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 at 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.