RESUMEN

The dentition is critical to animal survival and teeth are present in modern vertebrates including teleost fish, sharks, amphibians, mammals and reptiles. The developmental processes that give rise to teeth are not just preserved through evolution but also share high level of similarity with the embryogenesis of other ectodermal organs. In this review we go beyond the embryonic phase of tooth development to life-long tooth replacement. We will address the origins of successional teeth, the location of putative tissue-resident stem cells, how de novo tooth formation continues throughout life and how teeth are shed in a spatially and temporally controlled manner. We review the evidence that the dental epithelium, which is the earliest recognizable dental structure in the reptilian dentition, serves as a putative niche for tissue-resident epithelial stem cells and recent molecular findings from transcriptomics carried out in reptilian dentitions. We discuss how odontoclasts resorb the primary tooth allowing eruption of the successional tooth. The reptiles, particularly lizards, are emerging as some of the most accessible animals to study tooth replacement which has relevance to evolution of the dentition and human dental disorders.

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Tooth replacement rates of polyphyodont cartilaginous and bony fishes are hard to determine because of a lack of obvious patterning and maintaining specimens long enough to observe replacement. Pulse-chase is a fluorescent technique that differentially colours developing mineralized tissue. We present in situ tooth replacement rate and position data for the oral and pharyngeal detentions of Ophiodon elongatus (Pacific lingcod). We assessed over 10 000 teeth, in 20 fish, and found a daily replacement rate of about two teeth (3.6% of the dentition). The average tooth is in the dental battery for 27 days. The replacement was higher in the lower pharyngeal jaw (LPJ). We found no difference between replacement rates of feeding and non-feeding fish, suggesting feeding was not a driver of tooth replacement. Lingcod teeth have both a size and location fate; smaller teeth at one spot will not grow into larger teeth, even if a large tooth nearby is lost. We also found increased rates of replacement at the posterior of the LPJ relative to the anterior. We propose that lingcod teeth do not migrate in the jaw as they develop; their teeth are fated in size and location, erupting in their functional position.

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Many reptiles are able to continuously replace their teeth through life, an ability attributed to the existence of epithelial stem cells. Tooth replacement occurs in a spatially and temporally regulated manner, suggesting the involvement of diffusible factors, potentially over long distances. Here, we locally disrupted tooth replacement in the leopard gecko (Eublepharis macularius) and followed the recovery of the dentition. We looked at the effects on local patterning and functionally tested whether putative epithelial stem cells can give rise to multiple cell types in the enamel organs of new teeth. Second generation teeth with enamel and dentine were removed from adult geckos. The dental lamina was either left intact or disrupted in order to interfere with local patterning cues. The dentition began to reform by 1 month and was nearly recovered by 2-3 months as shown in µCT scans and eruption of teeth labeled with fluorescent markers. Microscopic analysis showed that the dental lamina was fully healed by 1 month. The deepest parts of the dental lamina retained odontogenic identity as shown by PITX2 staining. A pulse-chase was carried out to label cells that were stimulated to enter the cell cycle and then would carry BrdU forward into subsequent tooth generations. Initially we labeled 70-78% of PCNA cells with BrdU. After a 1-month chase, the percentage of BrdU + PCNA labeled cells in the dental lamina had dropped to 10%, consistent with the dilution of the label. There was also a population of single, BrdU-labeled cells present up to 2 months post surgery. These BrdU-labeled cells were almost entirely located in the dental lamina and were the likely progenitor/stem cells because they had not entered the cell cycle. In contrast fragmented BrdU was seen in the PCNA-positive, proliferating enamel organs. Homeostasis and recovery of the gecko dentition was therefore mediated by a stable population of epithelial stem cells in the dental lamina.

RESUMEN

The three major enamel matrix proteins (EMPs): amelogenin (AMEL), ameloblastin (AMBN), and enamelin (ENAM), are intrinsically linked to tooth development in tetrapods. However, reptiles and mammals exhibit significant differences in dental patterning and development, potentially affecting how EMPs evolve in each group. In most reptiles, teeth are replaced continuously throughout life, while mammals have reduced replacement to only one or two generations. Reptiles also form structurally simple, aprismatic enamel while mammalian enamel is composed of highly organized hydroxyapatite prisms. These differences, combined with reported low sequence homology in reptiles, led us to predict that reptiles may experience lower selection pressure on their EMPs as compared with mammals. However, we found that like mammals, reptile EMPs are under moderate purifying selection, with some differences evident between AMEL, AMBN, and ENAM. We also demonstrate that sequence homology in reptile EMPs is closely associated with divergence times, with more recently diverged lineages exhibiting high homology, along with strong phylogenetic signal. Lastly, despite sequence divergence, none of the reptile species in our study exhibited mutations consistent with diseases that cause degeneration of enamel (e.g. amelogenesis imperfecta). Despite short tooth retention time and simplicity in enamel structure, reptile EMPs still exhibit purifying selection required to form durable enamel.

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To study tooth cycling in polyphyodont animals, we chose to work on alligators. Alligators have teeth in three phases of development at each tooth location. This assembly of three teeth is called a tooth family unit. As part of the study, in order to study tooth cycling in alligators, we wanted to know the configuration of the tooth family unit in every tooth position. From the surface of the mouth, this is difficult to assess. Therefore, we decided to use MicroCT which can image X-ray dense materials providing a three-dimensional view. MicroCT provided us with valuable information for this study. The method described below can be applied to study tooth cycling in other vertebrate species.

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Alligators have robust regenerative potential for tooth renewal. In contrast, extant mammals can either renew their teeth once (diphyodont dentition, as found in humans) or not at all (monophyodont dentition, present in mice). Previously, the authors used multiple mitotic labeling to map putative stem cells in alligator dental laminae, which contain quiescent odontogenic progenitors. The authors demonstrated that alligator tooth cycle initiation is related to ß-catenin/Wnt pathway activity in the dental lamina bulge. However, the molecular circuitry underlying the developmental progression of polyphyodont teeth remains elusive. Here, the authors used transcriptomic analyses to examine the additional molecular pathways related to the process of alligator tooth development. The authors collected juvenile alligator dental laminae at different developmental stages and performed RNA-seq. This data shows that Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) pathways are activated at the transition from pre-initiation stage (bud) to initiation stage (cap). Intriguingly, the activation of Wnt ligands, receptors and co-activators accompanies the inactivation of Wnt antagonists. In addition, the authors identified the molecular circuitry at different stages of tooth development. The authors conclude that multiple pathways are associated with specific stages of tooth development in the alligator. This data shows that Wnt pathway activation may play the most important role in the initiation of tooth development. This result may offer insight into ways to modulate the genetic controls involved in mammalian tooth renewal.

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The canonical (ß-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble Wnt inhibitor dickkopf1 (dkk1) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of Wnt signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axin1, mimic overexpression of Wnt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LiCl had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical Wnt pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of Wnt signaling in tooth development in polyphyodonts.