Genetic Modifiers of MeCP2 Figure 2. MeCP2 overexpression leads to eye, wing and motor performance phenotypes. Light microscope images (A–E), and scanning electron microscope images (A’–D’’) of fly eyes from controls or animals expressing MeCP2 driven by GMR-Gal4 driver at either 27.5uC or 30uC. External eyes of control flies show normal ommatidial organization, while eyes from animals expressing any of four distinct MeCP2 alleles show disruption in the structured pattern of the eye the surface. Note increased severity of the phenotypes at the higher temperature. F–G. The C5-Gal4 driver was used to drive either UAS-lacZ or full-length MeCP2 throughout the wing pouch at 25uC. Compared to controls, MeCP2 expressing flies have extra vein tissue (arrowheads) near L3 and L5. H. The neuronal driver CHA-Gal4 was used to drive expression of either UAS-eGFP or full-length MeCP2 at 25uC. Each sample represents a group of 20 virgin females. Beginning at 3 days of age, a lower percent of MeCP2 expressing flies are able to climb to 7 cm in 18 seconds as compared to control flies (Repeated measures ANOVA p,0.001). Over time, both groups decrease in their ability to climb. Error bars represent the standard error. Genotypes: A-A’’, GMR-Gal4/+. B-B’’, GMR-Gal4:UAS-MeCP2FLM119-2M/+. C-C’’, GMR-Gal4:UAS-MeCP2 R106W/+. D-D’’, GMR-Gal4:UAS-MeCP2 D166/+. E-E’, GMR-Gal4:UAS-MeCP2 R294X/+. F, C5-Gal4/+. G, C5-Gal4:UAS-MeCP2 FLM119-1M/+. H, CHA-Gal4/UAS- eGFP and CHA-Gal4/UAS-MeCP2 FLM119-2M. doi:10.1371/journal.pgen.1000179.g002 Each mutant line carrying a candidate modifier was crossed to flies expressing the full-length MeCP2 allele from the GMR-Gal4 driver and screened for both enhancers and suppressors. The initial hits in this screen were then re-evaluated with an independent full-length MeCP2 transgenic line. Genes that modify the MeCP2 phenotypes across multiple strains and MeCP2 lines are the chromatin remodeling genes Additional sex combs (Asx), corto, osa, Sex combs on midleg (Scm), and trithorax (trx), the kinase tricornered (trc) and the UBE3A target pebble (pbl) (Table 1). Partial loss of function of Asx, corto, osa, pebble, or Scm suppress the eye phenotype induced by full-length MeCP2, while trc has a similar effect when it is overexpressed (Figure 4A–H, note improved ommatidial organization relative to MeCP2 control). In contrast, enhance- ment of the eye phenotype was observed in MeCP2 animals with either loss-of-function mutations in trx or overexpression alleles of Scm, osa, and pbl (Figure 4I-4R, Figure S1, note greater ommatidial disruption, loss of interommatidial bristles and, in some cases, reduction in eye size and eye depigmentation). To exclude the possibility that modifiers of the Gal4-UAS system may simply cause changes in expression of MeCP2, western blot analysis was PLoS Genetics | www.plosgenetics.org 4 September 2008 | Volume 4 | Issue 9 | e1000179