The cellular landscape of the developing OFT and its adult derivatives.

A. Experimental schematics. Nuclei isolated from two embryonic (CS 16-17) and two fetal (12pcw) OFTs and from three adult aortic valves (AV) were analyzed by snRNA-seq. Four cryo-sections including the OFT region of a 12pcw heart were used in spatial transcriptomics (Visium). B. Sample correlation visualized by unsupervised clustering and projected on a two-dimensional 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 visualised by UMAP. Nuclei are colored by cell type. See also Fig S1.

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 RSS score. 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. GREAT analysis of GATA6 high-confidence peaks (FE>10) in posterior pharyngeal arches and OFT at embryonic day (E) 11.5 (mouse). GATA6 peaks predominantly cluster around genes associated with cardiovascular terms, and specifically with outflow tract and artery development, as well as semilunar valve development (red arrows). G. Selected regulon genes associated with GATA6 binding in mouse embryo OFT and pharyngeal arches (see also Table S3) 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. H. 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. I. Spatial Transcriptomics of Aorta (Ao) and Pulmonary Artery (PA) (clockwise): Hematoxylin and eosin (H&E) staining of the tissue area, with asterisks marking the semilunar valves; spatial distribution of LTBP1, NOTCH2, MECOM. J. 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 sub-clustering of aggregated fetal and embryonic nuclei shown in Fig S2A. K. Expression signatures in embryonic mesenchymal (4, 20) and endothelial-like (7) clusters. Both cluster 7 and 4 express high levels of cardiac TFs (GATA4, TBX20) and HAPLN1, a marker of semilunar valves. In contrast cluster 20 nuclei exhibit higher expression of neural crest markers, HOXA3-B3 and PLXNA2.

Spatial distribution of mesenchymal clusters

A-F. Outflow tract valve formation and remodelling. 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 outflow tract has septated into the aorta (Ao) and pulmonary trunk (PT). The immature outflow tract 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. EF. At 11pcw 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). 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). J-JI. Cluster 9 (J) and smooth muscle lineage specific marker MYH11 (JI) map to the aortic walls as well as to the pulmonary artery. K-KI Cluster 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 Fig S3.

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 in two groups. C. Mesenchymal cell clusters of embryonic and fetal time points, projected on a two-dimensional tSNE and labelled 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 descendent of embryonic cluster 4, while cluster 6 and 9 are the most likely descendant of embryonic cluster 20, consistent with the use of gene modules in the mesenchymal subset of fetal nuclei in 3D.

Cellular constituents of the mature aortic valves.

A. Aortic valve sample association projected on a two-dimensional 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 descendent 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 descendent 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 cluster 4 and cluster 20, respectively. The same dot plot, previously shown in Fig 3C, has been included here to facilitate cross-comparison with Fig 6GH. G. Distribution of cluster 4 embryonic signature genes in adult nuclei clusters. Cluster 4, 7 and 5 express a highly significant fraction of embryonic cluster 4 genes. Top 30 DE genes in embryonic cluster 4 and cluster 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 cluster 4 and cluster 20 (shown in F) are highlighted by dots.

Genes mutated in congenital OFT defects.

A. H&E staining of spatial transcriptomics section (Fig 2Jc), and magnified view of aortic and pulmonary valve area (AI). The aorta and pulmonary trunk are indicated by blue and green arrowheads, respectively. B-D. 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 (BI, CI, DI). E. Genes identified as displaying spatially similar expression pattern to JAG1.