Molecular atlas of the adult mouse brain

Abstract

Brain maps are essential for integrating information and interpreting the structure-function relationship of circuits and behavior. We aimed to generate a systematic classification of the adult mouse brain based purely on the unbiased identification of spatially defining features by employing whole-brain spatial transcriptomics. We found that the molecular information was sufficient to deduce the complex and detailed neuroanatomical organization of the brain. The unsupervised (non-expert, data-driven) classification revealed new area- and layer-specific subregions, for example in isocortex and hippocampus, and new subdivisions of striatum. The molecular atlas further supports the characterization of the spatial identity of neurons from their single-cell RNA profile, and provides a resource for annotating the brain using a minimal gene set-a brain palette. In summary, we have established a molecular atlas to formally define the spatial organization of brain regions, including the molecular code for mapping and targeting of discrete neuroanatomical domains.

Fig. 1. ST to generate a whole-brain molecular atlas.

(A) Distribution of coronal sections used to generate the molecular atlas. Adjacent sections shown with light and dark red color. (B) Example of image processing showing the section outline in 3D (top left), the H&E-stained coronal section (top right), alignment with the ST array (bottom left), and alignment with the ABA mouse brain reference atlas (bottom right). (C) Transformation (light blue lines) of section shown in (B) to generate a common reference framework containing ST spot position and ABA neuroanatomical definitions. (D) 3D mapping of all spots color-coded according to main region identity. (E) Distribution of all spots according to main ABA region definitions. (F) Expression of three candidate genes with spatially discrete signals: in isocortex (Rasgrf2, green), striatum (Gpr88, blue), and thalamus (Rora, red). Left side, expression in the molecular atlas; right side, ISH signal from ABA. In 3D, right hemisphere shows spots with gene expression level above the 95th percentile. Left hemisphere shows striatum (light blue) and thalamus (light red) in ABA. Composite 3D image showing expression of all three genes including region definitions. Color scheme in (C) to (E), based on ABA reference color scheme.

Fig. 2. Computational approaches to extract new spatial domains from the molecular atlas.

(A) Examples of ICs (biological versus technical). Left side: IC signal (normalized score). Right side: Corresponding ABA reference section. IC scores above the 95th percentile in absolute value are shown in 3D. (B) 2D t-SNE with categorical color scheme to visualize spots in 181 molecular cluster (27 unique colors). (C) 3D t-SNE to visualize molecular similarity of clusters. (D) Mapping of all spots in 3D colored by molecular similarity. (E) 2D t-SNE showing spots according to molecular similarity coloring. (F) 2D t-SNE showing spots according to ABA neuroanatomical regions. (G) Hierarchical clustering of the 181 molecular clusters (fan plot). Molecular clusters annotated with a numerical identifier and a name according to correspondence to ABA subregion. Inner layer is color-coded using molecular similarity. Outer layer colored according to ABA subregions. (H) Example coronal sections from the molecular atlas. Left side: Position and molecular identity of spots for selected clusters (black lines, ABA region borders). Right side: The same clusters shown in the molecular atlas (black lines, molecular cluster borders). (I) Example coronal sections from the molecular atlas (left side, ABA reference atlas; right side, molecular clusters color-coded on the basis of molecular similarity). (J) Virtual sectioning of the molecular atlas (black line shows example of horizontal or a sagittal plane). Molecular similarity colors based on median coordinates of spots per cluster in the 3D t-SNE shown in (C) to (E), (G), and (I).

Fig. 3. New molecular definitions of subregions and layers in isocortex and striatum.

(A) t-SNE showing isocortical clusters (categorical color scheme). (B) t-SNE showing coloring of spots according to layer identity from registration in the ABA. (C) Circular phylogram showing layer identity of isocortical clusters. (D) Visualization of isocortical molecular clusters and gene expression (averaged z scored) for layer markers. (E) Visualization of all isocortical molecular clusters and their correspondence to major isocortical subregions. (F) Visualization of the isocortical clusters in coronal sections (AP axis). Left side: Individual spots belonging to molecular clusters. Right side: The corresponding smoothed clusters in the molecular atlas. (G to I) 3D visualization of molecular clusters found in different isocortical domains. (G) Molecular clusters in anterior isocortex (PL, ILA, ACA, and MO). (H) Molecular clusters in the central part of isocortex (SS, MOp, and PTLp). (I) Molecular clusters in the medial and posterior isocortex (RSP, POST, and PRE). (J) t-SNE showing striatal clusters. (K) t-SNE showing coloring of spots according to ABA reference. STRv, ventral striatum; STRd, dorsal striatum; sAMY, striatum-like amygdala nuclei; LSX, lateral septal complex. (L) Circular phylogram showing ABA reference for striatal clusters. (M) Visualization of the striatal molecular clusters and gene expression (averaged z scored) for reference markers. (N) Visualization of the striatal clusters in coronal sections of the molecular atlas. (O) 3D visualization of the striatal clusters. Left hemisphere, molecular atlas; right hemisphere, ABA striatum regions. Dot surface in (D), (E), and (M) corresponds to the absolute number of spots. Molecular clusters in (A), (E) to (J), and (L) to (O) are shown with the categorical color scheme. Molecular clusters in (B) to (D) show layer identity. Molecular clusters in (K) to (M) show striatal identity.

Fig. 4. Mapping the spatial origin of single cells using the molecular atlas.

(A) Schematic of the neural network (NN) organization. (B) Molecular atlas clusters that include ALM (blue outline) and VISp (red outline). Black lines show ABA reference. 3D visualization of the molecular clusters. (C) Top: Spatial mapping accuracy for single neurons (glutamatergic) and discarded cells. Bottom: Spatial mapping accuracy for single cells for the three main cell types. (D) Visualization of the spatial mapping of single neurons (scRNA-seq clusters from VISp, red outline; or ALM, blue outline). Black dots show spatial mapping of single neurons onto predicted cluster (clusters shown with categorical color scheme and ABA layers shown with back lines). Quantification of spatial mapping per region is shown for each scRNA-seq cluster with stacked bar plot. (E) Top row shows the accuracy of the spatial mapping of all cells using the NN predictions for each scRNA-seq cluster (133 clusters, 23,822 cells). Bottom row shows the proportion of mapped cells for each scRNA-seq cluster onto the molecular clusters. Color scheme of molecular clusters based on ABA reference colors.

Fig. 5. A reduced brain palette captures the whole-brain spatial organization.

(A) NMI used to select the gene set for a brain palette. NMI computed from either top IC loads (red) or top SVM weights (black). (B) t-SNE showing 181 molecular clusters based on the brain palette (266 genes). Clusters are colored on the basis of the best matching cluster in the full molecular atlas using the same categorical colors. (C) t-SNE showing individual spots colored according to neuroanatomical subregions (ABA reference colors). (D) Circular phylogram showing 181 molecular clusters in the brain palette atlas. The inner layer is color-coded using molecular similarity color scheme. The outer layer is colored according to ABA subregions. (E) Example of the similarity between the full molecular atlas (y axis) and the brain palette molecular atlas (x axis) for selected clusters. (F) Visualization of the brain palette molecular atlas (left side, pink background in the first section) and the full molecular atlas (right side, blue background in the first section) based on molecular similarity color scheme. (G) Visualization of isocortical, retrohippocampal, and olfactory clusters in the brain palette molecular atlas (left side, pink background in the first section) and the full molecular atlas (right side, blue background in the first section). (H) 3D visualization of selected isocortical clusters in the brain palette molecular atlas. Categorical color scheme is used to visualize the same clusters in (B), (E), (G), and (H).