This article has been updated


Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Change history

  • 20 June 2018

    In Fig. 1b of this Article, 'Csf1r' was misspelt 'Csfr1', and in Extended Data Fig. 11b, the genes in the first column had shifted down three rows. These errors have been corrected online.


Primary accessions

Gene Expression Omnibus


  1. 1.

    The pathobiology of vascular dementia. Neuron 80, 844–866 (2013)

  2. 2.

    , , & Establishment and dysfunction of the blood-brain barrier. Cell 163, 1064–1078 (2015)

  3. 3.

    , & Astrocyte–endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci. 7, 41–53 (2006)

  4. 4.

    The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 57, 178–201 (2008)

  5. 5.

    et al. Medulloblastoma genotype dictates blood brain barrier phenotype. Cancer Cell 29, 508–522 (2016)

  6. 6.

    & Translational aspects of blood-brain barrier transport and central nervous system effects of drugs: from discovery to patients. Clin. Pharmacol. Ther. 97, 380–394 (2015)

  7. 7.

    & Studies on inflammation. 1. The effect of histamine and serotonin on vascular permeability: an electron microscopic study. J. Biophys. Biochem. Cytol. 11, 571–605 (1961)

  8. 8.

    , & Segmental differentiations of cell junctions in the vascular endothelium. The microvasculature. J. Cell Biol. 67, 863–885 (1975)

  9. 9.

    et al. Bmx tyrosine kinase is specifically expressed in the endocardium and the endothelium of large arteries. Circulation 96, 1729–1732 (1997)

  10. 10.

    , & Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 93, 741–753 (1998)

  11. 11.

    et al. Vegfc/Flt4 signalling is suppressed by Dll4 in developing zebrafish intersegmental arteries. Development 136, 4001–4009 (2009)

  12. 12.

    et al. Differential endothelial transcriptomics identifies semaphorin 3G as a vascular class 3 semaphorin. Arterioscler. Thromb. Vasc. Biol. 31, 151–159 (2011)

  13. 13.

    & Differentiation of arterial and venous endothelial cells and vascular morphogenesis. Endothelium 13, 137–145 (2006)

  14. 14.

    , & Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev. Cell 21, 193–215 (2011)

  15. 15.

    , , , & What is a pericyte? J. Cereb. Blood Flow Metab. 36, 451–455 (2016)

  16. 16.

    et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014)

  17. 17.

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

  18. 18.

    et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science 352, 1326–1329 (2016)

  19. 19.

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

  20. 20.

    et al. Single-cell spatial reconstruction reveals global division of labour in the mammalian liver. Nature 542, 352–356 (2017)

  21. 21.

    et al. Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature 509, 503–506 (2014)

  22. 22.

    et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007)

  23. 23.

    et al. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulations. Nat. Methods 14, 935–936 (2017)

  24. 24.

    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)

  25. 25.

    et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci. Transl. Med. 4, 147ra111 (2012)

  26. 26.

    , , & Pericyte loss and microaneurysm formation in PDGF-B-deficient mice. Science 277, 242–245 (1997)

  27. 27.

    et al. Pericytes regulate the blood-brain barrier. Nature 468, 557–561 (2010)

  28. 28.

    , , & Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468, 562–566 (2010)

  29. 29.

    et al. Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. J. Exp. Med. 209, 1219–1234 (2012)

  30. 30.

    et al. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60 (2014)

  31. 31.

    et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 3, 301–313 (2008)

  32. 32.

    et al. A pericyte origin of spinal cord scar tissue. Science 333, 238–242 (2011)

  33. 33.

    et al. Perivascular fibroblasts form the fibrotic scar after contusive spinal cord injury. J. Neurosci. 33, 13882–13887 (2013)

  34. 34.

    et al. Regional blood flow in the normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not by capillary pericytes. Neuron 87, 95–110 (2015)

  35. 35.

    et al. Pericytes of multiple organs do not behave as mesenchymal stem cells in vivo. Cell Stem Cell 20, 345–359 (2017)

  36. 36.

    et al. Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev. Cell 26, 204–219 (2013)

  37. 37.

    et al. Sleep drives metabolite clearance from the adult brain. Science 342, 373–377 (2013)

  38. 38.

    et al. Bmx tyrosine kinase has a redundant function downstream of angiopoietin and vascular endothelial growth factor receptors in arterial endothelium. Mol. Cell. Biol. 21, 4647–4655 (2001)

  39. 39.

    et al. Analysis of the brain mural cell transcriptome. Sci. Rep. 6, 35108 (2016)

  40. 40.

    et al. Primary isolation of vascular cells from murine brain for single cell sequencing. Protoc. Exch. (2018)

  41. 41.

    , , & Isolation of vessel-associated pdgfra-H2BGFP positive cells from murine brain. Protoc. Exch. (2018)

  42. 42.

    , , & Preparation of single cell suspensions from the adult mouse lung. Protoc. Exch. (2018)

  43. 43.

    et al. Smart-seq2 for sensitive full-length transcriptome profiling in single cells. Nat. Methods 10, 1096–1098 (2013)

  44. 44.

    et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099 (2007)

  45. 45.

    et al. Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc. Natl Acad. Sci. USA 105, 1255–1260 (2008)

  46. 46.

    et al. Visualizing the cell-cycle progression of endothelial cells in zebrafish. Dev. Biol. 393, 10–23 (2014)

  47. 47.

    et al. Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish. Development 143, 1328–1339 (2016)

  48. 48.

    et al. Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration. Dev. Cell 20, 397–404 (2011)

  49. 49.

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

  50. 50.

    , & How to analyze gene expression using RNA-sequencing data. Methods Mol. Biol. 802, 259–274 (2012)

  51. 51.

    , & Efficient and comprehensive representation of uniqueness for next-generation sequencing by minimum unique length analyses. PLoS One 8, e53822 (2013)

  52. 52.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)

  53. 53.

    & Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995)

  54. 54.

    et al. Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature 525, 251–255 (2015)

  55. 55.

    et al. A promoter-level mammalian expression atlas. Nature 507, 462–470 (2014)

  56. 56.

    et al. Human blood-brain barrier receptors for Alzheimer’s amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer. J. Clin. Invest. 102, 734–743 (1998)

  57. 57.

    et al. Oatp1a4 and an l-thyroxine-sensitive transporter mediate the mouse blood-brain barrier transport of amyloid-β peptide. J. Alzheimers Dis. 36, 555–561 (2013)

  58. 58.

    et al. LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms. Neuron 43, 333–344 (2004)

  59. 59.

    et al. SRF and myocardin regulate LRP-mediated amyloid-beta clearance in brain vascular cells. Nat. Cell Biol. 11, 143–153 (2009)

  60. 60.

    et al. ABCG2 is upregulated in Alzheimer’s brain with cerebral amyloid angiopathy and may act as a gatekeeper at the blood-brain barrier for Aβ1–40 peptides. J. Neurosci. 29, 5463–5475 (2009)

  61. 61.

    et al. Active efflux of Dasatinib from the brain limits efficacy against murine glioblastoma: broad implications for the clinical use of molecularly targeted agents. Mol. Cancer Ther. 11, 2183–2192 (2012)

  62. 62.

    et al. β-Amyloid efflux mediated by p-glycoprotein. J. Neurochem. 76, 1121–1128 (2001)

  63. 63.

    et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 261, 921–923 (1993)

  64. 64.

    et al. Impaired amino acid transport at the blood brain barrier is a cause of autism spectrum disorder. Cell 167, 1481–1494 (2016)

  65. 65.

    et al. Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGFβ2 in vascular abnormalization. J. Pathol. 228, 378–390 (2012)

Download references


This study was supported by AstraZeneca AB (C.B., U.L.), the Swedish Research Council (C.B.: 2015-00550; U.L.: K2014-64X-20097-09-5), the European Research Council (C.B.: AdG294556), the Leducq Foundation (C.B., A.K.: 14CVD02), Swedish Cancer Society (C.B.:150735; U.L.:CAN 2016/271), Knut and Alice Wallenberg Foundation (C.B.: 2015.0030), Hjärnfonden (U.L.), Swiss National Science Foundation (A.K.: 31003A_159514/1) and the Synapsis Foundation (A.K.). We thank C. Olsson, H. Leksell, P. Peterson, J. Chmielniakova, K. Gaengel, BioVis (Uppsala), Center for Microscopy and Image Analysis (Zurich) and Eukaryotic Single Cell Genomics facility (Science for Life Laboratory) for technical help, and K. Alitalo, P. Soriano, D. Silver and L. Sorokin for reagents.

Author information

Author notes

    • Michael Vanlandewijck
    •  & Liqun He

    These authors contributed equally to this work.


  1. Karolinska Institutet/AstraZeneca Integrated Cardio Metabolic Centre (KI/AZ ICMC), Blickagången 6, SE-141 57 Huddinge, Sweden

    • Michael Vanlandewijck
    • , Elisabeth Raschperger
    •  & Christer Betsholtz
  2. Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin Neurological Institute, Key Laboratory of Post-Neuroinjury Neuro-Repair and Regeneration in Central Nervous System, Ministry of Education and Tianjin City, Tianjin 300052, China

    • Liqun He
  3. Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Dag Hammarskjölds väg 20, SE-751 85 Uppsala, Sweden

    • Michael Vanlandewijck
    • , Maarja Andaloussi Mäe
    • , Johanna Andrae
    • , Koji Ando
    • , Khayrun Nahar
    • , Thibaud Lebouvier
    • , Bàrbara Laviña
    • , Leonor Gouveia
    •  & Christer Betsholtz
  4. Department of Cell and Molecular Biology, Karolinska Institutet, Von Eulers väg 3, SE-171 77 Stockholm, Sweden

    • Francesca Del Gaudio
    •  & Urban Lendahl
  5. Inserm U1171, University of Lille, CHU, Memory Center, Distalz, F-59000 Lille, France

    • Thibaud Lebouvier
  6. Department of Bioinformatics, Zhongyuan Union Genetic Technology Co., Ltd., No.45, the 9th East Road, Tianjin Airport Economic Area, Tianjin 300304, China

    • Ying Sun
  7. Wihuri Research Institute and Translational Cancer Biology Program, Biomedicum Helsinki, University of Helsinki, Haartmaninkatu 8, P.O. Box 63, FI-00014 Helsinki, Finland

    • Markus Räsänen
  8. Division of Neurosurgery, Zürich University Hospital, Zürich University, Zürich, CH-8091, Switzerland

    • Yvette Zarb
    •  & Annika Keller
  9. Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka, Japan

    • Naoki Mochizuki
  10. AMED-CREST, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan

    • Naoki Mochizuki


  1. Search for Michael Vanlandewijck in:

  2. Search for Liqun He in:

  3. Search for Maarja Andaloussi Mäe in:

  4. Search for Johanna Andrae in:

  5. Search for Koji Ando in:

  6. Search for Francesca Del Gaudio in:

  7. Search for Khayrun Nahar in:

  8. Search for Thibaud Lebouvier in:

  9. Search for Bàrbara Laviña in:

  10. Search for Leonor Gouveia in:

  11. Search for Ying Sun in:

  12. Search for Elisabeth Raschperger in:

  13. Search for Markus Räsänen in:

  14. Search for Yvette Zarb in:

  15. Search for Naoki Mochizuki in:

  16. Search for Annika Keller in:

  17. Search for Urban Lendahl in:

  18. Search for Christer Betsholtz in:


M.V., L.H., N.M., U.L. and C.B. conceived and designed the project; M.V., M.A.M., J.A., K.A., F.D.G., K.N., T.L., B.L., E.R., L.G., Y.Z., M.R., A.K. and C.B. performed experiments; L.H. performed bioinformatic analysis; L.H. and Y.S. constructed the online database; C.B., L.H. and M.V. analysed the bioinformatic data; C.B. and U.L. wrote the manuscript with substantial input from M.V., L.H., M.A.M., J.A. and K.A. All authors reviewed the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Christer Betsholtz.

Reviewer Information Nature thanks D. McDonald and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    This file contains endothelial specific zonated genes.

  2. 2.

    Supplementary Table 2

    This file contains the top 500 endothelial specific differentially expressed genes.

  3. 3.

    Supplementary Table 3

    This file shows differential expression of mural cell specific genes.

  4. 4.

    Supplementary Table 4

    This file contains a list of used antibodies.


  1. 1.

    Localization of perivascular fibroblast-like cells

    A z-stack video taken perpendicular to the midsagittal plane visualizes the location of the pdgfra-H2BGFP positive perivascular cells. Note that, on larger vessels, the cells are inside of the AQP4-positive astrocyte end-feet (Red: AQP4), but outside of the vessel (White: CD31). Capillary-associated pdgfra-H2BGFP positive cells likely represent cells from the oligodendrocyte lineage, and are localized outside of the astrocyte end-feet. See Extended Data Figure 11b for high-resolution still images of the video.

About this article

Publication history





Further reading


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.