M. M. T. Mentink

Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras.

The endothelium of the coronary vascular system has been described in the literature as originating from different sources, varying from aortic endothelium for the main coronary stems, endocardium for the intramyocardial network, and sinus venosus lining for the venous part of the coronary system. Using an antibody against quail endothelial cells (alpha-MB1), we investigated the development of the coronary vascular system in the quail (Hamburger and Hamilton stages 15 to 35) and in a series of 36 quail-chicken chimeras. In the chimeras, pieces of quail epicardial primordium and/or liver tissue were transplanted into the pericardial cavity of a chicken host. The results showed that the coronary vascular endothelial distribution closely followed the formation of the epicardial covering of the heart. However, pure epicardial primordium transplants did not lead to endothelial cell formation, whereas a liver graft with or without an epicardial contribution did have this capacity. The first endothelial cells were seen to reach the heart at the sinus venosus region, subsequently spreading through the inner curvature to the atrioventricular sulcus and the outflow tract and, last of all, over the ventricular surfaces. At these sites, the precursor cells and small vessels were seen to invade the sinus venosus wall, the ventricular and atrial myocardium, and the mesenchymal border of the aortic orifice. Connections with the endocardium of the heart tube were only observed in the right ventricular outflow region. Initially, the connections with the aortic endothelium were multiple, but later in development only two of these connections persisted to form the proximal part of the two main coronary arteries. Connections to the pulmonary orifice were never observed. Our transplantation data showed that the entire coronary endothelial vasculature originated from an extracardiac source. Moreover, using the developing subepicardial layer as a matrix, we showed that the endothelial cells reached the heart from the liver region. Ingrowth into the various cardiac segments was also observed. Implications for the relation to specific congenital cardiac malformations are discussed.

The development of the coronary vessels and their differentiation into arteries and veins in the embryonic quail heart.

Research concerning the embryologic development of the coronary plexus has enriched our understanding of anomalous coronary vessel patterning. However, the differentiation of the coronary vessel plexus into arteries, veins, and a capillary network is still incomplete. Immunohistochemical techniques have been used for whole mounts and serial sections of quail embryo hearts to demonstrate endothelium, vascular smooth muscle cells, and fibroblasts. From HH35 onward, the lumen of the coronary plexus was visualized by injecting India ink into the aorta. In HH17, branches from the sinus venosus plexus expand into the proepicardial organ to reach the dorsal side of the atrioventricular sulcus. From HH25 onward, vessel formation proceeds toward the ventral side and the apex of the heart. After lumenized connections of the coronary vessels with the aorta and right atrium are established, a media composed of smooth muscle cells and an adventitia composed of procollagen‐producing fibroblasts are formed around the coronary arteries. In the early stage, bloodflow through the coronary plexus is possible, although connections with the aorta have yet to be established. After the coronary plexus and the aorta and right atrium are interconnected, coronary vessel differentiation proceeds by media and adventitia formation around the proximal coronary arteries. At the same time, the remodeling of the vascular plexus is manifested by disappearance of arteriovenous anastomoses, leaving only capillaries to connect the arterial and venous system. Dev. Dyn. 208:338–348, 1997.

The development of the myocardium and endocardium in mouse embryos

SummaryThe formation of the single heart tube by hypothetical fusion of two separately developed heart tubes is re-investigated, because this intricate process is ambiguously and often incompletely described. To gain a better insight into this problem ten mouse embryos ranging from 7.5 to 8.5 days of development (presomite to 6 somites) were serially sectioned (1 μm) and reconstructed graphically. Twenty mouse embryos of comparative ages, were studied by scanning electron microscopy. Two large embryonic mesodermal compartments, derived from the primitive streak, extend rostrally on either side of the embryonic axis, and meet in front of the buccopharyngeal membrane. In each compartment a coelomic cavity develops, splitting the mesoderm into a splanchnic and somatic layer. The splanchnic mesoderm differentiates into a layer of cuboidal splanchnic mesothelial cells (promyocardium) and a subjacent plexus of elongated endothelial cells (proendocardium). Before the 1-somite stage the left and right splanchnic mesoderm are separated in front of the buccopharyngeal membrane by a thickening of the yolk sac endoderm. The splanchnic mesoderm then fuses, forming a single horseshoe-shaped heart primordium consisting of a promyocardial layer and a subjacent vascular plexus. Until the 2-somite stage both coelomic cavities remain separated by a bilayer of squamous somatic mesothelial cells (‘mesocardium’). The plexus of endothelial cells that forms the proendocardium, also seems to be the source of the lining of the vitelline veins, the pharyngeal arch arteries and the dorsal aortae. The relatively close adherence of endoderm to the medial part of the horseshoeshaped heart primordium, combined with a bilateral accumulation of cardiac jelly, is suggestive of a double heart tube. However, promyocardium and proendocardium are both translocated as one horseshoe-shaped layer, thus fusion of the left and right parts of the heart primordium does not occur.

Development of the pharyngeal arch system related to the pulmonary and bronchial vessels in the avian embryo. With a concept on systemic-pulmonary collateral artery formation.

Background. The literature is ambiguous as to the question of the developmental background of systemic‐pulmonary collateral arteries. These are found in combination with various congenital heart malformations such as pulmonary atresia. From a clinical point of view, it is of interest to know whether we are dealing with the persistence of transient embryological vessels such as ventral segmental arteries or parts of pharyngeal arch arteries or with the prenatal or postnatal recruitment of the bronchial vasculature that normally supplies the lung. This study of the embryology of the extrapulmonary and intrapulmonary vasculature aims at a better understanding of the variations in origin, course, branching pattern, and histology of collateral arteries. Methods and Results. Serial sections of quail embryos ranging between stage HH11 and stage HH28 were incubated with a monoclonal antibody (&agr;MB1) against endothelial cells and their precursors. Additional series of chick embryos were injected with india ink to study the lumenized vascular patterns. A splanchnic plexus consisting of endothelial cells and precursors is present around the foregut before the lung buds develop. This plexus expands and gives rise to the pharyngeal arch arteries, the ventral pharyngeal veins, the pulmonary vessels, and the bronchial vessels, including the intrapulmonary vessel network. During two subsequent periods, the splanchnic plexus is transiently connected to the systemic arteries and veins. The bronchial arteries and veins develop in the second period from these transient vessels. The expansion and extension of the splanchnic plexus to many organs during the formation of the bronchial vessels explains the varying course and branching pattern of the bronchial vasculature. Conclusions. These results show that we are not dealing with two or more individual vascular systems that contribute to the developing vessels of the lungs but with one vascular plexus that normally gives rise to the pulmonary and bronchial vasculature but has the potential to give rise to other systemic‐pulmonary connections. (Circulation 1993;87:1306‐1319)

Coronary Artery and Orifice Development Is Associated With Proper Timing of Epicardial Outgrowth and Correlated Fas Ligand Associated Apoptosis Patterns

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.

Histologic studies on normal and persistent ductus arteriosus in the dog

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.

CYTOKERATINS AS A MARKER FOR EPICARDIAL FORMATION IN THE QUAIL EMBRYO

Several techniques have been used to visualize the migration pattern of the epicardial cells from the proepicardial organ over the myocardial surface. As the epicardial cells contain keratin tonofilament bundles, we have incubated 92 whole-mount quail hearts with an anti-keratin antibody. This immunohistochemical method showed that the complete epicardial covering of the embryonic heart is preceded by the formation of three epicardial rings. The epicardial rings are formed on the outer myocardial surface in the grooves that separate the cardiac segments from each other. We have also documented timing and patterning of isolated epicardial islands. They are not encountered at random over the myocardial surface, but only along the edge of the advancing epicardial front border and in two defined future epicardial ring areas on the ventral side of the outflow tract. The epicardial islands suggest that in the quail free-floating parts of epicardium can attach to the myocardium. Characteristics of the surface of the myocardium at the transitional zones between the cardiac segments, as well as the three-dimensional remodelling of the heart during cardiac morphogenesis seem to play a role in the pattern in which the epicardium eventually completely ensheaths the myocardial surface. Congenital heart defects are often related to malpositioned transitional zones that dictate the pattern of epicardial outgrowth. As the embryonic position of the epicardial rings is mirrored in the pattern of the main arterial stems, the coronary vascularization pattern might be altered in congenitally malformed hearts as well.

The formation of mesoderm and mesectoderm in 5- to 41-somite rat embryos cultured in vitro, using WGA-Au as a marker

SummaryThe formation of mesectodermal cells by the neural crest in 5- to 41-somite stage embryos was investigated experimentally in rat embryos cultured in vitro, using lectincoated colloidal gold as a probe. This method labelled all ectodermal cells, among them neural crest, surface ectodermal placodal and epiblastic (primitive streak) cells. The neural crest provides the mesodermal compartment of the entire head region with cells, including the primitive cranial ganglia and the branchial arches. In the head region migration of neural crest cells over a great distance (long-distance migration) was not observed. In the trunk region neural crest derived cells were mainly found to form the primitive spinal ganglia and the sympathetic trunk, once again without long-distance cell migration. Structures and tissues that supposedly were derived from the primitive streak were hardly labelled with colloidal gold. Surface ectodermal placodes were not only found at the expected sites (e.g. epibranchial placodes) but also in the ectoderm covering the transverse septum and lateral abdominal walls.

Vascular endothelial cells migrate centripetally within embryonic arteries.

SummaryMigration of vascular endothelial cells was traced in quail-chick chimeras. After heterospecific transplantations of quail limb bud pieces, or other tissues containing blood vessels, into the limbs or the coelomic cavity, the immunohistochemically stained endothelial cells of the quail were found to invade the chick host vessels, favouring the arteries. Within these vessels the endothelial cells regularly reach the host aorta, where they contribute to the endothelium on the ipsilateral side. It is concluded that the endothelial cells activity migrate, because microinjections of a synthetic peptide which contains the RGD-sequence and mimics fibronectin, stop the invasion of endothelial cells.

A SEM study on the development of the ventricular surface morphology in the diencephalon of the rat

SummaryThe morphogenesis of the ventricular surface of the diencephalon of the rat was studied using scanning electron microscopy, cryostat serial sections and direct observations under a dissection microscope. Based on these observations a description is given of the neuromeres present within the prosencephalon and of the termination of the sulcus limitans. Two conclusions are reached. First, three neuromeres are present in the prosencephalon. Neuromere I consists of the telencephalon, the hypothalamic regions and the parencephalon anterius. Neuromere II is the parencephalon posterius, neuromere III the synencephalon. Second, the sulcus limitans terminates ventrally in the parencephalon posterius and does not continue towards the preoptic recess. No exact termination point of the sulcus limitans could be delineated.