Three pathways to mature macrophages in the early mouse yolk sac.

The existence of macrophages (Mphi) of yolk-sac (YS) origin has been reported in all vertebrate models. However, the nature of their precursors and pathways of differentiation have not been elucidated. Phenotypic and differentiation potential analyses of YS at 7.5 to 10 postcoital days (dpc), performed in CX3CR1(GFP) embryos, allowed us to discern 3 independent Mphi populations. A first transient wave consisted of mature, maternal-derived Mphipresent as early as 7.5 to 8 dpc. A second wave of committed Mphi precursors arose at 8 dpc (2-4 somite stage) and was followed by a third wave of erythromyeloid precursors (4-6 somite stage). Both types of precursors displayed similar phenotypes and gave rise to CX3CR1/green fluorescent protein (GFP)-positive Mphi, but differed by their differentiation potential, at the clonal level. The combined data of phenotypic, gene-expression, and in situ analyses allowed us to conclude that the previously named "primitive Mphi" corresponded to a mixture of the first transient wave and committed Mphi precursors. Both YS-derived precursors followed a developmental pathway common to adult Mphi and could be qualified as definitive.

Characterization of purified intraembryonic hematopoietic stem cells as a tool to define their site of origin.

Little is known about hematopoietic stem cell (HSC) development from mesoderm. To gain more information on the intraembryonic HSC site of origin, we purified multipotent hematopoietic progenitors from the aorta-gonads-mesonephros (AGM) of mice. This population, expressing c-Kit, AA4.1, CD31, and CD41, but not Flk1, and mainly negative for CD45, proved capable of long-term reconstitution in sublethally irradiated Rag2gammac(-/-) recipients. We assigned the expression of GATA-2, GATA-3, and lmo2 to AGM-HSC, whereas erythromyeloid progenitors express only GATA-2. This unique combination of surface markers and transcription factors could be allocated in the AGM to the intraaortic clusters and the subaortic patches underlying aortic endothelial cells. Taken together, those data indicate that embryonic HSCs (i) differ from their fetal liver and adult counterpart by the low expression of CD45, (ii) do not colocalize with aortic endothelial cells as previously thought, and (iii) are localized, at 10.5 days postcoitum, in the splanchnic mesoderm underlying aortic endothelial cells, within GATA-3(+)CD31(+) cell clusters.