Single-cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation
- Centre for Inflammation Research, University of Edinburgh, United Kingdom
- MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, United Kingdom
Figures
Figure 1
Single-cell RNA sequence analysis identified mesenchymal cell populations unique to ‘repairing’ tissue in a mouse model of endometrial breakdown and repair (menstruation).
(A, i) Mouse model of endometrial tissue breakdown and repair, (ii) histological morphology of uterine tissues 0, 24, 48, and 72 hr progesterone withdrawal illustrating tissue breakdown, repair, …
Figure 2 with 1 supplement
Gene expression analysis identifies transcriptomic profiles of mesenchymal cells and the expression of genes associated with epithelial cell identity in repair-specific fibroblasts (cluster F4).
(A) Heatmap (yellow, high; purple, low) displaying differentially expressed genes per cluster when compared to all other clusters (logFC >0.5, pvalue <0.05, Wilcoxon rank-sum test) top is colour …
Figure 2—figure supplement 1
Gene expression data for mesenchyme subpopulations including identification of apoptosis related genes.
(A) Gene expression plots: expression of Pecam1 (CD31), Ptprc (CD45) and Pdgfrb (PDGFRβ) in the 8 mesenchymal cell clusters confirming that the population of cells isolated for the scRNAseq study …
Figure 3
Analysis of Pdgfrb-BAC-eGFP uterine tissue during endometrial tissue repair and remodelling identifies transient expression of GFP in EPCAM + epithelial cells.
(A) Immunohistochemical analysis of GFP reporter protein (green) and epithelial cell marker EPCAM (red) in uterine tissues 24, 48, and 72 hr following progesterone withdrawal. (i) In 24 hr tissues …
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Figure 3—source data 1
Summary statistics for flow cytometry analyses performed in Figure 3C.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig3-data1-v2.docx
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Figure 3—source data 2
One-way ANOVA with Sidak’s multiple comparisons test for Figure 3C.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig3-data2-v2.docx
Figure 4 with 1 supplement
In silico analysis of scRNAseq reveals transcriptomic similarity between repair-specific mesenchymal cells and subpopulations of EPCAM + endometrial epithelial cells.
(A) UMAP visualisation: GFP + mesenchymal cells and GFP-EPCAM + epithelial cells isolated from Pdgfrb-BAC-eGFP mouse endometrium (cycling/24/48 hr and cycling/48 hr, respectively) cluster into 16 …
Figure 4—figure supplement 1
Comparison between gene expression in F4 population and all other cell clustes confirms overlap with both fibroblast and epithelial cells.
(A) Expression of canonical markers by GFP + mesenchymal cells and GFP-EPCAM + epithelial cells (Pdgfrb-BAC-eGFP uterus) for (i) mesenchymal (Vim, Des, Thy1, Pdgfrb), (ii) perivascular (Mcam, Cspg4, …
Figure 5 with 2 supplements
Lineage tracing strategy using inducible cre recombinase system to target mesenchymal subpopulations in endometrium of adult mice.
(A) UMAP visualisation of trajectory analysis. (i) Monocle3 separated data into two ‘partitions’ placing fibroblasts and epithelial cells into the same trajectory. (ii) Monocle3 revealed a putative …
Figure 5—figure supplement 1
Trajectory Analysis.
(A) UMAP visualisation of scVelo analysis: RNA velocity vectors (grey arrows) superimposed on mesenchymal and epithelial clusters; arrow size = magnitude of residual from unspliced/spliced mRNA …
Figure 5—figure supplement 2
Optimisation of reporter gene expression and wash out following induction of transgenes with Tamoxifen.
(A) Expression of canonical pericyte marker CD146 in steady state/control uterine tissues from different transgenic models. (i) Two populations of cells are present in Pdgfrb-BAC-eGFP uterus: GFP + …
Figure 6
Lineage tracing of PDGFRα + cells identifies a population that undergoes MET.
(A) Analysis of tdTm reporter protein and canonical epithelial cell marker EPCAM in Pdgfra-creERT2;Rosa26-tdTm uterine tissues 24 hr following progesterone withdrawal when the tissue is undergoing …
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Figure 6—source data 1
Summary statistics for flow cytometry analyses performed in Figure 6C.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig6-data1-v2.docx
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Figure 6—source data 2
One-way ANOVA with Tukey’s multiple comparisons test of values in Figure 6C.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig6-data2-v2.docx
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Figure 6—source data 3
Summary statistics for flow cytometry analyses performed in Figure 6E.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig6-data3-v2.docx
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Figure 6—source data 4
One-way ANOVA with Tukey’s multiple comparisons test of values in Figure 6E.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig6-data4-v2.docx
Figure 7 with 1 supplement
Lineage tracing studies confirm that epithelial cells derived from PDGFRα+fibroblasts persist in the post repair luminal epithelium.
(A) Analysis of tdTm reporter protein expression in Pdgfra-creERT2;Rosa26-tdTm uterine tissues 48 hr following progesterone withdrawal when the tissue is remodelling and the luminal epithelium is …
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Figure 7—source data 1
Summary statistics for flow cytometry analyses performed in Figure 7E.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig7-data1-v2.docx
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Figure 7—source data 2
One-way ANOVA with Tukey’s multiple comparisons test for values in Figure 7C.
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig7-data2-v2.docx
Figure 7—figure supplement 1
Evaluation of cell fate in NG2creER/Rosa26-tdTM reporter mice.
(A) Analysis of tdTm reporter protein expression in NG2-CreERTM;Rosa26-tdTm uterine tissues 0, 24, 48, and 72 hr following progesterone withdrawal. Note tdTm + cells were located throughout the …
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Figure 7—figure supplement 1—source data 1
Sidak’s multiple comparisons test of data from expression of tdTm reporter protein by EPCAM +epithelial cells in mouse uterus following induction with PDGFRalpha or NG2 Cre (shown in D).
- https://cdn.elifesciences.org/articles/77663/elife-77663-fig7-figsupp1-data1-v2.docx
Additional files
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Supplementary file 1
Details of antibodies used in the study.
(a) Primary and secondary antibodies with associated working dilutions used to detect proteins in mouse uterine tissues. (b) Flow cytometry antibodies selected and optimised to interrogate mesenchymal cell populations in murine uterus.
- https://cdn.elifesciences.org/articles/77663/elife-77663-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/77663/elife-77663-mdarchecklist1-v2.docx
