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• W2807609377 abstract "•Brain signals induce and sustain homeostatic gene expression in microglia•Hematopoietic stem cell (HSC)-derived macrophages attain a microglia-like identity•Stable markers and gene signatures betray HSC origin•Macrophages with HSC origin markers are found in human neurodegeneration Microglia, the brain’s resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies. Microglia, the brain’s resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies. Microglia are vital residents of the brain parenchyma, where they play important but poorly understood roles in development, injury, and disease (Li and Barres, 2018Li Q. Barres B.A. Microglia and macrophages in brain homeostasis and disease.Nat. Rev. Immunol. 2018; 18: 225-242Crossref PubMed Scopus (822) Google Scholar, Salter and Stevens, 2017Salter M.W. Stevens B. Microglia emerge as central players in brain disease.Nat. Med. 2017; 23: 1018-1027Crossref PubMed Scopus (842) Google Scholar). Arising from yolk sac erythro-myeloid progenitors (YS EMPs) that colonize neural tissue during embryogenesis and undergo a distinct maturational process, microglia are unique among tissue macrophages in that they are thought to remain YS derived throughout life, without contribution from the fetal liver or definitive hematopoiesis (Ginhoux et al., 2010Ginhoux F. Greter M. Leboeuf M. Nandi S. See P. Gokhan S. Mehler M.F. Conway S.J. Ng L.G. Stanley E.R. et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (3172) Google Scholar, Hagemeyer et al., 2016Hagemeyer N. Kierdorf K. Frenzel K. Xue J. Ringelhan M. Abdullah Z. Godin I. Wieghofer P. Costa Jordão M.J. Ulas T. et al.Transcriptome-based profiling of yolk sac-derived macrophages reveals a role for Irf8 in macrophage maturation.EMBO J. 2016; 35: 1730-1744Crossref PubMed Scopus (84) Google Scholar, Hoeffel et al., 2015Hoeffel G. Chen J. Lavin Y. Low D. Almeida F.F. See P. Beaudin A.E. Lum J. Low I. Forsberg E.C. et al.C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.Immunity. 2015; 42: 665-678Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, Kierdorf et al., 2013Kierdorf K. Erny D. Goldmann T. Sander V. Schulz C. Perdiguero E.G. Wieghofer P. Heinrich A. Riemke P. Hölscher C. et al.Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways.Nat. Neurosci. 2013; 16: 273-280Crossref PubMed Scopus (918) Google Scholar, Gomez Perdiguero et al., 2015Gomez Perdiguero E. Klapproth K. Schulz C. Busch K. Azzoni E. Crozet L. Garner H. Trouillet C. de Bruijn M.F. Geissmann F. Rodewald H.R. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1336) Google Scholar, Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E.W. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (1724) Google Scholar). After insults such as experimental autoimmune encephalomyelitis, stroke, malignancy, or genetic depletion, however, microglia dramatically change their phenotype and are joined by infiltrating monocytes/macrophages from the circulation (Ajami et al., 2011Ajami B. Bennett J.L. Krieger C. McNagny K.M. Rossi F.M.V. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.Nat. Neurosci. 2011; 14: 1142-1149Crossref PubMed Scopus (760) Google Scholar, Varvel et al., 2012Varvel N.H. Grathwohl S.A. Baumann F. Liebig C. Bosch A. Brawek B. Thal D.R. Charo I.F. Heppner F.L. Aguzzi A. et al.Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells.Proc. Natl. Acad. Sci. USA. 2012; 109: 18150-18155Crossref PubMed Scopus (193) Google Scholar). These CNS-alien, hematopoietic stem cell (HSC)-derived occupants can resemble microglia in morphology and surface marker expression but appear to participate differently in disease pathogenesis, making it essential to further clarify their functions. Identification of a microglia-specific gene cassette has improved resolution of resident from infiltrating cells (Bennett et al., 2016Bennett M.L. Bennett F.C. Liddelow S.A. Ajami B. Zamanian J.L. Fernhoff N.B. Mulinyawe S.B. Bohlen C.J. Adil A. Tucker A. et al.New tools for studying microglia in the mouse and human CNS.Proc. Natl. Acad. Sci. USA. 2016; 113: E1738-E1746Crossref PubMed Scopus (1025) Google Scholar, Hickman et al., 2013Hickman S.E. Kingery N.D. Ohsumi T.K. Borowsky M.L. Wang L.-C. Means T.K. El Khoury J. The microglial sensome revealed by direct RNA sequencing.Nat. Neurosci. 2013; 16: 1896-1905Crossref PubMed Scopus (951) Google Scholar), but does not address whether infiltrating cells inherently lack the capacity to become microglia under appropriate conditions, nor whether microglia can irreversibly lose their identity in abnormal environments. Although circulating monocytes can effectively differentiate into tissue macrophages in the lung and liver (Scott et al., 2016Scott C.L. Zheng F. De Baetselier P. Martens L. Saeys Y. De Prijck S. Lippens S. Abels C. Schoonooghe S. Raes G. et al.Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells.Nat. Commun. 2016; 7: 10321Crossref PubMed Scopus (449) Google Scholar, van de Laar et al., 2016van de Laar L. Saelens W. De Prijck S. Martens L. Scott C.L. Van Isterdael G. Hoffmann E. Beyaert R. Saeys Y. Lambrecht B.N. Guilliams M. Yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages.Immunity. 2016; 44: 755-768Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar), the CNS environment is highly distinct and protected from exposure to circulating factors and cells by the blood-brain barrier (BBB) (Obermeier et al., 2013Obermeier B. Daneman R. Ransohoff R.M. Development, maintenance and disruption of the blood-brain barrier.Nat. Med. 2013; 19: 1584-1596Crossref PubMed Scopus (1333) Google Scholar). As such, the precise contributions of ontogeny and CNS environment, which have been highly implicated in microglial identity (Gosselin et al., 2014Gosselin D. Link V.M. Romanoski C.E. Fonseca G.J. Eichenfield D.Z. Spann N.J. Stender J.D. Chun H.B. Garner H. Geissmann F. Glass C.K. Environment drives selection and function of enhancers controlling tissue-specific macrophage identities.Cell. 2014; 159: 1327-1340Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, Lavin et al., 2014Lavin Y. Winter D. Blecher-Gonen R. David E. Keren-Shaul H. Merad M. Jung S. Amit I. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment.Cell. 2014; 159: 1312-1326Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar, Mass et al., 2016Mass E. Ballesteros I. Farlik M. Halbritter F. Günther P. Crozet L. Jacome-Galarza C.E. Händler K. Klughammer J. Kobayashi Y. et al.Specification of tissue-resident macrophages during organogenesis.Science. 2016; 353: aaf4238Crossref PubMed Scopus (467) Google Scholar), remain incompletely understood. Do microglia permanently change their identity following disease, or can they return to a homeostatic state? Like YS-derived cells, can macrophages derived from HSCs become microglia? The answers are critical not only to how microglia and infiltrating myeloid cells affect the brain, but also to the growing use of myeloid cell therapies such as HSC transplantation to treat brain disease in humans (Biffi et al., 2013Biffi A. Montini E. Lorioli L. Cesani M. Fumagalli F. Plati T. Baldoli C. Martino S. Calabria A. Canale S. et al.Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.Science. 2013; 341: 1233158Crossref PubMed Scopus (906) Google Scholar). Since our goal was to precisely measure how ontogeny and environment affect microglial identity, we aimed to create a system for transplantation of myeloid cells across development into the brain. We took advantage of Csf1r−/− mice, which lack microglia (Dai et al., 2002Dai X.-M. Ryan G.R. Hapel A.J. Dominguez M.G. Russell R.G. Kapp S. Sylvestre V. Stanley E.R. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects.Blood. 2002; 99: 111-120Crossref PubMed Scopus (826) Google Scholar, Ginhoux et al., 2010Ginhoux F. Greter M. Leboeuf M. Nandi S. See P. Gokhan S. Mehler M.F. Conway S.J. Ng L.G. Stanley E.R. et al.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (3172) Google Scholar, Liddelow et al., 2017Liddelow S.A. Guttenplan K.A. Clarke L.E. Bennett F.C. Bohlen C.J. Schirmer L. Bennett M.L. Münch A.E. Chung W.-S. Peterson T.C. et al.Neurotoxic reactive astrocytes are induced by activated microglia.Nature. 2017; 541: 481-487Crossref PubMed Scopus (3353) Google Scholar), and found that directly injected myeloid cells extensively engraft in the brain parenchyma, allowing study of donor populations with varied ontogeny. Transplantation into Csf1r−/− hosts offers several advantages. It can be used to study donor cells of diverse origin and developmental stage, and does not require conditioning irradiation or chemotherapy. It yields large numbers of donor-derived microglia-like cells (MLCs) that have been conditioned by the brain parenchyma to express microglial genes in the absence of potentially confounding host macrophages, overcoming limitations of prior foundational approaches to understanding microglial identity (Bennett et al., 2016Bennett M.L. Bennett F.C. Liddelow S.A. Ajami B. Zamanian J.L. Fernhoff N.B. Mulinyawe S.B. Bohlen C.J. Adil A. Tucker A. et al.New tools for studying microglia in the mouse and human CNS.Proc. Natl. Acad. Sci. USA. 2016; 113: E1738-E1746Crossref PubMed Scopus (1025) Google Scholar, Bruttger et al., 2015Bruttger J. Karram K. Wörtge S. Regen T. Marini F. Hoppmann N. Klein M. Blank T. Yona S. Wolf Y. et al.Genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system.Immunity. 2015; 43: 92-106Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, Mildner et al., 2007Mildner A. Schmidt H. Nitsche M. Merkler D. Hanisch U.-K. Mack M. Heikenwalder M. Brück W. Priller J. Prinz M. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.Nat. Neurosci. 2007; 10: 1544-1553Crossref PubMed Scopus (812) Google Scholar). By comparing multiple engrafted microglia types to MLCs from YS and HSC lineages, we found that microglial identity remains intact ex vivo, even following cell culture. We noted general similarity between MLCs derived from all donor lineages, but found striking ontogeny-dependent differences between HSC- and YS-derived populations, leading to discovery of durable markers of parenchymal macrophage ontogeny. We extended this approach to a humanized transplantation system and verified fundamental conclusions in human microglia and MLCs. In sum, we devised an experimental system to unravel the contributions of brain environment and ontogeny to macrophage identity in mouse and human. We recently demonstrated that cultured microglia have the capacity to engraft in the Csf1r−/− brain parenchyma, which otherwise lacks microglia, after intracerebral transplantation (ICT) (Bohlen et al., 2017Bohlen C.J. Bennett F.C. Tucker A.F. Collins H.Y. Mulinyawe S.B. Barres B.A. Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures.Neuron. 2017; 94: 759-773.e8Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). To further study intrinsic versus acquired properties of microglial identity, we compared three distinct microglia populations after ICT into the CNS between postnatal day 0 and 4 (P0–P4): (1) acutely isolated mature microglia (P21, ICT MG); (2) developmentally immature microglia (P5, ICT P5 MG), which lack expression of the full microglial gene cassette (Bennett et al., 2016Bennett M.L. Bennett F.C. Liddelow S.A. Ajami B. Zamanian J.L. Fernhoff N.B. Mulinyawe S.B. Bohlen C.J. Adil A. Tucker A. et al.New tools for studying microglia in the mouse and human CNS.Proc. Natl. Acad. Sci. USA. 2016; 113: E1738-E1746Crossref PubMed Scopus (1025) Google Scholar); and (3) cultured microglia (P18–P35, ICT Cultured MG), which undergo dramatic transcriptional changes in vitro including loss of expression of the microglial signature cassette (Bohlen et al., 2017Bohlen C.J. Bennett F.C. Tucker A.F. Collins H.Y. Mulinyawe S.B. Barres B.A. Diverse requirements for microglial survival, specification, and function revealed by defined-medium cultures.Neuron. 2017; 94: 759-773.e8Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, Gosselin et al., 2017Gosselin D. Skola D. Coufal N.G. Holtman I.R. Schlachetzki J.C.M. Sajti E. Jaeger B.N. O’Connor C. Fitzpatrick C. Pasillas M.P. et al.An environment-dependent transcriptional network specifies human microglia identity.Science. 2017; 356: 1-10Crossref Scopus (587) Google Scholar) (Figure 1A). By 14 days after intracerebral injection, all donor microglia types extensively engrafted and ramified in the brain parenchyma, often filling entire sagittal sections (Figures 1B and S1A). When normalized to area of engraftment, transplanted microglia reached a similar density to endogenous microglia in a wild-type Csf1r+/+ (WT) host (Figure S1B). By flow cytometric analysis, engrafted cells were CD45+CD11B+ and expressed WT levels of TMEM119 (Figures S1C–S1E). By immunostaining, 100% were TMEM119+ in sections from 4–7 biological and at least 5 technical replicates each across the brain. As in WT mice, we found no TMEM119 staining in the meninges and choroid plexus of ICT mice (data not shown). Extent of donor cell engraftment varied—by fluorescence-activated cell sorting (FACS), we retrieved fewer microglia from transplanted hosts than WT controls and occasionally observed minimal to no engraftment. Because the host strain for Csf1r−/− transplant experiments was FVB, for which no robustly expressed fluorescent reporters exist, we also verified that sorted engrafted microglia were WT at the Csf1r locus (Figure S1F). These data show that microglia from multiple developmental stages can occupy the postnatal brain, ramify, and express TMEM119 only when engrafted in the parenchyma. To better understand relationships between microglial ontogeny, environment, and transcriptional phenotype, we used optimized techniques to isolate RNA from highly pure parenchymal microglia after ICT into Csf1r−/− hosts based on TMEM119 immunoreactivity (Bennett et al., 2016Bennett M.L. Bennett F.C. Liddelow S.A. Ajami B. Zamanian J.L. Fernhoff N.B. Mulinyawe S.B. Bohlen C.J. Adil A. Tucker A. et al.New tools for studying microglia in the mouse and human CNS.Proc. Natl. Acad. Sci. USA. 2016; 113: E1738-E1746Crossref PubMed Scopus (1025) Google Scholar). Transcriptomic profiling by RNA sequencing (RNA-seq) showed that by 14 days in vivo, microglia that had either lost expression of signature genes in vitro (ICT Cultured MG) or not attained full maturity (ICT P5 MG) expressed mature microglial signature genes at nearly normal levels, including Tmem119, P2ry12, Olfml3, and Sall1 (Figure 1C). More broadly, ICT cultured, P5, and adult microglia were highly similar to each other and to their WT counterparts. Of 1,827 differentially expressed genes in in vitro microglia, all but 16 returned to within 2-fold of WT levels after re-engraftment of cultured microglia in the CNS (Figure S1G). Volcano plot overlays demonstrate that differences between cultured WT microglia are largely restored after re-engraftment in the brain (Figure 1D). While transplanted microglia have statistically meaningful differences in gene expression compared to untransplanted WT microglia, these changes likely represent an “engraftment signature” from donor cell isolation, culture, and the Csf1r−/− host environment. Gene expression changes were generally of small magnitude and included several chemokines, tetraspanins, and G protein-coupled receptors, but not a signature of reactivity or specific functional process (Table S1). These experiments show that the Csf1r−/− CNS is sufficient to sustain, induce, and re-induce microglial identity, and that microglial identity potential persists despite dramatic transcriptional perturbations induced ex vivo. Given stable microglial identity despite highly plastic gene expression between adult, P5, and cultured microglia, we appreciated that ICT could clarify relationships between brain macrophage ontogeny and environment. In particular, we wondered whether HSC- or YS-derived macrophages originating outside the developed brain could become microglia in a permissive CNS environment capable of supporting homeostatic microglia. Therefore, we individually transplanted whole tissues and sorted myeloid cells into the Csf1r−/− CNS at P0–P4, including YS-derived cells from yolk sac and fetal brain, HSC-derived cells from blood and bone marrow (BM), and monocytes from the fetal liver, which, at embryonic day 13 (E13)–E14, contains a mix of HSC- and YS-derived cells (Hoeffel et al., 2015Hoeffel G. Chen J. Lavin Y. Low D. Almeida F.F. See P. Beaudin A.E. Lum J. Low I. Forsberg E.C. et al.C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.Immunity. 2015; 42: 665-678Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, Gomez Perdiguero et al., 2015Gomez Perdiguero E. Klapproth K. Schulz C. Busch K. Azzoni E. Crozet L. Garner H. Trouillet C. de Bruijn M.F. Geissmann F. Rodewald H.R. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1336) Google Scholar). We observed extensive engraftment of ramified IBA1+/TMEM119+ MLCs using all tissue types tested across both embryonic and postnatal lineages (Figures 2A, 2B, S2A–S2C, and S2F), though YS-derived MLCs (YS-MLCs) had a consistently more ramified morphology than HSC-derived MLCs (HSC-MLCs). We verified donor origin of MLCs using a GFP reporter after backcrossing the Csf1r−/− allele to C57BL/6 (Figure 2B), and additionally noted extensive coverage of the spinal cord by donor cells delivered by supratentorial injection (Figure S2D). By flow cytometry, nearly all CD45+CD11B+ cells were TMEM119 immunoreactive, although HSC donor tissues consistently showed lower intensity staining than WT (Figure 2C). Since we saw a small TMEM119-negative population in some cases, we again confirmed by immunostaining that, as with transplanted microglia, all parenchymal but no other IBA1+ MLCs were TMEM119+ (Figure S2G). All donor tissues engrafted to similar densities as microglia in Csf1r−/− brains, except for fetal liver, which reached a significantly higher density (Figure S2E). FACS plots, engraftment levels, and percent TMEM119-positive values are further detailed in Figure S2 to provide potential users a realistic assessment of the robustness of this system. An inherent limitation of the Csf1r−/− model is poor host viability, which required us to measure the effects of CNS residence in ICT experiments after 14 days. To better study the trajectory of effects of longer incubation, we also created a chemotherapy- and irradiation-free peripheral BM transplantation system that allows study of long-term MLC engraftment. Whereas Csf1r−/− mice do not typically survive past weaning age (Dai et al., 2002Dai X.-M. Ryan G.R. Hapel A.J. Dominguez M.G. Russell R.G. Kapp S. Sylvestre V. Stanley E.R. Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects.Blood. 2002; 99: 111-120Crossref PubMed Scopus (826) Google Scholar, Li et al., 2006Li J. Chen K. Zhu L. Pollard J.W. Conditional deletion of the colony stimulating factor-1 receptor (c-fms proto-oncogene) in mice.Genesis. 2006; 44: 328-335Crossref PubMed Scopus (88) Google Scholar), simple intraperitoneal (i.p.) injection of WT BM “rescued” approximately 50% of pups, leading to prolonged survival, tooth eruption, occasional fertility, and engraftment of donor-derived myeloid cells in multiple tissues including the brain parenchyma and liver (Figures 3A–3D, S3A, and S3B). By 1 month, the brain parenchyma of rescued mice showed complete, uniform coverage by donor-derived cells (Figure 3A). We harvested well-appearing rescued mice up to 1 year after transplantation and observed stable occupancy of the brain parenchyma by MLCs (Figure 3B). Taken together, these studies show that the CNS niche readily hosts macrophages from multiple donor tissues, including for long periods using BM. The surprising observation that intraperitoneally injected BM populated the Csf1r−/− brain without preconditioning led us to further characterize how donor cells might enter the brain. Since a prior study of macrophage repopulation by peripheral cells found evidence for increased BBB permeability (Varvel et al., 2012Varvel N.H. Grathwohl S.A. Baumann F. Liebig C. Bosch A. Brawek B. Thal D.R. Charo I.F. Heppner F.L. Aguzzi A. et al.Microglial repopulation model reveals a robust homeostatic process for replacing CNS myeloid cells.Proc. Natl. Acad. Sci. USA. 2012; 109: 18150-18155Crossref PubMed Scopus (193) Google Scholar), we tested for the presence of increased levels of IgG and albumin in the brain, which are largely excluded from the parenchyma under homeostatic conditions. Quantitative region of interest (ROI) analysis of immunostained histological sections showed no evidence for increased albumin or IgG extravasation, and we did not observe focal areas of increased staining, suggesting “normal” BBB permeability (Figure S3C). Since multiple studies suggest that monocyte infiltration into the diseased or injured CNS is facilitated by CCR2 (Ajami et al., 2011Ajami B. Bennett J.L. Krieger C. McNagny K.M. Rossi F.M.V. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.Nat. Neurosci. 2011; 14: 1142-1149Crossref PubMed Scopus (760) Google Scholar, Dzenko et al., 2001Dzenko K.A. Andjelkovic A.V. Kuziel W.A. Pachter J.S. The chemokine receptor CCR2 mediates the binding and internalization of monocyte chemoattractant protein-1 along brain microvessels.J. Neurosci. 2001; 21: 9214-9223Crossref PubMed Google Scholar), we wondered whether engraftment of MLCs was similarly CCR2 dependent in Csf1r−/− hosts. We found that, as with WT BM, i.p. injection of CCR2 Rfp/Rfp (Ccr2 knockout) BM (Saederup et al., 2010Saederup N. Cardona A.E. Croft K. Mizutani M. Cotleur A.C. Tsou C.-L. Ransohoff R.M. Charo I.F. Selective chemokine receptor usage by central nervous system myeloid cells in CCR2-red fluorescent protein knock-in mice.PLoS ONE. 2010; 5: e13693Crossref PubMed Scopus (395) Google Scholar) into Csf1r−/− mice leads to robust TMEM119+ MLC engraftment at 2 weeks, meaning that CCR2 is dispensable in this system (Figure 3E). Interestingly, we observed a range of RFP fluorescence levels in engrafted cells (Figure S3D), suggesting that either brain signals suppress CCR2 expression or more than one population of BM cells (as distinguished by CCR2 reporter expression) is capable of brain engraftment. We also noted a preponderance of RFP+ cells in a periventricular distribution (Figure S3E). Finally, to determine whether myeloid progenitors or HSCs were strictly required to create MLCs, we also transplanted BM monocytes that were stringently depleted of progenitor populations (Figure S3F) and observed abundant TMEM119+ parenchymal MLCs by 2 weeks (Figure 3F). The ability of both HSC- and YS-derived donor cells to engraft in the brain parenchyma and express TMEM119 only when engrafted attests to the potency of programming signals from the brain parenchyma. To comprehensively measure the ability of diverse transplanted cells to adopt a microglial transcriptional program, we purified parenchymal TMEM119+ MLCs using identical methods to transplanted microglia, allowing highly specific isolation of parenchymal macrophages. We transcriptionally profiled MLCs derived from E8 yolk sac, E12–E13 fetal brain, E13–E14 fetal liver, adult blood, and BM, comparing them to each other and to transplanted microglia. Among these highly purified transcriptomes, we observed a striking degree of similarity between engrafted cell types (Figures 4A and S4A–S4C). MLCs, irrespective of ontogeny, expressed many microglia signature genes, including Tmem119, Fcrls, Hexb, and Olfml3, at near-microglial levels (Figure 4A). Gene expression in all MLC types was well correlated (Spearman coefficients >0.6–0.8), and in exploratory analyses combining published datasets, MLCs were more closely related to microglia than to other tissue macrophages, monocytes, or neutrophils (Figures S4C and S4D). These data confirm the strong programming effects of the brain parenchyma on macrophages, and the intrinsic ability of even CNS-alien macrophages to respond to programming signals by expressing microglial genes. Although grossly similar, we found major ontogeny-dependent differences between HSC- and YS-MLCs. Principal component analysis showed that the transcriptomes of transplanted microglia, YS-MLCs, and fetal brain MLCs overlap with each other, distinct from blood, BM, and fetal liver MLCs (Figure 4B). Unsupervised hierarchical clustering similarly showed that YS-derived (YS, fetal brain) MLC gene expression is more closely related to transplanted microglia than HSC-derived (blood, BM) and mixed-origin MLCs (fetal liver) (Figure 4C). To focus on ontogeny-specific gene expression patterns, we pooled gene expression data from YS- and HSC-MLC donor groups, excluding fetal liver MLCs since E13–E14 liver contains a mix of YS- and HSC-derived cells (Hoeffel et al., 2015Hoeffel G. Chen J. Lavin Y. Low D. Almeida F.F. See P. Beaudin A.E. Lum J. Low I. Forsberg E.C. et al.C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.Immunity. 2015; 42: 665-678Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, Gomez Perdiguero et al., 2015Gomez Perdiguero E. Klapproth K. Schulz C. Busch K. Azzoni E. Crozet L. Garner H. Trouillet C. de Bruijn M.F. Geissmann F. Rodewald H.R. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors.Nature. 2015; 518: 547-551Crossref PubMed Scopus (1336) Google Scholar). As a group, YS-lineage MLCs had 131 differentially regulated genes compared to transplanted microglia, while HSC-MLCs had 609 (Figure 4D). Volcano plot overlay further depicts the higher similarity of YS-MLCs to transplanted microglia at a whole-transcriptome level (Figure 4E). At the gene level, YS-MLCs expressed microglia signature genes more faithfully than their HSC-derived counterparts, including Slc2a5, Olfml3, Gpr34, Sparc, and P2ry12 (Figure S5A). Among microglia-enriched genes in TMEM119+ cells from a prior study (Bennett et al., 2016Bennett M.L. Bennett F.C. Liddelow S.A. Ajami B. Zamanian J.L. Fernhoff N.B. Mulinyawe S.B. Bohlen C.J. Adil A. Tucker A. et al.New tools for studying microglia in the mouse and human CNS.Proc. Natl. Acad. Sci. USA. 2016; 113: E1738-E1746Crossref PubMed Scopus (1025) Google Scholar), YS-MLCs were significantly closer to microglial expression levels in 30 of 32 measured genes (Table S2)" @default.
• W2807609377 created "2018-06-13" @default.
• W2807609377 creator A5012203120 @default.
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• W2807609377 date "2018-06-01" @default.
• W2807609377 modified "2023-10-16" @default.
• W2807609377 title "A Combination of Ontogeny and CNS Environment Establishes Microglial Identity" @default.
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