Early dental epithelial transcription factors distinguish ameloblastoma from keratocystic odontogenic tumor.

The aim of the study was to characterize the molecular relationship between ameloblastoma and keratocystic odontogenic tumor (KCOT) by means of a genome-wide expression analysis. Total RNA from 27 fresh tumor samples of 15 solid/multicystic intraosseous ameloblastomas and 12 sporadic KCOTs was hybridized on Affymetrix whole genome arrays. Hierarchical clustering separated ameloblastomas and KCOTs into 2 distinct groups. The gene set enrichment analysis based on 303 dental genes showed a similar separation of ameloblastomas and KCOTs. Early dental epithelial markers PITX2, MSX2, DLX2, RUNX1, and ISL1 were differentially overexpressed in ameloblastoma, indicating its dental identity. Also, PTHLH, a hormone involved in tooth eruption and invasive growth, was one of the most differentially upregulated genes in ameloblastoma. The most differentially overexpressed genes in KCOT were squamous epithelial differentiation markers SPRR1A, KRTDAP, and KRT4, as well as DSG1, a component of desmosomal cell-cell junctions. Additonally, the epithelial stem cell marker SOX2 was significantly upregulated in KCOT when compared with ameloblastoma. Taken together, the gene expression profile of ameloblastoma reflects differentiation from dental lamina toward the cap/bell stage of tooth development, as indicated by dental epithelium-specific transcription factors. In contrast, gene expression of KCOT indicates differentiation toward keratinocytes.

Current understanding of the process of tooth formation: transfer from the laboratory to the clinic.

Teeth are typical examples of organs in which genes determine the progress of development from initiation to the final shape, size and structure, whereas environmental factors play a minor role. Advances in gene technology over the last three decades have led to powerful novel methods to explore the mechanisms of embryonic development. Today we know a few hundred genes that regulate tooth development, and mutations in dozens of these genes have been shown to cause aberrations in tooth development in mice and/or humans. The functions of an increasing number of genes in tooth development have been discovered using genetically modified mouse models. We are now beginning to understand the 'programme' underlying the process of tooth formation. Key components of the programme are signals mediating communication between cells and complex gene regulatory networks in which the signal pathways are integrated. Understanding the mechanisms of tooth development at the level of genes, cells and molecules will lay the basis for new ways to prevent and treat dental defects and diseases. Over the last decade knowledge about dental stem cells has accumulated rapidly and novel stem cell technologies have been developed. Combining stem cell research with knowledge on the mechanisms of tooth development may open up novel possibilities for clinical tooth regeneration.

Novel Golgi protein, GoPro49, is a specific dental follicle marker.

UNLABELLED: GoPro49 is a recently identified, novel Golgi protein that is expressed in embryonic mesenchymal tissues, including dental follicle. In the present study, we have tested the hypothesis that the gene is a specific marker for the dental follicle, and examined its expression during the development of mouse incisors and molars. In situ hybridization showed that GoPro49 is expressed in dental follicles from bud to post-eruption stages. The expression is intense throughout the dental follicle during crown development, and persists in the root follicle during root development. In the forming periodontal ligament, GoPro49 expression is down-regulated upon differentiation of the follicle cells to cementoblasts and osteoblasts marked by Bsp1. In cultured dental follicle cells, the GoPro49 protein co-localizes with beta-COP, suggesting that GoPro49 may function in the secretory pathway. We conclude that GoPro49 is a novel, specific marker for the dental follicle and can be used to identify this tissue. ABBREVIATIONS: Bsp1, bone sialoprotein 1; GoPro49, Golgi protein 49 kDa; E16, embryonic day 16; HERS, Hertwig's epithelial root sheath; PDL, periodontal ligament; dpn, day post-natal.

Modulation of epithelial cell fate of the root in vitro.

Mouse molars are normally not capable of continuous growth. We hypothesized that the mouse molar has intrinsic potential to maintain the epithelial stem cell niche and assessed this potential by growth in vitro. Although the tooth germs flattened considerably, they developed a mineralized crown and a root. However, histologically, the root surface was composed of 3 structurally different regions affecting the fate of the dental epithelium. The anterior and posterior aspects maintained the morphological and molecular characteristics of the cervical loop of a continuously growing incisor, with a continuous layer of ameloblasts. The epithelium making contact with the supporting filter resembled Hertwig's epithelial root sheath. The top of the cultured molar exposed to air lacked epithelium altogether. We conclude that the fate of the epithelium is regulated by external cues influenced by culture conditions, and that the molar has the intrinsic capacity to grow continuously.

Activation of the Notch signaling pathway in response to pulp capping of rat molars.

Notch signaling is an evolutionarily conserved pathway that controls the developmental choices made by individual cells. Cells communicate via Notch receptors and their ligands, which direct decisions on the fate of stem cells according to the states of their neighbors. In this study we explored Notch signaling after the pulp capping of adult first upper rat molars. The wound was capped with calcium hydroxide. In situ hybridization revealed an increased expression of Notch signaling genes on day 1, which showed a tendency to decrease on day 3. Notch1 increased in the subodontoblast zone and close to the lesion limited to a few cells. Notch2 increased in pulp stroma surrounded by coronal odontoblasts. Notch1 and, especially, Notch3 expression increased, corresponding to perivascular cell groups. A low increase of ligand expression was observed near the injury with Delta1 expression along the dentin wall and Jagged1 in the stroma. Expression of the downstream target, Hes1, was observed along the lesion and adjacent dentin walls. Hes5 expression was not observed. The results indicate that Notch signaling is activated in response to injury and associated with the differentiation of pulp cells into perivascular cells and odontoblasts. The findings are consistent with the concept that the Notch pathway controls stem cell fate during pulp regeneration.

Runx2 (Cbfa1) inhibits Shh signaling in the lower but not upper molars of mouse embryos and prevents the budding of putative successional teeth.

Heterozygous mutations in the RUNX2 (CBFA1) gene cause cleidocranial dysplasia, characterized by multiple supernumerary teeth. This suggests that Runx2 inhibits successional tooth formation. However, in Runx2 knockout mice, molar development arrests at the late bud stage, and lower molars are more severely affected than upper ones. We have proposed that compensation by Runx3 may be involved. We compared the molar phenotypes of Runx2/Runx3 double-knockouts with those of Runx2 knockouts, but found no indication of such compensation. Shh and its mediators Ptc1, Ptc2, and Gli1 were down-regulated only in the lower but not the upper molars of Runx2 and Runx2/Runx3 knockouts. Interestingly, in front of the mutant upper molar, a prominent epithelial bud protruded lingually with active Shh signaling. Similar buds were also present in Runx2 heterozygotes, and they may represent the extension of dental lamina for successional teeth. The results suggest that Runx2 prevents the formation of Shh-expressing buds for successional teeth.

Expression of aquaporin isoforms during human and mouse tooth development.

Previously, we described the development of hyaluronan (HA) deposition in human tooth germ tissues that are consistent with water transport in different stages of tooth development. The aquaporins (AQP) constitute a family of membrane water channels that are expressed in many organs. However, there are no data available about the expression pattern of aquaporin water channels in dental structures. In the present study we have characterised the expression of six different aquaporin isoforms (AQP1-5, AQP-9) in developing human and mouse tooth germs by immunohistochemistry using isoform specific antibodies. In the "bell stage" AQP1 was expressed in endothelial cells of small vessels whereas no other structures of the tooth primordial were labeled. AQP2, AQP3 and AQP9 immunoreactivity was not observed in tooth germs, whereas strong AQP4 and AQP5 expression was observed in dental lamina, inner enamel epithelium, stratum intermedium, stellate reticulum and the outer enamel epithelium. Oral epithelium also exhibited AQP4 and AQP5 immunolabeling. During development of the matrices of the dental hard tissues AQP4 and AQP5 immunostaining was observed in the odontoblasts and their processes, as well as in the secretory ameloblast and their apical processes. Immunolabeling controls were negative. In conclusion, AQP4 and AQP5 are expressed in tooth germ tissues in early development in cells that previously have been shown to express HA and/or CD44, indicating that AQP water channels may play a role for ECM hydration during tooth development.

MSX1 gene is deleted in Wolf-Hirschhorn syndrome patients with oligodontia.

Abnormalities of the short arm of chromosome 4 cause multiple congenital malformations, including craniofacial, oral, and dental manifestations. A candidate gene for oral defects in this region is MSX1, which is mandatory for normal oral and tooth development. We examined the dentition and the presence of MSX1 in eight Finnish patients with abnormalities of 4p, including seven cases of Wolf-Hirschhorn syndrome. Five of the Wolf-Hirschhorn syndrome patients presented with agenesis of several teeth, suggesting that oligodontia may be a common (even though previously not well-documented) feature in Wolf-Hirschhorn syndrome. In fluorescence in situ hybridization (FISH) analysis, the five patients with oligodontia lacked one copy of MSX1, while the other three had two hybridization signals. One of these presented with the only case of cleft palate among the patients. Our result confirms that haploinsufficiency for MSX1 serves as a mechanism that causes selective tooth agenesis but, alone, is not enough to cause oral clefts.

Fgfr mRNA isoforms in craniofacial bone development.

Mutations in genes encoding for fibroblast growth factor receptors (FGFRs) have been identified as causes of both chondrodysplasias and craniosynostoses, both of which cause abnormalities in the growth and development of the craniofacial region. FGFRs form mRNA splicing isoforms, each with distinct ligand binding specificity and tissue distribution. These confer specific biological functions on these isoforms. Although it is known that FGFRs are expressed at numerous locations during early mouse development, including the craniofacial area, relatively little is known about the expression of the splicing isoforms during craniofacial bone development. To address this, we have performed a detailed survey to detect these genes in the developing mouse craniofacial region. We have analyzed the developing mouse mandible, calvaria, and cranial base, in particular the spheno-occipital synchondrosis, a key centre of craniofacial growth. Fgfr1c was detected weakly in osteoblastic cells in both the developing calvarial and mandibular bones. Fgfr3b and Fgfr3c were found chiefly in proliferating chondrocytes of the cranial base synchondroses and the mandibular condyle. Fgfr2b transcripts were most notably detected in the perichondria of the mandibular condyle and the cranial base. Fgfr2c transcripts were detected with high intensity in differentiating osteoblasts at the sutural osteogenic fronts of the calvarial bones. In addition, Fgfr2c was also expressed in the perichondria of the mandibular condyle and the cranial base. These expression patterns suggest both differing and similar functions for -b and -c isoforms. The former is exemplified by Fgfr1 transcripts, which show distinct differences in their distribution, being mutually exclusive. Similar functions are suggested by the overlapping expression patterns of the -b and -c isoforms of both Fgfr2 and Fgfr3. Fgfr4 transcripts were found in developing muscles. These data help to explain the disturbances in craniofacial growth exhibited by both patients and the growing number of transgenic mice carrying mutations in genes encoding FGFRs/Fgfrs.

Expression of bone morphogenetic proteins and Msx genes during root formation.

Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.

Signaling and subcellular localization of the TNF receptor Edar.

Tabby and downless mutant mice have identical phenotypes characterized by deficient development of several ectodermally derived organs such as teeth, hair, and sweat glands. Edar, encoded by the mouse downless gene and defective in human dominant and recessive forms of autosomal hypohidrotic ectodermal dysplasia (EDA) syndrome, is a new member of the tumor necrosis factor (TNF) receptor superfamily. The ligand of Edar is ectodysplasin, a TNF-like molecule mutated in the X-linked form of EDA and in the spontaneous mouse mutant Tabby. We have analyzed the response of Edar signaling in transfected cells and show that it activates nuclear factor-kappaB (NF-kappaB) in a dose-dependent manner. When Edar was expressed at low levels, the NF-kappaB response was enhanced by coexpression of ectodysplasin. The activation of NF-kappaB was greatly reduced in cells expressing mutant forms of Edar associated with the downless phenotype. Overexpression of Edar did not activate SAPK/JNK nor p38 kinase. Even though Edar harbors a death domain its overexpression did not induce apoptosis in any of the four cell lines analyzed, nor was there any difference in apoptosis in developing teeth of wild-type and Tabby mice. Additionally, we show that the subcellular localization of dominant negative alleles of downless is dramatically different from that of recessive or wild-type alleles. This together with differences in NF-kappaB responses suggests an explanation for the different mode of inheritance of the different downless alleles.

Characteristics of incisor-premolar hypodontia in families.

Nonsyndromic tooth agenesis is a genetically and phenotypically heterogenous condition. It is generally assumed that different phenotypic forms are caused by different mutated genes. We analyzed inheritance and phenotype of hypodontia and dental anomalies in 214 family members in three generations of 11 probands collected for genetic linkage study on incisor-premolar hypodontia (IPH). Our analysis confirms the autosomal-dominant transmission with reduced penetrance of IPH. The prevalence of hypodontia and/or peg-shaped teeth was over 40% in first- and second-degree relatives and 18% in first cousins of the probands. Four of nine noted obligate carriers of hypodontia gene had dental anomalies, including small upper lateral incisors, ectopic canines, taurodontism, and rotated premolars. These anomalies were also observed at higher than normal frequency in relatives affected with hypodontia. We conclude that incisor-premolar hypodontia is a genetic condition with autosomal-dominant transmission and that it is associated with several other dental abnormalities.

Tenascin-C in developing mouse teeth: expression of splice variants and stimulation by TGFbeta and FGF.

Tenascin-C is a protein of the extracellular matrix which has been suggested to regulate organogenesis. We have analysed the expression of tenascin-C mRNA during mouse tooth development. We show that it is transiently expressed during epithelial budding in the condensed dental mesenchyme, and that it reappears later in the dental papilla mesenchyme where it persists in the dental pulp but is downregulated in odontoblasts. Probes corresponding to the domains A4, B, and D of the differentially spliced and domain 7 of the constant region of the FNIII-like domain show similar patterns of hybridization. Dental epithelium has been shown to induce tenascin-C in early dental mesenchyme, and we show that growth factors in the transforming growth factor beta (TGFbeta) and fibroblast growth factor (FGF) families can mimic this effect. FGF-4, -8 and TGFbeta-1 proteins were applied locally by beads on dissected dental mesenchyme, and tenascin-C expression was analysed after 24 h culture by reverse transcriptase-polymerase chain reaction (RT-PCR) in situ hybridization, and immunohistochemistry. FGF-4 and TGFbeta-1 stimulated tenascin-C expression in E12 dental mesenchymes. RT-PCR showed induction of several tenascin-C isoforms by both TGFbeta-1 and FGFs. We conclude that several splice forms are expressed during mouse tooth development, and that TGFbeta- and FGF-family growth factors may act as epithelial signals inducing tenascin expression in the dental mesenchyme.

TNF signaling via the ligand-receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis.

Ectodermal dysplasia syndromes affect the development of several organs, including hair, teeth, and glands. The recent cloning of two genes responsible for these syndromes has led to the identification of a novel TNF family ligand, ectodysplasin, and TNF receptor, edar. This has indicated a developmental regulatory role for TNFs for the first time. Our in situ hybridization analysis of the expression of ectodysplasin (encoded by the Tabby gene) and edar (encoded by the downless gene) during mouse tooth morphogenesis showed that they are expressed in complementary patterns exclusively in ectodermal tissue layer. Edar was expressed reiteratively in signaling centers regulating key steps in morphogenesis. The analysis of the effects of eight signaling molecules in the TGFbeta, FGF, Hh, Wnt, and EGF families in tooth explant cultures revealed that the expression of edar was induced by activinbetaA, whereas Wnt6 induced ectodysplasin expression. Moreover, ectodysplasin expression was downregulated in branchial arch epithelium and in tooth germs of Lef1 mutant mice, suggesting that signaling by ectodysplasin is regulated by LEF-1-mediated Wnt signals. The analysis of the signaling centers in tooth germs of Tabby mice (ectodysplasin null mutants) indicated that in the absence of ectodysplasin the signaling centers were small. However, no downstream targets of ectodysplasin signaling were identified among several genes expressed in the signaling centers. We conclude that ectodysplasin functions as a planar signal between ectodermal compartments and regulates the function, but not the induction, of epithelial signaling centers. This TNF signaling is tightly associated with epithelial-mesenchymal interactions and with other signaling pathways regulating organogenesis. We suggest that activin signaling from mesenchyme induces the expression of the TNF receptor edar in the epithelial signaling centers, thus making them responsive to Wnt-induced ectodysplasin from the nearby ectoderm. This is the first demonstration of integration of the Wnt, activin, and TNF signaling pathways.

Identification of a nonsense mutation in the PAX9 gene in molar oligodontia.

Development of dentition is controlled by numerous genes, as has been shown by experimental animal studies and mutations that have been identified by genetic studies in man. Here we report a nonsense mutation in the PAX9 gene that is associated with molar tooth agenesis in a Finnish family. The A340T transversion creates a stop codon at lysine 114, and truncates the coded PAX9 protein at the end of the DNA-binding paired-box. All the affected members of the family were heterozygous for the mutation. The tooth agenesis phenotype involves all permanent second and third molars and most of the first molars and resembles the earlier reported phenotype that was also associated with a PAX9 mutation. The phenotype is presumably a consequence of haploinsufficiency of PAX9. In another Finnish family with molar tooth agenesis, we could not find similar sequence changes in PAX9.

Enamel knots as signaling centers linking tooth morphogenesis and odontoblast differentiation.

Odontoblasts differentiate from the cells of the dental papilla, and it has been well-established that their differentiation in developing teeth is induced by the dental epithelium. In experimental studies, no other mesenchymal cells have been shown to have the capacity to differentiate into odontoblasts, indicating that the dental papilla cells have been committed to odontoblast cell lineage during earlier developmental stages. We propose that the advancing differentiation within the odontoblast cell lineage is regulated by sequential epithelial signals. The first epithelial signals from the early oral ectoderm induce the odontogenic potential in the cranial neural crest cells. The next step in the determination of the odontogenic cell lineage is the development of the dental papilla from odontogenic mesenchyme. The formation of the dental papilla starts at the onset of the transition from the bud to the cap stage of tooth morphogenesis, and this is regulated by epithelial signals from the primary enamel knot. The primary enamel knot is a signaling center which forms at the tip of the epithelial tooth bud. It becomes fully developed and morphologically discernible in the cap-stage dental epithelium and expresses at least ten different signaling molecules belonging to the BMP, FGF, Hh, and Wnt families. In molar teeth, secondary enamel knots appear in the enamel epithelium at the sites of the future cusps. They also express several signaling molecules, and their formation precedes the folding and growth of the epithelium. The differentiation of odontoblasts always starts from the tips of the cusps, and therefore, it is conceivable that some of the signals expressed in the enamel knots may act as inducers of odontoblast differentiation. The functions of the different signals in enamel knots are not precisely known. We have shown that FGFs stimulate the proliferation of mesenchymal as well as epithelial cells, and they may also regulate the growth of the cusps. We have proposed that the enamel knot signals also have important roles, together with mesenchymal signals, in regulating the patterning of the cusps and hence the shape of the tooth crown. We suggest that the enamel knots are central regulators of tooth development, since they link cell differentiation to morphogenesis.