Discrete phosphorylated Retinoblastoma protein isoform expression in mouse tooth development

Objectives: It is widely accepted that Retinoblastoma protein (pRb) phosphorylation plays a central role in mediating cell cycle G1/S stage transition, together with E2 promoter-binding factors (E2F). The binding of pRb to E2F is controlled by the sequential and cumulative phosphorylation of pRb at various amino acids. In addition to the well characterized roles for pRb as a tumor suppressor, pRb has more recently been implicated in osteoprogenitor and other types of stem cell maintenance, proliferation and differentiation, thereby influencing the morphogenesis of developing organs.

Methods: In this study, immunohistochemical staining was performed on developmentally staged mouse molar tooth buds (E12.5, E14.5, E16.5, and P1) using antibodies against three phosphorylated pRb (ppRb) isoforms - ppRbS780, ppRbS795, and ppRbS807/811. Co-localization of individual ppRb isoforms and phospho-histone H3, a marker indicating cell cycle progression, was analyzed by double IF staining of developmentally staged mouse molar buds and P1 incisors.

Results: Our results revealed distinct developmental expression patterns for individual ppRb isoforms in differentiating dental epithelial and dental mesenchymal cells.  Most interestingly, ppRbS807/811 was only detected in the nuclei of pre-ameloblasts and not at all in differentiated ameloblasts, and in the cytoplasm and nuclei of differentiated odontoblasts, but not in pre-odontoblasts.

Conclusions: Our results suggest that each of the three pRb isoforms examined in this study may have discrete functions in tooth development.