Continuous Tooth Replacement Research Articles

Continuous tooth replacement: what can teleost fish teach us?

Most tooth-bearing non-mammalian vertebrates have the capacity to replace their teeth throughout life. This capacity was lost in mammals, which replace their teeth only once at most. Not surprisingly, continuous tooth replacement has attracted much attention. Classical morphological studies (e.g. to analyse patterns of replacement) are now being complemented by molecular studies that investigate the expression of genes involved in tooth formation. This review focuses on ray-finned fish (actinopterygians), which have teeth often distributed throughout the mouth and pharynx, and more specifically on teleost fish, the largest group of extant vertebrates. First we highlight the diversity in tooth distribution and in tooth replacement patterns. Replacement tooth formation can start from a distinct (usually discontinuous and transient) dental lamina, but also in the absence of a successional lamina, e.g. from the surface epithelium of the oropharynx or from the outer dental epithelium of a predecessor tooth. The relationship of a replacement tooth to its predecessor is closely related to whether replacement is the result of a prepattern or occurs on demand. As replacement teeth do not necessarily have the same molecular signature as first-generation teeth, the question of the actual trigger for tooth replacement is discussed. Much emphasis has been laid in the past on the potential role of epithelial stem cells in initiating tooth replacement. The outcome of such studies has been equivocal, possibly related to the taxa investigated, and the permanent or transient nature of the dental lamina. Alternatively, replacement may result from local proliferation of undifferentiated progenitors, stimulated by hitherto unknown, perhaps mesenchymal, factors. So far, the role of the neurovascular link in continuous tooth replacement has been poorly investigated, despite the presence of a rich vascularisation surrounding actinopterygian (as well as chondrichthyan) teeth and despite a complete arrest of tooth replacement after nerve resection. Lastly, tooth replacement is possibly co-opted as a process to expand the number of teeth in a dentition ontogenetically whilst conserving features of the primary dentition. That neither a dental lamina, nor stem cells appear to be required for tooth replacement places teleosts in an advantageous position as models for tooth regeneration in humans, where the dental lamina regresses and epithelial stem cells are considered lost.

Expression of secretory calcium-binding phosphoprotein (scpp) genes in medaka during the formation and replacement of pharyngeal teeth

BackgroundAnalyses of tooth families and tooth-forming units in medaka with regard to tooth replacement cycles and the localization of odontogenic stem cell niches in the pharyngeal dentition clearly indicate that continuous tooth replacement is maintained. The secretory calcium-binding phosphoprotein (scpp) gene cluster is involved in the formation of mineralized tissues, such as dental and bone tissues, and the genes encoding multiple SCPPs are conserved in fish, amphibians, reptiles, and mammals. In the present study, we examined the expression patterns of several scpp genes in the pharyngeal teeth of medaka to elucidate their roles during tooth formation and replacement.MethodsHimedaka (Japanese medaka, Oryzias latipes) of both sexes (body length: 28 to 33 mm) were used in this study. Real-time quantitative reverse transcription-polymerase chain reaction (PCR) (qPCR) data were evaluated using one-way analysis of variance for multi-group comparisons, and the significance of differences was determined by Tukey’s comparison test. The expression of scpp genes was examined using in situ hybridization (ISH) with a digoxigenin-labeled, single-stranded antisense probe.ResultsqPCR results showed that several scpp genes were strongly expressed in pharyngeal tissues. ISH analysis revealed specific expression of scpp1, scpp5, and sparc in tooth germ, and scpp5 was continually expressed in the odontoblasts of teeth attached to pedicles, but not in the osteoblasts of pedicles. In addition, many scpp genes were expressed in inner dental epithelium (ide), but not in odontoblasts, and scpp2 consistently showed epithelial-specific expression in the functional teeth. Taken together, these data indicate that specific expression of scpp2 and scpp5 may play a critical role in pharyngeal tooth formation in medaka.ConclusionWe characterized changes in the expression patterns of scpp genes in medaka during the formation and replacement of pharyngeal teeth.

Teeth outside the mouth: The evolution and development of shark denticles.

Vertebrate skin appendages are incredibly diverse. This diversity, which includes structures such as scales, feathers, and hair, likely evolved from a shared anatomical placode, suggesting broad conservation of the early development of these organs. Some of the earliest known skin appendages are dentine and enamel-rich tooth-like structures, collectively known as odontodes. These appendages evolved over 450 million years ago. Elasmobranchs (sharks, skates, and rays) have retained these ancient skin appendages in the form of both dermal denticles (scales) and oral teeth. Despite our knowledge of denticle function in adult sharks, our understanding of their development and morphogenesis is less advanced. Even though denticles in sharks appear structurally similar to oral teeth, there has been limited data directly comparing the molecular development of these distinct elements. Here, we chart the development of denticles in the embryonic small-spotted catshark (Scyliorhinus canicula) and characterize the expression of conserved genes known to mediate dental development. We find that shark denticle development shares a vast gene expression signature with developing teeth. However, denticles have restricted regenerative potential, as they lack a sox2+ stem cell niche associated with the maintenance of a dental lamina, an essential requirement for continuous tooth replacement. We compare developing denticles to other skin appendages, including both sensory skin appendages and avian feathers. This reveals that denticles are not only tooth-like in structure, but that they also share an ancient developmental gene set that is likely common to all epidermal appendages.

In ovo manipulation of Nile crocodile embryos: egg windowing and potential dental research applications.

Crocodilians exhibit continuous tooth replacement (i.e., polyphyodonty) and have been identified as suitable models for tooth regeneration research due to the similarity in dental cavity and tooth anatomy between these creatures and humans. Various studies reporting in ovo bird embryo manipulation exist, but such reports for reptiles are virtually non-existent. Egg windowing enables direct access to oviparous vertebrate embryos and is therefore an important component of in ovo embryo manipulation experiments. The aim of the present study was to window Nile crocodile, Crocodylus niloticus eggs and assess the potential of direct manipulations, targeting the pharyngeal region where the maxilla and mandible originates. Crocodylus niloticus eggs were successfully windowed, and a limited number of individuals survived the entire gestation period. The 1st trimester of gestation was the most sensitive, and 96.78% of the mortalities occurred within this period. Our data indicate the suitable window for embryo manipulation targeting the mandibular arch and maxillary process, without a risk of damaging the chorioallantoic membrane (CAM) (which may be fatal), was between day six and eight after laying for embryos incubated at 31°C. This data will be of use for future embryo-based experiments related to jaw and tooth development in crocodiles as well as human tooth regeneration research.

The Effects of Premature Tooth Extraction and Damage on Replacement Timing in the Green Iguana.

Reptiles with continuous tooth replacement, or polyphyodonty, replace their teeth in predictable, well-timed waves in alternating tooth positions around the mouth. This process is thought to occur irrespective of tooth wear or breakage. In this study, we aimed to determine if damage to teeth and premature tooth extraction affects tooth replacement timing long-term in juvenile green iguanas (Iguana iguana). First, we examined normal tooth development histologically using a BrdU pulse-chase analysis to detect label-retaining cells in replacement teeth and dental tissues. Next, we performed tooth extraction experiments for characterization of dental tissues after functional tooth (FT) extraction, including proliferation and β-Catenin expression, for up to 12 weeks. We then compared these results to a newly analyzed historical dataset of X-rays collected up to 7 months after FT damage and extraction in the green iguana. Results show that proliferation in the dental and successional lamina (SL) does not change after extraction of the FT, and proliferation occurs in the SL only when a tooth differentiates. Damage to an FT crown does not affect the timing of the tooth replacement cycle, however, complete extraction shifts the replacement cycle ahead by 4 weeks by removing the need for resorption of the FT. These results suggest that traumatic FT loss affects the timing of the replacement cycle at that one position, which may have implications for tooth replacement patterning around the entire mouth.

Articulated remains of the extinct shark Ptychodus (Elasmobranchii, Ptychodontidae) from the Upper Cretaceous of Spain provide insights into gigantism, growth rate and life history of ptychodontid sharks.

Due to their cartilaginous endoskeleton and the continuous tooth replacement, the chondrichthyan fossil record predominantly consists of isolated teeth, which offer diagnostic features for taxonomic identifications, but only provide very limited information of an organism's life history. In contrast, the calcified vertebral centra of elasmobranchs (sharks, skates and rays) yield important information about ecological and biological traits that can be utilized for constructing age-structured population dynamic models of extant species and palaeoecological reconstructions of such aspects in extinct groups. Here, we describe two large shark vertebrae from the Santonian (Upper Cretaceous) of Spain, which show a unique combination of characters (asterospondylic calcification pattern, with concentric lamellae and numerous parallel bands that are oriented perpendicular) that is only known from ptychodontid sharks, a distinct, extinct group of giant durophagous sharks of the Cretaceous era. Based on linear regression models for large extant sharks a total length between 430 and 707cm was estimated for the examined specimen. Our results indicate that ptychodontid sharks were large viviparous animals, with slow growth rates, matured very late and, therefore, show typical traits for K-selected species. These traits combined with a highly specialized feeding ecology might have played a crucial role for the success but also, eventually, extinction of this group.

Sox2 maintains epithelial cell proliferation in the successional dental lamina.

ObjectivesThe successional dental lamina is the distinctive structure on the lingual side of the vertebrate tooth germ. The aim of this study was to investigate the relationship among Sox2, Claudin10 and laminin5 and the role of Sox2 in successional dental lamina proliferation during vertebrate tooth development.Materials and MethodsTo understand the successional dental lamina, two types of successional tooth formation, that in geckos (with multiple rounds of tooth generation) and that in mice (with only one round of tooth generation), were analysed.ResultsUnique coexpression patterns of Sox2 and Claudin10 expression were compared in the successional dental lamina from the cap stage to the late bell stage in the mouse tooth germ and in juvenile gecko teeth to support continuous tooth replacement. Furthermore, Laminin5 expression was shown in the cap stage and decreased after the bell stage. Upon comparing the epithelial cell cycles and cell proliferation in successional dental lamina regions between mouse and gecko molars using BrdU and IdU staining and pulse‐chase methods, distinctive patterns of continuous expression were revealed. Moreover, Sox2 overexpression with a lentiviral system resulted in hyperplastic dental epithelium in mouse molars.ConclusionsOur findings indicate that the regulation of Sox2 in dental lamina proliferation is fundamental to the successional dental lamina in both species.

Dental microwear texture reflects dietary tendencies in extant Lepidosauria despite their limited use of oral food processing.

Lepidosauria show a large diversity in dietary adaptations, both among extant and extinct tetrapods. Unlike mammals, Lepidosauria do not engage in sophisticated mastication of their food and most species have continuous tooth replacement, further reducing the wear of individual teeth. However, dietary tendency estimation of extinct lepidosaurs usually rely on tooth shape and body size, which allows only for broad distinction between faunivores and herbivores. Microscopic wear features on teeth have long been successfully applied to reconstruct the diet of mammals and allow for subtle discrimination of feeding strategies and food abrasiveness. Here, we present, to our knowledge, the first detailed analysis of dental microwear texture on extant lepidosaurs using a combination of 46 surface texture parameters to establish a framework for dietary tendency estimation of fossil reptilian taxa. We measured dental surface textures of 77 specimens, belonging to herbivorous, algaevorous, frugivorous, carnivorous, ovivorous, insectivorous, molluscivorous, as well as omnivorous species. Carnivores show low density and shallow depth of furrows, whereas frugivores are characterized by the highest density of furrows. Molluscivores show the deepest wear features and highest roughness, herbivores have lower surface roughness and shallower furrows compared to insectivores and omnivores, which overlap in all parameters. Our study shows that despite short food-tooth interaction, dental surface texture parameters enable discrimination of several feeding strategies in lepidosaurs. This result opens new research avenues to assess diet in a broad variety of extant and extinct non-mammalian taxa including dinosaurs and early synapsids.

Disruption of continuous tooth replacement in squamates: Implications for tooth evolution and development

The ability for animals to continuously replace their teeth, or polyphyodonty, is a character deeply nested within Vertebrata. Tooth cycling in polyphyodont vertebrates occurs in a regular pattern, passing through alternating tooth positions in waves around the mouth. Although several hypotheses have been proposed for the establishment and maintenance of this phenomenon, it is not yet known if tooth patterning is controlled through global molecular signals in the jaw environment, signalling between adjacent teeth, or local signals within each tooth family, or how tooth size differences (heterodonty) may affect the regularity of replacement waves. To investigate the mechanisms of polyphyodonty, a historical dataset of unpublished reptile dental x‐rays collected in the 1950s and 60s was analysed. Thirty species of reptiles were x‐rayed monthly for up to two years. The dataset includes animals with heterodont dentitions, such as Alligator, Varanus, and Dracaena, and iguanas with homodont dentitions (teeth are the same size around the mouth) that underwent tooth removal experiments and were tracked with bi‐monthly x‐rays for up to seven months post surgery. Surgical experiments included functional tooth extraction, tooth bud extraction, and removal of portions of the dental lamina with and without tooth extraction.Results in all animals show predictable wave replacement patterns in the dentition over long time periods. In the homodont Iguana, the timing of tooth development and tooth replacement is tightly correlated. However, in the heterodont reptiles, the developmental time is different between teeth: smaller teeth take one or two months to develop before eruption, while larger teeth take up to five months. Tooth removal experiments in Iguana show disruptions in replacement timing after surgery, but near‐complete recovery after six months when developing teeth are removed and the dental lamina is left intact. Damage to the dental lamina creates a strong phenotype in tooth replacement timing and patterning that does not recover, with critical size defects as small as one tooth position. However, tooth replacement continues anterior and posterior to the surgical site, supporting a local rather than global mechanism governing tooth replacement in adult dentitions.These results suggest that tooth replacement in the adult is independently controlled within the tooth family and not through global signalling factors traveling along the dental lamina. Tooth development time is consistent at each tooth position, and does not change after minor disruption. This has implications for the evolution of dentitions, as discrete regions of the mouth can react differently to selection pressures, generating diversity in tooth size, shape, and function.Support or Funding InformationKB is funded by a Banting Postdoctoral Fellowship, a Killam Postdoctoral Fellowship, the Michael Smith Foundation for Health Research, and the American Association of Anatomists. Access to collections was provided by the Royal Ontario Museum.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tooth development, histology, and enamel microstructure in Changchunsaurus parvus: Implications for dental evolution in ornithopod dinosaurs.

The great diversity of dinosaurian tooth shapes and sizes, and in particular, the amazing dental complexity in derived ornithischians has attracted a lot of attention. However, the evolution of dental batteries in hadrosaurids and ceratopsids is difficult to understand without a broader comparative framework. Here we describe tooth histology and development in the "middle" Cretaceous ornithischian dinosaur Changchunsaurus parvus, a small herbivore that has been characterized as an early ornithopod, or even as a more basal ornithischian. We use this taxon to show how a "typical" ornithischian dentition develops, copes with wear, and undergoes tooth replacement. Although in most respects the histological properties of their teeth are similar to those of other dinosaurs, we show that, as in other more derived ornithischians, in C. parvus the pulp chamber is not invaded fully by the newly developing replacement tooth until eruption is nearly complete. This allowed C. parvus to maintain an uninterrupted shearing surface along a single tooth row, while undergoing continuous tooth replacement. Our histological sections also show that the replacement foramina on the lingual surfaces of the jaws are likely the entry points for an externally placed dental lamina, a feature found in many other ornithischian dinosaurs. Surprisingly, our histological analysis also revealed the presence of wavy enamel, the phylogenetically earliest occurrence of this type of tissue. This contradicts previous interpretations that this peculiar type of enamel arose in association with more complex hadrosauroid dentitions. In view of its early appearance, we suggest that wavy enamel may have evolved in association with a shearing-type dentition in a roughly symmetrically-enameled crown, although its precise function still remains somewhat of a mystery.

Developmental pathways of periodontal tissue regeneration: Developmental diversities of tooth morphogenesis do also map capacity of periodontal tissue regeneration?

Nothing is known on the impact of developmental divergence on periodontal tissue regeneration in vertebrate animals. Molecularly, the induction of tooth morphogenesis is highly conserved deploying across animal phyla a constant and reproducible set of gene pathways, which result in morphogenesis of multiple odontode forms and shapes. Genetic mutations positively affect animal speciation via evolving biting and masticatory forces as well as dietary habits selectively imprinted in animal phyla during evolutionary speciation. The geometry of the attachment apparatus of a tooth is important for the interpretation of the induction of cementogenesis with de novo Sharpey's fibres as in thecodonty, ie, a tripartite attachment of alveolar bone, periodontal ligament and cementum. This review addresses the tooth implantation in different animal clades from the fibrous attachment of the Elasmobranch Carcharinus obscurus dusky shark, reviewing the evolution and functional significance of cementum with functionally inserted Sharpey's fibres. In sharks there is a continuous tooth replacement mechanistically supported by the continuously erupting dental lamina. We show that the arching of the continuously erupting dental lamina, a critical step for the selachians' tooth differentiation, is prominently characterized by transforming growth factor-β3 (TGF-β3 ) expression not only within the dental lamina but also in cellular condensations in the mesenchymal tissues of the erupting tooth. Such findings indicate the pleiotropic multifaceted activity of a highly conserved mammalian gene across genera, masterminding tooth morphogenesis in both selachians and mammals as well as periodontal tissue induction in the non-human primate Papio ursinus. In P.ursinus, the induction of cementogenesis entails the expression of TGF-β3 and osteocalcin with fine-tuning and regulation of bone morphogenetic proteins BMP-2 and BMP-7, and upregulation of TGF-β3 . TGF-β3 autoinduction and upregulation during the induction of cementogenesis and osteogenesis in P.ursinus provide novel insights into the induction of cementogenesis. It is hypothesized that the evolutionary expression and upregulation of the TGF-β3 gene may provide the mechanistic insights into the induction of extensive cementogenesis as seen in stem mammals and the induction of trabecular-like cementum formation in mosasaurs' tooth attachment. Aspidin, the precursor of cementum, was reported to appear 310-330million years ago (Ma) in Odontostraci armoured fish. Studies showed that the differentiation of cementum with inserted Sharpey's fibres is also present in lower amniotes such as Diatectomorpha or Diadectidae, the first herbivorous tetrapods, 323Ma. In mosasaurs, 168-165Ma, there is the induction of extensive trabeculation of cementum though nothing is known on the phylogenetic temporo-spatial evolution of cementum before Diadectidae and stem mammals. The large trabeculations of cementum as seen in the attachment of extinct mosasaurs invocates a pleiotropic capacity of cemental growth previously unknown. The appearance of cementum facing a vascularized and innervated periodontal ligament space with Sharpey's fibres inserting on to mineralized cementum provides a multiform pleiotropic masticatory apparatus adapted to multiple biting and lacerating forces as well as finely tuned and controlled forces beyond mastication and deglutition. The remarkable cementogenesis as seen in stem mammals but particularly in mosasaurs with cemental trabeculations across the ligament space invocates the developmental capacity of cementum. The large cemental trabeculations as seen in mosasaurs and the cemental growth in stem mammals, together with regenerating scenarios in P.ursinus with large seams of cellular cementum and cementoid populated by contiguous cementoblasts indicate the continuous molecular cross-talk between cementum, newly formed cementoid matrix, cementoblasts and extracellular matrix soluble molecular signals. This molecular cross-talk may control the biomolecular homeostasis of both cementum and periodontal ligament, including angiogenesis. A further molecular scenario is invocated by the tight and exquisite anatomical relationships between the cementoid surfaces and the newly formed capillaries. The primitiveness of the craniate masticatory mineralized craniofacial apparatus has been controlled by several yet ancestral common genes not lastly the TGF-β3 gene. The TGF-β3 might have been responsible for the induction of cementogenesis not only in extant P.ursinus but also in Diatectomorpha and mosasaurs, thus providing continuous evolutionary mechanisms for the induction of tissue morphogenesis across animal phyla for almost a billion years of evolution, epitomizing Nature's parsimony in controlling tissue induction and morphogenesis. TGF-β receptor II regulates osterix expression via Smad-dependent pathways indicating that TGF-β signalling acts as an upstream regulator of osterix during cementoblast differentiation. The presence of morphogenetic signals within the cemental matrix capable of inducing bone formation needs now to be assigned: bone induction initiated by extracted and partially purified cemental matrices may be the result of a slow release of embryonic remnants of osteogenic signals required and deployed during cementogenesis. The cementum may thus rule the periodontal ligament space homeostasis, remodelling and repair by releasing sequestered morphogenetic signals that were deployed during embryogenesis.

The Permian reptile Opisthodontosaurus carrolli: amodel for acrodont tooth replacement and dental ontogeny.

Continuous tooth replacement is common for tetrapods, but some groups of acrodont lepidosaurs have lost the ability to replace their dentition (monophyodonty). Acrodonty, where the tooth attaches to the apex of the jawbone, is an unusual form of tooth attachment that has been associated with the highly autapomorphic condition of monophyodonty. Beyond Lepidosauria, very little is known about the relationship between acrodonty and monophyodonty in other amniotes. We test for this association with a detailed study of the dentition of Opisthodontosaurus, an unusual Early Permian captorhinid eureptile with acrodont dentition. We provide clear evidence, both histological and morphological, that there were regular tooth replacement events in the lower jaw of Opisthodontosaurus, similar to its captorhinid relatives. Thus, our study of the oldest known amniote with an acrodont dentition shows that acrodonty does not inhibit tooth replacement, and that many of the characteristics assigned to lepidosaurian acrodonty are actually highly derived features of lepidosaurs that have resulted secondarily from a lack of tooth replacement. In the context of reptilian dental evolution, we propose the retention of the simple definition of acrodonty, which only pertains to the relative position of the tooth at the apex of the jaw, where the jaw possesses equal lingual and labial walls. This definition of implantation therefore focuses solely on the spatial relationship between the tooth and the jawbone, and separates this relationship from tooth development and replacement.

Daughters of the Enamel Organ: Development, Fate, and Function of the Stratum Intermedium, Stellate Reticulum, and Outer Enamel Epithelium.

The tooth enamel organ (EO) is a complex epithelial cell assembly involved in multiple aspects of tooth development, including amelogenesis. The present study focuses on the role of the nonameloblast layers of the EO, the stratum intermedium, the stellate reticulum, and the outer enamel epithelium (OEE). The secretory stage stratum intermedium was distinguished by p63-positive epithelial stem cell marks, highly specific alkaline phosphatase labeling, as well as multiple desmosomes and gap junctions. At the location of the presecretory stage stellate reticulum, the pre-eruption EO prominently featured the papillary layer (PL) as a keratin immunopositive network of epithelial strands between tooth crowns and oral epithelium. PL cell strands contained numerous p63-positive epithelial stem cells, while BrdU proliferative cells were detected at the outer boundaries of the PL, suggesting that the stellate reticulum/PL epithelial cell sheath proliferated to facilitate an epithelial seal during tooth eruption. Comparative histology studies demonstrated continuity between the OEE and the general lamina of continuous tooth replacement in reptiles, and the outer layer of Hertwig's epithelial root sheath in humans, implicating the OEE as the formative layer for continuous tooth replacement and tooth root extension. Cell fate studies in organ culture verified that the cervical portion of the mouse molar EO gave rise to Malassez rest-like cell islands. Together, these studies indicate that the nonameloblast layers of the EO play multiple roles during odontogenesis, including the maintenance of several p63-positive stem cell reservoirs, a role during tooth root morphogenesis and tooth succession, a stabilizing function for the ameloblast layer, the facilitation of ion transport from the EO capillaries to the enamel layer, as well as safe and seamless tooth eruption.

Slow cycling cells in the continuous dental lamina of Scyliorhinus canicula: new evidence for stem cells in sharks

In the lesser spotted catshark (Scyliorhinus canicula), as in most non-mammalian vertebrates, the dentition renews throughout life. To contribute to our understanding of how continuous tooth replacement is achieved, we searched for evidence for the presence of stem cells in this species. Three-dimensional reconstructions of juvenile (2–3 weeks post-hatch) specimens showed that tooth families merge imperceptibly with so-called interdental zones within a continuous and permanent dental lamina. Interdental regions are composed of three layers, continuous with cervical loop, middle, and outer dental epithelium of the tooth families, respectively. A BrdU pulse-chase experiment revealed that cell proliferation is initiated in the lingual part of the dental lamina and the resulting population shifts one tooth position towards the oral epithelium in around four to five weeks. In the longest chase time (114 days) label-retaining and arguably non-differentiated cells were present at the lingual border of the dental lamina. These were found in the outer and middle dental epithelium, both within and between tooth families. This area of the dental lamina did not show expression or distribution of Sox2. Our data support the hypothesis that stem cells reside at the lingual border of the continuous dental lamina, more specifically in the middle dental epithelium at the level of the tooth families, and in its extension between the tooth families. To demonstrate their true stemness and their role in continuous tooth replacement, it remains to be shown that these cells have the potential to give rise to a complete new successor.

Osteoblast and osteoclast behaviors in the turnover of attachment bones during medaka tooth replacement

Tooth replacement in polyphyodont is a well-organized system for maintenance of homeostasis of teeth, containing the dynamic structural change in skeletal tissues such as the attachment bone, which is the supporting element of teeth. Histological analyses have revealed the character of tooth replacement, however, the cellular mechanism of how skeletal tissues are modified during tooth replacement is largely unknown. Here, we showed the important role of osteoblasts for controlling osteoclasts to modify the attachment bone during tooth replacement in medaka pharyngeal teeth, coupled with an osterix-DsRed/TRAP-GFP transgenic line to visualize osteoblasts and osteoclasts. In the turnover of the row of attachment bones, these bones were resorbed at the posterior side where most developed functional teeth were located, and generated at the anterior side where teeth were newly erupted, which caused continuous tooth replacement. In the cellular analysis, osteoclasts and osteoblasts were located at attachment bones separately, since mature osteoclasts were localized at the resorbing side and osteoblasts gathered at the generating side. To demonstrate the role of osteoclasts in tooth replacement, we established medaka made deficient in c-fms-a by TALEN. c-fms-a deficient medaka showed hyperplasia of attachment bones along with reduced bone resorption accompanied by a low number of TRAP-positive osteoclasts, indicating an important role of osteoclasts in the turnover of attachment bones. Furthermore, nitroreductase-mediated osteoblast-specific ablation induced disappearance of osteoclasts, indicating that osteoblasts were essential for maintenance of osteoclasts for the proper turnover. Taken together, our results suggested that the medaka attachment bone provides the model to understand the cellular mechanism for tooth replacement, and that osteoblasts act in the coordination of bone morphology by supporting osteoclasts.

The innervation of the zebrafish pharyngeal jaws and teeth.

Zebrafish (Danio rerio) teeth are increasingly used as a model to study odontogenesis in non-mammalians. Using serial semi-thin section histology and immunohistochemistry, the nerves innervating the pharyngeal jaws and teeth have been identified. The last pair of branchial arches, which are non-gill bearing but which carry the teeth, are innervated by an internal branch of a post-trematic ramus of the vagal nerve. Another, external, branch is probably responsible for the motor innervation of the branchiomeric musculature. Nerve fibres appear in the pulp cavity of the teeth only late during cytodifferentiation, and are therefore likely not involved in early steps of tooth formation. The precise role of the nervous system during continuous tooth replacement remains to be determined. Nonetheless, this study provides the necessary morphological background information to address this question.

Tooth Replacement in an Anatomical Context: Studies in the Zebrafish

Most non‐mammalians that possess teeth display lifelong tooth renewal. So far, studies aiming to dissect the mechanisms underlying continuous tooth replacement have focused mostly on the molecular cues that may regulate replacement. However, if we wish to fully understand this process, we also need to consider the cellular environment in which teeth develop.We have embarked on studies investigating the vasculature and innervation of the zebrafish pharyngeal dentition (there are no oral teeth), and their respective roles in tooth replacement, using light microscopy, 3D reconstructions and immunohistochemistry. First, we have demonstrated that, while the first‐generation teeth develop before the vasculature has reached the last pharyngeal arch on which these teeth develop, functional teeth become surrounded by an elaborate sinusoidal cavity. Capillaries branching off from this sinusoidal vessel supply replacement teeth at an advanced stage in their development, as they enter the pulp cavity only at late cytodifferentiation. Blocking VEGF, a major angiogenic factor, slows down replacement tooth development but does not prevent initiation of new teeth, suggesting that formation of new blood vessels does not trigger the replacement process. Like the more anterior, gill‐bearing arches, the dentigerous pharyngeal arch is innervated by posttrematic rami of the vagal nerve (NX). Sensory fibers enter the pulp cavity at a late stage of cytodifferentiation, shortly before attachment of the tooth. Autonomous fibers have not been identified so far.Whether nerves follow the vasculature in entering the pulp, as in mammalian teeth, and what the role is of the neurovascular supply in teeth that are renewed anyway, needs further studies.This study was supported by a grant of the IWT‐Vlaanderen to JC.

Imaging the zebrafish dentition: from traditional approaches to emerging technologies.

The zebrafish, a model organism for which a plethora of molecular and genetic techniques exists, has a lifelong replacing dentition of 22 pharyngeal teeth. This is in contrast to the mouse, which is the key organism in dental research but whose teeth are never replaced. Employing the zebrafish as the main organism to elucidate the mechanisms of continuous tooth replacement, however, poses at least one major problem, related to the fact that all teeth are located deep inside the body. Investigating tooth replacement thus relies on conventional histological methods, which are often laborious, time-consuming and can cause tissue deformations. In this review, we investigate the advantages and limitations of adapting current visualization techniques to dental research in zebrafish. We discuss techniques for fast sectioning, such as vibratome sectioning and high-resolution episcopic microscopy, and methods for in toto visualization, such as Alizarin red staining, micro-computed tomography, and optical projection tomography. Techniques for in vivo imaging, such as two-photon excitation fluorescence and second harmonic generation microscopy, are also covered. Finally, the possibilities of light sheet microscopy are addressed.

Tooth replacement without a dental lamina: the search for epithelial stem cells in Polypterus senegalus.

Most actinopterygians replace their teeth continuously throughout life. To address the question of where and how replacement teeth form in actinopterygians, it is advisable to investigate well-chosen representatives within the lineage. The African bichir, Polypterus senegalus, belongs to the earliest diverged group of the actinopterygian lineage with currently living representatives. Its well characterized dentition, together with its phylogenetic position, make this species an attractive model to answer following questions: (1) when and where does the replacement tooth form and how is it connected with the dental organ of the predecessor, and (2) is there any evidence for the presence of epithelial stem cells, hypothesized to play a role in replacement? Serial sections show that one tooth family can contain up to three members, which are all interconnected by dental epithelium. Replacement teeth develop without the presence of a successional dental lamina. We propose that this is the plesiomorphic condition for tooth replacement in actinopterygians. BrdU pulse-chase experiments reveal cells in the outer and middle dental epithelium, proliferating at the time of initiation of a new replacement tooth. It is tempting to assume that these cell layers provide a stem cell niche. The observed absence of label-retaining cells after long chase times (up to 8 weeks) is held against the light of divergent views on cell cycling properties of stem cells. At present, our data do not support, neither reject, the hypothesis on involvement of epithelial stem cells within the process of continuous tooth replacement.

Tooth replacement and putative odontogenic stem cell niches in pharyngeal dentition of medaka (Oryzias latipes)

The small-sized teleost fish medaka, Oryzias latipes, has as many as 1000 pharyngeal teeth undergoing continuous replacement. In this study, we sought to identify the tooth-forming units and determine its replacement cycles, and further localize odontogenic stem cell niches in the pharyngeal dentition of medaka to gain insights into the mechanisms whereby continuous tooth replacement is maintained. Three-dimensional reconstruction of pharyngeal epithelium and sequential fluorochrome labeling of pharyngeal bones and teeth indicated that the individual functional teeth and their successional teeth were organized in families, each comprising up to five generations of teeth and successional tooth germs, and that the replacement cycle of functional teeth was approximately 4 weeks. BrdU label/chase experiments confirmed the existence of clusters of label-retaining epithelial cells at the posterior end of each tooth family where the expression of pluripotency marker Sox2 was confirmed by in situ hybridization. Label-retaining cells were also identified in the mesoderm immediately adjacent to the posterior end of each tooth family. These data suggest the importance of existence of slow-cycling dental epithelial cells and Sox2 expressions at the posterior end of each tooth family to maintain continuous tooth formation and replacement in the pharyngeal dentition of medaka.