Single nuclei RNA-seq of mouse placental labyrinth development
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, United States
- Department of Urology, University of California, San Francisco, United States
Figures
Figure 1 with 1 supplement
Nuclear isolation and snRNA-seq of mouse placental cells (E9.5-E14.5).
(A) Schematic showing the main regions of the placenta – Labyrinth, Junctional Zone, Parietal TGC, and Decidua. Removal of the decidual stroma and the allantois is marked by scissors and cut lines. …
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Figure 1—source data 1
Marker genes for clusters in complete snRNA-seq data (E9.5-E14.5).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig1-data1-v2.csv
Figure 1—figure supplement 1
Quality control metrics andmarker genes for snRNA-se of mouse placentae (E9.5-E14.5).
(A) UMAP projection of all nuclei color coded by timepoint from which they were collected. (B) The number of transcripts identified per nucleus (left) and the number of unique genes identified …
Figure 2 with 3 supplements
Sub-clustering of the trophoblast nuclei identifies the cell types of the labyrinth and junctional zone.
(A) UMAP showing the 16,836 nuclei included in the trophoblast subset, clustered and plotted according to transcriptome similarity. Clusters were annotated according to canonical marker genes and …
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Figure 2—source data 1
Marker genes for clusters in subclustered trophoblast nuclei.
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig2-data1-v2.csv
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Figure 2—source data 2
Expression of the top 200 variable genes along the pseudotime ordering of cells from LaTP to SynTII (Figure 2C).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig2-data2-v2.csv
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Figure 2—source data 3
Expression of the top 200 variable genes along the pseudotime ordering of cells from LaTP2 to SynTI (Figure 2D).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig2-data3-v2.csv
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Figure 2—source data 4
Expression of the top 200 variable genes along the pseudotime ordering of cells from LaTP2 to S-TGC (Figure 2E).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig2-data4-v2.csv
Figure 2—figure supplement 1
Quality control metrics for snRNA-seq of subclustered trophoblast nuclei and dissection of LaTP populations by specific markers.
(A) Violin plots showing the number of unique genes (left), number of transcripts (middle), and the percent of reads mapping to mitochondrial genes (right) for each cluster identified in the …
Figure 2—figure supplement 2
Additional validation of the identities of Junctional Zone clusters.
(A) Nuclei along the differentiation from JZP1 to GCs were ordered by pseudotime using Slingshot. The nuclei included along each pseudotime axis shown at left. The expression of select genes …
Figure 2—figure supplement 3
Characterization of spongiotrophoblast and glycogen gell clusters by specific expression of prolactin genes.
(A) Table of prolactin gene expression in either spongiotrophoblast or glycogen cells from Simmons et al., 2008a and their expression in the snRNA-seq data. (B) Violin plots showing the expression …
Figure 3 with 1 supplement
Developmental time course and trajectory inference reveal details of lineage dynamics and commitment in trophoblast.
(A) UMAP projection of all nuclei captured at each gestational age. (B) Quantification of the proportion of each cluster captured at each developmental time point. (C) RNA velocity vectors of the …
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Figure 3—source data 1
Average RNA velocity magnitude split by developmental stage and cluster (Figure 3D).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig3-data1-v2.xlsx
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Figure 3—source data 2
S-TGC Differential Expression (E9.5 v E10.5).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig3-data2-v2.csv
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Figure 3—source data 3
S-TGC Differential Expression (E9.5 v E12.5).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig3-data3-v2.csv
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Figure 3—source data 4
S-TGC Differential Expression (E9.5 v E14.5).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig3-data4-v2.csv
Figure 3—figure supplement 1
Analysis of S-TGC across developmental time reveals a switch from proliferation to maturation.
(A) Expression of Mki67 projected in UMAP space at each time point (left). The dotted line outlines the progenitor populations LaTP, LaTP2, SynTI Precursor, SynTII Precursor, and JZP 1/2. …
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Figure 3—figure supplement 1—source data 1
S-TGC Gene Ontology results by developmental stage.
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
Defining distinct roles of the trophoblast subtypes at the gas exchange interface.
(A) Schematic showing the relative location of the cell types at the gas and nutrient exchange interface of the labyrinth – S-TGC, SynTI, SynTII, (sometimes referred to as trophoblast layer I, II, …
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Figure 4—source data 1
Differential expression between interface clusters (SynTI, SynTII, and S-TGC).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig4-data1-v2.csv
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Figure 4—source data 2
Gene Ontology results for differentially expressed genes between interface clusters (SynTI, SynTII, and S-TGC).
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig4-data2-v2.xlsx
Figure 4—figure supplement 1
Gene ontology (biological process) of differentially expressed genes SynTII (top), SynTI (middle), S-TGC (bottom).
Significantly differentially expressed genes are defined by adj. p-value<0.05 and logFC >0.5. GeneRatio is the of the number of genes found the given ontology divided by the number of genes found in …
Figure 5 with 2 supplements
Predicting Cell Signaling within the placental labyrinth.
(A) Heatmap showing all Wnt signaling interactions (top) and Vegf signaling (bottom) between the endothelium and labyrinth trophoblast populations. Left: schematics summarizing signaling …
Figure 5—figure supplement 1
All predicted receptor-ligand interactions between labyrinth populations.
(A) Heatmap of 139 ligand–receptor interactions predicted by CellPhoneDB (p-value<0.05 permutation test). The rows are ligand–receptor pairs and columns are cell–cell pairings. Strength of …
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Figure 5—figure supplement 1—source data 1
CellPhoneDB interaction raw data for trophoblast populations.
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig5-figsupp1-data1-v2.csv
Figure 5—figure supplement 2
Validation of predicted cell signaling events in the labyrinth.
(A–C) Immunofluorescence staining of sections from E12.5 placentas of (A) IGF1R (Purple) and VEGFA (Green); (B) STRA6 (Yellow) and HBEGF (Red); HBEGF (Red) and EGFR (Green). The white- dashed line …
Figure 6 with 1 supplement
Modeling transcription factor regulon activity identifies new candidate regulators of SynTII.
(A) UMAP projection derived from regulon activity predicted by SCENIC. The clusters are colored according to Seurat clustering of transcript data. The cluster identities of each of the five arms of …
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Figure 6—source data 1
SCENIC scaled regulon activity in trophoblast nuclei.
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig6-data1-v2.csv
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Figure 6—source data 2
SCENIC regulon predicted binding targets for all active regulons.
- https://cdn.elifesciences.org/articles/60266/elife-60266-fig6-data2-v2.txt
Figure 6—figure supplement 1
Additional validation of SCENIC and Gata1 expression and expression of genes with validated placental phenotypes in vivo.
(A) Heatmaps showing the Pearson correlation values between cluster averages of transcript expression (left) and SCENIC regulon activity (right). Clusters are ordered by hierarchical clustering with …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Mus musculus) | C57BL/6J Mice | https://www.jax.org/strain/000664 | 000664 | 6–12 weeks old |
| Commercial assay or kit | Nuclei isolation Kit: Nuclei EZ Prep | Sigma-Aldrich | NUC101-1KT | |
| Antibody | E-cadherin Monoclonal antibody (ECCD-2) | Thermofisher Scientific | 13–1900 | IF (1:250) |
| Antibody | Human/mouse NCAM-1/CD56 Polyclonal antibody | R and D Systems | AF2408-SP | IF (1:25) |
| Antibody | Anti-EpCAM Polyclonal antibody | Abcam | ab71916 | IF (1:1000) |
| Antibody | Anti-Stra6 Polyclonal antibody | Sigma-Aldrich | ABN1662 | IF (1:100) |
| Antibody | Anti-Igf1r Polyclonal antibody | R and D Systems | AF305 | IF (1:50 w/ antigen retrieval) |
| Antibody | Anti-Slco2a1 Polyclonal antibody | Atlas Antibodies | HPA013742 | IF (1:25 w/ antigen retrieval) |
| Antibody | Anti-Lepr Polyclonal antibody | R and D Systems | AF497 | IF (1:200) |
| Antibody | Anti-Pcdh12 Polyclonal antibody | Abcam | ab113720 | IF (1:25 w/ antigen retrieval) |
| Antibody | Anti-Podxl Polyclonal antibody | R and D Systems | AF1556 | IF (1:25) |
| Antibody | Anti-Pecam1 Polyclonal antibody | Abcam | ab23864 | IF (1:50) |
| Antibody | Anti-Vegfa Polyclonal antibody | Abcam | ab51745 | IF (1:50) |
| Antibody | Anti-Met Polyclonal antibody | R and D Systems | AF276 | IF (1:100) |
| Antibody | Anti-Egfr Polyclonal antibody | R and D Systems | AF1280 | IF (1:100) |
| Antibody | Anti-Gata1 Monoclonal antibody | Cell Signaling | 3535 | IF (1:100) |
| Antibody | Anti-Hbegf Polyclonal antibody | R and D Systems | AF8239 | IF (1:20) |
| Software, algorithm | R | https://www.r-project.org/ | ||
| Software, algorithm | ImageJ | ImageJ (http://imagej.nih.gov/ij/) | ||
| Software, algorithm | Seurat (3.1.3) | https://satijalab.org/seurat/ | ||
| Software, algorithm | cellranger (3.0.2) | https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/feature-bc | ||
| Software, algorithm | CellPhoneDB | https://www.cellphonedb.org/ | ||
| Software, algorithm | SCENIC | https://github.com/aertslab/SCENIC | ||
| Software, algorithm | ClusterProfiler | https://guangchuangyu.github.io/software/clusterProfiler/ | ||
| Software, algorithm | Slingshot | https://github.com/kstreet13/slingshot | ||
| Software, algorithm | scVelo | https://github.com/theislab/scvelo | ||
| Software, algorithm | FlowJo | https://www.flowjo.com |
Additional files
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Supplementary file 1
Sample information and processing.
Contains information of the number of nuclei captured at each timepoint and the processing information for each dataset (number of Principal Components and the resolution parameters used for cluster/integration)
- https://cdn.elifesciences.org/articles/60266/elife-60266-supp1-v2.xlsx
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Supplementary file 2
Number of nuclei captured per cluster complete dataset.
Breakdown of the number of nuclei collected at each timepoint for each cluster identified in the dataset used in Figure 1. Also, provided is the percent of the total nuclei assigned to each cluster captured at each timepoint. Finally, these data are normalized to the number of nuclei captured at each timepoint so that comparisons may be made with in a cluster across timepoints.
- https://cdn.elifesciences.org/articles/60266/elife-60266-supp2-v2.xlsx
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Supplementary file 3
Number of nuclei captured per cluster trophoblast dataset.
Breakdown of the number of nuclei collected at each timepoint for each cluster identified in the trophoblast dataset used in Figures 2–6. Also, provided is the percent of the total nuclei assigned to each cluster captured at each timepoint. Finally, these data are normalized to the number of nuclei captured at each timepoint so that comparisons may be made with in a cluster across timepoints.
- https://cdn.elifesciences.org/articles/60266/elife-60266-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60266/elife-60266-transrepform-v2.pdf
