Disruption of Nfic causes dissociation of odontoblasts by interfering with the formation of intercellular junctions and aberrant odontoblast differentiation

Abstract

We reported previously that Nfic-deficient mice exhibit short and abnormal molar roots and severely deformed incisors. The objective of this study is to address the mechanisms responsible for these changes using morphological, IHC, and RT-PCR analysis. Nfic-deficient mice exhibited aberrant odontoblasts and abnormal dentin formation in molar roots and the labial crown analog of incisors. The most striking changes observed in these aberrant odontoblasts were the loss of intercellular junctions and the decreased expression of ZO-1 and occludin. As a result, they became dissociated, had a round shape, and lost their cellular polarity and arrangement as a sheet of cells. Furthermore, the dissociated odontoblasts became trapped in dentin-like mineralized tissue, resembling osteodentin in the overall morphology. These findings suggest that loss of the Nfic gene interferes with the formation of intercellular junctions that causes aberrant odontoblast differentiation and abnormal dentin formation. Collectively, these changes in odontoblasts contributed to development of molars with short and abnormal roots in Nfic-deficient mice.

Figure 1

Light micrographs showing cross-section of incisors from P10 wild-type (WT) (A) and Nfic-deficient mice (B), and longitudinal section of molars from P18 WT (C) and Nfic-deficient mice (D). (A) A WT incisor showed circular dentin (D) with odontoblasts (OD) lining the inner surface. (B) An Nfic-deficient incisor showed an open area (asterisk) as a result of failure of dentin formation by abnormal odontoblasts. Note thick osteodentin (Otd) that contained numerous trapped cells (white arrowheads) and abnormal odontoblasts (black arrows) lining the inner surface of osteodentin. (C) The pulp side of a WT molar was covered with elongated and well-organized odontoblasts (black arrows). (D) The molar of an Nfic-deficient mouse showed a relatively short root, and the pulp side of Nfic-deficient mice was covered with polygonal and disorganized odontoblasts (black arrows). E, enamel; P, pulp. Hematoxylin and eosin (H&E) stain. Bar = 100 μm.

Figure 2

Light and electron micrographs showing differentiating odontoblasts and odontoblasts in longitudinal sections of mandibular incisors from P14 WT (A,B) and Nfic-deficient mice (C–E). (A) Differentiating odontoblasts (DO) in WT incisors were short in height but well organized and tightly arranged. (B) Fully differentiated odontoblasts (OD) were elongated and extremely well organized as a sheet of cells oriented perpendicular to the dentin (D) surface. (C) An Nfic-deficient incisor also showed short and well-organized differentiating odontoblasts (DO). (D) Abnormal odontoblasts showed a round shape (arrowheads), and many cells (OD) were embedded in osteodentin (arrows). Toluidine blue stain. (E) Abnormal odontoblasts (asterisks) that were embedded in osteodentin (Otd). Neither intercellular junctions nor terminal webs were observed between them. Uranyl acetate and lead citrate stain. Bar = 20 μm.

Figure 3

IHC localization of ZO-1 (A,B) occludin (C,D), and connexin-43 (Cx43) (E,F) showing cross-section of incisors from P18 WT and Nfic-deficient mice. (A) A WT incisor showed ZO-1 immunoreactivity in normal odontoblasts (arrows). (B) ZO-1 protein was barely expressed in abnormal odontoblasts (arrows) of Nfic-deficient mice. (C) A WT incisor showed occludin immunoreactivity in odontoblasts (arrows). (D) Occludin immunoreactivity was hardly observed in abnormal odontoblasts (arrows) of Nfic-deficient mice. (E) Cx43 immunoreactivity was observed at the junctional complexes between odontoblasts (arrows) from WT mice. (F) Cx43 protein was also weakly expressed in abnormal odontoblasts (arrows) of Nfic-deficient mice. E, enamel; Am, ameloblast; D, dentin; P, pulp. Bar = 100 μm.

Figure 4

Expression of E-cadherin, tubulin, vimentin, ZO-1, and Cx43 mRNA and protein by RT-PCR (A) and Western blot analysis (B). (A) Inactivation of Nfic results in a decrease in the expression of ZO-1 in MDPC-23 cells. ZO-1, tubulin, and E-cadherin mRNAs were expressed weakly in the primary pulp cells from Nfic-deficient mice compared with WT cells. (B) ZO-1 protein expression was decreased in the Nfic-inactivated MDPC-23 cells compared with normal. si NFIC, Nfic-inactivated MDPC-23 cells.

Figure 5

Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) peroxidase (POD) staining of incisors from P10 WT (A) and Nfic-deficient mice (B) and an electron micrograph of an abnormal odontoblast from P10 Nfic-deficient mice (C). (A) There were no TUNEL-positive cells in normal odontoblasts (OD) of WT mice. (B) In Nfic-deficient mice, apoptotic cells were evident in the pulp (arrows). Am, ameloblast; Otd, osteodentin; P, pulp. (C) Electron micrograph showing apoptotic cells. Uranyl acetate and lead citrate stain. Bar = 100 μm.