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 severe embryonic muscle loss or disruption. 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 the nautilus cDNA 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,in establishing the muscle founder and fusion competent myoblast populations,in regulating cell fusion, and in 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 will be determined by ChIP-Seq. 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 have generated a fly line with the highest affinity tag known joined to the carboxy terminus of the engodenous nautilus gene, a peptide sequence that can be biotinylated by E. coli biotin ligase expressed from the targeting vector. The selectivity of the biotin-avidin capture in ChIP is being evaluated using a known nautilus target gene, the 8-miR locus discussed below. Once the proper conditions are established, we will perform ChIP-Seq (Solexa 1G Genomiic Analyzer) to identify nautilus target DNA sequences. In addition, recent advances in gene targeting have enabled us to introduce an AttP site in the nautilus gene to study important DNA sequences involved in promoter function, nautilus TF activity, miRNA binding and enhancer function. Small 21bp RNAs known as micro RNAs (miRNAs) play a key role in gene regulation in development and disease. We have recently identified two miRNAs that regulate post transcriptional expression of nautilus in the embryo and the adult. The miRNA binding sites are conserved in the 3'UTR of the nautilus gene in multiple species of Drosophila. The nautilus 3'UTR alone can regulate reporter expression in response to the ectopic expression of these miRNAs in S2 cells. A profile of miRNA expression in the nautilus null revealed that the 8-miR locus, aka the 309-locus, a genomic region encoding 9 microRNAs, regulates the post transcriptional expression of greater than 4000 genes and is under the direct control of nautilus via two E-boxes in the 309-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 decrease Dmef2 RNA levels, a transcription factor required for muscle formation. Ectopic miR-3 expression also targets 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 gene regulation with clear implications for development and disease. AttP site insertion into the 8-miR locus as well now enables us to analyze each micro RNA in the cluster. 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 by the Dicer RNase III-related enzymes in response to the trigger dsRNA, were shown to act as primers to convert the target mRNA into new dsRNA which was then degraded again by Dicers in a cycle of amplification and degradation. This was one of the first biochemical results that could partially explain the potency of the silencing mechanism since very few molecules of dsRNA were able to inactivate hundreds of target mRNA molecules. RdRP is a highly conserved component in RNAi in C. elegans and lower eukaryotes and plays a role in heterochromatin maintenance as well. We identified the the Drosophila RdRP protein as elongator subunit 1, D-elp1, a highly conserved noncanonical RdRP present from S. pombe to humans. D-elp1 is involved in RNAi and transposon suppression and interacts with other key components of the RNAi machinery. A manuscript describing this important finding was published recently (Lipardi and Paterson, PNAS 2009). Importantly,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 identify the RdRP active site in D-elp1 as well as define domains essential for its role in RNAi.
| 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 |
