Fig. 1. Gene-Switch system drives targeted dFMRP expression in neurons. (A) Schematic of the Gene-Switch (GS) system. The GAL4 DNA-binding domain is fused to the p65 activation domain (p65AD) and a mutated progesterone receptor ligand-binding domain (PR-LBD). In the absence of RU486, the GS is `off'. In the presence of RU486, the hormone-responsive GAL4 drives dFMRP transcription downstream of the UAS regulatory sequence. This approach allows spatial and temporal control of dFMRP expression in the dfmr1-null background. (B) Western blot of isolated third instar Drosophila larval CNS. Genotypes as indicated: w¹¹¹⁸ (control), homozygous dfmr1^50M null allele (dfmr1) and dfmr1^50M, elav-GSG-301/dfmr1^50M, UAS-9557-3 (GS). Treatment as indicated: GS fed ethanol vehicle (GS+E) or RU486 (GS+RX, where X is the RU486 concentration in µg/mL). Blot was probed for dFMRP and -Tubulin, illustrating RU486 dosage responsiveness. (C) Quantification of western blot dFMRP levels. Individual band intensities were normalized to -Tubulin and expressed as a percentage of the control. Bars indicate mean±s.e.m. ^*P<0.05. (D) dFMRP immunohistochemistry of wandering third instar larvae CNS. Bottom row of panels shows higher magnification views of dFMRP staining in the VNC. Note the RU486 dosage dependence of dFMRP expression.