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H C Slavkin

Publications and source records attributed to H C Slavkin.

At least 163 records · Page 9Linked to original sources
PubMed1989

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.

Animals↗
PubMed1989

Splice of life: toward understanding genetic determinants of oral diseases.

The introduction of recombinant DNA technology has led to a rapid advancement of our knowledge of genes and genomic structure. Such technology, applied to the human genome, has provided valuable information concerning the nature and possible treatment of inherited disorders throughout the human life span. A number of oral diseases can be attacked by use of molecular biological techniques in conjunction with genetic linkage analysis. It will be possible to identify carriers of inherited diseases, to utilize prenatal and antenatal diagnosis when appropriate, and to pursue studies on the mode of inheritance of specific oral diseases. Present and projected advances in this dimension of dental research provide the knowledge and strategies for approaching and solving a number of major problems as we continue to pave the way for improvements in the diagnosis, treatment, and prevention of many oral and craniofacial diseases. This position paper examines a few of the promising areas and suggests future goals toward establishing a knowledge of the genetic determinants for oral diseases.

Humans↗
PubMed1989

Positional signalling and patterning for amelogenesis in mouse molar tooth development.

Oral mandibular epithelium determines the position and number of tooth organs. Cranial neural crest-derived ectomesenchyme provides inductive signals for (i) tooth shape, and (ii) sequential determination and expression of several different epithelial-derived enamel proteins. Inductive signalling follows a precise sequence, can be expressed in serumless, chemically-defined medium in vitro, and appears to be highly conserved during vertebrate evolution.

Amelogenesis↗
PubMed1989

Characterization of extracellular and nascent dentin phosphoproteins.

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.

Animals↗
PubMed1989

Of mice and men: anatomy of the amelogenin gene.

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.

Amelogenesis Imperfecta↗
PubMed1989

Early embryonic mouse mandibular morphogenesis and cytodifferentiation in serumless, chemically defined medium: a model for studies of autocrine and/or paracrine regulatory factors.

During craniofacial and mandibular development at least three interdependent processes become integrated: 1) regulation of time-dependent differential gene expression; 2) positional information resulting in pattern formations; and 3) morphogenesis. The present studies were designed to test the hypothesis that intrinsic and/or paracrine factors regulate the developmental program for embryonic mouse mandibular morphogenesis, histogenesis, and cytodifferentiation. Either E11 or E12 C57B110 (B10.A) strain mouse mandibular processes were cultured in serumless, chemically defined medium for periods up to 9 days in vitro. At selected stages of development 3H-thymidine incorporation into DNA was used to evaluate the mitotic labeling for selected tissue compartments. Macroscopic observations demonstrated that morphogenesis (shape/form) in vitro was comparable to that for in vivo controls. Histological results demonstrated that chondrogenesis, osteogenesis, tooth formation, tongue formation, lip formation, and epithelial differentiation with keratinization were expressed according to sequence, time, and positions comparable to those observed in controls. This experimental approach provided datasets to support the hypothesis that exogenous long-range factors are not required for embryonic mouse mandibular morphogenesis and further suggested that autocrine and/or paracrine factors mediate the timing and position of mandibular development.

Animals↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1988

Factors influencing the expression of dental extracellular matrix biomineralization.

The forming tooth organ provides a number of opportunities to investigate the cellular and molecular biology of cell-mediated extracellular matrix (ECM) biomineralization. Regulatory processes associated with tooth formation are being investigated by identifying when and where cell adhesion molecules (CAMs), substrate adhesion molecules (SAMs), dentine phosphoprotein and enamel gene products are expressed during sequential developmental stages. In vitro organotypic culture studies in serumless, chemically-defined medium, have shown that instructive and permissive signalling are required for both morphogenesis and cytodifferentiation. Intrinsic developmental instructions (autocrine and paracrine factors) act independently of long-range hormonal or exogenous growth factors and mediate morphogenesis from the initiation of the dental lamina to the crown stages of tooth development. This review summarizes the results of studies using experimental embryology, recombinant DNA technology and immunocytology to elucidate mechanisms responsive to instructive epithelial-mesenchymal interactions associated with ameloblast differentiation, odontoblast differentiation, and dentine and enamel ECM biomineralization.

Animals↗
PubMed1988

Molecular determinants of cranial neural crest-derived odontogenic ectomesenchyme during dentinogenesis.

Positional information on tooth morphogenesis is investigated by the identification of when and where phenotypic markers are expressed during odontogenesis. This temporal and positional information is correlated with the instructive and permissive signaling required for both dentinogenesis and amelogenesis. Of particular interest is the establishment of a map for the cranial neural crest-derived dental papilla ectomesenchyme and the odontoblast cell lineages. The expression of ectomesenchyme-derived cytotactin, dentin phosphoprotein, and epithelial-derived enamel proteins was studied in mice using embryonic, fetal, and postnatal mandibular first molar tooth organ development. This review summarizes the observations in the context of instructive epithelial-mesenchymal interactions and suggests that amelogenesis imperfecta and dentinogenesis imperfecta may in part be explained by alterations in these differentiation markers. Recombinant DNA methods should facilitate future investigations of these inherited dental disorders.

Animals↗
PubMed1988

Metabolic expression of intrinsic developmental programs for dentine and enamel biomineralization in serumless, chemically-defined, organotypic culture.

Biomineralization was investigated using embryonic mouse mandibular first molars (M1) cultured in the presence or absence of fetal calf serum. Metabolic features including cell division and Ca2+ and phosphate incorporation into dentine and enamel extracellular matrices were analyzed. The relative timing and magnitude of DNA synthesis for serumless cultures was comparable to in vivo controls. Isotopic calcium and phosphate incorporation into the mineral phase of dentine and enamel matrices, in the absence of serum, fluctuated during development. Molar tooth morphogenesis, cytodifferentiation, and extracellular matrix formation approximated late crown-stage development in serumless cultures. Von Kossa histochemical staining indicated calcium phosphate salt formation in serumless cultures. Analysis of anhydrous fixation-prepared enamel and dentine representing serumless cultured explants indicated that crystal size and orientation were comparable to in vivo enamel and dentine. In contrast, serum-supplemented cultures showed atypical crystal size and orientation. Calcium/phosphorous (Ca/P) ratio values for serumless cultures after 21 days showed Ca/P enamel values of 2.03 (SD +/- 0.04, p less than 0.025) and dentine values of 1.89 (SD +/- 0.01, p less than 0.025). Electron diffraction patterns of enamel and dentine formed in serumless cultures were principally those of highly-ordered crystalline hydroxyapatite. Our results suggest that tissue-specific dentine and enamel biomineralization is regulated by endogenous factors intrinsic to the developmental program of embryonic tooth organs during serumless culture.

Animals↗
PubMed1988

Embryonic mouse lung morphogenesis and type II cytodifferentiation in serumless, chemically defined medium using prolonged in vitro cultures.

The timing, position and mechanism(s) for determining type II cytodifferentiation during mammalian lung development are not known. To approach this problem, we have cultured Theiler stage 16 embryonic B10.A strain mouse lung primordia (12-days gestation, E12) in serumless, chemically defined medium in the presence or absence of dexamethasone (DEX) for periods up to 27 days in vitro. Morphogenesis and cytodifferentiation were evaluated by light and transmission electron microscopy and immunochemical techniques. Pulmonary surfactant-associated apoproteins (PSAP) were initially expressed by type II cells at 16.5-day gestation in vivo. DEX-supplementation to the culture medium resulted in the accelerated expression of PSAP; the apoprotein isoforms (A1, A2, and A3) produced in vitro were comparable to those synthesized during fetal and postnatal in vivo development by high resolution, two-dimensional gel electrophoresis coupled with immunoblot staining. Cultures without DEX produced PSAP A2 and A3 isoforms, but did not produce A1 (26-31 kDa, pI 5.2-5.3). DEX-treated cultures produced more lamellar bodies within type II cells than non-treated controls. The results demonstrate that long-term cultures of embryonic lung primordia express morphogenesis, cytodifferentiation and the synthesis and secretion of PSAP in the absence of exogenous hormones or growth factors. The data set further supports the hypothesis that morphogenesis and type II cytodifferentiation are regulated by autocrine and paracrine factors intrinsic to the embryonic lung developmental program and independent of exogenous hormone controls.

Adrenal Cortex Hormones↗
PubMed1988

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.

Animals↗
PubMed1988

1987 Kreshover lecture. Gene regulation in the development of oral tissues.

The regulatory processes associated with tooth formation are being investigated by the identification of when, where, and how cell adhesion molecules (CAMs), substrate adhesion molecules (SAMs), dentin phosphoprotein, enamel gene products, and intermediate cementum products are expressed during sequential developmental stages of morphogenesis, cytodifferentiation, dentin, enamel and cementum extracellular matrix (ECM) formation, and biomineralization. Instructive and permissive signaling is required for both morphogenesis and cytodifferentiation based upon in vitro organotypic culture studies in serumless, chemically-defined medium. Intrinsic developmental instructions, independent of exogenous growth factors, mediate tooth morphogenesis from the initiation of the dental lamina through crown and initial root development. Recent progress using recombinant DNA methods has advanced descriptions of several dental structural genes. The complete nucleic acid sequence for mouse amelogenin has been defined. This sequence is located on the mouse X chromosome and on the human X and Y chromosomes. This discussion summarizes recent results using experimental embryology, recombinant DNA technology, and immunocytology in the context of instructive epithelial-mesenchymal interactions associated with epithelial differentiation into ameloblasts, ectomesenchyme differentiation into odontoblasts, and dentin and enamel ECM biomineralization. The tooth organ provides opportunities at several levels of biological organization to investigate cellular, molecular, and developmental processes.

Animals↗
PubMed1988

Spatial- and temporal-restricted pattern for amelogenin gene expression during mouse molar tooth organogenesis.

Position- and time-restricted amelogenin gene transcription was analysed in developing tooth organs using in situ hybridization with asymmetric complementary RNA probes produced from a cDNA specific to the mouse 26 x 10(3) Mr amelogenin. In situ analysis was performed on developmentally staged fetal and neonatal mouse mandibular first (M1) and maxillary first (M1) molar tooth organs using serial sections and three-dimensional reconstruction. Amelogenin mRNA was first detected in a cluster of ameloblasts along one cusp of the M1 molar at the newborn stage of development. In subsequent developmental stages, amelogenin transcripts were detected within foci of ameloblasts lining each of the five cusps comprising the molar crown form. The number of amelogenin transcripts appeared to be position-dependent, being more abundant on one cusp surface while reduced along the opposite surface. Amelogenin gene transcription was found to be bilaterally symmetric between the developing right and left M1 molars, and complementary between the M1 and M1 developing molars; indicating position-restricted gene expression resulting in organ stereoisomerism. The application of in situ hybridization to forming tooth organ geometry provides a novel strategy to define epithelial-mesenchymal signal(s) which are believed to be responsible for organ morphogenesis, as well as for temporal- and spatial-restricted tissue-specific expression of enamel extracellular matrix.

Amelogenin↗
PubMed1987

Immunocytochemical and radioautographic evidence for secretion and intracellular degradation of enamel proteins by ameloblasts during the maturation stage of amelogenesis in rat incisors.

In the continuously erupting rat incisor the ameloblasts progress through distinct stages associated with the secretion and maturation of enamel. We have examined the possibility that the so-called "postsecretory" ameloblasts of the maturation stage of amelogenesis remain biosynthetically active and are engaged in the synthesis, secretion, and degradation of enamel gene products. The ultrastructural distribution of antigenic sites for enamel proteins was studied within enamel organ cells during the early maturation stage of amelogenesis in rat incisors by using the protein A-gold immunocytochemical technique and rabbit polyclonal antibodies developed against mouse amelogenins. All regions of amelogenesis from late secretion through the first complete modulation from ruffle-ended to smooth-ended ameloblasts were examined. Specific immunolabelling was found within the rough endoplasmic reticulum, Golgi saccules, secretory granules, and lysosomes of ameloblasts throughout these regions. The heaviest intracellular immunolabelling was found within secretory granules and lysosomes (multivesicular type). Quantitative analyses showed that the Golgi saccules and the multivesicular lysosomes of modulating ameloblasts were generally less immunoreactive compared to similar organelles in ameloblasts secreting the inner enamel layer. Radioautographic studies confirmed that ameloblasts of the maturation stage incorporated 3H-leucine and 3H-methionine and secreted labelled proteins into the enamel layer. Grain counts indicated that ameloblasts from the first ruffle-ended band incorporated about two-fold less 3H-methionine and secreted about tenfold less labelled proteins into the enamel compared to ameloblasts secreting the inner enamel layer. The results of this study confirm that ameloblasts do not terminate biosynthesis and secretion of enamel proteins once the final layer has been deposited on the surface of the developing enamel. They continue to form and release new proteins during the maturation stage which intermix with older proteins laid down initially during the secretory stage of amelogenesis. Secretory activity for enamel proteins has been detected in ameloblasts up to at least the second ruffle-ended phase of maturation, at which point the enamel matrix is partially soluble in EDTA.

Ameloblasts↗
PubMed1987

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.

Amelogenin↗