Early Stages Of Tooth Development Research Articles

Dynamic expression of IGFBP3 modulate dual actions of mineralization micro-environment during tooth development via Wnt/beta-catenin signaling pathway

BackgroundTooth development, as one of the major mineralized tissues in the body, require fine-tuning of mineralization micro-environment. The interaction between dental epithelium and mesenchyme plays a decisive role in this process. With epithelium–mesenchyme dissociation study, we found interesting expression pattern of insulin-like growth factor binding protein 3 (IGFBP3) in response to disruption of dental epithelium–mesenchyme interaction. Its action and related mechanisms as regulator of mineralization micro-environment during tooth development are investigated.ResultsExpressions of osteogenic markers at early stage of tooth development are significantly lower than those at later stage. BMP2 treatment further confirmed a high mineralization micro-environment is disruptive at early stage, but beneficial at later stage of tooth development. In contrast, IGFBP3's expression increased gradually from E14.5, peaked at P5, and decreased afterwards, demonstrating an inverse correlation with osteogenic markers. RNA-Seq and Co-immunoprecipitation showed that IGFBP3 regulates the Wnt/beta-catenin signaling pathway activity by enhancing DKK1 expression and direct protein–protein interaction. The suppression of the mineralization microenvironment effectuated by IGFBP3 could be reversed by the DKK1 inhibitor WAY-262611, further demonstrating that IGFBP3 exerted its influence via DKK1.ConclusionA deeper understanding of tooth development mechanisms is essential for tooth regeneration, which have great implications for dental care. The current study demonstrated that the IGFBP3 expression is regulated in accordance with the needs of the mineralization microenvironment during tooth development, and IGFBP3 exerts its modulating action on osteogenic/odontogenic differentiation of hDPSCs by DKK1-Wnt/ beta-catenin axis.

Progress in signal centers in early tooth development

At the early stage of tooth development, the tooth embryo underwent three successive stages: bud stage, cap stage and bell stage through the interaction between epithelium and mesenchymal cells. During these stages, especially during the interphase transition, the presence of signal centers was particularly crucial. Signal center was composed of a small group of oral epithelial cell collections which were formed in a specific sequence, playing a decisive role in signal expression, morphogenesis and cell differentiation by secreting growth factors and other signaling molecules to regulate developmental signaling pathways. This paper reviews the evolution of signal centers as well as the important signal molecules and transcription factors in signal centers since the formation of dental placode.

N6-methyladenosine (m6A) RNA methylation mediated by methyltransferase complex subunit WTAP regulates amelogenesis

N6-methyladenosine (m6A) RNA methylation, one of the most widespread posttranscriptional modifications in eukaryotes, plays crucial roles in various developmental processes. The m6A modification process is catalyzed by a methyltransferase complex that includes Wilms tumor 1-associated protein (WTAP) as a key component. Whether the development of dental enamel is regulated by m6A RNA methylation in mammals remains unclear. Here, we reveal that WTAP is widely expressed from the early stage of tooth development. Specific inactivation of Wtap in mouse enamel epithelium by the Cre/loxp system leads to serious developmental defects in amelogenesis. In Wtap conditional KO mice, we determined that the differentiation of enamel epithelial cells into mature ameloblasts at the early stages of enamel development is affected. Mechanistically, loss of Wtap inhibits the expression of Sonic hedgehog (SHH), which plays an important role in the generation of ameloblasts from stem cells. Together, our findings provide new insights into the functional role of WTAP-mediated m6A methylation in amelogenesis in mammals.

Mesocrystalline Ordering and Phase Transformation of Iron Oxide Biominerals in the Ultrahard Teeth of Cryptochiton stelleri

Biological organisms naturally synthesize complex, hierarchical, multifunctional materials through mineralization processes at ambient conditions and under physiological pH. One such example is the ultrahard and wear‐resistant radular teeth found in mollusks, which are used to scape against the rock to feed on algae. Herein, the biologically controlled structural development of the hard, outer magnetite‐containing shell of the chitin teeth is revealed. Specifically, the formation of a series of mesocrystalline iron oxide phases, templated by chitin‐binding proteins, is identified. The initial domains, consisting of ferrihydrite mesocrystals with a spherulite‐like morphology, undergo a solid‐state phase transformation to form magnetite while maintaining mesocrystallinity, likely via a shear‐induced solid‐state reaction, without any noticeable architectural changes. Subsequent growth via Ostwald ripening leads to nearly single‐crystalline rod‐like elements. In addition, an interpenetrating organic matrix is identified that, at early stages of tooth development, potentially contains iron‐binding proteins that guide the self‐assembly of the mesocrystalline mineral and influence the preferred orientation of the later‐formed magnetite nanorods, which ultimately determines the mechanical behavior of the mature chiton teeth.

Comparative transcriptional profiling of canine acanthomatous ameloblastoma and homology with human ameloblastoma

Ameloblastomas are odontogenic tumors that are rare in people but have a relatively high prevalence in dogs. Because canine acanthomatous ameloblastomas (CAA) have clinicopathologic and molecular features in common with human ameloblastomas (AM), spontaneous CAA can serve as a useful translational model of disease. However, the molecular basis of CAA and how it compares to AM are incompletely understood. In this study, we compared the global genomic expression profile of CAA with AM and evaluated its dental origin by using a bulk RNA-seq approach. For these studies, healthy gingiva and canine oral squamous cell carcinoma served as controls. We found that aberrant RAS signaling, and activation of the epithelial-to-mesenchymal transition cellular program are involved in the pathogenesis of CAA, and that CAA is enriched with genes known to be upregulated in AM including those expressed during the early stages of tooth development, suggesting a high level of molecular homology. These results support the model that domestic dogs with spontaneous CAA have potential for pre-clinical assessment of targeted therapeutic modalities against AM.

Characterization of Wolf‐Hirschhorn Syndrome Candidate 1 ( Whsc1 ) in Pitx2 ‐ miR‐23/24 ‐mediated Tooth Development

Objective Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate (CLP), hypodontia and taurodontism. However, the causative factors and underlying mechanisms of these oral anomalies are relatively unknown. Wolf-Hirschhorn syndrome candidate 1 (WHSC1) is a H3K36-specific methyltransferase that is frequently deleted in WHS. This gene has been associated with craniofacial defects including CLP and defects in occipital ossification. In our study, we aim to understand the role of WHSC1 in tooth development. Methods To characterize the role of Whsc1 in tooth development, we profiled the Whsc1 expression pattern during mouse tooth development by immunofluorescence staining (IF). To investigate the regulatory effects between Whsc1 and Pitx2, we overexpressed either Whsc1 or Pitx2 in oral epithelial cell line LS8 and detected their expression. Then, ChIP-PCR and luciferase assays were performed to confirm the binding on the promoter regions. To determine the negative regulation of Whsc1 by miR-23-3p and miR-24-3p, miRs were inhibited by our Plasmid-based microRNA inhibition system in PMIS-miR-23-27-24 embryos and LS8 cells followed by detecting the mRNA and protein level of Whsc1. Luciferase assays were also conducted to confirm the direct binding between miRs and Whsc1 3’UTR. Results Whsc1 expresses from early stage of tooth development and restricts to stem cell niches in homeostatic tooth germ. Whsc1 and Pitx2 reciprocally activate each other's expression through chromosomal and transcriptional regulation. miR-23-3p and miR-24-3p, two miRs that regulated by Pitx2, directly inhibit Whsc1. Conclusion Whsc1 expresses in the developing tooth germ and participates the Whsc1-Pitx2-miR23/24 regulatory network in oral epithelium cells. Our observations provide new insights into the potential role of Whsc1 in regulating tooth development and a possible causer of the dental defects in WHS.

The Spatiotemporal Expression Patterns of Mechanical-Stress Related Regulatory Proteins in Mouse Molar Development

In order to better understand the role of mechanical stress in early tooth development, we examined the spatiotemporal expression patterns of mechanical-stress related regulatory protein (actin filament, or F-actin), non-muscle myosin ⅡB (NMⅡB) and vinculin at different stages of tooth development in mice. Mouse first mandible molars were used as the research model. Immunofluorescence staining was performed to detect the expression patterns of F-actin, NMⅡB and Vinculin, the key molecules constituting the chemical mechanical system, at bud, cap, early bell and late bell stages of tooth. F-actin, NMⅡB and vinculin were all expressed in the tooth epithelium in an extensive or a limited way at different stages of tooth development, while F-actin was also expressed steadily in the mesenchymal cells. The quantitative analysis of fluorescence intensity showed that F-actin and NMⅡB exhibited significantly increase in the early stage of tooth development, but then dropped to their basal levels at the end of the late bell stage and the early bell stage respectively, with the differences of expression changes between successive developmental stages showing statistically significance ( P<0.05). Vinculin expression, however, remained at a relatively constant level except for the late bell stage when vinculin expression was slightly elevated compared to that of the early bell stage ( P<0.05). The findings suggest that mechanical stress is involved in early tooth development. F-actin may have an important role in dispersing and transmitting mechanical stress while NMⅡB may participate in tooth epithelial invagination and cusps formation. The findings also suggest that vinculin can respond to the mechanical stimuli and its interaction with cell adhesion molecules may play a role in tooth development. The mechanism of how actomyosin and cell adhesion interact with each other in regulating tooth development still needs further investigation.

Histology of Tooth Development in 3D Animation Video and Images - A Preliminary Report.

The process of tooth development is both, fascinating and well-described aspect of embryology. Although a big deal of the dental literature is being focused to the understanding of early stages of tooth development, still huge gap exist in our knowledge on how the dental hard tissues are formed, based on available images and descriptions. Tooth development process takes place in 3D form, inside our body. Therefore, histology should also be additionally explained with the help of additional 3D images and a video, which have not been reported so far. Methodology: Therefore, this brief article is a technical note and preliminary attempt to showcase 3D animation images and video of stages of tooth development which have been designed by the author herself using various the 3D animation softwares such as 3D max (Autodesk Media and Entertainment, San Rafael, California) and Adobe Premiere Pro 5.5 software which is a video-editing software (Adobe Systems, San Rafael, California).

In vitro tooth-shaped scaffold construction by mimicking late bell stage.

Neogenesis of osseous and ligamentous interfacial structures is essential for the regeneration of large oral or craniofacial defects. However, current treatment strategies are inadequate in renewing supporting tissues of teeth after trauma, chronic infections or surgical resection. Combined use of 3D scaffolds with stem cells became a promising treatment option for these injuries. Matching different scaffolding materials with different tissues can induce the correct cytokines and the differentiation of cells corresponding to that particular tissue. In this study, a hydroxyapatite (HA) based scaffold was used together with human adipose stem cells (hASCs), human bone marrow stem cells (hBMSCs) and gingival epithelial cells to mimic human tooth dentin-pulp-enamel tissue complexes and model an immature tooth at the late bell stage in vitro. Characteristics of the scaffold were determined via SEM, FTIR, pore size and density measurements. Changes in gene expression, protein secretions and tissue histology resulting from cross-interactions of different dental tissues grown in the system were shown. Classical tooth tissues such as cementum, pulp and bone like tissues were formed within the scaffold. Our study suggests that a HA-based scaffold with different cell lineages can successfully mimic early stages of tooth development and can be a valuable tool for hard tissue engineering.

MicroRNA-875-5p plays critical role for mesenchymal condensation in epithelial-mesenchymal interaction during tooth development

Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.

EVALUATION OF SOX2 AS A POTENTIAL STEM CELL MARKER IN BENIGN AND MALIGNANT ODONTOGENIC TUMORS

INTRODUCTION: Odontogenic tumors (OTs) constitute a wide range of lesions derived from the tooth forming apparatus and its remnants. These tumors are generally benign, yet some are aggressive and have locally invasive behavior with a high rate of recurrence and some are malignant. Tumor stem cells (TSCs) are a unique subpopulation of cells that possess the ability to initiate a neoplasm and sustain self-renewal. Epithelial stem cell (ESC) markers such as sex-determining region Y (SRY)-box 2 (SOX2) are capable of identifying these stem cells which are expressed during the early stages of tooth development. OBJECTIVES: To evaluate immunohistochemically the expression of the stem cell proliferating marker SOX2 in benign and malignant epithelial odontogenic tumors. MATERIAL AND METHODS: Immunohistochemical (IHC) study using the SOX2 antibody was done on 30 surgical specimens of benign and malignant epithelial OTs as study group and 5 normal tooth germ tissues as a control group. RESULTS: Malignant OTs biopsies showed intense immune-reactivity to SOX2, while aggressive benign OTs showed medium immunereactivity with variable intensity. Totally benign tumors were immune-negative. The IHC staining intensity was directly proportional to the state of malignancy of odontogenic tumors. The difference in the expression of SOX2 in malignant and benign OTs was statistically significant. However, the difference in the expression of SOX2 in OTs and control group was not statistically significant in mean of area percent, while it was statistically significant in mean of optical density. CONCLUSIONS: SOX2 is highly expressed in malignant OTs biopsies. Medium immune-reactivity with variable intensities is seen in aggressive benign OTs.

Ablation of Runx2 in Ameloblasts Suppresses Enamel Maturation in Tooth Development

Runt-related transcription factor 2 (Runx2) is involved in the early stage of tooth development. However, only few studies have reported the role of Runx2 in enamel development, which may be attributed to that Runx2 full knockout mice cannot survive after birth. In the present study, we successfully established a Runx2-deficient mouse model using a conditional knockout (cKO) method. We observed a significant reduction in the degree of mineralization and the decreased size of enamel rods in cKO mice. Histological analysis showed the retained enamel proteins in enamel layer at maturation stage in cKO molars. Further analysis by qRT-PCR revealed that the expressions of genes encoding enamel structure proteins, such as amelogenin (AMELX), ameloblastin (AMBN) and enamelin (ENAM), were increased in cKO enamel organs. On the other hand, the expression of kallikrein-related peptidase-4 (KLK4) at the mRNA and protein levels was dramatically decreased from late secretory stage to maturation stage in cKO enamel organs, while the expression of matrix metalloproteinase-20 (MMP-20) was not significantly altered. Finally, immunohistochemistry indicated that the uptake of amelogenins by ameloblasts was significantly decreased in cKO mice. Taken together, Runx2 played critical roles in controlling enamel maturation by increasing synthesis of KLK4 and decreasing synthesis of AMELX, AMBN and ENAM.

Cell cycle of the enamel knot during tooth morphogenesis.

Enamel knot (EK) is known to be a central organ in tooth development, especially for cusp patterning. To trace the exact position and movement among the inner dental epithelium (IDE) and EK cells, and to monitor the relationship between the EK and cusp patterning, it is essential that we understand the cell cycle status of the EK in early stages of tooth development. In this study, thymidine analogous (IdU, BrdU) staining was used to evaluate the cell cycle phase of the primary EK at the early casp stage (E13.0) and the gerbil embryo (E19) in a developing mouse embryo. The centerpiece of this study was to describe the cell cycle phasing and sequencing during proliferation in the IDE according to the expression of IdU and BrdU following their injection at calculated time points. The interval time between IdU injection and BrdU injection was set at 4h. As a result, the cell cycle in the IDE of the mouse and gerbil was found to be synchronous. Conversely, the cell cycle in primary EKs of mice was much longer than that of the IDE. Therefore, the difference of cell cycle of the IDE and the EK is related to the diversity of cusp patterning and would provide a new insight into tooth morphogenesis.

An Orthopantomographic study of hypodontia in permanent teeth of Iraqi patients

The congenital lack of one or more permanent teeth is a common anomaly in man .By definition, congenitally missing teeth are those that fail to erupt in the oral cavity and remain invisible in a radiograph, which implies this caused by disturbance during the early stages of tooth development.389 orthopantomograms of healthy patients were used the number of males were 194 and the number of females were 195.The patients has visited the hospital of college of dentistry of Baghdad university between 2006 and 2008.Thy were aged between 13 and 27years at the time the OPGs were taken .All OPGs were analyzed by viewer .The congenitally missing teeth were identify first clinically and confirm by the OPGsThe hypodontia mostly seen in upper lateral incisors, and lower second premolar, flowed by lower central incisors, upper central incisors, and lastly the lower lateral incisor, there were gender difference in hypodontia, but both genders show high percentage of upper lateral incisors hypodontia, 60.31%for males, and 72.30%for females. The hypodontia of lower second premolar in males 18.56%,and in females 14.5%.The hypodontia of upper central incisors in males 2.577%,and 0%in females. The hypodontia for lower central incisor in males 12.89%, and 13.3%in females. The hypodontia for lower lateral incisors in males 5.67%, and in females 0%.From these percentages it was clear that there were difference in hypodontia of upper and lower arches.To evaluate the frequency of hypodontia and compare its appearance between males and females and between upper and lower jaw.

Primordial odontogenic tumor: Subepithelial expression of Syndecan-1 and Ki-67 suggests origin during early odontogenesis.

Primordial odontogenic tumor (POT) is composed of variably cellular myxoid connective tissue, surrounded by cuboidal to columnar odontogenic epithelium resembling the inner epithelium of the enamel organ, which often invaginates into the underlying connective tissue. The tumor is delimited at least partially by a thin fibrous capsule. It derives from the early stages of tooth development. Syndecan-1 is a heparan sulfate proteoglycan that has a physiological role in several cellular functions, including maintenance of the epithelial architecture, cell-to-cell adhesion and interaction of cells with extracellular matrix, and with diverse growth factors, stimulating cell proliferation. Ki-67 is considered the gold standard as a cell proliferation marker. The aim of this study was to examine the expression of Syndecan-1 and Ki-67 proliferation index in POT and normal tooth germs to better understand the biological behavior of this tumor. Results showed that Syndecan-1 was more intensely expressed in subepithelial mesenchymal areas of POT, in a pattern that resembles the early stages of tooth development. The cell proliferation index (4.1%) suggests that POT is a slow growing tumor. Syndecan-1 expression in tooth germs in late cap and early bell stages was similar to POT, showing immunopositivity in subepithelial mesenchymal condensed areas. The immunohistochemical findings showed a pattern in which the population of subepithelial mesenchymal cells exhibited greater proliferative activity than the central portion of the dental papilla.

Combination of Runx2 and Cbfβ upregulates Amelotin gene expression in ameloblasts by directly interacting with cis‑enhancers during amelogenesis.

Amelotin (Amtn) is a recently identified enamel protein secreted by ameloblasts at late stage of enamel development. Runt‑related transcription factor 2 (Runx2) in combination with the coactivator core‑binding factor β (Cbfβ) regulates the early stages of tooth development. The aim of the present study was to investigate the role of Runx2 in the regulation of Amtn gene expression in ameloblasts. Immunohistochemistry was performed and the results revealed that Runx2 protein was predominantly expressed in the nuclei of ameloblasts during the transition stage and the maturation stage of enamel development, whereas Cbfβ was expressed in ameloblasts from the secretory stage to the maturation stage. Reverse transcription‑quantitative polymerase chain reaction results demonstrated that Runx2 knockdown decreased Amtn expression in ameloblast‑lineage cells and co‑expression of Runx2 and Cbfβ in ameloblast lineage cells induced an upregulation in Amtn gene expression. Two putative Runx2‑binding sites within the Amtn promoter were identified using bioinformatics analysis. Results of an electrophoretic mobility shift assay and chromatin immunoprecipitation indicated that Runx2/Cbfβ bound to specific DNA sequences. Site‑directed mutagenesis of the Runx2 binding sites within the Amtn promoter resulted in decreased basal promoter activity and did not affect the overexpressed Runx2/Cbfβ. The results of the present study suggest that Runx2 upregulates Amtn gene expression via binding directly to Runx2 sites within the Amtn promoter during amelogenesis.

Induction of Salivary Gland–Like Cells from Dental Follicle Epithelial Cells

The dental follicle (DF), most often associated with unerupted teeth, is a condensation of ectomesenchymal cells that surrounds the tooth germ in early stages of tooth development. In the present study, we aim to isolate epithelial stem-like cells from the human DF and explore their potential differentiation into salivary gland (SG) cells. We demonstrated the expression of stem cell–related genes in the epithelial components of human DF tissues, and these epithelial progenitor cells could be isolated and ex vivo expanded in a reproducible manner. The human DF-derived epithelial cells possessed clonogenic and sphere-forming capabilities, as well as expressed a panel of epithelial stem cell–related genes, thus conferring stem cell properties (hDF-EpiSCs). When cultured under in vitro 3-dimensional induction conditions, hDF-EpiSCs were capable to differentiate into SG acinar and duct cells. Furthermore, transplantation of hDF-EpiSC–loaded native de-cellularized rat parotid gland scaffolds into the renal capsule of nude mice led to the differentiation of transplanted hDF-EpiSCs into salivary gland–like cells. These findings suggest that hDF-EpiSCs might be a promising source of epithelial stem cells for the development of stem cell–based therapy or bioengineering SG tissues to repair/regenerate SG dysfunction.

FHL2 mediates tooth development and human dental pulp cell differentiation into odontoblasts, partially by interacting with Runx2.

The differentiation of mesenchymal cells in tooth germ and dental pulp cells into odontoblasts is crucial for dentin formation, and the transcription factor runt-related transcription factor (Runx2) is necessary for odontoblast differentiation. Our previous study demonstrated that four and a half LIM domains 2 (FHL2) may play an important role in tooth development and human dental pulp cell differentiation. This study aimed to determine whether FHL2 mediated the mesenchymal cells in tooth development and human dental pulp cell differentiation into odontoblasts by interacting with Runx2. The expression patterns of FHL2 and Runx2 were examined at the early stages of mouse molar development using double immunofluorescence staining. Western blot analysis and co-immunoprecipitation (Co-IP) were conducted for the preliminary study of the relationship between FHL2 and Runx2 in human dental pulp cell differentiation into odontoblasts. Results of double immunofluorescence staining showed that FHL2 and Runx2 exhibited similar expression patterns at the early stages of tooth development. Western blot analysis indicated that the expression patterns of FHL2 and Runx2 were synchronized on day 7 of induction, whereas those on day 14 differed. Co-IP analysis revealed positive bands of protein complexes, revealing the interaction of FHL2 and Runx2 on days 0, 7 and 14 of induction. Our data suggested that FHL2 might interact with Runx2 to mediate mesenchymal cell differentiation at the early stages of tooth development and human dental pulp cell differentiation.

OCT4 and SOX2 are reliable markers in detecting stem cells in odontogenic lesions

Context (Background): Stem cells are a unique subpopulation of cells in the human body with a capacity to initiate differentiation into various cell lines. Tumor stem cells (TSCs) are a unique subpopulation of cells that possess the ability to initiate a neoplasm and sustain self-renewal. Epithelial stem cell (ESC) markers such as octamer-binding transcription factor 4 (OCT4) and sex-determining region Y (SRY)-box 2 (SOX2) are capable of identifying these stem cells expressed during the early stages of tooth development. Aims: To detect the expression of the stem cell markers OCT4 and SOX2 in the normal odontogenic tissues and the odontogenic cysts and tumors. Materials and Methods: Paraffin sections of follicular tissue, radicular cyst, dentigerous cyst, odontogenic keratocyst, ameloblastoma, adenomatoid odontogenic tumor, and ameloblastic carcinoma were obtained from the archives. The sections were subjected to immunohistochemical assay by the use of mouse monoclonal antibodies to OCT4 and SOX2. Statistical Analysis: The results were evaluated by descriptive analysis. Results: The results show the presence of stem cells in the normal and lesional tissues with these stem cell identifying markers. SOX2 was found to be more consistent and reliable in the detection of stem cells. Conclusion: The stem cell expressions are maintained in the tumor transformation of tissue and probably suggest that there is no phenotypic change of stem cells in progression from normal embryonic state to its tumor component. The quantification and localization reveals interesting trends that indicate the probable role of the cells in the pathogenesis of the lesions.

Twist1 Is Essential for Tooth Morphogenesis and Odontoblast Differentiation

Twist1 is a basic helix-loop-helix-containing transcription factor that is expressed in the dental mesenchyme during the early stages of tooth development. To better delineate its roles in tooth development, we generated Twist1 conditional knockout embryos (Twist2(Cre) (/+);Twist1(fl/fl)) by breeding Twist1 floxed mice (Twist1(fl/fl)) with Twist2-Cre recombinase knockin mice (Twist2(Cre) (/+)). The Twist2(Cre) (/+);Twist1(fl/fl) embryos formed smaller tooth germs and abnormal cusps during early tooth morphogenesis. Molecular and histological analyses showed that the developing molars of the Twist2(Cre) (/+);Twist1(fl/fl) embryos had reduced cell proliferation and expression of fibroblast growth factors 3, 4, 9, and 10 and FGF receptors 1 and 2 in the dental epithelium and mesenchyme. In addition, 3-week-old renal capsular transplants of embryonic day 18.5 Twist2(Cre) (/+);Twist1(fl/fl) molars showed malformed crowns and cusps with defective crown dentin and enamel. Immunohistochemical analyses revealed that the implanted mutant molars had defects in odontoblast differentiation and delayed ameloblast differentiation. Furthermore, in vitro ChIP assays demonstrated that Twist1 was able to bind to a specific region of the Fgf10 promoter. In conclusion, our findings suggest that Twist1 plays crucial roles in regulating tooth development and that it may exert its functions through the FGF signaling pathway.