WNT5A Expression in Ameloblastoma and Its Roles in Regulating Enamel Epithelium Tumorigenic Behaviors

Odontogenic tumors originate from the remains of migrating enamel epithelium after the completion of normal tooth genesis. These enamel epithelium remnants exhibit the ability to recapitulate the events that occur during tooth formation. Several lines of evidence suggest that aberrance in the signaling pathways similar to the ones that are used during tooth development, including the WNT pathway, might be the cause of odontogenic tumorigenesis and maintenance. In this study we demonstrated that WNT5A expression was intense in both the epithelial component of ameloblastomas, the most common epithelial odontogenic tumor, and in this tumor's likely precursor cell, the enamel epithelium located at the cervical loop of normal developing human tooth buds. Additionally, when WNT5A was overexpressed in enamel epithelium cells (LS-8), the clones expressing high levels of WNT5A (S) exhibited characteristics of tumorigenic cells, including growth factor independence, loss of anchorage dependence, loss of contact inhibition, and tumor formation in immunocompromised mice. Moreover, overexpression of WNT5A drastically increased LS-8 cell migration and actin reorganization when compared with controls. Suppression of endogenous WNT5A in LS-8 cells (AS) greatly impaired their migration and AS cells failed to form significant actin reorganization and membrane protrusion was rarely seen. Taken together, our data indicate that WNT5A signaling is important in modulating tumorigenic behaviors of enamel epithelium cells in ameloblastomas

The role of amelogenin during enamel-crystallite growth and organization in vivo: Amelogenin and enamel-crystallite formation

Amelogenin is critical for enamel formation and human AMELX gene mutations cause hypoplastic and/or hypomaturation enamel phenotypes. The Amelx null (AKO) mouse has a severe hypoplastic phenotype. This study evaluated the effect of amelogenin loss on enamel formation and crystallite morphology. Enamel from AKO and wild type (WT) mice was used. AKO mice were mated with transgenic mice expressing the most abundant known amelogenin isoform TgM180-87 to rescue (KOM180-87) the enamel crystallite phenotype. Molar enamel was embedded, sectioned with a diamond microtome and photographed using transmission electron microscopy. Crystallite sizes from multiple sections were measured using Image J. Crystallite mean thicknesses were (WT = 26 nm, AKO = 16 nm, KOm180-87 = 25 nm) and the mean widths were (WT = 96 nm, AKO = 59 nm, KOm180-87 = 85 nm). Despite a complete loss of amelogenin in AKO mice, a mineralized enamel layer with well-defined and organized crystallites forms. Enamel crystallites forming in the absence of amelogenin were reduced in thickness and width. For the first time we show that introduction of the m180 amelogenin isoform into the AKO mouse through crossbreeding rescues the crystallite phenotype. We conclude that amelogenin is essential for the development of normal crystallite size

Rescue of the murine amelogenin null phenotype with two amelogenin transgenes

The amelogenin proteins are required for normal enamel development; the most abundant amelogenins expressed from alternatively spliced mRNAs are M180 and leucine rich amelogenin protein (LRAP). The Amelx null (KO) mouse has an enamel defect similar to human X-linked amelogenesis imperfecta. The disorganized enamel layer in KO mice is 10–20% the thickness of wild-type (WT) enamel and lacks prismatic structures. When the KO mice were mated with mice that express TgM180-87, partial rescue of the phenotype was observed such that enamel thickness, volume and density increased. A second transgene was introduced by mating the TgM180KO mice with TgLRAP mice, and male offspring were characterized for genotype and tooth phenotype was evaluated by SEM. TgM180LRAPKO molar enamel thickness further increased, and the structure was improved, with a more defined decussation pattern compared to singly rescued mice. We conclude that TgM180 provides significant rescue of the KO phenotype. Although the effectiveness of TgLRAP to rescue by itself is less obvious, the addition of TgLRAP to TgM180 in KO enamel leads to added improvement in both amount and structure and thus these transgenes function in a complementary manner. Together the two most abundant amelogenins lead to formation of obvious enamel decussation patterns

Mouse Genetic Background Influences the Dental Phenotype

Dental enamel covers the crown of the vertebrate tooth and is considered to be the hardest tissue in the body. Enamel develops during secretion of an extracellular matrix by ameloblast cells in the tooth germ, prior to eruption of the tooth into the oral cavity. Secreted enamel proteins direct mineralization patterns during the maturation stage of amelogenesis as the tooth prepares to erupt. The amelogenins are the most abundant enamel proteins, and are required for normal enamel development. Phenotypic differences were observed between incisors from individual Amelx (Amelogenin) null mice that had a mixed 129xC57BL/6J genetic background, and between inbred wld-type (WT) mice with different genetic backgrounds (C57BL/6J, C3H/HEJ, FVB/NJ). We hypothesized this could be due to modifier genes, as human patients with a mutation in an enamel protein gene causing the enamel defect amelogenesis imperfecta (AI) also can have varied appearance of dentitions within a kindred. Enamel density measurements varied for all WT inbred strains midway during incisor development. Enamel thickness varied between some WT strains and, unexpectedly, dentin density varied extensively between incisors and molars of all WT and Amelx null strains studied. WT FVB/NJ incisors were more similar to Amelx null than to the other WT strains in incisor height/weight ratio and pattern of enamel mineralization. Strain-specific differences led to the conclusion that modifier genes may be implicated in determining both normal development and severity of enamel appearance in AI mouse models and may in future studies be related to phenotypic heterogeneity within human AI kindreds reported in the literature

The Leucine-Rich Amelogenin Peptide Alters the Amelogenin Null Enamel Phenotype

The amelogenin proteins secreted by ameloblasts during dental enamel development are required for normal enamel structure. Amelx null (KO) mice have hypoplastic, disorganized enamel similar to that of human patients with mutations in the AMELX gene, and provide a model system for studies of the enamel defect amelogenesis imperfecta. Because many amelogenin proteins are present in developing enamel due to RNA alternative splicing and proteolytic processing, understanding the function of individual amelogenins has been challenging

An aortic aneurysm model for the evaluation of endovascular exclusion prostheses

Abstract Purpose: The purpose of this study was to develop an aortic aneurysm (AA) model with a predictable tendency for rupture for the evaluation of the efficacy of endovascular prostheses in preventing rupture and their long-term outcome after implantation. Methods: An infrarenal AA measuring two to three times the diameter of the proximal aorta was created in 18 dogs with a full-thickness patch of jejunum. Seven dogs were allowed to survive without aneurysm exclusion. In 11 dogs the aneurysm was immediately excluded with a stented 8 mm Dacron graft mounted in a 14F delivery system introduced through the femoral artery with aortographic guidance. The pressure differential between the aorta and the excluded aneurysm was measured, and angiography, necropsy, and histologic examination were performed at 3- and 6-month survival. Results: All animals survived aneurysm implantation. Without aneurysm exclusion, six dogs died of rupture within 1 to 6 days of surgery. In three dogs the exclusion failed because of graft-to-aorta size mismatch or misplacement demonstrated on angiography and by a low pressure differential between the aorta and the aneurysm (p < 0.023) in dogs with successfully excluded aneurysms (n = 6) compared with that in dogs without exclusion or with failed aneurysm exclusion (n = 7). Conclusion: This aneurysm model demonstrates that without effective aneurysm exclusion all animals die of rupture and that successfully placed endovascular prostheses can prevent AA rupture with long-term graft patency and stability. Endovascular aortic Dacron grafts in dogs undergo complete incorporation at 3 months from implantation. This aneurysm model is useful for the evaluation of endovascular devices designed for the treatment of AAs. (J VASC SURG 1995;22:306-15.

Enamelin Is Critical for Ameloblast Integrity and Enamel Ultrastructure Formation

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam−/− mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam−/− mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam−/− background did not fully recover enamel formation while a medium expresser in the Enam+/− background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation

<html>Human and Mouse Enamel Phenotypes Resulting from Mutation or Altered Expression of <i>AMEL, ENAM</i>, <i>MMP20</i> and <i>KLK4</i></html>

Amelogenesis imperfecta (AI) is caused by AMEL, ENAM, MMP20 and KLK4 gene mutations. Mice lacking expression of the AmelX, Enam and Mmp20 genes have been generated. These mouse models provide tools for understanding enamel formation and AI pathogenesis. This study describes the AI phenotypes and relates them to their mouse model counterparts. Human AI phenotypes were determined in a clinical population of AI families and published cases. Human and murine teeth were evaluated using light and electron microscopy. A total of 463 individuals from 54 families were evaluated and mutations in the AMEL, ENAM and KLK4 genes were identified. The majority of human mutations for genes coding enamel nonproteinase proteins (AMEL and ENAM) resulted in variable hypoplasia ranging from local pitting to a marked, generalized enamel thinning. Specific AMEL mutations were associated with abnormal mineralization and maturation defects. Amel and Enam null murine models displayed marked enamel hypoplasia and a complete loss of prism structure. Human mutations in genes coding for the enamel proteinases (MMP20 and KLK4) cause variable degrees of hypomineralization. The murine Mmp20 null mouse exhibits both hypoplastic and hypomineralized defects. The currently available Amel and Enam mouse models for AI exhibit enamel phenotypes (hypoplastic) that are generally similar to those seen in humans. Mmp20 null mice have a greater degree of hypoplasia than humans with MMP20 mutations. Mice lacking expression of the currently known genes associated with the human AI conditions provide useful models for understanding the pathogenesis of these conditions

Enamelin is critical for ameloblast integrity and enamel ultrastructure formation

Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam-/- mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam-/- mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam -/- background did not fully recover enamel formation while a medium expresser in the Enam+/- background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation. © 2014 Hu et al