The Ontogeny of Definitive Hematopoiesis in the Zebrafish.

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. The generation of definitive, or multilineage, hematopoietic precursors during embryogenesis remains less well understood. Here we show, using a combination of gene expression analyses, prospective isolation approaches, hematopoietic progenitor cultures, transplantation, and in vivo lineage tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of hematopoietic stem cells (HSCs). EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. By contrast, fate mapping studies of CD41:eGFP+ cells residing in the aorta/gonads/mesonephros (AGM) region demonstrate robust colonization of the pronephros and thymus. Using timelapse microscopy, we show that these sites of adult hematopoiesis are seeded by CD41+ cells that migrate along the pronephric ducts from the AGM. These studies provide phenotypic and functional analyses of the first hematopoietic stem and progenitor cells in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.