The developmental basis for scaling of mammalian tooth size

Mona M. Christensen, Outi Hallikas, Rishi Das Roy, Vilma Väänänen, Otto E. Stenberg, Teemu J. Häkkinen, Jean-Christophe François, Robert J. Asher, Ophir D. Klein, Martin Holzenberger, Jukka Jernvall

Preprint posted on 3 December 2022

Super-size me: how do mammals scale up their teeth

Selected by Alexa Sadier


From the teeny-tiny 30 mm long bumblebee bat to the giant 30 m long blue whale, mammals are extremely diverse in term of body size (Fig. 1). Despite this huge diversity in size, their overall body plan and many of their organs look the same, just at a different scale: some species look like miniature versions of others, and their organs are scaled to the organism body size. While many body parts exhibit such scaling in mammals, we still have a limited understating of the mechanisms by which species scale their organs while maintaining their overall organization and form. When does scaling happen during development? Are organs patterned first and then grow in size, or are they larger since the very first steps of their development? Are certain developmental steps more important than others for scaling body parts? And finally, how are bigger organs patterned with more cells and tissue?

Figure 1: Scaling animals
A: Mammalian sizes range from the tiny bumblebee bat to the blue whale
B: Molar scaling: from the tiny nectar bat to the enormous mastodon


To study some of these questions, the authors chose the first lower molar as a model system. Molars can be found in small and big mammals (e.g. bats, mice, elephants, opossums, dolphins, tigers), including model organisms (1). Thanks to the extensive body of research performed on mice molars (2), the different steps of tooth development, as well as the gene regulatory network controlling these steps are well understood, making it timely to study their variation in other species. Finally, thanks to museum collections rich in extant and extinct species, the ability to culture molars in vitro, and the existence of a computation model of tooth development, the results found in model organisms can be expanded to other mammals.


Key findings

To study the mechanisms behind scaling, the authors compared the development of the mouse and rat first lower molars, by combining in situ hybridization and 3D reconstruction from micro-computerised tomography (μCT) scans (Fig. 2). Mouse and rat molars exhibit a similar organization but rat molars are twice as big as mice molars.

The patterning of molars is similar between species of different sizes They found that tooth size starts to slightly diverge early during development, at the beginning of tooth formation and morphogenesis (from the “Bud” to “Cap” stages). Later, during the formation of the crown and the cusps, this difference increases despite both species developing at the same rate. Together, these results demonstrate that scaling happens during the whole patterning of teeth, right after the initiation stage, implying a scaling of the patterning process itself. In other words, the patterning of the molar remains similar in both species, independent of the amount of tissue to pattern.


Figure 2: Determining when teeth are scaled during development A, First lower molars of the mouse and the rat are similar in overall shape, but the rat molar is two times larger in linear dimensions. Occlusal views, anterior to the left, buccal to the top. Scale bar, 500 μm. B, When and how during tooth development the scaling process occurs is not known. Tooth development is regulated by the interactions between the epithelial (blue) and mesenchymal (yellow) tissues. After mineralization and eruption, crown shape cannot be remodeled.


Tooth size expansion and patterning can be explained by a higher expression of IGF1
To investigate the mechanisms by which tooth patterning scales, the authors used a combination of in situ hybridization, RNAseq, tooth culture experiments, and mouse mutants to respectively: follow the patterning of key structures of the teeth such as the cusps, identify candidate genes that could trigger the differences between mouse and rat development, and test these candidates functionally by adding or blocking specific pathways during tooth development in vitro and in vivo (Fig. 3).


Figure 3: Scaling of patterning involves changes in tooth size and spacing of signaling centers A, Secondary enamel knots visualized using in situ hybridization of Fgf4 expression in the mouse and rat molar. B, The rat molar is larger (size shown with white points, n = 8), and the secondary enamel knots are more spread apart (patterning area shown with grey points, n = 9) than in the mouse molar (n = 8 for both measurements). All p-values are 0.0001. Boxes enclose 50% of observations, the horizontal bar denotes the median, and whiskers extend to last values within 1.5 interquartiles. Anterior to the left, buccal to the top. Scale bar, 200 μm.


They found that tooth size expansion can be explained by a higher expression of IGF1 in rats compared to mice. When IGF1 is overexpressed in tooth cultures, not only the molar gets bigger, but the cusps become more widely spread. Further experiments and computer simulations revealed that IGF1 stimulates cell proliferation, explaining the tooth larger size, but inhibits cusp patterning by inhibiting the formation of cusps signaling centers, explaining how the patterning scales in larger developmental fields.

Scaling of teeth is controlled by IGF1 in other mammals
Finally, to extend these results to other mammals that possess a similar molar organization, the authors examined the differences between the “Cap” stage (in which morphological differences become noticeable) and the adult first lower molar in 14 mammalian species of varying final body sizes (Fig. 4). Interestingly, they found that while the tooth germ size remains relatively stable between species at the cap stage, later stages tend to scale up with the final tooth size, confirming the scaling of the patterning. This beautiful result has implications regarding the absolute size that a tooth germ can reach: by extrapolating the results of the “Cap” stage in multiple species, the authors concluded that there is a minimum limit for tooth size in mammals and larger teeth are achieved by a progressive increase of growth during pattering. As organs generally scale with body size, the authors suggest that similar scaling of patterning could happen in many other organs, potentially driven by IGF1.


Figure 4: Patterning scales across mammals A, Frontal sections of developing teeth of various mammals show similar bucco-lingual widths of tooth germs at early cap stage. The sections show dp4 (sheep), p4 (stoat), and dp3 (pig). The porpoise tooth identity cannot be determined. B, The cap stage widths (blue) do not show a marked increase with the final mineralized tooth widths (regression slope is 0.043 and the intercept is 2.095, r2 = 0.163), and the regression-line extrapolated minimum tooth width is 154 μm for single cusped teeth (arrow).

Conclusion – what I liked about this preprint

This work used a well-known model system – the mammalian molar – to assess a fundamental, unresolved, question about the organ size evolution. I particularly liked the elegant demonstrations mixing integrative approaches that allow the extension of the results from model organisms to multi-species comparisons. I also loved that this preprint tackles evolutionary questions from a developmental point of view to find general rules (here scaling) that explain the evolution of all organisms.


1. Berkovitz, B. K. B. & Shellis, R. P. The Teeth of Mammalian Vertebrates. (Academic Press, 2018).
2. Catón, J. & Tucker, A. S. Current knowledge of tooth development: patterning and mineralization of the murine dentition. J. Anat. 214, 502–515 (2009).

Tags: development, evo-devo, evolution, mammals, scaling, teeth

Posted on: 18 January 2023


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Author's response

Mona Christensen and Jukka Jernvall shared


Q:During tooth development, physical forces have been shown to play a role in modeling tooth shape, how do these forces scale during patterning?
A: Addressing this will requires future work. Because tooth patterning scales, our results suggest that also studies on the physical forces benefit from comparative approaches.

Q: Have you found other classical tooth genes that are known to play a role in tooth size (such as Edar) as downstream targets of IGF1?’
A: Only eight genes were significantly downregulated of the known tooth genes, but there are many more that showed more subtle changes (including Edar). The striking pattern is that the dominant direction of change is downregulation, even if some of the patterning genes themselves are involved in the promotion of cell proliferation.

Q:To what extent do you think this active retained scaling mechanism is shared between ectodermal appendages that use the same pathways for their development?
A: Although this will require future studies, the inverse regulation of growth and patterning by IGF could provide a general mechanism in scaling of organs, especially because it would also integrate paracrine and endocrine regulation of growth.

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