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 Rag2 promoter. 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.

Genome-wide chemical mutagenesis screens in the zebrafish (Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafish mpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, and L-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis. (Blood. 2001;98:643-651)

For decades immunologists have relied heavily on the mouse model for their experimental designs. With the realization of the important role innate immunity plays in orchestrating immune responses, invertebrates such as worms and flies have been added to the repertoire. Here, we discuss the advent of the zebrafish as a powerful vertebrate model organism that promises to positively impact immunologic research.

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...

Engineered zinc-finger nucleases (ZFNs) enable targeted genome modification. Here we describe Context-Dependent Assembly (CoDA), a platform for engineering ZFNs using only standard cloning techniques or custom DNA synthesis. Using CoDA ZFNs, we rapidly altered 20 genes in zebrafish, Arabidopsis, and soybean. The simplicity and efficacy of CoDA will enable broad adoption of ZFN technology and make possible large-scale projects focused on multi-gene pathways or genome-wide alterations.

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5-to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.microfluidics | cancer metastasis | CTC clusters | circulating tumor cell cluster microemboli | capillary microhemodynamics

Embryonal rhabdomyosarcoma (ERMS) is a devastating cancer with specific features of muscle differentiation that can result from mutational activation of RAS family members. However, to date, RAS pathway activation has not been reported in a majority of ERMS patients. Here, we have created a zebrafish model of RAS-induced ERMS, in which animals develop externally visible tumors by 10 d of life. Microarray analysis and cross-species comparisons identified two conserved gene signatures found in both zebrafish and human ERMS, one associated with tumor-specific and tissue-restricted gene expression in rhabdomyosarcoma and a second comprising a novel RAS-induced gene signature. Remarkably, our analysis uncovered that RAS pathway activation is exceedingly common in human RMS. We also created a new transgenic coinjection methodology to fluorescently label distinct subpopulations of tumor cells based on muscle differentiation status. In conjunction with fluorescent activated cell sorting, cell transplantation, and limiting dilution analysis, we were able to identify the cancer stem cell in zebrafish ERMS. When coupled with gene expression studies of this cell population, we propose that the zebrafish RMS cancer stem cell shares similar self-renewal programs as those found in activated satellite cells.[Keywords: Zebrafish; rhabdomyosarcoma; RAS; P53; transgenic] Supplemental material is available at http://www.genesdev.org.

Highlights d prkdc À/À , il2rga À/À zebrafish reared at 37 C engraft a wide array of human cancers d Growth and therapy responses can be dynamically visualized at single-cell resolution d Combination olaparib and temozolomide kill xenografted human rhabdomyosarcoma d Engrafting patient-derived cancers opens new avenues for personalized therapy Authors