Fig. 4.

Hyalocytes represent a long-living tissue-resident macrophage population. a Graphical scheme of the experimental setup. Six-week-old Cx3cr1^CreER:Rosa26-YFP mice were injected with tamoxifen (TAM) and retinal whole mounts subsequently analyzed by fluorescence microscopy at 2 and 26 weeks post-injection (p.i.). b Graphical scheme illustrating the rationale of the adult turnover approach. TAM administration leads to nuclear translocation of cytosolic Cre-ER fusion protein and subsequent Cre-mediated irreversible excision of a LoxP-site-flanked STOP-cassette in Cx3cr1-expressing cells. This causes a consistent level of YFP-expression under the control of the constitutively active Rosa26 promoter and labeling of CX₃CR1-positive cells and their progeny. c Confocal images of YFP⁺ and IBA1⁺ hyalocytes (asterisks) and microglia (arrowheads) in Cx3cr1^CreER:Rosa26-YFP at 2 weeks and 26 weeks after injection of TAM. Images are representative for eight animals (2 weeks) from three independent experiments and ten animals (26 weeks) from two independent experiments, respectively. Scale bar = 50 µm. d Percentages of YFP⁺ cells among IBA1⁺ hyalocytes and rMG in Cx3cr1^CreER:Rosa26-YFP mice 2 weeks (upper plot, N = 8, n.s., p > 0.05, paired t-test) and 26 weeks (lower plot, N = 10, n.s., p > 0.05, Wilcoxon signed-rank test) post-injection. Recombination efficiency, as determined by the percentage of YFP⁺ brain microglia using flow cytometry, was 90.38 ± 2.98% (2 weeks) and 97.17 ± 1.49% (26 weeks) among viable doublet-excluded CD45^loCD11b⁺ cells. Data are presented as mean $\pm$ S.E.M. e Representative confocal images of F4/80⁺YFP⁺ hyalocytes (asterisks) and F4/80⁻YFP⁺ rMG in retinal whole mounts from Cx3cr1^CreER:Rosa26-YFP at 2 weeks and 26 weeks after injection of TAM. Images are representative for four mice per timepoint. Scale bar = 50 µm