Innate Immunity

Innate immunity provides the first line of defense against pathogens. Teleosts, like mammals, are endowed with primary defense mechanisms against microbial agents. These mechanisms are so protective, that even Rag-1 -/- zebrafish (which have no adaptive immunity) survive disease-free in notoriously pathogen-infested fish water.

Receptors responsible for this process were first identified in the fruitfly, and belong to the family of Toll-like receptors (TLRs). Zebrafish have over 20 different TLRs, that are expressed on the surface of dendritic cells and macrophages and recognize repetitive pathogen associated molecular patterns (PAMPs) derived from bacteria or viruses. Following PAMP binding, TLRs transduce signals from the cell surface to the nucleus, a process mediated by adaptor molecules, that share a common Toll/interleukin-1 receptor (TIR) domain with TLRs. This process leads to activation of MAP kinase family members, translocation of NFkB to the nucleus and secretion of anti-infectious molecules. Many of the participants in these pathways have not yet been identified. In collaboration with the groups of Bob Finberg (U Mass, Worcester) and Carol Kim (University of Orono, Maine) we are establishing a transgenic line of zebrafish, where a downstream target of the TLR signaling pathway activates luciferase activity (see Figure 1) or a fluorescent marker (Figure 2). While luciferase has greater sensitivity, the fine resolution of fluorescence is superior, particularly in embryos (compare Figures 1 and 2). The read-out in this screen will be the fluorescent signal emitted by mutagenized embryos after stimulation either with synthetic stimulants or bacteria (Figure 3A). Alternatively, fluorescent bacteria can be used to infect mutagenized (non-transgenic) embryos, and their fate can be observed by fluorescent microscopy (Figure 3B). We expect to obtain mutants that show either constitutive activation or exaggerated response of the TLR pathway (gain of function, GOF, Fig. 3A and B), or defective signaling (loss of function, LOF). After rigorous retesting and exclusion of candidate genes, interesting mutants will be positionally cloned.