Exogenous fibroblast growth factor 8 rescues development of mouse diastemal vestigial tooth ex vivo.

Regression of vestigial tooth buds results in the formation of the toothless diastema, a unique feature of the mouse dentition. Revitalization of the diastemal vestigial tooth bud provides an excellent model for studying tooth regeneration and replacement. It has been previously shown that suppression of fibroblast growth factor (FGF) signaling in the diastema results in vestigial tooth bud regression. In this study, we report that application of exogenous FGF8 to the mouse embryonic diastemal region rescues diastemal tooth development. However, this rescue of diastemal tooth development occurs only in an isolated diastemal regions and not in the mandibular quadrant, which includes the incisor and molar germs. FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program, as evidenced by the expression of genes critical for normal tooth development. Our results also support the idea that the adjacent tooth germs contribute to the suppression of diastemal vestigial tooth buds by means of multiple signals.

Sprouty genes control diastema tooth development via bidirectional antagonism of epithelial-mesenchymal FGF signaling.

Unlike humans, who have a continuous row of teeth, mice have only molars and incisors separated by a toothless region called a diastema. Although tooth buds form in the embryonic diastema, they regress and do not develop into teeth. Here, we identify members of the Sprouty (Spry) family, which encode negative feedback regulators of fibroblast growth factor (FGF) and other receptor tyrosine kinase signaling, as genes that repress diastema tooth development. We show that different Sprouty genes are deployed in different tissue compartments--Spry2 in epithelium and Spry4 in mesenchyme--to prevent diastema tooth formation. We provide genetic evidence that they function to ensure that diastema tooth buds are refractory to signaling via FGF ligands that are present in the region and thus prevent these buds from engaging in the FGF-mediated bidirectional signaling between epithelium and mesenchyme that normally sustains tooth development.

Mesenchyme is responsible for tooth suppression in the mouse lower diastema.

Between the incisor and molars in each dental quadrant, mice have a toothless gap (diastema) that may contain vestigial tooth primordia. It is still not clear whether suppression of odontogenesis in the mouse lower diastema can be attributed to epithelium, mesenchyme, or both. Therefore, using recombination experiments with mouse tissues from E11.5 and E13.5 stages, we investigated whether the epithelium or mesenchyme is responsible for the suppression of odontogenesis. Five groups of recombinants were established and cultured under mouse kidney capsules. The results demonstrated that at E11.5, the lower diastemal epithelium and mesenchyme possessed odontogenic potential and competence, respectively; at E13.5, both the lower diastemal epithelium and mesenchyme had odontogenic competence, while the lower diastemal mesenchyme did not possess odontogenic potential. On the basis of comparison of the odontogenic capabilities between the lower diastemal and molar tooth primordia, we conclude that mesenchyme is responsible for tooth regression in the mouse lower diastema.

Origin and developmental fate of vestigial tooth primordia in the upper diastema of the field vole (Microtus agrestis, Rodentia).

OBJECTIVE: Odontogenesis in voles is a convenient model to test hypotheses on tooth development generated from investigations in the mouse. Similar to other rodents, the functional dentition of the vole includes a toothless diastema. At its mesial end, a vestigial tooth bud has been found in the upper jaw of vole embryos. The aim of this study was to analyse the developmental dynamics of vestigial tooth structures in the upper diastema of the field vole and to compare it with the situation in the mouse. DESIGN: The development of odontogenic structures in the upper diastema of the field vole was investigated using serial histological sections and three-dimensional (3D) computer-aided reconstruction. RESULTS: A transient continuous dental lamina in the upper diastema of the field vole extended mesially to the first molar primordium, but was not continuous with the dental lamina in the incisor region. At its mesial limit, a large vestigial tooth primordium was regularly present. A further distinct vestigial bud was located mesially to the first molar primordium. The segmentation of the dental lamina suggested a potential to give rise to further vestiges in the upper diastema of the vole. CONCLUSIONS: In the prospective diastema of the vole exists as in the mouse a continuous dental lamina. Beside the prominent vestigial tooth bud in the mesial diastema, a further large bud was transiently located in front of the molars. The incorporation of dental epithelium into the first upper molar (M(1)) primordium in the vole differs from that in the mouse.

Characteristic tissue interaction of the diastema region in mice.

Rodents have a toothless diastema between the incisor and the first molar, which may contain rudimentary tooth germs. In the lower diastema region of mice at E13, the rudimentary tooth germs, which developed into the bud stage before its removal by apoptosis, was found. The immunoreactivity to tenascin was observed in the condensed mesenchyme around the normal tooth bud and was detected in only the basement membrane in the diastema bud. This result shows that the relationship between mesenchymal condensation and tooth development. The similar patterns of Msx-1 and Msx-2 expression between the tooth bud and the diastema bud show that the diastema bud may have some other genetic mechanism in the developmental arrest of the rudimentary tooth germs rather than the Msx-1 and Msx-2 expression. Strikingly, the induction of the tooth formation was possible using tissue recombination between the oral epithelium of the diastema bud and the dental mesenchyme of the molar tooth bud, which indicates the potential capability of the diastema in the tooth formation. In conclusion, it is suggested that the condensed mesenchyme may be the key to tooth development.

Apoptosis is involved in the disappearance of the diastemal dental primordia in mouse embryo.

Three transient dental primordia (D1, D2 and D3) exist in the upper diastema in mouse embryos and their regression is associated with the presence of cell death. In order to specify the type of cell death and its temporo-spatial distribution, staining with hematoxylin, supravital staining with Nile Blue, TUNEL method, electron microscopic analysis and computer assisted 3-D reconstructions were performed. These data demonstrated that apoptosis is involved in the disappearance of the diastemal dental rudiments. Apoptosis occurred first with prevalence in the buccal part of the epithelium of the diastemal dental primordia and extended later to the whole epithelium of the dental rudiments and the dental lamina interconnecting them with the incisor and molar epithelia. Cell death occurred only sporadically in the adjacent mesenchyme. The prospective upper diastema in mouse embryos may provide a model for studies of developmental determination of toothless areas in the jaw as well as a tool for analyses of regulatory mechanisms of programmed cell death in morphogenesis.

Comparison of expression of the msx-1, msx-2, BMP-2 and BMP-4 genes in the mouse upper diastemal and molar tooth primordia.

The existence of transient putative tooth anlagen in the prospective mouse upper diastema region has been documented previously in morphological studies. By in situ hybridization we investigated the expression patterns of the msx-1, msx-2, BMP-2 and BMP-4 genes, supposed to regulate early tooth development, in day 10-14 mouse embryonic upper diastema and molar regions, using 49 series of frontal sections. On the basis of comparison of the temporo-spatial expression patterns in both diastemal and molar tooth primordia we conclude that each of the four genes was expressed at least for some period simultaneously and at a comparable developmental stage in the transient and persisting dental primordia. BMP-2 and BMP-4 expression was downregulated in the diastemal dental primordia during their regression starting at day 13. The temporo-spatial pattern of BMPs expression may be associated with the disappearance of diastemal rudiments. Contrary to the molar anlage, we did not detect msx-2 gene expression in the diastemal dental rudiments after the stage of epithelial thickening. The deficiency of the msx-2 gene products may play a role in the growth retardation of diastemal dental primordia resulting in their subsequent involution.

Mouse rudimentary diastema tooth primordia are devoid of peripheral nerve fibers.

The tooth is a well-defined peripheral target organ for trigeminal nerve fibers. However, only limited information is available regarding pioneer axon guidance to the developing tooth target field. In rodents there is a toothless diastema region between incisors and molars that in the mouse maxilla contains three rudimentary tooth anlagen. Their development stop at the early bud stage when the primary nerve axons grow towards the developing first molar tooth germs. In order to provide background information for studies of regulatory mechanisms of pioneer axon guidance to the developing tooth germs, we investigated the distribution of nerve fibers in the mouse diastema tooth buds, and compared it to the axon growth to the maxillary and mandibular first molar tooth germs by immunohistochemical localization of peripherin and PGP9.5. Analysis of serial sections showed that trigeminal nerve fibers emerging from the trigeminal maxillary and mandibular nerve trunks started to grow towards the developing molar tooth germ at the early bud stage, and subsequently they diverged into buccal and lingual branches next to the condensed dental mesenchyme. During the cap stage, nerve fibers were observed around the tooth germ in the dental follicle region. In contrast, no nerve fibers were located in the vicinity of the diastema tooth primordia at any stage studied, nor did any nerve fibers appear to grow towards this region. Our results show that the development and subsequent disappearance of the diastema tooth primordia takes place without peripheral trigeminal innervation. The diastema tooth primordia may therefore be a useful model system for future studies on molecular regulatory mechanisms of pioneer axon guidance to the tooth germs, and possibly also for evolutionary studies of peripheral axon guidance mechanisms.

Three-dimensional analysis of the early development of the dentition.

Tooth development has attracted the attention of researchers since the 19th century. It became obvious even then that morphogenesis could not fully be appreciated from two-dimensional histological sections. Therefore, methods of three-dimensional (3D) reconstructions were employed to visualize the surface morphology of developing structures and to help appreciate the complexity of early tooth morphogenesis. The present review surveys the data provided by computer-aided 3D analyses to update classical knowledge of early odontogenesis in the laboratory mouse and in humans. 3D reconstructions have demonstrated that odontogenesis in the early stages is a complex process which also includes the development of rudimentary odontogenic structures with different fates. Their developmental, evolutionary, and pathological aspects are discussed. The combination of in situ hybridization and 3D reconstruction have demonstrated the temporo-spatial dynamics of the signalling centres that reflect transient existence of rudimentary tooth primordia at loci where teeth were present in ancestors. The rudiments can rescue their suppressed development and revitalize, and then their subsequent autonomous development can give rise to oral pathologies. This shows that tooth-forming potential in mammals can be greater than that observed from their functional dentitions. From this perspective, the mouse rudimentary tooth primordia represent a natural model to test possibilities of tooth regeneration.

Proteome analysis of developing mice diastema region.

Different from humans, who have a continuous dentition of teeth, mice have only three molars and one incisor separated by a toothless region called the diastema in the hemi mandibular arch. Although tooth buds form in the embryonic diastema, they regress and do not develop into teeth. In this study, we evaluated the proteins that modulate the diastema formation through comparative analysis with molar-forming tissue by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteome analysis. From the comparative and semi-quantitative proteome analysis, we identified 147 up- and 173 down-regulated proteins in the diastema compared to the molar forming proteins. Based on this proteome analysis, we selected and evaluated two candidate proteins, EMERIN and RAB7A, as diastema tissue specific markers. This study provides the first list of proteins that were detected in the mouse embryonic diastema region, which will be useful to understand the mechanisms of tooth development.

Mouse embryonic diastema region is an ideal site for the development of ectopically transplanted tooth germ.

The anterior eye chamber and the kidney capsule of the mouse have been traditionally used for long-term culture of tooth germ grafts. However, although these sites provide an excellent growth environment, they do not represent real in situ sites for the development of a grafted tooth germ. Here, we describe a protocol to transplant a tooth germ into the mandibular diastema region of mouse embryos using exo utero surgery. Our results demonstrate that the mouse embryonic diastema region represents a normal physiological environment for the development of transplanted tooth germs. Transplanted tooth germs developed synchronically with and became indistinguishable from the endogenous ones. These ectopic teeth were vascularized and surrounded with nerve fibers, and were able to erupt normally. Thus, the exo utero transplantation approach will provide a new avenue to study tooth development and regeneration.

Dental lamina develops even within the mouse diastema.

In embryos of albino mice of ICR strain, collected between days 13 and 15, the epithelial lining within the future upper maxillary diastema was studied using frontal histological sections stained with hematoxylin-eosin and PAS methods. In embryos harvested on the 12th hr of day 13 (stage 13/12), a continuous epithelial rudiment of dentition was found in the anterior extension of the epithelial anlage of the first upper molar, up to the level of the lower anterior margin of the primary choana. In this stage the rudiment acquired, in the most anterior region of the future diastema, an arrangement typical for the dental lamina. In its dorsal extension there was found a distinct tooth anlage at the transitory stage lamina bud, which further (at stage 13/24) disintegrated into several segments. Starting with the day 14 (stage 14/12), the epithelial rudiment of dentition within the future upper diastema began to regress. From the stage 14/24 on, the anlage persisted only in its posterior terminal part where it merged with the epithelial lamina extending anteriorly from the anlage of the first upper molar. The existence of the dentition rudiment within the future mouse diastema constitutes the ontogenetic evidence that the diastema originates only secondarily--by regression. In some mutant strains of mice (tabby, crooked, sleek), the regression appears incomplete. The odontogenic potency of mouse diastema tissues should be considered when interpreting the results of in vitro experiments investigating the odontogenic inductive tissue interactions in mouse.

Gene expression patterns associated with suppression of odontogenesis in mouse and vole diastema regions.

Rodents have a toothless diastema region between the incisor and molar teeth which may contain rudimentary tooth germs. We found in upper diastema region of the mouse (Mus musculus) three small tooth germs which developed into early bud stage before their apoptotic removal, while the sibling vole (Microtus rossiaemeridionalis) had only a single but larger tooth germ in this region, and this developed into late bud stage before regressing apoptotically. To analyze the genetic mechanisms of the developmental arrest of the rudimentary tooth germs we compared the expression patterns of several developmental regulatory genes (Bmp2, Bmp4, Fgf4, Fgf8, Lef1, Msx1, Msx2, p21, Pitx2, Pax9 and Shh) between molars and diastema buds of mice and voles. In diastema tooth buds the expression of all the genes differed from that of molars. The gene expression patterns suggest that the odontogenic program consists of partially independent signaling cascades which define the exact location of the tooth germ, initiate epithelial budding, and transfer the odontogenic potential from the epithelium to the underlying mesenchyma. Although the diastema regions of the two species differed, in both species the earliest difference that we found was weaker expression of mesenchymal Pax9 in the diastema region than in molar and incisor regions at the dental lamina stage. However, based on earlier tissue recombination experiments it is conceivable that the developmental arrest is determined by the early oral epithelium.

DIASTEMA INTERINCISIVO CENTRAL SUPERIOR

La práctica de la Odontología debe estar siempre en manos del profesional odontólogo y que él por lo tanto debe ser capaz de brindar ese servicio, conocer la presencia del hueso premaxilar y la influencia de la sutura en la línea media como nueva causa para la presencia del diastema interincisivo central superior, prvocando un espacio antómico entre dos dientes vecinos que impide el apoyo coronario mesiodistal, cuando éstos se encuentran correctamente erupcionados y alineados formando el sector anterior del arco dentario. Lo que ha permitido precisar, como tema actual y necesario a investigar: "EL DIASTEMA INTERINCISIVO CENTRAL SUPERIOR". Sobre la base temática se determinó el siguiente: PROBLEMA: Cómo influye la fusión imperfecta en línea media de los maxilares en la etiología del diastema interincisivo central superior? OBJETO DE ESTUDIO: El sector alveolar anterior de los huesos maxilares superiores. OBJETIVO: Demostrar la presencia de fusión imperfecta en la línea media de los maxilares superiores como causa etiológica del diastema interincisivo central superior