The LDSB investigates the organization and activities of developmental regulatory networks using formation of the Drosophila embryonic heart and body wall muscles as a model system. The overarching goal of this work is to comprehensively identify and characterize the upstream regulators of cell fate specification, the downstream effectors of differentiation, and the complex functional interactions that occur among these components during organogenesis. To achieve this objective, we combine contemporary genome-wide experimental and computational approaches with classical genetics and embryology to generate mechanistic hypotheses that we then test at single cell resolution in the intact organism. In a separate project, we have discovered DNA sequence motifs that are preferentially bound by different homeodomain (HD) transcription factors, which suggests a novel mechanism for how HD-specific transcriptional responses are generated during embryonic development. We have tested the hypothesis that HD-preferred sites serve such a function using muscle founder cell (FC) enhancers as a model. In these studies, we determined that the FC identity HD (FCI-HD) protein Slouch (Slou) binds to unique 9-mers that are over-represented in putative enhancers located within the noncoding regions of Slou-responsive FC genes. One such Slou-preferred site occurs in each of two FC enhancers that we have previously characterized in detail. Moreover, we found that both of these Slou-preferred binding sites are essential for the normal activities of these two cis-regulatory modules (CRMs). In one case, Slou functions as an activator through a particular Slou-preferred binding site, and in the other it functions as a repressor by binding to a distinct Slou-preferred motif. Furthermore, neither a different Slou-preferred site nor a site that binds to all HDs is capable of substituting for the wild-type Slou-preferred sites in these enhancers. These findings indicate that the specific sequence of a HD binding site dictates which HD family member binds to and regulates a particular enhancer, a previously unrecognized mechanism for how cell type-specific transcription factors induce the distinct genetic programs of individual embryonic cells. We have also used our extensive HD protein binding microarray datasets to map the locations of each class of HD binding site in a number of mesodermal enhancers. Targeted mutagenesis of these sites then allowed us to assess the relative contribution of an individual HD transcription factor to the cell-specific activity of each regulatory element. Using this approach, we have undertaken a systematic analysis of the inputs of FCI-HD, NK-HD and Hox transcription factors to three enhancers that are active in FCs, cardiac mesoderm, visceral mesoderm and the differentiated heart. Analysis of mutant enhancers revealed that all three HD classes are required for the full activity of each mesodermal CRM, although the qualitative contribution of each HD varies with the particular regulatory element. Additional experiments are in progress to determine the precise effects of each HD class on the cell type specificity of mesodermal gene expression.