True enamel covering in teeth of the Australian lungfish Neoceratodus forsteri

Lungfish are a unique order of sarcopterygian fish cleidographically positioned between tetrapods and fish. An uninterrupted 400-million-year-old fossil record has documented lungfish skeletal elements to remain virtually unchanged since the Early Devonian. In the current study we investigated the enamel layer of lungfish teeth in order to determine whether there was evidence for higher vertebrate "true" enamel in the Australian lungfish. Juvenile lungfish from the Brisbane River were processed for light and electron microscopy and analyzed for parameters indicative of true enamel formation. Using anti-amelogenin primary antibodies for immunodetection and Western blots, enamel protein epitopes were detected in developing lungfish teeth. Using transmission electron microscopy and electron diffraction analysis, long and parallel-oriented hydroxyapatite crystals were observed in lungfish outer tooth coverings. Our findings indicate that Australian lungfish teeth are covered by a layer of true enamel. Based on the lungfish fossil record we conclude that features of true enamel formation may be as old as 400 million years. Based on taxonomic classification we confirm that true enamel is found not only in tetrapods but also in the sarcopterygian clade of the Gnathostomata.