ART ic LE S Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways Katrin Kierdorf1,2, Daniel Erny1, Tobias Goldmann1, Victor Sander1, Christian Schulz3,4, Elisa Gomez Perdiguero3,4, Peter Wieghofer1,2, Annette Heinrich5, Pia Riemke6, Christoph Hölscher7,8, Dominik N Müller9, Bruno Luckow10, Thomas Brocker11, Katharina Debowski12, Günter Fritz1, Ghislain Opdenakker13, Andreas Diefenbach14, Knut Biber5,15, Mathias Heikenwalder16, Frederic Geissmann3,4, Frank Rosenbauer6 & Marco Prinz1,17 Microglia are crucial for immune responses in the brain. Although their origin from the yolk sac has been recognized for some time, their precise precursors and the transcription program that is used are not known. We found that mouse microglia were derived from primitive c-kit+ erythromyeloid precursors that were detected in the yolk sac as early as 8 d post conception. + lo − + − + These precursors developed into CD45 c-kit CX3CR1 immature (A1) cells and matured into CD45 c-kit CX3CR1 (A2) cells, as evidenced by the downregulation of CD31 and concomitant upregulation of F4/80 and macrophage colony stimulating factor receptor (MCSF-R). Proliferating A2 cells became microglia and invaded the developing brain using specific matrix metalloproteinases. Notably, microgliogenesis was not only dependent on the transcription factor Pu.1 (also known as Sfpi), but also required Irf8, which was vital for the development of the A2 population, whereas Myb, Id2, Batf3 and Klf4 were not required. Our data provide cellular and molecular insights into the origin and development of microglia. Microglia are the tissue macrophages of the brain and scavenge dying have the ability to give rise to microglia and macrophages in vitro cells, pathogens and molecules using pattern recognition receptors and in vivo under defined conditions. During their journey into the and subsequent phagocytosis and endocytosis1–3. Unlike macroglia developing CNS, these cells acquired macrophage-specific markers (astrocytes and oligodendrocytes) and neurons, which are derived and finally settled in the target tissue, where they differentiated into from the neuroectoderm, microglia progenitors arise from the periph- definitive microglia with fine processes. Of note, this extremely com- eral mesodermal tissue4,5. Their origin from the yolk sac, which was plex developmental process required specific molecules that could © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature proposed in 1999 (ref. 6), was confirmed by two recent fate-mapping be identified at defined time points. Our results describe microglia studies7,8. One study used a tamoxifen-inducible Cre recombinase as a distinct tissue-specific macrophage population that is clearly under the control of Runx1, a transcription factor involved in the genetically different from circulating myeloid cells. npg development of all lineages of normal hematopoiesis7. In a more recent study, we found that microglia were derived from MCSF- RESULTS R–expressing yolk sac precursors that were present between 8.5 and c-kit+ erythromyeloid cells are the microglia progenitors 9.5 days post conception (dpc)8. However, these studies focused on To follow the fate of late myeloid cells in the brain during embryogen- GFP/wt already committed CX3CR1-expressing yolk sac myeloid cells and esis, we used Cx3cr1 mice, which have a GFP knock-in on one therefore considered immature macrophages to be microglia precur- allele of the Cx3cr1 gene. CX3CR1 is expressed in mature monocytes sors, thereby missing possible earlier progenitors that lack macro- and in a subset of peripheral mononuclear phagocytes. In the brain, phage markers such as F4/80 and CD11b. only microglia express this marker9,10. We found that uncommitted c-kit+ stem cells that have both eryth- We first addressed the question of the origin of Cx3cr1GFP/wt micro- roid and myeloid potential are the direct yolk sac–derived precur- glia progenitors in the mouse embryo. It has been shown that hemato- sors of microglia during early embryogenesis. These primitive cells poietic cells can be independently generated in two distinct sites. 1Department of Neuropathology, University of Freiburg, Freiburg, Germany. 2Faculty of Biology, University of Freiburg, Freiburg, Germany. 3Centre for Molecular and Cellular Biology of Inflammation, King’s College London, London, UK. 4Peter Gorer Department of Immunobiology, King’s College London, London, UK. 5Department of Psychiatry, University of Freiburg, Freiburg, Germany. 6Institute of Molecular Tumor Biology, University of Muenster, Muenster, Germany. 7Division of Infection Immunology, Research Center Borstel, Borstel, Germany. 8Cluster of Excellence, Inflammation at Interfaces (Borstel-Kiel-Lübeck-Plön), Kiel, Germany. 9Experimental and Clinical Research Center, Charité Medical Faculty and the Max-Delbrück Center for Molecular Medicine, Berlin, Germany. 10Klinikum der Universität München, Medizinische Klinik und Poliklinik IV, Arbeitsgruppe Klinische Biochemie, München, Germany. 11Institute for Immunology, Ludwig-Maximilians-Universität München, Munich, Germany. 12Cell Research Group, German Primate Center, Göttingen, Germany. 13Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Belgium. 14Department of Microbiology, University of Freiburg, Freiburg, Germany. 15Department of Neuroscience, University Medical Center Groningen, Groningen, The Netherlands. 16Institute of Virology, Technische Universität München/Helmholtz-Zentrum Munich, Munich, Germany. 17Center for Biological Signaling Studies, University of Freiburg, Freiburg, Germany. Correspondence should be addressed to M.P. ([email protected]). Received 6 November 2012; accepted 21 December 2012; published online 20 January 2013; corrected online 6 February 2013 (details online); doi:10.1038/nn.3318 NATURE NEUROSCIENCE VOLUME 16 | NUMBER 3 | MARCH 2013 273 ART ic LE S dpc 8.0 dpc 9.0 CX CR1+ a b Cell population 3 Ter119+ day 7 CD45+ day 7 M CD45+ c-kit– nd nd 4.9 A – – A1 A2 CD45 c-kit nd nd CD45 CD45– c-kit+ (EMP) 70–80% 5–10% 1.8 BC c-kit c-kit CX3CR1-GFP 78.2 CD45 Yolk sac macrophages dpc 10.5 A1 A2 FSC-A 5.3 CX3CR1-GFP Ter119 A A1 A2 56.5 3.9 CD45 Cell population CX CR1+ CX CR1+ BC c 3 3 CD45+ day 7 CD45+ day 28 c-kit CX3CR1-GFP c-kit c-kit + – CD45 CX3CR1 (A1) 5–15% >90% Head macrophages dpc 10.5 A1 A2 + + CD45 CX3CR1 (A2) nd nd A A1 A2 10.2 0.0 10.7 99.5 CD45 CD45 CD45 BC c-kit CX CR1-GFP c-kit c-kit 3 CX3CR1-GFP CX3CR1-GFP d Depleted EMP transplanted CD45+ c-kit– transplanted A1 transplanted A2 transplanted Iba-1 Iba-1 DAPI CX3CR1 Iba-1 CX3CR1 CX3CR1 CX3CR1 CX3CR1 CX3CR1 e EMP CD45+ c-kit– macrophages Adult microglia © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature EMP CD45+ c-kit– macrophages 15 µm 15 µm 13 µm Adult microglia npg 800 Dendrite length 80 Branching points 150 Dendrite segments 80 Dendrite m) terminal points µ 600 60 60 100 400 40 40 *** *** *** 50 *** Number of Number of 200 20 Number of 20 branching points dendrite segments Dendrite length ( 0 0 0 dendrite terminal points 0 Figure 1 Identification and characterization of the microglia progenitor. (a) Top, fractionation of 8.0-dpc yolk sac cells using CD45 and c-kit markers allowed for distinction of separate subsets of precursor cells by fluorescence-activated cell sorting (FACS, left). We analyzed 9.0-dpc yolk sac from a GFP/GFP wt/wt + male Cx3cr1 and female Cx3cr1 mating (right). Separation of CD45 cells allowed for discrimination of two subsets designated A1 (CX3CR1- − + + − GFP ) and A2 (CX3CR1-GFP ) and a maternal (M) CD45 CX3CR1-GFP population. Middle and bottom, characterization of myeloid precursors in the yolk sac and the head at 10.5 dpc. In the CD45-expressing cells in the brain, GFP+ and GFP− populations were found. The CD45 fraction in the yolk sac can be further subdivided according to different levels of c-kit expression, namely into CD45+ c-kit− and CD45+ c-kit+ in A1 and a single CD45+ c-kit− population in A2. Data are representative of four independent experiments. Percentages are indicated. (b) In vitro differentiation potential of 8.0-dpc yolk sac subsets defined in a. The hematopoietic potential was strictly ascribed to the CD45− c-kit+ subset that comprised myeloid and erythroid precursors, a characteristic feature of EMPs. Data are representative of three independent experiments. nd, not detectable. (c) In vitro differentiation potential of 9.0-dpc yolk sac populations shown in a. Only c-kit+ A1 cells differentiated into the myeloid lineage at the indicated time points. Representative dot plots are depicted. Morphology of the cultured cells is illustrated with Pappenheim staining. Data are representative of two independent experiments. (d) Differentiation of adoptively transferred yolk sac subsets on organotypic hippocampal brain slice cultures depleted of endogenous microglia before cell transfer. At 12 d in culture, transferred yolk sac cells from embryos of a male Cx3cr1GFP/GFP and female Cx3cr1wt/wt cross-breeding were analyzed using the macrophage marker Iba-1 and CX3CR1-GFP. Scale bars represent 200 µm (top panels) and 20 µm (bottom panels and insets). Data are representative of three independent experiments. (e) Three-dimensional reconstruction (top) and Imaris-based automatic quantification of cell morphology (bottom) of transferred cells. Data represent means ± s.e.m. of at least five cells per group. ***P < 0.001. Data are representative of two independent experiments. 274 VOLUME 16 | NUMBER 3 | MARCH 2013 NATURE NEUROSCIENCE ART ic LE S The first site, the yolk sac, generates erythroid cells and macrophages that could act as the direct microglia precursors. To test the potential starting at 7.5–8.0 dpc, that is, before microglia progenitors are first of yolk sac cells to differentiate into microglia cells in situ, we used detected in the neural folds11–13. To determine whether the yolk sac microglia-free organotypic hippocampal slice cultures (OHSCs) as contains progenitor cells with macrophage properties, we analyzed described previously15 (Fig. 1d).