Tooth induction in chick epithelium: expression of quiescent genes for enamel synthesis.

Intraocular grafts of chick epithelium combined with mouse molar mesenchyme produced a variety of dental structures including perfectly formed crowns with differentiated ameloblasts depositing enamel matrix. The results suggest that the loss of teeth in Aves did not result from a loss of genetic coding for enamel synthesis in the oral epithelium but from an alteration in the tissue interactions requisite for odontogenesis.

Effect of alpha, alpha'-dipyridyl on the basement membrane of tooth germs in vitro.

Dipyridyl inhibits the morphogenesis of tooth germs, but the germs recover when transferred to control medium. In this study, the effect of dipyridyl on basement membrane was investigated in vitro. The basal lamina was always present, but the subjacent collagen fibrils disappeared in the presence of dipyridyl and reappeared during recovery in control medium.

Differentiation of odontoblasts in grafted recombinants of murine epithelial root sheath and dental mesenchyme.

The epithelial root sheath (ERS) was isolated from first molars of 5-day-old post-natal CD-1 mice using trypsin. After isolation, ERS cells remained viable in vitro and immunohistochemical examination of cultures confirmed the epithelial phenotype and the absence of mesenchymal contamination. Recombinants of isolated ERS and dental papilla resulted in odontoblast differentiation within cells of the dental papilla, and the formation of root-like fragments of dentine after 2 weeks of intra-ocular grafting. These findings indicate the inductive influence of the ERS on dental papilla cells.

The induction of odontogenesis in non-dental mesenchyme combined with early murine mandibular arch epithelium.

First and second branchial arches were dissected from mouse embryos of 9-13 days gestational age. The epithelial and mesenchymal components were separated after enzymic digestion. Scanning electron microscopy did not reveal a dental lamina along the dental arches before day 12, after which the lamina was formed locally in the incisor and molar regions. There was no epithelial down-growth in the diastema region. Heterotypic recombinations of mandibular arch and second branchial arch tissues showed that early mandibular arch epithelia, before day 12, have odontogenic potential and can elicit the formation of a dental papilla in non-odontogenic, neural-crest-derived mesenchymal cells of the second arch. However, the mandibular mesenchyme must interact with mandibular epithelium in order to have the competence to induce teeth in non-odontogenic epithelium.

Enhancement of avian mandibular chondrogenesis in vitro in the absence of epithelium.

The roles of mandibular epithelium in chondrogenesis and growth of mandibular mesenchyme were examined in organ cultures. Epithelium and mesenchyme were separated from the mandibular arches of chick embryos at stages before and after the onset of chondrogenesis in vivo (stages 18-28). Isochronic and heterochronic tissue recombinations were prepared. Removal of the mandibular epithelium resulted in reduced growth of the explants and enhanced chondrogenesis, resulting in increased levels of mRNAs for type II collagen and aggrecan. The presence of mandibular epithelium promoted cell division in loosely arranged undifferentiated tissue from the mandibular mesenchyme and resulted in increased levels of type I collagen mRNA. Enhanced chondrogenesis was also observed in the mesenchyme isolated with basement membrane and isolated mesenchyme grown within Matrigel. These findings suggest that mandibular epithelium has mitogenic and chondrogenic-inhibitory effects on the underlying mesenchyme that are stage independent. Furthermore, the chondrogenic-inhibitory effect of mandibular epithelium on the underlying mesenchymal cells is not mediated by basement membrane.

Expression of epidermal growth factor mRNA in the developing mouse mandibular process.

Reverse transcription and cDNA amplification (polymerase chain reaction) of total RNA preparations were used to characterize the expression of EGF mRNA in the mandibular arch of day 9-17 mouse embryos. EGF mRNA was present in mandibles at day 9 and 10 but not at days 11-17. Separate RNA preparations from epithelium and mesenchyme at day 10 revealed EGF mRNA in both components.

Effects of retinol on the temporal expression of transforming growth factor-alpha mRNA in the embryonic mouse mandible.

Development of the mouse embryonic mandible from days 9 to 14 involves tissue interactions in the formation of bone, cartilage, salivary glands and teeth. Growth factors may play an important role in these interactions. Epidermal growth factor (EGF) mRNA expression has been characterized and its presence has been shown to be necessary for odontogenesis. In addition, retinol alters the pattern of dental lamina formation; this effect is correlated with an alteration of the expression of the mRNA for this mitogen (EGF). Transforming growth factor-alpha (TGF alpha) mRNA expression has now been characterized by polymerase chain reaction for this entire period of development (days 9-14). Although the mRNA is present at the same time as EGF (days 9 and 10 only), retinol does not alter the expression of this mitogen as it does EGF. This suggests that retinoids may act to control the proliferative pattern of the dental lamina through EGF expression and not TGF alpha expression, although mRNAs for both mitogens are present at the same time.

The correlation of temporal regulation of glycosaminoglycan synthesis with morphogenetic events in mouse tooth development.

The purpose of this study was to investigate the pattern of sulphated glycosaminoglycan synthesis during morphogenesis and cytodifferentiation in mouse tooth rudiments and to compare the results with those obtained in another study for salivary gland, a branched organ. Sulphated glycosaminoglycan was labelled by incubating molar rudiments from day 15 of gestation to day 1 post partum in medium containing [35S]-sodium sulphate. The rudiments were washed, homogenized and digested in pronase and then were sequentially digested by chondroitinase ABC and chemically degraded by nitrous acid oxidation. The fractions from each of these procedures were analysed by chromatography on Sephadex G-50 columns. The analysis revealed that, during morphogenesis, levels of chondroitin sulphate increased to a peak of 91% at day 18 and levels of heparan sulphate diminished to 8% during this period. As cytodifferentiation occurred, the level of chondroitin sulphate dropped to 64% and that of heparan sulphate increased to 35%. These results are similar to those reported for rat submaxillary gland, a branching organ. It appears that this pattern of sulphated glycosaminoglycan synthesis is not a unique feature of branching morphogenesis but may be one which marks the transition between morphogenesis and cytodifferentiation in non-branching rudiments as well.

Alteration of murine odontogenic patterning and prolongation of expression of epidermal growth factor mRNA by retinol in vitro.

Retinoids alter the patterning of murine odontogenesis in vivo and stimulate epithelial proliferation. Because odontogenesis is dependent on proliferation of mandibular epithelium, the effects of retinol on the patterning of odontogenic epithelium were studied. These experiments control for developmental stage, applied retinoid concentration and duration of exposure. Explants exposed for 24 h to 0.1 micrograms/ml of retinol exhibited enhanced odontogenesis. Day-9 mandibles exposed to retinol at 1-5 micrograms/ml had altered epithelial patterns consistent with those in previous in vivo experiments, including supernumerary epithelial buds in regions associated with supernumerary incisors in vivo. These changes were associated with a dose-dependent increase in epithelial proliferation and a prolonged expression of epidermal growth factor (EGF) mRNA. Altered expression of EGF mRNA may be responsible for the disrupted pattern of the dental lamina. This is the first report of a retinoid-induced alteration in EGF mRNA expression.

Interactions between cloned gingival or periodontal ligament cells and oral epithelial cells in vitro.

Interactions between epithelial cells and fibroblast clones were examined to determine whether various fibroblasts clones derived from gingival or periodontal tissues responded differently to oral epithelial cells. We provide evidence that epithelial root sheath (ERS) cells enhanced collagen type I (CI) expression in most periodontal clones, whereas ERS cells variably influenced CI expression of gingival fibroblast clones. Modulation of collagen type III (CIII) expression in both gingival and periodontal clones by ERS cells was less in magnitude and mostly suppressive in gingival clones. Fibronectin (Fn) expression in many gingival and periodontal clones was decreased in cells associated with ERS cells. On the other hand, the influence of gingival epithelial cells on fibroblast clones tended to be inhibitory, especially in periodontal clones. Thus, gingival epithelial (GE) cells suppressed the expression of collagen types I and III and Fn in most periodontal clones. Except for suppression of Fn expression, GE cells had less influence on CI and CIII expression in gingival clones. The modulations of fibroblast extracellular matrix components by ERS and GE have profound implications for the regulation of the development, repair and regeneration of the periodontal tissues.

Gene-environment interactions during tooth development.

The nature of the interaction between the environment and the genetic information of the organism has been examined vis-a-vis the local tissue environments in which the teeth are initiated. The directive role of the dental connective tissue, the papilla, and sac in initiating the tooth bud, controlling its shape, and maintaining its differentiation emphasize the importance of the mesenchymal connective tissue elements of tooth buds. The molecular character of this tissue interaction was described and the role of collagen as a mediator of developmental information was stressed. Finally these data from experimental studies suggest that the etiology of ectodermal dysplasia and inherited diseases involving the dentition may involve the function of the connective tissue of the dintition.