Lymphocyte differentiation in sea bass thymus: CD4 and CD8-α gene expression studies

Abstract

Different developmental stages (from eggs to 1-year-old juveniles) of the teleost fish Dicentrarchus labrax (L.) were assayed for CD4 gene expression. RT-PCR revealed the appearance of CD4 transcripts in postlarvae from 51 days post-hatching (dph). This finding overlaps the first detection of CD8-a mRNA. Real-time PCR with specific primers quantified CD4, CD8-a and TCR-b transcripts in larvae and postlarvae (25, 51, 75 and 92 dph) and 1-year-old thymus. At 92 dph, TcR-b and CD8-a transcripts were significantly higher (P < 0.001) than in previous stages, as CD4 transcripts compared with 51 dph (P < 0.01). High levels of TCR-b and CD8-a transcripts were found in the thymus, while CD4 transcripts were lower (P < 0.05 vs. TCR-b). In situ hybridization identified CD4 mRNAs at 51 dph, localized in thymocytes of the outer and lateral zones of the thymic glands. From 75 dph on the signal was mainly detected in the outer region, drawing a cortex–medulla demarcation. Developmental expression of CD4 and CD8-a almost coincided. In each adult thymic lobe CD4þ and CD8-aþ thymocytes filled the cortex. The expression patterns of CD4 and CD8-a largely overlap, except in the medulla, where CD4þ thymocytes were isolated, while CD8-aþ ones mainly arranged in cords. These results provide new information about the thymic compartmentalization and lymphocyte differentiation pathways in a teleost, almost demonstrating that double negative thymocytes fill the cortex giving rise to further selection in the medulla.