phenotypic outcomes can be compared in each case [17,20]. CAA expression transgenes appear to be present at a lower steady state level [17], possibly due to their inability to form stable secondary structures. Since it is possible that greater steady state RNA levels may be an outcome, rather than a cause, of pathogenic RNA- protein interactions, these lines were included nonetheless, along with other controls to ensure that robust comparisons were made in each case. Expanded repeat RNA transgenes, under UAS control, were expressed ubiquitously using the da-GAL4 driver [54]. Progeny expressing expanded CUG (4xrCUG,100), or CAG (4xrCAG,100) repeat RNA showed a variable reduction in adult viability compared to each of the controls. At the standard culture temperature of 25uC, a significant reduction was observed in one of two independent 4xrCUG,100 expressing lines compared to those expressing 4xrCAA,100 and the EV control (Table S1A). One of two independent 4xrCAG,100 expressing lines showed a significant reduction compared to 4xrCAA,100 expressing progeny, but not the EV control (Table S1A). When grown at 29uC, giving increased GAL4 activity and hence increased repeat expression levels, complete lethality was observed in 4xrCUG,100 and 4xrCAG,100 expressing progeny but not in either 4xrCAA,100 expressing progeny or EV controls (Table S1B). These results therefore demonstrate that ubiquitous expression of expanded CUG or CAG repeat RNA leads to pathology in this Drosophila model. Closer physical examination of adult flies revealed a phenotype in viable progeny expressing expanded CUG or CAG repeat RNA at 25uC whereby the tergites of the dorsal abdomen were not correctly formed. In wild-type adults, the abdomen contains a series of regularly arranged tergites (Figure 1A), while in repeat expressing flies one or more of the tergite bands is split down the midline so that the two sides do not meet at all, or meet only partially (Figure 1B). A strong phenotype was observed in both independent lines expressing 4xrCAG,100, with semi-lethality and the strongest phenotype in 4xrCAG,100 [line 1], and a moderate to strong phenotype in 4xrCAG,100 [line 2]. Both independent 4xrCUG,100 lines gave a more moderate phenotype. This phenotype was not observed with expression of independent 4xrCAA,100 lines, the EV control line, or in flies carrying each set of transgenes, in the absence of a GAL4 driver. Repeat expression via an independent ubiquitous driver, Actin5C-GAL4, gave an almost identical pheno- type. In this case, the most severe phenotypes were observed in the same lines which gave the strongest da-GAL4 phenotype (Table S2). Thus, ubiquitous expression of CUG or CAG repeat RNA gives rise to a specific morphological disrupted tergite phenotype. To further confirm and characterise the disrupted tergite phenotype in each repeat expression line, a method was established to quantify phenotype severity. Newly eclosed adult female flies were individually scored and placed into one of four categories based on the severity of disruption and the number of tergites affected (See Methods) (Figure 1C). To give an overall measure of phenotype severity for each genotype, data was tallied to determine the proportion of progeny within each phenotype category (Figure 1D, E, F, G). Using this method, control progeny expressing either EV or each of two independent 4xrCAA,100 transgenes were scored as having a phenotype at a frequency of only 0–2% (Figure 1D, E and Table S3). Similarly those carrying da-GAL4 alone, or each UAS construct in the absence of GAL4 driver expression, were scored as having a phenotype in less than 1% of progeny (Table S3). Progeny expressing 4xrCAG,100 had the most severe phenotype with independent transgenic lines showing at least 89% of progeny with a phenotype (Figure 1G and CUG and CAG Repeat RNA Pathogenesis in Drosophila Figure 1. Tergite phenotype caused by ubiquitous expression of CAG or CUG repeat RNA in Drosophila. A, wild-type flies show a regular arrangement of tergite bands along the dorsal abdomen (arrows). B, An example of the disrupted phenotype, whereby tergites do not fuse at all (white arrowheads), or fuse only partially (grey arrowheads). C, Phenotype severity was scored on a scale of 1–4, images show typical examples from each category, where category 1 is like wildtype; category 2, tergites disrupted but not split; category 3, one tergite split; and category 4, two or more tergites split. D-E, Graphs showing the proportion of progeny within each scoring category. D, da-GAL4 driven expression of the EV control line (n = 161) and, E, da- GAL4 driven expression of 4xrCAA,100 [line 1] (n = 148) gives no phenotype with almost all progeny like wild-type. F, da-GAL4 driven PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38516