Amphibian Teeth

Biol. Rev. (2007), 82, pp. 4981 doi:10.1111/j.1469-185X.2006.00003.
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Amphibian teeth: current knowledge, unanswered questions, and some directions for future research
al, Hideki Chisaka*, Sidney Delgado and Jean-Yves Sire Tiphaine Davit-Be
UMR 7138-Systematique, Adaptation, Evolution, Universite Pierre & Marie Curie-Paris 6 Case 7077, 7 Quai St-Bernard, Paris 75005, France (Received 12 December 2005; revised 31 August 2006; accepted 11 September 2006)
ABSTRACT Elucidation of the mechanisms controlling early development and organogenesis is currently progressing in several model species and a new field of research, evolutionary developmental biology, which integrates developmental and comparative approaches, has emerged. Although the expression pattern of many genes during tooth development in mammals is known, data on other lineages are virtually non-existent. Comparison of tooth development, and particularly of gene expression (and function) during tooth morphogenesis and differentiation, in representative species of various vertebrate lineages is a prerequisite to understand what makes one 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 available on diverse aspects of tooth biology in various species. The aim of this review is to evaluate current knowledge on amphibian teeth, principally concerning tooth development and replacement (including resorption), and changes in morphology and structure during ontogeny and metamorphosis. Throughout this review we highlight important questions which remain to be answered and that could be addressed using comparative morphological studies and molecular techniques. We illustrate several aspects of amphibian tooth biology using data obtained for the caudate Pleurodeles waltl. This salamander has been used extensively in experimental embryology research during the past century and appears to be one of the most favourable amphibian species to use as a model in studies of tooth development. Key words: lissamphibians, Anura, Caudata, Gymnophiona, tooth, odontogenesis. CONTENTS I. Introduction ...................................................................................................................................... II. Critical evaluation of the use of teeth in amphibian phylogeny ..................................................... (1) The origin of the lissamphibians ................................................................................................ (2) The significance of teeth for lissamphibian phylogeny ............................................................. (3) The significance of teeth for lissamphibian systematics ............................................................ III. Lissamphibians in the laboratory ..................................................................................................... IV. Overview of tooth morphology and structure in lissamphibians .................................................... (1) Enameloid .................................................................................................................................. (2) Dividing zone .............................................................................................................................. 50 51 51 51 53 53 54 55 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: sire@ccr.jussieu.fr).
Biological Reviews 82 (2007) 4981 2007 The Authors Journal compilation 2007 Cambridge Philosophical Society
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al and others Tiphaine Davit-Be

(3) Pedicel ......................................................................................................................................... V. Tooth development and replacement .............................................................................................. (1) Tooth morphogenesis and differentiation .................................................................................. (a ) Caudata ................................................................................................................................. (b ) Gymnophiona ........................................................................................................................ (c ) Anura (excluding Pipidae) ..................................................................................................... (d ) Pipidae ................................................................................................................................... (2) Relationships between tooth and bone support development .................................................. (3) Tooth replacement and resorption ............................................................................................. (4) Tooth replacement pattern ......................................................................................................... VI. Tooth changes ................................................................................................................................... (1) Monocuspid to bicuspid: the role of thyroxine at metamorphosis ........................................... (2) Bicuspid to monocuspid: the role of androgens ........................................................................ VII. Tooth regeneration ........................................................................................................................... VIII. Directions for future research ........................................................................................................... (1) Are pedicellate teeth homologous among lissamphibians? ....................................................... (2) Are the dentition pattern and development of the dental lamina important features for lissamphibian systematics? .......................................................................................................... (3) Do ameloblasts participate in enameloid formation in lissamphibian larvae? ......................... (4) How does the enameloid-enamel transition proceed through caudate ontogeny? .................. (5) How do the dividing zone and the pedicel appear during lissamphibian ontogeny? .............. (6) What mechanisms control the initiation of a replacement tooth in lissamphibians? ............... (7) Which mechanisms control the initiation of tooth resorption? ................................................. (8) What is the fate of the tooth tip in adult lissamphibians? ........................................................ (9) What mechanism controls the periodicity of lissamphibian tooth replacement? ..................... (10) How do thyroxine levels affect tooth shape in lissamphibian teeth? ........................................ IX. Conclusions ....................................................................................................................................... X. Acknowledgements ............................................................................................................................ XI. References ......................................................................................................................................... 56 60 60 60 64 64 65 66 67 70 71 71 71 71 72 72 72 72 73 73 73 73 74 74 74 74 75 75
I. INTRODUCTION Rapid recent progress in molecular biology and developmental genetics has allowed investigators in odontology to re-open doors that have remained closed since the end of the 1970s. Over the last two to three decades new tools have allowed investigations to extend from tissue and cellular integration to the molecular level, and understanding of mechanisms controlling tooth development is progressing rapidly using the mouse as a model species. The expression pattern of more than 120 genes during mammalian tooth patterning and development has been described: see http:// bite-it.helsinki.fi (Nieminen et al., 1998). We know that all animals share many of the same molecular processes, including regulatory genetic pathways. However, how these commonalities are used to make one tooth different from another is far from understood. This question can only be answered through comparison of gene expression (and function) during odontogenesis in various lineages or within multiple taxa in the same lineage. Answering this question would lead to an understanding of how teeth have changed during evolution in terms of initiation (time), position (space), type (morphology), mode of replacement, etc. In fact, studies of the evolutionary developmental biology of teeth are virtually non-existent, with the exception of some recent evolutionary work on rodent teeth (Jernvall, Keranen & Thesleff, 2000; Kangas et al., 2004). In particular, Kangas
et al. (2004) show correlated changes in dental characters as a function of quantitative changes in intercellular signalling, and conclude that most aspects of tooth shape could have the 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 selected species (Sire et al., 2002), research suffers from a lack of comparison of the genes involved (and of their function) with nonmammalian lineages. In parallel with detailed odontogenetic studies in the mouse, it is important to compare tooth development in species representative of other lineages (e.g. reptiles, amphibians, actinopterygian fishes, sharks) or in a lineage that includes taxa with variants, so that tooth evolution can be assessed in a phylogenetic framework. Among toothed vertebrates, mammals have either a single or two tooth generations (mono- or diphyodonty), while nonmammalian species renew their teeth continuously (polyphyodonty). The study of tooth development in polyphyodonts would seem to have several advantages for the biologist, and molecular studies have just begun. The first data are available for the zebrafish Danio rerio (Laurenti et al., 2004; Jackman, Draper & Stock, 2004; Borday-Birraux et al., 2006) but studies on tooth development are far from easy in this species which possesses teeth in the pharyngeal region only (Huysseune, Van der heyden & Sire, 1998; Van der
Amphibian tooth morphology and development

heyden, Huysseune & Sire, 2000). In addition, the zebrafish belongs to the actinopterygian lineage, which means more than 420 million years of evolution separate this teleost fish from the mouse; the tooth structure and late odontogenic phases in fish differ from those of mammals (Huysseune et al., 1998; Laurenti et al., 2004; Borday-Birraux et al., 2006). Therefore, to address some of the still unanswered questions in 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) several species (e.g. Pleurodeles waltl, Ambystoma mexicanum, Xenopus laevis, Silurana tropicalis, Eleutherodactylus coqui, and Bombina variegatus) are easy to breed in the laboratory (in contrast to many reptiles), and (iii) for almost a century a large amount of data has accumulated on various aspects of amphibian tooth biology. Numerous molecular tools (a large number of sequenced genes, possibility of transgenesis) are now available for X. laevis and S. tropicalis (its genome is currently being sequenced), which are used as models in embryological and developmental studies. Numerous genes have also been 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 knowledge accumulated during the past century on amphibian teeth with respect to development, replacement (including resorption), changes in morphology and structure in relation to growth and metamorphosis, and (ii) to highlight unanswered questions in amphibian tooth biology and tooth development.
51 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 number of characters in extant and extinct amphibian species (including skeleton and soft anatomy), the hypothesis of a common ancestry for the lissamphibians has nevertheless been retained (Szarski, 1962; Parsons & Williams, 1963; Laurin, 1998a, b, 2002; Schoch & Carroll, 2003). This hypothesis is also supported by molecular phylogenies showing the monophyly of lissamphibians: caecilians and salamanders being sister taxa, with frogs their outgroup (Hedges, Moberg & Maxson, 1990; Hedges & Maxson, 1993; Hay et al., 1995; Feller & Hedges, 1998). Fossil records indicate that the crown-group lissamphibians started diversifying by the end of the Permian (approximately 250 mya), before the breakup of Pangaea, and their diversity increased greatly during the Jurassic and Early Cretaceous periods (approximately 200-150 mya) (Schoch & Carroll, 2003). This was recently confirmed using a molecular phylogeny (San Mauro et al., 2005). Putative ancestors of salamanders are recognized from the Carboniferous-Permian boundary (Schoch & Carroll, 2003), a fossil caecilian possessing reduced limbs, Eocaecilia micropodia, has been discovered from 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), Lissamphibia 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 use the current standard taxonomic reference for the amphibian orders, i.e. node-based names defined on the basis of Recent taxa instead of stem-based names which include the fossil taxa: Anura instead of Salientia for frogs, Caudata instead of Urodela for salamanders, and Gymnophiona instead of Apoda for caecilians (e.g. Trueb & Cloutier, 1991; Cannatella & Hillis, 1993, 2004; Ford & Cannatella, 1993; Frost, 2004]. Fewer than ten species from each order have been examined so far with respect to tooth development (Fig. 1). (2) The significance of teeth for lissamphibian phylogeny In most stem-tetrapods and in extinct amphibians, teeth were haplodont (i.e. simple: conical and unicuspid) with some heterodonty (i.e., differing in general appearance throughout 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 sometimes pleurodont (i.e. attached to the labial side). Tooth structure has been studied in a few early tetrapods (e.g. temnospondyls) and lepospondyls (microsaurs, nectrideans) (e.g. Owen, 1842; Bystrow, 1938; Parsons & Williams, 1962; Peyer, 1968; Bolt, 1969, 1979). In general, the teeth were conical with a large base. The dentine shaft surrounded a pulp cavity and was covered by a thin enamel layer. Tooth structure was characterised by a typical folded arrangement of the dentine, called plicidentine (Fig. 2). Plicidentine is, however, not a typical feature of early amphibians, and
II. CRITICAL EVALUATION OF THE USE OF TEETH IN AMPHIBIAN PHYLOGENY (1) The origin of the lissamphibians Amphibians appeared by the end of the Devonian or the early Carboniferous [approximately 300 million years ago (mya)], when the two tetrapod lineages, reptiliomorphs (which include amniotes) and amphibians, separated from a tetrapod ancestor (Laurin, 1998a, b; Carroll, 1988). They comprise both living species and their extinct relatives, grouped into the lissamphibian clade (frogs, salamanders and caecilians), and several extinct lineages that have been grouped either into a large group including lepospondyls and temnospondyls (Trueb & Cloutier, 1991; Lombard & Sumida, 1992; Ahlberg & Milner, 1994), or into lepospondyls only (Laurin & Reisz, 1997; Laurin, 1998a, b). Lissamphibians are supposed to have originated at the onset of the Triassic period (approximately 250 mya), probably from a lepospondyl ancestor (Laurin & Reisz, 1997, 1999; Laurin, 2002). However, the fossil record has provided little evidence on the evolutionary origin of lissamphibians, and it is difficult to postulate which group among the Paleozoic lepospondyls is most closely related to them (Laurin, 1998a, b; Anderson, 2001). This explains why the question of the origin of the lissamphibians has been long debated in the literature (Romer, 1945; Holmgren, 1952; Eaton, 1959; Jarvik, 1960; Bolt, 1969, 1977, 1979, 1991),
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Fig. 1. Amphibian relationships with particular focus on the taxa investigated with respect to tooth development. indicates extinct taxa. After Larson & Dimmick (1993), Laurin & Reisz (1997), Feller & Hedges (1998), San Mauro et al. (2004a, b).
cannot be used as a strong phylogenetic argument: it is absent in several Paleozoic amphibians, in particular among microsaurs (Peyer, 1968), and it is widespread in basal sarcopterygian taxa (Schultze, 1969). Some temnospondyls possessed a branchial apparatus, in which small toothbearing plates occurred in the throat region (Hook, 1983;
Fig. 2. (A) Tooth of Palaeogyrinus, an extinct Embolomeri, a stem tetrapod sensu Laurin (1998a). (B) Transverse section of the crown showing the enamel. (C) Transverse section of the mid shaft showing the typical folded dentine, plicidentine. Modified from Miles & Poole (1967).
Coates, 1996), a location which is similar to the pharyngeal teeth described in a number of actinopterygians. The value of tooth characters as evidence of lissamphibian phylogeny has been investigated in depth by Parsons & Williams (1962, 1963). Although the three lissamphibian orders possess relatively few distinguishing characters (which explains the current debate on their relationships), the presence of bicuspid and pedicellate teeth has been widely accepted as strong support for their monophyly (see discussion 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 dental laminae are also of phylogenetic importance. Lissamphibian teeth are characterised by the division of the dentine shaft into a relatively short crown and a long pedicel, separated by an uncalcified (or poorly calcified) region resembling a ligament, called the dividing zone. Pedicellate teeth are present in fossil representatives of caudate, gymnophione and anuran lineages. However, in a few lissamphibian species, teeth lack a dividing zone, but this feature is considered a derived rather than a plesiomorphic character (Parsons & Williams, 1962; Parker & Dunn, 1964; Means, 1972). The presence of bicuspid teeth in adults also has been tentatively used to support close lissamphibian relationships, but such a character is not restricted to amphibians (Bolt, 1969). Bicuspid teeth are not primitive for tetrapods 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 primitive condition because it has been encountered in various stem-tetrapod lineages. In addition, some taxa have only monocuspid teeth in adults, such as pipids (e.g. Xenopus laevis: Cambray, 1976) or several gymnophione genera (e.g.

Dermophis, Gymnopis, Caecilia, Gegeneophis, Typhlonectes: Wake, 2003). The monocuspid condition in adults is, however, considered to be phylogenetically different from the monocuspid condition in larvae (Wake & Wurst, 1979; Greven, 1984; Beneski & Larsen, 1989a, b). Pedicellate teeth (in general bicuspid) are therefore the only dental character interpreted as evidence of close amphibian relationships (e.g. Schultze, 1969, 1970; Bolt, 1980; Lombard & Sumida, 1992). Nevertheless, pedicellate teeth with a distinct separation between the crown and the pedicel have been described in extinct temnospondyls (Doleserpeton from the Lower Permian: Bolt, 1969; Apateon: Schoch & Carroll, 2003; and other branchiosaurids: Boy, 1978). In contrast to the condition observed in mature stages, in Apateon larvae the teeth do not have a gap between the base and the crown (Schoch & Carroll, 2003). In modern salamanders, the firstgeneration teeth in larvae do not possess a pedicel, while pedicels are well formed in juvenile specimens (Wistuba, Greven & Clemen, 2002). (3) The significance of teeth for lissamphibian systematics Dentition pattern, dental lamina development, and crown morphology have been suggested to be important features to establish 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 detailed descriptions of the mouth cavity and the dentition pattern in numerous caudate species (Clemen, 1979a-c, 1988; Greven & Clemen, 1985, 1990; Clemen & Greven, 1988; Ehmcke & Clemen, 2000a). Such a large amount of data allows comparison of the development of the dentition pattern, the organisation of the dental lamina, and variations in tooth shape in relation to the location of the teeth in the oral cavity, among species and between sexes. It is beyond the scope of this review to summarise all these descriptions, but some interesting points are highlighted below. In the plethodontid salamander Bolitoglossa subpalmata (Boulenger, 1896), a direct developer, teeth are absent on the 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 their well-developed tongue to transport the small prey deep into the mouth, towards the vomerine dentition, while adults feed on larger prey (Wake & Deban, 2000). The teeth of the upper jaw are, therefore, of very little importance in young individuals (Ehmcke & Clemen, 2000a). Sirenids lack teeth on the upper jaw (Clemen & Greven, 1988), but this loss of upper jaw dentition may be secondary when considering the condition in a fossil sirenid (Habrosaurus dilatus), which bears teeth on the premaxillae and the maxillae (Estes, 1965). During the breeding season, in some plethodontids the males have a few, long (300 mm versus 200 mm in the interim
53 period), monocuspid teeth protruding from the upper lip (Noble, 1929; Stewart, 1958; Clemen & Greven, 2000; Ehmcke & Clemen, 2000a; Ehmcke et al., 2003). The males use such teeth to stimulate the female during courtship. The temporary monocuspidity (versus bicuspid teeth during the interim period) of these particular teeth in males is under the influence of androgens (Stewart, 1958). This suggests that 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 only be changed through tooth replacement this implies that the bicuspid teeth located in this region of the upper jaw are lost and replaced by monocuspid teeth during the breeding season (see also Section VI). It is known that metamorphosed caudates have bicuspid teeth, 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, monocuspidity in larvae must be regarded as a plesiomorphic condition as reported for first-generation teeth in actinopterygians (Sire et al., 2002). However, monocuspid teeth have been reported in some metamorphosed lissamphibians such as pipid anurans (Katow, 1979; Greven & Laumeier, 1987), some salamanders such as the plethodontid Aneides lugubris (Wake, Wake & Wake 1983) and several gymnophione genera (Taylor, 1968; Wake & Wurst, 1979; Greven & Clemen, 1980; Wake, 2003). Do they express a less derived condition in these species than in other lissamphibians? Greven (1984) points out that the monocuspid (spikelike) teeth in adult caudates are morphologically different from those (with sharp edges) observed in larvae, the former type being regarded as less derived. Such a careful distinction may be useful in understanding lissamphibian relationships. Wilkinson (1991) discussed whether monocuspid teeth are derived or not within Gymnophiona.
III. LISSAMPHIBIANS IN THE LABORATORY For more than a century (e.g. Owen, 1845) investigators have taken advantage of the relative ease with which lissamphibians can be reared in captivity from eggs or larvae caught in the wild to study the dentition of numerous species, especially frogs (e.g. Rana pipiens Schreber, 1782), and salamanders [Cynops pyrrhogaster Boie, 1826; Necturus maculosus (Rafinesque, 1818), Ambystoma mexicanum (Shaw & Nodder, 1798) and Pleurodeles waltl Michahelles, 1830]. Around the middle of the 20th Century experimental work concentrated on species from which numerous eggs could be obtained in the laboratory, either naturally (e.g. Ambystoma mexicanum and Pleurodeles waltl) or by artificial induction [Xenopus laevis (Daudin, 1802) and, recently, Silurana tropicalis (Gray, 1864)]. Appropriate breeding conditions 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
54 advances were obtained in the understanding of lissamphibian tooth development in larvae, mainly in A. mexicanum and P. waltl, and/or in juvenile and adult specimens (caudates and various anurans). Although studies dealing with tooth morphogenesis and differentiation in lissamphibians have declined since the end of the 1970s, the number of species bred in laboratories is increasing, and three model species (X. laevis, A. mexicanum and, to a lesser degree, P. waltl) are still used in laboratories studying early developmental processes, reproduction biology, and many other topics. Unquestionably, the most studied species is X. laevis, for which numerous developmental genes have been cloned. However, in X. laevis the teeth form late, at the end of the larval period, i.e. 2-3 months after hatching (Cambray, 1976; Shaw, 1979). By contrast, in Caudata the first teeth start to develop by the end of the embryonic period, similar to the situation in actinopterygian fish (Sire et al., 2002). A. mexicanum and P. waltl are, therefore, more appropriate model species to study tooth development in lissamphibians at the molecular level, particularly in an evolutionary perspective. Of these, P. waltl is preferred because A. mexicanum is generally neotenic and has a large genome. Recently, Silurana tropicalis has become a widespread model species in the lab. It has the advantages of being diploid (versus tetraploid in Xenopus laevis), growing more

rapidly (25C), having shorter generation time, and genome sequencing is already well advanced (see www.xenbase.org). In the near future this species will substitute X. laevis for most developmental studies, including odontogenesis.
IV. OVERVIEW OF TOOTH MORPHOLOGY AND STRUCTURE IN LISSAMPHIBIANS Adult lissamphibians possess an haplodont dentition, with conical or cylindrico-conical, generally homodont teeth, but some caudate and gymnophione species have an heterodont dentition (Greven, 1984, 1986; Wake, 1980; Wake et al., 1983). Teeth are restricted to the oral cavity. Lissamphibians, as most nonmammalian taxa, replace their teeth continuously during life, i.e. they are polyphyodont. Caudate and gymnophione teeth have a large diversity of size, shape (mono-, bi-, pluricuspid) (Fig. 3), and mode of attachment (pleurodont for most teeth, except for the palatal teeth, which are acrodont). This diversity contrasts with a number of well-conserved features, such as tooth structure (a pulp cavity surrounded by a dentine cone covered by enamel; a crown and a pedicel separated by a 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, Pleurodeles waltl. (A) First-generation tooth in a larva, stage 44. The tooth is monocuspid and the dividing zone is lacking. (B) Third- (left) and fourth- (right) generation tooth in a five-month-old, postmetamorphosed specimen. The teeth are bicuspid and the dividing zone is visible. (C) Detail of the tooth tip in an adult showing the two cusps. The main cusp is lingually oriented. (D, E, F) Teeth in Gymniophona. (D) Typical tooth morphology in an embryo of Geotrypetes seraphini (left) and in a foetus of Nectocaecilia petersi (right). (E) Adult tooth in Hypogeophis rostratus. (F) Adult tooth in Geotrypetes seraphini. (G) Adult tooth in the anuran Bombina 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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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.
replacement (lingual). In lissamphibians, the ameloblasts do not form Tomes processes (which are supposed to play a role in enamel crystal orientation: Carlson, 1990) and amphibian enamel differs from mammalian in being nonprismatic (Zaki, Yaeger & Gilllette, 1970; Zaki & MacRae, 1977, 1978; Kogaya, 1994). In post-metamorphic salamanders the presence of enamel matrix proteins has been identified using immunocytochemistry, especially revealing amelogenin-like proteins (Herold, Rosenbloom & Granovsky, 1989). The first deposited enamel matrix forms globular patches within which the enamel crystals are mostly radially arranged (Kallenbach & Piesco, 1978). In later stages of amelogenesis, a thick enamel layer is formed in which the enamel 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 have raised questions, still unanswered, with respect to the enameloid/enamel transition, the formation of the dividing zone and the nature of the pedicel. (1) Enameloid versus enamel The nature of the enamel-like material covering the teeth in larval and adult caudates has long been debated since the
pioneering studies of Owen (1845), Leydig (1867) and Hertwig (1874). Levi (1940), Kvam (1946, 1953, 1960) and Kerr (1960) believed that the external covering of the monocuspid teeth in larvae was a mesodermal enamel, i.e. a highly mineralised dentine, called durodentine, exclusively deposited by the dental papilla cells. Using polarized light, Schmidt (1957, 1958) considered the outer surface of the adult teeth to be durodentine. Later, he changed his view and suggested that this layer is an ectodermal enamel, i.e. deposited by the enamel organ (Schmidt, 1970). In a first attempt to study amelogenesis in the caudate Pleurodeles 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 transmission electron microscopic (TEM) observations have revealed its presence (Smith & Miles, 1971; Chibon, 1972; Roux & Chibon, 1973; Roux, 1973). In larvae, the dental papilla cells, the odontoblasts, deposit first a layer of a particular dentine that mineralises more strongly than regular dentine. This layer is now called enameloid, a term introduced by Poole (1967) and rvig (1967) to replace the confusing terms mesodermal enamel, vitrodentine and durodentine. In fact, the difficulty of recognising enameloid in larval teeth resides in the fact that the first
56 matrix deposited by odontoblasts at the tooth tip resembles predentine matrix in that it contains a relatively dense collagen network (Fig. 5). The pre-dentine-like matrix is secondarily converted into enameloid during the mineralisation and maturation process, which could be under the influence of the inner dental epithelial cells, the ameloblasts. These eventually deposit a thin layer of true enamel at the enameloid surface. The presence of a thin layer of true enamel covering enameloid in larval teeth has been confirmed in the first-generation teeth of the caudate Ambystoma mexicanum using scanning electron microscopy (SEM) (Bolte & Clemen, 1992). The layer of enameloid does not exist (or is extremely reduced and located at the dentine-enamel junction) in adult teeth, in which a thick layer of enamel covers the dentine directly (Chibon et al., 1971; Smith & Miles, 1971). In adults of some lissamphibian species, enamel is orange due to the presence of iron ions, which are concentrated within ferritin patches in the secretory ameloblasts (Randall, 1966). The formation of either enamel or enameloid is thought to be related to heterochrony in the secretion of ameloblasts and odontoblasts (Smith, 1995). (2) Dividing zone In the three orders of lissamphibians, adult teeth are usually composed 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 to the presence of undivided teeth in most larval stages. In most gymnophione genera, however, foetal teeth have a discrete pedicel (Wake, 1976, 1980). In their extensive study of lissamphibian 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 adult lissamphibians. There are, however, a few exceptions, in which the division within the teeth has been reported as sterdam, 1766 absent, such as in the caudate Siren lacertina O and in the anuran Xenopus laevis (e.g. Parsons & Williams, 1962; Means, 1972). In these cases the teeth are calcified from the crown to the base and anchored to the jawbone by attachment bone (Katow, 1979; Shaw, 1979). Tesche & Greven (1989) also report that the first-generation teeth in anurans are not pedicellate. There are no reports of any adult gymnophionans lacking the dividing zone. The dividing zone most commonly appears as a welldefined transverse region, resembling a suture between two bones (Fig. 6). This zone of weakness is revealed by the tendency for the crowns to fall off in jaws that have been vigorously cleaned. In such cases, because they are firmly fused to the bone support, the pedicels are left as hollow cylinders. Alizarin red staining has revealed that this zone of division is either not, or is only slightly, mineralised (Gillette, 1955). This low level of mineralisation of the collagen matrix was further confirmed by TEM studies (Wistuba et al., 2002). Although the functional significance of this zone is not known with certainty, it has been suggested that, in addition to providing a certain degree of flexibility as a ligament, it allows 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, Moury et al. (1985) concluded that this uncalcified region allows tooth flexion only in a posterior direction. In Pleurodeles waltl, most of the dividing zone looks like a ligament linking the crown to the pedicel (Fig. 6 C, D). In Ambystoma mexicanum, it is noteworthy that the odontoblasts facing the dividing zone lack 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. The dividing zone appears, therefore, to be a transition between the tubular dentine of the crown and the atubular dentine of the pedicel. (3) Pedicel The crown is universally agreed to be dentine, but the nature of the pedicel has long been the subject of discussion. Indeed, the lack of dentine tubules and the presence of some enclosed cells in this region have led authors to consider 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 of the jawbone. Hertwig (1874) was the first to assert that since the pedicel is formed within the epithelial sheath and is formed anew each time a tooth is replaced, it should be considered part of the tooth rather than a bony projection of the jaw. He also assigned the term cementum to the substance of the pedicel since it contains cell bodies. Cell bodies were never observed in the pedicel of young P. waltl we have studied (Fig. 7). Studying tooth replacement in the anuran Rana pipiens, Gillette (1955) was the first to conclude that the pedicel is composed of dentine laid down by odontoblasts, and that the cementum is located only at the base of the pedicel and at its outer surface. This finding was confirmed by Parsons & Williams (1962) in gymnophione teeth, and by Kerr (1960) and by us herein in caudate teeth (Fig. 7). However, despite their common origin, in adult caudates the crown and pedicel possess a different Ca/P der, 1980; Bolte, Krefting & ratio (Clemen, Greven & Schro Clemen, 1996). In Ambystoma mexicanum this ratio, which determines the hardness of the tooth region, is 45.9% in the central crown versus 38.4% in the central pedicel. This indicates 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 the tooth. However, the term pedicel is restricted to the mineralised cylinder of dentine which is located below the dividing zone, while the term tooth base includes the bone of attachment that links the pedicel to the bone support (Moury et al., 1987). The dentine of the pedicel lacks tubules, but it is clearly distinct from the bone of the jaw (Howes, 1978) (Fig. 7). The same conclusion that the pedicel/attachment bone is part of the tooth was reached for the bone of attachment of the teeth in lower vertebrates (Peyer, 1968; Sire & Huysseune, 2003). In fact, the base of the pedicel is linked to the bone support by a particular zone, which could be considered to be bone of attachment (Fig. 7A, D). The cement-like tissue is deposited on the
57
Fig. 5. Enameloid: a peculiar feature of the teeth in caudate larvae. (A) Schematic drawing of a developing tooth indicating the location of enameloid between dentine and enamel. (B) In this first-generation tooth of Pleurodeles waltl, the enameloid matrix has been recently deposited by the odontoblasts. Some cytoplasmic prolongations of the odontoblasts are visible in the enameloid matrix (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 hardly visible. The enameloid matrix is composed of thin collagen fibrils loosely organised, except along the tooth surface, where they run parallel to the cell surface. The first elements of the predentine matrix have been deposited below the enameloid. (C) Mineralisation stage. This sample was decalcified using ethylenediaminetetraacetic acid (EDTA); the narrow, empty space located between the enameloid and the ameloblast surface indicates that a thin layer of enamel was present at the enameloid surface, but removed during the decalcification process. Around the tooth tip the ameloblasts show numerous cell membrane folds, which characterize the postsecretory phase. Asterisks indicate cell prolongations from odontoblasts. (D) Enlargement of the tip of the tooth in C showing the ameloblasts located at the enameloid surface and their prominent folds. The foamy aspect of the enameloid matrix indicates that the mineralisation process has started. A modified from Smith & Miles, 1971; B, C, D original micrographs. Scale bars: B, C 1 mm; D 250 nm. am: ameloblasts; en: enamel; ena: enameloid; de: dentine; od: odontoblasts; pde: predentine.
58
Fig. 6. Dividing zone in the teeth of Pleurodeles waltl. (A, B) Scanning electron micrographs showing the dividing zone in a 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 dividing zone than elsewhere along the tooth shaft, and a large part of this matrix is not mineralised. (D) Detail of the dividing zone of the tooth in C. The mineralisation front is irregular. (E, F, G) Transmission electron micrographs. (E) General view of the structure of the dividing zone (lingual side). The arrows indicate the mineralisation front. (F, G) Detail of the dividing zone in the region facing the pulp (F) and facing the mesenchyme (G). (F) The surface of the dividing zone is irregular and covered by large, active odontoblasts, which deposit a matrix composed of thin, unmineralised collagen fibrils. (G) Flattened cells of the retracting enamel organ cover the tooth matrix, from which they are separated by a thin, barely visible basement membrane (arrow). Facing the cell the matrix is composed of a loose network of thin unmineralised collagen fibrils, which mostly run parallel to the tooth surface. At a 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.
59
Fig. 7. The pedicel, pulp cavity and cementum of the teeth of Pleurodeles waltl. (A-D) One-mm thick, transverse sections of the teeth in a larval stage 42 (A), and in three-month- (B), five-month- (C) and eight-month- (D) old specimen. (A) In larval teeth, the pulp cavity contains only a few cells. It was secondarily invaded by blood vessels. The dividing zone is hardly visible. Note that the tooth on the left is attached on one side onto the bone support and, on the other, to the attachment bone region of the adjacent tooth; in both 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 are well organized and polarised, while the centre of the pulp contains blood vessels and undifferentiated cells. (D) On the pulpal and mesenchymal side the pedicel surface of the functional teeth is lined by odontoblast- and osteoblast-like cells, respectively. At the pulp side, the odontoblasts are depositing predentine at the dentine surface (arrow). At the mesenchymal side the enamel organ (the so-called cervical loop) has retracted, and a reversal line is visible (arrowheads), delimiting the dentine matrix from a thin layer of cement, which has been secondarily deposited on the pedicel surface by osteoblast-like cells. The latter are more active at the pedicel base, where they deposit the attachment bone matrix. (E, F) Electron micrographs of the attachment zone. (E) 12-month-old specimen. 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.
60 dentine surface of the pedicel by osteoblast-like cells, in the region where Hertwigs sheath has retracted after the tooth has become functional. In Pleurodeles waltl the cementum is not deposited in the first-generation teeth.

matrix of the mineralised cap is entirely removed during the maturation process. This is clearly revealed by decalcification which leaves an empty space between the dentine and the ameloblast surface (see Fig. 5C). Predentine matrix is next deposited by the odontoblasts (Figs. 8B, 9B). All features indicate that these cells are the same as those that previously deposited the enameloid matrix, suggesting a switch in the functioning of odontoblasts from enameloid to dentine matrix production. The first deposited (immature) collagen fibrils of the predentine measure between 6 and 12 nm in diameter. Their diameter then increases to 30 nm (mature fibrils) reaching 60-80 nm prior to mineralisation. All the odontoblasts located along the dentine shaft possess long cytoplasmic processes, which penetrate the predentine matrix. The dentine shaft elongates towards the surface of the developing bone support, to which the tooth base will eventually fuse (Figs 8C-E, 9D-F). This primary type of tooth attachment in first-generation teeth differs from the secondary type of attachment of replacement teeth, in which the tooth base attaches to a pre-existing bone surface as indicated by the presence of a cementing line. The tooth pierces the oral epithelium and becomes functional. In the developing first-generation teeth, the pulp cavity is entirely occupied by odontoblasts. Most of them degenerate when the tooth becomes functional, while some remain active along the dentine surface. The pulp cavity is secondarily penetrated by a capillary blood vessel through a large pore located lingually at the interface between the dentine shaft and the bone surface. The development of subsequent generations of teeth is similar to that of first-generation teeth, except for those features described in Section IV: enamel progressively covers enameloid which disappears at metamorphosis (Fig. 5), there is a dividing zone separating the dentine shaft 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 the replacement teeth; this is discussed in Section V.3. (a ) Caudata Most caudates are oviparous, with rare exceptions such as some salamanders of the family Salamandridae (e.g. genera Salamandra, Lyciasalamandra, Mertensiella) which are livebearing (viviparous). Some species are neotenic (e.g. Ambystoma mexicanum, Necturus maculosus, some Triturus species, and some plethodontids such as some Gyrinophilus and Eurycea species). These do not metamorphose, and conserve most 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), and A. mexicanum by Smith & Miles (1971) and by Wistuba et al. (2002). Most of our knowledge of tooth development in caudates comes from studies at the light microscope and TEM level in larvae and adults of, mainly, P. waltl and A. mexicanum. These studies have focused either on general features of tooth development (Wistuba et al., 2002), or on various aspects of odontogenesis such as the
V. TOOTH DEVELOPMENT AND REPLACEMENT Since Hertwig (1874) established the first bases of knowledge on lissamphibian teeth, developmental events have been well documented in many species. Here, we review generalised lissamphibian tooth development (morphogenesis and differentiation), resorption and replacement patterns, and then discuss in detail the three orders, Caudata, Gymnophiona and Anura. Within the anurans we will describe the Pipidae separately. We pay particular attention to the salamander Pleurodeles waltl, a model species widely used in tooth development. A characteristic feature resulting from polyphyodonty in lissamphibians is that, at a given time, several replacement teeth can be found in a single specimen and, especially, in juvenile stages. This is a considerable advantage when studying tooth morphogenesis and differentiation. Indeed, all stages of tooth development can be found on a jaw, but early stages are barely visible at the light microscopical level. (1) Tooth morphogenesis and differentiation Tooth development in a generalised lissamphibian is schematically illustrated in figure 8 and micrographs of P. waltl detail 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 an epithelial invagination, two cell layers wide, into the mesenchyme. Then, in particular regions of the dental lamina and facing the mesenchyme, the basal epithelial cells differentiate into placodes. Mesenchymal cells, originating from the neural crest (e.g. Wagner, 1949; Chibon, 1966) concentrate at the level of the placodes. The basal layer cells of the dental lamina invaginate more or less deeply into the mesenchyme and develop into a cap. The dental epithelium differentiates into an enamel organ composed of two cell layers, the inner and the outer dental epithelium, while the facing 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 cells into odontoblasts. The first tooth matrix is produced by the odontoblasts 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). The enameloid is secondarily modified by the activity of the facing ameloblasts, which deposit a thin layer of enamel matrix on the outer surface of the enameloid. The ameloblasts next participate in the maturation process of both the enameloid and enamel matrices, resulting in the presence of a highly mineralised tooth cap. The organic
61
Fig. 8. Schematic representation of the development of a tooth and its successor in a generalised lissamphibian. Anterior is to the left. (A) Morphogenesis and early cytodifferentiation. Originating from the basal epidermal layer of the oral epithelium, the primary dental lamina extends into the subjacent mesenchyme. The distal region of the dental lamina interacts with mesenchymal cells and forms a cup. The epithelial cells differentiate into an enamel organ, which further differentiates into an inner and an outer dental epithelium. (B) Late cytodifferentiation. Mesenchymal cells have differentiated into odontoblasts, which deposit an unmineralised matrix, predentine. The latter mineralises to become dentine. Facing the latter the inner dental epithelium cells differentiate into preameloblasts. (C) The preameloblasts differentiate into ameloblasts and deposit the enamel matrix on the dentine surface. The dentine 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 base of the crown. The pedicel is separated from the dentine cone by an unmineralised region, the dividing zone. The secondary dental lamina has extended deeply into the mesenchyme. (E) The tooth has attached to the supporting bone through its pedicel and its tip has pierced the oral epithelium. The tooth is now functional and its replacement tooth has started to form. Note that in caudate larvae the development of the first-generation tooth differs in that enameloid is the first matrix deposited by the odontoblasts, before dentine. 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.
62
Fig. 9. Development and attachment of the first-generation teeth in Pleurodeles waltl larvae, 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 have formed a small dental papilla: this is the bud stage. (B) Early cytodifferentiation, stage 36. The ameloblasts, i.e. the inner dental epithelium cells, and the odontoblasts, i.e. the dental papilla cells, have differentiated. Tooth matrix has begun to be deposited by the 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 as a prolongation of the dentine shaft towards the surface of the supporting bone. (E) Stage 40. The tooth base is anchored to the dentary bone by means of attachment bone. A blood vessel has penetrated the pulp cavity and the odontoblasts have slowed down their activity. (F) Stage 55. Tooth recently attached to the dentary bone. The dentine crown, the pedicel and the attachment bone are 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.

differentiation of the dental epithelium (Smith & Miles, 1971), dentinogenesis (Roux, 1973), amelogenesis (Roux & Chibon, 1973), or on the characteristics of a particular region such as the enameloid or enamel cap (Kogaya et al., 1992; Kogaya, 1999), the tooth base (Moury et al., 1987), and the dividing zone (Moury et al., 1985; Greven & Clemen, 1990; Wistuba et al., 2002). The comparative analysis of this large amount of data leads to the conclusion that (i) tooth morphogenesis and differentiation in caudates are similar to that described in mammals, and (ii) in all species examined larvae and adults share similar features, with only a few differences, which relate mainly to tooth size and to the presence of enameloid in larvae versus enamel only in adults (see Section IV.1). In larvae, teeth are attached to the paired bones of the upper jaw (premaxillaries, maxillaries, prevomers and palatines) and the lower jaw (dentaries and coronoids) (Signoret, 1960) (Fig. 10). These bones form between stage 37 (Gallien & Durocher, 1957), i.e. 15 days post-fertilisation (dpf) and stage 55 (90 dpf). The last bones to be formed are the maxillaries, which ossify shortly before metamorphosis (see Section V.2 for comments on the relationships between teeth and bone supports). During metamorphosis, the
63 palatines disappear from the upper jaw and are replaced by the extension of the vomers. In the lower jaw, the coronoids disappear and only the dentaries remain (Corsin, 1966; Reilly, 1986) (Fig. 10). The first-generation teeth start to form in embryos from stage 33a (initiation, 9 dpf specimens). The first matrix is deposited at stage 35 (12 dpf). The teeth grow, attach to the bone support, pierce the buccal epithelium and become functional when the mouth opens, at hatching (stage 37, 15 dpf). At this stage, there are on average 23 teeth on the upper jaw: a row of eight teeth on the premaxillaries and two rows of seven and eight teeth on the vomers and the palatines, respectively. A row of 25 mandibular teeth is present on the lower jaw, supported by the dentaries and coronoids (Roux & Chibon, 1973). The teeth on the dentaries face the premaxillary teeth, while those on the coronoids face the vomerine and palatine teeth. Two or three tooth generations succeed the first during larval life and the number of tooth positions increases in each row. Although new teeth are added at each position, tooth resorption starts at stage 48 (50 dpf) only, suggesting the retention 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 of Pleurodeles waltl during ontogeny. (A, B) Larva, lower and upper jaws, respectively; (C, D) adult, lower and upper jaws, respectively. A, B are modified from Signoret (1960).
64 the second row considered replacement teeth (Roux & Chibon, 1973). The teeth in larvae are conical and monocuspid. The first-generation teeth are 100-150 mm tall. The height increases in replacement teeth to reach 500 mm after metamorphosis, during which bicuspid teeth replace monocuspid teeth (see Section VI.1). In direct developing species (e.g. many plethodontids) bicuspid teeth are 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, eight in old larvae and 16 in post-metamorphosed specimens. These experiments also indicated that some phases of odontogenesis (initiation, morphogenesis, early differentiation) proceed slowly while others are rapid (late cytodifferentiation, growth and eruption). (b ) Gymnophiona Caecilians possess numerous pedicellate teeth on the lower and upper jaw, which are usually arrayed in two rows. The dentition is generally homodont but exceptions exist, for instance in foetuses (Wake, 1980) and in adults of some species which have different degrees of bicuspidality on the upper jaw and monocuspid teeth on the lower jaw (e.g. Gegeneophis ramaswamii Taylor, 1964 (Greven, 1984). The tooth structure is known at the light microscopic level (Wake, 1976; Clemen & Opolka, 1990) and is similar to that described in caudates. Tooth morphology differs depending on whether the species are viviparous or oviparous, and on developmental stage (Fig. 3). In viviparous species, tooth development has been ril & Bibron, 1841) studied in Dermophis mexicanus (Dume by Wake (1976, 1980), and described from a single stage in ril, 1859) by Parker (1956), Parker Geotrypetes seraphini (Dume & Dunn (1964) and Wake (1976), and in Gymnopis multiplicata ril & Peters, 1874 and Typhlonectes compressicauda (Dume Bibron, 1841) by Wake (1976) and Hraoui-Bloquet & Exbrayat (1996). The growth of embryos continues in utero after the egg yolk has been exhausted; the foetuses develop through metamorphosis in the oviducts and possess particular teeth called foetal teeth. For Parker & Dunn (1964) foetal teeth are functionless (a relictual retention of a fish-like character). By contrast, Wake (1976, 1980, 1993) considers they function to aid ingestion of intra-oviductal nutrient material and to scrape the oviduct wall to stimulate secretion 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 they are specialised for scraping. This contrasts with the condition found in other vertebrate larvae in which the first-generation teeth are invariably conical and monocuspid, and considered an ancestral state for vertebrates (Sire et al., 2002). In gymnophione embryos, tooth buds appear first on the lower jaw at an early developmental stage, when the bone support is not yet formed (Wake, 1976). Teeth appear earlier in G. multiplicata than in T. compressicauda (Wake, 1976). In G. multiplicata foetuses the teeth possess two major cusps (a primary labial and a secondary lingual) and

several spike-like, minor cusps. The teeth are pedicellate and only the top of the crown pierces the buccal epithelium. In T. compressicauda, the foetal teeth first have two cusps, then other cusps appear during ontogeny, indicating that replacement has occurred during foetal life. The foetal teeth are arranged in several rows (in contrast to a single row in adults). This is explained by the retention of the replacement teeth at every position, as in caudate larvae. In foetuses, the height of the crown varies from 140 to 300 mm depending on species (and, probably, on generation time) and the pedicel is short (one third of the crown height) and fused to the bone support. The foetal teeth are entirely resorbed (or shed) at birth and replaced by bicuspid or monocuspid teeth such as found in adults (Wake & Wurst, 1979). This sudden transition is presumed to be a response to hormonal induction (Wake, 1993). In oviparous species, tooth development has been studied in Hypogeophis rostratus by Marcus (1920), Reuther (1931) and Lawson (1965a, b) and in Ichthyophis glutinosus by Clemen & Opolka (1990). In the latter species, the dental lamina of embyros is different from that in adults, which suggests that the dental lamina divides later in ontogeny (Clemen & Opolka, 1990). In these species, the embryonic teeth are arranged in a single row on the dentigerous bones and are monocuspid (Clemen & Opolka, 1990), while they are bicuspid and pedicellate in larvae and juveniles (Parker & Dunn, 1964). In both viviparous and oviparous species, the odontogenetic processes are broadly similar to those described above (Lawson, 1965a, b; Wake, 1976) (Figs 8, 9). In Hypogeophis rostratus, a thin cone of predentine is laid down by odontoblasts and major and minor cusps are formed. Mineralisation starts in the predentine when the first cusp is formed. A thin layer of enamel is deposited by the ameloblasts over the cusps (Lawson, 1965a). It seems that the enamel matrix mineralises rapidly after its deposition because no pre-enamel matrix was found in undecalcified specimens. Enamel is thicker over the cusps than elsewhere. Early during its development the tooth germ lies more or less at right angles to the position occupied by the functional tooth, and is attached to the buccal epithelium by the dental lamina. Then it rotates approximately 90 and the dentine cone elongates. A second mass of dentine is produced at a short distance from the base of the dentine cone and eventually forms the pedicel (Lawson, 1965a). The calcification of the pedicel starts when it has reached the base of the crown. The lingual side of the pedicel develops more quickly than the labial side, leading to a pleurodont type of attachment by ankylosis to the jawbone. During the functional 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 other frogs teeth are restricted to the upper jaw, with the exception of some hylids, in which teeth are also found on the dentary (Goin & Hester, 1961). On the upper jaw, the teeth are arranged in a single row on the paired premaxillaries,

the paired maxillaries and the paired vomers. In some species the dentary is ornamented by odontoid (tooth-like) structures, which are bony elements (Trueb, 1993). Teeth are small (less than 1 mm long in adult Rana pipiens), but very numerous, and their number and size varies with frog size. For instance, Gillette (1955) counted 89-95 functional teeth 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 average 368 teeth in its dentition (small specimens have approximately 140 teeth). Teeth are bicuspid and the dentition is homodont. Anurans are mostly oviparous, but several species are ovoviviparous and others are viviparous (Duellman & Trueb, 1986; McDiarmid & Altig, 1999). Their aquatic larvae, the tadpoles, are mostly herbivorous and detritivorous, and their dentition is not composed of true, mineralised teeth. Instead, there are horny labial teeth in the upper and lower beak, which are formed by keratinised epithelial cells organised into columns (Kaung, 1975; Takahama et al., 1987). The cell located at the upper extremity of the column forms the top of the tooth. The number of teeth and the size of the beak increase during larval life. At metamorphosis, the keratinized epithelial cells are destroyed by autolysis, a process similar to that observed in the tail. Hertwig (1874) was first to describe tooth development in frogs. Subsequent detailed accounts mainly confirmed his findings. To date, descriptions of tooth development are available for: Rana pipiens by Gillette (1955), Hyla cinerea (Schneider, 1799) by Goin & Hester (1961), R. temporaria Linnaeus, 1758 and R. esculenta Linnaeus, 1758 by Spinelli & Chibon (1973) and by Chibon (1977), and H. arborea (Linnaeus, 1758) and R. nigromaculata Hallowell, 1861 by Sato et al. (1986a, b). The first tooth germs appear at metamorphosis, by the end of hindlimb organogenesis, and the first teeth are functional after 25-26 days of growth. The developmental features are similar to those described above for a generalised lissamphibian. In R. pipiens, there are six separate dental laminae corresponding to the six dentigerous bones, and extending lingually to the dental process of each bone (Gillette, 1955). Predentine first calcifies in the lingual and then in the labial cusp. Concomitant with the first calcification of the dentine the ameloblasts differentiate, then deposit enamel matrix first on the labial sides of the cusps. The enamel mineralises rapidly, but slower than in caudates (Spinelli & Chibon, 1973). During pedicel formation the tooth progressively changes its orientation. The calcifying process stops abruptly at the crown-pedicel junction. Ankylosis of the pedicel to the bone is ensured by cellular cementum, which completely bridges the gap between the pedicel and the bone surface. Eruption is arrested when the cementum matrix calcifies. During the functional period more dentine is added to the crown and to the pedicel on the pulp side. Cellular cementum is deposited on the outer surface of the pedicel, when the epithelial root sheath (Hertwigs sheath) has retreated. In Hyla cinerea teeth develop similarly to those of Rana pipiens with only a slight difference in the mode of
65 attachment, which is related to the form of the dental process of the maxillary (Goin & Hester, 1961). (d ) Pipidae The Pipidae are a well-known anuran family thanks to Xenopus laevis, which is used as a model in developmental biology. X. laevis may live as long as 23 years in the laboratory (Deuchar, 1975). In their Developmental Table for X. laevis Nieuwkoop & Faber (1956) briefly commented on the development of the dentition. Cambray (1976) provided the first description of tooth development in larvae and adults, but his doctoral thesis remained unpublished. Subsequently, Shaw (1979, 1985, 1986, 1989) published reference data on tooth development and replacement in X. laevis. X. laevis differs from the other anuran taxa in that true teeth begin to develop on the upper jaw in tadpoles during the last stages of larval life (Fig. 11). However, teeth do not erupt until the end of metamorphosis; they are 225-250 mm tall (Shaw, 1979). In larvae, newly metamorphosed and adult specimens the teeth are morphologically similar, only differing in size. The dentition is homodont and the teeth form a single row on the upper jaw. The teeth are conical, monocuspid and slightly curved at the tip. In adults, only 50100 mm of the tooth tip projects into the mouth so that it is hard to ascribe an important function to them (Shaw, 1979). Tooth structure is known at the light microscopic level only (Shaw, 1979). Unlike in many other lissamphibians, the enamel organ is composed of three layers, a stellate reticulum being present between the inner and the outer dental epithelium. In addition, teeth are not pedicellate (there is no dividing zone) and there is no indication of a layer of cementum along their base (Katow, 1979). They are ankylosed to the premaxillaries and maxillaries by a short, ring-shaped bony pedicel (bone of attachment), which is continuous with the jaw bone in newly metamorphosed specimens but distinct from the underlying bone in the adults. All these features are typically those of first-generation teeth in nonmammalian vertebrates (Sire et al., 2002). The first-generation teeth develop at stage 55, approximately 40 days before metamorphosis (Shaw, 1979) (Fig. 11). Teeth appear in alternating (even and odd) positions, starting from the mid-line, to reach 22 positions on each side of the jaw at metamorphosis: eight on the premaxillary and 14 on the maxillary. Teeth at even positions develop first, 8-9 days before those at odd positions. From the second day after germ initiation, dentine deposition commences, followed shortly by enamel formation. We have not been able to identify enameloid unequivocally during early tooth development in Xenopus laevis (Fig. 11 and H. Chisaka, unpublished results), but this needs to be investigated further. Enamel formation and mineralisation 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 of rapid dentinogenesis, re-orientate to a more vertical position, and develop a dental lamina for their successor tooth. Attachment lasts from day 23 to 25. From day 27 after first tooth germ initiation, the tooth germs at odd positions begin their period of rapid growth. Osteoclastic
66
Fig. 11. Tooth development in an anuran, the pipid Xenopus 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 developed at 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) One mm-thick vertical sections of the upper jaw of tadpoles (stages 54, 58, 63 and 65, respectively) showing various developmental stages of 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: inner dental epithelium; mb: maxillary bone; od: odontoblast; oe: oral epithelium; pc: pulp cavity; pm: premaxillary bone.
resorption of the first even-numbered teeth begins at 32-33 days (i.e., 4-6 days post stage 65) (Shaw, 1979). The second generation teeth erupt five days later, i.e. slightly before the end of metamorphosis. In summary, in larvae a complete tooth cycle takes 33 days, with teeth being functional for only seven days, versus 59-71 days for the complete cycle and 24-29 days functional in adults. (2) Relationships between tooth and bone support development In nonmammalian species studied so far, the development of a first-generation tooth ends with the anchoring of the tooth base (the pedicel or attachment bone) to a bony support (premaxillaries, maxillaries, dentaries, vomers, palatines, pharyngeal bones, etc.). However, the matrix of the supporting bone is not present when the tooth is initiated (Sire et al., 2002). Osteogenesis and odontogenesis progress approximately simultaneously. This is achieved such that, in the dentigerous region, both the bone and the tooth matrix seem to converge towards each other. Eventually, both matrices (bone surface and base of the pedicel) merge, forming the socalled primary tooth attachment (versus secondary tooth attachment, which occurs when the bone support is already present when the tooth attaches). Such a process suggests the existence of coordination between the odontoblasts at the base of the pedicel and the osteoblasts at the bone surface facing the developing tooth (mediated by signalling molecules?), at least during the final stages of development of
the two elements. Further (molecular) studies will be necessary to understand the interactions between these cell populations. Although these observations suggest that teeth need the presence (or concomitant development) of a bony support in order to develop, experimental studies do not support this conclusion. With the aim of perturbing organogenesis in embryonic P. waltl, Signoret (1960) applied various concentrations of lithium chloride, a molecule known to induce morphogenetic perturbations. He found organ reduction (hypomorphy) in these embryos. In addition, tooth development was found to be very sensitive to lithium chloride: some bones which are normally dentigerous (dentaries, premaxillaries, palatines), developed normally, but without teeth. In addition, (i) teeth were found in regions devoid of bone support, and (ii) some bones which were normally not dentigerous (the angular and the parasphenoid) were found to bear teeth. These experiments demonstrated clearly that tooth development does not depend on the nature and location of the bone support. The relationship between a tooth and its surrounding bone may therefore be secondary, resulting from topographic conditions only. In the frog Rana pipiens, Howes (1977) transplanted teeth during the crown formation phase to an ectopic site (either in the anterior eye chamber or in a dorsal subcutaneous site). these transplanted teeth grew normally and formed a normal-sized pedicel area demonstrating that (i) once initiated the genetic programme is able to support complete odontogenesis, and (ii) the pedicel is purely odontogenetic in origin. Similarly, the ablation of part of the premaxilla in this species

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, and were even replaced by their successors although the supporting bone, which regenerated slowly, was still not formed. Such results confirm that complete tooth development does not require the presence of a bone support. Observations made in the armoured catfish Corydoras spp. and Hoplosternum littorale confirm the generality of these findings. On the upper jaw, the first-generation teeth develop long before the development of the maxillary. Each tooth forms a pedicel of attachment bone, and the pedicels of adjacent teeth merge to constitute a kind of dentigerous bone, which will later be connected to the developing maxillary (Huysseune & Sire, 1997b). Also, in some zebrafish (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 in a similar manner in anurans (including Xenopus laevis), gymnophiones and caudates. Resorption of a functional tooth is always related to the close presence of a growing replacement tooth, as illustrated in Pleurodeles waltl (Fig. 12).
67 A replacement tooth is first seen lingually as a bud from the region located at the limit between the dental organ and the dental lamina of the functional tooth (Fig. 12). The bud extends as a new dental lamina into the mesenchyme, with which it interacts to give rise to a new tooth. The replacement tooth grows and, once fairly well developed, its enamel organ generally contacts the lingual side of the functional tooth (Fig. 12C). This contact induces the recruitment of osteoclasts as a probable reaction to pressure forces acting on the external wall of the tooth. Most generations of replacement teeth in lissamphibians exhibit these general features, although the first generation of replacement teeth does not provoke the resorption of the first-generation teeth, resulting in their retention and hence the presence of two tooth rows on the upper rand lower jaws of young larvae. The first-generation teeth are very small (20-30 mm wide) thus there is probably enough space to accommodate two teeth from the same family. Such a condition has also been described in the zebrafish (Van der heyden & Huysseune, 2000). In P. waltl, the first signs indicating imminent resorption of the 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 decrease in cell number. Numerous cells are degenerative and some macrophages are present (Roux & Chibon, 1974). The latter
Fig. 12. Tooth replacement in Pleurodeles 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 two layers, 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 have formed 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 of the 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.
68 have secondarily invaded the pulp cavity and are involved in phagocytosis of necrotic cells. Macrophages could also be responsible for the destruction of Hertwigs sheath and the basal region of the dental lamina (Chibon, 1977). The first resorption features are observed at stage 48 (50 dpf), approximately two months before metamorphosis, when two tooth generations have formed. The first tooth is completely resorbed at stage 52 (64 dpf). The cells responsible for tooth resorption are typically multinucleated osteoclasts. There are also some mononucleated macrophages removing cell debris. First, the external surface of the dentine wall at the base of the pedicel is attacked by osteoclasts at the lingual side (Fig. 13A, E). Then, osteoclasts (also called odontoclasts) penetrate the pulp cavity and start to resorb the opposite wall, while the resorption extends labially and to the top of the tooth shaft (Fig. 13B, C, F). Simultaneously, cell necrosis is observed in the pulp cavity and Hertwigs sheath retracts. The dentine shaft is entirely resorbed as well as part of the adjacent supporting bone. The large, multinucleated cells responsible for the resorption of lissamphibian teeth share similar features with osteoclasts described in many vertebrate species (Fig. 13). However, some authors have called them odontoclasts with reference to their involvement in dentine resorption (e.g. Clemen & Greven, 1974, 1977; Bouvet & Chibon, 1976; Chibon & Bouvet, 1976; Wistuba, Bolte & Clemen, 2000). The first TEM description of these cells was for frogs (Yaeger & Kraucunas, 1969). Wistuba et al. (2000), working on Ambystoma mexicanum, provided additional details. Interestingly, activity of tartrate-resistant acid phosphatase (TRAP) has not been detected during the first stages of tooth resorption, whereas it was shown to reveal osteoclastic activity in other vertebrates, such as teleosts (Witten, 1997) and mammals (Sahara et al., 1998). The presence of clastic cells in A. mexicanum, a neotenic caudate, which lacks parathyroids, indicates that the production of parathyroid hormone (PTH) is not a prerequisite for the regulation of these cells. Instead, regulation through a pituitary factor has been suggested (Pang et al., 1980). In Pleurodeles waltl tooth replacement has been shown to be under 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 normal specimens (Dournon & Chibon, 1974). This is probably related to the role played by thyroxine in cell proliferation. The fate of the tooth tip during tooth resorption has given rise to some controversy. Most authors either state, or assume, that lissamphibian teeth become loose at their bases and 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 than the pedicel, provoking shedding of the crown, and explaining why most crowns are absent in teeth undergoing resorption, while pedicels remain, at least partially (Casey & Lawson, 1981). However, in their illustrations there is no histological section showing the absence of crown resorption. Contrary to this view, Shaw (1986, 1989) found it logical that the major part of a tooth in Xenopus laevis is absorbed rather than lost by shedding. Chibon (1977) reached a similar conclusion with P. waltl larvae where the tips of teeth in an advanced state of resorption were found entirely embedded in the oral epithelium. We confirm that the teeth in P. waltl are entirely resorbed, at least in larvae (Fig. 13H, I). However, in lissamphibians osteoclastic resorption of the enamel cap has not been reported to date in the literature, in contrast to some mammals, in which enamel resorption has been reported (Sahara et al., 1998). In adult caecilians [e.g. Hypogeophis rostratus (Cuvier, 1829), Grandisonia diminutiva Taylor, 1968] tooth replacement occurs as described in P. waltl. The process of resorption is supposed to be rapid because partially resorbed teeth are uncommon (Casey & Lawson, 1981). The pedicel is entirely removed by resorption. However, it is not clear how much of the crown is resorbed before shedding. The loss of a large number of crowns may represent a considerable long-term drain on calcium reserves. Indeed, in addition to its function in permitting tooth replacement to occur, resorption is considered a conservative process making tooth constituents (minerals and organic matrix) available for re-use. In the anuran Rana pipiens, Gillette (1955) found that the tooth resorption process is similar to that described in nez de la Pleurodeles waltl. In Hemiphractus proboscideus (Jime Espada, 1871), Shaw (1983) calculated that the volume of
Fig. 13. Tooth resorption in Pleurodeles waltl. (A-D) Scanning electron micrographs of teeth subjected to resorption, viewed from the lingual 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) Tenmonth-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 of teeth subjected to resorption. (E) Larva, stage 51. The surface of the pedicel located close to the enamel organ of the replacement tooth is subjected to resorption. (F) 12-month-old specimen. Most of the pedicel has been resorbed and two large, multinucleated osteoclasts are attacking the base of the dentine crown (arrows). (G) Eight-month-old specimen. An osteoclast has penetrated the pulp cavity and a large part of the dentine crown is resorbed. Note the decalcification of the dentine matrix prior to resorption. Another osteoclast is apposed onto the surface of the dentary bone. The cells of the enamel organ of the resorbed tooth have not retracted (arrow), while the dentine crown they were covering has been resorbed. (H) Larva, stage 48. The resorption of this firstgeneration tooth is well advanced. Note that a single osteoclast is involved in the resorption of the dentine cone and of the attachment bone, simultaneously. (I) Larva, stage 52. This first-generation tooth has been resorbed, but its tooth tip is still visible (arrow), entirely surrounded by an osteoclast. Scale bars: A, B, C 100 mm; D, H 20 mm; E, I 10 mm; F, G 50 mm. ab: attachment bone; bv: blood vessel; db: dentary bone; de: dentine; eo: enamel organ; oc: osteoclast; oe: oral epithelium; pc: pulp cavity; pe: pedicel; rt: replacement tooth.

the dental tissues (excluding enamel) represented 3.64% of the volume of the supporting bone, and that 96.36% of the volume of the dental tissues is resorbed (and thus re-used) rather than shed. In Xenopus laevis, multinucleated osteoclasts are first observed at 4-6 days post-stage 65 (i.e. by the end of metamorphosis), on the outer surface of the firstgeneration teeth, along the dentine wall and along the bone
69 of attachment (Shaw, 1983; H. Chisaka, personal observations). Then, osteoclasts penetrate the pulp cavity, where they start to break down the dentine, from base to tip. Most first-generation teeth are lost (they are apparently entirely resorbed, but the fate of the tooth tip remains uncertain) at 9-11 days post-stage 65. Therefore, the duration of the resorption process is five days (the dentine being destroyed
70 in about two days), representing only 15% of the total lifetime (33 days) of a first-generation tooth. Numerous lymphocytes and macrophages are observed at the tooth base during the resorption process. In adult X. laevis, the dentition (upper jaw) is maintained through the resorption and replacement of several hundred teeth per annum (Shaw, 1985). Shaw (1989) reported that the mean volume of the dentine of all teeth is about 23.5% of that of the supporting bones (premaxillaries and maxillaries), and that during tooth replacement the osteoclasts resorb up to 98% of the dentine. (4) Tooth replacement pattern Intuitively, the maintenance of both the number and sharpness of the teeth might be of great importance to toothed lissamphibians, which catch moving live prey. Indeed, if all the teeth were to be replaced simultaneously, there would be phases of toothlessness, which would represent a serious disadvantage for the animal. Therefore, logically the pattern of replacement along the tooth rows ensures that no toothless phases occur and that an efficient dentition is always present. However, toads are toothless and they are able to catch moving live prey. Data on tooth replacement are available in so few species (see below) that they do not yet permit conclusions for the entire class Lissamphibia. The pattern of tooth replacement has attracted attention for well over a century, but its complexity (i.e. how the developmental cycles of the many individual teeth are interwoven to produce a continuously functioning dentition) defied understanding until the studies of Edmund (1960, 1962, 1969) in reptiles pointed out underlying principles (the Zahnreihen model). Edmund postulated that waves of stimuli pass along the jaw from front to back at regular intervals and initiate tooth replacement in alternate tooth positions. This model was found to apply to most common tooth replacement patterns in reptiles, but increasing numbers of examples exist (notably in teleost fish and amphibians) where such replacement patterns cannot be identified (see review in Berkovitz, 2000), and evidence for the Zahnreihen model, at least for tooth replacement, is becoming less convincing (Smith, 2003; Huysseune & Witten, 2006). Among the numerous studies devoted to lissamphibian teeth, a number include data on the pattern of tooth replacement (e.g. Lawson, 1965b; Lawson et al., 1971; Miller & Rowe, 1973; Wake, 1976, 1980; Chibon, 1977; see also Berkowitz, 2000). Some of these studies were undertaken with the aim of discovering whether or not the regular patterns of tooth replacement proposed by Edmund (1960, 1962, 1969) exist in lissamphibians. In fact, the reptilian condition in which functional teeth alternate with non-functional teeth rarely applies to lissamphibians. The pattern of tooth replacement in lissamphibians tends to be obscured by the fact that, in general, each tooth locus possesses a mature, functional tooth (less than 25% are non-functional) compared to only 50% in reptiles. This means that the teeth are retained functionally for a long period compared to the rapid phases of development and resorption. In caudates, Lawson et al. (1971) reported the pattern of tooth replacement in Plethodon cinereus (Green, 1818) using

various techniques (Alizarin Red, radiographs, wax impressions). Miller & Rowe (1973) discussed that of Necturus maculosus, and Chibon (1977) that of Pleurodeles waltl. In Plethodon cinereus the tooth replacement pattern is consistent with the Zahnreihen theory, but with some variations. By contrast, in N. maculosus the replacement pattern is very irregular, varying seasonally and with respect to the bone support, and does not follow the Zahnreihen model. During the winter period of hibernation tooth replacement ceases while it is most active in late summer and autumn. In P. waltl, tooth replacement again does not seem to be regular either. In gymnophiones, the pattern of tooth replacement was studied in adult Hypogeophis rostratus by Lawson (1965b) and in foetuses and adult Gymnopis multiplicata, Typhlonectes compressicauda and Dermophis mexicanus by Wake (1976, 1980). By dividing the life cycle of teeth into a number of histological stages on the basis of morphological characters, Lawson (1965b) found that some developmental phases take place slowly and others more rapidly: the stages leading from tooth initiation to the first deposit of enamel as well as the resorption of the functional tooth are extremely rapid, whereas phases of tooth growth and dentine calcification, and of pedicel calcification and attachment are long. In the two jaws, a functional and a replacement tooth occupy each locus. In H. rostratus, the sequence of tooth replacement occurs, similarly to that described in reptiles by Edmund (1960): a comparison of the developmental stages in alternate tooth loci showed (i) that tooth replacement occurs in waves, which run craniad along the jaw and (ii) that a full replacement wave occupies six tooth positions (Lawson, 1965b). In embryos and foetuses of G. multiplicata, T. compressicauda and D. mexicanus new tooth loci are added both posteriorly along the jaw and between existing loci (Wake, 1976, 1980). Using three developmental stages in foetal teeth and by studying tooth replacement pattern on the lower and upper jaws (four rows), Wake (1980) demonstrated that tooth replacement proceeds alternately. During the transition from foetal to adult dentition, along the jaw tooth replacement along the jaw follows a pattern similar to that predicted by the Zahnreihen model, as in most adult lissamphibians. In anurans, the dynamics of continuous tooth succession was studied in Rana pipiens by Gillette (1955), in Hyla cinerea by Goin & Hester (1961), and in R. temporaria by Lawson (1966) and by Chibon (1977). Using Alizarin Red staining of tooth-bearing bones, Gillette (1955) was able to divide the developmental cycle of individual teeth in R. pipiens into eight equal time periods. A correlation was found between the developmental stage of (i) a given tooth and that of its successor, (ii) a given tooth and that of adjacent teeth, and (iii) teeth in alternate positions. The existence of this regular pattern indicates that the initiation of new teeth is not a random process. This is in conflict with the idea that tooth replacement occurs only when its functioning predecessor is lost or strenuously used (Hertwig, 1874). In fact the initiation of a new germ is due to a condition intrinsic to the dental lamina, and its development causes the resorption of the predecessor, clearing the way for its eruption. Gillette (1955) calculated that a tooth predecessor is 45 days ahead of its successor and that the entire cycle of a tooth takes 90 days, but this can vary with temperature,

season and age (size) of the frogs. One can question whether indefinite tooth succession is possible and the discovery of edentulous individuals in some frog species could be related to old age (Smith, 1883). However, working on several anuran species, and in particular H. cinerea, Goin & Hester (1961) could not define a regular pattern of replacement, even though waves of tooth replacement seem to exist in alternate tooth positions. They concluded that tooth succession is a haphazard process, which cannot be explained by Edmunds (1960) theory, but they probably misinterpreted the Zahnreihen concept of waves stimulating tooth replacement in alternate tooth positions. Indeed, Lawson (1966) showed in R. temporaria that teeth in alternate loci are usually at the same stage of development, suggesting that tooth replacement involves teeth in alternate loci. In Xenopus laevis, two or three new positions are added posteriorly to the row during metamorphosis. New positions are subsequently added in post-metamorphosed specimens and in adults to reach 30 positions on each side. The timescale of tooth replacement has been studied for firstgeneration teeth in newly metamorphosed X. laevis (Shaw, 1979) and in adults using impressions of the upper jaws on dental gold-casting wax (Shaw, 1985). In adults, the tooth replacement cycle was 38-42 days, functional life was 24-29 days and the gap period (between loss of a tooth and attachment of its successor) was 9-17 days. The dentition develops in alternating series, the teeth at even-numbered loci developing before the odd-numbered loci.
71 teeth are partially transformed, i.e. bicuspid on the upper jaw, monocuspid on the palatine and splenial, and of a mixed type on the dentaries, indicating differences in sensitivity of the dental laminae to metamorphic hormones (Clemen & Greven, 1977; Greven & Clemen, 1990; Bolte & Clemen, 1991, 1992). In general, paedomorphic species possess a mosaic of larval and metamorphic tooth features, which reflect their degree of paedomorphosis (Greven & Clemen, 1980; Clemen & Greven, 1988). Experiments where complete metamorphosis was induced led to fully transformed teeth (Clemen, 1988; Greven & Clemen, 1990). The cells of the dental organ could react differently (for example due to the presence or absence of appropriate receptors) because jaw teeth could be more sensitive to thyroxine than palatine teeth (Clemen & Greven, 1977; Clemen, 1988; Mutz & Clemen, 1992). Although many studies are currently devoted to elucidating the genetic mechanisms of thyroid hormone action through activation of its receptors during lissamphibian development and metamorphosis (mostly in Xenopus laevis), none are specifically related to tooth development (but see Rose, 1999 and Section VIII below). (2) Bicuspid to monocuspid: the role of androgens In some species of plethodontid salamanders, instead of the typical bicuspid teeth the males bear monocuspid teeth on the jaws during the mating season (Means, 1972; Ehmcke & Clemen, 2000b). These teeth play an important role during courtship (Duellman & Trueb, 1986). In Desmognathus fuscus (Rafinesque, 1820), castration revealed that the appearance of these monocuspid teeth is controlled by androgens (Noble, 1926; Noble & Davis, 1928; Noble & Pope, 1929). Recently, working on Bolitoglossa schizodactyla Wake & Brame, 1966, a plethodontid in which monocuspid teeth are restricted to the premaxillary bone, Ehmcke et al. (2003) showed that this was related to the restriction of androgen receptors to the cells of the premaxillary dental lamina, explaining the selective response of these cells to androgens.
VI. TOOTH CHANGES (1) Monocuspid to bicuspid: the role of thyroxine at metamorphosis Most metamorphosed caudates have bicuspid teeth, while teeth are monocuspid in larvae. It is known that bicuspidality is established during or immediately after metamorphosis, when the monocuspid larval teeth are replaced (Kerr, 1960; Chibon, 1972; Clemen & Greven, 1974, 1977, 1979; Beneski & Larsen, 1989a). Therefore, a clear relationship has been identified between tooth morphology and the increase of thyroid activity during metamorphosis. Two dental features which are substantially modified during metamorphosis seem to be sensitive to thyroxine: cusp morphology and enamel thickness. Both cuspidality and enamel production are controlled by ameloblasts in the dental epithelium. In the newt Triturus helveticus (Razoumovski, 1789), Chibon (1972) and Gabrion & Chibon (1972) showed that cusp modification and enamel thickness are under the control of thyroid hormonal secretion during the entire life. Indeed, in neotenic newts (i.e. specimens retaining larval characters beyond larval life) most teeth were of the larval type (monocuspid with a thin enamel layer). Experimental hypophysectomy or thyroid dysfunction in Pleurodeles waltl also resulted in the conservation of larval characters, even in old specimens. In P. waltl, enamel thickness depends on thyroid hormone levels: increased levels resulted in thicker enamel (Roux & Chibon, 1973). In Ambystoma mexicanum, a neotenic salamander, the
VII. TOOTH REGENERATION Most lissamphibians, and especially caudates, are able to regenerate large parts of the body such as the tail, limbs, etc. in larvae as well as in adults. This regeneration potential has long been known but it is less well known that jaws also can regenerate completely in larvae and adults (Goss & Stagg, 1958a, b; Goss, 1969). Lauga-Reyrel (1974) used this ability to study tooth regeneration in Pleurodeles waltl: after partial amputation the lower jaw regenerates and new teeth form. It is noteworthy that the teeth that form on these regenerated jaws start to develop in the buccal epithelium as soon as wound healing is finished, before the new cartilage and, therefore, long before the bone support appears. Tooth regeneration does not require the presence of the regenerated supporting cartilage, which needs the formation of a blastema. This shows that tooth regeneration depends only on the regeneration of the basal layer of the buccal
72 epithelium, from which the regenerated dental lamina will develop. However, this phenomenon is rather closer to a tooth replacement process than to de novo tooth ontogenesis, because of the presence of the regenerated dental lamina. Working on jaw regeneration in the newt Notophthalmus viridescens (Rafinesque, 1820), Graver (1973, 1974) reported that the dental lamina did not arise de novo from the oral epithelium, but that it could only regenerate from the residual lamina. Clemen (1979a) reached a similar conclusion for the vomerine dental lamina in Salamandra salamandra (Linnaeus, 1758). The complete amputation of the dental lamina of the vomer or of the palatine gives rise to toothless bones (Clemen, 1979b). Experiments dealing with repeated regeneration of the jaw have shown that the events, especially those involved in the repeated regeneration of the dental lamina, are the same as in normal regeneration but that the regrowth is more rapid (Graver, 1978). In the frog Rana pipiens, Howes (1978) has shown that the bones of the upper jaw regenerate slowly compared to the jaw tissues in which teeth form normally.

(2) Are the dentition pattern and development of the dental lamina important features for lissamphibian systematics? Several dental characters may represent useful tools to establish lissamphibian relationships, but it must be verified that these characters are independent and not developmentally correlated. Cusp morphology is unlikely to be of use because examples of convergent evolution are already known. A comprehensive analysis of the large body of data available on the dentition pattern and organisation of the dental lamina in lissamphibians (see Section V.4) may prove useful in resolving relationships within Lissamphibia. (3) Do ameloblasts participate in enameloid formation in lissamphibian larvae? During the deposition of the enameloid matrix by odontoblasts, the ameloblasts are differentiated and show characteristic features, such as a polarized organisation, the presence of a ruffled border, and the absence of the basement membrane (Fig. 5). These features do not prove that ameloblasts are involved in the deposition of enamel proteins, but suggest that they are at least involved in the maturation process of the enameloid (Smith & Miles, 1971). It would be interesting to determine whether enamel proteins are synthesized by ameloblasts prior to, during and after enameloid deposition. This could be achieved using in situ hybridisation techniques. Among the candidate proteins, amelogenin may be the best choice as it is the major enamel protein. The amelogenin gene is well known in numerous mammals (Delgado, Girondot & Sire, 2005), in three reptiles (Ishiyama et al., 1998; Toyosawa et al., 1998; Delgado et al., 2006) and two lissamphibians (Toyosawa et al., 1998; Wang et al., 2005). Amelogenin could be cloned in a caudate such as P. waltl, and its expression studied in early larvae during enameloid formation in first-generation teeth. Using the same technique, the presence of proteinases (principally metalloproteinases MMP-20, enamelysin, and KLK4, kallikrein 4) that are involved in enamel maturation could be investigated. However, to date genes coding for such proteinases are only known in mammals (Bartlett et al., 1997; Fukae et al., 1998; Bartlett & Simmer, 1999). In the anuran Xenopus laevis, dentine is described as forming first in the first-generation teeth, but it is not clear whether enameloid is present, even though it is known that these teeth are subsequently covered by a layer of enamel (Shaw, 1979). If enameloid is found, tooth development in this species would be similar to that in caudates. Observations of appropriate stages at the TEM level are therefore required and, particularly, a more detailed study of ameloblast functioning. In situ hybridization performed on larvae developing first-generation teeth should reveal whether or not ameloblasts are synthesizing amelogenin when the odontoblasts are depositing dentine-like material in this species.
VIII. DIRECTIONS FOR FUTURE RESEARCH Here we identify 10 questions that remain unanswered on various aspects of amphibian tooth biology, and we propose research avenues that could try to answer them. (1) Are pedicellate teeth homologous among lissamphibians? The presence of pedicellate teeth in most species of the three modern lissamphibian orders could indicate that this is an ancestral character although this can not be assessed from the palaeontological data due to the scarcity of fossil temnospondyls and Paleozoic tetrapods. However, in some teleost fish there is an uncalcified (or slowly mineralising) region, similar to the dividing zone of lissamphibian teeth, separating the dentine shaft from an elongated bone of attachment, similar to a pedicel. The length of this pedicel varies from short, as in armoured catfishes and cichlids, to long and morphologically similar to that found in lissamphibians, as in some osteoglossiforms (Huysseune & Sire, 1997a, b; Sire et al., 2002). The pedicel of lissamphibian teeth and this pedicellate structure in teleosts could have appeared independently in both sarcopterygian and actinopterygian lineages rather than being homologous, i.e. derived from pedicellate teeth in a common ancestral osteichthyan. Indeed, in teleosts the pedicellate bone of attachment is never covered by the dental epithelium (enamel organ), in contrast to the pedicel of lissamphibian teeth. Does the extent of the enamel organ along the tooth shaft define the boundary between the tooth proper (i.e. dentine shaft ] pedicel in lissamphibians) and the attachment bone? Comparative studies of tooth development in lissamphibians and actinopterygians could reveal typical features that would indicate whether the dividing zones in these lineages are homologous or have been acquired independently.

(4) How does the enameloid-enamel transition proceed through caudate ontogeny? The enameloid layer, which is present in the first-generation teeth in caudate larvae (and covered secondarily by a thin layer of enamel) is absent in the teeth of post-metamorphosed specimens, in which a thick enamel layer is present. More detailed study of tooth development in a growth series of a salamander like P. waltl using light microscopy and TEM is needed to understand the morphological changes and the fate of the enameloid through ontogeny. Does this layer disappear within one replacement cycle during metamorphosis or is there a gradual reduction over successive tooth generations during ontogeny? Answering this question would require careful sectioning to pass through the upper region of the 30-50 mm diameter tooth, and the collection of data for each tooth generation, from the first to the fourth or fifth, i.e. after metamorphosis. Such a study could also generate information on the formation and origin of the dentine-enamel junction. (5) How do the dividing zone and the pedicel appear during lissamphibian ontogeny? In Pleurodeles waltl and Ambystoma mexicanum, it has been reported that the first-generation teeth are undivided, whilst a dividing zone and a pedicel are present in subsequent teeth (Wistuba et al., 2002). The formation of the dividing zone is related to change in functioning of the odontoblasts: the absence of cytoplasmic prolongations, which are known to play an important role in dentinogenesis (Sasaki & Garant, 1986), is an indication of this chcange in behaviour. A detailed morphological comparison of the features of the odontoblasts lining the crown with those lining the dividing zone in a growth series could determine whether the dividing zone forms progressively through successive tooth generations or whether it appears suddenly in second-generation teeth. Such a comparison would also clarify how can a low-mineralised zone can be produced and maintained when surrounded by conventional, calcified dentine, and lined by a continuous layer of odontoblasts. It should be possible to identify the collagen type composing the fibrils, using both immunocytochemistry and in situ hybridization techniques. Indeed, it has been reported that the fibrils associated with this zone are smaller in diameter than those of the adjacent dentine regions in the crown and pedicel, which are mostly composed of type I collagen (Zaki et al., 1970; Smith & Miles, 1971). Type V collagen fibrils are known to be thinner than type I fibrils but it is not known whether they are present in the dividing zone. It would also be of interest to look for non-collagen proteins, such as proteoglycans, which are known to be involved in the mineralisation process. A detailed study of pedicel formation in teeth of successive generations should also provide data on the formation of cementum. Are the cementoblasts simply osteoblasts or are they cells originating from the dental sac? The role of Hertwigs sheath during the last stages of tooth development and after teeth have attached to the bone support also might be
73 investigated to determine the relationship between retraction of Hertwigs sheath and formation of the cementum. (6) What mechanisms control the initiation of a replacement tooth in lissamphibians? Our observations on replacement tooth initiation in numerous growth series of Pleurodeles waltl have shown that: (1) differentiation of the secondary dental lamina starts long before the previous tooth becomes functional, and the replacement tooth is well developed when the predecessor tooth attaches to the bone support. Thus, the initiation process is correlated neither with tooth attachment nor with eruption but presumably is initiated for all teeth in relation to a particular developmental step of the previous tooth. In reptiles, the regular pattern of tooth replacement strongly al & Sire, 2003), suggests this possibility (Delgado, Davit-Be however, the genetic mechanisms controlling this process have yet to be understood. (2) The secondary dental lamina forms from the upper region of the dental organ of the previous tooth and is always located lingually and slightly posterior to the latter. This suggests that the cells of this particular region of the outer dental epithelium have conserved the ability to differentiate into a dental lamina. In teleost fish, in which the replacement process is very similar to that in lissamphibians, Huysseune & Thesleff (2004) suggested that this region of the dental organ could be an epithelial stem cell niche. Further investigations could extend to the genetic level, and particularly to the molecular cascades underlying the regulation of such a putative stem cell niche. The role of nerves in tooth replacement also needs further study. In a cichlid fish (Tilapia mariae) subjected to unilateral denervation of the lower jaw through neurectomy of the ramus alveolaris trigemini, replacement teeth did not form; the tooth germs already initiated at the time of denervation continued to grow but no new germ was initiated when a functional tooth was lost (Tuisku & Hildebrand, 1994). This suggests that the secondary dental lamina is no longer initiated when the nerve is disrupted; similar studies on Lissamphibians are lacking.
(7) Which mechanisms control the initiation of tooth resorption? It is assumed that a functional tooth is subjected to resorption as a reaction to the presence of a developing replacement tooth, hence, the absence of replacement teeth would lead to unresorbed functional teeth. In cichlid fish in which replacement teeth were no longer initiated after unilateral denervation, some teeth were lost in the absence of replacement teeth but there were no data on the functional life of these teeth (Tuisku & Hildebrand, 1994). The first-generation teeth in Pleurodeles waltl are not resorbed when their successors are growing, while in all other generations the replacement tooth provokes tooth resorption al, Allizard & Sire, in press), suggesting and loss (Davit-Be that resorption occurs only when the replacement tooth develops close to the previous tooth. Again, detailed comparative work is required to investigate this further.
74 (8) What is the fate of the tooth tip in adult lissamphibians? While most authors agree that teeth are almost completely resorbed, the fate of the enamel at the tooth tip is less clear. Are osteoclasts able to resorb enamel? It is possible that teeth are entirely resorbed in larvae, but not in adults, where a large part of the crown can be shed when the dividing zone is undergoing resorption. Detailed studies of tooth resorption in a growth series, based on serial sections and on observations at the TEM level is necessary to understand this process. (9) What mechanism controls the periodicity of lissamphibian tooth replacement? To date, no experimental data are vailable on the control of tooth replacement patterns in nonmammalian species. Some authors, however, have speculated that the tissues capable of forming teeth could be programmed to produce buds in a given sequence as the field becomes progressively differentiated (e.g. Osborn, 1970, 1971, 1973; reviewed by Berkovitz, 2000). Once the initial sequence has been established it could be maintained by an intra-oral mechanism(s) including repression of the developing tooth by the functional tooth until a particular point. Such local control over replacement tooth formation is in conflict with earlier conclusions about a Zahnreihen model of waves of replacement (see Section V.4). A detailed and comparative study performed on various developmental stages could reveal whether there is a correlation between the initiation of a new replacement tooth and the developmental stage of the preceding tooth in al et al. (2006) the same family. In Pleurodeles waltl, Davit-Be found a correlation between the time the tooth becomes functional (i.e. eruption]attachment) and initiation of its successor. Study of the expression pattern of genes known to be involved in tooth initiation in mammals may help to clarify control mechanisms of tooth replacement in P. waltl. (10) How do thyroxine levels affect tooth shape in lissamphibian teeth? It is clear that thyroxine affects both the entire enamel organ (inner and outer dental epithelium), which determines the shape of the cusps when the first tooth tissues are deposited, and stimulates the ameloblasts, which are responsible for enamel formation. Thyroxine also stimulates the osteoclasts responsible for skeletal remodelling during metamorphosis. Thyroxine (T4 and its deiodinated form T3, which is more active) passes through the cell membrane, binds to nuclear receptors in immediate contact with DNA and triggers a shift in gene transcription causing, in competent tissues, a set of major changes through either cell alteration, proliferation, differentiation or migration (Jacobs, Michielsen & Kuhn, 1988). Thyroid hormone receptor genes (TR alpha and beta) have been sequenced in lissamphibians (Safi et al., 1997, 2004; Sachs et al., 2002). In Xenopus laevis, the gene sonic hedgehog (Xshh), which is known to be involved in tooth

morphogenesis in mammals, is activated as a direct response to thyroid hormone (Stolow & Shi, 1995). In lissamphibians, the larval thyroid gland contains thyroxine at hatching (Hanaoka et al., 1973), but its plasma concentration is below detectable levels: teeth are monocuspid and the enamel is thin. During metamorphosis, plasma T4 levels increase to reach 25-30 nmol l[1 (Larras-Regard, Taurog & Dorris, 1981): teeth become bicuspid and the enamel thickens. Understanding the expression of TR and shh genes in the enamel organ of larvae and metamorphosing Pleurodeles waltl could clarify the developmental action of thyroid hormone and its relationship with enamel thickness and cusp formation. That the enamel organ is also sensitive to androgens is shown by the transformation of bicuspid into monocuspid teeth during the breeding season in males of many plethodontid species (Stewart, 1958; Ehmcke & Clemen, 2000b; Ehmcke et al., 2003). Here, the effects of androgens are opposite (loss of a cusp) to those of thyroxine (gain of a cusp). It would be interesting to determine (i) how competition between T4 receptors and androgen receptors is regulated in tooth-forming cells, and particularly in ameloblasts, of these animals, and (ii) how this competition leads to the monocuspid condition typically a feature of larvae, in which levels of T4 and androgens are below detection limits detectable. To our knowledge, there are no data on the effects of thyroxine on tooth shape in mammals. For example, is the enamel knot a target of T4 or T3? This should be investigated in light of the involvement of these hormones in tooth shape changes in lissamphibians.
IX. CONCLUSIONS (1) This review is the first major summary of current knowledge on teeth in extinct and extant amphibians (Caudata, Gymnophiona, and Anura). A large amount of morphological data are already available on diverse aspects of tooth biology in various lissamphibian species, some of them being potentially good model animals given their easy breeding at the laboratory and their amenability to experimental studies. (2) To date, research has principally focused on tooth development and replacement, and on changes in morphology and structure during ontogeny and metamorphosis. Taken together these studies have established solid basis for further research aiming to elucidate tooth developmental mechanisms in lissamphibians, and particularly in looking for gene expression (and function) during tooth morphogenesis and differentiation. These future studies will prove to be important for understanding evolutionary developmental biology of teeth. Indeed, dental research in vertebrates suffers from a lack of comparison of genes involved in representative species of various nonmammalian lineages. Because all animals share many of the same molecular processes, such comparative studies are a prerequisite to understand, for instance, what makes one tooth different from another.

(3) This review highlights important questions which remain to be answered. They should be adressed using comparative morphological studies at the tissue and cell levels, and molecular techniques. Most questions which could be addressed in lissamphibians concern more generally tooth evolution and development in vertebrates. For instance, the enameloid-enamel transition, which has occurred in several lineages during vertebrate tooth evolution, could be studied in caudates using molecular markers for enamel proteins. Indeed, such a transition exists in caudates, in which larval teeth possess both enameloid and enamel, while teeth are only covered by enamel in postmetamorphosed animals. (4) Future research could also deal with the genetical mechanisms that control replacement tooth initiation, probably from the possible activation of stem cells. This is a hot subject when considering current investigations on tooth regeneration in mammals. Indeed, dissecting the genetic pathway(s) involved in periodically replacing teeth in lissamphibians could help much in understanding why teeth can no longer be continuously replaced in mammals. Similarly, the clear correlation that exists between the increase of thyroxine levels at metamorphosis and tooth shape change (monocuspid to bicuspid) seems to be a good opportunity to study in detail the role played by thyroid hormones in shaping the enamel organ. (5) Lissamphibians appear to be good candidates for such research because tooth structure is similar to that in mammals and teeth are renewed continuously during life. This allows to perform developmental studies not only in larvae but also in later stages, in larger specimens. The salamander Pleurodeles waltl, which has been used extensively in experimental embryology research during the past century, appears to be one of the most favourable lissamphibian to be used as a model species in further studies of tooth development.
75
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X. ACKNOWLEDGEMENTS We are grateful to Ann Huysseune (Ghent University, Belgium), and Marvalee and David Wake (University of California, Berkeley, USA) for critical reading of previous drafts of the manuscript, and to Michel Laurin (CNRS, Paris) for fruitful discussions. We thank Miss Franc xoise Allizard for excellent technical assistance. SEM and TEM work was carried out at the Service de Microscopie grative CNRS/ Electronique de lIFR de Biologie Inte Paris VI. Universite
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Biol. Rev. (2007), 82, pp. 4981 doi:10.1111/j.1469-185X.2006.00003.

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

al and others Tiphaine Davit-Be

Amphibian tooth morphology and development

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