Discrete phosphorylated retinoblastoma protein isoform expression in mouse tooth development

Abstract

Retinoblastoma protein (pRb) phosphorylation plays a central role in mediating cell cycle G1/S stage transition, together with E2F transcription factors. The binding of pRb to E2F is thought to be controlled by the sequential and cumulative phosphorylation of pRb at various amino acids. In addition to well characterized roles 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. In this study, we present data characterizing the expression of pRb and three phosphorylated pRb (ppRb) isoforms-ppRbS780, ppRbS795, ppRbS807/811-in developmentally staged mouse molar and incisor teeth. Our results reveal distinct developmental expression patterns for individual ppRb isoforms in dental epithelial and dental mesenchymal cell differentiation, suggesting discrete functions in tooth development.

Figure 1. Individual ppRb isoform expression in E12.5, E14.5 and E16.5 mouse tooth development

a. Schematic of the pRb protein, with short arrows indicating identified phosphorylation sites throughout the pRb protein and with long arrows indicating the three ppRb isoforms examined in this study. b. Individual ppRb isoforms exhibit discrete expression in developmentally staged mouse teeth (arrows). Abbreviations: am, ameloblast; de, dental epithelium; dm, dental mesenchyme; df, dental follicle; ide, inner dental epithelium; od, odontoblast; ode, outer dental epithelium; oe, odontal epithelium; hm, head mesenchyme. Scale bars indicate 100µm.

Figure 2. ppRb isoform expression in newborn (P1) mouse M1 molar teeth

Individual ppRb isoforms exhibit discrete expression patterns in cusp and cervical loop regions of newborn M1 molar teeth (arrows). Boxes embedded in each left hand panels are shown enlarged in adjacent three right hand panels. The schematic molar tooth (m) indicates areas of dental epithelial cell proliferation (solid black areas), and non-proliferative zones (solid white areas). Abbreviations: am, ameloblast; de, dental epithelium; dm, dental mesenchyme; ide, inner dental epithelium; od, odontoblast; ode, outer dental epithelium. Scale bars indicate 100µm.

Figure 3. ppRb isoform expression in newborn mouse incisor teeth

Discrete ppRb isoform expression was detected in P1 mouse incisor teeth (a, h, o). Boxed areas embedded in each bright field panel are shown enlarged to the right. Primed panels are double immunofluorescent images of phospho-Histone H3 and each corresponding ppRb isoform expression. Panel v. IF images of ppRbS807/811 demonstrate overlapping ppRbS807/811 and phospho-Histone H3 expression (arrows). Abbreviations: am, ameloblast; de, dental epithelium; dm, dental mesenchyme; LaCL, labial cervical loop; LiCL, lingual cervical loop; od, odontoblast. Scale bars indicate 100µm (A-C) or 10 µm (D).

Figure 4. Schematic of ppRb isoform expression in mouse molar and incisor teeth

Schematic colorized drawings of individual ppRb isoform expression in newborn mouse M1 molar and incisor teeth. ppRbS780 expression is indicated in red, ppRbS795 expression is shown in yellow, and ppRbS807/811 expression is indicated in blue. Overlapping expression of ppRbS780 and ppRbS795 is indicated in orange (Panel b, ameloblast, am). Abbreviations: am, ameloblast; bm, basement membrane; DE, dental epithelium; dp, dental papilla; ds, dental sac; Lab. CL, labial cervical loop; Lin. CL, lingual cervical loop; od, odontoblast; oee, outer enamel epithelium; pa, pre-ameloblasts; si, stellate reticulum; sr, stellate reticulum.