Transgenically ectopic expression of Bmp4 to the Msx1 mutant dental mesenchyme restores downstream gene expression but represses Shh and Bmp2 in the enamel knot of wild type tooth germ

Bmp4 is a downstream gene of Msx1 in early mouse tooth development. In this study, we introduced the Msx1-Bmp4 transgenic allele to the Msx1 mutants in which tooth development is arrested at the bud stage in an effort of rescuing Msx1 mutant tooth phenotype in vivo. Ectopic expression of a Bmp4 transgene driven by the mouse Msx1promoter in the dental mesenchyme restored the expression of Lef-1 and Dlx2 but neither Fgf3 nor syndecan-1 in the Msx1 mutant molar tooth germ. The mutant phenotype of molar but not incisor could be partially rescued to progress to the cap stage. The Msx1-Bmp4 transgene was also able to rescue the alveolar processes and the neonatal lethality of the Msx1 mutants. In contrast, overexpression of Bmp4 in the wild type molar mesenchyme down-regulated Shh and Bmp2 expression in the enamel knot, the putative signaling center for tooth patterning, but did not produce a tooth phenotype. These results indicate that Bmp4 can bypass Msx1 function to partially rescue molar tooth development in vivo, and to support alveolar process formation. Expression of Shh and Bmp2 in the enamel knot may not represent critical signals for tooth patterning.     

Fgfr2b mediated epithelial-mesenchymal interactions coordinate tooth morphogenesis and dental trigeminal axon patterning

Dental trigeminal nerve fiber growth and patterning are strictly integrated with tooth morphogenesis, but it is still unknown, how these two developmental processes are coordinated. Here we show that targeted inactivation of the dental epithelium expressed Fgfr2b results in cessation of the mouse mandibular first molar development at the degenerated cap stage and the failure of the trigeminal molar nerve to establish the lingual branch at E13.5 stage while the buccal branch develops properly. This axon patterning defect correlates to the histological absence of the mesenchymal dental follicle and adjacent Semaphorin3A-free dental follicle target field as well as appearance of ectopic Sema3A expression domain in the lingual side of the epithelial bud. Although the mesenchymal ligands for Fgfr2b, Fgf3 and -10 were present in the Fgfr2b(-/)(-) dental mesenchyme, mutant dental epithelium showed dramatically reduced proliferation and the lack of Fgf3. Tgfbeta1, which controls Sema3A was absent from the Fgfr2b(-/-) tooth germ, and Sema3A was specifically downregulated in the dental mesenchyme at the bud and cap stage. In addition, the epithelial primary enamel knot signaling center although being molecularly present neither was histologically detectable nor expressed Bmp4 and Fgf3 as well as Fgf4, which is essential for tooth morphogenesis and stimulates mesenchymal Fgf3 and Tgfbeta1. Fgf4 beads rescued Tgfbeta1 in the Fgfr2b(-/-) dental mesenchyme explants and Tgfbeta1 induced de novo Sema3A expression in the dental mesenchyme. Collectively these results demonstrate that epithelial Fgfr2b controls tooth morphogenesis and dental axon patterning, and suggests that Fgfr2b, by mediating local epithelial-mesenchymal interactions, integrates these two distinct developmental processes during odontogenesis.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

BMP4 rescues a non-cell-autonomous function of Msx1 in tooth development

The development of many organs depends on sequential epithelial-mesenchymal interactions, and the developing tooth germ provides a powerful model for elucidating the nature of these inductive tissue interactions. In Msx1-deficient mice, tooth development arrests at the bud stage when Msx1 is required for the expression of Bmp4 and Fgf3 in the dental mesenchyme (Bei, M. and Maas, R. (1998) Development 125, 4325-4333). To define the tissue requirements for Msx1 function, we performed tissue recombinations between wild-type and Msx1 mutant dental epithelium and mesenchyme. We show that through the E14.5 cap stage of tooth development, Msx1 is required in the dental mesenchyme for tooth formation. After the cap stage, however, tooth development becomes Msx1 independent, although our experiments identify a further late function of Msx1 in odontoblast and dental pulp survival. These results suggest that prior to the cap stage, the dental epithelium receives an Msx1-dependent signal from the dental mesenchyme that is necessary for tooth formation. To further test this hypothesis, Msx1 mutant tooth germs were first cultured with either BMP4 or with various FGFs for two days in vitro and then grown under the kidney capsule of syngeneic mice to permit completion of organogenesis and terminal differentiation. Previously, using an in vitro culture system, we showed that BMP4 stimulated the growth of Msx1 mutant dental epithelium (Chen, Y., Bei, M. Woo, I., Satokata, I. and Maas, R. (1996). Development 122, 3035-3044). Using the more powerful kidney capsule grafting procedure, we now show that when added to explanted Msx1-deficient tooth germs prior to grafting, BMP4 rescues Msx1 mutant tooth germs all the way to definitive stages of enamel and dentin formation. Collectively, these results establish a transient functional requirement for Msx1 in the dental mesenchyme that is almost fully supplied by BMP4 alone, and not by FGFs. In addition, they formally prove the postulated downstream relationship of BMP4 with respect to Msx1, establish the non-cell-autonomous nature of Msx1 during odontogenesis, and disclose an additional late survival function for Msx1 in odontoblasts and dental pulp.     

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.     

The primary enamel knot determines the position of the first buccal cusp in developing mice molars

The enamel knot (EK), which is located in the center of bud and cap stage tooth germs, is a transitory cluster of non-dividing epithelial cells. The EK acts as a signaling center that provides positional information for tooth morphogenesis and regulates the growth of tooth cusps by inducing secondary EKs. The morphological, cellular, and molecular events leading to the relationship between the primary and secondary EKs have not been described clearly. This study investigated the relationship between the primary and secondary EKs in the maxillary and mandibular first molars of mice. The location of the primary EK and secondary EKs was investigated by chasing Fgf4 expression patterns in tooth germ at some intervals of in vitro culture, and the relationship between the primary EK and secondary EK was examined by tracing the primary EK cells in the E13.5 tooth germs which were frontally half sliced to expose the primary EK. After 48 hr, the primary EK cells in the sliced tooth germs were located on the buccal secondary EKs, which correspond to the future paracone in maxilla and protoconid in mandible. The Bmp4 expression in buccal part of the dental mesenchyme might be related with the lower growth in buccal epithelium than in lingual epithelium, and the Msx2 expressing area in epithelium was overlapped with the enamel cord (or septum) and cell dense area. The enamel cord might connect the primary EK with enamel navel to fix the location of the primary EK in the buccal side during the cap to bell stages. Overall, these results suggest that primary EK cells strictly contribute to form the paracone or protoconid, which are the main cusps of the tooth in the maxilla or mandible.     

Molar tooth development in caspase-3 deficient mice

Tooth morphogenesis is accompanied by apoptotic events which show restricted temporospatial patterns suggesting multiple roles in odontogenesis. Dental apoptosis seems to be caspase dependent and caspase-3 has been shown to be activated during dental apoptosis.Caspase-3 mutant mice on different genetic backgrounds were used to investigate alterations in dental apoptosis and molar tooth morphogenesis. Mouse embryos at E15.5 were analyzed to reveal any changes in enamel knots, which are transient structures eliminated by apoptosis. In caspase-3(-/-) mice on the B57BL/6 background, disorganization of the epithelium was found in the original primary enamel knot area and confirmed by altered expression of Shh. Despite this early defect in molar tooth development, these mutants showed correct formation of secondary enamel knots as indicated by Fgf-4 expression. Analyses of adult molar teeth did not reveal any major alterations in tooth shape, enamel structure or pattern when compared to heterozygote littermates. In caspase-3(-/-) mice on the 129X1/SvJ background, no defects in tooth development were found except the position of the upper molars which developed more posteriorly in the oral cavity. This is likely, however, to be a secondary defect caused by a physical squashing of the face by the malformed brain. The results suggest that although caspase-3 becomes activated and may be essential for dental apoptosis, it does not seem fundamental for formation of normal mineralised molar teeth.     

Reiterative signaling and patterning during mammalian tooth morphogenesis

Mammalian dentition consists of teeth that develop as discrete organs. From anterior to posterior, the dentition is divided into regions of incisor, canine, premolar and molar tooth types. Particularly teeth in the molar region are very diverse in shape. The development of individual teeth involves epithelial-mesenchymal interactions that are mediated by signals shared with other organs. Parts of the molecular details of signaling networks have been established, particularly in the signal families BMP, FGF, Hh and Wnt, mostly by the analysis of gene expression and signaling responses in knockout mice with arrested tooth development. Recent evidence suggests that largely the same signaling cascade is used reiteratively throughout tooth development. The successional determination of tooth region, tooth type, tooth crown base and individual cusps involves signals that regulate tissue growth and differentiation. Tooth type appears to be determined by epithelial signals and to involve differential activation of homeobox genes in the mesenchyme. This differential signaling could have allowed the evolutionary divergence of tooth shapes among the four tooth types. The advancing tooth morphogenesis is punctuated by transient signaling centers in the epithelium corresponding to the initiation of tooth buds, tooth crowns and individual cusps. The latter two signaling centers, the primary enamel knot and the secondary enamel knot, have been well characterized and are thought to direct the differential growth and subsequent folding of the dental epithelium. Several members of the FGF signal family have been implicated in the control of cell proliferation around the non-dividing enamel knots. Spatiotemporal induction of the secondary enamel knots determines the cusp patterns of individual teeth and is likely to involve repeated activation and inhibition of signaling as suggested for patterning of other epithelial organs.