a, UMAP plot for astrocyte nuclei, coloured by astrocyte subtype or brain region of origin. b, Global breakdown and regional composition of astrocyte subtypes. c, Gene expression heat map for the top markers of each astrocyte subclass, averaged to sample by subtype and scaled to the row maximum (max.). d, RNAscope validation of GRM3 and LGR6 as markers of AQP4+ neocortical and TH astrocytes, respectively (bold markers in c). Representative images (left) showing AQP4 transcripts (blue puncta) and GRM3 or LGR6 transcripts (red puncta). Scale bars, 20 μm. Quantification (right) was performed using two-tailed unpaired Student’s t-tests; ****P < 0.0001. Each dot represents an individual cell, pooled from eight samples (four individuals; each with one PFC and one thalamus sample). GRM3: n = 37 (PFC) and n = 23 (TH) cells; LGR6: n = 17 (PFC) and n = 23 (TH) cells. e, The framework for detecting gene expression modules using scdemon. f, The number of modules enriched for each covariate across all module sets (hypergeometric test, P < 0.001). Bar plots are coloured by the covariate level for which the modules are enriched (or by the major cell type used for module discovery for cell subtype). Diag., diagnosis. g,h, Gene–gene network (g) and magnification of the indicated regions (h) for astrocyte modules, with insets for M19, a subtype identity module for LUZP2 astrocytes (h, left) and M17, a functional program involved in cholesterol biosynthesis (h, right). AA, amino acid. i, Contour plots on the astrocyte UMAP for module expression of five identity (top row) and five functional (bottom row) programs. Expression was smoothed on a 500 × 500 grid with a 2D Gaussian kernel (size = 25 × 25; σ = 1). j,k, Module contours showing regions of top expression on the astrocyte UMAP for selected identity modules (j) and corresponding module scores (k) for the 18 labelled representative cells across the astrocyte UMAP for selected identity and expression models, scaled to the maximal expression of each module. ER, endoplasmic reticulum; SVD, singular value decomposition.