New tools for studying microglia in the mouse and human CNS Mariko L. Bennetta,1,F.ChrisBennetta,b, Shane A. Liddelowa,c, Bahareh Ajamid, Jennifer L. Zamaniana, Nathaniel B. Fernhoffe,f,g,h, Sara B. Mulinyawea, Christopher J. Bohlena, Aykezar Adila, Andrew Tuckera, Irving L. Weissmane,f,g,h, Edward F. Changi, Gordon Lij, Gerald A. Grantj, Melanie G. Hayden Gephartj, and Ben A. Barresa,1 aDepartment of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA 94305; cDepartment of Pharmacology and Therapeutics, University of Melbourne, Melbourne, VIC, Australia, 3010; dDepartment of Neurology, Stanford University School of Medicine, Stanford, CA 94305; eInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; fLudwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA 94305; gStanford Cancer Institute, Stanford University School of Medicine, Stanford, CA 94305; hDepartment of Pathology, Stanford University School of Medicine, Stanford, CA 94305; iUniversity of California, San Francisco Epilepsy Center, University of California, San Francisco, CA 94143; and jDepartment of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305 Contributed by Ben A. Barres, January 12, 2016 (sent for review November 8, 2015; reviewed by Roman J. Giger and Beth Stevens) The specific function of microglia, the tissue resident macrophages and well-validated antibodies to known antigens yet exist to of the brain and spinal cord, has been difficult to ascertain because specifically and stably identify microglia. of a lack of tools to distinguish microglia from other immune cells, Therefore, we sought a molecular marker that would allow for thereby limiting specific immunostaining, purification, and manipu- the identification, isolation, and study of microglia across many lation. Because of their unique developmental origins and predicted applications. Whereas several studies have proposed potential functions, the distinction of microglia from other myeloid cells is microglia-specific markers (15–17), none have systematically critically important for understanding brain development and dis- validated these candidates, elucidated whether the markers ease; better tools would greatly facilitate studies of microglia function identified all microglia, or developed microglia-specific anti- in the developing, adult, and injured CNS. Here, we identify trans- bodies for use by the larger neuroscience community. Here we membrane protein 119 (Tmem119), a cell-surface protein of unknown identify and describe transmembrane protein 119 (Tmem119) as function, as a highly expressed microglia-specific marker in both a microglia-specific marker in both mouse and human CNS. We mouse and human. We developed monoclonal antibodies to its developed rabbit monoclonal antibodies against the intracellular intracellular and extracellular domains that enable the immuno- and extracellular domains of mouse Tmem119 for use in im- staining of microglia in histological sections in healthy and diseased munohistochemical identification and FACS isolation of brains, as well as isolation of pure nonactivated microglia by FACS. microglia, respectively. We adapted existing isolation methods to Using our antibodies, we provide, to our knowledge, the first generate what are, to our knowledge, the first ever RNA sequencing RNAseq profiles of highly pure mouse microglia during develop- profiles of pure, nonactivated microglia during development ment and after an immune challenge. We used these to demon- strate that mouse microglia mature by the second postnatal week Significance and to predict novel microglial functions. Together, we antici- pate these resources will be valuable for the future study and understanding of microglia in health and disease. Microglia are the tissue resident macrophages of the brain and spinal cord, implicated in important developmental, homeo- microglia | glia | developmental neuroscience | RNAseq | macrophage static, and disease processes, although our understanding of their roles is complicated by an inability to distinguish micro- glia from related cell types. Although they share many features icroglia are the resident parenchymal myeloid cells of the with other macrophages, microglia have distinct developmental MCNS, with important roles in development, homeostasis, origins and functions. Here we validate a stable and robustly disease, and injury (1). A major limitation to dissecting microglia- expressed microglial marker for both mouse and human, trans- specific contributions to these processes is an inability to distin- membrane protein 119 (Tmem119). We use custom-made anti- guish microglia from related cells, such as macrophages. The bodies against Tmem119 to perform deep RNA sequencing of importance of this distinction is increasingly clear, because micro- developing microglia, and demonstrate that microglia mature by glia not only have unique origins and developmental transcriptional the second postnatal week in mice. The antibodies, cell isolation programs, but are self-renewing and function differently than in- – methods, and RNAseq profiles presented here will greatly facilitate filtrating macrophages in CNS disease and injury (2 4). our understanding of microglial function in health and disease. Until recently, methods to distinguish microglia from other CNS cells relied on morphological distinctions (ramified vs. amoeboid), Author contributions: M.L.B., F.C.B., and B.A.B. designed research; M.L.B., F.C.B., S.A.L., relative marker expression by flow cytometry (the common B.A., N.B.F., S.B.M., C.J.B., A.A., and A.T. performed research; M.L.B., J.L.Z., I.L.W., E.F.C., G.L., leukocyte antigen CD45hi/lo) (5), or generating bone marrow G.A.G., and M.G.H.G. contributed new reagents/analytic tools; M.L.B., F.C.B., B.A., and (BM) chimeras (6, 7). Unfortunately, these approaches have B.A.B. analyzed data; M.L.B. and B.A.B. wrote the paper; and I.L.W. performed BMT exper- iments in his laboratory. inherent limitations. First, parameters such as morphology or Reviewers: R.J.G., University of Michigan; and B.S., Harvard Medical School Children’s CD45 expression may change with disease or injury. Second, Hospital. chimeric mouse generation leads to partial chimerism, causes The authors declare no conflict of interest. inflammatory damage, and can take many months (8, 9). More Freely available online through the PNAS open access option. recently, tools based on microglial expression of the fractalkine Data deposition: The sequence reported in this paper has been deposited in the NCBI receptor, Cx3cr1, overcame some of these limitations. The gen- BioProject, www.ncbi.nlm.nih.gov/bioproject (accession no. PRJNA307271). eration of knockin Cx3cr1-GFP (10) and Cx3cr1-CreERT (11, 12) See Commentary on page 3130. mice advanced the specificity and sophistication with which to 1 To whom correspondence may be addressed. Email: [email protected] or study microglial function. Cx3cr1, however, is also highly [email protected]. lo expressed by circulating monocytes (Ly6C ) and other tissue This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. resident macrophages (10, 13, 14). In addition, no widely available 1073/pnas.1525528113/-/DCSupplemental. E1738–E1746 | PNAS | Published online February 16, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1525528113 Downloaded by guest on September 23, 2021 and activated microglia following systemic inflammation. We Custom Anti-Tmem119 Monoclonal Antibodies Specifically Identify PNAS PLUS added these data to a user-friendly website (www.BrainRNAseq. Microglia. Tmem119 is a type IA single-pass transmembrane org). For human study, we identified and validated an anti-human protein with a reported role in osteoblast differentiation (23, 24). TMEM119 rabbit polyclonal antiserum that specifically stains Because no specific anti-mouse Tmem119 antibodies existed, we microglia in postmortem and surgical human brain sections. To- made two different monoclonal antibodies (mAbs) against the gether, the new microglial tools we developed have the potential to predicted extracellular (ECD) and intracellular (ICD) domains of mouse Tmem119 (SI Appendix, Fig. S2A). We stained cryo- broadly enable studies of microglia function in health and disease. + sections from Cx3cr1GFP/ × Tmem119 WT and knockout (KO) Results mice, as well as WT and KO lacking Cx3cr1-GFP. Both mAbs Tmem119 mRNA Expression Is Highly Enriched in the CNS and Specific demonstrated robust microglial staining in WT but not KO tis- SEE COMMENTARY to Most or all Microglia. To identify a microglia-specific marker, sues (Fig. 2 A and G and SI Appendix, Fig. S2B). The ICD mAb we generated a microglia-enriched gene expression profile of immunostained the microglial cell surface, revealing many fine + processes not apparent by classic markers (Fig. 2 B and C). CD45 immunopanned brain leukocytes from adult mice (18). + + Comparing these data with our datasets of highly pure CNS cells Staining was limited to parenchymal Cx3cr1-GFP and Iba1 and profiles of acutely purified immune cells (18–22), we iden- cells, and absent from meninges or choroid plexus, consistent with Tmem119 mRNA (Fig. 2 A–D and SI Appendix,Fig.S2C–G). tified seven highly expressed and enriched candidates: Tmem119, + By flow cytometry, ECD mAbs