The initiation knot is a signaling center required for molar tooth development

Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.