Outflow tract septation and the aortic arch system in reptiles: lessons for understanding the mammalian heart

Abstract

Background: Cardiac outflow tract patterning and cell contribution are studied using an evo-devo approach to reveal insight into the development of aorto-pulmonary septation.

Results: We studied embryonic stages of reptile hearts (lizard, turtle and crocodile) and compared these to avian and mammalian development. Immunohistochemistry allowed us to indicate where the essential cell components in the outflow tract and aortic sac were deployed, more specifically endocardial, neural crest and second heart field cells. The neural crest-derived aorto-pulmonary septum separates the pulmonary trunk from both aortae in reptiles, presenting with a left visceral and a right systemic aorta arising from the unseptated ventricle. Second heart field-derived cells function as flow dividers between both aortae and between the two pulmonary arteries. In birds, the left visceral aorta disappears early in development, while the right systemic aorta persists. This leads to a fusion of the aorto-pulmonary septum and the aortic flow divider (second heart field population) forming an avian aorto-pulmonary septal complex. In mammals, there is also a second heart field-derived aortic flow divider, albeit at a more distal site, while the aorto-pulmonary septum separates the aortic trunk from the pulmonary trunk. As in birds there is fusion with second heart field-derived cells albeit from the pulmonary flow divider as the right 6th pharyngeal arch artery disappears, resulting in a mammalian aorto-pulmonary septal complex. In crocodiles, birds and mammals, the main septal and parietal endocardial cushions receive neural crest cells that are functional in fusion and myocardialization of the outflow tract septum. Longer-lasting septation in crocodiles demonstrates a heterochrony in development. In other reptiles with no indication of incursion of neural crest cells, there is either no myocardialized outflow tract septum (lizard) or it is vestigial (turtle). Crocodiles are unique in bearing a central shunt, the foramen of Panizza, between the roots of both aortae. Finally, the soft-shell turtle investigated here exhibits a spongy histology of the developing carotid arteries supposedly related to regulation of blood flow during pharyngeal excretion in this species.

Conclusions: This is the first time that is shown that an interplay of second heart field-derived flow dividers with a neural crest-derived cell population is a variable but common, denominator across all species studied for vascular patterning and outflow tract septation. The observed differences in normal development of reptiles may have impact on the understanding of development of human congenital outflow tract malformations.

Keywords: Aorto-pulmonary septation; Bird; Cardiac development; Crocodile; Flow divider; Neural crest; Outflow tract cushions; Reptiles; Second heart field.

Fig. 1

Early development of the pharyngeal arches in chicken. a HH16 TFAP2α (brown staining) present in NCC, the negative core of pharyngeal arches is indicated (arrows). b, c HH22, Isl1 positive core of the pharyngeal arch (bold arrow); the NCC area of the arch and the arterial wall are negative (thin arrows). d–f HH27 from distal (d) to proximal (f), WT1 in the coelomic lining (arrowhead) and the mesenchyme are positive (arrowheads). Note NCC-derived arterial walls and condensed mesenchyme (* in f) are WT1 negative. Numbers 3, 4 and 6 indicate the various PAAs

Fig. 2

Development of the AP septum in chicken HH26–28. a The AP septum between Pu (6 + 6) and Ao channels (3 + 3 + 4 + 4). WT1 staining (brown) in the epicardium and mesenchymal second heart field cells. b slightly more distal, the Pu is separated into the left and right PAA6 and the Ao channel in the right PAA4 and separate PAA3. Note that the lumen of the left PAA4 has disappeared. c The area of apoptosis is continuous with the massive apoptosis in the AP septum (arrows and inset, see also Fig. 4e). d In HH28, the lumen of the left PAA4 (4L) is nearly occluded and the vascular smooth muscle cells (negative in this WT1 staining) are still visible. Note the elaborate WT1 staining (brown) between the vascular segments. e Amira reconstruction of HH28 seen from ventral showing the lumina of the arterial trunks. la left atrium, ra right atrium, myo ventr myocardium ventricle f Same embryo, the fused OFT cushions (blue) contain a groove for the condensed mesenchyme (*) running between PAA6 (6) and the stem of PAA4R (4R) and PAA3 (3). Note that the lumen of PAA4L (4L) is interrupted and does not reach the heart anymore. OFT cu fused outflow tract cushions, ventr lumen ventricle

Fig. 3

The OFT cushions in chicken HH28. The endocardial outflow tract (OFT) cushions from proximal to distal in adjacent sister sections. The septal (sc) and parietal cushion (pc) are indicated. a Both cushions appose each other in the proximal OFT. b, c They come close to each other, the Ao and Pu channels are indicated. d The sc and pc have fused showing here the NCC-derived condensed mesenchyme (c*). e The Ao channel is divided into the left visceral (vAo) and right systemic aortic (sAo) channels. f The pc shows 2 segments on both sides of the condensed mesenchyme, WT1-positive cells dorsally to the AP septum (arrow). g, h Chicken HH31. The left PAA4 has completely regressed from the OFT. Only both PAA3, PAA4R and PAA6 are present. Pu indicates the stem of both PAA6, of which PAA6R is present in g and PAA6L in h. WT1 staining reveals the mesenchyme cells surrounding the negative tunica media of the arteries. Thin arrows indicate the WT1-positive mesenchyme between the arteries. The different positions of the left and right PAA6 leave an asymmetric pericardial recesses dorsal to the arterial pole (bold arrow). Ao aortic trunk, AP aorto-pulmonary septum, AV atrioventricular cushions, FS folding septum, ic intercalated cushion, MB main bronchi, Oes oesophagus, pc parietal cushion, Pu pulmonary trunk, sAo right systemic aorta, sc septal cushion, T trachea, vAo left visceral aorta

Fig. 4

OFT development in the lizard Pogona HH22–29.5. a In the proximal OFT a thin cellularized septal cushion, flanking the folding septum and the equally indistinct parietal cushion are present. Bar = 100 µm. b The centrally located septal (sc) cushion is flanked by the parietal (pc) and intercalated (ic) cushions. Note that the condensed mesenchyme in the septal cushion is inconspicuous. c In HH28, the septal and parietal cushion have fused (*), separating the aortic and pulmonary channels. d At HH29.5, the aortic channel is not divided, yet, but a left visceral aorta (vAo) and a right systemic aorta (sAo) can be discerned. Bar = 50 µm. e Higher magnification shows some apoptotic cells in the septal cushion. f The aorto-pulmonary septum (*) separates the two aortic from the pulmonary channels, while a second separation (#) is present between both aortae. No condensed mesenchyme is visible in the AP septum. Abbreviations as in Fig. 3

Fig. 5

OFT development in the turtle Pelodiscus HH22–27. a TFAP2α+ cells (brown) in the pharyngeal mesoderm and the endocardiac jelly (arrow). Positive cells are only found over a short distance (<50 μm) into the heart. Bar = 100 µm. b In the same embryo, the periphery of the pharyngeal arch arteries also shows positive brown-stained cells (arrows). c HH27 TFAP2α in the wall of the truncus caroticus (PAA3, long arrow). There is no TFAP2a positivity in the OFT cushions beyond the border with PAA3 (arrowheads). TFAP2α-positive cells in the basis of the individual PAA3, but not in the sAo, the vAo or the PAA6. d, e Amira reconstruction of the ventricular lumen (red), OFT cushions (blue), NCC condensed mesenchyme (green*) and PAAs, indicated by colour and number (3, 4, 6). e Seen from cranial. OFT cu outflow tract cushions, ventr ventricular lumen, D dorsal, V ventral

Fig. 6

Development of the vascular segments in the turtle Pelodiscus. OFT cushions (from proximal to distal) and NCC contribution, movat and TFAP2α staining. a In the proximal OFT only the septal cushion is prominent between the cellularized cardiac jelly. b The septal cushion containing condensed mesenchyme becomes flanked by the other cushions. Here, a cell-rich whorl is present, pointed to the parietal cushion. c Magnification of an adjacent section, negatively stained for TFAP2α. d Another condensation (rich in SHF, #) separates both aortae. The myocardial wall on the dorsal side is retreating. e The three arterial trunks are separated. Note that a spur of myocardium (arrow) protrudes into the NCC area (*). f The carotid trunk (3 + 3) branches off the systemic aorta. The myocardial sleeve is not longer present. g Positive TFAP2α in the inner wall of the carotid trunk (3 + 3). h The inner media of the carotid trunk is rich in glycosaminoglycans compared to the adjacent arteries, giving it a spongy appearance. Abbreviations as in Fig. 3. i Cross section of the neck region of an embryo of Emys orbicularis showing the carotid trunk (3 + 3). The arterial wall is not spongy as in Pelodiscus (compare h). The Emys embryo belongs to material described earlier by our group [50]

Fig. 7

OFT development in Crocodylus. a In the proximal OFT the septal cushion flanking the folding septum (FS, yellow curve) is obvious. b–d Three consecutive sections in the distal OFT in which the OFT cushions are visible. The condensed mesenchyme of the aorto-pulmonary septum is indicated (*). In c at the arterial level the AP separates the pulmonary trunk from the aortic channels. The NCC (*) and SHF aorto-aortic flow divider (#) are indicated. At the ventral side, the myocardium protrudes inward (orange curve). d The arterial trunks are completely separated by the AP septum and the aortic flow divider (#). e TFAP2α-positive cells (NCC) in the wall of PAA3 (arrows), but not in the other PAAs (not shown). f 3D reconstruction of the fused OFT cushions and the PAAs (indicated by their number) including the AP septum (green*) between PAA3 + 4 and PAA6. Abbreviations as in Fig. 3

Fig. 8

Outflow tract septation in Crocodylus. Subsequent levels from the proximal (a–e) to the distal OFT. a The septal and parietal cushion are at the same level as the AV cushions (AV). b The OFT is individualized and the parietal and septal cushion form a semicircle. c In the distal OFT the sc presents with NCC condensed mesenchyme (*) adjacent to the first vacuoles of the foramen of Panizza (arrows). Here the sc is apposed to the intercalated cushion. d At this level fusion (#) has occurred between the sc, flanking the folding septum (FS) and the ic separating the systemic from the visceral aorta. e Separation of Pu and Ao is completed, here the ventral myocardial protrusion (Myo) meets the NCC-derived condensed mesenchyme and the two aortae are separated by the flow divider (#). Bar = 100 µm. f Slightly older embryo shows the elaborate vacuolar spaces (arrows) proximal to the SHF separation (#) of both aortae, there is not yet a connection with the sinus of Valsalva (sV). g Cartoon depicts the division of the 4 distal OFT cushions over the 3 main arterial trunks. Note that only the sAO has two cushion derivatives, while the vAo and the Pu have two large cushions plus one small cushion derivative each. The condensed mesenchyme (*) and aortic flow divider (#) are indicated. For clarity all endocardial cushions are shown in one level. Abbreviations as in Fig. 3

Fig. 9

Outflow tract septation in Crocodylus HH40. Consecutive sections from proximal to distal (a–h) stained as indicated. a The interventricular septum is indicated but left and right ventricles are not completely separated, yet. Bar = 200 µm. b, c Sc and pc in the proximal OFT, showing signs of chondrification (arrows). c Foramen of Panizza (arrowhead) is present in sc, flanking the folding septum and adjacent to the NCC condensed mesenchyme (*). d Fusion of sc and the ic results in separation of both aortae by the aortic flow divider (#). The facing sinus of Valsalva (red curve) are connected with each other through the foramen of Panizza in c. Bar = 100 µm. d, e The folding septum and the condensed mesenchyme extend into the lumen between Pu and vAo. Note that some NCC condensed mesenchyme still separates the protrusion of the ventral myocardium (Myo) from the dorsal folding septum [19]. Two adjacent coronary ostia are indicated (ca). f CTNI staining demonstrates that the central mass (brown) of d–e is indeed myocardium, compare with Fig. 8e. g The central mass of myocardium (Myo) is continuous with the ventral myocardium. The connection of both aortic valve leaflets is to the left-sided free wall (#). Two further coronary ostia (ca) are indicated. h The pulmonary semilunar valve leaflets are located more distal than the aortic leaflets. ca coronary ostium, ic intercalated cushion, FS folding septum, IVS interventricular septum, LV left ventricle, Myo myocardium, pc parietal cushion, Pu pulmonary trunk, RV right ventricle, sAo right systemic aorta, sc septal cushion, vAo left visceral aorta, * NCC condensed mesenchyme, # interaortic flow divider, arrowhead foramen of Panizza, red curved line: facing sinus of Valsalva in sc, connected by FOP

Fig. 10

Cartoon depicting the results of aorto-pulmonary septation and PAA remodelling in crocodilians, birds and mammals. a In reptiles, represented here by the crocodile, the visceral (left PAA4) and systemic aorta (right PAA4) are separated by the aorto-aortic flow divider (light yellow), while the pulmonary trunk (PAA6) is separated from both aortae by the AP septum (green, *). The pulmonary flow divider (dark yellow) is not involved in aorto-pulmonary separation. b In birds, the left PAA4 disappears and the aortic flow divider merges with the AP septum. c In mammals, both PAA4 persist, but the right PAA6 disappears and the pulmonary flow divider merges with the AP septum. In birds, the aorto-pulmonary septal complex results from the merging of the neural crest-derived AP septum with the aortic flow divider and in mammals from the AP septum with the pulmonary flow divider. *AP septum, 3, 4, 6 PAAs by number; cd carotid duct, dAo dorsal Aorta, pa pulmonary artery, sa subclavian artery, vAo visceral aorta; Left left side; Right right side