During this fiscal year, we have worked on three separate projects:? ? 1. Characterization of a runx1 knockout zebrafish model? ? Traditional and conditional knockout mouse models have shown that Runx1 is absolutely necessary for the emergence of hematopoietic stem cells (HSCs) during embryogenesis but not for their maintenance and differentiation during adult hematopoiesis. However, its role in the initiation of adult HSCs can not be addressed due to the embryonic lethality of the knockout and the presence of Runx1 during the critical embryonic period in the conditional knockout animals. Zebrafish provide an alternate model system to study hematopoiesis due to their optical clarity and ability to perform genetic screens. By TILLING (Targeting Induced Local Lesions IN Genomes) we identified a truncation mutation, W84X, in zebrafish runx1 gene. Homozygous null embryos develop normal primitive hematopoiesis but no definitive hematopoiesis during the larval stage. In contrast to the embryonic lethality of knockout mice, 20% of runx1 null zebrafish embryos survive to adulthood and sustain multilineage adult hematopoiesis. These data show that as suggested recently in mammals, definitive hematopoiesis in zebrafish also occurs in two stages: larval (equivalent to fetal) and adult. Using cd41-GFP, we demonstrate that HSCs initiate and migrate to the appropriate locations in the absence of runx1. This was a surprising finding and would not have been uncovered in the mouse models due to their stringent requirement for blood circulation during embryogenesis. ? ? 2. Truncation mutation of dhx8, a member of the DEAH family of RNA helicases, results in cell cycle and hematopoietic defects in zebrafish embryos? Embryonic hematopoiesis in both zebrafish and mammals is a tightly regulated process that is coordinated by a series of transcription factors. In order to identify novel genes required for embryonic hematopoiesis, particularly those affecting early stages as well as myelopoiesis, we conducted a whole mount RNA in situ hybridization screen of ENU-mutagenized F2 haploid embryos with antisense probes for cbfb and l-plastin, genes expressed in the hematopoietic stem cells and myeloid lineage cells, respectively. One mutant line, mummy (mmy), was identified from this screen that showed reduced scl, cbfb expression and total lack of l-plastin expression. In addition, mummy mutants have reduced gata1 and hemoglobin expression. As both erythroid and myeloid lineages are affected, these results suggested that the defect in mmy is at or above HSC level. The mmy mutant embryos also suffer from widespread cell death and die around 30 hpf. Genetic mapping and positional cloning have identified the mutated gene as dhx8, an RNA helicase of the DEAH family. Morpholino injection has confirmed that knock-down of dhx8 can produce a similar phenotype in wild-type embryos. The yeast homolog of dhx8, prp22, functions during mRNA splicing. Our results show that mmy mutants are defective in the splicing of some hematopoietic genes. In addition, when dhx8 was knocked-down in HeLa cells using shRNA, cells were observed with a multi-nucleated phenotype with visible signs of chromosome bridges connecting them clearly demonstrating a cell cycle defect. When stained with tubulin anti-body, mmy mutant embryos showed increased cell cycle defects as characterized by disorganized micro-tubules and multiple spindle poles formations. Together, our results suggest that dhx8 is not only involved in mRNA splicing, its expression is also essential for correct cell cycle progression. Loss of dhx8 expression can lead to cytokinesis defects in HeLa cells as well as cell cycle defects in mmy mutant embryos. These observations provide a mechanism by which loss of dhx8 expression can lead to hematopoietic defects and embryonic lethality in zebrafish embryos.? ? 3. Differential requirement for gata1 between primitive and definitive stages of hematopoiesis in the zebrafish? The GATA1 transcription factor plays an essential role in hematopoiesis. Gata1 knockout in mice results in blockage of erythroid and megakaryocytic developments. In humans, mutations in gata1 are associated with anemia and leukemia development in children with Down syndrome. To further investigate GATA1 function in hematopoiesis and leukemogenesis we generated fish carrying gata1 point mutations that led to either reduced (T301K) or complete loss (R339X) of DNA binding. Fish embryos homozygous for the T301K mutation had blood circulation and normal survival rate while embryos homozygous for the R339X mutation were bloodless and died around 11-15 days post fertilization (dpf). Embryos compound-heterozygous for these two mutations (gata1-T301K/R339X) lacked blood circulation initially but recovered circulation between 7-14 dpf, suggesting that primitive hematopoiesis requires higher GATA1 dosages than definitive hematopoiesis. In situ hybridization showed a GATA1 dosage-dependent expansion of myeloid marker l-plastin and the hematopoietic stem cell marker c-myb. Visual observations of gata1-T301K/R339X, gata1-R339X/R339X and wild type embryos carrying the green fluorecent protein (GFP) driven by either the cd41 (expressed in thrombocytes) or gata1 (marking erythroid lineage) promoter showed that the number of thrombocytes is reduced in gata1-T301K/R339X embryos initially and absent in gata1-R339X/R339X embryos, and that gata1 is required for maintenance and differentiation of the erythroid/thromboid progenitors but not for their initiation. FACS analysis of adult gata1-T301K/R339X whole kidney marrow showed an increase of the populations containing precursors and lymphocytes. We are generating a mouse model carrying a Gata1 mutation equivalent to the zebrafish T301K substitution and will use this mouse model to investigate if primitive hematopoiesis in zebrafish and mouse require similar GATA1 dosage.
| Sood, Raman; English, Milton A; Jones, MaryPat et al. (2006) Methods for reverse genetic screening in zebrafish by resequencing and TILLING. Methods 39:220-7 |
| Zhang, Jinghui; Wheeler, David A; Yakub, Imtiaz et al. (2005) SNPdetector: a software tool for sensitive and accurate SNP detection. PLoS Comput Biol 1:e53 |
| Nishikawa, Keizo; Kobayashi, Makoto; Masumi, Atsuko et al. (2003) Self-association of Gata1 enhances transcriptional activity in vivo in zebra fish embryos. Mol Cell Biol 23:8295-305 |
