A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function

Abstract

Understanding the cell-cell interactions that control CNS development and function has long been limited by the lack of methods to cleanly separate neural cell types. Here we describe methods for the prospective isolation and purification of astrocytes, neurons, and oligodendrocytes from developing and mature mouse forebrain. We used FACS (fluorescent-activated cell sorting) to isolate astrocytes from transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of an S100beta promoter. Using Affymetrix GeneChip Arrays, we then created a transcriptome database of the expression levels of >20,000 genes by gene profiling these three main CNS neural cell types at various postnatal ages between postnatal day 1 (P1) and P30. This database provides a detailed global characterization and comparison of the genes expressed by acutely isolated astrocytes, neurons, and oligodendrocytes. We found that Aldh1L1 is a highly specific antigenic marker for astrocytes with a substantially broader pattern of astrocyte expression than the traditional astrocyte marker GFAP. Astrocytes were enriched in specific metabolic and lipid synthetic pathways, as well as the draper/Megf10 and Mertk/integrin alpha(v)beta5 phagocytic pathways suggesting that astrocytes are professional phagocytes. Our findings call into question the concept of a "glial" cell class as the gene profiles of astrocytes and oligodendrocytes are as dissimilar to each other as they are to neurons. This transcriptome database of acutely isolated purified astrocytes, neurons, and oligodendrocytes provides a resource to the neuroscience community by providing improved cell-type-specific markers and for better understanding of neural development, function, and disease.

Figure 1.

Purification of astrocytes, neurons, and oligodendrocytes. A, Purification of astrocytes and neurons. A cell suspension of mouse forebrain was depleted of OPCs and OLs using immunopanning with OL lineage-specific antibodies. Astrocytes were then isolated from the remaining cells by FACS purifying the EGFP-positive astrocytes. The FACS-purified EGFP-negative cells were isolated and represent an enriched neuron population. B, Purification of OLs. A cell suspension of mouse forebrain was first depleted of microglia by panning on BSL1 lectin panning plates, and the remaining cells were then incubated on PDGFRα panning plates to purify and deplete OPCs, MOG panning plates to purify and deplete myelinating OLs, and a GalC panning plate to purify the remaining OLs. C–E, Expression levels of well described markers for astrocytes (C), OLs (D), and neurons (E) demonstrates high purity of each cell type. The y-axis represents the level of gene expression determined by MAS 5.0. Error bars represent ±SEM.

Figure 2.

Dendrogram and sample clustering of purified CNS cell types. Hierarchical clustering of highly purified CNS cell type samples from different developmental stages reveals three distinct clusters representing astrocytes, neurons, and oligodendrocytes. The similarity of gene expression between different samples is represented by the vertical distances on each branch of the dendrogram. Biological replicates show the highest degree of correlation within samples, represented by short vertical distances. Within each cell population, gene expression is more highly correlated between maturing and mature samples (Astros P7, Astros P17, OLs, Myelin OLs) than between immature and maturing samples (Astros P1, Astros P7, OPCs, OLs). Color bar and sample labels describe each individual sample type (green, astrocytes; yellow, neurons; orange–red, OL lineage cells; P, postnatal day, represented by different color shades; g, cerebral cortical gray matter astrocytes; n, neuron samples depleted of residual endothelial cells).

Figure 3.

Expression of astrocyte, oligodendrocyte, and neuron-specific genes. The top 40 cell-type-specific genes expressed by astrocytes (green bar), neurons (yellow bar), and oligodendrocytes (red bar) are depicted. Each individual gene expression level was normalized (see Materials and Methods) and plotted on a log₂ color scale, with blue representing low expression and red representing high expression. The fold enrichment can be estimated from the log₂ color bar scale, for example, the change from medium blue (−2) to red (3) represents a 32-fold difference in expression level.

Figure 4.

Validation of gene expression data by in situ hybridizations. A–C, Coronal brain sections showing ISH for genes identified by array data as having specific neuronal expression: A, Nov; B, Tmem130; C, Brunol4. D–L, Higher-magnification images corresponding to outlined region in A showing hippocampus, corpus callosum (cc), and the overlying cortex. D–F, Genes identified by array data as having specific neuronal expression, displaying expression in the hippocampus and cortex: D, Nov; E, Tmem130; F, Brunol4. G–I, Genes identified by array data as having astrocyte- enriched expression, showing fibrous, positive cells throughout the white and gray matter: G, Ntsr2; H, Aldh1L1; I, Acsbg1. J–L, ISH for genes identified by array data as having specific OL expression, showing white matter expression in the corpus callosum and the occasional positive cell in the overlying cortex: J, Fa2h; K, Tmem125/6330530A05Rik; L, Gpr62. All ISH performed on P17 mouse brains. Scale bars: A–C, 2 mm; D–L, 200 μm.

Figure 5.

Aldh1L1 is a specific pan-astrocyte marker. A–F, Immunohistochemical staining of P15 rat cortex shows Aldh1L1 is a cell-type-specific pan-astrocyte marker. A–C, Costaining of rat cortex for Aldh1L1 (red) and GFAP (green) staining. A, Aldh1L1 labels both the cell bodies and extensive processes of astrocytes in the cortex. B, GFAP labels the astrocyte intermediate filament cytoskeleton but not the finer processes that Aldh1L1 is capable of labeling. C, GFAP labels a subset of astrocytes: cells labeled by GFAP are also Aldh1L1 positive (white arrowheads), whereas Aldh1L1 labels many astrocytes not labeled by GFAP (black arrowheads). D–F, Aldh1L1 does not label neurons (D, Tuj1), OLs (E, MBP + CC1), or OPCs (F, NG2). G–I, The merge (I) of Aldh1L1 immunostaining (G) and strong BAC Aldh1L1-EGFP fluorescence (H) seen in the Aldh1L1-EGFP transgenic mouse cortex shows that all cells expressing the EGFP transgene also express the endogenous Aldh1L1 protein. Scale bars: A–F, 40 μm; G–I, 60 μm.

Figure 6.

Genes upregulated and downregulated during astrocyte and oligodendrocyte development. The top 60 genes most downregulated during astrocyte development (A, light green bar) and OL development (B, orange bar), and the top 60 genes most upregulated during astrocyte development (C, dark green bar) and OL development (D, red bar). The genes are plotted on a heat map to illustrate gene expression patterns in all CNS cell types at different developmental stages. The individual gene expression level for each cell type is normalized to the age averaged astrocyte expression (A, C) and the age averaged OL expression (B, D). The normalized values are plotted on a log₂ color scale, with blue representing low expression and red representing high expression. The fold enrichment can be estimated from the log₂ color bar scale. For example, the change from light blue (−1) to medium red (2) represents an eightfold difference in expression level. Note that, although few genes strongly downregulated during development (A, B) are expressed in a cell-type-specific pattern, the majority of genes strongly upregulated during development (C, D) are expressed in a cell-type-specific pattern.