MICROSCOPY RESEARCH AND TECHNIQUE 59:408 – 434 (2002)

First-Generation Teeth in Nonmammalian Lineages: Evidence for a Conserved Ancestral Character? JEAN-YVES SIRE,1* TIPHAINE DAVIT-BEAL,1 SIDNEY DELGADO,1 CHRISTINE VAN DER HEYDEN,2 ANN HUYSSEUNE2 1 2

AND

CNRS UMR 8570, Universite´ Paris, France Biology Department, Ghent University, B-9000 Gent, Belgium

KEY WORDS

vertebrates; larvae; hatchlings; dentition; first-generation teeth; tooth development; evolution

ABSTRACT The present study focuses on the main characteristics of first-generation teeth (i.e., the first teeth of the dentition to develop in a given position and to become functional) in representatives of the major lineages of nonmammalian vertebrates (chondrichthyans, actinopterygians, and sarcopterygians: dipnoans, urodeles, squamates, and crocodiles). Comparative investigations on the LM and TEM level reveal the existence of two major types of first-generation teeth. One type (generalized Type 1) is characterized by its small size, conical shape, atubular dentine, and small pulp cavity without capillaries and blood vessels. This type is found in actinopterygians, dipnoans, and urodeles and coincides with the occurrence of short embryonic periods in these species. The other type assembles a variety of first-generation teeth, which have in common that they represent miniature versions of adult teeth. They are generally larger than the first type, have more complex shapes, tubular dentine, and a large pulp cavity containing blood vessels. These teeth are found in chondrichtyans, squamates, and crocodiles, taxa which all share an extended embryonic period. The presence in certain taxa of a particular type of first-generation teeth is neither linked to their phylogenetic relationships nor to adult body size or tooth structure, but relates to the duration of embryonic development. Given that the plesiomorphic state in vertebrates is a short embryonic development, we consider the generalized Type 1 first-generation tooth to represent an ancestral character for gnathostomes. We hypothesize that an extended embryonic development leads to the suppression of tooth generations in the development of dentition. These may still be present in the form of rudimentary germs in the embryonic period. In our view, this generalized Type 1 first-generation teeth has been conserved through evolution because it represents a very economic and efficient way of building small and simple teeth adapted to larval life. The highly adapted adult dentition characteristic for each lineage has been possible only through polyphyodonty. Microsc. Res. Tech. 59:408 – 434, 2002. © 2002 Wiley-Liss, Inc. INTRODUCTION The dentitions of living vertebrates show an overwhelming diversity. Teeth can vary in their location (several bones in the oropharyngeal cavity can bear teeth), number (from several hundreds to some units only), size (from several cm down to less than 100 ␮m), shape (mono- to multicuspid, molariform, etc.), orientation (inclined forwards, backwards, etc.), structure (several types of dentines, enamel and enameloid, etc.), mode of attachment (ligamentously, ankylosed by spongious bone or cement, pedicellate, etc.) and replacement (polyphyodonty, diphyodonty, etc.). Clearly, the process of natural selection has endowed each taxon with the best possible adaptation to acquire its food in a particular environment. However large the diversity of vertebrate dentitions may be, it contrasts with the relative maintenance of tooth architecture and the conservation of early steps of tooth development. In all vertebrate taxa, teeth are basically built according to the same plan, i.e., a central pulp cavity (containing various types of cells, nerves, and blood vessels), surrounded by a dentine cone (a mineralized tissue of which the organic matrix is ©

2002 WILEY-LISS, INC.

mainly composed of collagen fibrils), itself covered by a hypermineralized cap (enamel or enameloid) and with an attachment tissue linking the tooth to a (bone) support. Modifications in the phenotypic outcome of the morphogenetic and differentiative processes that characterize tooth development have probably been permitted, provided this fundamental plan of the tooth was not modified. Given the conservation of developmental processes, and in particular those involved in organogenesis, such evolutionary changes have most likely been possible only through slight variations in the genetic control of developmental processes (Kim et al., 2000; Raff, 2000). It is this characteristic organization and way of development of teeth which also allows

*Correspondence to: Dr. Jean-Yves Sire, Equipe “Evolution & Developpement du Squelette dermique,” CNRS UMR 8570, Universite´ Paris 7, Case 7077, 2, place Jussieu, 75251 Paris cedex 05, France. E-mail: [email protected] Received 27 February 2002; accepted in revised form 18 July 2002 Contract grant sponsor: CNRS, UMR 8570, PICS 483 and the CRIOBE-UMR 8046 EPHE-CNRS. DOI 10.1002/jemt.10220 Published online in Wiley InterScience (www.interscience.wiley.com).

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discrimination between odontogenic and skeletogenic cell populations, irrespective of the possible occurrence of homoplasies (Sire and Arnulf, 2000; Sire and Huysseune, submitted). This conserved bauplan of teeth suggests that it is deeply rooted in vertebrate evolution and that it derives from a structure found in a common ancestor of all gnathostome lineages, i.e., a jawless vertebrate that lived more than 450 MY ago. This ancestral structural organization can be found in the odontodes (sensu Ørvig, 1977) which covered the body of jawless vertebrates (e.g., thelodonts). For a long time odontodes were held to be the precursors of teeth (see Peyer, 1968) and this hypothesis is still supported by several authors. Recently, however, some authors have suggested that teeth could have derived from oropharyngeal denticles present in agnathan ancestors (see reviews in Smith and Coates, 1998, reviews in Smith and Coates, 2000) or even conodont elements (see review in Donoghue and Aldridge, 2001). How then can this huge range of adaptations in adult dentitions be explained in terms of conserved mechanisms of tooth formation? We know from past work, and from literature data, that during ontogeny more or less important modifications can occur in location, number, orientation, size, shape, and mere occurrence of the teeth. Striking examples exist in mammals with a highly specialized diet (e.g., Xenarthra (anteaters), or Mysticeti (whales)), where teeth develop in embryos but never become functional (Grasse´ , 1955; Bourdelle and Grasse´ , 1955). The most numerous examples nevertheless come from teleost fish, which provide the major source of natural experiments in this field. Three examples illustrate such ontogenetic modifications. Adult callichthyid siluriforms (e.g., Corydoras, Hoplosternum, etc.) are edentulous. Their feeding apparatus is adapted to search the sandy bottom, from which they retain small organic particles. In contrast, the larvae are carnivorous and possess small, but welldeveloped conical teeth, well adapted to catch small prey (Huysseune and Sire, 1997a). In these species teeth are lost (rather, they are no longer replaced) in a juvenile stage, when the upper and lower jaws are transformed to serve the specialized adult feeding function. A second example is found in lepidophageous serrasalmid species. In adults, several teeth located at the anterior extremity of the jaws are directed forward. The fish use these teeth to grasp the flanks of the prey to remove their skin and scales. The larvae and juveniles of these species develop normally oriented teeth (Je´ gu and Dos Santos, 1990). Third, adults of the cichlid fish Astatoreochromis alluaudi can exhibit two phenotypes. The juveniles of this species feed on soft prey and possess slender pharyngeal jaws with small and fine teeth; adults develop stout pharyngeal jaws with large molariform teeth if they are forced to feed on snails, but retain the papilliform dentition if not (Huysseune, 1995, 2000). All these modifications would not have been possible in the absence of polyphyodonty, i.e., the condition in which teeth are continuously replaced throughout life. Polyphyodonty characterizes all fossil and extant nonmammalian vertebrate lineages. Because every tooth in a single position inevitably goes through the phases of morphogenesis and cytodifferen-

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Fig. 1. List of the species studied, placed in a cladogram of the vertebrates. Relationships between aquatic lineages were drawn from Nelson (1994).

tiation, renewed tooth formation can be an adequate substrate for ontogenetic changes. So, do the characteristics of adult dentitions reflect mere adult adaptations to specialized food conditions, made possible due to the existence of polyphyodonty, and is there a trace in early ontogeny of a conserved tooth shape and structure? The question whether larval and adult teeth resemble each other morphologically and structurally is thus of major importance from an evolutionary perspective but has never been addressed in a systematic way, covering a wide range of lineages and species. In the present work, we wished to test the hypothesis of the existence of a generalized phenotype (both in shape and in structure) of first-generation teeth in nonmammalian vertebrates. To address this question, we collected observations on first-generation teeth of selected taxa representative of different lineages of vertebrates (Fig. 1) and added these to the scarce literature data available. To enhance the potential value of this comparison, we compared 1) distantly related taxa (e.g., actinopterygians vs. urodele amphibians); 2) closely related species but with different adult body size (e.g., the cyprinids Danio rerio and Cyprinus carpio (30 mm vs. up to 80 cm adult size); 3) species known to possess a highly specialized adult dental structure (e.g., in the dentine, Gadus morhua); and 4) species with a highly specialized adult feeding apparatus (e.g., Scarus sp.). We have studied the structure (i.e., type of cells and tissues involved) of the first functional teeth in ontogeny at the light and, when possible, at the transmission electron microscope level. MATERIALS AND METHODS The materials used (larvae and hatchlings of different lineages) are presented in Table 1. Most were laboratory-bred specimens. Other specimens and/or sec-

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— 14.0 mm TL 53.0 & 85.0 mm LT — hatchling, stage 50 10 dPH, stage 39 Late embryo (stage 39) & 1 dPH 28, 34, 40, 50 & 70 embryonic days

L W L

D L L D

Hemichromis bimaculatus (Gill, 1862) Scarus sp. Scarus sp.

Neoceratodus forsteri (Krefft, 1870) Pleurodeles waltl Michahelles, 1830 Chalcides sexlineatus Steindachner, 1891 Crocodylus niloticus Laurenti, 1768

D: donated specimens; L: laboratory-bred specimens; W: wild-caught specimens; dPF: days postfertilization; dPH: days posthatching; EDTA ⫽ decalcified samples; EM ⫽ glutaraldehyde-paraformaldehyde fixation followed by osmium tetroxide postfixation.

4.2 & 4.5 mm SL 7.0 mm SL 8.0, 10.0 & 18.0 mm SL 5 & 6 dPH t0 t0 ⫹ 7d, t0 ⫹ 15d & t0 ⫹ 59d

L L L L D Danio rerio (Hamilton, 1822) Cyprinus carpio Linnaeus, 1758 Hoplosternum littorale (Hancock, 1828) Oncorhynchus mykiss (Walbaum, 1792) Gadus morhua Linnaeus, 1758

Formalin, paraffin EM, EDTA EM EM

EM EM EM, EDTA Formalin, EDTA, paraffin Alcohol, EM, EDTA 2.8 & 4.2 mm SL 5.0 mm SL 5.5, 16.0 & 22.0 mm SL 17.7 mm SL 7.0 mm SL 3 & 6 dPF 3 dPH — 12 dPH —

L D D Polypterus senegalus (Cuvier, 1829) Osteoglossum bicirrhosum (Cuvier, 1829) Anguilla australis Richardson, 1841

EM, EDTA EM, EDTA EM, EDTA

EM, EDTA Frozen, EM, EDTA Formalin, EM, EDTA 9.0 & 18.0 mm SL 35.0 mm SL 47.0 mm TL 5 & 24 dPH — —

EM, EDTA

Fixation TL/SL

108.0 mm TL

Age L

Source Species

Scyliorhinus canicula (Linnaeus, 1758)

Chondrichthyes Actinopterygii Cladistia Osteoglossiformes Anguilliformes Ostariophysi Cypriniformes Cypriniformes Siluriformes, Callichthyidae Salmoniformes Paracanthopterygii, Gadiformes Percomorpha Cichlidae Scaridae Scaridae Sarcopterygii Dipnoi Lissamphibia Squamata Crocodilia

TABLE 1. List of the vertebrate species used in the study

7 dPH

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tions were a kind gift (see Acknowledgments for the source of this material). In one case, wild-caught specimens were used. Living specimens were killed using an overdose of anesthetics, then fixed (entirely for small larvae or after dissection of the jaws for large specimens) for 2 hours at room temperature in a solution containing 1.5% glutaraldehyde and 1.5% paraformaldehyde in cacodylate buffer (0.1 M, pH 7.4) (⫽ EM fixative). Most of the samples were then decalcified for 7 days in the same fixative, to which 0.1 M EDTA was added. This solution was changed every 2 days. After a quick rinse in cacodylate buffer, the specimens were postfixed for 1 hour in osmium tetroxide, dehydrated in a graded ethanol series, and embedded in Epon 812. For light microscopy, serial transverse sections were cut with a diamond knife at a thickness of 1 ␮m and stained with Toluidine blue. For TEM, ultrathin sections were contrasted with lead citrate and uranyl acetate and examined in a Philips 201 EM operating at 80 kV. Donated specimens (either frozen or formalin- or alcohol-fixed) were rehydrated, postfixed in the EM fixative, decalcified, dehydrated, and embedded in Epon prior to sectioning. These specimens often showed poor preservation of cellular details, but the mineralized skeletal and dental tissues were, on average, well conserved. DESCRIPTION OF FIRST-GENERATION TEETH IN NONMAMMALIAN VERTEBRATES Throughout this study we have considered first-generation teeth to be the first teeth of the dentition to develop in a given position and to become functional. A tooth is considered functional when it has completed its development, is attached to the jaw mesenchyme (whether bone or fibrous connective tissue), and is erupted through the epithelium. In general, at hatching or at birth some of the first-generation teeth are already attached. They can be erupted or they will pierce the epithelium when food is processed for the first time. We have not considered first-generation teeth that form later in ontogeny, even though they may be the first teeth to develop in a new position. This restriction is acceptable from the viewpoint that these first-generation teeth appear in larger specimens, which often have a diet considerably different from that of the larvae or hatchlings. The teeth may therefore differ considerably in shape and structure when compared to larval first-generation teeth as defined above. In some cases, the difficulty of obtaining appropriate larval stages led us to include functional teeth that could have been preceded by other, true first-generation teeth. The implications of this choice will be discussed under the appropriate heading. Although the literature dealing with tooth structure and development in nonmammalian lineages is rich, data on the shape, structure, and development of firstgeneration teeth are rare. Most studies have focused on specific aspects of tooth morphogenesis and differentiation and have used replacement teeth, i.e., a material much more easily accessible because replacement teeth are available throughout the lifespan of these polyphyodont species. The information available on juveniles

FIRST-GENERATION TEETH IN VERTEBRATES

and adults, however, is useful in that it provides comparative material for the present study. Below, the description of first-generation teeth is preceded by a brief summary of the current knowledge available on the (adult) tooth structure and development in each of the major lineages that we have included in the present study (Fig. 1). Chondrichthyes Most sharks, skates, and rays have an extended developmental period in egg capsules or are ovoviviparous. In Heterodontus, the earliest teeth that develop in embryos are rudimentary teeth, which never become functional (Reif, 1976). Shortly before hatching these teeth are replaced so that hatchlings possess a row composed of several functional teeth on the upper and lower jaws. In addition, for every tooth position on the row there are several successional teeth in decreasing stages of development. Tooth development in elasmobranchs has been studied in adults and embryos of a large number of chondrichthyans (e.g., in rays: Raja erinacea by Prostak and Skobe [1988] and Prostak et al. [1990]; Dasyatis akajei and Urolophus aurantiacus by Sasagawa and Akai [1992]; and in various shark species: Grady [1970a], Sasagawa [1989, 1991], Risnes [1990]). Most descriptions concern aspects of tooth morphogenesis and differentiation and most studies have focused on enameloid cap formation and mineralization. The odontoblasts first deposit the tooth cap matrix, an amorphous ground substance containing fine odontoblast processes, then form dentine while the cap is mineralizing (e.g., Sasagawa, 1984; Prostak and Skobe, 1988). The dentine type is orthodentine and contains parallel tubules that radiate into the dentine and reach the enameloid (Kerr, 1955; Schmidt and Keil, 1971). Enameloid is more heavily mineralized than dentine (Reif, 1973). Dentine extends towards the tooth base and merges with the matrix of the basal plate that is deposited by differentiated mesenchymal cells, which resemble odontoblasts (Grady, 1970a). This mineralized basal plate is considered acellular bone by Moss (1970). The basal plates of adjoining teeth fuse to form a continuous layer, to which all the functional teeth are attached. In most cases, the shark dentition is characterized by important ontogenetic changes, from hatchlings having single-cusped teeth to older specimens with multicusped teeth (Peyer, 1968; Reif, 1982). In the 7-day-old specimen of Scyliorhinus canicula, some of the teeth present are already well-developed and functional, as are several odontodes (dermal denticles) located in the skin (Fig. 2a). Five generations of teeth are present at a distance from the cartilage surface. Tooth 1 is possibly the first-generation tooth in this position. Tooth 2 is already attached and erupted; this tooth seems ready to replace Tooth 1 (Fig. 2b). These functional teeth are linked to the mesenchyme by an acellular attachment tissue. Both pedestals lie close to one another but they are not fused. In addition to these functional teeth, several germs, in various stages of development, are linked to each other by a dental lamina (Fig. 2a). The dental organ is composed of two layers only, the inner and outer dental epithelium. The three well-formed teeth are similar in shape

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(Fig. 2b,c). They are lance-shaped (not conical), approximately 400 ␮m high and 150 ␮m in diameter, and their shape resembles that of the adult teeth. The oldest tooth has a large base and the dentine cone is well-developed and covered by a layer of enameloid (the matrix of which has been removed during decalcification). During development the 50 ␮m wide, 200 ␮m high pulp cavity is filled with a dense population of mesenchymal cells (Fig. 2c). The cells located along the dentine wall, the odontoblasts, deposit the collagenous dentine matrix, which is bordered along its external surface by the inner dental epithelium (Fig. 2d). In the tip of the dentine cone, odontoblast processes are embedded in the dentine matrix and some of them reach the dentine– enameloid junction (Fig. 2e,f); such prolongations are not visible in the 15–20-␮m-thick dentine shaft proper (Fig. 2d). When decalcified, the mature enameloid of the young teeth lacks most of its organic content and only some thin fibrils can be observed (Fig. 2f). In erupted teeth, the pulp cavity is seen to contain blood vessels (not illustrated). Actinopterygii Although extensive literature exists on aspects of development of larval and replacement teeth in actinopterygians, the teeth examined were rarely firstgeneration teeth. The only studies reporting some observations on first-generation teeth were done in Amia calva (Degener, 1924), Belone vulgaris (Moy-Thomas, 1934), Pagrus major (Higashi et al., 1983), and the rainbow trout, Oncorhynchus mykiss (Berkovitz, 1978), along with our recent studies on Hoplosternum littorale, Hemichromis bimaculatus, and Danio rerio, summarized in this article (Huysseune and Sire, 1997a b; Huysseune et al., 1998). Only rarely have the descriptions been compared to adult teeth in these taxa. Tooth development in actinopterygian fish is characterized by the absence of an initial series of rudimentary, nonfunctional teeth (Berkovitz, 2000). Teeth have an individual attachment to a bone support and are replaced individually (except in piranhas; see Roberts, 1975; Berkovitz, 1975, 1978; Berkovitz and Shellis, 1978). Enameloid is deposited first by odontoblasts that synthesize dentine beneath the enameloid (Shellis and Miles, 1976; Sasagawa and Igarashi, 1985; Prostak and Skobe, 1986a; Sasagawa and Ferguson, 1990; Sasagawa, 1992). Histochemical and autoradiographic evidence suggests that the inner dental epithelium cells in teleosts and ameloblasts in tetrapods secrete similar matrix proteins (Shellis and Miles, 1974). The only difference (but of importance) could be in the delay (heterochronous shift) in protein secretion by inner dental epithelium (ide) cells, the odontoblasts depositing dentine before the epithelial cells become active in enameloid maturation (e.g., Shellis, 1975). Further development leads to the calcification of both matrices and to attachment of the tooth to the underlying bone (various modes, see below). The pulp cavity is very cellular during tooth differentiation; then the cells decrease in number when the tooth is functional. Orthodentine is the most common type and is composed of an external layer called palleal dentine and an internal layer called circumpulpal dentine (Ørvig, 1951). The orthodentine matrix is composed of collagen fibrils oriented radially and longitudinally.

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Fig. 2.

FIRST-GENERATION TEETH IN VERTEBRATES

The teeth of, e.g., polypteriforms (e.g., Meinke, 1982), lepisosteids (e.g., Prostak et al., 1989), anguilliforms (Shellis and Miles, 1976), cichlids (e.g., Prostak and Skobe, 1986b), and tetraodontiforms (e.g., Prostak et al., 1993) fit the above description. In actinopterygians, dentine presents more architectural variations than do tetrapods. This is illustrated by the presence of vasodentine (in gadids; Peyer, 1968; Herold, 1970; Schaeffer, 1977) and trabecular osteodentine with features intermediate between bone and orthodentine (e.g., in the pike, Esox lucius; Herold and Landino, 1970; Herold, 1971). Dentine can be cellular as reported in Amia calva (Moss, 1964; Peyer, 1968). In most adult species the mineralized orthodentine contains embedded odontoblast processes constituting the typical dentine tubules. In adult teleosts, teeth show different modes of attachment. A review has been presented by Fink (1981). The way of tooth attachment can change through ontogeny, as in the cichlid Hemichromis bimaculatus (Huysseune and Sire, 1997b). Polypteriformes In the 5-day-old larvae of Polypterus senegalus several teeth are developing on the upper and lower jaws. The upper jaw teeth form in loose mesenchyme, in close contact to the buccal epithelium and at a distance from the bone support (Fig. 3a). The buccal epithelium has invaginated to form the dental organ and surrounds mesenchymal cells that constitute the dental papilla. The dental organ is composed of two layers, the inner and outer dental epithelium. Dentine-like matrix is deposited by differentiated odontoblasts along the epithelial-mesenchymal interface with a gradient from tip to base (Fig. 3b). The pulp cavity is one or two odontoblasts wide (10 –15 ␮m) only. Predentine and dentine

Fig. 2. Scyliorhinus canicula. Functional teeth in a 108-mm TL catshark, 1 week after hatching. Transverse sections of the left lower jaw. a– c: 1 ␮m-thick sections; d–f: thin sections. a: General view showing a functional tooth (1) and its successors (2–5) located in the oral mucosa and regularly spaced odontodes located in the epidermis (arrowheads). b: Enlarged view of the functional tooth (left, tooth 1 of a) and its successor (right, tooth 2). c: Section slightly posterior to that shown in a, showing tooth 3 in a final stage of differentiation. Enameloid ends its maturation while dentine is still deposited centripetally. Arrowheads indicate limit of enamel organ and boundary between dentine and attachment tissue. Squared regions 1, 2, 3 are detailed in d–f. d: Squared region 1 in c. The dentine shaft is composed of a woven-fibered matrix in the region facing the inner dental epithelium and of a parallel-fibered matrix in the region facing the pulp cavity. No cell prolongations are seen penetrating the matrix. A thin layer of granular material is located along the ide/dentine boundary. e: Squared region 2 in c. The cytoplasm of the ide cells shows a vacuolized aspect in the region facing the maturing enameloid; in the latter some granular and fibrillar material is visible. Note cell processes in the dentine matrix (arrows). f: Squared region 3 in c. After decalcification, a thin, fibrillar, loose material is seen to compose the enameloid matrix. The odontoblast processes reach the dentine/enameloid junction. Scale bars: a ⫽ 100 ␮m; b,c ⫽ 50 ␮m; d–f ⫽ 2 ␮m. Abbreviations used: ab ⫽ attachment bone; am ⫽ ameloblast; at ⫽ attachment tissue; bc ⫽ buccal cavity; cb ⫽ ceratobranchial cartilage; db ⫽ dentary bone; de ⫽ dentine; dl ⫽ dental lamina; dp ⫽ dental papilla; en ⫽ enamel/enameloid; eo ⫽ enamel organ; ide ⫽ inner dental epithelium; li ⫽ ligament; m ⫽ muscle; mc ⫽ Meckel’s cartilage; od ⫽ odontoblast; ode ⫽ outer dental epithelium; pc ⫽ pulp cavity; pde ⫽ predentine; phc ⫽ pharyngeal cavity; pmx ⫽ premaxillary bone; sde ⫽ secondarily deposited dentine; si ⫽ stratum intermedium; sr ⫽ stellate reticulum; tg ⫽ tooth germ.

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are composed of 30 – 40 nm-diameter collagen fibrils parallel to the long axis of the tooth (Fig. 3c,d). An electron-dense layer separates the inner dental epithelium cells from the dentine surface. Odontoblast processes penetrate the unmineralized predentin matrix (Fig. 3d). However, functional attached teeth lack odontoblast processes in the mature dentine (Fig. 3e,i). In a recently formed tooth the enameloid matrix is composed of 50 nm-diameter collagen fibrils parallel to the long axis of the tooth and separated by extrafibrillar spaces (Fig. 3g,h). The functional first-generation teeth are conical and small (approximately 100 ␮m high, 20 ␮m in diameter). In older specimens the functional teeth show a slightly enlarged (25 ␮m in diameter) pulp cavity, the dentine shaft is 5–10 ␮m thick and is ankylosed to the bone support (Fig. 3j). There are, however, no odontoblast processes embedded in the mature dentine. Osteoglossomorpha In the 35-mm SL specimen of Osteoglossum bicirrhosum, the teeth are already functional, i.e., erupted and attached to the bone support (Fig. 4a). Although it is a small specimen compared to the 60 cm of the adult, it is likely that the illustrated tooth is not the very first tooth in the lower jaw. It is conical in shape and firmly ankylosed to the bone support. The elongated dentine shaft (approximately 10 ␮m-thick) contains embedded odontoblast processes in its upper region only (Fig. 4c–f). Cell prolongations are obvious within the tip of the dentine cone and some cell processes even penetrate into the enameloid matrix (Fig. 4b). Within the dentine the collagen fibrils run largely parallel to the long axis of the tooth (Fig. 4b,c). The limit between dentine and attachment bone is not visible but the dentine matrix is embedded in an electron-dense substance in the basal region of the tooth (Fig. 4e,f). The pulp cavity is 50 ␮m in diameter. The absence of cells within the pulp cavity is due to poor preservation (frozen specimen). Elopomorpha In Anguilla australis the first-generation teeth develop in the mesenchyme, external to the bone (Fig. 5a). The functional teeth are conical and small (25 ␮m in diameter at the base and less than 100 ␮m in height). The pulp cavity is 2–3 cells wide, and lacks blood vessels and nerve endings. The tooth base is attached to the attachment bone by a large, unmineralized ligament (Fig. 5b,d) and the limit between the attachment bone and the dentary bone is not visible. The dentine shaft is 5 ␮m thick and it lacks embedded cell processes, although odontoblast prolongations penetrate the unmineralized predentine matrix composed of 60 – 80 nm diameter collagen fibrils (Fig. 5c–f). Ostariophysi Cypriniformes The zebrafish, Danio rerio: The development of the first-generation teeth has been described in detail by Huysseune et al. (1998). Only the main features will be reported below. In zebrafish larvae, the first functional teeth are small (50 –100 ␮m tall, 10 –20 ␮m in diameter) and have a curved, conical shape. The first tooth germs are present at hatching time (48 hours postfertilization).

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Fig. 3.

FIRST-GENERATION TEETH IN VERTEBRATES

Teeth develop in the mesenchyme, external to the bone. Once the pharyngeal epithelium has folded and formed a bilayered dental organ surrounding a well-organized dental papilla (Fig. 6a), a layer of dentine-like material is deposited along the entire epithelial–mesenchymal interface (Fig. 6b). At the tooth tip this material is identifiable as dentine (collagen-rich) rather than as a precursor of the enameloid. Mineralization starts in the upper region of the tooth, then extends down towards the tooth base (Fig. 6c). In the prolongation of the tooth base, attachment bone is deposited (Fig. 6d). The latter is continuous with the supporting bone and the limit between both matrices is not distinguishable. This type of attachment has been called primary attachment (Huysseune and Sire, 1997a). The tip of the functional teeth is covered by a tissue which has lost most of its organic matrix when mineralized; this tissue is interpreted as enameloid (Fig. 6e,f). Enameloid is covered by a thin electron-dense membrane (Fig. 6f). The dentine matrix is now well distinguishable from the enameloid and is composed of collagen fibrils oriented parallel to the long axis of the tooth. The dentine matrix does not contain odontoblast processes (Fig. 6g,h). The pulp cavity of a functional tooth is 2–3 cells wide only, is devoid of capillaries and nerves, and contains odontoblasts only. In older larvae, replacement teeth develop and attach close to the functional teeth, which, however, are not yet shedding. The only difference concerns their mode of attachment: the primary attachment type (characterized by the absence of a clear limit between the attachment bone and the bone support) is replaced by the secondary attachment type (characterized by the presence of a distinct reversal line between attachment bone and underlying bone due to a secondary

Fig. 3. Polypterus senegalus. a– e, g–i ⫽ 5-day-old larvae (9 mm SL); f,j ⫽ 24-day-old larvae (18 mm SL). Transverse sections of the upper (a– e) and lower (f–j) jaws. a,b,f,j: 1 ␮m-thick sections; c– e,g–i: thin sections. a: General view of the first-generation teeth developing on the upper jaw. Laterally, teeth (arrows) form in the vicinity of the supporting premaxillary bone; teeth located medially (arrowheads) develop at some distance from a bony support. b: Detail of a tooth forming close to the premaxillary bone. The dental papilla is 1–2 cells wide. Active osteoblasts are located at the bone surface facing the base of the forming tooth. c: Detail of b showing the organization of the dentine matrix. Note lack of cell processes within the matrix. d: Early stage of dentine deposition showing penetration of the collagen matrix by odontoblast processes. e: Dentine maturation in a well-formed tooth. The reversal line (arrowheads) indicates the arrest of primary dentine formation. It was followed by a secondary deposition of dentine. f: Tooth in the process of attachment to the dentary bone (arrows). A well-mineralized enameloid layer covers the tooth tip. The tooth has not yet erupted and the pulp cavity is occupied by odontoblasts only. g: Section through the upper region of a tooth in a developmental stage close to that shown in f. The inner dental epithelium (ide) cells surrounding the tooth matrix have a highly vacuolar cytoplasm. This suggests that the maturation process of the enameloid matrix has started. h: Detail of the enameloid matrix of the tip of the tooth in g. A thin collagenous, fibrillar matrix is oriented parallel to the long axis of the tooth with some interfibrillar spaces in between. i: Detail of the dentine matrix of the tooth in g. The collagen fibrils are oriented parallel to the long axis of the tooth and the matrix is devoid of cell processes. j: In this older specimen the tooth is erupted and ankylosed to the dentary bone surface by means of attachment bone. The odontoblasts show a reduced activity. Dentine, attachment bone, and dentary bone are hardly distinguishable from one another. Scale bars: a ⫽ 100 ␮m; b,f,j ⫽ 10 ␮m; c,h,i ⫽ 1 ␮m; d,e ⫽ 500 nm; g ⫽ 5 ␮m.

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deposition of the former). The pulp cavity enlarges and the first blood vessels appear at about 1 month after fertilization. The carp, Cyprinus carpio: In the 3-day posthatching larvae examined, the first functional teeth are approximately 100 ␮m in height and 30 – 40 ␮m in diameter (Fig. 7). One of them is attached to the outer surface of the perichondral bone surrounding the ceratobranchial cartilage at one side and to an apolamella originating from this bone at the other side (Fig. 7a,b). Medially, a second tooth is fixed adjacent to the base of the first tooth. The structure is similar for the two teeth except that the 3– 4 cells wide pulp cavity of the more medial tooth clearly contains a blood vessel. During development the dental papilla of the tooth germs is densely cellular and dentine-like matrix is deposited along nearly the entire interface between the dental papilla and the dental organ (Fig. 7c). Without TEM observations it is difficult to confirm the absence of odontoblast processes in the mature dentine. The limit between dentine and attachment bone is hardly distinguishable and indicated by the limit of the cervical loop tip only. The teeth are ankylosed to the bone support either in a primary or in a secondary attachment type. Siluriformes The development of the first-generation teeth in the armored catfish, the callichthyid Hoplosternum littorale, has been described by Huysseune and Sire (1997a). The main features are reported below to facilitate further comparisons. In contrast to other catfish, teeth in callichthyids are present in larvae and young juveniles only; they are replaced several times but disappear from 2-month-old specimens (Machado-Allison, 1986). Shortly after hatching the first teeth start to develop in the mesenchyme, close to the buccal epithelium and external to the bone (Fig. 8a). The first functional teeth are conical, approximately 100 ␮m tall and 20 ␮m in diameter. The dental papilla is 1–2 cells wide and lacks capillaries and nerve endings (Fig. 8a,b). The mature dentine is composed of collagen fibrils (30 – 40 nm in diameter) parallel to the long axis of the tooth and interrupted at intervals by radially oriented thin fibrils (25 nm thick); however, the dentine does not contain any odontoblast prolongations (Fig. 8c,f). The enameloid surface is covered by a thin electron-dense membrane (Fig. 8g). The limit between the dentine and attachment bone is not visible; its position is nevertheless revealed by the position of the cervical loop (Fig. 8a). Teeth are ankylosed to the bone support by a primary type of attachment (Fig. 8a,h). In older specimens, the dentine layer is still atubular (Fig. 8d,e) and the pulp cavity remains devoid of capillaries and nerves. Primary attachment still occurs but is progressively replaced by a secondary type of attachment (Fig. 8e). Salmoniformes The two sections of the jaws of Oncorhynchus mykiss larvae illustrate the main characteristics of the firstgeneration teeth: conical teeth, 100 ␮m tall and 25 ␮m in diameter; a narrow pulp cavity lacking capillaries and nerve endings; dentine lacking tubules; and the

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Fig. 4. Osteoglossum bicirrhosum. Larvae, 35 mm SL. Transverse sections through the left lower jaw. a: 1 ␮m-thick sections; b–f: thin sections. a: This tooth has erupted and is attached to the dentary bone. The enameloid cap is visible even after decalcification. The organization of the tooth matrix is detailed in b–f, from the top to the base of the dentine cone (numbered 1 to 5). b: Region 1. Enameloid/dentine junction. Some cell processes (arrowheads) are seen in this region and some fibrillar matrix is still visible in the enameloid. c: Region 2. Below the ide cells, the collagen fibrils are

parallel to the long axis of the tooth. d: Region 3. The fibrils are still parallel to the long axis of the tooth. Some odontoblast processes are embedded in the matrix (arrow). e: Region 4. In this basal region of the dentine cone the collagen fibrils are still parallel to the long axis of the tooth. They are embedded in an electron-dense, noncollagenous material. No odontoblast process is seen within the matrix. f: Region 5. Attachment region. The noncollagenous material is abundant and the dentine is devoid of cell processes. Scale bars: a ⫽ 50 ␮m; b ⫽ 2 ␮m; c–f ⫽ 1 ␮m.

presence of an enameloid cap in functional teeth (Fig. 9a,b).

The first dentary teeth start to form as early as 2 days posthatching (simultaneously with the opening of the mouth) in the mesenchyme, close to the buccal epithelium. Once functional, they are small conical teeth (approximately 30 ␮m tall, 12 ␮m in diameter). The pulp cavity is 1–2 cells wide and lacks capillaries and nerve endings. The enameloid matrix is deposited first and is composed of collagen fibrils deposited by the odontoblasts parallel to the long axis of the tooth (Fig. 11c,d); most of this collagenous matrix will disappear during the maturation process at the tooth tip. Predentine (Fig. 11a) is deposited after the enameloid and it mineralizes into dentine when enameloid maturation has started. Odontoblast prolongations penetrate the predentine (Fig. 11a,b) and are present before the maturation process at the enameloid–predentine junction (Fig. 11c) but they are not embedded in the mature dentine (Fig. 11b,e). The predominant orientation of the collagen fibrils in the dentine is parallel to the long axis of the tooth, whereas it is woven-fibered in the attachment bone (Fig. 11b,e). The limit between the

Paracanthopterygii Despite the poor preservation, the functional teeth in this larva of the gadiform Gadus morhua can be described as conical, approximately 40 ␮m tall and 20 ␮m in diameter. The narrow pulp cavity is only one cell wide. The dentine lacks odontoblast processes. The dentine base is linked to the attachment bone by means of a ligament and tooth attachment is of the secondary type as revealed by the presence of a cement line between the bone of attachment and the supporting bone (Fig. 10). Percomorpha Cichlidae A detailed description of the development of firstgeneration teeth in the cichlid Hemichromis bimaculatus has been published by Huysseune and Sire (1997b); below we briefly review the results relevant to the present article.

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Fig. 5. Anguilla australis. Larvae, 47 mm TL. Transverse section through the lower jaw. a,b: 1 ␮m-thick sections; c–f: thin sections. a: General view of the lower jaw showing 3 and 5 teeth located on the left and right sides, respectively. The teeth are sectioned at different levels of the dentine shaft. On both sides of the jaw one tooth is attached to the dentary bone surface by means of a ligament. b: Enlargement of the left side of the jaw. At the base of the attached tooth the pulp cavity communicates with the medullary cavity of the dentary bone. The distinct predentine layer in each of the three teeth suggests that these teeth have formed recently. c: Upper part of the attached tooth in b showing well-formed dentine, predentine, and

active odontoblasts with numerous cell processes penetrating the thick predentine layer. d: Base of the same tooth showing the large ligament linking the dentine base to the bone support. e: Recently deposited predentine in a transversely sectioned tooth. The collagen fibrils are parallel to the long axis of the tooth and the matrix is devoid of cell processes. f: Detail of the mature dentine of the tooth in c. A thin layer of granular material lies between the inner dental epithelium (ide) cell surface and the dentine (arrow). This material could represent enameloid matrix. Scale bars: a ⫽ 50 ␮m; b ⫽ 10 ␮m; c ⫽ 5 ␮m; d ⫽ 2 ␮m; e ⫽ 1 ␮m; f ⫽ 500 nm.

dentine and the attachment bone is distinguishable as a thin layer of loose collagenous matrix at the level of the cervical loop tip. The attachment to the bone support is of the primary type. Scaridae The scarids are characterized by their upper and lower oral jaws, which are transformed into a beak (hence their name of parrotfish). This beak is used to scrape algae covering rocks and coral skeletons. The major part of the beak is constituted by the bone of the jaws to which teeth are added. In Scarus, the teeth develop in the medullary cavity of the bone and grow to the bone surface, where they become ankylosed and reinforce the scraping surface (J.Y. Sire, pers. obs.). The pulp cavity of these functional teeth is entirely filled by secondarily deposited dentine, probably to resist abrasion. The teeth are not shed but entirely worn down and replaced by new teeth forming below.

Below, we will describe the teeth present in young Scarus larvae of unknown age, caught during their migration into the lagoon where they subsequently settle. Although their typical beaks have not yet differentiated at that time, the larvae are toothed. The functional teeth are conical and very small, approximately 30 ␮m tall and 10 ␮m in diameter (Fig.12a). The pulp cavity is 1–2 cells wide and contains nothing but densely packed mesenchymal cells. Functional teeth show a well-formed enameloid cap, the matrix of which has been removed during maturation. Only traces of the enameloid are visible after decalcification (Fig. 12a,d,f). In the developing teeth the collagen fibrils of the predentine are deposited parallel to the long axis of the tooth (Fig. 12b,c). Odontoblast prolongations penetrate the predentine (Fig. 12b,c) but they are absent from mature dentine (Fig. 12d,e). In specimens bred

Fig. 6. Danio rerio. a– b: Larvae, 3 days postfertilization; c– h: larvae, 6 days PF. Transverse sections through the pharyngeal region (undecalcified except g: partially demineralized). a,d,e: 1 ␮m-thick sections; b,c,f– h: thin sections. a: Tooth germ developing near the ceratobranchial cartilage. At the base of the tooth, the pulp cavity is 2–3 cells wide. Dentine deposition has started along the interface between the odontoblasts and the ide cells. b: Detail of the tooth germ in a showing the organization of the odontoblasts and of the ide cells. The dentine matrix facing the ide cells has started to mineralize. Deposition of the predentine matrix extends deep towards the base of the tooth (arrows). c: Advanced stage of dentinogenesis. An electrondense membrane borders the outer surface of the dentine. d: Basal region of two functional teeth (1, 2) ankylosed (at least at one side) to

the perichondral bone surrounding the ceratobranchial cartilage. The pulp cavity is 2–3 cells wide. e: Section through the upper region of tooth 1 in d. The tooth has erupted and it possesses an enameloid cap (arrow). f: Detail of the upper region of the erupted tooth shown in e. Note the presence of an electron-dense membrane surrounding the enameloid cap. Maturation of the enameloid matrix is not completed, as indicated by the remains of organic matrix. g: Section through the dentine shaft of a functional tooth. Secondary deposition of dentine is visible near the tip of the pulp cavity. h: Detail of the primary and secondarily deposited dentine. Note the absence of odontoblast processes and the difference of organization of the two dentine matrices. Scale bars: a ⫽ 10 ␮m; b ⫽ 5 ␮m; c,g ⫽ 1 ␮m; d,e ⫽ 25 ␮m; f,h ⫽ 500 nm.

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Fig. 7. Cyprinus carpio. Larvae, 3 days posthatching. Transverse sections through the pharyngeal region. One ␮m-thick sections. a: Three teeth (1, 2, 3) are seen on each side of the jaw; teeth 1 and 2 are functional and tooth 3 is developing. The functional teeth have a wide pulp cavity, in which blood vessels can be seen (arrowheads). At one side, teeth 1 and 2 share the same attachment bone (arrow). b: This functional tooth is attached to the perichondral bone surrounding the

ceratobranchial cartilage by means of attachment bone (arrow) and, at the other side, to a lamella of membrane bone extending from the perichondral bone (arrowhead). A thin cap of enameloid covers the upper region of the tooth. c: Section through a tooth germ showing the organization of the odontoblasts and of the enamel organ. Dentine matrix is being deposited. Scale bars: a ⫽ 50 ␮m; b,c ⫽ 10 ␮m.

7 days at the laboratory, the teeth have erupted and are attached to a support which seems to be formed out of the attachment bones of adjacent teeth (Fig. 12g). Later in ontogeny the transformation of the jaw has started and functional teeth are ankylosed to the lateral edge of the premaxillary bone, both in a primary and a secondary mode of attachment. Replacement teeth develop in the medullary cavity (Fig. 12h). In the oldest specimen studied the typical jaw of the parrotfish has formed (Fig. 12i).

there is only one row. In the three living genera, Neoceratodus, Protopterus, and Lepidosiren, the first teeth are small and conical, as reported by a number of authors (Parker, 1892; Semon, 1899; Kerr, 1903; MoyThomas, 1934; Lison, 1941; Bertmar, 1966). Another particularity for the dipnoan teeth is that the pulp cavity is secondarily (not in the youngest stages: Lison, 1941; Kemp, 1979) filled with petrodentine, a hypermineralized dentine that is deposited by a type of odontoblast called petroblasts (Smith, 1984). The first-generation teeth develop separate from the bone support and teeth developing in subsequent stages resemble the first teeth, but are continuous at their bases with the pedestal tissue of the previous tooth in each row (Smith, 1985). In the youngest stage of Protopterus aethiopicus (27.5 mm TL larvae), the central material is similar to that termed petrodentine in subsequent stages (Smith, 1985). Traces of enamel matrix surround the pallial dentine over the most coronal part of the tooth only. Pallial dentine forms first, then petrodentine, then dentine pedestal and bone of attachment (Smith, 1985). The fact that lungfish teeth are covered by enamel is now generally accepted

Sarcopterygii Dipnoi Juvenile and adult dipnoans share a unique developmental process in vertebrates in that teeth are not shed and that both the first and later generations of teeth are retained in the adult dentition; this process leads to the formation of tooth plates composed of tooth rows in the dentary, prearticular, pterygoid, and vomer (Kemp, 1977, 1979; 1995; Smith,1985; Smith and Krupina, 2001). In each bone, the early tooth pattern starts by a first tooth called either initiator or primordial tooth; new teeth are then added anterolaterally into divergent rows, except on the dentary, in which

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Fig. 8.

FIRST-GENERATION TEETH IN VERTEBRATES

Fig. 9. Oncorhynchus mykiss. Larvae, 17.7 mm SL. Transverse, 10 ␮m-thick sections through the lower (a) and upper (b) jaws (paraffin, improved trichrome staining). a: Dentary tooth in its final stage of development. Enameloid has matured and is well visible at the tip of the tooth. Dentine is mineralized in the upper region and predentine is still deposited in lower regions of the tooth. The pulp cavity is 2–3 cells wide and the odontoblasts are densely packed. b: Nearly functional premaxillary tooth; the enameloid and the attachment bone region are not visible. Most of the dentine is mineralized and the pulp cavity shows a rather loose aspect. Scale bars ⫽ 50 ␮m.

(Peyer, 1968; Schmidt and Keil, 1971; Ishiyama and Teraki, 1990; Satchell et al., 2000). The only section available to us of a hatchling of Neoceratodus forsteri shows the presence of four individual teeth (Fig. 13). The teeth are 100 ␮m high and 50 ␮m in diameter. The pulp cavity, where sectioned, is several cell diameters wide and loosely organized; the presence of capillaries cannot be excluded. The teeth are in a different stage of development: one erupted tooth is ankylosed to a bone support (primary attachment type), another erupted tooth has deposited dentine and petrodentine (a kind of secondary dentine) but

Fig. 8. Hoplosternum littorale. a– c,f– h: larvae, 5.5 mm SL; d: juvenile, 16.0 mm SL; e: juvenile, 22 mm SL. Transverse sections through the upper jaw. a,d,e: 1 ␮m-thick sections; b,c,f– h: thin sections. a: Early dentine deposition in a first-generation tooth germ. This tooth will become attached to the premaxillary bone more posteriorly. Two adjoining teeth are already attached to the premaxillary bone (arrows). b: Detail of a bell-shaped tooth germ as presented in a. Dentine is being deposited at the interface of enamel organ and dental papilla. c: Mineralized dentine constituting the shaft of a developing tooth near the tip of the pulp cavity. There are no odontoblast processes penetrating the dentine. d: Dentine deposition of a replacement tooth in a juvenile specimen. Although the tooth is taller than in the larval stage, the pulp cavity is still only two cells wide. The surface of the premaxillary bone has been slightly resorbed prior to the attachment of this new tooth. e: Functional teeth in an older juvenile showing the enameloid cap (arrows). Both primary attachment (left tooth) and secondary attachment (two teeth on the right) can be seen. Note that the teeth are not fully ankylosed to the bone support. f: Mature dentine near the tip of a functional tooth. The basal lamina has disappeared at the interface between inner dental epithelium (ide) cells and dentine. g: Detail of the matrix at tooth tip. A thin electrondense membrane (arrow) covers the enameloid. h: Primary attachment. The surface of the dentine base is not yet entirely fused to the attachment bone and patches of unmineralized material are visible in between. Scale bars: a,d,e ⫽ 25 ␮m; b ⫽ 5 ␮m; c,g ⫽ 500 nm; f ⫽ 1 ␮m; h ⫽ 2 ␮m.

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Fig. 10. Gadus morhua. Larvae, 10.0 mm SL. Transverse, 1 ␮m-thick section through the lower jaw. Small functional tooth; only one single odontoblast is visible within the pulp cavity. The dentine (arrow) is mineralized and a narrow layer of predentine is visible. Note the distinct cement line separating the attachment bone (arrowhead) from the dentary bone. Scale bar ⫽ 10 ␮m.

is not attached; the two other teeth are probably still depositing dentine (Fig. 13). Lissamphibia Tooth development has been studied in the larval stages of a number of salamanders (e.g., Kerr, 1960; Smith and Miles, 1971; Roux, 1973; Roux and Chibon, 1973), apodans (Wake, 1976; 1980), and Xenopus laevis, a pipiid anuran (Shaw, 1979), but these studies have mostly focused on the nature of the tooth enamel and the role of the inner dental epithelium in the formation of the upper layer. After a long-lasting controversy, Kerr (1960) and Smith and Miles (1971) have demonstrated, e.g., in the axolotl, Ambystoma mexicanum, that enameloid covers the teeth in larval specimens while the tooth of metamorphosed animals is capped with true enamel. This difference could be due to a change in cellular activity of the ide, which occurs during metamorphosis (Smith and Miles, 1971). However, the enameloid has been found to be overlapped by 0.2– 0.3 ␮m thin layer of enamel (Roux and Chibon, 1973). A cuticle covers this enamel layer (Smith and Miles, 1971; Roux and Chibon, 1973). In the larval Pleurodeles waltl, odontoblastic prolongations extend in the predentine and dentine (Kerr, 1960; Roux, 1973). In these larval salamanders, the pulp cavity contains a few cells only, but there are no reports on the presence or absence of blood vessels. In young larval salamanders teeth are undivided, meaning that the connection between the tooth and the tooth-bearing bone is totally calcified. In older larvae and after metamorphosis, teeth become divided, thus showing a nonmineralized bone of attachment (Parsons and Williams, 1962; Greven, 1989). This unpedicellate aspect of primary teeth has also been confirmed by Tesche and Greven (1989) and contrasts with the pedicellate condition in adults, i.e., teeth are attached to a cylindrical pedestal, or pedicel, projecting from the bone below (Kerr, 1960; Edmund, 1969). The nature of the pedicel (characterized by the absence of cells included in the bony matrix) has long been debated.

Fig. 11. Hemichromis bimaculatus. Larvae, 5 and 6 days posthatching. Transverse, thin sections through the lower jaw. a: Predentine is deposited by the odontoblasts, the prolongations of which penetrate the recently deposited matrix. b: Maturing dentine. The odontoblast processes no longer penetrate the dentine and extend into the unmineralized predentine only. c: Recently deposited enameloid matrix composed of loosely organized collagen fibrils with large interfibrillar spaces in between. Odontoblast processes are visible in the unmineralized matrix. A basement membrane separates the enam-

eloid surface from the covering inner dental epithelium (ide) cells. d: Advanced stage of enameloid formation. Most of the matrix is composed of collagen fibrils oriented parallel to the long axis of the tooth. The basement membrane is no longer visible and the membrane of the ide cells facing the enameloid shows a ruffled border. e: Attachment bone formation. The pulp cavity is one cell wide and the odontoblasts are very active. Arrows indicate the limit between the attachment bone and dentine. Scale bars: a ⫽ 1 ␮m; b,c ⫽ 500 nm; d,e ⫽ 2 ␮m.

FIRST-GENERATION TEETH IN VERTEBRATES

According to some authors it has a dental origin, while others estimate it to be a bony element (e.g., Parsons and Williams, 1962; Clemen and Greven, 1994). In adult amphibians dentine is typical orthodentine, referring to the presence of odontoblastic prolongations extending in the dentine (Peyer, 1968; Kerr, 1960). The pulp contains dispersed cells as well as capillaries and nerves (Roux, 1973; Chibon and Ricqle`s, 1995). In some adult amphibians such as Proteus and Discoglossus, the dental pulp is partly filled up with a calcified matrix with bony characteristics, while in Necturus and Ceratophrys, the pulp is completely occupied by such tissue (Chibon and Ricqle`s, 1995). In their review, Chibon and Ricqle`s (1995), reported that the adult dentine is probably identical as the larval dentine. In Rana pipiens, the odontoblastic prolongations never extend into the enamel matrix (Chibon and Ricqle`s, 1995). Larval teeth are very small and they differ in shape from the teeth in adult salamanders (Kerr, 1960, Sato et al., 1992; Clemen and Greven, 1994). Kerr (1960) remarked that the larval teeth have the typical “teleost” tooth form. In the axolotl, Ambystoma mexicanum, the tooth form changes from monocuspid. Monocuspid teeth are present in nonmetamorphosed animals and the transition from the monocuspid to the bicuspid dentition appears gradually (Kerr, 1960). In viviparous caecilians, the first-formed teeth constitute a fetal dentition that is functional, the fetuses using their teeth to scrape the oviducal epithelium (Wake, 1976, 1980). This fetal dentition is composed of well-differentiated teeth: they possess numerous short cusps, a pedicel, and are ankylosed to the jaw. Moreover, these teeth are replaced before birth. As in actinopterygians, a stellate reticulum and stratum intermedium are not present in the dental organ. In the larvae of the urodele Pleurodeles waltl of 10 days posthatching, several teeth are either functional or developing on the upper and lower jaws (Fig. 14). During the first stages of development (Fig. 14a,c,e) the tooth germs lie at some distance from the bone support; later they approach the bone surface to finally attach; their attachment is at least partially of the primary type (Fig. 14h,i). During development, the inner and outer dental epithelium adjoin each other without any intervening layer. The dental papilla is one or two odontoblasts (15–20 ␮m) wide only. Predentine matrix is deposited by differentiated odontoblasts along the epithelial–mesenchymal interface with a gradient from tip to base (Fig. 14a,c,e). The predentine matrix is composed of 30 – 40 nm-diameter collagen fibrils parallel to the long axis of the tooth. This unmineralized matrix is penetrated by odontoblast processes and numerous matrix vesicles are visible among the collagen fibrils (Fig. 14b). The odontoblast prolongations are no longer visible in the mature dentine of the dentine shaft, but they are present in the upper region of the tooth (Fig. 14f). The matrix vesicles are still visible in the mature dentine (Fig. 14d,g). In a recently formed tooth the enameloid matrix is composed of thick, 80 –90 nm-diameter collagen fibrils parallel to the long axis of the tooth and separated by interfibrillar spaces (Fig. 14f). In more developed, attached teeth the enameloid matrix is well mineralized and covers the tip of the teeth (Fig. 14h). A basement

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membrane separates the inner dental epithelium cells from the predentine and dentine surface (Fig. 14b,d,g) but it disappears along the enameloid surface when the maturation process starts (Fig. 14f). At the base of an attached tooth, the pulp cavity is slightly wider (approximately 50 ␮m in diameter) (Fig. 14i). The dentine shaft is about 5 ␮m thick and the teeth are ankylosed to the bone support through attachment bone. The limit between dentine and attachment bone is indicated by the cervical loop tip only (Fig. 14h–j). Reptiles Most studies on squamate and crocodile teeth have been concerned with the comparison of the dentition pattern and especially the question of the replacement waves (Edmund, 1960, 1969; Cooper, 1966; Cooper et al., 1970; Rieppel 1978; Rocek, 1980). The pattern of tooth replacement has generated a large debate in terms of evolutionary implications (Osborn, 1970, 1984; DeMar, 1972, 1974; Osborn and Crompton, 1973) and of embryological reality (e.g., Osborn, 1971, 1972; Westergaard, 1986; Westergaard and Ferguson, 1986). Histological studies of tooth development are either old (e.g., Harrison, 1901; Woederman, 1919) or have served as a tool to understand the pattern of developing teeth (e.g., Osborn, 1971). The first-generation teeth develop early in embryos (e.g., on days 16 –19 in Alligator mississipiensis: Westergaard and Ferguson, 1986) and are initiated from the oral epithelium before the dental lamina is formed. Most of the first-formed teeth are rudimentary (degenerative) teeth that are replaced before birth or soon thereafter (e.g., Ro¨ se, 1894; Woerdeman, 1919; Edmund, 1969; Osborn, 1971; Westergaard, 1986; Westergaard and Ferguson, 1986). In some of these teeth the enamel organ never differentiates entirely but a dental lamina develops and replacement teeth are formed in connection with it. All functional teeth in reptiles develop, however, from the lamina (Westergaard, 1986; Westergaard and Ferguson, 1987, 1990). Invariably, teeth start to develop after the epithelial cells of the dental lamina and a mesenchymal cell population have entered in contact and have differentiated into a dental organ and a dental papilla, respectively, at a short distance from the oral surface (Osborn, 1971; Ogawa, 1977). A thin layer of dentine is deposited first by the odontoblasts, starts to mineralize, and is covered by a thin layer of enamel matrix formed by the ameloblasts. Then the tooth elongates towards the supporting bone, to which it ankyloses by means of a bone of attachment, before the tooth tip pierces the buccal epithelium. The center of the pulp cavity is loose and some blood vessels are present. Reptilian enamel is prismless (see Sander, 2001, for a detailed review of reptile enamel) and orthodentine, containing dentinal tubules, is the only reported type of dentine. However, in the developing teeth of some squamates, and particularly in varanids, the dentine at the base of the tooth folds to form a typical tissue called plicidentine (e.g., Schultze, 1969). Unlike the teeth of most squamates, which are generally ankylosed to the inner side of the labial wall (⫽ pleurodont mode) (e.g., Edmund, 1969), crocodilian teeth are attached to the jaws by a periodontal ligament. Its development, and that of the root cementum,

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Fig. 12.

FIRST-GENERATION TEETH IN VERTEBRATES

Fig. 13. Neoceratodus forsteri. Stage 50. Transverse section through the buccal cavity. Four isolated teeth (1– 4) are visible. Tooth 1 is depositing the dentine layer, tooth 2 starts the deposition of petrodentine at the tip of the pulp cavity, tooth 3 shows mineralized dentine and possibly petrodentine, and tooth 4 is ankylosed to a bone support. Scale bar ⫽ 100 ␮m.

is similar to that in mammals (⫽ thecodont mode) (e.g., Miller, 1968; Berkovitz and Sloan, 1979; Owens and Ferguson, 1982). Squamata In prehatchlings of the scincid lizard Chalcides sexlineatus (1 or 2 weeks before birth) numerous developing germs are present on the upper and lower jaws (Fig. 15a– e). In hatchlings (1 day after birth) the teeth are well attached to the jaw in a pleurodont fashion (Fig. 15f). In the embryos, a bilayered dental lamina extends from the buccal epithelium deep into the jaw

Fig. 12. Scarus sp. a–f: Larvae, t0, 7.0 mm SL; g: t0⫹7 days, 8.0 mm; h: t0⫹15 days, 10.0 mm; i: t0⫹59 days, 18.0 mm. Transverse sections through the upper jaw. a,g–i: 1 ␮m-thick sections; b–f: thin sections. a: Several teeth are forming on each side of the jaw. The most lateral tooth on the left side (1) is in an advanced stage of formation and the location of the enameloid cap is visible after decalcification; in tooth 2 dentine is mineralizing; tooth 3 is depositing predentine; and, close to the midline of the jaw, tooth 4 is starting its differentiation. In all these teeth the dental papilla is 1–2 cells wide only. b: Tooth 3. Predentine is deposited by active odontoblasts at the interface with the nonpolarized inner dental epithelium (ide) cells. c: Detail of b. Cytoplasmic prolongations are present within the recently deposited predentine, separating the matrix into several bundles of collagen fibrils. d: Tooth 1. Enameloid is mature and dentine well mineralized. The odontoblasts are active and they deposit predentine. Polarized ide cells showing a ruffled cell membrane surround the tooth tip. e: Detail of d. The dentine matrix is composed of collagen fibrils oriented parallel to the long axis of the tooth. f: Detail of d showing the enameloid/dentine junction. There is no basement membrane between the ide cells and the tooth matrix. The organic matrix of the enameloid has mostly disappeared during the maturation process. g: Four wellformed teeth ankylosed to the premaxillary bone. These teeth have erupted and possess a well-developed enameloid cap. The pulp cavity is very narrow and seems to be almost entirely obliterated. h: Functional tooth showing primary attachment (below) and secondary attachment (above) to the premaxillary bone. A replacement tooth, which is larger than the functional tooth, is forming and its growth induces resorption of the bone (arrow). i: Typical, well-formed jaw of a parrotfish. A tooth is functional along the lower border of the jaw (arrow) while several replacement teeth (most of them sectioned through the enameloid region) are forming within the medullary cavity. Scale bars: a,g,h ⫽ 10 ␮m; b,d ⫽ 2 ␮m; c ⫽ 500 nm; e,f ⫽ 1 ␮m; i ⫽ 100 ␮m.

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mesenchyme (about 100 –200 ␮m). The first-generation teeth that have developed from this dental lamina are still connected to it by their dental organ. The enamel organ is composed of a distinct inner (ameloblast layer) and outer dental epithelium. Both layers are separated by a layer of approximately 2–3 cells thick, interpreted as a thin stellate reticulum (Ogawa, 1977). The leastdeveloped germs terminate enamel matrix deposition (Fig. 15a), while in the other teeth enamel is maturing (Fig. 15d,e). The ameloblasts are tall, polarized cells, perpendicular to the tooth surface. The enamel layer is 3– 4 ␮m thick and, except for the region of the dentine– enamel junction, the crystallites are oriented parallel to one another and perpendicular to the tooth surface (Fig. 15b). In the youngest teeth present at this stage the dentine matrix has started to mineralize while the predentine is deposited by odontoblasts located on top of the dental papilla (Fig. 15a). In a slightly more advanced stage, the dental organ has grown deeper into the mesenchyme (Fig. 15d). The odontoblasts differentiate into polarized cells and deposit the predentin matrix along the tooth shaft opposite well-differentiated inner epithelial cells (Fig. 15e). The pulp cavity is 50 ␮m in diameter. In the first stages of development it is occupied by densely packed mesenchymal cells, in which capillaries are not visible (Fig. 15a,d); however, next the center of the pulp becomes more loosely organized (Fig. 15e). In the tooth tip and shaft, the predentine and dentine matrix are penetrated by numerous odontoblast processes oriented mostly perpendicular to the tooth surface (Fig. 15c). Odontoblast prolongations are also seen close to the enamel– dentine junction (Fig. 15b). Each developing first-generation tooth is connected to the dental lamina through its outer dental epithelium. At the lingual side and at a short distance from the forming tooth, the dental lamina enters in interaction with the surrounding mesenchyme. Depending on the developmental stage of the tooth germ present, the replacement tooth is either in an initiation phase (Fig. 15a) or has started its differentiation (Fig. 15d). In functional, i.e., attached and erupted teeth, the pulp cavity has been invaded by blood vessels, the dentine shaft is 30 ␮m thick, and secondary dentine is deposited (Fig. 15f). The teeth are ankylosed to the bone support through attachment bone. The limit between dentine and attachment bone is not obvious and only indicated by the localization of the cervical loop tip (Fig. 15f). The limit between the attachment bone and the dentigerous bone proper, on the other hand, is recognizable because osteocytes are included in the bone matrix only. Crocodilia In the embryos of the Nile crocodile, Crocodylus niloticus, teeth are already seen to develop on the upper and lower jaws as early as 28 days after egg deposition, i.e., long before hatching (incubation time is approximately 80 days in the rearing conditions used). In C. niloticus embryos, the features of tooth development are roughly similar to those described for the lizard embryo. The first-generation teeth develop close to the buccal epithelium, to which they are linked by a bilayered dental lamina (Fig. 16). The developing teeth are surrounded by a dental organ connected to the dental lamina over a large area. The dental organ is composed

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Fig. 14.

FIRST-GENERATION TEETH IN VERTEBRATES

of four distinct layers: the inner dental epithelium, covering the tooth surface and composed of cells differentiated into polarized ameloblasts depositing enamel matrix; the stratum intermedium, covering the ide and composed of several layers of undifferentiated, flat cells; the stellate reticulum, a loose meshwork of epithelial cells; and a thin outer dental epithelium separating the dental organ from the surrounding mesenchyme and connecting the tooth germ to the dental lamina (Fig. 16a– d). Enamel and dentine matrix are deposited first at the tip of the tooth by typically differentiated ameloblasts and odontoblasts, respectively. The dentine matrix then gradually extends towards the tooth base, concomitant with the downgrowth of the dental organ (Fig. 16d,e). The pulp cavity is large (140 –200 ␮m in diameter) and the dentine wall is 25–30 ␮m thick. Along the dentine surface it is bordered by odontoblasts arranged in a pseudo-epithelial manner. The center of the pulp is occupied by loosely disposed, undifferentiated mesenchymal cells. Some capillaries penetrate into the pulp cavity. Mineralization of enamel and dentine starts at the interface of both tissues (Fig. 16e,f). Near the top of the tooth the enamel layer is 20 –30 ␮m thick and the crystallites are arranged parallel to each other and perpendicular to the tooth surface (Fig. 16f,g). Numerous odontoblast processes are embedded within the dentine and predentine matrix, some of them reaching the enamel– dentine junction (Fig. 16f,h). Shortly before hatching (70 days of incubation) the teeth are still not attached (Fig. 16e). Like in Chalcides, replacement teeth develop from the dental lamina lingual to the tooth germ already present.

Fig. 14. Pleurodeles waltl. Larvae, stage 39, 10 days after hatching. Transverse sections through the upper (a– d) and lower (e–j) jaw. a,c,e,h,i: 1 ␮m-thick sections; b,d,f,g,j: thin sections. a: Predentine deposition at the interface between the inner dental epithelium (ide) cells and the odontoblasts. The dental papilla is one cell wide and the odontoblasts are crescent-shaped. The cells of the ide are polarized. b: Detail of a. The predentine matrix is penetrated by odontoblast prolongations (arrow) and numerous matrix vesicles are visible (arrowheads). c: Section through the mid shaft of the tooth showing dentine starting to mineralize. d: Detail of c. The densely packed collagen fibrils of the dentine matrix are embedded within an electron-dense background substance. Some matrix vesicles are seen in the dentine (arrowheads) but odontoblast prolongations are absent. e: Enameloid matrix starts to mineralize at the tip of the tooth. The lower border of the enamel organ has come to lie in close vicinity to Meckel’s cartilage. f: Detail of the matrix at the tooth tip in e. After decalcification most of the enameloid matrix has persisted. It is composed of a rather dense collagenous network with large interfibrillar spaces. The collagen fibrils are thinner but more densely packed in the dentine than in the enameloid. g: Detail of the well-formed dentine of an attached tooth such as the ones illustrated in h and i. A basement membrane separates the ide cells from the dentine surface. Note numerous matrix vesicles within the dentine matrix and an electron-dense cement line (arrow) delimiting primary from secondarily deposited dentine. h: Section of a tooth recently attached to the dentary bone. There is no clear limit between the dentine and the attachment bone. Most of the section is through the dentine wall (dentine and predentine) and the pulp cavity is not visible except in the upper part of the tooth. i: Basal region of a well-attached tooth showing secondary attachment bone on the left side (immediately below the cervical loop tip, arrow) and primary attachment bone on the right side. The pulp cavity is 2–3 cells wide and no nerve and blood vessels are seen. j: Detail of the arrowed region in i. There is no distinct limit between the dentine and the attachment bone. Scale bars: a,c,e,h,i ⫽ 10 ␮m; b,d,f,g ⫽ 1 ␮m; j ⫽ 5 ␮m.

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The teeth in the youngest embryos studied, 28 and 34 days after egg-laying (Fig. 16a– c), although presenting characteristic features of developing teeth, appear to be slightly different from the teeth in older embryos. They are smaller (less than 100 ␮m tall vs. more than 200 ␮m in older embryos); their dental organ shows a more or less rounded shape (vs. elongated in older embryos); and replacement teeth have already initiated at the extremity of the dental lamina (whereas they are absent next to well-formed first-generation teeth in older embryos) (compare Fig. 16a– c to 16d,e). Indeed, in Figure 16a the replacement tooth has already formed its characteristic bell-shape and the cells are differentiating on both sides of the epithelial–mesenchymal interface; yet the predecessor has only deposited a small amount of enamel and dentine matrix (the odontoblasts are scarce and predentine is not visible). These features suggest that the teeth presented in Figure 16a,b are rudimentary teeth and will not become functional. DISCUSSION In this article we have attempted to collect data as much as possible on first-generation teeth in a number of major vertebrate lineages. In some cases the teeth studied were likely not the very first teeth to develop in a particular position in the dentition (e.g., in the shark specimen). However, from a structural viewpoint these “secondary” teeth appeared to fit the general trends emerging from our observations, which we present below. From the data presented, two major types of first functional teeth seem to emerge. The first type of teeth (called Type 1) is characterized by its small size (approximately 40 –100 ␮m tall and 20 ␮m in diameter) and conical shape (often called caniniform, or villiform, in the literature). The dentine wall is approximately 5 ␮m thick; the collagenous dentine matrix is oriented predominantly parallel to the long axis of the tooth and devoid of odontoblastic prolongations. The dentine tip of fully formed teeth is invariably covered by a highly mineralized tissue, the enameloid. The pulp cavity is narrow (2–3 cell diameters wide), contains only a few odontoblasts, and is devoid of capillaries and nerve endings. The attachment is variable. Teeth of the following taxa studied here fit this description: all the actinopterygians, Neoceratodus forsteri, and Pleurodeles waltl. The teeth of Osteoglossum bicirrhosum and Anguilla australis, despite probably not belonging to the first generation, fit this description as well. In addition, descriptions or illustrations of larval teeth, taken from the literature, allow us to attribute the following species to this type: Amia calva (Degener, 1924); Pagrus major (Higashi et al., 1983), and Belone vulgaris (Moy-Thomas, 1934). In passing, we would like to insist on the presence of enameloid at the tooth tip right from the first-generation onwards. Whereas an enameloid cap is usually prominent after completion of maturation, the unmineralized precursor of the enameloid is hardly recognizable as such during development of the tooth because it is indistinguishable from dentine. This adds further evidence to our hypothesis that the enameloid/dentine transition is the result of a mere switch in odontoblast activity.

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Fig. 15. Chalcides sexlineatus. a– e: Embryo, approximately 1 week before birth, left lower jaw; f: 1 day after birth, right lower jaw. Transverse sections (undecalcified). a,d–f: 1 ␮m-thick sections; b,c: thin sections. Lingual is to the right in a– e and to the left in f. a: A thick layer of enamel matrix has been deposited but is not well mineralized yet, while the dentine below is mineralized. The mineralization front separating the dentine from the predentine is represented by a dark line. A population of densely packed mesenchymal cells occupies the pulp cavity. b: Detail of the enamel matrix of the tooth in a; the crystallites are perpendicular to the tooth surface. The limit between dentine and enamel matrix is irregular. Patches of enamel matrix are seen within the collagen matrix of the dentine. c: Detail of the dentine of the tooth in a. Arrowheads indicate the boundary between the dentine and the predentine. Numerous odon-

toblast processes are embedded in the matrix. d: A tooth is in the process of enamel maturation while its replacement tooth is forming; both teeth are linked to the buccal epithelium and they form close to the surface of the dentary bone. e: Tooth in a further stage of differentiation. The enamel is now highly mineralized and a cusp is clearly visible (arrowhead). f: Section through the basal region of a functional tooth showing its mode of ankylosis to the bone support. In the labial region (to the right) the dentine cone is shorter than at the opposite, lingual side. The limit between dentine and attachment bone is not visible but is assumed to be located at the level of the cervical loop (arrows). The dentary bone is easy to identify because its matrix contains numerous osteocytes, whereas the attachment bone is acellular. Scale bars: a,d,e,f ⫽ 50 ␮m; b ⫽ 1 ␮m; c ⫽ 5 ␮m.

The second type (called Type 2) comprises the teeth of Scyliorhinus canicula, Crocodylus niloticus, and Chalcides sexlineatus. Although their first teeth do not resemble each other, they have been grouped on the basis of their large size and the fact that they much

better reflect the shape and structure of their adult counterparts. These teeth are usually 200 –500 ␮m tall and 50 –300 ␮m in diameter, lance-shaped in S. canicula, and conical in the two reptiles. The dentine wall is 20 –30 ␮m thick and the mineralized collagenous

FIRST-GENERATION TEETH IN VERTEBRATES

matrix of the dentine contains odontoblastic prolongations at least in the tip of the dentine cone. The pulp cavity is large and contains blood vessels. How, then, do these findings relate to the questions raised in the Introduction? 1) It is clear from our observations that distantly related taxa sometimes show remarkable similarities (compare for instance the (pharyngeal) tooth of the actinopterygian Cyprinus carpio (Fig. 7b) with the (oral) tooth of the lissamphibian Pleurodeles waltl (Fig. 14h) or the forming enameloid in the basal actinopterygian Polypterus senegalus (Fig. 3g,h) with that in the teleost Hemichromis bimaculatus (Fig. 11d)); this is in contrast to what one would expect from taxa that have diverged more than 400 and 300 MY ago, respectively (Pough et al., 2002). These resemblances can either point to shared ancestral characters or represent homoplasies. We will discuss this point further below. 2) Despite the large difference in adult body size (Cyprinus carpio can reach a size 20 times larger than Danio rerio), the first-generation teeth by no means differ in size to the same extent and their structure resembles each other very well (compare Fig. 6d of D. rerio with Fig. 7a of C. carpio). The presence of capillaries in the pulp cavity of carp teeth will be discussed below. 3) Whereas some of the species studied have a highly specialized adult dentine structure, their first-generation teeth fit the generalized Type 1 described above. E.g., the teeth of Gadus morhua larvae do not show any trace of the vasodentine described as typical of the adult teeth of gadids (e.g., Merluccius merluccius: Peyer, 1968; Urophycis tenuis: Herold, 1970). Vasodentine is characterized by the presence of canals which contain capillaries only and have no histogenetic relations to dentinal tubules (Peyer, 1968; Herold, 1970; Schaeffer, 1977). As another example, the nonshedding teeth of the larvae of Neoceratodus forsteri are first devoid of the typical petrodentine. Clearly, these adult characters develop only in later ontogenetic stages. This may hold for the other specialized dentine types such as the osteodentine described in the adult teeth of Esox lucius by Herold and Landino (1970) and Herold (1971). 4) Species with a highly specialized adult feeding apparatus exhibit a larval dentition composed of teeth of the generalized Type 1. This is well-illustrated in two examples in the present study: the parrotfish Scarus sp., which has an osseous beak with embedded teeth in the adult, and the armored catfish H. littorale, which loses its dentition altogether during ontogeny. In both, the highly specialized features developing later in ontogeny are preceded by a generalized type of teeth in larval stages. In summary, the larval teeth are never a miniature version of the adult teeth for those species where the generalized Type 1 is found. The absence of complex shape in the first-generation teeth can be explained from a developmental viewpoint by the low number of cells involved. With only a few cells in the dental organ, a complex folding of the enamel organ simply cannot be

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achieved. The size of these generalized Type 1 teeth might well be close to a minimal size for a tooth to develop, both in diameter and in height (30 – 40 ␮m high, 10 –15 ␮m diameter). A physiological explanation for the ubiquitous presence of Type 1 larval teeth is that it seems the most economic way to build a tooth. The fact that the dentine walls are so thin probably explains why odontoblastic processes are not required. That they are still present in the unmineralized predentine matrix may be related to a possible role in organic matrix deposition rather than a trophic (sensitive?) role. A similar explanation may be invoked for the absence of capillaries in the small pulp cavity. Orthodentine is the main type of dentine found in adult vertebrate teeth. The matrix of orthodentine is composed of a thick layer of collagen fibrils parallel to one another and delimited into bundles by thin radially oriented collagen bundles. In addition, during growth the odontoblast processes are surrounded by organic matrix and persist in the dentine to become embedded in the typical dentine tubules. Both the organization in bundles and the dentine tubules lack the generalized Type 1 larval teeth. We nevertheless consider the atubular dentine in the larval teeth as an odontogenic precursor of orthodentine, as discussed previously (Huysseune and Sire, 1997b). Interestingly, the vasodentine described in the adult gadid Urophycis tenuis is atubular (Herold, 1970). The vascular canals located in the dentine matrix render the dentine tubules unnecessary. This could be viewed as indirect evidence of a nutritive role of dentine tubules. In the small larval teeth of the sparid Pagrus major, the dentine has been called “homogeneous dentine” because of the absence of cell processes (Sasaki et al., 1982). Notwithstanding the general trends described above, there are some variations which merit further discussion. They concern the absence of a general mode of tooth attachment, the presence of capillaries reported in the first-generation teeth of carp, and the presence of matrix vesicles in the urodele teeth. First-generation teeth attach in various ways to the supporting bone (ankylosed, hinged, ligamentously connected). Fink (1981) has reviewed these attachment types from a phylogenetic perspective. Both primary and secondary attachment can be found for first-generation type; which type depends on the extent to which the supporting bone has grown at the moment the teeth attach. When the growth of the bone keeps pace with the development of teeth at its edge, primary attachment is found. When the tooth develops in a position where bone is already present, a reversal line marks the limit of attachment bone and supporting bone and the attachment is called secondary. We therefore postulate that the way of attachment is independent of the type of the first-formed teeth. Capillaries were found in the pulp cavity in one case: the carp. Studies dealing with species having both an oral and a pharyngeal dentition may reveal whether this feature is particular to pharyngeal teeth and/or whether the difference with zebrafish (where no capillaries are found) relates to the slightly larger size of its teeth. The anabantoid (gouramy) fish Colisa lalia (a perciform) has no teeth in the oral cavity but possesses pharyngeal teeth. In the early larvae well-formed teeth

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Fig. 16.

FIRST-GENERATION TEETH IN VERTEBRATES

are present in the pharyngeal region (upper and lower pharyngeal jaws). They resemble the cyprinid pharyngeal teeth in that they have a large pulp cavity, which contains only a few cells in functional teeth. The teeth are attached on the perichondral bone. However, no capillaries are present (JYS, pers. obs.). In the beloniform Belone vulgaris the pharyngeal teeth are also larger than the oral teeth and possess a wide pulp cavity (Moy-Thomas, 1934). The appearance of 30 – 80 nm diameter matrix vesicles containing electron-dense substances has been reported during enameloid and dentine formation in adult teleosts (thus in large teeth): in Anguilla anguilla by Shellis and Miles (1976), in the medaka Oryzias latipes by Yamada and Ozawa (1978), in a sparid by Sasaki et al. (1982), in a salmonid by Sasagawa and Igarashi (1985), in a cichlid, the carp, and a labrid by Sasagawa (1988). The matrix vesicles are produced by budding off from odontoblast processes and they may play an important role in initiating mineralization at the junction between the enameloid and dentine, but not elsewhere (Sasagawa, 1988). Matrix vesicles have been reported in the small teeth of sparid larvae by Sasaki et al. (1982) but Prostak and Skobe (1986a) did not find such vesicles in the forming enameloid in juvenile cichlids (although they were found in a cichlid by Sasagawa, 1988). From the comparison of the first-generation teeth in the main lineages of nonmammalian vertebrates we cannot deny the fact that the teeth which are grouped in Type 1 develop in species with a short embryonic period (thus yielding small larvae), whereas the teeth which belong to the other type occur in species with a long embryonic period. We therefore hypothesize that an extended embryonic development leads to the sup-

Fig. 16. Crocodylus niloticus. a,c: 34-day-old embryo; b: 28 days; d: 40 days; e: 70 days; f– h: 50 days. Transverse sections of the left lower jaw (undecalcified). a–f: 1 ␮m-thick sections; g– h: thin sections. a: A tooth is differentiating and enamel and dentine matrix are deposited. This tooth is linked to a young replacement tooth by means of the dental lamina. In the replacement tooth, the epithelial cells have differentiated and the dental organ has folded around a loose population of mesenchymal cells, which form the dental papilla. b: Dentine matrix is mineralized and predentine is deposited centripetally by the odontoblasts. The pulp cavity is 10 –15 cells wide and entirely occupied by densely packed mesenchymal cells. Polarized ameloblasts are depositing the first layer of enamel matrix (arrowhead). A dental lamina, connected to the surface of the dental organ, extends deep into the mesenchyme. c: The dental organ is well differentiated and surrounds a pulp cavity, in which odontoblasts have differentiated and deposited the first elements of the predentine matrix. Some small blood vessels have entered the pulp cavity. d: The pulp cavity is well delimited and the odontoblasts located in its upper region synthesize the predentine. The outer layer of predentine has mineralized to give dentine. Enamel matrix (arrowhead) has been deposited by the ameloblasts. The base of the tooth lies close to the dentary bone surface. e: The dentine layer has thickened and predentine is still deposited towards the base of the tooth. Both the dentine and the deeper region of the enamel are well mineralized, while the outer region of the enamel matrix is only slightly mineralized. The tooth is not yet attached to the dentary bone, which can be seen on both sides of the tooth. f: Upper region of a well-formed tooth at the start of enamel matrix maturation. g: Detail of the enamel matrix. The crystallites are perpendicular to the tooth surface. h: Detail of the dentine and predentine showing the presence of numerous cell processes embedded in the matrix. Scale bars: a– e ⫽ 100 ␮m; f ⫽ 10 ␮m; g ⫽ 1 ␮m; h ⫽ 5 ␮m.

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pression of tooth generation in the development of dentition. These may still be present in the form of rudimentary germs during the embryonic period. Strikingly, such rudimentary teeth are present in the three species considered here to belong to Type 2 (in sharks, Reif, 1976; in lizards, Osborn, 1971; in crocodiles, Westergaard and Ferguson, 1986). From this perspective it would be interesting to study the timing of appearance, shape, and structure of abortive teeth in these species. A lineage for which data are obviously missing are the coelacanths. In the adult coelacanth, Latimeria chalumnae, the teeth are composed of a cone of orthodentine covered by true enamel. Towards the tooth base the dentine merges into the attachment bone, which itself is fused to the bone support (Miller and Hobdell, 1968; Miller, 1969; Grady, 1970b; Castanet et al., 1975; Shellis and Poole, 1978; Smith, 1978, 1979). To our knowledge, larval and adult tooth development has not been studied. However, L. chalumnae is ovoviviparous (Smith et al., 1975). The old embryos resemble miniature adults and they are more than 30 cm TL, i.e., 20% of the adult length. The first-generation teeth are likely to be large and their structure is predicted to be close to that of the adult teeth, as in lizards and crocodiles. To further test this hypothesis on a linkage between embryo retention, the absence of a generalized Type 1 larval teeth and the presence of rudimentary teeth, it would be equally useful to compare the first-generation teeth in an actinopterygian lineage possessing taxa with both short (oviparous) and long (ovoviviparous) embryonic periods (e.g., cyprinodontids). Conversely, it would be interesting to know at what stage in ontogeny species with Type 1 convert their generalized first tooth generation into miniature adult teeth. In principle, such changes should be monitored by following teeth of successive tooth generations in a single position in the dentition. Wautier et al. (unpubl. obs.) found evidence for a sudden early switch from larval to miniature adult tooth shape in Danio rerio. Shape changes have also been demonstrated in the course of ontogeny for urodele amphibians (Beneski and Larsen, 1989; Clemen and Greven, 1994). What is the evolutionary significance of the generalized type described as Type 1? If we consider the distribution of the vertebrate lineages having a short embryonic period, this character is a plesiomorphy for vertebrates (hagfish and lampreys have short embryonic periods). We therefore propose that Type 1 firstgeneration teeth represent an ancestral character for gnathostomes. In our view, this condition has been conserved for over 450 MY because it is a very economic and efficient way of building small and simple teeth adapted to the role of food acquisition in the majority of larvae. This does not preclude the ancestor of gnathostomes to have had a highly adapted adult dentition. How, then, is it possible that a wide range of vertebrates can start with such a generalized tooth type, yet achieve during their ontogeny the complex and heritable shape that characterizes so many lineages? Clearly, ontogenetic changes of tooth shape can only be achieved through the existence of polyphyodonty. They require that the molecular control regulating the development of generalized Type 1 larval teeth is adjusted to produce more complex organs, both from a morphological and structural viewpoint, whereas the

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machinery is present all the time. We propose that shifts in the timing, duration, and/or spatial extent of expression of regulatory genes are responsible for the shifts in shape and structure of teeth during successive tooth generations, but that constraints limit the outcome in successive generations. A detailed study to reveal the timing of changes in tooth shape and structure, and to what developmental events these changes are linked, is strongly recommended if we wish to further unravel the evolutionary significance of the generalized Type 1 larval teeth in nonmammalian vertebrates. ACKNOWLEDGMENTS We thank Miss Franc¸ oise Allizard for excellent technical assistance; Prof. M.M. Smith (Guy’s Hospital, London) for the loan of Neoceratodus forsteri sections, Prof. Jacques Castanet (Univ. Paris 6) for the gift of embryos and juveniles of Chalcides sexlineatus, Dr. Rene´ Galzin (EPHE Moorea) and M. Yan Laurent (SPE Rangiroa) for valuable help in the collection of larval material (especially of Scarus sp.) in French Polynesia, Dr. Michel Hignette (Aquarium du Muse´ e des Arts Africains et Oce´ aniens, Paris) for the gift of frozen larvae of Osteoglossum bicirrhosum, M. Samuel Iglesias (MNHN, Concarneau, France) for the gift of egg capsules of Scyliorhinus canicula, Dr. Eckhard Witten (Zoological Institute and Zoological Museum, University of Hamburg) for the gift of Anguilla australis and Gadus morhua larvae, and M. Luc Fougeirol (Ferme aux crocodiles, Pierrelatte, France) for the gift of growth series of Crocodylus niloticus embryos. TEM work was carried out at the “Service de Microscopie Electronique de l’IFR de Biologie Inte´ grative – CNRS – Paris VI.” REFERENCES Beneski JT, Larsen JH. 1989. Ontogenetic alterations in the gross tooth morphology of Dicamptodon and Rhyacotriton (Amphibia, Urodela, and Dicamptodontidae). J Morphol 199:165–174. Berkovitz BKB. 1975. Observations on tooth replacement in piranhas (Characidae). Arch Oral Biol 20:53–56. Berkovitz BKB. 1978. Tooth ontogeny in the upper jaw and tongue of the rainbow trout (Salmo gairdneri). J Biol Buccale 6:205–215. Berkovitz BKB. 2000. Non-mammalian tooth replacement. In: Teaford MF, Smith MM, Ferguson MWJ, editors. Development, function and evolution of teeth. Cambridge, UK: Cambridge University Press. p 186 –200. Berkovitz BKB, Shellis RP. 1978. A longitudinal study of tooth succession in piranhas (Pisces: Characidae), with an analysis of the replacement cycle. J Zool Lond 184:545–561. Berkovitz BKB, Sloan P. 1979. Attachment tissues of the teeth in Caiman slerops (Crocodilia). J Zool Lond 187:179 –194. Bertmar G. 1966. The development of skeleton, blood vessels and nerves in the dipnoan snout with a discussion on the homology of the dipnoan posterior nostrils. Acta Zool 47:82–150. Bourdelle E, Grasse´ PP. 1955. Ordre des Ce´ tace´ s. In: Grasse´ PP, editor. Traite´ de zoologie, vol. 17(I). Paris: Masson. p 341– 450. Castanet J, Meunier FJ, Bergot C, Franc¸ ois Y, Francillon H, Tuan Phong DN, Ricqle`s Ade. 1975. Donne´ es pre´ liminaires sur les structures histologiques du squelette de Latimeria chalumnae. In: Proble`mes actuels de pale´ ontologie-e´ volution des Verte´ bre´ s. Coll Int CNRS 218:160 –167. Chibon P, Ricqle`s Ade. 1995. Dents et tissus dentaires des adultes. In: Delsol M, editor. Traite´ de zoologie. Amphibiens. Vol. 14(I-A). Paris: Masson. p 167–187. Clemen G, Greven H. 1994. The buccal cavity of larval and metamorphosed Salamandra salamandra: structural and developmental aspects. Mertensiella 4:83–109. Cooper JS. 1966. Tooth replacement in the slow worm (Anguis fragilis). J Zool Lond 150:235–248.

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