Tissue-specific expression of Idh-R195H, the Drosophila homolog to IDH1-R132H, results in D-2HG production. (A) Homology between human IDH1 (GI: 49168486), human IDH2 (GI: 28178832), and Drosophila Idh (CG7176-RK translated region). Human cancer hotspots, including IDH1 R132, IDH2 R140, and IDH2 R172, are shown in red with homologous residues in Drosophila Idh. (B) The UAS/Gal4 binary expression system to express Idh-R195H in targeted tissue lineages. (C) D-2HG in whole pupae, with expression of the indicated transgene driven by the Actin5c driver. Pupae were F1 from the cross of Actin5c-Gal4 and UAS-transgene flies. Data are expressed as mean ± standard deviation (n = 3). (D) Third instar wandering larva with Idh-R195H expressed in hemocytes demonstrating melanotic mass, genotype hml-Gal4, UAS-GFP/+; UAS-Idh-R195H/+ (hml>Idh-R195H). (E) Panneuronal expression of Idh-R195H using the Elav promoter leads to eclosion defect, locomotor defect, and early lethality. Adult Elav-Gal4/+; UAS-Idh-R195H/+ fly exhibiting a wing-expansion defect (top arrow) and cuticle-tanning defect (bottom arrow) is shown. (F) Flies expressing Idh-R195H in CCAP neurons demonstrate wing expansion defect (top arrow) and cuticle-tanning defect (bottom arrow) without obvious locomotor defects. Genotype: CCAP-Gal4/+; UAS-Idh-R195H/+. Flies were visualized on a Leica Kombistereo microscope, original magnification ×10. MLS, mitochondria localization signal; TSP, tissue-specific promoter.