Abstract
Astrocytes are the most abundant cell type in the brain, where they perform a wide array of functions, yet the nature of their cellular heterogeneity and how it oversees these diverse roles remains shrouded in mystery. Using an intersectional fluorescence-activated cell sorting–based strategy, we identified five distinct astrocyte subpopulations present across three brain regions that show extensive molecular diversity. Application of this molecular insight toward function revealed that these populations differentially support synaptogenesis between neurons. We identified correlative populations in mouse and human glioma and found that the emergence of specific subpopulations during tumor progression corresponded with the onset of seizures and tumor invasion. In sum, we have identified subpopulations of astrocytes in the adult brain and their correlates in glioma that are endowed with diverse cellular, molecular and functional properties. These populations selectively contribute to synaptogenesis and tumor pathophysiology, providing a blueprint for understanding diverse astrocyte contributions to neurological disease.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 /Â 30Â days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Barres, B.A. The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60, 430–440 (2008).
Ligon, K.L., Fancy, S.P., Franklin, R.J. & Rowitch, D.H. Olig gene function in CNS development and disease. Glia 54, 1–10 (2006).
Jiang, X. et al. Principles of connectivity among morphologically defined cell types in adult neocortex. Science 350, aac9462 (2015).
Kepecs, A. & Fishell, G. Interneuron cell types are fit to function. Nature 505, 318–326 (2014).
Miyoshi, G. et al. Prox1 regulates the subtype-specific development of caudal-ganglionic-eminence-derived GABAergic cortical interneurons. J. Neurosci. 35, 12869–12889 (2015).
Abbott, N.J., Rönnbäck, L. & Hansson, E. Astrocyte–endothelial interactions at the blood–brain barrier. Nat. Rev. Neurosci. 7, 41–53 (2006).
Chung, W.S., Allen, N.J. & Eroglu, C. Astrocytes control synapse formation, function, and elimination. Cold Spring Harb. Perspect. Biol. 7, a020370 (2015).
RamĂłn y Cajal, S. Histology of the Nervous System of Man and Vertebrates (Oxford University Press, New York, 1897).
Andriezen, W.L. The neuroglia elements in the human brain. BMJ 2, 227–230 (1893).
Kölliker, A. Handbuch der Gewebelehre des Menschen (Verlag von Wilhelm Engelmann, 1889).
Bayraktar, O.A., Fuentealba, L.C., Alvarez-Buylla, A. & Rowitch, D.H. Astrocyte development and heterogeneity. Cold Spring Harb. Perspect. Biol. 7, a020362 (2014).
Iwai, Y. et al. Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor, in the Drosophila embryonic CNS. Neuron 19, 77–89 (1997).
Hochstim, C., Deneen, B., Lukaszewicz, A., Zhou, Q. & Anderson, D.J. Identification of positionally distinct astrocyte subtypes whose identities are specified by a homeodomain code. Cell 133, 510–522 (2008).
Tsai, H.H. et al. Regional astrocyte allocation regulates CNS synaptogenesis and repair. Science 337, 358–362 (2012).
Anthony, T.E. & Heintz, N. The folate metabolic enzyme ALDH1L1 is restricted to the midline of the early CNS, suggesting a role in human neural tube defects. J. Comp. Neurol. 500, 368–383 (2007).
Cahoy, J.D. et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J. Neurosci. 28, 264–278 (2008).
Lovatt, D. et al. The transcriptome and metabolic gene signature of protoplasmic astrocytes in the adult murine cortex. J. Neurosci. 27, 12255–12266 (2007).
Bakheit, A.M., Brennan, A., Gan, P., Green, H. & Roberts, S. Anarchic hand syndrome following resection of a frontal lobe tumor. Neurocase 19, 36–40 (2013).
Verhaak, R.G. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98–110 (2010).
de la Iglesia, N. et al. Identification of a PTEN-regulated STAT3 brain tumor suppressor pathway. Genes Dev. 22, 449–462 (2008).
Xue, W. et al. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature 514, 380–384 (2014).
Chen, F., Rosiene, J., Che, A., Becker, A. & LoTurco, J. Tracking and transforming neocortical progenitors by CRISPR–Cas9 gene targeting and piggyBac transposase lineage-labeling. Development 142, 3601–3611 (2015).
Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57–70 (2000).
Dudek, F.E. Role of glial cells in seizures and epilepsy: intracellular calcium oscillations sn spatial buffering. Epilepsy Curr. 2, 137–139 (2002).
Ransom, B.R. & Sontheimer, H. The neurophysiology of glial cells. J. Clin. Neurophysiol. 9, 224–251 (1992).
Ran, X. et al. EpilepsyGene: a genetic resource for genes and mutations related to epilepsy. Nucleic Acids Res. 43, D893–D899 (2015).
Cuddapah, V.A., Robel, S., Watkins, S. & Sontheimer, H. A neurocentric perspective on glioma invasion. Nat. Rev. Neurosci. 15, 455–465 (2014).
Paw, I., Carpenter, R.C., Watabe, K., Debinski, W. & Lo, H.W. Mechanisms regulating glioma invasion. Cancer Lett. 362, 1–7 (2015).
Yeh, T.H., Lee, D.Y., Gianino, S.M. & Gutmann, D.H. Microarray analyses reveal regional astrocyte heterogeneity with implications for neurofibromatosis type 1 (NF1)-regulated glial proliferation. Glia 57, 1239–1249 (2009).
Bailey, M.S. & Shipley, M.T. Astrocyte subtypes in the rat olfactory bulb: morphological heterogeneity and differential laminar distribution. J. Comp. Neurol. 328, 501–526 (1993).
Cameron, R.S. & Rakic, P. Glial cell lineage in the cerebral cortex: a review and synthesis. Glia 4, 124–137 (1991).
Ge, W.P., Miyawaki, A., Gage, F.H., Jan, Y.N. & Jan, L.Y. Local generation of glia is a major astrocyte source in postnatal cortex. Nature 484, 376–380 (2012).
Tau, G.Z. & Peterson, B.S. Normal development of brain circuits. Neuropsychopharmacology 35, 147–168 (2010).
Jiang, X. & Nardelli, J. Cellular and molecular introduction to brain development. Neurobiol. Dis. 92 (Pt. A), 3–17 (2016).
Farmer, W.T. et al. Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling. Science 351, 849–854 (2016).
Molofsky, A.V. et al. Astrocytes and disease: a neurodevelopmental perspective. Genes Dev. 26, 891–907 (2012).
Dallas, N.A. et al. Endoglin (CD105): a marker of tumor vasculature and potential target for therapy. Clin. Cancer Res. 14, 1931–1937 (2008).
Wang, J. et al. CD133-negative glioma cells form tumors in nude rats and give rise to CD133-positive cells. Int. J. Cancer 122, 761–768 (2008).
Singh, S.K. et al. Identification of human brain tumor–initiating cells. Nature 432, 396–401 (2004).
Stiles, C.D. & Rowitch, D.H. Glioma stem cells: a midterm exam. Neuron 58, 832–846 (2008).
Patel, A.P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014).
Robel, S. & Sontheimer, H. Glia as drivers of abnormal neuronal activity. Nat. Neurosci. 19, 28–33 (2016).
Campbell, S.L. et al. GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor-associated epilepsy. Glia 63, 23–36 (2015).
Venkatesh, H.S. et al. Neuronal activity promotes glioma growth through neuroligin-3 secretion. Cell 161, 803–816 (2015).
Weston, M.C., Chen, H. & Swann, J.W. Multiple roles for mammalian target of rapamycin signaling in both glutamatergic and GABAergic synaptic transmission. J. Neurosci. 32, 11441–11452 (2012).
Saldanha, A.J. Java Treeview—extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).
Storey, J.D. & Tibshirani, R. Statistical significance for genome-wide studies. Proc. Natl. Acad. Sci. USA 100, 9440–9445 (2003).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).
Glasgow, S.M. et al. Mutual antagonism between Sox10 and NFIA regulates diversification of glial lineages and glioma subtypes. Nat. Neurosci. 17, 1322–1329 (2014).
Barkmeier, D.T. et al. High inter-reviewer variability of spike detection on intracranial EEG addressed by an automated multichannel algorithm. Clin. Neurophysiol. 123, 1088–1095 (2012).
Acknowledgements
We thank M. Brenner and M. Goodell for assistance with our cell-surface-marker antibody screen. This work was supported by grants from the Sontag Foundation (B.D.), the National Multiple Sclerosis Society (RG-1501-02756; B.D.), the Cancer Prevention Research Institute of Texas (RP510334 and RP160192 awarded to both B.D. and C.J.C.), the American Cancer Society (PF-15-220; K.Y.) and the US National Institutes of Health (NIH) (NS071153 and AG054111 (B.D.), NS089366 (B.D.), NS29709 (J.L.N.) and T32HL902332 (K.Y. and J.C.)). This project was also supported in part by the Genomic and RNA Profiling Core at Baylor College of Medicine with funding from the NIH–NCI grant (P30CA125123) and the expert assistance of L. White, the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the NIH (P30 AI036211, P30 CA125123 and S10 RR024574), the expert assistance of J. Sederstrom and by IDDRC grant number 1U54 HD083092 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
Author information
Authors and Affiliations
Contributions
C.-C.J.L., K.Y. and B.D. conceived the project, designed the experiments and wrote the manuscript; C.-C.J.L., K.Y., A.H., T.-W.H., H.K.L., J.C. and W.Z. performed the experiments; M.C.W., C.A.M., N.A., A.J. and B.R.A. provided essential reagents; F.C., Y.Z. and C.J.C. designed and executed the bioinformatics analysis; and J.L.N. and C.J.C. assisted in experimental design.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Representative FACS plots from Aldh1l1-GFP mouse cortex.
(A) Aldh1l1-GFP FACS plot from 14 week old mouse cortex. (B) Aldh1l1-GFP positive population was gated and the number of cells within this fraction that are CD51 and CD71 positive was determined. (C) The remaining Aldh1l1-GFP cells that were CD51/CD71 negative, were gated for CD63, which did not have any overlap with the Aldh1l1-GFP/CD51 or Aldh1l1-GFP/CD71 populations. (D) Work Flow of Antibody Screening from 83 candidate cell surface antibody to thee antibodies (CD51, CD71, and CD63) used in this study. Please see methods for details of the screening criteria.
Supplementary Figure 2 Astrocyte sub-population dynamics across brain regions and cortical development.
(A-F) Graphs comparing the relative abundance of each population across the diverse adult brain regions and spinal cord. The data is derived from Figure 1 and each graph represents the abundance of a given subpopulation across various regions. (G-K) Graphs comparing the relative abundance of each population during the P1-P28 developmental interval, in the cortex. The data is derived from Figure 2 and each graph represents the abundance of a given subpopulation across various developmental timepoints. Statistical analysis comparing the abundance of these populations across brain regions and development can be found in Table S3; the associated statistical analysis in Table S3 was performed using one-way ANOVA followed by Tukey's test.
Supplementary Figure 3 Representative gene expression heatmaps for control gene sets, unsupervised clustering, and global comparison with human astrocytes.
(A) Heat map of established astrocyte and neuronal genes and their relative expression across populations A-E, in the OB, cortex, and brainstem. (B) Unsupervised Clustering Analysis of Astrocyte Subpopulation RNA-Seq data. Note that “1” denotes OB, “2” denotes cortex, and “3” denotes brainstem. (C) Global correlation heatmap comparing mouse astrocyte subpopulations with human astrocyte RNA-Seq profiles (Brain-Seq dataset; Zhang, et al 2016). Note that the mouse astrocyte populations are strongly correlated with human astrocytes and not oligodendrocytes, neurons, or other cell populations in the human brain that were queried. Note that 1= Olfactory bulb, 2=cortex, and 3=brainstem.
Supplementary Figure 4 Marker Validation and Pop v. Negative Venn Analysis
(A-D) CD71 staining in Aldh1l1-GFP astrocytes in the adult cortex. Note that CD71 appears to be enriched in outer layer Aldh1l1-GFP astrocytes. (E-H) Immunostaining of candidate markers of populations in Aldh1l1-GFP astrocytes in the cortex or olfactory bulb. Filled arrows represent co-labeling and unfilled arrows represent no co-labeling between marker and Alldh1l1-GFP. All scale bars are 20um. (I) Complete GSEA table comparing gene expression profiles with synapse genes. See Figure 3. (J) Venn Diagram plot showing the overlapping and unique gene expression patterns between astrocyte subpopulations A-E. Note that there are a total of 31 possible intersection points, and due to the dimensional constraints, we are only able to represent 21 of these in the figure. The complete list of intersection points and the associated quantification can be found in Table S4, tab 11.
Supplementary Figure 5 Representative FACS plots from P70 CRISPR Glioma.
(A) P70 tumor was dissected, dissociated and analyzed for GFP+ cells. (B) Tumor was stained for both CD133 and CD31, both makers were placed in the same channel. After gating for GFP+ tumor cells, we then selected for the CD133-/CD31- fraction (arrow). (C) Within this population the number of CD51+ and CD71+ cells was determined. (D) Validation of CRISPR deletion of glioma-associated tumor suppressors. Schematic and FACS plots for isolating of GFP+ and GFP- fractions from brain for immunoblotting of NFI, p53, and PTEN. Representative of 3 individual tumors. For the purpose of presentation, the blot images were cropped from the original images.
Supplementary Figure 6 Gene Expression Heatmaps comparing Populations B/C with astrocyte counterparts and uncropped Western gels
(A) Heat map showing the relative expression of Astrocyte Population B enriched genes in glioma populations B-D. (B) Heat map showing the relative expression of Astrocyte Population C enriched genes in glioma populations B-D. (C) Heat map showing genes highlighted in Fig 2B that are associated with GO pathways (ion transport, glutatmate transport, extra cellular) and their relative expression in glioma populations B-D. (D) Uncropped Western Gel Image associated with Figure 7B. (E) Uncropped Western Gel image associated with Figure S5D.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 928 kb)
Supplementary Table 1
List of Cell Surface Antibodies; Statistical Analysis of Astrocyte Subpopulation Dynamics (XLSX 24 kb)
Supplementary Table 2
Astrocyte and Neuron Control Gene List (XLSX 139 kb)
Supplementary Table 3
RNA-Seq Statistical Analysis, Pop v Neg Gene Lists (XLSX 11035 kb)
Supplementary Table 4
Pop v Neg Gene List GO Analysis (XLSX 2439 kb)
Supplementary Table 5
RNA-Seq Statistical Analysis, Pop v Pop Gene Lists (XLSX 7673 kb)
Supplementary Table 6
Pop v Pop Gene List GO Analysis (XLSX 3119 kb)
Supplementary Table 7
GSEA derived Population C Synpase gene list and GO (XLSX 420 kb)
Supplementary Table 8
Glioma Population Specific Gene Lists (XLSX 144 kb)
Supplementary Table 9
Astrocyte Subpopulation Gene Expression in Glioma Populations; Epilepsy GSEA analysis gene list (XLSX 227 kb)
Supplementary Table 10
Subject Genders and Ages (XLSX 8 kb)
Supplementary Table 11
Antibody Information (XLSX 9 kb)
Rights and permissions
About this article
Cite this article
John Lin, CC., Yu, K., Hatcher, A. et al. Identification of diverse astrocyte populations and their malignant analogs. Nat Neurosci 20, 396–405 (2017). https://doi.org/10.1038/nn.4493
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nn.4493
This article is cited by
-
Distinct forebrain regions define a dichotomous astrocytic profile in multiple system atrophy
Acta Neuropathologica Communications (2024)
-
Crym-positive striatal astrocytes gate perseverative behaviour
Nature (2024)
-
Leveraging translational insights toward precision medicine approaches for brain metastases
Nature Cancer (2023)
-
A toolbox of astrocyte-specific, serotype-independent adeno-associated viral vectors using microRNA targeting sequences
Nature Communications (2023)
-
The neuroscience of cancer
Nature (2023)
