DEVELOPMENTAL DYNAMICS 230:727–733, 2004

PATTERNS & PHENOTYPES

Cellular Expression of eve1 Suggests Its Requirement for the Differentiation of the Ameloblasts and for the Initiation and Morphogenesis of the First Tooth in the Zebrafish (Danio rerio) Patrick Laurenti,1 Christelle Thae ¨ ron,2 Franc¸oise Allizard,3 Ann Huysseune,4 and Jean-Yves Sire3*

even-skipped-related (evx) genes encode homeodomain-containing transcription factors that are involved in a series of developmental processes such as posterior body patterning and neurodifferentiation. Although evx1 and evx2 were not reported to be expressed during mammalian tooth development, we present here evidence that eve1, the closest paralog of evx1 in the actinopterygian lineage, is expressed during pharyngeal tooth formation in the zebrafish, Danio rerio. We have performed whole-mount in situ hybridization on zebrafish embryos and larvae ranging from 24 to 192 hours postfertilization (hpf). A detailed analysis of serial sections through the pharyngeal region of whole-mount hybridized and control specimens indicates that only dental epithelial cells express eve1. eve1 transcription was activated at 48 hpf, in the placode of the first tooth (i.e., the initiation site of tooth 4V1), and maintained in the dental epithelium throughout morphogenesis. Then, by 72 hpf, eve1 expression was restricted to the differentiating ameloblasts of the enamel organ during early differentiation stage, and this expression decreased as soon as matrix was deposited. In subsequent primary teeth (3 V1 and 5 V1) as well as in their successors (replacement teeth 4V2, 3V2, and 5V2), eve1 expression was restricted to the differentiating ameloblasts and, again, disappeared when matrix was deposited. Therefore, in the zebrafish, eve1 expression in the pharyngeal region is correlated with two key steps of tooth development: initiation and morphogenesis of the first tooth, and ameloblast differentiation of all developing teeth. Developmental Dynamics 230:727–733, 2004. © 2004 Wiley-Liss, Inc. Key words: eve1; even-skipped; tooth initiation; ameloblast differentiation; tooth development; Danio rerio Received 5 November 2003; Revised 15 January 2004; Accepted 2 February 2004

INTRODUCTION During the past decade, extensive studies of mammalian tooth development have led to the accumulation of a large set of molecular data. To date, more than 120 genes are known to be expressed in the epithe-

lium and/or mesenchyme during the different steps of tooth formation (see Nieminen et al., 1998; and Web site: http://bite-it.helsinki.fi). Not only have gene expression patterns been characterized, but also functional studies have enabled us to depict the ge-

netic control of tooth development in mammals (Chen et al., 1996; Keranen et al., 1998; Kettunen et al., 2000; Stock, 2001). This amount of information stands in sharp contrast with the established fact that virtually nothing is known at the molecular level on

Populations, Ge´ne´tique et E´volution, Gif-sur-Yvette, France Neurobiologie Ge´ne´tique et Inte´grative, Gif-sur-Yvette, France 3 Equipe “Evolution et De´veloppement du Squelette”, Universite´ Paris 6, France 4 Biology Department, Ghent University, Belgium Grant sponsor: Centre National de la Recherche Scientifique; Grant sponsor: Ghent University; Grant number: 011V1298. Drs. Laurenti and Thae¨ron contributed equally to this work. *Correspondence to: Jean-Yves Sire, Universite´ Paris 6, CNRS FRE 2696, Case 7077, 7, Quai St-Bernard, 75251 Paris cedex 05, France. E-mail: [email protected] 1 2

DOI 10.1002/dvdy.20080 Published online 3 June 2004 in Wiley InterScience (www.interscience.wiley.com).

© 2004 Wiley-Liss, Inc.

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tooth development in nonmammalian species. In chondrichthyans, lungfishes, lissamphibians, sauropsids, and actinopterygians, all lineages in which teeth are replaced several times during the life span, the published data on tooth development are quite restricted to morphological descriptions (reviewed in Sire et al., 2002). The only two exceptions concern the report of parvalbumine3A (pvalb3a) expression in zebrafish (Danio rerio) tooth germs (Hsiao et al., 2002) and the demonstration of the functional requirement for Alk8 in zebrafish tooth development (Payne et al., 2001). Note that the expression of engrailedlike antigens reported by Hatta et al. (1991) have not been yet confirmed by transcript assays. This lack of data is probably due to the difficulties related to studying tooth development in zebrafish. Indeed, during evolution, cyprinids have lost their teeth in the buccal cavity, and their teeth are restricted to the pharyngeal region (attached to the fifth ceratobranchials). Moreover, the pharyngeal teeth are small, and they appear and replace quickly in complex patterns that have been only elucidated recently. They develop in three rows: the ventral (V), mediodorsal (MD), and dorsal (D) rows, with 5 (1V to 5V), 4 (1MD to 4 MD), and 2 teeth (1D and 2D), respectively (Huysseune et al., 1998; Van der Heyden and Huysseune, 2000). In the course of a detailed survey of the even-skipped-related (evx) genes expression in the zebrafish, some of us have noticed the presence, from 48 to 96 hr postfertilization (hpf), of an eve1 signal in bilateral cell clusters corresponding to tooth germs (Avaron et al., 2003). The evx gene family contains two paralogs in tetrapods (evx1 and evx2, linked to the HoxA and HoxB complexes, respectively; Bastian and Gruss, 1990; D’Esposito et al., 1991; Faiella et al., 1991; Bastian et al., 1992; Dush and Martin, 1992) and three paralogs in actinopterygians (evx1, evx2, and eve1) linked to the hoxaa, hoxd, and hoxba complexes, respectively (Joly et al., 1993; Sordino et al., 1996; Amores et al., 1998; Thaeron et al., 2000; Avaron et al., 2003). eve1 is phylogenetically more closely related to evx1 than to evx2 and was found only in acti-

nopterygians (Amores et al., 1998; Avaron et al., 2003). The finding of an evx gene expression in forming teeth was surprising. First, because evx genes are expressed in a wide array of developing structures such as posterior gastrula, the growth zone of the tail, the proctodeum, and posterior part of the gut, and several subsets of specific neurones entering their terminal differentiation (Bastian and Gruss, 1990; Dush and Martin, 1992; Joly et al., 1993; Dolle ´ et al., 1994; Thaeron et al., 2000; Ferrier et al., 2001; Moran-Rivard et al., 2001; Avaron et al., 2003), but no evx gene expression were ever reported in the dentigerous region of vertebrate species. Second, extensive studies were carried out to identify homeobox-containing genes involved in tooth development in the mouse and failed to detect an evx gene expression in teeth. With the aim to establish the relationships between the location of the eve1 transcripts and the level of cell and tissue differentiation during the various steps of tooth development, we combined detailed observations of serial sections of eve1-hybridized embryos, and nonhybridized embryos prepared for histological observation. Our study period (24 to 192 hpf) covers the development of the first three teeth and of their successors (replacement teeth). These teeth develop in three of the five ventral positions, starting with position 4V at 48 hpf (the tooth is thus called 4V1, superscript indicating the generation number of the tooth), followed by teeth 3V1 and 5V1 at 56 hpf. Next, the first replacement tooth appears in position 4V (4V2) at 80 hpf, followed by replacement teeth 3V2 and 5V2 at 144 hpf. The teeth at other positions appear later during ontogeny (12 days postfertilization onward; Van der Heyden and Huysseune, 2000). Between 48 hpf and 192 hpf, teeth 4V1, 3V1, and 5V1 fully develop and attach, while teeth 4V2, 3V2, and 5V2 fully form but do not yet attach. The development of each tooth progresses through phases that can be identified morphologically (for further details, see Huysseune et al., 1998). At 48 hpf, the pharyngeal epithelium forms a placode, a local thickening composed of high and cy-

lindrical polarized cells, that reveals the site of initiation of tooth 4V1. Note that the replacement teeth (4V2, 3V2, and 5V2) do not display a placode stage, and bud off directly from the reduced dental epithelium of their predecessors. All the teeth undergo a phase of morphogenesis, during which the dental epithelium invaginates into the underlying mesenchyme. It progressively forms a bellshaped enamel organ composed of two cell layers: the inner- and outerdental epithelium (IDE and ODE, respectively). This process is followed by a phase of early differentiation, during which the IDE cells (the ameloblasts) and the mesenchymal cells facing them (the odontoblasts) start their differentiation. During the late differentiation phase, the ameloblasts and the odontoblasts deposit the tooth matrices (enameloid and dentin, respectively). In the last phase, the deposition of matrix at the tooth base results in ankylosis of the tooth to the bone support, and the tooth becomes attached (phase of attachment). We show here that eve1 expression is activated in the placode of the first tooth to be formed (4V1) and that the transcription is maintained in its whole dental epithelium throughout the morphogenesis phase, but then is becoming restricted to the ameloblasts entering their terminal differentiation. eve1 transcription ceases as soon as these cells are fully differentiated. In the other teeth to be formed, i.e., new tooth positions (3V1 and 5V1) and replacement teeth (4V2, 3V2, and 5V2), eve1 is not expressed at early phases of odontogenesis, and expression is strictly restricted to the early differentiating ameloblasts. This cellular expression suggests that eve1 is required during two steps of odontogenesis in the zebrafish: initiation of the first tooth, and differentiation of the ameloblasts of this tooth and of all the subsequent teeth.

RESULTS AND DISCUSSION Overall Description of eve1 Expression in the Pharyngeal Region To characterize precisely the pattern of eve1 transcripts during tooth formation, we performed whole-mount

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in situ hybridization of 24-, 36-, 40-, 44-, 48-, 56-, 72-, 80-, 96-, 120-, 144-, 168-, and 192-hpf developing zebrafish embryos and larvae. For each stage, at least 30 specimens were carefully observed as wholemounts, and at least five specimens were selected for sectioning and further analysis (see below). We carefully checked for eve1 expression in the oral jaws, and we did not detect any signal, nor in whole-mount hybridized specimens, neither in sections of the mandibular region of 44to 80-hpf of hybridized specimens (not shown). No eve1 transcription was detected in the pharyngeal region of 24 – 44 hpf embryos, whereas an eve1 signal was observed in the tail, thus providing a valuable control (not shown). The transcription of eve1 was activated at approximately 48 hpf and was easily detectable in wholemounts up to 96 hpf. In older specimens, the signal was difficult to observe through the skin and, therefore, apparently weak. All 48 hpf embryos examined displayed a single, bilateral, and strongly stained eve1-expressing cell cluster in the pharyngeal region (Fig. 1A). This small and rounded cell cluster was located in the ventral part of the pharynx at the level where tooth 4V1 is developing. From 72 to 80 hpf, while the signal disappeared in the first eve1⫹ cell cluster, two similar-sized spots appeared in the locations where teeth 3V1 and 5V1 are developing (Fig. 2A).

Initiation, Morphogenesis, and Differentiation of the First Tooth: 4V1 To identify accurately the location of the eve1-expressing cells during tooth development, we performed serial sections of both whole-mount hybridized zebrafish and nonhybridized specimens of the same stages, and used the latter for morphological analysis. In the presumptive dentigerous region of 48 hpf larvae, eve1 transcription activated in a well-delimited region of the pharyngeal epithelium. The labeled cells were located on both sides of the midline, between the presumptive location of the pharyngeal lumen and the ventral basement mem-

brane separating the epithelium from the underlying mesenchyme (Fig. 1B). The population of labeled cells stretched for approximately 24 ␮m in anterior–posterior direction and approximately 20 ␮m in transverse direction, and strongly expressed eve1. No transcripts were identified elsewhere in the pharyngeal epithelium, nor in the surrounding mesenchyme or in any other area on the sections. Analyses of the nonhybridized specimens showed that the pharyngeal epithelium was approximately two to three cell layers thick. Ventrally, on both sides of the midline, a local thickening of the pharyngeal epithelium (called placode) was observed. The placode cells were cylindrical, polarized, and had a large nucleus. They could not be confused with the densely packed mesenchymal cells. This placode corresponded to the initiation site of tooth 4V1, and the eve1-expressing cells belong to this placode (Fig. 1B,C). However, the morphological analysis showed that the placode was slightly shorter (approximately 20 ␮m in an anterior– posterior direction), indicating that some eve1-expressing cells could be located outside of it. In 56 hpf specimens, a strong expression of eve1 was observed in a rounded cell cluster (24 –30 ␮m in diameter) associated with the ventral pharyngeal epithelium (not shown). In general, the cell cluster expressing eve1 did not reach the epithelial– mesenchymal boundary but was separated from it by a layer of unlabeled cells of the pharyngeal epithelium. No other region of the pharyngeal epithelium was labeled in any of the series examined at this stage. Analysis of the nonhybridized control specimens showed that the early differentiation phase of tooth 4V1 had started. The eve1-expressing cells corresponded to the cells of the whole dental epithelium of tooth 4V1, that was, in most cases, separated from the underlying mesenchyme by a layer of pharyngeal epithelium. Then, the IDE and ODE cell layers formed within the dental epithelium and the eve1 transcription ceased in the ODE cells. At 72 hpf, the eve1 expression progressively restricted to the IDE cells that had be-

gun their differentiation into ameloblasts (Fig. 1D,E), the cells partly responsible for the deposition of the enameloid matrix. By 80 hpf, the transcription of eve1 ceased in the ameloblasts of tooth 4V1 that had undergone their late phase of differentiation, as indicated by the amount of dental matrix deposited (Fig. 2B,E). At 72 hpf, medial and lateral to tooth 4V1, teeth 3V1 and 5V1 have started their morphogenesis and are present as young tooth germs in control specimens. It is striking that, unlike tooth 4V1, these second and third teeth did not express eve1 at the early morphogenesis stage. One possible explanation would be that the initiation of teeth 3V1 and 5V1 does not need an induction step. Accordingly, we did not observe distinct placodes for teeth 3V1 and 5V1 in nonhybridized specimens. Another possibility is suggested by the fact that the spatial extent of the eve1 signal is slightly larger than the extent of the 4V1 placode in controls. eve1-expressing cells on both sides of the 4V1 placode, therefore, would correspond to 3V1 and 5V1 initiation loci.

Differentiation of Subsequent (3V1 and 5V1) and Replacement (4V2, 3V2, and 5V2) teeth In some 72 hpf and in most 80 hpf specimens, two new, bilateral cell clusters expressed eve1 (Fig. 2A–E). These two cell populations were located ventral to the pharyngeal epithelium and lie approximately 10 –15 ␮m apart in an anteroposterior direction and 18 –21 ␮m apart in a dorsal–ventral direction. The most anterior eve1-expressing cells appeared bilaterally as a rounded cell cluster (6 to 10 cells in the section) medial to the tooth 4V1(Fig. 2B). On control sections, these cells corresponded to the early differentiating ameloblasts of tooth 3V1 (Fig. 2C). The more posterior, symmetrical cell population showed a strong signal, similar to that described in Figure 2B, but the two small patches of cells laid further apart from each other, more dorsally and lateral to tooth 4V1 (Fig. 2D). In control specimens,

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Fig. 1

these clusters corresponded to early differentiating ameloblasts of tooth 5V1, located dorsal and lateral to each tooth 4V1 (Fig. 2E). As described for teeth 3V1 and 5V1, we did not detect any eve1 transcripts during the morphogenesis stage of replacement teeth. At 120 hpf, tooth 4V1 had formed and attached (not shown). Both teeth 3V1 and 5V1 were in a phase of late differentiation (matrix has been deposited, but the teeth were not still attached), and no eve1 transcription was detected in these teeth

(Fig. 2F). However, a new signal was observed on both sides of the midline in the pharyngeal region (Fig. 2F). The eve1-expressing cells formed a crescent-shaped cell cluster, that corresponded to the early differentiating ameloblasts of the first replacement tooth, 4V2. There were no other epithelial or mesenchymal cells labeled in this area. In controls, tooth 4V2 had started its phase of differentiation, and some tooth matrix had even been deposited (Fig. 2G). At 168 hpf and 192 hpf, eve1 was no longer transcribed in late-

Fig. 1. eve1 expression in 4V1, the first tooth to develop in the zebrafish. A: 48 hours postfertilization (hpf), activation of the transcription of eve1 in tooth 4V1. Top: In situ hybridisation (ISH) with probe to eve1 (ISH eve1), whole-mount, lateral view. Bottom: Interpretative drawing. The first bilateral eve1-expressing cells cluster (approximately 20 ␮m in diameter) is located in the ventral part of the pharynx, just posterior and dorsal to the fifth branchial arch (arrow). Pharyngeal arches are numbered (I to V). B–E: Transverse sections through the pharyngeal region, at the level of the Vth branchial arch. Left panels: ISH eve1; middle panels: camera lucida drawing of the mirror image of the section; right panels: control, nonhybridised specimen. B,C: At 48 hpf. Placode stage of tooth 4V1 (initiation/early morphogenesis) (arrows). D,E: At 72 hpf. Early differentiation stage (differentiation of the inner dental epithelium cells) of tooth 4V1 (arrows). On controls, tooth 4V1 appears as a placode at 48 hpf (C, arrow). By 56 –72 hpf, the ameloblasts of tooth 4V1 differentiate (E, arrow). The arrowheads point to the regions in which teeth 3V1 and 5V1 start to form (out of focus). e, epidermis; nc, notochord; oc, otic capsule; phe, pharyngeal epithelium; y, yolk. Scale bars ⫽ 100 ␮m in A, 50 ␮m in B–E. Fig. 3. Schematic representation of eve1 expression during tooth development. In tooth 4V1, five phases can be morphologically distinguished: (1) initiation (⫽ placode stage), (2) morphogenesis, (3) early differentiation, (4) late differentiation, (5) attachment. Expression domains of eve1 are figured in blue. The transcription of eve1 is activated in the whole placode at the initiation stage (1), then it is maintained throughout morphogenesis in the whole dental epithelium (2). When the ameloblasts started to differentiate, eve1 expression becomes restricted to the inner dental epithelium (3). eve1 expression ceases as soon as the ameloblasts are fully differentiated (4). As shown by matrix deposition, ameloblasts differentiate in an apex to base wave. In some sections, the basal-most, thus not fully differentiated, ameloblasts still express eve1 (4). No sign of eve1 activity is detected at the attachment stage (5). In subsequent teeth, phase 1 is not distinguishable and eve1 expression is restricted to the early differentiating ameloblasts (phases 3 and 4). c.b.V, ceratobranchial Vth; d.e., dental epithelium; d.p, dental papilla; i.d.e, inner dental epithelium; me, mesenchyme; o.d.e, outer dental epithelium; p, placode; p.c, pulp cavity; ph.e, pharyngeal epithelium; t.m, tooth matrix; *, enameloid cap.

Fig. 3

Fig. 2. eve1 expression in differentiating ameloblasts of first generation (3V1 and 5V1) and replacement (4V2, 3V2 and 5V2) teeth. A–E: At 80 hours postfertilization (hpf). A: Transcription of eve1 in teeth 3V1 and 5V1. Top: ISH eve1, whole-mount, lateral view. Bottom: Interpretative drawing. While the transcription has ceased in tooth 4V1, two new bilateral eve1-expressing cell clusters appear that correspond to the forming teeth 3V1 and 5V1 (arrows). B–E: Transverse sections through the pharyngeal region at the level of the Vth branchial arch. Left panels: In situ hybridization (ISH) eve1; middle panels: camera lucida drawing of the mirror image of the section; right panels: control, nonhybridised specimen. Early differentiation stage of teeth 3V1 (B, C), 5V1 (D,E). The arrows point to tooth 4V1, in which dental matrix has accumulated. C,E: the arrowheads indicate the location of teeth 3V1 and 5V1, respectively. F,G: At 120 hpf. Early differentiation stage of replacement tooth 4V2. Left panel: ISH eve1; right panel: control, nonhybridised specimen. In G, the black arrows point to teeth 3V1, the white arrowheads to teeth 5V1, and the white arrows to tooth 4V2. H,I: At 168 hpf. Early differentiation stage of replacement teeth 3V2 and 5V2, respectively. Left panels: ISH eve1; right panels: camera lucida drawings of the mirror image of the section. pf, pectoral fin; other abbreviations as in Figure 1A. Scale bars ⫽ 100 ␮m in A, 50 ␮m in B–I.

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differentiating tooth 4V2, and teeth 3V1 and 5V1 were attached. Two new signals were observed on both sides of the midline in the pharyngeal region, and they succeed each other anterior–posteriorly. On control sections, the more anterior signal, medial to tooth 4V2 and close to the posterior side of tooth 3V1, corresponded to the location of early differentiating tooth 3V2 (Fig. 2H). The second signal stained the early differentiating ameloblasts of tooth 5V2 that was located more posteriorly in the series, and lateral to tooth 4V2 (Fig. 2I). In both cases, eve1 expression was detected in only a few cells.

Concluding Remarks Our study shows that eve1 is activated in the pharyngeal epithelium while the placode of tooth 4V1is forming. Expression is then maintained in the whole dental epithelium throughout morphogenesis but progressively becomes restricted to the early differentiating ameloblasts (Fig. 3). In all tooth germs developing after tooth 4V1, eve1 expression is strictly restricted to the differentiating IDE cells, i.e., the ameloblast layer facing the differentiating dental papilla cells in the mesenchyme. The surrounding cell layer, the outer dental epithelium (ODE), never shows any signal. Thus, except for the first tooth 4V1, eve1 expression is restricted to a particular phase of odontogenesis, namely cytodifferentiation, covering the period of ameloblast differentiation. Therefore, in the zebrafish, we can suppose that eve1 is involved during the differentiation process of the ameloblasts in all developing teeth, whatever their position and generation number (Fig. 3). In addition, there is a clear correlation between the expression domain of eve1 and the level of differentiation of the ameloblasts. Indeed, the entire IDE is not at the same stage of differentiation: the ameloblasts fully differentiate in a wave that spreads from the apex to the base of the IDE. eve1 is upregulated at the end of the phase of morphogenesis, at a moment when the IDE becomes clearly distinguishable from the ODE, and when the

IDE cells facing the dental papilla cells are starting their differentiation into ameloblasts. Then, as ameloblasts are fully differentiated, eve1 is down-regulated in the apex areas of the IDE where a thin layer of matrix has been deposited, whereas the expression is maintained at the base of the IDE, where ameloblasts are not fully differentiated (Fig. 3). Taken together, our results suggest that eve1 is required for the initiation and morphogenesis of tooth 4V1, and for the differentiation of the ameloblasts of all forming teeth, including replacement ones. The involvement of an evx gene in a developmental process during both the initiation and terminal differentiation phases is not surprising. The even-skipped (eve) gene of Drosophila, for instance, was first identified on the basis of its very early role in the initiation of the segmentation of the insect body (Nusslein-Volhard and Wieschaus, 1980). It was next demonstrated to control, in a later phase, terminal differentiation and axonogenesis of specific neurones (Doe et al., 1988). Similarly, evx1 and eve1 in zebrafish, and evx1 in mice were shown to be involved in the determination of the posterior part of the gastrula, in the initiation of the growth of the tail and in the terminal differentiation of specific interneurones (Bastian and Gruss, 1990; Dush and Martin, 1992; Joly et al., 1993; Barro et al., 1995; Thaeron et al., 2000; Moran-Rivard et al., 2001). In tetrapods, evx1 displays the ancestral pattern of evx1, while in the actinopterygian lineage, the ancestral pattern has been distributed between eve1 and evx1 (Avaron et al., 2003). It is thus somewhat surprising that no evx1 (nor evx2) expression has been reported during tooth development in mammals. It is unlikely that an evx signal in mouse teeth was missed in previous studies, because evx genes expression has been studied for more than a decade (Bastian and Gruss, 1990; Dush and Martin, 1992; Dolle ´ et al., 1994; Mansouri and Gruss, 1998; Moran-Rivard et al., 2001; Pierani et al., 2001), and because tooth development in mice has been extensively characterized at the molecular level, leading to the identification of a large

number of transcription factors involved in this developmental process (Nieminen et al., 1998). The absence of an evx gene expression in mammalian tooth development could lead to two evolutionary hypotheses: (1) oral and pharyngeal dentitions are derived from a common osteichthyan ancestor, different regulatory pathways have been selected to control ameloblast differentiation in tetrapod and actinopterygian lineages; or (2) despite their morphological resemblance, oral and pharyngeal teeth are not homologous elements. In this case, evx1 could have been only involved in pharyngeal tooth development in the common ancestor of the osteichthyans and not required for oral tooth formation.

EXPERIMENTAL PROCEDURES Biological Material Zebrafish (Danio rerio) were raised at a temperature of 28.5°C under a 14/10 day/night cycle. Developing embryos, larvae and juveniles were fixed at 24, 36, 40, 44, 48, 56, 72, 80 hpf, and every day from day 4 (96 hpf) to day 8 (192 hpf; a total of 13 stages). These stages were chosen to cover the most relevant steps of developing first-generation teeth as well as their successors according to the pattern described by Huysseune et al. (1998) and by Van der Heyden and Huysseune (2000).

Whole-Mount In Situ Hybridization For each stages, 30 specimens were fixed overnight at 4°C in a phosphate-buffered saline solution (PBS) containing 4% paraformaldehyde (PFA), and then stored at ⫺20°C in methanol. Whole-mount in situ hybridization (ISH) was performed as described by Thisse et al. (http:// www-igbmc.u-strasbg.fr/zf_info/ zfbook/chapt9/9.82.html). The eve1 probe consisted of a fragment containing the entire coding region and was used as described in Thaeron et al. (2000). After hybridization, the larvae were post-fixed in 4% PFA and photographed in lateral and ventral view, after removal of the yolk sac when necessary.

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Histological Preparations Whole-mount hybridized specimens were dehydrated, embedded in Epon, and 5 ␮m-thick serially sectioned by using a diamond knife. Sectioning was as perpendicular as possible to the anterior–posterior axis from the snout tip backward. The sections were mounted unstained and observed and photographed under Nomarski light. For each stage, control specimens (taken from the same batch as the corresponding ISH specimens) were immersed in 0.1 M cacodylate buffer containing 1.5% glutaraldehyde and 1.5% PFA for 2 hr at room temperature, post-fixed in osmium tetroxide, dehydrated, and embedded in Epon. One-micrometer-thick, transverse, serial sections were obtained in the pharyngeal region and stained with toluidine blue.

ACKNOWLEDGMENTS We thank Ve ´ ronique Borday and Didier Casane for their help, fruitful discussions, and critical reading of the manuscript. A.H. was funded by a grant from the ‘Bijzonder Onderzoeksfonds’ of Ghent University.

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