Hematopoietic stem cells are derived from totipotent embryonic mesodermal cells upon induction by spatially-localized signals. Although growth factors involved in hematopoietic progenitor maturation have been extensively studied, the molecular mechanisms responsible for the commitment of embryonic stem cells to hematopoiesis are poorly understood. Xenopus is an ideal organism to study hematopoiesis since development has been well characterized, fate maps (of the 32 cell embryo) have been constructed, multipotential embryonic stem cells can be easily obtained from embryos before blood island formation, and hematopoietic maturation occurs in a synchronous wave within 36 hours after fertilization. Whole embryo in situ analysis has demonstrated that the GATA-binding proteins and SCL are excellent early markers of the hematopoietic program and are initially expressed during gastrulation at 11 hours post-fertilization. Based on the fact that zygotic transcription starts at 7 hours of development, the induction of the hematopoietic stem cell is likely to occur between 7 and 11 hours. In an expression cloning screen for inducers of hematopoiesis that are expressed during this time period in the ventral axis of the Xenopus embryo, we isolated and characterized a homeobox gene, Mix. 1, that participates in early hematopoietic induction. In this proposal, six other positive cDNA pools will be sib-selected to obtain factors that cause excessive blood formation in whole embryos. Using a variety of assays, we plan to determine the role of these new factors and their relationship to BMP4, wnt, and FGF signaling cascades during the induction of blood. The function of these cascades and hematopoietic transcription factors will be studied by determining the effect of overexpression and dominant negative mutants on blood development in whole embryos and in embryonic explant assays. A novel plasmid-based expression cloning screen will be used to isolate cDNAs that directly induce hematopoietic stem cells from undifferentiated mesoderm. The proposed study will provide new insights into the biology of hematopoiesis in both normal and pathologic states such as anemia and hemoglobinopathies, and will be useful for bone marrow transplantation and gene therapy.
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