During this fiscal year, we have worked on three separate projects: A. Novel zebrafish runx1 mutant Recent studies showed that hemogenic endothelial cells in the aortic-gonado-mesonephros (AGM) region of the developing mouse embryo give rise to most fetal liver and bone marrow hematopoietic cells44,45. In addition, the earliest step of hematopoiesis, the emergence of hematopoietic stem cells (HSC) from the hemogenic endothelium, is controlled by Runx145,46. However, it is not clear whether Runx1 is required for the emergence of all definitive HSCs, both in the embryos and in the adult bone marrow. Through TILLING14, we developed a zebrafish line with a truncation mutation (W84X) in the runx1 gene (Paper #3)10. Characterization of this line showed that fetal (or larval in zebrafish) and adult hematopoiesis share a common stem cell pool, but runx1 is only required for the initiation of larval hematopoiesis. The embryos of the runx1W84X/W84X fish had complete block of larval (equivalent to fetal liver) hematopoiesis and 80% of the runx1W84X/W84X larvae died due to their bloodless phenotype. Strikingly, 20% runx1W84X/W84X larvae resumed blood circulation and grew up to fertile adults with multi-lineage adult hematopoiesis. We crossed the runx1W84X/W84X fish with a line with cd41 promoter driven GFP, Tg(cd41-GFP), which is expressed in both thrombocytes and hematopoietic stem cells47-49. The cd41-GFP+ hematopoietic stem cells were found to be retained in the Tg(cd41-GFP), runx1W84X/W84X embryos and migrated from the sites for larval hematopoiesis to the kidney, where adult hematopoiesis takes place. These findings suggest that even though fetal and adult hematopoiesis share common cd41-GFP+ precursors, their initiation requires distinct regulatory programs (Sood et al., submitted). B. Novel zebrafish gata1 mutants In vertebrates, hematopoiesis is separated into primitive and definitive stages, which take place at distinct locations. Previous studies showed that gata1 is required for the development of erythrocytes and megakaryocytes50, but it is not clear if it is equally required at all stages of hematopoiesis and whether its DNA binding domain in the C-finger is important. To answer these questions we analyzed zebrafish with mutant gata1 alleles that led to either reduced (gata1T301K, a novel mutation that we isolated by TILLING) or complete loss of (gata1R339X, also known as vlad tepes, or vlt) DNA binding and transactivation activities. As we reported before, gata1vlt/vlt embryos were bloodless and died around 11-15 days post fertilization (dpf)40. On the other hand, gata1T301K/T301K fish had essentially normal hematopoiesis. Interestingly the compound heterozygous gata1T301K/vlt embryos had nearly complete block of primitive hematopoiesis, but they resumed hematopoiesis between 7-14 dpf and grew to phenotypically normal fish with normal adult hematopoiesis. Our findings suggest that the impact of Gata1 on hematopoiesis correlates with its DNA-binding ability and that primitive hematopoiesis is more sensitive to reduction of Gata1 function than definitive hematopoiesis (Belele et al., Blood, in revision). C. Using zebrafish embryos as a model to test novel chemicals In collaboration with NIH Chemical Genomics Center we have conducted a screen of roughly 1 quarter million chemicals for inhibitors of RUNX1-CBFbeta interaction, which has been demonstrated to be a critical interaction for several types of human acute myeloid leukemia. Over 70 candidate compounds have been identified after the initial screen and subsequent confirmation/validation experiments. We have developed a zebrafish-based assay to determine if the candidate compounds block RUNX1-CBFbeta interaction in vivo. The assay is to incubate transgenic Tg(cd41-GFP) zebrafish embryos with the compounds and observe for hematopoietic defects as reflected by the number of circulating cd41-GFP+ cells, which are thrombocytes generated by larval stage definitive hematopoiesis. Circulating cd41-GFP+ cells are expected to decrease in the embryos if Runx1 function is blocked by the candidate compounds, since Runx1 is required for the larval stage definitive hematopoiesis (see Project A). Preliminary data showed that one of the 10 candidate compounds tested were able to reduce circulating cd41-GFP+ cells in the embryos, which essentially phenocopied the runx1W84X/W84X embryos (Project A), while otherwise the embryos looked normal.
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