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. 2021 Aug 25;218(10):e20210745. doi: 10.1084/jem.20210745

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© 2021 Gschwend et al.

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Figure 5. — Timed induction of Csf2 expression in nascent AT2s instructs AM differentiation in fetal lung monocytes. (A) Flow cytometry analysis of kinetics of tdTomato expression in perinatal lungs in Csf2^+/fl and Csf2^+/+ mice ranging from E14.5 to P4, gated on CD45⁻ cells. DOB, day of birth. (B) Percentage of EpCAM⁺tdTomato⁺ cells for time points represented in A. (C–I) Analysis of E17.5 lungs isolated from Csf2^fl and SPC^Cre/+;Csf2^fl mice. (C) Flow cytometry analysis of EpCAM⁺tdTomato⁺ populations, gated on CD45⁻ cells. (D) Flow cytometry analysis of primitive macrophages (G1; F4/80^high) and fetal monocytes (G2; F4/80^int), gated on CD45⁺Ly-6G⁻CD64⁺MHCII⁻ cells. (E) Quantification of primitive macrophages (G1) and fetal monocytes (G2). (F) Further flow cytometry analysis of Ly-6C and CD11b levels in the developing fetal monocytes population (G2). (G) Quantification of Ly-6C^high (G2.1), Ly-6C^int (G2.2), and Ly-6C^low (G2.3) fetal monocytes. (H) Flow cytometry analysis of Ki-67 expression, gated on CD45⁺ cells. (I) Ki67 expression levels in primitive macrophages (F4/80^high) and fetal monocytes (F4/80^int) for Csf2^fl (gray) and SPC^Cre/+;Csf2^fl (blue) mice. (A and B) Data representative of at least two independent experiments per time point. (B, E, and G) Data are pooled from at least two (B) or three (E and G) independent experiments. (C, D, F, H, and I) Data are from one experiment, representative of three (C, D, and F) or two (H and I) independent experiments. (E and G) ns, P ≥ 0.05; **, P 0.01–0.001; ***, P 0.0001–0.001; ****, P < 0.0001.