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Tooth Enamel Development

Enamel Development

Defining the boundaries between potency and commitment. The human body hosts a number of diverse stem cell populations that sustain continuous turnover and self-renewal of their respective host tissues and provide a reservoir for new tissue formation. Similar to differentiated cells that reside within target tissues, these “adult” stem cell populations are originally derived from embryonic stem cells, but unlike differentiated cells retain various degrees of pluripotency or multipotency. Among tissue-specific adult stem cells, orofacial stem cells as a group balance the stability and plasticity of their developmental states while at the same time sustaining tissue-specific cell renewal capabilities for alveolar bone, dentin, cementum and periodontal ligament tissues. As much as this fine-tuned equilibrium is essential for the physiological function of orofacial stem cells during the self-renewal of host tissues, offsetting this balance by manipulating signaling, genetic and epigenetic factors harbors tremendous therapeutic opportunities.

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B1. Structural Biology of Enamel matrix

Enamel Protein Function

Figure 3: 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).

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.

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Independence of Enamel Crystals from Adjacent Dentin Crystals.

Figure 6: 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.

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).

Architecture of the Mouse Amelogenin Gene

Figure 7: 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).

Scientists

Dr. Tianquan Jin uses nuclear magnetic resonance spectroscopy to understand the structure and function of enamel proteins in 3D. Yoshohiro Ito has developed a series of mouse models to further decipher developmental mechanisms of enamel formation. Caryn Reizl Ayarzagoitia is a biology student interested in the effect of loss of amelogenin on enamel formation. Marcella Schmidt is a bioengineering graduate student studying temporo-spatial events in enamel formation. UIC Collaborators on the enamel biomineralization project include Profs. Steven Guggenhein in the Department of Earth Sciences and Timothy Keiderling in the Department of Chemistry.

Dr. Tianquan Jin

Dr. Caryn Reizl Ayarzagoitia

Dr. Yoshihiro Ito

Related Publications

Jin, T., Ito, Y., Luan, X., Dangaria, S., Walker, C., Allen, M., Kulkarni A., Gibson, C., Braatz, R., Liao, X., and Diekwisch, T.G.H. (2009). Supramolecular compaction through polyproline motif elongation as a mechanism for vertebrate enamel evolution. PLoS Biology 7(12): e1000262. doi:10.1371/journal.pbio.1000262.

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.

Diekwisch, T.G.H. (1998). Subunit compartments of secretory stage enamel matrix. Conn. Tiss. Res. 38, 101-111.

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.

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.

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.

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.

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.

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.

Diekwisch, T.G.H., Marches, F., Spears, R., and Dechow, P. (1995). Effect of enamel protein expression on enamel crystal formation: a phylogenetic study. R.J. Radlanski and H. Renz (Eds.) Proc. 10th Int. Symp. Dent. Morph., Berlin 1995, pp. 82-87.

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.

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.