The emerging field of evo-devo studies, in part, how genotype connects to phenotype. This project seeks to test the function of a few candidate transcription factors in the development of butterfly eyespots. Several transcription factors and gene circuits that are known to be involved in wing and leg development in Drosophila are hypothesized to be involved in the development of eyespots in butterflies. In particular, when Distal-less (Dll) is ectopically activated in Drosophila it causes complete leg duplications, thus suggesting a master regulatory function. In Bicyclus, Dll is expressed in signaling cells in the eyespot's center at the earliest stages of eyespot development, and moreover, variation in Dll is linked with variation in eyespot size. Have butterflies co-opted the same "leg" developmental circuit in the formation of eyespots? An activated ligand receptor, phosphorylated Mothers Against Decapentaplegic, pMad, is expressed in the eyespot field during early pupal development. Is the morphogen Decapentaplegic (Dpp) being produced at the eyespot's center, diffusing to surrounding cells and differentiating a complete eyespot pattern? Another transcription factor, Engrailed (En), is expressed in a ring of epidermal cells that correlates with the pattern of gold colored scales on the eyespot of the adult Bicyclus wing. Does En cause the differentiation of gold colored scales in eyespots? The experiments outlined in this project test these hypotheses by ectopically expressing Dll, Dpp, and En on the developing wing and observing whether, in the first two cases, a complete eyespot pattern differentiates around the site of expression, or whether, in the second case, gold scales develop at the site of expression. Additional experiments will test whether over-expressing Dll and Dpp at the eyespot's center will lead to differences in eyespot size. To induce ectopic expression the lab has developed a laser-assisted heat-shocking technique that will allow the activation of these genes in a controlled spatial and temporal fashion on the developing wing of transgenic butterflies. The heat-shocking technique has been successfully tested with a fluorescent marker gene but now this marker will be substituted for the coding sequences of Dll, Dpp, and En. Establishing a causal relationship between these genes and differentiation of eyespots or colored scales will allow the later examination of the relationship between the evolution of these genes and the evolution of the phenotype. Intellectual merit: Much work in evo-devo has been dominated by inferring novel functions for genes based solely on their novel expression domains. This project intends to go beyond the correlational evidence and to really test whether "old" developmental genes belonging to known gene circuits have been functionally co-opted to specify a morphological novelty in evolution, butterfly eyespots. Once this functional co-option is established future work will attempt to track the evolution of eyespots, from their creation throughout their various levels of quantitative and qualitative modifications. This long-term study will dissect the evolution of regulatory genes with a demonstrated key role in differentiating these adaptive traits. This project aims to test whether a few candidate genes display that key role. In addition, this project will make available a series of transformation constructs of wide applicability across organisms, as well as develop important tools for functional genetics in non-model organisms. Broader Impacts: Dr. Monteiro has currently involved one postdoc and 3 graduate students in this project. One is a woman and a member of the Native American tribe Choctaw Nation of Oklahoma. Another is a minority graduate student in the Department of Electrical Engeneering at Buffalo. These two students are both recipients of NSF IGERT training grants in Biophotonics, and are truly working at the interface of Biology and Photonics via an established collaboration with the Department of Electrical Engineering at Buffalo. She will continue to train graduate and undergraduates students alike in Population Genetics, Phylogenetics, and Developmental Biology, irrespective of gender and ethnicity, in order to provide them with a broad multifaceted approach to Evolution and Development.
