Figure 3. Gata5-Cre-induced deletion of Alk2 function leads to defective development of aortic valves.

A–F: Histological comparison of representative control (A, C, E) and mutant (B, D, F) aortic valves at E14; approximately transverse sections of the same valves at three levels. A, B: lower magnification at the level of coronary orifices (red arrow; coronary vessel: red arrowhead), where formed, distal to aortic valve leaflets showing normal development and relationship of aortic-side structures relative to pulmonary valve. All three control aortic leaflet cushions show evidence of excavation forming sinuses (green *): left (L) in panel C, right (R) and non-coronary (N) in panel E. N cushion occupies a similar segment of the circumference (black lines) as each of R and L (in E). In mutant valve, L (in D) and R (in F) leaflet cushions show normal excavation. Cushion ‘R’, although in the same position as N in control, is continuous with tissue R (panel D), not simply apposed as in control (in C). Nearer the base of the leaflet cushions (in E, F), mutant R is larger than control R. A small cushion (‘N’) is present between L and ‘R’ (in D, F), but shows no evidence of excavation and occupies a much smaller segment of the circumference (black lines in F) than N in control, so the valve is functionally bicuspid (bifoliate), with two excavating leaflet cushions, L and R/‘R’, apposing one another distally (in D). G–R: Histological comparison of representative control (A, J, M, P) and two functionally bicuspid mutant (H, K, N, Q and I, L, O, R) aortic valves at E17; approximately transverse sections of the same valves at four levels. G-I, at the level of coronary orifices showing their consistent position above right and left coronary leaflets, and consistent positioning of the leaflets in relation to adjacent tissues, in control and mutants. All three control aortic leaflet walls lie adjacent to well-developed sinuses (green *) left (L) in panel J, right (R) and non-coronary (N) in panel M. N leaflet occupies a similar segment of the circumference (black lines) as R and L (in J). Two examples of functionally bicuspid mutant valves are shown to illustrate key details of their morphology at this stage. The example in H, K, N, Q has features consistent with a fusion of R and N leaflets. Distally (K), only two leaflets, R and L, appose each other, with no small leaflet between them in the non-coronary position, each occupying about half the circumference (black lines). More proximally, the sinus of the R leaflet is divided into two (green *) by a raphe (black arrowhead) in N, but the leaflet does not then divide into two separate bases (arrowhead, Q). Distally the mutant valve shown in I, L, O, R also consists of two leaflets (in panel L; as R and ‘R’ are joined more proximally) that are not separated by a small cushion, but more proximally, one (‘N’) present. An area representing an interleaflet triangle (red curved line) can be identified between L and R leaflets in control (in M) and mutant (K, L) valves, and between L and N (control, in M) but the bases of the R and N leaflets even in control remain adjacent (in P, red X, black arrow) though not continuous (unlike Q). S,T: 6 month old adult control aortic valve with normal left (L) right (R) and non-coronary (N) leaflets (black arrows) and three sinuses (green*), and partially dissected functionally bicuspid aortic valve in Alk2/Gata5-Cre mutant, demonstrating two leaflets (black arrows) which appose only with each other across the entire lumen, and two sinuses (green *). Aor Tr, Aortic trunk; Pul Val, Pulmonary Valve.