The Septation And Remodeling Of The Outflow Tract Of Embryonic Heart

The single tubular outflow tract of early embryonic heart connects primary ventricle with the aortic sac, which wall is composed of primary myocardium and endothelium with cardiac jelly in between. With development, the outflow tract undergoes complex septation and remodeling to form intrapericardial ascending aorta and pulmonary trunk and their valves and the outlets of the two ventricles. Many debates remain to be elucidated though there have been plenty of studies about the outflow tract development and its septation mechanism of embryonic heart of different species. The investigation about these questions could provide theory foundation for the congenital heart disease pathogenesis resulting from the abnormality of the outflow tract development and septation.The neural crest cells between the mid-otic placode and the third somite are called as cardiac neural crest, which can migrate into embryonic heart and play important role in the formation of the aorto-pulmonary septum and endocardial cushion and the outflow tract septation. Ablating the cardiac neural crest prior to its migration could lead to cardiac deficiency such as persistent truncus arteriosus. Thus the studies about cardiac neural crest distribution in embryonic heart and its function become one of the focuses. Because of the differences in the species and experimental methods, the debates still exist on the cardiac neural crest function during the septation of the outflow tract.The fusion of the outflow tract ridges to form mesenchymal septum is accompanied with its musculization. But the musculization mechanism remains to be elucidated. Some scholars reported that cardiomyocytes of the outflow tract wall progressively extended into the septum and recruited neighboring mesenchymal cells differentiating towards cardiomyocytes to complete the myocardiac septum formation. But at the same time, we also observed in situ differentiation of the mesenchymal cells in the outflow tract ridges and the septum except myocardialization and recruitment. So the formation mechanism of myocardiac septum is open to be explored.In this study, we observed the expression patterns ofα-SMA (α-smooth muscle actin),α-SCA (α-sarcomeric actin) and MHC (myosin heavy chain) in embryonic human heart from Carnegie stage 10 to Carnegie stage 19 to explore the formation of the outflow tract ridge and the aorto-pulmonary septum and the septation of the aortic sac and the outflow tract, and also to elucidateα-SMA expression significance in the myocardium and endocardial cushion of the outflow tract. We observed the distribution of PlexinA2 positive cells andα-SMA positive cells in the outflow tract of embryonic mouse heart from ED(embryonic day)10 to ED14 and the celluar ultrastructure change of the condensed mesenchymal cell masses during their fusion to explore the spacio-temporal exression patterns of PlexinA2 andα-SMA and their influence on the mesenchymal cell ultrastructure. We observed the myocardial septum formation of the outflow tract in ED12 to ED16 embryonic mouse heart to investigate the formation mechanism of the myocardial septum.Chapter I The septation of the outflow tract in the early embryonic human heartSerial sections of thirty-two human embryonic hearts from Carnegie stage 10 to Carnegie stage 19 (C10～C19, 22±1～47 postovulatory day) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA (α-sarcomeric actin) and MHC (myosin heavy chain) to observe the migrating route of theα-SMA positive cells to the outflow tract ridge, the development of the aortic sac and the septation mechanism of the aortic sac and the outflow tract. The results showed that from C10 to C13, the aortic sac was situated outside of the pericardial cavity and the demarcation between the aortic sac and the outflow tract was located at the reflection of the dorsal wall of pericardial cavity to the outflow tract. Between C14 and C15, the proximal part of the aortic sac began to progressively protrude into pericardial cavity and its wall gradully myocardialized to become the distal pole of the myocardial outflow tract leading to lengthening of the latter. At C16, the aortic sac at the fourth aortic arch level invaginated into the pericardial cavity, but its wall was not myocardialized. So the demarcation between the aortic sac and the myocardial outflow tract became to be located in the pericardial cavity. From C12 to C15,α-SMA positive cells aggregated in the endocardial cushion of the outflow tract and took part in forming the two opposite spiral ridges. At C16,α-SMA positive aorto-pulmonary septum could be observed and the outflow tract ridge shortened. The aortic sac was septated before protruding into the pericardial cavity at C14. In the pericardial cavity, although the outflow tract ridges were not fused, the aortic sac had been divided into the ascending aorta and pulmonary trunk by the aorto-pulmonary septum at C16. The valve anlagen could be seen at the distal pole of the myocardial outflow tract. At C19, the outflow tract ridges began to fuse with each other at the valve level to form the outlet septum and septate the outflow tract. Theα-SMA positive cells from the two ridges aggregated in the centre of the septum. The results suggest that the formation of the outflow tract ridge is earlier than the aorto-pulmonary septum. The distal and proximal ends of the ridge keep at the same level with the two poles of the myocardial outflow tract. The aortic sac has been septated when it is embeded in the pharyngeal mesenchyme. After descending into the pericardial cavity, the aortic sac is septated into the ascending aorta and pulmonary trunk by the aorto-pulmonary septum. The distal poles of the outflow tract ridges form the valve anlagen of the two arteries. The main part of the ridges fuses to septate the outflow tract under the valves.Chapter II The development of theα-SMA positive cells in the outflow tract of the early embryonic human heartSerial sections of twenty-nine human embryonic hearts from Carnegie stage 10 to Carnegie stage 16 (C10～C16, 22±1～37 postovulatory day) were stained immunohistochemically with antibodies againstα-SMA (α-smooth muscle actin),α-SCA (α-sarcomeric actin) and MHC (myosin heavy chain). It was observed that during C10 to C13, a few of the pericardial splanchnic epithelium cells showedα-SMA positive and expressedα-SCA, MHC weakly, which meant that the pericardial splanchnic epithelium progressively differentiated into myocardial cells and these new cardiomyocytes were added to the distal pole of the outflow tract to make it lengthen. The expression ofα-SMA of these cells was earlier than the expression ofα-SCA and MHC. At C14 and C15, the aortic sac wall was stainedα-SMA strongly when protruding into the pericardial cavity. But the expression ofα-SCA and MHC was strong only on the left wall, which suggested that the aortic sac was myocardializing. From C12 to C15,α-SMA positive cells gradually migrated into the endocardial cushion from the pharyngeal mesenchyme. At the same time, the endocardial cells of the outflow tract began to expressα-SMA and differentiate towards mesenchymal cells. Mesenchymal cells of different origins aggregated in the endocardial cushion to form the outflow tract ridge. Withα-SMA positive cells progressively migrating into the ridge, the ridge became larger. At C16, the outflow tract was shortened and most of the mesenchymal cells in the ridge expressedα-SMA weakly. But a few of positive cells expressedα-SMA strongly aligning with the myocardial cells of the outflow tract wall. The cardiomyocytes linked with theseα-SMA positive cells by their protrusion. We conclude thatα-SMA could be regarded as an early marker of cardiomyocytes differentiation. The endocardial cells of the outflow tract ridge expressα-SMA when differentiating towards mesenchymal cells.α-SMA positive cells in the ridge and the aorto-pulmonary septum take part in the septation of the aortic sac. Chapter III The development of the outflow tract ridge in the embryonic mouse heartSerial sections of ED(embryonic day)10 to ED14 mouse embryonic hearts were stained by monoclonal antibodies againstα-SMA (α-smooth muscle actin) andα-SCA (α-sarcomeric actin). In situ hybridization was performed on the sections of ED10 to ED14 embryos to visualize the migration and distribution pattern of PlexinA2 positive cells in the outflow tract. The outflow tract ridges fusion was observed by transmission electron microscope at ED12.5. The results showed that from ED10 to ED11, PlexinA2 positive cells were seen in the neural tube, the mesenchyme around it, the aortic sac and aortic arch. These cells also migrated into the outflow tract ridge along the aortic sac wall. Some of them expressedα-SMA. At ED12, PlexinA2 was expressed in the dorsal ganglia, the pharyngeal mesenchyme, the aorto-pulmonary septum and the ascending aorta and pulmonary trunk. The septum was shownα-SMA strongly positive. But only a few ofα-SMA positive cells were observed in the ascending aorta and pulmonary trunk. At ED12.5, two clusters of condensed mesenchymal cells in the outflow tract ridges formed and began to merge at the semilunar level expressing PlexinA2 weakly andα-SMA strongly. When the ridges began to fuse, the endothelial cells formed a cellular seam, which rapidly broke into pieces and disappeared and were replaced by the sparsed mesenchymal cells containing a few of microfilaments. Two clusters of condensed mesenchymal cells gradully moved to merge. It was noted that some mesenchymal cells contained plenty of microfilament bundles, mitochondria and unmatured focal contacts between them. Some mesenchymal cells only had a few of microfilaments and plasma membrane fusion was seen between them. Cell fusion of endothelial cells or mesenchymal cells was also observed to form double nuclus cells. From the results we conclude thatα-SMA positive cells in the outflow tract cushion may be derived from cardiac neural crest. The endothelial cells participate in the fusion of the outflow tract ridges by endothelial-mesenchymal transformation. Mesenchymal cells of the condensed cell mass contain plenty of microfilament bundles and focal contacts or membrane fusion, which increase connection firmness between cells and contribute to the ridges fusion. PlexinA2 down-regulation may facilitate the mesenchymal cell adhesion and aggregation.Chapter IV Musculization of the mensenchymal septum of the outflow tract in embryonic mouse heartSerial sections of mouse embryonic hearts from ED(embryonic day)12 to ED16 were stained by monoclonal antibodies againstα-SMA (α-smooth muscle actin), Desmin andα-SCA (α-sarcomeric actin) to investigate the mechanism of the myocardial septum formation in the outflow tract of embryonic mouse heart. Apoptosis was determined by TUNEL assay. We found that at ED12, the outflow tract ridges were not fused yet and a few ofα-SCA positive cells had been observed in one of the ridges. The cardiomyocytes of the outflow tract wall began to extend into the ridge. From ED12.5 to ED14, the outflow tract ridges fused to form the mensenchymal septum at and under the semilunar valve level.α-SCA positive cardiomyocytes could be observed progressively migrating from the wall of the outflow tract into the mesenchymal septum. It needed to be stressed that a few ofα-SMA andα-SCA positive cardiomyocytes independently existed in the centre of the septum. During ED13 to ED15,α-SMA positive cells aggregated to form the whorl in the centre of the septum. More mesenchymal cells in the septum were replaced by cardiomyocytes. Soα-SCA negative area only lied in the centre of the septum. At ED16, the septum was filled with cardiomyocytes to form the myocardial septum. From ED13 to ED15, the apoptosed cells in the centre of the septum gradually increased. The conclusions could be drawed that myocardialization is not the single mechanism to explain the transforming of the mesenchymal septum to the myocardial septum. Transdifferentiation and recruitment of the mesenchymal cells in the septum may also be reasons for it. The formation of the myocardial septum is companied by apoptosis of part of theα-SMA positive cells in the septum. Theα-SMA positive cells without being apoptosed may transdifferentiate into cardiomyocytes and take part in the formation of the myocardial septum.