Edward J. Kollar

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.

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.

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. Dev. Dyn. 1998;213:386–397.

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.

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 f 1(I) collagen in various tissues including odontoblasts.

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.

Effects of l-azetidine-2-carboxylic acid, a proline analogue, on the in vitro development of mouse tooth germs

Abstract Incisor and molar tooth germs from 14- and 15-day-old mouse embryos were cultured on agar-solidified Eagles basal medium. Such tooth germs showed typical histo- and cytodifferentiation during the 8-day culture period. However, development of tooth germs cultured on medium containing l -azetidine was suppressed. In the experimental cultures, some germs displayed slight regression and disorganization whereas others were merely arrested at the se of development at the time of explanation. On the other hand, tooth germs cultured on medium containing l -azetidine in combination with proline developed normally. Suppressed tooth germs which were removed from medium containing l -azetidine and placed on control medium or proline-rich medium resumed development, so that their stage of differentiation was equivalent to that of controls at the termination of the culture period. These results support the notion that the collagen component of the extracellular matrix influences epithelial-mesenchymal inductive interactions.

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.

Effects of retinol on the temporal expression of transforming growth factor-α 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.

Inhibition of Mouse Molar Morphogenesis in vitro by Hadacidin

The in vitro morphogenesis of mouse molar tooth germs is inhibited by the anti-tumor antibiotic, hadacidin (N-formyl hydroxyaminoacetic acid). Organ cultures prepared from 15- and 16-day-old C57BL/6J embryos were maintained on agar-solidified medium. Treatment with hadacidin resulted in smaller than normal, morphologically aberrant tooth germs. Additionally, mitotic activity of tooth germs was inhibited in treated explants. Similarly, oral epithelium did not undergo its normal in vitro histogenesis when treated by this drug. Extended periods of treatment or treatment with high concentrations of hadacidin damaged the mesenchymal component of the tooth germs to a greater extent than it damaged the epithelium. Reversal of the developmental and mitotic inhibitions initiated by hadacidin was achieved by allowing the explants to recover on control medium. Hadacidin has been shown to be a competitive inhibitor of adenylo-succinate synthetase, which participates in the de novo synthesis of adenosine monophosphate (AMP). AMP can be synthesized by an alternative salvage pathway. Prevention of mitotic inhibition in hadacidin-treated explants was achieved by the simultaneous application of adenine and adenosine. These observations suggest that salvage purine biosynthetic pathways can function in developing tooth germs.