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Abstract

Vertebrate blood originates from self-renewing hematopoietic stem cells (HSC) and their subsequent differentiation into all blood lineages. HSCs are derived from a bipotential precursor, the he- mangioblast, which has the potential to give rise to both blood and blood vessels. The known components of the molecular pathway of ventral mesoderm induction and subsequent HSC induction are conserved in all vertebrates. The molecules involved in the induc- tion of HSC, however, are largely unknown. Previous genetic screens in zebrafish identified only very few mutations affecting hemangioblast specification and differentiation. We have therefore undertaken a large-scale mutagenesis screen to genetically charac- terize HSC specification and early hematopoietic development. To date, we screened 2022 genome equivalents for mutations with altered expression pattern of the earliest blood-specific marker scl (stem cell leukemia factor) using RNA in situ hybridization. Thirty-two potential mutants were isolated which can be grouped into several classes: reduced or increased scl expression, ectopic scl expression, or mutants affecting the ventral mesoderm patterning which will ultimately give rise to the hemangioblast. The mutant HV001 shows reduced scl staining at the 5-somite stage when blood and endothelial precursors are still intermingled. At the 18-somite stage the endothelial-specific expression pattern of scl is absent, suggesting that this mutation specifically affects the endo- thelial development of the hemangioblast. Surprisingly, we have not been able to isolate mutants that lack scl expression com- pletely. Additionally, we screened 3452 genome equivalents for mutations with aberrant blood morphology at day 4 of embryonic development, followed by benzidine staining for hemoglobin, and identified 220 potential mutants. The majority of these are blood- less, and some have reduced amounts of erythrocytes or lack of hemoglobin production. Complementation analysis by mapping and single-pair matings will put the mutants in context to each other and to previously isolated mutants. The analysis of the hematopoietic mutants will ultimately define a genetic pathway leading to the specification of the hematopoietic lineage in zebrafish.