Experimental design to measure the landscape of gene expression in the postmortem human locus coeruleus (LC) using spatially-resolved transcriptomics (SRT) and single-nucleus RNA-sequencing (snRNA-seq).

(A) Brainstem dissections at the level of the LC were conducted to collect tissue blocks from 5 neurotypical adult human brain donors. (B) Inclusion of the LC within the tissue sample block was validated using RNAscope [36,37] for a pan-neuronal marker gene (SNAP25) and two NE neuron-specific marker genes (TH and SLC6A2). High-resolution H&E stained histology images were acquired prior to SRT and snRNA-seq assays (scale bars: 2 mm in H&E stained image; 20 μm in RNAscope images). (C) Prior to collecting tissue sections for SRT and snRNA-seq assays, tissue blocks were scored to enrich for the NE neuron-containing regions. For each sample, the LC region was manually annotated by visually identifying NE neurons in the H&E stained tissue sections. 100 μm tissue sections from 3 of the same donors were used for snRNA-seq assays, which included FANS-based neuronal enrichment prior to library preparation to enrich for neuronal populations. After all quality control (QC) steps, the final SRT and snRNA-seq datasets used for analyses consisted of samples from 4 and 3 donors, respectively.

Spatial gene expression in the human LC using SRT.

(A) Spots within manually annotated LC regions containing NE neurons (red) and non-LC regions (gray), which were identified based on pigmentation, cell size, and morphology from the H&E stained histology images, from donors Br2701 (top row) and Br8079 (bottom row). (B) Expression of two NE neuron-specific marker genes (TH and SLC6A2). Color scale indicates unique molecular identifier (UMI) counts per spot. Additional samples corresponding to A and B are shown in Supplementary Figures 1, 3A-B. (C) Boxplots illustrating the enrichment in expression of two NE neuron-specific marker genes (TH and SLC6A2) in manually annotated LC regions compared to non-LC regions in the N=8 Visium samples. Values show mean log-transformed normalized counts (logcounts) per spot within the regions per sample. Additional details are shown in Supplementary Figure 3C. (D) Volcano plot resulting from differential expression (DE) testing between the pseudobulked manually annotated LC and non-LC regions, which identified 32 highly significant genes (red) at a false discovery rate (FDR) significance threshold of 10-3 and expression fold-change (FC) threshold of 3 (dashed blue lines). Horizontal axis is shown on log2 scale and vertical axis on log10 scale. Additional details and results for 437 statistically significant genes identified at an FDR threshold of 0.05 and an FC threshold of 2 are shown in Supplementary Figure 8 and Supplementary Table 2. (E) Average expression in manually annotated LC and non-LC regions for the 32 genes from D. Color scale shows logcounts in the pseudobulked LC and non-LC regions averaged across N=8 Visium samples. Genes are ordered in descending order by FDR (Supplementary Table 2). (F-G) Cross-species comparison showing expression of human ortholog genes for LC-associated genes identified in the rodent LC [30,31] using alternative experimental technologies. Boxplots show mean logcounts per spot in the manually annotated LC and non-LC regions per sample in the human data.

Single-nucleus gene expression in the human LC using snRNA-seq.

We applied an unsupervised clustering workflow to identify cell populations in the snRNA-seq data. (A) Unsupervised clustering identified 30 clusters representing populations including NE neurons (red), 5-HT neurons (purple), and other major neuronal and non-neuronal cell populations (additional colors). Marker genes (columns) were used to identify clusters (rows). Cluster IDs are shown in labels on the right, and numbers of nuclei per cluster are shown in horizontal bars on the right. Percentages of nuclei per cluster are also shown in Supplementary Figure 10D. Heatmap values represent mean logcounts per cluster. (B) UMAP representation of nuclei, with colors matching cell populations from heatmap. (C) DE testing between neuronal clusters identified a total of 327 statistically significant genes with elevated expression in the NE neuron cluster, at an FDR threshold of 0.05 and FC threshold of 2. Heatmap displays the top 70 genes, ranked in descending order by FDR, excluding mitochondrial genes, with NE neuron marker genes described in text highlighted in red. The full list of 327 genes including mitochondrial genes is provided in Supplementary Table 4. Heatmap values represent mean logcounts in the NE neuron cluster and mean logcounts per cluster averaged across all other neuronal clusters (excluding ambiguous). (D-E) Cross-species comparison showing expression of human ortholog genes for LC-associated genes identified in the rodent LC [30,31] using alternative experimental technologies. Boxplots show logcounts per nucleus in the NE neuron cluster and all other neuronal clusters. Boxplot whiskers extend to 1.5 times interquartile range, and outliers are not shown. (F) DE testing between neuronal clusters identified a total of 361 statistically significant genes with elevated expression in the 5-HT neuron cluster, at an FDR threshold of 0.05 and FC threshold of 2. Heatmap displays the top 70 genes, ranked in descending order by FDR, with 5-HT neuron marker genes described in text highlighted in red. The full list of 361 genes is provided in Supplementary Table 7.

Summary of data resources providing access to datasets described in this manuscript.

All datasets described in this manuscript are freely accessible in the form of interactive web apps and downloadable R/Bioconductor objects.