AbstractErythropoiesis is characterized by 2 waves of production during mouse embryogenesis: a primitive one originating from the yolk sac (YS) and a definitive one produced from both the YS and the embryo proper. How the latter wave is generated remains unclear. To investigate our hypothesis that endothelial cells (ECs) could generate erythroid cells, we designed a method to label ECs at 10 days after coitus. This labeling method associates 2 techniques: an intracardiac inoculation that allows molecules to be delivered into the bloodstream followed by a whole-embryo culture period. DiI-conjugated acetylated low-density lipoproteins (Ac-LDL-DiI) were used to specifically tag ECs from the inside. One hour after inoculation, DiI staining was found along the entire endothelial tree. Fluorescence-activated cell sorter (FACS) analysis revealed that DiI+ cells were CD31+, CD34+, and CD45–, an antigen makeup characteristic of the endothelial lineage. Twelve hours after inoculation, 43% of DiI+ circulating cells belonged to the erythroid lineage. These cells expressed Ter119 and displayed an adult globin chain arrangement; thus they belonged to the definitive lineage as confirmed in erythroid colony formation. The remaining cells likely represent committed white blood cells or multipotent progenitors, as revealed by a mixed-colony formation. Beyond the 29-somite stage, the proportion of DiI+ erythroid cells gradually decreased. These results demonstrate the generation of hematopoietic cells from an endothelial intermediate, using in vivo tracing. We provide evidence for a release of these cells into the circulation and hypothesize that these cells are able to colonize the fetal liver and generate definitive erythrocytes in vivo.
Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling.
