Figure 2.

Fetal macrophages are already present in the skin rudiment of E12.5 embryos. (A and B) CD11b and F4/80 expression on gated DAPI⁻CD45⁺ cells isolated from indicated tissues of E12.5 embryos. (B) Expression of indicated cell surface markers or GFP reporters (red) compared with isotype/WT controls (blue) on DAPI⁻CD45⁺CD11b⁺F4/80⁺ cells from YS or skin (limb buds) isolated from WT, Cx3cr1^gfp/+, and Csf1r^gfp/+ embryos. Data are representative of 3 independent experiments (n = 6 mice). (C) E12.5 YS or limb bud macrophages were sorted based on their phenotype (DAPI⁻CD45⁺CD11b⁺F4/80⁺) and harvested onto cytospin slides for SEM microscopy (top; bar, 1 µm) or Wright-Giemsa staining (bottom; bar, 5 µm). Data are representative of two independent experiments. (D) Flow cytometric cell cycle analysis of CD45⁺F4/80⁺CD11b⁺ macrophages present in E10.5 and E12.5 limb buds. Data from two pooled experiments (n = 6 to 7). (E) Cross sections of developing skin obtained from E12.5 Cx3cr1^gfp/+ embryos were labeled with anti-F4/80 monoclonal antibody (red) and counterstained with DAPI that label nuclei (blue). Data are representative of 2 experiments (n = 2; bar, 30 µm). (F) High magnification image of CSF-1R^gfp cells (green) in Csf1r^gfp/+ E12.5 developing skin acquired by two photon microscopy. Evans blue stains the blood vessels (red) and DAPI (blue) stains the superficial layer of the epidermis. Top panel illustrates the side view of these 3D reconstructions, and top view snapshots at different depths are shown in the bottom panels (Bars: I, ∼30 µm; II, ∼60 µm; III, ∼90 µm). Bars: 80 µm (top); 30 µm (side). Data are representative of 2 experiments (n = 2). (G and H) CD11b and F4/80 expression on gated DAPI⁻CD45⁺ cells isolated from E12.5 Csf-1r^−/− or control littermate (Wt) FVB/NJ embryos. (H) Percentage fetal macrophages (F4/80⁺CD11b⁺) isolated from E12.5 Csf-1r^−/− or control littermate (Wt) mice. Pooled data from three separate experiments. **, P < 0.001.