Structure and development of first-generation teeth in the cichlid

formation, first-generation teeth show peculiarities compared with later tooth ... 1986b; Sasagawa, 1988) or collar enameloid formation ..... lysosomes (Fig. 12).
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Tissue & Cell, 1997 29 (6) 679-697 O 1997 Harcourt Brace & Co Ltd

Structure and development of first-generation teeth in the cichlid Hemichromis bimaculatus ('l'eleostei, Cichlidae) A. Huysseune 1, J.-Y. Sire 2

Abstract. In order to build a reference system to assess results of ongoing in vitro experiments on the study of epithelial-mesenchymal interactions during odontogenesis in actinopterygians, we have chosen to study the firstgeneration teeth of the cichlid Hemichromis bimaculatus from initiation until attachment both at the light and transmission electron microscopical level. Although their development follows the general pattern of teleost tooth formation, first-generation teeth show peculiarities compared with later tooth generations, including their size, bare emergence from the epithelium, absence of dentinal tubules and of nerves and capillaries in the pulp cavity, and organization of the outer dental epithelium. Four developmental stages (a to d) prior to attachment (stage e) have been distinguished. The oral epithelium invaginates into the underlying mesenchyme (stage a) and is later folded to form a bell-shaped dental organ (stage b) without any primordial thickening, or any other morphological indication of imminent invagination. Then, the collagenous enameloid matrix is laid down, most probably by the odontoblasts (early stage c), soon followed by predentine deposition and the beginning of enameloid mineralization (late stage c). With ongoing dentinogenesis, the enameloid matrix matures (stage d), i.e. the organic constituents are removed and the matrix further mineralizes. Finally (stage e), an annular collar of attachment bone is deposited to fix the tooth onto the underlying bone.

Keywords: Teeth, TEM, Cichlidae, Teleostei

Introduction Teeth are composed of a combination of mesenchymederived dentine and epithelium-derived enamel or enamellike substance, surrounding a pulp cavity. They are present in most classes of vertebrates. The structure and development of

'lnstituut voor Dierkunde, Universiteit Gent, BeLgium. 2URA CNRS 1137, Universit~ Paris 7, France. Received 17 March 1997 Accepted 29 July 1997 Correspondence to: Ann Huysseune, Instituut voor Dierkunde, Universiteit Gent, Ledeganckstraat 36, B-9O00 Gent, BeLgium.Tel: 32 9 2645229; Fax: 32 9 2645344; E-mail: [email protected]

teeth are particularly well known in mammals where they have long served as a model to study epithelialmesenchymal interactions (see reviews in Thesleff et al., 1995a, b). Mammalian teeth, however, are specialized compared with ancestral teeth and have many derived characters (e.g. reduction in number, heterodonty, diphyodonty). In order to reach a broader understanding of these interactions from the standpoint of comparative evolution, it would be useful to study them in a vertebrate where the teeth are numerous, of simple shape and undergo replacement throughout life (polyphyodonty). Such a condition is found in many actinopterygian fishes. To study the epithelial-mesenchymal interactions underlying odontogenesis in actinopterygians, our laboratories 679

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have engaged in in vitro experimental studies, focusing on the problem of pattern generation and the mechanism of tooth initiation. To build up a reference system to assess the results of such experiments both on larval and juvenile dentitions, a standard description of tooth development was required, of both first-generation and replacement teeth. Actinopterygian teeth have already been the subject of numerous studies, yet important gaps remain. Firstly, studies that have concentrated on structure and/or development have mostly focused on replacement teeth in large juveniles or in adult specimens (Herold, 1974; Shellis & Miles, 1976; Sasagawa, 1988, 1993, 1995a, b; Sasagawa & Ishiyama, 1988; Sasagawa & Ferguson, 1990). Yet replacement teeth differ in a number of aspects from firstgeneration teeth, notably in size of tooth germ, epithelial connectivity, relationship to the skeletal support, and some major morphological features (Huysseune & Sire. personal observations). In addition, most works deal with very specific aspects of tooth development, e.g. enameloid formation (Shellis & Miles, 1976; Sasagawa, 1984, 1995a, b; Prostak & Skobe, 1986a; Prostak et al., 1993), mineralization (Sasagawa & Igarashi, 1985; Prostak & Skobe, 1986b; Sasagawa, 1988) or collar enameloid formation (Sasagawa & Ishiyama, 1988). To our knowledge, few papers have covered the entire development of tooth germs, from initiation to attachment, in one species, while covering both light microscopic and ultrastructural aspects. A description of this kind has recently been made for callichthyid teeth (Huysseune & Sire, 1997) and odontodes located on the dermal scutes (Sire & Huysseune, 1995). However, it was concluded that many of the features related to the callichthyid dentition show a regression with respect to the primitive state and that this family possibly lacks true polyphyodonty. Consequently, these teeth were not representative of teleost, let alone actinopterygian teeth. For our purposes, Hemichromis bimaculatus has been selected as one of the experimental species. This cichlid, belonging to the highly evolved order of the Perciformes, has been a model in our laboratories for many years. Many studies have been devoted to the unravelling of the structure, development and evolutionary history of its dermal skeleton (e.g., Sire & Meunier, 1981; Sire, 1982; Sire & G6raudie, 1983, 1984; Sire & Huysseune, 1993). As part of these studies, we have investigated the appearance of the mandibular dentition (Huysseune, 1990) and the relationship of developing mandibular tooth germs to their skeletal support (Huysseune & Sire, 1992). Moreover, cichlids have already been used on several occasions in studies dealing with certain restricted aspects of tooth development in teleosts (Huysseune, 1983; Prostak & Skobe, 1985, 1986a, b; Prostak et al., 1989; Sasagawa, 1988, 1993, 1995a, b; Tuisku & Hildebrand, 1994). Hemichromis bimaculatus, like other cichlids, possesses teeth on four skeletal elements: the paired dentary bones (the anterior bones of the lower jaw, or mandible; see Huysseune, 1990), the paired premaxillary bones (teeth on dentaries and premaxillaries being labelled as oral teeth),

the paired upper and single lower pharyngeal jaws (i.e. pharyngeal teeth) (for a description of tooth-bearing skeletal elements in cichlids, see Barel et al., 1977, and Ismail, 1979). The number, as well as the shape, of the teeth shows considerable variation among cichlids. In adult H. bimaculatus, dentaries and premaxillaries bear a complete outer and an incomplete inner row composed of unicuspid teeth. In the pharyngeal jaws, both size and shape vary considerably according to the location; teeth are mainly bicuspid (Huysseune, personal observations). The tooth pattern on the dentaries of H. bimaculatus in early stages has been described in earlier papers (Huysseune, 1990; Huysseune & Sire, 1992) and has been used as a guide to trace the various developmental stages of tooth germs in this study. Briefly, dentary teeth start to form simultaneously with the opening of the mouth at approximately 2 days post-hatching. They develop in a lateral position slightly posterior to the symphysis of Meckel's cartilage. They continue to arise in a row (up to approximately 20 teeth in specimens about 1 month old), the more anterior germs lying lateral and the more posterior ones dorsal to the cartilage. In young postembryonic stages, at about 11 days post-hatching or slightly before, some germs appear to induce resorption of Meckel's cartilage, which subsequently disappears from the anterior part of the mandible. The first replacement teeth develop more than 1 month later. Because of the great structural discrepancies between larval and juvenile or adult teeth (Huysseune & Sire, personal observations), the description will be divided into two parts. This paper describes the development of first-generation teeth in the cichlid Hemichromis bimaculatus, both at a light and transmission electron microscopic (TEM) level. In a subsequent paper, the development of replacement teeth will be described. The present paper will mainly deal with the oral dentition. Whenever relevant, reference will be made to the pharyngeal dentition.

Materials and methods Materials For the description given below, we have chosen 5- and 6-day-old laboratory-reared specimens (approximately 4.2 to 4.5 mm standard length, SL; cf. Barel et al., 1977) since these possess tooth germs in various stages of development. Developmental stages are expressed as number of days after hatching (the embryonic period at 25°C lasts for 3 days). At 5 to 6 days, Meckel's cartilage in the dentigerous area is surrounded by a perichondral bone (the mentomeckelian bone) which is continuous with the parachondral, dermal dentary bone (Huysseune, 1990). Methods Animals were sacrificed by means of an overdose of the anaesthetic MS-222 (Sandoz, Basel, Switzerland) and processed for light and TEM observations as described previously. Specimens were fixed for 2 h, at room temperature, in

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a mixture containing 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4). Some specimens were decalcified for 7 days at 4°C in the same fixative with 0.1 M EDTA added. After rinsing for 1 night at 4°C in the same buffer, to which 10% sucrose was added, specimens were postfixed in 1% osmium tetroxide, in cacodylate buffer containing 8% sucrose and rinsed again. They were next dehydrated using a graded series of ethanol and then embedded in epon. Several specimens were processed into uninterrupted series of semithin (1 or 2 gm thick) sections cut transversely relative to the body axis, which were then stained with toluidine blue; in other specimens, the series were interrupted for ultrathin sectioning. Thin sections were contrasted with uranyl acetate and lead citrate and viewed in a Philips 201 transmission electron microscope operating at 80 kV.

Results Development and fine structure Before starting the detailed description, it may be useful to draw attention to a few problems related to the description of the first-generation teeth. Firs@, the initial teeth are very small (0.5% of the size of adult teeth); secondly, their development proceeds quickly (2 days from initiation until attachment); thirdly, the orientation of the germs can vary; and fourthly, the tooth germ is bent in such a way that it is impossible to visualize different regions of the germ on the same section. Given these difficulties it is sometimes hard to illustrate a specific region in a particular tooth germ simultaneously at the light and the electron microscopic level. Therefore, the subsequent description was only possible through the compilation of observations on many series of serial sections. Four stages of tooth development prior to attachment will be distinguished. They have been termed (a) to (d) in Huysseune (1983) and were inspired from the stages distinguished by Shellis & Miles (1976) in the eel. The drawings in Figure 1 present these four stages followed by a final stage in which attachment bone is deposited. It should be stressed that the process described below is continuous, and distinction into several stages is artificial. Indeed, whereas the distinction between stages (b) and (c) is clearcut (start of matrix deposition), the distinction between (c) and (d) is much more arbitrary. Furthermore, the tip forms earlier than the base of the teeth because their development is centripetal. Stage (a): initial tooth germ. This stage covers the development of the tooth germ from initiation up to a distinct epithelial invagination. At initiation, no epithelial invagination is yet visible. By the end of this stage, the aboral epithelial-mesenchymal junction in turn invaginates the epithelial mass of cells (covering bud stage in mammalian tooth formation) (Fig. 1a). Prior to the formation of the bud, the buccal epithelium in the dentigerous region can vary in thickness, mainly

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because of the presence of taste buds. No obvious morphological indications announce the subsequent development of a tooth germ until the buccal epithelium thickens locally at the presumptive site of tooth formation (Fig. 2, inset). At this level the buccal epithelium consists of about four layers of closely adjoined cells. The superficial cells are flattened and frequently interdigitate whereas the epithelial basal cells are plump and have a large nucleus with dispersed chromatin (Fig. 2). The cytoplasm of the latter cells mainly contains numerous free ribosomes and a few mitochondria. The basement membrane is fairly smooth. Below, the mesenchymal cells are not conspicuously aggregated. They are separated from the lamina densa of the basement membrane by an approximately 500-nm wide space which is filled with a loose network of thin fibrils (Fig. 2). The mesenchymal cells are separated by narrow intercellular spaces nearly devoid of extracellular matrix. These cells have an irregular outline with many cytoplasmic prolongations penetrating the intercellular spaces, fairly large nuclei with peripheral condensations of chromatin, and moderate amounts of organelles: mitochondria, cisternae of the rough endoplasmic reticulum (RER), and free ribosomes. Depending on the position, the tooth anlage can either lack any support (anterior dentary tooth germs), or develop close to bone (posterior dentary and premaxillary tooth germs). In a more advanced stage, the small epithelial invagination has grown into a considerable mass of epithelial cells which extends into the dermis and comes close to the dermal-epidermal junction at the opposite side of the jaw (Fig. 3, inset). The germ extends for nearly 20 gm in an anteroposterior direction and is about 35 gm high in its centre. On the dentaries, the connection of the germ with the epithelium (called the epithelial strand) is localized in the anterior part of the germ. Further development will take place in the centre. Thus, in further stages, deposition of the tooth matrix will take place posterior to where the epithelial connection is found. At the ultrastructural level, the dental epithelium (also called dental organ) is adjoined at its basis by an already well-delimited population of dermal cells, called dental papilla (Fig. 3). The two cell populations are clearly separated by a basement membrane. The dental organ is composed of a mass of epithelial cells (approximately 15 to 20 cells on one section) linked to the buccal epithelium by a short (one or two cells thick) strand of slightly more electron-dense epithelial cells. Cells of the dental organ are closely juxtaposed and are joined by sparse and short desmosomes. These cells show large nuclei with some patches of condensed chromatin, cisternae of the rough endoplasmic reticulum, mitochondria and free ribosomes (Fig. 3). Cells facing the dental papilla proper have a smooth basal surface and show a slight polarization perpendicular to the epithelial-mesenchymal boundary, whereas more superficial cells are organized more or less in two layers perpendicular to the former cells. The dental papilla cells, through their compact organization, are distinctly separated as a whole from the surrounding dermal cells by a narrow but obvious extracellular space (Fig. 3, inset). The

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ep

ep

(a)

(b)

ep

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late (c)

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Fig. 1

Diagrammatic representation of the stages of development of first-generation teeth on the mandible (dentary) in the cichlid Hemichromis bimaculatus. (a) Stage a, initial tooth germ. (b) Stage b, bell-shaped tooth germ. (e) Enameloid deposition (early) and mineralization (late) stage c. (d) Stage d, enameloid maturation and dentine deposition. (e) stage e, attachment bone deposition, ab, attachment bone; am, ameloblast, de. dentine; do, dental organ; dp, dental papilla; ds, dental sac; en, enameloid; ep, buccal epithelium; ide, inner dental epithelium; od, odontoblast: ode, outer dental epithelium; pd, predentine.

dental papilla cells are organized in two populations not yet separated by a sharp boundary; one forms the dental papilla proper, immediately facing the polarized epithelial cells, and the other forms a dental sac surrounding the dental organ (Fig. 3). On one section, there are about 10 more or less flattened cells constituting the dental papilla proper. They are organized in approximately three half-circles around a central cell. Their cytoplasmic content hardly differs from that of the epithelial cells. However, the cells facing the dental organ show an elaborate network of cytoplasmic prolongations filling the space between the dental papilla and the basement membrane (Fig. 4). Some of these prolongations are seen to contact the basement membrane.

Stage (b): bell-shaped tooth germ. This stage refers to the period from the beginning of re-invagination of the dental epithelium to the formation of a bell-shaped germ prior to the deposition of any dental matrix (covering cap and early bell stage in mammalian tooth formation) (Fig. lb). When the aboral end of the dental organ has folded into a bell shape, it consists of approximately two layers surrounding a well-organized dental papilla. The proximal margin of the dental organ (i.e. the side of the future tooth base, opposite to the future tooth tip) constitutes the cervical loop (Fig. 5, inset). No distinction can yet be made in the dental organ between an inner and an outer dental epithelium, and the number of cells has hardly increased. The cells facing the dental papilla are still polarized and show a similar stage of differentiation as in stage (a) (Fig. 5). The only noticeable difference is that the cells are tightly joined by fine interdigitations. As in the previous stage, the plasmalemma facing the dental papilla is smooth. The number of cells of the dental papilla proper, i.e., the future odontoblasts, has not markedly increased. One central cell is located deepest within the bell. It is surrounded by five or six ever-increasing semicircular layers of crescent-shaped cells, linked by scarce, short desmosomes. The central cell has a few cytoplasmic extensions, but it is rich in free ribosomes and possesses a large Golgi apparatus and numerous small, rounded vesicles (Fig. 6). The cells of the semicircular layers below have a centrally placed nucleus and tapering ends, but they still show the same organelles as in stage (a) (Fig. 5). Some of the cytoplasmic prolongations which occupy the space between the dental papilla and the dental organ are seen to contact the basement membrane (Figs 5 and 6). Stage (c): enameloid deposition. This stage starts with the deposition of the first enameloid matrix, followed by the

first predentine deposition, and ends at the beginning of matrix mineralization (Fig. lc). Concomitant with a deeper ingrowth of the invagination, the long axis of the tooth germ becomes deflected. Therefore, the events leading to enameloid matrix deposition take place at a slightly more anterior level compared to the location of the cervical loop tip. The description given below records the changes that take place at the apex of the dental papilla, where the first matrix becomes deposited. At the level of the tooth apex the dental organ is still seen to be linked to the buccal epithelium by a one-cell wide connection (Fig. 7, inset). The number of cells has increased in the dental organ, which is now well organized and can be separated into an inner (ide) and an outer (ode) dental epithelium (Fig. 7). Cells of the ide, from now on called ameloblasts, are slightly cylindrical, with their long axis perpendicular to the tooth matrix. In contrast, cells of the ode are rectangular, with their long axis parallel to the contour of the germ. In both layers, cell nuclei possess small patches of condensed chromatin. Mitotic figures can be observed in the ode only. The nearer the ameloblasts are to the tooth apex, the more they are differentiated, i.e. have their nucleus at the basal part of the cells and the organelles predominantly in the more distal cytoplasm close to the first deposited tooth matrix. Near the tooth tip the ameloblasts show moderate amounts of RER cisternae and mitochondria, but a well developed Golgi apparatus, numerous vesicles and free ribosomes (Fig. 8). The ameloblasts are closely adjoined without showing important interdigitations. The plasmalemma facing the tooth matrix is smooth except at the tooth tip where it has some folds. Over its entire surface, the tooth matrix is separated from the ameloblasts by a thin, 60-nm wide basement membrane. The matrix proper, i.e. the enameloid, is non-mineralized and composed of a loosely arranged network of typically striated collagen fibrils 20 to 30 nm in diameter, free of interfibrillar background substance. At the periphery, the fibrils are arranged predominantly parallel to the tooth axis whereas the more centrally placed fibrils are oriented perpendicularly (Fig. 8). Cytoplasmic prolongations derived from the most apical odontoblasts of the dental papilla penetrate the enameloid matrix. The dental papilla cells are still organized into concentric layers facing the dental organ. The dental sac consists of one layer of flattened, dense cells lining the ode (Fig. 7). In a more advanced stage, major changes have occurred at the level of the ide, which presents an increasingly vacuolized aspect towards the tooth apex (Fig. 9, inset). Ultrastructural observations reveal that this vacuolized aspect is due to the appearance of large intercellular spaces, 1 to 3 gm in diameter, between adjacent ameloblasts, the

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Figs 2-25 Ultrastructural features of various stages of tooth development in the first oral dentition of Hemichromis bimaculatus (5 and 6 days posthatching). The germ is represented with its apex towards the top of the figure, irrespective of its orientation in situ. Light micrographs of the corresponding region are shown in the insets, all of which are presented at the same magnification (bars = 10 gm). Unless stated otherwise, specimens have been demineralized. Fig. 2 Undemineralized. Slightly thickened buccal epithelium (ep) near the premaxillary bone at a site of presumptive tooth formation (arrow on inset). Notice the difference in organization of the basal versus the superficial epithelial cell layers. Below, mesenchymal cells (me) are separated from the lamina densa of the basement membrane (bm) by a narrow but distinct space (arrowhead), occasionally crossed by a cellular prolongation. Bar = 2 gm. Fig. 3 Dentarytooth germ in stage (a) (inset). The germ is connected to the buccal epithelium (ep) via a thin strand (es). The dental papilla (dp) is clearly delimited from the dental organ (do); part of its cells envelop the latter as a dental sac (ds). Bar = 2 gm. Fig. 4 Higher magnification of the epithelial-mesenchymaljunction indicated by an asterisk in Figure 3. The dental papilla cells (dp) adjoining the centralmost cell show an elaborate labyrinth of finger-like projections (arrow), some of which contact the lamina densa of the basement membrane (bm). do, dental organ. Bar = 1 gm. Fig. 5 Undemineralized.Dentary tooth germ in stage (b) close to Meckel's cartilage (mc) (inset). The ultrastructural picture shows part of the dental organ (do) and the dental papilla (dp). There are no striking differences between the inner and outer dental epithelium. The cells formerly adjacent to the dental papilla cell nearest the centre have now partially enveloped the latter, and still present an elaborated network of prolongations (arrow) facing the basement membrane. The asterisk indicates the cervical loop tip. ep, buccal epithelium. Bar = 2 gm. Fig. 6 Undemineralized.High magnification of the apical part of the dental papilla (dp) of the germ presented in Figure 5 showing details of the organization of the distal cytoplasm of the future odontoblasts. The inner dental epithelium (ide) cells (= future ameloblasts) are interdigitated (arrows/. Bar= 1 gm.

plasmalemmae of which are still linked by specialized cellular contacts, mainly gap junctions (Figs 9 and 10). The cell membrane facing the enameloid matrix shows numerous folds about 0.5 g m deep. The distal region of the cytoplasm in these ruffle-ended ameloblasts is characterized by the virtual absence of organelles, and it contacts the enameloid matrix without an intervening basement membrane. The rest of the cytoplasm is rich in small, rounded, vesicles, 50 to 100 nm in diameter (Fig. 10). The enameloid matrix is more electron-dense than before, mainly because the number of collagen fibrils has increased (Figs 9 and 10). The latter are still 20 to 30 nm in diameter and arranged parallel to one another and to the long axis of the tooth. The enameloid matrix is not sharply delimited from the predentine which starts to be deposited below (Fig. 9). In the tooth region facing the ruffle-ended ameloblasts the structure of the collagen fibrils looks disorganized, many of them having lost their striated pattern. At the tooth base, predentine is now clearly being synthesized (Fig. 11, inset). Opposite this newly synthesized predentine the ameloblasts are slightly columnar and polarized, their nucleus is more centrally placed, and the organelles are dispersed throughout the cytoplasm (Fig. 11). These include fairly large amounts of RER, well-developed Golgi areas, and moderate numbers of mitochondria and lysosomes (Fig. 12). Many cisternae are dilated and contain fine granular material. The cytoplasm also contains numerous small vesicles, some of which fuse with the apical as well as the lateral cell membranes. The plasm a l e m m a e of adjacent ameloblasts do not present important digitations, yet slightly enlarged intercellular spaces can be found. The distal p l a s m a l e m m a is smooth and faces an electron-dense layer corresponding to the lamina densa of the basement membrane (Figs 11 and 12). Except at the level of the cervical loop, the ode is highly vacuolized (Figs 11 and 13). The extremely convoluted cytoplasm of the ode

cells mainly contains a large number of small vesicles, many of which fuse with the cell membrane (Fig. 13). The dental sac is clearly visible as a single layer of flattened fibroblast-like cells enveloping the entire germ (Fig. 11). The dental papilla is arranged as before in a pyramidal shape, with a central pillar of cells surrounded by crescentic layers. The predentine consists of a layer of collagen fibrils (approximately 80 to 100 nm in diameter), orientated parallel to the longitudinal axis of the tooth (Fig. 14). Towards the odontoblast surface, the diameter of the fibrils decreases to 20 nm. The odontoblasts are rich in RER cisternae, Golgi areas and small vesicles (Figs 14 and 15). These cells are interconnected by desmosomes and have cytoplasmic prolongations which cross the predentine and run up to the dense layer facing the ameloblasts. This dense layer, further on called the limiting layer, is less well defined and thicker than the former lamina densa to which it corresponds (up to 100 nm versus 30 nm). A similar fine granular matrix penetrates between the superficial collagen fibrils of the predentine (Fig. 14). Between the limiting layer and the apical p l a s m a l e m m a of the ameloblasts, where small vesicles fuse with the latter, a fine granular material appears to have been deposited. W h e n the tooth germ is cut transversely, it is clear that the fibrils are arranged in longitudinal bundles, separated by radially projecting odontoblast processes (Fig. 15). In a transitional stage towards stage (d), i.e. before the mineralization of the enameloid is completed, predentine has started its maturation into dentine (Fig. 16). Throughout the entire thickness of the dentine (1 to 2 gin), the collagen fibrils are embedded in an electron-dense background substance. This substance extends towards the surface of the odontoblast processes; the predentine is reduced (Fig. 16). The apical odontoblasts present obvious prolongations which no longer appear to penetrate into the dentine (Fig. 16).

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Fig. 7 Premaxillary tooth germ in stage (c) with its connection (arrow) to the buccal epithelium (inset). The events leading to the first matrix deposition at the apical part of the dental papilla can not be visualized simultaneously with the changes taking place more proximally. At the level of enameloid (en) formation, the connection with the buccal epithelium can still be observed (arrows). Note an increasing polarization of the ide cells (ameloblasts) and an increased flattening of the ode cells towards the tooth apex. Mitoses (arrowheads) are visible both in the ode and in the mesenchymal cells constituting the dental sac (ds). dp, dental papilla; od, odontoblast. Bar = 5 p.m. Fig. 8 Detail of newly deposited enameloid (en) matrix. Note the loose organization of the matrix, the presence of a basement membrane (arrow), and the smooth distal plasmalemma of the aineloblasts (am). Odontoblastic prolongations (arrowheads) penetrate deeply into the enameloid. Bar = 500 nm.

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Fig. 9 Advanced stage of enameloid (en) formation in a premaxillary tooth (inset). Note that the germ contacts the buccal epithelium without a connection being visible at this level. Compared to the previous stage (see Fig. 7), the ide has become highly vacuolized through the appearance of large intercellular spaces (asterisks). The distal membrane of the ameloblasts presents conspicuous infoldings (arrow). The enameloid matrix has become much more compact; a basement membrane can no longer be observed, od, odontoblast process; pd, predentine. Bar = 2 gm.

Fig. 10 Detail of the raffle-ended ameloblasts (am) at the apex of the tooth shown in Figure 9. The area of the distal cytoplasm is virtually devoid of organelles (asterisks). Arrows show gap junctions between adjacent ameloblasts. Note the vesicles (arrowheads) fusing with the lateral cell membrane and apparently secreting their content into the distended intercellular spaces. The collagen fibrils of the enameloid (en) matrix have a frayed aspect. Bar -- 500 nm.

Stage (d): enameloid maturation and further dentine deposition. This stage represents the final stage of tooth formation and ends immediately prior to the deposition of attachment bone. During this phase enameloid becomes completely mineralized and dentine deposition is completed (Fig. ld). At the light microscopic level, this stage is characterized by the near absence of any vacuolization in the dental organ (Fig. 17, inset). Only a few vacuoles are discernible below the tooth apex. Stage (d) is furthermore distinguishable by the presence of a hypermineralized cap and the fact that the tooth tip has not emerged yet from the surface of the buccal epithelium. TEM observations at the level of the tooth apex show that the large intercellular spaces between the ameloblasts, responsible for the vacuolized aspect of the ide, are no longer present (Fig. 17). The ameloblasts in this area are closely adjoined and present important interdigitations. The apical cytoplasm of the ameloblasts immediately facing the enameloid is relatively poor in organelles. The rest of the cytoplasm contains very little RER, mitochondria and Golgi areas, but appears to be enriched in microfilaments, free ribosomes and dense granules. The enameloid is well mineralized with densely packed crystals (Fig. 18). The limits between individual crystals are not discernible at the magnifications used. The overall orientation of the mineral is parallel to the tooth surface and corresponds to the collagen fibril orientation in the enameloid matrix described earlier (cf. Fig. 9), although the fibrils themselves can no longer be observed. The orientated mineralized matrix is separated from the ameloblasts by a homogeneous layer of about 50 nm in thickness consisting of amorphous electrondense material (Fig. 18). A little closer to the tooth base, the enameloid matrix presents fuzzy bands of a fine granular substance (Fig. 19). The enameloid is separated from the ameloblast surface by a 25 nm thick electron-dense membrane. Closer to the tooth base, this membrane diminishes in thickness to disappear completely (without the limiting layer described previously) where the dentine directly adjoins the ameloblasts in a region located above the apical part of the pulp cavity (Fig. 20; compare with Fig. 15). The ameloblasts in this region resemble those described for stage (c) (cf. Fig. 9), yet without presenting a prominent ruffled end. They show a reduced vacuolization (Figs 20 and 21), and are deeply interdigitated. The cytoplasm is especially rich in small vesicles, many of which fuse with the plasmalemma along the enlarged intercellular spaces. The dentine has increased

in thickness and is penetrated by long cytoplasmic prolongations from the odontoblast nearest the apex. In this area, the collagen matrix contains small, electron-lucent granulated dots (Fig. 21).

Stage (e): deposition of attachment bone. The final stage starts when the first elements of the attachment bone are deposited, and ends at the eruption of the tooth (Fig. le). On all three elements examined (dentaries, premaxillafies, and pharyngeal jaws), the same attachment mode is found. At the time oral teeth are ready to become attached, they have reached a height of approximately 30 ~tm and a diameter at the base of about 12 ~m. At this stage, matrix is deposited in the prolongation of the tooth base to form the annular collar of attachment bone (Fig. 22, inset). Along the tooth, the ameloblasts are deeply interdigitated, with no enlarged intercellular spaces. They contain numerous mitochondria, large amounts of free ribosomes, microfilaments, and small vesicles filled with electron-dense fine granular material (Figs 22 and 23). A 125 nm thick layer of granular substance lines the dentine along its interface with the ameloblasts. The dentine is composed of collagen fibrils diminishing in diameter from about 60 nm in the outer dentine layer to 25 nm towards the pulp cavity. A distinct predentine layer is absent except at the dentine-attachment bone interface (Fig. 22). Major changes have occurred at the tooth base. Cells of the dental papilla have given rise to one continuous, pear-shaped population containing both the cells of the pulp cavity (odontoblasts proper) and the cells enveloped by the attachment bone. Only the latter have retained indications of intense secretory activity: a welldeveloped RER and numerous mitochondria. These features have been lost in the odontoblast nearest the apex, the cytoplasm of which is much more electron-lucent and loaded with polysomes (Fig. 24). The outer surface of the attachment bone is covered with secretory cells probably derived from the dental sac. The limit between dentine and attachment bone is clearly defined as an electron-lucent strip and neatly related to two morphological features: on the outer side this limit corresponds to the tip of the cervical loop of the dental organ; on the inner side, the dental papilla cells send cytoplasmic prolongations towards the interface of both matrices (Figs 22 and 25). Between dentine and attachment bone, the matrix is loose with a few fine collagen fibrils that lack any particular organization. Often, cells have been observed to face the dentine and the attachment bone simultaneously. The matrices of the basal region of the

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Fig. 11 Section at the level of the cervical loop tip in stage (c) of a premaxillary tooth where predentine (arrow) is being deposited (inset): a connection with the buccal epithelium (ep) is situated more anteriorly. Because of the oblique orientation of the germ, the ameloblasts (am) are hit in the proximal part of the germ only: the majority of the cells in the dental organ belonging to the ode. Both can be distinguished through the highly convoluted aspect of the ode cells. The dental papilla (dp) has retained much the same general aspect as in stage (b) (compare insets of Figs 5 and 11). An asterisk indicates the predentine, ds, dental sac. Bar = 5 gm. Fig. 12 High magnification of the epithelial-mesenchymal boundary in Figure 11, showing portions of ameloblasts (am) facing the predentine (pd). Note the presence of intercellular spaces (asterisk) and vesicles fusing with the cell membranes (arrow). Cisternae of the RER are dilated (arrowheads). Bar = 500 nm. Fig. 13 Detail of an ode cell from Figure 11. Arrowheads indicate vesicles fusing with the cell membrane. The arrow points to the basement membrane separating the dental organ from the mesenchyme. Bar = 500 nm. Fig. 14 Detail of the predentine (pd) and of the odontoblasts (od) shown in Figure 11. An asterisk marks the electron-dense outer layer of the predentine (= limiting layer) corresponding to the former lamina densa of the basement membrane. Above it a fine granular material can be seen (arrow). A cytoplasmic prolongation (arrowhead) from an odontoblast reaches the limiting layer. Bar = 500 nm. Fig. 15

Odontoblast (od) prolongations (arrows) penetrate the recently deposited predentine (pd) and divide it into bundles. Bar = 500 nm.

Fig. 16 Detail of ameloblasts (am) and of odontoblasts (od) on both sides of the maturating dentine (de). The odontoblast process (arrows) do not appear to penetrate the dentine matrix. Bar = 500 nm.

dentine and of the attachment bone resemble each other (Fig. 25). The most striking differences are: (i) The predominant organization of the fibrils in the dentine is parallel to the longitudinal axis of the tooth, whereas the matrix of the attachment bone is wovenfibre& (ii) Fibril size in the dentine is larger except along the inside of the very base of the tooth (60 nm in the dentine versus 25 nm in the attachment bone). (iii) Most importantly the dentine is characterized by the presence, on its outer surface, of an electron-dense limiting layer representing the continuation of the lamina densa of the basement membrane. Such a limiting layer is absent along the attachment bone; in contrast its outer surface is lined with an electronlucent layer composed of fine fibrils, constituting an osteoid matrix. The very apex of the tooth reaches the surface of the buccal epithelium, but only pierces it when the attachment bone itself has fused to the underlying dentigerous bone, and by no more than a few micrometres. Even when attachment bone has been formed, the pulp cavity is filled with odontoblasts only. In these first-generation teeth, the pulp cavity has never been seen to contain any capillaries or nerve endings.

Discussion The present paper is one of the first that describes the development of first-generation teeth in a teleost, covering all stages of development from initiation until attachment, as well as characterizing these both at a light and transmission electron microscopical level. Much of the literature dealing with teleost tooth development refers to the dentition of juveniles or adults, and many of the differences that we find may relate to ontogenetic rather than to interspecific

differences. This has become clear when comparing the results reported here with those for juvenile and adult teeth in the same species (Huysseune & Sire, personal observations) or in other cichlids (Prostak & Skobe, 1985, 1986a, b; Sasagawa, 1988, 1993, 1995a, b; Prostak et al., 1989). Initiation of tooth germs The term 'initiation' has two meanings, both of which have been used in this paper: in morphological terms, it refers to the sites in the epithelium where new germs will appear; in developmental terms, it refers to the mechanisms responsible for the commitment of presumptive dental epithelium and dental mesenchyme to an odontogenic fate. Morphological evidence suggesting imminent tooth germ formation on the dentaries or premaxillaries in H. bimaculatus is very poor. Indeed, despite our knowledge of the pattern of tooth initiation on the dentaries of Hemichromis (Huysseune, 1990) and on the pharyngeal jaws in Astatotilapia elegans, another generalized cichlid (Huysseune, 1983, 1989), it has proven impossible to predict, both in time and in space, the exact spot of epithelial invagination. This is not simply due to the variation that exists between individuals. There are no distinct features in the presumptive dental epithelium that announce imminent epithelial invagination, such as enlargement or polarization of the basal epithelial layer. The first epithelial thickening is local and already represents a separate tooth anlage. However, this does not preclude the existence of odontogenic potential over a larger field in the epithelium. In the presumptive dental mesenchyme, evidence for a clear aggregation is delayed until a distinct epithelial invagination exists. This is in contrast to the situation in rainbow trout hatchlings (Salmo gairdneri), where the tooth-related part of the oral epithelium can be distinguished by the more elongated appearance of its cells and by a concentration of underlying mesenchymal cells (Berkovitz, 1978). Irrespective of whether odontogenic potential is along a continuous strip, or only at specific sites of the epithelium,

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Fig. 17 Undemineralized. Dentary tooth germ in stage (d) close to Meckel's cartilage (mc) (inset). The hypermineralized cap enameloid (en) is surrounded by interdigitated ameloblasts (am). do, dental organ: ep, buccal epithelium; mf, microfilaments. Bar = 500 nm. Fig. 18 Undemineralized. Detail of the hypermineralized enameloid (en) adjacent to that shown in Figure 17. The mineral crystals are close to one another and parallel to the ameloblast (am) surface. The transverse bands are probably artefacts. The arrow points at the layer of amorphous electron-dense material at the ameloblast-enameloid interface. Bar = 250 nm. Fig. 19 Undemineralized. Same tooth shown in its more proximal region. An electron-dense membrane (arrow) separates the enameloid (en) from the ameloblasts (am). The electron-lucent aspect of the enameloid is probably due to partial demineralization after sectioning. Bar = 250 nm.

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Fig. 20 Undemineralized. Same tooth but cut through the dentine region. The dentine (de) is surrounded by interdigitated ameloblasts (am) showing a slight vacuolization (star). ode, outer dental epithelium. Bar = 2 gin. Fig. 21 Detail of Figure 20 showing the dentine (de) with electron-lucent, granulated dots (arrowheads), possibly representing initial sites of mineralization. and the apical portions of interdigitated ameloblasts (am). The arrows point to vesicles fusing with the plasmalemma. The electron-dense membrane shown in Figure 19 is no longer present at this level. Bar = 500 nm.

a true dental lamina (see Reif, 1982, 1984) does not develop. This is also true in trout larvae where tooth germs develop directly from the oral epithelium (Berkovitz, 1978). One could argue that the small strand that later ensures the connection of the epithelial mass to the superficial epithelium represents a dental lamina, since, as we will show in a subsequent paper, such a discontinuous and non-permanent dental lamina develops for the replacement teeth (Huysseune & Sire, personal observations). In developmental terms, much of the attention that is currently given to the problem of tooth initiation in mammals concentrates on the role of retinoids, genes encoding transcription factors (Msx-1, Lef-1) and growth factors (BMPs, FGFs), and their possible interplay in this process (see the reviews by Thesleff et al., 1995a, b, and Thesleff & Nieminen, 1996). Although the precise timing of tooth initiation in Hemichromis larvae is unknown, it occurs at a time when no cellular aggregation is visible in the mesenchyme. This suggests that an aggregation of committed, ectomesenchymal cells is not required to induce the overlying epithelium and that the signal(s) which is/are responsible for attributing odontogenic competence to the epithelium are probably different. It furthermore lends support to the hypothesis of epithelial primacy in tooth initiation (cf. Lumsden, 1988; Westergaard & Ferguson, 1990). A similar statement has recently been made for zebrafish scale initiation (Sire et al., 1997). Other elements possibly involved in tooth initiation are capillaries and nerve endings. Diametrically opposed to earlier reports claiming that innervation has no inductive role in initiating odontogenesis (e.g., Lumsden & Buchanan, 1986), Tuisku & Hildebrand (1994) showed that replacement tooth formation in the cichlid Tilapia mariae is inhibited after transection of the ramus alveolaris of the mandibular branch of the trigeminal nerve, the major nerve trunk that innervates the lower jaw (Holje et al., 1986). In their subsequent review (Hildebrand et al., 1995), they further elaborated on the role of innervation and in particular of neural cell adhesion molecules (N-CAM), and neurotrophins and their receptors. We have not yet mapped the fine terminal branches of the trigeminal innervation in the lower jaw in H. bimaculatus but presume that this is extremely difficult to perform even at the TEM level, considering the size of the jaws and the tooth germs. As to vascularization, capillaries may permit the transport of substances like hormones, growth factors etc. There is no doubt that the mandible is richly vascularized and that capillaries occur close to the sites of tooth initiation. However, so far we have not determined whether or not there is a precise spatial and temporal relationship between the occurrence of capillaries and the sites of tooth

initiation. Such an approach, similarly to a mapping of the innervation, would necessitate detailed 3D reconstructions, most likely at the TEM level. Therefore, it is clear that if we want to study the mechanisms that govern initiation in this particular fish species, we will have to study factors affecting the overall dental pattern rather than individual sites of tooth formation. The tooth germs of the first dental generation in Hemichromis develop in the dermis, external to the bone on each of the four elements that have been examined (dentaries, premaxillary bones, upper and lower pharyngeal jaws) (see also Huysseune, 1983, 1989; Huysseune & Sire, 1992). As will become clear in a forthcoming paper (Huysseune & Sire, personal observations), this situation changes for subsequent tooth generations: replacement teeth on each of these four elements develop in their medullary cavities. It is known that the position where teeth develop is variable among teleosts: external to the bone as in Salmo (Bergot, 1975b; Berkovitz, 1978), Lophius (Kerebel et al., 1979) and in some armoured catfish (Huysseune & Sire, 1997), or in bony crypts as in Serrasalmus (Berkovitz, 1975). It can even vary within a species, but to our knowledge, positions varying during ontogeny have never been reported. Ameloblast differentiation and enameloid formation The sequence of cellular events that characterizes the differentiation of ameloblasts from early dental epithelium cells can be summarized as follows. (1) The inner dental epithelium (ide) cells are only slightly polarized during stages (a) and (b) of tooth formation (Figs la and lb), but they become cylindrical and clearly polarized in the initial stage (c) of enameloid matrix deposition (Fig. lc); (2) During enameloid matrix deposition ameloblasts show features indicative of a moderate secretory activity, with moderate RER and mitochondria, a well developed Golgi apparatus, numerous vesicles, and free ribosomes. There are no important lateral interdigitations and the distal plasma membrane is smooth. (3) Very rapidly, the cells assume the features of mffieended ameloblasts, rich in small rounded vesicles, and separated from their neighbours by large intercellular spaces and gap junctions. (4) In stage (d), i.e. once dentine has been laid down and is being mineralized, the ameloblasts again present a smooth apical surface and intercellular spaces no longer persist, despite important interdigitations of the lateral cell membranes (Fig. ld).

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Fig. 22 Undemineralized. Dentary tooth in a stage of attachment bone formation (arrow on inset). The attachment bone (ab) is well formed on the left side of the figure only. Cells on the right belong to the dental organ (do) of an adjacent tooth germ. The limit between the tooth base and the attachment bone is clear (arrow), even at this low magnification. The arrowheads point to the cervical loop tips. An asterisk indicates the region shown in detail in Figure 23. am, ameloblasts; de, dentine; dp, dental papilla; ds, dental sac; ep, buccal epithelium; od, odontoblast; ode, outer dental epithelium. Bar = 2 gm. Fig. 23 Higher magnification of the dentine (de) and parts of the adjacent ameloblasts (am) shown in Figure 22. The black arrow points to the intervening granular layer, and the white arrow indicates a vesicle with an electron-dense, granular content. Bar = 500 nm.

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Fig. 24 Detail of the apicalmost odontoblast (od) and surrounding dentine (de) of Figure 22. Portions of ameloblasts (am) are visible on the left side of the figure. Bar = 500 nm. Fig. 25 Undemineralized. Junction between dentine (de) and attachment bone (ab) of the tooth in Figure 22. The arrowhead indicates cytoplasmic prolongations of dental papilla cells (dp) at the level of the dentine-attachment bone interface (arrow). am, ameloblast; bm, basement membrane; ob, osteoblast. Bar = 1 gin.

These cellular events take place in a few cells only and succeed each other very rapidly which undoubtedly is related to the small size of the teeth involved; the cellular characteristics of a particular stage therefore cannot be defined as easily as in adult cichlid teeth. Sasagawa (1995a, b) could distinguish three stages during enameloid matrix formation in adult Tilapia (early, middle and late stage), followed by enameloid mineralization and finally enameloid maturation. Ameloblasts showed secretory granules in the late stage of formation, changed from secretory to absorptive cells during the mineralization stage and became ruffle-ended during maturation. In first-generation teeth of Hemichromis only a brief secretory stage (phase (2)) and a somewhat more extended maturation stage (phase (3)) can be distinguished. Prostak & Skobe (1986a) studied enameloid matrix deposition in juvenile Cichlasoma and described columnar ameloblasts, 30 to 40 gm tall (i.e. the size of a functional first-generation tooth of Hemichromis), with elongated secretory granules containing cross-striated fibrillar material, resembling procollagen. We could not identify procollagen secretory granules in the ameloblasts, which suggests that the ide does not participate in the formation of the collagen matrix of the enameloid, although the cells are clearly engaged in the synthesis of products and their accumulation prior to their secretion. The secretory granules probably contain non-collagenous, as yet unidentified enameloid matrix proteins. Mammalian ameloblasts have traditionally been classified as presecretory, secretory and maturation stage ameloblasts, involved in respectively differentiation, production and release of enamel proteins, and resorption of matrix proteins and transportation of calcium ions (Nanci & Smith, 1992; Deutsch et al., 1995). During maturation stages, mammalian ameloblasts show modulation in that they repeatedly undergo ruffle-to-smooth-ended transitions, up to 3 times a day. The ameloblasts in first-generation teeth of Hemichromis do not appear to undergo different modulation cycles, although Prostak & Skobe (1986b) considered this possibility in Cichlasoma teeth. The hypermineralized substance that covers cichlid teeth, and supposedly all teleost teeth (Smith, 1978) has been identified as enameloid (Prostak & Skobe, 1985, 1986a, b; Sasagawa, 1988, 1993, 1995a, b; Prostak et al., 1989). The sequence of morphological features that characterizes the enameloid matrix during the formation of firstgeneration teeth in Hemichromis can be summarized as follows. (1) A loose woven-fibred matrix of small-sized collagen fibrils lacking any interfibrillar material; at this moment, the basal lamina is still present.

(2) Acquisition of a more electron-dense matrix due to accumulation of collagen, which is oriented predominantly in the longitudinal axis of the tooth. (3) Maturation of the enameloid, involving degradation of the fibrillar matrix (depolymerization of the collagen, probably through the action of proteinases) and mineralization; formation of the enameloid membrane. The basement membrane is present below the ameloblasts until these exhibit the apical ruffle-ended plasma membrane. This corresponds with observations by Sasagawa (1995a) on Tilapia tooth formation in which the basal lamina became gradually indistinct with the progress of mineralization at the surface layer. The presence of a basal lamina below the ameloblasts during deposition of the collagenous enameloid matrix, along with the lack of evidence for collagen secretion by ameloblasts, and the pronounced secretory aspect of the odontoblasts during enameloid formation, suggests that the bulk of the collagenous matrix of the enameloid is produced by the odontoblasts. This interpretation of enameloid formation conforms to the Shellis & Miles model (1974, 1976), elaborated by Schaeffer (1977), and further substantiated by Sasagawa (1995b). According to this model, the bulk of the enameloid matrix is synthesized by the odontoblasts; it reaches definitive size and shape before mineralization is initiated (centripetal growth) and it may contain dentinal tubules. According to the initial interpretation of Shellis & Miles (1974), the ide secretes a mammalian-like enamel matrix protein that interacts with the enameloid so that the matrix becomes labile during enameloid mineralization. Prostak & Skobe (1985, 1986a, b), on the other hand, regarded the collagen of the enameloid to be of epithelial origin, based on experiments with blocking agents and many TEM observations showing the resemblance of ameloblast secretory granules with procolIagen granules. The collagenous nature of the enameloid in first-generation teeth of Hemichromis conforms to what is generally known for actinopterygian enameloid (Shellis & Miles 1974, 1976; Sasagawa, 1984, 1988, 1993, 1995a, b; Prostak & Skobe, 1985, 1986a, b) and contrasts highly with chondrichthyan enameloid which has an essentially non-collagenous organic matrix consisting largely of tubular vesicles limited by a unit membrane (Prostak & Skobe, 1988; Risnes, 1990; Sasagawa 1993; Huysseune & Sire, in press). The enameloid membrane that we have observed is very similar to that found on the surface of the teeth (Huysseune & Sire, 1997) and of the odontodes (Sire & Huysseune, 1995) in callichthyid teleosts. At present, we do not know its composition, nor do we have an indication of its function.

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Possibly, the membrane may be involved in eruption. A superficial layer resembling this enameloid membrane has been described on various occasions and has usually, but not always (e.g. Chaetodon, Sparks et al., 1990), been interpreted in one of two ways: either as a superficial layer of ectodermal origin, or as a hypercalcified dentine giving rise to an enameloid layer (cf. Smith's (1978) interpretation of the cuticular layer in Serrasahnus, Shellis & Berkovitz, 1976). The former represents a primitive form of enamel (e.g., the superficial terminal membrane in Esox (Herold, 1974), Anguilla (Shellis & Miles, 1976), or Stephanolepis (Uehara & Miyoshi, 1987), all of which conform to the enamel cuticle reported for teleosts by Schmidt (1969). Smith (1978) refuted the existence of a superficial enamel layer in actinopterygians. Unfortunately, we lack images of early stages of mineralization of the enameloid, which again is no doubt because of the speed with which this process takes place, in relation to the small size of the teeth. In actinopterygians in general (Kerr, 1960; Yamashita & Ichijo, 1983) and in cichlids in particular (Prostak & Skobe, 1986b: Sasagawa, 1988; 1993, 1995b), enameloid mineralization in replacement teeth always appears to start at the dentine-enameloid junction (DEJ) and to proceed outwards to the surface. A slightly different picture is reported for replacement teeth in the Japanese parrotfish, Hoplognathus fasciatus, where enameloid mineralization takes place in 3 more or less successive steps: first at the DEJ, next at the enameloid surface, and finally in between (Isokawa et al., 1970; lnoue et al., 1973). It is clear from our observations that the features of the enameloid in the first-generation teeth are not as distinct as those described for adult cichlid teeth (Sasagawa, 1988, 1993, 1995a, b). The enameloid of first-generation teeth of Hemichromis resembles closely that covering Corydoras odontodes (Sire & Huysseune, 1995) and first-generation teeth in other catfish (Huysseune & Sire, 1997). This resemblance may well be linked to the small size of both odontodes and first-generation teeth (be it Corydoras or Hemichromis) and may represent a primitive character state. A notorious difference between first and later generation teeth concerns the organization of the outer dental epithelium (ode). Although the ode in first-generation teeth in Hemichromis presents a clearly labyrinthine organization at the stage of enameloid maturation, this aspect is not associated with an extensive vascularization of the surrounding mesenchyme, although it is in the replacement teeth (Huysseune & Sire, personal observations), just as in juvenile Cichlasoma replacement teeth (Prostak & Skobe, 1986b). Such a vascularization has been related to the high fluoride content in the enameloid of cichlid teeth (Prostak et al., 1993). Whether the absence in first-generation teeth is related to their fluoride content cannot be answered yet. However, it is clear that the open channels between the ode cells, as well as the folded invaginations of the apical cell membranes of the ide, reflect the involvement of the dental epithelium in rapid transport of ions and water (cf. Herold, 1974).

Sasagawa (1995b) described an intermediate layer between ide and ode in adult Tilapia teeth, though his figures showed it to be prominent at the tooth apex only. We have not been able to identify such intermediate cells in first-generation teeth of H. bimaculatus. Odontoblast differentiation and dentine formation

Our study has clearly demonstrated a dual fate for the mesenchymal condensation: on the one hand the cells contribute to a dental papilla, on the other hand a number of cells constitute a dental sac. The distinction is visible from early stages onwards. The dental papilla gives rise to the odontoblasts. The sequence of cellular changes that characterizes the odontoblasts in first-generation teeth can be summarized as follows. (1) During initial stages of differentiation, the odontoblasts have cellular contacts with the ameloblasts through prominent cellular prolongations. (2) At the onset of initial matrix deposition, i.e. the deposition of enameloid, the odontoblasts assume the aspect of strongly secretory cells. Contacts with the ameloblasts are lost. Cellular prolongations persist nevertheless in the first deposited enameloid. (3) At a given time, the matrix that is deposited assumes the characteristics of true dentine, with well oriented fibres of a larger size. Cellular prolongations are only found in the prolongation of the pulp cavity. (4) Finally, at the onset of attachment bone formation, the odontoblasts have lost the aspect of secretory cells. However, the cells of the dental papilla that form the continuation of this population in the prolongation of the tooth base show distinct features of secretory cells and are no doubt involved in the deposition of attachment bone (see below). These observations, and the lack of evidence for collagen secretion by the ameloblasts during initial stages of enameloid deposition, lead us to assume that odontoblasts are involved in the deposition of the enameloid. Considering the small size of the pulp cavity, and the low number of cells involved, we feel furthermore inclined to consider this population as consisting of only one cell type producing the enameloid and dentine in sequence, rather than of separate populations producing either enameloid or dentine. This view is furthermore reinforced by the frequent observation, in different taxa, of a single odontoblast apparently being involved in the production of different matrices (dentine, attachment bone and possibly even supporting bone) (Huysseune & Sire, 1997, and personal observations). Odontoblastic dimorphism is more likely to be the case in sharks where enameloid and dentine differ considerably (cf. Risnes, 1990). Throughout stages (b) to (d), the odontoblasts fill the entire pulp 'cavity'. This is in sharp contrast to the situation in replacement teeth and to what has been described or

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illustrated for other teleosts, where only the cells that face the ameloblasts, and later the cells that face the predentine, differentiate into odontoblasts (e.g., Kerr, 1960; Herold, 1974; Bergot, 1975b; Prostak & Skobe, 1986b; Sasagawa & Ishiyama, 1988; Sasagawa, 1993, 1995b). Clearly, this is linked to the size of the teeth, the pulp cavity being so narrow as to allow space for only a few cells. Similar observations were made in odontodes (Sire & Huysseune, 1995) and in teeth of callichthyid teleosts (Huysseune & Sire, 1997). The situation depicted here may be a common feature in larval dentitions (e.g. in the larval Belone, the teeth are 30 ~tm long and a single odontoblast occupies the pulp cavity; Moy-Thomas, 1934). Despite evidence for early odontoblastic prolongations, well formed dentine clearly lacks dentinal tubules. The absence of dentinal tubules stands in sharp contrast to the observations on replacement teeth, which do possess distinct dentinal tubules (Huysseune & Sire, personal observations). Therefore, the atubular dentine is an ontogenetic precursor of the tubule-containing dentine, i.e. orthodentine (see Orvig 1951, 1967; Peyer, 1968; Carlson, 1990). Earlier, we noticed the absence of tubules in larval teeth and in odontodes of armoured catfish (Sire & Huysseune, 1995; Huysseune & Sire, 1997) and suggested that the dentine lacks tubules because of the extreme thinness of the dentinal walls (2-5 gm thick), much as young cellular bone can lack osteocytes because of the minute thickness of the bone (Meunier, 1987; Huysseune, personal observations). The fact that other authors have reported similar observations (e.g., in salmon (Bergot, 1975a; Sasagawa & Igarashi, 1985), and in sea bream (Higashi et al., 1983)) may suggest that this character is far more common in teleosts than previously assumed. As with the lack of dentinal tubules, the absence of capillaries and nerves in the pulp cavity may be related to its small size, the pulp cavity hardly being large enough to contain one or a few odontoblasts. Part of the mesenchymal cell population constitutes the dental sac. A dental sac has not yet frequently been observed in teleosts. Its absence was noted in larval Pagrus pharyngeal teeth (Higashi et al., 1983), but its presence was reported in adult pike (Esox) replacement teeth (Herold, 1974). We think that this population is involved in later stages of odontogenesis, and its possible role will be discussed below.

Attachment and eruption Cichlid teeth, once they are well formed (mineralized dentine, mineralized enameloid), invariably become supported by an annular collar of attachment bone, to which they never become ankylosed since the interjacent matrix does not mineralize (attachment mode 2 of Fink, 1981). The time needed between epithelial invagination and attachment is very short; tooth development and attachment in certain loci can be completed in as little as 2 days (Huysseune,

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1990). Our observations suggest that two populations of cells are involved in the deposition of attachment bone: cells of the dental papilla along the inside of the collar, and of the dental sac along its outside. At the boundary between tooth base and attachment bone, there are no morphological or cellular indications which can help to distinguish between those cells which deposit the last elements of the dentine matrix from those which deposit the attachment bone matrix. Some cells indeed face both matrices. This raises the question as to what induces the formation of the attachment bone. At a given time, the cervical loop tip no longer elongates. The absence of the epithelium possibly allows dental sac cells to become activated, and may also act as a signal controlling the change of function of the dental papilla cells at the limit between dentine and future attachment bone (cf. Shellis, 1982). Signals transmitted to the cells of the dental papilla may be captured via the cell prolongations that have been observed at this level. Why this zone remains unmineralized and becomes a ligament in certain species, whereas the tooth becomes ankylosed in others, is not known. In addition to these queries, several teleost species are known to possess attachment modes that differ according to the bone considered (e.g., cod lower jaw teeth are ankylosed, upper jaw teeth are hinged, Herold, 1975) or to the position on a particular bone (e.g., the adult pike dentary possesses anterior teeth that are hinged and posterior teeth that are ankylosed (Herold & Landino 1970)); Lophius has hinged and ankylosed mandibular teeth (Kerebel et al., 1979). Attachment type may even show sexual dimorphism as in 0t~yzias (Parenti, 1987). Despite superficial resemblances between dentine and attachment bone matrix and the involvement of similar, and even the same cells, we have shown that cells of the dental sac are likely to be involved in the deposition of attachment bone and in fact assume the characteristics of osteoblasts. The attachment bone therefore, unlike a dentine, grows by accretion fi'om both sides. Based on these observations, as well as on the absence of dentinal tubules both in larval and adult fish, we regard the attachment bone as true bone, not as dentine (in disagreement with Hughes et al., 1994). Experiments using vital fluorescent marking have indeed shown that, in contrast to the dentine which grows and mineralizes centripetally, the attachment bone mineralizes both along the inside and the outside of the collar (Bergot, 1975b; Huysseune, 1989). Nevertheless, because of the intimate developmental relationship between tooth formation and attachment bone deposition, we agree with Shellis (1982) in considering the collar of attachment bone as part of the dental unit (cf. Huysseune, 1983; Smith & Hall, 1990). In addition to its likely role in the deposition of the attachment bone, the dental sac may perhaps play a role in eruption. First-generation teeth hardly protrude above the oral epithelium. This is the case too for first-generation teeth in trout (B erkovitz, 1978).

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Conclusion Having discussed the development and fine structure of the teeth in the early post-embryonic dentition of Hemichromis, it is clear that these follow the general pattern of teleost tooth formation (cf. Kerr, 1960; Bergot, 1975b; Huysseune, 1983) in that the epithelial downgrowth re-invaginates into a bell-shaped dental organ, that tooth matrices are subsequently deposited in a centripetal way, and that the tooth finally becomes attached. Yet the development of firstgeneration teeth shows peculiarities compared with later tooth generations (Huysseune & Sire, personal observations). These include a limited amount of enameloid having an indistinct boundary with the dentine during its formation, absence of dentinal tubules and of nerves and capillaries in the pulp cavity, and different organization of the ode. Whether or not such ontogenetic differences are common among teleosts remains to be explored. ACKNOWLEDGEMENTS Mrs F. Allizard and Mrs G. De Wever are gratefully acknowledged for expert technical assistance in preparing the sections. TEM work and preparation of the photographic prints have been carried out at the Centre Interuniversitaire de Microscopic Electronique (CIME), Paris 6 and 7. The authors gratefully acknowledge Prof. Dr A. de Ricqlbs and Dr M. Laurin (Universit6 Paris 7) and Prof. Dr F. Meunier (MNHN, Paris) for critically reading the manuscript. This work has been supported by an exchange programme between the Ministerie van de Vlaamse Gemeenschap (Belgium) and the Centre National de la Recherche Scientifique (France) and has received financial support from the Bijzonder Onderzoeksfonds of the University of Ghent. REFERENCES Barel, C.D.N., van Oijen, M.J.P., Witte, F. and Witte-Maas, E. 1977. An introduction to the taxonomy and morphology of the haplochromine Cichlidae from Lake Victoria. Neth. J. Zool., 27, 333-389. Bergot, C. 1975a. Mise en 6vidence d'un type particulier de dentine chez la truite (Salmo sp., Salmonid~s, Ttldost4ens). Bull. Soc. Zool. Fr., 100, 587-594. Bergot, C. 1975b. Morphogentse et structure des dents d'un ttltostten (Salmofario L.). J. Biol. Buccale, 3,301-324. Berkovitz, B.K.B. 1975. Observations on tooth replacement in piranhas (Characidae). Arch. Oral Biol., 20, 53-56. Berkovitz, B.K.B. 1978. Tooth ontogeny in the upper jaw and tongue of the rainbow trout (Salmo gairdneri). J. Biol. Buccale, 6, 205-215. Carlson, S.J. 1990. Vertebrate dental structures. In: Skeletal biomineralization: patterns, processes and evolutionary trends (ed. J.G. Carter). Van Nostrand Reinhold, New York, vol. 1, 531-556. Deutsch, D., Catalano-Sherman, J., Dafni, L., David, S. and Palmon, A. 1995. Enamel matrix proteins and ameloblast biology. Conn. Tissue Res., 32, 97-107. Fink, W.L. 1981. Ontogeny and phylogeny of tooth attachment modes in actinopterygian fishes. J. Morphol., 167, 167-184. Herold, R.C.B. 1974. Ultrastructure of odontogenesis in the pike (Esox lucius). Role of dental epithelium and formation of enameloid layer. J. Ultrastruct. Res., 48,435-454. Herold, R.C.B. 1975. Scanning electron microscopy of enameloid and dentine in fish teeth. Arch. Oral Biol., 20, 635-640. Herold, R.C.B. and Landino, L. 1970. The development and mature structure of dentine in the pike, Esox lucius, analyzed by bright field, phase and polarization microscopy. Arch. Oral Biol., 15,747-760.

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