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.     

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.     

Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis

Cleft palate, the most frequent congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development. Mutations in the MSX1 homeobox gene are associated with non-syndromic cleft palate and tooth agenesis in humans. We have used Msx1-deficient mice as a model system that exhibits severe craniofacial abnormalities, including cleft secondary palate and lack of teeth, to study the genetic regulation of mammalian palatogenesis. We found that Msx1 expression was restricted to the anterior of the first upper molar site in the palatal mesenchyme and that Msx1 was required for the expression of Bmp4 and Bmp2 in the mesenchyme and Shh in the medial edge epithelium (MEE) in the same region of developing palate. In vivo and in vitro analyses indicated that the cleft palate seen in Msx1 mutants resulted from a defect in cell proliferation in the anterior palatal mesenchyme rather than a failure in palatal fusion. Transgenic expression of human Bmp4 driven by the mouse Msx1 promoter in the Msx1(-/-) palatal mesenchyme rescued the cleft palate phenotype and neonatal lethality. Associated with the rescue of the cleft palate was a restoration of Shh and Bmp2 expression, as well as a return of cell proliferation to the normal levels. Ectopic Bmp4 appears to bypass the requirement for Msx1 and functions upstream of Shh and Bmp2 to support palatal development. Further in vitro assays indicated that Shh (normally expressed in the MEE) activates Bmp2 expression in the palatal mesenchyme which in turn acts as a mitogen to stimulate cell division. Msx1 thus controls a genetic hierarchy involving BMP and Shh signals that regulates the growth of the anterior region of palate during mammalian palatogenesis. Our findings provide insights into the cellular and molecular etiology of the non-syndromic clefting associated with Msx1 mutations.     

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.     

Enhanced BMP signaling results in supernumerary tooth formation in USAG-1 deficient mouse

Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist, and also modulates Wnt signaling. We previously reported that USAG-1 deficient mice have supernumerary teeth. The supernumerary maxillary incisor appears to form as a result of the successive development of the rudimentary upper incisor. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. We confirmed that BMPs were expressed in both the epithelium and mesenchyme of the rudimentary incisor at E14 and E15. BMP signaling in the rudimentary maxillary incisor, assessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was significantly enhanced. Wnt signaling as demonstrated by the nuclear localization of β-catenin was also up-regulated. Inhibition of BMP signaling rescues supernumerary tooth formation in E15 incisor explant culture. Based upon these results, we conclude that enhanced BMP signaling results in supernumerary teeth and BMP signaling was modulated by Wnt signaling in the USAG-1 deficient mouse model.     

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. Received: 1 February 1999 / Accepted: 30 March 1999     

Independent regulation of Dlx2 expression in the epithelium and mesenchyme of the first branchial arch

Dlx2, a member of the distal-less gene family, is expressed in the first branchial arch, prior to the initiation of tooth development, in distinct, non-overlapping domains in the mesenchyme and the epithelium. In the mesenchyme Dlx2 is expressed proximally, whereas in oral epithelium it is expressed distally. Dlx2 has been shown to be involved in the patterning of the murine dentition, since loss of function of Dlx1 and Dlx2 results in early failure of development of upper molar teeth. We have investigated the regulation of Dlx2 expression to determine how the early epithelial and mesenchymal expression boundaries are maintained, to help to understand the role of these distinct expression domains in patterning of the dentition. Transgenic mice produced with a lacZ reporter construct, containing 3.8 kb upstream sequence of Dlx2, led to the mapping of regulatory regions driving epithelial but not mesenchymal expression in the first branchial arch. We show that the epithelial expression of Dlx2 is regulated by planar signalling by BMP4, which is coexpressed in distal oral epithelium. Mesenchymal expression is regulated by a different mechanism involving FGF8, which is expressed in the overlying epithelium. FGF8 also inhibits expression of Dlx2 in the epithelium by a signalling pathway that requires the mesenchyme. Thus, the signalling molecules BMP4 and FGF8 provide the mechanism for maintaining the strict epithelial and mesenchymal expression domains of Dlx2 in the first arch.     

Wnt/beta-catenin signaling directs multiple stages of tooth morphogenesis

Wnt/beta-catenin signaling plays key roles in tooth development, but how this pathway intersects with the complex interplay of signaling factors regulating dental morphogenesis has been unclear. We demonstrate that Wnt/beta-catenin signaling is active at multiple stages of tooth development. Mutation of beta-catenin to a constitutively active form in oral epithelium causes formation of large, misshapen tooth buds and ectopic teeth, and expanded expression of signaling molecules important for tooth development. Conversely, expression of key morphogenetic regulators including Bmp4, Msx1, and Msx2 is downregulated in embryos expressing the secreted Wnt inhibitor Dkk1 which blocks signaling in epithelial and underlying mesenchymal cells. Similar phenotypes are observed in embryos lacking epithelial beta-catenin, demonstrating a requirement for Wnt signaling within the epithelium. Inducible Dkk1 expression after the bud stage causes formation of blunted molar cusps, downregulation of the enamel knot marker p21, and loss of restricted ectodin expression, revealing requirements for Wnt activity in maintaining secondary enamel knots. These data place Wnt/beta-catenin signaling upstream of key morphogenetic signaling pathways at multiple stages of tooth development and indicate that tight regulation of this pathway is essential both for patterning tooth development in the dental lamina, and for controlling the shape of individual teeth.     

Evidence for an early role for BMP4 signaling in thymus and parathyroid morphogenesis

The thymus and parathyroids are pharyngeal endoderm-derived organs that develop from common organ primordia, which undergo a series of morphological events resulting in separate organs in distinct locations in the embryo. Previous gene expression and functional analyses have suggested a role for BMP4 signaling in early thymus organogenesis. We have used conditional deletion of Bmp4 or Alk3 from the pharyngeal endoderm and/or the surrounding mesenchyme using Foxg1-Cre, Wnt1-Cre or Foxn1-Cre. Deleting Bmp4 from both neural crest cells (NCC) and early endoderm-derived epithelial cells in Foxg1-Cre;Bmp4 conditional mutants resulted in defects in thymus-parathyroid morphogenesis. Defects included reduced condensation of mesenchymal cells around the epithelium, partial absence of the thymic capsule, a delay in thymus and parathyroid separation, and failed or dramatically reduced organ migration. Patterning of the primordia and initial organ differentiation were not affected in any of the mutants. Deleting Bmp4 from NCC-derived mesenchyme or differentiating thymic epithelial cells (TECs) had no effects on thymus-parathyroid development, while loss of Alk3 from either neural crest cells or TECs resulted in only a mild thymic hypoplasia. these results show that the processes of cell specification and morphogenesis during thymus-parathyroid development are independently controlled, and suggest a specific temporal and spatial role for BMP4-mediated epithelial-mesenchymal interactions during early thymus and parathyroid morphogenesis.     

FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Fibroblast growth factor-10 (FGF-10), which is expressed in the mesenchyme near the distal tips, and bone morphogenetic protein 4 (BMP4), which is expressed in the most distal regions of the epithelium, are important molecules in lung morphogenesis. In the present study, we used two in vitro systems to examine the induction, growth, and differentiation of lung epithelium. Transfilter cultures were used to determine the effect of diffusible factors from the distal lung mesenchyme (LgM) on epithelial branching, and FGF-10 bead cultures were used to ascertain the effect of a high local concentration of a single diffusible molecule on the epithelium. Embryonic tracheal epithelium (TrE) was induced to grow in both culture systems and to express the distal epithelial marker surfactant protein C at the tips nearest the diffusible protein source. TrE cultured on the opposite side of a filter to LgM branched in a pattern resembling intact lungs, whereas TrE cultured in apposition to an FGF-10 bead resembled a single elongating epithelial bud. Examination of the role of BMP4 on lung bud morphogenesis revealed that BMP4 signaling suppressed expression of the proximal epithelial genes Ccsp and Foxj1 in both types of culture and upregulated the expression of Sprouty 2 in TrE cultured with an FGF-10 bead. Antagonizing BMP signaling with Noggin, however, increased expression of both Ccsp and Foxj1.     

Wnt/β-catenin signaling in the dental mesenchyme regulates incisor development by regulating Bmp4

Loss- and gain-of function approaches modulating canonical Wnt/β-catenin activity have established a role for the Wnt/β-catenin pathway during tooth development. Here we show that Wnt/β-catenin signaling is required in the dental mesenchyme for normal incisor development, as locally restricted genetic inactivation of β-catenin results in a splitting of the incisor placode, giving rise to two incisors. Molecularly this is first associated with down-regulation of Bmp4 and subsequent splitting of the Shh domain at a subsequent stage. The latter phenotype can be mimicked by ectopic application of the BMP antagonist Noggin. Conditional genetic inactivation of Bmp4 in the mesenchyme reveals that mesenchymal BMP4 activity is required for maintenance of Shh expression in the dental ectoderm. Taken together our results indicate that β-catenin together with Lef1 and Tcf1 are required to activate Bmp4 expression in order to maintain Shh expression in the dental ectoderm. This provides a mechanism whereby the number of incisors arising from one placode can be varied through local alterations of a mesenchymal signaling circuit involving β-catenin, Lef1, Tcf1 and Bmp4.     

Three-dimensional epithelial and mesenchymal cell co-cultures form early tooth epithelium invagination-like structures: expression patterns of relevant molecules

Epithelium invagination is the key feature of early tooth development. In this study, we built a three-dimensional (3D) model to represent epithelium invagination-like structure by tissue engineering. Human normal oral epithelial cells (OECs) and dental pulp stem cells (DPSCs) were co-cultivated for 2-7 weeks on matrigel or collagen gel to form epithelial and mesenchymal tissues. The histological change and gene expression were analyzed by HE staining, immunostaining, and quantitative real-time RT-PCR (qRT-PCR). After 4 weeks of cultivation, OECs-formed epithelium invaginated into DPSCs-derived mesenchyme on both matrigel and collagen gel. OEC-DPSC co-cultures on matrigel showed typical invagination of epithelial cells and condensation of the underlying mesenchymal cells. Epithelial invagination-related molecules, CD44 and E-cadherin, and mesenchymal condensation involved molecules, N-cadherin and Msx1 expressed at a high level in the tissue model, suggesting the epithelial invagination is functional. However, when OECs and DPSCs were co-cultivated on collagen gel; the invaginated epithelium was transformed to several epithelial colonies inside the mesenchyme after long culture period. When DPSCs were co-cultivated with immortalized human OECs NDUSD-1, all of the above-mentioned features were not presented. Immunohistological staining and qRT-PCR analysis showed that p75, BMP2, Shh, Wnt10b, E-cadherin, N-cadherin, Msx1, and Pax9 are involved in initiating epithelium invagination and epithelial-mesenchymal interaction in the 3D OEC-DPSC co-cultures. Our results suggest that co-cultivated OECs and DPSCs on matrigel under certain conditions can build an epithelium invagination-like model. This model might be explored as a potential research tool for epithelial-mesenchymal interaction and tooth regeneration.