DEGs in the combined CS16/19 cushion/valve cluster.

Genes are ordered from highest (most differentially expressed) to lowest. (XLSX)</p

S7 Fig -

IPA analysis on the unfiltered CS16/CS19 combined dataset showing all statistically significant A) canonical pathways, B) upstream transcriptional regulators and c) diseases and biological processes. A) canonical pathways, B) upstream transcriptional regulators and c) diseases and biological processes identified using Ingenuity Pathway Analysis (IPA) to scrutinise the CS16/19 combined dataset. No filtering was applied. This complements the data provided in Fig 6. (TIF)</p

Comparisons between the valve cluster and published datasets.

Sheet 1: comparison of top 250 DEGs from Queen data (this manuscript) to DeLaughter et al (2013). Shared genes are in red. Sheet 2: list of top 250 DEGs from Queen data (this manuscript) and Asp et al (2019) clusters 5 and 6. Sheet 3: comparison of top 30 DEGs from Queen data (this manuscript) to Asp et al (2019) clusters 5 and 6. Shared genes are in green. Sheet 4. comparison of top 250 DEGs from Queen data (this manuscript) to Asp et al (2019) clusters 5 and 6. Shared genes are in green. (XLSX)</p

Functional associations of valve cluster genes generated by filtered IPA analysis.

A) Top 20 regulators of genes in the valve cluster. B) Circos plot linking valve regulators to cellular processes. The strongest associations relate to cell movement and growth/proliferation but cardiovascular disease is also a string association for this group of valve regulatory genes. C) Following filtering for the terms “cardiac” and “development” regulation of EMT was the only pathway significantly upregulated in the valve dataset compared to the myocardial and blood clusters. Notch and Wnt were the likely upstream regulators. Pathways highlight the genes in the Notch and Wnt pathways that are positively associated regulators of EMT for the valve cluster (dark green genes are the most differentially upregulated, with mid green and light green progressively less so). D,E) IPA pathway analysis shows that for disease and biological processes, developmental terms such as development of trunk, genitourinary system, vasculature and neurons were strongly associated with the valve cluster. Negative associations were also found, the most striking being those to familial heart/cardiovascular disease, and congenital heart/cardiovascular disease/anomaly. These terms were positively associated with the myocardial cluster dataset (red box).</p

RBP1 plays a crucial role in the developing arterial valves.

A) RBP1 and CRABP2 expression in the human and mouse heart. White boxes denote the area covered by the high-power images (b,d,f,h). RBP1 protein is expressed (red) in the cells underlying the endocardium (arrowheads) of the developing aortic and pulmonary valves in human CS16 hearts, as well as at lower levels in the vessel walls. In contrast, it is expressed in the endocardium (arrowheads) itself in the mouse heart and is not found in the vessel wall. CRABP2 expression (red) localises to the wall surrounding the valve leaflets in both species at the same timepoints but is not found in the endocardium (arrowheads). B) RBP1 expression in a CS19 human embryo using RNAScope shows expression in the subendocardial region of the aortic valve leaflet (white arrowheads point to the endocardium). Expression can also be seen in the mesenchyme of the proximal outflow cushions and in the arterial wall. Boxed area shows the valve leaflets at higher magnification. C) Rbp1 protein is specifically localised to the endocardium of the developing valve leaflets and cardiac chambers at E13.5 in the mouse heart. It is not found in the interstitium of the developing valve leaflets (*). D) Table showing cardiac malformations found in Rbp1 knockout animals. E) Abnormalities of the aortic valve including BAV (in the E15.5 mutant; e) and valve dysplasia (arrow in P1 mutant; g) were seen in late fetal and neonatal RBP1 mutants. Rbp1 mutant neonates also have abnormalities on the ventricular myocardium including muscular ventricular septal defects (arrows in f,h). Stage-matched and oriented control aortic valves are shown for comparison. F) 3D reconstructions of the aortic valve of wild type and Rbp1 null mutants at E15.5 (a-d) and P1 (e,f). WT and Rbp1-/- mutants are matched for orientation. Red = non-coronary leaflet, yellow = left leaflet, green = right leaflet. Orange is a fused non-coronary and left leaflet. a-d) Three leaflets were seen in the aortic valve of WT at E15.5 (a,c) compared to two leaflets observed in a Rbp1-/- observed from above (a,b) and from the right side (c,d). e,f) three leaflets are seen in both the WT and Rbp1-/- at P1, although abnormalities in the shape and position of the leaflets means that the leaflets are not all the same level (arrows in e,f). Ao = aortic valve; L = left leaflet; L/N = left-non-coronary fused leaflet; LV = left ventricle; N = non-coronary leaflet; poftc = proximal outflow tract cushions; Pu = pulmonary valve; R = right leaflet, RV = right ventricle. Scale bar in A = 100μm in a,c,e,g, 35 μm in b,d,f,h; 150μm in B; in C = 75μm in a, 40μm in b, 50μm in c, 20μm in d; in E = 50μm in a,c,e,g, 150μm in b,f, 300μm in d,h.</p

Cluster analysis.

A) UMAP projection showing overlap of data between sections from the CS16 (orange dots) and CS19 (turquoise dots) embryos. B) UMAP projection showing clustering of combined CS16 and CS19 data, again showing three clusters (red, blue and orange dots). C) Heat map showing top 10 genes in each cluster, highlighting the gene expression differences between the three clusters. At this stage, based on the expressed genes, putative tissue types can be proposed as myocardium (orange), cushion (blue) and blood (red). D) Perfect marker analysis using recognised markers for specific cardiac cell types confirms the orange cluster as myocardium, the red cluster as blood cells, and shows that the putative cushion cluster has characteristics of cushions, bone and fibrous tissue, and thus should be better named as “valve” as this implies the entire structure, not just the developing leaflets. E) Mapping “perfect markers” back to the H&E sections shows that they localise to the expected areas (compare to Fig 1D). The H&E section shows the identity of the tissues and structures contained within the section. oft = outflow tract, oftw = outflow tract wall, rbc = red blood cells (in lumen), v = ventricular myocardium, vlp = valve leaflet primordia.</p

Mapping back of perfect markers to CS19 human valve sections.

Perfect marker genes (see Fig 2) for a range of cardiac progenitor cell types (neural crest cells), differentiated cardiac cell types (cardiomyocytes, cushion tissue, fibrous tissue, endocardium, smooth muscle cells), haematopoietic cell types that are known to be found in the heart (red blood cells, lymphocytes, macrophages) and those that are known to be similar to cushion tissue (bone, cartilage), were mapped back to the CS19 human valve sections used for the ST analysis. (TIF)</p

DEGs in the combined CS16/19 myocardial cluster.

Genes are ordered from highest (most differentially expressed) to lowest. (XLSX)</p

Comparison of “valve” genes to published ST/single cell RNASeq datasets.

A) Comparison of the top 20 genes in our valve cluster with the human embryonic “atrioventricular mesenchyme and valve” ST cluster from Asp et al (2019) reveals 5 shared genes. Similarly, comparison of our top 30 valve cluster genes with scRNASeq “VIC” cluster data from P7 and P30 mouse atrioventricular and arterial valves (Hulin et al, 2019) reveals 7 genes in common. Notably, LGALS1, which has not previously been reported to be specific to the arterial valves, is found in all 3 datasets. B) Immunohistochemistry for LGALS1 in human and mouse embryonic hearts. White boxes denote the area covered by the high-power images (b,d). LGALS1 is expressed at high level in the developing aorto-pulmonary septum and at lower level in the mesenchyme of the distal and proximal outflow tract cushions of the human CS16 heart. In the E11.5 mouse heart, Lgals1 is found at high level in the aortopulmonary septum and throughout the distal and proximal cushions. LGALS1/Lgals1 is found at only low level in the endocardium in both species (arrowheads). C) Circos plot showing the top 30 genes in our valve cluster mapped to GO terms. D) STX10, HES4 and MRXA5, none of which are found in the mouse genome, are expressed (red dots) in the developing outflow tract at CS16. White boxes denote the area covered by the high-power images (d,e,f). MXRA5 is expressed at high level in the aortopulmonary septum (APS) and the proximal cushions, whereas HES4 is restricted to the APS and STX10 is found at lower level in both tissues. STX10 and HES4 are strongly expressed in the forming walls of the arterial roots, whereas MXRA5 is found only at low level in this tissue. Whereas all three genes are found in the walls of the forming arterial roots, only STX10 and HES4 are also found in the endocardium in this region. Ao = aortic valve; APS = aortopulmonary septum; cm = cushion mesenchyme; doftc = distal outflow tract cushions; end = endocardium; oftw = outflow tract wall; poftc = proximal outflow tract cushions; Pu = pulmonary valve. Scale bar in B = 100μm in a,c, 20μm in b,d. Scale bar in D = 300μm in a-c, 40μm in d-f.</p

Identification of cluster genes.

A) Top 30 DEGs in each of the three clusters. In each case, the coloured genes are already documented to be expressed in the proposed cluster tissue, whereas genes in black font were not identified as being expressed in the relevant tissue by literature search. B) Expression of novel Cluster 1 (valve) genes in the developing E14.5 mouse heart (GenePaint images) Higher power images of the heart are included for each gene in the bottom left corner of each image. C) PDLIM3, ID4 and LINC00632 transcripts (red dots in all cases) are all found in the CS16/CS19 human arterial valves. White box denotes the area covered by the high-power image for each gene (b,d,f). PDLIM3 and ID4 are expressed at CS16 in the cushion mesenchyme, although ID4 is more restricted to the distal region close to where the valves will form. Neither are expressed at high level in the endocardium of the forming valve region (arrows). LINC00632 is also expressed in the valve leaflets and supporting mesenchyme at CS19 and is not expressed in the developing valve endocardial cells (arrows). D) Venn diagram showing the distribution of valve genes that are specific to CS16, expressed at both CS16 and CS19, or specific to CS19. The numbers reflect the number of genes in each category. E) Top 10 valve genes that are specific to CS16, expressed at both CS16 and CS19, or specific to CS19. Blue colour denotes genes that are already known to be expressed in the developing or mature valve. All of the genes in the CS16/19 overlap are also found in the top 30 most highly DEGs when the two datasets are integrated and analysed together (compare with (A). F) IPA shows that most of the genes fall into similar pathways for disease and biological processes. There are more differences for canonical signalling pathways, although the top two are related to fibrosis and are shared. The dot in some boxes denotes the result was not statistically significant. Ao = aortic valve; APS = aortopulmonary septum; EC = endocardium; poftc = proximal outflow tract cushions; Pu = pulmonary valve; VEC = valve endocardial cells; VIC = valve interstitial cells. Scale bar in C = 300μm in a,c, 40μm in b,d, 200μm in e, 66μm in f.</p