An amelogenin mutation leads to disruption of the odontogenic apparatus and aberrant expression of Notch1

Abstract

Background: Amelogenins are highly conserved proteins secreted by ameloblasts in the dental organ of developing teeth. These proteins regulate dental enamel thickness and structure in humans and mice. Mice that express an amelogenin transgene with a P70T mutation (TgP70T) develop abnormal epithelial proliferation in an amelogenin null (KO) background. Some of these cellular masses have the appearance of proliferating stratum intermedium, which is the layer adjacent to the ameloblasts in unerupted teeth. As Notch proteins are thought to constitute the developmental switch that separates ameloblasts from stratum intermedium, these signaling proteins were evaluated in normal and proliferating tissues.

Methods: Mandibles were dissected for histology and immunohistochemistry using Notch1 antibodies. Molar teeth were dissected for western blotting and RT-PCR for evaluation of Notch levels through imaging and statistical analyses.

Results: Notch1 was immunolocalized to ameloblasts of TgP70TKO mice, KO ameloblasts stained, but less strongly, and wild-type teeth had minimal staining. Cells within the proliferating epithelial cell masses were positive for Notch1 and had an appearance reminiscent of calcifying epithelial odontogenic tumor with amyloid-like deposits. Notch1 protein and mRNA were elevated in molar teeth from TgP70TKO mice.

Conclusion: Expression of TgP70T leads to abnormal structures in mandibles and maxillae of mice with the KO genetic background and these mice have elevated levels of Notch 1 in developing molars. As cells within the masses also express transgenic amelogenins, development of the abnormal proliferations suggests communication between amelogenin producing cells and the proliferating cells, dependent on the presence of the mutated amelogenin protein.

Figure 1

Histology and immunohistochemistry for Notch 1 in first molars from 3-day-old WT and transgenic/KO mice. (A, D) TgP70TKO male; (B, E) KO male; (C, F) WT. (A, B) hematoxylin and eosin (H&E) stain; (D–F) immunohistochemistry using Notch 1 antibody; (C) negative control lacking primary antibody. Ameloblasts are indicated with brackets, stratum intermedium with arrows and the secreted enamel and dentin developing layers by asterisks. Magnification bars are indicated.

Figure 2

Histology and immunohistochemistry for Notch 1 in consecutive sections through an abnormal cellular mass from a 4-week-old TgP70TKO mouse. (A) H&E stain; (B) immunohistochemistry using Notch 1 antibody; (C) negative control lacking primary antibody. Magnification bars are indicated.

Figure 3

The P70T transgene induces an aberrant odontogenic epithelial cell proliferation reminiscent of calcifying epithelial odontogenic tumor. (A) A dense sheet of epithelial cells is seen surrounding a developing tooth (H&E. 40x) within the left mandible of a TgP70Thet female mouse. (B and C) Proliferation of epithelial cells with interspersed Liesegang ring-like psammomatoid calcifications (arrow) and amorphous material resembling amyloid (arrowhead). (B) magnification. 100x; (C) magnification, 400x. (D) An island of epithelium containing aggregates of the amorphous material (400x), taken from a section of the right maxilla.

Figure 4

Introduction of mutant amelogenin disrupts normal odontogenic development. (A) Supernumerary teeth (arrows) were observed in the right maxilla of a male TgP70TKO mouse (H&E, 40x). (B) An aberrant benign epithelial proliferation (*) was noted overlying the crown of a developing tooth in a 4-week-old TgP70Thet female. Aggregates of calcified, enameloid-type material were scattered throughout the epithelium (40x). (C) Island of epithelium containing ghost cells (arrow) and the calcified substance (100x) from the mouse shown in B.

Figure 5

Western blot for comparison of Notch 1 protein in first molars. (A) Typical western blot in which identical amounts of extract from first mandibular molars were loaded onto the gel, and transfer to nitrocellulose was followed by probing with Notch 1 antibody. Subsequently the blot was probed with anti-actin antibody without stripping the membrane for normalization. Lane 1: TgP70TKO male; lane 2: KO male; lane 3: TgP70Thet female; lane 4: het female; lane 5, WT. (B) The histogram shows mean band intensities for Notch 1 in five experiments ± SEM. *A statistically significant difference between P70TKO male samples and the combined group of male and female wild-type. KO males and het females, lacking the transgene.