Supporting Text

Supporting Materials and Methods

RT-PCR Analysis of Fluorescence-Activated Cell Sorted ACS Cell Populations. GFP-positive and -negative fluorescence-activated cell sorted blood cell populations were obtained from the kidney and thymus of lck-GFP and rag2-GFP transgenic fish. RNA was extracted with TRIzol (GIBCO/BRL). Samples were treated with DNase to eliminate residual genomic DNA, and RNA was used in reverse transcription reactions with or without reverse transcriptase. cDNA samples were diluted (1:1, 1:10, 1:100, and 1:1,000) and used in semiquantitative PCR with primers specific for lck, Ig Light Chain 3 (IgLC3), rag2, and b -actin (Table 2). Both the lck and b -actin primers span introns, whereas the IgLC3 and rag2 primers do not. PCR cycling conditions varied between primer sets: for lck and rag2, cycling was completed at 94°C for 40 s, 60°C for 60 s, and 72°C for 60 s (35 and 30 cycles, respectively); and for IgLC3 and b -actin, it was completed at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s (30 cycles).

T Cell Ablation by g -Irradiation. Adult rag2-GFP transgenic fish were administered a whole-body dose of g -irradiation (20 or 23 Gy from a 137Cs source) and analyzed for GFP expression within the thymus at 2, 4, 8, 11, 14, 18, and 22 days posttreatment.

Supporting Results

Isolation of the Zebrafish lck cDNA. The full-length zebrafish lck cDNA encodes a 504-aa protein that shares high amino acid similarity with other vertebrate LCK proteins (Fig. 7A) as well as conserved domain structures with human LCK (Fig. 7B), especially the tyrosine kinase, SH3, and SH2 domains. The unique domain is the region that binds CD4 and CD8. Given that these two receptor proteins have diverged during evolutionary time, not surprisingly, the zebrafish unique domain is not well conserved when compared to human. Zebrafish lck shares 70% amino acid identity with the Fugu lck (Fig. 7C).

Generation of lck-GFP Transgenic Zebrafish. All four lck-GFP transgenic zebrafish lines exhibited strong GFP expression confined to the developing thymus by 5 days postfertilization (dpf) with variable levels of GFP expression detected in the trunk musculature. One line expressed GFP in the neuromast cells, head musculature, and nose, and a second expressed it in the dorsal musculature and thymus. The two other lck-GFP lines expressed low levels of GFP in the body musculature by 5 dpf, but not in adulthood, as demonstrated by fluorescence microscopy and anti-GFP immunostaining of paraffin-embedded sections. Both transgenic lines showed very high GFP expression in the thymus by 8 dpf (Fig. 1D). The GFP expression in neuromast cells and head musculature in the two lines most likely reflects transgene integration effects, increased transgene copy number, or both.

T Cells in Adult Fish Are Ablated by Irradiation. To assess T cell sensitivity to radiation, we irradiated 2-month-old rag2-GFP fish and monitored fish by fluorescence microscopy for T cell ablation and T cell reconstitution over time. GFP-positive T cells in the thymus were eliminated or greatly reduced by 2 days postirradiation but eventually repopulated the thymus over time. These results suggest that treating fish with 20-23 Gy is sufficient to ablate T cells and most thymic progenitors; however, a sufficient number of progenitor cells survive in the thymus or in the kidney to reconstitute the T cell lymphoid system.

T Cells Are Ablated by Dexamethasone Treatment of rag2-GFP Fish. In one set of experiments, all six rag2-GFP fish treated for 3 days with 250 m g•ml–1 dexamethasone lacked GFP-labeled T cells by 8 dpf, compared with only two of six fish given 25 m g•ml–1 dexamethasone. Some incompletely ablated fish in the 25 m g•ml–1 treatment group had fewer T cells than did ethanol-treated controls.

In a second set of experiments, we assessed the effects of administering dexamethasone for shorter times. Five-day-old fish were dosed with dexamethasone for 1, 2, and 3 days and analyzed at 8 dpf for GFP expression in T cells. Longer treatment with dexamethasone led to increased numbers of fish responding to treatment, as judged from the loss of GFP-labeled T cells in the thymus, whereas all fish treated with ethanol vehicle retained GFP-labeled T cells in the thymus (Table 3).

GFP-Labeled T Cells Home to the Thymus, Kidney, and Peripheral Sites of T Cell Accumulation in Transplanted Adult Fish. To confirm that GFP-labeled cells from lck-GFP fish identify mature T cell populations in the periphery, we transplanted irradiated adult fish with lck-GFP thymic and kidney marrow cells and analyzed paraffin-embedded sections (at 14 and 25 days posttransplantation) for GFP protein expression based on anti-GFP immunostaining. GFP-labeled T cells were found in the kidney, gastrointestinal tract, esophagus, ovary, olfactory bulb, and gills of irradiated recipients transplanted with either lck-GFP kidney marrow or thymus cells (data not shown). This finding suggests that transplanted cells were able to home to sites normally populated by T lymphocytes, and confirms that GFP-expressing cells identified in the lck-GFP stable transgenic fish were hematopoietic in origin.