Molecular Architecture of the Mouse Nervous System

Abstract

The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance. We discovered seven distinct, regionally restricted astrocyte types that obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system and enables genetic manipulation of specific cell types.

Keywords: RNA sequencing; cell type; classification; single-cell transcriptomics; transcriptomics.

Graphical abstract

Figure 1

Molecular Survey of the Mouse Nervous System Using Single-Cell RNA Sequencing (A) Schematic illustration of the sampling strategy. The brain was divided into coarse anatomical units, and in addition, we sampled from the spinal cord, dorsal root ganglia, sympathetic ganglion, and enteric nervous system. (B) Visualization of the single-cell data using gt-SNE embedding (see STAR Methods). Cells are colored by rank 3 taxonomy units indicated in the legend. (C) Dendrogram describing the taxonomy of all identified cell types. Main branches, corresponding to the taxonomy, are annotated with labels and colored background. The neurotransmitter used by each cell type is indicated below the leaves as colored circles. The lower panel indicates the developmental compartment of origin for each cell types. See also Figure S1 and Tables S1, S2, S3, S4, and S5.

Figure S1

Data Quality, Related to Figure 1 (A) Number of cells retained in analysis for each level of the pipeline. (B) Circle plots showing number of cells from each main class and each dissection region. (C) Cluster robustness and relatedness. The heatmap illustrates the performance of a random forest classifier, showing the average probability assigned to every cell type (rows) for each test cell of given type (columns). When the correct cell type (diagonal) has high probability, almost every test cell will be correctly classified. (D,E) Magnified view of heatmap as indicated in (C). (F) Distribution of Gene and UMI counts for individual Chromium samples (gray) and major cell classes (colored), shown for each of a representative selection of tissues. (G) Comparison of cell type fractions observed by osmFISH (single-molecule fluorescent in situ hybridization) and scRNA-seq. (H) Comparison of oligodendrocyte lineage clustering in the present paper and those previously published in Marques et al., 2016.

Figure 2

A Map of Neurogenesis in the Juvenile Mouse Brain (A) A cut-out from dendrogram of relevant cell types, including neuroblasts, radial glia-like cells, astrocytes, OPCs, and ependymal cells. (B) Sketch illustrating the locations where we found neurogenic activity. (C) gt-SNE embedding of all cells from the relevant cell types shown in (A). The dashed line suggests the border between glia-like cells and neuroblasts. (D) Expression distribution of individual key genes projected onto the gt-SNE embedding. See also Figure S2.

Figure S2

Markers and Validation of Neurogenesis and Astroependymal Cells, Related to Figure 2 (A) Additional marker genes for neurogenesis-related clusters, relevant clusters are indicated on the g-tSNE embedding. (B) Additional marker genes for various astroependymal cell types. The most enriched cluster is indicated. (C) Additional close-ups from validation using RNAscope. Genes and location indicated around the image. Scale bars: 500μm (CB, SVZ); 100μm (OB). (D) Composite image with colored dots representing cells, reconstructed from Allen Brain Atlas images, similar to Figure 3F but showing in situ hybridization. (E,F) Position of reference points used for alignment of multiple sagittal sections of RNAscope (E) and Allen Brain in situ hybridization (F) images. (G) In situ hybridization (Allen Brain Atlas) showing the extent of expression of Slc17a6 and Slc17a7, for comparison with astrocyte cell types.

Figure 3

Molecular and Spatial Diversity of the Astroependymal Cells in the CNS (A) Subtree describing the hierarchy of astroependymal cell types. (B) Schematic sagittal section showing the location of astroependymal cells. (C–E) gt-SNE embedding of all cells from the relevant clusters colored by cluster identity (C), tissue of origin (D), and patterning transcription factors (E). (F) Validation of spatial distribution of astrocytes cell types using multiplex ISH (RNAscope). Images from three consecutive sections were aligned and overlaid (see STAR Methods) to generate a composite with dots representing cells (upper panel). Below, high-magnification images show details of spatial location. Scale bars: top and bottom left, 500 μm; right, 1000 μm (cerebellum overview) and 100μm (olfactory bulb and cerebellum zoom-ins). (G) Gene expression of selected markers shown on the gt-SNE layout.

Figure 4

Convergence to a Common State at the Early Stages of Oligodendrocytes Lineage (A and B) gt-SNE embedding of the three first stages of the oligodendrocytes lineage OPC, COP, and NFOL colored by cluster identity (A) and tissue of origin (B). Edges in (A) connect nodes between mutual neighbors (k = 150), but only if they are from different clusters. (C) Gene expression of selected markers overlaid on gt-SNE embedding. (D) Patterning transcription factor analysis. Circles represent fraction of positive cells in each cluster and brain region. (E) Illustration of the proposed model of primary patterning, loss of regional identity and secondary diversification.

Figure 5

Diversity of the Vasculature and Neural-Crest-like Glia (A) Subtree describing the vasculature and neural-crest-like glia. (B) Expression dot plots for marker genes on log scale and jittered vertically in a uniform interval. Dots are colored only if the trinarization score is positive (posterior probability greater than 0.95), and colors represent the taxonomy rank 4 taxa. (C) The tissue contribution to each cluster represented by the circle size (enteric glia not shown). (D) Schematic illustration of the approximate position of vascular cell types and the meninges. See also Figure S3.

Figure S3

Markers of Neural-Crest-like Glia and Vascular Cell Types, Related to Figure 5 (A) Additional marker genes for the neural crest-like glia taxonomy unit, related to Figure 5. First panel on the left show the different clusters. Other panels show the expression (red high) distribution of marker genes. Black arrows indicate small clusters. (B) Similar to (A) but for the vasculature taxon. (C) Scatterplot showing differences between Schwann cells and mature oligodendrocytes (MOL) clusters. Values shown are log₂(x+1) transformed average molecule counts. The top 10 differentially expressed genes are shown in red and labeled.

Figure S4

Neurons of the Peripheral Nervous System, Related to Figure 7 (A) Hierarchical structure of the peripheral nervous system neuronal cell types. Neurotransmitters used by each cell types are indicated by the colored dots next to each leaf. (B) gt-SNE embedding of all related cells demonstrate the diversity and abundance of the different clusters. (C) Dot plots for marker genes along the PNS neurons. Dots show gene expression on log scale, and jittered vertically for clarity. Colors are shown only if the trinarization score is positive (posterior probability greater than 0.95 with f = 0.2).

Figure S5

Spatial Distribution of All Telencephalon Excitatory Projecting Neurons, Related to Figure 6 Dendrogram above shows the hierarchical structure as in Figure 1C. Left, reference atlas annotation (Allen Brain Atlas). Each column shows the expression map of an individual cluster, where dark brown is high and white is low correlation.

Figure 6

Neuronal Cell Types Are Spatially Restricted Examples of inferred spatial distributions for cell types across the brain. The left column shows reference images from the Allen Brain Atlas. Each row shows one coronal section, ordered rostrocaudally, and each column shows one cluster as indicated at the top. For every cluster and every voxel, the correlation coefficient is depicted by the colormap (dark high, white low). Labels indicate the top-scoring anatomical unit for each cluster. See also Figure S5.

Figure S6

Neurotransmitter Modularity, Related to Figure 7 (A) Co-expression of neurotransmitters. Rows and columns represent neurotransmitters. Circle size represents the number of cells in clusters with the indicated combination. Each circle shows (as a pie chart) the brain compartment in which we found the relevant clusters. (B) Analysis of pan-markers along the different ranks of the taxonomy. The heatmap represents the percentage of clusters with trinarization score greater than 90% within the taxonomy unit. Rows represent taxonomy units and columns genes. (C) Similar co-expression analysis as in (A) but with individual genes encoding neurotransmitters and neuropeptides. Upper half of the matrix shows pie charts representing only the compartment distribution. Lower part of the matrix represent the frequency each combination was found. (D) Summary diagram of the biosynthesis components and transporters used to define neurotransmitter phenotypes. Asterisks indicate the genes used separately (glutamatergic neurons) or jointly (all other) to identify each class of neurotransmitter.

Figure 7

Drivers of Cellular Diversity (A) Gene ontology analysis of the most highly enriched genes in CNS neuronal clusters. Each panel shows the significantly (false discovery rate [FDR] < 10%) enriched terms ranked by FDR. Bars show the percentage of all genes (belonging to each term) that were enriched and the FDR. Colors indicate major categories of terms, as indicated below the figure. (B) Gene expression of developmental patterning transcription factors is shown along the cell-type taxonomy. Each row represents one transcription factor, and columns represent clusters. Circles represent mean expression values proportional to area. Genes are sorted according to their expression pattern, with Hox genes sorted rostrocaudally. Labels on the right indicate the approximate anatomical extent of the expression of corresponding Hox genes. See also Figures S4, S6, and S7.

Figure S7

Expression Patterns of Transcription Factors, Related to Figure 7 Each section shows the expression of the indicated family of transcription factors, omitting genes that showed uniform or no expression. Circle areas are shown proportional to average gene expression in each cluster, normalized by row.