The homeodomain protein Fushi tarazu (Ftz) and its obligate cofactor Ftz-F1, an orphan nuclear receptor, cooperatively bind to DNA and co-regulate the transcription of genes responsible for segmentation in the early Drosophila embryo. Two interesting questions have arisen about these genes. The first question concerns the evolution of Ftz, which changed in arthropods from a Hox protein that likely bound its transcriptional targets with the cofactor Exd, to a pair-rule protein in Drosophila that synergistically binds DNA with its cofactor Ftz-F1. This change in function involved changes in both the expression and protein sequence of Ftz, which are being explored throughout arthropod lineages. To determine if the expression and function of ftz is conserved in Diptera, Ftz and related genes were examined in the mosquito Aedes aegypti using reverse transcriptase - PCR, in situ hybridization, and ectopic expression techniques. The second question probes the mechanisms underlying Drosophila Ftz/Ftz-F1 target site recognition in vivo. To date, direct computational attempts to identify downstream target genes and their enhancers have been inadequate. Towards this end, a microarray analysis was performed comparing wild type and ftz-f1 mutant embryos. This generated a list of Ftz/Ftz-F1 target genes whose expression was lower in ftz-f1 mutants than wild type. To identify genes among this group that are directly regulated by Ftz/Ftz-F1, potential Ftz/Ftz-F1 binding sites around these genes

were identified by combining Ftz in vivo ChIP data with a computational search for candidate Ftz-F1 binding sites. Next, to test whether these regions correspond to Ftz/Ftz-F1-dependent enhancers, enhancer-lacZ reporter genes were constructed and their expression was analyzed in wild type and ftz mutant embryos. Of 10 enhancers tested, 8 generated expression patterns that overlap with Ftz and Ftz-F1 expression in early embryos and were lost in ftz mutants. The enhancers found in this study, along with previously identified Ftz/Ftz-F1-dependent enhancers, were analyzed to identify binding motifs for additional transcription factors that might co-regulate gene expression with Ftz/Ftz-F1. Four transcription factors were identified that could potentially be involved in Ftz/Ftz-F1-dependent gene regulation: Deaf-1, Dichaete, Zeste, all transcriptional activators, and GAGA factor, a repressor. Together, these studies identified five new Ftz/Ftz-F1-dependent target genes and seven new Ftz/Ftz-F1-regulated enhancers, and they suggest that other transcription factors may also play roles in the pair-rule gene regulatory system.