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

Abstract

In 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 ddx18 hi1727/؉ 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. (Blood. 2011;118(4):903-915)

25 a Zebrafish Model of Myelodysplastic Syndrome Produced Through Tet2 Genomic Editing

“…In the last few years, several mouse models with targeted disruption of Tet2 in the hematopoietic system have been reported, and they have been used to study the role of Tet2-inactivating mutations in normal hematopoietic cell development, the evolution of clonal dominance, and the development of hematopoietic malignancies. Tet2-deficient mice predominantly developed CMML, followed by hematopoietic malignancies, such as MDS and MPN (23)(24)(25)(26). Similar to the results reported in murine models (23)(24)(25)(26), the tet2 m/m zebrafish we studied did not exhibit defects in embryonic hematopoiesis, remained viable and fertile, and developed clonal myelodysplasia of the kidney marrow as they aged, culminating in true MDS with anemia.…”

“…The zebrafish has been shown to provide a faithful model of vertebrate hematopoiesis in which both small-molecule and genetic screens have proven particularly advantageous. For example, small-molecule screens conducted in zebrafish embryos have yielded insights directly relevant to pathways regulating both human HSCs and more differentiated hematopoietic cells, leading to the discovery of drugs that augment engraftment, as shown in clinical trials of cord blood cell transplantation in patients (21)(22)(23)(24)(25)(26)(27)(28)(29)(30).…”

Ddx46 Is Required for Multi-Lineage Differentiation of Hematopoietic Stem Cells in Zebrafish

“…By genetic screening using zebrafish, two DExD/H-box genes, Ddx18 and Dhx8, have been identified thus far as novel genes that are essential for hematopoiesis [18,19]. A recent study has reported that Ddx18 is required for primitive hematopoiesis through the regulation of p53-dependent G1 cell-cycle arrest [18]. Moreover, a sequence variation in human DDX18, which acts as a dominant-negative, was identified in samples from patients with acute myeloid leukemia [18].…”

“…A recent study has reported that Ddx18 is required for primitive hematopoiesis through the regulation of p53-dependent G1 cell-cycle arrest [18]. Moreover, a sequence variation in human DDX18, which acts as a dominant-negative, was identified in samples from patients with acute myeloid leukemia [18]. A more recent report showed that a mutation in Dhx8, a zebrafish orthologue of the yeast splicing factor Prp22, led to defects in cell division, pre-mRNA splicing, and primitive hematopoiesis [19].…”

“…The DExD/H-box RNA helicases are known to function in all aspects of RNA metabolism such as pre-mRNA splicing, rRNA biogenesis, and transcription by using the energy derived from ATP hydrolysis [16,17]. By genetic screening using zebrafish, two DExD/H-box genes, Ddx18 and Dhx8, have been identified thus far as novel genes that are essential for hematopoiesis [18,19]. A recent study has reported that Ddx18 is required for primitive hematopoiesis through the regulation of p53-dependent G1 cell-cycle arrest [18].…”