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Supplementary Data 1-4, Table from Targeting Angiogenesis via a c-Myc/Hypoxia-Inducible Factor-1α–Dependent Pathway in Multiple Myeloma

Bone marrow angiogenesis is associated with multiple myeloma (MM) progression. Here, we report high constitutive hypoxia-inducible factor-1α (Hif-1α) expression in MM cells, which is associated with oncogenic c-Myc. A drug screen for anti-MM agents that decrease Hif-1α and c-Myc levels identified a variety of compounds, including bortezomib, lenalidomide, enzastaurin, and adaphostin. Functionally, based on transient knockdowns and overexpression, our data delineate a c-Myc/Hif-1α–dependent pathway mediating vascular endothelial growth factor production and secretion. The antiangiogenic activity of our tool compound, adaphostin, was subsequently shown in a zebrafish model and translated into a preclinical in vitro and in vivo model of MM in the bone marrow milieu. Our data, therefore, identify Hif-1α as a novel molecular target in MM and add another facet to anti-MM drug activity. [Cancer Res 2009;69(12):5082–90]

Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a "black box". Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO

Despite the common knowledge that the reticuloendothelial system is largely responsible for blood clearance of systemically administered nanoparticles, the sequestration mechanism remains a "black box". Using transgenic zebrafish embryos with cell type-specific fluorescent reporters and fluorescently labeled model nanoparticles (70 nm SiO2), we here demonstrate simultaneous three-color in vivo imaging of intravenously injected nanoparticles, macrophages, and scavenger endothelial cells (SECs). The trafficking processes were further revealed at ultrastructural resolution by transmission electron microscopy. We also find, using a correlative light-electron microscopy approach, that macrophages rapidly sequester nanoparticles via membrane adhesion and endocytosis (including macropinocytosis) within minutes after injection. In contrast, SECs trap single nanoparticles via scavenger receptor-mediated endocytosis, resulting in gradual sequestration with a time scale of hours. Inhibition of the scavenger receptors prevented SECs from accumulating nanoparticles but enhanced uptake in macrophages, indicating the competitive nature of nanoparticle clearance in vivo. To directly quantify the relative contributions of the two cell types to overall nanoparticle sequestration, the differential sequestration kinetics was studied within the first 30 min post-injection. This revealed a much higher and increasing relative contribution of SECs, as they by far outnumber macrophages in zebrafish embryos, suggesting the importance of the macrophage:SECs ratio in a given tissue. Further characterizing macrophages on their efficiency in nanoparticle clearance, we show that inflammatory stimuli diminish the uptake of nanoparticles per cell. Our study demonstrates the strength of transgenic zebrafish embryos for intravital real-time and ultrastructural imaging of nanomaterials that may provide mechanistic insights into nanoparticle clearance in rodent models and humans.

Acute myeloid leukemia (AML) occurs when multiple genetic aberrations alter white blood cell development, leading to hyperproliferation and arrest of cell differentiation. Pertinent animal models link in vitro studies with the use of new agents in clinical trials. We generated a transgenic zebrafish expressing human NUP98-HOXA9 (NHA9), a fusion oncogene found in high-risk AML. Embryos developed a preleukemic state with anemia and myeloid cell expansion, and adult fish developed a myeloproliferative neoplasm (MPN). We leveraged this model to show that NHA9 increases the number of hematopoietic stem cells, and that oncogenic function of NHA9 depends on downstream activation of meis1, the PTGS/COX pathway and genome hypermethylation through the DNA methyltransferase, dnmt1. We restored normal hematopoiesis in NHA9 embryos with knockdown of meis1 or dnmt1, as well as pharmacologic treatment with DNA (cytosine-5)-methyltransferase (DNMT) inhibitors or cyclo-oxygenase (COX) inhibitors. DNMT inhibitors reduced genome methylation to near normal levels. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of a histone deacetylase inhibitor plus either a DNMT inhibitor or COX inhibitor to block the effects of NHA9 on zebrafish blood development. Our work proposes novel drug targets in NHA9-induced myeloid disease, and suggests rational therapies by combining minimal doses of known bioactive compounds.

For 3D reconstructions of whole immune cells from zebrafish, isolated from adult animals by FAC-sorting we employed array tomography on hundreds of serial sections deposited on silicon wafers. Image stacks were either recorded manually or automatically with the newly released ZEISS Atlas 5 Array Tomography platform on a Zeiss FEGSEM. To characterize different populations of immune cells, organelle inventories were created by segmenting individual cells. In addition, arrays were used for quantification of cell populations with respect to the various cell types they contained. The detection of immunological synapses in cocultures of cell populations from thymus or WKM with cancer cells helped to identify the cytotoxic nature of these cells. Our results demonstrate the practicality and benefit of AT for high-throughput ultrastructural imaging of substantial volumes.

High-performance sensors for reactive oxygen species are instrumental to monitor dynamic events in cells and organisms. Here, we present HyPer-3, a genetically encoded fluorescent indicator for intracellular H(2)O(2) exhibiting improved performance with respect to response time and speed. HyPer-3 has an expanded dynamic range compared to HyPer and significantly faster oxidation/reduction dynamics compared to HyPer-2. We demonstrate this performance by in vivo imaging of tissue-scale H(2)O(2) gradients in zebrafish larvae. Moreover, HyPer-3 was successfully employed for single-wavelength fluorescent lifetime imaging of H(2)O(2) levels both in vitro and in vivo.

Prompt neutrophil arrival is critical for host defense immediately after injury [1-3]. Following wounding, a hydrogen peroxide (H(2)O(2)) burst generated in injured tissues is the earliest known leukocyte chemoattractant [4]. Generating this tissue-scale H(2)O(2) gradient uses dual oxidase [4] and neutrophils sense H(2)O(2) by a mechanism involving the LYN Src-family kinase [5], but the molecular mechanisms responsible for H(2)O(2) clearance are unknown [6]. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H(2)O(2)-consuming reaction [7, 8]. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H(2)O(2) concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H(2)O(2) concentrations and leukocytes [4, 9-11] and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H(2)O(2) burst, indicating that leukocytes themselves were required for H(2)O(2) downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H(2)O(2) concentrations despite similar numbers of arriving neutrophils. A local H(2)O(2)/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H(2)O(2) gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder [7, 12, 13], offering unique possibilities for investigating its clinical consequences.

Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H(2)O(2) during the early events of wound responses in zebrafish larvae expressing a genetically encoded H(2)O(2) sensor. This reporter revealed a sustained rise in H(2)O(2) concentration at the wound margin, starting approximately 3 min after wounding and peaking at approximately 20 min, which extended approximately 100-200 microm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H(2)O(2) pattern, and the first evidence that H(2)O(2) signals to leukocytes in tissues, in addition to its known antiseptic role.

Redox signals have emerged as important regulators of cellular physiology and pathology. The advent of redox imaging in vertebrate systems now provides the opportunity to dynamically visualize redox signaling during development and disease. In this review, we summarize recent advances in the generation of genetically encoded redox indicators (GERIs), introduce new redox imaging strategies, and highlight key publications in the field of vertebrate redox imaging. We also discuss the limitations and future potential of in vivo redox imaging in zebrafish and mice.

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.

12 páginas, 5 figuras, 1 tabla.-- et al., In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation., This work was supported by the Roche Research Foundation (F.L.) the Japanese Agency for Science and Technology (JST) (R.Q., M.C., S.W.), the Company of Biologists Ltd (F.D.B.), the Boehringer Ingelheim Foundation (C.G.), the Marie Curie Foundation (J-R. M-M.) and through grants from the DFG, EC (J.W) and HFSP (H.K, J.W.).

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.

We tested the Sleeping Beauty transposable element for its ability to efficiently insert transgenes into the genome of medaka (Oryzias latipes), an important model system for vertebrate development. We show that the SB transposon efficiently mediates integration of a reporter gene into the fish germ line. In pilot experiments, we established 174 transgenic lines with a transgenesis efficiency of 32%. Transgenes are stably transmitted to, and expressed in, subsequent generations. Interestingly, the transgenic lines show novel expression patterns with temporal and spatial specificity at a rate of 12% (21/174), likely due to both, enhancing and silencing position effects. Furthermore, promoter-dependent GFP expression in injected fish embryos is tightly correlated with germ line transmission, facilitating easy selection of founder fish. Thus, the SB transposon/transposase system provides a highly efficient tool for transgenesis in general and for the generation of novel reporter gene expression patterns in particular.

For 3D reconstructions of whole immune cells from zebrafish, isolated from adult animals by FAC-sorting we employed array tomography on hundreds of serial sections deposited on silicon wafers. Image stacks were either recorded manually or automatically with the newly released ZEISS Atlas 5 Array Tomography platform on a Zeiss FEGSEM. To characterize different populations of immune cells, organelle inventories were created by segmenting individual cells. In addition, arrays were used for quantification of cell populations with respect to the various cell types they contained. The detection of immunological synapses in cocultures of cell populations from thymus or WKM with cancer cells helped to identify the cytotoxic nature of these cells. Our results demonstrate the practicality and benefit of AT for high-throughput ultrastructural imaging of substantial volumes. Lay Description To look at immune cells from zebrafish we employed array tomography, a technique where arrays of serial sections deposited on solid substrates are used for imaging. Cell populations were isolated from the different organs of zebrafish involved in haematopoiesis, the production of blood cells. They were chemically fixed and centrifuged to concentrate them in a pellet that was then dehydrated and embedded in resin. Using a custom-built handling device it was possible to place hundreds of serial sections on silicon wafers as well ordered arrays. To image a whole cell at a resolution that would allow identifying all the organelles (i.e. compartments surrounded by membranes) inside the cell, stacks of usually 50–100 images were recorded in a scanning electron microscope (SEM). This recording was either done manually or automatically using the newly released Atlas Array Tomography platform on a ZEISS SEM. For the imaging of the sections a pixel size of about 5 nm was chosen, which defines membrane boundaries very well and allows segmentation of the membrane topology. After alignment of the images, cellular components were segmented to locate the individual organelles within the 3D reconstruction of the whole cell and also to create an inventory of organelles. Based on their morphologies we could identify specific cell types in the different hematopoietic organs. We could also quantify the proportion of each cell type in the whole population isolated from a given organ. Some of these specific cells from zebrafish were grown in a culture dish together with human cancer cells. By time-lapse light microscopy we observed that the fish cells attacked the cancer cells and killed them. From this we concluded that these cells must be similar to the cytotoxic cells from humans that play an important role in defence against spontaneously arising cancer cells in our bodies. They form special structures, called immunological synapses that we could also identify on our arrays and reconstruct in 3D. This is the first time the potential of zebrafish immune cells to form immunological synapses has been demonstrated. Our study is a good example for the practicality and benefit of array tomography in high-throughput ultrastructure imaging of substantial volumes, applicable to many areas of cell and developmental biology.

Neutrophils are the most abundant polymorphonuclear leukocytes, presenting the first line of defence against infection or tissue damage. To characterize the molecular changes on the protein level in neutrophils during sterile inflammation we established the chemically-induced inflammation (ChIn) assay in adult zebrafish and investigated the proteome dynamics within neutrophils of adult zebrafish upon inflammation. Through label-free proteomics we identified 48 proteins that were differentially regulated during inflammation. Gene ontology analysis revealed that these proteins were associated with cell cycle, nitric oxide signalling, regulation of cytoskeleton rearrangement and intermediate filaments as well as immune-related processes such as antigen presentation, leucocyte chemotaxis and IL-6 signalling. Comparison of protein expression dynamics with transcript expression dynamics suggests the existence of regulatory mechanisms confined to the protein level for some genes. This is the first proteome analysis of adult zebrafish neutrophils upon chemically-induced inflammation providing a valuable reference for future studies using zebrafish inflammation models.

The complete absence of eyes in the medaka fish mutation eyeless is the result of defective optic vesicle evagination. We show that the eyeless mutation is caused by an intronic insertion in the Rx3 homeobox gene resulting in a transcriptional repression of the locus that is rescued by injection of plasmid DNA containing the wild-type locus. Functional analysis reveals that Six3- and Pax6- dependent retina determination does not require Rx3. However, gain- and loss-of-function phenotypes show that Rx3 is indispensable to initiate optic vesicle evagination and to control vesicle proliferation, by that regulating organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye.

International audience; Abstract The second European Zebrafish Principal Investigator (PI) Meeting was held in March, 2012, in Karlsruhe, Germany. It brought together PIs from all over Europe who work with fish models such as zebrafish and medaka to discuss their latest results, as well as to resolve strategic issues faced by this research community. Scientific discussion ranged from the development of new technologies for working with fish models to progress in various fields of research such as injury and repair, disease models, and cell polarity and dynamics. This meeting also marked the establishment of the European Zebrafish Resource Centre (EZRC) at Karlsruhe that in the future will serve as an important focus and community resource for zebrafish- and medaka-based research.

NUP98-HOXA9 [t(7;11) (p15;p15)] is associated with inferior prognosis in de novo and treatment-related acute myeloid leukaemia (AML) and contributes to blast crisis in chronic myeloid leukaemia (CML). We have engineered an inducible transgenic zebrafish harbouring human NUP98-HOXA9 under the zebrafish spi1(pu.1) promoter. NUP98-HOXA9 perturbed zebrafish embryonic haematopoiesis, with upregulated spi1 expression at the expense of gata1a. Markers associated with more differentiated myeloid cells, lcp1, lyz, and mpx were also elevated, but to a lesser extent than spi1, suggesting differentiation of early myeloid progenitors may be impaired by NUP98-HOXA9. Following irradiation, NUP98-HOXA9-expressing embryos showed increased numbers of cells in G2-M transition compared to controls and absence of a normal apoptotic response, which may result from an upregulation of bcl2. These data suggest NUP98-HOXA9-induced oncogenesis may result from a combination of defects in haematopoiesis and an aberrant response to DNA damage. Importantly, 23% of adult NUP98-HOXA9-transgenic fish developed a myeloproliferative neoplasm (MPN) at 19-23 months of age. In summary, we have identified an embryonic haematopoietic phenotype in a transgenic zebrafish line that subsequently develops MPN. This tool provides a unique opportunity for high-throughput in vivo chemical modifier screens to identify novel therapeutic agents in high risk AML.

Precise regulatory mechanisms are required to appropriately modulate the cellular levels of transcription factors controlling cell fate decisions during blood cell development. In this study, we show that miR-126 is a novel physiological regulator of the proto-oncogene c-myb during definitive hematopoiesis. We show that knockdown of miR-126 results in increased c-Myb levels and promotes erythropoiesis at the expense of thrombopoiesis in vivo. We further provide evidence that specification of thrombocyte versus erythrocyte cell lineages is altered by the concerted activities of the microRNAs (miRNAs) miR-126 and miR-150. Both miRNAs are required but not sufficient individually to precisely regulate the cell fate decision between erythroid and megakaryocytic lineages during definitive hematopoiesis in vivo. These results support the notion that miRNAs not only function to provide precision to developmental programs but also are essential determinants in the control of variable potential functions of a single gene during hematopoiesis.

Mutations in the human nucleophosmin (NPM1) gene are the most frequent genetic alteration in adult acute myeloid leukemias (AMLs) and result in aberrant cytoplasmic translocation of this nucleolar phosphoprotein (NPMc+). However, underlying mechanisms leading to leukemogenesis remain unknown. To address this issue, we took advantage of the zebrafish model organism, which expresses 2 genes orthologous to human NPM1, referred to as npm1a and npm1b. Both genes are ubiquitously expressed, and their knockdown produces a reduction in myeloid cell numbers that is specifically rescued by NPM1 expression. In zebrafish, wild-type human NPM1 is nucleolar while NPMc+ is cytoplasmic, as in human AML, and both interact with endogenous zebrafish Npm1a and Npm1b. Forced NPMc+ expression in zebrafish causes an increase in pu.1+ primitive early myeloid cells. A more marked perturbation of myelopoiesis occurs in p53m/m embryos expressing NPMc+, where mpx+ and csf1r+ cell numbers are also expanded. Importantly, NPMc+ expression results in increased numbers of definitive hematopoietic cells, including erythromyeloid progenitors in the posterior blood island and c-myb/cd41+ cells in the ventral wall of the aorta. These results are likely to be relevant to human NPMc+ AML, where the observed NPMc+ multilineage expression pattern implies transformation of a multipotent stem or progenitor cell.

Bone marrow angiogenesis is associated with multiple myeloma (MM) progression. Here, we report high constitutive hypoxia-inducible factor-1α (Hif-1α) expression in MM cells, which is associated with oncogenic c-Myc. A drug screen for anti-MM agents that decrease Hif-1α and c-Myc levels identified a variety of compounds, including bortezomib, lenalidomide, enzastaurin, and adaphostin. Functionally, based on transient knockdowns and overexpression, our data delineate a c-Myc/Hif-1α–dependent pathway mediating vascular endothelial growth factor production and secretion. The antiangiogenic activity of our tool compound, adaphostin, was subsequently shown in a zebrafish model and translated into a preclinical in vitro and in vivo model of MM in the bone marrow milieu. Our data, therefore, identify Hif-1α as a novel molecular target in MM and add another facet to anti-MM drug activity. [Cancer Res 2009;69(12):5082–90]

Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences.

Nucleophosmin (NPM1) is a multi-functional ubiquitous phosphoprotein that shuttles between the nucleolus and cytoplasm. Located on chromosome 5q35 NPM1 is involved in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) both as a chimeric fusion partner and as a putative key haploinsufficient tumor suppressor. Heterozygous NPMc+ mutations have been identified in 30% of AML, usually with normal karyotype. Such mutations create new nuclear export signals and disrupt the normal nucleolar localization signal, resulting in re-localization of both mutant and heterodimeric wild-type (WT) NPM protein from the nucleolus to the cytoplasm. The zebrafish is a model organism ideally suited to genotypic and phenotypic analysis of myelopoiesis and leukemogenesis, with a proven track record for facilitating the discovery of novel pathogenetic pathways. Using in silico analysis we identified two homologues (a common finding in zebrafish due to genome duplication during piscine evolution) of the human nucleophosmin gene in zebrafish. These two genes have been designated znpm1a and znpm1b. znpm1a is annotated by the National Center for Biotechnology (NCBI) while znpm1b is a known protein-coding region located via blast search of the human NPM1 amino acid sequence at www.ensembl.org/Danio_rerio/index.html. Whilst znpm1b exhibits slightly less identity to human NPM1 (47%) than znpm1a (64%) it demonstrates clear synteny with human chromosome 5q35 and mouse chromosome 11 (Figure 1). We confirmed expression of both znpm1a and znpm1b in embryonic tissue and adult hematopoietic tissue of the major lineages by RT-PCR of Green Fluorescent Protein (GFP)-sorted cells in pu.1-GFP transgenic zebrafish embryos and in adult zebrafish kidney cells (sorted by forward and side scatter charactersistics). Morpholinos (stable, synthesized antisense oligonucleotides that specifically block gene expression when injected into embryos at the one-cell stage) were designed to inhibit znpm1a or 1b and injected into zebrafish embryos at the 1–4 cells stage to assess the effect of knockdown of znpm1a and 1b alone and in combination on hematopoiesis. Whole-mount in situ hybridization of 48 hours post-fertilization(hpf) injected embryos demonstrated a 50% reduction in the expression of myeloperoxidase (mpo) and a similar reduction in alpha globin (α-globin) expression as markers of myelo- and erythropoiesis. To investigate the mechanism of the reduction in hematopoietic cells we injected the znpm1a and 1b morpholinos into zebrafish carrying mutated p53 and observed partial rescue of the hematopoietic phenotype suggesting that loss of npm1 in zebrafish activates p53 dependent cell cycle arrest, senescence or cell death. Thus zebrafish npm1 proteins are required for normal hematopoiesis consistent with the role for NPM1 as a tumor suppressor in AML/MDS with loss of all or part of chromosome 5. Future studies using this model will address which pathways are disrupted by the loss of npm1 and thus may contribute to the pathogenesis of human AML/MDS and facilitate identification of potential therapeutic targets. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11. Figure 1. Zebrafish znpm1b exhibits synteny with human chromosome 5q35 and mouse chromosome 11.

The chromosomal translocation t(7;9) in human T-cell acute lymphoblastic leukaemia (T-ALL) results in deregulated expression of a truncated, activated form of Notch 1 (TAN1) under the control of the T-cell receptor-beta (TCRB) locus. Although TAN1 efficiently induces T-ALL in mouse models, t(7;9) is present in less than 1% of human T-ALL cases. The recent discovery of novel activating mutations in NOTCH1 in more than 50% of human T-ALL samples has made it clear that Notch 1 is far more important in human T-ALL pathogenesis than previously suspected.

Epigenetic therapy implies the use of drugs that target regulators of gene expression, such as DNA and histone methylation or histone acetylation, without altering the DNA coding sequence. Epigenetic mechanisms have been found to be perturbed in myeloid diseases, and in fact, the DNA demethylating agents, 5-azacytadine and decitabine are FDA approved drugs for myelodysplastic syndrome. However, use of epigenetic therapies in other types of myeloid disease including acute myeloid leukemia (AML) has been met with variable success, suggesting efficacy might be improved by preselecting subtypes of disease in which there is a high degree of epigenetic dysregulation. AML has recently been found to be associated with a host of epigenetic abnormalities including mutations in DNA methyltransferase 3A (DNMT3A), ten-eleven translocation-2 (TET2) and enhancer of zeste homolog 2 (EZH2). We generated a transgenic zebrafish model of high risk myeloid disease expressing the human NUP98-HOXA9 (NHA9) fusion oncogene, a genetic lesion for which epigenetic dysregulation has not previously been identified. Transgenic embryos exhibit an increase in immature myeloid cells at the expense of erythroid cells and adult fish develop a myeloproliferative neoplasm (MPN). Leveraging this model in a microarray screen, we identified 3-fold elevated levels of the epigenetic regulator, dnmt1, the major maintenance methyltransferase, for the first time in high risk AML. Decitabine specifically inhibits DNMT1 and treatment of NHA9 transgenic embryos with 75µM decitabine restored normal hematopoiesis, as evidenced by normal numbers of leukocytes and red cells. Moreover, using hematopoietic stem cell (HSC) reporter lines and whole mount in situ hybridization, we identified a 2-3 fold increase in this population in NHA9 embryos, suggesting the HSC as the cell of origin in this disease. Interestingly, 75µM decitabine therapy also restored normal HSC numbers. Strikingly, we discovered synergy when we combined sub-monotherapeutic doses of DNMT1 inhibitors, decitabine (10-25µM) or zebularine (100µM), plus histone deacetylase inhibitors, valproic acid (25-100µM) or trichostatin A (250nM), to inhibit the effects of NHA9 on hematopoiesis. To determine if NHA9 expression directly results in changes to DNA methylation, we performed MeDIP-Seq on genomic DNA from pools of untreated and decitabine-treated NHA9 or control embryos. Untreated NHA9 embryos displayed significantly higher methylation levels at the regions of gene promoters compared to control embryos, which were restored to control levels following treatment with 75µM decitabine. Similarly, combination therapy with 10µM decitabine and 25µM valproic acid significantly reduced methylation to near control levels following a 5 hour treatment, while prolonged exposures to these same doses resulted in profound global hypomethylation. These findings elucidate underlying mechanisms in the pathogenesis of NHA9-induced myeloid disease and propose novel actionable epigenetic drug targets. Furthermore, we highlight the opportunities inherent in the zebrafish model as a unique in vivo platform for the preclinical screening of epigenetic based combination therapy. Disclosures No relevant conflicts of interest to declare.

The metameric structure of the vertebrate trunk is generated by repeated formation of somites from the unsegmented presomitic mesoderm (PSM). We report the initial characterization of nine different mutants affecting segmentation that were isolated in a large-scale mutagenesis screen in Medaka (Oryzias latipes). Four mutants were identified that show a complete or partial absence of somites or somite boundaries. In addition, five mutations were found that cause fused somites or somites with irregular sizes and shapes. In situ hybridization analysis using specific markers involved in the segmentation clock and antero-posterior (A-P) polarity of somites revealed that the nine mutants can be compiled into two groups. In group 1, mutants exhibit defects in tailbud formation and PSM prepatterning, whereas A-P identity in the somites is defective in group 2 mutants. Three mutants (planlos, pll; schnelles ende, sne; samidare, sam) have characteristic phenotypes that are similar to those in zebrafish mutants affected in the Delta/Notch signaling pathway. The majority of mutants, however, exhibit somitic phenotypes distinct from those found in zebrafish, such as individually fused somites and irregular somite sizes. Thus, these Medaka mutants can be expected to provide clues to uncovering novel components essential for somitogenesis.

Publisher Summary This chapter focuses on highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Fish are excellent candidates for the production of transgenics for two important reasons. First, fish represent the largest and most diverse group of vertebrates and provide an advantageous system for in vivo studies of developmental processes to gain knowledge of gene regulation and the action of gene products in vertebrates. Second, conventional selective breeding of fish for improved growth or other characteristics is a very slow process. Transgenic fish technology has the potential to improve genetic traits such as increased growth potential, disease resistance, improved feed conversion efficiency, or other desirable genetic traits for aquaculture in one generation. The establishment of methods for successful transgenesis is one of the basic criteria for an organism to be referred to as model organism. Several endonucleases (meganucleases) encoded by introns and inteins have been shown to promote homing (lateral transfer) of their respective genetic elements into intron- or inteinless homologous allelic sites. By introducing sitespecific double-strand breaks (DSBs) in intronless alleles, these nucleases create recombinogenic ends that engage in gene conversion, resulting in duplication of the intron. The I-SceI system has been used as a tool in mammalian cells and Drosophila. The meganuclease can also be used in fish for comparative studies of cis-acting regulatory elements and homologous recombination (HR). Meganuclease transgenesis as described in this chapter could be further improved, including insulators upstream and downstream of the DNA of interest to protect the transgene from the influences of heterochromatin and epigenetic control of surrounding genomic sequences.

Erratum to: “I-SceI meganuclease mediates highly efficient transgenesis in fish” Mechanisms of Development, Volume 120, Issue 2, February 2003, Pages 267; The widespread use of fish as model systems is still limited by the mosaic distribution of cells transiently expressing transgenes leading to a low frequency of transgenic fish. Here we present a strategy that overcomes this problem. Transgenes of interest were flanked by two I-SceI meganuclease recognition sites, and co-injected together with the I-SceI meganuclease enzyme into medaka embryos (Oryzias latipes) at the one-cell stage. First, the promoter dependent expression was strongly enhanced. Already in F0, 76% of the embryos exhibited uniform promoter dependent expression compared to 26% when injections were performed without meganuclease. Second, the transgenesis frequency was raised to 30.5%. Even more striking was the increase in the germline transmission rate. Whereas in standard protocols it does not exceed a few percent, the number of transgenic F1 offspring of an identified founder fish reached the optimum of 50% in most lines resulting from meganuclease co-injection. Southern blot analysis showed that the individual integration loci contain only one or few copies of the transgene in tandem. At a lower rate this method also leads to enhancer trapping effects, novel patterns that are likely due to the integration of the transgene in the vicinity of enhancer elements. Meganuclease co-injection thus provides a simple and highly efficient tool to improve transgenesis by microinjection.

Abstract 2391 Acute myeloid leukemia (AML) results from multiple genetic lesions that alter white blood cell development, leading to hyperproliferation and a block in differentiation. A better understanding of these molecular pathways will enable the development of therapies that selectively target the specific abnormality in a leukemic cell with improved outcome and limited toxicity. Pertinent animal models serve as essential intermediaries between in vitro studies and the use of these new agents in clinical trials. The NUP98-HOXA9 (NHA9) fusion oncogene is found in high risk de novo AML, treatment related AML and chronic myeloid leukemia (CML) blast crisis. We previously generated a transgenic zebrafish model overexpressing human NHA 9 under the zebrafish pu.1 promoter (Forrester et al, BJH, 2012). Almost 25% of adult NHA9-transgenic fish develop a myeloproliferative neoplasm (MPN) in the kidney marrow, the site of adult hematopoiesis. Additionally, NHA9- transgenic embryos demonstrate an increase in immature myeloid cells (l-plastin1, 2.3-fold by in situ, P Microarray analysis found high expression of DNA (cytosine-5-)-methyltransferase 1 (dnmt1) in NHA9 embryos, which was confirmed by qRT-PCR (3.4-fold increase). The methylating activity of the DNMT1 enzyme is part of the epigenetic machinery that represses genes needed for terminal myeloid differentiation. Overexpression of human DNMT1 has been found in some cases of AML, but has not been previously linked with NHA9-induced disease. The overexpression of the zebrafish dnmt1 homolog in NHA9 embryos may keep cells trapped in an immature state, a hallmark of AML. Injecting NHA9 embryos with a gene-blocking dnmt1 morpholino restored normal hematopoiesis with wild-type expression levels of both l-plastin and gata1. Similarly, a dose-dependent return to normal proportions of myeloid and erythroid cells was achieved by bathing NHA9 embryos in 50–100 μM decitabine (5-aza-2'-deoxycytidine), a demethylating agent that specifically targets the DNMT1 enzyme. However, decitabine use as a monotherapy carries the risk of genomic instability due to wide-spread DNA hypomethylation. We therefore considered treating our NHA9 zebrafish embryos with combination therapy against multiple molecular targets. DNMT1 is part of a larger epigenetic machinery and works in parallel with histone deacetylation complexes (HDACs). Similar to decitabine, exposing NHA9 embryos to 150–250 μM of the HDAC inhibitor, valproic acid (VPA), lead to a dose-dependent return of normal hematopoiesis. In vitro studies suggest that combined treatment with a demethylating agent and an HDAC inhibitor may synergize to be more effective than either compound alone in combating myeloid disease, permitting use of lower drug doses, thus avoiding unnecessary toxicity. Indeed, we found that combination doses as low as 10 μM each of decitabine and VPA restored normal hematopoiesis as effectively as significantly higher doses of each monotherapy. This drug synergy identified in our transgenic zebrafish directly links NHA9-induced leukemia and epigenetic regulation and has set the stage for a new and exciting therapeutic approach for high risk AML. True to our goal, translating this treatment to clinical studies may ultimately improve outcome and minimize toxic side-effects, thereby increasing the long-term survival of patients with high-risk AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3622 High-risk forms of acute myeloid leukemia (AML) remain fatal for 40% of patients, both due to refractory disease and toxicity from traditional chemotherapeutic agents. Using zebrafish, we aim to discover new or repurposed drugs to combat high-risk leukemia that are less toxic and achieve better survival for human patients. The NUP98-HOXA9 (NHA9) mutation is a hallmark of high-risk AML, characterized by a failure of granulocyte maturation and increased cell division. The zebrafish is a robust model to study blood development and leukemia, with the inherent capacity to easily perform drug screens in embryos. We created an AML disease model using transgenic zebrafish that harbor the NHA9 mutation (Forrester et al., Br J Haematol, Aug 2 Epub). We are using these living models of cancer to study the genetic interactions that promote high-risk leukemia. Our model has already provided novel insights into the direct mechanisms of NHA9 oncogenesis. In response to DNA-damage, NHA9 failed to initiate cell cycle arrest and apoptosis, likely through upregulation of bcl2 rather than direct suppression of tumor protein 53 (tp53) signaling. Upon exposure to 16 Gy ionizing radiation, wild-type embryos displayed a 4.16-fold decrease of phospho-histone-H3 (pH3)-labeled cells, whereas NHA9 embryos only suffered a 1.63-fold decrease (relative difference of 2.55-fold, P NHA9 also perturbs blood cell development. In embryos, NHA9 produced a loss of red blood cells (gata1a, decreased expression 19.34-fold by qRT-PCR, P The cell division gene, β-catenin, has recently proven important for driving AML in mice, but co-operation with NHA9 is unknown. We are contrasting ‘stimulated' fish (NHA9 + β-catenin, using 20 μM prostaglandin E2 [PGE2]) with ‘inhibited' fish (NHA9 – β-catenin, using 20 μM indomethacin) to measure the combined effects of NHA9 and β-catenin on cell division and blood development. Similarly, overexpression of Meis1 accelerates the onset of NHA9 -induced leukemia in mice, but the underlying mechanism is unknown. We show that morpholino (MO) knockdown of zebrafish meis1 limits the myeloproliferative effects in NHA9 embryos by blocking myeloid cell development in the PBI (83% decreased expression of the myeloid marker, l-plastin /lcp1). Microarray analysis on these various cohorts of embryos will help to identify a subset of genes that are regulated by NHA9, β-catenin, and Meis1 together. These studies will help determine how normal blood cells are transformed into leukemia cells. Our ultimate goal is to use these NHA9 fish to discover new drugs that combat high-risk leukemia. Our model provides an excellent opportunity to perform chemical library screens for compounds that restore normal levels of red blood cells (measured by in situ for gata1a) in NHA9 embryos. We will present results of screening with compounds contained in the Biomol ICCB 480 library. Promising candidates may represent future therapies against NHA9 in human patients with AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3961 Poster Board III-897 The NUP98-HOXA9 fusion oncogene results from a t(7;11)(p15;p15) chromosomal translocation and is associated with inferior prognosis in de novo and treatment-related acute myeloid leukemia (AML), as well as blast crisis in chronic myeloid leukemia (CML). HOXA9 belongs to the highly-conserved HOX gene family of developmental transcription factors and is critical for vertebrate hematopoiesis. Upregulation of HOXA9 is a frequent phenomenon in human AML, implicating a central role in myeloid disease. Thus, elucidating the activity of oncogenic NUP98-HOXA9 may reveal universal mechanisms of leukemogenesis and lead to design of targeted therapies. The zebrafish is a robust model for studying vertebrate hematopoiesis and leukemogenesis, by virtue of its ex utero development, and conserved genetics and cell biology. We have engineered a Cre-lox inducible transgenic zebrafish harboring the human NUP98-HOXA9 fusion oncogene downstream of the zebrafish pu.1 promoter. NUP98-HOXA9 perturbed zebrafish embryonic hematopoiesis, leading to upregulated expression of myeloid-specific genes, pu.1, lysC, and l-plastin, and downregulation of the erythroid-specific gene, gata1. These changes in gene transcription affect both ‘primitive’ and ‘definitive’ waves of zebrafish hematopoiesis (including the recently-described erythro-myeloid progenitors [EMPs]), suggesting that NUP98-HOXA9 reprograms blood cell precursors to a predominantly myeloid fate. Preferential upregulation of pu.1 during ‘primitive’ hematopoiesis further suggests an impaired potential for terminal myeloid differentiation. These effects appear developmentally mediated, as cell proliferation was not increased as measured by phosphorylated histone-H3 (pH3) staining. However, following ionizing irradiation (IR), NUP98-HOXA9 expression preserved cell proliferation and suppressed apoptosis in the presence of DNA damage. Irradiated transgenic embryos displayed wild-type levels of pH3, suggesting inhibition of cell cycle checkpoints. Irradiated embryos also displayed reduced levels of the conserved apoptotic marker, activated caspase-3, as well as downregulation of the puma pro-apoptotic gene, and upregulation of bcl2 and bcl-xL anti-apoptotic genes. These data postulate suppression of caspase-3-dependent apoptosis as a mechanism for NUP98-HOXA9 -mediated oncogenesis in vivo. Incomplete phenotypic ‘rescue’ with bcl-xL morpholino suggests insufficiency of single-target therapy, with a need for multi-target interruptions to restore radiosensitivity. Between 20-24 months post fertilization, a proportion of these NUP98-HOXA9 transgenic zebrafish (n=9/19; 47%) presented with gross abdominal masses and/or abnormal swimming behavior. Thus far, histological sectioning has demonstrated enlarged kidneys and livers, with evidence of infiltrative, pleiomorphic, mitotically-active cells with myeloid morphology. This pathology is reminiscent of the myeloproliferative disease (MPD) observed in NUP98-HOXA9 transgenic mice. Taken together, these data provide insights into the mechanisms underlying NUP98-HOXA9-mediated leukemogenesis and provide an unprecedented opportunity for in vivo chemical modifier screens to identify promising therapeutic agents that restore a normal phenotype. Disclosures: No relevant conflicts of interest to declare.

Bone marrow (BM) angiogenesis has been associated with multiple myeloma (MM) progression. Here we demonstrate that oncogenic c-Myc, also prominently associated with adverse prognosis in MM, regulates Hif-1α promoter activity, Hif-1alpha protein levels, and consequently the expression of pro-angiogenic target genes such as VEGF in MM cells. As observed with c-Myc knockdown, anti-MM agents with known anti-angiogenic activity including bortezomib, lenalidomide, and enzastaurin also downregulate Hif-1alpha and VEGF. Moreover, tyrophostin adaphostin induces similar effects, associated with anti-angiogenic activity, in a zebrafish model. The correlation between c-Myc, Hif-1alpha and tumor angiogenesis was further confirmed in a xenograft mouse model of human MM. Mechanistically, our in vitro data show that oncogenic c-Myc stabilizes caspase-8, and thereby protects Hif-1alpha against caspase-8- dependent degradation and attenuation of VEGF secretion. These sequelae also contribute to enhanced VEGF secretion triggered by MM cell-bone marrow stromal cell binding. Indeed, conditioned media from adaphostin- treated co-cultures inhibit endothelial cell growth and tubule formation. In addition, HIF1alpha gene demonstrated significant increased expression (p=0.02, Mann Whitney test) in tumor cells versus plasma cells derived from healthy donors. Importantly, when tested on a dataset of 559 patients, a significant link between HIF1alpha overexpression and poor prognosis was found (p=0,0069). These data further point to the possible relevance of HIF1alpha in the progression of a subset of MM patients. In summary, our data provide strong evidence of a key role of Hif-1alpha in MM angiogenesis and the therapeutic potential of specific Hif-1alpha and c-Myc inhibitors. Moreover they demonstrate additional biologic sequelae of novel MM therapies.

Myelodysplasia (MDS) is a common condition characterized by ineffective and dysplastic hematopoiesis, peripheral blood cytopenias, and evolution to acute myeloid leukemia. Although the biology of MDS is heterogeneous, a common feature is the disruption of effective hematopoiesis, resulting from either reduced hematopoiesis (with hypocellular bone marrow) or ineffective hematopoiesis (with hypercellular bone marrow and increased intramedullary cell apoptosis). Our current understanding of the genetic pathways controlling human hematopoiesis, and how they are disrupted in MDS remains very limited. Non-random chromosomal deletions occur frequently i n patients with MDS. Such deletions are thought to inactivate tumor suppressor genes (TSGs) that contribute to the maintenance of normal myelopoiesis. Unfortunately, these inactivated TSGs remain largely unidentified. The zebrafish is a vertebrate model organism well suited to the phenotypic and genotypic investigation of MDS, since transcription factors and other proteins regulating hematopoiesis are conserved between zebrafish and humans. Genome-wide mutagenesis screens in zebrafish to assay for deficiencies in mature granulocytes can reveal novel genes involved in myelopoiesis and a subset of these genes may also be involved in the pathogenesis of MDS. The dead-box 18 protein (DDX18) is a member of the highly conserved dead-box family of DNA-dependant RNA helicases. DDX18 is a target of the MYC oncogene and the drosophila homologue pitchoune is involved in cell growth and proliferation. We conducted a recessive screen of viral insertional zebrafish mutants and found that the loss of ddx18 in zebrafish embryos significantly reduced numbers of myeloperoxidase- (mpo-) expressing cells at 2 days post-fertilization (dpf). We have confirmed that the myelopoietic phenotype results from the specific loss of ddx18 by using antisense morpholinos that disrupt transcription of ddx18. In addition to loss of mature myeloid cells, ddx18 mutant embryos showed reduced alpha-globin expression at 2dpf (as a marker of erythoid cells); as well as loss of expression of runx1 and c-myb at 36hpf, which are expressed in definitive hematopoietic stem cells (HSC) along the ventral wall of the zebrafish aorta. These data support the hypothesis that loss of myeloid and erythoid cells result from a reduction in hematopoietic stem cell numbers. To determine whether the reduction in mpo-expressing cells observed in ddx18 mutants resulted from diminished production or from increased cell death, we performed acridine orange and TUNEL staining. This demonstrated markedly increased cell death in ddx18 mutants. Flow cytometric assessment of whole embryo cell lysates for annexin V binding also confirmed increased numbers of apoptotic cells in ddx18 mutants at 2 dpf indicating that active cell death was occurring at this time point. Finally, injection of a morpholino to knockdown expression of p53 was able to rescue the reduction of mpo-expressing cells seen in ddx18 mutants and reverted myeloid cell numbers to that of WT siblings. Thus, loss of ddx18 results in p53-dependant cell death in hematopoietic cells. To address whether DDX18 in plays a role in human MDS/AML we sequenced the DDX18 gene in 22 human AML cell lines and patient samples from 72 patients. Three heterozygous nonsynonymous genomic DNA sequence alterations resulting in amino acid substitutions were detected in 2 MDS patients, C184Y and V371I in one patient and V621I in a second. None of these has been previously annotated as single nucleotide polymorphisms in any species. Two of the 3 alterations were located in regions conserved with the zebrafish ddx18 gene (C184 and V371). Our results demonstrate that ddx18 plays a critical role in developmental hematopoiesis in zebrafish. Infrequent DDX18 sequence alterations occur in human AML/MDS cells, suggesting that DDX18 disruption may have a role in the pathogenesis of AML/MDS in a subset of patients. Further studies will delineate the interaction between p53 and ddx18 in the zebrafish and address the functional consequences of the heterozygous mutations found in human AML/MDS samples to determine whether DDX18 can function as a novel tumor suppressor (and a potential therapeutic target) in MDS/AML.

The most frequent genetic alterations in adult cases of Acute Myeloid Leukemia (AML) are mutations in the human nucleophosmin (hNPM1) gene. In about 30% of AMLs this nucleolar phosphoprotein is aberrantly localized to the cytoplasm (hNPMc) due to these mutations affecting the protein’s nuclear shuttling. hNPMc AMLs exhibit distinctive clinical and biological features that have led to its WHO classification as a distinct myeloid neoplasm. Despite its prominent association with AML, in vitro and in vivo models of hNPMc transformation in myeloid cells are lacking and its role in this process remains poorly understood. To further our understanding of hNPM function we are using the zebrafish vertebrate model system that is ideally suited for the in vivo analysis of cellular function and development during embryonic hematopoiesis. Importantly, the wide variety of blood cell types and key cellular factors regulating hematopoiesis are highly conserved between mammals and zebrafish. To investigate the in vivo role of hNPMc in hematopoiesis, we injected mRNAs encoding hNPM1wt and the leukemia-associated mutant hNPMc into one-cell stage zebrafish embryos. By fusing GFP to these hNPM proteins, we were able to follow the developmental expression of hNPM1 and its subcellular localization during embryogenesis. Analysis using confocal microscopy showed that NPMc is localized to the cell cytoplasm, while NPM1wt is found in nucleoli, as in human and mouse cells. These studies demonstrate the conservation of the nuclear-cytoplasmic transport functions of the human proteins in the zebrafish and further support its validity as a model to analyze and determine hNPM function. We also observed that hNPMc is expressed at far lower levels than its wild-type counterpart and is almost undetectable at 36hpf while hNPMwt continues to be expressed. Unlike mammals, two endogenous zebrafish NPM1 proteins were identified and named, zNpm1a and zNpm1b. Both zNpm1a and zNpm1b proteins are ubiquitously expressed in the embryo and demonstrated nucleolar localization. Expression of hNPMwt resulted in its colocalization with endogenous zNpm while hNPMc was able to bring about the export of both zebrafish proteins to the cytoplasm through heterotypic interactions. Co-immunoprecipitation experiments confirmed the interaction between human and zebrafish NPM1 proteins and zNpm1a and zNpm1b were both able to bind and co-immunoprecipitate with hNPM1 and hNPMc. These experiments suggest that transient hNPMc expression during zebrafish hematopoiesis may mimic its function in human leukemic blasts and provide clues to its functional role in AML. Comparable protein levels of either hNPMwt or hNPMc were expressed in embryos, confirmed by western blot at 22–24 hpf, and analyzed by whole mount in situ hybridization (WISH) using antisense RNA markers of specific hematopoietic lineages. Expression of hNPMc caused an increase in cMYB expression at 36 hpf, indicating an increase in the hematopoietic stem cell compartment. Furthermore, myeloid precursors (PU.1 at 22 hpf) also showed an increase upon hNPMc expression; however, mature myeloid cell (MPO and L-plastin at 26 hpf) levels were not increased relative to those in control hNPMwt injected embryos. Interestingly, the expression of hNPMc in p53 mutant embryos resulted in elevated levels of both PU.1 and MPO expressing cells, suggesting that hNPMc in zebrafish can activate p53 dependent cell cycle arrest, senescence or cell death in PU.1 cells prior to differentiation. These in vivo studies of hNPMc function during zebrafish hematopoietic differentiation suggest that hNPMc expression may increase the stem cell/ myeloid precursor compartment and can activate a p53 dependent cell death response in myeloid cells. Taking advantage of the zebrafish system in these continuing studies will further address how hNPMc expression may contribute to leukemogenesis.

From among a plethora of various gene delivery methods, the researcher must choose the right one according to availability for a given species and the precise application the transgenic animal is intended for. Here we review the progress in meganuclease and Sleeping Beauty transposon mediated transgenesis over recent years with a focus on medaka and zebrafish. We present a side-by-side comparison of these two approaches based on their biologic properties and provide interesting perspectives for future experiments and applications, which are different for the two techniques because of their distinct modes of action.