The overall goal of this project is to understand the early development of the cells and organs of the immune system, for example, the nature of the progenitor cells from which different lymphocyte subsets are derived, and the interactions between lymphocytes and the thymic epithelium required for the maturation of each. It is proposed to use the small freshwater fish, Danio rerio (zebrafish), to address these questions. The zebrafish embryo is free-living and transparent for the first two weeks of life. Since most organs are formed by five days, their development can be followed by visual inspection. Large numbers of the small fish can be housed and bred. Assays of gene expression and function at early developmental stages can be carried out easily. The zebrafish also permits genetic analysis of developmental pathways on a scale not possible in the mouse. These advantages have propelled the zebrafish to center stage as a choice model for studies of vertebrate development. Basic information about the zebrafish immune system was not available before this project was initiated; some of the aims are directed at providing necessary infrastructure. Experiments directed toward these aims were begun in the last project period. The recombination activating genes were characterized and their expression analyzed to delineate the organs in which B and T lymphocytes differentiate. Immunoglobulin (Ig) and T cell receptor (TCR) genes have been identified and their expression patterns during development are being determined. The existence of two populations of T lymphocytes, bearing distinct TCR, will be evaluated. Mapping and sequencing the putative zebrafish TCR Calpha/Cdelta locus will establish to what degree the high degree of conservation of this locus between human and mouse is preserved in the teleost. The whn gene will be characterized; its expression in wild-type and pharyngeal-arch mutants will be followed as a guide to the development of the thymic epithelial primordium. Individual cells in the embryo will be labeled and tracked through early development; migration to the thymus would suggest that the labeled cells are lymphocytic progenitors.