It has long been recognized that the direction of evolution is determined not only by the environments to which organisms are exposed, but also by features of their genetic and developmental programs that constrain the ability to vary. A pattern indicative of such 'developmental constraint' is irreversible evolution, or the failure of lost structures to reappear when organisms return to ancestral environments or behaviors. This project employs an example of irreversible evolution, the reduction of tooth-bearing locations in cypriniform fishes, to characterize the genetic features underlying developmental constraint. Teeth in this group, which includes minnows and suckers, were lost from the mouth and upper throat over 50 million years ago and are retained only on the lower surface of the throat. The lost teeth have never returned, despite the adoption of ancestral feeding modes by multiple lineages of this diverse group. The project will identify genetic changes that occurred in association with loss of teeth in cypriniforms and test the hypothesis that irreversible evolution is the result of accumulated changes in multiple genes and developmental pathways. In addition, genetic engineering approaches will be applied to a common laboratory cypriniform, the Zebra Danio, in an attempt to restore lost teeth. The enhanced understanding of developmental constraint expected to result from the project is relevant to the practical question of whether species will be able to adapt to manmade environmental change or will instead become extinct. In addition to providing research training at the undergraduate, graduate, and post-graduate levels, the project will produce modified fish strains that will enhance courses in Fish Biology and Genetics at the University of Colorado, as well as an exhibit at its Museum of Natural History.