IADR Abstract Archives
Enamel-free Area is Responsible for Ameloblast Formation in Crown Cusps
Objectives: The enamel-free area (EFA), an example of position-specific ameloblast patterning, is unique to rodent molars, although its biological significance is largely unknown. The goal of this study was to investigate the contribution of the EFA to postnatal ameloblast formation and enamel production.
Methods: To define the cell fate of the enamel-free area, the ROSA26-tdTomato were crossed with Gli1-CreERT2 (expressed in the early progenitors) and activated by a one-time tamoxifen induction plus BrdU injections at P1. The animals were sacrificed at days 2, 6, 8 and 15, respectively. The combined approaches of in vivo cell lineage-tracing, histology, and immunohistochemistry, using various ameloblast protein markers, were used to determine the contributions of the Gli1+ cells.
Results: One day after Gli1 activation, there were many BrdU/Edu+-cells in EFA. Some of these cells overlapped with the Gli1 expression (red) +, supporting the idea that EFA cells are progenitors. Importantly, some of these Gli1+ cells started to differentiate into early ameloblasts in the cusp. By P4-8, these newly added “red” ameloblast cells were fully polarized, ameloblastin/enamelin+, formed enamel and covered the expanding cuspal surface. By P15 these red ameloblasts became cuboid-shaped (the protective stage). The EFA cells remained red with no sign of differentiation. Next, we showed that the EFA directly originated from the stratum intermedium (SI) using the cell-lineage tracing approach. Furthermore, we found evidence that the SI layer, which was positive for both Edu and BrdU, contributed to the formation of EFA.
Conclusions: Our findings support the notion that during cusp formation EFA cells (as a stem cell niche similar to the incisor cervical loop niche) “invade” into the existing ameloblast layer followed by differentiation into the newly expanded ameloblast layer to form enamel.
Methods: To define the cell fate of the enamel-free area, the ROSA26-tdTomato were crossed with Gli1-CreERT2 (expressed in the early progenitors) and activated by a one-time tamoxifen induction plus BrdU injections at P1. The animals were sacrificed at days 2, 6, 8 and 15, respectively. The combined approaches of in vivo cell lineage-tracing, histology, and immunohistochemistry, using various ameloblast protein markers, were used to determine the contributions of the Gli1+ cells.
Results: One day after Gli1 activation, there were many BrdU/Edu+-cells in EFA. Some of these cells overlapped with the Gli1 expression (red) +, supporting the idea that EFA cells are progenitors. Importantly, some of these Gli1+ cells started to differentiate into early ameloblasts in the cusp. By P4-8, these newly added “red” ameloblast cells were fully polarized, ameloblastin/enamelin+, formed enamel and covered the expanding cuspal surface. By P15 these red ameloblasts became cuboid-shaped (the protective stage). The EFA cells remained red with no sign of differentiation. Next, we showed that the EFA directly originated from the stratum intermedium (SI) using the cell-lineage tracing approach. Furthermore, we found evidence that the SI layer, which was positive for both Edu and BrdU, contributed to the formation of EFA.
Conclusions: Our findings support the notion that during cusp formation EFA cells (as a stem cell niche similar to the incisor cervical loop niche) “invade” into the existing ameloblast layer followed by differentiation into the newly expanded ameloblast layer to form enamel.
