expression of 4xrCUG,100 [line 1] (n = 271) or G, 4xrCAG,100 [line 2] (n = 343) gives a tergite disruption phenotype. H, Schematic (not to scale) showing the location of histoblast cells (black). Histoblasts proliferate and migrate to form the tergite bands (arrows). I, Expression within the histoblasts using T155-GAL4 gives wild-type tergites in EV control progeny (n = 56). J, T155-GAL4 expression of 4xrCAG,100 [line 1] (n = 25) gives a mild tergite phenotype, ***p,0.001 comparing the proportion with a phenotype in I and J. doi:10.1371/journal.pone.0038516.g001 Figure S1). Independent lines expressing 4xrCUG,100 showed a less severe phenotype, with at most 78% of progeny with a phenotype and a greater proportion within the mildest category, compared to 4xrCAG,100 expression (Figure 1F and Figure S1). Decreased dosage of rCAG,100, with ectopic expression from lines carrying two transgene copies, gave a more mild tergite phenotype than expression from the corresponding four transgene copy line (Figure S1). Thus, the phenotype is dependent on the number of transgene insertions being expressed, suggesting that increasing the dosage of available repeat-containing transcripts increases pheno- type severity. Together, these results confirm that similar tergite disruption phenotypes are caused by the expression of CUG or CAG repeat-containing transcripts. Although repeat RNA is ubiquitously expressed in this system, specific tergite disruption is observed, indicating that particular cells, or processes, may be sensitive to repeat RNA-mediated perturbation. The adult tergites are formed from the histoblast cells, a small population that is specified during early development and is located symmetrically, either side of the larvae [55]. During pupation these cells proliferate and migrate towards the midline, eventually giving rise to the tergite bands [56] (Figure 1H). We hypothesised that histoblast cells may be specifically perturbed by repeat expression, leading to the observed phenotype. To examine this possibility, repeats were expressed within histoblasts using the T155-GAL4 driver [57,58]. Expression from 4xrCAG,100 [line 1] gave a mild phenotype with T155-GAL4, while line 2 gave a phenotype in a very small, but significant proportion of progeny (Figure 1I&J and Figure S2). 4xrCUG,100 expression with T155- GAL4 did not give a significant phenotype in either independent transgenic line (Figure S2). The milder CAG phenotype, and absence of CUG phenotype, observed with this driver may be due to a lower level of expression than da-GAL4, or a requirement for repeat expression in neighbouring cell types to give a strong phenotype. Nonetheless, these results indicate that expression within histoblast cells is sufficient to cause tergite disruption, supporting the conclusion that cell specific perturbation is responsible for the phenotype. Tergite Disruption is not Enhanced by Reducing Muscleblind Levels Expanded CUG or CAG repeat RNA expression in this Drosophila model leads to common perturbation of specific cells, giving a tergite disruption phenotype. This provides a quantitative biological read-out, and hence a model system to investigate whether genetically altering specific pathways modifies the observed repeat-mediated tergite phenotype. Using this approach, experiments examined whether pathways known to contribute to other forms of repeat RNA-mediated pathology also contribute to the disrupted tergite phenotype. Muscleblind (mbl), the Drosophila orthologue of MBNL-1, was chosen as an initial candidate, as previous studies in Drosophila have found that altering this gene can modify repeat RNA-mediated pathology in the eye [14,15,51]. To determine whether reducing mbl levels modifies tergite pathology, repeats were expressed with CUG and CAG Repeat RNA Pathogenesis in Drosophila da-GAL4 in progeny carrying one copy of the mblE27 mutant allele [27]. We hypothesized that if Mbl protein sequestration contrib- utes to the phenotype, a further reduction in Mbl, in progeny heterozygous for the mutant allele, would lead to an enhanced phenotype. The severity of the disrupted tergite phenotype was compared to determine whether repeat-mediated phenotypes were modified in progeny carrying one copy of mblE27, compared to those with two wild-type mbl alleles (Figure 2A, B, C, D, S3 and Table S4). The proportion of progeny showing any phenotype (defined by progeny in all three phenotype categories) and the proportion showing a strong phenotype only (defined by progeny in the two most severe categories), were compared between genotypes (Figure 2E, F and Table S7). Statistical analysis revealed a significant (Fisher’s exact test) decrease in the proportion that show any phenotype with expression from two independent 4xrCUG,100 lines in the presence of mblE27 (Figure 2E and S3). However, no change was observed when comparing the proportion within the strongest two categories only, perhaps indicating that only those with the weakest phenotype are suppressed (Figure 2E and S3). Expression of 4xrCAG,100 in the presence of mblE27 led to no significant change in phenotype proportion compared to expres- sion of 4xrCAG,100 alone (Figure 2F and S3). Thus reducing Mbl levels did not enhance the tergite phenotype caused by either CUG or CAG repeat expression in this Drosophila system. Instead, suppression was observed that appeared to be specific to the phenotype caused by CUG expression only. Flies homozygous for mblE27 were not viable and thus it was not possible to examine the effect of complete Mbl loss-of-function in this system. However, these results do not support a role for Mbl sequestration as a common contributor to CUG or CAG RNA-mediated tergite phenotypes. Reduced Dicer-2 Processing is not Rate Limiting for the Tergite Phenotype Previously we reported that co-expression of complementary CAG and CUG repeat RNA transcripts leads to pathology that is suppressed by reducing Dcr-2 levels [20]. Based on this evidence, and the presence of 21nt repeat RNAs, it is proposed that complementary repeats form double-stranded repeat RNA that is processed by Dicer enzymes [20]. Pathology is associated with altered miRNA profiles, indicating that double-stranded repeat RNA may perturb normal Dicer processing pathways [20]. Single- stranded ‘hairpin’ RNA has also been reported to be a substrate for Dicer processing, and therefore could potentially perturb Dicer processing pathways in a similar manner [59]. Ectopic expression of CUG or CAG repeat transgenes separately does not lead to a phenotype in the eye, such that modification by reducing Dicer processing could not be tested [20,17]. However, as ubiquituous expression of each transgene gives tergite disruption, this phenotype can be used to genetically examine the role of Dicer processing in cellular perturbation caused by single-stranded CUG or CAG ‘hairpin’ repeat RNA, in the absence of a complementary transcript. Experiments first examined whether reducing Dcr-2 levels can modify the tergite phenotype. Each of the expanded repeat RNA expression lines, 4xrCAG,100, 4xrCUG,100 and 4xrCAA,100, were ubiquitously expressed via da-GAL4 in the presence of one copy of the Dcr-2L811fsX loss-of-function mutant allele (Figure 3A, B, C, D, S4 and Table S5). If Dcr-2 function is rate limiting for the phenotype then this reduction in Dcr-2 levels would be expected to result in suppression, as was previously observed with complementary repeat RNA expression [20]. In independent lines, 4xrCUG,100 and 4xrCAG,100 expression in the presence of PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38516