Fig 1.
Spatial situation of the myocardial conal walls.
(A) Right sagittal view of the cephalic portion of a stage 22HH chicken embryo showing the different regions that make up the heart and the location of the conal walls in the organ in situ. Arrowheads: yellow = dorsal wall; green = ventral wall; red = right wall; blue = left wall. (B) Cross-section of the middle zone of the conus at stage 22HH, haematoxylin-eosin stain. Digitally, colours were impressed to the conal walls myocardium as described in section A. Observe a black label immersed in the myocardium of the ventral conal wall. Abbreviations: As = atrial segment; C = conus; Ca = caudal; Ce = cephalic; D = dorsal; L = left; R = right; T = truncus; V = ventral; Vs = ventricular segment.
Table 1.
Embryonic hearts acquired and number of hearts used for each stage.
Fig 2.
Prospective fate of the right conal wall (classically denoted anterior conus).
(A) Representative images of embryonic heart at stage 22HH showing a black label inserted in the myocardium on the right surface of the cono-ventricular boundary (red arrowhead). (A’) Embryonic heart at stage 36HH. Visualization of small dark spots (red arrowheads) distributed in the myocardium of the apical ventricular region in relation to the anterior interventricular groove (AIVG). (B) Embryonic heart at stage 22HH with a black label inserted in the myocardium on the right surface of the conus-truncus border (red arrowheads). (B’) Embryonic heart at stage 36HH showing small dark spots (red arrowheads) distributed in the myocardium from the right region of the cardiac base to the subvalvular pulmonary region. The dotted white line indicates the limit of the right ventricular-free wall. Abbreviations: AIVG = anterior interventricular groove; As = atrial segment; C = conus; IVS = interventricular septum; LA = left atrium; LV = left ventricle; PA = pulmonary artery, RA = right atrium; RV = right ventricle; T = truncus, Vs = ventricular segment.
Fig 3.
Prospective fate of the myocardium of the ventral, dorsal and left conal walls.
(A) Embryonic heart at stage 22HH showing a black label inserted in the myocardium at the medial zone of the ventral conal wall (green arrowhead). (A’) Embryonic heart at stage 36HH with small dark granules (green arrowheads) distributed in the myocardium of the sub-valvular region of the pulmonary artery. (B) Embryonic heart at stage 22HH with a black label inserted in the myocardium of the dorsal conal wall (yellow arrowhead). (B’) Embryonic heart at stage 36HH showing small dark spots (yellow arrowheads) distributed in the myocardium along the middle third of the free wall of the right ventricle. (C) Embryonic heart at stage 22HH with a black label inserted in the myocardium of the medial zone of the left conal wall. (C’) Embryonic heart at stage 36HH. Visualization of some small dark spots (blue arrowheads) distributed in the myocardium between the acute border of the heart and the pulmonary infundibulum. The dotted white line indicates the limit of the right ventricular-free wall. Abbreviations: AIVG = anterior interventricular groove; Ao = aorta; As = atrial segment; C = conus; IVS = interventricular septum; LA = left atrium; LV = left ventricle; PA = pulmonary artery, RA = right atrium; RV = right ventricle; T = truncus, Vs = ventricular segment.
Fig 4.
Increase in conus length during cardiac septation.
Increase in conus length during cardiac septation was statistically significant between stages 22–36HH. Data are expressed as means (standard deviation) using *p ≤ 0.01 for comparisons.
Fig 5.
Conus gradual medial displacement during cardiac septation.
Frontal views of the embryonic heart. (A) Stage 22HH visualization of the right extra cardiac initial position of the conus. (B) Stage 28HH visualization of the conus completely integrated into the right ventricle, occupying a ventral inferior position. (C) Graphic. Data are expressed as means (standard deviation) using *p ≤ 0.01 for comparisons. Abbreviations: Ao = developing aorta; As = atrial segment; C = conus; LA = left atrium; LV = left ventricle; PA = developing pulmonary artery, RA = right atrium; RV = right ventricle; T = truncus, Vs = ventricular segment.
Fig 6.
Conal myocardium and mesenchymal remodelling.
Adjacent cephalo-caudal sections of the embryonic heart from stages 22HH to 26HH. (A) Stage 22HH. The lumen of the conus and the AV canal are side by side. (A’) Immunolocalization of the skeletal muscle myosin (green). Observe the tubular structure of the conal myocardium, the homogeneously distributed mesenchymal tissue (Mes), and the endocardium (red nucleus). (B, B’) Stage 24HH. The conus is in a slightly left and ventral position with respect to the AV canal. (B’). A small myocardial notch (*) is observed at the left-dorsal conal wall. (C, C’). Stage 26HH. Observe the loss of continuity at the thinned left-dorsal myocardial conal wall (*) and the incipient endocardial conal crest (X). Abbreviations: AVC = atrioventricular canal; Mes = mesenchymal tissue.
Fig 7.
Pulmonary vestibule and the aortic infundibulum develop from different embryonic components.
(A, B). Adjacent cephalo-caudal histological sections of the embryonic heart at stage 28HH. Observe a wider opening zone at the originally left-dorsal myocardial conal wall (*). The voluminous mesenchymal structure separating the incipient right (1) and left (2) atrioventricular orifices corresponding to the ventro-superior and the dorso-inferior cushions of the AV canal during the fusion process (3). (A’, B’) Skeletal muscle myosin immunolocalization (green) corroborating the left-dorsal myocardial conal wall loss of continuity (*). (C) Stage 30HH. Visualization of the well-developed dextrodorsal (4) and sinistroventral (5) crests within the conus (C). The incipient mural leaflet of the left AV valve (6) is also evident. Note the elongated canal (7) bordered by the dorsal surface of the conal crest (4 and 5) and the right surface of the incipient anteroseptal leaflet of the left AV valve (8). (D, E). Stage 31 and 32HH. Mature heart showing the fused conal crests forming the immature supraventricular crest (9). The single conal conduct corresponds to the pulmonary infundibulum (PI). The aortic vestibule (AoV) behind the developing anteroseptal leaflet of the left AV valve (8) is well developed. E’. Amplification of the red box in E. Observe traces from the label as some fine charcoal granules immersed in the myocardium of the pulmonary infundibulum (40X). Abbreviations: RAVV = Right atrioventricular valve; LAVV = Left atrioventricular valve.
Fig 8.
Apoptotic pattern at the embryonic outflow.
(A–E) Representative images of embryonic hearts (stages 24-32HH) that have been previously labelled at the conus truncus limit and treated with LysoTracker Red. Visualization of positive apoptotic cells, mainly at the level of the previously labelled conus-truncus border, dotted red line. (A’- E”) Histological transversal sections of embryonic hearts treated with LysoTracker Red at the conus level. In this case, the apoptotic cells correspond to the red dots with white arrowheads. (A’) Stage 24HH, apoptotic cells were observed in the conal mesenchyme. (B’) Stage 26HH, some apoptotic cells were observed in the myocardium of the ventral conal wall. No apoptotic cells were detected in the myocardium of the thinner left-dorsal conal wall between the conal mesenchyme (CM) and the atrioventricular cushions (AVC). (C’) Stage 28HH. Observe few apoptotic cells immersed in the myocardium of the ventral conal wall. (D’- E”) Stages 30 and 32HH. Visualization of apoptotic cells principally in the epicardium. Abbreviations: LAVV = left atrioventricular oprifice; RAVV = right atrioventricular orifice. 1. Dextrodorsal conal ridge; 2. sinistroventral conal ridge; 3. fused atrioventricular cushions; 4. septal leaflet of the mitral valve; 5. pulmonary infundibulum; 6. aortic vestibulum.
Fig 9.
Fate map of the conal walls based on the labelling experiments.
(A) Micrograph obtained using a scanning electron microscope of a 26HH chicken embryo heart dissection. The results show the partial displacement of the conus on the way to its final ventral position and the discontinuity of the dorsal-left wall conal myocardium (*). (B) Frontal view of the chicken embryo heart at stage 36HH, depicting the myocardial conal wall distribution exclusively throughout the right ventricle (RV). The white dotted line marks the edge of the free wall of the right ventricle. (C). Chicken postnatal heart dissection showing differential fate of each of the conal walls in the right ventricle below the arterial and atrioventricular valves. The black dotted line denotes the fibrous skeleton where the pulmonary valvular ring (PA) is excluded.