The zebrafish is a promising vertebrate model for the examination of basic processes in immunity and disease. Importantly, zebrafish possess blood cells similar to those of mammals, so that basic research using zebrafish can reveal insights about immunity and disease that have potential significance to human health, a broader scientific impact of this study. The overarching goal of this project is to examine B cell development and activation in zebrafish. B cells are white blood cells that produce antibodies to protect vertebrates from invading pathogens. Although mammals have four types of antibody-producing B cells, fish have just two types: those that produce IgM, which is observed in mammals, and those that produce IgZ, which is very different from mammalian antibodies. The specific goals of this project are thus to analyze when and where IgM+ and IgZ+ B cells develop in zebrafish and how these B cells protect fish from various pathogens. Results from these studies will have direct applications to fish health and aquaculture and to fish models of human diseases.
Genetic approaches will be used to create transgenic fish in which the different types of B cells are marked with fluorescent proteins. Advanced imaging techniques will be used to track the development and activation of the B cells. It is expected that the IgM+ and IgZ+ B cells will behave in distinct ways depending upon the age of the fish or the type of pathogen to which they are exposed. Results from these experiments will increase our knowledge of the fish immune system and will reveal basic information needed to understand the evolution of immune systems. Moreover, the important tool that will be developed in this project, transgenic zebrafish expressing fluorescent B cells, will be distributed to the zebrafish community for use in a wide variety of aquaculture and human disease models. This tool will enable and catalyze additional research using zebrafish, a significant expected outcome. Another important broader impact is to provide undergraduate research experiences for at least 14 students, including underrepresented students. Participation in research leads to a first-hand understanding of how science is conducted, and has been shown to increase the retention of students who then pursue graduate studies in the biological sciences.
The zebrafish is a promising model for the examination of immunity and disease, as they have the same populations of blood cells that mammals do. The advantages of zebrafish include their small size, rapid development, embryonic transparency, high fertility, and low cost of maintenance. Additionally, transgenic zebrafish, which have a gene added to their DNA, can be produced with high efficiency. For example, many transgenic zebrafish that have various populations of blood cells labelled with fluorescent proteins have already been produced. The fluorescent proteins in these zebrafish allow us to visualize the behavior of the blood cells in living animals. However, no fluorescent transgenic zebrafish existed that specifically allowed us to visualize B cells. B cells are the cells in the blood that produce antibodies, which are proteins that bind to pathogens and clear them from the system. It was predicted that zebrafish have two types of B cells, whereas mammals have five types of B cells. We therefore created transgenic reporter lines that marked these two B cell populations with different fluorescent proteins so as to study their function in the immune system. We first studied where B cells develop in the zebrafish and found that they arise between the blood vessels of the trunk (Figure 1) and also in the kidney. This is a novel finding that suggests that B cell development shifts from the trunk to the kidney as the fish age, similar to developmental shifts previously seen in mammals and chickens. Thus, our results in zebrafish suggest that shifting sites of B cell development are a feature of all jawed vertebrates. We also studied the immune response in zebrafish, and we found that it appears to be temporally delayed relative to mammals, where activated B cells appear only a few days after immunization. In contrast, we did not observe significant numbers of activated B cells in the zebrafish until two to four weeks after immunization (Figure 2). Overall, results from these experiments have increased our knowledge of both the zebrafish immune system and our knowledge of the evolution of the immune system from fish to mammals. Importantly, these B cell transgenic zebrafish can be used to study how fish B cells respond to different pathogens, which has direct applications to fish health and aquaculture, as well as to fish models of human diseases. We have already distributed these transgenic zebrafish to several laboratories who are using them as a model for the human cancers, leukemia and lymphoma. Finally, a broader impact of this work was to enhance undergraduate research experiences. Undergraduate participation in research is essential not only because it increases the retention of Biology students who then pursue graduate studies, but also because it creates a more accurate understanding of the nature of science than coursework does. Nineteen undergraduates participated extensively in this project, with most gaining 400-800 hours of research experience. In addition, 131 undergraduate students in an Immunology laboratory also participated in the project by designing and carrying out their own experiments based on materials that were made available to them through this project.
