Zebrafish drug screen identifies anti–T-cell compounds. Zebrafish larvae carrying the T-cell specific lck:EGFP transgene were used for screening of a small molecule library for anti–T-cell effect. (A) Dorsal (left) and lateral (right) views of 5 days post fertilization (dpf) normal healthy lck:EGFP larva (yellow arrow indicates eye; and arrowhead, thymus). (B) Three 5-dpf larvae per well in 96-well format were treated with compounds, DMSO (vehicle, yellow box) and dexamethasone (Dex; positive control, red box). Fluorescence emission was evaluated after 48 hours: no effect/normal fluorescence (dark green), strong effect (light green), toxic effect (black well/red x), and empty well (hatched). (C) Examples of strong (bottom) and no effect (top). Of 26 400 compounds screened, 387 compounds with weak reduction of thymus fluorescence were also identified. (D) Twenty-one compounds with strong effect on survival of immature T cells were tested for cell cycle effects in nonlymphoid cells. Four hours postfertilization (hpf) zebrafish larvae were incubated in candidate compounds for 20 hours, dissociated into single-cell suspensions, stained with propidium iodide (PI) and subjected to flow cytometry. (E) Representative cell cycle profile for DMSO vehicle-treated embryos. (F) Hydroxyurea-treated embryos show S-phase arrest (arrow) as well as sub-G₁ peak (arrowhead). (G) Lenaldekar (5μM) shows a cell cycle profile similar to that of control embryos. (H) Compound 4 shows S-phase arrest (arrow) and sub-G₁ peak (arrowhead); 2n indicates G₀/G₁ phase; 4n, G₂/M phase; and between 2n and 4n, S phase. (A-C) Images were acquired at room temperature using Olympus MVX10 microscope with MV PLAPO 1× lens (Olympus). Camera used was Diagnostic Instruments model 14.2 Color Mosaic Insight FireWire SPOT. Acquisition software used was SPOT Alias Version 4.6 software, 2000 ms exposure with no binning, γ = 1.0. Fluorescence excitation light source was EXFO X-Cite Series 120. (D) Microscope used was Nikon Eclipse E600 with Nikon Plan APO 4× lens at room temperature. Camera used was CRI Nuance multispectral imaging system model N-MSI-420-FL. SPOT Advanced Version 4.6 acquisition software was used.