Role In Tooth Development Research Articles

Neural Crest Deletion of Dlx3 Leads to Major Dentin Defects through Down-regulation of Dspp

During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.

Gene expression profiling of oral epithelium during tooth development

ObjectiveTooth development is regulated by the complex interplay of various regulatory molecules. To identify new regulatory genes released from the dental epithelium, gene expression profiling of dental epithelium was analysed. DesignICR mouse dental epithelia were isolated from the initiation (E10.5) and bell (E16.5) stages, and microarray analysis was performed using Affymetrix GeneChip®. Microarray data were validated using reverse transcriptase polymerase chain reaction (RT-PCR), and gene ontology and signalling network were analysed. ResultsDetection signals more than 300 and changes more than two folds were considered as positive signals and were further analysed. Expressions of 193 genes in the E10.5 epithelium and 582 genes in the E16.5 epithelium were significantly increased. Validation of the selected genes using RT-PCR showed a well correlation with microarray data. Subsequent signalling network analysis revealed that at E10.5 and 16.5, nine genes such as histones, signalling molecules and transcription factors were closely related with neighbouring molecules. Moreover, gene ontology analysis showed that seven growth factors/receptors or secreted proteins were highly expressed at E10.5, including the platelet-derived growth factor, C polypeptide (Pdgfc), insulin-like growth factor binding protein 2 (Igfbp2) and Igfbp5. At E16.5, nine growth factors/receptors or secreted proteins, including Igfbp3, Igfbp10/Cyr61 and heparin-binding EGF-like growth factor (Hbegf) were highly expressed. ConclusionsThese data suggest that the regulatory genes newly identified in this study may play significant roles in tooth development.

Osteoadherin Accumulates in the Predentin towards the Mineralization Front in the Developing Tooth

BackgroundProteoglycans (PG) are known to be involved in the organization and assembly of the extracellular matrix (ECM) prior to mineral deposition. Osteoadherin (OSAD), a keratan sulphate PG is a member of the small leucine-rich (SLRP) family of PGs and unlike other SLRPs, OSAD expression is restricted to mineralized tissues. It is proposed to have a high affinity for hydroxyapatite and has been shown to be expressed by mature osteoblasts but its exact role remains to be elucidated.Methodology/Principal FindingsWe investigated the protein distribution of OSAD in the developing mouse tooth using immunohistochemistry and compared its expression with other SLRPs, biglycan (BGN), decorin (DCN) and fibromodulin (FMD). OSAD was found to be specifically localized in the predentin layer of the tooth and focused at the mineralization front. These studies were confirmed at the ultrastructural level using electron microscopy (iEM), where the distribution of immunogold labeled OSAD particles were quantified and significant amounts were found in the predentin, forming a gradient towards the mineralization front. In addition, iEM results revealed OSAD to lie in close association with collagen fibers, further suggesting an important role for OSAD in the organization of the ECM. The expression profile of mineralization-related SLRP genes by rat dental pulp cells exposed to mineralization inducing factors, showed an increase in all SLRP genes. Indeed, OSAD expression was significantly increased during the mineralization process, specifically following, matrix maturation, and finally mineral deposition. Alizarin Red S staining for calcium deposition showed clear bone-like nodules, which support matrix maturation and mineralization.ConclusionsThese studies provide new evidence for the role of OSAD in the mineralization process and its specific localization in the predentin layer accumulating at the mineralization front highlighting its role in tooth development.

Effects of Wnt/β‐catenin signalling on proliferation and differentiation of apical papilla stem cells

The Wnt signalling pathway has been shown to play an important role in tooth development, however its effects with stem cells from the apical papilla (SCAP) have remained unclear. The purpose of this study was to determine effects of Wnt/β-catenin on proliferation and differentiation of SCAP in vitro. SCAP were obtained, identified and cultured. Cell proliferation, alkaline phosphatase (ALP) activity, mRNA expression of mineralization-related genes and mineralized nodule formation were measured in presence or absence of various concentrations of lithium chloride. MTT assay and flow cytometry demonstrated that Wnt/β-catenin activity could promote proliferation of SCAP. Real-time PCR analysis found that Wnt/β-catenin strongly upregulated expression of dentine sialophosphoprotein, osteocalcin and ALP in SCAP after incubation with mineralization induction medium, while ALP and alizarin red staining indicated that Wnt/β-catenin enhanced ALP activity and formation of mineralized nodules. Our results suggest that canonical Wnt/β-catenin signalling promotes proliferation and odonto/osteogenic differentiation of SCAP.

Expression patterns of histone acetyltransferases p300 and CBP during murine tooth development

The transcription coactivators p300 and CBP were reported to have indispensable roles in various tissues during the embryonic and fetal stages. Little is known about the activities of these coactivators during the tooth development, so we investigated the temporal expression patterns of p300 and CBP in developing mouse tooth germs. The first molars of fetal BALB/c mice were used to investigate p300 and CBP expression patterns. Immunohistochemistry was performed on a series of sections from embryonic Day(E) 11.5 to E19.5. Laser microdissection and real-time polymerase chain reactions were used to investigate the inner enamel epithelium, stellate reticulum, outer enamel epithelium, and dental papilla between E18.5 and E19.5. P300 had a presence in nearly every region of the tooth germ from E11.5 to E19.5. However, the expression of CBP was first detected at E12.5 and was relatively lower than the expression of p300 until E18.5. Both p300 and CBP expression levels peaked unexpectedly on DayE18.5. Additionally, the expression of p300 was distinctly lower than CBP at E19.5. In the late bell stage, p300 and CBP mRNA was highly expressed in the stellate reticulum and dental papilla, and there was a relatively low amount of CBP mRNA in the outer enamel epithelia and inner enamel epithelia from E18.5 to E19.5. The temporal patterns of p300 and CBP expression were identified during tooth development. The difference in the spatiotemporal expression of p300 and CBP indicated that they play different roles in tooth development.

The smallest teeth in the world are caused by mutations in the PCNT gene

We report a follow up study on two MOPD II Thai families with severe dental anomalies and hypoplastic alveolar bone. Striking dental anomalies comprise severe microdontia, opalescent and abnormally shaped teeth, and rootless molars. As a result of severe hypoplastic alveolar bone, most permanent teeth have been lost. Mutation analysis of PCNT revealed 2 novel mutations (p.Lys3154del and p.Glu1154X) and a recurrent mutation (p.Pro1923X). Teeth of the patient who carried a homozygous novel mutation of p.Glu1154X are probably the smallest ever reported. The sizes of the mandibular permanent incisors and all premolars were approximately 2-2.5 mm, mesiodistally. All previously reported, PCNT mutations have been described to cause premature truncation of the pericentrin protein. p.Lys3154del mutation was unique as it was pathogenic as a result of missing only a single amino acid. In situ hybridization of Pcnt shows its expression in the epithelium and mesenchyme during early stages of rodent tooth development. It is evident that PCNT has crucial role in tooth development. The permanent dentition is more severely affected than the one. This implies that PCNT appears to have more role in the development of the permanent dentition. As pericentrin is a critical centrosomal protein, the dental phenotype found in MOPD II patients is postulated to be the consequence of loss of microtubule integrity which leads to defective centrosome function.

Expression of fibroblast growth factors (Fgfs) in murine tooth development

Fgf signalling is known to play critical roles in tooth development. Twenty-two Fgf ligands have been identified in mammals, but expression of only 10 in molars and three in the incisor loop stem cell region have been documented in murine tooth development. Our understanding of Fgf signalling in tooth development thus remains incomplete and we therefore carried out comparative in situ hybridisation analysis of unexamined Fgf ligands (eight in molars and 15 in cervical loops of incisors; Fgf11-Fgf14 were excluded from this analysis because they are not secreted and do not activate Fgf receptors) during tooth development. To identify where Fgf signalling is activated, we also examined the expression of Etv4 and Etv5, considered to be transcriptional targets of the Fgf signalling pathway. In molar tooth development, the expression of Fgf15 and Fgf20 was restricted to the primary enamel knots, whereas Etv4 and Etv5 were expressed in cells surrounding the primary enamel knots. Fgf20 expression was observed in the secondary enamel knots, whereas Fgf15 showed localised expression in the adjacent mesenchyme. Fgf16, Etv4 and Etv5 were strongly expressed in the ameloblasts of molars. In the incisor cervical loop stem cell region, Fgf17, Fgf18, Etv4 and Etv5 showed a restricted expression pattern. These molecules thus show dynamic temporo-spatial expression in murine tooth development. We also analysed teeth in Fgf15(-/-) and Fgf15(-/-) ;Fgf8(+/-) mutant mice. Neither mutant showed significant abnormalities in tooth development, indicating likely functional redundancy.

Possible involvement of maspin in tooth development

Maspin is a 42 kDa serine protease inhibitor that possesses tumor suppressive and anti-angiogenic activities. Despite of a huge amount of data concerning the expression pattern of maspin in various tissues and its relevance to the biological properties of a variety of human cancer cells, little is known on the maspin expression in skeletal and tooth tissues. Recently, we reported that maspin may play an important role in extracellular matrix formation in bone by enhancing the accumulation of latent TGF-β in the extracellular matrix. This study was performed to elucidate the possible role of maspin in tooth development. First, an immunohistochemical analysis for human tooth germs at the late bell stage showed the expression of maspin by active ameloblasts and odontoblasts that were forming enamel and dentin, respectively. During rat tooth development, maspin expression was observed for the first time in inner and outer enamel epithelial cells and dental papilla cells at early bell stage. The neutralizing anti-maspin antibody inhibited the proper dental tissue formation in organ cultures of mandibular first molars obtained from 21-day-old rat embryos. In addition, the proliferation of HAT-7 cells, a rat odontogenic epithelial cell line, and human dental papilla cells were suppressed in a dose-dependent manner with anti-maspin antibody. Moreover, RT-PCR analysis showed that the expression of mRNA for tooth-related genes including dentin matrix protein 1, dentin sialophosphoprotein and osteopontin in human dental papilla cells was inhibited when treated with anti-maspin antibody. These findings suggest that maspin expressed in ameloblasts and odontoblasts plays an important physiological role in tooth development through the regulation of matrix formation in dental tissues.

Bmp2b and bmp4 are dispensable for zebrafish tooth development

Bone morphogenetic protein (Bmp) signaling has been shown to play important roles in tooth development at virtually all stages from initiation to hard tissue formation. The specific ligands involved in these processes have not been directly tested by loss-of-function experiments, however. We used morpholino antisense oligonucleotides and mutant analysis in the zebrafish to reduce or eliminate the function of bmp2b and bmp4, two ligands known to be expressed in zebrafish teeth and whose mammalian orthologs are thought to play important roles in tooth development. Surprisingly, we found that elimination of function of these two genes singly and in combination did not prevent the formation of mature, attached teeth. The mostly likely explanation for this result is functional redundancy with other Bmp ligands, which may differ between the zebrafish and the mouse.

MicroRNAs Play a Critical Role in Tooth Development

MicroRNAs are known to regulate gene function in many tissues and organs, but their expression and function, if any, in tooth development are elusive. We sought to identify them by microRNA screening analyses and reveal their overall roles by inactivating Dicer1 in the dental epithelium and mesenchyme. Discrete sets of microRNAs are expressed in molars compared with incisors as well as epithelium compared with mesenchyme. Conditional knockout (cKO) of Dicer1 (mature microRNAs) in the dental epithelium of the Pitx2-Cre mouse results in multiple and branched enamel-free incisors and cuspless molars, and change in incisor patterning and in incisor and molar size and shape. Analyses of differentiating dental epithelial markers reveal a defect in ameloblast differentiation. Conversely, the cervical loop (stem cell niche) is expanded in Dicer1 cKO. These results demonstrate that tooth development is tightly controlled by microRNAs and that specific microRNAs regulate tooth epithelial stem cell differentiation.

Disturbed tooth germ development in the absence of MINT in the cultured mouse mandibular explants

The Msx2-interacting nuclear target protein (MINT) is a nuclear matrix protein that regulates the development of many tissues. However, little is known regarding the role of MINT in tooth development. In this study, we prepared polyclonal antibodies against MINT, and found that that MINT was expressed in different cells at each stage of tooth germ development by immunohistochemistry. The role of MINT in tooth development was further illustrated by the misshapen and severely hypoplastic tooth organ in the cultured mandibular explants of MINT deficient mice. From the initiation to cap stage, the differences between mutants and wild-type molars were more and more distinguished histologically. In the MINT-deficient mandibular explants, the tooth germ was reduced in the overall size and lacked enamel knot, with abnormal dental lamina and collapsed stellate reticulum. Furthermore, the BrdU incorporation experiment showed that the proliferation activity was significantly reduced in MINT-deficient dental epithelium. Our results suggest that MINT plays an important role in tooth development, in particular, epithelial morphogenesis.

Differential expression of Notch receptors and their ligands in desmoplastic ameloblastoma

In mammals, the Notch gene family encodes four receptors (Notch1-4), and all of them are important for cell fate decisions. Notch signaling pathway plays an essential role in tooth development. The ameloblastoma, a benign odontogenic epithelial neoplasm, histologically recapitulates the enamel organ at bell stage. Notch has been detected in the plexiform and follicular ameloblastoma. Its activity in the desmoplastic ameloblastoma is unknown. Notch1-4 and their ligands (Jagged1, Jagged2 and Delta1) were examined immunohistochemically in 10 cases of desmoplastic ameloblastoma. Ameloblastoma tumor epithelium demonstrated positive expression for Notch1 (n = 5/10), Notch3 (n = 8/10), Notch4 (n = 10/10), Jagged1 (n = 6/10) and Delta1 (n = 5/10), but no reactivity for Notch2 (n = 10/10) and Jagged2 (10/10). Expression patterns were distinct with some overlap. Positive activity was detected largely in the cell membrane and cytoplasm of peripheral and central neoplastic epithelial cells, and sometimes in the nucleus. Staining score was highest for Notch4. Stromal components namely endothelial cells and fibroblasts showed overexpression for Notch4 but were mildly or non-reactive for the other Notch members and their ligands. These findings suggest that Notch receptors and their ligands may play differing roles during the development of the desmoplastic ameloblastoma with Notch4 probably playing a greater role in the acquisition of tissue-specific cellular characteristics in the desmoplastic ameloblastoma.

WNT5A inhibits human dental papilla cell proliferation and migration

WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

Effect of Wnt6 on Human Dental Papilla Cells In Vitro

Introduction The Wnt signaling pathway plays an important role in tissue development by acting on proliferation, differentiation, and cell fate decisions. Because the role of Wnt6 in tooth development was still unknown, the purpose of this study was to investigate the role of Wnt6 in tooth morphogenesis and dental tissue mineralization by elucidating its effect on human dental papilla cells (hDPCs) in vitro. Methods Human dental papilla cells were enzymatically separated from tooth germs. Recombinant adenovirus encoding full-length Wnt6 cDNA was constructed to overexpress Wnt6, and the biologic effects of Wnt6 on hDPCs were investigated. Wnt6-transduced changes in hDPC proliferation were examined by means of a 5-bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. Wnt6-transduced changes in hDPC differentiation were investigated by evaluating alkaline phosphatase (ALPase) activity, by a mineralization assay, and analysis of mineralization-related gene expression including ALP, type I collagen (Col I), osteonectin (ON), osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein-1 (DMP-1). Results Wnt6 overexpression had no significant effect on the proliferation of hDPCs by BrdU incorporation assay and flow cytometric analysis. Wnt6 enhanced differentiation of hDPCs into functional odontoblast-like cells with up-regulated activity of ALPase and the expression of mineralization-related genes such as ALP, Col I, ON, OPN, BSP, and DMP-1. Wnt6 overexpression also promoted the mineralization of hDPCs. Conclusions Our findings verified that Wnt6 plays an important role in tooth development by promoting hDPC differentiation, without significant effects on hDPC proliferation.

Ctip2/Bcl11b controls ameloblast formation during mammalian odontogenesis

The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also plays a key role in tooth development. Ctip2 is highly expressed in the ectodermal components of the developing tooth, including inner and outer enamel epithelia, stellate reticulum, stratum intermedium, and the ameloblast cell lineage. In Ctip2(-/-) mice, tooth morphogenesis appeared to proceed normally through the cap stage but developed multiple defects at the bell stage. Mutant incisors and molars were reduced in size and exhibited hypoplasticity of the stellate reticulum. An ameloblast-like cell population developed ectopically on the lingual aspect of mutant lower incisors, and the morphology, polarization, and adhesion properties of ameloblasts on the labial side of these teeth were severely disrupted. Perturbations of gene expression were also observed in the mandible of Ctip2(-/-) mice: expression of the ameloblast markers amelogenin, ameloblastin, and enamelin was down-regulated, as was expression of Msx2 and epiprofin, transcription factors implicated in the tooth development and ameloblast differentiation. These results suggest that Ctip2 functions as a critical regulator of epithelial cell fate and differentiation during tooth morphogenesis.

Hepatocyte Growth Factor Exerts Promoting Functions on Murine Dental Papilla Cells

Hepatocyte growth factor (HGF) is a potent mitogenic and angiogenic factor involved in the development of many tissues including the teeth. We previously reported that dental pulp cells express the HGF receptor c-met and that HGF can enhance cell proliferation and differentiation. The purpose of this study was to determine whether HGF could stimulate proliferation and/or differentiation of murine dental papilla cells (MDPCs). MDPCs were isolated from neonatal mice, and the mitogenic potential of HGF on MDPCs was measured by 3-(4, 5-dimethyl-thyazol-2-yl)-2, 5-diphenyltetrazolium and flow cytometry. Differentiation of MDPCs in the presence of HGF was characterized by mineralization assay, alkaline phosphatase (ALP) activity and reverse-transcription polymerase chain reaction. We reported that MDPCs expressed both HGF and c-met and that HGF enhanced proliferation of MDPCs by concentrations ranging from 5 ng/mL to 20 ng/mL, with 10 ng/mL as the optimal concentration (p < 0.05). HGF prolonged S phase and shortened G1 phase of MDPCs, increased ALP activity significantly over the control (p < 0.05), and enhanced the formation of mineral nodules. ALP, dentin sialophosphoprotein, and dentin matrix protein-1 expression were significantly increased in the presence of HGF (p < 0.05), as were the expression of Msx1, Runx2, and Pax9. Our data suggest that HGF plays an important role in tooth development, by promoting the proliferation and differentiation of MDPCs.

Immortalization of normal human gingival keratinocytes and cytological and cytogenetic characterization of the cells

Most in vitro studies of oral carcinogenesis in human cells are carried out with oral keratinocytes immortalized by human papillomavirus type 16 DNA. However, because various etiological factors for oral cancer are known, it is important to establish new human keratinocyte cell lines useful for studying the mechanism of oral carcinogenesis. Normal human gingival keratinocytes in secondary cultures grown in serum-free medium were either transfected with origin (-) SV40 DNA or sequentially transfected with origin (-) SV40 DNA and human c-fos. The transfected cells were continually passaged and analyzed for cytological and cytogenetic characterizations. Four immortal cell lines were grown for over 1100 days in culture and maintained a vigorous growth for over 250 population doublings. They expressed SV40 T antigen, cytokeratins 8 and 18, and E-cadherin, and overexpressed the c-Fos protein. The immortal cell lines had telomerase activity but lacked transformed phenotypes on soft agar or in nude mice. Each cell line had nonrandom chromosomal abnormalities and minisatellite alterations. One of the immortal cell lines, NDUSD-1, retained the capability to deposit calcium, which was also demonstrated in normal human gingival keratinocytes by alizarin red staining, indicating the possibility that NDUSD-1 cells may retain some natural characteristics of normal gingival keratinocytes. Because the oral ectoderm plays an important role in tooth development, these immortal cell lines may be useful in various experimental models for investigations of oral biology and oral carcinogenesis.

Polymorphism in the Msx1 gene associated with hypodontia in a Brazilian family

Tooth development is regulated by a reciprocal series of epithelial-mesenchymal interactions. With the large number of genes involved in the odontogenesis process, the opportunity for mutations to disrupt this process is high. Mutational analysis has revealed genes that are major causes of non-syndromic hypodontia. The most common permanent missing teeth are the third molars, second premolars, and maxillary lateral incisors. Although hypodontia does not represent a serious public health problem, it may cause masticatory and speech dysfunctions and esthetic problems. Msx1 (Muscle Segment Box) is believed to play an important role in tooth development. To further investigate the role of the gene in human hypodontia, we analyzed genotypes in a family with hypodontia using the SSCP assay. Examinations of all affected and unaffected members of the family studied indicated that 5 of the 10 family members had hypodontia, and it was possible to observe polymorphisms/mutation by SSCP as bands with an anomalous migration pattern in individuals with hypodontia. Our data suggest that Msx1 gene polymorphism is associated with hypodontia.

Identification and Functional Analysis of Two Novel PAX9 Mutations

The paired-domain transcription factor PAX9 plays a critical role in tooth development, as heterozygous mutations in PAX9 have been shown to be associated with human tooth agenesis. In this study, we report 2 novel missense mutations, gly6arg (G6R) and ser43lys (S43K), in the paired domain of PAX9 in Chinese patients with varying degrees of nonsyndromic tooth agenesis. Excluding third molars, the individual with the G6R mutation was missing 2 mandibular incisors and a maxillary premolar, while the phenotype of individuals with the S43K mutation consisted of peg-shaped upper lateral incisors and missing molars, premolars and canines. As these 2 mutations occur at highly conserved amino acids in the PAX gene family and between different species, we further analyzed the effects of the mutations on the function of the resulting proteins. Immunofluorescence and immunoblotting studies showed that the mutations did not alter nuclear localization in mammalian cells. Gel shift and super shift assays indicate that both mutant proteins bound DNA at a lower level than the normal protein, with G6R having a greater affinity for DNA than S43K. Likewise, the G6R protein was able to transcriptionally activate a Bmp4 promoter construct to a greater extent than S43K. Our finding that the severity of tooth agenesis in the patients was correlated to the DNA-binding capacity of the mutated PAX9 9proteins supports the hypothesis that DNA binding is responsible for the genetic defect.

Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides -97 and -72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.