A distinguishing TEM signature expressed by mouse embryonic/fetal macrophages. (A) Flow cytometry analysis of representative E14.5 Tie2-GFP transgenic embryos (> 8 embryos pooled together). Virtually all GFP⁺ cells express the Tie2 receptor. Whereas a small proportion of the Cd11b⁺ cells are GFP⁺, a substantial proportion of the F4/80⁺ cells are GFP⁺. The Cd11b⁻ or F4/80⁻, GFP⁺ cells mostly represent ECs. Similar results were obtained in E8.5 to E14.5 embryos. Results are representative of n = 11 litters analyzed. (B) Flow cytometric analysis of representative E12.5 Tie2-GFP transgenic embryos (> 8 embryos pooled together), analyzed as whole embryos (total cells), or embryos without fetal liver (total cells without fetal liver), or fetal liver alone (fetal liver). In whole embryos, the majority of Lyve1⁺ cells are F4/80⁺ macrophages. In embryos without fetal liver, virtually all the Lyve1⁺ cells are F4/80⁺ macrophages. In the fetal liver, the majority of the Lyve1⁺ cells are F4/80⁻ cells, probably representing lymphatic ECs. The Lyve1⁺F4/80⁺ macrophages (green gate in the dot plot at bottom left) display enhanced GFP expression (histogram at bottom right, open green line) compared with the Lyve1⁻ cells (red gate and filled red line). (C) A representative E12.5 wild-type embryo analyzed for Lyve1 (green), Tie2 receptor (red), and F4/80 (blue) expression. The dorsal area of the embryo is shown. Note the Lyve1⁺Tie2⁺F4/80⁺ macrophages scattered among Tie2⁺Lyve1⁻F4/80⁻ blood vessels. Scale bar represents 60 μm. Results are representative of n = 2 litters (each including 3 embryos) analyzed. For each embryo, at least 3 sections were analyzed. (D) Morphology (May-Grünwald-Giemsa staining) of Tie2-GFP⁻Cd11b⁺ (left) and Tie2-GFP⁺Cd11b⁺ (right) cells sorted from E13.5 Tie2-GFP transgenic embryos depleted of the fetal liver. Scale bar represents 30 μm. Photos are representative of n = 25 photos/sample. (E) A representative E12.5 Tie2-GFPmir142T transgenic embryo analyzed for GFP (green) and Mrc1 (red) expression. The spinal cord of the embryo is shown. Note that the Mrc1⁺ macrophages do not express GFP in this mouse model, whereas ECs do. The bottom right panel shows a high-power magnification of the inset indicated in the bottom left panel. Similar results were obtained in all analyzed embryos (E12.5-E15.5). Results are representative of n = 4 litters (each including 3 embryos) analyzed. For each embryo, at least 3 sections were analyzed. (F) Flow cytometric analysis of representative E12.5 Tie2-GFP and Tie2-GFPmir142T transgenic embryos (> 8 embryos pooled together). Note that in Tie2-GFPmit142T embryos (n = 8 litters analyzed at different stages) there are no Cd11b⁺ or F4/80⁺ cells expressing GFP.