Anatomical and single-cell transcriptional profiling of the murine habenular complex
- Howard Hughes Medical Institute, Harvard Medical School, United States
Figures
Figure 1 with 2 supplements
High-throughput single-cell transcriptomic profiling of the habenula.
(A) Schematic for scRNA-seq using the inDrop platform. Tissue containing the habenula was microdissected from acute coronal brain slices prepared from adult mice (1). Tissue chunks were digested in …
Figure 1—figure supplement 1
Comparison of cell-type composition across hemispheres and gene diversity, mitochondrial genes, and UMIs across cell types.
(A) t-SNE plot of the dataset with cells color-coded by the hemisphere from which the sample was acquired. (B) Bar plots showing the percentage of cells in each hemisphere that are categorized into …
Figure 1—figure supplement 2
Expression of genes known to be important for habenular microglial and astrocytic function.
Violin plots of gene expression (scaled and log normalized) for Tnf (A), Tnfrsf1a (TNF-receptor) (B), and Kcnj10 (Kir4.1) (C). Each point represents a single cell and the filled area is a …
Figure 2 with 11 supplements
MHb neuron subtypes can be distinguished transcriptionally.
(A) Location of MHb and ISH of Tac2 expression from the Allen Institute Database. Tac2 expression is restricted to cells in the MHb in this region. (B) Tac2 serves as an excellent marker for MHb …
Figure 2—figure supplement 1
Subclustering of MHb neurons before and after subtraction of heterogeneity introduced by elevated expression of activity-dependent genes (ADGs).
(A) t-SNE plot of subclustered MHb neurons extracted from cells in Figure 1B. (B) t-SNE plot showing three clusters of cells (top) that expressed elevated levels of several ADGs (Fos, Fosb, Egr1, …
Figure 2—figure supplement 2
Sample ISH images showing spatial distribution of selected differentially expressed genes in MHb.
(A–J) Sample ISH images from the Allen Institute Database showing selected differentially expressed genes for distinct transcriptionally defined neuronal subtypes in MHb. Gene name is in the upper …
Figure 2—figure supplement 3
Differentially expressed genes define distinct habenular subtypes.
Figure 2—figure supplement 4
Ion channel diversity in MHb and LHb neuronal subtypes.
Dot plot displaying the expression of all ion channels detected in MHb and LHb neurons. The color of each dot (blue to red) indicates the relative expression of each gene, whereas the dot size …
Figure 2—figure supplement 5
Ion channel diversity in MHb and LHb neuronal subtypes (part 2).
Dot plot displaying the expression of all ion channels detected in MHb and LHb neurons. The color of each dot (blue to red) indicates the relative expression of each gene, whereas the dot size …
Figure 2—figure supplement 6
Neurotransmitter receptor diversity in MHb and LHb neuronal subtypes.
Dot plot displaying gene expression of all small molecule neurotransmitter receptors detected in MHb and LHb neurons. The color of each dot (blue to red) indicates the relative expression of each …
Figure 2—figure supplement 7
Neurotransmitter receptor diversity in MHb and LHb neuronal subtypes (part 2).
Dot plot displaying gene expression of all small molecule neurotransmitter receptors detected in MHb and LHb neurons. The color of each dot (blue to red) indicates the relative expression of each …
Figure 2—figure supplement 8
GPCR diversity in MHb and LHb neuronal subtypes.
Dot plot displaying gene expression of all GPCRs detected in MHb and LHb neurons (Harding et al., 2018; Regard et al., 2008). Genes are grouped and listed alphabetically by receptor family name …
Figure 2—figure supplement 9
GPCR diversity in MHb and LHb neuronal subtypes (part 2).
Dot plot displaying gene expression of all GPCRs detected in MHb and LHb neurons (Harding et al., 2018; Regard et al., 2008). Genes are grouped and listed alphabetically by receptor family name …
Figure 2—figure supplement 10
GPCR diversity in MHb and LHb neuronal subtypes (part 3).
Dot plot displaying gene expression of all GPCRs detected in MHb and LHb neurons (Harding et al., 2018; Regard et al., 2008). Genes are grouped and listed alphabetically by receptor family name …
Figure 2—figure supplement 11
GPCR diversity in MHb and LHb neuronal subtypes (part 4).
Dot plot displaying gene expression of all GPCRs detected in MHb and LHb neurons (Harding et al., 2018; Regard et al., 2008). Genes are grouped and listed alphabetically by receptor family name …
Figure 3
Characterization of genes differentially expressed between LHb neuron subtypes.
(A) Location of LHb clusters and ISH Gap43 expression from the Allen Institute Database. Gap43 is highly expressed in neurons of the LHb and surrounding thalamus in this region, but excluded from …
Figure 4 with 3 supplements
FISH confirms that differentially expressed genes from LHb subclusters are nonoverlapping and confined to specific spatial locations of LHb.
(A) Left: Sample FISH of two differentially expressed LHb genes (Vgf (yellow) and Chrm3 (magenta)), with distinct spatial profiles (LHb outlined with gray dashed line). Right: Quantification of …
Figure 4—figure supplement 1
A map of habenula subregions based on single-cell transcriptomic profiling.
(A) Habenular subregions are outlined in black, MHb subregions are green and LHb subregions are magenta. The location of borders is a rough estimate of a boundary between transcriptionally defined …
Figure 4—figure supplement 2
Spatial distribution of gene expression patterns along the medial/lateral axis for selected LHb DEGs.
(A) Illustration of LHb subregions depicting the medial (0) and lateral (1) edges of the habenula used to describe the spatial distribution of gene expression. (B,C) (Top) Expression levels of Chrm3 …
Figure 4—figure supplement 3
FISH confirms that differentially expressed genes from the same LHb subcluster are overlapping and confined to similar spatial locations of LHb.
(A) Left: Sample FISH of two differentially expressed LHb genes (Chrm3 (yellow) and Rbfox1 (magenta)), from the same cluster (Oval/Medial) (LHb outlined with gray dashed line). Right: Quantification …
Figure 5 with 3 supplements
Distinct LHb neuron subtypes prefer different downstream targets, but all subtypes target both the VTA.
(A) Location of site for AAV helper viruses (AAV-FLEX-TVA-mCh and AAV-FLEX-RVG) and pseudotyped rabies virus (EnvA-RbV-GFP) injection into VTA. (B) Sample habenula FISH images for RbV-N and Chrm3 …
Figure 5—figure supplement 1
Cells from all four LHb subtypes project to both VGAT-IRES-Cre+ and DAT-IRES-Cre+ cells in the VTA.
(A) Quantification of fluorescence coverage of single cells for FISH of selected enriched genes in LHb neurons that were positive for RbV-N following monosynaptic retrograde tracing from …
Figure 5—figure supplement 2
Quantification and genetic characterization of VTA starter cells from monosynaptic retrograde tracing and controls for rabies virus.
(A) Left: Coronal section of injection site into VTA and starter cells location for Cre-dependent monosynaptic retrograde-tracing experiments. Right: FISH for RbV-N to demonstrate the location of …
Figure 5—figure supplement 3
Quantification and genetic characterization of LHb cells that project to the DRN using a nonpseudotyped (G-deleted) rabies virus.
(A) Location of site for nonpseudotyped (G-deleted) rabies virus (RbV-GFP) injection into DRN. (B) Sample habenula FISH images for RbV-N and Chrm3 (top), Vgf (middle), or Gpr151 (bottom) following …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Mus musculus) | C57Bl/6J | The Jackson Laboratory | Cat# JAX:000664 RRID: IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | VGAT-IRES-Cre | The Jackson Laboratory | Cat# JAX: 016962 RRID: IMSR_JAX:016962 | |
| Genetic reagent (Mus musculus) | DAT-IRES-Cre | The Jackson Laboratory | Cat# JAX: 006660 RRID: IMSR_JAX:006660 | |
| Genetic reagent (non-pseudotyped G-deleted rabies virus) | B19-SADdG- EGFP, RbV-GFP | Other | N/A | Generated in-house (see Materials and methods) 109 IU/mL |
| Genetic reagent (pseudotyped G-deleted rabies virus) | EnvA-RbV-GFP | Janelia Viral Tools facility | Addgene# 52487 RRID: Addgene_52487 | 108 IU/mL |
| Commercial assay, kit | RNAscope V1 fluorescent multiplex detection assay reagents | ACDBio | Cat#320851 | |
| Commerical assay, kit | RNAscope V1 fluorescent multiplex detection assay probes | ACDBio | Cat# 456781 Cat# 317321 Cat# 404631 Cat# 556241 Cat# 436381 Cat# 519911 Cat# 437701 Cat# 517421 Cat# 423321 Cat# 319171 Cat# 319191 Cat# 315441 | V-RABV-gp1 Gpr151 Sst Plch1 Pbx3 Rbfox1 Chrm3 Vgf Cre Slc17a6 Slc32a1 Slc6a3 |
| Software, algorithm | inDrops pipeline (Python) | PMID: 26000487 | N/A | https://github.com/indrops/indrops |
| Software, algorithm | R 3.4.4 | R project for statistical computing | RRID:SCR_001905 | https://cran.r-project.org/ |
| Software, algorithm | Seurat 2.3.4 | PMID: 29608179 | RRID:SCR_016341 | https://satijalab.org/seurat/ |
| Software, algorithm | MAST 1.4.1 | PMID: 26653891 | RRID:SCR_016340 | https://bioconductor.org/packages/release/bioc/html/MAST.html |
| Software, algorithm | MATLAB (R2015a) | MathWorks | RRID:SCR_001622 | |
| Software, algorithm | Fiji | PMID: 22743772 | RRID:SCR_002285 | https://imagej.net/Fiji |
Additional files
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Supplementary file 1
Average expression of all genes in all clusters.
- https://cdn.elifesciences.org/articles/51271/elife-51271-supp1-v1.csv
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Supplementary file 2
Differentially expressed genes from all clusters using MAST, dendrogram included for reference.
- https://cdn.elifesciences.org/articles/51271/elife-51271-supp2-v1.xlsx
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Supplementary file 3
Differentially expressed genes from MHb subclusters using MAST, dendrogram included for reference.
- https://cdn.elifesciences.org/articles/51271/elife-51271-supp3-v1.xlsx
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Supplementary file 4
Average expression of all genes from MHb and LHb subclusters.
- https://cdn.elifesciences.org/articles/51271/elife-51271-supp4-v1.csv
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Supplementary file 5
Differentially expressed genes from LHb subclusters using MAST, dendrogram included for reference.
- https://cdn.elifesciences.org/articles/51271/elife-51271-supp5-v1.xlsx
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Supplementary file 6
Statistical comparisons for retrograde labeling experiments shown in Figure 5 and Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/51271/elife-51271-supp6-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/51271/elife-51271-transrepform-v1.docx
