Development of the arterial roots and ventricular outflow tracts.

The separation of the outflow tract of the developing heart into the systemic and pulmonary arterial channels remains controversial and poorly understood. The definitive outflow tracts have three components. The developing outflow tract, in contrast, has usually been described in two parts. When the tract has exclusively myocardial walls, such bipartite description is justified, with an obvious dogleg bend separating proximal and distal components. With the addition of non-myocardial walls distally, it becomes possible to recognise three parts. The middle part, which initially still has myocardial walls, contains within its lumen a pair of intercalated valvar swellings. The swellings interdigitate with the distal ends of major outflow cushions, formed by the remodelling of cardiac jelly, to form the primordiums of the arterial roots. The proximal parts of the major cushions, occupying the proximal part of the outflow tract, which also has myocardial walls, themselves fuse and muscularise. The myocardial shelf thus formed remodels to become the free-standing subpulmonary infundibulum. Details of all these processes are currently lacking. In this account, we describe the anatomical changes seen during the overall remodelling. Our interpretations are based on the interrogation of serially sectioned histological and high-resolution episcopic microscopy datasets prepared from developing human and mouse embryos, with some of the datasets processed and reconstructed to reveal the specific nature of the tissues contributing to the separation of the outflow channels. Our findings confirm that the tripartite postnatal arrangement can be correlated with the changes occurring during development

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

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

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

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

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

PANTHER designation of Top250 genes in valve cluster based on A) protein class and B) biological process.

Pie charts showing A) the most common gene/protein classes and B) associated biological processes for the genes in the combined CS16/CS19 valve dataset determined using PANTHER. Gene Ontology (GO) codes are in brackets. (TIF)</p

Top 100 DEGs in the valve cluster linked to cardiovascular phenotypes in human patients and in mouse mutants.

Dark yellow suggests an arterial valve phenotype, lighter yellow reflects a cardiovascular phenotype. (XLSX)</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

3D reconstructions of WT and <i>Rbp1-/-</i> aortic valves in position within the aortic root.

3D reconstructions of the aortic valve of wild type and Rbp1 null mutants at E15.5 and P1 placed within the aortic root (grey). 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. At E15.5, three leaflets were seen in the aortic valve of WT at E15.5. In comparison, two leaflets observed in a Rbp1-/- mutant (Mut 1) observed from the right side. In the other mutant (Mut 2) shown, three leaflets were observed although two were fused along the majority of their length. In this latter case, it is clear that the proximal extent of the leaflets is not the same (arrows). Three leaflets are seen in both the WT and Rbp1-/- at P1, although abnormalities in the shape and position of the leaflets are apparent. (TIF)</p