All multicellular organisms, from lizards to humans, must be able to repair and regrow damaged tissue. This includes not only healing after an injury, but also replacing parts of the body that suffer wear and tear. For example, many animals shed and replace worn out teeth throughout their life, but the number of times this occurs varies greatly between species.

Much of the understanding about how teeth grow and develop has come from researching mice. However, mice only develop one set of teeth, making them a poor ‘model’ for studying how species such as fish and reptiles can re-grow and replace their teeth. Recent studies of these species has shown that regenerating teeth relies on a specialised structure known as the dental lamina. In mice, the dental lamina forms but then quickly disappears, preventing new sets of teeth from developing. In most animals that regrow their teeth, however, the dental lamina keeps growing beyond the most recently produced tooth to create an area where its replacement will emerge. Now, Salomies et al. have identified other strategies involved in tooth replacement from studying the bearded dragon lizard, a rare example of an animal that continuously regenerates some, but not all, of its teeth.

Analysing the cells in different parts of the re-growing teeth from bearded dragon lizards revealed new features of the dental lamina. Specifically, Salomies et al. found that a previously uncharacterized set of genes within the dental lamina could determine whether or not a tooth will be replaced. Further experiments using microscope imaging revealed that bearded dragon lizards use two distinct groups of stem cells – specialised cells that have the potential to develop into various cell types in the body – to re-grow their teeth. These experiments demonstrate how the bearded dragon lizard uses a previously unknown mechanism to regenerate its teeth, combining elements used by gecko lizards and sharks.

These findings are an important step towards understanding the different strategies animals can use to maintain and regenerate their teeth. The knowledge gained could one day help design better therapies for patients suffering from inherited dental disorders or tooth loss.

Exposed to the oral cavity of most jawed vertebrates, teeth are continually worn down through food acquisition, a process that must be compensated to maintain the oral apparatus functional (Berkovitz and Shellis, 2016). The most common, and likely ancestral, solution to this problem is polyphyodonty, or continuous replacement of the dentition, through shedding of functional teeth (Jernvall and Thesleff, 2012; Tucker and Fraser, 2014). Whereas polyphyodonty prevails in the majority of vertebrate taxa, mammals show a substantial reduction in the number of tooth generations, a phenomenon often associated with increased dental complexity in this group (Kielan-Jaworowska et al., 2004). Particularly, the vast majority of mammals display a diphyodont dentition, producing only two sets of teeth—deciduous or ‘milk’ teeth and permanent replacement teeth. Rodents such as Mus musculus, one of the most commonly used vertebrate animal models, are even more extreme and develop only one generation of teeth, a condition known as monophyodonty (Jernvall and Thesleff, 2012). As a result, although mouse developmental studies of the past decades have revealed many of the signaling pathways controlling primary tooth development, relatively little is known about the molecular mechanisms underlying lifelong tooth regeneration in vertebrates.

Recent studies in polyphyodont and diphyodont species from all major vertebrate groups have shown that, with the exception of some teleost fish such as salmonids (Fraser et al., 2006; Jernvall and Thesleff, 2012; Vandenplas et al., 2016), tooth replacement is generally dependent on a specialized epithelial structure, the dental lamina (DL). This structure first emerges as a sheet of epithelial cells that invaginates from the oral epithelium (OE), and plays a crucial role in the developmental events contributing to primary tooth formation. The DL is maintained throughout the lifetime of polyphyodont species, and continues to grow beyond the previously generated tooth to create a free margin, the successional dental lamina (SDL), which constitutes the initiation site of replacement teeth. In monophyodont or diphyodont species such as mouse (Dosedělová et al., 2015), minipig (Buchtová et al., 2012), ferret (Jussila et al., 2014), and chameleon (Buchtová et al., 2013), both the DL and associated SDL degenerate relatively rapidly following formation of either one or two sets of teeth, thus highlighting the importance of maintaining these epithelial structures for tooth replacement. Coherent with this, the DL is considered to be the main source of putative odontogenic stem cells required for long-term tooth renewal (Huysseune and Thesleff, 2004; Smith et al., 2009; Jernvall and Thesleff, 2012; Juuri et al., 2013; Thiery et al., 2017), and label-retaining cells (LRCs) have been initially mapped to this region in the polyphyodont leopard gecko (Handrigan et al., 2010). More recently, putative stem/progenitor populations have been identified in the distal DL bulge of the American alligator (Wu et al., 2013), in the SDL and OE of sharks (Martin et al., 2016), as well as in the superficial DL of pufferfish (Thiery et al., 2017), thus indicating variations in dental regenerative strategies among polyphyodont species. Molecularly, compelling evidence from a number of reptilian species, teleost fish, shark, and ferret has revealed a conserved set of transcription factors and signaling molecules exhibiting similar expression patterns in the dental apparatus. In particular, the canonical Wnt pathway has emerged as a strong candidate signaling pathway associated with tooth replacement, because of its local activation (as revealed by the expression of Wnt/β-catenin target genes) in most studied polyphyodont and diphyodont species (Handrigan and Richman, 2010a; Fraser et al., 2013; Gaete and Tucker, 2013; Wu et al., 2013; Jussila et al., 2014; Rasch et al., 2016), but also because of its capacity to produce a large number of supernumerary teeth when activated in snakes and transgenic mice (Järvinen et al., 2006; Wang et al., 2009; Gaete and Tucker, 2013). Furthermore, stabilizing Wnt/β-catenin signaling was recently shown to induce formation of replacement tooth germs in the normally degenerating mouse SDL, thus providing further support for the important role of this pathway in controlling tooth induction and regeneration (Popa et al., 2019). However, only low-scale, single- or cross-species candidate gene approaches based on mouse expression patterns have been used so far to characterize polyphyodont dentitions, and the complete molecular signature of vertebrate tooth regeneration is still unknown.

In this study, we examined one agamid lizard model, the bearded dragon (Pogona vitticeps), which offers an excellent model system to study the events that define tooth development and regeneration because of its uncommon dentition with distinct modes of implantation and regenerative capacities along the same jaw—acrodont, monophyodont teeth posteriorly and pleurodont, polyphyodont teeth anteriorly (Cooper et al., 1970; Di-Poï and Milinkovitch, 2016). This lizard is one of the rare extant vertebrate species with such marked heterodonty, which allows direct comparisons of extreme types of vertebrate dentition in a single model. Unlike the dentition of other polyphyodont reptiles investigated so far, our data further indicate that bearded dragons possess a relatively slow replacement process (based on a ‘one-for-one’ strategy) as well as a permanent, proliferating SDL that only initiates replacement at anterior polyphyodont locations, making this model extremely attractive for elucidating the key importance of DL and SDL epithelial structures in vertebrate tooth initiation and regeneration. Indeed, we show here that the early degradation of the SDL is not the only mechanism restricting vertebrate tooth replacement (Buchtová et al., 2012; Dosedělová et al., 2015). Instead, our developmental data and large-scale transcriptomic analysis of microdissected dental tissues unveil the critical importance of SDL patterning at various levels, including directional growth but also gene expression levels, dynamics, and regionalization, for tooth regeneration. Particularly, we identify a large number of yet uncharacterized developmental genes associated with dental patterning in regenerating and non-regenerating teeth, so our lizard model is also uniquely suited for revealing new signaling pathways critical for tooth replacement in vertebrates. Additionally, our bearded dragon findings reveal a novel mechanism of continuous tooth replacement in vertebrates, which shares with the leopard gecko a source of putative odontogenic progenitor/stem cells in the DL but closely parallels the shark by the presence of a separate second source of cells originating from the OE. This novel dental replacement strategy definitely demonstrates that the bearded dragon provides a rare opportunity to expand our understanding of tooth developmental dynamics and diversity, while providing a unique model system to study the function and specialization of epithelial structures and dental progenitor/stem cell niches present in distinct groups of vertebrates. Altogether, and given the well-established history in captivity and key advantages of the bearded dragon lizard for research (Ollonen et al., 2018) this emerging model organism offers a powerful system to elucidate the developmental and genetic basis of both evolutionary novelty and tooth regeneration, thus allowing to answer both developmental and evolutionary questions critical for the design of more appropriate therapies targeted at congenital dental disorders and tooth loss in humans.

The lack of lifelong replacement of oral dentition in many different lineages including classical model organisms has hampered the analysis of tooth regeneration in vertebrates. Furthermore, studies investigating polyphyodont species have so far largely focused on single- or cross-species comparisons of selected candidate genes based on mouse expression patterns, and the exact molecular basis of tooth renewal is still unclear. Here, we show that the bearded dragon, one of the rare vertebrate species with both polyphyodont and monophyodont teeth, constitutes a key model for filling this gap, allowing direct comparison of different dentition types. As already shown in non-teleost polyphyodont species, our findings first confirm the critical importance of both DL and SDL structures in tooth renewal. However, in contrast to a previous hypothesis (Buchtová et al., 2012; Dosedělová et al., 2015), the early degradation of the SDL is not the only mechanism restricting vertebrate tooth replacement. Indeed, bearded dragons maintain a proliferative epithelial SDL structure even in non-replacing teeth, indicating that the SDL maintenance is independent of and not strictly associated with tooth renewal. These data also contrast with previous observations made in acrodont teeth from chameleons, the second family of acrodont lizards, where the SDL was shown to diminish in size at postnatal stages independently of increased apoptosis (Buchtová et al., 2012). The bearded dragon posterior dentition might thus represent an intermediary stage between polyphyodonty and complete monophyodonty, at least at the cellular level. In favor of that, and in contrast to chameleons, the implantation mode in most agamids is similar to intermediate pleurodont-acrodont dentition patterns observed in extinct acrodont iguanian lizards (Averianov et al., 1996; Simões et al., 2015). Haridy even recently proposed that the posterior tooth phenotype in bearded dragons might result from pleurodont to acrodont remodeling changes through ontogeny (Haridy, 2018). The latter hypothesis is coherent with our developmental data, as no clear morphological variations were observed in the two types of developing teeth, except for the length of the embryonic primary DL. The origin of such difference in the developing DL is unclear but might be linked to the delayed initiation of pleurodont teeth, when compared to acrodont teeth, which would extend the timing of DL proliferation. Importantly, differences were also noticed in the directional growth of SDL at hatchling and postnatal stages, with the pleurodont and acrodont SDL growing lingually or projecting more downwards towards the jaw bone, respectively. This differential directional SDL growth likely constitutes a key process to accommodate the replacement tooth, and was accompanied by an asymmetric expression pattern of conserved dental genes such as LEF1 and PITX2, but also of proliferation, towards the pleurodont SDL tip. Interestingly, focal localization of LEF1 expression and/or Wnt signaling activity was already noticed in the squamate SDL (Handrigan et al., 2010; Gaete and Tucker, 2013), alligator DL bulge (Wu et al., 2013), shark SDL (Rasch et al., 2016), and ferret SDL (Jussila et al., 2014). However, our large-scale transcriptomic analysis now indicates that a large number of yet uncharacterized developmental genes and signaling pathways are, in fact, likely associated with SDL outgrowth and/or induction. Furthermore, besides differences in gene expression dynamics and regionalization between pleurodont and acrodont dentitions, our data show drastic changes in gene expression levels in both SDL and surrounding mesenchymal tissues, indicating the importance of multi-level dental organization for initiation of tooth replacement. Along this line, the overall conserved expression levels of Wnt signaling members in our comparative pleurodont-acrodont transcriptome analysis suggest that this conserved pathway might primarily regulate SDL growth, a process maintained in bearded dragon acrodont dentition, rather than directly induce tooth replacement. This hypothesis is consistent with our observations that separate processes regulate SDL growth and initiation of replacement teeth, further indicating that SDL maintenance is not causatively associated with tooth replacement. Among newly identified developmental factors differentially regulated between monophyodont and polyphyodont dentitions, several genes encoding homeobox transcription factors were highly upregulated in regenerating teeth. For example, ALX1, a regulator of cartilage and craniofacial development in vertebrates (Zhao et al., 1993; Zhao et al., 1996; Dee et al., 2013), was one of the most highly upregulated genes in both pleurodont SDL and mesenchymal tissues, and it is likely that the severe and relatively early craniofacial phenotypes of ALX1 mutations (frontonasal dysplasia) before initiation of odontogenesis have hampered identification of this gene as a regulator of tooth development and/or replacement in mammals. Similarly, SIX3 plays crucial roles in the development of the vertebrate sensory system (Oliver et al., 1995; Loosli et al., 1999; Del Bene et al., 2004), but this gene has not yet been detected in the developing mouse dentition (Nonomura et al., 2010). Finally, ISL1 has been reported to be selectively expressed in the dental epithelium of mouse incisor, while being absent from molar epithelium (Mitsiadis et al., 2003), but this tooth-specific patterning has never been linked with continuous tooth growth or replacement in previous vertebrate studies. Based on the dynamic expression pattern observed in the developing epithelial and/or mesenchymal tissues, further investigation of these genes should provide key information on the signaling pathways controlling odontogenesis, including SDL outgrowth and/or initiation of replacement teeth.

As already suggested by several studies in polyphyodont species (Handrigan et al., 2010; Wu et al., 2013; Martin et al., 2016; Thiery et al., 2017), our identification of bearded dragon slow-cycling LRCs co-expressing stem/progenitor markers provides further evidence of the importance of epithelial stem/progenitor cell populations in regulating tooth renewal in vertebrates (Figure 7). However, the mechanism of continuous tooth replacement identified in our model differs from the leopard gecko, the first inspected lizard species (Handrigan et al., 2010), by the presence of two separate sources of putative stem/progenitor cells in the DL and OE (Figure 7), thus indicating alternative regenerative strategies and some specialization of stem/progenitor niches in squamates. Particularly, we show that migration from SOX2+ LRCs at the DL/OE junction contributes to the proliferative growth of DL and SDL structures, a process shared by both acrodont and pleurodont teeth (Figure 7). These findings support our hypothesis about the intermediate phenotype of bearded dragon acrodont dentition and closely parallel the regenerative dental strategy of sharks, which exhibit a similar LRC population at the taste/tooth junction (Figure 7). It is then tempting to speculate that SOX2+ OE cells of bearded dragons could also be bipotent, contributing to both taste and dental lineages (Martin et al., 2016). In support of this, SOX2 has been recently identified as a conserved marker of dental progenitors but also of taste bud and non-gustatory epithelial fate in vertebrates (Ohmoto et al., 2017). Furthermore, similarly to sharks, the OE of bearded dragons contains a heterogeneous population of cells, consisting of self-renewing epithelial progenitors and taste buds in close proximity to the DL (Figure 3A and data not shown). However, anatomical studies have revealed strong heterogeneity in the presence and location of taste buds in lizards and snakes (Uchida, 1980; Schwenk, 1985), indicating that this potential bipotency may not be a general mechanism conserved across squamates. It is also still unclear if the newly identified OE LRC population exists and contributes to tooth replacement in other reptiles, as the OE has not been analyzed in previous lizard and snake studies despite the strong SOX2 expression observed in this region (Juuri et al., 2013; Gaete and Tucker, 2013). The presence of one OE LRC population might also be directly linked to a specific tooth replacement rate and/or strategy in squamates, in a similar way as the heterogeneity in DL reported in teleosts with different replacement states (Fraser et al., 2006; Jernvall and Thesleff, 2012). Indeed, replacement of pleurodont teeth in agamid lizards is relatively slow (Cooper et al., 1970) and this study) and based on a ‘one-for-one’ strategy (a single replacement tooth is formed at any one time for a single tooth position), whereas many squamates exhibit a ‘many-for-one’ replacement state with several replacement teeth formed ahead of function at a single tooth position. In this context, the conservation of only one putative stem/progenitor cell population in the DL might constitute a more efficient positioning strategy for generating multiple tooth generations, as exemplified by the leopard gecko (Handrigan et al., 2010). Our characterization of the second source of LRCs in the bearded dragon pleurodont DL indicates striking similarities with the leopard gecko in terms of location, directly beside the lingually protruding SDL in the DDL, but also in terms of stem/progenitor marker expression profile, suggesting evolutionary conservation of this cell population at least among lizards (Figure 7). However, the exact function of putative stem/progenitor niches has remained unclear since their discovery in the leopard gecko. Our pleurodont-acrodont comparisons in bearded dragons now provide the first evidence of functional specialization of putative dental stem/progenitor populations in vertebrates. Specifically, we show that the DL population is likely strictly necessary for tooth replacement, as it is not detected in monophyodont bearded dragon teeth. The altered proliferation pattern, including lack of asymmetry and increased presence of BrdU-retaining cells, as well as differences in gene expression levels and location in the acrodont SDL suggest a key role for the DL population in the complex SDL organization required for initiation of tooth replacement (Figure 7). In contrast, the maintained OE population in the whole bearded dragon dentition contributes to SDL maintenance and growth, a process that is not causatively associated with initiation of tooth replacement.

Figure 7

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In conclusion, our results demonstrate that the various key dental features of the emerging bearded dragon model offer a powerful system to elucidate the developmental and genetic basis of evolutionary novelty and tooth regeneration in vertebrates. Particularly, the direct comparative analysis of pleurodont and acrodont dentitions indicates the importance of SDL patterning, in addition to maintenance, for vertebrate tooth replacement. This patterning process happens at various levels, including directional growth but also gene expression levels, dynamics, and regionalization, and involves a large number of yet uncharacterized developmental genes and signaling pathways. Finally, the two separate putative stem/progenitor populations identified in bearded dragon pleurodont teeth reveal a new vertebrate dental regenerative strategy associated with cell migration and functional specialization of putative stem/progenitor cells.

All Illumina reads have been deposited on Dryad Digital Repository under the link https://doi.org/10.5061/dryad.k66jn2s. Primers used for qPCR and ISH probes are available in the Key Resources Table. All other data generated or analyzed during this study are included in the manuscript and Supplementary file 1.

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Author details

1. Lotta Salomies

   Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1518-153X

2. Julia Eymann

   Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8147-9161

3. Imran Khan

   Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland

   Contribution

   Formal analysis, Funding acquisition, Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Nicolas Di-Poï

   Program in Developmental Biology, Institute of Biotechnology, University of Helsinki, Helsinki, Finland

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Investigation, Methodology, Project administration, Writing—review and editing

   For correspondence

   nicolas.di-poi@helsinki.fi

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3313-3016

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