Transgenic analysis of Dlx regulation in fish tooth development reveals evolutionary retention of enhancer function despite organ loss

it has been considered a law that a lost structure cannot reappear in evolution. The common explanation, that genes required for the development of the lost structure degrade by mutation, remains largely theoretical, however. Additionally, the extent to which this mechanism applies to systems of repeated parts, where individual modules are likely to exhibit few unique aspects of genetic control, is unclear. We investigated reversibility of evolution in one such system, the vertebrate dentition, using as a model loss of oral teeth in cypriniform fishes, which include the zebrafish. This evolutionary event, which occurred > 50 million years ago, has not been reversed despite subsequent diversification of feeding modes and retention of pharyngeal teeth. We asked whether the cis-regulatory region of a gene whose expression loss parallels cypriniform tooth loss, DIx2b, retains the capacity for expression in oral teeth. We first created a zebrafish reporter transgenic line that recapitulates endogenous dix2b expression. We then showed that this zebrafish construct drives reporter expression in oral teeth of the related characiform Astyanax mexicanus. This result, along with our finding that Dlx genes are required for normal tooth development, suggests that changes in trans-acting regulators of these genes were responsible for loss of cypriniform oral teeth. Preservation of oral enhancer function unused for > 50 million years could be the result of pleiotropic function in the pharyngeal dentition. If enhancers of other genes in the tooth developmental pathway are similarly preserved, teeth lost from specific regions may be relatively easy to reacquire in evolution.