We have made substantial progress in understanding the role of nautilus in Drosophila myogenesis. The highly organized and segmentally reiterated muscle pattern in the Drosophila embryo is prefigured by the arrangement of a sub-population of mesodermal cells called founder myoblasts. We had shown earlier that the expression of nautilus, the only MyoD-related gene in Drosophila, is initiated at stage 9 in a stereo-specific pattern in a subset of mesodermal cells that become incorporated into every somatic muscle in the embryo. Targeted ricin toxin ablation of these cells resulted in the loss of embryonic muscle. We now know that at stage 11 these same cells begin to express a later founder cell-specific marker, duf (rP298LacZ) thus nautilus is the earliest marker for the critical founder myoblast population. We inactivated the nautilus gene using both homology-directed gene targeting and a novel gal4-inducible nautilus RNAi transgene to determine if any aspect of founder cell function required nautilus gene activity. An earlier study in our lab, using the injection of nautilus dsRNA to induce gene silencing by RNAi, indicated loss of nautilus function resulted in a range of phenotypes from no muscle disruption to severe embryonic muscle loss and disruption (30% of the embryos). Both gene targeting and the gal4-inducable nautilus RNAi resulted in a range of defects that included severe embryonic muscle disruption, reduced viability and female sterility. All these phenotypes were rescued by a hsp70 nautilus cDNA transgene in the absence of heat shock in independent transgenic lines. More importantly, the highly organized founder cell pattern that is needed to establish the proper embryonic muscle organization was disrupted in nautilus null embryos prior to MHC expression and the disruption prefigured the subsequent embryonic muscle defects observed at later stages in development. Tinman, a marker for mesodermal cells that give rise to the dorsal vessel or heart, was expressed normally in the nautilus null embryo. Although nautilus does not specify the myogenic cell lineage, it has a cell autonomous role in establishing the correct muscle organization in the embryo through its regulation of the founder cell pattern. This work has been published recently in PNAS (Wei et al). We are currently carrying out experiments to identify nautilus target genes. To identify nautilus target genes we have used two approaches. First we have just completed a transcriptome comparison between mutant and wild-type embryos using the Solexa HiSeq2000 Genomic Analyzer with more that 30Gb of filtered reads for each sample that are currently under analysis by NCI informatics and other collaborators. Nautilus is expressed in only 0.1% of the cells in the embryo (800 cells). In order to capture gene loci that interact with nautilus, we used gene targeting to generated a fly line to express C-terminal biotinylated nautilus protein. The selectivity of the biotin-avidin capture has been evaluated using a recently identified nautilus target gene, the miR-309 micro RNA cluster. Biotin selected DNA from 9-13 hour wild-type and nau-biotin embryos has been isolated and sequenced on the Solexa 1G Genomic Analyzer to give 16-17 million filtered, good quality reads. We have preliminary data on 3000 peaks across the genome that are under analysis with the help of Dr. Sameet Mehta, an informatics expert in Dr. Shiv Grewal's lab. We have also targeted an AttP site into the nautilus gene in order to determine the role of possible regulatory DNA sequences in the promoter and 3' UTR. We have also identified six intra-genic enhancer regions that modulate the nautilus expression pattern in the embryo using insulated enhancer reporter transgenes. Selective removal of these regions will determine their impact on muscle formation. micro RNAs (miRNAs) play a key role in gene regulation in development and disease. A miRNA expression profile in the nautilus null revealedthat expression from the miR-309 locus is down regulated and is dependent upon two E-boxes in the miR-309 promoter/enhancer. miR-3 in the locus fine tunes nautilus expression in the embryo in a negative feedback loop involving the nau 3'-UTR. Deletion of the miR-309 cluster or ectopic expression of miR-3 also decrease Dmef2 RNA levels, a transcription factor required for muscle formation. Ectopic miR-3 expression enhances output from the miR-310 locus encoding 7 micro RNAs, four of which target the 3'UTR of the essential myogenic regulatory factor, Dmef2. The convergence of these miRNA regulatory pathways points to a previously unappreciated complexity in nautilus gene regulation of Drosophila myogenesis and the complex miRNA circuitry buffering the myogenic transcriptome. Targeted deletion of the mirs in the miR-309 cluster using an attP site as well as removal of the mir-3 binding site in the nau 3'-UTR via the nau attP are under way. We have used a specific mir-3 sponge transgene to determine the effects of mir-3 reduction during development. Loss or gain of mir-3 result in severe muscle disruption in 10-15% of the embryos. Importantly, nautilus direct regulation of the miR-309 cluster and mir-3 output, coupled to mir-3 activation of the miR-310 cluster which regulates dMef2, the notch pathway and Jak-Stat signaling, all affect Drosophila myogenesis. Transcriptome buffering by microRNA circuits under the control of tissue-sspecific transcription factors give insight to cellular homeostasis and a basis for disease mechanisms. Recently we have used the CRISPR system to delete the mir-3 binding site in the nautilus gene which leads to misregulation of nautilus expression, miR-309/310 output and a massive disruption of the muscle pattern. We are currently adding a carboxy terminal eGFP tag to the endogenous nautilus gene to reduce background on ChIP-SEQ experiments to identify nautilus target genes more precisely using eGFP single chain nanobodies with nanomolar affinity. We hope to assemble a transcriptome network for embryonic muscle development.