We have made substantial progress in understanding the role of nautilus in myogenesis in the fly embryo. 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 of which were rescued by a nautilus transgene. 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. We are currently carrying out experiments to identify nautilus target genes that are important for cell-cell recognition, cell movement and migration-issues important to understanding the regulation of normal cell growth and patterning in human disease and development. 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. Many of the genes involved in the regulation of muscle development that are involved in determining the myogenic field in the mesoderm, establishing the muscle founder and fusion competent myoblast populations, regulating cell fusion, and the activation of the muscle identity genes, are measurably down regulated in the nautilus null e.g. Snail, Twist, Lethal of scute, Notch, Delta, Enhancer of split, Numb, Inscuteable, Tinman, Drop (msh),Kruppel, Vestigial, Apterous, Lame Duck, Rols/Ants,rolling pebbles, Rst, Crk, Collier, Kruppel, Ladybird, grip, and hibris. The genes linked to founder myoblast (FM) and fusion competent myoblast (FCM) fusion are of interest with regard to the nautilus null phenotype due to their critical role in muscle fiber formation. Rolling pebbles, Crk, Rst and wasp all show decreased expression in the null, while Duf, rost, blow and kette are unchanged and sns and mbc are slightly up regulated. Expression patterns for genes involved in myotube targeting are also altered in the null: Perdido (kon) shows decreased expression while Grip expression is increased. By contrast, certain genes expressed in muscle fibers representing structural proteins, actin-binding proteins, ion channels, excitation-contraction coupling components, calcium binding proteins, and synaptic vesicle movement are misregulated 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 0.1% of the cells in the embryo, stringent capture methods must be employed to identify target genes. In order to capture gene sequences that interact with nautilus, we are generating a fly line with a novel tag targeted to the engodenous nautilus gene, a peptide sequence that can be biotinylated by E. coli biotin ligase that is expressed from the targeting vector. The efficiency and stringency of biotin-avidin capture will enable us to perform ChIP-Seq (Solexa 1G Genomiic Analyzer) to identify nautilus target DNA sequences. Target gene candidates will be analyzed in vivo using available mutant stocks or by inducible RNAi stocks made in the lab or obtained from the Vienna Drosophila RNAi Center. We have also 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 four 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 8-miR locus, a genomic region encoding 8 microRNAs potentially regulating the post transcriptional expression of >3000 genes, is under the control of nautilus, and that miR3 in the locus fine tunes nautilus expression in the embryo in a feedback loop. This regulatory pathway points to a previously unappreciated complexity of gene regulation with regard to development and disease. miRNA expression from the 8-miR locus is lost in the nautilus null and a deletion of the locus has a muscle phenotype similar to the nautilus null. This study will be submitted for publication shortly. 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 termed degradative PCR. This was the first biochemical evidence to shed light on the role of the siRNAs in RNAi and provided a basis to explain the potentcy of the mechanism in post transcriptional gene silencing since very few molecules of dsRNA were able to inactivate hundreds of target mRNA molecules. RdRP is a highly conserved key component in RNAi in C. elegans and lower eukaryotes and plays a role in heterochromatin maintenance as well. We have now identified the the RdRP protein, the first example of a highly conserved noncanonical RdRP in eukaryotes conserved from S. pombe to humans. The RdRP is involved in RNAi and transposon suppression and interacts with other key components of the RNAi machinery. A manuscript describing this important find is in press in Proc. Nat. Acad Sci. USA (Sept. 2009). Importantly,a mutation in the human gene produces a truncated RdRP protein and is correlated with the neurological disease Familial Dysautonomia. A fly model of the [summary truncated at 7800 characters]
| 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 |
