Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: ( A ) Spots within manually annotated LC regions containing norepinephrine (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 and . ( 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 . ( 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 log 2 scale and vertical axis on log 10 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 and . ( 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 . ( F–G ) Cross-species comparison showing expression of human ortholog genes for LC-associated genes identified in the rodent LC ( ; ) 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.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Staining, Expressing, Marker, Transformation Assay, Quantitative Proteomics, Comparison

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: For each of the n=9 Visium capture areas (hereafter referred to as samples), the spots were manually annotated as being within the LC regions (red) or within the non-LC regions (gray) based on spots containing NE neurons, which were identified by pigmentation, cell size, and morphology on the H&E stained histology images.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Staining

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: ( A ) Hematoxylin and eosin (H&E) stained histology images for the n=9 Visium samples. Higher-resolution images can also be viewed through the Shiny web app ( https://libd.shinyapps.io/locus-c_Visium/ ). ( B ) Estimated number of cells per spot within annotated LC regions in 6 Visium samples, based on application of cell segmentation software (VistoSeg, ). Boxplots show medians, first and third quartiles, whiskers extending to the furthest values no more than 1.5 times the interquartile range from each quartile, and outliers.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Staining, Software

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: ( A-B ) Spot-plot visualizations of NE neuron marker gene expression ( TH and SLC6A2 , A and B, respectively) in the n=9 Visium samples. Color scale shows UMI counts per spot. One sample (Br5459_LC_round2) did not show clear expression of the NE neuron marker genes. This sample was excluded from subsequent analyses, leaving n=8 Visium capture areas (samples) from 4 out of the 5 donors. The annotated LC regions are shown in . ( C ) Enrichment of NE neuron marker gene expression ( TH and SLC6A2 ) within manually annotated LC regions compared to non-LC regions in the n=8 Visium samples that passed QC. Boxplots show values as mean log-transformed normalized counts (logcounts) per spot within each region per sample, with samples represented by shapes.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Marker, Gene Expression, Expressing, Transformation Assay

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: We applied a batch integration tool (Harmony, ) to remove technical variation in the molecular measurements between the n=8 Visium samples from 4 donors. The integrated measurements were subsequently used as the input for spatially-aware clustering using BayesSpace . ( A ) Principal component analysis (PCA) (top 2 PCs) calculated on molecular expression measurements, with spots labeled (left to right) by donor ID, round ID, and sample ID, without applying any batch integration. ( B ) Harmony embeddings (top 2 Harmony embedding dimensions) after applying Harmony batch integration on sample IDs, with spots labeled (left to right) by donor ID, round ID, and sample ID, demonstrating that the technical variation has been reduced.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Expressing, Labeling

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: We applied a spatially-aware unsupervised clustering algorithm (BayesSpace, ) to investigate whether the LC and non-LC regions in each Visium sample could be annotated in a data-driven manner. ( A ) Using BayesSpace with k =5 clusters, we clustered spots from the n=8 Visium samples using the Harmony batch-integrated molecular measurements (clustering performed across samples). Cluster 4 (red) corresponds most closely to the manually annotated LC regions. The annotated LC regions are shown in . ( B ) BayesSpace clustering performance evaluated in terms of concordance between cluster 4 (red) and the manually annotated LC region in each sample. Clustering performance was evaluated in terms of precision, recall, F1 score, and adjusted Rand index (ARI) (see Methods for definitions).

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques:

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: ( A ) We manually annotated individual Visium spots (black) overlapping with NE neuron cell bodies within the previously manually annotated LC regions (red), based on pigmentation, cell size, and morphology from the H&E stained histology images, in the n=8 Visium samples. ( B ) We observed relatively low overlap between spots with expression of the NE neuron marker gene TH (≥2 observed UMI counts per spot) and the set of annotated individual spots. The differences included both false positives (annotated spots that were not TH +) and false negatives ( TH + spots that were not annotated). Therefore, we did not use the spot-level annotations for subsequent analyses, and instead used the LC region-level annotations for all further analyses.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Staining, Expressing, Marker

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: We applied nnSVG , a method to identify spatially variable genes (SVGs), in the Visium SRT samples. We ran nnSVG within each contiguous tissue area containing a manually annotated LC region (13 tissue areas in the n=8 Visium samples) and calculated an overall ranking of top SVGs by averaging the ranks per gene from each tissue area. ( A ) The top 50 ranked SVGs from this analysis included a subset (11 out of 50) of genes that were highly ranked in samples from only one donor (Br8079, genes highlighted in maroon). We determined that this was due to the inclusion of a section of the choroid plexus adjacent to the LC for this donor. Bars show the number of times (out of 13 tissue areas) each gene was included within the top 100 SVGs. Rows are ordered by overall average ranking in descending order. ( B ) Spatial expression of CAPS , a choroid plexus marker gene, in the n=8 Visium samples. ( C ) Histology image showing the two tissue areas for sample Br8079_LC_round3. ( D ) In order to focus on LC-associated SVGs, we calculated an overall average ranking of SVGs that were each included within the top 100 SVGs in at least 10 out of the 13 tissue areas, which identified 32 highly-ranked, replicated LC-associated SVGs. Boxplots show the ranks in each tissue area. Rows are ordered by the overall average ranking in descending order.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Expressing, Marker

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: ( A-B ) We visualized the spatial expression of 5-HT (5-hydroxytryptamine or serotonin) neuron marker genes ( TPH2 and SLC6A4 ) in the n=9 initial Visium SRT samples within the Visium SRT samples, which showed that the population of 5-HT neurons was distributed across both the LC and non-LC regions. The annotated LC regions are shown in . ( C ) Enrichment of 5-HT neuron marker gene expression ( TPH2 and SLC6A4 ) within manually annotated LC regions compared to non-LC regions in the n=8 Visium SRT samples that passed QC (see ). Boxplots show values as mean log-transformed normalized counts (logcounts) per spot within each region per sample, with samples represented by shapes.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Expressing, Marker, Gene Expression, Transformation Assay

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: We visualized the spatial expression of ( A-B ) additional marker genes for 5-HT neurons ( SLC18A2 , FEV ) and ( C-D ) 5-HT autoreceptor genes ( HTR1A , HTR1B ) in the n=9 initial Visium samples. SLC6A18 is not shown since we observed zero expression of this gene in the Visium samples. Color scale shows UMI counts per spot.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Expressing, Marker

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: We applied a spot-level deconvolution algorithm (cell2location, ) to integrate the snRNA-seq and SRT data by estimating the cell abundance of the snRNA-seq populations, which are used as reference populations, at each spatial location (spot) in the Visium SRT samples. While this approach mapped ( A ) NE neurons (cluster 6) and ( B ) 5-HT neurons (cluster 21) to the spatial regions where these populations were previously identified based on expression of marker genes ( and ), the overall mapping performance was relatively poor. We note that these are relatively rare populations, with relatively subtle expression differences compared to other neuronal populations, and NE neurons are characterized by large size and high transcriptional activity, which may have affected performance of the algorithm. The annotated LC regions are shown in .

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Expressing, Marker, Activity Assay

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: We visualized the spatial expression of DA neuron marker genes ( A ) SLC6A3 (encoding the dopamine transporter), ( B ) ALDH1A1 , and ( C ) SLC26A7 in the n=9 initial Visium SRT samples, which showed that these genes were not strongly expressed within the annotated LC regions. Color scale shows UMI counts per spot. The annotated LC regions are shown in .

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Expressing, Marker

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: We visualized the spatial expression of cholinergic marker genes ( A ) SLC5A7 and ( B ) ACHE in the n=9 initial Visium SRT samples, which showed that these genes were expressed both within and outside the annotated LC regions. Color scale shows UMI counts per spot. The annotated LC regions are shown in .

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: Expressing, Marker

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: 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.

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques:

Journal: eLife

Article Title: The gene expression landscape of the human locus coeruleus revealed by single-nucleus and spatially-resolved transcriptomics

doi: 10.7554/eLife.84628

Figure Lengend Snippet: All datasets described in this manuscript are freely accessible via interactive web apps and downloadable R/Bioconductor objects (see for details). ( A ) Screenshot of Shiny web app providing interactive access to Visium SRT data. ( B ) Screenshot of iSEE web app providing interactive access to snRNA-seq data. For instructions on how to use the web apps to search for and display individual genes, see and .

Article Snippet: For tissue blocks included in the study, we cut additional 10 μm tissue sections, which were used for gene expression profiling at spatial resolution using the 10x Genomics Visium SRT platform ( ; ).

Techniques: