Embryologic Origin of Functional Granulopoiesis

The first progenitors with granulocyte potential arise in the murine yolk sac beginning at embryonic day 8.5 (E8.5), 48 hours before hematopoietic stem cells (HSCs) are formed. These granulocyte progenitors are part of a wave of definitive erythro-myeloid progenitors (EMPs) that display a unique immunophenotype. By E10.5, we observe EMPs in the bloodstream and enriched in the liver, the site of fetal hematopoiesis, consistent with previous reports of GM-CFC presence in the early embryo. HSCs begin to colonize the fetal liver between E11.5 and E12.5, where they subsequently expand and differentiate. Thus, the presence of maturing blood cells at this time-point likely represent the output of EMP that colonize the fetal liver before HSCs. In vitro culture of purified EMPs results in the complete myeloid repertoire found in the adult, including neutrophils, basophils, eosinophils and mast cells. To see which of these potentials is actually realized in the embryo, we examined myelopoiesis in the liver at E11.5- E12.5. There was no morphological evidence of eosinophils, basophils or mast cells in the early fetal liver, and there were no lineage specific transcripts for these cells types (EPX, FCepsilonR) detected by qPCR before E15.5. However, rare cells with neutrophil morphology were found in the fetal liver and in the bloodstream at E12.5. We utilized flow cytometry to enumerate granulocytes (GF1+, Mac1+) in both liver and the bloodstream during early embryogenesis. In order to rule out contamination from maternal cells, we analyzed embryos generated from GFP+ male mice mated with wild-type females. Per embryo equivalent, we consistently found a small number of granulocytes already present in both fetal liver and circulation at E11.5. The number of granulocytes increases to over one hundred at E12.5 and thousands by E13.5. We used imaging flow cytometry to examine the maturational state of the granulocytes in the fetal liver. Consistent with their recent differentiation, fetal liver granulocytes were predominately at the most immature stages as compared to bone marrow samples. Interestingly, at E11.5 and E12.5, a large proportion the circulating granulocytes were maternal derived (GFP-). The presence of these maternal granulocytes could not be accounted for by contamination of maternal blood given the levels of maternal RBCs in the samples. These data indicate that both maternal- and embryonic EMP-derived neutrophils co-circulate in the early embryo. Furthermore, when fetal blood was stimulated with bacteria-like BioParticles, fetal derived (GFP+) and maternal granulocytes (GFP-) each responded with oxidative bursts. Taken together, these data indicate that the early mammalian embryo utilizes both yolk sac-derived transient definitive progenitors and maternal granulocytes to provide host defense before HSC-derived hematopoiesis is established.