Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in PHF6, encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established in vitro models. Our findings reveal that PHF6 levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of NOTCH1 and its downstream target genes. This functional interaction in developing thymocytes was confirmed in vivo using a phf6-deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced phf6 or notch1 activation. In summary, our work reveals that appropriate control of PHF6 expression is important for normal human hematopoiesis and provides clues towards the role of PHF6 in T-ALL development.

The TP53 tumor-suppressor gene is mutated in >50% of human tumors and Li-Fraumeni patients with germ line inactivation are predisposed to developing cancer. Here, we generated tp53 deleted zebrafish that spontaneously develop malignant peripheral nerve-sheath tumors, angiosarcomas, germ cell tumors, and an aggressive Natural Killer cell-like leukemia for which no animal model has been developed. Because the tp53 deletion was generated in syngeneic zebrafish, engraftment of fluorescent-labeled tumors could be dynamically visualized over time. Importantly, engrafted tumors shared gene expression signatures with predicted cells of origin in human tissue. Finally, we showed that tp53(del/del) enhanced invasion and metastasis in kRAS(G12D)-induced embryonal rhabdomyosarcoma (ERMS), but did not alter the overall frequency of cancer stem cells, suggesting novel pro-metastatic roles for TP53 loss-of-function in human muscle tumors. In summary, we have developed a Li-Fraumeni zebrafish model that is amenable to large-scale transplantation and direct visualization of tumor growth in live animals.

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg-deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg-like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.

Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.

Abstract Cell transplantation into immune compromised mice has transformed our understanding of cancer and is now the gold standard for assessing therapeutic responses in vivo. However, mouse models are expensive and engraftment is often difficult to visualize directly. To overcome these challenges, we have developed immune compromised zebrafish (ICZ) in the transparent casper background using genome editing techniques. We have successfully targeted genes required for immune cell function and are well known to cause immune deficiency in human and mice. To date, we have developed homozygous viable mutants for recombination-activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), janus kinase 3 (jak3), interleukin 2 receptor gamma (Il2rg), zeta-chain (TCR) associated protein kinase 70 (zap70), and forkhead box N1 (foxn1/nude). Gene expression analysis of marrow cells using RNAseq has identified novel transcript changes correlated with loss of specific cell types, and in conjunction with large-scale single cell transcriptional profiling, has identified specific cellular defects associated with T, B, and NK cell loss. For example, homozygous prkdc (SCID) mutant fish lack mature T and B cells, but have intact NK cell signaling. By contrast, il2rg-deficient zebrafish lack T and NK cells. Importantly, these ICZ models accurately recapitulate known human severe combined immune deficiencies and established mouse models that are commonly used for cell transplantation. Thus, it is not unexpected that a subset of zebrafish mutants have reduced immune cell function, permitting engraftment of normal hematopoietic and muscle satellite cells from allogeneic donors. Additionally, we have demonstrated robust and persistent engraftment of fluorescently labeled leukemia, rhabdomyosarcoma, neuroblastoma, and melanoma from a wide range of zebrafish strains. Because mutations have been created in optically-clear, casper-strain zebrafish and cancers are fluorescently labeled, we now have unprecedented access to directly visualize tumor cells at single cell resolution in live animals. To date, we have optimized our models to visualize neovascularization, intratumoral cell heterogeneity, clonal evolution and metastisis. The ability to transplant non-immune matched cell types will likely revolutionize the types and scale of cell transplantation experiments performed in the zebrafish and will likely permit engraftment of mouse and human cells into compound mutant ICZ models in the near future. Citation Format: John C. Moore, Qin Tang, Nora Torres Yordan, Timothy Mulligan, Finola E. Moore, Riadh Lobbardi, Ashwin Ramakrishnan, Anthony Anselmo, Ruslan Sadreyev, Jason Berman, Robert Liwski, Brant Weinstein, John Rawls, David M. Langenau. Dynamic visualization of cancer cell engraftment into immune compromised zebrafish. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4177.

Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4(+) cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2(E450fs) mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4(+) cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2(E450fs) mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4(+)/CD8(+) cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia.

Abstract The clinical challenges associated with T-cell acute lymphoblastic leukemia (T-ALL) are two-fold: 1) the recently improved cure rate for primary T-ALL is largely attributed to highly toxic chemotherapy regimens that have both short- and long-term adverse effects in patients, and 2) chemotherapy is often ineffective against relapsed T-ALL, which has a dismal 5-year survival rate of <30% in children and <10% in adults. The development of new and better chemotherapies requires a detailed understanding of the genes and pathways that drive T-ALL malignancy. We have completed an unbiased cell transplantation screen using a zebrafish model of T-ALL and in excess of 6,000 adult recipient animals to identify molecular targets that promote T-ALL progression and relapse. Single leukemic cells were grown in syngeneic recipient fish and animals were assessed for differences in growth, leukemia propagating cell frequency, and therapy resistance. Serial transplantation experiments then followed evolution within 48 single-cell clones, identifying 6 clones that evolved increased LPC frequency and/or elevated growth potential. Comparative genomic hybridization arrays were used to identify recurrent amplifications associated with clonal evolution, and identified Protein Tyrosine Phosphatase 4A3 (PRL3) as being genomically amplified in 30% of clones with elevated LPC frequency and growth. Real-time quantitative PCR showed that 90% of clones with high LPC frequency and growth expressed high levels of PRL3, suggesting additional genetic pathways likely activate PRL3. PRL-3 was also genomically amplified in a subset of human T-ALL and highly expressed in 58% of primary T-ALL patient samples, suggesting that this phosphatase is an important and previously undefined driver of human T-ALL. PRL3 has not been previously associated with leukemia progression or survival, although it is expressed in Multiple Myeloma and Acute Myelogenous Leukemia. PRL3 knock-down in human T-ALL significantly reduced the viability of cell lines in vitro and in xenograft models (p<0.0001). Additionally, a specific PRL3 inhibitor strongly induced apoptosis of PRL3-expressing T-ALL cells in a dose-dependent manner. PRL3 inhibition also killed T-ALL cell lines that were resistant to dexamethasone, the standard chemotherapy for T-ALL, suggesting that therapies that inactivate PRL3 will be useful for the treatment of refractory and relapse disease. We have also identified several FDA-approved, general phosphatase inhibitors that have potent anti-PRL3 activity and are capable of killing T-ALL cells in vitro. Current work is focused on moving these inhibitors into pre-clinical testing using patient-derived xenografts. Phospho-profiling approaches are also being used to identify the substrates of PRL3 tyrosine phosphatase activity that are critical to T-ALL survival and may represent new, tractable drug targets for the treatment of T-ALL and other types of leukemias. Note: This abstract was not presented at the meeting. Citation Format: Jessica S. Blackburn, Aleksey Molodstov, Riadh Lobbardi, Finola Moore, David Langeau. The tyrosine phosphatase PRL3 as a novel drug target in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2015-1725

Abstract The aggressive and unpredictable behavior of T-cell acute lymphoblastic leukemia (T-ALL) presents a major clinical challenge in both the pediatric and adult setting. The challenges associated with T-ALL are two-fold: 1) the recently improved cure rate for primary T-ALL is largely attributed to highly toxic chemotherapy regimens that have both short- and long-term adverse effects in patients, and 2) chemotherapy is often ineffective against relapsed T-ALL, which has a dismal 5-year survival rate of <30% in children and <10% in adults. The development of new and better chemotherapies requires a detailed understanding of the genes and pathways that drive T-ALL malignancy. We have completed an unbiased cell transplantation screen using a zebrafish model of T-ALL and in excess of 6,000 adult recipient animals to identify molecular targets that promote T-ALL progression and relapse. Single leukemic cells were grown in syngeneic recipient fish and animals were assessed for differences in growth, leukemia propagating cell frequency, and therapy resistance. Serial transplantation experiments then followed evolution within 48 single-cell clones, identifying 6 clones that evolved increased LPC frequency and/or elevated growth potential. Comparative genomic hybridization arrays were used to identify recurrent amplifications associated with clonal evolution, and identified Protein Tyrosine Phosphatase 4A3 (PRL3) as being genomically amplified in 30% of clones with elevated LPC frequency and growth. Real-time quantitative PCR showed that 90% of clones with high LPC frequency and growth expressed high levels of PRL-3, suggesting additional genetic pathways likely activate PRL3. PRL-3 was also genomically amplified in a subset of human T-ALL and highly expressed in 58% of primary T-ALL patient samples, suggesting that this phosphatase is an important and previously undefined driver of human T-ALL. PRL3 has not been previously associated with leukemia progression or survival, although it is expressed in Multiple Myeloma and Acute Myelogenous Leukemia. PRL3 knock-down in human T-ALL significantly reduced the viability of cell lines in vitro and in xenograft models (p<0.0001). Additionally, the specific PRL3 inhibitor 1-(2-bromobenzyloxy)-4-bromo-2-benzylidene rhodanine strongly induced apoptosis of PRL3-expressing T-ALL cells in a dose-dependent manner. PRL3 inhibition also killed T-ALL cell lines that were resistant to dexamethasone, the standard chemotherapy for T-ALL, suggesting that therapies that inactivate PRL3 will be useful for the treatment of refractory and relapse disease. We have also identified several FDA-approved, general phosphatase inhibitors that have potent anti-PRL3 activity and are capable of killing T-ALL cells in vitro. Current work is focused on moving these inhibitors into pre-clinical testing using patient-derived xenografts. Phospho-profiling approaches are also being used to identify the substrates of PRL3 tyrosine phosphatase activity that are critical to T-ALL survival and may represent new, tractable drug targets for the treatment of T-ALL and other types of leukemias. Citation Format: Jessica Blackburn, Aleksey Molodtsov, Riadh Lobbardi, Finola Moore, David Langenau. The tyrosine phosphatase PRL3 as a novel drug target in T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A16.

Abstract Plant Home domain Finger 6 (PHF6) is a tumor suppressor of unknown function for blood malignancies such as T-cell Acute Lymphoblastic Leukemia (T-ALL), Acute Myeloid leukemia (AML), and Chronic Myeloid Leukemia (CML). PHF6 contains two zinc finger-like PH domains and interacts with the Nucleosome Remodeling and Deacetylation (NuRD) complex, suggesting a role in chromatin remodeling. Although PHF6 loss-of-function mutations are found in nearly 40% of T-ALL patients, little is known about how PHF6 mutations contribute to blood development and leukemogenesis. To understand the function of PHF6, beginning with its role in hematopoiesis, we have undertaken developmental studies in zebrafish to discover how loss of phf6 affects blood development and to determine which pathways are regulated by phf6. Zebrafish will be used to study hematopoiesis due to the remarkable conservation of molecular pathways that regulate blood development, genetic tractability, and ability to observe embryonic development over a short window of time. RNA in situ hybridization studies of zebrafish embryos showed that phf6 is expressed broadly during zebrafish development, and especially in the dorsal aorta, a site analogous to the aorta-gonad-mesonephros (AGM) in mammals, from which hematopoietic stem cells (HSCs) arise. Further, phf6 is highly expressed in lymphocytes of adult zebrafish, reminiscent of the expression patterns found in human and mouse. To determine the effect of phf6 loss on hematopoiesis, phf6 expression was knocked down by morpholino injection. We find that phf6 morphants have increased numbers of HSCs by RNA in situ hybridization of runx1/cmyb in the AGM and caudal hematopoietic tissue ((CHT) analogous to mammalian fetal liver), sites of HSC emergence and migration. Later in development, phf6 morphants demonstrate increased lymphocytes by RNA in situ hybridization of rag1 at the thymus. Similar phenotypes were observed in homozygous phf6-null mutants generated by TALEN-mediated knockout. Phf6-null mutant zebrafish survive to Mendelian ratios and are fertile as adults. In total, we found a new role for phf6 in regulation of HSC formation. Citation Format: Finola E. Moore, Virginie Esain, Riadh Lobbardi, Jessica S. Blackburn, Trista E. North, David M. Langenau. Role for the tumor suppressor phf6 in hematopoiesis. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A33.

Clonal evolution and intratumoral heterogeneity drive cancer progression through unknown molecular mechanisms. To address this issue, functional differences between single T cell acute lymphoblastic leukemia (T-ALL) clones were assessed using a zebrafish transgenic model. Functional variation was observed within individual clones, with a minority of clones enhancing growth rate and leukemia-propagating potential with time. Akt pathway activation was acquired in a subset of these evolved clones, which increased the number of leukemia-propagating cells through activating mTORC1, elevated growth rate likely by stabilizing the Myc protein, and rendered cells resistant to dexamethasone, which was reversed by combined treatment with an Akt inhibitor. Thus, T-ALL clones spontaneously and continuously evolve to drive leukemia progression even in the absence of therapy-induced selection.

Abstract Leukemia progression and relapse are driven by molecularly distinct and often-rare cancer cells called leukemia-propagating cells (LPCs). If LPCs are retained following treatment, they will ultimately initiate relapse disease. Despite the substantial number of genetic lesions that have been identified in relapse samples and the contention that these mutations likely modulate response to therapy, acquired mutations that increase the overall frequency of tumor propagating cells following continued clonal evolution have not been reported in any cancer to date. Here, we have developed a transgenic zebrafish model where single fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) cells are transplanted into genetically identical recipient fish and functionally assessed for differences in leukemia propagating cell frequency, growth, and dexamethasone resistance. While subclonal variation was observed within single cells from the same primary leukemia, a subset of clones continued to evolve genetic lesions and epigenetic modifications to enhance growth and overall LPC frequency. A majority of evolved clones acquired activated AKT signaling, which simultaneously increased the number of leukemia propagating cells through activating the mTORC1 pathway, enhanced growth by stabilizing Myc protein levels, and rendered T-ALL cells resistant to dexamethasone, which was reversed by combined treatment with an AKT inhibitor. These results were confirmed using large-scale transgenic epistasis experiments and limiting-dilution cell transplantation studies. In total, our data suggest that diagnosis clones can stochastically acquire mutations necessary to survive treatment and drive relapse even before a patient begins treatment, with acquired mutations being independently selected based on important cancer phenotypes including elevated growth rate and leukemia propagating potential. Moreover, our work also identifies combination therapies that utilize dexamethasone and AKT inhibitor can kill LPCs in a subset of refractory T-ALL. Finally, these are the first studies performed in any model to follow single cell evolution as it relates to relapse, utilizing in excess of 6,000 transplant recipient animals and opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. This abstract is also presented as Poster A08. Citation Format: Jessica Blackburn, Sali Liu, Kimberly Dobrinski, Sarah Martinez, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR06.

This abstract is being presented as a short talk in Session 3: Mechanisms of Resistance. A full abstract is printed in the Proffered Abstracts section (PR06) of the Conference Proceedings. Citation Format: Jessica Blackburn, Sali Liu, Kimberly Dobrinski, Sarah Martinez, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A08.

This paper describes immunocompromised rag2 mutant zebrafish, which allow efficient and robust cell transplantations in adult zebrafish, thereby facilitating stem cell, cancer and regeneration research. Cell transplantation into adult zebrafish has lagged behind mouse models owing to the lack of immunocompromised strains. Here we have created rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft muscle, blood stem cells and various cancers. rag2E450fs mutant zebrafish are the first immunocompromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer.

Leukemia progression and relapse are driven by molecularly distinct and often-rare cancer cells called leukemia-propagating cells (LPCs). If LPCs are retained following treatment, they will ultimately initiate relapse disease. Despite the substantial number of genetic lesions that have been identified in relapse samples and the contention that these mutations likely modulate response to therapy, acquired mutations that increase the overall frequency of tumor propagating cells following continued clonal evolution have not been reported in any cancer to date. Here, we have developed a transgenic zebrafish model where single fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) cells are transplanted into genetically identical recipient fish and functionally assessed for differences in leukemia propagating cell frequency, growth, and dexamethasone resistance. While subclonal variation was observed within single cells from the same primary leukemia, a subset of clones continued to evolve genetic lesions and epigenetic modifications to enhance growth and overall LPC frequency. A majority of evolved clones acquired activated AKT signaling, which simultaneously increased the number of leukemia propagating cells through activating the mTORC1 pathway, enhanced growth by stabilizing Myc protein levels, and rendered T-ALL cells resistant to dexamethasone, which was reversed by combined treatment with an AKT inhibitor. These results were confirmed using large-scale transgenic epistasis experiments and limiting-dilution cell transplantation studies. In total, our data suggest that diagnosis clones can stochastically acquire mutations necessary to survive treatment and drive relapse even before a patient begins treatment, with acquired mutations being independently selected based on important cancer phenotypes including elevated growth rate and leukemia propagating potential. Moreover, our work also identifies combination therapies that utilize dexamethasone and AKT inhibitor can kill LPCs in a subset of refractory T-ALL. Finally, these are the first studies performed in any model to follow single cell evolution as it relates to relapse, utilizing in excess of 6,000 transplant recipient animals and opening new and exciting avenues of study to uncover genetic pathways that drive cancer malignancy. This abstract is also presented as Poster B23. Citation Format: Jessica Blackburn, Sali Liu, Sarah Martinez, Kimberly Dobrinski, Finola Moore, Riadh Lobbardi, David Langenau. Clonal evolution enhances leukemia-propagating cell activity in T-cell acutelymphoblastic leukemia through AKT/mTORC1 pathway activation. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR05.

Zinc Finger Nucleases (ZFNs) made by Context-Dependent Assembly (CoDA) and Transcription Activator-Like Effector Nucleases (TALENs) provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

Zebrafish have emerged as a powerful model of development and cancer. Human, mouse, and zebrafish malignancies exhibit striking histopathologic and molecular similarities, underscoring the remarkable conservation of genetic pathways required to induce cancer. Zebrafish are uniquely suited for large-scale studies in which hundreds of animals can be used to investigate cancer processes. Moreover, zebrafish are small in size, optically clear during development, and amenable to genetic manipulation. Facile transgenic approaches and new technologies in gene inactivation have provided much needed genomic resources to interrogate the function of specific oncogenic and tumor suppressor pathways in cancer. This manuscript focuses on the unique attribute of labeling leukemia cells with fluorescent proteins and directly visualizing cancer processes in vivo including tumor growth, dissemination, and intravasation into the vasculature. We will also discuss the use of fluorescent transgenic approaches and cell transplantation to assess leukemia-propagating cell frequency and response to chemotherapy.