In vivo tracking of T cell development, ablation, and engraftment in transgenic zebrafish

Abstract

Transgenic zebrafish that express GFP under control of the T cell-specific tyrosine kinase (lck) promoter were used to analyze critical aspects of the immune system, including patterns of T cell development and T cell homing after transplant. GFP-labeled T cells could be ablated in larvae by either irradiation or dexamethasone added to the water, illustrating that T cells have evolutionarily conserved responses to chemical and radiation ablation. In transplant experiments, thymocytes from lck-GFP fish repopulated the thymus of irradiated wild-type fish only transiently, suggesting that the thymus contains only short-term thymic repopulating cells. By contrast, whole kidney marrow permanently reconstituted the T lymphoid compartment of irradiated wild-type fish, suggesting that long-term thymic repopulating cells reside in the kidney.T he zebrafish has emerged as an important vertebrate genetic model of development. Zebrafish embryos are transparent and develop rapidly ex utero, and most organ systems are fully developed by 5 days postfertilization (dpf). Forward genetic screens in the zebrafish have been instrumental in identifying gene mutations that affect development (1-3). Such genetic approaches have been used successfully to identify genes involved in patterning, regeneration, and the development of organs, including heart, eye, and blood (4, 5). Additionally, small-molecule-based screens in the zebrafish have identified chemicals that perturb normal development (6, 7), and drug screens have been proposed to identify compounds that suppress cancer-associated phenotypes (8-10).Comparisons of fish and mammalian hematopoiesis indicate that the genetic programs underlying vertebrate blood development have been highly conserved throughout evolution (5). Like their mammalian counterparts, fish B cells express Ig proteins, and T cells express T cell receptor components (11); both of which require gene rearrangements mediated by the rag1 and rag2 proteins (12). Moreover, many homologs of the mammalian lymphocyte receptors have been identified in fish, including CD3 (13) and the CD8 coreceptor (14). Taken together, these findings suggest that T and B cell development in the fish relies on many of the same molecules and pathways used in mammalian lymphopoiesis (15,16).The development and functional anatomy of the thymus are also remarkably similar between fish and humans. The zebrafish thymic rudiment is completely developed by 60 h postfertilization (hpf) and by 68 hpf is colonized by T lymphocyte progenitors, which begin to transcribe rag1 and rag2 by 72 hpf (15,(17)(18)(19). As in mice and humans, mature T cells localize to the medulla of the adult zebrafish thymus, whereas immature T cells localize to the cortex (20).The genetics underlying the development of the zebrafish lymphoid system is under active investigation, but functional studies of the zebrafish immune system have been limited by the lack of tools to facilitate the isolation and characterization of different lymphoid cell populations. For example, a...

The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

“…To assess the effect of the reduced furinA expression on hematopoiesis in the furinA td204e/ϩ fish, we studied their blood cell composition with flow cytometry (Fig. 1B and C) (31). The flow cytometric analysis revealed no marked differences in blood cell precursor, erythrocyte or lymphocyte populations in furinA td204e/ϩ zebrafish compared to WT controls.…”

“…The development of hematopoietic cells in zebrafish is highly similar to that in humans (31,37). To assess the effect of the reduced furinA expression on hematopoiesis in the furinA td204e/ϩ fish, we studied their blood cell composition with flow cytometry (Fig.…”

“…The data were analyzed with the FlowJo program (v7.5; Tree Star, Inc., Oregon, USA). Hematopoietic cell types were identified based on granularity (side scatter [SSC]) and particle size (forward scatter [FSC]) (31). Granulocytes and lymphocytes for furinA expression analyses were purified from WT AB zebrafish kidneys by using flow cytometric sorting with a FACSAria I apparatus (Becton Dickinson).…”

Catch of the day: zebrafish as a human cancer model

“…These animals would greatly facilitate real-time imaging of the tumor-induced vascular remodeling events and the dynamic immune cell movements that occur in the tumor microenvironment. Many transgenic lines have already been developed including T cell, macrophage and neutrophil reporter animals (Langenau et al, 2004;Mathias et al, 2006;Hall et al, 2007).…”

Immunological tools: Engaging students in the use and analysis of flow cytometry and enzyme‐linked immunosorbent assay (ELISA)

“…The first laboratory exercise, which was performed during laboratory sessions 1 and 2, required students to identify the cell type expressing enhanced green fluorescent protein (EGFP) in a transgenic zebrafish line by comparing forward [25][26][27][28][29][30].…”