Collapsin Response Mediator Protein 1, a Novel Marker Protein for Differentiated Odontoblasts

Abstract

We previously reported that the terminal differentiation of odontoblasts was inhibited in Runx2 transgenic {Tg(Col1a1-Runx2)} mice under the control of the 2.3-kb Col1a1 promoter. Odontoblasts in Tg(Col1a1-Runx2) mice lose their characteristic long cellular processes, and show marked reductions in the protein levels of markers for odontoblasts, such as dentin sialophosphoprotein, nestin, and microtubule-associated protein tau (Mapt). We herein demonstrated that collapsin response mediator protein 1 (CRMP1), a neuronal phosphoprotein that participates in various aspects of neuronal development, was specifically expressed in the differentiated odontoblasts of wild-type, but not Tg(Col1a1-Runx2) tooth germs by comparing expression profiles in wild-type and Tg(Col1a1-Runx2) mouse molars using microarray and immunohistochemical analyses. CRMP1 expression was detected at a slightly later differentiation stage in odontoblasts than type 1 collagen, nestin, and Mapt expression, which was observed from the onset of dentinogenesis. Among these proteins, CRMP1 was the most specifically localized in odontoblasts in the tooth germ. In erupted molars, odontoblast-specific CRMP1 expression decreased with age. These results indicate that CRMP1 is a novel marker protein for differentiated odontoblasts in mouse tooth germs, and suggest that CRMP1 participates in the morphogenesis of functioning odontoblasts.

Keywords: Runx2; collapsin response mediator protein 1 (CRMP1); odontoblasts.

Fig. 1.

Real-time RT-PCR analysis of CRMP1 in tooth germs of 2-week-old mice. The value in wild-type (wt) mice was set as one and the relative level of Tg(Co1a1-Runx2) (tg) mice is shown. Data are the mean ± SD of seven wild-type and six tg mice. *P < 0.01.

Fig. 2.

Expression of the CRMP1 protein in tooth germs of first molars in wild-type (wt) (a, b) and Tg(Col1a1-Runx2) (tg) (c, d) mice at 7 days of age. Boxed regions in a and c are magnified in b and d, respectively. CRMP1 expression was exclusively detected in differentiated odontoblasts in wild-type mice, whereas it was negligibly expressed in the odontoblasts of Tg(Col1a1-Runx2) mice. Bars = 200 μm (a and c), 40 μm (b and d).

Fig. 3.

Comparison of the expression of CRMP1 (a), Col1a1 (b), nestin (c), and Mapt (d) in tooth germs of first molars in 7-day-old wild-type mice. The expression of Col1a1, nestin, and Mapt in odontoblasts appeared with the onset of dentinogenesis (thick arrows), while CRMP1 expression in odontoblasts appeared after the onset of dentinogenesis (arrow head). Nerve fibers were also positive for CRMP1 and Mapt (thin arrows). The expression of Col1a1 was also observed in osteoblasts and collagen fibers, while that of nestin and Mapt was detected in dental pulp cells under the odontoblast layer (asterisks). Bars = 40 μm.

Fig. 4.

Expression of the CRMP1 protein in erupted first molars in wild-type mice at 3 (a), 6 (c), and 10 weeks (e) and 6 months (g) of age. Cervical regions indicated by boxes in a, c, e and g are magnified in b, d, f and h, respectively. CRMP1 expression was down-regulated with age, starting from the coronal region, and was not detected in the whole region at 6 months of age. Bars = 200 μm (a, c, e and g), 20 μm (b, d, f and h).