Fate mapping analysis reveals that adult microglia derive from primitive macrophages

Abstract

Microglia are the resident macrophages of the central nervous system and are associated with the pathogenesis of many neurodegenerative and brain inflammatory diseases; however, the origin of adult microglia remains controversial. We show that postnatal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain. In contrast to many macrophage populations, we show that microglia develop in mice that lack colony stimulating factor-1 (CSF-1) but are absent in CSF-1 receptor-deficient mice. In vivo lineage tracing studies established that adult microglia derive from primitive myeloid progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically derived microglial progenitors for the treatment of various brain disorders.

Fig. 1

Microglia arise during early embryonic life. (A) Left image, schematic of the imaging field. Right image, three-dimensional rendering of E10.5 brain rudiment from Cx3cr1^gfp/+ mice. DAPI (blue) stains the ectoderm. Representative data of two experiments. (B and C) Flow-cytometric analysis of the expression of CD11b and GFP (CX3CR1) on gated 4´,6´-diamidino-2-phenylindole (DAPI)–CD45⁺ brain (B) and yolk sac (C) cells isolated from Cx3cr1^gfp/+ mice at different stages during development. Histograms show F4/80 (red) or isotype control (blue) on gated cells. Representative data of three experiments.

Fig. 2

Microglia and yolk sac macrophages are absent in Csf-1r^−/− mice. (A) Flow-cytometric analysis of CSF-1R expression (red) on microglia and yolk sac macrophages (blue, isotype control). Representative data of three experiments. (B and C) Percentage of microglia (B) and yolk sac macrophages (C) in Csf-1r^−/− (black squares) or control littermate (Wt) (white squares) FVB/NJ mice. Pooled data from three separate experiments. **P < 0.001; ***P < 0.0001. (D) Coronal sections of 3-week-old Wt, Csf-1^op/op, and Csf-1r^−/− brains of region boxed in the schematic stained for the microglial marker Iba1. DG indicates dentate gyrus; Cx, cerebral cortex; CA3, CA3 region of the hippocampus. Mean number of Iba1⁺ cells per field from three different brain regions is shown. Average of six fields (0.5 mm²) per region per genotype. Error bars represent mean ± SD of data from two pooled experiments. *P < 0.05; ****P < 0.00001.

Fig. 3

Microglia arise from primitive myeloid progenitors. Runx1^Cre/wt:Rosa26^R26R-eYFP mice were treated with 4′OHT to induce Cre-mediated recombination at E7.25 to E7.5 and analyzed at E10.5 [(A) to (C)] or at 8 weeks postbirth [(D) and (E)]. Controls are nontreated mice. (A) Flow-cytometric analysis from one representative embryo showing the percent recombination among yolk sac macrophages and microglial progenitors. (B and D) Pooled data from two experiments showing the percent recombination among yolk sac macrophages and microglial progenitors cells in embryos (B), and among monocytes and microglia in adult mice (n = 10) (D). (C) Correlation and regression analysis between the percent recombination in microglial progenitors and yolk sac macrophages. r², coefficient of regression. (E) Percent recombination among monocytes, lung macrophages, and microglia in adult mice activated at different embryonic age. Error bars represent mean ± SEM of pooled data from two experiments (n = 8 to 16). Gating strategy for each leukocyte population is detailed in fig. S8.

Fig. 4

Runx1⁺ yolk sac progenitors seed the brain between E8.5 and E9.5 through blood circulation. (A and B) Runx1^Cre/w:Rosa^26R26R-LacZ embryos activated at E7.25 to E7.5 were isolated at E8.25 to E8.5 (A) or E9.25 to E9.5 (B) and processed for whole-mount LacZ staining as described in the materials and methods section. At E8.25 to E8.5, labeled cells are detected in the yolk sac but not in the brain rudiment or in the neural tube (A), whereas labeled cells infiltrate the brain rudiment of E9.25 to E9.5 embryos (B). (C to E) Yolk sac and brain rudiment tissues were isolated from E10.0 to E10.5 Ncx1^−/− embryos or control littermates and processed for flow cytometry analysis as described in the materials and methods section. Dot plots show the presence of yolk sac macrophages in Ncx1^−/− embryos and control littermates (C), whereas microglia were present in control but not in Ncx1^−/− embryos (D). (E) The percentage ± SEM of hematopoietic cells (CD45⁺) in control littermates (white bars, n = 4) and Ncx1^−/− embryos (black bars, n = 3).