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 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 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. 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 undertaken a transcriptome analysis of mutant and wild-type embryos using the Solexa 1G Genomic Analyzer, a so-called deep sequence approach. Genes involved in determining the myogenic field in the mesoderm, establishing the muscle founder and fusion competent myoblast populations, regulating cell fusion, and establishing muscle identity are measurably down regulated in the nautilus null. Expression patterns for genes involved in myotube positioning are also altered in the null. By contrast, certain genes representing muscle structural proteins, actin-binding proteins, ion channels, excitation-contraction coupling components, calcium binding proteins, and synaptic vesicle movement are 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 roles in myogenesis. These genes may be direct targets for nautilus regulation and this is being determined with the application of a novel ChIP-Seq strategy. Since nautilus is expressed in only 0.1% of the cells in the embryo, stringent ChIP conditions must be employed to identify target genes. In order to capture gene sequences that interact with nautilus we generated a fly line with a biotinylatable peptide tag joined to the carboxy terminus of the engodenous nautilus gene, a peptide that can be biotinylated by E. coli biotin ligase expressed from the targeting vector. The selectivity of the biotin-avidin capture in ChIP has now been evaluated using a known nautilus target gene, the 8-miR locus discussed below. The proper ChIP conditions have been established and we are performing ChIP-Seq (Solexa 1G Genomiic Analyzer) to identify nautilus target DNA sequences in a temporal fashion. We have also introduced an AttP site in the nautilus gene to determine the role of DNA sequences involved in promoter function, nautilus transcription factor activity, miRNA binding and enhancer function. We have identified six enhancer regions that modulate the nautilus expression pattern. Selective removal of these regions will determine their impact on muscle formation. Small 21bp RNAs known as micro RNAs (miRNAs) play a key role in gene regulation in development and disease. Using in silico methods we identifed two miRNAs that were confirmed to regulate nautilus expression in the embryo and the adult. The nautilus 3'UTR with the mir target sites can also regulate a reporter in S2 cells in response to the ectopic expression of these miRNAs. A miRNA expression profile in the nautilus null revealed that the 8-miR locus encoding 9 microRNAs is affected and is under the direct control of nautilus via two E-boxes in the 8miR-locus promoter. miR3 in the locus fine tunes nautilus expression in the embryo in a negative feedback loop. Loss of the 8-miR locus impacts miR-1 and miR-184 levels, essential micro RNAs for myogenesis and egg laying, respectively. 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. This work has been submitted 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.
|Lipardi, Concetta; Paterson, Bruce M (2010) Identification of an RNA-dependent RNA polymerase in Drosophila establishes a common theme in RNA silencing. Fly (Austin) 4:30-5|
|Lipardi, Concetta; Paterson, Bruce M (2009) Identification of an RNA-dependent RNA polymerase in Drosophila involved in RNAi and transposon suppression. Proc Natl Acad Sci U S A 106:15645-50|