ABSTRACT: The dentition of elasmobranchs (sharks, skates and rays) is uniquely productive, capable of both rapid and continuous, lifelong regeneration. Elasmobranchs represent an important group of vertebrates with a deep evolutionary history, possessing several ancient and basal characters, i.e., the continuously regenerative dentition from a specialized dental lamina. The dental lamina is an expanded component of the oral epithelia that is responsible for initiating and producing new teeth among all toothed-vertebrates. In sharks, this dynamic epithelial unit is permanent and continuous – meaning it extends to cover the entirety of each jaw (jaw-wide) and develops early during embryogenesis and retained to produce teeth for the life of the shark. It is rare for a truly embryonic vertebrate tissue to be retained for its original function for the life of the organism. The dental lamina in sharks is unique and houses teeth in a developmental series from the deepest part, where teeth are initiated, through stages of tooth development in the form of a related, family of teeth to eruption and functionality of the advanced teeth at the jaw margin. How teeth are made and regenerated is an important question in vertebrate biology; here we investigated this question in the small spotted catshark (Scyliorhinus canicula), a new model in the field of developmental biology. Specifically, we divided the shark dental lamina into stage-compartments as follows: (i) the initiation site – the successional lamina (SL); (ii) the early developing teeth (ET); (iii) the late stage developing teeth (LT); (iv) the tooth-taste junction between the superficial oral and dental epithelium at the jaw margin that separates the taste territory and the dental lamina proper (TTJ); and basi-hyal oral epithelium that is strictly non-dental and only contains taste buds (BHTB). These 5 compartments each house both a shared and unique signature of gene transcripts. This study aims to understand the transcriptomic basis of continuous tooth regeneration in the shark. In this study we combine X-ray computed tomography, classic histology, insitu hybridization, immunohistochemistry, and functional assays of novel markers, and de novo and genome guided transcriptome assemblies for each of these 5 dental lamina compartments of the hatchling (stage 34) catshark (S. canicula). Overall design: We produced both a de novo and genome-guided transcriptome assembly and expression analysis of the 5 dental lamina compartments (SL, ET, LT, TTJ and BHTB) of developing dentition in the hatching-stage (stage 34) small-spotted catshark (Scyliorhinus canicula).
Project description:Teeth and denticles belong to a specialized class of mineralizing epithelial appendages called odontodes. Although homology of oral teeth in jawed vertebrates is well supported, the evolutionary origin of teeth and their relationship with other odontode types is less clear. We compared the cellular and molecular mechanisms directing development of teeth and skin denticles in sharks, where both odontode types are retained. We show that teeth and denticles are deeply homologous developmental modules with equivalent underlying odontode gene regulatory networks (GRNs). Notably, the expression of the epithelial progenitor and stem cell marker sex-determining region Y-related box 2 (sox2) was tooth-specific and this correlates with notable differences in odontode regenerative ability. Whereas shark teeth retain the ancestral gnathostome character of continuous successional regeneration, new denticles arise only asynchronously with growth or after wounding. Sox2+ putative stem cells associated with the shark dental lamina (DL) emerge from a field of epithelial progenitors shared with anteriormost taste buds, before establishing within slow-cycling cell niches at the (i) superficial taste/tooth junction (T/TJ), and (ii) deep successional lamina (SL) where tooth regeneration initiates. Furthermore, during regeneration, cells from the superficial T/TJ migrate into the SL and contribute to new teeth, demonstrating persistent contribution of taste-associated progenitors to tooth regeneration in vivo. This data suggests a trajectory for tooth evolution involving cooption of the odontode GRN from nonregenerating denticles by sox2+ progenitors native to the oral taste epithelium, facilitating the evolution of a novel regenerative module of odontodes in the mouth of early jawed vertebrates: the teeth.
Project description:Teeth and taste buds are iteratively patterned structures that line the oro-pharynx of vertebrates. Biologists do not fully understand how teeth and taste buds develop from undifferentiated epithelium or how variation in organ density is regulated. These organs are typically studied independently because of their separate anatomical location in mammals: teeth on the jaw margin and taste buds on the tongue. However, in many aquatic animals like bony fishes, teeth and taste buds are colocalized one next to the other. Using genetic mapping in cichlid fishes, we identified shared loci controlling a positive correlation between tooth and taste bud densities. Genome intervals contained candidate genes expressed in tooth and taste bud fields. sfrp5 and bmper, notable for roles in Wingless (Wnt) and bone morphogenetic protein (BMP) signaling, were differentially expressed across cichlid species with divergent tooth and taste bud density, and were expressed in the development of both organs in mice. Synexpression analysis and chemical manipulation of Wnt, BMP, and Hedgehog (Hh) pathways suggest that a common cichlid oral lamina is competent to form teeth or taste buds. Wnt signaling couples tooth and taste bud density and BMP and Hh mediate distinct organ identity. Synthesizing data from fish and mouse, we suggest that the Wnt-BMP-Hh regulatory hierarchy that configures teeth and taste buds on mammalian jaws and tongues may be an evolutionary remnant inherited from ancestors wherein these organs were copatterned from common epithelium.
Project description:The successional dental lamina (SDL) plays an essential role in the development of replacement teeth in diphyodont and polyphyodont animals. A morphologically similar structure, the rudimental successional dental lamina (RSDL), has been described in monophyodont (only one tooth generation) lizards on the lingual side of the developing functional tooth. This rudimentary lamina regresses, which has been proposed to play a role in preventing the formation of future generations of teeth. A similar rudimentary lingual structure has been reported associated with the first molar in the monophyodont mouse, and we show that this structure is common to all murine molars. Intriguingly, a lingual lamina is also observed on the non-replacing molars of other diphyodont mammals (pig and hedgehog), initially appearing very similar to the successional dental lamina on the replacing teeth. We have analyzed the morphological as well as ultrastructural changes that occur during the development and loss of this molar lamina in the mouse, from its initiation at late embryonic stages to its disappearance at postnatal stages. We show that loss appears to be driven by a reduction in cell proliferation, down-regulation of the progenitor marker Sox2, with only a small number of cells undergoing programmed cell death. The lingual lamina was associated with the dental stalk, a short epithelial connection between the tooth germ and the oral epithelium. The dental stalk remained in contact with the oral epithelium throughout tooth development up to eruption when connective tissue and numerous capillaries progressively invaded the dental stalk. The buccal side of the dental stalk underwent keratinisation and became part of the gingival epithelium, while most of the lingual cells underwent programmed cell death and the tissue directly above the erupting tooth was shed into the oral cavity.
Project description:In Lake Malawi cichlids, each tooth is replaced in one-for-one fashion every ?20 to 50 d, and taste buds (TBs) are continuously renewed as in mammals. These structures are colocalized in the fish mouth and throat, from the point of initiation through adulthood. Here, we found that replacement teeth (RT) share a continuous band of epithelium with adjacent TBs and that both organs coexpress stem cell factors in subsets of label-retaining cells. We used RNA-seq to characterize transcriptomes of RT germs and TB-bearing oral epithelium. Analysis revealed differential usage of developmental pathways in RT compared to TB oral epithelia, as well as a repertoire of genome paralogues expressed complimentarily in each organ. Notably, BMP ligands were expressed in RT but excluded from TBs. Morphant fishes bathed in a BMP chemical antagonist exhibited RT with abrogated shh expression in the inner dental epithelium (IDE) and ectopic expression of calb2 (a TB marker) in these very cells. In the mouse, teeth are located on the jaw margin while TBs and other oral papillae are located on the tongue. Previous study reported that tongue intermolar eminence (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants exhibited dysmorphic invagination. We used these mutants to demonstrate altered transcriptomes and ectopic expression of dental markers in tongue IE. Our results suggest that vertebrate oral epithelium retains inherent plasticity to form tooth and taste-like cell types, mediated by BMP specification of progenitor cells. These findings indicate underappreciated epithelial cell populations with promising potential in bioengineering and dental therapeutics.
Project description:In classical theory, teeth of vertebrate dentitions evolved from co-option of external skin denticles into the oral cavity. This hypothesis predicts that ordered tooth arrangement and regulated replacement in the oral dentition were also derived from skin denticles. The fossil batoid ray Schizorhiza stromeri (Chondrichthyes; Cretaceous) provides a test of this theory. Schizorhiza preserves an extended cartilaginous rostrum with closely spaced, alternating saw-teeth, different from sawfish and sawsharks today. Multiple replacement teeth reveal unique new data from micro-CT scanning, showing how the 'cone-in-cone' series of ordered saw-teeth sets arrange themselves developmentally, to become enclosed by the roots of pre-existing saw-teeth. At the rostrum tip, newly developing saw-teeth are present, as mineralized crown tips within a vascular, cartilaginous furrow; these reorient via two 90° rotations then relocate laterally between previously formed roots. Saw-tooth replacement slows mid-rostrum where fewer saw-teeth are regenerated. These exceptional developmental data reveal regulated order for serial self-renewal, maintaining the saw edge with ever-increasing saw-tooth size. This mimics tooth replacement in chondrichthyans, but differs in the crown reorientation and their enclosure directly between roots of predecessor saw-teeth. Schizorhiza saw-tooth development is decoupled from the jaw teeth and their replacement, dependent on a dental lamina. This highly specialized rostral saw, derived from diversification of skin denticles, is distinct from the dentition and demonstrates the potential developmental plasticity of skin denticles.
Project description:For a dentition representing the most basal extant gnathostomes, that of the shark can provide us with key insights into the evolution of vertebrate dentitions. To detail the pattern of odontogenesis, we have profiled the expression of sonic hedgehog, a key regulator of tooth induction. We find in the catshark (Scyliorhinus canicula) that intense shh expression first occurs in a bilaterally symmetrical pattern restricted to broad regions in each half of the dentition in the embryo jaw. As in the mouse, there follows a changing temporal pattern of shh spatial restriction corresponding to epithelial bands of left and right dental fields, but also a subfield for symphyseal teeth. Then, intense shh expression is restricted to loci coincident with a temporal series of teeth in iterative jaw positions. The developmental expression of shh reveals previously undetected timing within epithelial stages of tooth formation. Each locus at alternate, even then odd, jaw positions establishes precise sequential timing for successive replacement within each tooth family. Shh appears first in the central cusp, iteratively along the jaw, then reiteratively within each tooth for secondary cusps. This progressive, sequential restriction of shh is shared by toothed gnathostomes and conserved through 500 million years of evolution.
Project description:In contrast to mammals, most reptiles constantly regenerate their teeth. In the snake, the epithelial dental lamina ends in a successional lamina, which proliferates and elongates forming multiple tooth generations, all linked by a permanent dental lamina. To investigate the mechanisms used to control the initiation of new tooth germs in an ordered sequential pattern we utilized the polyphodont (multiple-generation) corn snake (Pantherophis guttatus). We observed that the dental lamina expressed the transcription factor Sox2, a multipotent stem cell marker, whereas the successional lamina cells expressed the transcription factor Lef1, a Wnt/?-catenin pathway target gene. Activation of the Wnt/?-catenin pathway in culture increased the number of developing tooth germs, in comparison to control untreated cultures. These additional tooth germs budded off from ectopic positions along the dental lamina, rather than in an ordered sequence from the successional lamina. Wnt/?-catenin activation enhanced cell proliferation, particularly in normally non-odontogenic regions of the dental lamina, which widely expressed Lef1, restricting the Sox2 domain. This suggests an expansion of the successional lamina at the expense of the dental lamina. Activation of the Wnt/?-catenin pathway in cultured snake dental organs, therefore, led to changes in proliferation and to the molecular pattern of the dental lamina, resulting in loss of the organised emergence of tooth germs. These results suggest that epithelial compartments are critical for the arrangement of organs that develop in sequence, and highlight the role of Wnt/?-catenin signalling in such processes.
Project description:Vertebrate dentitions originated in the posterior pharynx of jawless fishes more than half a billion years ago. As gnathostomes (jawed vertebrates) evolved, teeth developed on oral jaws and helped to establish the dominance of this lineage on land and in the sea. The advent of oral jaws was facilitated, in part, by absence of hox gene expression in the first, most anterior, pharyngeal arch. Much later in evolutionary time, teleost fishes evolved a novel toothed jaw in the pharynx, the location of the first vertebrate teeth. To examine the evolutionary modularity of dentitions, we asked whether oral and pharyngeal teeth develop using common or independent gene regulatory pathways. First, we showed that tooth number is correlated on oral and pharyngeal jaws across species of cichlid fishes from Lake Malawi (East Africa), suggestive of common regulatory mechanisms for tooth initiation. Surprisingly, we found that cichlid pharyngeal dentitions develop in a region of dense hox gene expression. Thus, regulation of tooth number is conserved, despite distinct developmental environments of oral and pharyngeal jaws; pharyngeal jaws occupy hox-positive, endodermal sites, and oral jaws develop in hox-negative regions with ectodermal cell contributions. Next, we studied the expression of a dental gene network for tooth initiation, most genes of which are similarly deployed across the two disparate jaw sites. This collection of genes includes members of the ectodysplasin pathway, eda and edar, expressed identically during the patterning of oral and pharyngeal teeth. Taken together, these data suggest that pharyngeal teeth of jawless vertebrates utilized an ancient gene network before the origin of oral jaws, oral teeth, and ectodermal appendages. The first vertebrate dentition likely appeared in a hox-positive, endodermal environment and expressed a genetic program including ectodysplasin pathway genes. This ancient regulatory circuit was co-opted and modified for teeth in oral jaws of the first jawed vertebrate, and subsequently deployed as jaws enveloped teeth on novel pharyngeal jaws. Our data highlight an amazing modularity of jaws and teeth as they coevolved during the history of vertebrates. We exploit this diversity to infer a core dental gene network, common to the first tooth and all of its descendants.
Project description:The lifelong tooth replacement in elasmobranch fishes (sharks, rays and skates) has led to the assemblage of a great number of teeth from fossil and extant species, rendering tooth morphology an important character for taxonomic descriptions, analysing phylogenetic interrelationships and deciphering their evolutionary history (e.g. origination, divergence, extinction). Heterodonty (exhibition of different tooth morphologies) occurs in most elasmobranch species and has proven to be one of the main challenges for these analyses. Although numerous shark species are discovered and described every year, detailed descriptions of tooth morphologies and heterodonty patterns are lacking or are only insufficiently known for most species. Here, we use landmark-based 2D geometric morphometrics on teeth of the tiger shark Galeocerdo cuvier to analyse and describe dental heterodonties among four different ontogenetic stages ranging from embryo to adult. Our results reveal rather gradual and subtle ontogenetic shape changes, mostly characterized by increasing size and complexity of the teeth. We furthermore provide the first comprehensive description of embryonic dental morphologies in tiger sharks. Also, tooth shapes of tiger sharks in different ontogenetic stages are re-assessed and depicted in detail. Finally, multiple cases of tooth file reversal are described. This study, therefore, contributes to our knowledge of dental traits across ontogeny in the extant tiger shark G. cuvier and provides a baseline for further morphological and genetic studies on the dental variation in sharks. Therefore, it has the potential to assist elucidating the underlying developmental and evolutionary processes behind the vast dental diversity observed in elasmobranch fishes today and in deep time.
Project description:Wnt/beta-catenin signaling plays key roles in tooth development, but how this pathway intersects with the complex interplay of signaling factors regulating dental morphogenesis has been unclear. We demonstrate that Wnt/beta-catenin signaling is active at multiple stages of tooth development. Mutation of beta-catenin to a constitutively active form in oral epithelium causes formation of large, misshapen tooth buds and ectopic teeth, and expanded expression of signaling molecules important for tooth development. Conversely, expression of key morphogenetic regulators including Bmp4, Msx1, and Msx2 is downregulated in embryos expressing the secreted Wnt inhibitor Dkk1 which blocks signaling in epithelial and underlying mesenchymal cells. Similar phenotypes are observed in embryos lacking epithelial beta-catenin, demonstrating a requirement for Wnt signaling within the epithelium. Inducible Dkk1 expression after the bud stage causes formation of blunted molar cusps, downregulation of the enamel knot marker p21, and loss of restricted ectodin expression, revealing requirements for Wnt activity in maintaining secondary enamel knots. These data place Wnt/beta-catenin signaling upstream of key morphogenetic signaling pathways at multiple stages of tooth development and indicate that tight regulation of this pathway is essential both for patterning tooth development in the dental lamina, and for controlling the shape of individual teeth.