A Genetic Screen for Zebrafish Mutants Affecting Early Hematopoiesis.

Hematopoiesis, or blood cell formation, starts from hematopoietic stem cells and goes through many rounds of cell proliferation and a number of commitment steps that progressively restrict cells to a particular lineage. The generation and maintenance of stem cell pool and the regulation of lineage commitment are poorly understood. Right now there is only one reported zebrafish mutant, cloche, that has defects in early hematopoiesis. We have undertaken a chemical mutagenesis screen using ENU in the zebrafish to uncover mutants in early hematopoiesis. Haploid embryos from F1 females were screened by RNA in situ hybridization using a stem cell marker, core binding factor ? (cbfb), and a myeloid-specific marker, leukocyte-specific plastin (l-plastin). We have screened 510 F1 females and 47 putative mutants have been identified. Of these, 22 showed decreased expression of l-plastin, 3 for cbfb, 2 for both l-plastin and cbfb and the remainder were mutated for l-plastin and other non-hematopoietic markers. Right now, we have focused our efforts on two mutants.

The first mutant line, L32, displayed a blood-less phenotype and showed a significant decrease in the expression of the hemangioblast marker scl. In addition to the marked reduction in the expression of the myeloid specific markers l-plastin, c/ebp1 and mpo, these embryos also showed decreased expression of the erythroid specific marker gata-1. Linkage analysis has placed this mutant to the proximal region of linkage group 12. Currently, we are in the process of testing candidate genes to identify the mutated gene. The second mutant line, L63 also has a blood less phenotype and showed decreased expression of both l-plastin and mpo. Like L32, L63 homozygous embryos have significant developmental defects and die 3 dpf. Low resolution mapping has placed this mutant to LG 8. Through this approach, we hope to identify novel genes that play critical roles in the developmental process regulating early hematopoiesis.