Z.A. Yuan

AN AMELOGENIN GENE DEFECT ASSOCIATED WITH HUMAN X-LINKED AMELOGENESIS IMPERFECTA

Dental enamel is a product of ameloblast cells, which secrete a mineralizing organic matrix, composed primarily of amelogenin proteins. The amelogenins are thought to be crucial for development of normal, highly mineralized enamel. The X-chromosomal amelogenin gene is a candidate gene for those cases of amelogenesis imperfecta, resulting in defective enamel, in which inheritance is X-linked. In this report, a kindred is described that has a C to A mutation resulting in a pro to thr change in exon 6 of the X-chromosomal amelogenin gene in three affected individuals, a change not found in unaffected members of the kindred. The proline that is changed by the mutation is conserved in amelogenin genes from all species examined to date.

Physiological implications of DLX homeoproteins in enamel formation

Tooth development is a complex process including successive stages of initiation, morphogenesis, and histogenesis. The role of the Dlx family of homeobox genes during the early stages of tooth development has been widely analyzed, while little data has been reported on their role in dental histogenesis. The expression pattern of Dlx2 has been described in the mouse incisor; an inverse linear relationship exists between the level of Dlx2 expression and enamel thickness, suggesting a role for Dlx2 in regulation of ameloblast differentiation and activity. In vitro data have revealed that DLX homeoproteins are able to regulate the expression of matrix proteins such as osteocalcin. The aim of the present study was to analyze the expression and function of Dlx genes during amelogenesis. Analysis of Dlx2/LacZ transgenic reporter mice, Dlx2 and Dlx1/Dlx2 null mutant mice, identified spatial variations in Dlx2 expression within molar tooth germs and suggests a role for Dlx2 in the organization of preameloblastic cells as a palisade in the labial region of molars. Later, during the secretory and maturation stages of amelogenesis, the expression pattern in molars was found to be similar to that described in incisors. The expression patterns of the other Dlx genes were examined in incisors and compared to Dlx2. Within the ameloblasts Dlx3 and Dlx6 are expressed constantly throughout presecretory, secretory, and maturation stages; during the secretory phase when Dlx2 is transitorily switched off, Dlx1 expression is upregulated. These data suggest a role for DLX homeoproteins in the morphological control of enamel. Sequence analysis of the amelogenin gene promoter revealed five potential responsive elements for DLX proteins that are shown to be functional for DLX2. Regulation of amelogenin in ameloblasts may be one method by which DLX homeoproteins may control enamel formation. To conclude, this study establishes supplementary functions of Dlx family members during tooth development: the participation in establishment of dental epithelial functional organization and the control of enamel morphogenesis via regulation of amelogenin expression. J. Cell. Physiol. 216: 688–697, 2008,

Analysis of amelogenin mRNA during bovine tooth development

The amelogenins are highly conserved enamel-matrix proteins that are essential for proper mineral formation. Transcriptionally active genes encoding the bovine amelogenin proteins reside on both the X and Y chromosomes. Comparison of relative levels of amelogenin mRNAs at various stages of development indicated that the X-chromosomal amelogenin message is at least six fold more abundant than the Y. Alternative splicing generates at least seven messages, five from the X primary transcript, and two from the Y. The two most abundant X-chromosomal amelogenin messages are approx. 850 and 450 nucleotides long, and nearly 10-fold more 850-nucleotide mRNA can be measured than 450 nucleotide, which has lost most of exon 6 by splicing. The predominant small message encodes leucine-rich amelogenin protein (LRAP), and amounts of LRAP message are relatively constant during development. However, the amelogenin message from which exon 3 has been spliced declines approximately 2.3-fold, when compared to total X chromosomal amelogenin transcripts, suggesting differential regulation of alternative splicing. In addition, a new exon was identified within genomic DNA, which was shown to be expressed by the use of reverse transcriptase-polymerase chain reaction, and the exons were renamed accordingly. This new exon-4 sequence is unusual in that it is not highly conserved between species.

A new frameshift mutation encoding a truncated amelogenin leads to X-linked amelogenesis imperfecta

The amelogenin proteins are the most abundant organic components of developing dental enamel. Their importance for the proper mineralization of enamel is evident from the association between previously identified mutations in the X-chromosomal gene that encodes them and the enamel defect amelogenesis imperfecta. In this investigation, an adult male presenting with a severe hypoplastic enamel phenotype was found to have a single base deletion at the codon for amino acid 110 of the X-chromosomal 175-amino acid amelogenin protein. The probands mother, who also has affected enamel, carries the identical deletion on one of her X-chromosomes, while the father has both normal enamel and DNA sequence. This frameshift mutation deletes part of the coding region for the repetitive portion of amelogenin as well as the hydrophilic tail, replacing them with a 47-amino acid segment containing nine cysteine residues. While greater than 60% of the protein is predicted to be intact, the severity of this phenotype illustrates the importance of the C-terminal region of the amelogenin protein for the formation of enamel with normal thickness.

The Small Bovine Amelogenin LRAP Fails to Rescue the Amelogenin Null Phenotype

Amelogenins are the most abundant secreted proteins in developing dental enamel. These evolutionarily-conserved proteins have important roles in enamel mineral formation, as mutations within the amelogenin gene coding region lead to defects in enamel thickness or mineral structure. Because of extensive alternative splicing of the primary RNA transcript and proteolytic processing of the secreted proteins, it has been difficult to assign functions to individual amelogenins. To address the function of one of the amelogenins, we have created a transgenic mouse that expresses bovine leucine-rich amelogenin peptide (LRAP) in the enamel-secreting ameloblast cells of the dental organ. Our strategy was to breed this transgenic mouse with the recently generated amelogenin knockout mouse, which makes none of the amelogenin proteins and has a severe hypoplastic and disorganized enamel phenotype. It was found that LRAP does not rescue the enamel defect in amelogenin null mice, and enamel remains hypoplastic and disorganized in the presence of this small amelogenin. In addition, LRAP overexpression in the transgenic mouse (wildtype background) leads to pitting in the enamel surface, which may result from excess protein production or altered protein processing due to minor differences between the amino acid compositions of murine and bovine LRAP. Since introduction of bovine LRAP into the amelogenin null mouse does not restore normal enamel structure, it is concluded that other amelogenin proteins are essential for normal appearance and function.

The Amelogenin C-Terminus Is Required for Enamel Development

The abundant amelogenin proteins are responsible for generating proper enamel thickness and structure, and most amelogenins include a conserved hydrophilic C-terminus. To evaluate the importance of the C-terminus, we generated transgenic mice that express an amelogenin lacking the C-terminal 13 amino acids (CTRNC). MicroCT analysis of TgCTRNC29 teeth (low transgene number) indicated that molar enamel density was similar to that of wild-type mice, but TgCTRNC18 molar enamel (high transgene number) was deficient, indicating that extra transgene copies were associated with a more severe phenotype. When amelogenin-null (KO) and TgCTRNC transgenic mice were mated, density and volume of molar enamel from TgCTRNCKO offspring were not different from those of KO mice, indicating that neither TgCTRNC18 nor TgCTRNC29 rescued enamel’s physical characteristics. Because transgenic full-length amelogenin partially rescues both density and volume of KO molar enamel, it was concluded that the amelogenin C-terminus is essential for proper enamel density, volume, and organization.

Transgenic Mice that Express Normal and Mutated Amelogenins

Amelogenin proteins are secreted by ameloblasts within the enamel organ during tooth development. To better understand the function of the 180-amino-acid amelogenin (M180), and to test the hypothesis that a single proline-to-threonine (P70T) change would lead to an enamel defect similar to amelogenesis imperfecta (AI) in humans, we generated transgenic mice with expression of M180, or M180 with the proline-to-threonine (P70T) mutation, under control of the Amelx gene regulatory regions. M180 teeth had a relatively normal phenotype; however, P70T mineral was abnormally porous, with aprismatic regions similar to those in enamel of male amelogenesis imperfecta patients with an identical mutation. When Amelx null females were mated with P70T transgenic males, offspring developed structures similar to calcifying epithelial odontogenic tumors in humans. The phenotype argues for dominant-negative activity for the P70T amelogenin, and for the robust nature of the process of amelogenesis.

Comparison of upstream regions of X- and Y-chromosomal amelogenin genes

The amelogenin genes encode abundant enamel proteins that are required for the development of normal tooth enamel. These genes are active only in enamel-forming ameloblasts within the dental organ of the developing tooth, and are part of a small group of genes that are active on both sex chromosomes. The upstream regions of the bovine X- and Y-chromosomal and the sole murine X-chromosomal amelogenin genes have been cloned and sequenced, and conservation at nearly 60% is found in the 300 bp upstream of exon 1 for the 3 genes. A region of the bovine X-chromosomal gene that has inhibitory activity when assayed by gene transfer into heterologous cells includes motifs that have a silencing activity in other genes, and may be important to the mechanism that represses amelogenin expression in non-ameloblast cells in vivo. A comparison of sequences from three genes has led to the identification of several regions with conserved motifs that are strong candidates for having positive or negative regulatory functions, and these regions can now be tested further for interaction with nuclear proteins, and for their ability to regulate expression in vivo.

Model system for evaluation of alternative splicing: exon skipping.

Alternative splicing of the primary RNA transcript is a common mechanism for generating protein diversity. A model system was developed to study this process in vitro that is useful for evaluation of splicing of transcripts expressed in cells that do not grow well in culture. The system was used to analyze skipping of exon 4 of the amelogenin message, normally expressed in ameloblast cells for a short interval during tooth enamel development. Amelogenins are highly conserved proteins resulting from extensive alternative splicing, with domains involved in a range of functions, including mineral formation and intercellular signaling. In the bovine gene, the very short intron 4 was predicted to inhibit inclusion of exon 4, because in murine ameloblasts, exon 4 is detectably included in mRNA, and intron 4 is longer than the bovine counterpart. Bovine intron 4 was lengthened, and this size increase enhanced exon 4 inclusion sixfold to eightfold, although splice site selection was inaccurate. Intron length, therefore, is not the sole determinant controlling amelogenin exon 4 inclusion, and cis-acting inhibitory elements may also be involved in exon skipping. This vector system allows evaluation of splicing of a tissue-specific RNA by focusing on exons of interest through transfection of heterologous cultured cells without complications attributable to background transcription of the gene being evaluated.

Analysis of the regulatory region of the bovine X-chromosomal amelogenin gene.

The amelogenin proteins, which are crucial for normal enamel mineral formation, are secreted by ameloblasts during development of tooth enamel. In order to better understand the mechanisms involved in regulation of expression of the amelogenin genes, the bovine X-chromosomal amelogenin gene was cloned and a 3.5 KB fragment upstream of exon 1 was inserted into a beta galactosidase (beta gal) expression vector for production of transgenic mice. When tissues from these mice were treated with Xgal, a substrate for beta gal, only ameloblasts and some of the adjacent stratum intermedium cells contained blue stain. To obtain further information concerning regulation of expression, the 3.5 KB amelogenin gene fragment was evaluated in transfection experiments. Nonoverlapping 1.9 and 1.5 KB fragments of the upstream region were subcloned separately into a vector that contains the SV40 promoter and the CAT reporter gene. Each amelogenin gene fragment was able to suppress CAT activity driven by the heterologous SV40 promoter in transfected HeLa cells. We theorize that each of these gene fragments contains regulatory elements important for the tissue-specific and developmentally-regulated pattern of expression of the X-chromosomal amelogenin gene.