Gain ofcis-regulatory activities underlies novel domains ofwinglessgene expression inDrosophila

Changes in gene expression during animal development are largely responsible for the evolution of morphological diversity. However, the genetic and molecular mechanisms responsible for the origins of new gene-expression domains have been difficult to elucidate. Here, we sought to identify molecular events underlying the origins of three novel features ofwingless(wg) gene expression that are associated with distinct pigmentation patterns inDrosophila guttifera. We compared the activity ofcis-regulatory sequences (enhancers) across thewglocus inD. guttiferaandDrosophila melanogasterand found strong functional conservation among the enhancers that control similar patterns ofwgexpression in larval imaginal discs that are essential for appendage development. For pupal tissues, however, we found three novelwgenhancer activities inD. guttiferaassociated with novel domains ofwgexpression, including two enhancers located surprisingly far away in an intron of the distantWnt10gene. Detailed analysis of one enhancer (the vein-tip enhancer) revealed that it overlapped with a region controllingwgexpression in wing crossveins (crossvein enhancer) inD. guttiferaand other species. Our results indicate that one novel domain ofwgexpression inD. guttiferawings evolved by co-opting pre-existing regulatory sequences governing gene activity in the developing wing. We suggest that the modification of existing enhancers is a common path to the evolution of new gene-expression domains and enhancers.