In many vertebrates, one function of the innate immune system is carried out by cytotoxic cells that recognize and destroy virally infected or cancerous cells. The molecular mechanisms involved in these processes are well studied in mammalian natural killer (NK) cells, but very little is known about them in other species. A family of Novel Immune Type Receptor (NITR) genes has recently been identified in bony fish with characteristics similar to the mammalian NK receptors. Thus, it has been hypothesized that these genes play a role in cytotoxic cell function in fish. This project will determine whether zebrafish NITR gene families are expressed in cell lineages known to possess cytotoxic properties and whether zebrafish NITR-expressing cells exhibit cytotoxic activity. First, tissue-specific expression patterns will be determined for multiple NITR gene families by RT-PCR and RNA blot analysis. Second, expression of NITR genes will be examined by RNA in situ hybridization in cell lineages known to be cytotoxic. Third, transgenic lines of zebrafish will be generated and analyzed in which the Green Fluorescent Protein will serve as an in vivo fluorescent biomarker for NITR-expressing cells, which will then be isolated and tested for cytotoxic activity. Knowledge gained from this project will provide new insight into the function of cytotoxic signaling pathways and elucidate the molecular mechanisms of innate immune reactivity in fish. The results of this project will contribute to a better understanding of immune function in fish, including those of agricultural importance and will have implications for understanding the molecular mechanisms of cytotoxic defense in organisms that occupy vastly different environmental niches and encounter diverse pathogens. This project will involve multiple graduate and undergraduate students who will gain expertise in various genetic, molecular and biochemical techniques, while being trained in experimental design and the scientific process.
