Transgenic rescue of enamel phenotype in Ambn null mice

Abstract

Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin (AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.

Figure 1.

Ameloblastin expression levels and associated enamel phenotypes. (A) SDS-PAGE stained with Coomassie brilliant blue (CBB), and Western blots immunostained with an antibody raised against recombinant mouse amelogenin (Amel-179), or against an ameloblastin peptide absent from the smaller ameloblastin expressed by the Ambn knockout (Ambn-89). The apparent molecular weights (in kDa) of protein bands in the samples are indicated on the left. Equal fractions of enamel proteins extracted from day 5 molars from Ambn wild-type (lanes ++), Ambn heterozygotes (lanes +-), Ambn heterozygotes expressing an ameloblastin transgene (lanes +-T), Ambn homozygous knockout mice (lanes—), and Ambn knockout mice expressing an ameloblastin transgene (lanes—T) were applied to each lane. The yield of enamel proteins from the Ambn^-/- mice was always much lower than that from other samples because of the virtual absence of an enamel layer. Except for the wild-type (++) lanes, all samples on a single gel or blot are from littermates. The only ameloblastin band reacting against the Ambn-89 antibody migrated at 17 kDa. The ladder of immunopositive bands in Western for Line 14 is due to aggregation of the 17-kDa ameloblastin cleavage product when loaded at the high concentrations expressed. (B) Oral photographs at 7 wks of the incisors in situ and a lingual view of molars following removal of soft tissue. The molars and incisors from the Ambn^-/-Tg++ mice held up to occlusal forces, did not chip or abrade, and could not be distinguished from teeth from wild-type mice.

Figure 2.

Exposed mandibular incisors. These photos were taken through a dissecting microscope and show the labial surfaces of mandibular incisors at 7 wks. The incisors from the transgenic line expressing somewhat higher than normal amounts of the ameloblastin transgene (Tg++) appeared similar to those of the wild-type mouse when expressed in either the Ambn heterozygous or Ambn null mice.

Figure 3.

Scanning electron micrographs of mandibular incisors at 9 wks. All images are of cross-sections obtained at the level of the buccal alveolar crest. Row 1: Low-magnification view of polished cross-section showing the entire enamel layer. The Ambn^+/+ and Ambn^+/- mice show no enamel defects. The Ambn^-/- mice have no true enamel, but a thin, rough crust of mineral covers the surface of dentin. The enamel phenotype is recovered in the central region when ameloblastin transgenes provide less than (Tg+) and more than (Tg+++) optimal amounts of ameloblastin, but the distal enamel is thinner than normal and has a rough surface. Row 2: Higher-magnification view of polished enamel showing the enamel thickness in the central region. The line spanning the enamel layer is where measurements of enamel thickness were made. The enamel layer in the Tg++ mice was the same thickness as in the wild-type. Row 3: High magnification of polished, cut, lightly etched enamel showing the underlying rod architecture. Only the Ambn^-/- mice show a complete absence of enamel rods. Rod and interrod enamel is clearly distinguished in the Tg++ mice. Row 4: The enamel layer after being fractured reveals the decussating patterns of enamel rods. Although a normal pattern of decussating enamel rods is evident in the mice expressing different levels of the ameloblastin transgene, the fractured surface of the mice expressing the smaller Ambn from the knockout gene was not as well-defined as the wild-type, possibly due to an increased level of, or residual, enamel protein.