Coronary blood vessels from distinct origins converge to equivalent states during mouse and human development

  1. Ragini Phansalkar
  2. Josephine Krieger
  3. Mingming Zhao
  4. Sai Saroja Kolluru
  5. Robert C Jones
  6. Stephen R Quake
  7. Irving Weissman
  8. Daniel Bernstein
  9. Virginia D Winn
  10. Gaetano D'Amato  Is a corresponding author
  11. Kristy Red-Horse  Is a corresponding author
  1. Department of Genetics, Stanford University School of Medicine, United States
  2. Department of Biology, Stanford University, United States
  3. Division of Pediatric Cardiology, Department of Pediatrics, Stanford University School of Medicine, United States
  4. Stanford Cardiovascular Institute, Stanford University School of Medicine, United States
  5. Department of Bioengineering and Department of Applied Physics, Stanford University, United States
  6. Chan Zuckerberg Biohub, United States
  7. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, United States
  8. Department of Obstetrics and Gynecology, Stanford University School of Medicine, United States
9 figures, 5 tables and 1 additional file

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 …

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…

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 …

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 …

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 …

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
.

(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 genesSV-specific genes
Cdkn1cCldn11
Tmem108Aplnr
NrkAgr2
Fabp5Bmp4
Cdh11Hoxa5
Cd81Gm13889
Irx5Zfp503
H19Hotairm1
Hand2Rassf9
Adgrg6Mmrn1
Maged2Pcdh17
Ccnd2Tox
PlvapSlco3a1
Igf2Tspan13
DokNr2f2
Col13a1Fst
Tm4sf1Cd36
Igf2rCldn5
Gm1673Aqp1
BlvrbHoxb4
Gsta4Khdrbs3
Rap2aKitl
GatmWnt16
Olfml3Limch1
SdprAhr
Ece1Edn1
Plagl1Lamp5
Prr15Cav1
Igfbp4Ddah1
Ccnd3Tbx5
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-valueAverage log-fold change% Endo cells expressing% SV cells expressingAdjusted p-valueHigher fold change in Cap1 versus Cap2 comparison?
tdTomato1.91E-2380.393785110.45205.35E-234
Gt(ROSA)26Sor2.43E-153–0.6348019420.5680.8376.81E-149
Anxa14.24E-1090.5539339540.8040.531.19E-104
Igf26.74E-93–0.3571915790.990.9991.89E-88
Timp41.28E-88–0.6327102280.4470.7663.58E-84Yes
Fmo18.34E-70–0.3517548060.2310.5112.34E-65Yes
Txnip2.09E-67–0.371319150.8510.9335.85E-63Yes
Aplnr5.42E-67–0.401259810.6770.8631.52E-62Yes
Car41.68E-65–0.5956628240.3470.624.70E-61Yes
Aqp73.33E-62–0.3688236150.3450.639.31E-58Yes
Apoe1.19E-600.3028620960.3070.093.34E-56
Cd368.58E-59–0.3085655770.970.9972.40E-54Yes
Igfbp51.84E-570.46021950.2580.0645.15E-53
Fabp56.53E-540.308616220.99911.83E-49Yes
1810011O10Rik1.08E-52–0.3402492850.9070.9633.04E-48Yes
Aqp19.79E-47–0.4719137910.540.7292.74E-42Yes
Gap437.23E-380.344186510.5230.3312.02E-33Yes
Sat11.99E-370.3941438160.7530.635.59E-33Yes
Cd633.81E-360.330083010.7580.6111.07E-31Yes
Igfbp33.45E-340.6402088490.6340.4689.66E-30Yes
Tm4sf11.38E-320.3022077930.9820.943.87E-28
Maged24.03E-320.3238855270.8450.7571.13E-27
Ly6c11.32E-31–0.3016964790.8450.933.70E-27Yes
Rbp11.26E-150.4323936950.7570.693.52E-11Yes
Table 3
Ratio of Erg+ Car4+/Erg+ cells in adult mouse hearts.
LVSeptum
heart10.8740.853
heart20.8640.819
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 resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus)BmxCreERMouse Genome InformaticsMGI: 5513853; RRID: IMSR_TAC:14081
Strain, strain background (Mus musculus)RosatdTomatoThe Jackson LaboratoryStock No: 007909; RRID: IMSR_JAX:007909
Strain, strain background (Mus musculus)CD1Charles River LaboratoriesStrain code: 022; RRID: IMSR_CRL:022
Biological sample (Homo sapiens)Primary fetal heart tissueCollected at Stanford from elective terminations
AntibodyRat anti- APC/Cy7 Cd45 (rat monoclonal)BiolegendCat #: 103116; RRID: AB_312981(1:50)
AntibodyRat anti- APC Pecam1 (rat monoclonal)BiolegendCat #: 102410; RRID: AB_312905(1:50)
AntibodyRat anti- APC-Cy7 Ter119 (rat monoclonal)BiolegendCat #: 116223; RRID: AB_2137788(1:50)
AntibodyMouse anti- Pacific Blue CD235a (mouse monoclonal)BiolegendCat #: 349107; RRID: AB_11219199(1:50)
AntibodyMouse anti- FITC CD36 (mouse monoclonal)BiolegendCat #: 336204; RRID: AB_1575025(1:50)
AntibodyMouse anti- APC-Cy7 PECAM1 (mouse monoclonal)BiolegendCat #: 303119; RRID: AB_10643590(1:50)
AntibodyMouse anti- Pacific Blue CD45 (mouse monoclonal)BiolegendCat #: 304021; RRID: AB_493654(1:50)
AntibodyMouse anti- PerCP-Cy5.5 CD235a (mouse monoclonal)BiolegendCat #: 349110; RRID: AB_2562706(1:50)
AntibodyMouse anti-PerCP-Cy5.5 CD45 (mouse monoclonal)BiolegendCat #: 304028; RRID: AB_893338(1:50)
AntibodyRabbit anti-Erg (rabbit monoclonal)AbcamCat #: ab92513; RRID: AB_2630401(1:200)
AntibodyGoat anti-Car4 (goat polyclonal)R&DCat #: AF2414; RRID: AB_2070332(1:200)
AntibodyRabbit anti-Smmhc (rabbit polyclonal)ProteintechCat #: 21404–1-AP; RRID: AB_10732819(1:100)
AntibodyMouse anti-Cldn5 (mouse monoclonal)InvitrogenCat #: 35–2500; RRID: AB_2533200(1:200)
Peptide, recombinant proteinCollagenase IVWorthingtonCat #: LS004186
Peptide, recombinant proteinDispaseWorthingtonCat #: LS02100
Peptide, recombinant proteinDNase IWorthingtonCat #: LS002007
Commercial assay or kitRNEasy Mini KitQiagenCat #: 74,104
Commercial assay or kitiScript Reverse Transcription SupermixBio-RadCat #: 1708840
Commercial assay or kitRoche DIG RNA Labeling kitMillipore SigmaCat #: 11175025910
Commercial assay or kitRNAscope Multiplex Fluorescent V2 assay kitAdvanced Cell DiagnosticsCat #: 323,100
Commercial assay or kitClick-iT EdU Imaging kitThermo Fisher ScientificCat #: C10086
Chemical compound, drugTamoxifenSigma-AldrichCat #: T5648
Chemical compound, drug4-OH TamoxifenSigma-AldrichCat #: H6278
Software, algorithmSeurat v3https://doi.org/10.1016/j.cell.2019.05.031RRID: SCR_007322
Software, algorithmbcl2fastqilluminaRRID: SCR_015058
Software, algorithmcutadapt 2.7https://doi.org/10.14806/ej.17.1.200RRID: SCR_011841
Software, algorithmCell Ranger v3.1.010× GenomicsRRID: SCR_017344
Software, algorithmSTAR v2.7.1ahttps://doi.org/10.1093/bioinformatics/bts635RRID: SCR_004463
Software, algorithmSubread v1.6.0https://doi.org/10.1093/nar/gkt214RRID: SCR_009803
Software, algorithmheatmaplyhttp://dx.doi.org/10.1093/bioinformatics/btx657https://github.com/talgalili/heatmaply (Galili, 2021)
Software, algorithmbiomaRt10.18129/B9.bioc.biomaRtRRID: SCR_019214
Software, algorithmPAGAhttps://doi.org/10.1186/s13059-019-1663-xhttps://github.com/theislab/paga (Thesis Lab, 2019)
Software, algorithmRNA velocity10.1038/s41586-018-0414-6RRID: SCR_018168; RRID: SCR_018167
Software, algorithmSlingshothttps://doi.org/10.1186/s12864-018-4772-0RRID: SCR_017012
Software, algorithmSCENIC10.1038/nmeth.4463RRID: SCR_017247
Software, algorithmFIJIdoi:10.1038/nmeth.2019RRID: SCR_002285
Software, algorithmQuPathhttps://doi.org/10.1038/s41598-017-17204-5RRID: SCR_018257
Software, algorithmPrism 8GraphPad SoftwareRRID: SCR_002798
OtherTINAGL1 RNA probeAdvanced Cell DiagnosticsCat #: 857221-C2

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