Enamel Development

Tooth enamel development begins with the differentiation of cells of the oral epithelium. Oral epithelium cells thicken and protrude into the underlying mesenchyme while forming the inner enamel epithelium. Following a series of epithelial-mesenchymal interactions, these cells further develop and differentiate to form pre-ameloblasts which in turn become fully differentiated ameloblasts. Ameloblasts are organized into a layer of highly prismatic cells that secrete enamel proteins such as amelogenin. They are also involved in the transport of mineral ions such as calcium and phosphate into the enamel matrix. The process by which enamel proteins such as amelogenin mediate the formation of hydroxyapatite crystals from calcium and phosphate is called enamel biomineralization.

Figure 1. This figure illustrates the localization of the amelogenin protein in the ameloblast cells and their secretory vesicles as well as in the newly formed enamel layer of developing teeth. The amelogenin protein is labeled in red color.
Figure 1
Figure 2 Figure 3
Figures 2 and 3. Enamel crystals are some of the most astounding structures in nature featuring extremely long and parallel organized hydroxyapatite crystals organized in bundles which are called prisms. Rows of enamel prisms are often organized perpendicular to each other. For orientation purposes, the enamel prisms containing bundles of hydroxyapatite crystals in the figures above are colorized. Fig. 2 is a Scanning Electron Micrograph and Fig. 3 is a Transmission Electron Micrograph.

Enamel Protein Function

Figure 4

Tooth enamel is a wonderful biomineral that evolves as the result of highly controlled interactions between specialized enamel proteins and tooth minerals. Prior to 1993, it was not known how enamel proteins interact with the tooth mineral and whether they had any function at all. In our landmark 1993 publication, we were the first to establish a concrete relation between amelogenin enamel protein function and hydroxyapatite crystal habit (Diekwisch et al. 1993). Specifically, we demonstrated that both enamel crystal size and enamel matrix subunit dimensions were significantly reduced following amelogenin function inhibition via antisense oligonucleotides. Our studies on the functional alteration of enamel matrix dimensions provided the basis for the "nanosphere theory" of enamel development (Fincham et al. 1994). In 1998, we were able to identify the nature of the protein nanospheres in the developing enamel matrix using Atomic Force Microscopy and specialized electron microscopic imaging techniques (Diekwisch 1998). Most recently, we were the first to be able to crystallize amelogenin enamel proteins. This work is supported by the NIDCR grant DE13378.

Figure 4 . Following antisense inhibition of amelogenin translation, the supra-organizational pattern of enamel proteins changes via reduction of sub-unit compartment size. As a consequence, hydroxyapatite enamel crystal growth is inhibited (Diekwisch et al. 1993).
Figure 5 Figure 6
Figures 5 and 6. In the developing enamel matrix, enamel proteins are organized into spherical substructures, which were illustrated here for the first time in vivo using Atomic Force Microscopy. From: Diekwisch (1998). Subunit compartments of secretory stage enamel matrix. Conn. Tiss. Res. 38, 101-111.
Independence of Enamel Crystals from Adjacent Dentin Crystals.

The dogma that enamel forms on dentin and therefore enamel crystals form as extension of dentin crystals goes back to the German microscopists at the end of the 19th century and has found entry into most standard textbooks. As recently as 1988, Arsenault and Robinson published a study in support of this theory. A number of experiments demonstrating the uniqueness of enamel tissue-specific biomineralization prompted us to question the established doctrine. We therefore performed a set of studies to test whether enamel crystals grew independent from adjacent dentin mineral (Diekwisch et al. 1995). Our 1995 paper in Cell and Tissue Research demonstrating enamel crystals growing independently from mineralized dentin has meanwhile been confirmed by several other authors (Takano et al. 1996, Dong and Warshawsky 1996, Cuisinier et al. 2000, and others). Other papers on the localization of proteins in the developing enamel matrix have dealt with tuftelin (Diekwisch et al. 1997), proteoglycans (Thieberg et al. 1999), and enamel proteases (Satchell et al., in press).

Figure7
Figure 7. As this transmission electron micrograph of an anhydrously prepared specimen demonstrates, enamel crystals grow in tight proximity to the adjacent dentin. However, careful analysis reveals that enamel crystals grow independently from adjacent dentin crystals. From: Diekwisch et al. (1995). Initial enamel crystals are spatially not associated with mineralized dentine. Cell & Tissue 279, 149-167.
Architecture of the Mouse Amelogenin Gene.
Figure 8
Figure 8 demonstrates important segments of the major enamel matrix protein, amelogenin. The colored dot indicates the position of a functionally significant amino acid: the singular phosphorylation site in Ser16. The horizontal bar diagram illustrates important domains, including the tyrosyl binding motif between codons 34 and 45, the metalloproteinase cleavage sites in codons 151-170, and the hydrophilic domain of the carboxy-terminal 13 amino acids. Protein binding studies have identified two highly conserved domains, the N-terminal A-domain and the C-terminal B-domain. A third functionally important domain, the C-domain, is encoded by exon fragments 6b and 6c and contains the evolutionarily important tandem repeats that are the focus of this study. The exon structure bar graph illustrates the expressed margins of exons 2 and 7 as well as exons 3, 5, and 6. Exon 4 has only been detected in a few species at low levels. Exon 1 is situated 5' of the start-codon and thus never expressed. Exon 6 alone accounts for 145 amino acids, which is roughly 80% of the entire expressed sequence. The two fragments of exon 6, which encode the important tandem repeats, namely 6b and 6c (C-domain), amount for 97 amino acids altogether, which is still more than half of the entire protein. The position of the amelogenin polypeptides LRAP and TRAP is indicated below. The M59 "Pre-LRAP" is encoded by an mRNA generated by exon skipping and its translation product has been detected as "A-4" in dentin and pulp. M59 has not been found as a translated enamel matrix protein product since it is cleaved into a 46 amino acid polypeptide (LRAP - leucine-rich amelogenin peptide) by removal of the C-terminal 13 amino acids as soon as it enters the developing enamel matrix. Similar to LRAP, which is most likely cleaved by MMP20 metalloproteinases, TRAP (tyrosine-rich amelogenin peptide) is a proteinase degradation product, most likely generated by the proteolytic activity of EMSP1 (enamel matrix serin proteinase 1).
1 Wang, X., Fan, J.-L., Ito, Y., Luan, X., and Diekwisch, T.G.H. (2006). Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana. J. Exp. Zool. Mol. Dev. Evol. 305B in press.
2 Diekwisch, T.G.H., Wang, X., Fan, J.-L., Ito, Y., and Luan, X. (2006). Expression
and characterization of a Rana pipiens amelogenin protein. Eur. J. Oral Sci. in press.
3 Wang, X., Ito Y., Luan, X., Yamane, A., and Diekwisch, T.G.H. (2005). Amelogenin
sequence and enamel biomineralization in Rana pipiens. J. Exp. Zool. Mol. Dev. Evol. 304B:1-10.
4 Diekwisch, T.G.H., Berman, B.J., Anderton, X., Gurinsky, B., Ortega A.J., Satchell P.G., Williams, M., Arumugham C., Luan X., McIntosh J.E., Yamane A., Carlson, D.S., Sire, J.-Y., Simmer J.P., and Shuler, C.F. (in press). Membranes, Minerals, and Proteins of Developing Vertebrate Enamel. Microscopy Research Technique.
5 Satchell, P.G., Anderton, X., Ryu, O.H., Luan, X., Ortega, A.J., Opamen, R., Berman, B.J., Witherspoon, D.E., Gutmann, J.L., Yamane, A., Zeichner-David, M., Simmer, J.P., Shuler, C.F., and Diekwisch, T.G.H. (in press). Conservation and variation in enamel protein distribution during tooth development across vertebrates. Mol. Dev. Evol.
6 Thieberg, R.H., Yamauchi, M., Satchell, P.G., and Diekwisch, T.G.H. (1999). Sequential distribution of keratan sulfate and chondroitin sulfate epitopes during ameloblast differentiation. Histochem. J. 31, 573-578.
7 Diekwisch, T.G.H. (1998). Subunit compartments of secretory stage enamel matrix. Conn. Tiss. Res. 38, 101-111.
8 Zeichner-David, M., Vo. H., Tan, H., Diekwisch, T., Berman, B., Thiemann, F., Alcocer, M.D., Hsu, P., Wang, T., Reyna, J., Caton, J., Slavkin, H.C., and MacDougall, M. (1997). Timing of the expression of enamel gene products during mouse tooth development. Int. J. Dev. Biol. 41, 27-38.
9 Diekwisch, T.G.H., Ware, J., Fincham, A.G., and Zeichner-David, M. (1997).
Immunohistochemical similarities and differences between amelogenin and tuftelin gene products during tooth development. J. Histochem. Cytochem. 40, 859-866.
10 Fincham, A.G., Moradian-Oldak, J., Diekwisch, T.G.H., Lyaruu, D.M., Wright, J.T., Bringas, P., Jr., and Slavkin, H.C. (1995). Evidence for amelogenin "nanospheres" as functional components of secretory-stage enamel matrix. J. Struct. Biol. 115, 50-59.
11 Moradian-Oldak, J., Simmer, J.P., Lau, E.C., Diekwisch, T., Slavkin, H.C., and Fincham, A.G. (1995). A review of the aggregation properties of a recombinant amelogenin. Connect. Tiss. Res. 32, 125-130.
12 Zeichner-David, M., Diekwisch, T., Fincham, A., Lau, E., Mac Dougall, M., Moradian-Oldak, J., Simmer, J., Snead, M., and Slavkin, H.C. (1995). Control of Ameloblast Differentiation. J.-V. Ruch (Ed.), Odontogenesis: Embryonic dentition as a tool for developmental biology. Int. J. Dev. Biol. 39, 69-92.
13 Diekwisch, T.G.H., Berman, B.J., Gentner, S., and Slavkin, H.C. (1995). Initial enamel crystals are spatially not associated with mineralized dentine. Cell & Tissue 279, 149-167.
14 Fincham, A.G., Moradian-Oldak, J. Simmer, J.P., Sarte, P., Lau, E.C., Diekwisch, T., and Slavkin, H.C. (1994). Self-assembly of a recombinant amelogenin protein generates supramolecular structures. J. Struct. Biol. 112, 103-109.
15 Diekwisch, T., David, S., Bringas, P., Santos, V. and Slavkin, H.C. (1993). Antisense inhibition of AMEL translation demonstrates supramolecular controls for enamel HAP crystal growth during embryonic mouse molar development. Development 117, 471-482.
16 Diekwisch, T. (1989). Localization of microfilaments and microtubules during dental development in the rat. Acta histochemica 37, 209-212.