The induction of hematopoietic stem cells from undifferentiated mesoderm occurs during gastrula stages of embryonic development. Few studies have examined the factors that regulate this process, largely due to an inability to obtain tissue samples from this developmental period in higher vertebrate species. The zebrafish is an ideal organism to study hematopoietic mesoderm induction. The organism is transparent, developmental stages are well-characterized, and genetic mutants for embryonic blood formation can be obtained. We have isolated cDNAs encoding zebrafish GATA-1 and GATA-2, and have studied their expression pattern during embryogenesis. Zebrafish hematopoietic progenitors that express GATA-1 and GATA-2 initially occupy an extra-embryonic position on the yolk sac, similar to higher vertebrates. As development proceeds, these cells enter the embryo and form a distinct structure known as the hematopoietic intermediate cell mass. Two bloodless mutations in the zebrafish, bloodless and spadetail, have a deficiency in ability to express GATA-1 and GATA-2 during embryonic hematopoiesis. The bloodless mutation was obtained in an enhancer trap screen in which the lacZ gene integrated at the locus. In this proposal, the gene encoding bloodless will be isolated by cloning the integration site. The regulation and function of the bloodless gene will be studied with respect to hematopoietic mesoderm induction. The isolation of additional bloodless mutations and a genetic epistasis analysis will determine individual steps required for normal hematopoietic induction. Ultimately, the mammalian homologues of important regulatory genes will be isolated. The outcome of these studies may lead to an enhanced ability to manipulate the hematopoietic stem cell in vitro, which would be useful for bone marrow transplantation and gene therapy.
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