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 interdependencies, 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.

The origin of the turtle shell over 200 million years ago greatly modified the amniote body plan, and the morphological plasticity of the shell has promoted the adaptive radiation of turtles. The shell, comprising a dorsal carapace and a ventral plastron, is a layered structure formed by basal endochondral axial skeletal elements (ribs, vertebrae) and plates of bone, which are overlain by keratinous ectodermal scutes. Studies of turtle development have mostly focused on the bones of the shell; however, the genetic regulation of the epidermal scutes has not been investigated. Here, we show that scutes develop from an array of patterned placodes and that these placodes are absent from a softshelled turtle in which scutes were lost secondarily. Experimentally inhibiting Shh, Bmp or Fgf signaling results in the disruption of the placodal pattern. Finally, a computational model is used to show how two coupled reaction-diffusion systems reproduce both natural and abnormal variation in turtle scutes. Taken together, these placodal signaling centers are likely to represent developmental modules that are responsible for the evolution of scutes in turtles, and the regulation of these centers has allowed for the diversification of the turtle shell.

One of the fascinating aspects of the history of life is the apparent increase in morphological complexity through time, a well known example being mammalian cheek tooth evolution. In contrast, experimental studies of development more readily show a decrease in complexity, again well exemplified by mammalian teeth, in which tooth crown features called cusps are frequently lost in mutant and transgenic mice. Here we report that mouse tooth complexity can be increased substantially by adjusting multiple signalling pathways simultaneously. We cultured teeth in vitro and adjusted ectodysplasin (EDA), activin A and sonic hedgehog (SHH) pathways, all of which are individually required for normal tooth development. We quantified tooth complexity using the number of cusps and a topographic measure of surface complexity. The results show that whereas activation of EDA and activin A signalling, and inhibition of SHH signalling, individually cause subtle to moderate increases in complexity, cusp number is doubled when all three pathways are adjusted in unison. Furthermore, the increase in cusp number does not result from an increase in tooth size, but from an altered primary patterning phase of development. The combination of a lack of complex mutants, the paucity of natural variants with complex phenotypes, and our results of greatly increased dental complexity using multiple pathways, suggests that an increase may be inherently different from a decrease in phenotypic complexity.

Despite considerable advances in knowledge of the anatomy, ecology and evolution of early mammals, far less is known about their physiology. Evidence is contradictory concerning the timing and fossil groups in which mammalian endothermy arose. To determine the state of metabolic evolution in two of the earliest stem-mammals, the Early Jurassic Morganucodon and Kuehneotherium, we use separate proxies for basal and maximum metabolic rate. Here we report, using synchrotron X-ray tomographic imaging of incremental tooth cementum, that they had maximum lifespans considerably longer than comparably sized living mammals, but similar to those of reptiles, and so they likely had reptilian-level basal metabolic rates. Measurements of femoral nutrient foramina show Morganucodon had blood flow rates intermediate between living mammals and reptiles, suggesting maximum metabolic rates increased evolutionarily before basal metabolic rates. Stem mammals lacked the elevated endothermic metabolism of living mammals, highlighting the mosaic nature of mammalian physiological evolution.

No abstract

To understand the limitations occurring during enzymatic hydrolysis of cellulosic materials in renewable energy production, we used wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), X-ray microtomography, and transmission electron microscopy (TEM) to characterize submicrometer changes in the structure of microcrystalline cellulose (Avicel) digested with the Trichoderma reesei enzyme system. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures, similar elongated and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results of rewetted samples suggested a slight change in the structure in scale of 10-20 nm, whereas the WAXS results confirmed that the degree of crystallinity and the crystal sizes remained unchanged. This indicates that the enzymes act on the surface of cellulose bundles and are unable to penetrate into the nanopores of wet cellulose.

Epithelial morphogenesis generates the shape of the tooth crown. This is driven by patterned differentiation of cells into enamel knots, root-forming cervical loops and enamel-forming ameloblasts. Enamel knots are signaling centers that define the positions of cusp tips in a tooth by instructing the adjacent epithelium to fold and proliferate. Here, we show that the forkhead-box transcription factor Foxi3 inhibits formation of enamel knots and cervical loops and thus the differentiation of dental epithelium in mice. Conditional deletion of Foxi3 (Foxi3 cKO) led to fusion of molars with abnormally patterned shallow cusps. Foxi3 was expressed in the epithelium, and its expression was reduced in the enamel knots and cervical loops and in ameloblasts. Bmp4, a known inducer of enamel knots and dental epithelial differentiation, downregulated Foxi3 in wild-type teeth. Using genome-wide gene expression profiling, we showed that in Foxi3 cKO there was an early upregulation of differentiation markers, such as p21, Fgf15 and Sfrp5. Different signaling pathway components that are normally restricted to the enamel knots were expanded in the epithelium, and Sostdc1, a marker of the intercuspal epithelium, was missing. These findings indicated that the activator-inhibitor balance regulating cusp patterning was disrupted in Foxi3 cKO. In addition, early molar bud morphogenesis and, in particular, formation of the suprabasal epithelial cell layer were impaired. We identified keratin 10 as a marker of suprabasal epithelial cells in teeth. Our results suggest that Foxi3 maintains dental epithelial cells in an undifferentiated state and thereby regulates multiple stages of tooth morphogenesis.

Different diets wear teeth in different ways and generate distinguishable wear and microwear patterns that have long been the basis of palaeodiet reconstructions. Little experimental research has been performed to study them together. Here, we show that an artificial mechanical masticator, a chewing machine, occluding real horse teeth in continuous simulated chewing (of 100 000 chewing cycles) is capable of replicating microscopic wear features and gross wear on teeth that resemble wear in specimens collected from nature. Simulating pure attrition (chewing without food) and four plant material diets of different abrasives content (at n ¼ 5 tooth pairs per group), we detected differences in microscopic wear features by stereomicroscopy of the chewing surface in the number and quality of pits and scratches that were not always as expected. Using computed tomography scanning in one tooth per diet, absolute wear was quantified as the mean height change after the simulated chewing. Absolute wear increased with diet abrasiveness, originating from phytoliths and grit. In combination, our findings highlight that differences in actual dental tissue loss can occur at similar microwear patterns, cautioning against a direct transformation of microwear results into predictions about diet or tooth wear rate.