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 is currently under analysis by NCI informatics. A previous analysis suggested genes involved in the determination of the mesodermal myogenic field, establishment of the muscle founder and fusion competent myoblast populations, regulation of myoblast fusion, and the establishment of muscle identity are measurably down regulated in the nautilus null. Expression patterns for genes involved in myotube positioning are also altered in the null. In addition, certain genes representing muscle structural proteins, actin-binding proteins, ion channels, excitation-contraction coupling components, calcium binding proteins, and synaptic vesicle movement appear mis-regulated and are expressed at somewhat higher levels in the nautilus null embryo. More that 2000 genes are unaffected in the mutant. Trends apparent in the transcriptome analysis have identified groups of genes that are negatively affected in the null, consistent with their role in myogenesis. These genes may be direct targets for nautilus regulation and this is being determined with the application of a novel biotin ChIP-Seq strategy. Since nautilus is expressed in only 0.1% of the cells in the embryo (800 cells), stringent ChIP conditions must be employed to identify target genes. In order to capture gene sequences that interact with nautiluswe generated a fly line with a biotinylatable 14 amino acid peptide tag joined in frame to the carboxy terminus of the engodenous nautilus gene. The tag is biotinylated by a 3'cis- element containing E. coli biotin ligase (BirA)fused to mCherry, the latter serving as a marker for gene targeting. The selectivity of the biotin-avidin capture in ChIP has now been evaluated using two recently identified nautilus target genes, the 8-miR micro RNA locus, discussed below, and the promoter of the collier gene. 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 selected DNA sequences in the promoter and 3'UTR. We have identified six enhancer regions that modulate the nautilus expression pattern 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 revealed that expression from the 8-miR locus is down regulated and is dependent upon two E-boxes in the 8-miR promoter. 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 8-miR 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 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 8miR cluster using the attP site we targeted to the *-miR cluster as well as removal of the mir-3 binding site in the nau 3'-UTR via the nau attP site will culminate in the submission of this work for publication.In our efforts to gain insight into the molecular basis of RNAi-induced gene silencing, we identified a novel mechanism in Drosophila that appeared to involve an RNA-dependent RNA polymerase (RdRP) activity in RNA target degradation. siRNAs produced in Drosophila embryo extract by Dicer cleavage of dsRNA were converted to dsRNA in a reaction dependent upon cognate mRNA which was then cleaved again by dicer. This degradative mechanism could explain how very few molecules of dsRNA could inactivate hundreds of target mRNAs. RdRP is a highly conserved component in RNAi in C. elegans and most lower eukaryotes and also has a role in the maintenance of heterochromatin. We identified elongator subunit 1, D-elp1, highly conserved from S. pombe to humans, as a potential RdRP but this has since been retracted. However, D-elp1 is involved in RNAi and transposon suppression but not miRNA function and interacts with other components of the RNAi machinery. A manuscript describing this important finding was published (Lipardi &Paterson, PNAS 2009). A mutation in the human homologue of D-elp1 produces a truncated protein correlated with the neurological disease Familial Dysautonomia (FD)that affects predominately the Ashkenazi Jewish population. A fly model of the mutation is being generated using a targeted AttP site in the gene. We intend to study the FD phenotype and determine the role of D-elp1 in RNAi and transposon suppression since the latter phenomenon may impact endogenous virus regulation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005258-33
Application #
8552567
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
33
Fiscal Year
2012
Total Cost
$1,073,835
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
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State
Country
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