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Science 330 (6005): 841-845

Copyright © 2010 by the American Association for the Advancement of Science

Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive Macrophages

Florent Ginhoux1,2,*, Melanie Greter1, Marylene Leboeuf1, Sayan Nandi3, Peter See2, Solen Gokhan4, Mark F. Mehler4,5, Simon J. Conway6, Lai Guan Ng2, E. Richard Stanley3, Igor M. Samokhvalov7, and Miriam Merad1,*

1 Department of Gene and Cell Medicine and the Immunology Institute, Mount Sinai School of Medicine, 1425 Madison Avenue, New York, NY 10029, USA.
2 Singapore Immunology Network (SIgN), 8A Biomedical Grove, IMMUNOS Building Nos. 3-4, BIOPOLIS, 138648, Singapore.
3 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
4 Institute for Brain Disorders and Neural Regeneration, Rose F. Kennedy Center for Research on Intellectual and Developmental Disabilities, and Department of Neurology, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA.
5 Departments of Neuroscience, Psychiatry, and Behavioral Sciences, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA.
6 Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202, USA.
7 Laboratory for Stem Cell Biology, Center for Developmental Biology (CDB), RIKEN Kobe, Kobe 6500047, Japan.


Figure 1
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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 Cx3cr1gfp/+ 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 Cx3cr1gfp/+ mice at different stages during development. Histograms show F4/80 (red) or isotype control (blue) on gated cells. Representative data of three experiments.

 

Figure 2
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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-1op/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.

 

Figure 3
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Fig. 3. Microglia arise from primitive myeloid progenitors. Runx1Cre/wt:Rosa26R26R-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.

 

Figure 4
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Fig. 4. Runx1+ yolk sac progenitors seed the brain between E8.5 and E9.5 through blood circulation. (A and B) Runx1Cre/w:Rosa26R26R-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).

 


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