Epithelial cytodifferentiation and extracellular matrix formation in enamel-free areas of the occlusal cusp during development of mouse molars: light and electron microscopic studies

Mandibular first molars in mice ranging in age from 18 days prenatal to 5 days postnatal were used for light and electron microscopic examinations of the enamel-free area (EFA) during development of the occlusal cusp (mesiobuccal cusp). Notable morphological changes in the inner enamel epithelium and the cells of the stratum intermedium were observed. At prenatal age of 18 days, the inner enamel epithelium of the EFA (EFA epithelium) was composed of a layer of columnar cells and covered by the cells of the stratum intermedium. Two days after birth, the EFA epithelium was made up largely of preameloblasts, with mitochondria located in the proximal side of the cells toward the stratum intermedium. The cells of the stratum intermedium were irregularly shaped, with wide intercellular spaces between them. At a postnatal age of 3 days, most of the EFA epithelial cells resembled maturation-stage ameloblasts, being short and columnar in shape and having nuclei located in their proximal side. Distal cell membranes were folded, and mitochondria were scattered throughout the cytoplasm. In 4-day-old mice, the EFA epithelium was found to be formed of short columnar or cuboidal cells with distinct intercellular spaces. The cells of the stratum intermedium could no longer be detected, and cells of the EFA epithelium could not be distinguished from those of the stellate reticulum. Odontoblasts of the EFA were arranged and polarized parallel to the basal lamina, and odontoblastic processes extended toward the cusp tip. The orientation of thin and thick collagen fibers within predentin and dentin was also parallel to the basal lamina. Even after dentin mineralization, disrupted basal lamina and long, aperiodic, fine fibrils were found between the epithelium and the dentin. Following the disappearance of the basal lamina and fine fibrils, stippled material and crystals appeared on the dentin surface. The mineralized matrix, which x-ray microanalytical energy peaks identified as containing calcium and phosphorus, was continuous with enamel in the distal slope of the cusp at the cusp tip. Thus, the inner enamel epithelium of the EFA differentiated into secretory cells capable of enamel-like matrix formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Identification of four extracellular-matrix enamel proteins during embryonic-rabbit tooth-organ development.

1. Investigations were designed to identify the proteins which characterize the ameloblast phenotype, and to determine to what extent these extracellular-matrix proteins were degraded as a function of enamel matrix mineralization and maturation. 2. The identification of enamel proteins was based on comparisons between the electrophoretic patterns of enamel-containing and non-enamel-containing matrix extracts isolated from specific regions within 26-day embryonic New Zealand White rabbit incisor and molar tooth organs. 3. Since enamel proteins become mineralized on secretion, matrix specimens were demineralized in cold 5% (w/v) trichloroacetic acid, extracted with buffered 6M-urea and reduced with mercaptoethanol, and then the solubilized proteins were fractionated by urea/polyacrylamide-gel electrophoresis. 4. Three enamel-specific electrophoretic components were identified in newly secreted enamel-matrix specimens and this number increased as a function of mineralization and maturation. 5. Antibodies were prepared against embryonic rabbit extracellular matrix containing enamel. Comparison between immunoelectrophoretic patterns demonstrated that two of the three enamel components were antigenic. 6. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate was used to identify four enamel proteins of mol.wts. (1) 65 000 (2) 58000 (3) 22 000 and (4) 20 000, localized within enamel matrix. Enamel proteins (1) and (3) were phosphorylated, whereas (2) and (4) did not contain detectable phosphate. Labelled proline, leucine, tryptophan and glucosamine were incorporated into each of the four enamel proteins extracted from tooth explants incubated in the presence of radioactive precursors for 6 h. Whereas four proteins were identified in newly secreted enamel matrix, the concentrations of high-molecular-weight proteins (1) and (2) were found to decrease and the number (greater than 10) and concentration of low-molecular-weight polypeptides increased as a function of advanced enamel-matrix mineralization and maturation.     

Immunohistochemical localization of LIM mineralization protein 1 during mouse molar development

LIM mineralization protein 1 (LMP-1) is an essential positive regulator of osteoblast differentiation, maturation and bone formation. Our previous investigations on the distribution of LMP-1 in mature human teeth indicated that LMP-1 might play a role in the odontoblast differentiation and dentin matrix mineralization. The aim of the present study was to use immunohistochemistry to determine the expression of LMP-1 during tooth development in mouse molars. In embryonic and postnatal Kunming mice, LMP-1 protein was expressed during molar development, but the expression levels and patterns differed at various developmental stages. At embryonic day 13.5 (E13.5), LMP-1 was found in the enamel organ. At E14.5, LMP-1 was detected in the entire enamel organ and in the underlying mesenchyme. At E16.5, LMP-1 was observed in the inner and outer enamel epithelium and the stratum intermedium. The expression also converged at the cusps in the dental papilla. At E18.5 and postnatal day 2.5 (P2.5), LMP-1 was restricted to the stratum intermedium, in differentiating dental papilla cells at cusps, while it disappeared in terminal differentiated ameloblasts and odontoblasts. At P13.5, no positive staining was detected in the odontoblasts or in the dental pulp cells. Therefore, LMP-1 showed spatiotemporal expression patterns during molar development and might participate in molar crown morphogenesis and odontoblast differentiation at late molar development.     

The alterations of mouse plasma proteins during septic development

A fundamental issue for sepsis therapy is to control the development of inflammation at an early stage. With cecal ligation and puncture (CLP) surgery, the mouse model has clearly shown the septic signs triggered by chronic insult. To monitor the plasma proteomic responses to sepsis, the mouse blood was collected at intervals after sham and CLP surgery followed by the sample treatment to remove high abundance serum albumin. The treated mouse plasma proteins were well resolved by two-dimensional electrophoresis (2-DE). The image analysis revealed that these 2-DE spots observed from the sham and the CLP samples 4 h after surgery were comparable, whereas more than 30 different spots appeared on the 2-DE gels between the sham and CLP mouse plasma 24 h after surgery, indicating that some plasma proteins responded to the inflammatory development. These differential spots were verified by MALDI-TOF/TOF MS, resulting in 13 unique sepsis-responsive proteins. More importantly, most of them exhibited multiple spots as difference on the 2-DE gels. Furthermore, these isospots were incubated with PNGase F to eliminate N-linked oligosaccharides on proteins and then evaluated by Western blot as well as mass spectrometry. The results of PNGase F digestion suggested that most sepsis-associated proteins remained in N-glycosylation status but changed their N-glycans during septic development.     

Expression patterns of follistatin and two follistatin-related proteins during mouse development

We compared the expression patterns of follistatin and two follistatin-related proteins (FRP and m7365) during early mouse development. m7365 is expressed continuously during preimplantation development, in contrast to FRP and follistatin. At early postimplantation stages, follistatin and 7365 are expressed from E6.0, while FRP is detected from E7.5 onwards. Although there is some overlap between the expression of these genes in the primitive streak and somites, their overall expression patterns are distinct.     

Brief communication: dental development and enamel thickness in the Lakonis Neanderthal molar

Developmental and structural affinities between modern human and Neanderthal dental remains continue to be a subject of debate as well as their utility for informing assessments of life history and taxonomy. Excavation of the Middle Paleolithic cave site Lakonis in southern Greece has yielded a lower third molar (LKH 1). Here, we detail the crown development and enamel thickness of the distal cusps of the LKH 1 specimen, which has been classified as a Neanderthal based on the presence of an anterior fovea and mid-trigonid crest. Crown formation was determined using standard histological techniques, and enamel thickness was measured from a virtual plane of section. Developmental differences include thinner cuspal enamel and a lower periodicity than modern humans. Crown formation in the LKH 1 hypoconid is estimated to be 2.6-2.7 years, which is shorter than modern human times. The LKH 1 hypoconid also shows a more rapid overall crown extension rate than modern humans. Relative enamel thickness was approximately half that of a modern human sample mean; enamel on the distal cusps of modern human third molars is extremely thick in absolute and relative terms. These findings are consistent with recent studies that demonstrate differences in crown development, tissue proportions, and enamel thickness between Neanderthals and modern humans. Although overlap in some developmental variables may be found, the results of this and other studies suggest that Neanderthal molars formed in shorter periods of time than modern humans, due in part to thinner enamel and faster crown extension rates.     

Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins.

Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs). IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I. Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity. IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage. None of the crosslinked IGF-BPs exhibit O-linked side chains. Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr. Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity. This is the first report of IGF-BP degrading activity in a cell culture system. These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.     

Aspects of cell proliferation kinetics of the inner dental epithelium during mouse molar and incisor morphogenesis: a reappraisal of the role of the enamel knot area.

First lower E-14 and E-16 mouse molars and E-13 lower incisors were cultured in vitro and either sequentially or continuously labelled with BrdU (5-bromo-2'-deoxyuridine). The behaviour of the non-cycling inner dental epithelial cells emerging from the enamel knot area of the molars was analysed by 3D (three dimensional) reconstructions of serial sections. These cells, as well as slow cycling cells underwent a coordinated temporo-spatial patterning leading to their patchy segregation at the tips of the forming cusps. In incisors (in vitro and in vivo), non-cycling cells were also present in the inner dental epithelium of the enamel knot area. However, these cells were not redistributed during incisor morphogenesis. These non-dividing inner dental epithelium cells of the enamel knot area which are either redistributed or not according to the tooth type specific morphogenesis might represent the organizers of morphogenetic units (OMU), the cusps.     

C-terminal Src kinase controls development and maintenance of mouse squamous epithelia

Carboxy-terminal Src kinase (Csk) is a negative regulator of Src family kinases, which play pivotal roles in controlling cell adhesion, migration, and cancer progression. To elucidate the in vivo role of Csk in epithelial tissues, we conditionally inactivated Csk in squamous epithelia using the keratin-5 promoter/Cre-loxP system in mice. The mutant mice developed apparent defects in the skin, esophagus, and forestomach, with concomitant hyperplasia and chronic inflammation. Histology of the mutant epidermis revealed impaired cell-cell adhesion in basal cell layers. Analysis of primary keratinocytes showed that the defective cell-cell adhesion was caused by cytoskeletal remodeling via activation of the Rac1 pathway. Mutant keratinocytes also showed elevated expression of mesenchymal proteins, matrix metalloproteinases (MMPs), and the proinflammatory cytokine TNF-alpha. Inhibition of the expression of TNF-alpha and MMP9 by the anti-inflammatory reagent FK506 could cure the epidermal hyperplasia, suggesting a causal link between inflammation and epidermal hyperplasia. These observations demonstrate that the Src/Csk circuit plays crucial roles in development and maintenance of epithelia by controlling cytoskeletal organization as well as phenotypic conversion linked to inflammatory events.     

Swiss 3T3 mouse embryo fibroblasts transfected with a human prepro-GRP gene synthesize and secrete pro-GRP rather than GRP

A prepro-gastrin-releasing peptide (GRP) gene was introduced into Swiss 3T3 mouse embryo fibroblasts by DNA transfection in an attempt to establish autocrine growth stimulation. Clonal transfectants expressed varying amounts of GRP encoding mRNA. They synthesized and secreted a ～ 15-kd pro-GRP hormone but not fully processed 2.8-kd GRP. Accordingly, no changes in growth properties were associated with GRP gene expression. We postulate that Swiss 3T3 fibroblasts lack the enzymes necessary to process significantly pro-GRP into biologically active peptides and that this deficiency may be responsible for the failure to establish autocrine growth stimulation in the transfected cells.     

Distribution of glycoconjugates localized by peanut and Maclura pomifera agglutinins during mouse molar root development.

Glycolipids, glycoproteins, glycosaminoglycans and sialoglycoproteins have all been implicated in a number of developmentally significant processes related to complex interactions between cell surfaces and the extracellular matrix. The present study was designed to localize glycoconjugates recognized by peanut agglutinin (PNA) and Maclura pomifera (MPA) lectins during mouse molar root development. Postnatal ICR mice at 10, 15, 21, 28 and 42 days were used. Lower jaws were dissected, fixed in 4% paraformaldehyde, decalcified in 5% EDTA and embedded in paraffin. Serial sections were made and stained with FITC-conjugated PNA or MPA. beta-Lactose was used as an inhibitory sugar for PNA, and alpha-D-melibiose for MPA. PNA specifically stained Hertwig's epithelial root sheath (HERS), whereas MPA stained a number of tissues. The outermost layer of root dentin, forming cellular cementum, alveolar bone and HERS showed positive reactions with MPA. Glycoconjugates localized by the lectins may be functionally related to molecules which contribute to root formation and cemento-genesis.     

Histochemical demonstration of succinic dehydrogenase activity during sequential molar development in the Swiss albino mouse

Summary Applying the ditetrazolium salt (Nitro-BT) method for succinic dehydrogenase, murine molar teeth were studied sequentially from the cap stage of development through the appositional stages of odontogenesis. Reaction-distribution and intensity varied relative to the developmental stage as well as the zone of maturation within a given stage. The peripheral cells of the parent dental lamina exhibited some activity, as did the outer enamel epithelium of the bell stage. During the period of matrix apposition, components of the stratum intermedium, ameloblastic zone and odontoblastic layer region of the dental papilla demonstrated intense enzymatic activity. Cells actively engaged in enamel matrix production demonstrated activity in the basal and distal cell segments. High activity continued in the papillary layer of the enamel organ, as well as in the cells of the dental sac during the postamelogenic period.Supported by PHS Grant No. 2800-02, National Institute of Dental Research, National Institutes of Health.     

Matrix metalloproteinase inhibition impairs the processing, formation and mineralization of dental tissues during mouse molar development

Organotypic cultures of embryonic mouse tooth germs were used to investigate the developmental expression and roles of MMPs in the formation and mineralization of dentin and enamel matrices. The spatially and temporally regulated expression of MMP-2, MMP-9 and MMP-20 in developing mouse tooth germs in vivo was maintained in culture. The inhibition of metalloproteinases activity by marimastat altered the morphogenesis and mineralization of the tooth germs associated with an inhibition of the activation of both MMP-20 and MMP-2. MMP inhibition deregulated the molecular processing of two major dental matrix proteins, amelogenin and dentin sialoprotein (DSP). This coincided with their accumulation and the loss of their normal distribution within the extracellular matrix, resulting in a defective mineralization of dentin and enamel matrices. These findings demonstrate the critical role of MMPs in the processing and maturation of the dental matrix.