Center for Craniofacial Molecular Biology

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional 10-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific genes.

Experiments were designed to detect and determine the biosynthetic behavior of enamel proteins in Syrian Golden hamsters. Enamel matrix proteins were extracted from 3-day-old postnatal first molar tooth organs. Labeling pulse/chase experiments with [35S]-methionine followed by light microscopic autoradiography, or polyacrylamide slab gel electrophoresis and fluorography, showed the synthesis of epithelial-specific gene products. Synthesis and secretion of enamel proteins required approximately 30 min under these in vitro organ culture conditions; both enamelin and amelogenin proteins were synthesized and secreted into the forming extracellular matrix. Amelogenin proteins were secreted initially and rapidly degraded into increasingly smaller polypeptides. In contrast, enamelin proteins were secreted at a slower rate and remained more or less stable over the duration of the experiment. The specific activities of both classes of proteins increased over a 6-hour synthesis period, indicating the accumulation of both proteins into the forming extracellular matrix. Comparisons of the kinetics of formation and posttranslational processing of enamelin and amelogenin are consistent with the presence of possibly two different gene products in hamster secretory ameloblasts.

Mammalian enamel matrix is composed of two principal proteins, the enamelins and amelogenin. Recombinant complementary DNA (cDNA) molecules for the predominant mouse amelogenin have been identified, characterized by direct determination of the DNA sequence, and used as a specific hybridization probe. The spatial- and temporal-restricted pattern for amelogenin gene expression within developing mouse molars has been traced at the level of a single cell using in situ hybridization. The mouse genome has been shown to contain only one copy of the amelogenin (AMEL) gene which is not amplified or rearranged during ameloblast determination. In contrast, the human genome contains two copies of the AMEL gene, one residing on the X chromosome and one upon the Y chromosome. These observations, the availability of specific enamel gene probes coupled with the application of new techniques in molecular biology now afford unique opportunities for the analysis of the molecular basis of inherited defects of human enamel such as amelogenesis imperfecta. Recent advances towards obtaining a physical map and the complete nucleotide sequence for the human genome, as well as the documented developmental biology, defined genetics and transgenic capability of the mouse, suggest that mouse and man are the most relevant and potentially informative models for analysis of normal and abnormal enamel biomineralization.

Experiments were designed to compare extracellular dentin phosphoprotein (DPP) and nascent DPP prior to post-translational modifications from several vertebrate species. Dental matrix proteins were extracted with acetic acid, followed by GuHCl-EDTA, and precipitated with CaCl2. Cross-reactivity of the DPPs with a mouse DPP antibody was determined by a dot-immunobinding assay. To analyze nascent DPP, mRNA was isolated from developing tooth organs and the mRNA-directed translation products were immunoprecipitated with the DPP specific antibody. All DPP components identified in the species which contained a DPP were shown to cross-react with the polyclonal mouse DPP antibody. The extracellular matrix DPPs were found to exhibit as much as a 30 kDa size difference using the same SDS PAGE system. In contrast, nascent DPPs were found to be the same size for all species examined. Our results indicate that differences in the molecular weight size of DPPs between species may be due to the degree of post-translational modifications such as phosphorylation.

Epithelial differentiation is a complex process which requires an integrated synthesis of DNA along with synthesis of a full complement of unique mRNAs and their respective proteins characteristic for each cell type. The time of initial transcription of enamel protein mRNAs and subsequent translation of proteins characteristic for secretory ameloblasts is not known. In order to determine when enamel protein mRNAs appear during New Zealand White rabbit molar tooth organogenesis, and when nascent enamel proteins are first translated, we analyzed early cap stages through late crown stages of molar tooth formation (i.e., 21-days gestation through 2-days postnatal). The biochemical phenotype which characterized rabbit ameloblasts were the acidic glycoproteins termed enamelins. Polyclonal antibodies were produced against the major fetal rabbit enamelin of approximately 70,000 daltons. Immunoprecipitation of enamelins from mRNA-directed translation products in a reticulocyte cell-free system, was used to characterize enamelin mRNAs. Enamelin mRNAs were first detected during bell stages (circa 23-days gestation), and persisted till crown stage (circa 28-days gestation). Indirect immunofluorescent localization of enamelin antigen showed staining over the extracellular enamel organ matrix by 23-days gestation. Neither enamelin mRNAs or polypeptides were detected during early or late cap stages of odontogenesis. Transcription of enamelin mRNAs coding for two enamelins of 65 an 58 kd (kilodaltons) appeared to be closely coupled with the translation of these enamel proteins. We assume that close-range ectomesenchyme- derived instructions mediate the biochemical differentiation of ameloblasts between 21-days and 23-days gestation during fetal rabbit development.

We have established the time and position of expression for multiple enamel proteins during the development of the mouse molar tooth organ. Using high-resolution two-dimensional gel electrophoresis coupled with immunoblotting and immunocytochemistry, a 46-kDa enamel protein (pI, 5.5) was detected during late cap stage (18-days gestation, E18d) within differentiation-zone-II inner enamel epithelia associated with an intact basal lamina. At E19d a second enamel polypeptide of 72 kDa (pI, 5.8) was identified at the time and position of initial biomineralization in differentiation zone V. At 20 days, differentiation-zone-VI ameloblasts without basal lamina (late bell stage) expressed 46- and 72-kDa enamel proteins and, in addition, expressed a relatively more basic 26-kDa enamel protein (pI, 6.5-6.7); detected after initial formation of calcium hydroxyapatite crystals. Antibodies raised against chemically synthesized enamel peptides cross-reacted with both the 72-kDa and 26-kDa polypeptides, but did not cross-react with the 46-kDa enamel polypeptide. The sequential expression of multiple enamel proteins suggests several functions: (a) the anionic enamel proteins may provide an instructive template for calcium hydroxyapatite crystal formation; (b) the more neutral proteins possibly serve to regulate size, shape and rates of enamel crystal formation. We suggest that initial expression of enamel gene products during mouse tooth development possibly recapitulates ancestral features of amelogenesis documented in prereptilian vertebrates. These results imply that multiple instructive signals may be responsible for mammalian enamel protein induction and that the sequential expression of a family of enamel proteins reflects the evolutionary acquisition of a more complex genetic program for amelogenesis.

Previous results from our laboratory indicated that rabbit enamel high molecular weight proteins have the same amino-terminal sequence that amelogenins, thus suggesting the possibility that this domain is shared by both, enamelins and amelogenins. To determine if this is true for other species, enamel proteins and mRNA were extracted from rabbit and hamster developing teeth and analyzed using probes targeted towards the N-terminal sequence of the amelogenins. Our results strongly suggest that both, enamelins and amelogenins share the same amino-terminal amino acid sequence.

The developmental problem of how dental epithelia and/or dental papilla ectomesenchyme induce and/or up- or down-regulate tooth formation are as yet unresolved issues. We have designed studies to map the synthesis and fate pathways of secreted amelogenin proteins from Kallenbach differentiation zones II-IV during in vivo and in vitro mouse mandibular first molar tooth development (M1). Tooth organs from cap, bell, and crown stages were processed for reverse transcriptase/polymerase chain reaction (RT-PCR) and high resolution Protein A immunocytochemistry using anti-amelogenin and anti-peptide antibodies. Cap stage M1 were cultured for periods ranging from 10-21 days in vitro using either serum-less, or 15% fetal calf sera-supplemented, chemically-defined medium. Amelogenin transcripts are expressed in the mouse embryonic molar from E15 through early postnatal development. Amelogenin antigens were first detected in Kallenbach's differentiation zone II. Amelogenin proteins secreted from preameloblasts were identified along cell processes and cell surfaces of odontoblasts adjacent to forming mantle dentine extracellular matrix (ECM) prior to biomineralization. Amelogenin proteins were restricted to forming endocytotic vesicles, clathrin-coated vesicles, and lysosomes within odontoblasts. At later stages (e.g. 2 days postnatal development), enamel proteins were not identified in odontoblasts or predentine matrix following mineralization. Comparable observations for stages of development were noted for in vitro cultured tooth explants. Preameloblasts synthesize and secrete amelogenin proteins which bind to odontoblast cell surfaces possibly through the process of receptor-mediated endocytosis. We conclude that amelogenin proteins secreted from preameloblasts, prior to the initiation of biomineralization, were translocated to odontoblasts to serve as yet unknown biological functions.

Insulin and insulin-like growth factors (IGF-I and IGF-II) are considered pleiotropic, acting as both mitogen and differentiation factors. Several investigators have demonstrated the expression of insulin, IGFs, their cognate receptors and IGF binding proteins during tooth morphogenesis. Previous work done in our laboratory indicated that exogenous insulin and IGFs induce the accumulation of enamel extracellular matrix on mouse mandibular molars cultured in a serumless, chemically defined medium. In order to determine the level of control of these factors in the induction of enamel biomineralization, we designed experiments to quantitate mRNAs for enamel specific-gene products. Mandibular first molars (MI) obtained from E15 Swiss Webster mice were placed in organ culture in the presence of insulin (1,000 ng/ml), IGF-I (100 ng/ml) or IGF-II (100 ng/ml) for 6, 12 and 18-days. At termination date, the RNA was extracted and the concentration of mRNAs for amelogenin, tuftelin and ameloblastin were determined using a quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) technique (PCR mimic). Our results showed that after 6-days in culture; treatment with insulin, IGF-I and IGF-II increased the synthesis of amelogenin and ameloblastin. In contrast, the expression of tuftelin mRNA was not affected by either factor. In conclusion, our studies showed that the increase in enamel matrix formation by overexpression of IGFs is the result of transcriptional regulation of enamel specific proteins like amelogenin and ameloblastin but not tuftelin. These studies also suggest that the regulatory mechanisms controlling tuftelin gene expression are different than the mechanisms regulating ameloblastin and amelogenin transcription.