Membranes, minerals, and proteins of developing vertebrate enamel.

Developing tooth enamel is formed as organized mineral in a specialized protein matrix. In order to analyze patterns of enamel mineralization and enamel protein expression in species representative of the main extant vertebrate lineages, we investigated developing teeth in a chondrichthyan, the horn shark, a teleost, the guppy, a urodele amphibian, the Mexican axolotl, an anuran amphibian, the leopard frog, two lepidosauria, a gecko and an iguana, and two mammals, a marsupial, the South American short-tailed gray opossum, and the house mouse. Electron microscopic analysis documented the presence of a distinct basal lamina in all species investigated. Subsequent stages of enamel biomineralization featured highly organized long and parallel enamel crystals in mammals, lepidosaurians, the frog, and the shark, while amorphous mineral deposits and/or randomly oriented crystals were observed in the guppy and the axolotl. In situ hybridization using a full-length mouse probe for amelogenin mRNA resulted in amelogenin specific signals in mouse, opossum, gecko, frog, axolotl, and shark. Using immunohistochemistry, amelogenin and tuftelin enamel proteins were detected in the enamel organ of many species investigated, but tuftelin epitopes were also found in other tissues. The anti-M179 antibody, however, did not react with the guppy and axolotl enameloid matrix. We conclude that basic features of vertebrate enamel/enameloid formation such as the presence of enamel proteins or the mineral deposition along the dentin-enamel junction were highly conserved in vertebrates. There were also differences in terms of enamel protein distribution and mineral organization between the vertebrates lineages. Our findings indicated a correlation between the presence of amelogenins and the presence of long and parallel hydroxyapatite crystals in tetrapods and shark.

Caiman periodontium as an intermediate between basal vertebrate ankylosis-type attachment and mammalian "true" periodontium.

The teeth of many fish, amphibia, and reptiles are attached to the alveolar bone via ankylosis. In contrast, mammalian periodontia are characterized by a gomphosis, an attachment of the tooth root in the alveolar bone socket via periodontal ligament fibers. Among the reptiles, the crocodilians are the only group featuring a gomphosis-type connection between tooth root and alveolar bone, while in other reptiles tooth-root and jawbone are connected via ankylosis. The purpose of the present study was to compare several key features of the crocodilian periodontium with those of the mammalian and noncrocodilian reptile periodontium. As experimental models for our study we chose the periodontium of newborn geckos (Hemidacylus turcicus), juvenile caimans (Caiman crocodilus crocodilus), and 10-day-postnatal Swiss-Webster mice (Mus musculus) as representative models for noncrocodilian reptiles, crocodilian reptiles, and mammals. The caiman periodontium emerged as an intermediary between the mineral-free mouse ligament and the mineralized gecko ankylosis-type attachment. Caiman ligament fibers were less organized than mouse ligament fibers but featured distinct fasciae surrounding ligament fiber bundles. Caiman Hertwig's epithelial root sheath (HERS) was similarly perforated as mouse HERS and distinctly different from the continuous gecko HERS. Both caiman and mouse HERS covered the entire tooth root length, while in the gecko HERS was limited to the coronal portion of the root, allowing for cementoid-mediated ankylosis at the apical tip of the root. We interpret our data to indicate distinct differences in mineral distribution, periodontal ligament fiber organization, and HERS distribution between noncrocodilian reptiles, crocodilian reptiles, and mammals. Mineral deposits in the caiman ligament may reflect an evolutionary position of the caiman periodontium between ankylosis and gomphosis.

Conservation and variation in enamel protein distribution during vertebrate tooth development.

Vertebrate enamel formation is a unique synthesis of the function of highly specialized enamel proteins and their effect on the growth and organization of apatite crystals. Among tetrapods, the physical structure of enamel is highly conserved, while there is a greater variety of enameloid tooth coverings in fish. In the present study, we postulated that in enamel microstructures of similar organization, the principle components of the enamel protein matrix would have to be highly conserved. In order to identify the enamel proteins that might be most highly conserved and thus potentially most essential to the process of mammalian enamel formation, we used immunoscreening with enamel protein antibodies as a means to assay for degrees of homology to mammalian enamel proteins. Enamel preparations from mouse, gecko, frog, lungfish, and shark were screened with mammalian enamel protein antibodies, including amelogenin, enamelin, tuftelin, MMP20, and EMSP1. Our results demonstrated that amelogenin was the most highly conserved enamel protein associated with the enamel organ, enamelin featured a distinct presence in shark enameloid but was also present in the enamel organ of other species, while the other enamel proteins, tuftelin, MMP20, and EMSP1, were detected in both in the enamel organ and in other tissues of all species investigated. We thus conclude that the investigated enamel proteins, amelogenin, enamelin, tuftelin, MMP20, and EMSP1, were highly conserved in a variety of vertebrate species. We speculate that there might be a unique correlation between amelogenin-rich tetrapod and lungfish enamel with long and parallel crystals and enamelin-rich basal vertebrate enameloid with diverse patterns of crystal organization.