Jan E. Kronmiller

Blockade of the initiation of murine odontogenesis in vitro by citral, an inhibitor of endogenous retinoic acid synthesis

Endogenous retinoids are present in the embryonic mouse mandible and reach a concentration peak immediately before the formation of the dental lamina. Because exogenous retinoids alter the pattern of the dental lamina and the expression of epidermal growth factor mRNA (a transcript necessary for initiation of odontogenesis), the role of retinoic acid in the initiation of odontogenesis was studied here. Citral (3,7-dimethyl-2,6-octadienal), a known inhibitor of retinoic acid synthesis, was used to block the endogenous synthesis of retinoic acid in the mouse embryonic mandible before the formation of the dental lamina (gestational day 9). A 24-h exposure to citral totally blocked tooth formation in 7/10 mandibles. Reductions of endogenous retinoic acid concentrations were confirmed by high-performance liquid chromatography. Tooth formation was restored by simultaneous treatment with all-trans retinoic acid or 9-cis retinoic acid during the citral exposures (first 24 h of culture). Endogenous retinoic acid is necessary for the initiation of odontogenesis.

Development of asymmetries

One of the goals of the management of the developing occlusion and the correction of malocclusion is the establishment of symmetry within the individual arches (arch form) and the occlusal relationships between the maxillary and mandibular arches. The symmetry in arch form is related to the axial inclinations and rotations of the individual teeth. The occlusion is affected not only by the positions of the teeth, but also by the patterns of skeletal growth. Although these two components are interrelated, this discussion attempts to address them independently.

Spatial and temporal distribution of Sonic hedgehog mRNA in the embryonic mouse mandible by reverse transcription/polymerase chain reaction and in situ hybridization analysis

Hedgehog genes have recently been implicated in the control of pattern formation in many developing organ system. Vertebrate homologues of the Drosophila hedgehog have been identified in mouse and rate embryos. The temporal regulation of sonic hedgehog (mouse homologue) has previously been studied by Northern analysis of whole embryos with varying results. Sonic hedgehog transcript expression in the mouse mandibular process was now characterized using polymerase chain reaction (PCR) an in situ hybridization techniques. PCR analysis revealed transcripts at gestational days 10 and 11, before the formation of the dental lamina, but not at days 12-14, after tooth buds have formed. Transcripts were localized to, primarily, the epithelium in the presumptive incisor region of the mandibular midline at gestational day 10. No mRNA was detected by in situ hybridization techniques in the presumptive molar regions of odontogenic epithelium. Sonic hedgehog expression may be involved in the regulation of pattern formation through establishment of an incisor-molar axis of polarity.

Spatial distribution of epidermal growth-factor transcripts and effects of exogenous epidermal growth factor on the pattern of the mouse dental lamina

The initiation of odontogenesis is characterized by the site-specific proliferation of mandibular epithelium in the formation of the dental lamina. The epidermal growth factor (EGF) gene is expressed in the developing mandible immediately before the appearance of the dental lamina. This expression is necessary for the formation of the dental lamina and subsequent development of teeth. Previous work has demonstrated that retinoids and EGF may interact in the establishment of the pattern of the dentition. In the present study explanted mandibles that were treated with exogenous EGF (40 ng/ml of medium) contained supernumerary buds of mandibular epithelium in the diastema region. These pattern changes were the same as in previous retinoid-treated explants. These results, in addition to the previously reported effects of retinoids on the expression of the EGF gene, support the hypothesis that retinoids and EGF interact in controlling, at least in part, the pattern of the dentition by affecting the pattern of the dental lamina. The spatial distribution of EGF transcripts was also characterized. The location of EGF transcripts in the mesenchyme adjacent to the mandibular epithelium suggests a paracrine mechanism in the stimulation of epithelial proliferation in the formation of the dental lamina.

Instruction by retinoic acid of incisor morphology in the mouse embryonic mandible

Endogenous retinoids are present in the embryonic mouse mandible and reach a peak in concentration at the time of the formation of the dental lamina. All-trans retinoic acid is present in a 10-fold higher concentration in the future incisor region of the mouse embryonic mandible at day 11.5. It was found here that exogenous all-trans retinoic acid has pleiotropic effects on the pattern of odontogenesis when applied before the formation of the dental lamina. These effects include a change in the pattern of the dental lamina, supernumerary buds and incisors in the diastema region, and replacement of molars with incisors in the molar region. Thus retinoic acid appears to instruct incisor morphology in the mouse embryonic mandible.

Spatial distribution of endogenous retinoids in the murine embryonic mandible.

Retinoids play an important part in pattern formation during embryonic development. Exogenous retinoids alter the pattern of skeletal, neural and odontogenic tissues. Endogenous retinoids have been demonstrated previously in the murine embryonic mandible, reaching a concentration peak during the initiation of odontogenesis. It was now found that endogenous retinoids are present in a concentration gradient in the embryonic mouse mandible at the time of the initiation of the dental lamina. All-trans-retinoic acid was more concentrated in the incisor region and retinol in the molar region. These results, and the fact that exogenous retinoids produce supernumerary incisors and missing molars, suggest that all-trans-retinoic acid may instruct incisor morphology.

Effects of the intermediate retinoid metabolite retinal on the pattern of the dental lamina in vitro

Retinoids have important roles in pattern formation during embryonic development and might act as endogenous morphogens. They are necessary for normal odontogenesis and excess retinol alters the pattern of odontogenesis producing supernumerary buds of the dental lamina in the diastema region of the mouse mandible. Because the metabolism of retinoids in the developing mandible is unknown, the effects of retinal (an intermediate metabolite in the local conversion of retinol to retinoic acid) on the patterning of odontogenesis were examined. Retinal produces supernumerary buds and enhanced epithelial proliferation in day-9 mandibles in vitro. The endogenous levels of retinal in the mandible at the time of initiation of odontogenesis were also measured by high-performance liquid chromatography. Retinal was detected only at day 10 and not at later stages of development. Local metabolism of this intermediate retinoid may be a rate-determining step in the production of active retinoid metabolites that may control the pattern of the dentition, which is established at the time of the appearance of the dental lamina at embryonic day 12.

Temporal distribution of endogenous retinoids in the embryonic mouse mandible.

Retinoids play an important part in embryonic pattern formation. They are necessary for normal differentiation of odontogenic tissues and, in excess, disrupt the pattern of tooth formation. Excess retinoids produce supernumerary buds of the dental lamina in the diastema region of the mouse embryonic mandible where teeth do not normally form. This effect is coincident with an increase in epithelial proliferation and an alteration in epidermal growth factor mRNA expression (a gene product necessary for tooth formation). It was found by high-performance liquid chromatography that endogenous retinoids are present in the developing murine mandible and that concentrations of some retinoids reach a peak at the time of the initiation of odontogenesis (dental lamina formation).

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.

Spatial and temporal distribution ofIndian hedgehog mRNA in the embryonic mouse mandible

Hedgehog genes are involved in pattern formation during embryonic development. A recent report showed that Sonic hedgehog is expressed in the mouse mandible in the presumptive incisor region. In the present study, Indian hedgehog (Ihh) transcripts were present from gestational day 9 to 14 in the mouse mandible (reverse transcription/polymerase chain reaction analysis). Ihh mRNA was present in the dental lamina in both incisor and molar regions and in the developing whiskers (in-situ hybridization). Ihh may be involved in the site-specific proliferation of mandibular epithelium during the formation of the dental lamina. This is consistent with the observation that endogenous synthesis of retinoic acid is necessary for the initiation of odontogenesis and that retinoic acid induces hedgehog expression.