Human developing enamel proteins exhibit a sex-linked dimorphism.

The amelogenin protein of developing dental enamel is generally accepted to mediate the regulation of the form and size of the hydroxyapatite crystallites during enamel biomineralization (1). A genetic disorder of enamel development (amelogenesis imperfecta) has been linked to the amelogenin gene AMEL(2-3), and loci regulating enamel thickness and tooth size have been mapped to the human sex chromosomes (4). In the human genome there are two AMEL loci with one copy of the gene on each of the sex chromosomes (AMELX and AMELY), whereas in the mouse only an AMELX locus is present (5). It is presently unknown if human AMELY is transcriptionally active. These observations prompted us to examine specimens of human developing enamel for sexual dimorphism at the protein level. We report here, for the first time, a diagnosis of differences in human enamel proteins which permits the distinction of specimens according to the sex of the individual.

Epidermal growth factor regulates gene expression of both epithelial and mesenchymal cells in mouse molar tooth organs in culture.

Epidermal growth factor and cis-hydroxyproline specifically inhibited synthesis of type 1 collagen, a major gene product of the differentiated dental mesenchymal cells (odontoblasts). In tandem, synthesis of enamel proteins, specific gene products of differentiated dental epithelial cells (ameloblasts), was also inhibited. Under these culture conditions, total protein synthesis in tooth organs was not inhibited but rather increased. Inhibition curves of the gene products specific for epithelial and mesenchymal phenotypes were quite similar, indicating coordinate and intimately associated regulation of gene expression under conditions that perturb cytodifferentiation.

Cartilage, bone and tooth induction during early embryonic mouse mandibular morphogenesis using serumless, chemically-defined medium.

Studies were designed to test the hypothesis that plasma- and serum-deprived embryonic cells and tissues in vitro are capable of producing growth regulating factors which augment cartilage, bone and tooth induction during mouse mandibular process development. Embryonic mouse first branchial arch-derived mandibular processes (E11-E12, Theiler stages 18-19) or cap stage molar tooth (M1) organs (E15-E16, Theiler stage 23) expressed morphogenesis, histogenesis and cytodifferentiation (e.g., Meckel's cartilage and mandibular bone) when cultured as explants in permissive serumless and chemically-defined BGJB medium for periods up to 31 days in vitro. Organ cultures of early mandibular process explants in serumless conditions showed DNA synthesis comparable to the time- and position-restricted patterns characteristic for control in vivo development. As a paradigm for embryonic cell expression of putative growth factors, sense and antisense oligodeoxynucleotide probes corresponding to amino acids 1070-1081 for preproEGF, and antibodies directed against amino acids 348-691 of preproEGF, were used to identify and localize mRNA transcripts and translation products. Our preliminary evidence suggests that odontogenic epithelial and ectomesenchyme cells produce EGF-like products during instructive phases of tooth development. We suggest that plasma- and serum-deprived cells and tissues in vitro produce autocrine and/or paracrine growth factors which mediate embryonic mandibular morphogenesis, histogenesis and cytodifferentiation.

Localization of epidermal growth factor precursor in tooth and lung during embryonic mouse development.

The murine epidermal growth factor (EGF) precursor is a 1217 amino acid protein which contains mature EGF (amino acid residues 977-1029) as well as eight EGF-like repeats. Although the highest levels of EGF are found in the adult male mouse submandibular gland, the results of in situ hybridization studies and mRNA analyses suggest that EGF precursor mRNA is synthesized in several adult mouse tissues including the lung and the incisor. To determine if EGF precursor gene expression is intrinsic to the developmental program for either embryonic tooth or lung organogenesis, sense and antisense oligodeoxyribonucleotide probes corresponding to amino acids 1070-1081 of the precursor were used to localize cellular sites of synthesis of EGF precursor mRNA by in situ hybridization. Antibodies directed against amino acid residues 348-691 of the precursor were used in immunodetection techniques to identify either EGF precursor protein or processed derivatives. In contrast to earlier reports indicating that embryonic mouse tissues do not synthesize EGF precursor mRNA, we found that EGF precursor mRNA is present in clusters of ectoderm-, mesoderm-, and ectomesenchyme-derived cells associated with embryonic teeth and lung organs. Moreover, epitopes common to the EGF precursor were immunolocalized in both the epithelial and mesenchymal tissues of embryonic mouse tooth and lung organs. These results suggest that the EGF precursor and/or motifs contained within the precursor molecule, including mature EGF, may play an instructive or permissive role in epithelial-mesenchymal interactions pursuant to organogenesis.

Amelogenesis in vitro: a model for studies of epithelial postsecretory processing during tissue-specific extracellular matrix biomineralization.

The extracellular matrix (ECM) of developing mammalian enamel comprises a complex of unusual epithelial-derived proteins, which appear to function in concert to initiate and propagate tissue-specific biomineralization. Following enamel protein synthesis by ameloblast cells within the enamel organ, the subsequent steps of posttranslational modification, secretion, postsecretory processing and eventual removal of these proteins from forming enamel are largely unknown. To address this issue we have designed studies to investigate the hypothesis that enamel proteins are removed from enamel and translocated into the vasculature as relatively high-molecular-weight components. We examined enamel proteins recovered from serumless medium during prolonged organ culture of mouse capstage mandibular first molars. By 21 days in vitro the tooth crown formed and dentine and enamel biomineralization were apparent. At 31 days, explants retained metabolic activity and the enamel matrix showed extensive transformation. Immunologically identified enamel proteins of 26-18 k Da were produced by cultured tooth organs, translocated from tooth explants to the culture medium, recovered from the medium and then compared to control enamel protein from in vivo preparations. Comparable postsecretory processing of the 26-k Da amelogenin protein was observed in vitro and in vivo. We speculate that the pathway reported in the present studies is comparable to the processing of the enamel protein polypeptides of the maturing enamel which occurs in vivo. The in vitro organ culture model described in this report provides an approach with which to investigate the molecular events associated with epithelial-derived postsecretory processing of ECM molecules associated with tissue-specific biomineralization.

Human and mouse cementum proteins immunologically related to enamel proteins.

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.

Hertwig's epithelial root sheath differentiation and initial cementum and bone formation during long-term organ culture of mouse mandibular first molars using serumless, chemically-defined medium.

Studies were designed to test the hypothesis that Hertwig's epithelial root sheath (HERS) synthesizes and secretes enamel-related proteins that participate in the process of acellular cementum formation. Our experimental strategy was to examine sequential root development of the mouse mandibular first molar in vivo and in long-term organ culture in vitro using serumless, chemically-defined medium. Using anti-amelogenin, anti-enamelin and anti-peptide antibodies, enamel-related antigens were localized within intermediate cementum during HERS differentiation and root formation in vivo. Cap stage molars maintained for periods of up to 31 days in organ culture expressed morphogenesis and cytodifferentiation as identified by tooth crown and initial root, cementum and bone formation. Metabolically-labeled HERS products were analyzed by immunodetection using enamel-related antibodies and one- and two-dimensional SDS gel electrophoresis. A 72 kDa and 26 kDa polypeptide were identified in forming mouse cementum. Both of these root putative cementum proteins yield similar (identical) amino acid compositions; however, both proteins differed from the compositions of either mouse crown enamelin or amelogenin proteins. This approach provides a new and novel in vitro model towards understanding HERS differentiation and functions related to root and bone formation. The data support the hypothesis that HERS cells synthesize polypeptides related to but also different from canonical crown enamel proteins.

Biosynthesis and characterization of rabbit tooth enamel extracellular-matrix proteins.

Tooth enamel biomineralization is mediated by enamel proteins synthesized by ameloblast cells. Two classes of proteins have been described: enamelins and amelogenins. In lower vertebrates the absence of amelogenins is believed to give rise to aprismatic enamel; however, rabbit teeth, which apparently do not synthesize amelogenins, form prismatic enamel. The present study was designed to characterize the enamel proteins present in rabbit tooth organs and to gain an insight into the process of biomineralization. Rabbit enamel extracellular-matrix proteins were isolated and characterized during sequential stages of rabbit tooth organogenesis. The biosynthesis of enamel proteins was analysed by metabolic 'pulse-chase' experiments as well as mRNA-translation studies in cell-free systems. Our results indicated that rabbit enamel extracellular matrix contains 'amelogenin-like' proteins. However, these proteins are not synthesized as typical amelogenins, as in other mammalian species, thus suggesting that they are the processing products of higher-molecular-mass precursors. An N-terminal amino acid sequence of 29 residues, considered characteristic of mammalian amelogenins, was present in the rabbit 'amelogenin-like' proteins. By using anti-peptide antibodies to this region, similar epitopes were detected in all nascent enamel proteins, including enamelins. These studies suggest that the N-terminal sequence might be characteristic of all enamel proteins, not only amelogenins.

Sequential expression and differential function of multiple enamel proteins during fetal, neonatal, and early postnatal stages of mouse molar organogenesis.

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.

Amelogenin antigenic domain defined by clonal epitope selection.

To experimentally examine the participation of amelogenins in controlled mineral-phase maturation of mammalian enamel, the identification of the individual proteins and their corresponding gene(s) is required. For this purpose, cDNAs were constructed from polyadenylated RNA from 2-day postnatal murine teeth, molecularly cloned into lambda-gt11 expression vectors and transfected into E. coli. The cDNA library was screened for amelogenin gene(s) by using either antibody or nucleic acid probes. An amelogenin cDNA clone encoding 79 carboxy-terminal amino acid residues and 100 nucleotides of the 3' noncoding sequence was demonstrated to contain a major antigenic site for amelogenin protein by immunostaining of specific amelogenin proteins from total extracted enamel protein blots using clonal epitope selected antibody. This is the first report linking amelogenin epitope(s) to a defined DNA sequence, and consequently a defined portion of the amino acid sequence for amelogenins. Secondary structure analysis, based on the relative average linear hydropathy of the amino acid sequence of amelogenin, predicted epitopes in the amino terminus of the molecule rather than the carboxy terminus. Our present data suggest that the carboxy terminus of the amelogenins is sufficiently externalized to be an antigenic domain. These data may be useful in subsequent structural analysis of amelogenin proteins and enhancing our understanding of their physicochemical participation in biomineralization.

Immunochemical homology between elasmobranch scale and tooth extracellular matrix proteins in Cephaloscyllium ventriosum.

Studies were designed to test the hypothesis that homologous proteins are expressed in elasmobranch scale, tooth enameloid, and mammalian enamel. Using indirect immunohistochemistry and high-resolution two-dimensional gel electrophoresis with immunoblotting, mouse enamel proteins were compared with placoid scale and enameloid proteins from the swell shark, Cephaloscyllium ventriosum. Swiss Webster mouse molar teeth show a characteristic enamel protein pattern consisting of two anionic enamel proteins of 72 kDa (pI 5.8) and 46 kDa (pI 5.5) and several more basic and lower-molecular-weight enamel polypeptides. Both anionic and basic classes of enamel proteins cross-reacted with either antiamelogenin or antienamelin antibodies. Placoid scale and tooth enameloid contained two anionic proteins identified as 58 kDa (pI 5.7) and 46 kDa (pI 5.5), which cross-reacted with either antimouse amelogenin or antihuman enamelin IgG antibodies. A minor antigenically related protein of 43 kDa (pI 6.2) was detected. Immunochemical staining showed localization within placoid scale, swell shark inner enamel epithelia, enameloid, and mouse inner enamel epithelia and enamel. We interpret these results to suggest that both placoid scale and enameloid proteins share epitopes and that these epitopes are also shared with mammalian enamel proteins. Based on molecular weights, isoelectric pH values, and amino acid compositions, placoid scale and enameloid ECM proteins do not contain amelogenin proteins. We suggest that enamelinlike proteins are highly conserved during vertebrate evolution and that these relatively anionic macromolecules may serve a primary function in the initiation of calcium hydroxyapatite formation during enameloid biomineralization.

Total poly(A+)-messenger RNA from bovine lens cofractionates with sucrose purified fiber cell plasma membrane.

Poly(A+)-messenger RNA was isolated from bovine lens as well as poly(A+)-mRNA from crude and sucrose-purified plasma membrane. Bovine lens MP26 antisera was also propagated in rabbits, from which anti-MP26 IgG was partially purified and used to assay for the specific synthesis of MP26 during in vitro translation of the isolated poly(A+)-mRNAs. We found that membrane-associated poly(A+)-mRNA supported the synthesis of proteins which were identical to those translated from total lens fiber cell poly(A+)-mRNA. These proteins included MP26, as assayed by immunoprecipitation of [35S]-labeled MP26.

De novo gene expression detected by amelogenin gene transcript analysis.

Reciprocal epithelial-mesenchymal interactions are responsible for mouse molar tooth organogenesis. Only dental ectomesenchymal cells are capable of instructing adjacent epithelial cells to become determined to synthesize and secrete enamel-specific proteins termed the amelogenins. To identify when inner enamel epithelial cells first express enamel specific gene products, cytoplasmic RNA has been analyzed from developing teeth by hybridization to a cloned cDNA probe to one of the amelogenins. It is reported that the de novo expression of amelogenin-encoding RNA as well as immunoprecipitated amelogenin polypeptides are first detected at Theiler stage 27. These data indicate that ectomesenchymal-mediated induction of inner enamel organ epithelia results in both the nascent transcription of amelogenin RNA and subsequent translation of amelogenin polypeptides, which are first detected at birth.

Basal lamina persistence during epithelial-mesenchymal interactions in murine tooth development in vitro.

Numerous investigations have demonstrated the necessity of mesenchymal instruction for epithelial differentiation during epidermal organogenesis. In the specific case of tooth formation, cap-stage tooth organ mesenchyme instructs epithelial differentiation into ameloblasts with production of enamel extracellular matrix. The "instructive event" is presumed to be direct cell contact. Mesenchyme-mediated cell contact with adjacent epithelia is assumed to "instruct" epithelial differentiation into ameloblasts. If this were true, basal lamina removal and mesenchyme cell contact with epithelia would be prerequisites for epithelial cytodifferentiation and morphogenesis in the developing tooth system. To test this hypothesis, we designed experiments to evaluate basal lamina stability during epithelial differentiation into ameloblasts. Our studies utilized cap-stage murine molar tooth organs, a serumless and chemically defined medium (PYMS), metabolic isotopic labeling of basal lamina constituents, biochemical methods to analyze macromolecular stability throughout 10 days of organ culture in vitro, and immunological methods to localize the distribution of laminin and fibronectin. Our results indicate that (3H)glucosamine is incorporated into basement membranes present in Theiler stage 25 mandibular mouse molar tooth organ. At this stage, the isotope was incorporated into high molecular weight macromolecules. Specific enzyme methods coupled with electrophoresis and fluorography demonstrated that (3H)glucosamine was incorporated into proteoglycans containing chondroitin sulfates, dermatan sulfate, and hyaluronate. After 10 days in vitro the radiolabeled material remained localized in these same molecules, indicating stability of these constituents within basement membranes. Ultrastructural observations indicated that the basal lamina was not removed during ameloblast differentiation in vitro using PYMS medium. Laminin and fibronectin were localized in the basement membranes during cap stages and did not disappear during subsequent morphogenesis and differentiation. Mesenchymal cells appear to mediate epithelial differentiation in vitro using PYMS medium without a removal of the basal lamina.

Enamel gene products during murine amelogenesis in vivo and in vitro.

Epithelial-mesenchymal interactions regulate determination and differentiation of amelogenesis. Our attention has focused on identification of ameloblast gene products, the regulation of enamel mRNA synthesis, and subsequent translation into enamel proteins in vivo and in vitro. Enamel proteins are the most abundant gene products synthesized in fully-differentiated ameloblasts. Our experimental strategy has been to isolate major proteins, produce antibodies, localize enamel protein antigens during tooth development in vivo as well as in vitro (using serumless, chemically-defined medium), develop an immunoprecipitation assay, isolate poly(A)-products in a cell-free translation system, and then initiate molecular cloning of the corresponding murine enamel gene(s). The major murine enamel mRNA appears to code for a predominant polypeptide of approximately 20,000 MW. Inner-enamel epithelial cells differentiate into ameloblasts, and synthesize and secrete enamel proteins within six d when cap-stage molar tooth organs are cultured in serumless, chemically-defined medium. The regulation of epithelial differentiation under these experimental conditions indicates that epithelial-mesenchymal interactions determine and maintian ameloblast differentiation in vitro.

Spatiotemporal patterns of fibronectin distribution during embryonic development. I. Chick limbs.

It has been suggested that an extracellular matrix - and cell surface - associated glycoprotein, fibronectin, plays a role in the positioning of cells in morphogenesis and in the maintenance of orderly tissue organization. In the present study the appearance and distribution of fibronectin during in ovo chick limb development has been investigated by indirect immunofluorescence techniques in H.H. stages 20-30. Fibronectin is not detectable until just prior to the transition from the morphogenetic to the cytodifferentiation phase of development. Beginning at H.H. stage 25, successive nonrandom patterns of fibronectin detection and distribution, which resemble the subsequent cartilaginous elements, precede overt chondrogenesis as detected by Alcian blue staining. This corresponds to the onset of the cytodifferentiation phase of limb development. As the accumulation of acidic proteoglycan increases in the cartilage matrix and the mesenchymal cells become more round in appearance, the presence of detectable fibronectin decreases and is ultimately seen only in the perichondria and basement membrane. However, predigestion of developed cartilage tissue with testicular hyaluronidase, prior to fibronectin staining, indicated that fibronectin remains a major constituent of cartilage matrix and is apparently masked by cartilage-specific proteoglycans. This study of chick limb development is consistent with the hypothesis that fibronectin may be a molecule that facilitates the spatial organization of cartilaginous primordia cytodifferentiation.

Possible functions of mesenchyme cell-derived fibronectin during formation of basal lamina.

Epithelial synthesis and secretion of basal lamina has been considered to be a general feature of various vertebrate epithelium-mesenchyme interacting systems (e.g., salivary gland, mammary gland, feather, hair, and tooth morphogenesis). It has been repeatedly assumed that embryonic ectoderm and ectodermal derivatives, such as epithelial tissues associated with tooth morphogenesis, synthesize and secrete basal lamina. Basal lamina of embryonic mouse tooth organs contain laminin, type IV collagen, glycosaminoglycans, and possibly fibronectin. Ectodermally derived epithelia produce laminin, collagens, and glycosaminoglycans but they do not appear to produce fibronectin. Mesenchyme can effect basal lamina formation in vitro by releasing mesenchyme-derived fibronectin. Theiler stage 25 molar tooth mesenchymal and epithelial tissues were enzymatically separated and cultured in chemically defined media without serum, embryonic extracts, or antibiotics for periods not exceeding 24 hr. Isolated epithelia did not reconstitute a basal lamina. Mesenchymepreconditioned media, fibronectin substrata, or addition of 10% fetal calf serum induced reconstitution of epithelium-derived basal lamina. Dental mesenchyme-preconditioned medium contained, as a major component, a protein of M(r) approximately 2.3 x 10(5) identified as fibronectin by the criteria of gelatin binding and subunit molecular weight. Fibronectin was not produced by isolated epithelia. These results support the hypothesis that basal lamina ultrastructural organization results from supramolecular interactions between epithelium-derived macromolecules (e.g., type IV collagen, proteoglycans, glycosaminoglycans, and laminin) with mesenchyme-derived cell surface fibronectin.