EXPRESSION OF ZEBRAFISH RAG GENES DURING EARLY DEVELOPMENT IDENTIFIESTHE THYMUS

Recent experiments have demonstrated that zebrafish is a vertebrate in which it is possible to carry out large-scale mutagenic screens to id entify genes involved in specific developmental pathways. To follow de velopment of the immune system in zebrafish, we have analyzed the expr ession of the recombination activating genes, rag1 and rag2, which we have previously isolated and characterized. These genes catalyze the r earrangement of immunoglobulin genes in immature B lymphocytes and of T eel receptor genes in immature T lymphocytes and are therefore appro priate markers to follow the development of organs containing these ce lls. By whole-mount in situ hybridization, we detected expression of b oth rag genes in a paired organ in the head, beginning on the fourth d ay after fertilization. Histological examination of this organ indicat ed that it corresponds to the thymus, as described for other fish, an organ that has not previously been identified in zebrafish. By histolo gical analysis, the thymus primordium appears at 54 hr but does not en large significantly until 30 hr later. The thymus continues to enlarge and reaches its mature histological organization at 1 month. The pron ephros, the major hematopoietic organ in the adult fish, begins to dev elop hematopoietic tissue about 2 weeks after fertilization. By 1 mont h, mature lymphocytes are distinguishable in the tissue surrounding re nal tubules. Lymphocytes appear in the kidney too late for screening b y whole-mount in situ hybridization; however, the pattern of rag1 expr ession in the thymus forms the basis of an assay for mutations affecti ng development of the thymus or its constituent lymphocytes. (C) 1997 Academic Press.