shown). However, in a specific subset of cells, identified by morphology as larval muscle cells, multiple speckled foci were observed throughout the nucleus (Figure 5C). Identical staining was observed in independent samples and in independent transgenic lines (Figure S6). Furthermore, no signal was observed in lines carrying the repeat expression transgene but no GAL4 driver, confirming that RNA foci are dependent on transgene expression (Figure S6). 4xrCAG,100 expression from independent transgenic lines gave up to four sites of RNA accumulation in all cell types, with multiple speckled foci not observed in muscle cells (Figure 5D, S7). Unexpectedly 4xrCAA,100 expression gave a staining pattern almost identical to that observed with 4xrCAG,100. Up to four sites of RNA accumulation were observed in both muscle and non-muscle nuclei from independent transgenic lines expressing 4xrCAA,100, suggesting that sites of RNA accumulation are not ‘hairpin’-specific (Figure 5E, S8). These sites may be the sites of transcription, or processing, of the repeat containing transgenes, as the one to four sites observed correlate with the four transgenes being expressed. In each case, no staining was observed in nuclei carrying the transgene, but no GAL4 driver, suggesting that the observations are dependent on the transgene being transcribed (Figure S6, S7, S8). RNA localisation results were confirmed by expressing each expanded repeat RNA sequence within the 59UTR of the transcript encoding green fluorescent protein (GFP). In this case a probe complementary to the GFP sequence was used (Figure 5F), eliminating any effects specific to the binding dynamics of each repeat sequence. Lines carrying four transgenes were generated for each expanded repeat sequence: 4xrCUG,100-GFP, 4xrCAG,100- GFP and 4xrCAA,100-GFP. Identical results were obtained to those observed initially, with 4xrCUG,100-GFP expression giving mul- tiple foci within muscle cells (Figure 5H). However, 4xrCAG,100- GFP and 4xrCAA,100-GFP expression within a GFP encoding transcript gave only one to four sites of RNA concentration in all cell types (Figure 5I, J). Although each of the lines carried four independent transgene insertions, the number of sites of RNA concentration observed was between one and four for each cell. It is possible that the variation in this number is dependent on the focal plane observed for any given cell. In order to confirm this, a control line expressing a single GFP transgene, with no repeat sequence was examined. In this case nuclei showed a single site of RNA concentration in all cells, indicating that these RNA localisation experiments are detecting sites of transgene expression (Figure 5K). In this model, ubiquitously expressed CUG, but not CAG or CAA-containing transcripts are able to undergo interactions sufficient to result in the formation of nuclear foci in specific cells. Thus, the mechanism that gives rise to cell-specific foci appears to be specific to CUG, but not CAG repeat RNA ‘hairpins’ in this system. Discussion Recent evidence suggests that repeat RNA transcripts may contribute to dominantly inherited human pathology through multiple pathways [19,20]. However, the specific mechanisms involved, particularly the way in which different repeat sequences give rise to similar pathology, is not fully understood. In this study we characterise a new system in which to study common CUG or CAG repeat RNA-mediated pathology. Reduced viability and a disrupted tergite phenotype were observed with ubiquitous expression of ‘hairpin’-forming CUG or CAG repeat RNA in this Drosophila model. Expression of either ‘hairpin’ repeat sequence gives similar tergite disruption, likely involving the same CUG and CAG Repeat RNA Pathogenesis in Drosophila cell type, suggesting that a common pathway is responsible for cellular perturbation. In contrast, co-expressing both complemen- tary repeat sequences in the same Drosophila system, to give double- stranded repeat RNA led to complete lethality [20]. Thus, single- stranded hairpin RNA may make a milder, more specific contribution to pathology, perturbing a particular pathway or cellular process and giving rise to susceptibility in certain cell types only. In support of this, expression of repeat RNA transcripts in histoblast cells was sufficient to cause mild tergite pathology. Repeat RNA expression phenotypes observed in this study are not expected to mirror human pathology, but rather provide a tissue specific biological read-out of repeat-mediated cellular perturbation. The Drosophila eye has been used in a similar manner to show that key disease mechanisms identified in vertebrates appear to be conserved in flies [7,8,13–15]. The tergite phenotype provides an independent system in which to explore repeat RNA-mediated pathology. As distinct cell types, and biological processes, are predominant in each model tissue, an independent system may extend our knowledge of the genetic pathways that may contribute to pathology in human disease. The nature of the tergite phenotype itself suggests that processes involving cytoskeletal regulation, required for histoblast migration and intercalation, may be candidate pathogenic pathways [56]. The cytoskeleton is important for neuronal function and transcriptional changes in genes involved in cytoskeletal regulation were previously identified in microarray studies of neuronal repeat RNA expression in Drosophila [17]. While perturbation in neurons likely gives a different cell biological outcome than in histoblasts, the molecular pathways involved may be the same in each case. Thus, this Drosophila system provides a way to study these pathways using a quantitative morphological read-out, that would likely not be observed with neuronal perturbation. Furthermore, the tergite phenotype appears to perturb cells that are proliferating and migrating, and thus such processes that are not occurring in the Drosophila eye could be specifically examined in this system. In our study, a modifier gene approach was used to examine whether pathways shown to alter repeat RNA-mediated pathology in the eye [14,20] could also modify the tergite phenotype. Initially we examined whether perturbation of Mbl, a known target of repeat RNA-mediated sequestration and contributor to pathology, contributes to the tergite disruption phenotype. If the phenotype was caused by Mbl sequestration, a further reduction in Mbl would be expected to enhance pathology, however, this was not observed with either CUG or CAG expression phenotypes. Thus, Mbl sequestration does not appear to be a major contributor to the common pathway that leads to similar CUG or CAG-mediated tergite disruption. Interestingly, in both CUG expressing lines, the proportion of progeny showing a milder phenotype was reduced when Mbl levels were reduced, while the proportion with a stronger phenotype was not significantly changed. Pathways involving Mbl sequestration may therefore make a mild, or indirect, contribution to CUG specific pathology, perhaps through the role of Mbl in RNA processing and stability [15,62]. Structural studies show that each repeat sequence has distinct binding abilities that may account for the CUG specificity in this case [63]. We previously reported that co-expression of complementary CAG and CUG repeat RNA expression transgenes in the eye gives a strong phenotype, that is suppressed by reducing Dcr-2 levels [20]. However, expression of either CAG or CUG repeat RNA transgenes alone, in the absence of a complementary transcript and thus acting as a single-stranded ‘hairpin’ RNA only, gives no eye phenotype [20]. The tergite phenotype therefore enabled us, for the first time, to examine whether single-stranded ‘hairpin’ RNA-mediated cellular perturbation is also suppressed by PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38516