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Ameloblasts Express Type I Collagen during Amelogenesis N. Assaraf-Weill, B. Gasse, J. Silvent, C. Bardet, J.-Y. Sire and T. Davit-Béal J DENT RES 2014 93: 502 originally published online 25 February 2014 DOI: 10.1177/0022034514526236 The online version of this article can be found at: http://jdr.sagepub.com/content/93/5/502
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research-article2014
JDR
93510.1177/0022034514526236
Research Reports Biological
N. Assaraf-Weill1, B. Gasse1, J. Silvent1, C. Bardet1,2, J.-Y. Sire1, and T. Davit-Béal1*
Ameloblasts Express Type I Collagen during Amelogenesis
1
UMR 7138-SAE, Research Group “Evolution & Development of the Skeleton”, Université Pierre et Marie Curie, 7 quai St-Bernard, Case 5, 75005 Paris, France; and 2EA 2496, Faculté de Chirurgie Dentaire, Université Paris Descartes, 1 rue Maurice Arnoux, 92120 Montrouge, France; *corresponding author, [email protected] J Dent Res 93(5):502-507, 2014
Abstract
Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while postmetamorphic teeth possess enamel only. In firstgeneration teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.
KEY WORDS: amphibian, Pleurodeles waltl,
odontogenesis, enameloid, amelogenin, in situ hybridization.
DOI: 10.1177/0022034514526236 Received October 14, 2013; Last revision January 13, 2014; Accepted February 9, 2014
Introduction
T
he nature, evolutionary origin, and relationships of the highly mineralized tissues covering teeth in vertebrates, enameloid and enamel, have long been debated. Some authors considered that enamel was present in first vertebrates (Smith, 1992), but most scientists believed that enamel derived from enameloid (Poole, 1967; Kawasaki et al., 2005). This controversy could explain why enameloid formation – and whether it is only a mesodermal or an ectodermal-mesodermal tissue – attracted authors’ attention. In living vertebrates, enameloid is present in chondrichthyans (Sasagawa, 2002) and in actinopterygians (Shellis and Miles, 1974; Kawasaki et al., 1987), while in sarcopterygians it is found only in larval stages of caudate amphibians (Poole, 1967; Davit-Béal et al., 2007). Enamel has been documented in the sarcopterygian lineage (Schmidt, 1970; Sander, 2001; Kemp, 2002), and also described in a few actinopterygian lineages (Ishiyama et al., 2001; Sasagawa et al., 2012). In contrast to enameloid, which is recognizable by its loose network of collagen fibrils, enamel matrix is devoid of collagen and is composed mainly of enamel matrix proteins (EMPs): amelogenin (AMEL), enamelin (ENAM), and ameloblastin (AMBN) (Sire et al., 2007; Moradian-Oldak, 2012). Enamel is only synthesized by ameloblasts. Given the presence of collagen fibrils, enameloid has been considered a unique product of odontoblasts, but a contribution from ameloblasts was proposed in chondrichthyans (Sasagawa, 2002), actinopterygians (Sasagawa et al., 2006), and caudate larvae (Davit-Béal et al., 2006, 2007). These hypotheses were supported by similarities in mineralization and maturation processes, which imply the presence of EMPs and proteases, and also included a direct contribution from ameloblasts to collagen synthesis. In caudates, prior to metamorphosis, enameloid, followed by enamel, forms in larval teeth, while only enamel is present in post-metamorphic juvenile teeth. These amphibians offer unique possibilities for the study of the enameloid-to-enamel transition, a process that has probably occurred similarly during vertebrate evolution. Previously, in the newt Pleurodeles waltl, we demonstrated that the disappearance of enameloid was the result of a slowing of odontoblast activity, whereas enamel progressively thickened through increasing ameloblast activity (Davit-Béal et al., 2007). Here, using P. waltl, we wondered whether (i) enameloid contained EMPs in addition to collagen, and (ii) ameloblasts participated in collagen synthesis.
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
Materials & Methods
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See the Appendix for a detailed description of materials and methods.
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J Dent Res 93(5) 2014 503 Ameloblasts Express Type I Collagen during Amelogenesis Biological Material Pleurodeles waltl is routinely bred in our laboratory. One hundred specimens, ranging from embryos to 13-month-old juveniles, were used for our study. All specimens were deeply anaesthetized (MS222) and sacrificed according to the guidelines of the French Ethics Committee.
Histology The materials and histological procedures for obtaining 12-µm-thick, then ultrathin, sections for transmission electron microscopic (TEM) observations were previously described (Davit-Béal et al., 2007). Also see the Appendix.
PCR Total RNAs were extracted from specimens at stages 30, 33, 34, and 36 (whole embryo) and from juveniles (mandibular jaw). cDNAs obtained by RT-PCR were amplified and sent to GATC Biotech AG (Konstanz, Germany) for sequencing.
Real-time PCR (qPCR) qPCR was performed with AMEL primers and specific primers for the housekeeping GAPDH gene (Bascove and Frippiat, 2010).
In situ Hybridization (ISH) Antisense and sense (control) AMEL and COL1A1 RNA probes were synthesized as described in the Appendix. Briefly, the samples were fixed in Formoy solution, demineralized in acetic acid, embedded in Paraplast, and sectioned (10 µm thick). The sections were de-waxed, digested in proteinase K, fixed in paraformaldehyde, and hybridized overnight. They were then rinsed and labeled with Digoxigenin overnight. Finally, the samples were rinsed, the signal was revealed, and the slides were mounted and photographed.
Results Serial jaw-sectioning allowed for the identification of various stages of tooth development, i.e., enameloid and/or enamel cap formation, dentin cone formation, and attachment to bone support (Fig. 1A).
PCR and qPCR Using PCR and qPCR, we monitored only AMEL expression through successive steps of enameloid and enamel formation. These experiments would not be relevant for COL1A1, since it is expressed in various jaw tissues. We studied AMEL expression only in first-generation teeth, which initiate in embryos and become functional in early larvae, because all teeth were from the same generation and all possessed well-developed enameloid. We also performed PCR and qPCR in a post-metamorphic juvenile, because all teeth possessed enamel only.
Figure 1. Monitoring of AMEL expression in Pleurodeles waltl (Pw) during development of a first-generation tooth (tooth 1) and in a juvenile tooth. (A) Schematic drawings of tooth 1 odontogenesis and of a well-developed bicuspid juvenile tooth (4-month-old specimen). Stage 33 (Pw33), tooth bud prior to matrix deposition; Pw34, enameloid deposition; and Pw36, well-formed tooth, shortly before attachment. (B) PCR of AMEL and GAPDH (housekeeping gene). (C) Real-time PCR. AMEL expression in the developmental stages illustrated in (A). Pw30 (stage 30): no teeth in the mandibular and maxillary jaws. *p < .05. De, dentin; Dp, dental papilla; En, enamel; Eno, enameloid; Eo, enamel organ.
PCR revealed that AMEL was not expressed before tooth 1 initiation (stage 30). The expression was weak during ameloblast differentiation (stage 33), and strong in further developmental stages (Fig. 1B). qPCR indicated that (i) a few transcripts were present before tooth initiation, (ii) expression was weak prior to enameloid deposition, (iii) expression increased 10-fold when ameloblasts and odontoblasts had differentiated and enameloid matrix was deposited, and (iv) transcripts increased three-fold when the thin enamel layer was deposited on top of enameloid (stage 36) (Fig. 1C). However, these values remained 10-fold lower than those obtained in post-metamorphic juveniles.
ISH AMEL and COL1A1 expression was monitored from tooth 1 to tooth 6. In teeth 1 to 3, it is worth noting that we never identified a basement membrane between the ameloblasts and the depositing enameloid matrix, even at the ultrastructural level (Fig. 2A).
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J Dent Res 93(5) 2014
Figure 2. AMEL and COLIA1 expression during enamel/enameloid formation in Pleurodeles waltl before (teeth 1 to 3) and after (tooth 4) metamorphosis. In situ hybridization of mandibular jaw sections with DIG-labeled AMEL and COL1A1 antisense (B-D, F-H, J-L, N-Q) and control sense probes (I, M). B-H, J = transverse sections (oral cavity on the top); I-Q = sagittal sections (anterior to the left, oral cavity on the top). No basement membrane was identified at the ultrastructural level between the ameloblasts and the enameloid matrix during its deposition (A). Note the typical collagen banding of the fibrils close to the ameloblast surface (inset). During amelogenesis and throughout ontogeny, the enamel organ exists in the outer dental epithelium (Ode) composed of flattened cells, and in the inner dental epithelium (Ide), as a layer of polarized ameloblasts facing the forming tooth matrix. Neither stellate reticulum nor stratum intermedium is present between Ide and Ode. In embryos and larvae, teeth 1 to 3 are first capped with enameloid, then covered with a thin enamel layer at the end of amelogenesis. In post-metamorphic specimens, from tooth 4 onward, only enamel is found. COLIA1 expression is strong in ameloblasts during enameloid deposition and maturation in tooth 1 (B), then decreases in tooth 2 (F) and tooth 3 (J). It is no longer detected in ameloblasts during late-stage maturation (D, H, L), nor during amelogenesis of tooth 4 (N). In contrast, COLIA1 expression is strong in odontoblasts from early enameloid deposition (B, F, J) to dentin formation and attachment to the bone support (D, H, L, P, Q). AMEL is expressed during late formation of enameloid in larvae (C, G, K) and during enamel formation in juveniles (O). (E): Schematic drawing of (F). Am, ameloblast; En, enamel; Eno, enameloid; Ide, inner dental epithelium; OE, oral epithelium; MC, Meckel’s cartilage; OC, oral cavity; Od, odontoblast; Ode, outer dental epithelium. B-D, tooth 1; E-H, tooth 2; J-L, tooth 3; I, M-Q, tooth 4. A, B, stage 34; C, stage 36; D, stage 36; E, F, stage 38; G, stage 40; H, stage 42; I, juvenile; J, stage 48; K, stage 50; L, stage 54; M-Q, juvenile. Scale bars: A = 1 µm, inset = 500 nm; B, I, K-M, P, Q = 25 µm; C-H, J, N, O = 10 µm. Downloaded from jdr.sagepub.com at ANADOLU UNIV on May 7, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(5) 2014 505 Ameloblasts Express Type I Collagen during Amelogenesis Tooth 1 The small, conical, and monocuspid first-generation teeth develop in 6 days in embryos. COL1A1 expression was detected first at stage 33b (beginning of enameloid deposition) in the upper region of developing teeth, in both ameloblasts and odontoblasts. At stage 34, a strong COL1A1 expression was observed in the ameloblasts surrounding the enameloid cap, in the single odontoblast at the inner surface of the enameloid, and in odontoblasts depositing on the predentin matrix (Fig. 2B). Later (stage 36), when the dentin cone is finished and bone attachment has begun, a thin enamel layer is deposited at the enameloid surface. Ameloblasts expressed AMEL (Fig. 2C) but no longer COL1A1 (Fig. 2D). COL1A1 transcripts are still well-labeled in the odontoblasts located in the pulp cavity, and in those producing the base of the tooth (Fig. 2D).
Tooth 2 The second-generation teeth are larger but display the same structural and developmental features as teeth 1. They develop in stage 36 and attach to bone 15 days later (stage 42). At the end of enameloid deposition (stage 38), the ameloblasts around the tooth tip express COL1A1 and AMEL (Figs. 2F, 2G). COL1A1 transcripts can be clearly identified in the odontoblasts (Fig. 2F). At stage 40, the ameloblasts strongly express not COL1A1 but rather AMEL. The latter is not detected in the IDE cells located along the tooth shaft (Fig. 2G). Prior to tooth attachment, AMEL is no longer expressed in ameloblasts, while the odontoblasts synthesizing the dentin still express COL1A1 (Fig. 2H).
Tooth 3 Teeth 3 are larger, but still conical and monocuspid. They develop in stage 44 and become functional at stage 55a, a few days before metamorphosis. During enameloid deposition, ameloblasts express COL1A1 (Fig. 2J), but more weakly than in teeth 1 and 2 (see Figs. 2B, 2F). During enameloid maturation, COL1A1 expression eventually disappears in ameloblasts, in which AMEL transcripts can be detected (Fig. 2K). COL1A1 expression was stronger in odontoblasts depositing enameloid matrix than in those located along the dentin cone prior to attachment bone formation (Figs. 2J, 2L).
Tooth 4 These teeth are tall and bicuspid, with a major lingual cusp and a minor labial cusp. They develop over a wide period after metamorphosis, and bone attachment occurs in 5-month-old juveniles. No enameloid is identified between dentin and enamel matrix. Ameloblasts expressed AMEL (Fig. 2O) but not COL1A1 (Fig. 2N). COL1A1 transcripts were well-detected in odontoblasts during dentin formation, until teeth are fully formed (Figs. 2P, 2Q). During the formation of teeth 5 and 6, COL1A1 and AMEL expression was similar to that described in tooth 4 (Figs. 2N2Q) (data not shown). It is worth noting that (i) neither COL1A1 nor AMEL transcripts were identified in the outer dental epithelium cells, and (ii) neither odontoblasts nor cells located at the outer surface of the dentin shaft in the attachment region were labeled with the
AMEL probe. As expected, COL1A1 transcripts were detected in various cells of the mandibular and maxillary jaws, and especially in the osteoblasts.
Discussion Ameloblasts and Odontoblasts Express COL1A1 during Enameloid Formation Monitoring expression of COL1A1 during amelogenesis in larval Pleurodeles waltl revealed, for the first time in a tetrapod, that both odontoblasts and ameloblasts are responsible for collagen fibril deposition in enameloid. Our finding of COL1A1 expression in newt odontoblasts during enameloid formation is not surprising, because odontoblasts are known to synthesize type I collagen during dentin formation. In addition, the presence of collagen fibrils in caudate enameloid led previous authors to propose that enameloid was produced by odontoblasts (Kogaya, 1999). Less expected was the high expression level of COL1A1 in the ameloblasts surrounding the enameloid matrix in tooth 1 (to a lesser degree in teeth 2 and 3), a level which strongly suggested that the protein was expressed. The lack of a basement membrane between enameloid matrix and ameloblasts also strengthens this finding (Davit-Béal et al., 2007). During amelogenesis, COL1A1 expression decreased from secretory to mineralization and maturation stages, corresponding to the rise of AMEL expression in the same ameloblasts. During ontogeny, expression decreased from tooth 1 to tooth 3, in relation to the progressive reduction of enameloid matrix until post-metamorphic juvenile teeth, in which COL1A1 transcripts are no longer detected in ameloblasts. The collagenous enameloid matrix of larval caudate teeth is therefore produced by both ectomesenchymal cells (odontoblasts) and epithelial cells (ameloblasts). A dual origin for enameloid was previously proposed, but most authors considered that odontoblasts were responsible for collagen deposition and ameloblasts for enamel matrix protein and/or protease synthesis (Prostak and Skobe, 1985; Prostak et al., 1993). Collagen synthesis by ameloblasts, however, was suggested in teleosts and chondrichthyans, in which well-differentiated ameloblasts face tooth caps composed entirely of enameloid (Sasagawa, 2002; Sasagawa et al., 2006). In Atlantic salmon and zebrafish, COL1A1 expression was demonstrated in ameloblasts during enameloid deposition, then expression is down-regulated at the maturation stage (Huysseune et al., 2008; Kawasaki, 2009). To date, there is no report concerning COL1A1 expression in chondrichthyan ameloblasts. Collagen expression by epithelial cells is not an exception in vertebrates. In chicken, corneal epithelial cells secrete type I collagen (Hay and Revel, 1969). In zebrafish, epidermal basal layer cells express COL1A2 during early formation of the dermis (Le Guellec et al., 2004).
Ameloblasts Express AMEL during Enameloid Formation In tooth 1, qPCR revealed that AMEL was expressed in ameloblasts during enameloid formation, which means that AMEL and COL1A1 transcripts are simultaneously present in the same
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cells. The fact that AMEL expression was detected by ISH in ameloblasts only at the end of enameloid formation is probably related to both the rapid formation of tooth 1 and its small size. qPCR performed on whole-jaw extracts therefore appears to be more efficient in revealing that AMEL transcripts were less numerous during enameloid formation than during the final deposit of the thin enamel layer. In teeth 2 and 3, the late AMEL expression in the ameloblasts lining enameloid could be related to the progressive reduction of enameloid thickness vs. thickening of enamel matrix. This finding indicates that AMEL is probably secreted in the enameloid matrix during its late formation stage. These last 40 years, the hypothesis that EMPs (including AMBN and ENAM) were present in the enameloid matrix has been tested. During enameloid formation in teleosts, Shellis and Miles (1974) showed that ameloblasts synthesized a proline-rich protein. This protein could indeed be an EMP but also type I collagen, because all are proline-rich proteins. Using immunohistochemistry, several authors demonstrated that AMEL was lacking in the enameloid matrix of various actinopterygian species (Herold et al., 1989; Ishiyama et al., 2001; Sasagawa et al., 2006). Herold et al. (1989), however, suggested that enamelins were present in fish enameloid. In chondrichthyans, Ishiyama and colleagues (1994) did not identify EMPs in the enameloid matrix, while mouse AMEL antibody reacted positively (Diekwisch et al., 2002; Satchell et al., 2002). In caudate larvae, mammalian antibodies did not label AMEL during enameloid matrix formation stage but detected its presence in the outermost surface layer of enameloid and in the thin covering enamel layer (Kogaya, 1999). Here, using specific AMEL and COL1A1 probes, we validated previous hypotheses and demonstrated that both ameloblasts and odontoblasts are involved in these processes.
Newt Amelogenesis and Enameloid-to-Enamel Transition in Vertebrates In a previous study, we showed that enameloid-to-enamel transition occurred progressively prior to metamorphosis and was related to a slowing in enameloid deposition by odontoblasts concomitant with an increased enamel deposition by ameloblasts (Davit-Béal et al., 2007). By monitoring COL1A1 and AMEL expression during amelogenesis and throughout ontogeny, we are able to propose a scenario for enameloid-to-enamel transition. Modulation of odontoblast and ameloblast synthetic activity is a key factor in this process. The two cell populations are responsible for setting up the loose collagen network of enameloid in the first three tooth generations. This collagenous matrix could be impregnated with AMEL (and probably other EMPs and proteinases) synthesized by ameloblasts. For a stillunknown reason, while odontoblasts continue to deposit a collagenrich matrix, ameloblasts reduce their own collagen synthesis and increase AMEL production, a process which results in the formation of a collagen-free enamel matrix at the enameloid surface. Metamorphosis then coincides with the complete arrest of collagen synthesis by ameloblasts, which deposit enamel matrix only. Therefore, we postulate that enameloid-to-enamel transition could be due to a switch in ameloblast activity, the determination of which remains thus far unknown. This hypothesis could
J Dent Res 93(5) 2014 imply that sustained production of type I collagen by ameloblasts is required for enameloid formation, a process which could explain why enameloid is still the only highly mineralized covering tissue found in chondrichthyans and teleost fish. Although COL1A1 expression has not yet been reported in chondrichthyan ameloblasts, the demonstration of its expression in teleost (Huysseune et al., 2008; Kawasaki, 2009) and caudate ameloblasts suggests that a similar process occurs in sharks and rays. The presence of enameloid in chondrichthyans, actinopterygians, and sarcopterygians indicates that this tissue was certainly present in the last common ancestor of these lineages, then secondarily replaced with enamel. Taken together, our findings and analysis of the literature data strongly suggest that: (i) when enameloid is the only covering tooth tissue (chondrichthyans, teleost fish), ameloblasts synthesize type I collagen throughout amelogenesis; (ii) when enameloid is present only in larval teeth (caudate larvae), ameloblasts lose the capability to produce type I collagen after metamorphosis; and (iii) when enameloid is no longer present, even in juveniles (other tetrapods), ameloblasts lose the capacity for type I collagen synthesis in the concerned lineages. The transitory presence of enameloid in larval caudates could illustrate, in a living lineage, an intermediate step in enameloid-to-enamel transition, similar to that which occurred during tetrapod evolution. The ancestral ability of vertebrate ameloblasts to produce type I collagen, along with AMEL and certainly other EMPs, has probably greatly favored enameloidto-enamel transition.
Acknowledgments We thank Marie-Claire Lajarille for her technical assistance in sectioning. This study was financially supported by grants from CNRS and Université Pierre & Marie Curie. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
References Bascove M, Frippiat JP (2010). Molecular characterization of Pleurodeles waltl activation induced cytidine deaminase. Mol Immunol 47:16401649. Davit-Béal T, Allizard F, Sire JY (2006). Morphological variations in a tooth family through ontogeny in Pleurodeles waltl (Lissamphibia, Caudata). J Morphol 267:1048-1065. Davit-Béal T, Allizard F, Sire JY (2007). Enameloid/enamel transition through successive tooth replacements in Pleurodeles waltl (Lissamphibia, Caudata). Cell Tissue Res 328:167-183. Diekwisch TG, Berman BJ, Anderton X, Gurinsky B, Ortega AJ, Satchell PG, et al. (2002). Membranes, minerals and proteins of developing vertebrate enamel. Microsc Res Tech. 59:373-395. Hay ED, Revel JP (1969). Fine structure of the developing avian cornea. In: Monographs in developmental biology. Vol. 1. Wolski A, Chen PS, editors. Basel, Switzerland: Karger, pp. 1-144. Herold R, Rosenbloom J, Granovsky M (1989). Phylogenetic distribution of enamel proteins: immunohistochemical localization with monoclonal antibodies indicates the evolutionary appearance of enamelins prior to amelogenins. Calcif Tissue Int 45:88-94. Huysseune A, Takle H, Soenens M, Taerwe K, Witten PE (2008). Unique and shared gene expression patterns in Atlantic salmon (Salmo salar) tooth development. Dev Genes Evol 218:427-437.
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J Dent Res 93(5) 2014 507 Ameloblasts Express Type I Collagen during Amelogenesis Ishiyama M, Inage T, Shimokawa H, Yoshie S (1994). Immunocytochemical detection of enamel proteins in dental matrix of certain fishes. Bull Inst Oceanogr (Monaco) 14(Suppl 1):175-182. Ishiyama M, Inage T, Shimokawa H (2001). Abortive secretion of an enamel matrix in the inner enamel epithelial cells during an enameloid formation in the gar-pike, Lepisosteus oculatus (Holostei, Actinopterygii). Arch Histol Cytol 64:99-107. Kawasaki K (2009). The SCPP gene repertoire in bony vertebrates and graded differences in mineralized tissues. Dev Genes Evol 219:147157. Kawasaki K, Shimoda S, Fukae M (1987). Histological and bio-chemical observations of developing enameloid of the sea bream. Adv Dent Res 1:191-195. Kawasaki K, Suzuki T, Weiss KM (2005). Phenogenetic drift in evolution: the changing genetic basis of vertebrate teeth. Proc Natl Acad Sci USA 102:18063-18068. Kemp A (2002). Unique dentition of lungfish. Microsc Res Tech 59:435448. Kogaya Y (1999). Immunohistochemical localisation of amelogenin-like proteins and type I collagen and histochemical demonstration of sulphated glycoconjugates in developing enameloid and enamel matrices of the larval urodele (Triturus pyrrhogaster) teeth. J Anat 195(Pt 3):455-464. Le Guellec D, Morvan-Dubois G, Sire JY (2004). Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio). Int J Dev Biol 48:217-231. Moradian-Oldak J (2012). Protein-mediated enamel mineralization. Front Biosci 17:1996-2023. Poole DF (1967). Phylogeny of tooth tissue: enameloid and enamel in recent vertebrate, with a note on the history of cementum. In: Structural and chemical organization of teeth. Miles AE, editor. New York: Academic Press, pp. 111-149.
Prostak K, Skobe Z (1985). The effects of colchicine on the ultrastructure of the dental epithelium and odontoblasts of teleost tooth buds. J Craniofac Genet Dev Biol 5:75-88. Prostak KS, Seifert P, Skobe Z (1993). Enameloid formation in two tetraodontiform fish species with high and low fluoride contents in enameloid. Arch Oral Biol 38:1031-1044. Sander PM (2001). Prismless enamel in amniotes: terminology, function and evolution. In: Development, function and evolution of teeth. Teaford M, Ferguson MW, Smith MM, editors. New York, NY: Cambridge University Press, pp. 92-106. Sasagawa I (2002). Mineralization patterns in elasmobranch fish. Microsc Res Tech 59:396-407. Sasagawa I, Ishiyama M, Akai J (2006). Cellular influence in the formation of enameloid during odontogenesis in bony fishes. Mat Sci Eng C 26:630-634. Sasagawa I, Yokosuka H, Ishiyama M, Mikami M, Shimokawa H, Uchida T (2012). Fine structural and immunohistochemical detection of collar enamel in the teeth of Polypterus senegalus, an actinopterygian fish. Cell Tissue Res 347:369-381. Satchell PG, Anderton X, Ryu OH, Luan X, Ortega AJ, Opamen R, et al. (2002). Conservation and variation in enamel protein distribution during vertebrate tooth development. J Exp Zool 294:91-106. Schmidt WJ (1970). Der Zahnschmelz urodeler und anurer Amphibien. Z Zellforsch 104:295-300. Shellis RP, Miles AE (1974). Autoradiographic study of the formation of enameloid and dentine matrices in teleost fishes using tritiated amino acids. Proc R Soc Lond [Biol] 185:51-72. Sire JY, Davit-Béal T, Delgado S, Gu X (2007). The origin and evolution of enamel mineralization genes. Cells Tissues Organs 186:25-48. Smith MM (1992). Microstructure and evolution of enamel amongst osteichthyan fishes and early tetrapods. In: Structure, function and evolution of teeth. Smith P, Tchernov E, editors. London, UK: Freund, pp. 73-101.
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Ameloblasts Express Type I Collagen during Amelogenesis N. Assaraf-Weill, B. Gasse, J. Silvent, C. Bardet, J.-Y. Sire and T. Davit-Béal J DENT RES 2014 93: 502 originally published online 25 February 2014 DOI: 10.1177/0022034514526236 The online version of this article can be found at: http://jdr.sagepub.com/content/93/5/502
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
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research-article2014
JDR
93510.1177/0022034514526236
Research Reports Biological
N. Assaraf-Weill1, B. Gasse1, J. Silvent1, C. Bardet1,2, J.-Y. Sire1, and T. Davit-Béal1*
Ameloblasts Express Type I Collagen during Amelogenesis
1
UMR 7138-SAE, Research Group “Evolution & Development of the Skeleton”, Université Pierre et Marie Curie, 7 quai St-Bernard, Case 5, 75005 Paris, France; and 2EA 2496, Faculté de Chirurgie Dentaire, Université Paris Descartes, 1 rue Maurice Arnoux, 92120 Montrouge, France; *corresponding author, [email protected] J Dent Res 93(5):502-507, 2014
Abstract
Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while postmetamorphic teeth possess enamel only. In firstgeneration teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution.
KEY WORDS: amphibian, Pleurodeles waltl,
odontogenesis, enameloid, amelogenin, in situ hybridization.
DOI: 10.1177/0022034514526236 Received October 14, 2013; Last revision January 13, 2014; Accepted February 9, 2014
Introduction
T
he nature, evolutionary origin, and relationships of the highly mineralized tissues covering teeth in vertebrates, enameloid and enamel, have long been debated. Some authors considered that enamel was present in first vertebrates (Smith, 1992), but most scientists believed that enamel derived from enameloid (Poole, 1967; Kawasaki et al., 2005). This controversy could explain why enameloid formation – and whether it is only a mesodermal or an ectodermal-mesodermal tissue – attracted authors’ attention. In living vertebrates, enameloid is present in chondrichthyans (Sasagawa, 2002) and in actinopterygians (Shellis and Miles, 1974; Kawasaki et al., 1987), while in sarcopterygians it is found only in larval stages of caudate amphibians (Poole, 1967; Davit-Béal et al., 2007). Enamel has been documented in the sarcopterygian lineage (Schmidt, 1970; Sander, 2001; Kemp, 2002), and also described in a few actinopterygian lineages (Ishiyama et al., 2001; Sasagawa et al., 2012). In contrast to enameloid, which is recognizable by its loose network of collagen fibrils, enamel matrix is devoid of collagen and is composed mainly of enamel matrix proteins (EMPs): amelogenin (AMEL), enamelin (ENAM), and ameloblastin (AMBN) (Sire et al., 2007; Moradian-Oldak, 2012). Enamel is only synthesized by ameloblasts. Given the presence of collagen fibrils, enameloid has been considered a unique product of odontoblasts, but a contribution from ameloblasts was proposed in chondrichthyans (Sasagawa, 2002), actinopterygians (Sasagawa et al., 2006), and caudate larvae (Davit-Béal et al., 2006, 2007). These hypotheses were supported by similarities in mineralization and maturation processes, which imply the presence of EMPs and proteases, and also included a direct contribution from ameloblasts to collagen synthesis. In caudates, prior to metamorphosis, enameloid, followed by enamel, forms in larval teeth, while only enamel is present in post-metamorphic juvenile teeth. These amphibians offer unique possibilities for the study of the enameloid-to-enamel transition, a process that has probably occurred similarly during vertebrate evolution. Previously, in the newt Pleurodeles waltl, we demonstrated that the disappearance of enameloid was the result of a slowing of odontoblast activity, whereas enamel progressively thickened through increasing ameloblast activity (Davit-Béal et al., 2007). Here, using P. waltl, we wondered whether (i) enameloid contained EMPs in addition to collagen, and (ii) ameloblasts participated in collagen synthesis.
A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental.
Materials & Methods
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See the Appendix for a detailed description of materials and methods.
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J Dent Res 93(5) 2014 503 Ameloblasts Express Type I Collagen during Amelogenesis Biological Material Pleurodeles waltl is routinely bred in our laboratory. One hundred specimens, ranging from embryos to 13-month-old juveniles, were used for our study. All specimens were deeply anaesthetized (MS222) and sacrificed according to the guidelines of the French Ethics Committee.
Histology The materials and histological procedures for obtaining 12-µm-thick, then ultrathin, sections for transmission electron microscopic (TEM) observations were previously described (Davit-Béal et al., 2007). Also see the Appendix.
PCR Total RNAs were extracted from specimens at stages 30, 33, 34, and 36 (whole embryo) and from juveniles (mandibular jaw). cDNAs obtained by RT-PCR were amplified and sent to GATC Biotech AG (Konstanz, Germany) for sequencing.
Real-time PCR (qPCR) qPCR was performed with AMEL primers and specific primers for the housekeeping GAPDH gene (Bascove and Frippiat, 2010).
In situ Hybridization (ISH) Antisense and sense (control) AMEL and COL1A1 RNA probes were synthesized as described in the Appendix. Briefly, the samples were fixed in Formoy solution, demineralized in acetic acid, embedded in Paraplast, and sectioned (10 µm thick). The sections were de-waxed, digested in proteinase K, fixed in paraformaldehyde, and hybridized overnight. They were then rinsed and labeled with Digoxigenin overnight. Finally, the samples were rinsed, the signal was revealed, and the slides were mounted and photographed.
Results Serial jaw-sectioning allowed for the identification of various stages of tooth development, i.e., enameloid and/or enamel cap formation, dentin cone formation, and attachment to bone support (Fig. 1A).
PCR and qPCR Using PCR and qPCR, we monitored only AMEL expression through successive steps of enameloid and enamel formation. These experiments would not be relevant for COL1A1, since it is expressed in various jaw tissues. We studied AMEL expression only in first-generation teeth, which initiate in embryos and become functional in early larvae, because all teeth were from the same generation and all possessed well-developed enameloid. We also performed PCR and qPCR in a post-metamorphic juvenile, because all teeth possessed enamel only.
Figure 1. Monitoring of AMEL expression in Pleurodeles waltl (Pw) during development of a first-generation tooth (tooth 1) and in a juvenile tooth. (A) Schematic drawings of tooth 1 odontogenesis and of a well-developed bicuspid juvenile tooth (4-month-old specimen). Stage 33 (Pw33), tooth bud prior to matrix deposition; Pw34, enameloid deposition; and Pw36, well-formed tooth, shortly before attachment. (B) PCR of AMEL and GAPDH (housekeeping gene). (C) Real-time PCR. AMEL expression in the developmental stages illustrated in (A). Pw30 (stage 30): no teeth in the mandibular and maxillary jaws. *p < .05. De, dentin; Dp, dental papilla; En, enamel; Eno, enameloid; Eo, enamel organ.
PCR revealed that AMEL was not expressed before tooth 1 initiation (stage 30). The expression was weak during ameloblast differentiation (stage 33), and strong in further developmental stages (Fig. 1B). qPCR indicated that (i) a few transcripts were present before tooth initiation, (ii) expression was weak prior to enameloid deposition, (iii) expression increased 10-fold when ameloblasts and odontoblasts had differentiated and enameloid matrix was deposited, and (iv) transcripts increased three-fold when the thin enamel layer was deposited on top of enameloid (stage 36) (Fig. 1C). However, these values remained 10-fold lower than those obtained in post-metamorphic juveniles.
ISH AMEL and COL1A1 expression was monitored from tooth 1 to tooth 6. In teeth 1 to 3, it is worth noting that we never identified a basement membrane between the ameloblasts and the depositing enameloid matrix, even at the ultrastructural level (Fig. 2A).
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J Dent Res 93(5) 2014
Figure 2. AMEL and COLIA1 expression during enamel/enameloid formation in Pleurodeles waltl before (teeth 1 to 3) and after (tooth 4) metamorphosis. In situ hybridization of mandibular jaw sections with DIG-labeled AMEL and COL1A1 antisense (B-D, F-H, J-L, N-Q) and control sense probes (I, M). B-H, J = transverse sections (oral cavity on the top); I-Q = sagittal sections (anterior to the left, oral cavity on the top). No basement membrane was identified at the ultrastructural level between the ameloblasts and the enameloid matrix during its deposition (A). Note the typical collagen banding of the fibrils close to the ameloblast surface (inset). During amelogenesis and throughout ontogeny, the enamel organ exists in the outer dental epithelium (Ode) composed of flattened cells, and in the inner dental epithelium (Ide), as a layer of polarized ameloblasts facing the forming tooth matrix. Neither stellate reticulum nor stratum intermedium is present between Ide and Ode. In embryos and larvae, teeth 1 to 3 are first capped with enameloid, then covered with a thin enamel layer at the end of amelogenesis. In post-metamorphic specimens, from tooth 4 onward, only enamel is found. COLIA1 expression is strong in ameloblasts during enameloid deposition and maturation in tooth 1 (B), then decreases in tooth 2 (F) and tooth 3 (J). It is no longer detected in ameloblasts during late-stage maturation (D, H, L), nor during amelogenesis of tooth 4 (N). In contrast, COLIA1 expression is strong in odontoblasts from early enameloid deposition (B, F, J) to dentin formation and attachment to the bone support (D, H, L, P, Q). AMEL is expressed during late formation of enameloid in larvae (C, G, K) and during enamel formation in juveniles (O). (E): Schematic drawing of (F). Am, ameloblast; En, enamel; Eno, enameloid; Ide, inner dental epithelium; OE, oral epithelium; MC, Meckel’s cartilage; OC, oral cavity; Od, odontoblast; Ode, outer dental epithelium. B-D, tooth 1; E-H, tooth 2; J-L, tooth 3; I, M-Q, tooth 4. A, B, stage 34; C, stage 36; D, stage 36; E, F, stage 38; G, stage 40; H, stage 42; I, juvenile; J, stage 48; K, stage 50; L, stage 54; M-Q, juvenile. Scale bars: A = 1 µm, inset = 500 nm; B, I, K-M, P, Q = 25 µm; C-H, J, N, O = 10 µm. Downloaded from jdr.sagepub.com at ANADOLU UNIV on May 7, 2014 For personal use only. No other uses without permission. © International & American Associations for Dental Research
J Dent Res 93(5) 2014 505 Ameloblasts Express Type I Collagen during Amelogenesis Tooth 1 The small, conical, and monocuspid first-generation teeth develop in 6 days in embryos. COL1A1 expression was detected first at stage 33b (beginning of enameloid deposition) in the upper region of developing teeth, in both ameloblasts and odontoblasts. At stage 34, a strong COL1A1 expression was observed in the ameloblasts surrounding the enameloid cap, in the single odontoblast at the inner surface of the enameloid, and in odontoblasts depositing on the predentin matrix (Fig. 2B). Later (stage 36), when the dentin cone is finished and bone attachment has begun, a thin enamel layer is deposited at the enameloid surface. Ameloblasts expressed AMEL (Fig. 2C) but no longer COL1A1 (Fig. 2D). COL1A1 transcripts are still well-labeled in the odontoblasts located in the pulp cavity, and in those producing the base of the tooth (Fig. 2D).
Tooth 2 The second-generation teeth are larger but display the same structural and developmental features as teeth 1. They develop in stage 36 and attach to bone 15 days later (stage 42). At the end of enameloid deposition (stage 38), the ameloblasts around the tooth tip express COL1A1 and AMEL (Figs. 2F, 2G). COL1A1 transcripts can be clearly identified in the odontoblasts (Fig. 2F). At stage 40, the ameloblasts strongly express not COL1A1 but rather AMEL. The latter is not detected in the IDE cells located along the tooth shaft (Fig. 2G). Prior to tooth attachment, AMEL is no longer expressed in ameloblasts, while the odontoblasts synthesizing the dentin still express COL1A1 (Fig. 2H).
Tooth 3 Teeth 3 are larger, but still conical and monocuspid. They develop in stage 44 and become functional at stage 55a, a few days before metamorphosis. During enameloid deposition, ameloblasts express COL1A1 (Fig. 2J), but more weakly than in teeth 1 and 2 (see Figs. 2B, 2F). During enameloid maturation, COL1A1 expression eventually disappears in ameloblasts, in which AMEL transcripts can be detected (Fig. 2K). COL1A1 expression was stronger in odontoblasts depositing enameloid matrix than in those located along the dentin cone prior to attachment bone formation (Figs. 2J, 2L).
Tooth 4 These teeth are tall and bicuspid, with a major lingual cusp and a minor labial cusp. They develop over a wide period after metamorphosis, and bone attachment occurs in 5-month-old juveniles. No enameloid is identified between dentin and enamel matrix. Ameloblasts expressed AMEL (Fig. 2O) but not COL1A1 (Fig. 2N). COL1A1 transcripts were well-detected in odontoblasts during dentin formation, until teeth are fully formed (Figs. 2P, 2Q). During the formation of teeth 5 and 6, COL1A1 and AMEL expression was similar to that described in tooth 4 (Figs. 2N2Q) (data not shown). It is worth noting that (i) neither COL1A1 nor AMEL transcripts were identified in the outer dental epithelium cells, and (ii) neither odontoblasts nor cells located at the outer surface of the dentin shaft in the attachment region were labeled with the
AMEL probe. As expected, COL1A1 transcripts were detected in various cells of the mandibular and maxillary jaws, and especially in the osteoblasts.
Discussion Ameloblasts and Odontoblasts Express COL1A1 during Enameloid Formation Monitoring expression of COL1A1 during amelogenesis in larval Pleurodeles waltl revealed, for the first time in a tetrapod, that both odontoblasts and ameloblasts are responsible for collagen fibril deposition in enameloid. Our finding of COL1A1 expression in newt odontoblasts during enameloid formation is not surprising, because odontoblasts are known to synthesize type I collagen during dentin formation. In addition, the presence of collagen fibrils in caudate enameloid led previous authors to propose that enameloid was produced by odontoblasts (Kogaya, 1999). Less expected was the high expression level of COL1A1 in the ameloblasts surrounding the enameloid matrix in tooth 1 (to a lesser degree in teeth 2 and 3), a level which strongly suggested that the protein was expressed. The lack of a basement membrane between enameloid matrix and ameloblasts also strengthens this finding (Davit-Béal et al., 2007). During amelogenesis, COL1A1 expression decreased from secretory to mineralization and maturation stages, corresponding to the rise of AMEL expression in the same ameloblasts. During ontogeny, expression decreased from tooth 1 to tooth 3, in relation to the progressive reduction of enameloid matrix until post-metamorphic juvenile teeth, in which COL1A1 transcripts are no longer detected in ameloblasts. The collagenous enameloid matrix of larval caudate teeth is therefore produced by both ectomesenchymal cells (odontoblasts) and epithelial cells (ameloblasts). A dual origin for enameloid was previously proposed, but most authors considered that odontoblasts were responsible for collagen deposition and ameloblasts for enamel matrix protein and/or protease synthesis (Prostak and Skobe, 1985; Prostak et al., 1993). Collagen synthesis by ameloblasts, however, was suggested in teleosts and chondrichthyans, in which well-differentiated ameloblasts face tooth caps composed entirely of enameloid (Sasagawa, 2002; Sasagawa et al., 2006). In Atlantic salmon and zebrafish, COL1A1 expression was demonstrated in ameloblasts during enameloid deposition, then expression is down-regulated at the maturation stage (Huysseune et al., 2008; Kawasaki, 2009). To date, there is no report concerning COL1A1 expression in chondrichthyan ameloblasts. Collagen expression by epithelial cells is not an exception in vertebrates. In chicken, corneal epithelial cells secrete type I collagen (Hay and Revel, 1969). In zebrafish, epidermal basal layer cells express COL1A2 during early formation of the dermis (Le Guellec et al., 2004).
Ameloblasts Express AMEL during Enameloid Formation In tooth 1, qPCR revealed that AMEL was expressed in ameloblasts during enameloid formation, which means that AMEL and COL1A1 transcripts are simultaneously present in the same
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cells. The fact that AMEL expression was detected by ISH in ameloblasts only at the end of enameloid formation is probably related to both the rapid formation of tooth 1 and its small size. qPCR performed on whole-jaw extracts therefore appears to be more efficient in revealing that AMEL transcripts were less numerous during enameloid formation than during the final deposit of the thin enamel layer. In teeth 2 and 3, the late AMEL expression in the ameloblasts lining enameloid could be related to the progressive reduction of enameloid thickness vs. thickening of enamel matrix. This finding indicates that AMEL is probably secreted in the enameloid matrix during its late formation stage. These last 40 years, the hypothesis that EMPs (including AMBN and ENAM) were present in the enameloid matrix has been tested. During enameloid formation in teleosts, Shellis and Miles (1974) showed that ameloblasts synthesized a proline-rich protein. This protein could indeed be an EMP but also type I collagen, because all are proline-rich proteins. Using immunohistochemistry, several authors demonstrated that AMEL was lacking in the enameloid matrix of various actinopterygian species (Herold et al., 1989; Ishiyama et al., 2001; Sasagawa et al., 2006). Herold et al. (1989), however, suggested that enamelins were present in fish enameloid. In chondrichthyans, Ishiyama and colleagues (1994) did not identify EMPs in the enameloid matrix, while mouse AMEL antibody reacted positively (Diekwisch et al., 2002; Satchell et al., 2002). In caudate larvae, mammalian antibodies did not label AMEL during enameloid matrix formation stage but detected its presence in the outermost surface layer of enameloid and in the thin covering enamel layer (Kogaya, 1999). Here, using specific AMEL and COL1A1 probes, we validated previous hypotheses and demonstrated that both ameloblasts and odontoblasts are involved in these processes.
Newt Amelogenesis and Enameloid-to-Enamel Transition in Vertebrates In a previous study, we showed that enameloid-to-enamel transition occurred progressively prior to metamorphosis and was related to a slowing in enameloid deposition by odontoblasts concomitant with an increased enamel deposition by ameloblasts (Davit-Béal et al., 2007). By monitoring COL1A1 and AMEL expression during amelogenesis and throughout ontogeny, we are able to propose a scenario for enameloid-to-enamel transition. Modulation of odontoblast and ameloblast synthetic activity is a key factor in this process. The two cell populations are responsible for setting up the loose collagen network of enameloid in the first three tooth generations. This collagenous matrix could be impregnated with AMEL (and probably other EMPs and proteinases) synthesized by ameloblasts. For a stillunknown reason, while odontoblasts continue to deposit a collagenrich matrix, ameloblasts reduce their own collagen synthesis and increase AMEL production, a process which results in the formation of a collagen-free enamel matrix at the enameloid surface. Metamorphosis then coincides with the complete arrest of collagen synthesis by ameloblasts, which deposit enamel matrix only. Therefore, we postulate that enameloid-to-enamel transition could be due to a switch in ameloblast activity, the determination of which remains thus far unknown. This hypothesis could
J Dent Res 93(5) 2014 imply that sustained production of type I collagen by ameloblasts is required for enameloid formation, a process which could explain why enameloid is still the only highly mineralized covering tissue found in chondrichthyans and teleost fish. Although COL1A1 expression has not yet been reported in chondrichthyan ameloblasts, the demonstration of its expression in teleost (Huysseune et al., 2008; Kawasaki, 2009) and caudate ameloblasts suggests that a similar process occurs in sharks and rays. The presence of enameloid in chondrichthyans, actinopterygians, and sarcopterygians indicates that this tissue was certainly present in the last common ancestor of these lineages, then secondarily replaced with enamel. Taken together, our findings and analysis of the literature data strongly suggest that: (i) when enameloid is the only covering tooth tissue (chondrichthyans, teleost fish), ameloblasts synthesize type I collagen throughout amelogenesis; (ii) when enameloid is present only in larval teeth (caudate larvae), ameloblasts lose the capability to produce type I collagen after metamorphosis; and (iii) when enameloid is no longer present, even in juveniles (other tetrapods), ameloblasts lose the capacity for type I collagen synthesis in the concerned lineages. The transitory presence of enameloid in larval caudates could illustrate, in a living lineage, an intermediate step in enameloid-to-enamel transition, similar to that which occurred during tetrapod evolution. The ancestral ability of vertebrate ameloblasts to produce type I collagen, along with AMEL and certainly other EMPs, has probably greatly favored enameloidto-enamel transition.
Acknowledgments We thank Marie-Claire Lajarille for her technical assistance in sectioning. This study was financially supported by grants from CNRS and Université Pierre & Marie Curie. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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