Embryonic hematopoietic stem cells are derived from totipotent stem cells through activation of a coordinated cascade of spatially-expressed inductive, proliferative and differentiating signals. Although growth factors involved in hematopoietic progenitor maturation have been extensively studied, the mechanisms responsible for the commitment of stem cells to hematopoiesis are poorly understood. The proposed studies are aimed at (1) characterization of the expression and function of hematopoietic transcription factors in defining the embryonic hematopoietic axis; (2) isolation and characterization of the signals involved in hematopoietic stem cell determination. The process of hematopoiesis is well conserved throughout vertebrate evolution. Xenopus is an ideal organism to study hematopoiesis since development has been extensively studied, cell fate maps have been constructed from the 32 cell embryo, multipotential embryonic stem cells can be easily obtained from staged embryos before blood island formation, and hematopoietic maturation occurs rapidly within 36 hours after fertilization. cDNA clones have been isolated which encode the Xenopus homologs of transcription factors expressed early in the hematopoietic program including GATA-1, 2, and 3, and SCL. In situ analysis of whole embryos has demonstrated that GATA-1 and 2 are expressed in the ventral region before blood is histologically evident, indicating that the GATA-binding proteins are involved in the determination of the embryonic hematopoietic axis. By studying the expression of these DNA-binding proteins in multipotential stem cells incubated with factors which affect mesoderm induction, we hope to define the environmental conditions necessary for hematopoiesis. Our results to date indicate that a signal derived from the ventral region of the embryo functions to induce hematopoietic development and maintain GATA-1 expression. This signal does not appear to be a known mesoderm inducing factor such as activin or fibroblast growth factor. This early hematopoietic signal will be isolated and characterized. The proposed study will provide new insights into the developmental biology of hematopoiesis in both normal and pathologic states such as anemia and hemoglobinopathies.
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