A transient wave of T cells is in situ generated from the PBI. (A and B) Outline of the infrared-mediated temporal–spatial cell labeling strategy. The T cell reporter Tg(lck:loxP-DsRedx-loxP-GFP) fish are outcrossed with Tg(hsp70:mCherry-T2a-CreER^T2) line. The double-transgenic embryos are heat-shocked at restricted regions by infrared (IR) laser irradiation to induce temporal–spatial restricted expression of CreER. After 4-hydroxytamoxifen (4-OHT) treatment, CreER-mediated loxP recombination will remove DsRedx, resulting in GFP expression. The RBI region is irradiated at 14–16 hpf with three spots, the PBI region is irradiated at 20–22 hpf with two spots, and the AGM region is irradiated at 26–28 hpf with four spots. The target regions are indicated by red dots, and each dot represents one heat shock spot. (C) Images of lck:GFP⁺ cells in the thymus of the RBI-, PBI-, and AGM-irradiated fish at 5 dpf, 40 dpf, and adulthood. Dashed lines depict the thymus of 5-dpf embryos. The signals in the thymus of 5-dpf embryos are captured directly by fluorescent microscope, whereas the signals in the thymus of 40-dpf juveniles and adult fish are imaged after anti-GFP staining of the cryosection. (D) Quantification of lck:GFP⁺ cells in the thymus of 5-dpf embryos. AGM (n = 25), PBI (n = 25), and RBI (n = 25) represent embryos that are irradiated by IR and treated with 4-OHT, whereas 4-OHT control (n = 25) are embryos treated with 4-OHT only. Data are represented as mean ± SD. Unpaired, two-tailed t test was performed to determine significance. ****, P < 0.0001. See also Figs. S1, S2, and S3.