Search

The proepicardial organ provides differentiated cell types to the myocardial wall and facilitates coronary development. Ingrowth of the coronary arteries into the aorta has recently been linked to apoptosis. This study was set up to examine the effect of an inhibition of epicardial outgrowth on apoptotic patterning and coronary development. Epicardial outgrowth was blocked at HH15–17 in quail embryos, which survived until HH25–35 (n=33). Embryos with complete inhibition of outgrowth did not survive after HH29. These embryos presented with thin compact myocardium, devoid of vessels. In embryos with delayed epicardial outgrowth the phenotype was less severe, and surviving embryos were studied up to HH35. In these embryos, myocardial vascularization was poor and apoptosis in the peritruncal region at HH30 was diminished. Embryos at HH35 displayed an abnormal coronary network and absent coronary orifices. In a further set of experiments (n=10), outgrowth was inhibited in chicken embryos at HH15, followed by transplantation of a quail proepicardial organ into the pericardial cavity to rescue cardiac phenotype. These chimeras were studied at HH29 and HH35. Myocardial development was restored; however, in 3 of 4 embryos (HH35), the coronary orifices were absent. Examination of double stainings of quail-chicken chimeras revealed that EPDCs produce Fas ligand as an apoptotic inductor at sites of coronary ingrowth. In the absence of proper timing of epicardial outgrowth, myocardial development and vascularization are disturbed. Also apoptosis in the peritruncal region is diminished. During later development, this leads to defective or absent connections of the coronary system to the systemic circulation.

The beta-geo (LacZ) reporter gene encodes for beta-galactosidase (beta-gal) in all cells of the ROSA26 mouse during embryonic development. As such, beta-gal activity constitutes an excellent marker for in situ labeling of expressing cells. However, the intracellular distribution of beta-gal differs between cells, and changes during embryonic development. Therefore, we studied LacZ-encoded beta-gal using light and electron microscopy in the heart, lung, liver, and small intestine on days 13 and 16 of gestation, and the kidney on day 16 of gestation in ROSA26 mice. The Bluo-gal method was carried out under standardized conditions, including fixation, washing, and incubation procedures. Intracellular beta-gal staining is encountered in a combination of membranous compartments, including the nuclear envelope, the endoplasmic reticulum, and the plasma membrane. Its exact localization depends on the cell type and is regulated during development. Therefore, one must take the compartmental transition of intracellular beta-gal staining into consideration when interpreting results obtained from experiments using ROSA26 mice.

Previous research has revealed that cells contributing to coronary vascular formation are derived from the dorsal mesocardium, however, the fate of these cells during consecutive stages of heart development is still unclear. We have conducted a study regarding the recruitment of vascular components and the subsequent differentiation into mature vessel wall structures with the aid of immunohistochemical markers directed against endothelium, smooth muscle cells, and fibroblasts. The proepicardial organ including an adhering piece of primordial liver of quail embryos (ranging from HH15 to HH18) was transplanted into the pericardial cavity of chicken embryos (ranging from HH15 to HH18). The chicken-quail chimeras (n=16) were harvested from the early stage of endothelial tube formation (HH25) to the late stage of mature vessel wall composition (HH43). Before HH32 endothelial cells have invaded the myocardium to give rise to yet undifferentiated coronary vessels. These endothelial cells are not accompanied by other non-endothelial cells. The superficial epicardial layer changes from a squamous mesothelium into a cuboid epithelium preceding media and adventitia formation. Subsequently, a condensed area of mesenchymal cells delaminates from the cuboidal lining extending toward the vessel plexus. Around the coronary arteries, these mesenchymal cells differentiate into smooth muscle cells or fibroblasts as shown by immunohistochemical markers. We conclude that epithelial-mesenchymal transformation of the epicardial lining delivers the smooth muscle cells and fibroblasts of the coronary arterial vessel wall. Molecules involved in epithelial transformation processes elsewhere in the embryo are also expressed within the subepicardial layer, and are considered to participate in inducing this process.

Background —Abnormal atrial automaticity in young patients with structurally normal hearts is often located around the pulmonary veins and in sinus venosus–related parts of the right atrium. We hypothesize that these ectopic pacemaker sites correspond to areas of embryonic myocardium with an early phenotypic differentiation, as indicated by differences in antigen expression during normal cardiac development. Methods and Results —In human embryos ranging in age from 42 to 54 days of gestation, the development of the cardiac conduction system was studied with the use of HNK-1 immunohistochemistry. HNK-1 stains the developing atrioventricular conduction system, ie, the bundle branches, His bundle, right atrioventricular ring, and retroaortic ring. In addition, the myocardium around the common pulmonary vein showed transient HNK-1 antigen expression. In the right atrium, 3 HNK-1–positive connections were demonstrated between the sinoatrial node and the right atrioventricular ring. An anterior tract through the septum spurium connects the sinoatrial node with the anterior right atrioventricular ring, and 2 posterior tracts connect the sinoatrial node with the posterior right atrioventricular ring through the right venous valve (future crista terminalis) and sinus septum, encircling the coronary sinus. The medioposterior part of the right atrioventricular ring connected to the His bundle and the medioanterior part form 2 node-like structures. Conclusions —In patients with abnormal atrial automaticity, the distribution of left and right atrial pacemaker foci correspond to areas of the embryonic myocardium that temporarily express the HNK-1 antigen.

Objective: The differentiation of the coronary vasculature was studied to establish in particular the formation of the coronary venous system. Methods: Antibody markers were used to demonstrate endothelial, smooth muscle, and fibroblastic cells in serial sections of embryonic quail hearts. The anti- β myosin heavy chain and the neuronal marker HNK-1 were added to our incubation protocol. Results: In HH32, the coronary vascular network has developed into a circulatory system with connections to the sinus venosus, the aorta and the right atrium. The connections between the aorta and the right atrium allow for direct arteriovenous shunting. Subsequently, differentiation into coronary arteries and veins occurs with an interposed capillary network. The smooth muscle cells of the coronary arterial media derive from the subepicardial layer, whereas the subepicardially located cardiac veins recrute atrial myocardium, as these cells express the β -myosin heavy chain antigen. Ganglia are located in the subepicardium close to the vessels, while nerve fibres tend to colocalize with the formed vessel channels. Conclusions: A new finding is presented in which the subepicardial coronary veins have a media that consists of myocardial cells. The close positional relationship of neural tissue and coronary vessels that penetrate the heart wall is explained as inductive for vessel wall differentiation, but not for invasion into the heart.

In the present study, we investigated the modulatory role of the epicardium in myocardial and coronary development. Epicardial cell tracing experiments have shown that epicardium-derived cells are the source of interstitial myocardial fibroblasts, cushion mesenchyme, and smooth muscle cells. Epicardial outgrowth inhibition studies show abnormalities of the compact myocardial layer, myocardialization of cushion tissue, looping, septation, and coronary vascular formation. Lack of epicardial spreading is partly compensated by mesothelial outgrowth over the conotruncal region. Heterospecific epicardial transplant is able to partially rescue the myocardial development, as well as septation and coronary formation.

The process of anatomic closure of the ductus arteriosus was studied at the ultrastructural level in 15 normal beagles (age 0 hour to 13 days) and in 18 specimens from a strain of dogs with hereditary persistent ductus arteriosus (age 4 hours to 27 days). Normal ductal closure takes place from the pulmonary artery to the aortic end. It is accompanied by a series of histologic changes: 1) separation of the endothelial cells from the internal elastic lamina resulting in a wide region of subendothelial edema; 2) ingrowth and infolding of endothelial cells and migration of undifferentiated smooth muscle cells from the inner media into the subendothelial region; 3) apposition of endothelial cells bordering the lumen; and 4) degenerative changes. In persistent ductus arteriosus, these changes do not occur. The endothelial cells remain closely adhered to the internal elastic lamina and the underlying media is abnormal in structure. In the case of partial persistent ductus arteriosus (ductus diverticulum), both the normal and the abnormal type of wall are found in a single ductus arteriosus. The histologic features of the normal and the persistent ductus arteriosus in the dog resemble those of the normal and the persistent ductus arteriosus in humans, suggesting a similar pathogenesis.