To investigate the respective contributions of the yolk sac and HSCs to the macrophage pool, the authors first used reporter mice that expressed green fluorescent protein (GFP) from the CX3C-chemokine receptor 1 locus (Cx3cr1gfp/+ mice). CD45+CX3CR1hiF4/80hi yolk sac-derived macrophages could be detected in most tissues from embryonic day 10.5 (E10.5). From E12.5, a distinct CD45+CX3CR1+F4/80lowCD11bhi population appeared. The dependence of this second population on the transcription factor MYB indicated that these cells arise from HSCs. By contrast, yolk sac macrophages developed normally in Myb−/− mice but failed to develop in mice lacking PU.1. In Myb−/− mice, yolk sac macrophages accounted for nearly all of the macrophages in the skin, spleen, pancreas, kidneys, brain and lungs, where they were found closely associated with epithelial structures. The E10.5 yolk sac macrophages shared a gene expression signature with previously described populations of E16.5 F4/80hi cells that was distinct from that of F4/80low macrophages. Expression of the growth factor receptor FMS-related tyrosine kinase 3 (FLT3) was restricted to F4/80low macrophages, whereas transcripts encoding colony-stimulating factor 1 receptor (CSF1R) were more abundant in F4/80hi macrophages.
Next, fate-mapping analyses of macrophages were performed using the induction of yellow fluorescent protein (YFP) in FLT3-expressing cells. After birth, the expression of YFP was high in F4/80low macrophages (the typical progeny of adult HSCs), but low in the putative yolk sac-derived macrophages. A second approach involved transplanting limb buds from Cx3cr1gfp/+ E10.5 mouse embryos onto the chorioallantoic membrane of chicken embryos. After 12 days, CX3CR1+ yolk sac-derived mouse macrophages were found colonizing the dermis of growing limb-like structures. A third approach involved the induction of YFP expression in utero in CSF1R-expressing cells. After birth, YFP-labelled F4/80hi macrophages were present in all tissues examined, but YFP expression was not found in the typical progeny of adult HSCs, such as blood leukocytes.
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