Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development
- Department of Genetics, Stanford University School of Medicine, United States
- Department of Biology, Stanford University, United States
- Division of Pediatric Cardiology, Department of Pediatrics, Stanford University School of Medicine, United States
- Stanford Cardiovascular Institute, Stanford University School of Medicine, United States
- Department of Bioengineering and Department of Applied Physics, Stanford University, United States
- Chan Zuckerberg Biohub, United States
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, United States
- Department of Obstetrics and Gynecology, Stanford University School of Medicine, United States
Figures
Figure 1 with 5 supplements
Single-cell RNA sequencing (ScRNAseq) of lineage-traced coronary endothelial cells (ECs) at three stages reveals capillary heterogeneity during embryonic development.
(A and B) Overview of lineage tracing and scRNAseq approach in embryonic and adult mice. (C–K) Unbiased clustering of embryonic coronary ECs at the indicated time points and the contribution of …
Figure 1—figure supplement 1
Localization and expression of recombinant markers.
(A and B) tdTomato localization in sections BmxCreER;RosatdTomato hearts at e17.5 (A) and adult (B). Dashed yellow lines indicate the borders of the tissue sections. Dashed red line indicates the …
Figure 1—figure supplement 2
Selection of coronary vascular endothelial cells (ECs) from e12 and e17.5 datasets.
(A and B) Uniform Manifold Approximation and Projections (UMAPs) showing expression of selected EC subtype markers in e12 ECs (A) and e17.5 ECs (B). (C and D) Breakdown of cell types as percentage …
Figure 1—figure supplement 3
Coronary endothelial cell (EC) subtype markers.
(A, B, and C) Uniform Manifold Approximation and Projections (UMAPs) showing expression of selected coronary EC subtype markers in coronary ECs at e12 (A), e17.5 (B) and adult (C). Scale bar from (C)…
Figure 1—figure supplement 4
Cell cycle regression in e17.5 coronary endothelial cells (ECs).
(A) Uniform Manifold Approximation and Projection (UMAP) showing unbiased clustering of e17.5 mouse coronary ECs before the removal of cycling cells. (B) UMAP showing unbiased clustering of e17.5 …
Figure 1—figure supplement 5
Expression of marker genes adult mouse coronary endothelial cell (EC) dataset.
(A) Uniform Manifold Approximation and Projections (UMAPs) showing expression of selected coronary EC subtype markers in the adult coronary EC dataset from Figure 1.
Figure 2 with 2 supplements
Expression of endocardium (Endo) and sinus venosus (SV) genes in coronary endothelial cells (ECs).
(A) Heatmap showing expression of the top 30 (by p-value) Endo-defining genes (enriched in the Endo compared to the SV) and the top 30 (by p-value) SV-defining genes (enriched in the SV compared to …
Figure 2—figure supplement 1
Expression of selected endocardium (Endo)- and sinus venosus (SV)-defining genes.
(A) Uniform Manifold Approximation and Projection (UMAP) showing expression of canonical Endo (Cdh11) and SV (Vwf, Bmp4) markers in heart endothelial cells (ECs) at e12. (B, C, and D) Expression of …
Figure 2—figure supplement 2
Expression of e12 Cap1- and Cap2-specific genes in a dataset of e12.5 sinus venosus (SV)-derived endothelial cells (ECs).
(A) Uniform Manifold Approximation and Projection (UMAP) showing expression of selected coronary EC subtype markers in a previously published dataset. (B and C) Expression in e12 dataset of genes …
Figure 3 with 2 supplements
Gene expression and localization of e17.5 capillary clusters.
(A) Heatmap showing expression of selected genes enriched in either Cap1 or Cap2 at e17.5 (E = coronary cells from endocardium [Endo]-enriched sample, S = coronary cells from sinus venosus …
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Figure 3—source data 1
Counts of Car4+ and Car4- endothelial cells in sections of BmxCreER hearts at e17.5.
- https://cdn.elifesciences.org/articles/70246/elife-70246-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Expression of flow-induced genes.
(A) Uniform Manifold Approximation and Projections (UMAPs) showing expression of selected flow-induced genes from Kumar et al., 2014, in e17.5 coronary endothelial cells (ECs). (B) Quantification of …
Figure 3—figure supplement 2
Expression of endocardium (Endo)- and sinus venosus (SV)-defining genes at e17.5.
(A) Uniform Manifold Approximation and Projections (UMAPs) showing expression of Endo-enriched genes manually determined to be expressed in a higher percentage of proposed septum cells (as shown in F…
Figure 4
Comparison of injury responses of endocardium (Endo)- and sinus venosus (SV)-derived coronary endothelial cells (ECs).
(A) Overview of lineage tracing and ischemia-reperfusion (I/R) injury approach in adult mice. (B) Example of how EdU localization highlights mid-myocardial injury region. Yellow arrowheads indicate …
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Figure 4—source data 1
Counts of proliferating and non-proliferating endothelial cells in sections of adult BmxCreER hearts after injury.
- https://cdn.elifesciences.org/articles/70246/elife-70246-fig4-data1-v2.xlsx
Figure 5 with 2 supplements
Single-cell RNA sequencing (ScRNAseq) of coronary endothelial cells (ECs) from human fetal hearts.
(A) Overview of scRNAseq approach for three human fetal hearts. (B and C) Uniform Manifold Approximation and Projections (UMAPs) of all major PECAM1+ EC subtypes collected (B) and the non-cycling …
Figure 5—figure supplement 1
Additional analysis of developing human coronary endothelial cells (ECs).
(A) Immunofluorescence for CD36 and Erg in a section from a 14-week human fetal heart. (B) Uniform Manifold Approximation and Projections (UMAPs) showing expression of CD36 in human coronary ECs as …
Figure 5—figure supplement 2
Analysis of developing human coronary endothelial cells (ECs) separated by stage.
(A) Uniform Manifold Approximation and Projections (UMAPs) showing unbiased clustering of cells isolated from each individual human fetal heart. (B) Trajectory analysis of human coronary EC at each …
Figure 6
Trajectory analysis of developing human coronary arteries.
(A) Reference mapping showed that e17.5 mouse endothelial cells (ECs) from Figure 5g were assigned to all three human artery subsets. (B) Trajectory analysis of human coronary ECs using …
Figure 7
Gene expression in developing human coronary arteries.
(A) Heatmap showing expression of selected genes enriched in human artery clusters. (B and C) Regulon scores from SCENIC analysis for TFs enriched in all human and mouse artery clusters (B) and for …
Author response image 1
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(A) UMAP plots showing clustering of e17.5 dataset at multiple resolutions up to 0.6 as well as expression of the top 5 DEGs (by average log-fold change) between newly divided clusters. The clusters …
Author response image 2
Tables
Table 1
Differentially expressed genes between endocardium (Endo) and sinus venosus (SV) (from heatmap in Figure 2A).
| Endo-specific genes | SV-specific genes |
|---|---|
| Cdkn1c | Cldn11 |
| Tmem108 | Aplnr |
| Nrk | Agr2 |
| Fabp5 | Bmp4 |
| Cdh11 | Hoxa5 |
| Cd81 | Gm13889 |
| Irx5 | Zfp503 |
| H19 | Hotairm1 |
| Hand2 | Rassf9 |
| Adgrg6 | Mmrn1 |
| Maged2 | Pcdh17 |
| Ccnd2 | Tox |
| Plvap | Slco3a1 |
| Igf2 | Tspan13 |
| Dok | Nr2f2 |
| Col13a1 | Fst |
| Tm4sf1 | Cd36 |
| Igf2r | Cldn5 |
| Gm1673 | Aqp1 |
| Blvrb | Hoxb4 |
| Gsta4 | Khdrbs3 |
| Rap2a | Kitl |
| Gatm | Wnt16 |
| Olfml3 | Limch1 |
| Sdpr | Ahr |
| Ece1 | Edn1 |
| Plagl1 | Lamp5 |
| Prr15 | Cav1 |
| Igfbp4 | Ddah1 |
| Ccnd3 | Tbx5 |
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Table 1—source data 1
List of all differentially expressed genes between e12 endocardium (Endo) and sinus venosus (SV) sorted by Wilcoxon rank sum test.
- https://cdn.elifesciences.org/articles/70246/elife-70246-table1-data1-v2.xlsx
Table 2
Differentially expressed genes between all endocardium (Endo)-enriched and all sinus venosus (SV)-enriched capillaries.
Bolded genes are also differentially expressed between Cap1 and Cap2.
| Raw p-value | Average log-fold change | % Endo cells expressing | % SV cells expressing | Adjusted p-value | Higher fold change in Cap1 versus Cap2 comparison? | |
|---|---|---|---|---|---|---|
| tdTomato | 1.91E-238 | 0.39378511 | 0.452 | 0 | 5.35E-234 | |
| Gt(ROSA)26Sor | 2.43E-153 | –0.634801942 | 0.568 | 0.837 | 6.81E-149 | |
| Anxa1 | 4.24E-109 | 0.553933954 | 0.804 | 0.53 | 1.19E-104 | |
| Igf2 | 6.74E-93 | –0.357191579 | 0.99 | 0.999 | 1.89E-88 | |
| Timp4 | 1.28E-88 | –0.632710228 | 0.447 | 0.766 | 3.58E-84 | Yes |
| Fmo1 | 8.34E-70 | –0.351754806 | 0.231 | 0.511 | 2.34E-65 | Yes |
| Txnip | 2.09E-67 | –0.37131915 | 0.851 | 0.933 | 5.85E-63 | Yes |
| Aplnr | 5.42E-67 | –0.40125981 | 0.677 | 0.863 | 1.52E-62 | Yes |
| Car4 | 1.68E-65 | –0.595662824 | 0.347 | 0.62 | 4.70E-61 | Yes |
| Aqp7 | 3.33E-62 | –0.368823615 | 0.345 | 0.63 | 9.31E-58 | Yes |
| Apoe | 1.19E-60 | 0.302862096 | 0.307 | 0.09 | 3.34E-56 | |
| Cd36 | 8.58E-59 | –0.308565577 | 0.97 | 0.997 | 2.40E-54 | Yes |
| Igfbp5 | 1.84E-57 | 0.4602195 | 0.258 | 0.064 | 5.15E-53 | |
| Fabp5 | 6.53E-54 | 0.30861622 | 0.999 | 1 | 1.83E-49 | Yes |
| 1810011O10Rik | 1.08E-52 | –0.340249285 | 0.907 | 0.963 | 3.04E-48 | Yes |
| Aqp1 | 9.79E-47 | –0.471913791 | 0.54 | 0.729 | 2.74E-42 | Yes |
| Gap43 | 7.23E-38 | 0.34418651 | 0.523 | 0.331 | 2.02E-33 | Yes |
| Sat1 | 1.99E-37 | 0.394143816 | 0.753 | 0.63 | 5.59E-33 | Yes |
| Cd63 | 3.81E-36 | 0.33008301 | 0.758 | 0.611 | 1.07E-31 | Yes |
| Igfbp3 | 3.45E-34 | 0.640208849 | 0.634 | 0.468 | 9.66E-30 | Yes |
| Tm4sf1 | 1.38E-32 | 0.302207793 | 0.982 | 0.94 | 3.87E-28 | |
| Maged2 | 4.03E-32 | 0.323885527 | 0.845 | 0.757 | 1.13E-27 | |
| Ly6c1 | 1.32E-31 | –0.301696479 | 0.845 | 0.93 | 3.70E-27 | Yes |
| Rbp1 | 1.26E-15 | 0.432393695 | 0.757 | 0.69 | 3.52E-11 | Yes |
Table 3
Ratio of Erg+ Car4+/Erg+ cells in adult mouse hearts.
| LV | Septum | |
|---|---|---|
| heart1 | 0.874 | 0.853 |
| heart2 | 0.864 | 0.819 |
Table 4
Genes unique to human coronary endothelial cells (ECs).
| SLC14A1 |
| GABBR2 |
| NRN1 |
| EPHA3 |
| ADMTSL1 |
| A2M |
| PRND |
| GRIA2 |
| KCNN3 |
| SERPINE2 |
| LPCAT2 |
| LGALS3 |
| APOA1 |
| SYNJ2 |
| OCIAD2 |
| PRICKLE2 |
| CTNND1 |
| IFITM2 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | BmxCreER | Mouse Genome Informatics | MGI: 5513853; RRID: IMSR_TAC:14081 | |
| Strain, strain background (Mus musculus) | RosatdTomato | The Jackson Laboratory | Stock No: 007909; RRID: IMSR_JAX:007909 | |
| Strain, strain background (Mus musculus) | CD1 | Charles River Laboratories | Strain code: 022; RRID: IMSR_CRL:022 | |
| Biological sample (Homo sapiens) | Primary fetal heart tissue | Collected at Stanford from elective terminations | ||
| Antibody | Rat anti- APC/Cy7 Cd45 (rat monoclonal) | Biolegend | Cat #: 103116; RRID: AB_312981 | (1:50) |
| Antibody | Rat anti- APC Pecam1 (rat monoclonal) | Biolegend | Cat #: 102410; RRID: AB_312905 | (1:50) |
| Antibody | Rat anti- APC-Cy7 Ter119 (rat monoclonal) | Biolegend | Cat #: 116223; RRID: AB_2137788 | (1:50) |
| Antibody | Mouse anti- Pacific Blue CD235a (mouse monoclonal) | Biolegend | Cat #: 349107; RRID: AB_11219199 | (1:50) |
| Antibody | Mouse anti- FITC CD36 (mouse monoclonal) | Biolegend | Cat #: 336204; RRID: AB_1575025 | (1:50) |
| Antibody | Mouse anti- APC-Cy7 PECAM1 (mouse monoclonal) | Biolegend | Cat #: 303119; RRID: AB_10643590 | (1:50) |
| Antibody | Mouse anti- Pacific Blue CD45 (mouse monoclonal) | Biolegend | Cat #: 304021; RRID: AB_493654 | (1:50) |
| Antibody | Mouse anti- PerCP-Cy5.5 CD235a (mouse monoclonal) | Biolegend | Cat #: 349110; RRID: AB_2562706 | (1:50) |
| Antibody | Mouse anti-PerCP-Cy5.5 CD45 (mouse monoclonal) | Biolegend | Cat #: 304028; RRID: AB_893338 | (1:50) |
| Antibody | Rabbit anti-Erg (rabbit monoclonal) | Abcam | Cat #: ab92513; RRID: AB_2630401 | (1:200) |
| Antibody | Goat anti-Car4 (goat polyclonal) | R&D | Cat #: AF2414; RRID: AB_2070332 | (1:200) |
| Antibody | Rabbit anti-Smmhc (rabbit polyclonal) | Proteintech | Cat #: 21404–1-AP; RRID: AB_10732819 | (1:100) |
| Antibody | Mouse anti-Cldn5 (mouse monoclonal) | Invitrogen | Cat #: 35–2500; RRID: AB_2533200 | (1:200) |
| Peptide, recombinant protein | Collagenase IV | Worthington | Cat #: LS004186 | |
| Peptide, recombinant protein | Dispase | Worthington | Cat #: LS02100 | |
| Peptide, recombinant protein | DNase I | Worthington | Cat #: LS002007 | |
| Commercial assay or kit | RNEasy Mini Kit | Qiagen | Cat #: 74,104 | |
| Commercial assay or kit | iScript Reverse Transcription Supermix | Bio-Rad | Cat #: 1708840 | |
| Commercial assay or kit | Roche DIG RNA Labeling kit | Millipore Sigma | Cat #: 11175025910 | |
| Commercial assay or kit | RNAscope Multiplex Fluorescent V2 assay kit | Advanced Cell Diagnostics | Cat #: 323,100 | |
| Commercial assay or kit | Click-iT EdU Imaging kit | Thermo Fisher Scientific | Cat #: C10086 | |
| Chemical compound, drug | Tamoxifen | Sigma-Aldrich | Cat #: T5648 | |
| Chemical compound, drug | 4-OH Tamoxifen | Sigma-Aldrich | Cat #: H6278 | |
| Software, algorithm | Seurat v3 | https://doi.org/10.1016/j.cell.2019.05.031 | RRID: SCR_007322 | |
| Software, algorithm | bcl2fastq | illumina | RRID: SCR_015058 | |
| Software, algorithm | cutadapt 2.7 | https://doi.org/10.14806/ej.17.1.200 | RRID: SCR_011841 | |
| Software, algorithm | Cell Ranger v3.1.0 | 10× Genomics | RRID: SCR_017344 | |
| Software, algorithm | STAR v2.7.1a | https://doi.org/10.1093/bioinformatics/bts635 | RRID: SCR_004463 | |
| Software, algorithm | Subread v1.6.0 | https://doi.org/10.1093/nar/gkt214 | RRID: SCR_009803 | |
| Software, algorithm | heatmaply | http://dx.doi.org/10.1093/bioinformatics/btx657 | https://github.com/talgalili/heatmaply (Galili, 2021) | |
| Software, algorithm | biomaRt | 10.18129/B9.bioc.biomaRt | RRID: SCR_019214 | |
| Software, algorithm | PAGA | https://doi.org/10.1186/s13059-019-1663-x | https://github.com/theislab/paga (Thesis Lab, 2019) | |
| Software, algorithm | RNA velocity | 10.1038/s41586-018-0414-6 | RRID: SCR_018168; RRID: SCR_018167 | |
| Software, algorithm | Slingshot | https://doi.org/10.1186/s12864-018-4772-0 | RRID: SCR_017012 | |
| Software, algorithm | SCENIC | 10.1038/nmeth.4463 | RRID: SCR_017247 | |
| Software, algorithm | FIJI | doi:10.1038/nmeth.2019 | RRID: SCR_002285 | |
| Software, algorithm | QuPath | https://doi.org/10.1038/s41598-017-17204-5 | RRID: SCR_018257 | |
| Software, algorithm | Prism 8 | GraphPad Software | RRID: SCR_002798 | |
| Other | TINAGL1 RNA probe | Advanced Cell Diagnostics | Cat #: 857221-C2 |
