A cell atlas of the developing human outflow tract of the heart and its adult aortic valve derivatives
- Faculty of Biology, Medicine and Health, University of Manchester, United Kingdom
- Translational and Clinical Research Institute, Newcastle University, United Kingdom
- College of Medicine & Health, University of Birmingham, United Kingdom
- Newcastle University Biosciences Institute, Faculty of Medical Sciences, United Kingdom
Figures
Figure 1 with 1 supplement
The cellular landscape of the developing outflow tract (OFT) and its adult derivatives.
(A) Experimental schematics. Nuclei isolated from two embryonic (CS 16–17) and two fetal (12 pcw) OFTs and from three adult aortic valves (AV) were analyzed by snRNA-seq. Four cryo-sections including the OFT region of a 12 pcw heart were used in spatial transcriptomics (Visium). Section is shown within the Visium capture area (~6 mm × 6 mm; spot diameter 55 µm, centre-to-centre spacing 100 µm), which defines spatial scale. (B) Sample correlation visualized by unsupervised clustering and projected on a two-dimensional uniform manifold approximation and projection (UMAP). Nuclei are colored by sample, with embryonic (blue, orange), fetal (green, red), and adult (pink, purple, and brown). (C) Cell clusters visualized by the same UMAP as in B. Nuclei are colored by cluster. (D) Cluster composition in each sample, presented as percentage of nuclei. (E) Dotplot shows the mean expression levels of top differential genes across clusters and identifies five main cell types: cardiac, endothelial, mesenchymal (including valve interstitial), neural, and immune. (F) Cell types in E visualized by UMAP. Nuclei are colored by cell type. See also Figure 1—figure supplement 1.
Figure 1—figure supplement 1
Assessment of batch effects and lineage- and stage-specific markers.
Confusion matrix of Louvain clusters’ distribution across samples before (A) and after (B) Harmony batch correctionValues (number of nuclei in each cluster) are scaled by min max scaling. (A) Clusters are composed of time point matched biological replicates, showing that our datasets are not impacted by technical batch effects and that the variability we observe reflects meaningful changes across time. The only exception is cluster 11 (immune): immune cells are relatively homogenous across populations, and their assignments to different samples across two time points, fetal and adult, indicate batch effect is not creating undue artifacts in our data integration. (B) Most clusters include samples from different time points, which indicates a loss of biological variability across time after batch correction. (C) Cell types in Figure 1E labeled using established lineage markers. DCN identifies fibroblast populations, MYH11 labels SMCs, PECAM1 is a marker for endothelial cells, TNNT2 is a marker for cardiac cells, and PDGFRA and PDGFRB mark mesenchymal cells before differentiation. Each nucleus is colored based on the scaled expression of the indicated marker. (D) NEAT1 expression. Uniform manifold approximation and projection (UMAP) visualization of samples aggregated by stage: adult aortic valves (E, dark gray), embryonic (F, red), fetal (G, blue).
Figure 2 with 1 supplement
Characterization of embryonic mesenchymal nuclei.
Mesenchymal cell clusters (A) and sample projection (B) of fetal and embryonic samples following subclustering, visualized on a two-dimensional tSNE. Nuclei are colored by cluster (A) and sample (B). (C) Embryonic clusters (blue contour) do not express fibroblast (DCN) or smooth muscle (MYH11) markers, which are present in fetal nuclei (green contour). Nuclei are colored according to their scaled expression. (D) Top 10 regulons in fetal clusters based on Regulon Specificity Score (RSS). (E) Gene ontologies associated with the GATA6 regulon highlighted terms related to arterial and pulmonary valve morphogenesis. Functional annotation clustering of top 400 genes enriched GATA6 regulon was performed using DAVID and −log10(Pv) was plotted in Excel. (F) Hematoxylin and eosin (H&E)-stained section showing the aortic and pulmonary arteries with their respective valves (left) and a corresponding map of the spatial expression of the GATA6 regulon (right). (G) GREAT analysis of GATA6 high-confidence peaks (FE >10) in posterior pharyngeal arches and outflow tract (OFT) at embryonic day (E) 11.5 (mouse). GATA6 peaks predominantly cluster around genes associated with cardiovascular terms, and specifically with OFT and artery development, as well as semilunar valve development (red arrows). (H) Selected regulon genes associated with GATA6 binding in mouse embryo OFT and pharyngeal arches (see also Supplementary file 3) and associated with OFT-related abnormalities. Yellow genes are associated with human disease; light green genes cause mouse phenotypes; dark green genes are associated with both human and mouse defects. (I) UCSC tracks of H3K27Ac ChIP-seq, GATA6 ChIP-seq (boxed in red) in posterior pharyngeal arches and OFT at E11.5 (mouse) and mammal sequence conservation at MECOM (top), LTBP1 (middle) and NOTCH2 (bottom) loci. (J). Spatial Transcriptomics of Aorta (Ao) and Pulmonary Artery (PA) (clockwise): H&E staining of the tissue area, with asterisks marking the semilunar valves; spatial distribution of LTBP1, NOTCH2, and MECOM. (K) Trajectory inference of future state of embryonic nuclei (CS16–17) showing mesenchymal (4, 20), endothelial-like (7), and cardiac (2, 17) clusters. Embryonic clusters derive from subclustering of aggregated fetal and embryonic nuclei shown in Figure 2—figure supplement 1A. (L) Expression signatures in embryonic mesenchymal (4, 20) and endothelial-like (7) clusters. Both cluster 7 and 4 express high levels of cardiac TFs (GATA4 and TBX20) and HAPLN1, a marker of semilunar valves. In contrast, cluster 20 nuclei exhibit higher expression of neural crest markers, HOXA3-B3 and PLXNA2.
Figure 2—figure supplement 1
Re‑clustering and characterisation of embryonic and fetal samples.
(A, B) Louvain 0.4 re-clustering of embryonic and fetal samples (without adult samples), presented by tSNE. Nuclei are colored by sample (A) and cluster (B). Embryonic and fetal mesenchymal clusters are highlighted by blue and green contours, respectively. (C) Spatial Transcriptomics of Aorta (Ao) and Pulmonary Artery (PA) (clockwise): ROBO1, SLIT2, MYH10, and COL1A2. (D) Expression of GATA6 (log normalized values) in CS16-17 nuclei, projected onto the RNA velocity plot shown in Figure 2K. (E) Expression of GLI3 (Log normalized values) in CS16–17 nuclei, projected onto the RNA velocity plot shown in Figure 2K. (F) Gene ontologies associated with distinctive markers of embryonic endothelial cells (cluster 7). Functional annotation clustering of top 200 genes enriched in embryonic endothelial cluster 7 (relative to fetal endothelial clusters 9 and 13) was performed using DAVID and −log10(Pv) was plotted in Excel. Genes are listed in Supplementary file 3.
Figure 3 with 1 supplement
Spatial distribution of mesenchymal clusters.
(A–F) Outflow tract (OFT) valve formation and remodeling. Images were prepared using high-resolution episcopic microscopy at embryonic stages (A–D) and by micro-CT at the fetal stage (EF). (A) By CS16 the OFT has septated into the aorta (Ao) and pulmonary trunk (PT). The immature OFT cushions are visible: the septal (yellow), parietal (green), and intercalated (purple) cushions. (B) Neural crest cells (asterisks) contribute to valve formation. CD. At CS20 the cushions have begun to remodel to form the three leaflets of the aortic and pulmonary semilunar valves. (E, F) At 11 pcw, the valves have transformed into the leaflets that control the unidirectional flow of blood from the heart. Boxed regions in (A, C, E) are shown at higher magnification in (B, D, F). (G) Heart alignment for sectioning, with the OFT region marked in red (left). Hematoxylin and eosin (H&E) staining of OFT cryo-sections used for spatial transcriptomics from the base of the OFT (a) to the pulmonary valves (d). The aorta and pulmonary trunk are indicated by blue and green arrowheads, respectively. (H) H&E section ‘c’ annotated to show major structures. (I, II, J, K) Spatial distribution of mesenchymal clusters (purple and yellow). (III, JI, KI) Spatial distribution of lineage-specific markers (white and red). (I–III) Clusters 3 (I) and 6 (II) largely overlap with fibroblast lineage marker DCN (III). Cluster 9 (J) and smooth muscle lineage-specific marker MYH11 (JI) map to the aortic walls as well as to the pulmonary artery. luster 12 (K) and valve-specific marker HAPLN1 (KI) are mainly found in the valves at the base of the aorta and pulmonary artery. See also Figure 3—figure supplement 1.
Figure 3—figure supplement 1
Mapping of cell types from transcriptomics data to spatial location using Cell2location.
(A) Cardiac muscle cells in the atria and ventricle. (B) Mesenchymal cells consisting of smooth muscle cells inside the lumen of the aorta, fibroblast layer around the vessels, and in the aortic and pulmonary semilunar valves. (C) Endothelial cells in the aortic semilunar valves. (D) Immune cells. (E) Neuronal cells. (F) Cluster 15 is restricted to the right ventricle and was removed from the analysis of outflow tract (OFT) mesenchymal clusters.
Figure 4 with 1 supplement
Lineage deconvolution of embryonic and fetal nuclei.
(A) Pairwise differential gene expression of the two embryonic mesenchymal clusters; genes chosen for gene modules are marked by asterisks. (B) Heatmap using embryonic gene modules, obtained using k-means clustering, separates embryonic mesenchymal nuclei into two groups. (C) Mesenchymal cell clusters of embryonic and fetal time points, projected on a two-dimensional tSNE and labeled using gene modules. Fetal clusters derive from separate ‘blue’ and ‘red’ embryonic lineages. (D) Heatmap of embryonic gene modules and cell type marker genes using k-means clustering identifies three main groups of fetal nuclei. (E) Lineage trajectories of embryonic and fetal nuclei. Using the entire fetal datasets, cluster 3 and 12 nuclei are identified as descendants of embryonic cluster 4, while clusters 6 and 9 are the most likely descendants of embryonic cluster 20, consistent with the use of gene modules in the mesenchymal subset of fetal nuclei in 3D.
Figure 4—figure supplement 1
Gene ontologies associated with distinctive developmental signatures of mesenchymal subtypes.
Functional annotation clustering of top 100 genes enriched in embryonic cluster 4 (A) and cluster 20 (B) was performed using DAVID and −log10(Pv) was plotted in Excel. Cluster 4 (A) is enriched in cardiac-like markers and cluster 20 (B) in neural crest markers. Genes are listed in Supplementary file 4. (C) Lineage relationships between embryonic and differentiated fetal mesenchymal cells. Cluster 4 derives from the secondary heart field (SHF) with a contribution of SHF-derived endocardial cells. It gives rise to arterial valves and fibroblasts. Cluster 4 derives from SHF and cardiac neural crest; cells in cluster 4 are the progenitors of smooth muscle cells and fibroblasts.
Figure 5 with 3 supplements
Cellular constituents of the mature aortic valves.
(A) Aortic valve sample association projected on a two-dimensional uniform manifold approximation and projection (UMAP). Nuclei are colored by sample. (B) Nuclei clusters visualized by unsupervised clustering. Nuclei are colored by cluster. (C) Cell lineages identified using established lineage-specific markers. Each nucleus is colored based on the scaled expression of the indicated marker. (D) Top differentially expressed genes in clusters identify known lineage markers. (E) Overview of the method used to trace adult descendants of embryonic nuclei. The first step is the identification of distinctive signatures in embryonic progenitors; for this, we used the top 100 differentially expressed (DE) genes in our chosen progenitor populations, cluster 4 (blue) and cluster 20 (red). The second step is the identification of the top 5000 marker genes for each adult population; this is done by comparing each cluster with the rest of the dataset. Finally, we search for the 100 DE embryonic genes in the marker genes of adult clusters. Adult clusters with top hits are identified as the descendants of the embryonic lineage; the statistical significance is calculated using a hypergeometric test. (F) Dotplot displaying the 30 top DE genes (mean expression values) in embryonic clusters 4 and 20, respectively. The same dotplot, previously shown in Figure 3C, has been included here to facilitate cross-comparison with Figure 6G, H. (G) Distribution of cluster 4 embryonic signature genes in adult nuclei clusters. Clusters 4, 7, and 5 express a highly significant fraction of embryonic cluster 4 genes. Top 30 DE genes in embryonic clusters 4 and 20 (shown in F) are highlighted by dots. (H) Distribution of cluster 20 embryonic signature genes in adult nuclei clusters. Cluster 1 expresses a highly significant fraction of embryonic cluster 20 genes. Top 30 DE genes in embryonic clusters 4 and 20 (shown in F) are highlighted by dots.
Figure 5—figure supplement 1
Characterisation of adult cell populations.
(A) Differential gene expression heatmap of adult clusters. (B, C) Cluster composition in each adult sample, presented as percentage of nuclei with (A) and without (B) cluster 1. (D) ECM encoding transcripts in interstitial cells. Each nucleus is colored based on the scaled expression of the indicated marker. (E) Proportions of nuclei in each adult cluster.
Figure 5—figure supplement 2
Embryonic gene signatures in adult cell clusters.
(A, B) Dotplots showing the distribution of cluster 4 embryonic signature genes in individual samples, AV1 and AV3. Cluster 0 in AV1 (A) and cluster 3 in AV3 (B) express a highly significant fraction of embryonic cluster 4 genes. (C, D) Cluster correlation between adult aggregate nuclei and individual sample nuclei. AV1 nuclei in cluster 0 contain nuclei from aggregate clusters 4 and 7. Similarly, AV3 cluster 3 nuclei contain the majority of aggregate cluster 4 and 7 nuclei. (E, F) Relative expression of embryonic signature genes in embryonic (E, 4; F, 20) and adult clusters (E, 4; F, 1), which are lineage related. Genes were ranked by expression values, relative to all the genes expressed in each cluster. In descendant adult nuclei, most embryonic genes (65% in adult cluster 4) are not represented in the top 1000 expressed genes. In contrast, embryonic genes are among the top 1000 expressed genes in embryonic nuclei (73% in embryo cluster 4).
Figure 5—figure supplement 3
Expression (log normalized values) of 10 representative genes from the 100-gene embryonic signature lists of cluster 4 (A) and cluster 20 (B) projected onto the tSNE shown in Figure 4E.
Figure 6 with 1 supplement
Genes mutated in congenital OFT defects.
(A) Hematoxylin and eosin (H&E) staining of spatial transcriptomics section (Figure 2J), and magnified view of aortic and pulmonary valve area (AI). The aorta and pulmonary trunk are indicated by blue and green arrowheads, respectively. JAG1 (B), GATA5 (C), and NR2F2 (D) gene expression patterns on the same section. (BI–DI) Genes as in BD with corresponding magnification of valve area. (E) Genes identified as displaying spatially similar expression patterns to JAG1.
Figure 6—figure supplement 1
GATA4 and GATA6 expression in the outflow tract.
(A) GATA6 and (B) GATA4. GATA4 and GATA6 are broadly distributed across the outflow tract (OFT) and surrounding cardiac tissue. Mutations in both genes cause bicuspid aortic valve (BAV); the position of the valves is highlighted by HAPLN1 (C).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (human embryonic tissue) | Human embryonic outflow tract (CS16–17) | Human Developmental Biology Resource (HDBR) | Staged by HDBR guidelines; male | |
| Biological sample (human fetal tissue) | Human fetal outflow tract (12 pcw) | Human Developmental Biology Resource (HDBR) | Male | |
| Biological sample (human adult tissue) | Human aortic valve | Newcastle Institute of Transplantation Tissue Biobank | Female (age 55–70) | |
| Commercial assay or kit | Chromium Next GEM Single Cell 3′ Kit v3.1 | 10x genomics | CG000315 | RNA-seq library construction |
| Commercial assay or kit | Visium spatial gene expression – Spatial transcriptomics | 10x genomics | CG000239 | Fetal heart spatial analysis |
| Peptide, recombinant protein (bovine serum albumin) | Ultrapure BSA | Invitrogen | AM2616 | Nuclei resuspension |
| Peptide, recombinant protein (Protector RNAse inhibitor) | RNAse inhibitor | Roche | 3335399001 | Nuclei extraction and sorting |
| Software | Cell ranger – Pipeline for 10x data | 10x genomics V3.1.0, v6.1.2 | RRID:SCR_017344 | Alignment and UMI quantification |
| Software | Scanpy – Single cell data analysis | Scanpy v.1.9.5 | RRID:SCR_018139 | Normalization and integration |
| Algorithm | scVelo – RNA-velocity analysis | scVElo V0.2.4 | RRID:SCR_018168 | Analysis of embryonic stage dynamics |
| Algorithm | SCENIC – Gene regulatory network inference | SCENIC | RRID:SCR_017247 | Regulon activity scores |
| Algorithm | Cell2location – Spatial deconvolution | Cell2location v.0.1.3 | RRID:SCR_024859 | Maps snRNA-seq signatures to spatial spots |
Additional files
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Supplementary file 1
Accession numbers of single-cell experiments and spatial transcriptomics.
- https://cdn.elifesciences.org/articles/107748/elife-107748-supp1-v1.xlsx
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Supplementary file 2
Marker genes (top 100) for each of the 18 clusters (0–17) in Figure 1.
- https://cdn.elifesciences.org/articles/107748/elife-107748-supp2-v1.xlsx
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Supplementary file 3
GATA6 regulon genes associated with GATA6 binding in the OFT and pharyngeal arches in E11.5 mouse embryos.
Association was established using GATA6 peaks with a fold enrichment cutoff >10 and GREAT standard association rules.
- https://cdn.elifesciences.org/articles/107748/elife-107748-supp3-v1.xlsx
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Supplementary file 4
Genes enriched in cluster 7 (embryonic endothelial) relative to clusters 9–13 (fetal endothelial).
- https://cdn.elifesciences.org/articles/107748/elife-107748-supp4-v1.xlsx
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Supplementary file 5
Embryonic cluster 4 and 20 top 100 differentially expressed genes.
- https://cdn.elifesciences.org/articles/107748/elife-107748-supp5-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107748/elife-107748-mdarchecklist1-v1.pdf
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A cell atlas of the developing human outflow tract of the heart and its adult aortic valve derivatives
eLife 14:RP107748.
https://doi.org/10.7554/eLife.107748.3
