HSC-derived and HSC-independent T cells develop in successive waves. (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 of the AGM (red dot) and the PBI (red dot) flowed by 4-hydroxytamoxifen (4-OHT) treatment. At 5 dpf, the irradiated embryos are separated according to the presence (5-dpf-GFP⁺) or absence (5-dpf-GFP⁻) of lck:GFP⁺ cells in the thymus. The presence of lck:GFP⁺ cells in the thymus and their dynamic behavior are monitored at the indicated stages. (B) Images of lck:GFP⁺ cells in the thymus of 5-dpf-GFP⁻ group fish at various stages. Most of the AGM-irradiated 5-dpf-GFP⁻ fish begin to acquire GFP⁺ T cells from 8 dpf onward and continue to maintain GFP⁺ T cells to adulthood. The PBI-irradiated 5-dpf-GFP⁻ fish do not contain GFP⁺ T cells at all the time window examined. Dashed line depicts the thymus of 5- and 12-dpf embryos. (C) Percentage of GFP⁺ fish of each group at various stages. Control group (black line), AGM-irradiated 5-dpf-GFP⁻ group (red line), AGM-irradiated 5-dpf-GFP⁺ group (orange line), PBI-irradiated 5-dpf-GFP⁻ group (light blue line), and PBI-irradiated 5-dpf-GFP⁺ group (dark blue line) are shown. Three independent experiments are conducted (experiment 1: anterior AGM, n = 20; PBI, n = 25; and 4-OHT control, n = 22; experiment 2: anterior AGM, n = 25; PBI, n = 17; and 4-OHT control, n = 17; experiment 3: anterior AGM, n = 61; PBI, n = 13; and 4-OHT control, n = 28). Data are represented as mean ± SD. (D) Expression of cd4-1 and cd8α in the PBI- and AGM-derived lck:GFP⁺ T cells at various stages detected by RT-PCR. elf1a is used as internal control.