JACKMAN, W.R.*; STOCK, D.W.; University of Colorado at Boulder: Transgenic analysis of the mechanism of cypriniform oral tooth loss

We are interested in the developmental genetic mechanisms responsible for evolutionary change in fish dentition, and are specifically investigating the mechanism of oral tooth loss in cypriniform fishes. In a survey of genes with known tooth expression in mammals, we found that Dlx2 ortholog expression is missing from the oral epithelium of the cypriniform zebrafish. Such expression is present in fish species retaining oral teeth, however, such as the relatively closely-related characiform Astyanax mexicanus, as well as the more distantly related Japanese medaka. To test whether a cis-regulatory mutation rendered cypriniform Dlx2 orthologs incapable of oral expression, we created a reporter transgenic containing 4 kb of zebrafish genomic sequence 5′ of the dlx2b transcription start attached to GFP. We found in both transient injections of this construct and a stable transgenic line, that GFP is expressed in zebrafish pharyngeal tooth epithelium but not in the oral region. Injection of the same construct into A. mexicanus drives reporter expression in the epithelium of both pharyngeal and oral teeth. This indicates that evolution of dlx2b expression is the result of changes in trans-acting factors, rather than of changes to the gene itself. We have previously reported that Fibroblast growth factor (Fgf) signaling is required for Dlx2 ortholog expression in zebrafish pharyngeal teeth. Application of an Fgf pathway inhibitor to the dlx2b:GFP zebrafish transgenic line similarly eliminates GFP expression in pharyngeal teeth, indicating that this element requires Fgf signaling for its dental expression. We are currently investigating the Fgf pathway as a candidate trans-acting factor responsible for loss of oral Dlx2 ortholog expression in cypriniforms and perhaps loss of oral teeth as well.