Abstract
All tissue-resident macrophages of the central nervous system (CNS)—including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2,3,4,5,6,7—are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8,9,10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11,12,13,14,15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession codes GSE194432, GSE194433, GSE195437 and GSE192510. All other data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.
Code availability
The code for the scRNA-seq analysis of yolk sac macrophage progenitors is available at https://github.com/rsankowski/Taka_A1_A2_only.
Change history
21 September 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41586-022-05361-1
References
Van Hove, H. et al. A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment. Nat. Neurosci. 22, 1021–1035 (2019).
Kierdorf, K., Masuda, T., Jordao, M. J. C. & Prinz, M. Macrophages at CNS interfaces: ontogeny and function in health and disease. Nat. Rev. Neurosci. 20, 547–562 (2019).
Herz, J., Filiano, A. J., Smith, A., Yogev, N. & Kipnis, J. Myeloid cells in the central nervous system. Immunity 46, 943–956 (2017).
Prinz, M., Jung, S. & Priller, J. Microglia biology: one century of evolving concepts. Cell 179, 292–311 (2019).
Prinz, M., Erny, D. & Hagemeyer, N. Ontogeny and homeostasis of CNS myeloid cells. Nat. Immunol. 18, 385–392 (2017).
Colonna, M. & Butovsky, O. Microglia function in the central nervous system during health and neurodegeneration. Annu. Rev. Immunol. 35, 441–468 (2017).
Prinz, M., Masuda, T., Wheeler, M. A. & Quintana, F. J. Microglia and central nervous system-associated macrophages-from origin to disease modulation. Annu. Rev. Immunol. 39, 251–277 (2021).
Santisteban, M. M. et al. Endothelium–macrophage crosstalk mediates blood–brain barrier dysfunction in hypertension. Hypertension 76, 795–807 (2020).
Faraco, G. et al. Perivascular macrophages mediate the neurovascular and cognitive dysfunction associated with hypertension. J. Clin. Invest. 126, 4674–4689 (2016).
Pires, P. W. et al. Improvement in middle cerebral artery structure and endothelial function in stroke-prone spontaneously hypertensive rats after macrophage depletion. Microcirculation 20, 650–661 (2013).
Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016).
Gomez Perdiguero, E. et al. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature 518, 547–551 (2015).
Kierdorf, K. et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat. Neurosci. 16, 273–280 (2013).
Ginhoux, F. & Guilliams, M. Tissue-resident macrophage ontogeny and homeostasis. Immunity 44, 439–449 (2016).
Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).
Hagemeyer, N. et al. Transcriptome-based profiling of yolk sac-derived macrophages reveals a role for Irf8 in macrophage maturation. EMBO J. 35, 1730–1744 (2016).
Jordao, M. J. C. et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363, eaat7554 (2019).
Masuda, T. et al. Novel Hexb-based tools for studying microglia in the CNS. Nat. Immunol. 21, 802–815 (2020).
Mrdjen, D. et al. High-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease. Immunity 48, 380–395 (2018).
Masuda, T. et al. Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution. Nature 566, 388–392 (2019).
Hammond, T. R. et al. Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes. Immunity 50, 253–271 (2019).
Dasgupta, K. & Jeong, J. Developmental biology of the meninges. Genesis 57, e23288 (2019).
Munro, D. A. D. et al. CNS macrophages differentially rely on an intronic Csf1r enhancer for their development. Development 147, dev194449 (2020).
Zhao, Z., Nelson, A. R., Betsholtz, C. & Zlokovic, B. V. Establishment and dysfunction of the blood–brain barrier. Cell 163, 1064–1078 (2015).
Vanlandewijck, M. et al. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475–480 (2018).
Langen, U. H., Ayloo, S. & Gu, C. Development and cell biology of the blood–brain barrier. Annu. Rev. Cell Dev. Biol. 35, 591–613 (2019).
Corada, M. et al. Sox17 is indispensable for acquisition and maintenance of arterial identity. Nat. Commun. 4, 2609 (2013).
Zahr, A. et al. Endomucin prevents leukocyte-endothelial cell adhesion and has a critical role under resting and inflammatory conditions. Nat. Commun. 7, 10363 (2016).
Werner, Y. et al. Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke. Nat. Neurosci. 23, 351–362 (2020).
Ydens, E. et al. Profiling peripheral nerve macrophages reveals two macrophage subsets with distinct localization, transcriptome and response to injury. Nat. Neurosci. 23, 676–689 (2020).
Bertrand, J. Y. et al. Three pathways to mature macrophages in the early mouse yolk sac. Blood 106, 3004–3011 (2005).
Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol. 36, 411–420 (2018).
Utz, S. G. et al. Early fate defines microglia and non-parenchymal brain macrophage development. Cell 181, 557–573 (2020).
Tay, T. L. et al. A new fate mapping system reveals context-dependent random or clonal expansion of microglia. Nat. Neurosci. 20, 793–803 (2017).
Michael, M. & Parsons, M. New perspectives on integrin-dependent adhesions. Curr. Opin. Cell Biol. 63, 31–37 (2020).
Calderwood, D. A., Campbell, I. D. & Critchley, D. R. Talins and kindlins: partners in integrin-mediated adhesion. Nat. Rev. Mol. Cell Biol. 14, 503–517 (2013).
Mastorakos, P. & McGavern, D. The anatomy and immunology of vasculature in the central nervous system. Sci. Immunol. 4, eaav0492 (2019).
Irmady, K., Zechel, S. & Unsicker, K. Fibroblast growth factor 2 regulates astrocyte differentiation in a region-specific manner in the hindbrain. Glia 59, 708–719 (2011).
Shoneye, T. et al. Differential proliferation and maturation of subcortical astrocytes during postnatal development. Front. Neurosci. 14, 435 (2020).
Daneman, R., Zhou, L., Kebede, A. A. & Barres, B. A. Pericytes are required for blood–brain barrier integrity during embryogenesis. Nature 468, 562–566 (2010).
Wang, Q., Zhao, N., Kennard, S. & Lilly, B. Notch2 and Notch3 function together to regulate vascular smooth muscle development. PLoS ONE 7, e37365 (2012).
Henshall, T. L. et al. Notch3 is necessary for blood vessel integrity in the central nervous system. Arterioscler. Thromb. Vasc. Biol. 35, 409–420 (2015).
Domenga, V. et al. Notch3 is required for arterial identity and maturation of vascular smooth muscle cells. Genes Dev. 18, 2730–2735 (2004).
Han, H. et al. Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision. Int. Immunol. 14, 637–645 (2002).
Wirth, A. et al. G12-G13–LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat. Med. 14, 64–68 (2008).
Fouillade, C. et al. Transcriptome analysis for Notch3 target genes identifies Grip2 as a novel regulator of myogenic response in the cerebrovasculature. Arterioscler. Thromb. Vasc. Biol. 33, 76–86 (2013).
Sankowski, R., Monaco, G. & Prinz, M. Evaluating microglial phenotypes using single-cell technologies. Trends Neurosci. 45, 133–144 (2022).
de Boer, J. et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol. 33, 314–325 (2003).
Petrich, B. G. et al. Talin is required for integrin-mediated platelet function in hemostasis and thrombosis. J. Exp. Med. 204, 3103–3111 (2007).
Dono, R., Texido, G., Dussel, R., Ehmke, H. & Zeller, R. Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice. EMBO J. 17, 4213–4225 (1998).
Mitchell, K. J. et al. Functional analysis of secreted and transmembrane proteins critical to mouse development. Nat. Genet. 28, 241–249 (2001).
Potocnik, A. J., Brakebusch, C. & Fassler, R. Fetal and adult hematopoietic stem cells require β1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity 12, 653–663 (2000).
Feng, J. et al. IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools. J. Immunol. 186, 1458–1466 (2011).
Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).
Yu, W. M. et al. A Gata3-Mafb transcriptional network directs post-synaptic differentiation in synapses specialized for hearing. eLife 2, e01341 (2013).
Hashimshony, T. et al. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-seq. Genome Biol. 17, 77 (2016).
Herman, J. S., Sagar, & Grun, D. FateID infers cell fate bias in multipotent progenitors from single-cell RNA-seq data. Nat. Methods 15, 379–386 (2018).
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).
Baker, S. C. et al. The External RNA Controls Consortium: a progress report. Nat. Methods 2, 731–734 (2005).
Grun, D., Kester, L. & van Oudenaarden, A. Validation of noise models for single-cell transcriptomics. Nat. Methods 11, 637–640 (2014).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Harrow, J. et al. GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 22, 1760–1774 (2012).
Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).
Paterson, N & Lämmermann, T. Macrophage network dynamics depend on haptokinesis for optimal local surveillance. eLife 11, e75354 (2022).
R Core Team. R: A Language and Environment for Statistical Computing http://www.R-project.org/ (R Foundation for Statistical Computing, 2020).
Acknowledgements
We thank E. Barleon and M. Fukuzaki for technical assistance and D. Grün and Sagar for their support with the preparation of the scRNA-seq data. We thank the Lighthouse Core Facility (LCF) for their support with cell sorting. LCF is funded in part by the Medical Faculty, University of Freiburg (Project numbers 2021/A2-Fol; 2021/B3-Fol) and the DFG (Project number 450392965). We are also grateful to R. Fässler and D. Critchley for providing mouse strains. T.M. was supported by AMED (JP20gm6310016 (PRIME), JP21wm0425001), JSPS (KAKENHI JP20K22687, JP21H02752, JP21H00204), the Kanae Foundation, the Naito Foundation, the Inamori Foundation and the Takeda Science Foundation. M.P. was supported by the Sobek Foundation, the Ernst Jung Foundation, the Novo Nordisk Prize, the DFG SFB 992 (Project ID 192904750), SFB1160 (Project ID 256073931), Reinhart-Koselleck-Grant (Project ID 279642606) and Gottfried Wilhelm Leibniz Prize, the Alzheimer Forschung Initiative e.V. (AFI) and the Ministry of Science, Research and Arts, Baden-Wuerttemberg (Sonderlinie ‘Neuroinflammation’). This study was supported by the DFG under Germany’s Excellence Strategy (CIBSS – EXC-2189 – Project ID 390939984). J.P. was supported by the UK DRI Momentum and Programme Leader Awards and the DFG SFB/TRR265. K.K. was supported by the Fritz Thyssen Foundation, by the DFG project grant (Project ID 432207796). K.K. and M.P. were supported by the DFG project grants within the CRC1479 (Project ID 441891347). K.K., K.-P.K., J.P., E.N., T.L. and M.P. were supported by the DFG-funded CRC/TRR167 ‘NeuroMac’, Project ID 259373024. K.-P.K. was supported by the DFG GRK 2606 (Project ID 423813989). C.B. and U.L. were supported by the Swedish Brain Foundation and Erling-Persson Foundation. N.P. was supported by the DFG-funded project ID 89986987 (SFB 850).
Author information
Authors and Affiliations
Contributions
T.M. and L.A. designed experiments, analysed data and prepared the figures. G.M., R.S., O.S., M.K., F.D.G., L.H., M. Frosch, M. Fliegauf, A.Y. and L.F.P.B. performed experiments and analysed the data. E.N., N.P., F.F.-K., N.H., D.S., H.U., C.D., M.T., K.K., C.G., A.J., C.B., U.L., J.P., K.-P.K., R.D. and T.L. analysed the data and provided mice. J.P. and K.K. contributed to conception and critical revision. T.M. and M.P. supervised the project and wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature thanks the anonymous reviewers for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Preferential localization of perivascular macrophages in the arterial perivascular space during development in mice and humans.
(a) Electron microscopy (EM) showing the establishment of a narrow PVS at P3 in the brains of wildtype mice. Blue: outer vascular and glial basal laminas. Representative images out of 6 mice investigated are shown. SAS: subarachnoid space; E: endothelium; SM: smooth muscle cell; L: vascular lumen. (b, c) P2RY12 (blue) and LYVE1 (magenta) and Col IV (red) immunofluorescence in the cortex of Cx3cr1GFP/+ (green) mice. Arrowheads indicate double positive (filled) or single positive (blank) cells. Pictures representative out of at least three investigated mice per time point are shown. Scale bars: 50 μm. (d) Quantification thereof. Symbols represent individual mice. Bars indicate means ± s.e.m. (n = 4: E14.5 and P10 P2RY12, n = 3: E14.5 LYVE1, n = 5: P10 LYVE1). (e) Top: Immunofluorescence of CD206 (white) and laminin (blue, basement membranes) in the cortex of adult mice. Bottom: Most of the CD206+ perivascular macrophages (pvMΦ) are located in alpha smooth muscle actin (aSMA, red)+ transferrin receptor (TfR, green)– arteries/arterioles, with a minor population in aSMA-TfR+ veins/venules. Typical pictures out of 6 mice (3 male and 3 female) investigated are shown. A, artery/arteriole. Cap, capillary. V, vein/venule. Scale bar: 200 μm. (f) High-magnification images of dashed frames shown in d. Arrowheads indicate CD206+ pvMΦ. Scale bar: 50 μm. (g) Distribution of CD206+ pvMΦ in different vessels types (mean ± s.e.m., n = 3 mice for both genders). (h) Left: Immunofluorescence of CD206 (magenta or green), SOX17 (blue) and aSMA (red) in the cortex of adult Cx3cr1GFP/+ (green) mice. Pictures representative of three mice investigated. Scale bar: 50 μm. Right: Frequency of CD206+ CX3CR1–GFP+ pvMΦ within aSMA+ vessels (n = 3 mice). Three sections per mouse were quantified (means ± s.e.m.). (i) Left: Immunofluorescence of CD206 (green), endomucin (blue) CD31 (red) in the cortex of adult Cx3cr1GFP/+ (green) mice. Pictures representative of three mice investigated. Scale bar. 50 μm. Right: quantification of CD206+ CX3CR1–GFP+ pvMΦ within endomucin- CD31+ vessels (n = 3 mice). Three sections per mouse were quantified (means ± s.e.m.). (j) Representative images showing CX3CR1–GFP+ pvMΦ in endomucin- Col IV+ vessels at different developmental time points. Arrowheads indicate CX3CR1–GFP+ pvMΦ located in the endomucin- vessels. Images representative of 3 mice for each timepoint investigated. Scale bar: 50 μm. (k) EM illustrating that pvMΦ (yellow) are preferentially localized in PVS of arterial vessels of wild-type mice at P21. Vascular smooth muscle cells (blue) are lacking in venous vessels. Images representative of 6 mice investigated. L: lumen, N: neuropil, E: endothelial cell. Arrows: glial basement membrane. Asterisks: processes of fibroblasts. (l) Distribution of pvMΦ around arteries and veins based on quantification in EM images in cortical sections of P21 mouse brains. Multiple non-consecutive sections per mouse were quantified. Symbols represent individual mice (n = 6, mean ± s.e.m.). (m) Immunohistochemistry for CD206 (brown) combined with Elastica-Van-Gieson (EvG) staining in human cortical brain. Asterisks: pvMΦ, arrowheads: EvG-positive arteries. Images representative of one sample per time point.
Extended Data Fig. 2 Kinetics of microglia, meningeal and perivascular macrophages as a HSC-independent cell population during development.
(a) Left: representative images showing Ki-67+ CX3CR1–GFP+ cells in the parenchyma of Cx3cr1GFP/+ mice at P10. Arrowheads indicate double positive cells. Representative pictures out of four mice investigated are shown. Scale bar. 50 μm. Right: density (cells / mm2) and frequency (ratio of Ki-67+ cells out of all CX3CR1–GFP+ cells) of Ki-67+ CD206− CX3CR1–GFP+ cells in the parenchyma of Cx3cr1GFP/+ mice during development (n=3 mice for E16.5, P3, P5, P21, P28; n = 4 mice for E12.5, E14.5, P0, P7, P10, P14). Three sections per mouse were quantified (means ± s.e.m.). (b) Left: representative images showing Ki-67+ cells in the Col IV+ perivascular compartment of Cx3cr1GFP/+ mice at P10. Filled arrowheads: CD206+ CX3CR1–GFP+ Ki-67+ cells, blank arrowheads: single Ki-67+ cell. Images representative of four mice investigated. Scale bar: 50 μm. Right: density and frequency of Ki-67+ CD206+ CX3CR1–GFP+ or Ki-67+CD206−CX3CR1–GFP+ cells in the perivascular compartment of Cx3cr1GFP/+ mice (n=3 mice for E16.5, P3, P5, P21, P28; n = 4 mice for E12.5, E14.5, P0, P7, P10, P14). Three sections/mouse were quantified (means ± s.e.m.). (c) Left, representative images showing Ki-67+ or Ki-67− cells in the Col IV+ brain surfaces of Cx3cr1GFP/+ mice at E14.5, P3, and P21. Filled arrowhead indicate CD206+CX3CR1–GFP+Ki-67+ cells, and blank arrowhead indicate CD206+CX3CR1–GFP+Ki-67− cells. Representative pictures out of four mice investigated are shown. Scale bar: 50 μm. Right, density and frequency of CD206+CX3CR1–GFP+Ki-67+ or CD206− CX3CR1–GFP+Ki-67+ cells in the brain surfaces of Cx3cr1GFP/+ mice during development (n=3 mice for E16.5, P5, P14, P21, P28; n = 4 mice for E12.5, E14.5, P0, P3, P7, P10, P56). Three sections per mouse were quantified (means ± s.e.m.). (d) Upper, representative images showing active-caspase 3 (Cas3)− CX3CR1–GFP (GFP)+ cells in the perivascular compartment of Cx3cr1GFP/+ mice at E14.5. Filled arrowhead indicates GFP+Cas3− cell, and blank arrowhead indicates single Cas3+ cell. Representative pictures out of four mice investigated are shown. Scale bar: 50 μm. Bottom, quantification of Cas3+ CX3CR1–GFP+ cells in the perivascular compartment of Cx3cr1GFP/+ mice during embryogenesis (n = 4 for all timepoints). N.d., not detected. Three sections per mouse were quantified. (e) Scheme of experimental set-up. Pregnant Cx3cr1CreERT2/+R26YFP/YFP females were injected with 4OH-TAM at E9.5, and the embryos or the offspring were then analysed at E14.5, P0, or P14, respectively. (f) Representative immunofluorescence images showing YFP-expressing IBA1+ cells, in combination with Col IV+ basal membrane, in the parenchyma, perivascular compartment, and brain surface of Cx3cr1CreERT2/+R26YFP/YFP embryos (E14.5) or offspring (P0, P14). Filled yellow, filled white, blank white arrowheads indicate double positive cells in perivascular compartment, double positive cells in the parenchyma or meninges, or single positive cells, respectively. Representative pictures out of five mice investigated are shown. Scale bar: 50 μm. (g) Quantification thereof. Each symbol represents one mouse (n = 5). Means ± s.e.m. are shown. (h) Illustration of the experimental procedure for tamoxifen injection (TAM) and subsequent analysis of adult Cxcr4CreERT2/+R26tdT/+ mice. (i) Frequency of tdTomato (tdT)+ Ly6Clo and Ly6Chi monocytes in the blood of Cxcr4CreERT2/+R26tdT/+ mice after tamoxifen injection (n = 5 mice for all timepoints tested). Data are depicted as mean ± s.e.m. (j) Typical images showing that tdT (filled white arrowheads) is not expressed in IBA1+ microglia (green), CD206+ pvMΦ (green) or CD206+ meningeal macrophages (mMΦ, green), but in IBA1+ choroid plexus macrophages (cpMΦ, green) and in the lumen of CD31+ endothelial cells (green) in the brains of Cxcr4CreERT2/+R26tdT/+ mice 8 weeks after TAM injection. Yellow and blank arrowheads indicate double positive or single positive cells, respectively. EC, endothelial cell. Representative images out of 5 mice investigated are shown. Scale bars: 50 μm. (k) Frequency of tdT positive microglia, pvMΦ, and mMΦ in the cortex, and cpMΦ of Cxcr4CreERT2/+R26tdT/+ mice after TAM treatment (n = 5 mice for all timepoints tested). Data are depicted as mean ± s.e.m.
Extended Data Fig. 3 Gene expression profiling reveals a stepwise development of microglia, meningeal and perivascular macrophages.
(a) Heat map of the top 10 differentially regulated genes for each respective cell-type and time point. (b–e) Bar graphs depicting expression levels during development of microglia signature genes (b), CAM signature genes (c), transcription factors known to be expressed in macrophages (d) and Notch3 (e). Means ± s.d. are shown. Each dot represents an individual sample. N= 3 for E14.5, P3 and P10 microglia, E14.5, P3, P10, P21 and P56 mMΦ, P10 pvMΦ; n = 4 for P21 and P56 pvMΦ; n = 5 for P21 and P56 microglia.
Extended Data Fig. 4 Generation and comparative analysis of Mrc1CreERT2 mice.
(a) Targeting schemes. The T2A-CreERT2 cassette was knocked in into the Mrc1 locus just before the stop codon. From the resulting polycistronic RNA, a fusion protein is translated, which gives rise to MRC1 (CD206) and the CreERT protein through the activity of the self-cleaving T2A peptide. (b) Representative gating strategy for FACS-sorting of CD206+ CAMs from adult Mrc1+/+, Mrc1CreERT2/+ or Mrc1CreRT2/CreERT2 mice for RNA-expression analysis and for measuring CD206 surface expression levels. (c) Heat map of the top 1,000 differentially regulated genes across the respective genotypes. (d) Volcano plots depicting differentially regulated genes between the groups (14,639 variables) (n = 3 for Mrc1+/+ and Mrc1CreERT2/+, n = 4 for Mrc1CreERT2/ CreERT2). NS = not significant. (e) Left: Representative histograms showing CD206 surface expression levels on CAMs isolated using the gating strategy depicted in (b). Right: Quantification geometric mean fluorescence intensities (gMFI). Symbols represent individual mice (means ± s.e.m. are shown). N = 4 mice per genotype. One-way ANOVA with Tukey’s multiple comparisons test. (f) Immunofluorescence images showing IBA1+ (white) microglia, CD206+ (red) pvMΦ and CD206+ mMΦ in the cortex of Mrc1+/+, Mrc1CreERT2/+ or Mrc1CreERT2/CreERT2 mice. Images representative of 4 mice per genotype. Scale bar: 20 μm (g) Bar graphs depicting the expression levels of CAM signature genes. Dots represent individual mice (mean ± s.d.). (n = 3 for Mrc1+/+ and Mrc1CreERT2/+, n = 4 for Mrc1CreERT2/ CreERT2). (h) Quantification of microglia, pvMΦ and mMΦ in the cortex of adult Mrc1+/+ (black circles), Mrc1CreERT2/+ (grey circles) or Mrc1CreERT2/CreERT2 (white circles) mice. Each symbol represents one mouse (n = 4 for all genotypes). Three sections per mouse were quantified (means ± s.e.m.). One-way ANOVA with Tukey’s multiple comparisons test. (i) Scheme of the experimental set-up. (j) Direct fluorescence microscopic visualization reveals tdT signals (red) in CD206+ pvMΦ and mMΦ (green), but not in microglia (IBA1, green), astrocytes (SOX9, green), neurons (NeuN, green), or oligodendrocyte (APC, green) in the cortex of adult Mrc1CreERT2/CreERT2R26tdT/tdT mice. Col IV is shown in blue. Filled arrowheads indicate double positive pvMΦ and mMΦ, empty triangles show single positive cells. Images representative of three mice. Scale bars: 20 µm. (k) Quantification of recombination efficacy in several CNS cells of Mrc1CreERT2/CreERT2R26tdT/tdT and Mrc1CreERT2/+R26tdT/tdT mice (with or without TAM), respectively. Each symbol represents one mouse (Mrc1CreERT2/+R26tdT/tdT with TAM, n = 7; Mrc1CreERT2/+R26tdT/tdT without TAM, n = 2; Mrc1CreERT2/ CreERT2R26tdT/tdT with TAM, n = 3; Mrc1CreERT2/ CreERT2R26tdT/tdT without TAM, n = 3). Three sections per mouse were quantified (means ± s.e.m.). (l) Flow cytometry-based quantification of tdT-labelled blood immune cells. Each symbol represents one mouse (Mrc1CreERT2/+R26tdT/tdT, n = 6; Mrc1CreERT2/CreERT2R26tdT/tdT, n = 3. Data are shown as mean ± s.e.m.
Extended Data Fig. 5 An Mrc1+ A2 population in the yolk sac gives rise to both microglia and CAMs.
(a) Gating strategies for yolk sac macrophage progenitors. c-KitloCD45+ cells (R1, blue) isolated from the wild-type yolk sac at E9.5 were used for scRNA-seq shown in Fig. 3a–d. CX3CR1–GFP−c-Kit+ A1 and CX3CR1–GFP+c-Kit− A2 cells isolated from the yolk sac of Cx3cr1GFP/+ mice were used for the analysis shown in Fig. 3e, f. Representative plots out of three mice are shown. (b) UMAP plots representing the core signature genes for monocytes (left) and granulocytes (right) in the yolk sac. (c) UMAP plots of genes that are enriched in clusters C2 and C4 (Slc2a3 and Apoa2), or C1, C5 and C6 (Psat1) are shown (483 yolk sac cells). Each dot represents a single cell. (d) UMAP plot of Ccnd2 gene enriched in cluster C3 is shown (483 yolk sac cells). Each dot represents a single cell. (e) Gating strategies for EMPs are shown. c-Kit+AA4.1+CD45neg-loF4/80− cells isolated from the Cx3cr1GFP/+ yolk sac at E8.5 were used for the analysis shown in Fig. 3e, f. Representative plots out of four mice are shown. (f) Representative immunofluorescence images depicting CD206− c-Kit+ EMPs in the yolk sac at E8.5 of Cx3cr1GFP/+ pregnant females. Immunolabelling was performed for c-Kit (red), CD206 (blue) and CD31 for yolk sac endothelia cells (white), respectively. White filled arrowhead indicates CD206− c-Kit+ EMPs, and blank white arrowheads indicate CD206+ yolk sac cells. Representative pictures out of four yolk sac investigated are shown. Scale bars: 50 μm, 20 μm (inset). (g) Scheme of the experimental set-up. (h) Representative gating strategy for EMPs, A1 and A2 cells isolated from the yolk sac of Mrc1CreERT2/CreERT2R26tdT/tdT embryos at E10.5. Plots show six individual yolk sacs that were concatenated for representation. (i) Quantification of tdT-expressing EMPs, A1 and A2 cells isolated from the yolk sac of Mrc1CreERT2/CreERT2R26tdT/tdT embryos at E10.5 (n = 6). (j–l) Representative gating strategy (j) for the quantification of tdT-expressing CD206– and CD206+ macrophages isolated from the brains of Mrc1CreERT2/CreERT2R26tdT/tdT embryos at E16.5 (k: n = 6) or E17.5 (l: n = 6). (m–o) Representative gating strategy (m) for the quantification of tdT-expressing lineage-restricted progenitors (LRP), short-term (LT-) hematopoietic stem cells (HSC) and long-term (LT-) HSC isolated from the fetal livers of Mrc1CreERT2/CreERT2R26tdT/tdT embryos at E16.5 (n: n = 6) or E17.5 (o: n = 6). (p) Scheme of the experimental set-up. (q) Quantification of tdT+ microglia, pvMΦ and mMΦ in Mrc1CreERT2/+R26tdT/tdT mice. Each symbol represents one mouse (n = 4 mice). Three sections/mouse were quantified (means ± s.e.m.).
Extended Data Fig. 6 No contribution of bone-marrow-derived cells or microglia to the pool of perivascular macrophages during development, but dependence on the transcription factors Irf8 and Mafb.
(a) Scheme of the experimental set-up. (b) Flow cytometry-based quantification of tdT-labelled Ly6Chi or Ly6clo blood monocytes in Cxcr4CreERT2/+R26tdT/+ mice (n = 3). Symbols represent individual mice. Data are shown as mean ± s.e.m. (c) Left, representative immunofluorescence images of tdT signals (red) in vessel cells, but not in pvMΦ (CD206, green) and microglia (IBA1, cyan), combined with immunofluorescence for Col IV (blue), from Cxcr4CreERT2/+R26tdT/tdT mice at P14. Blank white, and blank yellow arrowheads indicate single positive microglia or single positive pvMΦ, respectively. Representative images from three mice are depicted. Scale bars: 50 µm. Right, quantification of tdT-expressing microglia (MG, IBA1), pvMΦ (CD206) and mMΦ (CD206) in Cxcr4CreERT2/+R26tdT/tdT mice. Each symbol represents one mouse (n = 3). Three sections per mouse were quantified (means ± s.e.m.). (d) Scheme of experimental set-up. (e) Flow cytometry-based quantification of YFP-labelled Ly6Chi or Ly6clo blood monocytes in HexbCreERT2/CreERT2R26YFP/YFP mice (n = 7). Each symbol represents one mouse. Data are shown as mean ± s.e.m. (f) Left, representative immunofluorescence images of YFP signals (green) in microglia (IBA1, purple), but not in pvMΦ (CD206, red) and mMΦ (CD206, red), combined with immunofluorescence for Col IV (blue), from HexbCreERT2/CreERT2R26YFP/YFP mice at P14. Filled white and blank yellow arrowheads indicate double positive microglia or single positive pvMΦ/mMΦ, respectively. Representative images from seven mice are depicted. Scale bars: 50 µm. Right, quantification of YFP-expressing microglia (MG, IBA1+), pvMΦ (CD206+) and mMΦ (CD206+) in HexbCreERT2/CreERT2R26YFP/YFP mice. Each symbol represents one mouse (n = 7). Three sections per mouse were quantified. Means ± s.e.m. (g) Scheme of experimental set-up. (h) Quantitative PCR of Irf8 mRNA levels in sorted microglia and CAMs from Cx3cr1CreERT2/+Irf8fl/fl and controls at P6. Each symbol represents one mouse (n = 6 for control, n = 10 for Cx3cr1CreERT2/+Irf8fl/fl). Two-sided unpaired t-test. (i) Representative immunofluorescence images from the cortex of Cx3cr1CreERT2/+Irf8fl/fl and control mice for CD11b (red), CD206 (green) and laminin (blue) depicting microglia (blank arrowheads) and pvMΦ (filled arrowheads). Scale bars: 50 µm. (j) Quantification of microglia (MG) and pvMΦ. Each symbol represents one mouse (Irf8fl/fl, n = 6; Cx3cr1CreERT2/+Irf8fl/fl, n = 7). Three sections per mouse were quantified. Means ± s.e.m. Two-sided unpaired t-test. (k) Histological pictures from the meninges of Cx3cr1CreERT2/+Irf8fl/fl and control mice for CD11b (red), CD206 (green) and laminin (blue) showing microglia (blank arrowheads) and mMΦ (filled arrowheads). Scale bars: 50 µm. (l) Quantification thereof. Each symbol represents one mouse (Irf8fl/fl, n = 6; Cx3cr1CreERT2/+Irf8fl/fl, n = 7). Three sections per mouse were quantified. Means ± s.e.m. Two-sided unpaired t-test. (m) Scheme of experimental set-up. (n) Quantitative PCR of Mafb mRNA levels in sorted microglia and CAMs from Cx3cr1CreERT2/+Mafbfl/fl (n = 9 mice) and controls (n = 10 mice) at P6. Each symbol represents one mouse. Two-sided unpaired t-test. (o) Typical histological images from the cortex of Cx3cr1CreERT2/+Mafbfl/fl and control mice for CD11b (red), CD206 (green) and laminin (blue) depicting pvMΦ (filled arrowheads). Scale bars: 50 µm. (p) Quantification of microglia (MG) and pvMΦ. Each symbol represents one mouse (n = 7 per genotype). Three sections per mouse were quantified. Means ± s.e.m. Two-sided unpaired t-test. (q) Histological pictures from the meninges of Cx3cr1CreERT2/+Mafbfl/fl mice for CD11b (red), CD206 (green) and laminin (blue) showing mMΦ (filled arrowheads). Scale bars: 50 µm. (r) Quantification thereof. Each symbol represents one mouse (n = 7 per genotype). Three sections per mouse were examined. Means ± s.e.m. Two-sided unpaired t-test.
Extended Data Fig. 7 Different roles of integrin signalling in the development of microglia, meningeal and perivascular macrophages.
(a) Illustrative electron microscopy images depicting the intersection of a brain-penetrating arterial blood vessel with the leptomeninges. Left: overview, 1: higher magnification of the area indicated in the overview image and a consecutive section (1*) of the same area. Arrowheads depicting a gap (in 1) and a direct contact (in 1*) between processes of pial cells that cover the PVS. Yellow: pia mater, Blue: arachnoid mater, Asterisk: PVS, L: vessel lumen, N: neuropil. Representative images out of 6 mice investigated are shown. (b) Representative images showing IBA1+ or CD206+ cells in the parenchyma, the perivascular compartment, or the brain surface of either control Tlnfl/fl or Cx3cr1CreTlnfl/fl embryos at E14.5. Col IV staining (red) was also included. Representative images from five (Tlnfl/fl) or six mice (Cx3cr1CreTlnfl/fl) are depicted. Scale bars: 50 µm. (c) Quantification thereof. Each symbol represents one embryo (n = 5 for Tlnfl/fl; n = 6 for Cx3cr1CreTlnfl/fl). Three sections/mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test. Ns: not significant. (d) Representative images showing claudin 5 (Cldn5, cyan), fibrinogen (red) and laminin (grey) immunoreactivity in the cortex of Cx3cr1Cre/+Tln1fl/fl and Tln1fl/fl controls. Arrowheads indicate fibrinogen immunofluorescence within the vessel lumen. Representative images from four (Tlnfl/fl) or six mice (Cx3cr1CreTlnfl/fl) are depicted. Scale bar: 50 µm. (e) Quantification of the area covered by Cldn5 or fibrinogen immunofluorescence, respectively. Each symbol represents one mouse (n = 4 for Tlnfl/fl; n = 6 for Cx3cr1CreTlnfl/fl). Three sections per mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test. Ns: not significant. (f) Left, representative immunofluorescence images of IBA1+ microglia (red) and CD206+ pvMΦ (green), combined with immunofluorescence for Col IV (blue), in control Itgb1fl/fl and VavCreItgb1fl/fl mice at P14. Representative images from four (Itgb1fl/fl) or seven (VavCreItgb1fl/fl) mice are depicted. Scale bar: 50 μm. Right, quantification thereof (n = 4 for Itgb1fl/fl, n = 7 for VavCreItgb1fl/fl). Three sections per mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test. (g) Left, representative immunofluorescence images of CD206+ mMΦ green) with Col IV+ basement membranes (blue) in control Itgb1fl/fl and VavCre Itgb1fl/fl mice at P14. Representative images from four (Itgb1fl/fl) or seven (VavCreItgb1fl/fl) mice are depicted. Scale bar: 50 μm. Right, quantification thereof (n = 4 for Itgb1fl/fl, n = 7 for VavCreItgb1fl/fl). Three sections per mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test.
Extended Data Fig. 8 Distribution of microglia, meningeal and perivascular macrophages in the brains of Fgf2−/−or Pdgfrb−/F7 mice.
(a, b) Representative immunofluorescence of IBA1+ microglia (red), CD206+ pvMΦ (green) and Col IV+ basement membranes (white) in the cortex of Fgf2+/+ or Fgf2−/− mice (a), and Pdgfrb+/+ or Pdgfrb−/F7 mice (c) at P14. Images representative of 3 (Fgf2+/+ or Fgf2−/−), 4 (Pdgfrb+/+) or 5 (Pdgfrb−/F7) mice. Scale bar: 30μm. (b, d) Representative immunofluorescence of IBA1+ (red) CD206+ (green) mMΦ and Col IV+ meninges (white) in the cortical surfaces of Fgf2+/+ or Fgf2−/− mice (b), and Pdgfrb+/+ or Pdgfrb−/F7 mice (d) at P14. Images representative of 3 (Fgf2+/+ or Fgf2−/−), 4 (Pdgfrb+/+) or 5 (Pdgfrb−/F7) mice. Scale bar: 30 μm. (e) Upper: Representative images from the cortex of Pdgfrb+/+ or Pdgfrb−/F7 mice at P14. Yellow arrowheads depict IBA1+ (red) P2ry12+ (green) CD206− (grey) microglia and cyan arrowheads reveal IBA1+ P2ry12− CD206+ pvMΦ. Images are representative of four mice per genotype. Lower: The amount of P2ry12+ microglia was quantified. Symbols represent individual moice (n = 4 for both genotypes). Three sections per mouse were quantified (means ± s.e.m.). Two-sided unpaired t-test. Ns: not significant. (f, h, j, l, n) Immunofluorescence of microglia (IBA1, green) and mMΦ (CD206, white), in combination with Col IV+ basal membrane (blue), in Notch3+/− (f), Notch3−/− (h), Rbpjfl/fl (j), SMMHCCreERT2Rbpjfl/fl (l), and Cx3cr1CreERT2/+Rbpjfl/fl mice (n), respectively. Representative images from 8 (Notch3+/−, Notch3−/−), 6 (Rbpjfl/fl), 7 (SMMHCCreERT2Rbpjfl/fl), and 4 (Cx3cr1CreERT2/+Rbpjfl/fl) mice are depicted. Scale bar: 50 μm. (g, i, k, m, o) Immunofluorescence of pvMΦ (CD206, white) in the aSMA+(red) TfR-(green) artery in combination with laminin for basement membranes (blue) in Notch3+/− (g), Notch3−/− (i), Rbpjfl/fl (k), SMMHCCreERT2Rbpjfl/fl (m), and Cx3cr1CreERT2/+Rbpjfl/fl mice (o), respectively. Representative images from 8 (Notch3+/−, Notch3−/−), 6 (Rbpjfl/fl), 7 (SMMHCCreERT2Rbpjfl/fl), and 4 (Cx3cr1CreERT2/+Rbpjfl/fl) mice are shown. Scale bar: 50 μm. (p) Representative images of Ki-67 (green) and cleaved-caspase3 (Cas3, magenta) immunoreactivity in CD206+ (white) mMΦ (top) or CD206+ pvMΦ (bottom) in Notch3+/− and Notch3−/− mice at P14 (n = 4). Scale bars: 50 µm, 10 µm (insets). (q) Quantification of Ki-67+ or Cas3+ mMΦ (top) and pvMΦ (bottom) in the cortex of Notch3+/+ (adult: n = 3), Notch3+/− (P14: n = 4) and Notch3−/− (P14: n = 4, adult: n = 3) mice at indicated time points. Three sections per mouse were quantified (means ± s.e.m.). Each symbol represents one mouse. Ns: not significant.
Extended Data Fig. 9 Microglia and CAM transcriptomes are not affected by the loss of Notch3.
(a) Scheme of the experimental set-up for the scRNA-seq analysis of brain macrophages from Notch3 mice. (b) UMAP plot of 1523 individual cells isolated from P14 Notch3+/+, Notch3+/– or Notch3–/– mice. Each dot represents an individual cell. Microglia and CAM clusters (0, 2, 3, 9 and 12) are framed with a dashed line. (c) Heat map of the top 10 differentially expressed genes for each cluster. (d) Feature plots depicting the expression of the typical brain macrophage genes Csf1r and Cx3cr1. (e, f) Feature plots showing the expression of microglial core genes (d) CAM core genes (e). (g–j) Re-analysis of the microglia and CAM clusters. Feature plots depicting the expression of microglial core genes (g) or CAM core genes (h). UMAP plot showing the individual clusters (i) and heat map (j) of the top 10 differentially expressed genes for each cluster. (k) Scheme of the experimental set-up for the scRNA-seq analysis of vascular smooth muscle cells from Notch3+/+SM22aCreERT2R26tdT or Notch3−/−SM22aCreERT2R26tdT mice. (l) Feature plots showing the expression of the arterial vascular smooth muscle cell core genes Acta2 and Aft3.
Extended Data Fig. 10 Graphical abstract of experimental findings.
Upper illustration depicts developmental trajectories of microglia and CAMs. Lower image shows stages of development in respective cell-types and blood brain barrier (BBB). EMPs: erythro-myeloid progenitors, mMΦ: meningeal macrophages, pvMΦ: perivascular macrophages, aVSMC: arterial vascular smooth muscle cells, pv: perivascular, E: embryonic days, P: postnatal days.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1.
Source data
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Masuda, T., Amann, L., Monaco, G. et al. Specification of CNS macrophage subsets occurs postnatally in defined niches. Nature 604, 740–748 (2022). https://doi.org/10.1038/s41586-022-04596-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-022-04596-2
This article is cited by
-
Understanding immune microenvironment alterations in the brain to improve the diagnosis and treatment of diverse brain diseases
Cell Communication and Signaling (2024)
-
Role of meningeal immunity in brain function and protection against pathogens
Journal of Inflammation (2024)
-
Granulocyte macrophage colony-stimulating factor-induced macrophages of individuals with autism spectrum disorder adversely affect neuronal dendrites through the secretion of pro-inflammatory cytokines
Molecular Autism (2024)
-
Human CNS-associated macrophages decoded in time and space
Nature Medicine (2024)
-
Analysis of brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke
Nature Immunology (2024)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
