An epithelial signalling centre in sharks supports homology of tooth morphogenesis in vertebrates
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Evaluation Summary:
Thiery et al. propose that the development of shark teeth employ a similar embryonic signaling center as the development of mammalian teeth. The implication is that the regulatory logic of tooth development is an ancient, shared feature among vertebrates. The research will be of interest to the developmental as well as evolutionary biology readers.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
Development of tooth shape is regulated by the enamel knot signalling centre, at least in mammals. Fgf signalling regulates differential proliferation between the enamel knot and adjacent dental epithelia during tooth development, leading to formation of the dental cusp. The presence of an enamel knot in non-mammalian vertebrates is debated given differences in signalling. Here, we show the conservation and restriction of fgf3, fgf10 , and shh to the sites of future dental cusps in the shark ( Scyliorhinus canicula ), whilst also highlighting striking differences between the shark and mouse. We reveal shifts in tooth size, shape, and cusp number following small molecule perturbations of canonical Wnt signalling. Resulting tooth phenotypes mirror observed effects in mammals, where canonical Wnt has been implicated as an upstream regulator of enamel knot signalling. In silico modelling of shark dental morphogenesis demonstrates how subtle changes in activatory and inhibitory signals can alter tooth shape, resembling developmental phenotypes and cusp shapes observed following experimental Wnt perturbation. Our results support the functional conservation of an enamel knot-like signalling centre throughout vertebrates and suggest that varied tooth types from sharks to mammals follow a similar developmental bauplan. Lineage-specific differences in signalling are not sufficient in refuting homology of this signalling centre, which is likely older than teeth themselves.
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Evaluation Summary:
Thiery et al. propose that the development of shark teeth employ a similar embryonic signaling center as the development of mammalian teeth. The implication is that the regulatory logic of tooth development is an ancient, shared feature among vertebrates. The research will be of interest to the developmental as well as evolutionary biology readers.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript prepared by Alexandre P. Thiery et al. is focused on enamel knot (EK) formation in the shark teeth. The authors brought here evidence of the existence of an enamel knot-like structure in this non-mammalian species showing its role on regulation of the tooth shape and cusps formation similarly to the mammalian EK. Shark tooth development has been previously used as an alternative model for odontogenesis by authors and there is no doubt that it offers valuable tools for research on the tooth regeneration through the formation of successional generations of teeth as well as on serial organ formation (activation and inhibition processes during tooth development). In silico modelling is properly used by authors in this present work and it serves here as a valuable tool documenting the experimental …
Reviewer #1 (Public Review):
The manuscript prepared by Alexandre P. Thiery et al. is focused on enamel knot (EK) formation in the shark teeth. The authors brought here evidence of the existence of an enamel knot-like structure in this non-mammalian species showing its role on regulation of the tooth shape and cusps formation similarly to the mammalian EK. Shark tooth development has been previously used as an alternative model for odontogenesis by authors and there is no doubt that it offers valuable tools for research on the tooth regeneration through the formation of successional generations of teeth as well as on serial organ formation (activation and inhibition processes during tooth development). In silico modelling is properly used by authors in this present work and it serves here as a valuable tool documenting the experimental results and confirming formulated conclusions.
This manuscript brings very interesting results showing that EK formation is highly conserved in the animal species, and it is not a structure unique only for the mammalian tooth development. The authors mapped the expressions of mammalian EK markers in details in the shark teeth to compare the expressions of genes in both mammalian and non-mammalian signalling centres. Interestingly they documented the expression of Fgf10 gene expressed in several epithelial cells at the tip of the dental germ in the catshark. Since Fgf10 is not expressed in EK in the mouse but it has been shown to be expressed in opossum, this could document the phylogenetic adaptation of EK on the way to the mammalian dentition. The manuscript also showed similarities of the enamel knot-like structure morphology with the mammalian EK (eg. the presence of non-proliferative cells detected by PCNA in this structure). In contrast to mammalian EK no apoptosis has been detected in the enamel-knot-like structure in the shark, what is however not preventing the formation of tooth cusps at all.
The authors furtherly focused on the important role of Wnt signalling during tooth cusps formation. Using experimental manipulation of the canonical Wnt pathway by small molecule activators and inhibitors they documented the role of Wnt signalling during tooth-crown morphogenesis, concretely in the cusp number determination. Upregulated Wnt signalling caused a higher number of cusps in the shark teeth and downregulation in contrast lead to the lower number of cusps or to unicusped teeth formation. The experiments were modelled using in silico model of tooth development (ToothMaker). The biomodelling confirmed the data obtained using manipulation experiments.
Based on these approaches the authors concluded that the enamel knot-like structure in the shark shows a role on regulation of the tooth shape and cusps formation similarly to the mammalian EK. According to obtained results the authors have stated that phylogenetically EK seems to serve as a general organizer of the shape (morphology) maintaining its function throughout the species evolution. They also suggest here an introduction of the term "apical enamel knot" as a more general signalling centre present not only in teeth.
The present manuscript does not have any substantial weaknesses. However, few points need to be addressed and discussed furtherly.
The authors claimed a difference between early and new generations of teeth in the catshark during development. The early generation develops more superficially in contrast to deeper ingrowing germs of additional generation of teeth. Interestingly, in the lower mouse incisor region it has been documented that the first and initial tooth placode at early stages of the development with its own signalling centre is also formed more superficially in contrast to the subsequently appearing pEK located deeper in the epithelium of the incisor germ. The authors should compare the expressions of especially epithelial markers shown to play a role in the first row of teeth with later appearing teeth in the catshark and discuss this comparison with respect to the knowledge of these two signalling centres with different roles in the mouse.
The authors used the computational in silico model, where the regulation of the inhibitor and activator of the tooth stems from its EK signalling centre. The authors claim in the manuscript that the tooth size and number of cusps is increasing in the new generations of dentition in the catshark, what they relate to the increasing initial site of the tooth formation and what also corresponded with the stimulated model situation. This could be probably connected to the growth of the jaws which gives more space between single signalling centres. Following this space expansion, the changes of the activation and inhibition (of activators and inhibitors levels defunding between neighbouring teeth in the tooth-row as well as between the tooth generations) could be the cause of this change in size and shape. This should be discussed more with respect to what is known in the mouse.
The authors documented that Shh, which is a key marker of EK in mammals including mice, is expressed within the apical dental epithelium in the catshark. However, Shh expression was downregulated between cusps within the inter-cusp dental epithelium. According to the Figures E, F the Shh expression seems to be region specific, and it changes antero-posteriorly. It could reflect the antero-posterior appearance of the tooth germs. The posterior ones would be less advanced and thus Shh expression in the anterior part of the tooth-row would be upregulated in fact. This should be discussed.
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Reviewer #2 (Public Review):
The "enamel knot signaling center" is an organizer of tooth morphogenesis first characterized in mammalian teeth. The so-called "primary enamel knot" corresponds to non-cycling epithelial cells sitting at the tip of the first forming cusp. "Secondary enamel knots" are found at the tip of the other cusps in multicuspidated teeth. There is a debate in the community whether homologous structures exist or not in non-mammalian vertebrates. The authors looked for an "enamel knot signaling center" in the developing teeth of catshark. The bauplan of catshark teeth resemble some mammalian teeth, like seal teeth: a primary, more or less central and tall cusp, is arranged on a line with smaller secondary cusps (3-cusps teeth), and sometimes with even smaller tertiary cusps (5-cusps teeth).
The authors first report the …
Reviewer #2 (Public Review):
The "enamel knot signaling center" is an organizer of tooth morphogenesis first characterized in mammalian teeth. The so-called "primary enamel knot" corresponds to non-cycling epithelial cells sitting at the tip of the first forming cusp. "Secondary enamel knots" are found at the tip of the other cusps in multicuspidated teeth. There is a debate in the community whether homologous structures exist or not in non-mammalian vertebrates. The authors looked for an "enamel knot signaling center" in the developing teeth of catshark. The bauplan of catshark teeth resemble some mammalian teeth, like seal teeth: a primary, more or less central and tall cusp, is arranged on a line with smaller secondary cusps (3-cusps teeth), and sometimes with even smaller tertiary cusps (5-cusps teeth).
The authors first report the expression pattern in catshark developing teeth for a selection of signaling genes. They emphasize Fgf3 and Fgf10, which they found expressed in the epithelial cells sitting at the tip of the primary and secondary cusps. This reminds the enamel knot expression of Fgf3 in mouse and opossum and Fgf10 in opossum only. BrdU staining showed that this cusp-tip expression coincides with proliferation arrest of epithelial cells from cusp tip down into the valleys, as seen in mammals. Because Wnt signaling is important for enamel knot formation in mammals, they perturbed this pathway with pharmacological treatments. Inhibiting the pathway reduced tooth size and cusp number whereas activating it increased tooth size and cusp number. The authors also played with a model built for seal teeth, in which activation-inhibition mechanisms rule the formation of enamel knots, and morphogenesis and differentiation proceeds tip-down starting from these enamel knot. Catshark teeth have a similar organization like seal teeth, as mentioned above, and a similar development: tip-down, with the primary cusp developing first and the secondary cusp developing in a second time. The model built for seal teeth therefore not surprisingly can form catshark-looking teeth, and playing with activation-inhibition parameters superficially recapitulates the increase or decrease in cusp number seen in experimental perturbations as well as the variability in tooth shape seen at different positions and/or lifetime in catshark. Shark-specific aspects of perturbed teeth are however not recapitulated by the model. The authors conclude that catshark teeth possess a signaling center homologous to the enamel knots in mammals and based on gene expression similarities at the tip of developing dermal denticles (notably of Fgf3), propose a deep homology with an apical signaling center preceding tooth evolution.
Strengths:
- The authors combined experiments: gene expression pattern for many genes, BrdU incorporation proliferation pattern, functional perturbation of a key pathway, some are tricky in such non-model species.
- The experiments combining both over-activation and reduction of Wnt signaling pathway clearly demonstrate a role in promoting tooth growth and cusp formation.
- The effect of treatments was quantified with 2D-outline analysis and statistics.
- Using a mammalian model of tooth development is interesting, but it has to be dissected and challenged in its principles to make sure its use is correct and not based on superficial similarities.
Weaknesses:
- Clearly, there is distinctive gene expression at the tip (even though it tends to then progress tip-down for several markers, rather than staying expressed only at the tip), proliferation arrest goes tip-down, and normal Wnt activity is necessary to reach proper cusp number and tooth morphology. But there is no functional demonstration that signaling from the tip controls cusp number, tooth morphology nor tip-down proliferation arrest. As a consequence, the data support the tip-down development of shark teeth with some critical involvement of the Wnt pathway more than the development of shark teeth from an apical signaling center.
- The model only very superficially recapitulates the experimental perturbation: reducing Wnt activity produces a very short unicuspid tooth, much shorter than the wild type primary tooth; the model produces a single large cusp, larger than in the wild type. Increasing Wnt activity produces a tiny accessory cusp, as predicted by the model but also large cusps, that do not fit at all (figure F). It is thus very unclear if the principles of the model hold true for shark, beyond the general principle of sequential and -down development. Moreover it is also not sufficient to state that the Wnt pathway plays the role of the activator/inhibitor, and this role only. Overall, too many shortcuts are taken, from a superficial resemblance of perturbed teeth to the Wnt pathway as "the activator", and from Wnt signaling as the activator, to a Wnt-centered view of tooth shape variation across tooth position and developmental time.
- Before looking for homology, it is necessary to define precisely the criteria that should be met, and especially those that can make the difference between homology and homoplasy. Such clear definitions are lacking. Mammalian teeth and shark teeth both develop on a tip-down principle: is it surprising that proliferation arrest goes tip-down, or would this be seen in any structure developing tip-down, including by pure homoplasy instead of shared ancestrality? What would be the minimal specifications to be met by an homologous apical signaling center (in terms of signaling, proliferation state, effect on tissue around...)? What similarities would be unsufficient? This is not clearly stated.
- When comparing developing organs in different species, it is common to find a mosaic of conserved and divergent patterns. It is therefore important to decide in advance, what are the most relevant genes, and what would be the minimal requirement (quantitative and/or qualitative) to decide that a given, well-defined, developmental process is homologous.
- Apical signaling centers organizing morphogenesis are found not only in dermal denticles, but are also found in many epithelial organs. It is therefore necessary to identify precise criteria to distinguish deep homology of dermal denticles and teeth from deep homology of developmental principles.
In conclusion, the authors provide a nice piece of data, including functional experiments not easily done in a non-standard model like catshark. These data suggest that teeth are developing tip-down and that signaling molecules are found at the tip of the primary and secondary cusps. However, it is not always clear how far these molecules are specifically emitted at the tip or are simply expressed by a developmentally more advanced tissue (e.g. with expression later progressing tip-down as differentiation proceeds). Moreover the functional experiments do not directly demonstrate that this putative signaling center, or the mechanisms patterning this signaling center, is/are responsible for cusp patterning and tooth morphogenesis. The link is done through the use of a mammalian model of tooth morphogenesis, which only superficially recapitulates perturbed teeth morphology. This strongly bias interpretations towards homology, and alternative scenarios are not explored. A more precise theoretical and experimental framework would be needed to support the conclusion and reach a broad public.
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Reviewer #3 (Public Review):
Perhaps because teeth are so widely distributed among the vertebrates, there has been considerable interest in the evolutionary origin of teeth. A related question is the regulatory logic, or developmental basis of tooth development. It is well established that tooth development from fish to mammals uses largely the same set of developmental genes. Similarly, early stages of development are quite similar among all the epithelial organs (e.g., teeth hair, glands, scales). In this context the novelty of the new work is in asking whether even the regulatory logic of teeth is a shared, evolutionary ancient feature. Thiery et al. address this question by studying multicusped teeth of sharks. Multicusped, complex tooth morphologies are prevalent in mammals, and their development is thought to require precise …
Reviewer #3 (Public Review):
Perhaps because teeth are so widely distributed among the vertebrates, there has been considerable interest in the evolutionary origin of teeth. A related question is the regulatory logic, or developmental basis of tooth development. It is well established that tooth development from fish to mammals uses largely the same set of developmental genes. Similarly, early stages of development are quite similar among all the epithelial organs (e.g., teeth hair, glands, scales). In this context the novelty of the new work is in asking whether even the regulatory logic of teeth is a shared, evolutionary ancient feature. Thiery et al. address this question by studying multicusped teeth of sharks. Multicusped, complex tooth morphologies are prevalent in mammals, and their development is thought to require precise regulation by epithelial signaling centers, called the enamel knots. Previously enamel knots have been thought to be restricted to mammals, but Thiery et al. argue that also developing shark teeth have them. Their evidence to support the presence of enamel knots in sharks are 1) gene expression patterns of several genes known to be dynamically expressed in the mammalian enamel knots and teeth, 2) experimental modulation of Wnt-signaling in developing sharks to see the phenotypic changes in tooth shape, and 3) computational modeling to test whether shark tooth development obeys similar regulatory logic as mammalian teeth. My overall inference from the evidence provided is that the basic thesis is quite convincing, and definitely worth publishing. There are, however, quite a few in individual components in the work that require clarification, and some statements are simply over the top.
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