Diversification of the dentition has been instrumental in the success of vertebrates. The dentition has become highly adapted to its respective environmental niche, and as a result has given rise to a plethora of unusual dental forms (Evans et al., 2007; Fraser et al., 2008; Hulsey et al., 2020; Jernvall and Thesleff, 2012; Kolmann et al., 2019; Thiery et al., 2017; Tucker and Fraser, 2014). It is generally observed that there is an overall increase in dental morphological complexity throughout evolution, culminating in mammals which possess multiple regionalised tooth types (heterodonty) (Jernvall and Thesleff, 2012; Kavanagh et al., 2007; Tucker and Fraser, 2014). A trade-off between tooth morphological complexity and dental regenerative ability is thought to exist within the mammalian lineage, with precise occlusion favoured at the expense of dental regeneration (Jernvall and Thesleff, 2012). The study of dental development has mostly focussed on transcriptional regulators, which mediate growth, morphogenesis, and cellular differentiation. Dental development is tightly regulated via interactions between the oral epithelium and underlying neural-crest derived mesenchyme (Zhang et al., 2005). Canonical Wnt, fibroblast growth factor (Fgf), hedgehog (Hh), bone morphogenetic protein (Bmp), and Notch signalling pathways have all been implicated as important regulators of tooth development and morphogenesis (Thesleff and Sharpe, 1997). Importantly, a unifying characteristic for the progression of vertebrate tooth development is the high level of cellular expression of developmental keystone genes (Hallikas et al., 2021). This suggests an essential core gene set (Fraser et al., 2010; Rasch et al., 2016) and their interactive signalling might be robust evolutionarily and highly conserved, capable of shape modifications of a conserved core vertebrate unit – the tooth.

Teeth begin their development from an initial epithelial placode, which proceeds to proliferate and grow in size during the subsequent bud stage. Following the bud stage, the tooth enters the cap stage, which marks the onset of morphogenesis. Morphogenesis of epithelial appendages coincides with a change in shape of the epithelial placode. This process can be driven via differential proliferation rates (i.e., teeth Jernvall et al., 1994; Salazar-Ciudad and Jernvall, 2010), cell shape changes (i.e., intestinal crypt Sumigray et al., 2018), or cell migration (i.e., hair Ahtiainen et al., 2014). Signalling molecules drive reciprocal epithelial to mesenchymal signalling and regulate the morphogenesis of epithelial appendages (Mustonen et al., 2002; Thesleff and Sharpe, 1997).

During morphogenesis, the tooth acquires its defining morphological feature: the dental cusp. In mammals, dental cusps are regulated by an epithelial signalling centre found at the tip of the first cusp, known as the primary enamel knot (EK). The EK is molecularly identifiable as non-proliferative and expresses Wnt, Bmp, Fgf, and Hh markers (Jernvall and Thesleff, 2012). Fgf signalling drives differential proliferation between the EK and rapidly proliferating adjacent dental epithelium, leading to folding of the epithelium at the site of dental cusps and the formation of the final tooth shape (Jernvall et al., 1998; Jernvall et al., 1994; Salazar-Ciudad and Jernvall, 2010). During the final stages of dental morphogenesis, the EK dramatically reduces in size as cells undergo apoptosis (Jernvall et al., 1998). In multi-cuspid teeth, such as mammalian molars, subsequent signalling centres termed secondary enamel knots (SEK) form at the site of each extra cusp and are thought to be induced by the EK. Folding of the dental epithelium between primary and secondary EKs leads to formation of cervical loops in-between each cusp (Jernvall et al., 1994; Salazar-Ciudad, 2012). Most of our understanding of tooth shape comes directly from the study of mammalian molar cusp development. Whilst it is known that EK signalling centres are found throughout mammals, their presence in other vertebrates is less clear.

The Wnt/β-catenin signalling pathway appears to govern a variety of events during tooth development from initiation to morphogenesis. Canonical Wnt signalling is active in major cellular contributions of the developing tooth, with varying roles in the mesenchyme and epithelium. For example, epithelial activation of Wnt/β-catenin signalling results in continuous initiation of new tooth units in mice; however, activation of Wnt signalling in the dental mesenchyme has an opposite effect, inhibiting sequential tooth emergence (Järvinen et al., 2018). Whilst over a dozen markers have been described within the mammalian EK, Wnt signalling appears to be a primary upstream regulator of EK patterning (Ahtiainen et al., 2016; Järvinen et al., 2018). Given this association with Wnt signalling and the control of tooth shape, including EK patterning, it seems appropriate to investigate further upstream effects of Wnt perturbation in alternative vertebrate models (Fraser et al., 2013; Richman and Handrigan, 2011). Lef1-/- mutants exhibit arrested tooth development during bud stage, with the tooth cap and associated cusps failing to both form and express EK markers, including Fgf4, Shh, and Bmp4 (Kratochwil et al., 2002). Fgf4 is capable of rescuing tooth development in Lef1-/- mutants (Kratochwil et al., 2002). Furthermore, the inhibition of Wnt signalling during early bud stage via ectopic expression of the Wnt antagonist Dickkopf-related protein 1 (Dkk1) leads to blunted cusps and a reduction in Bmp4 signalling (Liu et al., 2008). These results suggest that Wnt signalling falls upstream of Fgf and Bmp signalling in the EK. However, more complex feedback loops are involved, as mesenchymal-specific knock-down of Bmp4 (Bmp4ncko/ncko) also leads to a reduction in Lef1 expression (Jia et al., 2016).

In the subset of reptiles studied to date, there appears to be no clear histologically definable EK (Buchtová et al., 2008; Weeks et al., 2013), although in some species there is a thickening of the inner dental epithelium which leads to the asymmetric deposition of enamel and the formation of cusps (Zahradnicek et al., 2014). Furthermore, whilst in reptiles, cells of the inner dental epithelium are non-proliferative, this region is not as highly restricted to the cusp as in mammalian molars (Buchtová et al., 2008; Handrigan and Richman, 2011). However, there is conservation of signalling within an EK-like signalling centre (Richman and Handrigan, 2011). Similar EK-like signalling centres have been described in teleosts, with chemical perturbation of these signalling pathways resulting in shifts in cusp number (Fraser et al., 2013). Sharks, which are basal crown gnathostomes, also possess a variety of tooth types, with clearly defined cusps. In sharks and rays, there is a region of non-proliferative cells within the apical dental epithelium thought to be associated with the primary cusp (i.e., the primary enameloid knot; Rasch et al., 2020; Rasch et al., 2016), however the presence of a definitive EK has not yet reached a consensus (Debiais-Thibaud et al., 2015), and some have even argued that the EK is uniquely a mammalian innovation (Handrigan and Richman, 2011; Handrigan and Richman, 2010; Richman and Handrigan, 2011; Weeks et al., 2013).

Sharks and rays (elasmobranchs) represent two major subdivisions of the cartilaginous fishes, and house oral teeth in both the upper and lower jaws, and also possess tooth-like structures embedded within the skin, made of dentine and enamel-like (enameloid) tissues (Figure 1A). The diversity in tooth shape in the elasmobranchs is vast, ranging from flattened tooth units associated with crushing prey items, to multi-cuspid and serrated tooth units necessary for cutting (Jambura et al., 2020). The presence of multiple cusps as a standard tooth form for most sharks suggests conserved developmental control of tooth cusp morphogenesis across vertebrates. Given the conservation of general tooth development among vertebrates (Fraser et al., 2004; Martin et al., 2016; Rasch et al., 2020; Rasch et al., 2016; Thiery et al., 2017; Weeks et al., 2013), it seems appropriate that the epithelial and mesenchymal signals regulating dental morphogenesis would also be conserved, including the EK signalling centre. This would imply that this crucial tooth signalling centre is older than the mammalian clade. Therefore, due to the vast array of dental phenotypes in sharks and their position in the wider context of vertebrate phylogeny, we employ the shark as a developmental model for non-mammalian tooth morphogenesis. The small spotted catshark (Scyliorhinus canicula) has recently become a popular model for the study of developmental biology, including tooth and skin appendage development (Berio et al., 2021; Cooper et al., 2018; Cooper et al., 2017; Debiais-Thibaud et al., 2015; Martin et al., 2016; Rasch et al., 2016). In addition to the oral dentition (Figure 1B, C), sharks house a unique set of tooth-like denticles embedded in the skin (Figure 1A; Cooper et al., 2018), which also display a vast diversity of cusp shapes (Ankhelyi et al., 2018; Gabler-Smith et al., 2021; Sibert and Rubin, 2021).

Figure 1

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In order to determine the extent of signalling conservation between mammals and sharks during dental morphogenesis, we have documented the expression of mammalian EK markers in the small-spotted catshark, S. canicula. Furthermore, given the importance of Wnt signalling during dental morphogenesis and cusp formation in mammals, we sought to perturb Wnt signalling and determine its function during catshark tooth development. Following small molecule Wnt signalling perturbation, we identify shifts in tooth shape, size, and cusp number, and model the resulting phenotypes in silico using the ‘ToothMaker’ programme (Salazar-Ciudad and Jernvall, 2010; Savriama et al., 2018). Our results reveal that despite differences in molecular signalling between the catshark and mammals, the fundamental components of the EK signalling centre are highly conserved. Therefore, EK signalling at the apex of the developing tooth cusp is a vertebrate innovation and contributes to the vast range of vertebrate dental phenotypes.

Overall, our results demonstrate that signalling centres comparable to mammalian EKs are present in the shark during tooth morphogenesis. Our data implies that these dental signalling centres function to generate cusps in the shark. Although, how functionally similar these are to mammalian EKs requires further investigation. We identify restricted expression of Fgf markers within the non-proliferative apical dental epithelium corresponding to the same patterns of expression in the mammalian EK. We highlight shifts in cusp number and tooth shape following canonical Wnt manipulation, with similar results simulated via in silico modelling. Of course, care should be taken when interpreting the results of a model compared to real biological signalling, where a model cannot simply account for the complexity of a single pathway. Overall our data imply that a common set of developmental principles account for tooth morphogenesis between mammals and sharks. These common principles include the tip-down development of teeth from an apical signalling centre, and we suggest that Wnt signalling may take part in the activation-inhibition mechanisms that influence cusp patterning related to this signalling centre. Therefore, we suggest that the basic developmental principles of mammalian tooth morphogenesis are shared with sharks, and that perhaps shark tooth shape variation can be modelled with the same developmental principles. Future work will address whether there are indeed shared functional relationships between the mammalian and shark dental signalling centres.

Sharks possess a comparable EK-like signalling centre to the mammalian EK

Low levels of proliferation at the apical tip of the developing tooth have hinted at the presence of an EK in sharks (Rasch et al., 2016), although 3D restriction of signalling molecules within a distinct signalling centre is yet to be described. We note the expression of fgf10 and the canonical Wnt inhibitor dkk1 within the non-proliferative dental epithelium (Figure 4). Furthermore, our whole mount in situ hybridisation data reveals restricted upregulation of fgf3 and fgf10 within both primary (Figure 2) and secondary cusp regions (Figure 5) and therefore the presence of a spatially restricted dental epithelial signalling centre. In mammals, the expression of EK markers precedes dental shape change (Vaahtokari et al., 1996). Similarly, we observe the restricted expression of fgf3 (Figure 3F), fgf10 (Figure 4A), and shh (Figure 2), very early during dental morphogenesis, prior to the establishment of the overall tooth shape. This suggests that signalling precedes dental shape change and suggests that Fgf and Hh signalling may be driving this process in sharks, as in mammals (Jernvall et al., 1994; Kettunen et al., 2000).

Even at the earliest initiation of teeth in S. canicula, an initial, restricted expression pattern of these three markers (fgf3, fgf10, and shh) demarcates the emergence of the superficial first-generation tooth in the shark (Figure 2; Figure 10). This superficial and specific unit of epithelial expression appears to resemble initiation of the incisor tooth in the mouse, especially at stages E12.5–13.5 (Ahtiainen et al., 2016; Du et al., 2017; Hovorakova et al., 2016). The similarity between the superficial expression of fgf3, fgf10, and shh in the first-generation tooth in the shark (Figure 2) compared to the initiation knot (IK), in the incisor region of the mouse, is striking. In the mouse, the IK is an initial signalling centre similar to the primary EK, that marks a defining position between what will become the epithelial prominences of the vestibular lamina and the dental lamina (i.e., the incisor tooth bud; Du et al., 2017; Hovorakova et al., 2016). Soon after the expression of Shh related to the IK in mice, the primary EK forms separately at the cap stage in the incisor tooth bud (Du et al., 2017). A vestibular lamina (the division between the epithelial tissues of the lips and teeth in mammals) does not form in the shark, at least not in S. canicula, however, other fish species, for example pufferfish, do have a cleft that separates teeth and fleshy lips (Thiery et al., 2017). In the shark, the superficial and initial EK-like structure in the first tooth generation appears similar to the superficial IK in mice. Further investigation of EK markers prior to dental morphogenesis is key to discerning the role and regulation of EK-driven dental morphogenesis in the shark.

Figure 10

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Importantly, the mammalian EK itself is unable to respond to the Fgf signals, which it emits as it lacks Fgf receptors (Kettunen et al., 1998). Instead, these receptors are found within adjacent dental epithelial cells, and are important for the induction of differential proliferation between the EK and surrounding epithelial tissue (Kettunen et al., 2000). Further research is needed to identify whether this lack of receptors predates the evolution of the mammalian dentition and whether it is fundamentally required for the formation of cusped teeth. Given the conservation of localised Fgf signalling within the shark EK, we hypothesise that a similar lack of receptors drives differential proliferation between the EK and surrounding dental epithelium in the basal gnathostome lineage.

Various developmental characteristics have been used to define the presence of an EK, including spatial restriction of the signalling centre, a lack of cell proliferation, and apoptosis of the epithelial cells within the EK. Apoptosis has been implicated as an important developmental process in regulating the silencing of embryonic signalling centres (Kettunen et al., 2000; Vaahtokari et al., 1996). Apoptosis has been described in both primary and secondary mammalian EK through TUNEL assays and the expression of the pre-apoptotic marker, p21 (Jernvall et al., 1998; Vaahtokari et al., 1996). Such assays have so far revealed a lack of apoptosis within the inner dental epithelium of reptiles (Buchtová et al., 2008; Handrigan and Richman, 2010) and the catshark (Debiais-Thibaud et al., 2015) – this has been used to refute the presence of an EK altogether (Debiais-Thibaud et al., 2015). However, it has also been shown that mice develop cusps following the loss of apoptosis in caspase-3 deficient mice (Matalova et al., 2006). Although morphological defects are identified in non-apoptotic mouse molars (Matalova et al., 2006), these results suggest that apoptosis is not fundamentally required in the formation of dental cusps (Richman and Handrigan, 2011).

Whilst there are observable differences in signalling between the mammalian and chondrichthyan EKs (Debiais-Thibaud et al., 2015), this does not refute the presence of an EK in sharks. Instead, these differences highlight potential lineage-specific modifications which have led to the diversification of vertebrate dental morphology. Given the presence of enameloid, as opposed to ‘true’ enamel within the Chondrichthyes (Gillis and Donoghue, 2007), it has been proposed to term this signalling centre in sharks the ‘enameloid knot’ (Rasch et al., 2016). Our results reveal a complete lack of bmp4 expression within the non-proliferative apical dental epithelium. bmp4 expression in surrounding tissues suggests that unlike in mammals, where Bmp4 is secondarily upregulated within the EK, bmp4 may play a role in restricting the expression of other signalling molecules to the EK in the shark. Furthermore, Shh is a key marker of the EK in mammals. Although we note its expression within the apical dental epithelium, there is little downregulation of its expression within the inter-cusp dental epithelium. Asymmetric Shh expression is thought to regulate polarised growth of the developing feather bud (Ting-Berreth and Chuong, 1996) as it does in the zone of polarising activity within the vertebrate limb bud (Riddle et al., 1993). It is possible that shh is playing a similar role in teeth of sharks; shh may be regulating polarised growth of the tooth along the oral/aboral axis, but not the medial/lateral axis along which the cusps form. Despite differences in the gene-specific expression patterns of the mammalian and chondrichthyan EKs, representative markers of canonical Wnt, Fgf, Bmp, and Hh signalling are all found expressed within the apical dental epithelium.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Alexandre P Thiery

   1. Department of Animal and Plant Sciences, University of Sheffield, Sheffield, United Kingdom
   2. Department of Craniofacial Development and Stem Cell Biology, King’s College London, London, United Kingdom

   Contribution

   Conceptualization, Investigation, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Ariane SI Standing

   Department of Biology, University of Florida, Gainesville, United States

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

3. Rory L Cooper

   1. Department of Animal and Plant Sciences, University of Sheffield, Sheffield, United Kingdom
   2. Department of Genetics and Evolution, University of Geneva, Geneva, Switzerland

   Contribution

   Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0172-4708

4. Gareth J Fraser

   Department of Biology, University of Florida, Gainesville, United States

   Contribution

   Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Visualization, Writing – review and editing

   For correspondence

   g.fraser@ufl.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7376-0962

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