Whether macroevolutionary patterns result from natural selection or developmental constraints has long been debated. The proposed research seeks to explain in terms of such external or internal forces an evolutionary trend toward tooth reduction in vertebrates. Loss of oral teeth in cypriniform fishes (e.g. minnows) serves as a model of this trend. No member of the group has regained oral teeth, despite diverse feeding modes and retention of throat teeth, suggesting tooth loss as a constraint on evolution. Characterizing the developmental mechanism of this loss will test the constraint hypothesis; identifying a simple, experimentally-reversible mechanism would argue against it. Previous comparisons of gene expression in the cypriniform zebrafish (Danio rerio) and related groups with oral teeth identified an association between absence of teeth and of Dlx2 transcription factor expression. This result will be extended to further characterize modifications of developmental genetic pathways resulting in cypriniform tooth loss. Objectives are to 1) identify the mechanism of Dlx expression evolution, 2) identify consequences of loss and gain of Dlx function, 3) investigate Fibroblast growth factor (Fgf) signaling pathways as potential causes of loss of Dlx expression and oral teeth, and 4) identify any changes in expression of additional Dlx family members associated with tooth loss and evolution of gene family number. The primary species to be employed are the zebrafish and the Japanese medaka (Oryzias latipes), the latter of which retains oral teeth. Transgenic approaches will be used to compare regulation of Dlx2 gene expression in the two species and to misexpress native or modified Dlx and Fgf proteins. Expression of the entire complement of Dlx genes (some of which remain to be cloned) will be examined in the zebrafish, medaka and Mexican tetra (Astyanax mexicanus). These species exhibit differences in dentition and probably in the number of Dlx genes present in their genomes as well. Characterization of the mechanism of cypriniform oral tooth loss will allow comparison with other instances of vertebrate dental reduction, in addition to elucidating fundamental mechanisms of tooth development.
Transgenic lines and constructs generated will be valuable for studying the role of Dlx and Fgf genes in the development of other structures. The project will have significant educational value as well. Results and materials will be incorporated into an existing course in Evolutionary Developmental Biology, while several undergraduate students (recruited through universty programs), two graduate students, and a post-doctoral researcher will receive research training while executing the project.
