The AGM in situ generates HSC-independent T cells. (A) A schematic diagram of the experimental design. 22- to 24-hpf double-transgenic Tg(hsp70:mCherry-T2a-CreER^T2;lck:loxP-DsRedx-loxP-GFP) embryos are irradiated in the anterior (somite 7), middle (somite 11), and posterior (somite 15; red dot) of the AGM region and subsequently treated with 4-hydroxytamoxifen. At 5 dpf, embryos containing lck:GFP⁺ cells in the thymus are selected and survived to 40 dpf and adulthood for further analysis. IR, infrared. (B–D) Images of lck:GFP⁺ cells in the thymus of category I fish, in which GFP⁺ T cells are maintained from embryonic stage to adulthood. Dashed line depicts the thymus of 5-dpf embryos. (E–G) Images of lck:GFP⁺ cells in the thymus of category II fish, in which GFP⁺ T cells are present at 5 dpf but absent at 40 dpf and adulthood. Dashed line depicts the thymus of 5 dpf embryos. (H) Percentage of category I and category II fish in total fish irradiated in the PBI and in the anterior, middle, and posterior of the AGM. Three independent experiments are conducted [(experiment 1: anterior AGM, n = 4; middle AGM, n = 8; posterior AGM, n = 13; and PBI, n = 14); (experiment 2: anterior AGM, n = 16; middle AGM, n = 19; posterior AGM, n = 20; and PBI, n = 16); (experiment 3: anterior AGM, n = 12; middle AGM, n = 7; posterior AGM, n = 12; and PBI, n = 14)]. Data are represented as mean ± SD. See also Fig. S5.