Generation of Zebrafish Lines with New Gata1 Mutations and Their Characterization with a Novel In Vitro Colony-Forming Assay.

Abstract Gata1 is a transcription factor critical for erythroid and megakaryocyte differentiation. We have shown previously that the lethal bloodless phenotype of the zebrafish mutant vlad tepes (vlt) is due to a nonsense mutation in gata1, suggesting that hematopoietic regulation is conserved between zebrafish and mammals. We have now generated fish with 2 novel missense mutations, T301K and K333R, identified by sequencing exons 5 and 6 in 1235 F1 fish derived from ENU-mutagenized males. Both mutations change conserved residues in the C-terminal zinc finger domain of gata1. Embryos homozygous for either T301K or K333R mutation showed normal circulation and survived to adulthood. Reduced hemoglobin levels were observed in T301K homozygous embryos compared to K333R homozygotes and wildtype by o-dianisidine staining. We crossed both mutations to vlt carrier fish and generated compound heterozygotes for further evaluation. T301K/vlt compound heterozygous embryos lacked circulation and hemoglobin staining, whereas K333R/vlt embryos had normal circulation and hemoglobin staining. These data suggest that the T301K mutation acts as a hypomorphic allele, having stronger phenotype in the presence of a null allele. Fish with both T301K/vlt and K333R/vlt genotypes survive to adulthood in expected Mendelian ratios. Time course observations show that T301K/vlt fish regain circulation around day 14, which is the time when most vlt/vlt fish die. These data suggest that during definitive hematopoiesis cells may be less sensitive to gata1 deficiency. In addition, we observed reduced number of circulating platelets in vlt/vlt embryos but normal in T301K/vlt embryos, suggesting that megakaryocyte maturation is regulated by gata1 in zebrafish. The in vitro culture of hematopoietic progenitors has been a powerful tool to study mammalian hematopoiesis, but similar techniques have not been available in the zebrafish. We hypothesized that the use of the zebrafish orthologs of Stem Cell Factor (scf) and Erythropoietin (epo) in semi soild medium would allow the growth and enumeration of colonies derived from zebrafish kidney cells. Based on homology to mammalian proteins, we identified zebrafish scf and epo cDNA clones (40.8% and 49.7% similarities to human SCF and EPO respectively), and expressed them in 293 cells. Methylcellulose medium containing conditioned medium from transfected cells was mixed with 105 to 106 adult kidney cells. No colonies developed in cultures with mock transfected 293 conditioned medium. In contrast, small erythroid colonies appeared between 2 to 6 days in cultures containing epo and/or scf conditioned medium. Larger erythroid colonies were detected in 8 to12 days. In the presence of scf, additional distinct colonies comprising of monocytes, neutrophils and/or erythroid cells were observed. Cultures of adult kidney cells from T301K/vlt and T301K/T301K fish showed 2–6 fold reduction (p=0.01 and 0.006 respectively) in the number of colonies. These results are consistent with the reduced number of hematopoietic cells observed in kidney sections of adult T301K homozygotes. In conclusion, we have generated gata1 mutant fish that revealed a conserved role of gata1 in zebrafish erythroid and megakaryocyte developments. The viable gata1 mutants and our novel in vitro differentiation system will be useful for studying the role of gata1 in adult hematopoiesis and in leukemogenesis. The mutants will also serve as an ideal system for genetic modifier screens.