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.
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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.
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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.
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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)
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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
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DOI: https://doi.org/10.1038/nn.4493
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