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.

Fig. 1.

lck-GFP transgenic zebrafish. Diagrams of the genomic DNA sequence comprising the lck promoter (A) and the GFP construct (B) are shown. Enzyme digest sites used for cloning and restriction mapping of the minimal promoter are shown. lck-GFP transgenic fish expressing the 5.5-kb EcoRI-NotI fragment (C) are shown at 8 dpf (D), 45 dpf (E), and 80 dpf (F). Arrowheads denote GFP-labeled cells in the thymus (T). The views are lateral with anterior to the left.

Fig. 2.

FACS analysis of cells from the kidney (A-D) and thymus (E-H) of rag2-GFP and lck-GFP transgenic zebrafish. Gated populations of erythrocytes (red), lymphocytes (blue), granulocytes and monocytes (green), and blood cell precursors (purple) are outlined. Populations of cells within each gate are described as percentages of total live cells (±1 SD; n = 24 for A; n = 18 for C; and n = 8 for B, D, and E-H). Cell size is represented by forward scatter (FSC), and granularity is represented by side scatter (SSC). GFP-positive cells in the progenitor gate in thymus samples became confined to the lymphoid gate upon reanalysis, confirming that GFP-labeled populations in the progenitor gate are predominately lymphoid in origin.

Fig. 3.

Semiquantitative RT-PCR analysis of FACS-sorted blood cell populations in the kidney (K) and thymus (T) of lck-GFP and rag2-GFP transgenic fish. GFP-positive (+) and -negative (-) blood cell populations are shown. Results for “No RT Control” show absence of genomic DNA contamination in samples. The β-actin PCR control was completed on genomic DNA. Because the β-actin primers span an intron, PCR amplifies a 100-bp larger fragment than seen in RT samples.

Fig. 4.

GFP-labeled thymic T cells obtained from adult lck-GFP transgenic fish home to the thymus of transplanted embryos. (A) GFP fluorescent microscopic images of a 4-day-old transplanted embryo (anterior to the left and dorsal to the top) are shown. (B and C) Enlarged views of the head and tail region, respectively. Arrowheads denote GFP-labeled cells in the thymus (T) and in the tail region. Asterisks denote autofluorescence of the yolk sac.

Fig. 5.

GFP-labeled T cells in 8-day-old lck-GFP fish are ablated in response to γ-irradiation or dexamethasone treatment. (A) Nonirradiated control fish. (B) Fish 2 days postirradiation. (C) Control fish with 0.4% ethanol. (D) Fish 3 days after treatment with 100 μg·ml^-1 dexamethasone (DEX). Asterisks denote autofluorescence of the yolk sac. Arrowheads denote GFP-labeled cells in the thymus (T). The views are lateral with anterior to the left.

Fig. 6.

Lymphopoiesis is fully reconstituted in irradiated adult recipients transplanted with lck-GFP kidney marrow. Transplants consisted of thymus cells (1.5 × 10⁶ cells, A and B) or whole kidney marrow (3 × 10⁵ cells, C and D) at 15 or 24 days posttransplantation. The views are lateral with anterior to the left. Arrowheads denote the location of the thymus (T).