Col1a1-GFP transgene expression in developing incisors.

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of alpha 1(I) collagen in various tissues including odontoblasts.

Odontogenic epithelium induces similar molecular responses in chick and mouse mandibular mesenchyme.

Previous observations have shown that, during the initiation phase of odontogenesis, signals from mouse odontogenic epithelium can elicit teeth in non-odontogenic but neural crest-derived mesenchyme isolated from ectopic sites including chick mandibular mesenchyme. In the present study the formation of ectopic tooth buds and dental mesenchyme in chick mandibular mesenchyme was examined using heterospecific recombinations between E11 mouse odontogenic epithelium and stage 23 chick lateral mandibular mesenchyme. Both morphological criteria and chick-specific probes for Msx-1, Msx-2, and Bmp-4 mRNAs were used as markers for early dental mesenchyme. Our results demonstrated that interactions of mouse odontogenic epithelium with chick mandibular mesenchyme induce early changes in the chick mandibular mesenchyme including the appearance of a translucent zone, cell proliferation, and induction of expression of Msx-1, Msx-2, and Bmp-4, which have been shown to be associated with the formation of dental mesenchyme. In addition, tooth bud-like structures that resemble E13 tooth buds in vivo both morphologically and in their patterns of gene expression formed after 6 days in the heterospecific recombinations. The tooth bud-like structures consist of invaginated mouse mandibular epithelium and condensed chick mandibular mesenchyme expressing high levels of Msx-1 and Bmp-4, but undetectable levels of Msx-2. Unlike the induction of Msx-1, Msx-2, and Bmp-4 in the underlying mesenchyme, which is specific for signals derived from odontogenic epithelium, the induction of a translucent zone and cellular proliferation in the underlying mesenchyme may be related to the growth-promoting potential of embryonic epithelia and not be specific to signals derived from the odontogenic epithelium. Similar to mouse odontogenic epithelium, agarose beads soaked in recombinant BMP-4 induced a translucent zone, cellular proliferation, and expression of Msx-1, Msx-2, and Bmp-4 in chick mandibular mesenchyme after 24 hours. These observations together showed that avian mandibular mesenchyme has odontogenic potential that is expressed upon interactions with inductive signals from mouse odontogenic epithelium. Similar to odontogenesis in vivo, formation of dental mesenchyme in chick mandibular mesenchyme is mediated by the activation of Msx-1, Msx-2, and Bmp-4.

EGF does not induce Msx-1 and Msx-2 in dental mesenchyme.

Previous heterospecific tissue recombinations indicate that mandibular epithelium exerts the first known inductive signal for odontogenesis in mouse embryos. BMP-4 and EGF are two growth factors implicated as signaling molecules mediating the initial inductive epithelial-mesenchymal interactions during odontogenesis. The purpose of the present study was to examine and compare the effects of these growth factors and mouse mandibular epithelium on expression of Msx-1 and Msx-2 genes in molar-forming mesenchyme. Agarose beads soaked in growth factors or pieces of mouse mandibular epithelium (E11) were placed in contact with E11 molar-forming mesenchyme and cultured for 24 h. Whole-mount in situ hybridization analysis revealed that, in contrast to mouse mandibular epithelium and BMP-4-releasing beads, EGF-releasing beads did not induce the expression of Msx-1 and Msx-2 in E11 molar-forming mesenchyme. These observations suggest that whereas BMP-4 may be involved in activation of Msx-1 and Msx-2 in the underlying mesenchyme, EGF may regulate events involved in the formation of dental lamina.

Experimental analysis of Msx-1 and Msx-2 gene expression during chick mandibular morphogenesis.

Homeobox-containing genes are thought to be involved in regulating pattern formation in a variety of tissues during embryogenesis. We have examined the expression of the homeobox-related genes Msx-1 and Msx-2 during the development of the chick mandibular arch. Northern blot hybridization indicates that transcripts for both Msx-1 (1.6 Kb) and Msx-2 (3 Kb) are present in the mandibular arch as early as stage 18. The levels of both transcripts in the whole mandible decrease as cartilage is formed in vivo and in vitro. Using in situ hybridization, transcripts of Msx-1 were localized in high amounts to the mesenchyme of the mesial tips of the arches. Msx-2 transcripts were localized in high amounts to medial regions of the arches. Little or no hybridization of either probe was detected in the chondrogenic and myogenic regions of the arches. Transcripts of both genes were also excluded from calcified bone and cartilage. Our results further demonstrate that the mesial tip mesenchyme expressing Msx-1 includes areas of highly proliferative cells and has in vitro chondrogenic potential. The region of mesenchymal cells expressing the Msx-2 gene overlap with areas of developmentally programmed cell death which also contain very few proliferative cells and lack chondrogenic potential in vitro. These results are consistent with the possibility that Msx-1 may be involved in the outgrowth of the mandibular arch and Msx-2 may be involved in both developmentally programmed cell death and delineating the non-chondrogenic region of the medial part of the mandibular arch.

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.

Epithelial cell-fibroblast interactions: modulation of extracellular matrix proteins in cultured oral cells.

A model system involving co-cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival epithelial cells was used to study epithelial cell-fibroblast interactions. Double-labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular matrix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2-3 days of co-culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibited enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co-culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co-cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular matrix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.

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.

EGF antisense oligodeoxynucleotides block murine odontogenesis in vitro.

The initiation of odontogenesis depends on the site-specific proliferation of mandibular epithelium beginning at Day 11 in embryonic mice. We have previously reported that the local expression of epidermal growth factor mRNA in the murine mandible is developmentally regulated, expressed at Days 9 and 10 immediately prior to the initiation of tooth bud formation at Day 11. Exposure of Day 9 mandibular explants to antisense oligomers of epidermal growth factor blocks the initiation of odontogenesis. These results are the first demonstration of the involvement of epidermal growth factor in the inductive specification of a complex epithelial derivative.

Role of the early epithelium in the patterning of the teeth and Meckel's cartilage.

Embryonic epithelium from the mandibular branchial arch organizes the dentition and the deposition of Meckel's cartilage. During 9-11 days of gestation mandibular arch epithelium can induce teeth in nondental ectomesenchyme in both mice and birds. In addition, the deposition of Meckel's cartilage as a rod of cartilage in the middle of the first branchial arch is under the control of the epithelium. The epithelium inhibits chondrogenesis; if it is removed, large amorphous masses of cartilage are found instead of the narrow rod typical of Meckel's cartilage.

Temporal and spatial expression of genes for cartilage extracellular matrix proteins during avian mandibular arch development.

We have examined the temporal expression of genes for extracellular matrix proteins (type I collagen, type II collagen, and the cartilage specific proteoglycan core protein) during the development of the avian mandibular arch. We detected low levels of type II collagen mRNA in the mandibular arch as early as stage 15. Type II collagen mRNA remained low but increased slightly as development progressed from stage 15 to stage 25. More dramatic increases occurred after stage 25 coincident with overt chondrogenesis. In contrast, mRNA for the core protein of cartilage specific proteoglycan was not detected prior to the onset of chondrogenesis, appeared at stage 25, and increased thereafter. Type I collagen mRNA was also present as early as stage 15 and dramatically increased after stage 28/29, coincident with initiation of osteogenesis. Using in situ hybridization, we found that type II collagen mRNA became detectable in the center of the mandible around stage 24/25 coincident with the initiation of chondrogenesis. At later stages (26-32) type II collagen mRNA was localized in the cartilaginous rudiment. The pattern of hybridization observed with the proteoglycan core protein probe at later stages of development was essentially identical to that observed with the type II collagen probe. In contrast, the probe for the alpha 1 (I) collagen mRNA was localized over the perichondrium, over differentiated bone, and in areas within the mandibular arch where bone formation had been initiated.

Stage-related chondrogenic potential of avian mandibular ectomesenchymal cells.

We have examined the in vitro stage-related chondrogenic potential of avian mandibular ectomesenchymal cells using micromass cultures. Our results indicate that mandibular ectomesenchymal cells as early as stage 16, soon after the formation of the mandibular arches and well before the initiation of in vivo chondrogenesis, have chondrogenic potential which is expressed in micromass culture. There is an increase in the total area of the cultures occupied by cartilage when cells from increasing stages of development are used. The nodular pattern of chondrogenesis in these cultures indicates that mandibular ectomesenchymal cells are a heterogenous population from the time of mandibular arch formation. In addition, we studied the temporal expression of the genes for extracellular matrix proteins during in vitro chondrogenesis and correlated the morphological changes with the pattern of gene expression. Low levels of type II collagen mRNA are present in the cultures prior to detection of any stainable cartilage matrix and increase 5 fold just before the onset of chondrogenesis in vitro. On the other hand mRNA for cartilage proteoglycan core protein was not detected until the second day of culture when stainable cartilage matrix was present and progressively increased thereafter. Messenger RNA for type I collagen was present at the time of initiation of cultures and continuously increased during the culture period. Our experiments also indicated that embryonic epithelia can inhibit the in vitro chondrogenesis of mandibular ectomesenchymal cells and that the inhibitory effect of embryonic epithelia is independent of its age and site of origin.

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.

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.

Retrovirus-induced insertional mutation in Mov13 mice affects collagen I expression in a tissue-specific manner.

In the Mov13 mouse mutant, transcription of the alpha 1 (1) collagen gene is blocked by a retroviral insert in the first intron. We now report that teeth derived from homozygous embryos produce a dentin layer containing normal amounts of collagen 1. In situ hybridization and RNAase protection experiments indicate that the mutant allele is efficiently transcribed in odontoblasts, in contrast to other cell types. Correct splicing of the primary transcript containing the viral sequence results in a functional alpha 1 (1) collagen mRNA. The absence of a mutagenic effect in odontoblasts, as opposed to fibroblasts, suggests that the retroviral insert interferes with tissue-specific transcriptional control of the alpha 1 (1) collagen gene, most likely by inactivating cell-type-specific cis-acting regulatory elements.

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.