Hematopoietic Stem and Progenitor Cell Biology in the Zebrafish.

Over the past decade, the development of forward genetic approaches in the zebrafish system has provided unprecedented power in understanding the molecular basis of vertebrate blood development. Establishment of cellular and hematological approaches to better understand the biology of resulting blood mutants, however, has lagged behind these efforts. We have recently developed the means to identify zebrafish hematopoietic stem cells (HSCs), transgenic lines to mark hematopoietic precursors and their progeny, and the assays to test these populations functionally. Like other vertebrates, zebrafish demonstrate differential waves of hematopoiesis during embryogenesis. These waves can be visualized directly by fluorescent transgenesis in living embryos. The earliest blood-forming cells in the zebrafish embryo express the scl and lmo2 genes. By directing expression of GFP to early blood precursors using the lmo2 promoter, we have isolated early hematopoietic cells by flow cytometry and tested them functionally by transplantation. Transplantation of lmo2::GFP+ cells isolated from embryos at 14 hours post-fertilization (hpf) resulted in only transient reconstitution of erythrocytes, suggesting that the earliest identifiable blood-forming cells are committed to the erythroid lineage. Later in embryogenesis, lmo2:GFP+ GATA-1:dsRED+ cells are found in the posterior blood island (PBI) from approximately 30–60 hpf. Molecular and functional characterization of these cells suggests that they possess limited myeloid and erythroid, but not lymphoid differentiation potentials. This suggests that committed progenitors with definitive hematopoietic potential arise in the embryo before HSCs can be identified. Additional studies have suggested that the first multipotent HSCs are born later in the zebrafish aorta/gonad/mesonephros (AGM) region. We have visualized putative HSCs in the AGM by their expression of the lmo2 and cd41 transgenes. Using confocal timelapse imaging in living embryos, lmo2::GFP+ cells have been observed to emigrate from the AGM region into circulation. Transplantation studies are underway to test putative HSC populations for repopulation activity. Taken together, our findings suggest that at least three independent waves of blood cell precursors are formed during zebrafish embryogenesis.