RESEARCH REPORTS Biological

S. Delgado1, M.-L. Couble2, H. Magloire2, and J.-Y. Sire1* 1 UMR 7138-Systématique, Adaptation, Evolution, Université Paris 6, 7, quai St-Bernard, 75005 Paris, France; and 2EA 1892-Laboratoire du Développement des Tissus Dentaires, Faculté d'Odontologie, Lyon, France; *corresponding author, [email protected]

Cloning, Sequencing, and Expression of the Amelogenin Gene in Two Scincid Lizards

J Dent Res 85(2):138-143, 2006

ABSTRACT Our knowledge of the gene coding for amelogenin, the major enamel protein, is mainly based on mammalian sequences. Only two sequences are available in reptiles. To know whether the snake sequence is representative of the amelogenin condition in squamates, we have studied amelogenin in two scincid lizards. Lizard amelogenin possesses numerous conserved residues in the N- and C-terminal regions, but its central region is highly variable, even when compared with the snake sequence. This rapid evolution rate indicates that a single squamate sequence was not representative, and that comparative studies of reptilian amelogenins might be useful to detect the residues which are really important for amelogenin structure and function. Reptilian and mammalian enamel structure is roughly similar, but no data support amelogenin being similarly expressed during amelogenesis. By performing in situ hybridization using a specific probe, we showed that lizard ameloblasts express amelogenin as described during mammalian amelogenesis. However, we have not found amelogenin transcripts in odontoblasts. This indicates that full-length amelogenin is specific to enamel matrix, at least in this lizard. KEY WORDS: lizard, amelogenesis, amelogenin, in situ hybridization, cDNA sequence analysis.

INTRODUCTION melogenin, the principle protein of the developing enamel matrix, is A thought to play an essential role in the control of crystal deposition/growth and prism formation (Moradian-Oldak et al., 2003; Paine et al., 2003; Snead, 2003), and may also act as a signaling molecule (Veis et al., 2000). The crucial function of amelogenin is indirectly highlighted by mutations leading to amelogenesis imperfecta (review in Hart et al., 2002). However, the precise relationship between amelogenin structure and function, particularly the role of specific amino acid residues and domains, remains unclear. In mammals, comparative studies of the primary structure of amelogenin have proved to be useful indicators of the potential importance of specific amino acid residues (Delgado et al., 2005a; Sire et al., 2005). However, to improve the analysis, further data are needed in other lineages possessing well-structured enamel. Unfortunately, only four amelogenin sequences are available in non-mammalian lineages: a crocodile, a snake, and two frogs. Are these sequences representative of the amelogenin condition in these lineages? In lizards, amelogenesis was recently shown to be roughly similar to that in mammals, with respect to the typical features of secretory ameloblasts and the maturation process (Delgado et al., 2005b). However, in contrast to mammals, mature enamel in lizards lacks prisms (Sander, 2001), and immature enamel matrix is deposited on the dentin matrix with a slight delay, and is completed prior to maturation (Delgado et al., 2005b). Are these differences in enamel architecture and maturation related to differences in the timing of deposition and/or to the structure of the amelogenin? To try to answer these questions, we have cloned amelogenin in 2 scincid lizards, Chalcides viridanus and a related species, C. sexlineatus, and compared the lizard sequences with the other available sequences. Taking advantage of the fact that lizard teeth are replaced continuously throughout life, we next studied amelogenin gene expression during amelogenesis in replacement teeth developing in juveniles and adults (Delgado et al., 2003).

MATERIALS & METHODS Animals

Received July 3, 2004; Last revision August 23, 2005; Accepted September 9, 2005 A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.

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Several specimens of Canarian scincids were used: (for amelogenin cDNA cloning) Chalcides viridanus, juvenile, 5 mos old, and C. sexlineatus, female, 60 mos old; (for light microscopy) C. viridanus, juvenile, 1 mo old; and (for in situ hybridization) C. viridanus, 2 juveniles, 12 mos old. All animals used in this study were maintained at room temperature (agreement No. A-75-05-11) and were killed according to the guidelines of the French Ethics Committee.

Methods For a more detailed description of the methods, see the APPENDIX.

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Histological Analysis The anterior region of the lower jaw was fixed in a mixture of glutaraldehyde and paraformaldehyde, decalcified in EDTA, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in epon. One- ␮ mthick serial sections were stained with toluidine blue.

Amelogenin cDNA Cloning The lower jaw was immersed in liquid nitrogen and reduced to a powder. RNAs were purified, and mRNAs were isolated and converted into cDNA by RT-PCR. The following primers were designed: 5⬘ exon 2 - sense, 5⬘-CTGGAC TTTGGTTATGTGCC-3⬘; 3⬘ exon 6 - antisense, 5 ⬘ -CACTTCTTCTT GCTTGGTCTT-3⬘. cDNA (1 ␮L) was amplified by PCR, in the presence of primers and Red Hot polymerase. Amplification was performed, and a 600-bp cDNA fragment was amplified. 3 ⬘ RACE: Two runs of PCR were necessary, with an oligo(dT)18 (antisense) primer coupled successively with two (sense) primers: 5 ⬘- G G A C A C C A G T A C C C ACGTTAT-3⬘; 5⬘-TATGAACCTAT GGGAGGATGG-3⬘. One microgram of PCR product was ligated to pCR 2.1-TOPO plasmid vector by the TA-cloning method, then used to transform competent E. coli TOP10F bacteria. The plasmids were purified and sequenced. Amelogenin cDNA sequences were aligned by Clustal X (UBC Bioinformatics Centre, Vancouver, Canada), and the putative amino acid sequences were deduced with DNA strider 1.2 (Bio-web/Software).

Figure 1. Nucleotide and deduced amino acid sequences of the amelogenin in Chalcides viridanus and C. sexlineatus. The non-coding exon 7 sequence at the 3 ⬘ end is shown only for C. viridanus. Underlined = 3⬘ region of the signal peptide; boxed = proteolytic sites of TRAP43 and 45; bold = remarkable amino acids; 2ⱍ3 = limit between exon 2 and exon 3. (-) = nucleotide/residue identical; (*) = lack of nucleotide/residue.

In situ Hybridization The lower jaws were fixed in Formoy’s solution, then placed in a mixture of paraformaldehyde and acetic acid to complete decalcification. The jaws were dehydrated, embedded in paraffin, and cut into 10- ␮ m-thick sections, which were transferred to coated slides. The slides were air-dried, deparaffinized, rehydrated, fixed in paraformaldehyde, washed twice, then dehydrated and air-dried.

Probe Preparation and Labeling An asymmetric, linear amplification was utilized. The 600-bp amelogenin fragment was added to the amplification solution containing Taq polymerase and antisense primer. Sense primer was used for the synthesis of the control probe. A single-stranded DNA probe with a specific activity of about 2.8 x 106 dmp/pmol was obtained.

In situ Hybridization The sections were pre-hybridized, rapidly rinsed, dehydrated, and air-dried, then covered with the hybridization solution, rinsed, dehydrated, and air-dried. For autoradiography, the slides were immersed in LM-1 emulsion (Amersham, Buckinghamshire, England). The sections were stained with Masson’s hemalun, airdried, and mounted.

RESULTS Chalcides Amelogenin PCR amplification of cDNA from the 2 Chalcides species yielded a fragment homologous to exon2-exon6 regions of mammalian amelogenin. Using 3⬘ RACE, we obtained a fragment homologous

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Figure 2 continues on next page Figure 2. Amino acid alignments of lizard amelogenin with other amelogenin sequences. (A) Alignment of tetrapod amelogenin sequences. (top to bottom) The putative ancestral amelogenin sequence in mammals (from Delgado et al., 2005a); crocodile, Paleosuchus palpebrosus (GenBank access number, AF118568); ratsnake, Elaphe quadrivirgata (AF095568); lizard, Chalcides viridanus (present work); and three lissamphibians—the pipid frog, Xenopus laevis A, X. laevis B (AF095569; AF095570), and the bullfrog Rana pipiens (from Wang et al., 2005). Amino acid alignment is easy in the N- and C-ter regions, while it is difficult in the central region of exon 6 (in gray), due to numerous amino acid substitutions. Boxed = TRAP proteolytic sites; underlined = remarkable amino acids; 2ⱍ3 = limit between exon 2 and exon 3. LRAP = locus of intra-exonic alternative splicing in mammals. (-) = identical amino acid; (*) = either lack or insertion of amino acid. (B-E) Amino acid alignment of the amelogenin sequence of Chalcides viridanus with the snake, crocodile, ancestral mammal, and lissamphibian sequences taken separately. Alignment of the variable region (in gray) is still difficult to obtain, even between the closest species, lizard and snake (B).

to exon6-exon7 regions. The putative amino acid (aa) sequences were aligned, and the amelogenin organization was deduced from the sequences already published (Fig. 1). The 2 amelogenin sequences were found to be similar (92.9% similarity), but 7 nucleotides differed, 5 of them changing the amino acid. Two residues were lacking in C. sexlineatus amelogenin exon 6, for a total of 181 aa vs. 183 in C. viridanus. Five exons were identified (exons 2, 3, 5, 6, 7). Exons 4, 8, and 9, known in some mammalian species only and generally subjected to alternative splicing, were not found. Important sites detected in previous studies dealing with mammalian amelogenins were present in both lizards: serine 16, threonine 21, and proline 41; the 3 tyrosines (Y34, 37, and 39); and glycine 43 and tryptophane 45. Histidine 47 was substituted by an arginine. The LRAP (leucine-rich amelogenin peptide) intraexonic splice site in the C-terminus region of mammalian amelogenin exon 6 was not present in Chalcides.

Comparison with Other Amelogenin Sequences The amino acid sequence of Chalcides amelogenin was aligned with the 5 non-mammalian sequences (crocodile, snake, Xenopus

J Dent Res 85(2) 2006 A and B, and frog) and the putative ancestral mammalian sequence (Delgado et al., 2005a) (Figs. 2A2E). The N- and C-terminal regions (aa 1-48 and 185-216, respectively, in our alignment) showed high sequence conservation: 78.1% similarity with snake; 75.3% with crocodile; 78.1% with ancestral mammal; and 65.7% to 58.9% with frogs. In contrast, in exon 6 (aa 49 to 184), the numerous substitutions did not permit correct alignment, even when the lizard sequence was aligned with each of the other sequences (Figs. 2B-2E). We found less than 30% similarity with mammalian and frog exon 6. The number of variations observed within reptile lineages (31.1% similarity with snake; 38.3% with crocodile) was quite surprising. Lizard and crocodile sequences had a similar length (183 vs. 184 aa), while the snake sequence presented numerous deletions (162 aa). This indicated that snake amelogenin differentiated more rapidly than lizard amelogenin.

Amelogenin Expression during Enamel Formation

In serially sectioned jaws, the developmental stages of various tooth germs were identified, and the appropriate section levels were selected for the study of amelogenin expression (Fig. 3). The negative result obtained with the control probe (Figs. 3a, 3b) indicated that in situ hybridization signals corresponded to amelogenin transcripts. Amelogenesis began after predentin matrix deposition. Immature enamel matrix was easily recognizable by a strong toluidine blue metachromasia at the tooth tip, in the region facing the first deposited predentin matrix, and it extended as a thin layer on the tooth sides (Fig. 3c). The first enamel matrix was deposited at the level of the main cusp. Strong amelogenin expression was detected in the whole ameloblast layer covering the recently deposited immature enamel matrix (Fig. 3d). Next, dentin mineralized in the upper region of the tooth, and enamel maturation, identified indirectly by a decrease in toluidine blue metachromasia, started in the regions where the enamel had reached its full thickness, i.e., at the level of the cusp (Fig. 3e). Predentin was still being deposited at the pulpal side along the tooth shaft and covered by newly deposited enamel matrix. Enamel maturation progressively extended along the shaft surface from the tip to the base of the tooth. It started in the enamel region close to the dentin-enamel junction, and extended throughout the entire enamel thickness. While enamel maturation was well-advanced at the tooth tip, enamel matrix was still being deposited along the tooth base (Fig.

J Dent Res 85(2) 2006 3e). Amelogenin transcription was progressively down-regulated in the ameloblasts facing maturing enamel, while it was still strong in the ameloblasts facing the immature enamel surface (Fig. 3f). In a subsequent phase, amelogenin gene expression was no longer detected in the ameloblasts facing mature enamel in the upper region of the tooth, whereas it was still strong at the tooth base of wellformed replacement teeth (Figs. 3g-3j). In C. viridanus, the mode of attachment was pleurodont, and the dentin shaft was longer at the lingual than at the labial side. Therefore, the enamel covering extended deeper along the shaft surface at the lingual side, explaining the stronger signal observed. Amelogenin expression was never seen in functional teeth (Fig. 3a), and transcripts were not detected in the odontoblasts, or in other cells of the dental organ, or in osteoblasts at the bone surface (Fig. 3).

Lizard Amelogenin

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Figure 2. (Continued from previous page)

DISCUSSION Tetrapod Amelogenin Among the numerous conserved residues in the N- and C-terminal regions of lizard amelogenin, there are some noteworthy amino acids, the presence of which may be important for amelogenin function in mammals: serine 16, a phosphorylation site (Fincham et al., 1991); most residues of the alpha helices region (aa 19-29) (Toyosawa et al., 1998); most residues of the 'tyrosyl motif' that binds to the N-acetyl-D-glucosamine (aa 33-45) (Ravindranath et al., 1999); and glycine 43 and tryptophane 45, known in mammals to be proteolytic sites giving rise to TRAP43 and TRAP45 (Fincham and Moradian-Oldak, 1993). In addition, two sites of mutations leading to amelogenesis imperfecta in humans, threonine 21 and proline 41, are present (Hart et al., 2002). However, histidine 47, which also leads to amelogenesis imperfecta when substituted by a leucine, is replaced by an arginine in the lizard. A similar substitution was described in dolphin amelogenin (Delgado et al., 2005a). This confirms that arginine, which belongs to the same basic group of amino acids as histidine, can be substituted at this position without any negative consequence for the enamel structure. Residue conservation indicates a slow evolutionary rate, which is related to important functional constraints. This study points to numerous conserved residues that probably have an important

function, which remain to be found. In mammals, the C-terminal region possesses a remarkably conserved intra-exonic alternative splicing, responsible for the formation of LRAP fragments (Moradian-Oldak et al., 1994). Such a splicing site is lacking in lizard amelogenin, but four QP (Glu-Pro) positions in this region could be alternative splicing sites. Alignment of several squamate sequences could provide more precise information. During the last 250 million years of evolution, the variable region of amelogenin has been subjected to more substitutions in squamates than in mammals. Alignment of this region is not easy, not even within the reptile lineage, as shown when comparing the lizard sequence with that of either the snake or the crocodile. This high substitution rate is probably related to the more rapid evolution of the squamate genome, compared with the other

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J Dent Res 85(2) 2006 analysis of amelogenin evolution. Indeed, the regions that are permissive for substitutions, which do not interfere with amelogenin structure and function, will show more frequent changes than in other lineages. The only "important" sites are thus conserved. However, a comparative analysis of more sequences, in various representative species of this lineage, is necessary before we can understand the way in which lizard amelogenin is evolving in this region. The only 2 squamate sequences constrain our ability to find the triplet repeats P-X-X, revealed in the mammalian amelogenin and considered to be at the origin of exon 6 formation (Delgado et al., 2005a). The rapid evolution in squamates has probably blurred the phylogenetic information. Finally, the variable region does not show the large insertions and deletions (the socalled 'hot spot' of mutation) described in some mammals (Delgado et al., 2005a). The absence of such a region in the lizard could support our hypothesis of the novelty for this region of the amelogenin in mammals. In conclusion, the high substitution rate observed in amelogenin exon 6 proves that the snake sequence was not representative of squamate amelogenin. Our comparison of amelogenin sequences of 2 squamates, 1 crocodile, 2 frogs, and the putative mammalian ancestor does not provide sufficient information to understand amelogenin evolution in tetrapods. More data from squamate, crocodile, and amphibian species are necessary.

Amelogenin Expression during Amelogenesis For the first time in a non-mammalian species, we have used in situ hybridization with a specific probe to demonstrate that reptilian ameloblasts express amelogenin Figure 3. Chalcides viridanus. Different steps of amelogenesis during replacement tooth development. during amelogenesis. Until now, the Amelogenin gene (amelogenin) expression is revealed by in situ hybridization on sections (dark stippled possible existence of amelogenin in reptile 33 regions in a,d,f,h,j), with P-labeled amelogenin probe. (c,e,g,i) One-␮m-thick sections of stages of teeth was supported by indirect enamel formation in replacement teeth similar to those shown in d-j (toluidine blue staining). Labial to the observations: development and structure right. (a) Low magnification showing 3 tooth positions in the left lower jaw arcade sectioned longitudinally. Anterior to the right. Amelogenin is not expressed in functional teeth, while numerous of enamel roughly similar to that in transcripts are detected in the enamel organ of the replacement teeth below. (b) Negative control for (a), mammals (Delgado et al., 2005b); positive with the sense probe. (c-j) Enamel matrix deposition in replacement teeth, from early deposition (c,d) to cross-reaction with mammalian antibodies the end of enamel maturation (i,j). Transverse sections of the lower jaw. In situ hybridization reveals (Herold et al., 1989; Ishiyama et al., amelogenin transcripts in the enamel organ (ameloblasts). Transcripts are abundant in the ameloblasts 1998); and cloning of the amelogenin gene facing the recently deposited immature enamel matrix; they are less numerous in the ameloblasts facing the maturing enamel, and are absent in ameloblasts facing the mature enamel. Scale bars: a,b = 100 in a snake (Ishiyama et al., 1998) and in a ␮m; c-j = 50 ␮m. Abbreviations: am, ameloblasts; be, buccal epithelium; d, dentin; db, dentary bone; crocodile (Toyosawa et al., 1998). Here, dej, dentin-enamel junction; e, enamel; eo, enamel organ; ft, functional tooth; od, odontoblasts; ode, we clearly demonstrate that amelogenin is outer dental epithelium; pd, predentin; rt, replacement tooth; sr, stellate reticulum. specifically expressed in the ameloblasts from the moment at which the first lineages (Hughes and Mouchiroud, 2001), but it also implies that immature enamel matrix is deposited and during the secretory this region of the amelogenin is not subjected to phase. The expression decreases at the onset of the maturation functional/structural constraints. This is an advantage in the process, and completely disappears in post-secretory ameloblasts

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Lizard Amelogenin

facing the mature enamel. This is similar to previous descriptions of amelogenin gene expression during mammalian amelogenesis (Snead et al., 1988; Inage et al., 1996; Bleicher et al., 1999). We never detected amelogenin transcripts in the odontoblasts at any stage of their development. This confirms previous studies showing that full-length amelogenin is specific to enamel matrix (Snead et al., 1988; Inage et al., 1996; Bleicher et al., 1999). Recently, amelogenin expression was described in odontoblasts during mantle dentin deposition in pigs (Oida et al., 2002) and rats (Papagerakis et al., 2003). However, the amelogenin was expressed at a low level and during a specific stage of odontoblast differentiation, suggesting a possible role in epithelial-mesenchymal interactions (Papagerakis et al., 2003). Indeed, some amelogenin splicing products have been shown to act in vitro as signaling molecules during mineralized tissue formation (Veis et al., 2000), and particularly during cementogenesis (Boabaid et al., 2004). In conclusion, the amelogenin expression pattern during amelogenesis in the scincid lizard is similar to that described in mammals, and the architectural (prism) and structural (slight delay in maturation) differences do not seem to be related to variations in the timing of amelogenin deposition. However, given the large differences observed in the primary structure of the variable region of the protein, one can ask whether they would not result in variations in enamel microstructure and, as a consequence, in differences in resistance of the enamel to wear and injury.

ACKNOWLEDGMENTS We are grateful to Prof. Ann Huysseune (Ghent University, Belgium) for helpful criticism of the manuscript, and to two referees for their constructive remarks. Mrs. F. Allizard is acknowledged for her excellent technical assistance in preparing the sections. This project has been financially supported by the CNRS (UMR 7138), the "GIS-Institut des maladies rares", and a grant to S.D from the "Institut français pour la Recherche odontologique, IFRO".

REFERENCES Bleicher F, Couble ML, Farges JC, Couble P, Magloire H (1999). Sequential expression of matrix protein genes in developing rat teeth. Matrix Biol 18:133-143. Boabaid F, Gibson CW, Kuehl MA, Berry JE, Snead ML, Nociti FH Jr, et al. (2004). Leucine-rich amelogenin peptide: a candidate signaling molecule during cementogenesis. J Periodontol 75:1126-1136. Delgado S, Davit-Béal T, Sire JY (2003). Dentition and tooth replacement pattern in Chalcides (Squamata; Scincidae). J Morphol 256:146-159. Delgado S, Girondot M, Sire JY (2005a). Molecular evolution of amelogenin in mammals. J Mol Evol 60:12-30. Delgado S, Davit-Béal T, Allizard F, Sire JY (2005b). Tooth development in a scincid lizard, Chalcides viridanus (Squamata), with particular attention to enamel formation. Cell Tissue Res 319:71-89. Fincham AG, Moradian-Oldak J (1993). Amelogenin post-translational modifications: carboxy-terminal processing and the phosphorylation of bovine and porcine "TRAP" and "LRAP" amelogenins. Biochem Biophys Res Commun 197:248-255. Fincham AG, Hu Y, Lau EC, Slavkin HC, Snead ML (1991). Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth. Arch Oral Biol 36:305-317.

143

Hart PS, Hart TC, Simmer JP, Wright JT (2002). A nomenclature for X-linked amelogenesis imperfecta. Arch Oral Biol 47:255-260. 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. Hughes S, Mouchiroud D (2001). High evolutionary rates in nuclear genes of squamates. J Mol Evol 53:70-76. Inage T, Shimokawa H, Wakao K, Sasaki S (1996). Gene expression and localization of amelogenin in the rat incisor. Adv Dent Res 10:201-207. Ishiyama M, Mikami M, Shimokawa H, Oida S (1998). Amelogenin protein in tooth germs of the snake Elaphe quadrivirgata, immunohistochemistry, cloning and cDNA sequence. Arch Histol Cytol 61:467-474. Moradian-Oldak J, Simmer JP, Sarte PE, Zeichner-David M, Fincham AG (1994). Specific cleavage of a recombinant murine amelogenin at the carboxy-terminal region by a proteinase fraction isolated from developing bovine tooth enamel. Arch Oral Biol 39:647-656. Moradian-Oldak J, Iijima M, Bouropoulos N, Wen HB (2003). Assembly of amelogenin proteolytic products and control of octacalcium phosphate crystal morphology. Connect Tissue Res 44(Suppl 1):58-64. Oida S, Nagano T, Yamakoshi Y, Ando H, Yamada M, Fukae M (2002). Amelogenin gene expression in porcine odontoblasts. J Dent Res 81:103-108. Paine ML, Luo W, Zhu DH, Bringas P Jr, Snead ML (2003). Functional domains for amelogenin revealed by compound genetic defects. J Bone Miner Res 18:466-472. Papagerakis P, MacDougall M, Hotton D, Bailleul-Forestier I, Oboeuf M, Berdal A (2003). Expression of amelogenin in odontoblasts. Bone 32:228-240. Ravindranath RM, Moradian-Oldak J, Fincham AG (1999). Tyrosyl motif in amelogenins binds N-acetyl-D-glucosamine. J Biol Chem 274:2464-2471. Sander PM (2001). Primless enamel in amniotes: terminology, function and evolution. In: Development, function and evolution of teeth. Teaford M, Ferguson MWJ, Smith MM, editors. New York: Cambridge University Press, pp. 92-106. Sire JY, Davit-Beal T, Delgado S, Van Der Heyden C, Huysseune A (2002). First-generation teeth in nonmammalian lineages: evidence for a conserved ancestral character? Microsc Res Tech 59:408-434. Sire JY, Delgado S, Fromentin D, Girondot M (2005). Amelogenin: lessons from evolution. Arch Oral Biol 50:205-212. Snead ML (2003). Amelogenin protein exhibits a modlar design: implications for form and function. Connect Tissue Res 44(Suppl 1):47-51. Snead ML, Luo W, Lau EC, Slavkin HC (1988). Spatial- and temporal-restricted pattern for amelogenin gene expression during mouse molar tooth organogenesis. Development 104:77-85. Toyosawa S, O'hUigin C, Figueroa F, Tichy H, Klein J (1998). Identification and characterization of amelogenin genes in monotremes, reptiles, and amphibians. Proc Natl Acad Sci USA 95:13056-13061. Veis A, Tompkins K, Alvares K, Wei K, Wang L, Wang XS, et al. (2000). Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo. J Biol Chem 275:41263-41272. Wang X, Ito Y, Luan X, Yamane A, Diekwisch TG (2005). Amelogenin sequence and enamel biomineralization in Rana pipiens. J Exp Zoolog B Mol Dev Evol 304:177-186.