Induction of human keratinocytes into enamel-secreting ameloblasts

Mammalian tooth development relies heavily on the reciprocal and sequential interactions between cranial neural crest-derived mesenchymal cells and stomadial epithelium. During mouse tooth development, odontogenic potential, that is, the capability to direct an adjacent tissue to form a tooth, resides in dental epithelium initially, and shifts subsequently to dental mesenchyme. Recent studies have shown that mouse embryonic dental epithelium possessing odontogenic potential is able to induce the formation of a bioengineered tooth crown when confronted with postnatal mesenchymal stem cells of various sources. Despite many attempts, however, postnatal stem cells have not been used successfully as the epithelial component in the generation of a bioengineered tooth. We show here that epithelial sheets of cultured human keratinocytes, when recombined with mouse embryonic dental mesenchyme, are able to support tooth formation. Most significantly, human keratinocytes, recombined with mouse embryonic dental mesenchyme in the presence of exogenous FGF8, are induced to express the dental epithelial marker PITX2 and differentiate into enamel-secreting ameloblasts that develop a human-mouse chimeric whole tooth crown. We conclude that in the presence of appropriate odontogenic signals, human keratinocytes can be induced to become odontogenic competent; and that these are capable of participating in tooth crown morphogenesis and differentiating into ameloblasts. Our studies identify human keratinocytes as a potential cell source for in vitro generation of bioengineered teeth that may be used in replacement therapy.     

Sonic hedgehog signalling inhibits palatogenesis and arrests tooth development in a mouse model of the nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant or spontaneous disorder characterized by multiple cutaneous basal cell carcinomas, odontogenic keratocysts, skeletal anomalies and facial dysmorphology, including cleft lip and palate. Causative mutations for NBCCS occur in the PTCH1 gene on chromosome 9q22.3-q31, which encodes the principle receptor for the Hedgehog signalling pathway. We have investigated the molecular basis of craniofacial defects seen in NBCCS using a transgenic mouse model expressing Shh in basal epithelium under a Keratin-14 promoter. These mice have an absence of flat bones within the skull vault, hypertelorism, open-bite malocclusion, cleft palate and arrested tooth development. Significantly, increased Hedgehog signal transduction in these mice can influence cell fate within the craniofacial region. In medial edge epithelium of the palate, Shh activity prevents apoptosis and subsequent palatal shelf fusion. In contrast, high levels of Shh in odontogenic epithelium arrests tooth development at the bud stage, secondary to a lack of cell proliferation in this region. These findings illustrate the importance of appropriately regulated Hedgehog signalling during early craniofacial development and demonstrate that oro-facial clefting and hypodontia seen in NBCCS can occur as a direct consequence of increased Shh signal activity within embryonic epithelial tissues.     

Sonic hedgehog expression during early tooth development in Suncus murinus

Tooth development is a highly organized process characterized by reciprocal interactions between epithelium and mesenchyme. However, the expression patterns and functions of molecules involved in mouse tooth development are unclear from the viewpoint of explaining human dental malformations and anomalies. Here, we show the expression of sonic hedgehog (Shh), a potent initiator of morphogenesis, during the early stages of tooth development in Suncus murinus. Initially, symmetrical, elongated expression of suncus Shh (sShh) was observed in the thin layer of dental epithelial cells along the mesial-distal axis of both jaws. As the dental epithelium continued to develop, sShh was strictly restricted to the predicted leading parts of the growing, invaginating epithelium corresponding to tooth primordia and enamel knots. We propose that some aspects of Shh function in tooth development are widely conserved in mammalian phylogeny.     

Waking-up the sleeping beauty: recovery of the ancestral bird odontogenic program

Recent advances in molecular and developmental genetics have provided tools for understanding evolutionary changes in the nature of the epithelial-mesenchymal interactions regulating the patterned outgrowth of the tooth primordia. Tissue recombination experiments in mice have identified the oral epithelium as providing the instructive information for the initiation of tooth development. Teeth were lost in birds for more than 80 million years ago, but despite their disappearance, a number of gene products and the requisite tissue interactions needed for tooth formation are found in the avian oral region. It is believed that the avian ectomesenchyme has lost the odontogenic capacity, whilst the oral epithelium retains the molecular signaling required to induce odontogenesis. In order to investigate the odontogenic capacity of the neural crest-derived mesenchyme and its potential activation of the avian oral epithelium, we have realized mouse neural tube transplantations to chick embryos to replace the neural crest cells of chick with those of mouse. Teeth are formed in the mouse/chick chimeras, indicating that timing is critical for the acquisition of the odontogenic potential by the epithelium and, furthermore, suggesting that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.     

Vertebrate dentitions at the origin of jaws: when and how pattern evolved

New evidence shows that teeth evolved with a greater degree of independence from jaws than previously considered. Pharyngeal denticles occur in jawless fish and also in early gnathostomes and precede jaw teeth in phylogeny. Many of these denticles form joined polarized sets on each branchial arch; these resemble whorl-shaped tooth sets on the jaws of stem and crown gnathostomes and are proposed as homologous units. Therefore, the source of patterning of these pharyngeal denticle and tooth sets is conserved from jawless conditions. It is proposed that developmental regulatory systems, responsible for all such tooth patterns on the jaws, are co-opted from the pharyngeal region and not from the skin as classically understood. This strongly implicates embryonic endoderm as opposed to ectoderm in the genetic control of dentition patterning. New interpretations of ontogenetic data on patterning dentitions of extant sharks are proposed, together with those of osteichthyan fish. Two entirely fossil groups, placoderms and acanthodians, at the base of gnathostome phylogeny are reassessed on the basis of a new model. It is concluded that within stem group and crown group gnathostomes several different strategies, unique to each taxon, were adopted to produce different developmental models of dentition patterning from pharyngeal denticles. One shared developmental pattern is that of initiation from primordial tooth sites, independently in each dentate zone of the jaws. The new model is proposed as a framework for data on evolutionary developmental genetics.     

Growth factors and tooth development

The effects of various growth factors on tooth development were studied in organ cultures of mouse embryonic tooth germs. Transferrin was shown to be a necessary growth factor for early tooth morphogenesis. Transferrin was required for the development of bud- and early cap-staged teeth, and it was shown to be the only serum protein that was needed by early cap-staged teeth in organ culture. Promotion of tooth morphogenesis and dental cell differentiation was shown to be based on the stimulation of cell proliferation. The roles of polypeptide growth factors in tooth development were studied by adding these factors to the transferrin-containing chemically-defined culture medium which supports early tooth morphogenesis and cell differentiation. Fibroblast growth factor or platelet-derived growth factor did not affect cell proliferation or morphogenesis of tooth germs in culture. On the contrary, epidermal growth factor (EGF) stimulated cell proliferation in tooth explants, but at the same time inhibited tooth morphogenesis and dental cell differentiation. Autoradiographic localization of proliferating cells revealed that dental tissues responded to EGF with different proliferation rates. The responsiveness to EGF was stage-dependent, early cap-staged teeth were sensitive to EGF but late cap-staged and bell-staged teeth developed normally in the presence of EGF in the culture medium. The presence and distribution of receptors for both transferrin and EGF were studied in mouse embryonic teeth at various developmental stages by incubating freshly-separated tooth germs with 125Iodine-labeled transferrin or EGF, and then processing the tissues for autoradiography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular expression of neurotrophin mRNAs during tooth development

. Target-derived neurotrophins support and sustain peripheral sensory neurons during development. In addition, it has been suggested that these growth factors could have developmental functions in non-neuronal tissues. To further elucidate the possible roles of neurotrophins in tooth morphogenesis and innervation, we have used in-situ hybridization to determine the specific sites of neurotrophin gene activity in pre- and postnatal rat jaws from E16 to P7. All four neurotrophins were expressed during tooth development with specific temporospatial patterns. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs were mainly detected in the dental papilla/pulp in postnatal animals, and the pattern of expression correlated with the onset of dental innervation. In contrast, neurotrophin 3 (NT3) and neurotrophin 4 (NT4) mRNA expression patterns were predominantly epithelial and were strongest during early developmental stages when teeth are not yet innervated. Dental papilla NGF-mRNA expression was first seen in both epithelium and mesenchyme and later shifted to the odontoblast layer and the subodontoblast zone. BDNF-mRNA labeling was present in low levels in the early dental organ, but increased in the pulp and in the odontoblast cell layer of the developing teeth at later developmental stages. Both NT3 and NT4 mRNA were observed in the prenatal oral epithelium and the inner dental epithelium. NT3-mRNA labeling was seen mainly in the cervical loop region, fissure system depressions and cuspal tops, while NT4 mRNA was more evenly distributed in the dental epithelium. At P7, NT3-mRNA labeling was below detection level and NT4 mRNA expression was lower than at prior stages. Complementary to reports on the presence of low-affinity neurotrophin receptor (LANR), trkB and trkC mRNA in the developing teeth, our results suggest that neurotrophins may have multiple functions during tooth morphogenesis. Neurotrophins might participate in epithelial-mesenchymal interactions in early tooth morphogenetic events such as proliferation and differentiation of epithelial and mesenchymal cells. In addition, based on mRNA localization in postnatal animals, we also suggest that NGF and BDNF (beside glial cell line-derived neurotrophic factor) might participate in establishing and maintaining the innervation of the teeth, thus acting as classical neurotrophic factors.     

The role of effectors of the activin signalling pathway, activin receptors IIA and IIB, and Smad2, in patterning of tooth development.

The gene for activin betaA is expressed in the early odontogenic mesenchyme of all murine teeth but mutant mice show a patterning defect where incisors and mandibular molars fail to develop but maxillary molars develop normally. In order to understand why maxillary molar tooth development can proceed in the absence of activin, we have explored the role of mediators of activin signalling in tooth development. Analysis of tooth development in activin receptor II and Smad2 mutants shows that a similar tooth phenotype to activin betaA mutants can be observed. In addition, we identify a novel downstream target of activin signalling, the Iroquois-related homeobox gene, Irx1, and show that its expression in activin betaA mutant embryos is lost in all tooth germs, including the maxillary molars. These results strongly suggest that other transforming growth factor beta molecules are not stimulating the activin signalling pathway in the absence of activin. This was confirmed by a non-genetic approach using exogenous soluble receptors to inhibit all activin signalling in tooth development, which reproduced the genetic phenotypes. Activin, thus, has an essential role in early development of incisor and mandibular molar teeth but this pathway is not required for development of maxillary molars.     

BMP9-initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/β-catenin signalling activity

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/β-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down β-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down β-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.     

Levels and patterns of 125I-labeled transferrin binding in mouse embryonic teeth and kidneys at various developmental stages

The iron-transporting serum glycoprotein, transferrin, is necessary for the cell proliferation, morphogenesis, and differentiation of mouse embryonic teeth and kidneys in organ culture. The stimulatory effect of transferrin is mediated by the binding of transferrin to its specific cell-surface receptor and by receptor-mediated endocytosis. Since, in both teeth and kidneys, the requirement for and responsiveness to transferrin depend on the developmental stage of the organ, we studied the binding of transferrin at various stages of tooth and kidney development by incubating tissues with 125I-labeled transferrin. The amount of bound transferrin was determined by measuring the tissue-incorporated radioactivity, and the binding sites were localized by autoradiography. During tooth development in vitro, the requirement for exogenous transferrin is lost as the teeth proceed from the early cap stage to the bell stage. The level of transferrin binding was found to decrease simultaneously, and in bell-stage teeth, the transferrin receptors were concentrated in the areas of most active cell proliferation. In kidneys, the number of transferrin receptors was highest at the stage during which the undifferentiated kidney mesenchyme becomes responsive to transferrin. These receptors were located in both the ureter epithelium and the metanephric mesenchyme, and they dramatically decreased in number with advancing kidney differentiation. The results of the present study indicate that, during the embryonic development of teeth and kidneys, the amount and localization of transferrin binding are correlated with cell proliferation. The number of transferrin receptors is highest during the developmental stages when cell proliferation is most active, and decreases with advancing differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observations on the dental anatomy of piranhas (Characidae) with special reference to tooth structure

A study of the tissues of the teeth and jaws in piranhas, using the scanning electron microscope and various techniques of light microscopy, revealed many dental adaptations related to the specialized feeding habits of these carnivorous fishes. The dentition is primarily sectorial, although some anterior teeth may be used in grasping. The scissor-like rows of teeth are maintained by the specialized pattern of tooth replacement. The bones of the jaws and the tooth attachment support the teeth very firmly. In its structural organization, the enameloid covering the teeth closely resembles that on the sectorial teeth of sharks and is adapted to the probable stress patterns set up in biting.     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

Dental histology of Coelophysis bauri and the evolution of tooth attachment tissues in early dinosaurs

Studies of dinosaur teeth have focused primarily on external crown morphology and thus, use shed or in situ tooth crowns, and are limited to the enamel and dentine dental tissues. As a result, the full suites of periodontal tissues that attach teeth to the jaws remain poorly documented, particularly in early dinosaurs. These tissues are an integral part of the tooth and thus essential to a more complete understanding of dental anatomy, development, and evolution in dinosaurs. To identify the tooth attachment tissues in early dinosaurs, histological thin sections were prepared from the maxilla and dentary of a partial skull of the early theropod Coelophysis bauri from the Upper Triassic (Rhaetian‐ 209–201 Ma) Whitaker Quarry, New Mexico, USA. As one of the phylogenetically and geologically oldest dinosaurs, it is an ideal candidate for examining dental tissues near the base of the dinosaurian clade. The teeth of C. bauri exhibited a fibrous tooth attachment in which the teeth possessed five tissues: enamel, dentine, cementum, periodontal ligament (PDL), and alveolar bone. Our findings, coupled with those of more recent studies of ornithischian teeth, indicate that a tripartite periodontium, similar to that of crocodilians and mammals, is the plesiomorphic condition for dinosaurs. The occurrence of a tripartite periodontium in dinosaurs adds to the growing consensus that the presence of these tissues is the plesiomorphic condition for the major amniote clades. Furthermore, this study establishes the relative timing of tissue development and growth directions of periodontal tissues and provides the first comparative framework for future studies of dinosaur periodontal development, tooth replacement, and histology. J. Morphol. 277:916–924, 2016. © 2016 Wiley Periodicals, Inc.     

DEVELOPMENTAL BASIS OF TOOTHLESSNESS IN TURTLES: INSIGHT INTO CONVERGENT EVOLUTION OF VERTEBRATE MORPHOLOGY

The tooth is a major component of the vertebrate feeding apparatus and plays a crucial role in species survival, thus subjecting tooth developmental programs to strong selective constraints. However, irrespective of their functional importance, teeth have been lost in multiple lineages of tetrapod vertebrates independently. To understand both the generality and the diversity of developmental mechanisms that cause tooth agenesis in tetrapods, we investigated expression patterns of a series of tooth developmental genes in the lower jaw of toothless turtles and compared them to that of toothed crocodiles and the chicken as a representative of toothless modern birds. In turtle embryos, we found impairment of Shh signaling in the oral epithelium and early‐stage arrest of odontoblast development caused by termination of Msx2 expression in the dental mesenchyme. Our data indicate that such changes underlie tooth agenesis in turtles and suggest that the mechanism that leads to early‐stage odontogenic arrest differs between birds and turtles. Our results demonstrate that the cellular and molecular mechanisms that regulate early‐stage arrest of tooth development are diverse in tetrapod lineages, and odontogenic developmental programs may respond to changes in upstream molecules similarly thereby evolving convergently with feeding morphology.     

Rudiment incisors survive and erupt as supernumerary teeth as a result of USAG-1 abrogation

The term "supernumerary teeth" describes production of more than the normal number of teeth in the primary or permanent dentitions. Their aetiology is not understood. Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist that plays important roles in the local regulation of BMP signaling by binding and neutralizing BMP activities, and also serves as a modulator of Wnt signaling. We report here that USAG-1 deficient mice have supernumerary teeth. The supernumerary maxillary incisor appears to form as a result of the successive development of the rudimentary upper incisor tooth. We confirmed that the USAG-1 expression is localized to the epithelium and mesenchyme of the rudimentary maxillary incisor tooth organ formation. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells. Based upon these results, we conclude that USAG-1 controls the number of teeth in the maxillary incisor region by regulating apoptosis.