The amelogenin proteins regulate enamel mineral formation in the developing tooth. The human AMELX gene, which encodes the amelogenin proteins, is located within an intron of the Arhgap 6 gene. ARHGAP 6 encodes a Rho GAP, which regulates activity of Rho A, a small G protein involved in intracellular signal transduction. Mice were generated in which the entire ARHGAP 6 gene was deleted by Cre-mediated recombination, which also removed the nested Amel X gene. Enamel from these mice appeared chalky white, and the molars showed excessive wear. The enamel layer was hypoplastic and non-prismatic, whereas other dental tissues had normal morphology. This phenotype is similar to that reported for Amel X null mice, which have a short deletion that removed the region surrounding the translation initiation site, and resembles some forms of X-linked amelogenesis imperfecta in humans. Analysis of the enamel from the Arhgap 6/Amel X-deleted mice verifies that the Amel X gene is nested within the murine Arhgap 6 gene and shows that removal of the entire Amel X gene leads to a phenotype similar to the earlier Amel X null mouse results, in which no amelogenin protein was detected. However, an unusual layer of aprismatic enamel covers the enamel surface, which may be related to the 1.1-Mb deletion, which included Arhgap 6 in these mice.
"This strategy may avoid lethality as the Cre recombinase, under control of a tissue specific promoter , may be expressed later in development and in only the target tissues. Using the Cre-lox approach, a deletion was generated in the ARHGAP6 gene which also removed the amelogenin gene localized to an ARHGAP6 intron, leading to an enamel defect (Prakash et al., 2005). A mouse that expressed the Cre recombinase under control of the Amelogenin regulatory sequences was mated with mice with a floxed TGFβ receptor II gene to generate enamel pathology due to deletion of receptor activity (Cho et al., 2013). "
[Show abstract][Hide abstract] ABSTRACT: A primary goal of enamel research is to understand and potentially treat or prevent enamel defects related to amelogenesis imperfecta (AI). Rodents are ideal models to assist our understanding of how enamel is formed because they are easily genetically modified, and their continuously erupting incisors display all stages of enamel development and mineralization. While numerous methods have been developed to generate and analyze genetically modified rodent enamel, it is crucial to understand the limitations and challenges associated with these methods in order to draw appropriate conclusions that can be applied translationally, to AI patient care. We have highlighted methods involved in generating and analyzing rodent enamel and potential approaches to overcoming limitations of these methods: (1) generating transgenic, knockout, and knockin mouse models, and (2) analyzing rodent enamel mineral density and functional properties (structure and mechanics) of mature enamel. There is a need for a standardized workflow to analyze enamel phenotypes in rodent models so that investigators can compare data from different studies. These methods include analyses of gene and protein expression, developing enamel histology, enamel pigment, degree of mineralization, enamel structure, and mechanical properties. Standardization of these methods with regard to stage of enamel development and sample preparation is crucial, and ideally investigators can use correlative and complementary techniques with the understanding that developing mouse enamel is dynamic and complex.
Frontiers in Physiology 09/2014; 5:313. DOI:10.3389/fphys.2014.00313 · 3.53 Impact Factor
"Despite the possibility that defects in ARHGAP6 could modify the AMELX null phenotype, ARHGAP6 defects alone apparently do not cause enamel malformations. Arhgap6 null mice showed no enamel defects . AMELX mutations are consistently found in human kindreds with X-linked AI, suggesting there is no second X-linked gene that causes AI, as this would lead to the accumulation of unsolved X-linked AI cases without AMELX mutations, which is not observed. "
[Show abstract][Hide abstract] ABSTRACT: Amelogenesis imperfecta (AI) is a group of inherited conditions featuring isolated enamel malformations. About 5% of AI cases show an X-linked pattern of inheritance, which are caused by mutations in AMELX. In humans there are two, non-allelic amelogenin genes: AMELX (Xp22.3) and AMELY (Yp11.2). About 90% of amelogenin expression is from AMELX, which is nested within intron 1 of the gene encoding Rho GTPase activating protein 6 (ARHGAP6). We recruited two AI families and determined that their disease-causing mutations were partial deletions in ARHGAP6 that completely deleted AMELX. Affected males in both families had a distinctive enamel phenotype resembling "snow-capped" teeth. The 96,240 bp deletion in family 1 was confined to intron 1 of ARHGAP6 (g.302534_398773del96240), but removed alternative ARHGAP6 promoters 1c and 1d. Analyses of developing teeth in mice showed that ARHGAP6 is not expressed from these promoters in ameloblasts. The 52,654 bp deletion in family 2 (g.363924_416577del52654insA) removed ARHGAP6 promoter 1d and exon 2, precluding normal expression of ARHGAP6. The male proband of family 2 had slightly thinner enamel with greater surface roughness, but exhibited the same pattern of enamel malformations characteristic of males in family 1, which themselves showed minor variations in their enamel phenotypes. We conclude that the enamel defects in both families were caused by amelogenin insufficiency, that deletion of AMELX results in males with a characteristic snow-capped enamel phenotype, and failed ARHGAP6 expression did not appreciably alter the severity of enamel defects when AMELX was absent.
PLoS ONE 12/2012; 7(12):e52052. DOI:10.1371/journal.pone.0052052 · 3.23 Impact Factor
"Severe enamel phenotypes have been demonstrated in mice with defective amelogenin (Amel) [Gibson et al., 2001; Prakash et al., 2005], enamelin (Enam) [Masuya et al., 2005; Hu et al., 2008] or ameloblastin (Ambn) [Fukumoto et al., 2004] genes. In humans, defects in AMELX cause X-linked amelogenesis imperfecta, while ENAM mutations cause autosomal dominant amelogenesis imperfecta [Hu and Yamakoshi, 2003; Stephanopoulos et al., 2005; Kim et al., 2006]. "
[Show abstract][Hide abstract] ABSTRACT: Enamel development requires the strictly regulated spatiotemporal expression of genes encoding enamel matrix proteins. The mechanisms orchestrating the initiation and termination of gene transcription at each specific stage of amelogenesis are unknown. In this study, we identify cis- regulatory regions necessary for normal enamelin (Enam) expression. Sequence analysis of the Enam promoter 5'-noncoding region identified potentially important cis-regulatory elements located within 5.2 kb upstream of the Enam translation initiation site. DNA constructs containing 5.2 or 3.9 kb upstream of the Enam translation initiation site were linked to an LacZ reporter gene and used to generate transgenic mice. The 3.9-kb Enam-LacZ transgenic lines showed no expression in ameloblasts, but ectopic LacZ staining was detected in osteoblasts. In contrast, the 5.2-kb Enam-LacZ construct was sufficient to mimic the endogenous Enam ameloblast-specific expression pattern. Our study provides new insights into the molecular control of Enam cell- and stage-specific expression.
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