Genome sequences contain the information needed for the development and function of a given species. Exploring conservation of DNA sequence between species is a tool for identifying critical code that is likely to be important for functions shared between species. Genomic information is deployed by transcription and differential expression may well be important for phenotypic distinctions among closely related species. Comparative transcription has not been extensively explored. We are performing deep sequencing experiments (RNA-Seq) to probe expression in several of the newly sequenced Drosophila species. These expression data provide the only biological evidence for most of the predicted genes in these species. Conserved gene expression is outstanding evidence of function. Therefore, these data will also be used to validate gene models in Drosophila melanogaster. We are looking at the transcriptional response to altered gene dose in aneuploid tissue culture cells, during X chromosome dosage compensation, and in a series of flies heterozygous for a tiling path of deletions (DrosDel). These data allow us to assign a buffering or feedback value for each gene. Additionally, we are mapping the propagating dosage effect elsewhere in the genome. Preliminary data suggest that these propagating effects are coherent (genes with altered expression in the diploid region are connected to genes in the hemizygous region), and that altered gene expression is dampened as network distance increases (propagation is less than three nodes). These data are providing important validation of connectivity maps and will allow us to add directionality to network edges. Doublesex (Dsx) DMRT transcription factors direct gonadal development in nematodes, insects, and vertebrates, but little is known about the gene networks they regulate. We are interested in the identification of functional DSX transcription factor target genes. Current work involves using ChIP-Seq and DamID-Seq to determine the in vivo occupancy of DSX using tagged and native proteins in tissue culture cells and in Drosophila tissues. We are simultaneously examining expression profiles in wild type and mutant conditions (especially those where we change the isoform of Dsx being expressed and measure the response over time) by RNA-Seq. We are also performing knockdown experiments to test for the function of candidate DSX targets using RNAi (UAS-shRNAs driven by Dsx-Gal4 and other Gal4 lines).