Cell Tissue Res (2000) 302:205–219 DOI 10.1007/s004410000180

REGULAR ARTICLE

C. Van der heyden · A. Huysseune · J.-Y. Sire

Development and fine structure of pharyngeal replacement teeth in juvenile zebrafish (Danio rerio) (Teleostei, Cyprinidae) Received: 29 April 1999 / Accepted: 22 December 1999 / Published online: 11 October 2000 © Springer-Verlag 2000

Abstract Teeth are commonly used model systems for the study of epithelial-mesenchymal interactions during organogenesis. We describe here the ultrastructural characteristics of developing pharyngeal replacement teeth in juvenile zebrafish, an increasingly important model organism for vertebrate development. Replacement teeth develop in close association with the dental organ of a functional tooth. Morphogenesis is well advanced prior to the start of cytodifferentiation. Fibrillar enameloid matrix is formed first, followed by the deposition of predentine. Initial mineralization of the enameloid proceeds quickly; maturation involves the presence of ruffledbordered ameloblasts. Dentine mineralization is inotropic and is mediated by matrix vesicles. Woven-fibred attachment bone matrix is deposited before completion of dentine mineralization. Eruption of fully ankylosed teeth is a fast process and may involve degenerative changes in the pharyngeal epithelium. Mononucleated osteoclasts and clastic cells located in the pulp cavity intervene in tooth resorption prior to shedding. Structural differences with larval, first-generation zebrafish teeth include the presence of dentinal tubules and the absence of an electron-dense covering membrane. Part of these differences may relate to size differences of the teeth. Others, like the site of the replacement tooth bud, suggest that initiaThe research performed by C. Van der heyden was financed by a specialization grant from the Flemish Institute for the Advancement of Scientific-Technological Research in Industry (IWT). The work benefited from a grant from the Bijzonder Onderzoeksfonds of the University of Ghent (no. 01102995) and from an FWO grant (G.0109.99) and was also supported by an exchange program between the Ministerie van de Vlaamse Gemeenschap, Belgium, and the Centre National de Recherche Scientifique, France (C97.004) C. Van der heyden (✉) · A. Huysseune Instituut voor Dierkunde, Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium e-mail: [email protected] Tel.: +32 9 264 52 18, Fax: +32 9 264 53 44 J.-Y. Sire Université Paris 7 – Denis Diderot, CNRS UMR 8570, Case 7077, 2 Place Jussieu, 75251 Paris Cedex 05, France

tion may take place in already committed epithelium from the first initiation event in the larval stage. Keywords · Dentition · Tooth development · Transmission electron microscopy · Zebrafish, Danio rerio (Teleostei)

Introduction Vertebrate teeth provide an experimental model preferred by many researchers for the study of cell signalling and developmental regulatory processes. During the evolution of gnathostomes the structural and developmental characteristics of teeth were largely conserved (see the recent review by Huysseune and Sire 1998). However, mammals and non-mammalian vertebrates differ in the way their dentition is established: the former have a di(or even mono-) phyodont dentition, while the latter are polyphyodont. The dentition in polyphyodont species presents an interesting model with which to study the mechanisms underlying repeated initiation and subsequent development of teeth throughout the animal’s life. The zebrafish (Danio rerio) is a small teleost fish, belonging to the cyprinids. Like other non-mammalian vertebrates, zebrafish replace their teeth throughout life, but, in contrast to most, zebrafish lack teeth in the buccal cavity. Teeth are only associated with the fifth ceratobranchials, also termed the pharyngeal jaws (Fig. 1). This species has become, for several practical reasons, an important model organism widely used in morphological, genetic and molecular biological studies, and for which an important array of molecular techniques has been developed (Ekker and Akimenko 1991; Lele and Krone 1996; Roush 1996). In mammals, teeth are initiated at well-defined locations corresponding to incisor, canine, premolar and molar regions, respectively. The pattern of the dentition is species specific and to a large extent genetically controlled. Several hypotheses to explain the dental patterning have been proposed (e.g. the field theory, Butler

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Fig. 1 A Drawing of the zebrafish cranium showing the location of the pharyngeal jaws (grey) as observed from a cleared and stained specimen. B Dorsal view of the pharyngeal jaws and their teeth. C Scanning electron micrograph showing a medial view of an adult pharyngeal jaw. The three tooth rows are indicated (arrows). Two teeth of the ventral row are lacking (C caudal, D dorsal, R rostral, V ventral). Bars 500 µm (A), 250 µm (B), 100 µm (C)

1939; and the clone model, Osborn 1978; see also the reviews by Stock et al. 1997 and Thesleff and Sharpe 1997). Sharpe (1995) proposed an updated version of the clone model, suggesting that tooth shape and position are specified by the combinatorial action of different homeobox genes. Indeed, expression of several homeoboxcontaining genes (e.g. Dlx-1 and Dlx-2, gsc, Msx-1 and Barx-1) is restricted to clearly specified domains, implying the existence of a homeobox code (Sharpe 1995; Thomas et al. 1997; Thomas and Sharpe 1998; Thomas et al. 1998; Tucker et al. 1998a, 1998b). It has long been debated whether the tooth-inducing capacity resides in the epithelium or in the mesenchyme. The latest (molecular) results appear to identify the oral epithelium as the source of tooth-inducing signals in mammals (e.g. through the Bmp-4 pathway, Tucker et al. 1998a). In addition to the epitheliomesenchymal interactions, innervation may also be required for tooth initiation, as demonstrated in the teleost, Tilapia mariae (Tuisku and Hildebrand 1994). Studies on the innervation of rat teeth, however, show that peripheral nerves are not required for tooth initiation and morphogenesis but that local neuronal cells may participate in tooth formation (Luukko 1998). Results on monophyodont and heterodont mammals such as rodents cannot, however, be generalized for “vertebrates”. Studying the dentition of the zebrafish, a polyphyodont model organism, can therefore help to establish a clearer view on ongoing tooth initiation. To set up and interpret experiments (in vitro cultures and in situ hybridizations) aiming at a better understanding of the mechanisms involved in repeated tooth initiation in the zebrafish, knowledge of the establishment of the tooth pattern and of the histological development of the tooth is crucial. In particular, can histological descriptions of both first-generation teeth and replacement teeth provide us with a clue as to how developmental

mechanisms may differ between primordial initiation events in dentition patterning and those of ongoing initiation of new replacement tooth buds? The histological features of first-generation teeth have already been described both at the light-microscopic (LM) and the transmission electron-microscopic (TEM) level (Huysseune et al. 1998). It was clear that larval teeth differ in a number of aspects from juvenile and adult teeth (e.g. the epithelial connectivity between the teeth). The present study describes the histological features of the developing replacement teeth both at the LM and the TEM level, using specimens of approximately 30 dPF (SL ca. 9 mm). To the best of our knowledge, this is the first report in which the development of zebrafish replacement teeth is described and in which differences from first-generation teeth are discussed.

Materials and methods All specimens used in this study were laboratory reared at 25°C. A total of five specimens, ranging in age from 24 to 32 days postfertilization (dPF) [standard lengths (SL) ranging from 8.9 to 9.6 mm] and one individual of unknown age (SL 13.0 mm), were sacrificed by means of an overdose of the anaesthetic MS-222 (Sandoz, Basel) in accordance with the Belgian law on the protection of laboratory animals (Koninklijk Besluit, dd. 14 November, 1993). They were fixed for 2 h, at room temperature, in a mixture of 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4), rinsed in the same buffer, to which 10% sucrose was added, 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 embedded in Epon. The heads were cut with a diamond knife into series of transverse 1-µm-thick sections which were stained with toluidine blue. At intervals, the series were interrupted for ultrathin sectioning. Thin sections were contrasted with uranyl acetate and lead citrate and viewed under a Philips 201 transmission electron microscope operating at 80 kV.

Results The juvenile dentition in zebrafish consists of a ventral, a mediodorsal and a dorsal rostrocaudal tooth row (Fig. 1). The description mostly holds for tooth germs and teeth in the ventral row. Differences from dorsal teeth will be mentioned where appropriate.

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In the previous paper (Huysseune et al. 1998), three partially overlapping stages of tooth development were distinguished: initiation and morphogenesis, cytodifferentiation, and attachment. In juveniles, however, in which the teeth are larger, we were able to subdivide these three phases into more detailed developmental stages. Initiation Initation of a replacement tooth occurs in the contact zone between the dental organ of a functional tooth and the pharyngeal epithelium proper. At the LM level, this site is thickened and consists of densely packed cells with no mucous cells. The cells of the undulated basalmost layer are polarized (Fig. 2). They have a cytoplasm poor in organelles, irregular processes directed towards the mesenchyme, and a basal nucleus (Fig. 3). Medially positioned cells are obliquely oriented, whereas more laterally the cells are perpendicular. A few, slightly differentiated mesenchymal cells face the epithelial thickening and are separated from the basal lamina by a 0.75-µm-wide space containing a loose meshwork of thin collagen fibrils. Fingerlike processes are directed towards, and contact, the basal lamina (Fig. 3). Morphogenesis Early bud stage The first phase of morphogenesis is represented by a shallow epithelial downgrowth of desmosome-linked cells (Fig. 4). The cells of the proximal side of the invagination are still polarized but not well aligned, creating an undulating albeit smooth interface. An approximately 0.7-µm-wide space still separates the basal lamina from the few mesenchymal cells present. As before, occasional mesenchymal processes make contact with the basal lamina.

body axis, the latter towards the lateral side of the body (Fig. 5). Medial and lateral sides of the germ develop from the proximal epithelium. Intercellular spaces become a little more prominent in the dental organ; yet threadlike cytoplasmic processes persist and are highly imbricated (Fig. 6). The proximal cells of the dental organ retain their polarized aspect. Medial cells are obliquely oriented whereas more laterally cells are perpendicularly oriented and are more plump. Throughout the germ, the epithelial cells appear to be slightly more differentiated. In contrast to the early bud, the peripheral cells of the dental organ present numerous basal fingerlike processes (Fig. 6). There is still no clearly distinguishable accumulation of mesenchymal cells opposite the epithelial invagination. However, collagen fibrils have accumulated in a zone approximately 1 µm wide, facing the elaborate folds of the epithelial cells. Occasionally, mesenchymal cell processes come into close proximity to the basal lamina (Fig. 6). Early bell stage The skewed epithelial bud of the previous stage has now given rise to a crescent-shaped germ (Fig. 7). The dental lamina consists of two or three layers of flattened cells parallel to the superficial epithelium and resembling the epithelial cells of the late bud (Fig. 8). The cells of the lateral part of the germ are highly interdigitated and show a large amount of caveolae (Fig. 9). An accumulation of mesenchymal cells, collectively called the dental papilla, becomes visible opposite the lateral side of the germ. The cells at the future tooth tip are plump with more, electron-lucent, cytoplasm compared to the flattened surrounding fibroblasts but only have a small amount of organelles. They bear distinct cytoplasmic processes directed towards the basal lamina usually without making contact. These cells are preodontoblasts (Fig. 9). Late bell stage

Late bud stage The epithelial downgrowth (dental organ) contains a larger number of cells and is linked to the superficial epithelium by an epithelial connection of about three cells wide. The germ is skewed towards the future attachment site (Fig. 5), a difference from dorsal tooth germs. With regard to the pharyngeal epithelium, we will refer to distal as the side of the germ facing the superficial epithelium and proximal to that facing the opposite side (Fig. 5). The distal side constitutes the connection of the tooth germ to the superficial epithelium and is called dental lamina. With regard to the skewed shape of the germ proper, we will distinguish the medial and lateral sides. The former is directed towards the mediosagittal

The larger tooth germ has retained its skewed appearance (Fig. 10). The less advanced state of differentiation is the lateral side. The dental lamina is reduced in thickness and consists of two layers of extremely flat cells. The crescent-shaped dental organ of the early bell stage now envelopes a large dental papilla and has become distinctly bilayered. The cells of the layer facing the mesenchyme (called inner dental epithelium, IDE) are cuboidal at the future tooth tip while the superficial cells (outer dental epithelium, ODE) are more flattened. The cell membranes facing the basal lamina have become smooth, except at the cervical loop tip. The medial IDE cells show an increased amount of cytoplasm with many mitochondria and a few cisternae of the rough endoplasmic reticulum (RER). Both IDE and ODE cells

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contain numerous caveolae (Fig. 10). The cervical loop tip is formed by the process of one IDE or ODE cell (Fig. 10). The number of dental papilla cells has considerably increased. They are all plump with little cytoplasm and inconspicuous cytoplasmic processes between adjacent cells (Fig. 11). The intercellular spaces contain moderate amounts of 50-nm-diameter collagen fibrils sometimes packed in small bundles. The peripheral cells (preFig. 2 First morphologically discernible indications of odontogenesis (32 dPF zebrafish). Light micrograph (LM). Note the different orientation of the dental epithelial cells (arrows) with respect to the adjacent, non-odontogenic pharyngeal epithelium (PE) (FT functional tooth, M mesenchyme). Bar 50 µm Fig. 3 Initiation (32 dPF). Transmission electron micrograph (TEM). The epithelial cells of the basalmost layer (arrows) are polarized. Some mesenchymal cells are in contact with the basal lamina (arrowhead) (PE pharyngeal epithelium). Bar 5 µm Fig. 4 Early bud (28 dPF). LM. The early bud consists of a shallow epithelial downgrowth into the mesenchyme (arrowhead) (FT functional tooth). Bar 25 µm Fig. 5 Slightly more advanced invagination (30 dPF). TEM. Note the cytoplasmic processes of the epithelial basal cells (arrowheads) directed towards the basal lamina (arrow). The mesenchymal cells (M) surrounding the dental organ (DO), from which they are separated by a 1-µm-thick zone with collagen, clearly differ from the fibroblasts (F) (d distal, m medial, l lateral, p proximal). Bar 10 µm Fig. 6 Detail of the dental organ shown in Fig. 5. TEM. Note the different orientation of respectively medial (left) and proximal (right) cells as well as the presence of several interdigitations between the dental organ cells. The arrowhead points towards the basal lamina. Bar 5 µm Fig. 7 Crescent-shaped tooth germ (30 dPF). LM. A few mesenchymal cells aggregate to form the dental papilla (DP) (DO dental organ, PE pharyngeal epithelium). Bar 25 µm Fig. 8 Detail of the same tooth germ as in Fig. 7. TEM. Note the persistence of cellular processes along those epithelial cells facing the dental papilla (DP). The asterisk indicates the dental lamina (DO dental organ). Bar 5 µm Fig. 9 Detail of the lateral part of the tooth germ shown in Figs. 7 and 8. Note the cytoplasmic processes of both lateral dental organ cells and dental papilla cells directed towards the basal lamina (arrowheads) as well as the striking differences between future odontoblasts (OB) and ordinary, non-differentiated fibroblasts (F). Arrows point to interdigitations of the dental organ cells. Bar 2 µm Fig. 10 Late bell stage (13 mm standard length, age unknown). TEM. Note the decreasing level of differentiation of both inner (IDE) and outer dental epithelium (ODE) from medial (*) to lateral (**). The pulp is densely crowded and the cervical loop tip (arrowhead) at the proximal side of the germ is formed by one elaborately folded cell of the dental organ. The arrow (top of figure) points towards the thin dental lamina, connecting the tooth germ to the pharyngeal epithelium (PE). Bar 5 µm Fig. 11 Detail of the pulp in the late bell stage. The arrowheads point towards the several cytoplasmic processes interconnecting the dental papilla cells, leaving large intercellular spaces containing collagen fibrils (small arrows) (asterisk dental organ). Bar 2 µm Fig. 12 Detail of pre-odontoblastic processes (asterisks) making contact with the basal lamina (arrowhead). TEM (IDE inner dental epithelium). Bar 250 nm

odontoblasts) have distinct cytoplasmic processes which contact the basal lamina (Fig. 12), especially facing the most differentiated cells of the IDE (Fig. 10). Cytodifferentiation Enameloid formation The present description is given for a dorsal tooth germ (Fig. 13). At the tooth tip the dental organ is not connected to the pharyngeal epithelium. Cells of the IDE, called ameloblasts, have become more or less pear shaped with the broader part housing the nucleus and tapering towards the basal lamina (Fig. 14). They contain some mitochondria, moderate amounts of RER cisternae and have strongly interdigitated lateral cell membranes connected with desmosomes (Fig. 15). Often, such processes form the actual lining along the intact, smooth basal lamina (Fig. 15). The matrix (enameloid) is moderately dense and consists of fibrils of various diameters (Fig. 16). The thickest fibrils follow the axis of the tooth; the thinner fibrils are less abundant and are often oriented perpendicular to the thicker fibrils. Some of the thinner fibrils appear to anchor within, others to cross, the basal lamina. Although cells of the dental papilla do not extend up to this level, a cytoplasmic process may occasionally be observed within this matrix. Early dentinogenesis The pre-odontoblasts located medially have differentiated into odontoblasts, with a slightly more differentiated cytoplasm and prominent processes directed towards the basal lamina (Fig. 17). In between these processes, the first, loose predentine matrix is deposited. The collagen fibrils of the predentine are homogeneously distributed and preferentially oriented along the long axis of the tooth. They are of variable thickness, measuring from 50 to 100 nm in diameter. Thinner fibrils occasionally anchor in the basal lamina (Fig. 17). Late dentinogenesis The IDE cells facing the predentine differ from those involved in enameloid formation (Fig. 18, cf. Figs. 14, 15). Their fairly abundant cytoplasm contains numerous mitochondria and a moderately developed RER. Lateral cell membranes are not interdigitated and closely adjoin each other. The basal lamina is still present and smooth. The odontoblasts in this area have further differentiated (Fig. 18). Their cytoplasm has notably increased, they have many mitochondria and their RER is well developed with numerous cisternae. Cytoplasmic processes within the predentine appear to be less numerous and thinner than before. Occasionally, some of them come

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into close vicinity of the basal lamina (Fig. 18). Intercellular spaces between the differentiated odontoblasts have nearly disappeared. The predentine, in its most developed part, is approximately 2 µm thick. The homogeneously distributed collagen fibrils still have the same Fig. 13 Start of enameloid matrix formation at the tip of a dorsal tooth germ (30 dPF). LM. The tip is surrounded by the dental organ (DO) only, which is not connected to the pharyngeal epithelium (PE) at this level (arrow). Due to the curved shape of the germ, the tooth tip in transverse sections of the dentition is usually cut perpendicular to the main axis of the tooth, whereas towards the base the germ is cut longitudinally. Bar 25 µm Fig. 14 Unmineralized enameloid of the dorsal tooth germ in Fig. 13. TEM. The basal lamina is still present and separates the enameloid (EN) from the pear-shaped ameloblasts (AB). Their lateral cell membranes are broadly interdigitated (arrowheads) and are interconnected with desmosomes (ODE outer dental epithelium). Bar 5 µm Fig. 15 Detail of the same tooth germ as shown in Figs. 13 and 14. Note the presence of the interdigitations (arrow) of the ameloblasts (AB). The arrowhead points towards a desmosome linking the ameloblasts (EN enamel). Bar 2 µm Fig. 16 Detail of the unmineralized enameloid showing collagen fibrils of various diameters. TEM. Note the difference in orientation between the thickest (arrow) and the thinnest fibrils (asterisk). An arrowhead points to a thinner fibril apparently anchoring within the basal lamina (BL) (AB ameloblast, EN enameloid). Bar 200 nm Fig. 17 Early predentine formation, medial part of the germ (30 dPF). The dental papilla cells, now odontoblasts (OB), have well-developed processes directed towards the basal lamina with which they make contact (arrows). The collagen fibrils (arrowheads) are homogeneously distributed in the predentine (PD) while thinner fibrils are oriented perpendicular to the tooth axis and anchor in the basal lamina (asterisk) (BL basal lamina, IDE inner dental epithelium). Bar 500 nm Fig. 18 Medial part of a tooth germ with a thin layer of predentine (PD) (30 dPF). TEM. The inner dental epithelium cells (IDE) are rather well differentiated and contain mitochondria and moderately developed rough endoplasmic reticulum. The odontoblasts (OB) show fewer processes (arrowhead). The collagen fibrils in the predentine are a little more packed than previously. The arrow points towards the basal lamina. Bar 2 µm Fig. 19 Detail of the predentine matrix (PD) showing the thin collagen fibrils (arrowhead) anchoring in the basal lamina (BL) and the thicker collagen fibrils (arrow) which are oriented following the longitudinal axis of the tooth. TEM (IDE inner dental epithelium). Bar 500 nm Fig. 20 Detail of the predentine matrix showing tight clusters of collagen fibrils (asterisks), surrounded by predentine (PD) (30 dPF). Note the presence of the intact basal lamina (arrowhead) (IDE inner dental epithelium). Bar 1 µm Fig. 21 Highly polarized odontoblasts (OB) producing the predentine (PD) (30 dPF). TEM. Note the presence of gap junctions (arrowheads) and of desmosomes (arrow) linking the odontoblasts. Large intercellular spaces are seen (OBP odontoblastic process). Bar 2 µm. Inset: Detail of gap junctions (arrows) interlinking odontoblasts. Bar 500 nm Fig. 22 Tooth tip of an EDTA-demineralized functional tooth (32 dPF). TEM. The enameloid (EN) contains tubules (arrow) apparently without cellular process. The collagen fibrils, originally constituting the enameloid, have been modified during the mineralization process while they are still present in the demineralized dentine (D). Bar 1 µm

diameter (50–100 nm) but are more densely packed. A larger number of thin fibrils are radially arranged and anchor in the basal lamina (Fig. 19). During further dentinogenesis, changes in the IDE cells include reduction of the RER and appearance of numerous free ribosomes. The basal lamina retains its smooth aspect (Fig. 20). The odontoblasts become more elongated and polarized perpendicularly to the matrix which they are depositing (Fig. 21). Their nucleus is away from the secretory surface, and their cytoplasm is abundant with a well-developed RER with numerous parallel cisternae, large Golgi areas and many mitochondria. The odontoblasts are closely adjoined and present numerous cellular contacts by means of several gap junctions (Fig. 21, inset) and desmosomes. Odontoblastic processes are dispersed in the predentine matrix in which the density of collagen fibrils has considerably increased. However, the fibrils are still embedded in an electron-lucent ground substance (Fig. 21). In some areas, collagen fibrils appear to cluster in bundles of roughly 1 µm diameter separated by areas of ordinary predentine of approximately 0.25 µm wide. The fibrils in these bundles show the same preferential orientation as elsewhere in the predentine (Fig. 20). Enameloid mineralization and maturation During early enameloid mineralization, the organic matrix largely persists. It is a fairly dense, granular and electron-lucent matrix in which fibrillar components can hardly be recognized. The enameloid contains several tubules, apparently without cellular content (Fig. 22). The limit with the subjacent dentine is rather blurred; collagen fibrils of the dentine appear to fan out into the enameloid. In pronounced bicuspid dorsal teeth, the characteristics of the ameloblasts differ depending on whether they lie on the concave or the convex side of the tooth tip. During enameloid maturation, the ameloblasts facing the concave side are polarized, highly columnar cells (Fig. 23). Their nucleus is turned away from the mineralized tissue while the cell membrane facing the enameloid is folded into a ruffled border poor in organelles. The cytoplasm is abundant with a few dilated cisternae of RER, a fair number of mitochondria and extensive Golgi areas (Fig. 24). Ameloblastic interdigitations are closely adjoined and linked by desmosomes and gap junctions (Fig. 24). The ameloblasts along the convex side, although polarized, are less columnar with a nucleus oriented accordingly (Fig. 23). A basal lamina can no longer be distinguished. The mineral crystals immediately adjoin the ruffled border (Fig. 24). Separate crystals can hardly be distinguished, and because the organic matrix is no longer discernible, their orientation with respect to the fibrils cannot be determined. Odontoblastic processes in the enameloid are surrounded by a thin mineral-free area.

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213 Fig. 23 Enameloid maturation in a dorsal tooth germ forming the minor cusp (arrow) (30 dPF). TEM. The highly polarized ameloblasts (AB) at the level of the maturing enameloid present a ruffled border (asterisk). The basal lamina disappears at the border of the maturing enameloid (arrowhead). Note the flattened shape of the IDE cells down the shaft (IDE) (DP dental papilla, MF mineralization front, OB odontoblast, ODE outer dental epithelium, PD predentine). Bar 5 µm Fig. 24 Detail of an ameloblast (AB) located at the concave side of a dorsally positioned tooth germ during enameloid maturation (30 dPF). TEM. The ruffled border (RB) closely adjoins the maturing enameloid (EN). The abundant cytoplasm contains many mitochondria as well as extensive Golgi areas (asterisks). Neighbouring ameloblasts are interconnected with desmosomes and gap junctions (arrowhead), leaving small intercellular spaces. Bar 1 µm Fig. 25 Tooth shaft (30 dPF). TEM. A layer of predentine (PD) lines the inner surface of the dentine (D). Note the absence of the basal lamina and the difference in differentiation state between odontoblasts (OB) and inner dental epithelium cells (IDE). Bar 2 µm Fig. 26 Mineralizing dentine along the shaft and preceding the mineralization front in the same tooth as shown in Fig. 25. TEM. The orientation of the mineral crystals coincides with that of the collagen fibrils. The arrowhead points to predentinal (PD) matrix vesicles containing mineral crystals. Note the mineral crystals extending into the lamina densa of the basal lamina (arrow) (IDE inner dental epithelium). Bar 500 nm. Inset Detail of a matrix vesicle containing a mineral crystal (arrow). Bar same as in Fig. 12 Fig. 27 Dentine with an odontoblastic process (arrow) containing several transversely sectioned intermediate filaments (30 dPF). TEM. Note the zone of unmineralized matrix surrounding the cellular process (asterisk) (MD mineralized dentine). Bar 500 nm Fig. 28 Developing bone of attachment (30 dPF). TEM. The cervical loop tip (CLT) indicates the limit between tooth (dentine, D) and bone of attachment (BOA). The dental papilla cells (DPC) opposite the tooth proper and opposite the attachment bone in general show the same cellular characteristics, with the exception of the presence of several, long and relatively thick cytoplasmic processes (arrow) at the level of the attachment bone (M ordinary mesenchyme). Bar 5 µm Fig. 29 Detail of the limit between bone of attachment (BOA) and (pre-)dentine (D). TEM. The difference between the orientation of the collagen fibrils in both matrices is clearly demonstrated. Note the difference in thickness and length of the cytoplasmic processes (arrows) of the odontoblasts (OB) on the one hand and the dental papilla cells opposite the bone of attachment on the other hand. The arrowhead points towards the basal lamina (CLT cervical loop tip). Bar 1 µm Fig. 30 Base of the developing bone of attachment (BOA) with its randomly distributed collagen fibrils (30 dPF). TEM. The cytoplasm-rich dental papilla cells (DPC) show a large amount of RER in comparison to the normal mesenchymal cells (asterisk) lining the external side of the attachment bone. The small arrows point towards a thin layer of unmineralized matrix lining the bone of attachment. Bar 5 µm Fig. 31 Detail of the mineralizing bone of attachment shown in Fig. 30. TEM. The woven-fibred collagen fibrils are less densely packed compared to the predentine. The arrows show several sites of starting mineralization while the arrowheads point to matrix vesicles. Bar 500 nm Fig. 32 Supporting bone (SB fifth ceratobranchial) (30 dPF). TEM. New bone matrix (asterisk) has been deposited in the prolongation of the attachment bone. This unmineralized bone matrix also contains a large amount of woven-fibred collagen fibrils. Note the presence of large pulpal intercellular spaces bridged by cytoplasmic processes (arrowhead). Bar 2 µm

Dentine mineralization The shaft appears to be formed in predentine well before mineralization progresses into it. In the upper part of the shaft, the mineralized dentine is a homogeneous layer of approximately equal thickness (2 µm), leaving a layer of about 1 µm of unmineralized predentine along its inner surface (Fig. 25). The IDE cells in this area are rather flattened, with a nucleus oriented parallel to the tooth surface and a cytoplasm with numerous mitochondria and a moderately developed RER. These IDE cells are interconnected with desmosomes only, while both desmosomes and gap junctions link them to the ODE cells. A basal lamina can no longer be distinguished. The ongoing deposition of predentine is reflected in the highly differentiated state of the odontoblasts facing this matrix, and especially their extremely well developed RER with abundant, parallel cisternae. Downwards along the shaft, the mineralization front is preceded by mineralized patches over a considerable distance (Fig. 26). In this area, the predentine contains numerous matrix vesicles, some of which house mineral crystals (Fig. 26, inset). Towards the outer tooth surface, irregular patches of mineralized dentine are dispersed in the matrix extending up to the basal lamina. Some of these extend well into the lamina densa of the basal membrane (Fig. 26). The crystals are aligned with the collagen fibrils. Odontoblastic processes rich in intermediate filaments (Fig. 27) extend into the mineralized dentine. Mineralization around these processes appears to be delayed with respect to overall mineralization (Fig. 27). Attachment Well before mineralization has reached the cervical loop tip, a loose matrix layer about 2–3 µm wide is deposited in the prolongation of the basalmost predentine (Fig. 28). The dental papilla cells involved in this deposition are distinguished from the odontoblasts proper by the presence of conspicuous cell processes penetrating into this matrix. This early attachment bone matrix is slightly less dense than the predentine; its collagen fibrils are approximately 30–35 nm in diameter and randomly oriented (Fig. 29). With ongoing attachment bone formation, the dental papilla cells are no longer polarized perpendicularly to the matrix surface but retain their differentiated state (Fig. 30). The mesenchymal cells along the external surface of the attachment bone are elongated and well differentiated. The matrix of the attachment bone presents a more woven aspect compared to that of the predentine. Mineralization of the attachment bone starts well before completion of predentine mineralization. Patches of granular, electron-dense background substance appear in between the collagen fibrils, but these fibrils do not reach the density observed in the predentine (Figs. 30, 31). Most of the mineral clusters are found within these patches. As in dentine, the crystals are oriented along the

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Fig. 33 Tooth just before eruption (24 dPF). LM. The tooth tip (asterisk) is still surrounded by the dental organ (arrow). The arrowhead points towards the tip of a newly initiated tooth germ (M mesenchyme, PC pharyngeal cavity). Bar 25 µm Fig. 34 Same tooth as shown in Fig. 33, but in a 4 days older zebrafish (28 dPF). LM. The tooth has now erupted and its tip (asterisk) lies freely in the pharyngeal cavity. The tooth germ (arrowhead) is further developed than in Fig. 33 (M mesenchyme, PC pharyngeal cavity, PE pharyngeal epithelium). Bar 25 µm Fig. 35 Functional tooth ready to be shed (FT) (30 dPF). LM. The tooth base has already partly been eroded (arrowhead). A tooth germ (TG) is developing in the vicinity of the tooth base (SB supporting bone). Bar 100 µm

tics (arrows) (BOA bone of attachment, CLT cervical loop tip, D dentine, DP dental papilla). Bar 2 µm Fig. 37 Detail of an osteoclast (asterisk) at the outer surface of the tooth base shown in Fig. 36. This osteoclast is attacking the bone of attachment (BOA) and resorbing collagen fibrils and mineral crystals (arrowheads). TEM. Bar 500 nm Fig. 38 Detail of the tip of an assumed clastic cell (asterisk) in the pulp cavity of the tooth shown in Fig. 36 apparently attacking the dentine (D). TEM. Note the granular cytoplasm of these pulpal clastic cells and the absence of a ruffled border, a clear zone and cytoplasmic vesicles. Bar 500 nm

Fig. 36 Base of the same tooth as in Fig. 35. TEM. Osteoclasts are present at the outer tooth surface (arrowhead) while the inner surface is attacked by clastic cells which show other characteris-

Fig. 39 Detail of the ruffled border of another osteoclast (OC) attacking the supporting bone (SB) (30 dPF). TEM. The collagen fibrils (arrowheads) are observed deep in the folds of the osteoclast membrane while the mineral crystals appear to be dissolved more superficially. Bar 500 nm

collagen fibrils. Elsewhere, crystals also appear to be associated with matrix vesicles. Once mineralization of the attachment bone is well advanced, unmineralized matrix persists both along its inner and outer surface (Fig. 30). The dental papilla cells and the outer mesenchymal cells remain in a differentiated state. Concomitant with progressing attachment bone formation and mineralization at the tooth base, osteogenesis takes place at the surface of the supporting bone in the prolongation of the attachment bone (Fig. 32). The den-

tal papilla cells (internally) and the mesenchymal cells (externally) continue towards the supporting bone by cells resembling those involved in attachment bone formation. They surround a spicule of bone lined by unmineralized matrix which shows the same characteristics as the attachment bone: a fairly loose, woven-fibred matrix in which patches of electron-dense ground substance announce imminent crystal deposition. Eventually, the attachment bone fuses to this newly formed bone of the pharyngeal jaw. Once mineralization

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of the attachment bone is completed, the matrix of dentine, attachment bone and supporting bone can no longer be distinguished (Figs. 35, 36). Only the level of the cervical loop tip indicates the dentine/attachment bone boundary. Eruption Once the tooth is attached and has reached its final size, eruption occurs. Ultrastructural details of a tooth in the process of eruption are lacking. At the LM level, however, we were able to distinguish only two stages: one in which some epithelial cells, probably of the dental organ, still surround the tip (Fig. 33); the next in which the tip lies freely, and for a considerable distance, in a crypt of the pharyngeal epithelium (Fig. 34). Intermediate phases were not found. Shedding After a functional lifetime of about 8 days in young juveniles, the tooth is eventually shed. Resorption occurs along both the outer and the inner tooth surface (Figs. 35, 36). At the outer surface, the cells show typical osteoclastic features, i.e. the presence of a ruffled border and a clear zone, abundant mitochondria and several cytoplasmic vesicles sometimes containing collagen particles with associated crystals (Fig. 37). These cells are apparently mononucleated and sometimes lie in between the dentinal surface and dental organ cells (Fig. 36). In the pulp cavity, the cells apparently involved in resorption are moderately electron-dense with fairly abundant cytoplasm, rich in mitochondria and free ribosomes grouped in polysomes (see Figs. 36, 38 for detail). They are poor in RER, they appear to be mononucleated and no cytoplasmic vesicles are observed. Where cytoplasmic processes make contact with the mineralized tissue, the collagen fibrils of the dentine look disorganized (Fig. 38). Both in the pulp cavity and on the external wall of the tooth, the clastic or the supposedly clastic cells form a continuous lining along the dentine and the attachment bone (the limit between both being indicated by the cervical loop tip). The supporting bone is eroded by apparently mononuclear osteoclasts sealed to the bone surface by means of an organelle-free clear zone. The basal cell membrane is folded into a ruffled border, separated by a space of about 0.5 µm wide (Fig. 39). Within the folds of the ruffled border, fine collagen fibrils can be observed along with individual mineral crystals.

Discussion Many aspects of embryonic development of the zebrafish have received attention; yet few studies have focused on postembryonic development or on regeneration

in this model organism (e.g. Ferretti and Géraudie 1995; Cubbage and Mabee 1996; Sire et al. 1997a, 1997b; Huysseune et al. 1998). Only the latter paper has dealt with its developing dentition. Ultrastructural studies on tooth development in teleosts are scarce. Most deal with non-cyprinids (Higashi et al. 1983; Huysseune and Sire 1997a, 1997b; Sasagawa 1984, 1988, 1995, 1997), and only some with cyprinids (Cheprakova 1958, Sasagawa 1988, Sauvage 1972; Sauvage and Follenius 1973). To the best of our knowledge, this paper is the first ultrastructural description of the development of replacement teeth in juvenile zebrafish. The epithelial cells – ameloblasts – enameloid Replacement teeth in zebrafish develop from an epithelial thickening at the transition zone between the (possibly reduced) dental organ of a functional tooth and the pharyngeal epithelium proper. Independent development from the pharyngeal epithelium, as seen in first-generation teeth (Huysseune et al. 1998), is absent in developing replacement teeth. There is no permanent or a continuous dental lamina (sensu Reif 1982) from which new germs bud off. The close vicinity of the dental organ of a functional tooth points to its possible involvement in the onset of the formation of its successor and suggests that initiation may take place in already committed epithelium in the first initiation event in the larval stage. In situ hybridizations currently carried out in our laboratory must help to clarify this possible distinction in initiation events. The developing tooth remains connected to the superficial epithelium by epithelial cells which can be considered as a non-permanent dental lamina. In addition, functional teeth and their successors in different positions have no epithelial connection. This would imply the existence of separate tooth families for each functional tooth (C. Van der heyden, personal observation). As in other teleosts, IDE and ODE remain in close apposition, without an intervening stratum intermedium or stellate reticulum (Kerr 1960; Bergot 1975; Prostak and Skobe 1986a; Sasagawa 1995). Sauvage (1972), however, has reported the presence of a stratum intermedium and of a reticulum stellatum in tooth germs of the goldfish Carassius auratus. A large number of desmosomes link the dental organ cells throughout morphogenesis and cytodifferentiation. These junctions are known to provide mechanical integrity to the tissue and may also be involved in tissue remodelling during development (Garrod et al. 1996; Green and Jones 1996). In the teeth of larval zebrafish, desmosomes were not reported (Huysseune et al. 1998). The apparent absence of these junctions in larval teeth might be related to the small number of dental organ cells in these smaller, first-generation teeth. In addition, the ameloblasts of the first-generation teeth in cichlids are connected to each other by gap junctions (Huysseune and Sire 1997a). These specialized connections apparently do not interconnect the IDE cells of zebrafish re-

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placement teeth, while they connect the IDE cells with the ODE cells, suggesting a transfer of ions between IDE and ODE cells. During enameloid formation, the IDE cells, now ameloblasts, show features of secreting cells (extended RER, increased number of mitochondria, etc.). These findings are in accordance with literature reports on the ameloblasts in other teleosts (e.g. Shellis 1978; Prostak and Skobe 1986a; Sasagawa 1995; Huysseune and Sire 1997a) and in mammals (e.g. reviews in Deutsch et al. 1995; Smith and Nanci 1995). The nature of the secretory products is unknown but it is speculated that ameloblasts may be involved in the secretion of non-collagenous enamel proteins. In contrast to larval teeth (Huysseune et al. 1998), replacement teeth possess a clearly distinguishable unmineralized precursor of enameloid. The enameloid matrix consists of moderately densely packed bundles of fibrils of various diameters, probably collagen. The orientation of the fibrils appears to be in accordance to their thickness. Whether the ameloblasts or the odontoblasts form the collagen of the enameloid has long been debated. In view of the presence of a well-distinct basal lamina when the fibrillar enameloid matrix is deposited, we are inclined to regard the enameloid as a matrix of predominantly odontoblastic origin. However, the presence of thin collagen fibrils occasionally anchoring within or even crossing the basal lamina suggests an ameloblastic involvement as well. For further discussion, we refer to the review by Huysseune and Sire (1998). In spite of many observations on tooth germs with incipient matrix production, the stage of starting enameloid mineralization could not been found, suggesting that it is a brief phase. This would be in accordance with the findings of Sauvage (1972) on the goldfish. The fishes used in other studies on enameloid mineralization (Isokawa et al. 1970; Inoue et al. 1973; Prostak and Skobe 1986b; Sasagawa 1988, 1997) were strikingly larger (10–35 cm SL) than juvenile (and even adult) zebrafish (ca. 1 cm SL for the juveniles used in the present study). It may well be that small tooth size in zebrafish entails the rapid progression of the mineralization process into the enameloid layer. At the moment of enameloid maturation, the ameloblasts show the same features in zebrafish as in other teleosts. The ameloblasts, as in other teleosts, become ruffled bordered. This ruffled border is probably in relationship to the breakdown of the organic components of the enameloid matrix (Shellis 1978). Ruffled-bordered ameloblasts have also been reported by Sasagawa (1984, 1997) and Huysseune and Sire (1997a). In mammals, their function is related to enamel mineralization and maturation (see the review by Deutsch et al. 1995). Unlike in first-generation teeth (Huysseune et al. 1998), the mature enameloid in zebrafish replacement teeth lacks a covering electron-dense membrane. The enameloid contains odontoblastic tubules, which lack cytoplasmic processes, suggesting a retraction of the odontoblastic process during odontogenesis. In goldfish, how-

ever, Sauvage (1972) found dentinal tubules totally filled up with cytoplasmic processes. In addition, we were not able to observe patterns of crystallite arrangement similar to what was observed in goldfish “enamel”. A dentine-like hypermineralized substance, enameloid rather than true enamel, covers the zebrafish tooth tip (firstgeneration and juvenile – adult). The hypermineralized cap covering the tooth tip in goldfish is claimed to be enamel (Sauvage 1972; Sauvage and Follenius 1973), but this statement is based on the detailed study of a single odontogenetic stage. Moreover by stating that the goldfish has true enamel, Sauvage (1972) encountered difficulties explaining the presence of tubules containing odontoblastic processes extending into the enamel. Therefore, we suppose that the goldfish, like the zebrafish and other teleosts, has enameloid instead of enamel. Dental mesenchyme – odontoblasts – dentine On the mesenchymal side, odontogenesis starts at the initiation stage by a moderate accumulation of a few, slightly differentiated cells. Some of their cytoplasmic processes contact the basal lamina, which could indicate the occurrence of epithelial-mesenchymal interactions. These contacts persist until late dentinogenesis and were also described for zebrafish larvae (see Huysseune et al. 1998 for further discussion). Desmosomes as well as gap-like junctions interconnect the predentine-producing odontoblasts. The presence of numerous gap junctions in the odontoblasts at the onset of dentinogenesis was also reported in other teleosts, notably cichlids, such as the tilapia (Tilapia nilotica) (Sasagawa 1995) and Hemichromis bimaculatus (Huysseune and Sire 1997a) and could probably be linked to the intercellular transportation of calcium ions. Indeed, a delicate intracellular Ca2+ ion balance is maintained in mammalian odontoblasts (Linde and Lundgren 1995). Progression of dentine mineralization in juvenile zebrafish occurs in a similar way as in larval zebrafish teeth (Huysseune et al. 1998): (1) mineral crystals follow clearly the orientation of collagen fibrils (inotropic mineralization); (2) mineralization occurs in one front; and (3) some patches of crystals precede the mineralization front. Goldberg et al. (1995) and Linde and Lundgren (1995) stated that dentine phosphoproteins (DPPs) are believed to act as mineral nucleators in mammalian teeth. The presence of nucleation sites in zebrafish dentine suggests the action of PP. To the best of our knowledge, no zebrafish PP homologues have already been found. Matrix vesicles are present in the mineralizing dentine. It is known that matrix vesicles are associated with initiation of mineralization (Anderson 1995). The possible involvement of matrix vesicles in the mineralization process during odontogenesis in teleosts was reported by Sasagawa and Igarashi (1985) and Sasagawa (1988). The dentine in juvenile zebrafish is characterized by the presence of several odontoblastic processes that

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come into close vicinity of the tooth surface. An outer layer of atubular dentine described in the teeth of adult apodans (Casey and Lawson 1981) and of humans (Linde and Goldberg 1993) could therefore not be found in juvenile zebrafish. In contrast, the dentine of first-generation teeth of zebrafish is entirely atubular (Huysseune et al. 1998), as in the armoured catfish (Huysseune and Sire 1997b) and the dog salmon (Sasagawa and Igarashi 1985). The odontoblastic processes in juvenile zebrafish are surrounded by collagen-containing dentine, which lacks mineralization. We are not sure whether or not this is due to a possibly different composition or to retarded mineralization of the dentine surrounding the tubule. In many mammalian species, including humans, odontoblastic processes are surrounded by mineralized, peritubular dentine lacking collagenic components (Goldberg et al. 1995). Huysseune et al. (1998) reported dentine remodelling along the tooth shaft in larval zebrafish. Evidence of such a remodelling has been observed in a number of replacement teeth at the LM level (C. Van der heyden, personal observation), but has not been further explored in this study. Attachment, eruption and shedding The attachment bone in juvenile zebrafish is formed, at least partly, by cells resembling those producing the dentine, as described for larvae (Huysseune et al. 1998). Both dentine and attachment bone matrix are indistinguishable once mineralized, but their unmineralized precursor differs: (1) the collagen fibrils have a different orientation (in dentine: densely packed and mostly following the tooth axis; in attachment bone: less densely packed and more randomly spread); (2) an osteoid matrix is present at both inner and outer sides of the attachment bone (in dentine: predentine only at the inner surface); and (3) patches of electron-dense background substance appear prior to mineralization in the bone of attachment (dentine: without these patches). Comparable differences have been described for larval zebrafish (Huysseune et al. 1998), for cichlids (Huysseune and Sire 1997a), and for callichthyids (Huysseune and Sire 1997b). In all these species, only the cervical loop tip indicates the limit between the mineralized dentine and the mineralized attachment bone. Cheprakova (1958) was also unable to distinguish attachment bone from dentine in first-generation and replacement teeth in several cyprinid species, again indicating the morphological similarity between the different mineralized tissues. In adult (decalcified) zebrafish teeth a difference in toluidine blue coloration distinguishes supporting bone and attachment bone/dentine (C. Van der heyden, personal observation), suggesting that the attachment bone is formed anew with every tooth and is fused to the dentigerous (jaw) bone only afterwards. Despite numerous observations, we found no ultrastructural indications on how eruption could occur. By

measuring the tooth size in juvenile zebrafish immediately prior to and after eruption, we could exclude tooth growth as a factor in eruption (C. Van der heyden, personal observation). Furthermore, the tooth is fully ankylosed to the supporting bone, just before eruption occurs. Therefore, we suggest that, as in larvae (Huysseune et al. 1998), eruption may occur through a remodelling of the pharyngeal epithelium. We refer the reader to Huysseune et al. (1998) for further discussion. After a certain time of functionality, the tooth is shed. This action occurs after resorption of dentine, attachment bone and part of the supporting bone. In this paper, we also demonstrate that resorption of the tooth base occurs at both inner and outer surfaces. Often the area of resorption is closely adjoined by the developing germ of the successor. It is not excluded that the developing germ acts as a trigger for resorption. In the anterior part of the mandible in cichlids, developing teeth were thought to trigger resorption of the adjacent Meckel’s cartilage (Huysseune and Sire 1992). The cells attacking the outer surface of the teeth and their attaching structures are mononucleated osteoclasts with features described by Sire et al. (1990). Mononucleated osteoclasts occur in larval zebrafish as well (Huysseune et al. 1998). In other cyprinids, multinucleate cells have been reported to resorb both dentine and bone (Cheprakova 1958). There are some other literature reports of tooth destruction by clastic cells in other teleosts (e.g. Bergot 1975; Berkovitz 1978; Berkovitz and Shellis 1978). In the tooth pulp, a particular type of cell has been found with likely clastic activity. It is not clear whether these cells are originally pulpal cells or whether they have invaded the dental papilla from the surrounding mesenchyme. In mammals and humans, clastic cells attacking the dentinal hard tissues (cementum, dentine and enamel) have been referred to as odontoclasts or dentinoclasts and are active in the tooth pulp (Sasaki et al. 1989; Matsuda 1992; Domon et al. 1994; Sahara et al. 1992, 1994, 1996). Mammalian odontoclasts are characterized by a ruffled border and a clear zone. In general, they contain numerous mitochondria, moderate amounts of RER, stacks of Golgi membranes, lysosomes and numerous polyribosomes (Sasaki et al. 1989; Domon et al. 1994; Sahara et al. 1994, 1996). Exceptions to the cellular contents occur, but this might be related to their state of differentiation. Both mononucleated (Sasaki et al. 1989; Domon et al. 1994; Sahara et al. 1996) and multinucleated (Sasaki et al. 1989; Domon et al. 1994; Sahara et al. 1992, 1994, 1996) odontoclasts occur in mammals, the mononucleated clasts probably being the precursors of multinucleated ones (Sahara et al. 1996). In all cases, both mononuclear and multinucleated odontoclasts were active in tooth shedding. The pulpal cells involved in tooth resorption in zebrafish clearly differ morphologically from mammalian odontoclasts. Summarizing, replacement teeth in zebrafish differ from first-generation teeth in a number of significant aspects. Most importantly, the different site of origin of the germ suggests different underlying developmental mech-

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anisms involved in initiation. Structural differences include the presence of dentinal tubules, of pulpal blood vessels (C. Van der heyden, personal observation), distinct dentinal collagen bundles, the presence of a distinct enameloid precursor, all of which possibly relate to the larger size of the teeth. Other structural attributes are similar but less pronounced, such as the polarization of the basal epithelial cells prior to invagination. Finally, some structural features deserve further attention notably the occurrence of the peculiar clastic cells in the pulp cavity and the occurrence of internal remodelling of the dentine. This ultrastructural description of developing zebrafish replacement teeth provides the necessary morphological knowledge with which to plan and interpret experiments on the molecular control of tooth development in this model species. Acknowledgements Mrs. G. De Wever and Mrs. F. Allizard are gratefully acknowledged for their expert technical assistance in preparing the sections. Dr. D. Adriaens is thanked for drawing the zebrafish cranium (Fig. 1A). The TEM work and preparation of the photographic prints were carried out at the Centre Interuniversitaire de Microscopie Electronique (CIME) Jussieu, Paris 6 and 7. The paper has benefited from discussions within the framework of the COST action B8 on Odontogenesis, and from the suggestions of two anonymous reviewers.

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