Replaying evolutionary transitions from the dental fossil record


The evolutionary relationships of extinct species are ascertained primarily through the analysis of morphological characters. Character inter-dependencies can have a substantial effect on evolutionary interpretations, but the developmental underpinnings of character inter-dependence remain obscure because experiments frequently do not provide detailed resolution of morphological characters. Here we show experimentally and computationally how gradual modification of development differentially affects characters in the mouse dentition. We found that intermediate phenotypes could be produced by gradually adding ectodysplasin A (EDA) protein in culture to tooth explants carrying a null mutation in the tooth-patterning gene Eda. By identifying development-based character inter-dependencies, we show how to predict morphological patterns of teeth among mammalian species. Finally, in vivo inhibition of sonic hedgehog signalling in Eda null teeth enabled us to reproduce characters deep in the rodent ancestry. Taken together, evolutionarily informative transitions can be experimentally reproduced, thereby providing development-based expectations for character-state transitions used in evolutionary studies.

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Figure 1: Gradual dosage effects of EDA on Eda null mutant first lower molars (m1).
Figure 2: Computational modelling of gradual changes in signalling on cusp patterns.
Figure 3: Differential sensitivities of tooth crown regions to EDA.
Figure 4: Testing developmental predictions on evolutionary patterns.
Figure 5: Engineering mouse teeth to have basal character states.


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We thank M. Fortelius, J. Eronen, I. Thesleff, P. Munne for discussions; S. Alto, M. Mäkinen, R. Santalahti, R. Savolainen, and M. Christensen for technical assistance; P. Schneider for the Fc-EDA-A1-protein; F. de Sauvage for HhAntag compound; Hou Yemao for tomography; and the Finnish Museum of Natural History (Helsinki, Finland), Museum of Natural History (Stockholm, Sweden), and Museum für Naturkunde (Berlin, Germany) for specimen loans. This work was supported by the Academy of Finland to J.J., M.M., I.S.-C., R01-DE021420 (NIH/NIDCR) and an NIH Director’s New Innovator Award DP2-OD007191 to O.D.K., an Australian Research Council Future Fellowship to A.R.E, and the Major Basic Research Projects (2012CB821904) of MST to Z.-Q.Z. Data are presented in the Supplementary and Extended Data Tables, available in the MorphoBrowser database ( and from the authors, and models can be accessed at (

Author information




E.H., J.J. and O.D.K. designed the project and wrote the initial manuscript. E.H. and E.R. performed culturing experiments and K.S. mouse experiments. E.H., E.R., A.K. and J.J. performed measurements and prepared images. I.J.C., A.R.E. and J.J. analysed evolutionary data. I.S.-C. constructed the computational model and T.H. the ToothMaker. M.L.M., Z.-Q.Z. provided materials, observations and scientific interpretations. O.D.K. and J.J. coordinated the study. All authors discussed the results and provided input on the manuscript.

Corresponding authors

Correspondence to Ophir D. Klein or Jukka Jernvall.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Primary enamel knot size predicts cusp number.

Size of the primary enamel knot (day 2) and cusp number (day 7) for Eda null, Eda null + 10 ng ml−1 EDA, Eda null + 50 ng ml−1 EDA, and wild-type teeth. Reduced major axis regression for square root of µm2 are 0.0533 x −0.791, r2 = 0.613, P < 0.0001, n = 46. Enamel knot size does not increase substantially with higher EDA concentrations.

Extended Data Figure 2 The ToothMaker modelling interface and morphodynamic model of tooth development.

The model parameters can be changed manually or scanned automatically (Options-menu). For descriptions of parameters, see Methods. The figures illustrate the growth factor concentration (secreted from the enamel knots) showing the future cusp areas. Initial activator concentration (Ina) is not used in the model. The model can be downloaded at (

Extended Data Figure 3 Scanning parameter space to produce gradual changes.

a, Parameters producing variation in cusp number were scanned at 10 percent intervals up to 90 percent change from the wild-type (WT) mouse. Growth factor domains, produced by enamel knots, were used to tabulate cusps numbers (threshold = 0.04). b, Simulated teeth show the minimum number of cusps that can be produced by changing each parameter. Only parameter Act produced single-cusped morphology. Plus and minus signs after each parameter denote to the direction of parameter change that produced a decrease in cusp number. Act = activator auto-activation, Da = activator diffusion, Int = inhibitor production threshold, Inh = Activator inhibition by inhibitor, Di = inhibitor diffusion, Set = growth factor production threshold, Sec = growth factor secretion rate, Ds = growth factor diffusion, Dff = differentiation rate, Egr = epithelial proliferation rate, Mgr = mesenchymal proliferation rate. All simulations were run for a fixed number of iterations (14,000).

Extended Data Figure 4 Simulating EDA effects.

a, Simulated shapes produced by changing the activator parameter (Act) from 0.1 to 1.6 at 0.1 interval. b, The size of inhibitor domain (at arbitrary threshold 0.85) at iteration 1,000 and corresponding cusp number at iteration 14,000 approximates the relationship between primary enamel knot size and cusp number in real teeth.

Extended Data Figure 5 Simulating reduction of inhibition in Eda null teeth.

Reducing activator inhibition by inhibitor (Inh) or diffusion of inhibitor (Di) results in formation of multiple cusps in simulated Eda null molar. The effects are variable depending on the parameter values, and the lability of the system appears to be corroborated in the in vitro experiments.

Extended Data Figure 6 Rescuing cusps in Eda null teeth by inhibiting SHH.

Eda null teeth cultured with SHH antagonist show variable morphologies with tightly packed cusps (arrowheads). In addition, in roughly half of the cases (n = 4 of 11 teeth) portions of the first molar appear to form from the fusion with the developing second molar (two bottom rows). Scale bar, 500 µm.

Extended Data Figure 7 In vivo inhibition of SHH in Eda null embryos causes the formation of separate cusps without crests.

Obliquely anterior views and tomography sections (along the plane of the dotted line) of second molars show the lack of a crest (metalophid, arrowheads) in treated Eda null and Tribosphenomys minutus (V10775). Enamel in sections shown in blue colour except in Tribosphenomys fossil which did not allow segmentation of enamel due to high degree of mineralization. Scale bar, 500 µm.

Extended Data Table 1 Comparison of talonid height and cusp number in rodents
Extended Data Table 2 Comparison of talonid height and cusp number in carnivorans
Extended Data Table 3 Reduced major axis regression analyses between talonid height and talonid cusp numbers and complexity

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Harjunmaa, E., Seidel, K., Häkkinen, T. et al. Replaying evolutionary transitions from the dental fossil record. Nature 512, 44–48 (2014).

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