Developmental systems may preferentially produce certain types of variation and, thereby, bias phenotypic evolution. This is a central issue in evolutionary developmental biology, albeit somewhat understudied. Here we focus on the shape of the first upper molar which shows a clear, repeated tendency for anterior elongation at different scales from within mouse populations to between species of the Mus genus. In contrast, the lower molar displays more evolutionary stability. We compared upper and lower molar development of mouse strains representative of this fine variation (DUHi: elongated molars and FVB: short molars). Using a novel quantitative approach to examine small-scale developmental variation, we identified temporal, spatial and functional differences in tooth signaling centers between the two strains, likely due to different tuning of the activation-inhibition mechanisms ruling signaling center patterning. Based on the spatio-temporal dynamics of signaling centers and their lineage tracing, we show an intrinsic difference in the fate of signaling centers between lower and upper jaw of both strains. This can explain why variations in activation-inhibition parameters between strains are turned into anterior elongation in the upper molar only. Finally, although the 'elongated' DUHi strain was inbred, first molar elongation was variable in adults, and we found high levels of intra-strain developmental variation in upper molar development. This is consistent with the inherent developmental instability of the upper molar system enabling the morphological variability of the tooth phenotype. In conclusion, we have uncovered developmental properties that underlie the molar's capacity for repeated phenotypic change, or said differently, that underlie a 'line of least resistance'. By focusing on the developmental basis of fine phenotypic variation, our study also challenges some common assumptions and practices in developmental and evolutionary developmental biology.

Developmental variability drives mouse molar evolution along an evolutionary line of least resistance