Epithelial–Mesenchymal Interactions are Not Required for Postnatal Molar Cusp Formation

Objectives: Epithelial–mesenchymal interactions are required for initial ameloblast and odontoblast formation during embryonic development. However, molar cusp expansion occurs mainly after birth with enamel and dentin layers preventing further epithelial–mesenchymal interactions. The goal of this study was to investigate how molar cusps are formed and expanded without direct epithelial-mesenchymal interactions.
Methods: To define the fate of epithelial and mesenchymal cells in postnatal molar cusp formation, the ROSA26^-tdTomato mice were crossed with the Gli1-Cre^ERT2 (expressed in both epithelia and mesenchymal progenitor cells) with one-time tamoxifen induced at P3 and animals were sacrificed at different time points from one-day to 28-days separately. Gli1-lacZ knock-in mice were also used for tracing Gli1 signal in epithelial cells and their daughter cells. The combined approaches of in vivo cell lineage-tracing, histology, confocal microscopy, and immunohistochemistry with different ameloblast and odontoblast markers were used to determine the contributions of progenitor cells for ameloblasts and odontoblasts during molar cusp formation.
Results: In molar cusp enamel formation, the Gli1-lacZ signal (indicating epithelial progenitors) was initially identified in cells of the enamel free area, which progressively appeared in pre-ameloblasts and then ameloblasts in two days. The tomato signal induced by tamoxifen, which reflects Gli1 expressions, was further confirmed in all these cells. During the cusp dentin formation, the Gli1 signal was first observed in pulp cells with few Gli1 signals in pre-odontoblasts and odontoblasts. Gradually, more Gli1⁺ odontoblasts and their processes were detected, which counted for >90% odontoblasts and dentin tubules in molar cusps. Co-immunostain assays using OSX, DMP1, and nestin for odontoblasts showed the Gli1⁺ odontoblasts and their processes co-expressed these markers in the molar cusps. Finally, a lack of signal interaction between both progenitors was confirmed.
Conclusions: Epithelial–mesenchymal interactions are not required for postnatal molar cusp formation.