Expression of the immunity-related ikaros and rag1 genes during development signals the onset of lymphopoiesis in vertebrates. Partial sequences of ikaros and rag1 in Epinephelus malabaricus were cloned by degenerate primer-mediated reverse transcriptase polymerase chain reaction (RT-PCR). Their expression profiles in the thymus, head kidney, trunk kidney, spleen, intestine, brain, liver and pancreas, and the onset of expression at various developmental stages, were examined by RT-PCR using identical primers. Expressions of both ikaros and rag1 genes were detected as early as 3 days post fertilization (dpf). The thymus is thought to be the first organ of lymphopoiesis to develop in fish; its histogenesis in E. malabaricus was examined and used for comparison. To avoid the potential influence of environmental factors on different hatches, both age and larval morphology were used as indicators of maturation to define the stage of development. A bud-like thymus was observed at 9 dpf; lymphopoietic cells appeared at 19 dpf; and further development, such as cortex/medulla differentiation and the appearance of lymphocytes, occurred by 26 dpf. Trabecula development was detected at 41 dpf. Based on the histological evidence, the lymphoid cell expressed rag1 before the thymus developed, suggesting that extra-thymic lymphopoiesis takes place in E. malabaricus, and that a functional immune system might develop early in the metamorphosis stage.
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