A nonsense mutation in zebrafish <i>gata1</i> causes the bloodless phenotype in <i>vlad tepes</i>

Abstract

Vlad tepes ( vlt m651 ) is one of only five “bloodless” zebrafish mutants isolated through large-scale chemical mutagenesis screening. It is characterized by a severe reduction in blood cell progenitors and few or no blood cells at the onset of circulation. We now report characterization of the mutant phenotype and the identification of the gene mutated in vlt m651 . Embryos homozygous for the vlt m651 mutation had normal expression of hematopoietic stem cell markers through 24 h postfertilization, as well as normal expression of myeloid and lymphoid markers. Analysis of erythroid development revealed variable expression of erythroid markers. Through positional and candidate gene cloning approaches we identified a nonsense mutation in the gata1 gene, 1015C → T (Arg-339 → Stop), in vlt m651 . The nonsense mutation was located C-terminal to the two zinc fingers and resulted in a truncated protein that was unable to bind DNA or mediate GATA-specific transactivation. A BAC clone containing the zebrafish gata1 gene was able to rescue the bloodless phenotype in vlt m651 . These results show that the vlt m651 mutation is a previously uncharacterized gata1 allele in the zebrafish. The vlt m651 mutation sheds new light on Gata1 structure and function in vivo , demonstrates that Gata1 plays an essential role in zebrafish hematopoiesis with significant conservation of function between mammals and zebrafish, and offers a powerful tool for future studies of the hematopoietic pathway.