Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo

Abstract

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel.

Keywords: amelogenin; enamel development; knockout; mineralization; transgenic.

Figure 1

Relative amelogenin transgene DNA and protein expression in double and triple transgenic/AmelxKO mice. (A) Relative transgene copy number of homozygous vs. hemizygous M180Tg, CTRNCTg, and LRAPTg as determined by qPCR analyses with transgene-specific probes designed and tested by Transnetyx (Cordova, TN) (^**indicates significant difference, p < 0.0001). (B–D) Western blot of Amelx protein expression using anti-Amelogenin FL-191 (Santa Cruz), with β-actin control (Sigma) in transgenic 5 day-old developing molars. (B) Relative transgene protein expression of homozygous vs. hemizygous M180Tg, CTRNCTg, and LRAPTg as determined by Western blot analyses as measured by ImageJ using β-actin to normalize. Since we could not differentiate between M180Tg and CTRNCTg in lanes 1, 3, 4, and 8, M180Tg and CTRNCTg expression intensities were pooled when quantifying. Due to the lack of Mmp20 in lanes 5, 6, and 7, we were able to differentiate between M180Tg and CTRNCTg. (n = 3 blots, ^**indicates significant difference, p < 0.005). (C) Relative transgene protein expression for different genotypes as determined by Western blot analyses as measured by ImageJ using β-actin to normalize. Lane 1, WT; Lane 2, AmelxKO; Lane 3, M180Tg/CTRNCTg/LRAPTg/KO++; Lane 4, M180Tg/CTRNCTg/KO++; Lane 5, M180Tg/CTRNCTg/KO+ (Mmp20KO background control); Lane 6, M180Tg/CTRNCTg/LRAPTg/KO++ (Mmp20KO background control); Lane 7, M180Tg/CTRNCTg/LRAPTg/KO+ (Mmp20KO background control); Lane 8, M180Tg/CTRNCTg/KO++; Lane 9, M180Tg/LRAPTg/KO+. (D) Western blot with primary antibodies anti-Amelogenin and anti-β-actin, with lane numbers corresponding to genotypes in (C). In lane 1, molars had a smear of amelogenin protein between 17 and 20 kD representing most of the WT splice variants and cleavage products expressed during the secretory stage, which are absent in the AmelxKO lane 2. The M180Tg band is visible at ~25 kD in all lanes except lane 2. The CTRNCTg is visible ~22 kD in lanes 5, 6, and 7, since the absence of Mmp20 prevented its proteolytic degradation. The LRAPTg band is visible ~7 kD in lanes 3, 6 7, and 9, but below the detection level in lane 1. The β-actin loading control bands is visible in all lanes ~40 kD.

Figure 2

MicroCT analyses of adult molar and incisor enamel. First column: first molars in mid sagittal plane. Middle column: Incisor enamel in coronal plane through the distal root of the first molar, representing early maturation stage. Last column: Incisor enamel in coronal plane adjacent and mesial to the first molar, representing maturation stage enamel. White arrowheads point to mineralized enamel layers visible in molars and incisors. WT seen in last row (S–U); (P–R) AmelxKO with little enamel on molars and no enamel in the incisor enamel. (A–C) homozygous triple transgene M180Tg/CTRNCTg/LRAPTg/KO++, showing the thinnest enamel layer compared to all samples shown on both molars and incisors. (D–F) Hemizygous triple transgene M180Tg/CTRNCTg/LRAPTg/KO+ and (M–O) hemizygous double transgene M180Tg/LRAPTg/KO+ with thicker enamel than all other transgene phenotypes shown, but thinner than WT. (G–I) double transgene M180Tg/CTRNCTg/KO++ with a thin layer of enamel as is also seen in (J–L) M180Tg/CTRNCTg/KO+. Ectopic depositions (orange arrowheads) were visible in incisors of homozygous transgenes M180Tg/CTRNCTg/LRAPTg/KO++ and M180Tg/CTRNCTg/KO++ (A–C, G–I). Scale bars, 500 μm.

Figure 3

SEM analysis of double and triple transgenic/KO incisor and molar enamel. Polished and etched sections through mandibular incisor enamel and first molars imaged by scanning electron microscopy with secondary electron detection. (A–C) Homozygous triple transgenic M180Tg/CTRNCTg/LRAPTg/KO++, (D–F) hemizygous triple transgenic M180Tg/CTRNCTg/LRAPTg/KO+, (G–I) homozygous double transgenic M180Tg/CTRNCTg/KO++, (J–L) hemizygous double transgenic M180Tg/CTRNCTg/KO+ enamel, and (M–O) hemizygous double transgenic M180Tg/LRAPTg/KO+, (P–R) AmelX null, (S–U) WT enamel. First column: Incisors, scale bars 10 μm. Second column: Molars, scale bars 10 μm. Third column: Molars, scale bars 10 μm. Yellow triangles: LR White resin; turquoise dashed lines: DEJ; magenta colored circles: organic matrix in forming enamel.

Figure 4

Toluidine Blue staining of LR White embedded samples processed further after SEM analyses. (A,B) Homozygous triple transgenic enamel M180Tg/CTRNCTg/LRAPTg/KO++, (C,D) hemizygous triple transgenic M180Tg/CTRNCTg/LRAPTg/KO+, (E,F) homozygous double transgenic M180Tg/CTRNCTg/KO++, (G,H) hemizygous double transgenic M180Tg/CTRNCTg/KO+ enamel, (I,J) AmelX null, (K,L) WT enamel. Coronal sections through incisors (first column) and molars (second column). Nuclei and organic matter stained blue and marked by arrows, DEJ highlighted in yellow. Scale bars, 10 μm. Magnification 400X.