Novel Hexb-based tools for studying microglia in the CNS

An Author Correction to this article was published on 11 August 2020

This article has been updated

Abstract

Microglia and central nervous system (CNS)-associated macrophages (CAMs), such as perivascular and meningeal macrophages, are implicated in virtually all diseases of the CNS. However, little is known about their cell-type-specific roles in the absence of suitable tools that would allow for functional discrimination between the ontogenetically closely related microglia and CAMs. To develop a new microglia gene targeting model, we first applied massively parallel single-cell analyses to compare microglia and CAM signatures during homeostasis and disease and identified hexosaminidase subunit beta (Hexb) as a stably expressed microglia core gene, whereas other microglia core genes were substantially downregulated during pathologies. Next, we generated HexbtdTomato mice to stably monitor microglia behavior in vivo. Finally, the Hexb locus was employed for tamoxifen-inducible Cre-mediated gene manipulation in microglia and for fate mapping of microglia but not CAMs. In sum, we provide valuable new genetic tools to specifically study microglia functions in the CNS.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: scRNA-seq during de- and remyelination and FNX reveals Hexb as a stably expressed microglia core gene.
Fig. 2: Steady high expression of Hexb in microglia but not CAMs during homeostasis and autoimmune neuroinflammatory conditions.
Fig. 3: Generation and characterization of HexbtdTomato mice.
Fig. 4: Continuous detection of HexbtdT microglia during neurodegeneration and autoimmune demyelination.
Fig. 5: In vivo dynamics of HexbtdT microglia.
Fig. 6: Generation and comparative analysis of HexbCreERT2 mice.
Fig. 7: Fate mapping following CNS macrophage depletion using HexbCreERT2/CreERT2 R26yfp/yfp mice.
Fig. 8: Effects of Csf1r deletion on microglia but not CAMs.

Data availability

Raw data for new scRNA-seq or bulk RNA-seq have been deposited in the Gene Expression Omnibus, and are available at the following accession numbers: GSE148405 (scRNA-seq) and GSE148413 (bulk RNA-seq). All other data are published previously or are available from the corresponding authors on reasonable request.

Change history

  • 11 August 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 1.

    Colonna, M. & Butovsky, O. Microglia function in the central nervous system during health and neurodegeneration. Annu. Rev. Immunol. 35, 441–468 (2017).

    CAS  PubMed  Google Scholar 

  2. 2.

    Li, Q. & Barres, B. A. Microglia and macrophages in brain homeostasis and disease. Nat. Rev. Immunol. 18, 225–242 (2018).

    CAS  PubMed  Google Scholar 

  3. 3.

    Herz, J., Filiano, A. J., Smith, A., Yogev, N. & Kipnis, J. Myeloid cells in the central nervous system. Immunity 46, 943–956 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Priller, J. & Prinz, M. Targeting microglia in brain disorders. Science 365, 32–33 (2019).

    CAS  PubMed  Google Scholar 

  5. 5.

    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).

    CAS  PubMed  Google Scholar 

  6. 6.

    Prinz, M., Erny, D. & Hagemeyer, N. Ontogeny and homeostasis of CNS myeloid cells. Nat. Immunol. 18, 385–392 (2017).

    CAS  PubMed  Google Scholar 

  7. 7.

    Prinz, M., Jung, S. & Priller, J. Microglia biology: one century of evolving concepts. Cell 179, 292–311 (2019).

    CAS  PubMed  Google Scholar 

  8. 8.

    Ueno, M. et al. Layer V cortical neurons require microglial support for survival during postnatal development. Nat. Neurosci. 16, 543–551 (2013).

    CAS  PubMed  Google Scholar 

  9. 9.

    Hagemeyer, N. et al. Microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood. Acta Neuropathol. 134, 441–458 (2017).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    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).

    CAS  PubMed  Google Scholar 

  12. 12.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Ajami, B. et al. Single-cell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models. Nat. Neurosci. 21, 541–551 (2018).

    CAS  PubMed  Google Scholar 

  14. 14.

    Bottcher, C. et al. Human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry. Nat. Neurosci. 22, 78–90 (2019).

    PubMed  Google Scholar 

  15. 15.

    Jordao, M. J. C. et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363, eaat7554 (2019).

    CAS  PubMed  Google Scholar 

  16. 16.

    Masuda, T. et al. Spatial and temporal heterogeneity of mouse and human microglia at single-cell resolution. Nature 566, 388–392 (2019).

    CAS  PubMed  Google Scholar 

  17. 17.

    Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79–91 (2013).

    CAS  PubMed  Google Scholar 

  18. 18.

    Goldmann, T. et al. A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation. Nat. Neurosci. 16, 1618–1626 (2013).

    CAS  PubMed  Google Scholar 

  19. 19.

    Zeisel, A. et al. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347, 1138–1142 (2015).

    CAS  Google Scholar 

  20. 20.

    Mildner, A. et al. Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat. Neurosci. 10, 1544–1553 (2007).

    CAS  PubMed  Google Scholar 

  21. 21.

    Butovsky, O. et al. Identification of a unique TGF-β-dependent molecular and functional signature in microglia. Nat. Neurosci. 17, 131–143 (2014).

    CAS  PubMed  Google Scholar 

  22. 22.

    Zhang, Y. et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34, 11929–11947 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Hickman, S. E. et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. 16, 1896–1905 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    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).

    CAS  PubMed  Google Scholar 

  25. 25.

    Li, Q. et al. Developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell RNA sequencing. Neuron 101, 207–223 (2019).

    CAS  PubMed  Google Scholar 

  26. 26.

    Sango, K. et al. Mouse models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism. Nat. Genet. 11, 170–176 (1995).

    CAS  PubMed  Google Scholar 

  27. 27.

    Paolicelli, R. C. et al. Synaptic pruning by microglia is necessary for normal brain development. Science 333, 1456–1458 (2011).

    CAS  PubMed  Google Scholar 

  28. 28.

    Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178 (2007).

    CAS  PubMed  Google Scholar 

  29. 29.

    Inoue, S., Inoue, M., Fujimura, S. & Nishinakamura, R. A mouse line expressing Sall1-driven inducible Cre recombinase in the kidney mesenchyme. Genesis 48, 207–212 (2010).

    CAS  PubMed  Google Scholar 

  30. 30.

    Buttgereit, A. et al. Sall1 is a transcriptional regulator defining microglia identity and function. Nat. Immunol. 17, 1397–1406 (2016).

    CAS  PubMed  Google Scholar 

  31. 31.

    Chappell-Maor, L. et al. Comparative analysis of CreER transgenic mice for the study of brain macrophages: a case study. Eur. J. Immunol. 50, 353–362 (2020).

    CAS  PubMed  Google Scholar 

  32. 32.

    Bruttger, J. et al. Genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system. Immunity 43, 92–106 (2015).

    CAS  PubMed  Google Scholar 

  33. 33.

    Elmore, M. R. et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron 82, 380–397 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Zhan, L. et al. Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain. PLoS Biol. 17, e3000134 (2019).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Huang, Y. et al. Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion. Nat. Neurosci. 21, 530–540 (2018).

    CAS  PubMed  Google Scholar 

  36. 36.

    Van Hove, H. et al. Identifying the variables that drive tamoxifen-independent CreERT2 recombination: implications for microglial fate mapping and gene deletions. Eur. J. Immunol. 50, 459–463 (2020).

    CAS  PubMed  Google Scholar 

  37. 37.

    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).

    CAS  PubMed  Google Scholar 

  38. 38.

    Fuger, P. et al. Microglia turnover with aging and in an Alzheimer’s model via long-term in vivo single-cell imaging. Nat. Neurosci. 20, 1371–1376 (2017).

    PubMed  Google Scholar 

  39. 39.

    Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    Mildner, A., Huang, H., Radke, J., Stenzel, W. & Priller, J. P2Y12 receptor is expressed on human microglia under physiological conditions throughout development and is sensitive to neuroinflammatory diseases. Glia 65, 375–387 (2017).

    PubMed  Google Scholar 

  41. 41.

    Zrzavy, T. et al. Loss of ‘homeostatic’ microglia and patterns of their activation in active multiple sclerosis. Brain 140, 1900–1913 (2017).

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Bennett, M. L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl Acad. Sci. USA 113, E1738–E1746 (2016).

    CAS  PubMed  Google Scholar 

  43. 43.

    Kaiser, T. & Feng, G. Tmem119-EGFP and Tmem119-CreERT2 transgenic mice for labeling and manipulating microglia. eNeuro. 6, ENEURO.0448-18.2019 (2019).

  44. 44.

    Wieghofer, P., Knobeloch, K. P. & Prinz, M. Genetic targeting of microglia. Glia 63, 1–22 (2015).

    PubMed  Google Scholar 

  45. 45.

    Dighe, A. S. et al. Tissue-specific targeting of cytokine unresponsiveness in transgenic mice. Immunity 3, 657–666 (1995).

    CAS  PubMed  Google Scholar 

  46. 46.

    Zehntner, S. P., Brisebois, M., Tran, E., Owens, T. & Fournier, S. Constitutive expression of a costimulatory ligand on antigen-presenting cells in the nervous system drives demyelinating disease. FASEB J. 17, 1910–1912 (2003).

    CAS  PubMed  Google Scholar 

  47. 47.

    Siao, C. J., Fernandez, S. R. & Tsirka, S. E. Cell type-specific roles for tissue plasminogen activator released by neurons or microglia after excitotoxic injury. J. Neurosci. 23, 3234–3242 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Rong, L. L. et al. RAGE modulates peripheral nerve regeneration via recruitment of both inflammatory and axonal outgrowth pathways. FASEB J. 18, 1818–1825 (2004).

    CAS  PubMed  Google Scholar 

  49. 49.

    Jung, S. et al. In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens. Immunity 17, 211–220 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Heppner, F. L. et al. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat. Med 11, 146–152 (2005).

    CAS  PubMed  Google Scholar 

  51. 51.

    Hashimshony, T. et al. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-seq. Genome Biol. 17, 77 (2016).

    PubMed  PubMed Central  Google Scholar 

  52. 52.

    Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    CAS  PubMed  Google Scholar 

  53. 53.

    Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 41, e108 (2013).

    PubMed  PubMed Central  Google Scholar 

  54. 54.

    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).

    CAS  PubMed  Google Scholar 

  55. 55.

    Goldmann, T. et al. USP18 lack in microglia causes destructive interferonopathy of the mouse brain. EMBO J. 34, 1612–1629 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56.

    Raasch, J. et al. IκB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NF-κB in the central nervous system. Brain 134, 1184–1198 (2011).

    PubMed  PubMed Central  Google Scholar 

  57. 57.

    Dann, A. et al. Cytosolic RIG-I-like helicases act as negative regulators of sterile inflammation in the CNS. Nat. Neurosci. 15, 98–106 (2012).

    CAS  Google Scholar 

  58. 58.

    Prinz, M. et al. Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system. Immunity 28, 675–686 (2008).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank E. Barleon for excellent technical assistance and the Center for Animal Resource and Development (CARD, Kumamoto University) for providing research material. T. Masuda was supported by the KANAE Foundation for the Promotion of Medical Science and the Japan Society for the Promotion of Science (JSPS) as the JSPS Postdoctoral Fellow for Research Abroad. M.P. was supported by the Sobek Foundation, the Ernst-Jung Foundation, the German Research Foundation (DFG) (grant nos. SFB 992, SFB1160, SFB/TRR167, Reinhart-Koselleck-Grant and the Gottfried Wilhelm Leibniz-Prize) and the Ministry of Science, Research and Arts, Baden-Wuerttemberg (Sonderlinie ‘Neuroinflammation’). This study was supported by the DFG under Germany’s Excellence Strategy (grant no. CIBSS—EXC-2189, Project ID390939984). J.P. was supported by the DFG (grant nos. SFB/TRR167 B05 and B07), and the UK DRI Momentum Award. C.B. was supported by the DFG (grant no. SFB/TRR167 B05).

Author information

Affiliations

Authors

Contributions

T. Masuda, L.A., P.E., M.L., N.S., T. Misgeld, R.S., O.S., M.J.C.J., C.B., K.K., D.G., A.V. and K.P.K. conducted experiments and analyzed the data. M.P., K.P.K., S.J., M.M.L. and J.P. analyzed the data, contributed to the in vivo studies and provided mice or reagents. T. Masuda and M.P. supervised the project and wrote the manuscript, J.P. edited the manuscript.

Corresponding authors

Correspondence to Takahiro Masuda or Marco Prinz.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Editor recognition statement Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–14.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–13.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Masuda, T., Amann, L., Sankowski, R. et al. Novel Hexb-based tools for studying microglia in the CNS. Nat Immunol 21, 802–815 (2020). https://doi.org/10.1038/s41590-020-0707-4

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing