The long term goal of this project is to elucidate the molecular and cellular events underlining the initiation of hematopoiesis in the mammalian embryo. Mounting evidence suggests that definitive cells arise from pre-liver intra-embryonic progenitors. However, the special localization and developmental origin of these precursors is not known. The P.I. hypothesizes that mesoderm cells become committed to a hematopoietic fate as they migrate through the primitive streak and appose endoderm to form extraembryonic yolk sac and intra-embryonic splanchnopleure. In the first aim of this proposal, hematopoietic development in the embryo proper will be traced by in situ hybridization, using transcription factors integral to primitive (SCL, RBTN-1, GATA-2) and definitive (c-myb, AML-1) hematopoiesis. In the second aim of this proposal, the origin of hematopoietic potential during gastrulation in normal mouse embryos will be delineated. Specifically, the ability of isolated, staged yolk sac and intra-embryonic tissues to give rise to primitive and/or definitive hematopoietic colonies, as well as earlier multipotential progenitors will be examined. To identify other regulators of pre-liver hematopoiesis, the P.I. has isolated homeobox (Hox a 10) and zinc finger (EZF-1) genes from presomite yolk sac mesoderm-enriched c-DNAs. In the third aim of this proposal, the role of these genes in embryonic hematopoiesis will be analyzed. The function of Hox a 10, implicated in normal and malignant myelopoiesis, will be investigated in individually explanted yolk sacs from Hox a 10 minus embryos. EZF-1, a novel zinc finger gene expressed in yolk sac, in fetal liver and in leukemic cell lines, will be further characterized by cloning full length CDNAS and analyzing in vitro function in EZF-1 null ES cells. The P.I. proposed that a better understanding of the initiation of the mammalian hematopoiesis will provide insights into the ontogeny, regulation and expansion of hematopoietic stem cells, as well as the origin of leukemias and bone marrow failure syndromes.
