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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 post-natal hematopoietic
progenitors do not significantly contribute to microglia
homeostasis in the adult brain. In contrast to many
macrophage populations, we demonstrate 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.
Although microglial ontogeny is an extensive area of
research, much controversy remains regarding the nature of
microglial progenitors (1, 2). The most consensual hypothesis
to date is that embryonic and peri-natal hematopoietic waves
of microglial recruitment and differentiation occur in the
central nervous system (CNS) (1, 2). The exact contribution
of embryonic and post-natal hematopoietic progenitors to the
adult microglial pool in the steady state, however, remains
unclear. Here we examined the contribution of primitive and
definitive hematopoiesis to the adult microglial population
that populates the CNS during normal development. Our
results provide direct evidence that adult microglia derive
from primitive myeloid progenitors that arise before
embryonic age E8.0 and for the predominant contribution of
primitive myeloid progenitors to an adult hematopoietic
To address the contribution of peri-natal circulating
hematopoietic precursors to microglial homeostasis, we
reconsttuted sub-lethally -irradiated C57BL/6 CD45.2+
newborns with hematopoietic cells isolated from CD45.1+
congenic mice. Although more than 30% circulating
leukocytes and tissue macrophages were of donor origin 3
months after transplant (fig. S1A), 95% of adult microglia
remained of host origin at this time-point (fig. S1, A and B).
These results suggest that in contrast to previous reports (3,
4), peri-natal circulating hematopoietic precursors, including
monocytes, do not substantially contribute to adult microglial
homeostasis. Using adult congenic bone marrow chimera
models, evidence in favour of (5–7) and against (8, 9) the
contribution of circulating hematopoietic cells to microglial
homeostasis has been proposed. We found consistently that
10 to 20% microglia in the brain parenchyma are of donor
origin at 10, 15 and 21 months post-transplant (fig. S1C).
Parabiotic mice, which share the same blood circulation,
provide a means to follow the turnover of adult circulating
hematopoietic precursors without the need for exposure to
radiation injuries. Although the mixing of the myeloid lineage
is less efficient than the mixing of the lymphoid lineage (10),
an average of 30% of monocytes and tissue macrophages
were donor-derived at 1 month and 12 months post-parabiosis
Fate Mapping Analysis Reveals That Adult Microglia Derive from Primitive
Florent Ginhoux,1,2,3 Melanie Greter,1,2 Marylene Leboeuf,1,2 Sayan Nandi,4 Peter See,3 Solen Gokhan,5,6,7 Mark F. Mehler,5,6,7,8,9
Simon J. Conway,10 Lai Guan Ng,3 E. Richard Stanley,4 Igor M. Samokhvalov,11 Miriam Merad1,2
1Department of Gene and Cell Medicine, Mount Sinai School of Medicine, 1425 Madison Ave., New York, NY 10029, USA.
2The Immunology Institute, Mount Sinai School of Medicine, 1425 Madison Ave., New York, NY 10029, USA. 3Singapore
Immunology Network (SIgN), 8A Biomedical Grove, IMMUNOS Building #3-4, BIOPOLIS, 138648, Singapore. 4Department
of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461,
USA. 5Institute for Brain Disorders and Neural Regeneration, , Albert Einstein College of Medicine, 1410 Pelham Parkway
South, Bronx, NY 10461, USA. 6The Rose F. Kennedy Center for Research on Intellectual and Developmental Disabilities, ,
Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA. 7Department of Neurology, Albert
Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA. 8Department of Neuroscience, Albert
Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA. 9Department of Psychiatry and Behavioral
Sciences, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, NY 10461, USA. 10Herman B Wells
Center for Pediatric Research, Indiana University School of Medicine, 1044 West Walnut Street, Indianapolis, IN 46202, USA.
11Laboratory for Stem Cell Biology, Center for Developmental Biology, RIKEN Kobe, Kobe 6500047, Japan.
*To whom correspondence should be addressed. E-mail: Miriam.Merad@mssm.edu (M.M.); Florent_ginhoux@immunol.a-
(fig. S1D and (11)). In contrast, less than 5% of microglia
were donor-derived at these time-points (fig. S1D), in
agreement with a previous report in 5 month-old parabionts
(8). Consistent with previous reports (8, 9, 12), these results
suggest that the recruitment of bone marrow-derived cells to
the brain of chimeric animals is dependent on radiation-
induced brain injuries that followed the transplantation
regimen. These results also suggest that post-natal microglia
are maintained independently of circulating monocytes
throughout life and are maintained by local radio-resistant
precursors that colonize the brain prior to birth.
Next we examined the origin of microglia during
development. In mouse embryos, the first wave of
hematopoietic progenitors appears in the extra-embryonic
yolk sac and leads to the production of primitive
hematopoiesis, which takes place between E7.0 and E9.0 (13,
14). An independent wave of hematopoiesis termed
“definitive hematopoiesis” is initiated within the embryo
proper in the aorta, gonads and mesonephros (AGM) region
(13, 14). Around E10.5, hematopoietic progenitors start to
colonize the fetal liver, which serves as a major
hematopoietic organ after E11.5, while later during
development, hematopoiesis takes place in the spleen and
bone marrow (14). Tissue macrophages in the adult are
thought to derive from bone marrow monocytes and the
contribution of primitive hematopoiesis to adult tissue
macrophages remains unclear (15). To determine the
developmental stage at which the seeding of myeloid cells
occurs in the brain, we used Cx3cr1gfp/+ knock-in mice (16) as
the fractalkine receptor (CX3CR1) is a marker of early
myeloid progenitors (17) and microglia (18). Consistent with
previous results (19), myeloid cells expressing the
hematopoietic marker CD45 and the adult
macrophage/microglia markers CD11b, F4/80 and CX3CR1
were detectable in the developing brain starting from E9.5
(Fig. 1, A and B and fig S2). At E10.5, microglia cells were
present in both the cephalic mesenchyme and the
neuroepithelium, although at a lower density in the latter (Fig.
1A, fig. S2 and movies S1 and S2). The phenotype of
microglial cells resembled that of yolk sac macrophages
throughout embryonic development (Fig. 1, B and C and fig.
S3). Analysis of the DNA content of microglia and in vivo
live imaging indicated that they were highly proliferative
throughout embryonic life (fig. S4 and movie S3).
The differentiation of most macrophage populations in
adult mice is controlled by colony stimulating factor-1 (CSF-
1) and its receptor (CSF-1R) (20) but the role of the CSF-1
and CSF-1R in the development of yolk sac primitive
macrophages and microglia is unknown. We found that CSF-
1R was expressed in similar amounts on yolk sac
macrophages and microglia at E9.5 (Fig. 2A). CSF-1R
expression on microglia was maintained throughout
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development (Fig. 2A and fig. S5). Consistent with a
requirement for CSF-1R expression, absence of CSF-1R
greatly reduced the development of microglia (Fig. 2B and
fig. S6A) and yolk sac macrophages (Fig. 2C and fig. S6B),
whereas circulating monocytes were present in these mice
(fig. S6, C and D). Furthermore, microglia remained largely
absent throughout life in CSF-1R–deficient mice (Fig. 2B and
fig. S6A). Altogether, these results suggest that the
development of yolk sac macrophages and microglia, but not
monocytes, is strongly dependent on CSF-1R. Importantly
and in contrast to many tissue macrophages, adult microglia
can still form, albeit at reduced levels in Csf1op/op mice, which
carry a natural null mutation of the Csf1 gene (21) but are
absent in mice that lack the CSF-1R (Fig. 2D). A second
CSF-1R ligand, interleukin 34 (IL-34), has recently been
identified in mice, humans (22) and birds (23). The
expression of IL-34 mRNA in the brain is much higher than
the expression of CSF-1 mRNA during early post-natal
development and in the adult (24), consistent with an
important role for IL-34 in the regulation of microglial
homeostasis and with the increased severity of the microglial
phenotype in Csf1r-/- compared with Csf1op/op mice.
To examine the potential contribution of primitive myeloid
precursors to adult microglia in vivo, we performed lineage
tracing studies using mice expressing the tamoxifen-inducible
MER-Cre-MER recombinase gene under the control of one of
the endogenous promoters of the runt-related transcription
factor 1 (Runx1) locus. Runx1 expression is first seen at E6.5
and is strongly up-regulated around E7.5 in the proximal
visceral yolk sac region and until E8.0, Runx1+ cells are
restricted to the extra-embryonic yolk sac and are absent from
the embryo proper or allantois (25). We crossed the Runx1-
MER-Cre-MER mice (Runx1Cre/wt) with the Cre-reporter
mouse strain Rosa26R26R-eYF/R26R-eYFP and induced
recombination by a single injection of 4-Hydroxytamoxifen
(4’OHT) into pregnant females at different days of gestation.
Active recombination in these knock-in mice occurs in a
small time window that does not exceed 24 hrs post-injection
(25) and leads to the irreversible expression of the enhanced
yellow fluorescent protein (eYFP) reporter gene in Runx1+
cells and their progeny. At E10.5, embryos activated at
E7.25-E7.5 exhibited a similar proportion of labeled yolk sac
macrophages and microglia (31% +/- 10.8 for yolk sac
macrophages and 32% +/- 10.7 for brain progenitors, n=10)
(Fig. 3, A and B). The proportion of brain-infiltrating
hematopoietic cells and microglia was similar in control
littermate and heterozygote Runx1Cre/wt embryos, suggesting
that microglia homeostasis was not perturbed in Runx1Cre/wt
embryos (fig. S7, A and B). Furthermore, the proportion of
eYFP+ microglia was similar in E10.5 and E13.5 embryos
activated at E7.25-E7.5 (fig. S7C) and within individual
embryos, the proportions of eYFP+ yolk sac macrophages and
eYFP+ microglia were highly correlated (Fig. 3C). The partial
labeling of Runx1+ yolk sac cells is inherent to in vivo
labeling techniques and likely results from the insufficient
expression of MER-Cre-MER and limited availability of the
ligand in target cells (25). Thus our model likely
underestimates the contribution of Runx1+ precursors to adult
microglia homeostasis, although we cannot exclude the
potential contribution of non-labeled precursors to this
process. Nevertheless, these results strongly suggest that yolk
sac macrophages and microglia have the same origin and that
the first wave of microglia is specified prior the end of E8.0.
Strikingly, in adult mice activated at E7.25¬-E7.5 (n=10 from
3 litters), 32.11% +/- 18.0 of microglia were also eYFP+ (Fig.
3D and fig. S8), a proportion similar to that of yolk sac
macrophages and microglia found in E10.5 and E13.5
embryos activated at E7.25-E7.5 (Fig. 3C and fig. S7). In
contrast, less than 3% of blood circulating and tissue
macrophages including dermal and lung macrophages, as well
as circulating T cells, B cells and granulocytes were eYFP+ in
these mice (Fig. 3E, fig. S8 and fig. S9). We also examined
the earliest stage at which Runx1+ progenitors contributed to
the adult microglial pool. In adult mice activated at E6.5-
E7.0, less than 3.77% +/- 3.54 microglia were eYFP+,
whereas 29.6% +/- 10 microglia were eYFP+ in adult mice
activated at E7.0-E7.25 (Fig. 3E). In contrast 0.19% +/- 0.26
and 0.09% +/- 0.05 circulating leukocytes were eYFP+ in
adult mice activated at E6.5-E7.0 and E7.0-E7.25,
respectively (fig. S9). These results establish that primitive
myeloid progenitors that arise prior to E7.5 contribute
significantly to adult microglial homeostasis in the healthy
brain, but have limited potential to give rise to adult blood
To determine at which stage, Runx1+ precursors or their
progeny seed the brain during development, we injected
Runx1Cre/wt : Rosa26R26R-LacZ pregnant females with a single
dose of 4’OHT at E7.25-E7.5 and traced the apparition of
labeled cells into the brain rudiment at different time-points
after injection. A large number of Lac-Z+ cells populated the
yolk sac of E8.5 embryos, whereas no labeled cells were
detectable in the brain rudiment at this time-point (Fig. 4A
and fig. S10A). Brain-infiltrating cells appeared only when
blood circulation developed and a significant proportion of
Lac-Z+ cells appeared associated with blood vessels and
infiltrated the brain rudiment in E9.5 conceptus (Fig. 4B and
fig. S10, B and C). These results are consistent with prior
findings showing that CSF-1R+ cells first accumulate in the
yolk sac around E8.0 and infiltrate the embryo proper when
blood vessels develop around E9.0 (26). To address whether
the development of functional blood vessels was required for
the recruitment of myeloid precursors into the brain rudiment,
we used Ncx-1–/– animals that lack a heartbeat and functional
blood circulation due to a defect in the sodium calcium
/ www.sciencexpress.org / 21 October 2010 / Page 3 / 10.1126/science.1194637
exchanger 1 (27). We found that E9.5-E10.5 Ncx-1–/–
embryos have yolk sac macrophages levels comparable or
higher than control littermates (Fig. 4, C and E). In contrast,
Ncx-1–/–embryos have no detectable microglia in the brain,
whereas Ncx-1+/+ control littermates already have a
substantial number of microglia in the brain at this time-point
(Fig. 4, D and E). Altogether, these results suggest that
Runx1+ progenitors migrate from the yolk sac into the brain
through blood vessels between E8.5 and E9.5.
To examine the contribution of definitive hematopoiesis to
microglial homeostasis, we injected 4’OHT at E8.5, E9.5 and
E10.5. The proportion of eYFP+ leukocytes known to derive
from definitive hematopoiesis was much higher in mice
activated at E8.5 and E9.5 compared to mice activated at
E7.25-E7.5 (up to 40% versus less than 3%, n=10) (Fig. 3E
and fig. S9). In contrast, few eYFP+ microglia were detected
in the brains of adult mice activated after E8.5 and onwards
(Fig. 3E). The sharp descending contribution levels between
E7.5 and E8.5 argue against the contribution of post-E7.5
Runx1+ anatomic locations to the labeling of the adult
microglia lineage. Altogether, these data suggest minimal, if
any, contribution of definitive hematopoiesis to the
development of adult microglia.
Our results provide evidence that primitive myeloid
precursors give rise to microglia residing in the adult CNS in
the steady state. Primitive macrophages differentiate in the
yolk sac of mammals, birds and zebrafish prior to the onset of
blood circulation (13). Studies in zebrafish revealed that yolk
sac-derived macrophages spread in the cephalic mesenchyme
before invading the brain through the pial surfaces and the
fourth ventricle (28–30); findings consistent with the
observation that microglia in humans are formed in the pia
mater (31). The conservation of primitive macrophages
throughout evolution implies that they serve an important role
in the early embryo (13), most likely related to the clearance
of apoptotic bodies and the normal remodeling of brain
tissues. In contrast to most adult tissue macrophages,
microglia are maintained throughout life independently of
any blood input and can resist high dose γ-ray irradiation.
Whether these primitive macrophages are uniquely suited to
reducing the risk of inflammation-induced injuries and
maintaining the CNS integrity throughout adult life will be
important to determine.
The results of this study should help unravel the regulatory
program that controls microglial differentiation and function
in vivo and identify new means to manipulate microglia for
the treatment of neural diseases.
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31. M. A. Cuadros, J. Navascues, Prog Neurobiol 56, 173
32. We thank Dr S. Nishikawa, Dr. H. Snoeck and Dr. P.S.
Frenette for intellectual input in the study, the RIKEN
CDB Laboratory for Animal Resources and Genetic
Engineering for providing the Runx1-MER-Cre-MER
mice, Dr. G. Hoeffel, Dr. X.H. Zong, R. Basu and H.
Ketchum for technical assistance; Dr. L. Robinson for
critical review and editing of the manuscript. This work
/ www.sciencexpress.org / 21 October 2010 / Page 4 / 10.1126/science.1194637
was supported by National Institutes of Health grants
CA112100, HL086899 and AI080884 to M.M, CA32551
and CA26504 to E.R.S and MH66290 and NS38902 to
M.F.M. I.M.S. is supported by a grant from RIKEN
Strategic Programs for Research and Development
(President’s Fund). M.G. is supported by the National
Science Foundation of Switzerland. Runx1-MER-Cre-MER
mice have CDB Acc. No. CDB0524K:
Supporting Online Material
Materials and Methods
Figs. S1 to S10
Movies S1 to S3
06 July 2010; accepted 07 October 2010
Published online 21 October 2010; 10.1126/science.1194637
Fig. 1. Microglia arise during early embryonic life. (A) Left
panel, schematic of the imaging field. Right panel, three-
dimensional rendering of E10.5 brain rudiment from
Cx3cr1gfp/+ mice. DAPI (blue) stains the ectoderm.
Representative data of 2 experiments (n=2). (B-C) Flow
cytometric analysis of the expression of CD11b and GFP
(CX3CR1) on gated 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 3 experiments
Fig. 2. Microglia and yolk sac macrophages are absent in
Csf1r–/– mice. (A) Flow cytometric analysis of CSF-1R
expression (red) on microglia and yolk sac macrophages
(blue: isotype control). Representative data of 3 experiments
(n=3). (B-C) Percentage of microglia (B) and yolk sac
macrophages (C) in Csf1r–/– (black squares) or control
littermate (Wt) (white squares) FVB/NJ mice. Pooled data
from 3 separate experiments. **, P<0.001; ***, P<0.0001.
(D) Coronal sections of 3 week-old Wt, Csf1op/op and Csf1r-/-
brains of region boxed in the schematic stained for the
microglial marker Iba (DG, Dentate Gyrus; Cx, Cerebral
cortex; CA3, CA3 region of the hippocampus). Mean number
of Iba1+ cells/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 2
pooled experiments. *, P<0.05; ****, P<0.00001 .
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-E7.5 and
analyzed at E10.5 (A-C) or at 8 weeks post-birth (D-E).
Controls are non-treated mice. (A) Flow cytometric analysis
from one representative embryo showing the %
recombination among yolk sac macrophages and microglial
progenitors. (B and D) Pooled data from 2 experiments
showing the % 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 %
recombination in microglial progenitors and yolk sac
macrophages. r2, coefficient of regression. (E) %
recombination among monocytes, lung macrophages and
microglia in adult mice activated at different embryonic age.
Error bars represent mean ± SEM of pooled data from 2
experiments (n= 8-16). Gating strategy for each leukocyte
population is detailed in fig. S8.
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Fig. 4. Runx1+ yolk sac progenitors seed the brain between
E8.5 and E9.5 through blood circulation (A-B) Runx1Cre/wt :
Rosa26R26R-LacZ embryos activated at E7.25-E7.5 were isolated
at E8.25-E8.5 (A) or E9.25-E9.5 (B) and processed for whole
mount LacZ staining as described in the material and method
section. At E8.25-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-
E9.5 embryos (B). (C-E) Yolk sac and brain rudiment tissues
were isolated from E10.0-E10.5 Ncx1–/– embryos or control
littermates and processed for flow cytometry analysis as
described in the material and method 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) Graph shows the
percentage +/- SEM of hematopoietic cells (CD45+) in
control littermates (white bars, n=4) and Ncx1–/– embryos
(back bars, n=3).