Figure 3. Ontogeny of sciatic nerve macrophages and brain microglia is completely different.

A. Scheme of the TAM induced recombination in either macrophages or monocytes in the Cx3cr1^CreERT2:R26-YFP and Cxcr4^CreERT2: R26- Tomato reporter mouse lines. B. YFP expression in yolk sac derived macrophages was induced by 4-OH TAM injection in pregnant Cx3cr1^CreERT2:R26-YFP mice at embryonic day (E)9.5. The percentage of YFP⁺ brain microglia (MG), onMacs and sciatic nerve macrophages (snMac) was quantified by flow cytometry at postnatal day (p)42. C. YFP expression was induced by TAM injection in six weeks (6W) old Cx3cr1^CreERT2:R26-YFP mice. The percentage of YFP⁺ brain MG, onMacs, and snMacs was quantified by flow cytometry. D. YFP expression in embryonic macrophages was induced by 4-OH TAM injection in pregnant Cx3cr1^CreERT2:R26-YFP mice at E16.5. The percentage of YFP⁺ brain MG, onMacs and snMacs was quantified by flow cytometry. E. F. Tomato expression was induced by injecting TAM at 6W of age (E) or at postnatal days (p)1+3 (F) in Cxcr4^CreERT2:R26-Tomato mice. The percentage of Tomato⁺ brain MG, onMacs, Ly6C^hi blood monocytes (Ly6C^hi) and snMacs was quantified by flow cytometry. G. YFP expression in yolk sac derived macrophages was induced by 4-OH TAM injection in pregnant Csf1r^MERcreMER:R26-YFP mice at E8.5. The percentage of YFP⁺ brain MG, ON MG, Ly6C^hi and snMacs was quantified by flow cytometry at p42. H. The percentage of YFP⁺ brain MG, Kupffer cells, snMacs and liver monocytes was quantified by flow cytometry in adult S100A4^Cre:R26-YFP mice. I. Parabiotic mice were generated by suturing together CD45.1+ WT and CD45.2+CCR2-/- mice. The percentage of cells of CD45.1+ WT donor origin was determined in the macrophages and monocytes from indicated tissues from the CD45.2+CCR2-/- parabionts. Data represent two independent experiments involving four independent parabionts. J. K. CD45.2⁺ recipient mice underwent full-body irradiation (J) or partial-body irradiation with one leg protected by a lead shield (K). Mice were reconstituted with CD45.1⁺ donor BM and percentage of CD45.1⁺ brain MG, snMacs and blood monocytes was quantified by flow cytometry. All data are shown as mean ±SEM. Symbols represent individual animals. (B) Pooled data coming from 3 independent experiments (8 mice in total). (C) Pooled data from 3 independent experiments (at least 9 mice in total), except for week 36 (2 independent experiments with at least 3 mice). (D) Data from 2 pregnant females. Offspring divided over 2 time points (p4 (5 mice), p42 (4 mice). (E) Pooled data from 2 independent experiments (with at least 6 mice), except for week 12 (1 experiment with 4 mice). (F) P14 time point: 1 experiment with 3 mice; p42: data pooled from 2 independent experiments with 6 mice in total. (G) Pooled data from 2 independent experiments with 16 mice in total. (H) Pooled data from 2 independent experiments with 9 mice in total. (I) Data represent two independent experiments involving four independent parabionts. (J) Pooled data of two independent experiments with 8 mice in total. (K) One experiment shown of two independent experiments with 4 mice per experiment.