Normal and abnormal development of the intrapericardial arterial trunks in humans and mice

Abstract

Aims: The definitive cardiac outflow channels have three components: the intrapericardial arterial trunks; the arterial roots with valves; and the ventricular outflow tracts (OFTs). We studied the normal and abnormal development of the most distal of these, the arterial trunks, comparing findings in mice and humans.

Methods and results: Using lineage tracing and three-dimensional visualization by episcopic reconstruction and scanning electron microscopy, we studied embryonic day 9.5-12.5 mouse hearts, clarifying the development of the OFTs distal to the primordia of the arterial valves. We characterize a transient aortopulmonary (AP) foramen, located between the leading edge of a protrusion from the dorsal wall of the aortic sac and the distal margins of the two outflow cushions. The foramen is closed by fusion of the protrusion, with its cap of neural crest cells (NCCs), with the NCC-filled cushions; the resulting structure then functioning transiently as an AP septum. Only subsequent to this closure is it possible to recognize, more proximally, the previously described AP septal complex. The adjacent walls of the intrapericardial trunks are derived from the protrusion and distal parts of the outflow cushions, whereas the lateral walls are formed from intrapericardial extensions of the pharyngeal mesenchyme derived from the second heart field.

Conclusions: We provide, for the first time, objective evidence of the mechanisms of closure of an AP foramen that exists distally between the lumens of the developing intrapericardial arterial trunks. Our findings provide insights into the formation of AP windows and the variants of common arterial trunk.

Figure 1. Changes occurring in the composition of the developing mouse (A,C-L) and human (B) OFT, as revealed by Amira reconstructions.

Extrapericardial aortic arch arteries are labeled in red, myocardium in silver, and the non-myocardial intrapericardial mural components in green. The images on the right are from Wnt1-cre;R26R embryos, with NCC stained blue. In C,F,I,L the myocardium is labeled orange/brown by the MF20 antibody. The dashed lines in C,F,I show the level of the pericardial reflections, with the non-myocardial lateral walls of the distal outflow between the dashed lines and arrows. Supplementary Movie 2 shows animated sequences illustrating the arrangement of the aortic arch arteries comparable to the stages shown. A,C=28somites, B=CS12, D-F=35 somites, H-I=41 somites, J-L=52 somites. Scale bar in: C=410μm, F=500μm, I=620μm, L=630μm. ao=aorta; as=aortic sac; paa4=fourth pharyngeal arch artery; pt=pulmonary trunk.

Figure 2

A-F) Episcopic images showing the reduction is size and eventual closure of the AP foramen in E11.5 mouse hearts. All are shown from the right side, having removed the parietal wall of the cardiac structures. The insets show the foramen in detail. The arrow in panel A marks the protrusion of the dorsal wall of the aortic sac. G-I) Show the remodeling of the junction of the OFT with the aortic sac, comparing early (G), middle (H) and late (I) stages of E11.5. The developing aortic channel is shown in red, along with the third and fourth arch arteries, while the pulmonary channel and sixth arch arteries are shown in deep blue, with the pulmonary arteries arrowed. The circle in H shows the closing aortopulmonary foramen.

Figure 3. Episcopic images showing the rotation of the boundary of the distal OFT with the pharyngeal mesenchyme.

A) Shows the aortic sac with cranial and caudal components at early E11.5, which give rise symmetrically to the fourth and sixth arch arteries, respectively. The asterisk shows the intermediate part of the sac, with the dorsal wall separating the systemic and pulmonary parts of the sac. B) Is from a CS14 human embryo showing a comparable arrangement. C,D) Show views of a mid E11.5 OFT cut transversely through the closing AP foramen (arrow in D), close to the aortic sac, as shown in the inset in C. The oblique orientation of the protruding dorsal wall of the aortic sac is shown as the dashed line in D. E) Is an over view of an E11.5 heart, showing the orientation of section of A and F. F) Is a view made by removing the parietal wall of the aorta, as in Fig 2F, just prior to closure of the AP foramen. The dotted line shows the border between the intra- and extrapericardial aortic components. The upper double headed arrow shows the walls distal to the foramen, derived from the protrusion, while the lower double headed arrow shows the adjacent wall of the aorta proximal to the foramen, derived from the fused distal cushions.

Figure 4. Scanning electron micrographs of E10.5 and 11.5 mouse embryos.

A) (E10.5) and B-D) (E11.5) show the junction of the OFT and the mediastinal wall, viewed inferiorly, with the transected foregut and bilateral cardinal veins visible beneath. Note the progressive asymmetric bulging of the left and right 6th aortic arches (labelled 6 in B,C,D,E) into the pericardial cavity. This, together with excavation at the pericardial border, gives the impression that the OFT is being twisted anticlockwise in this ventral view. In D), the OFT has been transected in the plane indicated by the white line in C. This shows that the lumen at the site of the cut is made up of a large right-sided cranial portion that will be the intrapericadial aorta (ipa), and a small left-sided caudal portion that will be the pulmonary trunk (ipp). The double-headed white arrow shows the oblique orientation of the AP foramen and the protrusion. E) shows an OFT that has been sectioned longitudionally (transverse to the embryo, at the level shown by the white line in D) and is viewed superiorly. Note the narrowness of the junction (arrows) between OFT and body. The septal cushion (SC) is visible in the inferior portion of the OFT. F) shows a late E11.5 junction of the OFT and the mediastinal wall, viewed superiorly, showing the pronounced remolding between the ipa and ipp. aa – aortic arch.

Figure 5. The panels show the lineage of the components involved in separating the initially common lumen of the distal OFT.

A-D) are mid-E11.5, while E,F) are late E11.5. A) shows NCC stained blue from a Wnt1-cre;R26R embryo. The upper arrow shows the cells in the ventral protrusion, while the lower arrows show the NCC in the cushions. B,C) show a section stained for Isl1 antibody, and a comparable section from an Isl1cre;R26EYFP embryo. The brown tissues show the Isl1-positive non-myocardial parietal tongues (small arrows) and the ventral protrusion (long arrow). The surface of the protrusion is negative for Isl1, but as shown in panel A, is positive for NCC. D) shows an episcopic dataset reconstructed from an Isl1-lacZ reporter embryo, illustrating the same features, with the surface of the protrusion shown by the red arrow. E,F have been stained using an aSMA antibody. In E, the fusion site of protrusion and cushions has arterialized, forming the site of the so-called whorl (arrow). At a slightly later stage, as seen in F, the surface of the protrusion remains aSMA positive, but the core of the protrusion is negative, being replaced by connective tissue between the developing adjacent walls of the intrapericardial arterial trunks (arrow). Scale bar represents: A=950μm, B,C=1040μm, E,F=815 μm

Figure 6. The panels compare the situation in mouse mutants (A-F) with human hearts showing AP window (apw – G) and common arterial trunk (cat - H,I).

Lp/Lp are shown in A-D, Sp^2H/Sp^2H in E,F. A) shows formation of the ventral protrusion in an E11.5 Lp/Lp (asterisk), but failure of fusion of the protrusion with the cushions. B,C) show an AP window, with separate formation of aortic (ao) and pulmonary (pt) valvar leaflets (arrowheads). The arrow shows the AP window. D) shows common arterial trunk, but with obvious separation of the intrapericardial aortic and pulmonary components. In E,F), the Sp^2H/Sp^2H have common arterial trunk, with absence of any protrusion of the dorsal wall of the aortic sac in E. The arrows in F show the forming valves. G) shows an AP window in a human seen through the aorta (ao). Note the separate channels of the aorta and pulmonary trunk (pt) proximal to the window. In the common arterial trunk shown in H (arrow) there is no separation of the intrapericardial trunk, which is essentially aortic (aoc), the pulmonary arteries (pa) arising from the leftward and dorsal part of the trunk. The heart in I), in contrast, has separate formation of the intrapericardial aortic and pulmonary components (aoc, pc), with the pulmonary component continuing to supply the descending aorta through a persistently patent arterial duct. Scale bar represents: A,E=770μm, B,C =455μm, D=500 μm, F=400μm.