A nonsense mutation in zebrafish gata1 causes the bloodless phenotype in vlad tepes

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 3 T (Arg-339 3 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.

LSD1/KDM1A promotes hematopoietic commitment of hemangioblasts through downregulation of Etv2

“…For genotyping lsd1 it627 , genomic DNA at the mutation sites was amplified by PCR using the primers 5′-ctggcagaggagggacaaactgcacggccggcggcagct and 5′-gcagtcatctgactgtgcg, and was digested with PvuII. Genotyping of gata1 m651 was carried out as described previously (16). Tranylcypromine was purchased from Enzo Life Sciences.…”

“…Gata1 encodes a transcription factor that transactivates a variety of erythropoietic genes. Gata1 gene KO mice and mutant zebrafish have been demonstrated to be lethally anemic because of drastic defects in erythropoiesis, and Gata1 has been considered to be a key factor required for erythropoiesis (14)(15)(16). Interestingly, the expression of Gata1 appears to define the hematopoietic commitment stage, because Gata1-positive cells display hematopoietic, but not endothelial, differentiation potential (17).…”

“…In lsd1 it627 embryos, the gata1 expression at 12 hpf was similar to the gata1 expression in WT siblings, but its up-regulation after 13 hpf was impaired, suggesting that LSD1 is not required for the initiation of gata1 but is required for its up-regulation. To examine whether defects of gata1 up-regulation by the lsd1 mutation affect the expression of Gata1 downstream genes (16,32,33), the expression of hbbe1, alas2, and klfd was analyzed and demonstrated to be greatly reduced in lsd1 it627 embryos ( Fig. 2A).…”

Modeling human hematopoietic and cardiovascular diseases in zebrafish

“…There are both primitive and definitive waves of differentiation in the zebrafish, which produce primitive erythrocytes and macrophages, followed by definitive erthyrocytes, B cells, T cells, monocytes, granulocytes, and thrombocytes, respectively (Orkin and Zon, 1997;Trede and Zon, 1998;Herbomel et al, 1999;Willett et al, 1999;Bennett et al, 2001;Lieschke et al, 2001). Many zebrafish orthologs of blood-specific genes demonstrated to be important in mouse and human development have been isolated, including cmyb, gata1, gata2, globin, hhex, ikaros, lmo2, pu1, rag1, runx1, scl, and vegf (Hansen et al, 1997;Willett et al, 1997;Gering et al, 1998;Liao et al, 2000a;Liang et al, 2001;Burns et al, 2002;Lyons et al, 2002;Brownlie et al, 2003). Although the genetic program appears to be highly conserved during hematopoietic development in the vertebrate embryo, the site of hematopoiesis is not as consistent.…”

N- and C-terminal Transactivation Domains of GATA1 Protein Coordinate Hematopoietic Program

“…In addition, this region is phylogenetically conserved in the human and mouse. On the contrary, it has been reported that the truncated type of GATA1 lacking the C-terminal region could not sustain erythropoiesis in zebrafish mutant Vlad tepes (27). A nonsense mutation at codon 339 in zebrafish GATA1 gene gave rise to massive deletion of GATA1 from the C-finger tail to the C terminus (Fig.…”