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. 2021 Feb 8;40(6):e105123. doi: 10.15252/embj.2020105123

Figure 4. Contribution of definitive hematopoiesis to eye macrophage subsets during steady‐state.

Figure 4

  1. Scheme of a fate mapping experiment using adult Cx3cr1CreERT2:Rosa26‐YFP mice. Tamoxifen (TAM) injection was performed at postnatal day 42 (P42). Mice were evaluated at 2, 12, and 24 weeks post‐injections. Administration of TAM leads to the excision of a stop sequence flanked by loxP sites (gray triangles) in Cx3cr1 expressing cells in the eye which causes stable YFP expression under the control of the Rosa26 promotor.
  2. Flow cytometric measurement of the persistence of YFP+ retinal microglia (rMG), cliliary body (cb) MΦ, and corneal (c) MΦ in adult Cx3cr1CreERT2:Rosa26‐YFP mice. Doublets and dead cells were excluded by FSC‐W and viability dye. Representative flow cytometry plots from two independent experiments with at least six mice are displayed.
  3. Kinetics of YFP labeling in macrophages of the healthy eye. Symbols represent means ± s.e.m. rMG are shown as squares (2 weeks: n = 10 mice, 12 weeks: n = 9 mice, 24 weeks: n = 12 mice, Kruskal–Wallis ns P > 0.05), cbMΦ are depicted as triangles (2 weeks: n = 4 samples from eight mice, 12 weeks: n = 3 samples from six mice, 24 weeks: n = 6 samples from twelve mice, Kruskal–Wallis ns P > 0.05) and cMΦ as circles (2 weeks: n = 6 mice, 12 weeks: n = 8 mice, 24 weeks: n = 12 mice, one‐way ANOVA ***P < 0.0001). Data were obtained from two (cMΦ: 2 weeks, cbMΦ: 2 and 12 weeks), three (rMG: 2 weeks, cbMΦ: 24 weeks), or four (rMG: 12 and 24 weeks, coMΦ: 12 and 24 weeks) independent experiments.
  4. Sketch of the Flt3‐dependent Cre‐mediated recombination system with excision of the loxP‐flanked stop‐sequences leading to expression of YFP under the control of the Rosa26 promotor in Flt3Cre:Rosa26‐YFP mice.
  5. Left: Representative flow cytometric characterization of rMG by CD45 and CD11b and cbMΦ and cMΦ by CD45, CD11b, CD64, and F4/80 in Flt3Cre:Rosa26‐YFP mice. Doublets and dead cell were excluded. Right: representative flow cytometric images depicting YFP expression in eye tissue macrophages of 12‐ or 52‐week‐old Flt3Cre:Rosa26‐YFP mice. Typical images were taken from two independent experiments with six to seven mice.
  6. Quantification of the percentage of YFP+ eye macrophages at 12 and 52 weeks of age. rMG are shown as squares (12 weeks: n = 7 mice, 52 weeks: n = 6 mice), cbMΦ as triangles (12 weeks: n = 3 samples from six mice, 52 weeks: n = 3 samples from six mice), and cMΦ as circles (12 weeks: n = 7 mice, 52 weeks: n = 6 mice). Data are presented as means ± s.e.m. and were acquired in two independent experiments.