AML1-ETO induces generation of ROS⁺, Wg⁺ hemocyte precursors, and increased proliferation of hemocytes in larval circulation. (A) AML1-ETO, AML1, ETO, and Lz were expressed under control of the hemocyte specific driver hml^Δ-Gal4, using Gal4/UAS system. Hemocytes from 3rd instar larva were extracted and counted for each genotype. hml^Δ-Gal4,UAS-GFP heterozygous were used as wild-type control (WT). AML1-ETO causes a robust increase in the number of hemocytes in larval circulation compared with wild-type and overexpression of the other proteins. (B) Hemocyte precursors, identified as cells that do not express maturation markers: P1, Pxn, PPO, and L1 (all green) are rarely seen in wild-type (rare examples shown by arrow), but are significantly elevated upon AML1-ETO expression (arrows). (C) Quantitation of the data in panel B (n = 10, P < .001). (D) Wg is highly expressed in rare precursor cells that are hml negative (arrowhead) in circulation of wild-type larvae (WT). Number of Wg⁺ hemocytes is significantly increased in AML1-ETO mutant background. (E) Quantitation of the data in panel D (n = 10, P < .001). (F) ROS is expressed in hml^low(1) and rarely at low levels in some hml⁺(2) hemocytes in circulation of wild-type larva (WT). Number of cells expressing high level of ROS is increased in mutant background (AML1-ETO). (G) Quantitation of the data in panel F (n = 10, P < .001). (H) ROS⁺ cells (indicated by arrowhead) in WT and AML1-ETO backgrounds fail to phagocytose FluoSpheres (FS, blue) microparticles, suggesting they are precursors. Hemocyte markers, ROS, ToPro-3, and microspheres dyes color-coded in left panels. Scale bars, 5 μm.