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 rapidly generates erythrocytes and macrophages through monopotent precursors for immediate use in the developing embryo. Definitive hematopoiesis arises later and generates multipotent precursors, including hematopoietic stem cells (HSCs). We have recently performed the first prospective isolation and functional characterization of hematopoietic stem and progenitor cells in the zebrafish and shown that definitive hematopoiesis generates two distinct precursor types during embryogenesis. First to arise are erythromyeloid progenitors (EMPs) in the posterior blood island (PBI), cells that possess robust but transient proliferation potential but lack self-renewal and lymphoid differentiation capacities. Next to develop are HSCs in the aorta/gonad/mesonephros (AGM) region. Unlike EMPs, HSCs colonize the developing thymus to initiate T-lymphopoiesis and seed the pronephros, the site of adult hematopoiesis. 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 of CD41:eGFP+ cells residing in the AGM region demonstrate robust colonization of the pronephros and thymus. Using time-lapse microscopy, we have observed directly the behaviors of the first HSCs to arise in the embryo. AGM HSCs, marked by a CD41:eGFP or c-myb:eGFP transgene, enter circulation to seed the thymic anlage and migrate along the pronephric tubules to seed the pronephros. We are currently performing retrospective time-lapse analyses to determine where in the early embryo HSC and EMP precursors are born. These data will be informative in analyzing where instructive ligands are expressed, including the Delta genes that provide paracrine signals to cells expressing the Notch receptor. We have demonstrated that Notch signaling is necessary for the generation of HSCs but dispensable for EMP formation. We have utilized new and existing Notch reporter lines to determine more precisely where and when HSC precursors receive Notch signals. Together, these studies highlight the power of the zebrafish system in combining genetic approaches with the direct imaging of hematopoietic stem and progenitor cells in living embryos.