Mutagenesis Screen In Zebrafish Research Articles

Myron Gordon Award Lecture 2023: Painting the neural crest: How studying pigment cells illuminates neural crest cell biology.

It has been 30 (!!) years since I began working on zebrafish pigment cells, as a postdoc in the laboratory of Prof. Christiane Nüsslein-Volhard. There, I participated in the first large-scale mutagenesis screen in zebrafish, focusing on pigment cell mutant phenotypes. The isolation of colourless, shady, parade and choker mutants allowed us (as a postdoc in Prof. Judith Eisen's laboratory, and then in my own laboratory at the University of Bath since 1997) to pursue my ambition to address long-standing problems in the neural crest field. Thus, we have studied how neural crest cells choose individual fates, resulting in our recent proposal of a new, and potentially unifying, model which we call Cyclical Fate Restriction, as well as addressing how pigment cell patterns are generated. A key feature of our work in the last 10 years has been the use of mathematical modelling approaches to clarify our biological models and to refine our interpretations. None of this would have been possible without a hugely talented group of laboratory members and other collaborators from around the world-it has been, and I am sure will continue to be, a pleasure and privilege to work with you all!

Shutdown corner, a large deletion mutant isolated from a haploid mutagenesis screen in zebrafish.

Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened ∼100 genomes and isolated shutdown corner (sco), a mutant that exhibits multiple tissue defects and harbors a ∼10-Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner [Df(Chr05:sco)z207] could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or studying many genes lost in the mutant.

Knockout of SORBS2 Protein Disrupts the Structural Integrity of Intercalated Disc and Manifests Features of Arrhythmogenic Cardiomyopathy.

Background Sorbs2b (sorbin and SH3 domain‐containing 2b) was recently identified as a cardiomyopathy gene from a zebrafish mutagenesis screen. However, cardiac functions of its mammalian ortholog remain elusive.Methods and ResultsWe conducted a detailed expression and subcellular localization analysis of Sorbs2 ortholog in mice and a phenotypic characterization in Sorbs2 knockout mice. Sorbs2 is highly expressed in the mouse heart and encodes an adhesion junction/desmosome protein that is mainly localized to the intercalated disc. A mutation with near complete depletion of the Sorbs2 protein in mice results in phenotypes characteristic of human arrhythmogenic cardiomyopathy (ACM), including right ventricular dilation, right ventricular dysfunction, spontaneous ventricular tachycardia, and premature death. Sorbs2 is required to maintain the structural integrity of intercalated disc. Its absence resulted in profound cardiac electrical remodeling with impaired impulse conduction and action potential derangements. Targeted sequencing of human patients with ACM identified 2 rare splicing variants classified as likely pathogenic were in 2 unrelated individuals with ACM from a cohort of 59 patients with ACM.ConclusionsThe Sorbs2 knockout mouse manifests several key features reminiscent of human ACM. Although the candidacy of SORBS2 as a new ACM‐susceptibility gene is supported by preliminary human genetics study, future validation in larger cohorts with ACM is needed.

Molecular genetics of maternally-controlled cell divisions.

Forward genetic screens remain at the forefront of biology as an unbiased approach for discovering and elucidating gene function at the organismal and molecular level. Past mutagenesis screens targeting maternal-effect genes identified a broad spectrum of phenotypes ranging from defects in oocyte development to embryonic patterning. However, earlier vertebrate screens did not reach saturation, anticipated classes of phenotypes were not uncovered, and technological limitations made it difficult to pinpoint the causal gene. In this study, we performed a chemically-induced maternal-effect mutagenesis screen in zebrafish and identified eight distinct mutants specifically affecting the cleavage stage of development and one cleavage stage mutant that is also male sterile. The cleavage-stage phenotypes fell into three separate classes: developmental arrest proximal to the mid blastula transition (MBT), irregular cleavage, and cytokinesis mutants. We mapped each mutation to narrow genetic intervals and determined the molecular basis for two of the developmental arrest mutants, and a mutation causing male sterility and a maternal-effect mutant phenotype. One developmental arrest mutant gene encodes a maternal specific Stem Loop Binding Protein, which is required to maintain maternal histone levels. The other developmental arrest mutant encodes a maternal-specific subunit of the Minichromosome Maintenance Protein Complex, which is essential for maintaining normal chromosome integrity in the early blastomeres. Finally, we identify a hypomorphic allele of Polo-like kinase-1 (Plk-1), which results in a male sterile and maternal-effect phenotype. Collectively, these mutants expand our molecular-genetic understanding of the maternal regulation of early embryonic development in vertebrates.

Human Semaphorin 3 Variants Link Melanocortin Circuit Development and Energy Balance

SummaryHypothalamic melanocortin neurons play a pivotal role in weight regulation. Here, we examined the contribution of Semaphorin 3 (SEMA3) signaling to the development of these circuits. In genetic studies, we found 40 rare variants in SEMA3A-G and their receptors (PLXNA1-4; NRP1-2) in 573 severely obese individuals; variants disrupted secretion and/or signaling through multiple molecular mechanisms. Rare variants in this set of genes were significantly enriched in 982 severely obese cases compared to 4,449 controls. In a zebrafish mutagenesis screen, deletion of 7 genes in this pathway led to increased somatic growth and/or adiposity demonstrating that disruption of Semaphorin 3 signaling perturbs energy homeostasis. In mice, deletion of the Neuropilin-2 receptor in Pro-opiomelanocortin neurons disrupted their projections from the arcuate to the paraventricular nucleus, reduced energy expenditure, and caused weight gain. Cumulatively, these studies demonstrate that SEMA3-mediated signaling drives the development of hypothalamic melanocortin circuits involved in energy homeostasis.

The DEAD-box RNA helicase Ddx39ab is essential for myocyte and lens development in zebrafish.

RNA helicases from the DEAD-box family are found in almost all organisms and have important roles in RNA metabolism, including RNA synthesis, processing and degradation. The function and mechanism of action of most of these helicases in animal development and human disease remain largely unexplored. In a zebrafish mutagenesis screen to identify genes essential for heart development we identified a mutant that disrupts the gene encoding the RNA helicase DEAD-box39ab (ddx39ab). Homozygous ddx39ab mutant embryos exhibit profound cardiac and trunk muscle dystrophy, along with lens abnormalities, caused by abrupt terminal differentiation of cardiomyocyte, myoblast and lens fiber cells. Loss of ddx39ab hindered splicing of mRNAs encoding epigenetic regulatory factors, including members of the KMT2 gene family, leading to misregulation of structural gene expression in cardiomyocyte, myoblast and lens fiber cells. Taken together, these results show that Ddx39ab plays an essential role in establishment of the proper epigenetic status during differentiation of multiple cell lineages.

The Sec domain protein Scfd1 facilitates trafficking of ECM components during chondrogenesis

Chondrogenesis in the developing skeleton requires transformation of chondrocytes from a simple mesenchymal condensation to cells with a highly enriched extracellular matrix (ECM). This transition is in part accomplished by alterations in the chondrocyte protein transport machinery to cope with both the increased amount and large size of ECM components. In a zebrafish mutagenesis screen to identify genes essential for cartilage development, we uncovered a mutant that disrupts the gene encoding Sec1 family domain containing 1 (scfd1). Homozygous scfd1 mutant embryos exhibit a profound craniofacial abnormality caused by a failure of chondrogenesis. Loss of scfd1 was found to hinder ER to Golgi transport of ECM proteins and is accompanied with activation of the unfolded protein response in chondrocytes. We further demonstrate a conserved role for Scfd1 in differentiation of mammalian chondrocytes, in which loss of either SCFD1 or STX18, a SLY1 interacting t-SNARE, severely impair transport of type II collagen. These results show that the existence of a specific export pathway, mediated by a complex containing SCFD1 and STX18 that plays an essential role in secretion of large ECM proteins during chondrogenesis.

Defects of the Glycinergic Synapse in Zebrafish.

Glycine mediates fast inhibitory synaptic transmission. Physiological importance of the glycinergic synapse is well established in the brainstem and the spinal cord. In humans, the loss of glycinergic function in the spinal cord and brainstem leads to hyperekplexia, which is characterized by an excess startle reflex to sudden acoustic or tactile stimulation. In addition, glycinergic synapses in this region are also involved in the regulation of respiration and locomotion, and in the nociceptive processing. The importance of the glycinergic synapse is conserved across vertebrate species. A teleost fish, the zebrafish, offers several advantages as a vertebrate model for research of glycinergic synapse. Mutagenesis screens in zebrafish have isolated two motor defective mutants that have pathogenic mutations in glycinergic synaptic transmission: bandoneon (beo) and shocked (sho). Beo mutants have a loss-of-function mutation of glycine receptor (GlyR) β-subunit b, alternatively, sho mutant is a glycinergic transporter 1 (GlyT1) defective mutant. These mutants are useful animal models for understanding of glycinergic synaptic transmission and for identification of novel therapeutic agents for human diseases arising from defect in glycinergic transmission, such as hyperekplexia or glycine encephalopathy. Recent advances in techniques for genome editing and for imaging and manipulating of a molecule or a physiological process make zebrafish more attractive model. In this review, we describe the glycinergic defective zebrafish mutants and the technical advances in both forward and reverse genetic approaches as well as in vivo visualization and manipulation approaches for the study of the glycinergic synapse in zebrafish.

Kinesin family member 6 (kif6) is necessary for spine development in zebrafish.

Idiopathic scoliosis is a form of spinal deformity that affects 2-3% of children and results in curvature of the spine without structural defects of the vertebral units. The pathogenesis of idiopathic scoliosis remains poorly understood, in part due to the lack of a relevant animal model. We performed a forward mutagenesis screen in zebrafish to identify new models for idiopathic scoliosis. We isolated a recessive zebrafish mutant, called skolios, which develops isolated spinal curvature that arises independent of vertebral malformations. Using meiotic mapping and whole genome sequencing, we identified a nonsense mutation in kinesin family member 6 (kif6(gw326) ) unique to skolios mutants. Three additional kif6 frameshift alleles (gw327, gw328, gw329) were generated with transcription activator-like effector nucleases (TALENs). Zebrafish homozygous or compound heterozygous for kif6 frameshift mutations developed a scoliosis phenotype indistinguishable from skolios mutants, confirming that skolios is caused by the loss of kif6. Although kif6 may play a role in cilia, no evidence for cilia dysfunction was seen in kif6(gw326) mutants. Overall, these findings demonstrate a novel role for kif6 in spinal development and identify a new candidate gene for human idiopathic scoliosis.

An ENU Mutagenesis Screen in Zebrafish for Visual System Mutants Identifies a Novel Splice-Acceptor Site Mutation inpatched2that Results in Colobomas

To identify recessive mutations affecting development and/or maintenance of the zebrafish visual system. A three-generation ENU (N-Nitroso-N-ethylurea)-based forward genetic screen was performed. F3 embryos were screened visually from 1 to 5 days postfertilization (dpf) for ocular abnormalities, and 5 dpf embryos were fixed and processed for cryosectioning, after which eye sections were screened for defects in cellular organization within the retina, lens, and cornea. A combination of PCR and DNA sequencing, in situ hybridization, and pharmacological treatments were used to clone and characterize a coloboma mutant. A total of 126 F2 families were screened, and, from these, 18 recessive mutations were identified that affected eye development. Phenotypes included lens malformations and cataracts, photoreceptor defects, oculocutaneous albinism, microphthalmia, and colobomas. Analysis of one such coloboma mutant, uta(1), identified a splice-acceptor mutation in the patched2 gene that resulted in an in-frame deletion of 19 amino acids that are predicted to contribute to the first extracellular loop of Patched2. ptch2(uta1) mutants possessed elevated Hedgehog (Hh) pathway activity, and blocking the Hh pathway with cyclopamine prevented colobomas in ptch2(uta1) mutant embryos. We have identified 18 recessive mutations affecting development of the zebrafish visual system and we have characterized a novel splice-acceptor site mutation in patched2 that results in enhanced Hh pathway activity and colobomas.

G.P.3 Modeling congenital muscle diseases in zebrafish

Primary inherited disorders of muscle include both dystrophies and non-dystrophic congenital myopathies. These disorders, characterized by muscle weakness and impaired locomotion, form a heterogeneous group of hereditary diseases affecting both children and adults. Their complexity is highlighted by occasional involvement of associated symptoms in brain, eyes, heart or respiratory system. Owing to disease heterogeneity, variable penetrance, and mortality, genetic studies to determine the basis for these conditions in humans are often problematic. The zebrafish, Danio rerio, is a powerful developmental and genetic model system for dissection of skeletal muscle disorders and diseases. Several features of zebrafish, such as high degrees of synteny and sequence homology, conserved structure of skeletal muscles, transparency at embryonic stages, and rapid ex vivo development, make them ideal for skeletal muscle biology research. To identify novel genes causing muscular disorders, we conducted an ENU mutagenesis screen in zebrafish that led to the identification of 12 unique neuromuscular mutants. One of these represents a model of the dystroglycanopathies (dag1) and another, of congenital muscular dystrophy (lama2). Another mutant identified in this screen, osoi, displays impaired motility behavior and skeletal muscle myotonia. These mutant fish show skeletal muscle hypotrophy with extensive central nucleation. Ultrastructural studies revealed sarcomeric disorganization in osoi fish. Genetic mapping of the osoi locus has identified a loss of function mutation in a novel RNA binding protein implicated in RNA splicing. Preliminary studies point towards a role for this gene in the disease pathogenesis of myotonic dystrophy. Through an understanding of the abnormal skeletal muscle molecular pathways in osoi fish, we can better understand human neuromuscular diseases and begin to develop corrective therapies.

Macrophage‐stimulating protein and calcium homeostasis in zebrafish

To systematically identify novel gene functions essential for osteogenesis and skeletal mineralization, we performed a forward genetic mutagenesis screen in zebrafish and isolated a mutant that showed delayed skeletal mineralization. Analysis of the mutant phenotype in an osterix:nuclear-GFP transgenic background demonstrated that mutants contain osterix-expressing osteoblasts comparable to wild-type embryos. Positional cloning revealed a premature stop mutation in the macrophage-stimulating protein (msp) gene, predicted to result in a biologically inactive protein. Analysis of the embryonic expression pattern for the receptor for Msp, Ron, shows specific expression in the corpuscles of Stannius, a teleost-specific organ that produces stanniocalcin, a pivotal hormone in fish calcium homeostasis. Knockdown of Ron resulted in identical phenotypes as observed in msp mutants. Msp mutant embryos could be rescued by excess calcium. Consistent with a role for Msp/Ron in calcium homeostasis, calcium-regulating factors, such as pth1, pth2, stc1l, and trpv5/6 were significantly affected in msp mutant larvae. While Msp and Ron have previously been shown to play a critical role in a wide variety of biological processes, we introduce here the Msp/Ron signaling axis as a previously unappreciated player in calcium homeostasis and embryonic skeletal mineralization.

Incomplete splicing, cell division defects, and hematopoietic blockage in dhx8 mutant zebrafish.

Vertebrate hematopoiesis is a complex developmental process that is controlled by genes in diverse pathways. To identify novel genes involved in early hematopoiesis, we conducted an ENU (N-ethyl-N-nitrosourea) mutagenesis screen in zebrafish. The mummy (mmy) line was investigated because of its multiple hematopoietic defects. Homozygous mmy embryos lacked circulating blood cell types and were dead by 30 hr post-fertilization (hpf). The mmy mutants did not express myeloid markers and had significantly decreased expression of progenitor and erythroid markers in primitive hematopoiesis. Through positional cloning, we identified a truncation mutation in dhx8 in the mmy fish. dhx8 is the zebrafish ortholog of the yeast splicing factor prp22, which is a DEAH-box RNA helicase. mmy mutants had splicing defects in many genes, including several hematopoietic genes. mmy embryos also showed cell division defects as characterized by disorganized mitotic spindles and formation of multiple spindle poles in mitotic cells. These cell division defects were confirmed by DHX8 knockdown in HeLa cells. Together, our results confirm that dhx8 is involved in mRNA splicing and suggest that it is also important for cell division during mitosis. This is the first vertebrate model for dhx8, whose function is essential for primitive hematopoiesis in developing embryos.

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML

AbstractIn a zebrafish mutagenesis screen to identify genes essential for myelopoiesis, we identified an insertional allele hi1727, which disrupts the gene encoding RNA helicase dead-box 18 (Ddx18). Homozygous Ddx18 mutant embryos exhibit a profound loss of myeloid and erythroid cells along with cardiovascular abnormalities and reduced size. These mutants also display prominent apoptosis and a G1 cell-cycle arrest. Loss of p53, but not Bcl-xl overexpression, rescues myeloid cells to normal levels, suggesting that the hematopoietic defect is because of p53-dependent G1 cell-cycle arrest. We then sequenced primary samples from 262 patients with myeloid malignancies because genes essential for myelopoiesis are often mutated in human leukemias. We identified 4 nonsynonymous sequence variants (NSVs) of DDX18 in acute myeloid leukemia (AML) patient samples. RNA encoding wild-type DDX18 and 3 NSVs rescued the hematopoietic defect, indicating normal DDX18 activity. RNA encoding one mutation, DDX18-E76del, was unable to rescue hematopoiesis, and resulted in reduced myeloid cell numbers in ddx18hi1727/+ embryos, indicating this NSV likely functions as a dominant-negative allele. These studies demonstrate the use of the zebrafish as a robust in vivo system for assessing the function of genes mutated in AML, which will become increasingly important as more sequence variants are identified by next-generation resequencing technologies.

Cpsf1 is required for definitive HSC survival in zebrafish

AbstractA comprehensive understanding of the genes and pathways regulating hematopoiesis is needed to identify genes causally related to bone marrow failure syndromes, myelodysplastic syndromes, and hematopoietic neoplasms. To identify novel genes involved in hematopoiesis, we performed an ethyl-nitrosourea mutagenesis screen in zebrafish (Danio rerio) to search for mutants with defective definitive hematopoiesis. We report the recovery and analysis of the grechetto mutant, which harbors an inactivating mutation in cleavage and polyadenylation specificity factor 1 (cpsf1), a gene ubiquitously expressed and required for 3′ untranslated region processing of a subset of pre-mRNAs. grechetto mutants undergo normal primitive hematopoiesis and specify appropriate numbers of definitive HSCs at 36 hours postfertilization. However, when HSCs migrate to the caudal hematopoietic tissue at 3 days postfertilization, their numbers start decreasing as a result of apoptotic cell death. Consistent with Cpsf1 function, c-myb:EGFP+ cells in grechetto mutants also show defective polyadenylation of snrnp70, a gene required for HSC development. By 5 days postfertilization, definitive hematopoiesis is compromised and severely decreased blood cell numbers are observed across the myeloid, erythroid, and lymphoid cell lineages. These studies show that cpsf1 is essential for HSC survival and differentiation in caudal hematopoietic tissue.

Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice

AbstractHeterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow–transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit+/lineage−/Sca-1+ (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.

Zebrafish muscular disease models towards drug discovery

Background: Zebrafish is an amenable vertebrate model useful for the study of development and genetics. Small molecule screenings in zebrafish have successfully identified several drugs that affect developmental process. Objective: This review covers the basics of zebrafish muscle system such as muscle development and muscle defects. It also reviews the potential use of zebrafish for chemical screening with regards to muscle disorders. Conclusion: During embryogenesis, zebrafish start to coil their body by contracting trunk muscles 17 h postfertilization, indicating that a motor circuit and skeletal muscle are functionally developed at early stages. Mutagenesis screens in zebrafish have identified many motility mutants that display morphological or functional defects in the CNS, clustering defects of acetylcholine receptors at the neuromuscular junctions or pathological defects of muscles. Most of the muscular mutants are useful as animal models of human muscle disease such as muscle dystrophy. As zebrafish live in water, pharmacological drugs are easily assayable during development, and thus zebrafish may be used to determine novel drugs that mitigate muscle disease.

Three Heritable T Cell Malignancy Models Established Using a Forward Genetic Mutagenesis Screen in Zebrafish.

T cell cancers are common in pediatric oncology, and include the clinical entities T cell acute lymphoblastic leukemia (T-ALL) and the Non-Hodgkin Lymphoma, T cell lymphoblastic lymphoma (T-LBL). These diseases carry worse prognoses than their B cell counterparts, and the regimens used in their treatment confer significant short- and long-term morbidities. Unlike many pediatric malignancies, which have characteristic chromosomal translocations, most T cell-derived cancers carry no such cytogenetic hallmark. Because such straightforward oncogenic lesions are typically absent, insights into the genetic underpinnings and molecular pathogenesis of these T cell diseases are also less comprehensive. To address these deficiencies, we have performed a forward genetic screen for T cell malignancies using the vertebrate model organism, zebrafish (Danio rerio). Although others have used transgenic zebrafish to show that known mammalian oncogenes can induce zebrafish leukemias, to date, this powerful animal model has not been used as a discovery tool to elucidate novel genetic mutations underlying de novo T cell malignancies. Zebrafish share ontogeny with other vertebrates, and possess an adaptive immune system strikingly similar to mammals. However, unlike other vertebrate models, zebrafish are amenable to forward genetic approaches, where animals are randomly mutagenized, and then screened using high-throughput strategies to detect rarely-occurring phenotypes. Using a zebrafish line with T lymphocyte-specific expression of green fluorescent protein (GFP), we have conducted a germline mutagenesis screen to identify animals that develop T cell malignancy. Thus far, we have isolated three independent lines with a heritable predisposition to developing T cell cancers. In all three lines, age of onset, patterns of spread, and morphologic appearance resemble human T-ALL/-LBL. In addition, we have compiled histologic and immunohistochemical evidence further characterizing these diseases. Analysis of T cell receptor (TCR) β gene sequences from malignant tissue has verified the monoclonality of malignant cells, based on identical VDJ rearrangements. Malignant cells have also been successfully transplanted serially into sub-lethally irradiated allogeneic recipients, using as few as 2500 donor cells to transfer disease. Furthermore, we have shown the malignancies themselves to be radiation sensitive, like their human correlates. Soon after radiation-induced remissions are achieved, however, high rates of aggressive disease relapse are seen, modeling recurrent human T-ALL/-LBL. In summary, we have identified three independent zebrafish mutant lines, which each recapitulate human T lymphocyte malignancy, and that are all heritably transmissible from one generation to the next. The positional cloning of the underlying genetic mutation of each line will provide exciting candidates for study in these human diseases. Moreover, using these lines as subjects in ongoing investigations, we are examining the genomic, epigenetic, and transcriptional profiles of these malignant cell populations, comparing pre- and post-transplant samples, pre- and post-irradiation isolates, and following other accepted and investigational therapeutic interventions. Finally, these lines are now poised for utilization in additional high-throughput screening approaches, including forward genetic suppressor and enhancer mutagenesis projects, as well as small molecule drug screens. Collectively, these new vertebrate models of T-ALL/-LBL should provide vital experimental platforms for studies of this important class of human cancers.

The Role of RNA Helicase Dead Box 18 in Zebrafish Hematopoiesis and Human MDS

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.

A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP) s843 transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.