A genetic screen identified ecdysone receptors as modifiers of AML1-ETO induced blood disorder. (A) Schema of modifier genetic screen: flies of synthesized genetic background containing hml^Δ-Gal4,UAS-GFP;UAS-AML1-ETO/TM6,Tb were crossed with mutant alleles. Larvae from these crosses were scored under direct (top panels) and fluorescent (bottom panels) illumination (Zeiss SteREO Lumar.V12 Stereomicroscope, 12× magnification); from left to right: normal density of GFP⁺ hemocytes in wild-type (WT) larvae of hml^Δ-Gal4,UAS-GFP background. Numbers of GFP⁺ hemocytes and black melanotic tumors are robustly elevated in hml^Δ-Gal4,UAS-GFP;UAS-AML1-ETO larvae (AML1-ETO). Examples of enhancer and suppressor modifiers of AML1-ETO phenotype: single copy deficiencies [Df(3L)ZN47] or [Df(3L)pbl-X1] cause a dramatic increase and decrease, respectively, in the number of circulating hemocytes and melanotic tumors in hml^Δ-Gal4, UAS-GFP; UAS-AML1-ETO larvae. These deficiencies include hundreds of genes, a majority of which remain yet uncharacterized. Analysis of available mutant alleles of genes belonging to these genomic intervals has not yet identified single loci that can modify the phenotype. (B) EcR-B1 is required for induction of hemocyte proliferation by AML1-ETO. EcR-B1 and EcR-A were inactivated in hemocytes of hml^Δ-Gal4,UAS-GFP;UAS-AML1-ETO background (hml > AML1-ETO). AML1-ETO induced proliferation of hemocytes was dramatically suppressed by EcR hemizygosity (alleles KG04522, q50st) and dsRNA of EcR-B1. In contrast,the oncogene induced proliferation of hemocytes was very mildly increased by dsRNA allele of EcR-A. hml^Δ-Gal4,UAS-GFP heterozygous were used as wild-type control (WT control). (C) As controls for (B), hemizygosity for EcR-B1 or inactivation of either EcR-B1 or EcR-A with corresponding dsRNAs in normal hemocytes [hml^Δ-Gal4,UAS-GFP(hml)] does not affect proliferation of otherwise hemocytes. (D) Inactivation of EcR-A causes increase in circulating hemocytes expressing EcR-B1. EcR-B1 is highly expressed in nuclei of a population of circulating hemocytes in wild-type larvae (hml/w¹¹¹⁸). Expression of EcR-B1 is significantly reduced in EcR ^KG04522 heterozygous (hml/EcR^KG04522) animals, while the number of cells expressing high level of EcR-B1 is significantly increased upon dsRNA-mediated inactivation of EcR-A (hml/EcR-A^dsRNA). (E) Nuclear localization of AML1-ETO in hemocytes is not affected by deficiency of EcR-B1 (EcR ^KG04522) or EcR-A (EcR-A^dsRNA) in AML1-ETO mutant background. (G) Number of ROS⁺ hemocyte precursors is significantly reduced upon inactivation of EcR-B1 in AML1-ETO mutant larvae (hml > AML1-ETO). Abbreviations of genotypes indicated on top of each panel, cell markers are color coded. Scale bars, 5 μm.