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Myc-Induced T Cell Leukemia in Transgenic Zebrafish

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Science  07 Feb 2003:
Vol. 299, Issue 5608, pp. 887-890
DOI: 10.1126/science.1080280

Abstract

The zebrafish is an attractive model organism for studying cancer development because of its genetic accessibility. Here we describe the induction of clonally derived T cell acute lymphoblastic leukemia in transgenic zebrafish expressing mouse c-myc under control of the zebrafish Rag2promoter. Visualization of leukemic cells expressing a chimeric transgene encoding Myc fused to green fluorescent protein (GFP) revealed that leukemias arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into skeletal muscle and abdominal organs. Leukemic cells homed back to the thymus in irradiated fish transplanted with GFP-labeled leukemic lymphoblasts. This transgenic model provides a platform for drug screens and for genetic screens aimed at identifying mutations that suppress or enhance c-myc– induced carcinogenesis.

The zebrafish (Danio rerio) is a potentially valuable vertebrate system in which to elucidate novel molecular pathways of oncogenesis. In particular, this model organism develops an array of benign and malignant tumors resembling those in humans (1, 2) and is amenable to large-scale forward genetic screens that could be targeted to conserved cancer pathways (3, 4). However, it has not been possible to establish stable lines of transgenic tumor-bearing fish. Here we report the induction of T cell leukemia in zebrafish, using mouse c-myc (mMyc) and chimericEGFP-mMyc transgenes (5) under control of the zebrafish Rag2 promoter (zRag2), which targets gene expression specifically to lymphoid cells (6). This transgenic strategy is based on the demonstrated role ofMYC in the pathogenesis of human T and B cell leukemias and lymphomas and its ability to induce T and B lymphoid malignancies when aberrantly expressed in transgenic mice [reviewed in (7,8)].

We microinjected wild-type zebrafish embryos with thezRag2-mMyc or zRag2-EGFP-mMyc transgene at the one-cell stage of development (9), generating F0founder fish mosaic for expression of the transgene (10). Of 215 mosaic F0 zebrafish embryos injected withzRag2-mMyc, 11 (5%) developed tumors, as did 7 (6%) of 122 injected with zRag2-EGFP-mMyc. These results are similar to the proportion of embryos expressing green fluorescent protein (GFP) in developing thymocytes after injection of the controlzRag2-GFP reporter construct, suggesting that cancer develops in nearly every mosaic fish whose lymphoid progenitors carry a functional mMyc transgene. The mean latencies of tumor development in zRag2-mMyc+ andzRag2-EGFP-mMyc + fish were 44 and 52 days (range, 30 to 131 days), respectively.

The affected zebrafish had distended abdominal cavities and splayed eyes due to retro-orbital infiltration by malignant cells (compare Fig. 1, A and B, with Fig. 1, C and D). Some fish had growths protruding from under the operculum adjacent to the thymus (fig. S1, A and B), whereas some had tumors at the base of the pectoral fin (Fig. 1, C and D). Many of the fish showed extensive subcutaneous infiltration by transformed cells (Fig. 1F), resulting in progressive pallor.

Figure 1

External and histological features of leukemiczRag2-mMyc F0 mosaic fish. (A andB) Wild-type fish and (C and D)zRag2-EGFP-mMyc fish with leukemic infiltration into the retro-orbital soft tissue, olfactory region, and pectoral fins. Transverse sections of (E and G) wild-type fish and (F and H) leukemic fish with massive infiltration of lymphoblasts throughout the body [(E) and (F)] and into the kidney [(G) and (H)]. E, eye; F, fin; G, gut; K, kidney; M, muscle; O, olfactory region; and S, skin. Arrowheads indicate sites of leukemic cells. Scale bars in (E) and (F), 1 mm; scale bars in (G) and (H), 100 μm.

To establish the extent of leukemic cell infiltration, we studied tissue sections from seven diseased fish. Lymphoblasts almost completely effaced the kidney marrow [the site of definitive hematopoiesis in the fish (Fig. 1, G and H)] and were found between the fibers of skeletal muscle (fig. S1, E and F). Leukemic cell invasion was also observed in the gut, gills, and fins, and it was especially prominent in the region adjacent to the olfactory bulb. Although zebrafish Rag genes are expressed at the apical surface of the olfactory placode (6, 11), malignant cells in the olfactory region did not express α-keratin and exhibited lymphoblast morphology, demonstrating that they were lymphoid and not epithelial in origin.

To analyze the gene expression patterns in transformed lymphoblasts, we applied RNA in situ hybridization to paraffin-embedded sections from seven leukemic F0 fish, testing for expression of the T cell–specific gene zLck, the B cell–specific immunoglobulin heavy-chain (zIgM) and light-chain (zIgLC) genes (12), the early lymphoid geneszRag1 and zRag2, and mMyc. Each of the leukemias expressed mMyc (fig. S3, A and E),zRag2 (fig. S3, B and F), zRag1 (13), and zLck (fig. S3, C and G), but not zIgM orzIgLC, confirming expression of the transgene and the derivation of these leukemias from cells of the T lymphoid lineage.

We further investigated the clonality and lineage of the lymphoblasts by Southern blot analysis of restriction enzyme–digested leukemic cell DNAs, using radiolabeled probes for zTcr-α (12) and zIgM constant regions. Of the three leukemias analyzed, one had monoclonal and one had oligoclonalzTcr-α gene rearrangements (Fig. 2A). The third showed a germ linezTcr-α configuration, but very strong zLck RNA expression, verifying the thymic origin of the lymphoblasts and suggesting that transformed thymocytes in this fish were developmentally arrested at a stage before zTcr-α gene rearrangement. None of the leukemias had rearranged zIgMgenes. In addition, flow cytometric measurements of the DNA content of tumor cells from a zRag2-EGFP-mMyc fish showed that this Myc-induced tumor was clonally aneuploid (Fig. 2B). Taken together, these data indicate that Myc-induced leukemias in the zebrafish represent the clonal expansion of transformed T lymphocyte precursors and suggest that additional mutations are needed to produce the malignant phenotype.

Figure 2

Clonality of mMyc-induced leukemias. (A) Southern analysis of the zTcr-α and zIgMconstant regions in leukemic (T1 through T3) and wild-type (WT1 through WT4) fish. The presence of either two or four restriction fragments hybridizing to the zIgM probe results from a Bgl II polymorphic site within the gene, not gene rearrangement. (B) DNA content of leukemic cells from azRag2-EGFP-mMyc fish, as measured by flow cytometry. The DNA index (DI) of 1.15 for G1-phase leukemic cells indicates clonal aneuploidy. 2N, DNA content of the normal diploid zebrafish genome. Bars indicate the postion of modal DNA content peaks corresponding to the indicated G1 or G2/M cell populations.

To quantify the extent of leukemic cell infiltration of the kidney and spleen, we analyzed hematopoietic cells from these tissues by fluorescence-activated cell sorting (FACS) (14). In wild-type zebrafish, the kidney (n = 7) contained (8.4 ± 3.7) × 105 (mean ± SD) blood cells, and the spleen (n = 6) contained (8.2 ± 6.3) × 104 blood cells; these wild-type values were about one-sixth that of leukemic fish [kidney (n = 5), (4.95 ± 3.3) × 106 blood cells; spleen (n = 4), (4.64 ± 1.5) × 105blood cells]. The leukemic blasts comprised ∼87% of the kidney marrow cells and had a granularity similar to that of normal lymphocytes (y axis in Fig. 3), although they were slightly larger in size (x axis in Fig. 3). This analysis of the kidney marrow, the equivalent of mammalian bone marrow (15–17), supports the histopathological classification of these Myc-induced cancers as T cell acute leukemia rather than disseminated lymphoma, in which the blast cells account for <30% of the total cells in the marrow.

Figure 3

FACS analysis of cells from kidney (top) and spleen (bottom) from (A and C) wild-type samples and (B and D) leukemic fish. Gated populations are as follows: erythrocytes (red), lymphocytes (blue), granulocytes and monocytes (green), and blood cell precursors in wild-type samples (purple). Populations of cells within each gate are described as mean percentages of total cells (±1 SD). Cell size is represented by forward scatter (FSC), and granularity is represented by side scatter (SSC).

To assess the transplantability of zebrafish leukemic cells, we first injected the zRag2-mMyc transgene into fertilized eggs from a stable transgenic zRag2-GFP line, so that the lymphoblasts would be GFP positive when leukemia developed. GFP-positive lymphoblasts from a leukemic 62-day-old fish were then transplanted intraperitoneally into eight irradiated wild-type adult zebrafish. Leukemic cells were apparent at the site of transplantation within 7 days after injection (Fig. 4A) and had begun to spread throughout the peritoneal cavity within 14 days after injection (Fig. 4B). Lymphoblast homing to the thymus occurred relatively early in the disease process (fig. S6B), with GFP-positive cells becoming evident at this site in one fish within 14 days after injection and in six fish within 26 days after injection. There was also prominent leukemic cell infiltration into the region adjacent to the olfactory bulb in two fish within 14 days after injection and in all eight within 26 days after injection (fig. S6, C and D). These experiments establish the transplantability of Myc-induced leukemias in zebrafish, indicate that the disseminated spread of leukemic cells proceeds along anatomically defined pathways, and suggest that the region adjacent to the olfactory bulb is a preferred site for the homing of immature T cells.

Figure 4

Transplantability of zebrafish leukemias and germline transmission of zRag2-EGFP-mMyc. GFP-positive leukemic cells transplanted into sublethally irradiated adult fish were detected (A) at the site of injection into the peritoneal cavity at 14 days after injection, (B) in the peritoneal cavity at 24 days after injection, and (C) in the head region at 24 days after injection. After germline transmission of thezRag2-EGFP-mMyc transgene, GFP-positive leukemia progressed from (D) thymic enlargement, to (E) local infiltration of leukemic cells outside the thymus, to (F) extensive infiltration of the head. All panels are oriented with anterior to the right [lateral view, dorsal on top in (A) and (C) through (F) and ventral view in (B)]. AF, anal fin; E, eye; I, site of injection; OB, olfactory bulb; PF, pectoral fin; and T, thymic masses. Arrowheads indicate accumulations of leukemic cells. Autofluorescence is indicated with an asterisk.

The power of the zebrafish model lies in its ability to accommodate “forward-genetic” screens to identify modifier genes that influence the development of Myc-induced leukemia, an application requiring a stable transgenic zebrafish line that expresses the mMyctransgene. Thus, mosaic zRag2-EGFP-mMyc F0 fish injected as single-cell embryos were mated, and the F1offspring of one fish developed leukemia with a mean latency of 32 days (range of 21 to 44 days). GFP fluorescence was readily detectable in the thymic lymphoblasts of these fish (Fig. 4, D through F), demonstrating germline transmission and expression of the chimeric transgene.

The rapid onset of leukemia after germline transmission of the transgene made it necessary to propagate this stable line by in vitro fertilization (IVF). Hence, we killed 90-day-old leukemic males and used their sperm to fertilize eggs from normal females. The resultant progeny showed expansion of GFP-positive leukemic cells from the thymus by 1 month of age. Technology to render the expression of transgenic myc alleles that are regulatable with estrogen has been developed for in vivo use in the mouse (18), and we are currently testing this approach for its capacity to generate large numbers of transgenic fish for further genetic manipulations, thus eliminating the need for IVF procedures.

Modifier screens in the mouse have identified retrovirally activated genes that accelerate Myc-induced oncogenesis (19), and breeding studies with genetically manipulated mice have identified other critical interacting oncogenes and tumor suppressors (20,21). More recent studies in murine models have exploited tissue-specific, regulatable expression of myc alleles to show that myc expression is essential not only for establishment of the leukemic phenotype, but also for maintenance of the malignant phenotype (22, 23). Despite these advances, it has been difficult to conduct unbiased screens in the mouse to identify modifier genes whose inactivation delays or prevents the ability of Myc to generate a malignant phenotype. This obstacle could be overcome by conducting genomewide zebrafish screens for germline mutations induced by either ethylnitrosourea or retroviral integration. Such strategies would also be especially attractive for identifying tumor suppressor genes, as the mutant fish would carry inactivating mutations of single tumor suppressor alleles throughout development, increasing the likelihood of acquiring inactivating mutations or deletions affecting both alleles of the same gene. Such mutations may shed new light on the molecular mechanisms that drive T cell leukemia and other human cancers to which Myc has been linked (24). Others have demonstrated the utility of living zebrafish as subjects of screens for small molecules that perturb normal development (25,26). Thus, our transgenic zebrafish model may also provide the opportunity to conduct large-scale screens for new drugs for the prevention and treatment of leukemia.

Supporting Online Material

www.sciencemag.org/cgi/content/full/299/5608/887/DC1

Materials and Methods

Supporting Online Text

Figs. S1 to S6

References and Notes

  • * To whom correspondence should be addressed. E-mail: thomas_look{at}dfci.harvard.edu

REFERENCES AND NOTES

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