion of seven amino acid residues at this cleavage site (Fig. 7). This may explain the resistance of pherophorin I to this specific processing. Pherophorin I1 processing causing the liberation of the pheromone-homologous domain was discussed as part of a signal amplification mechanism that would explain the exquisite sensitivity of the sexinducing pheromone. Inhibition of pherophorin I1 processing indeed correlates with a suppression of sexual induction (Godl et al., 1995). However, direct proof that the C-terminal domain of pherophorin I1 has sex-inducing activity is still lacking.

b. Pherophorin S Application of the sex-inducing pheromone not only induces a modulation of the C Z of the ECM but also leads to substantial changes in the chemical composition of the deep zone. At least two novel ECM glycoproteins are synthesized by somatic cells and targeted to the deep zone in response to the pheromone. Very recently, one of these pheromone-dependent glycoproteins with an apparent molecular mass of 110 kDa was biochemically characterized in detail (Godl et al., 1997). Unexpectedly, this protein of the deep zone turned out to be a true member of the pherophorin family (Fig. 7) (now denoted as pherophorin S) although it exhibits a completely novel set of properties. Besides its different location within the ECM, pherophorin S remains a highly soluble component of the deep zone. Main structural differences from other members of the family include the presence of a poly-hydroxyproline spacer in pherophorin S, 88 amino acid residues in length, that separates the N- and C-terminal domains and, in addition, the presence of incorporated phosphate. This phosphate-containing element that is part of a saccharide attached to the

FIG. 7 Alignment of the amino acid sequences of pherophorin S and pherophorin 1-111. The polyproline stretches are given on gray background. Gaps (-) are introduced to allow maximal alignment. The C-terminal part of the pherophorins found to be homologous to the sexinducing pheromone is boxed. The site of pherophorin I1 and 111 cleavage is marked by an arrowhead. Mature polypeptides are given; for pherophorin S, the N terminus of the mature protein has not been experimentally determined.

70


poly-hydroxyproline spacer again turned out to be identical to the phosphodiester originally discovered in SSG 185 (see Sections III.B.l and 1V.D) which also contains an extended stretch of hydroxyprolines as a spacer element. Since SSG 185 and pherophorin S are located in completely different zones of the ECM, it is unlikely that this very special spacer element provides the signal for targeting pherophorin S to the deep zone. There are a few additional changes in the ECM composition that have been reported to be caused by the sex-inducing pheromone. A corresponding ECM protein, denoted as FSG, will be mentioned only briefly, because as yet only limited biochemical data are available. Synthesis of FSG (-280 kDa) is initiated within 20-30 min after the addition of the pheromone (Wenzl and Sumper, 1982). FSG is a sulfated glycoprotein: Arabinose, xylose, mannose, and galactose (-4.5:0.2:0.2:1.0) are the predominant neutral sugars. FSG is continued to be synthesized by somatic cells until the early stages of embryogenesis.

C. ECM Modifications Induced by Stress 1. Arylsulfatase (Sulfur Deprivation)

Arylsulfatase activity is found in many organisms and plays an important role in mineralization of sulfate (Speir and Ross, 1978). Volvox synthesizes arylsulfatase in response to sulfur deprivation. Enzyme activity remains associated with the spheroid and is not secreted into the culture medium. However, in a Volvox mutant strain called “dissociator” (Dauwalder et al., 1980), which is unable to produce a structurally intact ECM and consequently dissociates into single cells shortly after the end of embryogenesis, large amounts of enzyme activity are detectable in the culture medium. These facts indicate deposition of the arylsulfatase within the ECM of the wild-type strain, but no information is available about the subzone involved. Properties of the Volvox arylsulfatase (like its inducible synthesis and its extracellular location) indicate that mineralization of sulfate in response to sulfur deprivation seems to be the function of this enzyme. The inducible arylsulfatase of Volvox has been purified to homogeneity and the corresponding gene and cDNA have been cloned (Hallmann and Sumper, 1994a). The gene is composed of 16 introns and 17 exons that encode a 649-aminoacid polypeptide chain. The presence of a hydrophobic leader sequence together with the existence of seven potential N-glycosylation sites suggests glycosylation of the polypeptide. A novel posttranslational protein modification has recently been described in two human sulfatases, by which a cysteine is replaced by a serinesemialdehyde (2-amino-3-oxopropionic acid) residue (Schmidt et al.,


71

1995). Recently, the presence of this modification was also demonstrated in Volvox arylsulfatase (Selmer et al., 1996). The evolutionary conservation of this novel protein modification between sulfatases of Volvox and man supports the assumption that this modification is required for the catalytic activity of sulfatases and may be present in all sulfatases of eukaryotic origin (Selmer et al., 1996). Because the arylsulfatase enzyme is readily assayed using chromogenic substrates, but is not detectable in cells grown in sulfate-containing medium, the genes encoding arylsulfatase are useful reporter genes both in Chlamydomonas (Davies et al., 1992; Davies and Grossman, 1994; Quinn and Merchant, 1995) and in V. carteri (Hallmann and Sumper, 1994b; God1 et al., 1997). A remarkable property of the arylsulfatase from Volvox is its insensitivity toward detergents like dodecyl sulfate. Concentrations as high as 1%SDS do not affect enzyme activity. This property enables easy detection of the enzyme even in crude cell lysates (Hallmann and Sumper, 1994a). In addition, the highly regulated promoter of the arylsulfatase gene is a tool for producing inducible expression vectors for cloned genes.

2. Responses to Wounding By subtractive cDNA technology, a number of genes were recently identified that are induced in Volvox by wounding (Haas, Amon, and Sumper, unpublished results). Two of these cDNA clones have been characterized in detail (Amon et al., 1997) and turned out to encode a member of the chitin-binding proteins and a chitinaseAysozyme. In higher plants, these proteins have been implicated as part of a defense mechanism against fungi (Boller, 1988; Bowles, 1990; Linthorst, 1991). The first clone encodes a polypeptide with 309 amino acid residues including a typical signal sequence. The N-terminal half is composed of two nearly perfect repeats of a 48-amino-acid sequence motif. These repeats are separated by a typical spacer element (SGGGGSTPTSTAPPAR). Similar repeats located in phage lysozymes (Paces et al., 1986; Garvey et al., 1986), in a muramidase (Chu et al., 1992), and in an autolysin from Streptococcus faecalis (Beliveau et al., 1991) were identified as top-scoring hits in a BLASTP search (Altschul et al., 1990) of the Swiss-Prot Protein Sequence Database. The region of identities covers nearly the total length of the 48-amino-acid repeat. This repeat is diagnostic of enzymes of the lysozyme family (Joris et aZ., 1992) that also includes vacuolar and secreted chitinases from plants (Holm and Sander, 1994). A repeating unit structure is also a striking feature of the deduced amino acid sequence of the second clone. Three repeats of a domain, 48 amino acid residues in length, constitute the C-terminal part. Proline-rich se-

72


quences with Ser-(Pro), motifs typical of extensins separate these domains from each other. Again, a homology search revealed a close relation of the repeating unit to a well-known protein family, namely, to chitin-binding proteins (Raikhel et al., 1993). The chitin-binding domains of this Volvox polypeptide show extensive identity to the corresponding domains of other proteins like the class I chitinases (Linthorst et al., 1990), hevein (Broekaert et al., 1990), and the wound-induced WIN proteins from potato (Stanford et al., 1989). The N-terminal sequence of the Volvox chitin-binding protein shares high homology with a human cysteine protease (31% identity and 69% similarity over 241 amino acid residues) (Fuchs and Gassen, 1989). This remarkable combination of a protease domain, extensin-like spacer elements, and several chitin-binding domains suggests that this polypeptide is specialized for the degradation of chitin-linked proteins. A similar domain combination, however, with different types of spacer elements, has been described for a prokaryotic enzyme produced by Streptomyces griseus (Sidhu et al., 1994). In Section VI the domain organization of these Volvox ECM proteins induced by wounding is schematically illustrated. Both of these proteins were detected by Western blot experiments as components of the deep zone of the ECM.

D. Crosslinking The sulfated glycoprotein SSG 185 is the monomeric precursor of a highly insoluble polymer of Volvox ECM (CZ3). The phosphodiester found in SSG 185 has been investigated by methylation analysis, I3C-NMR, enzymatic assays, and mass spectrometry and identified as ~-Araf-5-phospho5’-~-Araf(Fig. 8). This phosphodiester bridge most probably crosslinks

/0

’

‘

P

O

H

HO-P=O

I I

OH

0

OH FIG. 8 Structure of the phosphodiester of arabinose isolated from the ECM glycoproteins SSG 185, pherophorin S, and HRGP-290 of Volvox carteri.

BIOCHEMISTRY OF THE !&ox

73

ECM

different carbohydrate chains either intra- or intermolecular in SSG 185 (Holst et af., 1989). There is indirect evidence for an intermolecular crosslink. Polymeric SSG 185 can easily be degraded to monomeric polypeptide chains by the action of anhydrous hydrogen fluoride at O"C, a procedure that selectively cleaves 0-glycosidic linkages (Mort and Lamport, 1977). This fact indicates the existence of intermolecular crosslinks between saccharide chains and excludes intermolecular crosslinks of the exceedingly stable diphenyl ether linkage of isodityrosine (Fry, 1982). Originally, this type of linkage was favored as an intermolecular crosslink in extensins from higher plants, but more recent evidence favors an intramolecular crosslink rigidifying the polypeptide backbone (Kieliszewski and Lamport, 1994). Isodityrosine was also detected in hydrolysates from Chlamydomonas W2 cell wall glycoprotein (Waffenschmidt et at., 1993), but again, proof for the existence of intermolecular crosslinks is lacking. Recently, the HRGPs present in the deep zone of Volvox ECM were found to contain many copies of the same phosphodiester of arabinose. Both of these novel glycoproteins are converted to polymeric structures, as is the case for SSG 185. A remarkable fact is the presence of the diester even in the monomeric precursors, which stimulates speculation about the chemical mechanism of crosslink formation. Without any energy requirement, an intramolecular phosphodiester bridge could be converted into an intermolecular bridge simply by a transesterification reaction (Fig. 9). This reaction would act in both directions. In principle, this chemistry is precisely what is required to remodel or rearrange a glycoprotein network in the

Ho-Ara

-1

A ra

1 A r a - O "

HO-Ara

Ara -@-

I

HO-Ara

I

Ara

Ara

A

+

Ara

FIG. 9 A speculative model for crosslink formation. Without any energy requirement, an intramolecular phosphodiester bridge could be converted into an intermolecular bridge simply by a transesterification reaction. This would be a reversible process.

74


wall to accommodate the insertion of new monomers and therefore permit extension. A comparison with the phosphodiesters of riboses in self-splicing RNAs drives speculations even further: Perhaps this transesterification works autocatalytically. Experiments are currently under way to check this possibility. E. ECM Lysis The liberation of Volvox daughters from the mother spheroid is effected by an enzymatic process, by which the sheath of the parental spheroid is lysed locally so that each daughter spheroid leaves through a hole. In Chlamydomonas, enzymes have been known for some time that are involved in the process of wall degradation (Claes, 1971; Schlosser, 1976, 1981). In C. reinhardtii there are at least two wall-degrading enzymes that function at specific developmental stages: a gamete lytic enzyme (GLE) and a vegetative lytic enzyme (VLE). GLE is a Zn2+-containingmetalloprotease with a molecular mass of 62 kDa (Matsuda et al., 1984, 1985). Its primary structure is homologous to those of mammalian collagenases (Kinoshita et al., 1992). VLE was partially purified and reported to be a 34-kDa glycoprotein (Jaenicke and Waffenschmidt, 1981; Jaenicke et al., 1987; Spessert and Waffenschmidt, 1990). However, the enzyme was recently purified to homogeneity and shown to be a serine protease with a molecular mass of 130 kDa (Matsuda et al., 1995), whereas the 34-kDa protein turned out to be a main impurity of the crude extract. Possibly, homologous proteases have also been described from V. carteri (Jaenicke and Waffenschmidt, 1979, 1981; Waffenschmidt et al., 1990). The lysins of Chlamydomonas and of Volvox degrade the cell wall/ECM in a species-specific manner. From the subtractive cDNA Volvox library mentioned above (Section IV.C.2), the existence of a Volvox homologue of Chlamydomonas GLE could recently be deduced exhibiting a remarkable mosaic structure (schematically illustrated in Section VI) (Haas, Godl, and Sumper, unpublished results). Like in SSG 185 and pherophorin S, a long stretch of prolines (hydroxyprolines?), -120 residues in length, is a central part of the polypeptide. N-terminally located is the sequence homologous to the metalloprotease GLE (38% identity and 74% similarity over 92 amino acid residues, including all active site residues), whereas the C-terminal part of this polypeptide consists of two modules, each repeated twice, of unknown function. V. Relationship to Higher Plant ECMs HRGPs found in the ECM of higher plants include extensins, repetitive proline-rich proteins (RPRPs), some nodulins, gum arabic glycoprotein

BIOCHEMISTRY OF THE Vdvox ECM

75

(GAGP), arabinogalactanproteins (AGPs), and chimeric proteins such as potato lectin which contain an extensin module fused to a lectin. The extensins, a large multigene family of higher plant HRGPs (Showalter and Varner, 1989), are the best characterized and probably the most abundant structural proteins of dicot cell walls (Cooper et al., 1984; Varner and Lin, 1989). Higher plant HRGPs are assumed to play key functions in cell wall self-assembly and cell extension. Their repetitive peptide motifs and the site-specific posttranslational modifications singly or in combination are believed to constitute functional sites involved in various aspects of cell wall biogenesis, as, for instance, self-assembly, adhesion, and crosslinking (Kieliszewski and Lamport, 1994). Based on their similar chemistry, it is reasonable to hypothesize that higher plant and VoZvox/Ch/arnydornonas HRGPs share similar functions in ECM assembly and architecture. Recent reviews concerning different aspects of higher plant ECM include Knox (1995), Brownlee and Berger (1995), Joseleau et al. (1994), Roberts (1994), Albersheim et a/. (1994), Lane (1994), Varner and Ye (1994), Gibeaut and Carpita (1994), Showalter (1993), Fry et al. (1993) and Levy and Staehelin (1992).

VI. Conclusions In this review, the structures of more than 10 ECM proteins from a single species of the multicellular alga Volvox are compared. For 10 ECM proteins, the complete primary structures have been deduced from their genes and the corresponding structures are summarized in the stylized drawing of Fig. 10. Together with structural data from cell wall glycoproteins of the unicellular relative C. reinhardtii, this pool of characterized proteins should allow recognition of common biochemical and structural features. Due to the high content of hydroxyproline, these proteins are usually denoted as HRGPs and are regarded as representing products of one giant supergene family. However, the sequence data do not support such a view. Rather, a typical feature of all algal ECM proteins is a striking modular composition, where hydroxyproline-rich sequences are strictly confined to the rod-shaped domains. Many other modules found in these ECM proteins are completely devoid of hydroxyproline residues. The multiple functions of a complex ECM can hardly be met on the basis of repetitive hydroxyproline-rich sequences. Instead of defining a HRGP family, it appears more appropriate to define a H R module family that can be combined with other modules to yield chimeric and multifunctional polypeptides. A particularly striking example for the confusion introduced by the term “HRGP” is found among the Vdv ox pherophorin protein family. By se-

Volvox ECM-protein

modular composition

localization I property

reference

SSG 185

CZ Imain component of the CZ; contains phosphodiester bridges

Wenzl and Sumper, Iga2; Wenzl et a/., 984; Ertl eta/., 1989

Pherophorin I

CZ Imain component of the CZ

~

Pherophorin II

CZ I C-terminal domain is liberated under the influence of the sex pheromone

sumper a/., 1993;

Pherophorin 111

cz

Godl et a/., 1995

PherophorinS

DZ Ithe only member of pherophorins Godl eta/., 1997 targeted lo DZ; contains phosphcdiester bridges

Algal-CAM

cell-cell contacts I cell adhesion molecule; homology to fasciclin I

IS0

BZ I synthesized for less than

chitin-binding protein

DZ I stress-induced; synthesized in response to wounding

Amon eta/., 1997

chitiflase/ lysozyme

DZ Istress-induced; synthesized in response to wounding

Amon eta/.,1997

~

p

~

~

~

~

Godl et af., 1995

,

= A-type domain

~

;

hydroxyproline-rich ~ = ~(HR)-module Q 3 ~

+rn -

= globular ISG domain = cysteine proteinase

Huber and Sumper' I

GLE4ike protein

10 min in invertingembryos

/homology to Chlamydomonas GLE

fascrclin I-like domain

Wenzl and Sumper, 1982; Schlipfenbacher eta/., 1986; Ertl ef a/. , 1992

chitin binding domain

= lysozvme domain

= OLE-like domain

3

(unpublished)

FIG.10 Domain organization of known Vofvox ECM glycoproteins.

@

= :Z'i?ikperties

-

domain with unknown properties

BIOCHEMISTRY

OF THE Vo/vox ECM

77

quence alignment (Fig. 7), all members are closely related; however, pherophorin I, 11, and 111 only contain a very short hydroxyproline-rich spacer that does not allow classification as a HRGP. In contrast, the amino acid composition of pherophorin S clearly qualifies this protein for membership in the HRGP family. But it is only the insertion of a long hydroxyprolinerich module between the N- and the C-terminal domains that converts this member of the pherophorins to the HRGP family. Most probably, the rodshaped HR modules have a structural function and serve as building blocks to create the defined framework of the ECM. This is supported by the unusually high variability of the HR modules found even among closely related ECM proteins. For instance, the Chlamydomonas homologue of the Volvox ECM protein ISG, denoted as VSP-3, replaces the typical Volvox H R module with numerous Ser-(Hyp)4-6 repeats by a different module with (Ser-Hyp), repeats. An even more striking example has been detected in cell wall proteins (denoted as frustulins) from diatoms which also exhibit a conspicuous modular composition (Kroger et al., 1994, 1996). Frustulins are composed of several repeats of a highly conserved module (ACR domains) which are separated by HR modules if isolated from the diatom Cylindrotheca fusiformis. In a highly homologous frustulin isolated from Navicula pelliculosa these ACR domains are separated by polyglycine modules. Probably, the purely structural task of the HR modules can be met by a large spectrum of rapidly evolving sequence variants, inserted in otherwise highly conserved ECM proteins. It has repeatedly been proposed that HRGPs should make excellent phylogenetic markers for all plants, because of the central role these proteins play in organizing cell and plant morphology. The HR modules within these proteins, however, do not appear to be suitable markers for assessing long-term evolution. In Table I the HR modules found so far in Volvox and Chlamydomonas cell wall/ECM proteins are compared. Where analyzed in more detail, these modules are found to be targets for extensive posttranslational modifications. Among the modifications found are 0-glycosylations with oligoarabinosides, introduction of phosphodiester bridges between arabinose residues, and, in a single case, the attachment of a highly sulfated polysaccharide. The mosaic structures of volvocine ECM proteins known so far appear to provide another example of the combinatorial advantage of shuffling modules, as it is so evident in the evolution of the metazoan ECM proteins (Doolittle, 1995). A high combinatorial potential may not only be a prerequisite for establishing the transition from a cell wall to the complex ECM of a multicellular organism, but also to achieve subsequent species diversification. Remarkably, the transition from unicellularity to multicellularity independently happened several times within the Volvocales and is, accord-

TABLE I HR Modules Known from Volvox and Chlamydomonas Cell WalllECM Proteins (Hydroxy)Proline-rich module Organism

ECM protein

Characteristic repeat

Volvox carteri Vo1vo.x carteri

SSG 185 Pherophorin I

Ser-(Pro)2.17 and (Pr0)2-4 -

Volvox carteri Volvox carteri Volvox carteri Volvox carteri Volvox carteri Chlamydomonas reinhardtii Chlarnydomonas reinhardtii Chlnmydomonas eugametos

Pherophorin S Algal CAM ISG Chitin-binding protein GLE-like protein

Ser-(Pro), Ser-(Pro)3-6 Ser-(Pr~)~_~ Ser-(Pro)z-4 Ser-(Pro)5

Zygote wall protein VSP-3 WP6

(Pro)j and (SerPro) (SerPro) (SerPro),

Maximum length of poly-(hydroxy)proline stretch (Pro) I X (Pro) 10 (pro)26 (Pro)6 (Pro17 (Pro17 (Proh

Data library accession no. X51616 X69801 YO7752 X80416 X65165

S44199 L29029 L29028

BIOCHEMISTRY OF THE Vdvox ECM

79

ing to rRNA sequence data, a very recent evolutionary event in contrast to textbook statements about Volvox (Rausch er al., 1989; Larson er al., 1992). In a stimulating essay on the origins of eukaryotic sex, Goodenough (1985) stressed the possible role of cell wall molecules in the evolution of eukaryotic sex. Derivatives of these molecules designed for self-assembly could have been recruited for promoting cell fusion, and, due to their specificity, for limiting such fusions to genetically appropriate partners. The structural properties of the sexual agglutinins from Chlurnydornonas (Adair and Snell, 1990) support this idea. Sexual speciation requires molecules that are, in addition, designed for variation. Again, the modular organization of ECM proteins may provide this important property. The sexual reproduction system of Volvox adds a new and unexpected aspect. Before entering the sexual reproductive cycle, asexually growing organisms have to be triggered to develop male and female organisms. This signal is provided by the sex-inducing pheromone (a glycoprotein). It was a great surprise that main compartments of the Volvox ECM contain proteins (pherophorins) that bear modules with homology to the sex-inducing pheromone. Possibly, the highly potent sex-inducing pheromone is evolutionarily derived from a member of the pherophorin family that originally served a structural function within the ECM. Sexual speciation again requires modulation of pheromone molecules. It is interesting to note that the pherophorins represent a large protein family, and, in particular, pherophorin 11-like proteins are represented by about 10 genes, in tandem arrangement, that encode similar but not identical sequences. Homologous recombination events at this repetitively organized DNA locus would create variability. Pherophorins are located in the CZ, and as mentioned earlier it is exactly this zone that exhibits species-specificity, whereas the BZ is highly conserved among different Volvocales. Volvox,the simplest multicellular organism, responds to environmental stimuli and wounding in much the same way as that observed in higher plants. These developmental processes depend on changes that take place in the structure of the ECM. Recent progress in the application of powerful molecular genetic techniques like transformation and gene replacement should support further advances in understanding the function of ECM proteins in these important mechanisms of plant biology.

Acknowledgment We are indebted to Dr. David L. Kirk for critical reading of the manuscript.

References Adair, W. S., and Appel, H. (1989). Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlumydomonus reinhurdtii and two other Volvocales. Pluntu 179,381-386.

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Adair, W. S., and Apt, K. E. (1990). Cell wall regeneration in Chlamydomonas: Accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins. Proc. Natl. Acad. Sci. USA 87,7355-7359. Adair, W. S., and Snell, W. J. (1990). The Chlamydomonas reinhardtii cell wall: Structure, biochemistry, and molecular biology. In “Organization and Assembly of Plant and Animal Extracellular Matrix” (W. S. Adair and R. P. Mecham, eds.), pp. 15-84. Academic Press, San Diego. Adair, W. S., Steinmetz, S. A., Mattson, D. M., Goodenough, U. W., and Heuser, J. E. (1987). Nucleated assembly of Chlamydomonas and Volvox cell walls. J. Cell Biol. 105,2373-2382. Albersheim, P., An, J., Freshour, G., Fuller, M. S., Guillen, R., Ham, K.-S., Hahn, M. G., Huang, J., O’Neill, M., Whitcombe, A., Williams, M. V., York, W. S., and Darvill, A. (1994). Structure and function studies of plant cell wall polysaccharides. Biochem. Soc. Trans. 22,374-378. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410. Amon, P., Haas, E., and Sumper, M. (1997). The sexual pheromone and wounding triggers the same set of genes in the multicellular green alga Volvox. Submitted for publication. Beliveau, C., Potvin, C., Trudel, J., Asselin, A., and Bellemare, G. (1991). Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecab autolysin. J. Bacteriol. 173, 5619-5623. Boller, T. (1988). Ethylene and the regulation of antifungal hydrolases in plants. In “Surveys of Plant Molecular and Cell Biology” (B. J. Miflin, ed.), Vol. 5. pp. 145-174. Oxford Univ. Press, London. Bowles, D. (1990). Defense-related proteins in higher plants. Annu. Rev. Biochem. 59,873-907. Broekaert, I., Lee, H. I., Kush, A., Chua, N. H., and Raikhel, N. (1990). Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis). Proc. Natl. Acad. Sci. USA 87, 7633-7637. Brownlee, C., and Berger, F. (1995). Extracellular matrix and pattern in plant embryos: On the lookout for developmental information. Trends Genet. 11,344-348. Catt, J. W., Hills, G. J., and Roberts, K. (1976). A structural glycoprotein, containing hydroxyproline, isolated from the cell wall of Chlamydomonas reinhardii. Planta 131, 165-171. Catt, J. W., Hills, G. J., and Roberts, K. (1978). Cell wall glycoproteins from Chlamydomonas reinhardii, and their self-assembly. Planta 138, 91-98. Chu, C. P., Kariyama, R., Daneo-Moore, L., and Shockman, G . D. (1992). Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae. J . Bacteriol. 174,16191625. Claes, H. (1971). Autolyse der Zellwand bei den Gameten von Chlamydomonas reinhardii. Arch Mikrobiol. 108,221-229. Cooper, J. B., Chen, J. A., and Varner, J. E. (1984). The glycoprotein component of plant cell walls. In “Structure, Function, and Biosynthesis of Plant Cell Walls” (W. M. Dugger and S. Bartnicki-Garcia, eds.), pp. 75-88. Waverly Press, Baltimore. Dauwalder, M., Whaley, W. G., and Starr, R. C. (1980). Differentiation and secretion in Volvox. J. Ultrastruct. Res. 10, 318-335. Davies, J. P., and Grossman, A. R. (1994). Sequences controlling transcription of the Chlamydomonas reinhardtii /3 2-tubulin gene after detlagellation and during the cell cycle. Mol. Cell. Biol. 14, 5165-5174. Davies, J. P., Weeks, D. P., and Grossman, A. R. (1992). Expression of the arylsulfatase gene from the p 2-tubulin promoter in Chlarnydomonas reinhardfii. Nucleic Acids Res. 20,2959-2965. Debuchy, R., Purton, S., and Rochaix, J. D. (1989). The argininosuccinate lyase gene of Chlarnydomonas reinhardtii: An important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARC7 locus. EMBO J. 8,2803-2809.

BIOCHEMISTRY OF THE V o h x ECM

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Desnitski, A. G. (1995). A review on the evolution of development in Volvoxt Morphological and physiological aspects. Eur. J. Protist. 31, 241-247. Doolittle, R. F. (1995). The multiplicity of domains in proteins. Annu. Rev. Biochem. 64, 287-314. Elkins, T., Hortsch, M., Bieber, A. J., Snow, P. M., and Goodman, C. S. (1990). Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin I11 can mediate cell sorting. J. Cell Biol. 110, 182551832, Englund, P. T. (1993). The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu. Rev. Biochem. 62,121-138. Ertl, H., Mengele, R., Wenzl, S., Engel, J., and Sumper, M. (1989). The extracellular matrix of Volvox carteri; Molecular structure of the cellular compartment. J. Cell Biol. 109,3493-3501. Ertl, H., Hallmann, A., Wenzl, S., and Sumper, M. (1992). A novel extensin that may organize extracellular matrix biogenesis in Volvox carteri. EMBO J. 11,2055-2062. Fry, S. C. (1982). Isodityrosine, a new cross-linking amino acid from plant cell-wall glycoprotein. Biochem. J. 204, 449-455. Fry, S. C., Aldington, S., Hetherington, P. R., and Aitken, J. (1993). Oligosaccharides as signals and substrates in the plant cell wall. Pfant Physiot. 103, 1-5. Fuchs, R., and Gassen, H. G. (1989). Nucleotide sequence of human preprocathepsin H, a lysosomal cysteine proteinase. Nucleic Acids Res. 17, 9471. Garvey, K., Saedi, M. S., and Ito, J. (1986). Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15: Homology of gene 15 with other phage lysozymes. Nucleic Acids Res. 14, 10001-10008. Gibeaut, D. M., and Carpita, N. C. (1994). Biosynthesis of plant cell wall polysaccharides. FASEB J. 8,904-915. Gilles, R., Gilles, C., and Jaenicke, L. (1983). Sexual differentiation of the green alga Volvox carteri: Involvement of extracellular phosphorylated proteins. Nafurwissenschaffen 70, 571-572. Gilles, R., Gilles, C., and Jaenicke, L. (1984). Pheromone-binding and matrix-mediated events in sexual induction of Volvox carteri. Z . Naturforsch. 39c, 5&1-592. Godl, K., Hallmann, A., Rappel, A., and Sumper, M. (1995). Pherophorins: A family of extracellular matrix glycoproteins from Volvox structurally related to the sex-inducing pheromone. Planfa 196,781-787. Godl, K., Hallmann, A,, Wenzl, S., and Sumper, M. (1997). Differential targeting of closely related ECM glycoproteins: The pherophorin family from Volvox. EMBO J. 16, 25-34. Goodenough, U. W. (1985). An essay on the origins and evolution of eukaryotic sex. In “The Origin and Evolution of Sex” (H. 0. Halvorson and S. Segal, eds.), pp. 123-140. A. R. Liss, New York. Goodenough, U. W., and Heuser, J. E. (1985). The Chlamydomonas cell wall and its constituent glycoproteins analyzed by the quick-freeze, deep-etch technique. J. Cell B i d . 101,1550-1568. Goodenough, U. W., and Heuser, J. E. (1988). Molecular organization of cell-wall crystals from Chlamydomonas reinhardtii and Volvox carferi.J. Cell Sci. 90, 717-734. Goodenough, U. W., Gebhart, B., Mecham, R. P., and Heuser, J. E. (1986). Crystals of the Chlamydomonas reinhardtii cell wall Polymerization, depolymerization, and purification of glycoprotein monomers. J. Cell Biol. 103,405-417. GOrlach, C. (1994). “Biochemische Charakterisierung einer Sexualpheromon-kontrollierten Komponente der Extrazellullren Matrix von Volvox carteri f: nagariensis.” Ph.D. thesis, Univ. Regensburg, Regensburg, Germany. Green, K. J., and Kirk, D. L. (1981). Cleavage patterns, cell lineages, and development of a cytoplasmic bridge system in Volvox embryos. J. Cell Biol. 91, 743-755. Gumpel, N. J., Rochaix, J. D., and Purton, S. (1994). Studies on homologous recombination in the green alga Chlamydornonas reinhardtii. Curr. Genet. 26, 438-442.

a2

MANFRED SUMPER AND ARMIN HALLMANN

Hallmann, A,, and Sumper, M. (1994a). A n inducible arylsulfatase of Volvox carteri with properties suitable for a reporter-gene system. Purification, characterization and molecular cloning. Eur. J. Biochem. 221, 143-150. Hallmann, A,, and Sumper, M. (1994b). Reporter genes and highly regulated promoters as tools for transformation experiments in Volvox carreri. Proc. Natl. Acad. Sci. USA 91, 11562-11566. Hallmann, A,, and Sumper, M. (1996). The Chlorella hexose/H+ symporter is a useful selectable marker and biochemical reagent when expressed in Volvox. Proc. Natl. Acad. Sci. USA 93,669-673. Hallmann, A , , Rappel, A,, and Sumper, M. (1997). Gene replacement by homologous recombination in the multicellular green alga Volvox carteri. Proc. Natl. Acad. Sci. USA 94,74697474. Hills. G. J., Philipps, J. M., Gay, M . R., and Roberts, K. (1975). Self-assembly of a plant cell wall in vitro. J. Mol. Biol. 96, 431-441. Holm, L., and Sander. C. (1994). Structural similarity of plant chitinase and lysozymes from animals and phage. An evolutionary connection. FEES Lett. 340, 129-132. Holst, O., Christoffel, V., Frund, R., Moll, H., and Sumper, M. (1989). A phosphodiester bridge between two arabinose residues as a structural element of an extracellular glycoprotein of Volvox carteri. Eur. J. Biochem. 181,345-350. Huber, O., and Sumper, M. (1994). Algal-CAMS: Isoforms of a cell adhesion molecule in embryos of the alga Volvox with homology to Drosophila fasciclin I. EMBO J . 13,4212-4222. Huskey, R. J. (1979). Mutants affecting vegetative cell orientation in Volvox carteri. Dev. Biol. 72, 236-243. Jaenicke, L., and Waffenschmidt, S. (1979). Matrix-lysis and release of daughter spheroids in Volvox carteri-A proteolytic process. FEBS Lett. 107, 250-253. Jaenicke, L., and Waffenschmidt, S. (1981). Liberation of reproductive units in Volvox and Chlamydomonas: Proteolytic processes. Ber. Dtsch. Bot. Ges. 94, 375-386. Jaenicke, L., Kuhne, W., Spessert, R., Wahle, U., and Waffenschmidt, S . (1987). Cell-wall lytic enzymes (autolysins) of Chlamydornonas reinhardtii are (hydroxy)proline-specific proteases. Eur. J. Biochem. 170,485-491. Joris, B., Englebert, S . , Chu, C. P., Kariyama, R., Daneo-Moore, L., Shockman, G. D., and Ghuysen, J. M. (1992). Modular design of the Enterococcus hirae muramidase-2 and Streptococcus faecalis autolysin. FEMS Microbiol. Lett. 70, 257-264. Joseleau, J.-P., Chambat, G., Cortelazzo, A , , Faik, A,,and Ruel, K. (1994). Putative biological action of oligosaccharides on enzymes involved in cell-wall development. Biochem. Soc. Trans. 22,403-407. Kieliszewski, M. J., and Lamport, D. T. A. (1994). Extensin: Repetitive motifs, functional sites, posttranslational codes, and phylogeny. Plant J. 5, 157-172. Kindle, K. L. (1990). High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc. Narl. Acad. Sci. USA 87, 1228-1232. Kindle, K. L., Schnell, R. A., Fernandez, E., and Lefebvre, P. A. (1989). Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J . Cell Biol. 109, 2589-2601. Kinoshita, T., Kukuzawa, H., Shimada, T., Saito, T., and Matsuda, Y. (1992). Primary structure and expression of a gamete lytic enzyme in Chlamydomonas reinhardtii; Similarity of functional domains to matrix metalloproteases. Proc. Natl. Acad. Sci. USA 89, 4693-4697. Kirk, D. L. (1988). The ontogeny and phylogeny of cellular differentiation in Volvox. Trends Genet. 4, 32-36. Kirk, D. L., and Harper, J. F. (1986). Genetic, biochemical and molecular approaches to Volvox development and evolution. Int. Rev. Cyrol. 99, 217-293. Kirk, D. L., and Kirk, M. M. (1986). Heat shock elicits production of sexual inducer in Volvox. Science 231, 51-54.


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Kirk, D. L., Birchem, R., and King, N. (1986). The extracellular matrix of Volvox: A comparative study and proposed system of nomenclature. J. Cell Sci. 80, 207-231. Knox, P. (1995). Developmentally regulated proteoglycans and glycoproteins of the plant cell surface. FASEB J. 9, 1004-1012. Kroger, N.,Bergsdorf, C., and Sumper, M. (1994). A new calcium binding glycoprotein family constitutes a major diatom cell wall component. EMBO J . 13, 4676-4683. Kroger, N., Bergsdorf, C., and Sumper, M. (1996). Frustulins: Domain conservation in a protein family associated with diatom cell walls. Eur. J. Biochem. 239, 259-264. Lane, B. G. (1994). Oxalate, germine, and the extracellular matrix of higher plants. FASEB J. 8, 294-301. Larson, A,, Kirk, M. M., and Kirk, D. L. (1992). Molecular phylogeny of the volvocine flagellates. Mol. Biol. Evol. 9, 85-105. Levy, S., and Staehelin, L. A. (1992). Synthesis, assembly and function of plant cell wall macromolecules. Curr. Opin. Cell Biol. 4, 856-862. Linthorst, H. J. M. (1991). Pathogenesis-related proteins of plants. Crit. Rev. Plant Sci. 10, 123-150. Linthorst, H. J. M., Van Loon, L. C., Van Rossum, C. M. A., Mayer, A., Bol, J. F., Van Roekel, J. S. C., Meulenhoff, E. J. S., and Cornelissen, B. J. C. (1990). Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco. Mo1.-Plant Microb. Interact. 3,252-258. Mages, H.-W., Tschochner, H., and Sumper, M. (1988). The sexual inducer of Volvox carteri. Primary structure deduced from cDNA sequence. FEBS Lett. 234,407-410. Matsuda, Y., Yamasaki, A,, Saito, T., and Yamaguchi, T. (1984). Purification and characterization of cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii. FEBS Lett. 166,293-297. Matsuda, Y., Saito, T., Yamaguchi, T., and Kawase, H. (1985). Cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardrii is a metalloprotease and digests the sodium perchlorate-insoluble component of cell wall. J. Biol. Chem. 260, 6373-6377. Matsuda, Y., Saito, T., Yamaguchi, T., Koseki, M., and Hayashi, K. (1987). Topography of cell wall lytic enzyme in Chlamydomonas reinhardtii: Form and location of the stored enzyme in vegetative cell and gamete. J . Cell Biol. 104, 321-329. Matsuda, Y., Koseki, M., Shimada, T., and Saito, T. (1995). Purification and characterization of a vegetative lytic enzyme responsible for liberation of daughter cells during the proliferation of Chlamydomonas reinhardtii. Plant Cell Physiol. 36, 681-689. Mengele, R. (1988). “Versuche zur strukturellen Charakterisierung eines Glykoproteins aus der Extrazellularen Matrix von Volvox carteri.” Ph.D. thesis, Univ. Regensburg, Regensburg, Germany. Miller, D. H., Mellman, I. S., Lamport, D. T. A., and Miller, M. (1974). The chemical composition of the cell wall of Chtamydomonas gymnogama and the concept of a plant cell wall protein. J. Cell Biol. 63,420-429. Mitchell, B. A. (1980). “Evidence for Polyhydroxyproline in the Extracellular Matrix of Volvox.” M.S. thesis, Michigan State University, East Lansing, MI. Mort, A. J., and Lamport, D. T. A. (1977). Anhydrous hydrogen fluoride deglycosylates glycoproteins. Anal. Biochem. 82,289-309. Nelson, J. A,, and Lefebvre, P. A. (1995). Targeted disruption of the NIT8 gene in Chlamydomonas reinhardtii. Mol. Cell. Biol. 15, 5762-5769. Nozaki, H. (1996). Morphology and evolution of sexual reproduction in the Volvocaceae (Chlorophyta). J . Plant Res. 109,353-361. Paces, V., Vlcek, C., and Urbanek, P. (1986). Nucleotide sequence of the late region of Bacillus subtilis phage PZA, a close relative of phi 29. Gene 44, 107-114. Pommerville, J., and Kochert, G. (1982). Effects of senescence on somatic cell physiology in the green alga Volvox carteri. Exp. Cell Res. 140, 39-45.

84


Quinn, J. M., and Merchant, S. (1995). Two copper-responsive elements associated with the Chlamydomonas Cyc6 gene function as targets for transcriptional activators. Plant Cell 7 , 623-628. Raikhel, N. V., Lee, H. I., and Broekaert, W. F. (1993). Structure and function of chitinbinding proteins. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44,591-615. Rausch, H., Larsen, N., and Schmitt. R. (1989). Phylogenetic relationships of the green alga Volvox carteri deduced from small-subunit ribosomal RNA comparisons. J. Mol. Evol. 29,255-265. Roberts, K. (1974). Crystalline glycoprotein cell walls of algae: Their structure, composition and assembly. Philos. Trans. R. Soc. London, Ser. B 268, 129-146. Roberts, K. (1979). Hydroxyproline: Its asymmetric distribution in a cell wall glycoprotein. Planta 146,275-279. Roberts, K. (1994). The plant extracellular matrix: In a new expansive mood. Curr. Opin. Cell Biol. 6, 688-694. Roberts, K., Gay, M. R., and Hills, G. J. (1980). Cell wall glycoproteins from Chlamydomonas are sulphated. Physiol. Plant. 49, 421-424. Roberts, K., Hills, G. J., and Shaw, P. J. (1982). The structure of algal cell walls. In “Electron Microscopy of Proteins’’ (J. R. Harris, ed.), Vol. 3, pp. 1-40. Academic Press, New York. Roberts, K., Grief, C., Hills, G. J., and Shaw, P. J. (1985). Cell wall glycoproteins: Structure and function. J. Cell Sci. Suppl. 2, 105-127. Schiedlmeier, B., Schmitt, R., Muller, W., Kirk, M. M., Gruber, H., Mages, W., and Kirk, D. L. (1994). Nuclear transformation of Volvox carteri. Proc. Natl. Acad. Sci. USA 91,5080-5084. Schlipfenbacher, R., Wenzl, S., Lottspeich, F., and Sumper, M. (1986). An extremely hydroxyproline-rich glycoprotein is expressed in inverting Volvox embryos. FEBS Lett. 209,57-62. Schlosser, U. G. (1976). Entwicklungsstadien- und sippenspezifische Zellwandautolysine bei der Freisetzung von Fortpflanzungszellen in der Gattung Chlamydomonas. Ber. Dtsch. Bot. Ges. 89, 1-56. Schlosser, U. G. (1981). Algal wall-degrading enzymes-Autolysins. In “Encyclopedia of Plant Physiology New Series” (W. Tanner and F. A. Loewus, eds.), pp. 333-351. SpringerVerlag, Berlin. Schlosser, U. G. (1984). Species-specific sporangium autolysins (cell-wall dissolving enzymes) in the genus Chlamydomonas. In “The Systematics of Green Algae” (D. E. G. Irvine and D. Johns, eds.), pp. 409-418. Academic Press, London. Schmidt, B., Selmer, T., Ingendoh, A., and von Figura, K. (1995). A novel amino acid modification in sulfatases that is defective in multiple sulfatase deficiency. Cell 82, 271-278. Schmitt, R., Fabry, S., and Kirk, D. L. (1992). In search of molecular origins of cellular differentiation in Volvox and its relatives. Int. Rev. Cytol. 139, 189-265. Selmer, T., Hallmann, A., Schmidt, B., Sumper, M., andvon Figura, K. (1996).The evolutionary conservation of a novel protein modification, the conversion of cysteine to serinesemialdehyde in arylsulfatase from Volvox carteri. Eur. J. Biochem. 238, 341-345. Showalter, A. M. (1993). Structure and function of plant cell wall proteins. Plant Cell 5,9-23. Showalter, A. M., and Varner, J. E. (1989). Plant hydroxyproline-rich glycoproteins. In “The Biochemistry of Plants” (P. K. Stumpf and E. E. Conn, eds.), Vol. 15, pp. 485-520. Academic Press, New York. Sidhu, S. S., Kalmar, G. B., Willis, L. G., and Borgford, T. J. (1994). Streptomyces griseus protease C. A novel enzyme of the chymotrypsin superfamily. J. Biol. Chem. 269,2016720171. Snell, W. J. (1983). Characterization of the flagellar collar. Cell Motil. 3, 273-280. Sodeinde, 0.A., and Kindle, K. L. (1993). Homologous recombination in the nuclear genome of Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 90, 9199-9203. Speir, T. W., and Ross, D. J. (1978). Soil phosphatase and sulfatase. In “Soil enzymes” (R. G. Burns, ed.), pp. 197-250. Academic Press, London.

BIOCHEMISTRY OF

THE Vo/vox ECM

85

Spessert, R., and Waffenschmidt, S. (1990). Studies on the vegetative autolysin during the vegetative life cycle in Chlamydvmvnas. Eur. J. Cell Biol. 51, 17-22. Stanford, A,, Bevan, M., and Northcote, D. (1989). Differential expression within a family of novel wound-induced genes in potato. Mvf. Gen. Genet. 215,200-208. Starr, R. C. (1969). Structure, reproduction, and differentiation in Volvvx carterif: nagariensis Iyengar, strains HK9 & 10. Arch. Prvtistenk. 111,204-222. Starr, R. C . (1970). Control of differentiation in Vvlvvx. Dev. Biof. Suppl. 4, 59-100. Stevens, D. R., Rochaix, J. D., and Purton, S . (1996). The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlurnydvrnvnas. Mol. Gen. Genet. 251,23-30. Sumper, M. (1979). Control of differentiation in Vvlvvx carteri: A model explaining pattern formation during embryogenesis. FEES Lett. 107, 241-246. Sumper, M., Berg, E., Wenzl, S., and Godl, K. (1993). How a sex pheromone might act at a M.EMBO J. 12, 831-836. concentration below Tschochner, H., Lottspeich, F., and Sumper, M. (1987). The sexual inducer of Vvlvvx carferi: Purification, chemical characterization and identification of its gene. EMBO J. 6,2203-2207. Varner, J. E., and Lin, L. (1989). Plant cell wall architecture. Cell 56, 231-239. Varner, J. E., and Ye, Z . (1994). Tissue printing. FASEB J . 8, 378-384. Waffenschmidt, S., Knittler, M., and Jaenicke, L. (1990). Characterization of a sperm lysin of Vvlvvx carteri. Sex. Plant Reprvd. 3, 1-6. Waffenschmidt, S., Woessner, J. P., Beer, K., and Goodenough, U. W. (1993). Isodityrosine crosslinking mediates insolubilization of cell walls in Chlarnydomvnas. Plant Cell 5,809-820. Wenzl, S., and Sumper, M. (1982). The occurrence of different sulphated cell surface glycoproteins correlates with defined developmental events in Vvlvvx. FEES Letr. 143,311-315. Wenzl, S., and Sumper, M. (1986). Early event of sexual induction in Vvlvox: Chemical modification of the extracellular matrix. Dev. B i d . 115, 119-128. Wenzl, S., Thym, D., and Sumper, M. (1984). Development-dependent modification of the extracellular matrix by a sulphated glycoprotein in Volvox carferi. EMBO J. 3,739-744. Woessner, J. P., Molendijk, A. J., van Egmond, P., Klis, F. M., Goodenough, U. W., and Haring, M. A. (1994). Domain conservation in several volvocacean cell wall proteins. Plant Mol. B i d . 26,947-960. Zinn, K., McAllister, L., and Goodman, C. S. (1988). Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drvsophila. Cell 53, 577-587.

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Cell Biology of the Basophil Ann M. Dvorak Departments of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

The cell biology of basophils, based on published studies spanning 1990-1997, is reviewed. These rarest cells of granulocyte lineages are now available in sufficient numbers for such studies to be done, based on new methods for isolating and purifying the cells from peripheral blood and organ sources and for their derivation in growth factorcontaining cultures from their precursors de now. These studies are dependent on electron microscopy for the accurate identification of basophils, studies which have recently established the presence of basophils in two new species-mice and monkeys. Secretory, endocytotic and storage properties of basophils constitute their mechanistic role@)in human disease; their role@)in health is, however, obscure. Development of immunoaffinity and enzyme-affinity ultrastructural labeling techniques to image the Charcot-Leyden crystal protein and histamine in human basophils, coupled with ultrastructuralanalysis of kinetic samples of cells obtained after stimulation with diverse secretogogues, has provided insight into the role of vesicles in secretory transport mechanisms in human basophils as well as the definition of key ultrastructural phenotypes of secreting basophils. KEY WORDS: Basophil, Electron microscopy, Histamine, Charcot-Leyden crystal protein, Vesicle transport, Allergy, Cytokines, Chemokines, Interleukin-3, Histaminereleasing factor, Monocyte chemotactic protein-1, Inflammation.

1. Introduction Basophils, granulocytes produced in the bone marrow, are cells that circulate as mature cells and have the capacity to invade tissues. They are the rarest of all circulating cell lineages, a fact that has hampered their study following their identification in humans in 1879 (Ehrlich, 1879). The historical, morphological, and immunological characterization of human basophils Inrernorional Review of Cylology. Vol. 180

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from 1879 to 1985 was recently reviewed (Dvorak, 1988a). Highlights roughly spanned three eras of development. Between 1879 and the late 1960s, early structural and histochemical studies utilized more easily obtained populations of malignant human basophils rather than rare and difficult-to-obtain normal human basophils. The participation of basophils in inflammatory exudates, definition of two forms of immunity [cellmediated immunity (CMI) and immediate hypersensitivity), recognition of the histamine content and releasability of basophils, and identification of a new immunoglobulin class (IgE) responsible for reaginic activity in serum-mediated hypersensitivity were all elucidated in the early era (Dvorak, 1988a). In the middle era (1970-1979), advances in the structural and cytochemical definitions of human basophils, documentation of basophils in a variety of cell-mediated and inflammatory events, description of a slow release reaction [termed piecemeal degranulation (PMD)] in these in vivo events, and extensive studies of the biochemistry of immediate hypersensitivity release reactions [termed anaphylactic degranulation (AND)] involving human basophils were accomplished (Dvorak, 1988a). The years 1980-1985 showed a rapid progress in technical developments allowing purification of large numbers of normal peripheral blood basophils and stimulation of human basophil growth from precursors by growth factors. These new sources of normal basophils facilitated ultrastructural analyses of AND of human basophils and of human basophil development (Dvorak, 1988a). Additional reviews have covered many aspects of the biology of basophils of humans and guinea pigs (Dvorak and Dvorak, 1972, 1973, 1974, 1975, 1979,1993;Dvorak et al., 1974a, 1979a, 1980a,f, 1981a, 1983a,b;1986;Galli et al., 1984; Dvorak, l978,1986,1987,1988b, l989,1991,1992a, l993,1994a,b, 1995a, 1997; Dvorak and Ishizaka, 1995; Monahan and Dvorak, 1985; Orenstein et al., 1981; MacGlashan et al., 1982a). The topics of some of these reviews are as follows: (i) basophils of man and guinea pigs in CMI (Dvorak, 1992a; Dvorak and Dvorak, 1972, 1973; Dvorak et al., 1974a, 1980f, 1986; Galli et al., 1984); (ii) a special form of CMI, termed cutaneous basophil hypersensitivity (CBH), based on the extensive tissue infiltration of basophils (Dvorak, 1992a; Dvorak and Dvorak, 1974, 1993; Dvorak et al., 1979b;Galli et aL., 1984); (iii) descriptions of PMD by human and guinea pig basophils, of uptake and granule storage of exogenous proteins, and presentation of a general degranulation model for basophil degranulation (Dvorak, l978,1988a, 1991,1992a, 1993; Dvorak et al., 1980a; Dvorak and Dvorak, 1975; Galli et al., 1984); (iv) ultrastructure of AND by guinea pig basophils (Dvorak, 1978, 1991; Dvorak and Dvorak, 1979; Dvorak et al., 1983b; Galli et al., 1984); (v) plasminogen activator, a surface protease on guinea pig basophils (Dvorak et al., 1979a); (vi) identification and ultrastructure of malignant basophils in granulocytic leukemias (Dvorak et al., 1981a;

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Monahan and Dvorak, 1985); (vii) proteoglycans in guinea pig basophils (Orenstein et af.,1981);(viii) purification of human and guinea pig basophils (MacGlashan et af., 1982a;Dvorak, 1988a, 1991);(ix) ultrastructural criteria for identification of basophils of multiple species (Dvorak, 1986, 1988b, 1989, 1991, 1994a,b; Dvorak et al., 1983a); (x) ultrastructural comparison of human granulocytic myelocytes (Dvorak, l987,1988a, 1994a); (xi) development of human basophils in vitro (Dvorak and Ishizaka, 1995); (xii) ultrastructure of AND by human basophils (Dvorak, 1988a,b, 1991,1993, 1994a, 1997; Dvorak et af., 1983b). In this review, progress in the cell biology of basophils, published in 1985-1997, will be emphasized. Human basophils continued to be a source for newer studies, and basophils in two new species (mouse and monkey) were identified (Dvorak et af., 1982a, 1989a, 1993a, 1994a; Seder et al., 1991a). Cytokines as growth factors, as cell activators, and as cell products were characterized, and the relevance of chemokines as potent secretogogues of basophils was established (Dvorak and Ishizaka, 1995; MacDonald, 1993; Grant et al., 1991; Kaplan et af., 1991; Ishizaka et al., 1989a,b). New sources of sufficient numbers of basophils for cell-biological studies were realized for human basophils in the development from cytokinestimulated precursors in vitro (Dvorak and Ishizaka, 1995; Ishizaka et al., 1989a,b),application of sophisticated cell-sorting methods for collection of mouse basophils from several organ sources (Seder et al., 1991a; Dvorak el al., 1993a, 1994a), and in vivo administration of interleukin (1L)-3 to obtain large numbers of circulating monkey basophils (Dvorak et af., 1989a). In-depth kinetic analyses, using a coordinated biochemical-morphological approach, of two different secretogogues, formyl methionyl leucyl phenylalanine (FMLP) and tetradecanoyl phorbol acetate (TPA), was accomplished (Dvorak et af., 1991a, 1992a; Warner et al., 1989; Schleimer et af., 1981, 1982), providing a solid anatomic foundation for subsequent studies of subcellular distributions of the Charcot-Leyden crystal (CLC) protein (Dvorak et al., 1996a, 1997a,b) and histamine (Dvorak, 1997; Dvorak et al., 1994b, 1995a, 1996f) in activated human basophils. Each of these new areas of study required the development of immunogold (Dvorak et al., 1988; Dvorak and Ackerman, 1989) and enzyme-affinity-gold (Dvorak, 1995a, 1997; Dvorak et al., 1993b, 1995b) ultrastructural methods for visualizing CLC protein and histamine. In aggregate, these new tools have facilitated better understanding of basophil secretion and recovery from secretion (Dvorak, 1991,1993,1994q 1995a, 1996,1997; Dvorak and Ishizaka, 1995; Dvorak et af., 1991a, 1992a, 1993a, 1995a, 1996a,b,f,1997a,b). Specifically,documentation of a degranulation continuum, originally proposed in 1975 as a general degranulation model for basophils (Dvorak and Dvorak, 1975), and of the key role for vesicular transport in effecting this process was accomplished (Dvorak,

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1995b; Dvorak et al., 1994b, 1996a,e,f, 1997a,b). Also, extension of the principle for uptake and storage of soluble exogenous proteins in basophil secretory granules to other secretory cells reinforced the general importance of this biological mechanism (Dvorak, 1991; Dvorak and Monahan-Earley, 1995a,b; Dvorak et al., 1985a,b). Finally, the morphometric analysis of activated, labeled human basophils revealed distinctive morphologic phenotypes (Dvorak et al., 1997a), giving rise to the realization that asynchronous events stimulated in this extremely rare blood granulocyte provide an anatomic basis for the daunting task of interfering with the regulation of these events to understand more fully basophil function in health and disease.

II. Basophil Identification Basophils were first identified by Paul Ehrlich, based on the affinity of their cytoplasmic granules for basic dyes (Ehrlich, 1879), a property shared by a similar cellular lineage, mast cells, also identified by Ehrlich (1877). The standard way for identifying basophils and mast cells for many years was based on this metachromatic dye-binding capacity of basophils and mast cells. Ehrlich and other early anatomists (Ehrlich, 1879; Jolly, 1900; Maximow, 1913; Michaelis, 1902; Zimmermann, 1908) clearly distinguished basophils from mast cells in man based upon morphologic criteria and dyebinding, using light microscopy. Despite recurrent confusion regarding whether these are two distinct cell types or maturational phases of a single cell lineage, the balance of anatomic data favors the original view of these early anatomists, i.e., that basophils and mast cells are indeed two separate cell lineages (Dvorak, 1988a). The basic ultrastructure of basophils and mast cells elucidates their individual, unique morphologies and changes superimposed on them by maturational and functional programs (Dvorak, 1986, 1988a,b, 1989, 1991, 1994a; Dvorak and Ishizaka, 1995; Dvorak er al., 1983a, 1985b; Kepley et al., 1994; Hastie, 1990; Eguchi, 1991). Use of metachromatic stains to identify basophils by light microscopy has certain pitfalls that are not widely recognized when predominantly immature cells are assessed (Dvorak, 1988a). In humans, large basophilic myelocytes packed with metachromatically staining granules could easily be confused either with mast cells of similar size or with eosinophilic myelocytes in which many immature granules also bind metachromatic dyes. By electron microscopy, myelocytes of the basophil and eosinophil lineages are readily identified, based on specific anatomic and cytochemical criteria (Dvorak, l986,1987,1988a, 1991,1994a; Dvorak and Ishizaka, 1995;Dvorak et al., 1982a, 1993a, 1994a).


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A. Electron Microscopy Mature human basophils (Fig. 1) typically contain a granulocyte nucleus, that is, a polylobed nucleus with extensive chromatin condensation. Cytoplasmic contents include mitochondria, cytoskeletal elements, glycogen, vesicles, and granules. Golgi structures are inconspicuous, and small amounts of free or membrane-bound ribosomes are present. Granule content is predominantly particulate but may be subcompartmentalized by multiple dense membranous arrays. Cell surface architecture is characterized by multiple broad surface processes that are irregularly spaced. A numerically minor small granule subset contains homogeneously dense material and resides near nuclear lobes (Dvorak, 1988a; Hastie, 1974,1990; Nichols and Bainton, 1973). Charcot-Leyden crystals form within the particulate matrix of the numerically predominant large secretory granules of

FIG. 1 Human basophil. Note the polylobed nucleus, irregular, broad surface processes, and secretory granules filled with particles. One small granule is also present. It does not contain particles (arrow). The cell surface is stained with cationized ferritin (with permission, from Dvorak, 1988b). Bar: 1.2 Fm.

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human basophils (Dvorak, 1988a, 1994a; Dvorak and Ackerman, 1989; Dvorak and Monahan, 1982).

1. Basophils in New Species a. Mouse Basophils The ultrastructural morphology of mature mouse basophils and immature mouse basophilic myelocytes was described and differentiated from the ultrastructural morphology of mature and immature mouse mast cells, mature eosinophils and neutrophils, and immature mouse eosinophilic myelocytes and neutrophilic myelocytes (Dvorak, 1986, 1991, 1994b, 1995b; Dvorak and Ishizaka, 1995; Dvorak et af., 1982a, 1983a,c, 1993a, 1994a; Seder et al., 1991a; Galli et al., 1983a, 1984). Mature mouse basophils have polylobed granulocytic nuclei with heavily condensed chromatin and small numbers of homogeneously dense secretory granules. Other cytoplasmic organelles include vesicles, mitochondria, cytoskeletal structures, small Golgi structures, glycogen, and free and membrane-bound ribosomes; the cell surface has irregular, broad processes (Dvorak et af., 1982a). Mouse basophils were discovered in large numbers, using these ultrastructural criteria, in sorted samples of mouse spleen and bone marrow non-B, non-T cells which expressed high-affinity Fc, receptors and produced interleukin-4 (IL-4) in response to crosslinkage of Fc, receptors, Fc, receptors, or to exposure to ionomycin (Seder et aL, 1991a). Thus, an association of IL-4 production with mouse basophils was first established (Seder et af., 1991a). Mouse basophils were identified ultrastructurally in non-B, non-T cells sorted from normal mouse bone marrow or spleen and were substantially increased when mice were previously injected with goat anti-mouse IgD antibodies (Dvorak et af.,1993a;Conrad et al., 1990). Of the granulated cells present in Fc,R+ non-T, non-B cells sorted from spleen or bone marrow of goat anti-mouse IgD-injected animals, -90% were in the basophil lineage (Dvorak et aL, 1993a). Of these, 97% were mature basophils in the spleen samples. By contrast, 31% of the cells in the basophil lineage in bone marrow samples were basophilic myelocytes (Dvorak et al., 1993a). Shortterm cultures of mouse bone marrow cells containing IL-3, with or without stem cell factor (SCF), were examined for mouse basophils (Dvorak et aZ., 1994a). Basophils did not develop increased granules and underwent apoptosis in cultures containing both factors, whereas mast cells thrived and did develop increased granule numbers. Basophils were Fc,R+ and ckit- when sorted after culture in IL-3 and SCF. Thus, mouse basophils, identified by electron microscopy, expressed little or no c-kit receptor on their cell surface, nor did they survive for long periods in SCF-supplemented cultures (Dvorak et af., 1994a).

b. Monkey Basophils Ultrastructural and cytochemical studies of monkey peripheral blood samples were performed after recombinant human

93


(rh) IL-3 was infused (Dvorak et al., 1989a). rhIL-3 stimulated a delayed granulocytosis (Donahue et af., 1988) primarily characterized by numerous mature basophils and fewer neutrophils and eosinophils (Dvorak et al., 1989a). Mature basophils were identified by electron microscopy (Dvorak et al., 1989a). They were found to be typical granulocytes with polylobed nuclei containing condensed chromatin. Numerous secretory granules were filled with homogeneously dense contents. Other cytoplasmic organelles included mitochondria, vesicles, cytoskeletal elements, glycogen, small Golgi structures, and minor amounts of free and membrane-bound ribosomes. Irregularly spaced broad surface processes were evident. Cytochemical and routine ultrastructural criteria aIIowed monkey basophils to be distinguished from other granulocytes and mast cells in this species (Dvorak et al., 1989a; Ts’ao et al., 1976; Patterson et al., 1980). Confirmation that IL-3 is a basophilopoietin in monkeys has appeared from the work of numerous laboratories (Mayer et al., 1989; Wagemaker et al., 1990; VolcPlatzer et al., 1991; Wognum et al., 1995; van Gils et al., 1995). 2. Basophils in New Circumstances-Human in Vitro

Basophils

We recently reviewed our experience in the ultrastructural analysis of a variety of culture systems of human cord blood mononuclear cells, spanning a 10-year period (Dvorak and Ishizaka, 1995). Suspension cultures of human cord blood mononuclear cells reliably gave rise to selective growth of human basophils (Ishizaka et al., 1985a) when supplemented with a fraction of culture supernatant of phytohemagglutinin-stimulated human T cells which lacked IL-2 (Ishizaka et al., 1985a; Ogawa et aL, 1983). These cells contained amounts of histamine similar to those in normal human peripheral blood basophils and had functional Fc, receptors of high affinity (Ishizaka et al., 1985a). Sequentially prepared cultures demonstrated the development of large, immature basophilic myelocytes that matured into small, mature basophils (Dvorak et al., 1985b), a process generally completed in -3 weeks. Similar cultures supplemented with either rhI1-3 or IL-5 (but not IL-4) gave rise primarily to eosinophils and basophils (IL-3) or eosinophils (IL-5) but not to mast cells (Ishizaka et al., 1989a,b; Saito et al., 1988; Dvorak et aZ., 1989b). Small numbers of mature human basophils were present in similar suspension cultures supplemented with the c-kit ligand, i.e., SCF, in its recombinant or naturally occurring form (Dvorak et al., 1993~).The basophils contained immunoreactive gold label for the CLC protein, whereas the more numerous mast cells developing therein did not (Dvorak et al., 1994~).Additional reports emphasize the utilization of electron-microscopic analysis to identify developing human basophils in a variety of culture systems (Eguchi, 1991; Eguchi et al., 1985,1989; Tanno et al., 1987;Fishman

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et al., 1985; Suda et al., 1985; Rimmer and Horton, 1986; Hermine et al., 1992; Boyce et al., 1995; Sorensen et al., 1988; Seldin et al., 1986); some of these reports concern primarily development of eosinophils (Saito et al., 1988; Eguchi et al., 1985; Rimmer and Horton, 1986; Hermine et al., 1992; Boyce et al., 1995; Sorensen et al., 1988; Seldin et al., 1986). Earlier reports

(i,e., prior to 1985) of utilization of electron microscopy to identify human basophils developing in vitro are summarized in Dvorak (1988a).

6. Cell Markers Considerable progress in characterizing cell markers that are displayed by human basophils has revealed commonalities with other cell lineages as well as several unique markers. These studies, recently reviewed (Valent and Bettelheim, 1992; Valent et al., 1990a), provide additional tools with which basophils can be identified in mixed cellular populations. Basophils and mast cells bind IgE on their surfaces via high-affinity Fc, receptors (Ishizaka et al., 1970; Ishizaka and Ishizaka, 1975; Sullivan et al., 1971; Becker et al., 1973), whereas basophils lack significant low-affinity Fc,RII (CD23) molecules (Stain et al., 1987). Basophils also have low-affinity binding sites for IgG (Valent et al., 1990a; Stain et al., 1987; Ishizaka et al., 1979; Nakagawa and de Weck, 1983), receptors for complement (Stain et al., 1987; Valent et al., 1990a; Olsen et al., 1988; Glovsky et al., 1979; de Boer and Roos, 1986) for the cytokines JL-3 (Lopez et al., 1990; Valent et al., 1989), IL-4 (Valent et al., 1990b), IL-2 (Stain et al., 1987; Stockinger et al., 1990), GM-CSF (Lopez et al., 1990), NGF (Valent et al., 1990a), and for histamine (Lichtenstein and Gillesie, 1973). Cell surfaces of basophils express HLA class I antigen (de Boer and Roos, 1986; Reshef and MacGlashan, 1987; Bochner et al., 1989), intercellular adhesion molecule-1 (Bochner et al., 1989), leukocyte common antigen (CD45) (Stain et al., 1987; de Boer and Roos, 1986), and the Bsp-1 antigen, a specific marker for human basophils (Valent et al., 1990a;Bodger and Newton, 1987;Bodger etal., 1987). A unique marker for human basophils, consistent with a granule location, has been reported (Kepley et al., 1995). The expression of surface integrins on human basophils includes B1 and B2 integrins and differs from the integrin pattern expressed by human mast cells (Sperr et al., 1992). Quantitative comparison of myeloid antigens on individual mature peripheral blood lineages reveals that these lineages can be distinguished based on this approach to cell identification with surface markers (Terstappen et al., 1990).A panel of 60 monoclonal antibodies (used to distinguish circulating basophils from eosinophils and neutrophils) has produced an antigenic membrane profile useful for basophil identification (Stain et al., 1987).


95

Similarly, this approach has revealed antigenic membrane profiles useful for distinguishing basophils from mast cells (Valent and Bettelheim, 1992).

C. Cell Contents

In man, histamine is present in the granules of two cells-mast cells and basophils (Dvorak 1991; Dvorak et al., 1993b, 1994b; Riley and West, 1953; Lagunoff et al., 1961; Pruzansky and Patterson, 1967; Ehrich, 1953). Basophils are smaller cells, containing an average of 1.3 pg of histamine per cell (Schroeder and Hanrahan, 1990), whereas larger mast cells are a rich source of histamine, containing 3.74 pgkell (Dvorak et al., 1985~).Proteoglycans are glycoconjugates composed of covalently linked glycosaminoglycans and proteins. These materials are responsible for the metachromasia displayed by basophils. Human (Galli et al., 1979; Ishizaka et al., 1985b; Metcalfe et al., 1984), guinea pig (Orenstein et al., 1978), rabbit (Sue and Jaques, 1974), and rat (Metcalfe et al., 1980) basophils contain chondroitin sulfates as their proteoglycan. Ultrastructural autoradiographic localization of radiolabeled sulfur to basophil granules (Orenstein et al., 1978;Galli etal., 1984) indicates that sulfur-containing granule proteoglycans are responsible for the metachromasia displayed by granules in appropriately stained smears of basophils. Mast cells, in contrast, generally contain heparin as the major proteoglycan (Metcalfe et al., 1979). Tryptase and chymase have been used to differentiate human mast cells in various anatomic sites (Irani et al., 1989) and to rule out basophil participation in mixed populations of human cells (Furitsu et al., 1989; Mitsui et al., 1993). Basophils lack chymase but contain extremely small amounts of tryptase (0.05 pgkell) (Castells et al., 1987), whereas mast cells contain as much as 35 pgkell of tryptase (Schwartz et al., 1987). Several eosinophil granule proteins are also found in basophilic leukocytes. Among these are eosinophil-derived neurotoxin (Abu-Ghazaleh et al., 1992), major basic protein (MBP) (Abu-Ghazaleh et al., 1992;Ackerman et al., 1983), and the CLC protein (Dvorak, 1996; Dvorak and Ackerman, 1989; Dvorak et a[.,1994c, 1996a, 1997a,b;Abu-Ghazaleh et a[., 1992;Ackerman et al., 1982). In contrast, MBP (Leiferman et al., 1986) and CLC protein (Dvorak, A.M. et al., 1994c; Leiferman et al., 1986) are absent from mast cells. It has recently been recognized that basophils (and mast cells and eosinophils) produce cytokines (Seder et al., 1991a; Burd et al., 1989; Galli et al., 1991). Initial studies in mice revealed that splenic non-B, non-T cells were associated with IL-4 production (Conrad et al., 1990; Ben-Sasson et al., 1990; Le Gros et al., 1990; Seder et al., 1991b; Paul et al., 1993) and that these Fc,R-positive cells were basophils (Seder et al., 1991a; Dvorak et aL, 1993a, 1994a). Ample evidence now exists showing that human basophils

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also produce IL-4 (Brunner et al., 1993; Arock et al., 1993; Schroeder et al., 1994,1996;MacGlashan etal., 1994a;Mueller et al., 1994; Ochensberger et aZ., 1995). In one of these reports, immunoreactive IL-4 was localized to Fc,RI-positive, activated peripheral blood basophils derived from normal donors, providing direct evidence that IL-4 is produced by activated human basophils (Mueller et al., 1994). More recently, immunoreactive IL-13, a cytokine closely resembling IL-4, has been localized in human basophils by immunocytochemistry (Li et al., 1996). D. Secretogogues and Secreted Cellular Products Biochemical studies of human basophil secretogogues and identification of secreted products are extensive. The unique patterns of secretogogue releasability and secretory products induced from human basophils allow identification of responding cells by analysis of fluids that bathe inflammatory and immunologicprocesses. For example, in late antigen-induced rhinitis, the mixture of inflammatory mediators present does not include prostaglandin D2 which is a mast cell product, thus implicating basophils in this immunologically mediated inflammatory process (Naclerio et al., 1985). Failure to detect LTB4,a leukotriene, in inflammatory fluids also implicates basophils and not mast cells, since basophils do not generate LTB4 (Warner et al., 1987). The ability to analyze such complex in uiuo events is based on a large background of biochemical studies of release reactions and activation of purified populations of human basophils and mast cells (Warner et d., 1987; MacGlashan and Guo, 1991; MacGlashan and Warner, 1991; MacGlashan and Lichtenstein, 1980; MacGlashan et al., 1982b, 1983; Peters et al., 1984; Lichtenstein et af., 1983). In these studies, it is clear that histamine is released from both basophils and mast cells, albeit in different amounts and sometimes with different kinetics. Histamine-releasing secretogogues can differ between these two cellular lineages, and in man, secretogogues for the release of histamine from mast cells derived from different organ sources (e.g., lung, skin, synovium, heart, uterus, gut) can have a different activation profile. Lipid products of basophils and mast cells (and subsets of mast cells) may also differ (MacGlashan and Warner, 1991; MacGlashan etaZ., 1982b;Peters etal., 1984;Lichtenstein et al., 1983;Warner et al., 1987; Naclerio et al., 1985). This group of inflammatory mediators is not performed; rather, it is generated from cellular lipids after stimulation. Analysis of lipid products of either cyclooxygenase or lipoxygenase metabolism of arachidonic acid produced by purified cellular populations is helpful in understanding contributions of individual cells to inflammatory exudates in uivo. Some of the secretogogues useful for examining these events in human basophils include those operating via an IgE-mediated mechanism


97

(Warner ef al., 1989; Lichtenstein, 1971, 1975; Findlay et al., 1980; KageySobotka et af., 1981), the calcium ionophore A23187 (Findlay et al., 1980; Lichtenstein, 1975; Warner et al., 1989), complement (Warner et aL, 1989; Siraganian and Hook, 1976; Hook and Siraganian, 1977; Hook et al., 1975; Grant et al., 1975, 1976, 1977; Farnam et al., 1985; Findlay et af., 1980), tumor-promoting phorbol esters (Schleimer et al., 1981, 1982; Warner et al., 1989), concanavalin A (Siraganian and Siraganian, 1974, 1975), or the bacterial peptide FMLP (Hook et al., 1976; Siraganian and Hook, 1977; Warner et al., 1989). Profiles of released mediators and kinetics of mediator release differ when purified human basophils are stimulated with different secretogogues. For example, anti-IgE elicited release of both histamine and LTC4 from human basophils within 15-30 min; FMLP released histamine and LTC4 in 2-5 min; C5a rapidly released histamine only; A23187 caused extensive release of histamine and LTC,, but TPA released only histamine and did so with slow kinetics-45-60 min (Warner et al., 1989). Thus, complex characteristics of these regulated secretov events in human basophils can be expected to impact differently on the pathogenesis of disease, where basophils prevail.

111. Basophils in Disease In 1970, we demonstrated in guinea pigs extensive infiltration of mature basophils from blood into tissues during cellular immunity of delayed onset mediated by lymphocytes (Dvorak et af., 1970). We named such reactions cutaneous basophil hypersensitivity (CBH) to distinguish them from classical delayed hypersensitivity (DH). Subsequently, invasion of tissues by basophils in a variety of circumstances and in numerous species was identified (Dvorak, 1988a; Dvorak and Dvorak, 1972,1973,1974,1975; Dvorak et af., 1973, 1974a,b, 1976a,b; 1979b,c, 1980f; Galli and Dvorak, 1995; Galli et al., 1984). Two recent reviews summarize the current status of knowledge regarding the tissue migration of circulating basophils in disease (Dvorak and Dvorak, 1993; Dvorak, 1992a). Thus, extensive circumstantial evidence exists implicating basophils in delayed-onset, cell-mediated disease and reactions such as chronic inflammation and graft rejection. Basophils have the capacity to produce fatal disease when systemic anaphylaxis is produced secondary to basophil degranulation (Bochner and Lichtenstein, 1991). These massive, often fatal events are examples of antibody-mediated immediate hypersensitivity (IH). Other IH disorders with a basophil-mediated component include hay fever (Creticos et aL, 1985), rhinitis (Naclerio et al., 1985; Bascom et al., 1988; Hastie et al., 1979;

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Iliopoulos et al., 1992), eczema (deShazo et al., 1979; Mitchell et al., 1982), and asthma (Lichtenstein and Bochner, 1991). A role for basophils in inflammation has been suggested (Lichtenstein et al., 1978). In order for basophils to participate in inflammatory processes, they need to migrate from the blood into tissues, as they must do in D H or CBH reactions. Cell migration is facilitated by complex adhesions between circulating cells and endothelia. Recently, endothelial and basophil adhesion molecules have been the subject of numerous studies (Bochner and Schleimer, 1994). That basophils participate in inflammatory responses to bacterial and viral pathogens in infectious diseases is suggested by the large number of studies showing that these pathogens can stimulate histamine secretion from basophils. Among the pathogens associated with secretion from basophils are staphylococci (Martin and White, 1969; Marone et al., 1982; Espersen et al., 1984), Escherichia coli (Norn et al., 1986),Salmonella (Norn et al., 1986), Candida albicans (Pedersen et al., 1987), Herpes simplex (Pedersen et al., 1987), influenza A (Clementsen et al., 1988a,b; Busse et al., 1983), and Vibrio cholera (Clementsen et al., 1988a).

IV. Basophils as Secretory Cells A. Analogies of Basophil Secretary Granules to Secretory Granules of Endocrine, Exocrine, and Mast Cells Basophils, like endocrine, exocrine, and mast cells, store their specific secretory products in membrane-bound containers in the cytoplasm. These storage organelles participate in secretion by exocytosis-i.e., fusion of granule and plasma membranes with extrusion of their contents to the extracellular space (Dvorak, 1988a,b, 1989, 1991, 1992b; Dvorak and Monahan-Earley, 1992, 1995c,d). Secretory granules, by routine ultrastructural examination, are filled with electron-dense contents and vary in size, shape, number, and substructural intragranular patterns. Some of these variations are typical for individual cells or for the same cells in different species. Yet all mature granules termed secretory granules (in the absence of PMD) are filled with secretory materials. Secretory granules are often referred to in the literature as “vesicles,” but we choose not to use this term for bona fide granules (see Vesicle Transport and PMD sections) in order to avoid confusion with processes that make use of vesicles to transport materials in and out of cells. The substructural patterns of electron-dense secretory granules are known for mature basophils in a number of species (Dvorak, 1992a). Since mast cells are the cells most often confused with basophils, we list their substructural patterns for the same species in Table I. Thus, mature basophil

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CELL BIOLOGY OF THE BASOPHIL TABLE I SubstructuralArchitecture of Human, Guinea Pig, Mouse, Rat, Monkey, and Rabbit Basophil and Mast Cell Granules Species Human (Dvorak, 1988a,b, 1989, 1991; Dvorak and Ackerman, 1989; Dvorak and Kissell, 1991; Dvorak and Monahan, 1982; Dvorak ef al., 1976a, 1980b, 1984a, 1989b, 1993c, 1994c; Eguchi, 1991; Eguchi et al., 1985, 1989; Fedorko and Hirsch, 1965; Fox et a1.,1981, 1984; Hastie, 1974,1990; Ishizaka etal., 1985a; Kobayasi eral., 1968; Orr, 1977; Parmley er al., 1976; Ts'ao et al., 1977; Wetzel, 1970; ZuckerFranklin, 1967)

Mature basophil granule patterns 1. Particles 2. Multiple concentric membrane arrays 3. CLCS 4. Homogeneously dense

Mature mast cell granule patterns

Scrolls Crystals Particles Concentric and tangled thick threads 5. Homogeneously dense

1. 2. 3. 4.

1. Crystals Guinea pig (Chan and Yoffey, 1960; Dvorak 1978,1986,1991; Dvoraketnl., 1981b;Fedorko 2. Finely granular and Hirsch, 1965; Hebert and Lindberg, 1982; 3. Homogeneously dense Murata and Spicer, 1974; Pearce et al., 1977; Stock et a!., 1989; Taichman, 1970,1971; Terry et al., 1969; Watanabe, 1954; Wetzel, 1970; Winqvist, 1960, 1963)

1. Crystals 2. Particles 3. Homogeneously dense 4. Irregular thick threads 5. Regular arrays of 12-nm tubules

1. Homogeneously dense


Rat (Bentfeld et al., 1977; Bentfeld-Barker and 1. Homogeneously Bainton, 1980; Combs, 1966; Hoenig and dense Levine, 1974; Login et al., 1987; Wetzel, 1970)


Monkey (Barrett and Metcalfe, 1985; Dvorak et al., 1989a; Patterson et al., 1980; Ts'ao et al., 1976; Wetzel, 1970)


1. Particles 2. Concentric and irregular threads 3. Homogeneously dense

1. Particles


Mouse (Combs, 1971; Dvorak, 1991; Dvorak et al., 1982a; Galli er al., 1987; Hammel et aL, 1987; Wetzel, 1970)

Rabbit (Benveniste ef a1.,1972; Dvorak, 1992a; Hardin and Spicer, 1971; Horn and Spicer, 1964; Komiyama and Spicer, 1974 Wetzel et al., 1967)

2. Concentric and

tangled thick threads 3. Finely granular 4. Homogeneously dense

Note. Modified (with permission) from Dvorak (1992a).

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granules in humans contain particles, multiple concentric membranous arrays, or are homogeneously dense (Dvorak, 1988a, 1991; Dvorak and Ackerman, 1989; Dvorak and Monahan, 1982; Dvorak et al., 1976a, 1980b, 1985b, 1989b, 1991a, 1992a, 1993c, 1994b,c, 1995a, 1996a,b,e, 1997a,b; Ishizaka et al., 1985a; Hastie, 1974,1990; Eguchi, 1991; Fox et al., 1984; ZuckerFranklin, 1967; Parmley et al., 1976; Wetzel, 1970). In guinea pigs, mature basophil granules display crystals and are finely granular or homogeneously dense (Wetzel, 1970; Fedorko and Hirsch, 1965; Dvorak, 1991; Dvorak and Monahan, 1985; Dvorak et al., 1970, 1981b, 1987; Winqvist, 1960, 1963; Terry et al., 1969; Murata and Spicer, 1974; Chan and Yoffey, 1960; Watanabe, 1954; Hebert and Lindberg, 1982). The electron-dense material is homogeneous without substructural patterns in mature basophils of mice (Wetzel, 1970; Dvorak, 1991; Dvorak et al., 1982a, 1983c, 1993a, 1994a; Seder et al., 1991a; Galli et al., 1983a), rats (Wetzel, 1970; Hoenig and Levine, 1974; Palade, 1955; Bentfeld-Barker and Bainton, 1980), and monkeys (Wetzel, 1970; Dvorak ef al., 1989a). In rabbits, mature basophil granules contain particles, concentric and tangled thick threads, and are finely granular or homogeneously dense (Wetzel et al., 1967; Benveniste et al., 1972; Hardin and Spicer, 1971; Komiyama and Spicer, 1974; Horn and Spicer, 1964; Dvorak, 1992a). 8 . Degranulation of Basophils

1. Anaphylactic Degranulation Anaphylactic d e g r a d a t i o n is the general term used to describe the rapid regulated secretory events of which basophils are capable (Fig. 2B). It corresponds to granule extrusion by exocytosis, a regulated secretory process common to secretory cells in general. AND in basophils is the coordinated secretion of granule mediators, accompanied by the visible extrusion, or solubilization within specially constructed intracytoplasmic degranula-

FIG.2 Purified human peripheral blood basophils, stimulated with FMLP for 20 s, show PMD (A) and AND (B). In A, a polymorphonuclear (N) basophil devoid of full granules shows extraordinary enlargement of discrete, empty granule containers (G) in the cytoplasm. In B, a polymorphonuclear (N) basophil shows extrusion of membrane-free granules (solid arrowheads) which are located in cul-de-sacs closely associated with the cell surface at multiple points around the cell. Markedly elongated, complex surface processes are noted. Focal collections of released, dense concentric membranes (open arrowheads) are associated with the complex cell surfaces of this basophil. The cytoplasm contains numerous aggregates of dense glycogen and elongated tubules of smooth endoplasmic reticulum (arrow) (with permission, from Dvorak et al., 1991a). Bars: (A) 1.4 pm; (B) 0.8 pm.

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tion chambers, of typical secretory granules, stimulated by IgE-mediated mechanisms (Dvorak, 1988a, 1991). Thus, this is an explosive and rapid secretory event in human basophils which is completed within minutes of stimulation. In contrast, slow emptying of cytoplasmic granules characterizes PMD of human basophils. AND, then, is a special type of regulated secretion, of which all granule-containing secretory cells, including basophils and mast cells, are capable. Important anatomical findings associated with AND in appropriately stimulated human basophils include extrusion of CLCs and dense concentric membranes in concert with granule particulate contents through multiple pores in the cell membrane; shedding of multiple membranes and portions of cell processes; surface amplification by externalization of granule containers; formation of intracytoplasmic degranulation chambers or sacs by fusion of multiple granule membranes; decreased granule numbers; decreased numbers of cytoplamic vesicles; and completely degranulated, viable basophils. AND has not been identified in human basophils in vivo for several reasons. These include the rarity of these cells, the sampling problem inherent in electron-microscopic evaluation of tissues into which basophils have migrated, and the rapidity of the reaction. The clinical condition caused by the massive release of histamine, known as anaphylaxis, would surely be accompanied by evidence of AND in human basophils and mast cells, if samples were available. However, ultrastructural studies have not been carried out in these life-threatening circumstances. A large number of studies have been performed, however, using isolated blood basophils (MacGlashan and Lichtenstein, 1980). Various secretogogues have been used to stimulate AND in isolated, partially purified human blood basophils from allergic and nonallergic donors. Our early ultrastructural studies are summarized in Dvorak (1988a). The secretogogues studied include antigen (Dvorak et al., 1980b), anti-IgE (Ishizaka et al., 1985a), complement (Dvorak et al., 1981c), histaminereleasing activity (HRA) from cultured mononuclear cell supernatants (Dvorak et al., 1984b), and mannitol (Findlay et al., 1981). Although many anatomical findings are similar in these studies, important differences have been established. Initially, we examined AND induced by antigen E in basophils obtained from allergic donors (Dvorak et al., 1980b). These studies showed that the main untrastructural event was the extrusion of membrane-free granules through multiple pores in the plasma membrane around the circumference of the cell. Rarely, several granules fused before extrusion. Maximum granule and histamine release occurred together over 15 min after antigen stimulation. Granule containers were also exteriorized, leading to an extensively amplified cell surface in cells with maximal release of granules. Mature basophils developing in growth factor-containing cultures of human cord


103

blood cells underwent similar AND when passively sensitized cells were stimulated with anti-IgE (Ishizaka et al., 1985a). Release of histamine by hyperosmolar materials was studied when peripheral blood basophils were exposed to mannitol (Findlay et al., 1981). In addition to AND, as described above, some of these basophils developed large, central, intracytoplasmic d e g r a d a t i o n sacs containing membranefree granules analogous to those that regularly develop during AND induced in guinea pig basophils (Dvorak et al., 1981b). The mononuclear cell product, HRA, also stimulates histamine release from human basophils (Thueson et al., 1979a,b). Several time points after stimulation with HRA were examined by electron microscopy (Dvorak et al., 1984b). Typical AND characterized the anatomy associated with histamine release. Some cells failed to undergo AND but formed prominent motile structures, or uropods, instead. Stimulation of human basophils with the complement factor C5a resulted in rapid histamine release, accompanied by the morphology of AND (Dvorak et al., 1981~).Additionally, we noted minute openings between the plasma membrane and underlying unaltered granules which readily admitted the electron-dense tracer, cationized ferritin. These were accompanied by plasma membrane invaginations. These events preceded the widening of the pores and extrusion of granules to cul-de-sacs among elongated surface processes. Extensive generation of intragranular concentric dense membranes did not occur in these C5a peptide-stimulated cells. 2. Piecemeal Degradation

Piecemeal d e g r a d a t i o n is a term introduced to explain the ultrastructural finding of partially and completely empty cytoplasmic granule containers, in the absence of intergranule fusions or granule fusions to the plasma membrane and subsequent extrusion of granule contents to the microenvironment (Fig. 2A). It occurs in human basophils participating in large numbers in experimentally induced and sequentially biopsied contact allergy lesions in human skin (Dvorak and Dvorak, 1975; Dvorak et al., 1974b, 1976a; Galli et al., 1984). These mature basophils are also characterized by large numbers of cytoplasmic vesicles, some of which are attached to granules. Visible particles like those in granules, homogeneously dense contents, or apparently empty (electron-lucent) interiors prevail among these smooth membrane-bound, small cytoplasmic vesicles. Therefore, PMD, simply stated, defines the release of granule materials, in the absence of typical granule extrusion, from basophils. It is a process mediated by vesicular transport that develops slowly over days in skin biopsies of evolving contact allergies (Dvorak et al., 1976a). This form of stimulated secretory activity is to be distinguished from AND.

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PMD of basophils has been identified in a number of experimentally induced or naturally occurring circumstances (reviewed in Dvorak, 1992a). Initially, we performed a kinetic analysis of experimentally produced lesions of contact allergy (Dvorak et aL, 1974b, 1976a) and sequential biopsies taken during skin graft rejection in humans (Dvorak et al., 1979c, 1980g). Basophils migrated into involved tissues from blood vessels and over time developed piecemeal losses of their particulate granule content. Quantification of skin contact allergy lesions over 6 days revealed significant increases in basophils undergoing PMD. Subsequent to these experimental studies of human skin contact allergy, we examined participating basophils in the peripheral blood and tissues from patients with Crohn’s disease, an inflammatory bowel disease of uncertain etiology (Dvorak et al., 1980~).Again, we were able to identify PMD as the predominant morphological expression of these reactive basophils. More recently, we have identified PMD in human basophils in uninvolved tissues of the small intestine in patients with ulcerative colitis, another inflammatory bowel disease (Dvorak et al., 1992b). Basophils migrate into tissues and body fluid from the blood in several human diseases (Dvorak, 1992a). In many of these circumstances, we have identified PMD of these participating cells (Dvorak, 1988a, 1991; Dvorak et al., 1982b; Fox et al., 1984), and PMD is evident in published electron micrographs (Collin and Allansmith, 1977; Glasser et al., 1976).

3. A Degranulation Model Proposed in 1975 In 1975, we proposed a general model of basophil d e g r a d a tio n (Fig. 3), based on our studies of guinea pig and human basophils, to account for the varied rates of granule substance release occurring in a variety of physiological and pathological circumstances (Dvorak and Dvorak, 1975). The model holds that PMD occurs by means of exocytotic vesicles which bud from the granule membrane, carrying with them small quanta of intact or dissolved granule material which flow to the cell surface, where they fuse with the plasma membrane and discharge their contents into the extracellular space. Glycogen aggregates that are closely associated with granules at sites of vesicle attachment may afford an energy source for vesicle budding. Once separation of vesicle from the granule membrane is complete, transport of vesicles may proceed at random. A net flux of granule contents out of the cell will result from vesicle movement if, for reasons of chemical structure, vesicles are able to fuse only with each other and with the membranes of other granules and the plasma membrane but not with the membranes limiting other cellular organelles. It is useful to postulate a closely coupled transport of endocytotic vesicles migrating from the cell surface to the cytoplasmic granules associated with vesicular exocytosis. Coupled exocytosis is necessary to account for the

BASOPHIL DEGRANULATION FIG. 3 Schematic diagram of a degranulation model in basophils. (A) Anaphylactic degranulation of guinea pig basophils. (1) Increased cytoplasmic vesicles 1 min after triggering with concanavalin A. Vesicles have fused with some granules; one has formed a bridge joining adjacent granules. (2) At 5-20 min, membranes of granules and cytoplasmic vesicles have fused with each other and with the plasma membrane, forming a “degranulation sac” containing membrane-free granules that open to the cell exterior by a narrow pore. Only occasional granules have escaped outside the cell, and many of these remain adherent to the cell surface. (3) Filaments have become prominent in the cytoplasmic processes embracing the degranulation sac. Over a period of hours, these processes retract, as indicated by arrows, presumably powered by the filaments, opening the degranulation sac to the exterior and permitting granule escape. (B) Anaphylactic degranulation of human basophils. (1, 2) Fusion of membranes of individual or joined granules directly with plasma membrane, with resulting discharge of granule contents. (3) Empty granule (EG) membranes eventually become part of plasma membrane as in 4. Note that portions of granule matrix remain adherent to plasma membrane. (C) Piecemeal degranulation of human (or guinea pig) basophils as in certain cell-mediated immune reactions. (1) Endocytotic vesicles travel from plasma membrane through the cytoplasm to basophilic granules, with which they fuse. (2) Exocytotic vesicles, containing bits of granule matrix, bud from granule membrane, pass through the cytoplasm, and fuse with the plasma membrane, discharging their contents to the extracellular space. (3) Same as 2, but budding proceeds at a faster rate. (4) Still more rapid exocytosis, in which vesicles bud from granule at so fast a rate that they do not separate from each other and form a channel joining the granule with the extracellular space. This is equivalent to anaphylaxis. N, nucleus (with permission, from Orenstein et al., 1981).

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undiminished (often slightly increased) size of granules which are releasing their contents and, hence, continuously losing portions of their enveloping membranes. An imbalance in the amount of vesicular traffic would result in changes in granule size. For example, excess efflux would produce smaller granules, and excess influx would produce larger granules. The extracellular fluid transported to the granules by endocytotic vesicles may provide solvent for the parital dissolution of granules that is regularly observed, for example, in the basophils infiltrating the reactions of allergic contact dermatitis (Dvorak et al., 1976a). Conceivably, the extra membranes accumulating within these basophil granules may be derived from those of the endocytotic vesicles, also indicating an imbalance between vesicular influx and efflux. In our vesicular transport model, the release of granule contents is postulated to proceed at a rate governed by the frequency of discharge of microvesicles from the granule membrane. The frequency of exocytosis is possibly determined by the rate of endocytosis which, in turn, may be controlled and modified by agents (antibodies, and probably other materials, such as cytokines, lymphokines, interleukins, bacterial peptides, etc.) acting at the cell surface. Based on the occasional granule alterations observed in “normal” circulating basophils, it is likely that a slow release of granule substance occurs under physiological conditions. In delayed-type cell-mediated immunologic reactions, degranulation apparently proceeds at a substantially greater pace. At still faster rates of degranulation, a threshold would eventually be reached, above which there would be insufficient time between successive discharges to permit complete separation of individual vesicles from the plasma or granule membranes or from each other. Under these conditions, endocytotic and/or exocytotic vesicles budding from the plasma or granule membranes would not form discrete, spherical structures; rather, they would coalesce to form continuous channels. Better developed channels might link the cell surface with granules and/or interconnect neighboring granules, depending on random collisions with either the plasma or the granule membranes. Continuous channels of this sort would lead to fusion of granule membranes with each other and with the plasma membrane, characteristic features of AND. It is clear that basophils have a role in mediating immediate hypersensitivity reactions or allergic diseases. It also seems clear that PMD of basophils may provide materials important to the onset, sustenance, or healing of a wide variety of disorders collectively involved with cell-mediated immunity. It is not clear, however, what role(s) these cells have in normal physiology, ie., in health. Secretion biologists categorize release mechanics from the various secreting cells in the body (the normal functions for most of which are known) as regulated or constitutive (Lacy, 1975; Orci et al., 1973; Palade, 1975;


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Jamieson and Palade, 1971; Kelly, 1985). Regulated secretion, put simply, means an episodic, triggered release of stored cellular synthetic products; constitutive secretion means a continuous flow of synthetic cellular products. Regulated secretion utilizes classic exocytosis of membrane-free granules; constitutive secretion occurs by vesicular transport. AND of basophils, then, is an example of regulated secretion; PMD of basophils is similar to, and perhaps an example of, “up-regulated” constitutive secretion.

C. Basophil Secretory Granules as Storage Organelles for Exogenous Proteins Classically, secretory granules were not considered to be storage repositories €orexogenous proteins. Rather, their contents were generally considered to derive from the synthetic capacities of individual cell lineages, such contents being held in reserve until appropriate secretogogues initiated their secretion by exocytosis of entire granules. So, too, were the granular contents of basophils considered. However, in 1972,we published our initial studies which showed that basophils stored an electron-dense exogenous tracer protein, horseradish peroxidase (Fig. 4A), in their secretory granules (Dvorak et al., 1972), thus setting the stage for release of such exogenous proteins when appropriately stimulated. In certain circumstances, internalization of the cellular products of another lineage, such as eosinophil peroxidase (EPO) (Fig. 4B), could render the cytochemical profile of basophils as mimicking that of eosinophils (Dvorak et aL, 1985a,b) in inflammatory reactions rich in EPO released from reactive eosinophils. Our initial studies of this general cell-biological mechanism in purified guinea pig basophils (Dvorak et al., 1974c, 1980d, 1985a), and later in human basophils arising in cultures replete with eosinophils undergoing EPO secretion (Dvorak et al., 1985b), have further documented this mechanism. Substantial studies of other cell lineages now provide greater generality €or this event (Handagama et al., 1987,1989, 1993; Harrison et al,, 1990; Hill et al., 1996; Wagner et al., 1996).

1. Uptake and Granule Storage of Exogenous Proteins by Basophils We noted a uniquely rich complement of small (100 to 150 nm) smooth vesicles in the cytoplasm of guinea pig bone marrow and peripheral blood basophils in situ (Dvorak et al., 1972; Dvorak and Dvorak, 1975). These structures suggested the possibility that basophils were engaged in endocytosis and perhaps secretion, using these vesicles as transport vehicles. Initially, we demonstrated the uptake function of these structures using an

FIG. 4 Guinea pig basophils prepared to demonstrate peroxidase activity by a cytochemical technique after exposure to HRP for 30 min at 37°C (A) or EPO for 15 min at 37°C (B). Note the large number of HRP-filled, dense, black cytoplasmic vesicles adjacent to a nonperoxidase-containing guinea pig basophil granule with a mixture of longitudinal and hexagonal arrays in A. The basophil in B contains EPO-loaded, dense, black vesicles and granules that are completely filled with dense. EPO, partially filled with EPO, or do not contain any EPO (arrow). N, nucleus (with permission, from Dvorak and Dvorak, 1993). Bars: (A) 0.6 pm; (B) 1.8 pm.


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intravenous tracer, horseradish peroxidase, which is rendered electrondense by a cytochemical procedure (Dvorak et al., 1972). These studies clearly showed uptake and transport of this protein to granules in viva Subsequently, using purified guinea pig basophils in v i m and wash-out experiments, we were able to show transport of horseradish peroxidase in vesicles in the reverse direction, such as cellular secretion might utilize in certain circumstances (Dvorak et al., 1980d). These studies of vesicular transport were extended to include uptake and transport to granules of exogenous EPO by guinea pig basophils, mouse mast cells, and mouse granular lymphocytes displaying NK activity in vitro, thereby documenting a role for vesicular transport of a biologically important protein released in eosinophil-rich inflammatory reactions and extending this uptake mechanism to mast cells and NK cells (Dvorak, 1991; Dvorak et al., 1985a). In studies of human basophils arising in factor-supplemented, eosinophilrich cultures, we similarly demonstrated endocytosis, vesicle attachment, and discharge into secretory granules in basophils of EPO released by the eosinophils (Dvorak et al., 1985b).

2. Uptake and Granule Storage of Exogenous Proteins by Other Secretory Cell Lineages Handagama et al. (1987, 1989, 1993) confirmed our findings in basophils by demonstrating the ability of megakaryocytes (MK) and platelets to store horseradish peroxidase in their alpha granules. Additionally, they showed that intravenously injected albumin, IgG, and fibrinogen were stored in guinea pig MK and platelet alpha granules. Platelet alpha granule contents include those proteins synthesized by precursor MK as well as numerous exogenous proteins that are synthesized elsewhere and are taken up by platelets from the plasma. Thus, platelet alpha granule endogenous proteins include platelet factor 4 and P-thromboglobulin, whereas exogenous proteins resembling plasma factors include albumin, factor V, thrombospondin, fibronectin, fibrinogen, and vitronectin (Harrison et al., 1990). Patients with congenital afibrinogenemia do not have fibrinogen in their platelets, suggesting that all fibrinogen in normal platelets is obtained by internalization of plasma stores (Harrison et al., 1990). Similarly, the platelets of patients with Glanzmann’s thrombasthenia are devoid of the fibrinogen receptor, GPIIb/IILa, and also do not have fibrinogen stores in their platelets (Harrison et al., 1990). These two illustrations show that circulating fibrinogen is necessary in order for platelets to store fibrinogen in alpha granules (Harrison et al., 1990), and, if platelets are devoid of their fibrinogen receptor, they also cannot internalize and store fibrinogen taken up from the plasma (Harrison et al., 1990). More recently, a unique disintegrin, which is a specific antagonist of the fibrinogen receptor GPIIb-IIIa, was used to

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demonstrate that all endocytotic uptake of plasma fibrinogen by platelet and MK alpha granules was inhibited (Handagama et al., 1993). Thus, platelet fibrinogen stored in alpha granules is derived entirely by endocytosis and not by synthesis in platelet precursor MK (Handagama et al., 1993). Similarly, a human megakaryocytic cell line was used to show that vitronectin is not a synthetic product of MKs but is endocytosed from serumderived vitronectin and stored in secretory granules where it was colocalized with an MK secretory protein, type 1 plasminogen activator inhibitor (Hill et al., 1996). In another study, it was determined that haptoglobin was not synthesized de novo by neutrophils. Rather, this ubiquitous mammalian serine protein (Javid, 1978) was internalized and concentrated in cytoplasmic granules and, in turn, was secreted following neutrophil activation (Wagner et al., 1996). Thus, at least two additional secretory cells, neutrophils and megakaryocytes (and their progeny, platelets), as well as basophils, store exogenous proteins in secretory granules.

V. Development of Tools for Advanced Cell-Biological Studies of Basophils A. Early Tools For -90 years following Ehrlich’s discovery of human basophils (Ehrlich, 1879), studies were seriously impeded by the inability to obtain cells for study. Since only 0.5% of the peripheral blood cells are basophils, most studies utilized malignant basophils, which increase in myelogenous leukemia (Dvorak, 1988a; Dvorak et al., 1981a; Dvorak and Dvorak, 197.5; Galli et al., 1983b; Maloney and Lange, 19.54) and were of light-microscopic preparations. Immunologists determined that histamine correlated with basophils, of all formed elements of blood (Graham et al., 1955), and that histamine could be released from sensitized basophils-reactions termed immediate hypersensitivity (Dvorak, 1988a). A new immunoglobulin, IgE, was found to be responsible for this release reaction (Ishizaka et al., 1970; Ishizaka and Ishizaka, 1975). In the 1970s,improved light-microscopic methods of Giemsa-stained plastic sections and electron microscopy contributed to the recognition that basophils were an important component of the inflammatory exudate in cell-mediated immunity (Dvorak 1987, 1988a; Dvorak et al., 1970; Monahan and Dvorak, 1985), and biochemical studies of AND and mediator release accelerated (Dvorak, 1988a). In the early 1980s, ultrastructural analysis of AND stimulated by several triggers was facilitated by the development of reliable methods for purifying peripheral

CELL BIOLOGY

OF THE BASOPHIL

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blood basophils (Dvorak, 1988a; MacGlashan and Lichtenstein, 1980; MacGlashan et al., 1982a; Dvorak et al., 1974~).

6. Newer Tools 1. Tools That Increase Basophil Supplies

In addition to earlier methods for purifying peripheral blood basophils (Dvorak, 1988a), new methods produced basophils for studies. Mouse basophils were successfully sorted from bone marrow and spleen on the basis of the presence or absence of cell surface markers (Seder et al., 1991a; Dvorak et al., 1993a, 1994a). Specific cytokines, which were determined to be basophilopoietins, successfully produced immature and mature human basophils in various culture systems (Dvorak and Ishizaka, 1995) and increased circulating basophils in monkeys in vivo (Dvorak et al., 1989a). The cytokines useful for this purpose are available in recombinant forms. Thus, IL-3 induced human basophils to develop in vitro (but not mast cells) (Dvorak et al., 1989b; Saito er aZ., 1988); SCF induced human mast cells to develop in vitro (small numbers of basophils also developed in this suspension culture system) (Dvorak and Ishizaka, 1995); IL-3 induced circulating basophils in monkeys (Dvorak et al., 1989a). Neither IL-3 nor SCF alone (or in combination) induced mouse basophils to develop or survive in vitro, but each factor supported mouse mast cells in vitro (Dvorak et al., 1994a). Identification of these various lineages was accomplished by routine ultrastructural methods. 2. Ultrastructural Kinetic Models of Basophil Secretion

We performed extensive ultrastructural analyses of human basophil secretion based on the kinetics of mediator secretion induced with two secretogogues (Dvorak et al., 1991a, 1992a). The two releasing agents were FMLP-a bacterial peptide with rapid biochemical release kinetics (Warner et al., 1989)-and TPA-a tumor-promoting phorbol diester with slow biochemical release kinetics (Schleimer et al., 1981, 1982). Morphologic changes, ascertained with routine ultrastructural methods, provided a solid basis for future studies with specific ultrastructural tags and tracers.

a. FMLP-Induced D eg r a d a tio n of Human Basophils We examined the kinetics of morphological change induced by stimulation of human basophils with FMLP, using purified cells from normal donors (Dvorak et al,, 1991a). Samples were prepared for electron microscopy at 0,10,20, and 30 s and 1,2,5, and 10 min poststimulation with FMLP. The ultrastructural

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morphology of basophils induced by FMLP stimulation was unique. FMLPstimulated basophils, for example, initially emptied granules, the containers of which remained in the cytoplasm in situ. Many of these empty containers enlarged dramatically before accumulated intragranular membranes and vesicles were released along with the extrusion of the empty container membranes. These early events (occurring 0-20 s poststimulation) were morphologically analogous to PMD of human basophils in viva In both cases, large numbers of cytoplasmic vesicles were present, and in the FMLPstimulated model, vesicle numbers increased in conjunction with acquisition of the morphology of PMD and preceded the development of the morphology of granule extrusions. Vesicle numbers decreased dramatically in cells displaying the morphology of AND. The extrusion of full granules (and the membranes of granules previously emptied by PMD) coincided with the half-maximum histamine release reported for this model at 1.3 min poststimulation (Warner et al., 1989). Shedding of intragranular concentric dense membranes and vesicles, surface membranes and processes, and extrusion of formed, intragranular CLCs accompanied the exocytosis of granules. Extraordinary membrane shifts occurred and persisted over the 10-min kinetic interval examined poststimulation and coincided primarily with the later time frame (3.5 min) within which LTC, was generated and released from human basophils (Warner et al., 1989). Granule exocytosis, which peaked at 1 min in this model, extended from 20 s to 2 min in the ultrastructural samples studied after FMLP stimulation. The extrusion of granules took place through multiple pores in the plasma membrane; extruded intragranular materials remained associated with cul-de-sacs formed by the irregular surfaces of releasing basophils. These materials included rounded, granule-shaped, membranefree aggregates of granule particles, spherically shaped homogeneous structures identical to intragranular CLCs, and masses of concentric dense membranes. Elongated cell processes, cells actively shedding membranous surface materials, and completely degranulated, granule-free cells were prevalent between 20 and 120 s. Thus, a degranulation continuum of morphologic change occurred when human basophils were stimulated with FMLP. Essentially, in morphologic kinetic studies, a continuum of PMD occurred early and progressed to AND at later time points, coincident with the rapid release of histamine.

b. TPA-InducedDegranulation ofHuman Basophils We also performed an ultrastructural kinetic analysis of tumor-promoting phorbol diesterinduced degranulation of human basophils (Dvorak et al., 1992a), a substance known to elicit histamine release (but not LTC, release) (Schleimer el al., 1981). Unlike the rapid kinetics associated with IgE-mediated histamine release (15 min) or FMLP-mediated histamine release (2 min), the hista-


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mine release stimulated from human basophils by the phorbol ester TPA is slow, reaching a maximum by 1 h (Schleimer eta/., 1981). We prepared, for ultrastructural analysis, TPA-stimulated and control samples of human basophils at multiple intervals, which were selected to precede and include maximum histamine release for this secretogogue. We found that, as in biochemical studies (Schleimer et aL, 1981, 1982), TPA was a unique ultrastructural secretogogue for human basophils. For example, extensive PMD was evident in multiple samples, achieving -50% granule alteration by 45 min poststimulation. This evidence of empty granules was associated with, and preceded by, a rapid, extensive, and sustained elevation in particlecontaining cytoplasmic vesicles, compared to buffer-incubated controls at all time points examined (p

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