Article Figures & Data
Figures
- Figure 1.
scRNA-seq identifies distinct populations of cortical brain interface versus bone-associated cells. Also see Extended Data Figures 1-1, 1-2, and 1-3. A, B, UMAP visualization of all transcriptomes identified in the cortical surface-associated (CSA; A) or cortical bone-associated (BA; B) datasets as analyzed by scRNA-seq. Shown are the mergers of four independent surface-associated runs (A) and two independent bone-associated runs (B). The merged CSA dataset in (A) was batch corrected using one iteration of Harmony. Transcriptionally distinct clusters are color coded, and plots are annotated for cell types corresponding to these clusters, as determined by analysis of well-characterized marker genes. VSMCs, vascular smooth muscle cells; Lepto, leptomeningeal cells; Dural, dural cells. C, D, Violin plots showing expression of Pdgfra, Pdgfrb, and Col1a1 in leptomeningeal (C; colored blue) and dural (D; colored blue) cells relative to the Pdgfra-negative pericytes (colored red) and VSMCs (colored green). Dots represent expression levels in individual cells. E, F, Pdgfra-positive cell transcriptomes were subsetted from the surface-associated and bone-associated datasets shown in A and B and merged together. Panel E shows the cluster UMAP, where transcriptionally distinct clusters are colored and numbered and F shows the dataset of origin for each transcriptome. CSA, cortical surface associated; BA, bone associated. G, Gene ontology was performed on the differentially expressed genes identified in the comparison between leptomeningeal and dural cells as defined in F (>1.3-fold average change, padj < 0.05; Extended Data Figs. 1-2 and 1-3). Shown are selected categories (y-axis) and the adjusted p value (x-axis). The peach-colored bars are categories enriched in leptomeningeal cells and the turquoise enriched in dural cells. H, Single-cell heatmap of select mRNAs that were differentially expressed (Extended Data Fig. 1-2) in the comparison between the leptomeningeal cells in cluster 0 and the dural cells in cluster 1 from the UMAPs in E and F. Every row represents expression in an individual cell, and levels are color coded as per the adjacent key. I, Violin plots showing relative expression levels of two mRNAs, Ptgds and Igfbp7, that were identified in the differential gene expression analysis as highly enriched in leptomeningeal (colored red) versus dural (colored turquoise) cells, respectively. Dots represent expression levels in individual cells. J, Transcriptional signatures for the putative leptomeningeal and dural cells were defined using the 50 most differentially expressed mRNAs in the leptomeningeal versus dural cell comparison (Extended Data Fig. 1-2). Shown is a UMAP as in E highlighting cells expressing a transcriptional signature score of >0.5 for one or the other cell type.
- Figure 2.
Identification of transcriptionally distinct border astrocytes by scRNA-seq analysis. Also see Extended Data Figures 2-1, 2-2, and 2-3. A, B, Astrocyte transcriptomes were subsetted from four different murine scRNA-seq datasets: cortical surface-associated cells as in Figure 1A, P60 whole cortex and P60 cortical gray matter from Dennis et al. (2024) (GEO GSE255405; Extended Data Fig. 2-1A,B), and whole-brain astrocytes from Hasel et al. (2021) (GEO GSE148611). In the latter case, only astrocytes enriched for Myoc and Gfap were extracted (Extended Data Fig. 2-1C,D). These astrocyte transcriptomes were merged and batch corrected using one iteration of Harmony. The resultant merged dataset is shown as UMAP visualizations with the transcriptionally distinct clusters numbered and color coded in A and datasets of origin shown in B. C, UMAPs as in A and B overlaid for expression of two pan-astrocyte mRNAs, Slc1a3 and Cxcl14, and two brain interface-enriched mRNAs, Myoc and Gfap. The putative border astrocytes are circled. Expression levels are color coded as per the adjacent keys. D, Single-cell heatmap of select mRNAs that were differentially expressed (Extended Data Fig. 2-2) in the comparison between the putative border astrocytes (cluster 2 in panel A) and cortical gray matter astrocytes (cluster 1 in panel A). Every row represents expression in an individual cell, and levels are color coded as per the adjacent key. E, Gene ontology was performed on the differentially expressed genes identified in the comparison between border astrocytes and cortical gray matter astrocytes (>1.3-fold average change, p adj < 0.05; Extended Data Figs. 2-2, 2-3). Shown are selected categories (y-axis) and the adjusted p value (x-axis). F, A transcriptional signature for the putative border astrocytes (cluster 2 in A) was defined using the 20 most differentially expressed mRNAs in the border versus cortical gray matter astrocyte comparison (Extended Data Fig. 2-2). Shown is a UMAP as in A with cells expressing a border astrocyte transcriptional signature score >0.5 highlighted in purple.
- Figure 3.
Lineage tracing and immunostaining identify the leptomeninges and the dura at the cortical brain interface. Also see Extended Data Figure 3-1. A, Schematic showing the region of the cortex and overlying skull that were analyzed in the lineage tracing and immunostaining studies. B, Ten-week-old Pdgfra-CreERT2;R26RtdT mice were treated with tamoxifen and tissues collected 10 d after the final administration. The brain and skull were decalcified, and sagittal cortical sections were immunostained for GFAP (white) and counterstained with DAPI (blue). Shown is a representative confocal image of the brain interface and skull showing TdTomato (red), GFAP (white), and DAPI (blue). The boxed region is shown at higher magnification to the right without the DAPI. Lepto, leptomeninges; Astro, border astrocytes. C, Representative confocal image of a sagittal wild-type mouse brain interface section prepared as in B, immunostained for GFAP (white) and counterstained with DAPI (blue). Lepto, leptomeninges. D, Violin plots showing the relative expression of Gjb6, Gjb2, Lama1, Tmem119, and Matn4 in the leptomeningeal (colored red) versus dural (colored turquoise) cells shown in Figure 1E,F. Dots represent individual transcriptomes. E, Ten-week-old Tmem119CreErt2;R26RtdT mice were treated with tamoxifen and tissues collected 10 d later. The brain and skull were decalcified and sagittal cortical sections immunostained for GFAP (white) and counterstained with DAPI (blue). The left panel shows a low magnification image of the skull, dura, and cortical interface showing TdTomato (red) and DAPI (blue). The space between the leptomeninges and dura is a processing artifact. The two right panels are high magnification confocal images of the same section showing TdTomato (red) and GFAP immunostaining (white) in the dura and skull (top right) and the brain interface (bottom right). Both top and bottom right panels come from the same field of view. Lepto, leptomeninges; Astro, border astrocytes. F, Representative images of sagittal sections through the decalcified skull and cortex, immunostained for GFAP (white) and MATN4 (yellow) and counterstained with DAPI (blue). The left panel shows a low magnification image of the skull, dura and cortical interface. The space between the leptomeninges and dura is a processing artifact. The two right panels are high magnification confocal images of a similar section from the same brain showing MATN4 (yellow) and GFAP (white) immunostaining in the dura and skull (top right) and the brain interface including the leptomeninges (bottom right). Both top and bottom right panels come from the same field of view. Lepto, leptomeninges; Astro, border astrocytes. G, High magnification confocal image of a sagittal adult cortical interface section immunostained for GFAP (white) and Laminin (yellow) and counterstained with DAPI. Note that the Laminin immunoreactivity is localized between the border astrocytes and the leptomeningeal cells. Lepto, leptomeninges; Astro, border astrocytes. H, High magnification confocal image of a sagittal adult cortical interface immunostained for GFAP (white) and CX26 (yellow) and counterstained with DAPI. The hatched line delineates the border between the leptomeninges and the dura. Lepto, leptomeninges; Astro, border astrocytes. I, High magnification confocal image of a sagittal adult cortical interface section immunostained for GFAP (white) and CX30 (yellow) and counterstained with DAPI. Lepto, leptomeninges; Astro, border astrocytes. For all panels, scale bars are 10 µm except for the leftmost images in E and F, where scale bars are 100 μm.
- Figure 4.
Single-cell multiplexed in situ gene expression analysis with two different probesets identifies the spatial location of cortical interface and layer 1 cell types. Also see Extended Data Figures 4-1, 4-2, and 4-3. Coronal adult mouse cortex sections at the levels shown in Extended Data Fig. 4-1A,B were analyzed by Xenium-based single-cell multiplexed in situ gene expression analysis with either a brain-targeted probeset targeting 347 genes or a mesenchymal probeset targeting 480 genes (Extended Data Fig. 4-3). The ROI that was analyzed is shown in A. Datasets from different sections and conditions were then merged and cell types identified by marker gene expression. A, Schematic of a coronal section through the adult cortex in the midline region showing the ROI that was analyzed (outlined with hatched black lines). B, C, UMAP cluster visualization of the merged transcriptomes resulting from analysis with the brain probeset (B) or the mesenchymal probeset (C), annotated for cell types. Each dot represents a single cell. VSMC, vascular smooth muscle cell; Oligos, oligodendrocytes; SST, somatostatin; Pvalb, parvalbumin; VIP, vasoactive intestinal peptide. D, E, Spatial plots of the midline and adjacent cortical interface and layer 1 from the ROI of representative sections analyzed using the brain (D) or mesenchymal (E) probesets, showing all cell types except neurons, color coded as per the legend. The boxed regions are also shown in enlarged views. L1 denotes cortical layer 1. Lepto, leptomeninges; B. Ast, border astrocytes. F, G, Spatial plots as in D and E from the same representative sections analyzed using the brain (F) or mesenchymal (G) probesets showing the leptomeninges and all of the neurons that were identified, color coded. L1 denotes cortical layer 1. Low magnification spatial plots in D–G were generated using Seurat with D and F showing the same ROI and E and G the same ROI. Each dot represents the centroid of one cell.
- Figure 5.
Single-cell spatial transcriptomic analysis defines the cortical brain interface structure and cell types. Also see Extended Data Figure 5-1. Coronal adult mouse cortex sections at the levels shown in Extended Data Fig. 4-1A,B were analyzed by Xenium-based single-cell multiplexed in situ gene expression analysis with either a brain probeset targeting 347 genes or a mesenchymal probeset targeting 480 genes (Extended Data Fig. 4-3). The ROI and UMAPs of the resultant merged datasets are shown in Figure 4A–C. A, Spatial plots of the cortical interface analyzed with the mesenchymal (MP, left) or brain (BP, right) probesets showing leptomeningeal cells (pink), border astrocytes (yellow), border macrophages (dark blue), and dural cells (green, mesenchymal probeset only). B, Spatial plot of the cortical interface and midline analyzed with the mesenchymal probeset showing dural (green) and leptomeningeal (pink) cells. C, High-resolution Xenium Explorer image of the cortical interface region analyzed with the mesenchymal probeset (MP) showing expression of H19 mRNA (orange dots) and Gjb2 mRNA (bright blue dots) relative to the leptomeningeal cells (pink) and dural cells (green). Nuclei of other cell types (white) are also shown. D, Spatial plots of the dural mRNA H19 (top) or the leptomeningeal mRNA Gjb2 (bottom) in all cells at the cortical interface, midline, and layer 1 analyzed using the mesenchymal probeset (MP). Relative mRNA expression levels are coded as per the adjacent keys. E, High-resolution Xenium Explorer images of the cortical interface region analyzed with the mesenchymal (MP, top) or brain (BP, bottom) probesets showing the spatial arrangement of leptomeningeal cells (pink), border astrocytes (yellow), border macrophages (blue), and dural cells (green, mesenchymal probeset only). Nuclei are also shown (white/gray). F, Spatial plots of the cortical interface and layer 1 analyzed with the mesenchymal (MP, top) or brain (BP, bottom) probesets showing border astrocytes (yellow) and parenchymal astrocytes (brown). G, Spatial plots of Myoc and Gfap mRNA expression in all cells of the full ROI analyzed using the brain probeset (BP). Relative expression levels are coded as per the adjacent keys. H, High-resolution Xenium Explorer image of the cortical interface analyzed with the mesenchymal (MP, left) or brain (BP, right) probesets showing expression of Myoc mRNA (light blue dots) relative to the leptomeningeal cells (pink) and border astrocytes (yellow). Nuclei of other cell types (white) are also shown. I, Spatial plots of the cortical interface and layer 1 analyzed with the mesenchymal (MP, left) or brain (BP, right) probesets showing border macrophages (blue) and microglia (tan). J, High-resolution Xenium Explorer image of the cortical interface and layer 1 analyzed with the mesenchymal probeset (MP) showing expression of Mrc1 mRNA (bright blue dots) and Sall1 mRNA (bright green dots) relative to the border macrophages (dark blue), microglia (tan), leptomeningeal cells (pink), and dural cells (darker green). Nuclei of other cell types (white) are also shown. Low magnification spatial plots in A, B, D, F, G, and I were generated using Seurat and show part of the ROI centered on the cortical midline region from one representative section each, with each dot representing the centroid of one cell. High-resolution spatial plots in C, E, H, and J were generated using Xenium Explorer. Scale bars, 10 μm.
- Figure 6.
Distinguishing the arachnoid and pial leptomeningeal layers using scRNA-seq and single-cell spatial transcriptomics. Also see Extended Data Figures 6-1 and 6-2. A–D, scRNA-seq-derived mesenchymal cell transcriptomes from Pietilä et al. (2023) (GEO GSE227713; shown in Extended Data Fig. 6-1A) were merged with the meningeal cell transcriptomes from Figure 1E and batch effects minimized using two iterations of Harmony. A UMAP of the total merged, annotated dataset is shown in Extended Data Figure 6-1B. A, B, UMAP visualizations of the leptomeningeal cells from the merged total meningeal cell scRNA-seq dataset (Extended Data Fig. 6-1B) showing the potential pial and arachnoid cell clusters (A) and their datasets of origin (B). C, UMAP as in A, overlaid for two pan-leptomeningeal mRNAs, Slc22a6 and Gjb6. Gene expression levels are color coded as per the adjacent keys. D, Violin plots showing relative expression of two mRNAs, Lama1 and Ppp1r1a, in cluster 0 versus 4 (the potential pial versus arachnoid cells) in the UMAP shown in A. Dots represent individual transcriptomes. E, Spatial expression plots of mRNAs differentially enriched in the cluster 0 putative pial cells from A (also see Extended Data Fig. 6-2), in the leptomeningeal cells identified by Xenium using either the mesenchymal probeset (MP) or the brain probeset (BP; as shown in Fig. 4F,G). Also shown is a similar spatial plot for the pial mRNA Lama1. Relative mRNA expression levels are coded as per the adjacent keys. F, Spatial expression plots of mRNAs differentially enriched in the cluster 4 putative arachnoid cells from A (also see Extended Data Fig. 6-2), in the leptomeningeal cells identified by Xenium using either the mesenchymal probeset (MP) or the brain probeset (BP; as shown in Fig. 4F,G). Also shown is a similar spatial plot for the arachnoid mRNA Ppp1r1a. Relative mRNA expression levels are coded as per the adjacent keys. G, UMAP cluster visualization of the leptomeningeal transcriptomes from the Xenium brain probeset data (Fig. 4B), shown at higher resolution and annotated for potential pial versus arachnoid cells. The transcriptionally distinct clusters (4, 6, and 8) are color coded as per the adjacent key. H, Violin plots showing relative expression levels of two pial mRNAs, Lama1 and Cyp1b1, and two arachnoid mRNAs, Slc47a1 and Dpp4, in the potential arachnoid versus pial leptomeningeal cells as annotated in G. Purple and green denote arachnoid and pial cells, respectively. Each dot corresponds to expression in an individual cell. I, Spatial plots of the cortical interface and midline showing spatial distribution of the cluster 6 pial cells (green) and the clusters 4 and 8 arachnoid cells (turquoise and purple, respectively), as annotated in G. Note that the pial but not arachnoid cells extend down the midline. J, High-resolution Xenium Explorer image of the cortical interface (all panels) and midline (left panel) analyzed with the brain probeset (BP) showing expression of the arachnoid mRNAs Cdh1 (red dots), Dpp4 (yellow dots), and Slc47a1 (pink dots) relative to the arachnoid and pial cells (turquoise and green, respectively) as annotated in G. Nuclei of other cell types (white) are also shown. Note that the pial cells extend down the midline (left panel). Low magnification spatial plots in E, F, and I were generated using Seurat, and each dot represents the centroid of one cell. High-resolution spatial plots in J were generated using Xenium Explorer. Scale bars, 10 μm.
- Figure 7.
Single-cell spatial transcriptomic analysis defines distinct proximal cellular neighborhoods for the cortical brain interface and layer 1. Coronal adult cortical sections were analyzed by Xenium-based single-cell multiplexed in situ gene expression analysis with either a brain probeset targeting 347 genes or a mesenchymal probeset targeting 480 genes (Extended Data Fig. 4-3). The ROI and UMAPs of the resultant merged datasets are shown in Figure 4A–C. A, Spatial plots of the cortical interface and layer 1 analyzed with the mesenchymal (MP, left) and brain (BP, right) probesets showing oligodendrocytes (pink), microglia (tan), parenchymal astrocytes (dark brown), leptomeninges (dark pink), OPCs (orange), border astrocytes (yellow), dural cells (green, mesenchymal probeset only), and border macrophages (dark blue). B, Spatial plots of the cortical interface and layer 1 analyzed with the mesenchymal (MP, left) and brain (BP, right) probesets showing pericytes (turquoise), endothelial cells (red), VAMCs (blue), dural cells (green, mesenchymal probeset only), and leptomeninges (dark pink). C, Spatial plot of the cortical interface and layer 1 analyzed with the brain probeset (BP) showing the leptomeninges (dark pink) and interneurons expressing Lamp5 (lime green, Lamp5IN), Vip (blue-green, VIPIN), Somatostatin (gray, SSTIN), or Parvalbumin (black, PvalbIN). D, Proximity analysis showing the relative strength of statistically significant interactions between different cell types located in the cortical brain interface and adjacent layer 1, as analyzed from the datasets obtained using either the brain probeset (left) or the mesenchymal probeset (right). The nodes are color coded and annotated for cell type, and the weight of the line indicates the strength of the interaction. Black lines indicate significant interactions based on a permutation test, comparing our data to a null distribution created from the random permutation of cell labels with fixed positions (see Materials and Methods for details). Green lines indicate interactions that were not statistically significant. Excitatory neuron-1 and excitatory neuron-2 denote transcriptionally distinct excitatory neuron clusters. Cell centroid information was used to perform this analysis, with a proximal interaction being defined as one where a cell centroid was within 70 µm of another cell centroid. This unsupervised analysis identifies two neighborhoods, the cortical brain interface and layer 1, as well as a partial neighborhood centered around the layer 2 excitatory neurons at the boundary of layer 1. Oligos, oligodendrocytes. Low magnification spatial plots (A–C) were generated using Seurat, and each dot represents the centroid of one cell. The proximity maps in D were generated using Giotto.
- Figure 8.
Leptomeningeal cells express ligands predicted to regulate border astrocytes and border macrophages. Also see Extended Data Figures 8-1 to 8-4. A–D, Leptomeningeal cells, border astrocytes, and border macrophages were analyzed for their expression of ligand and ligand receptor mRNAs using the scRNA-seq data (Extended Data Figs. 8-2, 8-3, and 8-4). Ligand or receptor mRNAs were included if they were expressed in ≥5% of the relevant cell type. The data were used to generate predictive models of the bioactive ligand environment within the brain interface neighborhood. Each box includes a ligand known to bind to a corresponding receptor expressed by at least one of the brain interface cell types. A, B, All of the ligands made by the brain interface cells that have a receptor expressed by at least one of the interface cell types. In A, these are color coded to show the cells that express these ligands, while in B, they are color coded to show the cells that express the relevant receptor and thus are predicted to respond to the ligands. C, D, Models with ligands expressed by the leptomeningeal cells that have corresponding receptors on either border macrophages (C) or border astrocytes (D). E, Dot plot showing expression levels of Igf2, Sema3a, Bmp6, and Bmp7 mRNAs in leptomeningeal cells, border astrocytes, and border macrophages, as determined from the scRNA-seq data. The size of the dot indicates the percentage of cells detectably expressing the mRNA, and the color indicates relative expression level, coded as per the adjacent keys. F, Spatial plots of Bmp7, Bmp6, Igf2, Sema3a, and Igf1r mRNAs in all brain interface and layer 1 cells analyzed used the brain probeset (BP), all shown on the same representative section. Relative mRNA expression levels are coded as per the adjacent keys. G, High-resolution Xenium Explorer images of the cortical interface region analyzed with the brain probeset (BP) showing expression of Bmp6 (light pink dots), Igf2 (blue dots), Bmp7 (green dots), Igf2 (blue dots), and Sema3a (white dots) mRNAs relative to the leptomeningeal cells (pink), border astrocytes (yellow), and border macrophages (dark blue). The same field of view is shown in each panel, and arrows denote the same leptomeningeal cell detectably coexpressing multiple ligand mRNAs. Nuclei of other cell types (white) are also shown. H, High-resolution Xenium Explorer images of the cortical interface region analyzed with the brain probeset (BP) showing expression of Igf2 (blue dots) and Igf1r (yellow dots) mRNAs relative to the same cell types as in G. Nuclei of other cell types (white) are also shown. Low magnification spatial plots in F were generated using Seurat and the high-resolution spatial plots in G and H with Xenium Explorer. Scale bars, 10 μm.
Extended Data
Figure 1-1
scRNA-seq to analyze adult murine cortical leptomeninges and dura-associated cell types. (A) Schematic of methods used to generate cells for the scRNA-seq datasets shown in Fig. 1A and B. The top illustrates the brain surface dissection and digestion and the bottom the bone-associated dissection. (B) UMAP visualization of merged transcriptomes from 4 independent cortical surface-associated (CSA) scRNA-seq runs (CSA1-4). Transcriptomes from each run are colored as per the adjacent legend. The top panel shows the cluster UMAP without Harmony batch correction, and the bottom with one iteration of Harmony. The bottom UMAP is also annotated for cell types. CP = choroid plexus, EC = endothelial cells, Lepto = leptomeninges, Ast/OPC = astrocytes and oligodendrocyte precursor cells, Neu = neurons, VSMC = vascular smooth muscle cells. (C) The batch-corrected, merged dataset in (B, bottom panel) was overlaid for expression of marker genes specific to different cell types. Expression levels are color-coded as per the adjacent keys. (D) UMAP visualization of merged transcriptomes from 2 independent bone-associated scRNA-seq runs (BA1, BA2). Transcriptomes from each run are colored as per the adjacent legend. The UMAP is also annotated for cell types. EC = endothelial cells. (E) The merged bone-associated dataset in (D) was overlaid for expression of marker genes specific to different cell types. Expression levels are color-coded as per the adjacent keys. (F, G) Pdgfrb-positive mesenchymal cells were subsetted from the bone-associated and cortical surface-associated datasets shown in (B) and (D), and reanalyzed. The annotated UMAPs (upper left panels) show the subsetted transcriptomes colored by cluster, and annotated for Pdgfra-positive dural or leptomeningeal cells (Dural in F, Lepto in G), and for Pdgfra-negative pericytes and vascular smooth mucle cells (VSMCs). The remainder of the panels show expression overlays for genes characteristic of each of the different mesenchymal cell types. Expression levels are color-coded as per the adjacent keys. (H) Pearson correlation analysis of averaged expression of each detected gene in leptomeningeal (y-axis) versus dural (x-axis) cell transcriptomes from the merged dataset shown in Fig. 1E. (I) Violin plots showing relative expression levels of select mRNAs that were identified in the differential gene expression analysis as being highly-enriched in leptomeningeal (colored blue) versus dural (colored orange) cells, respectively. Dots represent expression levels in individual cells. Download Figure 1-1, TIF file.
Figure 1-2
Genes differentially expressed in leptomeningeal and dural mesenchymal cells. Differential gene expression analysis was performed on the merged Pdgfra-positive leptomeningeal and dural clusters shown in Fig. 1E. Differentially expressed genes were defined as those expressed in >10% of cells in either group with a Bonferroni adjusted p-value < 0.05 (Adj. p-value) and ≥ 1.3 average log2 fold change (Avg log2FC). *Denotes genes included in the dural cell signature. **Denotes genes included in the leptomeningeal cell signature. Download Figure 1-2, XLS file.
Figure 1-3
Gene ontology for genes differentially enriched in leptomeningeal and dural cells as shown in Figure 1-2. g:Profiler was used to identify gene ontology terms that were overrepresented in the differentially enriched leptomeningeal versus dural mRNAs shown in Figure 1-2. Enriched terms from the Molecular Function (MF), Biological Process (BioP), and Cell Components (CC) categories are all included. Download Figure 1-3, XLS file.
Figure 2-1
scRNA-seq to analyze adult cortical and border astrocytes. (A, B) UMAP visualizations of transcriptomes from previously-published (Dennis et al., 2024; GEO GSE255405) scRNA-seq datasets of dissected postnatal day 60 (P60) cortical grey matter (A) or total cortex (B) tissue. Transcriptomes were reanalyzed and cell types were identified using well-characterized marker genes. The transcriptionally-distinct clusters are color-coded, and different cell types annotated and denoted by the hatched lines. (C) UMAP visualization of transcriptomes from a previously-published (Hasel et al., 2021; GEO GSE148611) whole brain astrocyte scRNA-seq dataset. Transcriptomes were reanalyzed and transcriptionally-distinct clusters are color-coded. The cluster most enriched for Myoc and Gfap expression (outlined) was subsetted and used for subsequent analyses. (D) Violin plots showing relative expression levels of Myoc and Gfap mRNAs in the putative whole brain border astrocytes (BorderAst) from the cluster outlined in (C), versus all other astrocytes (Ast) from the dataset shown in (C). Download Figure 2-1, TIF file.
Figure 2-2
Genes differentially expressed in border astrocytes versus cortical grey matter astrocytes. Differential gene expression analysis was performed on the merged astrocyte dataset shown in Fig. 2A, comparing the border astrocytes (cluster 2) and the cortical grey matter astrocytes (cluster 1). Differentially expressed genes were defined as those expressed in >10% of cells in either group with a Bonferroni adjusted p-value < 0.05 (Adj. p-value) and ≥ 1.3 average log2 fold change (Avg log2FC). *Denotes genes included in the border astrocyte gene signature. Download Figure 2-2, XLS file.
Figure 2-3
Gene ontology for genes differentially enriched in border astrocytes versus cortical grey matter astrocytes as shown in Figure 2-2. g:Profiler was used to identify gene ontology terms that were overrepresented in the differentially enriched border astrocyte versus cortical grey matter astrocyte mRNAs shown in Figure 2-2. Enriched terms from the Molecular Function (MF), Biological Process (BioP), and Cell Components (CC) categories are all included. Download Figure 2-3, XLS file.
Figure 3-1
Characterization of the cortical brain interface by immunostaining. (A) High magnification confocal images of a sagittal section through the brain interface and skull, immunostained for PDGFRα (yellow) and GFAP (white), and counterstained with DAPI (blue). The left panel shows the merge and the right two immunostaining for PDGFRα or GFAP separately. Lepto = leptomeninges, Astro = border astrocytes. Scale bar = 10 μm. (B, C) The border astrocyte transcriptomes from cluster 2 in Fig. 2A and the leptomeningeal and dural cell transcriptomes from Fig. 1E were merged and reanalyzed. Shown are UMAPs with the clusters color-coded and annotated (B) or the datasets of origin indicated as per the adjacent color legend (C). CSA = cortical surface-associated dataset, BA = bone-associated dataset. (D) Gene expression overlays of the merged astrocyte plus meningeal mesenchymal cell dataset shown in (B, C) for selected genes that distinguish the different cell types. Expression levels are color-coded as per the adjacent keys. Download Figure 3-1, TIF file.
Figure 4-1
Analysis of the cortical interface and layer one cells using single cell multiplexed in situ gene expression analysis with the brain probeset. (A, B) Coronal cortical sections were analyzed by Xenium-based single cell multiplexed in situ gene expression analysis. (A) shows a representative Xenium Explorer image of a rostral brain section at the level used for all mesenchymal probeset analyses and for 2 of the 5 brains analyzed with the brain probeset. (B) shows a representative Xenium Explorer image of the more caudal brain sections used to generate the brain probeset data previously published in Willis et al. (2025; GEO GSE266689, sections GSM8647390, GSM8647391, GSM8647392). The ROI that was analyzed for all sections is shown in Fig. 4A, and the annotated UMAP cluster visualizations of the resultant merged transcriptomes are shown in Fig. 4B and C. (C) UMAPs of the merged brain probeset data showing either the annotated clusters (bottom; the same UMAP as in Fig. 4B) or the section of origin (top) for each of the transcriptomes. Brain1-5 denotes 5 sections from 5 different mice. Each dot represents a single transcriptome. VSMCs = vascular smooth muscle cells, Oligos = oligodendrocytes, SST = somatostatin, Pvalb = parvalbumin, VIP = vasoactive intestinal peptide. (D) Expression overlays for selected marker genes on the UMAPs shown in (C). Expression levels are color-coded as per the adjacent keys. Space bars = 2500 μM. Download Figure 4-1, TIF file.
Figure 4-2
Analysis of the cortical interface and layer one cells using single cell multiplexed in situ gene expression analysis. (A, B) Coronal cortical sections as shown in Fig. 4-1A were analyzed by Xenium-based single cell multiplexed in situ gene expression analysis with the mesenchymal probeset. The ROI that was analyzed is shown in Fig. 4A. (A) UMAPs of the merged mesenchymal probeset data showing either the annotated clusters (bottom; the same UMAP as in Fig. 4C) or the section of origin (top) for each of the transcriptomes. MSC1-6 denotes 6 sections from 5 difference mice. Each dot represents a single transcriptome. VSMCs = vascular smooth muscle cells, Oligos = oligodendrocytes. (B) Expression overlays for selected marker genes on the UMAPs shown in (A). Expression levels are color-coded as per the adjacent keys. (C) Coronal adult mouse cortex sections at the level shown in Fig. 4-1B were analyzed by Xenium-based single cell multiplexed in situ gene expression analysis with the brain probeset. The ROI that was analyzed is shown in Fig. 4A. Shown are spatial plots of the midline and adjacent cortical interface and layer one from the ROI of a representative section illustrating all cell types except neurons (left) or leptomeninges and neurons (right), color-coded as per the legends. L1 denotes cortical layer one. Download Figure 4-2, TIF file.
Figure 4-3
Xenium probesets. Shown are the mesenchymal custom probeset, comprised of probes targeting 480 genes, as well as the custom add-on brain probeset targeting 100 genes that was used in conjunction with the 10X predesigned 247 gene mouse brain panel. The numbers of probes per gene were tuned for each Xenium custom add-on panel to ensure robust detection and isoform coverage, while avoiding optical crowding. Probe number selection was informed by the 10X Xenium panel designer. Download Figure 4-3, XLS file.
Figure 5-1
Genes differentially expressed in border macrophages versus cortical microglia. Differential gene expression analysis was performed on the border macrophages and microglia from the cortical surface-associated and bone-associated datasets in Fig. 1A and B. Differentially expressed genes were defined as those expressed in >10% of cells in either group with a Bonferroni adjusted p-value < 0.05 (Adj. p-value) and ≥ 1.3 average log2 fold change (Avg log2FC). Download Figure 5-1, XLS file.
Figure 6-1
Spatially defining arachnoid and pial cells at the cortical brain interface. (A) Two adult mouse brain surface scRNA-seq datasets from Pietilä et al. (2023; GEO GSE227713) were run through our pipeline, and analyzed and annotated as in the original publication (see Materials and Methods). Shown is the resultant cluster UMAP and annotations, with BFB4 and BFB5 corresponding to brain fibroblast1 and brain fibroblast5. The clusters labelled leptomeninges correspond to BFB1-3 in Pietilä et al. (2023; Fig. 1D in that paper). (B, C) The transcriptomes shown in (A) were merged with the leptomeningeal and dural cell transcriptomes from Fig. 1E, run through our pipeline, and two rounds of Harmony batch correction performed. Shown are the resultant UMAPs, annotated for cell types (B) or for dataset of origin (C). Leptomeningeal clusters 0 and 4 are annotated based upon analyses included in this manuscript. The other clusters are comprised of transcriptomes from Pietilä et al. (2023), and are thus annotated as in that paper. (D) Violin plot showing the number of transcripts per cell (nCount) in each of the datasets included in the merged dataset in (B, C). Each dot represents an individual transcriptome. (E) UMAP as in (B) overlaid for expression of two mRNAs, Aldh1a2, and Crabp2 known to be expressed in the developing leptomeninges. Expression levels are color-coded as per the adjacent keys. (F) UMAP cluster visualization of the leptomeningeal transcriptomes from the Xenium mesenchymal probeset dataset (Fig. 4C), shown at higher resolution and annotated for potential pial versus arachnoid cells. (G) Violin plots showing relative expression levels of two pial mRNAs, Lamc3 and Bmp2 and two arachnoid mRNAs, Bnc2 and Dpp4 in the pial versus arachnoid cells as annotated in (F). Red and green denote arachnoid and pial cells, respectively. Each dot corresponds to expression in an individual cell. (H) Spatial plots of the cortical interface and midline showing distribution of the pia and arachnoid cells annotated in (F). Note that pial cluster 9 cells (red) but not arachnoid cluster 6 or 11 cells (blue and black) extend down the midline. Download Figure 6-1, TIF file.
Figure 6-2
Genes differentially expressed in pial versus arachnoid leptomeningeal cells. Differential gene expression analysis was performed on the arachnoid and pial cell shown in Fig. 6A. Differentially expressed gene were defined as those expressed in >10% of cells in either group with a Bonferroni adjusted p-value < 0.05 (Adj. p-value) and ≥ 1.3 average log2 fold change (Avg log2FC). M = Mesenchymal Panel, B = Brain Panel. Download Figure 6-2, XLS file.
Figure 8-1
Specific expression of leptomeningeal ligands, as analyzed by scRNA-seq. UMAPs showing the cortical surface-associated dataset from Fig. 1A, overlaid for expression of four ligands that are highly-enriched in leptomeningeal cells, Sema3a, Bmp6, Bmp7 and Igf2. Expression levels are color-coded as per the adjacent keys. Download Figure 8-1, TIF file.
Figure 8-2
Analyses of leptomeningeal cell, border macrophage and border astrocyte ligand mRNA expression. Shown are ligand mRNAs expressed in leptomeningeal mesenchymal cells, border macrophages and border astrocytes at the cortical interface as determined by scRNA-seq, as well as the percentage of a given cell type that detectably expresses that ligand. For leptomeningeal cells and border macrophages ligands were extracted from the cortical surface-associated and bone-associated scRNA-seq datasets shown in Fig. 1A and B, and for border astrocytes from the merged astrocyte scRNA-seq dataset shown in Fig. 2A based on a curated ligand and associated receptor database (Toma et al., 2020). Ligands were only considered for further analysis (such as in the ligand-receptor modeling in Figure 8-4) if they were detectably expressed in at least 5% of the relevant cell type. Download Figure 8-2, XLS file.
Figure 8-3
Analyses of leptomeningeal cell, border macrophage and border astrocyte receptor mRNA expression. Shown are receptor mRNAs expressed in leptomeningeal mesenchymal cells, border macrophages and border astrocytes at the cortical interface as determined by scRNA-seq, as well as the percentage of a given cell type that detectably expresses that ligand. For leptomeningeal cells and border macrophages receptor mRNAs were extracted from the cortical surface-associated and bone-associated scRNA-seq datasets shown in Fig. 1A and B, and for border astrocytes from the merged astrocyte scRNA-seq dataset shown in Fig. 2A based on a curated ligand and associated receptor database (Toma et al., 2020). Receptors were only considered for further analysis (such as in the ligand-receptor modeling shown in Figure 8-4) if they were detectably expressed in at least 5% of the relevant cell type. Download Figure 8-3, XLS file.
Figure 8-4
Details of ligand-receptor communication models. Predicted ligand and receptor communication models were generated using the leptomeningeal, border macrophage and border astrocyte ligand and receptor mRNA lists shown in Figure 8-2 and 8-3. Shown are ligand-receptor communication models of (8-4i) ligands from all cells predicted to act on leptomeningeal, border macrophage or border astrocyte receptors, (8-4ii) leptomeningeal ligands predicted to act on border macrophage receptors, and (8-4iii) leptomeningeal ligands predicted to act on border astrocyte receptors. Download Figure 8-4, XLS file.
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