Spatially patterned excitatory neuron subtypes and projections of the claustrum
- Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Canada
- Neuroscience and Mental Health Institute, University of Alberta, Canada
- Janelia Research Campus, Howard Hughes Medical Institute, United States
- Department of Physiology, University of Alberta, Canada
- Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Canada
- School of Biomedical Engineering, University of British Columbia, Canada
Figures
Figure 1 with 4 supplements
Excitatory claustrum-occupying cells are separable into discrete transcriptomic populations.
(A) UMAP dimensionality reduction of single-cell transcriptomes. Points denote individual cells, with coloring denoting cluster identity obtained by graph-based clustering. (B) Violin plots …
Figure 1—figure supplement 1
Consistency and reproducibility of scRNA-seq data.
(A) t-SNE dimensionality reduction of scRNA-seq dataset, illustrating separation of clusters. Cells are colored according to cluster identity as in Figure 1. (B) Cells colored according to location …
Figure 1—figure supplement 2
Comparison of new and published scRNA-seq datasets.
(A) scRNA-seq analysis, integrating the new (‘Erwin’) dataset with published data from Zeisel et al., 2018 and Saunders et al., 2018 (‘Zeisel’ and ‘Saunders,’ respectively). Cells in the integrated …
Figure 1—figure supplement 3
Nnat and Pcp4 differentiate similar cells across datasets.
(A) Clusters of neurons obtained at a relatively fine clustering resolution, encompassing Nnat-expressing claustrum shell neurons from Erwin dataset, as well as similar neurons from published …
Figure 1—figure supplement 4
Pcp4 and Nnat differentiate deep cortical layers from claustrum shell.
(A) Chromogenic in situ hybridization showing Pcp4 expression in a coronal section of the mouse brain. Scale bar: 500 µm. (B, C) Expansion of the regions highlighted in (A). Note that Pcp4 exhibits …
Figure 2 with 5 supplements
Multiplexed fluorescent in situ hybridization analysis of the claustrum.
(A) Overview of anterior (left), intermediate (middle), and posterior (right) sections of the claustrum. Inset shows expansion of anterior section. Probe list provided at bottom middle, with atlas …
Figure 2—figure supplement 1
scRNA-seq profiles of multiplexed fluorescent in situ hybridization-targeted genes.
(A) scRNA-seq expression profiles of excitatory neuronal markers Slc17a7 and Slc17a6. Expression depicted in UMAP embedding, plotted as counts per million with color scale provided. (B) As in (A), …
Figure 2—figure supplement 2
Representative expansions of RNA signals detected via multiplexed fluorescent in situ hybridization (mFISH).
(A) Overview of signals associated with the 12 genes targeted by mFISH. Scale bar: 500 μm. (B) For each gene, expansions of the areas shown in (A). Scale bar: 50 μm.
Figure 2—figure supplement 3
Expression of core, shell, and layer 6 marker genes across the anterior-posterior axis.
(A) Expression of Synpr (green), Nnat (red), and Ctgf (blue) in anterior sections, as well as three-color overlay. Scale bar: 500 μm. (B, C) As in (A), but for intermediate (B) and posterior (C) …
Figure 2—figure supplement 4
Overview of cellular phenotyping across sections and mice.
(A) Cellular phenotyping across relatively anterior (left), intermediate (middle), and posterior (right) sections for a replicate animal, with cells colored according to cluster identity as in Figure…
Figure 2—figure supplement 5
Comparison of gene expression of putative core and shell populations across scRNA-seq and multiplexed fluorescent in situ hybridization (mFISH).
Top row: box plots illustrating expression of core-associated marker genes in scRNA-seq and mFISH datasets for core and shell clusters. Middle row: as in top row, but for shell-associated marker …
Figure 3 with 1 supplement
Claustrum transcriptomic subtypes are associated with different projections.
(A) Projections to the retrosplenial cortex (RSC; magenta) and lateral entorhinal cortex (LEC; green) emanate from different spatial locations. Atlas schematic denotes coronal section location, …
Figure 3—figure supplement 1
Multiplexed fluorescent in situ hybridization (mFISH)-derived gene expression profiles for retrosplenial cortex (RSC)- and lateral entorhinal cortex (LEC)-projecting neurons.
Box plots illustrating expression of genes in mFISH dataset according to projections to either the RSC or LEC. Plots grouped according to scRNA-seq-derived expression profiles.
Author response image 1
Similar expression of Pcp4 in the cortical cell cluster across datasets.
Using cortical cells from our dataset (Erwin et al.), we identified the corresponding cluster from the integrated analysis including other published datasets (Saunders t al., Zeisel et al.). Pcp4 …
Videos
Video 1
Example multiplexed fluorescent in situ hybridization image across fine and coarse spatial scales.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Sequence-based reagent | Cdh9 ISH probe | Advanced Cell Diagnostics | 443221-T1 | mFISH |
| Sequence-based reagent | Ctgf ISH probe | Advanced Cell Diagnostics | 314541-T2 | mFISH |
| Sequence-based reagent | Slc17a6 ISH probe | Advanced Cell Diagnostics | 319171-T3 | mFISH |
| Sequence-based reagent | Lxn ISH probe | Advanced Cell Diagnostics | 585801-T4 | mFISH |
| Sequence-based reagent | Slc30a3 ISH probe | Advanced Cell Diagnostics | 496291-T5 | mFISH |
| Sequence-based reagent | Gfra1 ISH probe | Advanced Cell Diagnostics | 431781-T6 | mFISH |
| Sequence-based reagent | Spon1 ISH probe | Advanced Cell Diagnostics | 492671-T7 | mFISH |
| Sequence-based reagent | Gnb4 ISH probe | Advanced Cell Diagnostics | 460951-T8 | mFISH |
| Sequence-based reagent | Nnat ISH probe | Advanced Cell Diagnostics | 432631-T9 | mFISH |
| Sequence-based reagent | Synpr ISH probe | Advanced Cell Diagnostics | 500961-T10 | mFISH |
| Sequence-based reagent | Pcp4 ISH probe | Advanced Cell Diagnostics | 402311-T11 | mFISH |
| Sequence-based reagent | Slc17a7 ISH probe | Advanced Cell Diagnostics | 416631-T12 | mFISH |
| Software, algorithm | R | https://www.r-project.org | SCR_001905 | - |
| Software, algorithm | Seurat | https://satijalab.org/seurat/ | SCR_007322 | - |
| Software, algorithm | Fiji | https://imagej.net/Fiji | RRID:SCR_002285 | - |
| Other | rAAV2-retro-CAG-GFP | Janelia Viral Core | - | scRNA-seq |
| Other | pAAV-CAG-GFP | Addgene | RRID:Addgene_37825 | mFISH |
| Other | pAAV-CAG-tdTomato | Addgene | RRID:Addgene_59462 | mFISH |
Additional files
-
Supplementary file 1
List of core-enriched genes and enrichment properties.
- https://cdn.elifesciences.org/articles/68967/elife-68967-supp1-v1.txt
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Supplementary file 2
List of shell-enriched genes and enrichment properties.
- https://cdn.elifesciences.org/articles/68967/elife-68967-supp2-v1.txt
-
Supplementary file 3
List of layer 6-enriched genes and enrichment properties.
- https://cdn.elifesciences.org/articles/68967/elife-68967-supp3-v1.txt
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68967/elife-68967-transrepform1-v1.pdf
