Hematopoietic stem cells (HSC) derived early during normal development populate distinct sites in the embryo and adult, and maintain the peripheral blood lineages for the life of the organism. In an effort to define the molecular events that regulate the induction of the HSC, we have utilized the zebrafish as a forward genetic and developmental system. The zebrafish egg is externally fertilized and transparent, allowing for an easy evaluation of the circulating blood. Moreover, the ability to house large numbers of zebrafish (currently 60,000 at our institution) and the high fecundity (300-500 embryos per female per week) establishes the genetics of the system. 26 complementation groups of mutants with defects in hematopoiesis have been isolated and we are utilizing positional and candidate cloning to isolate the affected genes. Some of the isolated mutant genes are novel regulators of hematopoiesis, whereas several mutants represent zebrafish models of human diseases. Bone morphogenetic signaling (BMP) regulates the earliest induction of HSC, and subsequently the stem cell leukemia gene (SCL) is a critical regulator of HSC and vascular tissue. In this proposal, we plan to use genetics and functional genomics to define molecular events downstream of BMP signaling, but upstream or parallel to SCL. Cloche, a mutant with both hematopoietic and vascular defects, can be rescued by SCL expression, thus establishing a pathway in which SCL acts downstream or parallel to the cloche gene. In addition to the cloche-SCL pathway, we have recently uncovered a novel role for lateral plate mesoderm that regulates the differentiation of HSC in vivo. The zebrafish mutants spadetail (a T box transcription factor), moonshine (a putative chromatin factor), and bloodless (yet uncloned) have defects in lateral plate mesoderm and are bloodless. The bloodless and cloche genes will be isolated and characterized. The genetic relationships of these factors will be determined by forced expression studies of each factor in spt, mon, bloodless, and cloche mutants, as well as other early dorsalized mutants that lack blood such as swirl (BMP-2b gene). In an effort to further define the induction of the stem cell, an in situ hybridization-based mutagenesis screen will be done for mutants with abnormal expression of SCL. This screen, which should approach saturation, will genetically define key steps in HSC derivation during embryogenesis. These isolated genes may be of therapeutic value for patients with anemia or leukemia, as well as for patients undergoing marrow transplantation or gene therapy.
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