Amphibian Teeth

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    Biol. Rev. (2007), 82, pp. 4981 49doi:10.1111/j.1469-185X.2006.00003.x

    Amphibian teeth: current knowledge,

    unanswered questions, and some

    directions for future researchTiphaine Davit-Beal, Hideki Chisaka*, Sidney Delgado and Jean-Yves Sire

    UMR7138-Systematique, Adaptation, Evolution, UniversitePierre & Marie Curie-Paris6 Case7077, 7 Quai St-Bernard,

    Paris75005, France

    (Received12 December2005; revised31 August2006; accepted11 September2006)

    ABSTRACT

    Elucidation of the mechanisms controlling early development and organogenesis is currently progressing inseveral model species and a new field of research, evolutionary developmental biology, which integratesdevelopmental and comparative approaches, has emerged. Although the expression pattern of many genesduring tooth development in mammals is known, data on other lineages are virtually non-existent. Comparisonof tooth development, and particularly of gene expression (and function) during tooth morphogenesis anddifferentiation, in representative species of various vertebrate lineages is a prerequisite to understand what makesone tooth different from another. Amphibians appear to be good candidates for such research for several reasons:tooth structure is similar to that in mammals, teeth are renewed continuously during life ( polyphyodonty),some species are easy to breed in the laboratory, and a large amount of morphological data are already availableon diverse aspects of tooth biology in various species. The aim of this review is to evaluate current knowledge onamphibian teeth, principally concerning tooth development and replacement (including resorption), and changesin morphology and structure during ontogeny and metamorphosis. Throughout this review we highlightimportant questions which remain to be answered and that could be addressed using comparative morphologicalstudies and molecular techniques. We illustrate several aspects of amphibian tooth biology using data obtainedfor the caudatePleurodeles waltl. This salamander has been used extensively in experimental embryology researchduring the past century and appears to be one of the most favourable amphibian species to use as a model instudies of tooth development.

    Key words: lissamphibians, Anura, Caudata, Gymnophiona, tooth, odontogenesis.

    CONTENTS

    I. Introduction ...................................................................................................................................... 50II. Critical evaluation of the use of teeth in amphibian phylogeny ..................................................... 51

    (1) The origin of the lissamphibians ................................................................................................ 51

    (2) The significance of teeth for lissamphibian phylogeny ............................................................. 51(3) The significance of teeth for lissamphibian systematics ............................................................ 53III. Lissamphibians in the laboratory ..................................................................................................... 53IV. Overview of tooth morphology and structure in lissamphibians .................................................... 54

    (1) Enameloid .................................................................................................................................. 55(2) Dividing zone .............................................................................................................................. 56

    * Present address: Department of Anatomy, Nihon University School of Dentistry at Matsudo, 870-1, Sakaecho, Nishi-2, Matsudo,Chiba 271-8587, Japan

    Address for correspondence: (Tel/Fax: 33-1-44-27-35-72; E-mail: [email protected]).

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    (3) Pedicel ......................................................................................................................................... 56V. Tooth development and replacement .............................................................................................. 60

    (1) Tooth morphogenesis and differentiation .................................................................................. 60(a) Caudata ................................................................................................................................. 60(b) Gymnophiona ........................................................................................................................ 64(c) Anura (excluding Pipidae) ..................................................................................................... 64(d) Pipidae ................................................................................................................................... 65

    (2) Relationships between tooth and bone support development .................................................. 66

    (3) Tooth replacement and resorption ............................................................................................. 67(4) Tooth replacement pattern ......................................................................................................... 70

    VI. Tooth changes ................................................................................................................................... 71(1) Monocuspid to bicuspid: the role of thyroxine at metamorphosis ........................................... 71(2) Bicuspid to monocuspid: the role of androgens ........................................................................ 71

    VII. Tooth regeneration ........................................................................................................................... 71VIII. Directions for future research ........................................................................................................... 72

    (1) Are pedicellate teeth homologous among lissamphibians? ....................................................... 72(2) Are the dentition pattern and development of the dental lamina important features for

    lissamphibian systematics? .......................................................................................................... 72(3) Do ameloblasts participate in enameloid formation in lissamphibian larvae? ......................... 72(4) How does the enameloid-enamel transition proceed through caudate ontogeny? .................. 73(5) How do the dividing zone and the pedicel appear during lissamphibian ontogeny? .............. 73(6) What mechanisms control the initiation of a replacement tooth in lissamphibians? ............... 73(7) Which mechanisms control the initiation of tooth resorption? ................................................. 73(8) What is the fate of the tooth tip in adult lissamphibians? ........................................................ 74(9) What mechanism controls the periodicity of lissamphibian tooth replacement? ..................... 74

    (10) How do thyroxine levels affect tooth shape in lissamphibian teeth? ........................................ 74IX. Conclusions ....................................................................................................................................... 74X. Acknowledgements ............................................................................................................................ 75

    XI. References ......................................................................................................................................... 75

    I. INTRODUCTION

    Rapid recent progress in molecular biology and develop-mental genetics has allowed investigators in odontology tore-open doors that have remained closed since the end of the1970s. Over the last two to three decades new tools haveallowed investigations to extend from tissue and cellularintegration to the molecular level, and understanding ofmechanisms controlling tooth development is progressingrapidly using the mouse as a model species. The expressionpattern of more than 120 genes during mammalian toothpatterning and development has been described: see http://bite-it.helsinki.fi (Nieminen et al., 1998). We know that allanimals share many of the same molecular processes,including regulatory genetic pathways. However, how thesecommonalities are used to make one tooth different from

    another is far from understood. This question can only beanswered through comparison of gene expression (andfunction) during odontogenesis in various lineages or withinmultiple taxa in the same lineage. Answering this questionwould lead to an understanding of how teeth have changedduring evolution in terms of initiation (time), position(space), type (morphology), mode of replacement, etc. Infact, studies of the evolutionary developmental biology ofteeth are virtually non-existent, with the exception of somerecent evolutionary work on rodent teeth (Jernvall, Keranen& Thesleff, 2000; Kangaset al., 2004). In particular, Kangas

    et al. (2004) show correlated changes in dental characters asa function of quantitative changes in intercellular signalling,

    and conclude that most aspects of tooth shape could havethe potential for independent changes during evolution.Although tooth diversity (e.g. shape, location, structure) is

    well known in numerous species (including extinct ones)from the main vertebrate lineages (Huysseune & Sire, 1998),and tooth development has been compared in selectedspecies (Sire et al., 2002), research suffers from a lack ofcomparison of the genes involved (and of their function) withnonmammalian lineages. In parallel with detailed odonto-genetic studies in the mouse, it is important to comparetooth development in species representative of other lineages(e.g. reptiles, amphibians, actinopterygian fishes, sharks) orin a lineage that includes taxa with variants, so that toothevolution can be assessed in a phylogenetic framework.

    Among toothed vertebrates, mammals have either a singleor two tooth generations (mono- or diphyodonty), whilenonmammalian species renew their teeth continuously(polyphyodonty). The study of tooth development in poly-phyodonts would seem to have several advantages for thebiologist, and molecular studies have just begun. The firstdata are available for the zebrafishDanio rerio(Laurentiet al.,2004; Jackman, Draper & Stock, 2004; Borday-Birrauxet al.,2006) but studies on tooth development are far from easy inthis species which possesses teeth in the pharyngeal regiononly (Huysseune, Van der heyden & Sire, 1998; Van der

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    heyden, Huysseune & Sire, 2000). In addition, the zebrafishbelongs to the actinopterygian lineage, which means morethan 420 million years of evolution separate this teleost fishfrom the mouse; the tooth structure and late odontogenicphases in fish differ from those of mammals (Huysseuneet al.,1998; Laurenti et al., 2004; Borday-Birraux et al., 2006).Therefore, to address some of the still unanswered questionsin tooth biology, studies of species belonging to the tetrapod

    lineage, such as amphibians and reptiles, may be useful.In the context of tooth evolutionary developmental

    biology, amphibians have several features of interest: (i)their teeth have a similar structure to mammals, (ii) severalspecies (e.g. Pleurodeles waltl, Ambystoma mexicanum, Xenopuslaevis, Silurana tropicalis, Eleutherodactylus coqui, and Bombinavariegatus) are easy to breed in the laboratory (in contrast tomany reptiles), and (iii) for almost a century a large amountof data has accumulated on various aspects of amphibiantooth biology. Numerous molecular tools (a large number ofsequenced genes, possibility of transgenesis) are nowavailable forX. laevisand S. tropicalis (its genome is currentlybeing sequenced), which are used as models in embryolo-gical and developmental studies. Numerous genes have alsobeen sequenced for various other species (e.g. A. mexicanum,A. tigrinum, E. coqui;see http://www.ncbi.nlm.nih.gov).

    The aims of this review are (i) to evaluate the knowledgeaccumulated during the past century on amphibian teeth withrespect to development, replacement (including resorption),changes in morphology and structure in relation to growth andmetamorphosis, and (ii) to highlight unanswered questions inamphibian tooth biology and tooth development.

    II. CRITICAL EVALUATION OF THE USE OFTEETH IN AMPHIBIAN PHYLOGENY

    (1) The origin of the lissamphibians

    Amphibians appeared by the end of the Devonian or theearly Carboniferous [approximately 300 million years ago(mya)], when the two tetrapod lineages, reptiliomorphs(which include amniotes) and amphibians, separated froma tetrapod ancestor (Laurin, 1998a,b; Carroll, 1988). Theycomprise both living species and their extinct relatives,grouped into the lissamphibian clade (frogs, salamandersand caecilians), and several extinct lineages that have beengrouped either into a large group including lepospondylsand temnospondyls (Trueb & Cloutier, 1991; Lombard &Sumida, 1992; Ahlberg & Milner, 1994), or into lepospondylsonly (Laurin & Reisz, 1997; Laurin, 1998a, b). Lissamphi-bians are supposed to have originated at the onset of theTriassic period (approximately 250 mya), probably froma lepospondyl ancestor (Laurin & Reisz, 1997, 1999;Laurin, 2002). However, the fossil record has providedlittle evidence on the evolutionary origin of lissamphibians,and it is difficult to postulate which group among thePaleozoic lepospondyls is most closely related to them(Laurin, 1998a, b; Anderson, 2001). This explains why thequestion of the origin of the lissamphibians has been longdebated in the literature (Romer, 1945; Holmgren, 1952;Eaton, 1959; Jarvik, 1960; Bolt, 1969, 1977, 1979, 1991),

    and why debate continues (e.g. Milner, 1988, 1993, 2000;Trueb & Cloutier, 1991; Laurin, 1998a, 1998b, 2002;Schoch & Carroll, 2003). After re-examination of a numberof characters in extant and extinct amphibian species(including skeleton and soft anatomy), the hypothesis ofa common ancestry for the lissamphibians has neverthelessbeen retained (Szarski, 1962; Parsons & Williams, 1963;Laurin, 1998a, b, 2002; Schoch & Carroll, 2003). This hy-

    pothesis is also supported by molecular phylogenies showingthe monophyly of lissamphibians: caecilians and salaman-ders being sister taxa, with frogs their outgroup (Hedges,Moberg & Maxson, 1990; Hedges & Maxson, 1993; Hayet al., 1995; Feller & Hedges, 1998). Fossil records indicatethat the crown-group lissamphibians started diversifying bythe end of the Permian (approximately 250 mya), before thebreakup of Pangaea, and their diversity increased greatlyduring the Jurassic and Early Cretaceous periods (approx-imately 200-150 mya) (Schoch & Carroll, 2003). This wasrecently confirmed using a molecular phylogeny (SanMauro et al., 2005). Putative ancestors of salamanders arerecognized from the Carboniferous-Permian boundary(Schoch & Carroll, 2003), a fossil caecilian possessingreduced limbs, Eocaecilia micropodia, has been discoveredfrom the Jurassic period in Arizona (Jenkins & Walsh, 1993),and frogs are also known from the Triassic and Jurassic(Estes & Rieg, 1973; Roelants & Bossuyt, 2005).

    Today (AmphibiaWeb database, Nov. 2005), Lissamphi-bia contains 5953 species distributed into three orders:Gymnophiona (caecilians) with 171 species; Anura (frogs,including pipids, and toads) with 5230 species; and Caudata(salamanders and newts) with 552 species. Note that we usethe current standard taxonomic reference for the amphibianorders, i.e. node-based names defined on the basis of Recenttaxa instead of stem-based names which include the fossiltaxa: Anura instead of Salientia for frogs, Caudata instead of

    Urodela for salamanders, and Gymnophiona instead ofApoda for caecilians (e.g. Trueb & Cloutier, 1991; Canna-tella & Hillis, 1993, 2004; Ford & Cannatella, 1993; Frost,2004]. Fewer than ten species from each order have beenexamined so far with respect to tooth development (Fig. 1).

    (2) The significance of teeth for lissamphibianphylogeny

    In most stem-tetrapods and in extinct amphibians, teethwere haplodont (i.e. simple: conical and unicuspid) withsome heterodonty (i.e., differing in general appearancethroughout the mouth but mainly in size) in a few species.Tooth attachment to the bone support was in general

    subthecodont (i.e. partially set in a socket), and sometimespleurodont (i.e. attached to the labial side). Tooth structurehas been studied in a few early tetrapods (e.g. temnospond-

    yls) and lepospondyls (microsaurs, nectrideans) (e.g. Owen,1842; Bystrow, 1938; Parsons & Williams, 1962; Peyer,1968; Bolt, 1969, 1979). In general, the teeth were conicalwith a large base. The dentine shaft surrounded a pulpcavity and was covered by a thin enamel layer. Toothstructure was characterised by a typical folded arrangementof the dentine, called plicidentine (Fig. 2). Plicidentine is,however, not a typical feature of early amphibians, and

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    cannot be used as a strong phylogenetic argument: it isabsent in several Paleozoic amphibians, in particular amongmicrosaurs (Peyer, 1968), and it is widespread in basalsarcopterygian taxa (Schultze, 1969). Some temnospondylspossessed a branchial apparatus, in which small tooth-bearing plates occurred in the throat region (Hook, 1983;

    Coates, 1996), a location which is similar to the pharyngealteeth described in a number of actinopterygians.

    The value of tooth characters as evidence of lissamphi-bian phylogeny has been investigated in depth by Parsons &Williams (1962, 1963). Although the three lissamphibianorders possess relatively few distinguishing characters(which explains the current debate on their relationships),the presence of bicuspid and pedicellate teeth has beenwidely accepted as strong support for their monophyly (seediscussion in Laurin, 1998a; Schoch & Carroll, 2003;Schoch & Milner, 2004). In a series of investigations on the

    morphology of the mouth cavity of caudates, H. Greven,G. Clemen and others (see, e.g. Greven & Clemen, 1979,1980, 1985; Clemen & Greven, 1977, 1979, 1980, 1988,2000) have shown that the number and course of dentallaminae are also of phylogenetic importance. Lissamphibianteeth are characterised by the division of the dentine shaftinto a relatively short crown and a long pedicel, separatedby an uncalcified (or poorly calcified) region resembling aligament, called the dividing zone. Pedicellate teeth arepresent in fossil representatives of caudate, gymnophioneand anuran lineages. However, in a few lissamphibianspecies, teeth lack a dividing zone, but this feature isconsidered a derived rather than a plesiomorphic character(Parsons & Williams, 1962; Parker & Dunn, 1964; Means,

    1972). The presence of bicuspid teeth in adults also hasbeen tentatively used to support close lissamphibianrelationships, but such a character is not restricted toamphibians (Bolt, 1969). Bicuspid teeth are not primitive fortetrapods and originated more than once in early tetrapods,which may or may not be true of pedicellate teeth (Bolt,1980). Pedicellate teeth is probably a more primitivecondition because it has been encountered in variousstem-tetrapod lineages. In addition, some taxa have onlymonocuspid teeth in adults, such as pipids (e.g. Xenopus laevis:Cambray, 1976) or several gymnophione genera (e.g.

    Fig. 1. Amphibian relationships with particular focus on the taxa investigated with respect to tooth development.

    indicatesextinct taxa. After Larson & Dimmick (1993), Laurin & Reisz (1997), Feller & Hedges (1998), San Mauro et al. (2004a, b).

    Fig. 2. (A) Tooth of Palaeogyrinus, an extinct Embolomeri,a stem tetrapod sensu Laurin (1998a). (B) Transverse section ofthe crown showing the enamel. (C) Transverse section of themid shaft showing the typical folded dentine, plicidentine.Modified from Miles & Poole (1967).

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    Dermophis, Gymnopis, Caecilia, Gegeneophis, Typhlonectes: Wake,2003). The monocuspid condition in adults is, however,considered to be phylogenetically different from themonocuspid condition in larvae (Wake & Wurst, 1979;Greven, 1984; Beneski & Larsen, 1989a,b). Pedicellate teeth(in general bicuspid) are therefore the only dental characterinterpreted as evidence of close amphibian relationships(e.g. Schultze, 1969, 1970; Bolt, 1980; Lombard & Sumida,

    1992). Nevertheless, pedicellate teeth with a distinctseparation between the crown and the pedicel have beendescribed in extinct temnospondyls (Doleserpeton from theLower Permian: Bolt, 1969; Apateon: Schoch & Carroll,2003; and other branchiosaurids: Boy, 1978). In contrast tothe condition observed in mature stages, in Apateon larvaethe teeth do not have a gap between the base and the crown(Schoch & Carroll, 2003). In modern salamanders, the first-generation teeth in larvae do not possess a pedicel, whilepedicels are well formed in juvenile specimens (Wistuba,Greven & Clemen, 2002).

    (3) The significance of teeth for lissamphibian

    systematicsDentition pattern, dental lamina development, and crownmorphology have been suggested to be important features toestablish relationships within the lissamphibian orders,mostly Caudata (e.g. Laurent, 1947; Regal, 1966; Clemen,1978a, b) and Gymnophiona (e.g. Wake & Wurst, 1979;Clemen & Opolka, 1990; Wilkinson, 1991). However,several studies have revealed intraspecific and ontogenetic

    variations in tooth morphology (e.g. Wake, 1980; Beneski &Larsen, 1989a, b). During the last 25 years, G. Clemen,H. Greven and colleagues have published a series of detaileddescriptions of the mouth cavity and the dentition pattern innumerous caudate species (Clemen, 1979a-c, 1988; Greven

    & Clemen, 1985, 1990; Clemen & Greven, 1988; Ehmcke &Clemen, 2000a). Such a large amount of data allowscomparison of the development of the dentition pattern,the organisation of the dental lamina, and variations in toothshape in relation to the location of the teeth in the oral cavity,among species and between sexes. It is beyond the scope ofthis review to summarise all these descriptions, but someinteresting points are highlighted below.

    In the plethodontid salamander Bolitoglossa subpalmata(Boulenger, 1896), a direct developer, teeth are absent onthe upper jaw in young individuals, but present in adults.This feature has been correlated to the different diet in

    juveniles and adults. Juveniles and young adults use theirwell-developed tongue to transport the small prey deep into

    the mouth, towards the vomerine dentition, while adultsfeed on larger prey (Wake & Deban, 2000). The teeth of theupper jaw are, therefore, of very little importance in youngindividuals (Ehmcke & Clemen, 2000a). Sirenids lack teethon the upper jaw (Clemen & Greven, 1988), but this lossof upper jaw dentition may be secondary when consideringthe condition in a fossil sirenid (Habrosaurus dilatus), whichbears teeth on the premaxillae and the maxillae (Estes,1965).

    During the breeding season, in some plethodontids themales have a few, long (300 mmversus200 mm in the interim

    period), monocuspid teeth protruding from the upper lip(Noble, 1929; Stewart, 1958; Clemen & Greven, 2000;Ehmcke & Clemen, 2000a; Ehmckeet al., 2003). The malesuse such teeth to stimulate the female during courtship. Thetemporary monocuspidity (versus bicuspid teeth during theinterim period) of these particular teeth in males is underthe influence of androgens (Stewart, 1958). This suggeststhat the premaxillary dental lamina only reacts to the rising

    androgen levels at the beginning of the breeding season(Ehmcke & Clemen, 2000b). Because tooth shape can onlybe changed through tooth replacement this implies that thebicuspid teeth located in this region of the upper jaw arelost and replaced by monocuspid teeth during the breedingseason (see also Section VI).

    It is known that metamorphosed caudates have bicuspidteeth, while the teeth are monocuspid in the larvae;bicuspidity being established during, or immediately after,metamorphosis (Kerr, 1960; Chibon, 1972; Clemen &Greven, 1974, 1977, 1979). As a consequence, monocus-pidity in larvae must be regarded as a plesiomorphiccondition as reported for first-generation teeth in actino-pterygians (Sire et al., 2002). However, monocuspid teethhave been reported in some metamorphosed lissamphibianssuch as pipid anurans (Katow, 1979; Greven & Laumeier,1987), some salamanders such as the plethodontid Aneideslugubris (Wake, Wake & Wake 1983) and several gymno-phione genera (Taylor, 1968; Wake & Wurst, 1979; Greven& Clemen, 1980; Wake, 2003). Do they express a lessderived condition in these species than in other lissamphi-bians? Greven (1984) points out that the monocuspid (spike-like) teeth in adult caudates are morphologically differentfrom those (with sharp edges) observed in larvae, the formertype being regarded as less derived. Such a carefuldistinction may be useful in understanding lissamphibianrelationships. Wilkinson (1991) discussed whether mono-

    cuspid teeth are derived or not within Gymnophiona.

    III. LISSAMPHIBIANS IN THE LABORATORY

    For more than a century (e.g. Owen, 1845) investigatorshave taken advantage of the relative ease with whichlissamphibians can be reared in captivity from eggs orlarvae caught in the wild to study the dentition of numerousspecies, especially frogs (e.g. Rana pipiens Schreber, 1782),and salamanders [Cynops pyrrhogaster Boie, 1826; Necturusmaculosus (Rafinesque, 1818), Ambystoma mexicanum (Shaw &Nodder, 1798) and Pleurodeles waltl Michahelles, 1830].

    Around the middle of the 20th Century experimental workconcentrated on species from which numerous eggs couldbe obtained in the laboratory, either naturally (e.g.Ambystoma mexicanum and Pleurodeles waltl) or by artificialinduction [Xenopus laevis (Daudin, 1802) and, recently,Silurana tropicalis (Gray, 1864)]. Appropriate breedingconditions and developmental Tables were published:Taylor & Kollros (1946) for R. pipiens; Nieuwkoop & Faber(1956) for X. laevis; Gallien & Durocher (1957) for P. waltl;and Bordzilovskaya & Dettlaff (1979) for A. mexicanum.Thanks to these experimental model species major

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    advances were obtained in the understanding of lissamphi-bian tooth development in larvae, mainly in A. mexicanumand P. waltl, and/or in juvenile and adult specimens(caudates and various anurans). Although studies dealingwith tooth morphogenesis and differentiation in lissamphi-bians have declined since the end of the 1970s, the numberof species bred in laboratories is increasing, and three modelspecies (X. laevis,A. mexicanumand, to a lesser degree,P. waltl)

    are still used in laboratories studying early developmentalprocesses, reproduction biology, and many other topics.Unquestionably, the most studied species is X. laevis, forwhich numerous developmental genes have been cloned.However, in X. laevisthe teeth form late, at the end of thelarval period, i.e. 2-3 months after hatching (Cambray,1976; Shaw, 1979). By contrast, in Caudata the first teethstart to develop by the end of the embryonic period, similarto the situation in actinopterygian fish (Sire et al., 2002).A. mexicanum and P. waltlare, therefore, more appropriatemodel species to study tooth development in lissamphibiansat the molecular level, particularly in an evolutionaryperspective. Of these, P. waltl is preferred becauseA. mexicanum is generally neotenic and has a large genome.

    Recently, Silurana tropicalis has become a widespreadmodel species in the lab. It has the advantages of beingdiploid (versus tetraploid in Xenopus laevis), growing more

    rapidly (25C), having shorter generation time, and genomesequencing is already well advanced (see www.xenbase.org).In the near future this species will substitute X. laevis formost developmental studies, including odontogenesis.

    IV. OVERVIEW OF TOOTH MORPHOLOGY

    AND STRUCTURE IN LISSAMPHIBIANS

    Adult lissamphibians possess an haplodont dentition, withconical or cylindrico-conical, generally homodont teeth, butsome caudate and gymnophione species have an heterodontdentition (Greven, 1984, 1986; Wake, 1980; Wake et al.,1983). Teeth are restricted to the oral cavity. Lissamphi-bians, as most nonmammalian taxa, replace their teethcontinuously during life, i.e. they are polyphyodont.Caudate and gymnophione teeth have a large diversity ofsize, shape (mono-, bi-, pluricuspid) (Fig. 3), and mode ofattachment (pleurodont for most teeth, except for thepalatal teeth, which are acrodont). This diversity contrastswith a number of well-conserved features, such as toothstructure (a pulp cavity surrounded by a dentine conecovered by enamel; a crown and a pedicel separated bya dividing zone) (Fig. 4), orientation (often lingual), and

    Fig. 3. Examples of tooth morphology in lissamphibians. (A, B, C) Tooth shape throughout ontogeny in the caudate, Pleurodeleswaltl. (A) First-generation tooth in a larva, stage 44. The tooth is monocuspid and the dividing zone is lacking. (B) Third- (left) andfourth- (right) generation tooth in a five-month-old, postmetamorphosed specimen. The teeth are bicuspid and the dividing zoneis visible. (C) Detail of the tooth tip in an adult showing the two cusps. The main cusp is lingually oriented. (D, E, F) Teethin Gymniophona. (D) Typical tooth morphology in an embryo ofGeotrypetes seraphini(left) and in a foetus ofNectocaecilia petersi(right).(E) Adult tooth inHypogeophis rostratus. (F) Adult tooth inGeotrypetes seraphini.(G) Adult tooth in the anuranBombina bombina (Linnaeus,1761). D modified from Parker & Dunn (1964); E, F from Wake & Wurst (1979): G from Clemen & Greven (1980). Scale bars: A, B,D-G 100 mm; C 10 mm.

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    replacement (lingual). In lissamphibians, the ameloblasts donot form Tomes processes (which are supposed to playa role in enamel crystal orientation: Carlson, 1990) andamphibian enamel differs from mammalian in being non-prismatic (Zaki, Yaeger & Gilllette, 1970; Zaki & MacRae,1977, 1978; Kogaya, 1994). In post-metamorphic salaman-ders the presence of enamel matrix proteins has beenidentified using immunocytochemistry, especially revealingamelogenin-like proteins (Herold, Rosenbloom & Granovsky,1989). The first deposited enamel matrix forms globularpatches within which the enamel crystals are mostly radiallyarranged (Kallenbach & Piesco, 1978). In later stages ofamelogenesis, a thick enamel layer is formed in which theenamel crystals are oriented perpendicularly to the tooth

    surface (Kogaya et al., 1992; Kogaya, 1994).Three particular regions of lissamphibian teeth have

    been the subject of many discussions in the past, and haveraised questions, still unanswered, with respect to theenameloid/enamel transition, the formation of the dividingzone and the nature of the pedicel.

    (1) Enameloid versus enamel

    The nature of the enamel-like material covering the teeth inlarval and adult caudates has long been debated since the

    pioneering studies of Owen (1845), Leydig (1867) andHertwig (1874). Levi (1940), Kvam (1946, 1953, 1960) andKerr (1960) believed that the external covering of themonocuspid teeth in larvae was a mesodermal enamel,i.e. a highly mineralised dentine, called durodentine,exclusively deposited by the dental papilla cells. Usingpolarized light, Schmidt (1957, 1958) considered the outersurface of the adult teeth to be durodentine. Later, hechanged his view and suggested that this layer is anectodermal enamel, i.e. deposited by the enamel organ(Schmidt, 1970). In a first attempt to study amelogenesis inthe caudatePleurodeles waltl,Chibon, Roux & Spinelli (1971)did not find enamel covering the teeth until metamorphosis.In fact, in larval teeth the thin enamel layer is hardly visible

    at the light microscopical level, and only transmissionelectron microscopic (TEM) observations have revealed itspresence (Smith & Miles, 1971; Chibon, 1972; Roux &Chibon, 1973; Roux, 1973). In larvae, the dental papillacells, the odontoblasts, deposit first a layer of a particulardentine that mineralises more strongly than regulardentine. This layer is now called enameloid, a termintroduced by Poole (1967) and rvig (1967) to replacethe confusing terms mesodermal enamel, vitrodentineand durodentine. In fact, the difficulty of recognisingenameloid in larval teeth resides in the fact that the first

    Fig. 4. Schematic drawings showing the tooth structure and the relations to the supporting bone in adult lissamphibians. (A)Generalised lissamphibian tooth. (B) Proteid (caudate) Necturus maculosus. (C) Salamandrid (caudate)Salamandra salamandra. (D) Ranid(anuran) Rana pipiens. (E) Hylid (anuran) Hyla cinerea. (F) Gymnophione Hypogeophis rostratus. A modified from Casey & Lawson(1981); B from Kerr (1960); C-F from Lawson (1966). db: dentary bone; de: dentine; dl: dental lamina; dz: dividing zone; en:enamel; Hs: Hertwigs sheath; mb: maxillary bone; n: nerve; oe: oral epithelium; pc: pulp cavity; pde: predentine; pe: pedicel.

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    matrix deposited by odontoblasts at the tooth tip resemblespredentine matrix in that it contains a relatively densecollagen network (Fig. 5). The pre-dentine-like matrix issecondarily converted into enameloid during the mineral-isation and maturation process, which could be under theinfluence of the inner dental epithelial cells, the ameloblasts.These eventually deposit a thin layer of true enamel at theenameloid surface. The presence of a thin layer of true

    enamel covering enameloid in larval teeth has beenconfirmed in the first-generation teeth of the caudateAmbystoma mexicanum using scanning electron microscopy(SEM) (Bolte & Clemen, 1992).

    The layer of enameloid does not exist (or is extremelyreduced and located at the dentine-enamel junction) inadult teeth, in which a thick layer of enamel covers thedentine directly (Chibon et al., 1971; Smith & Miles, 1971).In adults of some lissamphibian species, enamel is orangedue to the presence of iron ions, which are concentratedwithin ferritin patches in the secretory ameloblasts (Randall,1966). The formation of either enamel or enameloid isthought to be related to heterochrony in the secretion ofameloblasts and odontoblasts (Smith, 1995).

    (2) Dividing zone

    In the three orders of lissamphibians, adult teeth are usuallycomposed of two distinct regions: a proximal (basal) pedicel(or pedestal) and a distal crown, separated by a well-marked,transverse zone of weakness (Leydig, 1867; Gillette, 1955;Parsons & Williams, 1962; Means, 1972). This is in contrast tothe presence of undivided teeth in most larval stages. In mostgymnophione genera, however, foetal teeth have a discretepedicel (Wake, 1976, 1980). In their extensive study oflissamphibian tooth structure (42 Caudata, 8 Gymnophiona,118 Anura), Parsons & Williams (1962) noted that this

    separation into a crown and a pedicel is present in most adultlissamphibians. There are, however, a few exceptions, inwhich the division within the teeth has been reported asabsent, such as in the caudateSiren lacertinaOsterdam, 1766and in the anuran Xenopus laevis (e.g. Parsons & Williams,1962; Means, 1972). In these cases the teeth are calcified fromthe crown to the base and anchored to the jawbone byattachment bone (Katow, 1979; Shaw, 1979). Tesche &Greven (1989) also report that the first-generation teeth inanurans are not pedicellate. There are no reports of any adultgymnophionans lacking the dividing zone.

    The dividing zone most commonly appears as a well-defined transverse region, resembling a suture between twobones (Fig. 6). This zone of weakness is revealed by the

    tendency for the crowns to fall off in jaws that have beenvigorously cleaned. In such cases, because they are firmlyfused to the bone support, the pedicels are left as hollowcylinders. Alizarin red staining has revealed that this zone ofdivision is either not, or is only slightly, mineralised (Gillette,1955). This low level of mineralisation of the collagen matrixwas further confirmed by TEM studies (Wistuba et al., 2002).

    Although the functional significance of this zone is notknown with certainty, it has been suggested that, in additionto providing a certain degree of flexibility as a ligament, itallows the tip of the tooth to break off without damage to the

    underlying bone (Larsen & Guthrie, 1975; Moury, Curtis &Pav, 1985, 1987). Working on Plethodon cinereus, Mouryet al.(1985) concluded that this uncalcified region allows toothflexion only in a posterior direction. InPleurodeles waltl, mostof the dividing zone looks like a ligament linking the crownto the pedicel (Fig.6 C, D). In Ambystoma mexicanum, it isnoteworthy that the odontoblasts facing the dividing zonelack cytoplasmic processes and that, therefore, this region is

    devoid of dentine tubules (Wistuba et al., 2002). In P. waltl,indeed, odontoblast processes are hardly visible in this zone(Fig. 6E-G). The pedicel also lacks dentine tubules. Thedividing zone appears, therefore, to be a transition betweenthe tubular dentine of the crown and the atubular dentine ofthe pedicel.

    (3) Pedicel

    The crown is universally agreed to be dentine, but thenature of the pedicel has long been the subject of discussion.Indeed, the lack of dentine tubules and the presence ofsome enclosed cells in this region have led authors toconsider the pedicel either as composed of cement (e.g.Hertwig, 1874) or of bone (e.g. Oltmanns, 1952; Schmidt,1957). Sirena (1872) considered that the pedicel was part ofthe jawbone. Hertwig (1874) was the first to assert that sincethe pedicel is formed within the epithelial sheath and isformed anew each time a tooth is replaced, it should beconsidered part of the tooth rather than a bony projectionof the jaw. He also assigned the term cementum to thesubstance of the pedicel since it contains cell bodies. Cellbodies were never observed in the pedicel of youngP. waltlwe have studied(Fig. 7). Studying tooth replacement in theanuranRana pipiens, Gillette (1955) was the first to concludethat the pedicel is composed of dentine laid down byodontoblasts, and that the cementum is located only at the

    base of the pedicel and at its outer surface. This finding wasconfirmed by Parsons & Williams (1962) in gymnophioneteeth, and by Kerr (1960) and by us herein in caudate teeth(Fig. 7). However, despite their common origin, in adultcaudates the crown and pedicel possess a different Ca/Pratio (Clemen, Greven & Schroder, 1980; Bolte, Krefting &Clemen, 1996). In Ambystoma mexicanum this ratio, whichdetermines the hardness of the tooth region, is 45.9% in thecentral crown versus 38.4% in the central pedicel. Thisindicates that the pedicel is less hard than the crown (the Ca/P ratio is 53.9% in enamel and 34.9% in the bone support).

    It is now well established that the pedicel is part of thetooth. However, the term pedicel is restricted to themineralised cylinder of dentine which is located below

    the dividing zone, while the term tooth base includes thebone of attachment that links the pedicel to the bonesupport (Moury et al., 1987). The dentine of the pedicellacks tubules, but it is clearly distinct from the bone of the

    jaw (Howes, 1978) (Fig. 7). The same conclusion that thepedicel/attachment bone is part of the tooth was reachedfor the bone of attachment of the teeth in lower vertebrates(Peyer, 1968; Sire & Huysseune, 2003). In fact, the base ofthe pedicel is linked to the bone support by a particularzone, which could be considered to be bone of attachment(Fig. 7A, D). The cement-like tissue is deposited on the

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    Fig. 5. Enameloid: a peculiar feature of the teeth in caudate larvae. (A) Schematic drawing of a developing tooth indicating thelocation of enameloid between dentine and enamel. (B) In this first-generation tooth ofPleurodeles waltl, the enameloid matrix hasbeen recently deposited by the odontoblasts. Some cytoplasmic prolongations of the odontoblasts are visible in the enameloidmatrix (arrowheads). No basement membrane is visible between the ameloblast surface and the enameloid (arrows). The cytoplasm

    of the ameloblasts shows large, dilated vacuoles and numerous small vesicles, but a rough endoplasmic reticulum network is hardlyvisible. The enameloid matrix is composed of thin collagen fibrils loosely organised, except along the tooth surface, where they runparallel to the cell surface. The first elements of the predentine matrix have been deposited below the enameloid. (C) Mineralisationstage. This sample was decalcified using ethylenediaminetetraacetic acid (EDTA); the narrow, empty space located between theenameloid and the ameloblast surface indicates that a thin layer of enamel was present at the enameloid surface, but removedduring the decalcification process. Around the tooth tip the ameloblasts show numerous cell membrane folds, which characterizethe postsecretory phase. Asterisks indicate cell prolongations from odontoblasts. (D) Enlargement of the tip of the tooth in Cshowing the ameloblasts located at the enameloid surface and their prominent folds. The foamy aspect of the enameloid matrixindicates that the mineralisation process has started. A modified from Smith & Miles, 1971; B, C, D original micrographs. Scalebars: B, C 1 mm; D 250 nm. am: ameloblasts; en: enamel; ena: enameloid; de: dentine; od: odontoblasts; pde: predentine.

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    Fig. 6. Dividing zone in the teeth of Pleurodeles waltl. (A, B) Scanning electron micrographs showing the dividing zone ina developing (A) and a functional (B) tooth in an adult. (C) Five-month-old specimen. One mm-thick, vertical section of a functional

    tooth showing the dividing zone (arrows) separating the crown from the pedicel. The matrix is thicker at the level of the dividingzone than elsewhere along the tooth shaft, and a large part of this matrix is not mineralised. (D) Detail of the dividing zone of thetooth in C. The mineralisation front is irregular. (E, F, G) Transmission electron micrographs. (E) General view of the structure ofthe dividing zone (lingual side). The arrows indicate the mineralisation front. (F, G) Detail of the dividing zone in the region facingthe pulp (F) and facing the mesenchyme (G). (F) The surface of the dividing zone is irregular and covered by large, activeodontoblasts, which deposit a matrix composed of thin, unmineralised collagen fibrils. (G) Flattened cells of the retracting enamelorgan cover the tooth matrix, from which they are separated by a thin, barely visible basement membrane (arrow). Facing the cellthe matrix is composed of a loose network of thin unmineralised collagen fibrils, which mostly run parallel to the tooth surface. Ata distance from the cell, the collagen fibrils are thicker. Scale bars in A, B 100 mm; C 50 mm; D 10 mm; E 2 mm; F, G 1 mm. de: dentine; dz: dividing zone; eo: enamel organ; od: odontoblasts; oe: oral epithelium; pc: pulp cavity; pe: pedicel.

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    Fig. 7. The pedicel, pulp cavity and cementum of the teeth ofPleurodeles waltl.(A-D) One-mm thick, transverse sections of the teethin a larval stage 42 (A), and in three-month- (B), five-month- (C) and eight-month- (D) old specimen. (A) In larval teeth, the pulpcavity contains only a few cells. It was secondarily invaded by blood vessels. The dividing zone is hardly visible. Note that the toothon the left is attached on one side onto the bone support and, on the other, to the attachment bone region of the adjacent tooth; inboth locations attachment bone is deposited on each surface (arrows). (B) In juveniles the pulp cavity of the developing teeth

    contains a large number of more or less organised cells. (C) During growth the odontoblasts that deposit the predentine matrix arewell organized and polarised, while the centre of the pulp contains blood vessels and undifferentiated cells. (D) On the pulpal andmesenchymal side the pedicel surface of the functional teeth is lined by odontoblast- and osteoblast-like cells, respectively. At thepulp side, the odontoblasts are depositing predentine at the dentine surface (arrow). At the mesenchymal side the enamel organ (theso-called cervical loop) has retracted, and a reversal line is visible (arrowheads), delimiting the dentine matrix from a thin layer ofcement, which has been secondarily deposited on the pedicel surface by osteoblast-like cells. The latter are more active at thepedicel base, where they deposit the attachment bone matrix. (E, F) Electron micrographs of the attachment zone. (E) 12-month-oldspecimen. Odontoblasts depositing predentine along the pulpal side of the pedicel of a functional tooth (arrow in D). (F) Larva,stage 55. Osteoblast-like cells ( cementoblasts) depositing a thin collagenous matrix on the outer surface of the pedicel. Scale bars:A, B, D 10 mm; C 50 mm; E, F 1 mm. ab: attachment bone; bv: blood vessel; ce: cement; db: dentary bone; de: dentine; eo:enamel organ; ob: osteoblast; od: odontoblast; oe: oral epithelium; pc: pulp cavity; pde: predentine; pe: pedicel.

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    dentine surface of the pedicel by osteoblast-like cells, in theregion where Hertwigs sheath has retracted after the toothhas become functional. In Pleurodeles waltlthe cementum isnot deposited in the first-generation teeth.

    V. TOOTH DEVELOPMENT AND

    REPLACEMENT

    Since Hertwig (1874) established the first bases ofknowledge on lissamphibian teeth, developmental eventshave been well documented in many species. Here, wereview generalised lissamphibian tooth development (mor-phogenesis and differentiation), resorption and replacementpatterns, and then discuss in detail the three orders,Caudata, Gymnophiona and Anura. Within the anuranswe will describe the Pipidae separately. We pay particularattention to the salamander Pleurodeles waltl, a model specieswidely used in tooth development.

    A characteristic feature resulting from polyphyodonty inlissamphibians is that, at a given time, several replacementteeth can be found in a single specimen and, especially, in

    juvenile stages. This is a considerable advantage whenstudying tooth morphogenesis and differentiation. Indeed,all stages of tooth development can be found on a jaw, butearly stages are barely visible at the light microscopicallevel.

    (1) Tooth morphogenesis and differentiation

    Tooth development in a generalised lissamphibian isschematically illustrated in figure 8 and micrographs ofP. waltldetail specific stages in a caudate (Fig. 9).

    The initiation of the first-generation teeth, in which the

    dental lamina develops directly from the oral epithelium,begins at stage 34 (11 dpf). The dental lamina consists of anepithelial invagination, two cell layers wide, into themesenchyme. Then, in particular regions of the dentallamina and facing the mesenchyme, the basal epithelial cellsdifferentiate into placodes. Mesenchymal cells, originatingfrom the neural crest (e.g. Wagner, 1949; Chibon, 1966)concentrate at the level of the placodes. The basal layer cellsof the dental lamina invaginate more or less deeply into themesenchyme and develop into a cap. The dental epitheliumdifferentiates into an enamel organ composed of two celllayers, the inner and the outer dental epithelium, while thefacing mesenchymal cells differentiate into a dental papilla(Figs. 8A, 9A,B). The cells of the inner dental epithelium

    differentiate into ameloblasts and the dental papilla cellsinto odontoblasts. The first tooth matrix is produced by theodontoblasts at stage 35 (12 dpf). It consists of enameloid,a dentine-like matrix composed of thin collagen fibrils(20 nm in diameter) (see Section IV.1, and Fig. 5). Theenameloid is secondarily modified by the activity of thefacing ameloblasts, which deposit a thin layer of enamelmatrix on the outer surface of the enameloid. Theameloblasts next participate in the maturation process ofboth the enameloid and enamel matrices, resulting in thepresence of a highly mineralised tooth cap. The organic

    matrix of the mineralised cap is entirely removed during thematuration process. This is clearly revealed by decalcifica-tion which leaves an empty space between the dentine andthe ameloblast surface (see Fig. 5C). Predentine matrix isnext deposited by the odontoblasts (Figs. 8B, 9B). Allfeatures indicate that these cells are the same as those thatpreviously deposited the enameloid matrix, suggestinga switch in the functioning of odontoblasts from enameloid

    to dentine matrix production. The first deposited (imma-ture) collagen fibrils of the predentine measure between6 and 12 nm in diameter. Their diameter then increases to30 nm (mature fibrils) reaching 60-80 nm prior to minerali-sation. All the odontoblasts located along the dentine shaftpossess long cytoplasmic processes, which penetrate thepredentine matrix. The dentine shaft elongates towards thesurface of the developing bone support, to which the toothbase will eventually fuse (Figs 8C-E, 9D-F). This primarytype of tooth attachment in first-generation teeth differsfrom the secondary type of attachment of replacementteeth, in which the tooth base attaches to a pre-existingbone surface as indicated by the presence of a cementingline. The tooth pierces the oral epithelium and becomesfunctional. In the developing first-generation teeth, the pulpcavity is entirely occupied by odontoblasts. Most of themdegenerate when the tooth becomes functional, while someremain active along the dentine surface. The pulp cavity issecondarily penetrated by a capillary blood vessel througha large pore located lingually at the interface between thedentine shaft and the bone surface.

    The development of subsequent generations of teeth issimilar to that of first-generation teeth, except for thosefeatures described in Section IV: enamel progressivelycovers enameloid which disappears at metamorphosis(Fig. 5), there is a dividing zone separating the dentineshaft into a crown and a pedicel (Fig. 6), and cementum is

    deposited at the outer surface of the pedicel base (Fig. 7).Another difference concerns the initiation process of thereplacement teeth; this is discussed in Section V.3.

    (a) Caudata

    Most caudates are oviparous, with rare exceptions such assome salamanders of the family Salamandridae (e.g. generaSalamandra, Lyciasalamandra, Mertensiella) which are live-bearing (viviparous). Some species are neotenic (e.g.Ambystoma mexicanum, Necturus maculosus,someTriturusspecies,and some plethodontids such as some Gyrinophilus andEurycea species). These do not metamorphose, and conservemost larval features during their life, but they can reproduce.

    Tooth development was described, in greater or lesser detail,in Triturus alpestris (Laurenti, 1768) by Wagner (1954),N. maculosus by Kerr (1960), Pleurodeles waltl by Chibon(1966, 1967, 1970), T. vulgaris(Linnaeus, 1758) by Smith &Miles (1971), andA. mexicanumby Smith & Miles (1971) andby Wistuba et al. (2002). Most of our knowledge of toothdevelopment in caudates comes from studies at the lightmicroscope and TEM level in larvae and adults of, mainly,P. waltland A. mexicanum. These studies have focused eitheron general features of tooth development (Wistuba et al.,2002), or on various aspects of odontogenesis such as the

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    Fig. 8. Schematic representation of the development of a tooth and its successor in a generalised lissamphibian. Anterior is to theleft. (A) Morphogenesis and early cytodifferentiation. Originating from the basal epidermal layer of the oral epithelium, the primarydental lamina extends into the subjacent mesenchyme. The distal region of the dental lamina interacts with mesenchymal cells andforms a cup. The epithelial cells differentiate into an enamel organ, which further differentiates into an inner and an outer dentalepithelium. (B) Late cytodifferentiation. Mesenchymal cells have differentiated into odontoblasts, which deposit an unmineralisedmatrix, predentine. The latter mineralises to become dentine. Facing the latter the inner dental epithelium cells differentiate intopreameloblasts. (C) The preameloblasts differentiate into ameloblasts and deposit the enamel matrix on the dentine surface. Thedentine cone elongates due to the activity of the odontoblasts and the pulp cavity starts to form. A secondary dental lamina,originating from the upper region of the outer dental epithelium of the enamel organ at the posterior side of the tooth, extends into

    the mesenchyme. (D) The tooth has elongated and its tip is close to the oral epithelium. The pedicel has started to form at the baseof the crown. The pedicel is separated from the dentine cone by an unmineralised region, the dividing zone. The secondary dentallamina has extended deeply into the mesenchyme. (E) The tooth has attached to the supporting bone through its pedicel and its tiphas pierced the oral epithelium. The tooth is now functional and its replacement tooth has started to form. Note that in caudatelarvae the development of the first-generation tooth differs in that enameloid is the first matrix deposited by the odontoblasts, beforedentine. Modified from Kerr (1960) and Casey & Lawson (1981). am: ameloblast; de: dentine; dl: dental lamina; dz: dividing zone;en: enamel; ide: inner dental epithelium; od: odontoblast; ode: outer dental epithelium; oe: oral epithelium; pc: pulp cavity; pde:predentine; pe: pedicel; rt: replacement tooth; sb: supporting bone.

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    Fig. 9. Development and attachment of the first-generation teeth in Pleurodeles waltllarvae, from stage 36 to stage 55. (A) Initiation,stage 36. The cells of the basal oral epithelium have differentiated into a dental organ. Facing them, some mesenchymal cells haveformed a small dental papilla: this is the bud stage. (B) Early cytodifferentiation, stage 36. The ameloblasts, i.e. the inner dentalepithelium cells, and the odontoblasts, i.e. the dental papilla cells, have differentiated. Tooth matrix has begun to be deposited bythe odontoblasts. (C) Late cytodifferentiation, stage 36. The crown has elongated and the enamel organ forms a typical bell shape.Tooth matrix has started to mineralise, while predentine is deposited. (D) Tooth growth, stage 36. The pedicel has started to form asa prolongation of the dentine shaft towards the surface of the supporting bone. (E) Stage 40. The tooth base is anchored to thedentary bone by means of attachment bone. A blood vessel has penetrated the pulp cavity and the odontoblasts have slowed downtheir activity. (F) Stage 55. Tooth recently attached to the dentary bone. The dentine crown, the pedicel and the attachment boneare clearly visible, and the dividing zone is distinct. Scale bars: A-E 10 mm; F 50 mm. ab: attachment bone; am: ameloblast;bv: blood vessel; db: dentary bone; de: dentine; dp: dental papilla; dz: dividing zone; eo: enamel organ; ide: inner dental epithelium;ob: osteoblast; od: odontoblast; oe: oral epithelium; pc: pulp cavity; pe: pedicel.

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    differentiation of the dental epithelium (Smith & Miles,1971), dentinogenesis (Roux, 1973), amelogenesis (Roux &Chibon, 1973), or on the characteristics of a particularregion such as the enameloid or enamel cap (Kogaya et al.,1992; Kogaya, 1999), the tooth base (Moury et al., 1987), andthe dividing zone (Moury et al., 1985; Greven & Clemen,1990; Wistubaet al., 2002).

    The comparative analysis of this large amount of data

    leads to the conclusion that (i) tooth morphogenesis anddifferentiation in caudates are similar to that described inmammals, and (ii) in all species examined larvae and adultsshare similar features, with only a few differences, whichrelate mainly to tooth size and to the presence of enameloidin larvae versusenamel only in adults (see Section IV.1).

    In larvae, teeth are attached to the paired bones of theupper jaw (premaxillaries, maxillaries, prevomers andpalatines) and the lower jaw (dentaries and coronoids)(Signoret, 1960) (Fig. 10). These bones form between stage37 (Gallien & Durocher, 1957), i.e. 15 days post-fertilisation(dpf) and stage 55 (90 dpf). The last bones to be formed arethe maxillaries, which ossify shortly before metamorphosis(see Section V.2 for comments on the relationships betweenteeth and bone supports). During metamorphosis, the

    palatines disappear from the upper jaw and are replacedby the extension of the vomers. In the lower jaw, thecoronoids disappear and only the dentaries remain (Corsin,1966; Reilly, 1986) (Fig. 10).

    The first-generation teeth start to form in embryos fromstage 33a (initiation, 9 dpf specimens). The first matrix isdeposited at stage 35 (12 dpf). The teeth grow, attach to thebone support, pierce the buccal epithelium and become

    functional when the mouth opens, at hatching (stage 37, 15dpf). At this stage, there are on average 23 teeth on theupper jaw: a row of eight teeth on the premaxillaries andtwo rows of seven and eight teeth on the vomers and thepalatines, respectively. A row of 25 mandibular teeth ispresent on the lower jaw, supported by the dentaries andcoronoids (Roux & Chibon, 1973). The teeth on thedentaries face the premaxillary teeth, while those on thecoronoids face the vomerine and palatine teeth. Two orthree tooth generations succeed the first during larval lifeand the number of tooth positions increases in each row.

    Although new teeth are added at each position, toothresorption starts at stage 48 (50 dpf) only, suggesting theretention of previous-generation teeth at a particular locus.This results in the presence of two rows, with the teeth of

    Fig. 10. Tooth location and bone changes in the oral cavity ofPleurodeles waltlduring ontogeny. (A, B) Larva, lower and upper jaws,respectively; (C, D) adult, lower and upper jaws, respectively. A, B are modified from Signoret (1960).

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    the second row considered replacement teeth (Roux &Chibon, 1973). The teeth in larvae are conical andmonocuspid. The first-generation teeth are 100-150 mmtall. The height increases in replacement teeth to reach500 mm after metamorphosis, during which bicuspid teethreplace monocuspid teeth (see Section VI.1). In directdeveloping species (e.g. many plethodontids) bicuspid teethare also found in some prehatching larvae (Ehmcke &

    Clemen, 2000a).In P. waltl, the rate of growth of larval teeth was

    calculated using tritiated proline labelling (Chibon, 1977).Five days are needed in young larvae to form a tooth, eightin old larvae and 16 in post-metamorphosed specimens.These experiments also indicated that some phases ofodontogenesis (initiation, morphogenesis, early differentia-tion) proceed slowly while others are rapid (late cytodiffer-entiation, growth and eruption).

    (b) Gymnophiona

    Caecilians possess numerous pedicellate teeth on the lowerand upper jaw, which are usually arrayed in two rows. Thedentition is generally homodont but exceptions exist, forinstance in foetuses (Wake, 1980) and in adults of somespecies which have different degrees of bicuspidality on theupper jaw and monocuspid teeth on the lower jaw (e.g.Gegeneophis ramaswamii Taylor, 1964 (Greven, 1984). Thetooth structure is known at the light microscopic level(Wake, 1976; Clemen & Opolka, 1990) and is similar to thatdescribed in caudates. Tooth morphology differs dependingon whether the species are viviparous or oviparous, and ondevelopmental stage (Fig. 3).

    In viviparous species, tooth development has beenstudied in Dermophis mexicanus (Dumeril & Bibron, 1841)by Wake (1976, 1980), and described from a single stage in

    Geotrypetes seraphini(Dumeril, 1859) by Parker (1956), Parker& Dunn (1964) and Wake (1976), and in Gymnopis multiplicataPeters, 1874 and Typhlonectes compressicauda (Dumeril &Bibron, 1841) by Wake (1976) and Hraoui-Bloquet &Exbrayat (1996). The growth of embryos continues in uteroafter the egg yolk has been exhausted; the foetuses developthrough metamorphosis in the oviducts and possessparticular teeth called foetal teeth. For Parker & Dunn(1964) foetal teeth are functionless (a relictual retention ofa fish-like character). By contrast, Wake (1976, 1980, 1993)considers they function to aid ingestion of intra-oviductalnutrient material and to scrape the oviduct wall to stimulatesecretion during gestation. Indeed, the highly specialised(spatula-like) shape of the foetal teeth strongly suggests not

    only that they serve a purpose in food uptake, but that theyare specialised for scraping. This contrasts with thecondition found in other vertebrate larvae in which thefirst-generation teeth are invariably conical and monocus-pid, and considered an ancestral state for vertebrates (Sireet al., 2002). In gymnophione embryos, tooth buds appearfirst on the lower jaw at an early developmental stage, whenthe bone support is not yet formed (Wake, 1976). Teethappear earlier in G. multiplicata than in T. compressicauda(Wake, 1976). InG. multiplicatafoetuses the teeth possess twomajor cusps (a primary labial and a secondary lingual) and

    several spike-like, minor cusps. The teeth are pedicellateand only the top of the crown pierces the buccal epithelium.InT. compressicauda, the foetal teeth first have two cusps, thenother cusps appear during ontogeny, indicating thatreplacement has occurred during foetal life. The foetalteeth are arranged in several rows (in contrast to a singlerow in adults). This is explained by the retention of thereplacement teeth at every position, as in caudate larvae. In

    foetuses, the height of the crown varies from 140 to 300 mmdepending on species (and, probably, on generation time)and the pedicel is short (one third of the crown height) andfused to the bone support. The foetal teeth are entirelyresorbed (or shed) at birth and replaced by bicuspid ormonocuspid teeth such as found in adults (Wake & Wurst,1979). This sudden transition is presumed to be a responseto hormonal induction (Wake, 1993).

    In oviparous species, tooth development has been studied inHypogeophis rostratus by Marcus (1920), Reuther (1931) andLawson (1965a, b) and in Ichthyophis glutinosusby Clemen &Opolka (1990). In the latter species, the dental lamina ofembyros is differentfrom that in adults, which suggests that thedental lamina divides later in ontogeny (Clemen & Opolka,1990). In these species, the embryonic teeth are arranged ina single row on the dentigerous bones and are monocuspid(Clemen & Opolka, 1990), while they are bicuspid andpedicellate in larvae and juveniles (Parker & Dunn, 1964).

    In both viviparous and oviparous species, the odontoge-netic processes are broadly similar to those described above(Lawson, 1965a, b; Wake, 1976) (Figs 8, 9). In Hypogeophisrostratus, a thin cone of predentine is laid down byodontoblasts and major and minor cusps are formed.Mineralisation starts in the predentine when the first cusp isformed. A thin layer of enamel is deposited by theameloblasts over the cusps (Lawson, 1965a). It seems thatthe enamel matrix mineralises rapidly after its deposition

    because no pre-enamel matrix was found in undecalcifiedspecimens. Enamel is thicker over the cusps than elsewhere.Early during its development the tooth germ lies more orless at right angles to the position occupied by the functionaltooth, and is attached to the buccal epithelium by the dentallamina. Then it rotates approximately 90 and the dentinecone elongates. A second mass of dentine is produced ata short distance from the base of the dentine cone andeventually forms the pedicel (Lawson, 1965a). Thecalcification of the pedicel starts when it has reached thebase of the crown. The lingual side of the pedicel developsmore quickly than the labial side, leading to a pleurodonttype of attachment by ankylosis to the jawbone. During thefunctional life of the tooth the crown dentine thickens and

    the pulp cavity is reduced.As in caudates, some phases of odontogenesis in

    gymnophiones take place slowly while others are rapid.

    (c) Anura (excluding Pipidae)

    Bufonids (toads) do not possess teeth. In pipids and otherfrogs teeth are restricted to the upper jaw, with the ex-ception of some hylids, in which teeth are also found on thedentary (Goin & Hester, 1961). On the upper jaw, the teethare arranged in a single row on the paired premaxillaries,

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    the paired maxillaries and the paired vomers. In somespecies the dentary is ornamented by odontoid (tooth-like)structures, which are bony elements (Trueb, 1993). Teethare small (less than 1 mm long in adult Rana pipiens), but

    very numerous, and their number and size varies with frogsize. For instance, Gillette (1955) counted 89-95 functionalteeth on each half of the upper jaw in large adult R. pipiens.Each tooth position is occupied by a functional and

    a replacement tooth, giving such a specimen on average368 teeth in its dentition (small specimens have approxi-mately 140 teeth). Teeth are bicuspid and the dentition ishomodont.

    Anurans are mostly oviparous, but several species areovoviviparous and others are viviparous (Duellman &Trueb, 1986; McDiarmid & Altig, 1999). Their aquaticlarvae, the tadpoles, are mostly herbivorous and detritivo-rous, and their dentition is not composed of true,mineralised teeth. Instead, there are horny labial teeth inthe upper and lower beak, which are formed by keratinisedepithelial cells organised into columns (Kaung, 1975;Takahama et al., 1987). The cell located at the upperextremity of the column forms the top of the tooth. Thenumber of teeth and the size of the beak increase duringlarval life. At metamorphosis, the keratinized epithelial cellsare destroyed by autolysis, a process similar to that observedin the tail.

    Hertwig (1874) was first to describe tooth development infrogs. Subsequent detailed accounts mainly confirmed hisfindings. To date, descriptions of tooth development areavailable for: Rana pipiens by Gillette (1955), Hyla cinerea(Schneider, 1799) by Goin & Hester (1961), R. temporariaLinnaeus, 1758 and R. esculentaLinnaeus, 1758 by Spinelli& Chibon (1973) and by Chibon (1977), and H. arborea(Linnaeus, 1758) and R. nigromaculata Hallowell, 1861 bySato et al. (1986a, b).

    The first tooth germs appear at metamorphosis, by theend of hindlimb organogenesis, and the first teeth arefunctional after 25-26 days of growth. The developmentalfeatures are similar to those described above for a general-ised lissamphibian. InR. pipiens, there are six separate dentallaminae corresponding to the six dentigerous bones, andextending lingually to the dental process of each bone(Gillette, 1955). Predentine first calcifies in the lingual andthen in the labial cusp. Concomitant with the firstcalcification of the dentine the ameloblasts differentiate,then deposit enamel matrix first on the labial sides of thecusps. The enamel mineralises rapidly, but slower than incaudates (Spinelli & Chibon, 1973). During pedicelformation the tooth progressively changes its orientation.

    The calcifying process stops abruptly at the crown-pediceljunction. Ankylosis of the pedicel to the bone is ensured bycellular cementum, which completely bridges the gapbetween the pedicel and the bone surface. Eruption isarrested when the cementum matrix calcifies. During thefunctional period more dentine is added to the crown andto the pedicel on the pulp side. Cellular cementum isdeposited on the outer surface of the pedicel, when theepithelial root sheath (Hertwigs sheath) has retreated.

    In Hyla cinerea teeth develop similarly to those of Ranapipiens with only a slight difference in the mode of

    attachment, which is related to the form of the dentalprocess of the maxillary (Goin & Hester, 1961).

    (d) Pipidae

    The Pipidae are a well-known anuran family thanks toXenopus laevis, which is used as a model in developmentalbiology.X. laevismay live as long as 23 years in the laboratory

    (Deuchar, 1975). In their Developmental Table for X. laevisNieuwkoop & Faber (1956) briefly commented on thedevelopment of the dentition. Cambray (1976) provided thefirst description of tooth development in larvae and adults,but his doctoral thesis remained unpublished. Subsequently,Shaw (1979, 1985, 1986, 1989) published reference data ontooth development and replacement in X. laevis.

    X. laevis differs from the other anuran taxa in that trueteeth begin to develop on the upper jaw in tadpoles duringthe last stages of larval life (Fig. 11). However, teeth do noterupt until the end of metamorphosis; they are 225-250 mmtall (Shaw, 1979). In larvae, newly metamorphosed and adultspecimens the teeth are morphologically similar, onlydiffering in size. The dentition is homodont and the teethform a single row on the upper jaw. The teeth are conical,monocuspid and slightly curved at the tip. In adults, only 50-100 mm of the tooth tip projects into the mouth so that it ishard to ascribe an important function to them (Shaw, 1979).

    Tooth structure is known at the light microscopic levelonly (Shaw, 1979). Unlike in many other lissamphibians, theenamel organ is composed of three layers, a stellate retic-ulum being present between the inner and the outer dentalepithelium. In addition, teeth are not pedicellate (there is nodividing zone) and there is no indication of a layer of ce-mentum along their base (Katow, 1979). They are ankylosedto the premaxillaries and maxillaries by a short, ring-shapedbony pedicel (bone of attachment), which is continuous with

    the jaw bone in newly metamorphosed specimens butdistinct from the underlying bone in the adults. All thesefeatures are typically those of first-generation teeth innonmammalian vertebrates (Sire et al., 2002).

    The first-generation teeth develop at stage 55, approx-imately 40 days before metamorphosis (Shaw, 1979)(Fig. 11). Teeth appear in alternating (even and odd)positions, starting from the mid-line, to reach 22 positionson each side of the jaw at metamorphosis: eight on thepremaxillary and 14 on the maxillary. Teeth at evenpositions develop first, 8-9 days before those at oddpositions. From the second day after germ initiation,dentine deposition commences, followed shortly by enamelformation. We have not been able to identify enameloid

    unequivocally during early tooth development in Xenopuslaevis(Fig. 11 and H. Chisaka, unpublished results), but thisneeds to be investigated further. Enamel formation andmineralisation encompass a fairly short period (eight days).Dentinogenesis is relatively slow during the first 20 days,then the tooth germs at even positions start a period ofrapid dentinogenesis, re-orientate to a more verticalposition, and develop a dental lamina for their successortooth. Attachment lasts from day 23 to 25. From day 27after first tooth germ initiation, the tooth germs at oddpositions begin their period of rapid growth. Osteoclastic

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    resorption of the first even-numbered teeth begins at 32-33days (i.e., 4-6 days post stage 65) (Shaw, 1979). The secondgeneration teeth erupt five days later, i.e. slightly before theend of metamorphosis. In summary, in larvae a complete

    tooth cycle takes 33 days, with teeth being functional foronly seven days, versus 59-71 days for the complete cycleand 24-29 days functional in adults.

    (2) Relationships between tooth and bonesupport development

    In nonmammalian species studied so far, the development ofa first-generation tooth ends with the anchoring of the toothbase (the pedicel or attachment bone) to a bony support (pre-maxillaries, maxillaries, dentaries, vomers, palatines, pha-ryngeal bones, etc.). However, the matrix of the supportingbone is not present when the tooth is initiated (Sire et al.,2002). Osteogenesis and odontogenesis progress approxi-

    mately simultaneously. This is achieved such that, in thedentigerous region, both the bone and the tooth matrix seemto converge towards each other. Eventually, both matrices(bone surface and base of the pedicel) merge, forming the so-called primary tooth attachment (versus secondary toothattachment, which occurs when the bone support is alreadypresent when the tooth attaches). Such a process suggeststhe existence of coordination between the odontoblasts atthe base of the pedicel and the osteoblasts at the bone surfacefacing the developing tooth (mediated by signalling mole-cules?), at least during the final stages of development of

    the two elements. Further (molecular) studies will benecessary to understand the interactions between these cellpopulations. Although these observations suggest that teethneed the presence (or concomitant development) of a bony

    support in order to develop, experimental studies do notsupport this conclusion. With the aim of perturbingorganogenesis in embryonicP. waltl,Signoret (1960) applied

    various concentrations of lithium chloride, a molecule knownto induce morphogenetic perturbations. He found organreduction (hypomorphy) in these embryos. In addition, toothdevelopment was found to be very sensitive to lithiumchloride: some bones which are normally dentigerous(dentaries, premaxillaries, palatines), developed normally,but without teeth. In addition, (i) teeth were found in regionsdevoid of bone support, and (ii) some bones which werenormally not dentigerous (the angular and the parasphenoid)were found to bear teeth. These experiments demonstratedclearly that tooth development does not depend on the nature

    and location of the bone support. The relationship betweena tooth and its surrounding bone may therefore be secondary,resulting from topographic conditions only.

    In the frogRana pipiens, Howes (1977) transplanted teethduring the crown formation phase to an ectopic site (either inthe anterior eye chamber or in a dorsal subcutaneous site).these transplanted teeth grew normally and formed a nor-mal-sized pedicel area demonstrating that (i) once initiatedthe genetic programme is able to support complete odonto-genesis, and (ii) the pedicel is purely odontogenetic in origin.Similarly, the ablation of part of the premaxilla in this species

    Fig. 11. Tooth development in an anuran, the pipidXenopus laevis. In contrast to most anuran species, the teeth develop long before

    metamorphosis. (A, B) Tadpole, stage 59, ventral and lateral views, respectively. The first-generation teeth are already well developedat this stage. (C) Tooth pattern in a tadpole, stage 59. As in most anuran species teeth are only present on the upper jaw. (D-G) Onemm-thick vertical sections of the upper jaw of tadpoles (stages 54, 58, 63 and 65, respectively) showing various developmental stagesof first-generation teeth, from initiation (D) to attachment (G). The arrow in E indicates to the first deposition of the tooth matrix.The arrows in F and G indicate the previous location of the enamel in these samples which were decalcified with EDTA.Developmental stages are as in Nieuwkoop & Faber (1956). A, B modified from Nieuwkoop & Faber (1956), C from Cambray (1976).Scale bars: C 250 mm; D 10 mm; E-G 50 mm. de: dentine; do: dental organ; dp: dental papilla; eo: enamel organ; ide: innerdental epithelium; mb: maxillary bone; od: odontoblast; oe: oral epithelium; pc: pulp cavity; pm: premaxillary bone.

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    did not prevent teeth from growing in the regenerated jaw(see Section VII) in the absence of a bone support (Howes,1978). These teeth developed to normal size and shape, andwere even replaced by their successors although thesupporting bone, which regenerated slowly, was still notformed. Such results confirm that complete tooth develop-ment does not require the presence of a bone support.

    Observations made in the armoured catfishCorydorasspp.

    and Hoplosternum littorale confirm the generality of thesefindings. On the upper jaw, the first-generation teethdevelop long before the development of the maxillary. Eachtooth forms a pedicel of attachment bone, and the pedicelsof adjacent teeth merge to constitute a kind of dentigerousbone, which will later be connected to the developingmaxillary (Huysseune & Sire, 1997b). Also, in some zebra-fish (Danio rerio) mutants that do not form pharyngeal arches,teeth develop in the absence of the pharyngeal bone support(Schilling, Walker & Kimmel, 1996).

    (3) Tooth replacement and resorption

    The processes of tooth replacement and resorption occur ina similar manner in anurans (including Xenopus laevis),gymnophiones and caudates. Resorption of a functionaltooth is always related to the close presence of a growingreplacement tooth, as illustrated in Pleurodeles waltl(Fig. 12).

    A replacement tooth is first seen lingually as a bud fromthe region located at the limit between the dental organ andthe dental lamina of the functional tooth (Fig. 12). The budextends as a new dental lamina into the mesenchyme, withwhich it interacts to give rise to a new tooth. Thereplacement tooth grows and, once fairly well developed,its enamel organ generally contacts the lingual side of thefunctional tooth (Fig. 12C). This contact induces the

    recruitment of osteoclasts as a probable reaction to pressureforces acting on the external wall of the tooth. Mostgenerations of replacement teeth in lissamphibians exhibitthese general features, although the first generation ofreplacement teeth does not provoke the resorption of thefirst-generation teeth, resulting in their retention and hencethe presence of two tooth rows on the upper rand lower

    jaws of young larvae. The first-generation teeth are verysmall (20-30 mm wide) thus there is probably enough spaceto accommodate two teeth from the same family. Sucha condition has also been described in the zebrafish (Vander heyden & Huysseune, 2000).

    InP. waltl, the first signs indicating imminent resorption ofthe first-generation teeth are identified at larval stage 44,long after the first-replacement teeth have been functional.The pulp becomes more loosely organised due to a decreasein cell number. Numerous cells are degenerative and somemacrophages are present (Roux & Chibon, 1974). The latter

    Fig. 12. Tooth replacement inPleurodeles waltl. (A) Larva, stage 56. A secondary dental lamina, originating from the upper region (*)of the outer dental epithelium of the previous tooth, has extended into the mesenchyme. This dental lamina is composed of twolayers, the cells of which are differently arranged: flat and elongated at the posterior side and tall and polarized at the anterior side.(B) Larva, stage 56. The cells located at the anterior side and at the extremity of the dental lamina have proliferated and haveformed a cup, which surrounds mesenchymal cells (arrow). The asterisk indicates the origin of the secondary dental lamina.(C) Larva, stage 49. A replacement tooth is well formed, but still attached to the functional first-generation tooth by means ofthe secondary dental lamina (*). Scale bars: A 10 mm; B, C 20 mm. db: dentary bone; de: dentine; dl: dental lamina; ide:inner dental epithelium; ode: outer dental epithelium; oe: oral epithelium; pc: pulp cavity; pe: pedicel.

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    have secondarily invaded the pulp cavity and are involved inphagocytosis of necrotic cells. Macrophages could also beresponsible for the destruction of Hertwigs sheath and thebasal region of the dental lamina (Chibon, 1977). The firstresorption features are observed at stage 48 (50 dpf),approximately two months before metamorphosis, whentwo tooth generations have formed. The first tooth iscompletely resorbed at stage 52 (64 dpf). The cells

    responsible for tooth resorption are typically multinucleatedosteoclasts. There are also some mononucleated macro-phages removing cell debris. First, the external surface of thedentine wall at the base of the pedicel is attacked byosteoclasts at the lingual side(Fig. 13A, E). Then, osteoclasts(also called odontoclasts) penetrate the pulp cavity and startto resorb the opposite wall, while the resorption extendslabially and to the top of the tooth shaft (Fig. 13B, C, F).Simultaneously, cell necrosis is observed in the pulp cavityand Hertwigs sheath retracts. The dentine shaft is entirelyresorbed as well as part of the adjacent supporting bone.

    The large, multinucleated cells responsible for theresorption of lissamphibian teeth share similar featureswith osteoclasts described in many vertebrate species(Fig. 13). However, some authors have called themodontoclasts with reference to their involvement in dentineresorption (e.g. Clemen & Greven, 1974, 1977; Bouvet &Chibon, 1976; Chibon & Bouvet, 1976; Wistuba, Bolte &Clemen, 2000). The first TEM description of these cells wasfor frogs (Yaeger & Kraucunas, 1969). Wistuba et al. (2000),working on Ambystoma mexicanum, provided additionaldetails. Interestingly, activity of tartrate-resistant acidphosphatase (TRAP) has not been detected during the firststages of tooth resorption, whereas it was shown to revealosteoclastic activity in other vertebrates, such as teleosts(Witten, 1997) and mammals (Sahara et al., 1998). Thepresence of clastic cells inA. mexicanum, a neotenic caudate,

    which lacks parathyroids, indicates that the production ofparathyroid hormone (PTH) is not a prerequisite for theregulation of these cells. Instead, regulation througha pituitary factor has been suggested (Pang et al., 1980).

    InPleurodeles waltltooth replacement has been shown to beunder the influence of the thyroid hormone, thyroxine.When this hormone is absent (or inhibited), or at low

    concentration, teeth are replaced more slowly than in normalspecimens (Dournon & Chibon, 1974). This is probablyrelated to the role played by thyroxine in cell proliferation.

    The fate of the tooth tip during tooth resorption hasgiven rise to some controversy. Most authors either state, orassume, that lissamphibian teeth become loose at their basesand are shed into the mouth (e.g. Gillette, 1955; Lawson,1965b; Lawson, Wake & Beck, 1971; Clemen & Greven,

    1980). For instance, it is supposed that the weak,unmineralised dividing zone is destroyed more rapidly thanthe pedicel, provoking shedding of the crown, and explain-ing why most crowns are absent in teeth undergoingresorption, while pedicels remain, at least partially (Casey &Lawson, 1981). However, in their illustrations there is nohistological section showing the absence of crown resorp-tion. Contrary to this view, Shaw (1986, 1989) found itlogical that the major part of a tooth in Xenopus laevis isabsorbed rather than lost by shedding. Chibon (1977)reached a similar conclusion with P. waltl larvae where thetips of teeth in an advanced state of resorption were foundentirely embedded in the oral epithelium. We confirm thatthe teeth in P. waltlare entirely resorbed, at least in larvae(Fig. 13H, I). However, in lissamphibians osteoclasticresorption of the enamel cap has not been reported to datein the literature, in contrast to some mammals, in whichenamel resorption has been reported (Sahara et al., 1998).

    In adult caecilians [e.g.Hypogeophis rostratus (Cuvier, 1829),Grandisonia diminutiva Taylor, 1968] tooth replacementoccurs as described in P. waltl. The process of resorption issupposed to be rapid because partially resorbed teeth areuncommon (Casey & Lawson, 1981). The pedicel is entirelyremoved by resorption. However, it is not clear how muchof the crown is resorbed before shedding. The loss of a largenumber of crowns may represent a considerable long-termdrain on calcium reserves. Indeed, in addition to its function

    in permitting tooth replacement to occur, resorption isconsidered a conservative process making tooth constituents(minerals and organic matrix) available for re-use.

    In the anuran Rana pipiens, Gillette (1955) found that thetooth resorption process is similar to that described inPleurodeles waltl. In Hemiphractus proboscideus (Jimenez de laEspada, 1871), Shaw (1983) calculated that the volume of

    Fig. 13. Tooth resorption inPleurodeles waltl. (A-D) Scanning electron micrographs of teeth subjected to resorption, viewed from thelingual side. Note the numerous, well-delimited lacunae at the resorption sites, revealing the location of the osteoclasts. (A) Adult.Resorption has started at the level of the pedicel. (B) Adult. Resorption has extended to the whole surface of the pedicel. (C) Ten-month-old specimen. The pedicel surface is highly resorbed as well as the base of the crown, where the pulp cavity has been

    opened. (D) Adult. The tooth has been entirely resorbed, but most of the pedicel remains. (E-I) One mm-thick, vertical sections ofteeth subjected to resorption. (E) Larva, stage 51. The surface of the pedicel located close to the enamel organ of the replacementtooth is subjected to resorption. (F) 12-month-old specimen. Most of the pedicel has been resorbed and two large, multinucleatedosteoclasts are attacking the base of the dentine crown (arrows). (G) Eight-month-old specimen. An osteoclas