The amelogenin C-terminus is required for enamel development

Abstract

The abundant amelogenin proteins are responsible for generating proper enamel thickness and structure, and most amelogenins include a conserved hydrophilic C-terminus. To evaluate the importance of the C-terminus, we generated transgenic mice that express an amelogenin lacking the C-terminal 13 amino acids (CTRNC). MicroCT analysis of TgCTRNC29 teeth (low transgene number) indicated that molar enamel density was similar to that of wild-type mice, but TgCTRNC18 molar enamel (high transgene number) was deficient, indicating that extra transgene copies were associated with a more severe phenotype. When amelogenin-null (KO) and TgCTRNC transgenic mice were mated, density and volume of molar enamel from TgCTRNCKO offspring were not different from those of KO mice, indicating that neither TgCTRNC18 nor TgCTRNC29 rescued enamel's physical characteristics. Because transgenic full-length amelogenin partially rescues both density and volume of KO molar enamel, it was concluded that the amelogenin C-terminus is essential for proper enamel density, volume, and organization.

Figure 1.

Western blot and RT-PCR of extracts of TgCTRNCKO and WT teeth. (A) Western blot showing transgene expression in molars from a TgCTRNC18KO male (lane 1), several bands in a het female (lane 3), and none in a KO male (lane 2), as expected. The Western blot was probed with rat anti-amelogenin antibody, which recognized transgenic and WT amelogenins (arrowhead). (B) Western blot of amelogenin proteins. TgCTRNC protein in molars (lane 1) and incisors (lane 2) of a TgCTRNCKO male and WT molars and incisors (lanes 3, 4). (C) Gel with Amelx RT-PCR products from molar and incisor RNA. Lane 1: WT molar. 2: WT incisor. 3: TgCTRNCKO molar. 4: TgCTRNCKO incisor. 5-6: empty. 7-10: same RNA samples as lanes 1-4 with β-actin primers. Arrowheads indicate principal amelogenin and β-actin PCR products.

Figure 2.

Histology and immunohistochemistry of 4-day-old TgCTRNC18KO teeth. (A) Histology of a section through a molar showing a phenotypically normal ameloblast layer. (B) Immunohistochemistry of TgCTRNC18KO molar with amelogenin localization in ameloblasts and enamel layer. (C) Negative control. Arrows indicate Tomes’ process region of ameloblasts.

Figure 3.

Photographs, SEM, and microCT of TgCTRNC18 and WT teeth. Gross photographs of WT incisor (A), molars (B), and SEM of normal molar (C); photographs of TgCTRNC18 incisor (D), molars showing rough surfaces (E), and SEM showing rough surfaces (F). MicroCT density (G) and volume (H); incisor enamel indicated by solid bars and molar enamel by striped bars. See Appendix Table 1 for N and estimates of variability.

Figure 4.

SEM and microCT of teeth. SEM from fractured teeth: WT incisor (A), WT molar (B), KO molar (C), TgCTRNC18KO molar (D). Magnification bars = 10 µm; brackets on B, C, and D indicate the enamel thickness from the enamel surface to the dentin-enamel junction. MicroCT analysis for density (E) and volume (F) of incisor enamel (solid bars) and molar enamel (striped bars). See Appendix Table 2 for N and estimates of variability.