The zebrafish represents a powerful model for the study of vertebrate diseases, particularly those of the hematopoietic and immune systems. The primary objective of this proposal is to generate new animal models of primary immunodeficiency (PID) in the zebrafish. The broader experimental goal is to use these models to gain a deeper understanding of the molecular basis of PID and to identify novel genes or known genes with novel functions that direct normal functioning of adaptive immunity. To achieve these goals our strategy is based on a novel forward genetic screen using a transgenic line of zebrafish we have previously created. In this line the zebrafish T cell specific p56lck promoter drives eGFP expression (lck:eGFP), allowing for in vivo detection of absence of T cells at any stage of development. This provides a great advantage over previous screens where larvae had to be formalin fixed for detection of lymphocyte-specific genes by in situ hybridization. For example, on the lck:eGFP background mutant fish can be identified in vivo, permitting infection assays and examination throughout their life-span for phenotypic reversion and infectious or autoimmune complications. This screen has already been initiated in our laboratory and we have analyzed the first 150 families. This has led to the identification of 6 mutant lines with T cell or thymus defects that occur at different stages of development. We propose to characterize these mutants in more detail to deepen our understanding of normal and aberrant pathways in T cell and thymus development. We will carry out cell- autonomy studies to determine if the defect lies in T cells or thymic epithelial cells, and will go on to map the most interesting mutants to identify candidate genes. We also propose to continue screening more mutant families. We have previously identified the mutant ceylon (cey) with the unique combination of absence of T cells but normal thymus size. In addition, cey lacks hematopoietic stem cells at the fetal liver equivalent stage of development. We have mapped the cey locus with closely linked polymorphic markers and propose to study it in detail. Cloning of the cey mutant promises to reveal a gene with previously unknown function. The work laid out in this proposal will provide a novel set of immunodeficient vertebrates that will be instrumental in elucidating immunological and molecular characteristics of PIDs. In the future a series of investigations can be initiated, including: evaluating response to different pathogenic challenges, and studying the influence of genetic modifiers on disease outcomes in fish models of human disease. The use of PID models coupled with the utility of zebrafish as a particularly accessible developmental model system will yield new insights into the molecular mechanisms underlying human PIDs, ultimately benefiting patients afflicted with these life- threatening diseases.
To further our understanding of primary immunodeficiencies, and to develop better diagnostic tools, relevant small animal models need to be developed. The zebrafish is a small vertebrate animal that produces immune cells much like humans and can therefore be used to imitate human immunodeficiencies by mutating its genes. In this proposal we intend to produce mutant zebrafish lines that serve as models for immunodeficiency, and will study these models to improve the diagnosis and our understanding of human immunodeficiency disorders.
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