Cellular evolution of the hypothalamic preoptic area of behaviorally divergent deer mice
- Department of Molecular & Cellular Biology, Harvard University, United States
- Department of Organismic and Evolutionary Biology, Harvard University, United States
- Howard Hughes Medical Institute, Harvard University, United States
Figures
Figure 1 with 9 supplements
Overview of Peromyscus hypothalamus single-cell atlas.
(A) Images of Peromyscus maniculatus and Peromyscus polionotus and summary of their social behavioral differences. (B) Schematic of the preoptic area of the hypothalamus. Dotted boxes indicate the area dissected. (C) Schematic of pooling and sample assignment strategy used for single-nucleus RNA-sequencing (snRNA-seq). (D) UMAP visualization of all 52,121 neuronal nuclei collected across 6 males and 6 females from each of the P. maniculatus and P. polionotus species, colored by cell cluster. (E) UMAP visualization of all neuronal nuclei, colored by the assigned Mus cell type label. Only Peromyscus clusters for which a homologous Mus cell type label could be found are colored; remaining nuclei are shown in gray. (F) Heatmap of the proportion of each Peromyscus cell cluster (rows) that are assigned to each Mus cell type label (columns). Only Peromyscus clusters for which a homologous Mus cell type label could be found are shown.
Image credit: Joel Sartore / Photo Ark.
Figure 1—figure supplement 1
Schematic illustrating pipeline for singlet and multiplet assignment of pooled single-nucleus RNA-sequencing (snRNA-seq) data.
Figure 1—figure supplement 2
Sample assignment for pooled RNA-seq data.
Scatterplots of the number of reads that map to P. maniculatus (P. man, x-axis) and P. polionotus (P. pol, y-axis) for each droplet (dots) across six replicates. Color indicates the final sample assignment made by demuxlet (orange: P. maniculatus female; dark orange: P. maniculatus male; blue: P. polionotus female; dark blue: P. polionotus male; gray: multiplet).
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Figure 1—figure supplement 2—source data 1
Related to Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
Statistics of demultiplexed single-nucleus RNA-sequencing (snRNA-seq) data.
(A) Barplot of the number of droplets assigned to each sample. (B) Violin plot of the numbers of genes detected (>0 uniquely mapped read) per droplet across all samples (white dots: median). (C) Boxplot of the read counts of female-specific gene, Xist, across all samples.
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Figure 1—figure supplement 3—source data 1
Related to Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig1-figsupp3-data1-v1.xlsx
Figure 1—figure supplement 4
Clustering of single-nucleus RNA-sequencing (snRNA-seq) data into major cell classes.
(A) UMAP visualization of all clustered nuclei. (B) Dotplot of percentage of cells within each cluster expressing known marker genes (size of dots) and average expression level of marker genes (color of dots). Gene markers (x-axis) are colored by the cell class they represent (see C for key). (C, D) UMAP visualization and dotplot of same data, grouped by cell class as inferred by expression of marker genes.
Figure 1—figure supplement 5
UMAP visualizations of all neuronal nuclei, separated by sex and species.
Figure 1—figure supplement 6
Expression level and spatial distribution of six known cell type-specific genes.
UMAP visualizations of all neuronal nuclei. Color bars indicate the expression level of Avp (red), Oxt (pink), Vip (purple), Nms (green), Grp (brown), and Pvalb (blue).
Figure 1—figure supplement 7
Classification of inhibitory and excitatory cell clusters.
UMAP visualization of all neuronal nuclei (gray) and expression level of inhibitory markers Gad1 and Gad2 (blue) and excitatory marker Slc17a6 (orange).
Figure 1—figure supplement 8
Homology mapping of Mus cell type labels to Peromyscus cell clusters.
Heatmap of the proportion of each Peromyscus cell cluster (rows) that are assigned to each Mus cell type label (columns) for inhibitory (A) and excitatory (B) cell clusters.
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Figure 1—figure supplement 8—source data 1
Related to Figure 1—figure supplement 8.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig1-figsupp8-data1-v1.xlsx
Figure 1—figure supplement 9
Sequencing metrics across cell clusters.
Violin plots of number of reads mapped (top) and genes found (bottom) in each cell cluster. Bolded cluster numbers are clusters where Mus homology could be identified.
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Figure 1—figure supplement 9—source data 1
Related to Figure 1—figure supplement 9.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig1-figsupp9-data1-v1.xlsx
Figure 2 with 1 supplement
Differential abundance of cell types across species and sex.
(A) Schematic of procedure used to test for differential abundance of cell clusters. (B) Barplots of log2(fold change) across species (P. pol / P. man, left) and across sex (male/female, right) for each cell cluster. Bars are colored by -log10(FDR). (C) Boxplots of cell abundances (y-axis) of four cell types differentially abundant across species and (D) of two cell types differentially abundant across sex. Cell abundances across samples are normalized using TMM normalization (Methods).
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Figure 2—source data 1
Related to Figure 2B.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Galanin (Gal) expression across cell clusters.
(A) Read count of Gal in each neuronal nucleus, visualized by UMAP. (B) Dot plot of normalized expression of Gal (color intensity) and the percent of nuclei for which Gal is expressed (dot size) in each neuronal cell cluster.
Figure 3 with 1 supplement
Immunostaining and cell counts of regional populations of differentially abundant cell types.
(A) Representative images of immunoreactive (ir) staining of AVP+ neurons in the paraventricular (PVN) (top) and supraoptic (SON) (bottom) of P. maniculatus and P. polionotus. Scale bar represents 200 μm. (B) Boxplots of AVP+ neuron number counts in the PVN (left) and SON (right) of females and males of P. maniculatus (P. man) and P. polionotus (P.pol): *** p<0.001 (one-sided Mann Whitney test). (C, D) Same as A and B, but of OXT+ neurons. (E) Representative images of ir staining of CALB1+ neurons in the BNST (top) and SDN-POA (bottom) of a female and male P. maniculatus and P. polionotus. Scale bar represents 200 μm. (F) Boxplots of the area of CALB1+ in the bed nucleus of the stria terminalis (BNST) (left) or CALB1+ neuron number counts in the sexually dimorphic nucleus of the preoptic area (SDN-POA) (right) of females and males of P. maniculatus and P. polionotus: **p<0.01; *p<0.05; ns: not significant (one-sided Mann Whitney test).
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Figure 3—source data 1
Related to Figure 3B.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig3-data1-v1.xlsx
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Figure 3—source data 2
Related to Figure 3D.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig3-data2-v1.xlsx
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Figure 3—source data 3
Related to Figure 3F.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Arginine vasopressin (AVP) and oxytocin (OXT) neuron counts across the entire hypothalamus.
(A) Representative image of immunoreactive staining of AVP + neurons and subregion definitions. AHA: Anterior hypothalamus area; BNST: Bed nucleus of the stria terminalis; PVN: Paraventricular nucleus; SON: Supraoptic nucleus. Scale bar represents 200 μm. (B) Boxplots of AVP+ (left) and OXT+ (right) neuron number counts in each subregion of the hypothalamus of P. maniculatus and P. polionotus. ***p<0.001; ns: not significant (one-sided Mann Whitney test).
Figure 4
Differential gene expression across species.
(A) Schematic of procedure used to pseudobulk gene expression counts and test for differential gene expression across species. (B) Barplot of the number of differentially expressed (DE) genes for each cell cluster (bars) colored by P. maniculatus or P. polionotus bias. (C) Scatter plot of cell cluster abundance (x-axis) and number of differentially expressed genes (y-axis) identified in each cell cluster (dots). Line of best fit (gray line) and 95% confidence interval (gray shading) are shown. (D) Left: Volcano plot of log2(fold change) (x-axis) and log10(FDR) (y-axis) of DE genes across species. Negative values of log2(fold change) indicate P. maniculatus bias and positive values indicate P. polionotus bias. For genes DE in more than one cell type, the cell type with the highest expression level is shown. Genes are colored by gene categories listed on the right. Select outlier genes are labeled. Right: Barplot of enrichment scores (Methods) for each gene category. Dotted line at 1.0 indicates no enrichment or depletion. ***FDR <0.001; **FDR <0.01; *FDR <0.05; ns: not significant.
Figure 5 with 2 supplements
Differential gene expression across sex.
(A) Barplots of the number of sex-biased genes for each cell cluster (bars) in P. maniculatus (top) and P. polionotus (bottom), colored by female- or male-bias. (B) Barplot of the number of female- and male-biased genes aggregated across all cell clusters in P. maniculatus (orange), P. polionotus (blue), or shared across both species (gray). (C) Scatter plots of cell cluster abundance (x-axis) and number of differentially expressed (DE) genes (y-axis) identified in each cell cluster (dots) for P. maniculatus (left) and P. polionotus (right). Line of best fit (gray line) and 95% confidence interval (gray shading) are shown in each scatter plot.
Figure 5—figure supplement 1
Cell clusters 5 and 9 are enriched for sex-biased genes and sex steroid receptors.
(A) Scatter plots of cell cluster abundance (x-axis) and number of differentially expressed (DE) genes (y-axis) identified in each cell cluster (dots) for P. maniculatus (top) and P. polionotus (bottom) using data downsampled so that cell abundance was equal across sexes. Line of best fit (gray line) and 95% confidence interval (gray shading) are shown in each scatter plot. (B) Dot plot of normalized expression of sex steroid receptors Esr1, Ar, Prir, and Pgr (color intensity) and the percent of nuclei in which they are expressed (dot size) in each neuronal cell.
Figure 5—figure supplement 2
Expression levels of sex steroid receptors by species and sex.
Boxplots of gene expression level in log2(counts per million) (y-axis) of Esr1, Ar, Prlr, and Pgr in cell cluster 5 (top) and 9 (bottom) across females and males of P. maniculatus and P. polionotus. Significance represent p-values before correction for multiple hypothesis testing. ***p<0.001; **p<0.01; *p<0.05.
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Figure 5—figure supplement 2—source data 1
Related to Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/103109/elife-103109-fig5-figsupp2-data1-v1.xlsx
Figure 6
Enrichment of neuropeptides in sex-biased genes.
(A) Left: Volcano plot of log2(fold change) (x-axis) and log10(FDR) (y-axis) of sex-biased genes in P. maniculatus. Negative values of log2(fold change) indicate female bias and positive values indicate male bias. For genes sex-biased in more than one cell type, the cell type with the highest expression level is shown. Genes are colored by gene categories listed on the right. Select outlier genes are labeled. Right: Barplot of enrichment scores for each gene category. Dotted line at 1.0 indicates no enrichment or depletion. ***FDR <0.001; *FDR <0.05. (B) Same as A but for P. polionotus. (C) Venn diagrams of female- (top) and male-biased (bottom) neuropeptides, categorized by whether they are P. maniculatus specific, P. polionotus specific, or shared. Some neuropeptides (e.g. Nts, Tac1, Oxt, Gnrh1) are female-based in some cell types and male-biased in others. Neuropeptides that share sex-bias in the same cell type across species are starred and listed in more detail in.
Tables
Table 1
Neuropeptides conserved in sex-bias in same cell types across P. maniculatus and P. polionotus.
| Neuropeptide | Sex-bias | Cell Type | P. maniculatus fold change | P. maniculatus FDR | P. polionotus fold change | P. polionotus FDR |
|---|---|---|---|---|---|---|
| Kiss1 | female | i16:Gal/Th (cluster 20) | 26.0 | 1.2E-54 | 14.9 | 6E-49 |
| Tac1 | female | i18:Gal/Tac2 (cluster 5) | 2.1 | 3.3E-21 | 1.4 | 1.9E-03 |
| Gal | female | i18:Gal/Tac2 (cluster 5) | 1.6 | 3.4E-10 | 1.6 | 3.4E-08 |
| Tac1 | female | cluster 3 | 1.8 | 2.0E-09 | 1.5 | 8.9E-04 |
| Tac1 | male | i20:Gal/Moxd1 (cluster 9) | 3.5 | 1E-30 | 1.5 | 9.8E-03 |
| Cck | male | i20:Gal/Moxd1 (cluster 9) | 1.7 | 1.08E-05 | 2.3 | 7.5E-16 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Peromyscus maniculatus bairdii) | Dissections of preoptic area | Peromyscus Genetic Stock Center (Columbia, SC, USA) | Freshly isolated from Peromyscus maniculatus bairdii | |
| Biological sample (Peromyscus polionotus subgriseus) | Dissections of preoptic area | Peromyscus Genetic Stock Center (Columbia, SC, USA) | Freshly isolated from Peromyscus polionotus subgriseus | |
| Antibody | anti-Avp (Rabbit polyclonal) | ImmunoStar | Cat# 20069 RRID:AB_572219 | IHC (1:4000) |
| Antibody | anti-Oxt (Mouse monoclonal) | EMD Millipore | Cat# MAB5296 | IHC (1:2000) |
| Antibody | anti-Calb1 (Mouse monoclonal) | Millipore Sigma | Cat# C9848 | IHC (1:1000) |
| Commercial assay or kit | 10 X Genomics 3’ V3 Chip | 10 x Genomics | Nuclei were loaded at a target of 20,000 nuclei/run | |
| Software | CellRanger v5.0.0 | 10 x Genomics | RRID:SCR_023221 |
Additional files
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Supplementary file 1
10 x sequencing statistics.
- https://cdn.elifesciences.org/articles/103109/elife-103109-supp1-v1.xlsx
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Supplementary file 2
Cell abundance of all clusters across all replicates.
- https://cdn.elifesciences.org/articles/103109/elife-103109-supp2-v1.xlsx
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Supplementary file 3
Differential expression across species.
- https://cdn.elifesciences.org/articles/103109/elife-103109-supp3-v1.xlsx
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Supplementary file 4
Differential expression across sex.
- https://cdn.elifesciences.org/articles/103109/elife-103109-supp4-v1.xlsx
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Supplementary file 5
Gene lists.
- https://cdn.elifesciences.org/articles/103109/elife-103109-supp5-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/103109/elife-103109-mdarchecklist1-v1.docx
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Cellular evolution of the hypothalamic preoptic area of behaviorally divergent deer mice
eLife 13:RP103109.
https://doi.org/10.7554/eLife.103109.3
