Deletion of Trps1 regulatory elements recapitulates postnatal hip joint abnormalities and growth retardation of Trichorhinophalangeal syndrome in mice.

Trichorhinophalangeal syndrome (TRPS) is a genetic disorder caused by point mutations or deletions in the gene-encoding transcription factor TRPS1. TRPS patients display a range of skeletal dysplasias, including reduced jaw size, short stature, and a cone-shaped digit epiphysis. Certain TRPS patients experience early onset coxarthrosis that leads to a devastating drop in their daily activities. The etiologies of congenital skeletal abnormalities of TRPS were revealed through the analysis of Trps1 mutant mouse strains. However, early postnatal lethality in Trps1 knockout mice has hampered the study of postnatal TRPS pathology. Here, through epigenomic analysis we identified two previously uncharacterized candidate gene regulatory regions in the first intron of Trps1. We deleted these regions, either individually or simultaneously, and examined their effects on skeletal morphogenesis. Animals that were deleted individually for either region displayed only modest phenotypes. In contrast, the Trps1Δint/Δint mouse strain with simultaneous deletion of both genomic regions exhibit postnatal growth retardation. This strain displayed delayed secondary ossification center formation in the long bones and misshaped hip joint development that resulted in acetabular dysplasia. Reducing one allele of the Trps1 gene in Trps1Δint mice resulted in medial patellar dislocation that has been observed in some patients with TRPS. Our novel Trps1 hypomorphic strain recapitulates many postnatal pathologies observed in human TRPS patients, thus positioning this strain as a useful animal model to study postnatal TRPS pathogenesis. Our observations also suggest that Trps1 gene expression is regulated through several regulatory elements, thus guaranteeing robust expression maintenance in skeletal cells.

Methylation analysis of DCC gene in saliva samples is an efficient method for non-invasive detection of superficial hypopharyngeal cancer.

BACKGROUND: Advances in upper gastrointestinal endoscopic technology have enabled early detection and treatment of hypopharyngeal cancer. However, in-depth pharyngeal observations require sedation and are invasive. It is important to establish a minimally invasive and simple evaluation method to identify high-risk patients. METHODS: Eighty-seven patients with superficial hypopharyngeal cancer and 51 healthy controls were recruited. We assessed the methylation status of DCC, PTGDR1, EDNRB, and ECAD, in tissue and saliva samples and verified the diagnostic accuracy by methylation analyses of their promoter regions using quantitative methylation-specific PCR. RESULTS: Significant differences between cancer and their surrounding non-cancerous tissues were observed in the methylation values of DCC (p = 0.003), EDNRB (p = 0.001), and ECAD (p = 0.043). Using receiver operating characteristic analyses of the methylation values in saliva samples, DCC showed the highest area under the curve values for the detection of superficial hypopharyngeal cancer (0.917, 95% confidence interval = 0.864-0.970), compared with those for EDNRB (0.680) and ECAD (0.639). When the cutoff for the methylation values of DCC was set at ≥0.163, the sensitivity to detect hypopharyngeal cancer was 82.8% and the specificity was 90.2%. CONCLUSIONS: DCC methylation in saliva samples could be a non-invasive and efficient tool for early detection of hypopharyngeal cancer in high-risk patients.

GATA3 is essential for separating patterning domains during facial morphogenesis.

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

Direct observation of epoxy resin blocks for renal biopsy by low-vacuum scanning electron microscopy.

To improve the resolution of low-vacuum scanning electron microscopy (LVSEM), the epoxy resin block for the transmission electron microscopy (TEM) was observed directly with LVSEM. After observing ultrathin sections from renal biopsies of IgA nephropathy, membranous nephropathy, lupus nephritis, diabetic nephropathy (DM), thin basement membrane disease (TBMD), Alport's syndrome, Fabry's disease, and renal amyloidosis, the epoxy resin blocks of the same sites were observed by LVSEM and compared. The LVSEM image of the epoxy resin block corresponds to the negative of the TEM image, and when the gradation is reversed, the LVSEM image was comparable to the TEM image. At a low magnification of 100 ×, the entire specimen, including the glomerulus, was obtained. LVSEM at 5000 × magnification was sufficient to identify paramesangial deposits in IgA nephropathy and subepithelial electron-dense deposits (EDD) and spikes in membranous nephropathy. Glomerular basement membrane thickening in DM and thinning in TBMD could be sufficiently diagnosed with LVSEM at 6000 ×. Accumulation of ceramide in Fabry's disease was easily identified, but amyloid fibril could not be identified by LVSEM. LVSEM of renal biopsy epoxy resin blocks can replace TEM up to moderate magnification.

Endoscopic and Histological Gastritis in University Students with Helicobacter pylori Infection.

Objective Although the characteristics of Helicobacter pylori infection have been extensively reported, there is a lack of consensus regarding its characteristics in young adults. The present study examined the endoscopic and histological characteristics of young adults who underwent eradication therapy for H. pylori infection. Methods We examined the H. pylori infection status of first-year students at Okayama University School of Medicine and Dentistry between 2014 and 2020. A total of 152 (6.8%) students who were positive for H. pylori antibody or pepsinogen tests were enrolled in the study. Among them, 107 students underwent endoscopy, and their biopsy samples were investigated. Seventy-five students were diagnosed with H. pylori infections. Results Of 75 H. pylori-positive patients, 57 (76.0%) had endoscopic atrophic gastritis, and 42 (56.0%) had histological atrophy. A few patients had severe atrophic gastritis. All 65 patients who underwent an eradication assessment were successfully treated. After successful eradication, 26 patients underwent endoscopic follow-up. The mean follow-up period was 32.9 months. A histological evaluation revealed that gastric antrum atrophy had subsided in 11 of 14 patients, and atrophy in the lesser curvature of the gastric body had subsided in 7 of 8 patients. Conclusion More than half of young adults with H. pylori infection had atrophic gastritis. We found mild atrophy in young adults, which subsided shortly after eradication treatment. This study provides a foundation for future studies to evaluate the validity of eradication therapy in preventing gastric cancer in patients.

Lithium carbonate accelerates the healing of apical periodontitis.

Apical periodontitis is a disease caused by bacterial invasions through the root canals. Our previous study reported that lithium chloride (LiCl) had a healing effect on apical periodontitis. The aim of this report is to investigate the healing properties and mechanism of lithium ion (Li+) for apical periodontitis using rat root canal treatment model. 10-week-old male Wistar rat's mandibular first molars with experimentally induced apical periodontitis underwent root canal treatment and were applied lithium carbonate (Li2CO3) containing intracanal medicament. Base material of the medicament was used as a control. Subject teeth were scanned by micro-CT every week and the periapical lesion volume was evaluated. The lesion volume of Li2CO3 group was significantly smaller than that of the control group. Histological analysis showed that in Li2CO3 group, M2 macrophages and regulatory T cells were induced in the periapical lesion. In situ hybridization experiments revealed a greater expression of Col1a1 in Li2CO3 group compared with the control group. At 24 h after application of intracanal medicament, Axin2-positive cells were distributed in Li2CO3 group. In conclusion, Li2CO3 stimulates Wnt/ß-catenin signaling pathway and accelerate the healing process of apical periodontitis, modulating the immune system and the bone metabolism.

Evaluation of safety and efficacy of autologous oral mucosa-derived epithelial cell sheet transplantation for prevention of anastomotic restenosis in congenital esophageal atresia and congenital esophageal stenosis.

BACKGROUND: We performed the first autologous oral mucosa-derived epithelial cell sheet transplantation therapy in a patient with refractory postoperative anastomotic stricture in congenital esophageal atresia (CEA) and confirmed its safety. In this study, patients with CEA and congenital esophageal stenosis were newly added as subjects to further evaluate the safety and efficacy of cell sheet transplantation therapy. METHODS: Epithelial cell sheets were prepared from the oral mucosa of the subjects and transplanted into esophageal tears created by endoscopic balloon dilatation (EBD). The safety of the cell sheets was confirmed by quality control testing, and the safety of the transplantation treatment was confirmed by 48-week follow-up examinations. RESULTS: Subject 1 had a stenosis resected because the frequency of EBD did not decrease after the second transplantation. Histopathological examination of the resected stenosis revealed marked thickening of the submucosal layer. Subjects 2 and 3 did not require EBD for 48 weeks after transplantation, during which time they were able to maintain a normal diet by mouth. CONCLUSIONS: Subjects 2 and 3 were free of EBD for a long period of time after transplantation, confirming that cell sheet transplantation therapy is clearly effective in some cases. In the future, it is necessary to study more cases; develop new technologies such as an objective index to evaluate the efficacy of cell sheet transplantation therapy and a device to achieve more accurate transplantation; identify cases in which the current therapy is effective; and find the optimal timing of transplantation; and clarify the mechanism by which the current therapy improves stenosis. TRIAL REGISTRATION: UMIN, UMIN000034566, registered 19 October 2018, https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000039393 .

Trps1 is necessary for normal temporomandibular joint development.

Mutation of the human TRPS1 gene leads to trichorhinophalangeal syndrome (TRPS), which is characterized by an abnormal development of various organs including the craniofacial skeleton. Trps1 has recently been shown to be expressed in the jaw joints of zebrafish; however, whether Trps1 is expressed in the mammalian temporomandibular joint (TMJ), or whether it is necessary for TMJ development is unknown. We have analyzed (1) the expression pattern of Trps1 during TMJ development in mice and (2) TMJ development in Trps1 knockout animals. Trps1 is expressed in the maxillo-mandibular junction at embryonic day (E) 11.5. At E15.5, expression is restricted to the developing condylar cartilage and to the surrounding joint disc progenitor cells. In Trps1 knockout mice, the glenoid fossa of the temporal bone forms relatively normally but the condylar process is extremely small and the joint disc and cavities do not develop. The initiation of condyle formation is slightly delayed in the mutants at E14.5; however, at E18.5, the flattened chondrocyte layer is narrowed and most of the condylar chondrocytes exhibit precocious chondrocyte maturation. Expression of Runx2 and its target genes is expanded toward the condylar apex in the mutants. These observations underscore the indispensable role played by Trps1 in normal TMJ development in supporting the differentiation of disc and synoviocyte progenitor cells and in coordinating condylar chondrocyte differentiation.

The Wnt/ß-catenin signaling pathway has a healing ability for periapical periodontitis.

Various disease-related genes have recently been identified using single nucleotide polymorphisms (SNPs). This study identified disease-related genes by analyzing SNP using genomic DNA isolated from Japanese patients with periapical periodontitis. Results showed that the SNP in LRP5 demonstrated a significant genotypic association with periapical lesions (Fisher's exact test, P < 0.05). We constructed an in vivo murine periapical periodontitis model to confirm the Wnt/ß-catenin signaling pathway's role in developing and healing periapical periodontitis. We observed that administration of the Wnt/ß-catenin signaling pathway inhibitor enlarged the periapical lesion. Moreover, applying lithium chloride (LiCl) to root canals accelerated periapical periodontitis healing. Histological analysis demonstrated that the expression levels of Col1a1 and Runx2 increased in the LiCl application group compared to that in the control group. Furthermore, many CD45R-positive cells appeared in the periapical lesions in the LiCl application group. These results indicated that LiCl promoted the healing of periapical periodontitis by inducing bone formation and immune responses. Our findings suggest that the Wnt/ß-catenin signaling pathway regulates the development of periapical periodontitis. We propose a bioactive next-generation root canal treatment agent for this dental lesion.

Quantitative morphometric analysis of molar teeth and alveolar bone using micro-computed tomography in aged mice.

OBJECTIVES: Irreversible morphological regressions of the teeth or related structures in older people can diminish their overall health. However, research on human aging in dentistry is complicated by several confounding factors. In this study, we conducted a morphometric analysis of the mandibular second molars and surrounding alveolar bone in C57BL/6 mice to evaluate age-related changes in the oral cavity. METHODS: The animals were divided into five groups based on their age: 4 weeks (juvenile mice; n = 5); 20 weeks (n = 7), 50 weeks (n = 5), 77 weeks (n = 7), and 100 weeks (n = 5); changes were evaluated using micro-computed tomography. RESULTS: The molars of juvenile mice had sharp and pointed cusps and presented maximum heights. With age and occlusal wear, the cusp heights demonstrated a significant decrease (up to 75%) until the last stage of life. Conversely, apparent lesions were not observed on the basal portion of the crown, even in the most heavily worn teeth. The roots of the molars continued to grow in length at 4 weeks of age. Alveolar bone resorption begins to occur in middle age and continues throughout life. The proportion of vertical bone loss reached approximately 40% of the entire root length, demonstrating a remarkable increase between weeks 77 and 100. CONCLUSIONS: Overall, these morphological changes were similar to those observed in humans. Therefore, it might be appropriate to use aged mice as an experimental model for basic and clinical research in geriatric dentistry.

Senescence-accelerated mouse prone 8 (SAMP8) mice exhibit reduced entoconid in the lower second molar.

OBJECTIVE: The position and size of the major cusps in mammalian molars are arranged in a characteristic pattern that depends on taxonomy. In humans, the cusp which locates distally within each molar is smaller than the mesially located cusp, which is referred to as "distal reduction". Although this concept has been well-recognized, it is still unclear how this reduction occurs. Current study examined whether senescence-accelerating mouse prone 8 (SAMP8) mice could be a possible animal model for studying how the mammalian molar cusp size is determined. DESIGN: SAMP8 mice were compared with parental control (SAMR1) mice. Microcomputed tomography images of young and aged mice were captured to observe molar cusp morphologies. Cusp height from cement-enamel junction and mesio-distal length of molars were measured. The statistical comparison of the measurements was performed by Mann-Whitney U test. RESULTS: SAMP8 mice showed reduced development of the disto-lingual cusp (entoconid) of lower second molar when compared with SAMR1 mice. The enamel thickness and structure was disturbed at entoconid, and aged SAMP8 mice displayed severe wear of the entoconid in lower second molar. These phenotypes were observed on both sides of the lower second molar. CONCLUSIONS: In addition to the general senescence phenotype observed in SAMP8 mice, this strain may genetically possess molar cusp phenotypes which is determined prenatally. Further, SAMP8 mice would be a potential model strain to study the genetic causes of the distal reduction of molar cusp size.

Retinoic acid affects craniofacial patterning by changing Fgf8 expression in the pharyngeal ectoderm.

Retinoic acid signaling plays important roles in establishing normal patterning and cellular differentiation during embryonic development. In this study, we show that single administration of retinoic acid at embryonic day 8.5 causes homeotic transformation of the lower jaw into upper jaw-like structures. This homeosis was preceded by downregulation of Fgf8 and Sprouty expression in the proximal domain of the first pharyngeal arch. Downregulation of mesenchymal genes such as Dlx5, Hand2, Tbx1 and Pitx2 was also observed. The oropharynx in retinoic acid-treated embryos was severely constricted. Consistent with this observation, Patched expression in the arch endoderm and mesenchyme was downregulated. Thus, retinoic acid affects the expression of subsets of epithelial and mesenchymal genes, possibly disrupting the regional identity of the pharyngeal arch.

Semaphorin 4D inhibits collagen synthesis of rat pulp-derived cells.

OBJECTIVE: Semaphorins are a group of secreted and membrane-associated molecules that play important roles in axon navigation. Several semaphorin family molecules are expressed in the pharyngeal arches and tooth germs. We analysed the expression of membrane-associated Semaphorin 4D (Sema4D) in tooth germs, and examined its potential role in regulating the differentiation of rat incisor pulp-derived cells in vitro. DESIGN: mRNA expression was examined by in situ hybridisation. The effects of Sema4D on rat pulp-derived cells were examined by adenovirus-mediated overexpression in vitro. RESULTS: Both epithelial and mesenchymal cells of tooth germs expressed Sema4D at the early bell stage. Later, the odontoblasts predominantly expressed Sema4D, while the epithelial expression greatly decreased. The overexpression of Sema4D in rat incisor pulp-derived cells strongly inhibited mineralisation. This inhibition was preceded by a reduction of collagen fibre production at the level of mRNA synthesis. CONCLUSIONS: These results indicate that Sema4D is expressed in both epithelial and mesenchymal cells of the tooth germs. Sema4D represses collagen synthesis of pulp-derived cells, indicating it might negatively regulate odontoblast differentiation.

Fate of cranial neural crest cells during craniofacial development in endothelin-A receptor-deficient mice.

Most of the bone, cartilage and connective tissue of the lower jaw is derived from cranial neural crest cells (NCCs) arising from the posterior midbrain and hindbrain. Multiple factors direct the patterning of these NCCs, including endothelin-1-mediated endothelin A receptor (Edn1/Ednra) signaling. Loss of Ednra signaling results in multiple defects in lower jaw and neck structures, including homeotic transformation of lower jaw structures into upper jaw-like structures. However, since the Ednra gene is expressed by both migrating and post-migrating NCCs, the actual function of Ednra in cranial NCC development is not clear. Ednra signaling could be required for normal migration or guidance of NCCs to the pharyngeal arches or in subsequent events in post-migratory NCCs, including proliferation and survival. To address this question, we performed a fate analysis of cranial NCCs in Ednra-/- embryos using the R26R;Wnt1-Cre reporter system, in which Cre expression within NCCs results in permanent beta-galactosidase activity in NCCs and their derivatives. We find that loss of Ednra does not detectably alter either migration of most cranial NCCs into the mandibular first arch and second arch or their subsequent proliferation. However, mesenchymal cell apoptosis is increased two fold in both E9.5 and E10.5 Ednra-/- embryos, with apoptotic cells being present in and just proximal to the pharyngeal arches. Based on these studies, Ednra signaling appears to be required by most cranial NCCs after they reach the pharyngeal arches. However, a subset of NCCs appear to require Ednra signaling earlier, with loss of Ednra signaling likely leading to premature cessation of migration into or within the arches and subsequent cell death.

Temporomandibular joint loading generated during bilateral static bites at molars and premolars.

The aim of this study was to investigate the features of the loading vectors of the temporomandibular joint (TMJ) generated during bilateral static bites at the molars and at the premolars, and to determine the major factors affecting the difference between the two loading vectors. We computed the subjects' estimated and theoretical minimum TMJ loadings under the two different bite conditions by applying the subjects' bite-force and electromyographic (EMG) data to a two-dimensional (2D) standard model of the jaw based on a rigid-body spring model of the TMJ. For a molar bite, (1) the estimated loading vector was not equal to its theoretical minimum; (2) the TMJ-loading/bite-force ratio, describing the proportion of TMJ loading, was relatively small, 0.477 on average; and (3) the estimated loading vector pointed in the direction of the central part of the articular disk's intermediate zone. For a premolar bite, on the other hand, (1) the estimated loading vector was nearly equal to its theoretical minimum; (2) the TMJ-loading/bite-force ratio was relatively large, 0.904 on average; and (3) the estimated loading vector pointed at the superior portion of the articular disk's intermediate zone. The differences between the TMJ-loading vectors for molar and premolar bites originated primarily from changes in the bite-point location.

Fibronectin accelerates the growth and differentiation of ameloblast lineage cells in vitro.

During tooth development, the growth and differentiation of ameloblast lineage (AL) cells are regulated by epithelial-mesenchymal interactions. To examine the dynamic effects of components of the basement membrane, which is the extracellular matrix (ECM) lying between the epithelium and mesenchyme, we prepared AL cells from the epithelial layer sheet of mandibular incisors of postnatal day 7 rats and cultured them on plates coated with type IV collagen, laminin-1, or fibronectin. The growth of AL cells was supported by type IV collagen and fibronectin but not by laminin-1 in comparison with that on type I collagen as a reference. Clustering and differentiation of AL cells were observed on all matrices examined. AL cells showed normal growth and differentiation at low cell density on fibronectin but not on type I collagen. Furthermore, the population of cytokeratin 14-positive cells on fibronectin was lower than that on other ECM components, suggesting that fibronectin may be a modulator to accelerate the differentiation of AL cells. After the cells had been cultured for 9 days on fibronectin, crystal-like structures were observed. These structures overlaid the cell clusters and were positive for von Kossa staining. These findings indicate that each matrix component has a regulative role in the proliferation and differentiation of AL cells and that fibronectin causes the greatest acceleration of AL cell differentiation.

Activation of the Wnt/ß-catenin pathway and tissue inhibitor of metalloprotease 1 during tertiary dentinogenesis.

Tertiary dentin is deposited inside teeth after various stimuli and serves as a major defensive wall to preserve pulp cells. However, the molecular mechanisms of the activation of quiescent odontoblasts, immature pulp cells and tertiary dentin formation are still unclear. Therefore, we performed a comprehensive gene expression analysis of pulp cells after cavity preparation of 9-week-old rat molars to clarify the critical molecules in tertiary dentinogenesis. As a result, mRNA expression of various molecules was up- or down-regulated. Notably, several members of the matrix metalloprotease family and their endogenous inhibitors were up-regulated after cavity preparation. In situ hybridization showed that tissue inhibitor of metalloprotease 1 (Timp1) was widely and continuously distributed in the pulp beneath the cavity in vivo. We also observed accumulation of ß-catenin in the pulp cells beneath the cavity by fluorescence immunohistochemistry. Furthermore, Timp1 transcription was repressed by a dominant-negative TCF4 in immature undifferentiated mesenchymal cells, but not altered in mature odontoblast-like cells. These results indicate that cavity preparation may activate the Wnt/ß-catenin pathway and the Wnt/ß-catenin pathway and Timp1 may be correlatively involved in pulp repair. Timp1 might play crucial roles in reactivation of immature pulp cells for tertiary dentinogenesis.

Hydrogel-based biomimetic environment for in vitro modulation of branching morphogenesis.

The mechanical properties of the cellular microenvironment dramatically alter during tissue development and growth. Growing evidence suggests that physical microenvironments and mechanical stresses direct cell fate in developing tissue. However, how these physical cues affect the tissue morphogenesis remains a major unknown. We explain here that the physical properties of the cell and tissue microenvironment, biomimetically reproduced by using hydrogel, guide the tissue morphogenesis in the developmental submandibular gland (SMG). In particular, the softer gel enhances the bud expansion and cleft formation of SMG, whereas the stiffer gel attenuates them. These morphological changes in SMG tissue are led by soluble factors (FGF7/10) induction regulated by cell traction force derived from the tissue deformation. Our findings suggest that cells sense the mechanics of their surrounding environment and alter their properties for self-organization and the following tissue morphogenesis. Also, physically designed hydrogel material is a valuable tool for producing the biomimetic microenvironment to explore how physical cues affect tissue morphogenesis and to modulate tissue morphogenesis for in vitro tissue synthesis.

Bone morphogenetic protein 4 is involved in cusp formation in molar tooth germ of mice.

In order to clarify the role of BMP4 in the development of the tooth crown, we employed the antisense technique on molar tooth germs removed from the mandibles of embryonic 13.5-d-old mice. In the tooth germ explants incubated for 14 d with antisense oligodeoxynucleotide (AS-ODN) against Bmp4 (a) cusps were not formed, whereas dentin matrix was secreted in the whole region of the crown, (b) inner enamel epithelial (IEE) cells remained in the undifferentiated state in the occlusal region of the crown, though they differentiated in the proximal region (lateral surface region of tooth crown), and (c) insufficient growth of the dental papilla was observed. A 5-bromo-2'-deoxyuridine (BrdU) uptake experiment showed that, although a site-specific proliferation of IEE cells occurred in the occlusal region in the control explants, it was not found in the AS-ODN-treated explants. In the proximal region, however, the proliferation of IEE cells was detected evenly in all explants treated with or without AS-ODNs. These results suggest that AS-ODN against Bmp4 inhibited the differentiation and the site-specific proliferation of IEE cells in the occlusal region of molar tooth germs, resulting in the suppression of cusp formation. Our data thus suggest that BMP4 is involved in cusp formation and differentiation of ameloblasts in the occlusal region of molars.

Tooth-type specific expression of dHAND/Hand2: possible involvement in murine lower incisor morphogenesis.

dHAND/Hand2 is a basic helix-loop-helix transcription factor required for the development of the heart, pharyngeal arches, and vasculature and is expressed during embryogenesis. However, there are no reports on the involvement of the dHAND gene in tooth development. In the present study, the expression of dHAND was examined in developing tooth germs of mice. The dHAND gene was expressed in the mesenchyme of the presumptive incisor region of the lower jaw at an early stage and in the mesenchyme of the lower incisor tooth germ at a later stage. However, the dHAND gene was not expressed in the upper incisor region or the upper and lower molar regions during jaw development. Treatment of tooth germ explants of lower incisors with antisense oligodeoxinucleotide (ODN) against dHAND prevented the differentiation of tooth germ cells, including ameloblasts and odontoblasts, the formation of dentin and enamel, and the proliferation of tooth germ cells and increased the apoptosis of tooth germ cells, suggesting that dHAND is essential for these cells during development. On the other hand, the treatment of tooth germ explants of upper incisor and upper or lower molars did not induce severe effects on their development. Treatment of the explants with basic fibroblast growth factor in association with antisense ODN partially rescued them from the effects of antisense ODN. The present results suggest that the dHAND gene plays important roles in type-specific development of lower incisors, and that basic fibroblast growth factor is involved downstream of the dHAND pathway in tooth germ cells.