Smoothelin expression during chicken embryogenesis: detection of an embryonic isoform.

Two isoforms of a novel smooth muscle cell (SMC) -specific cytoskeletal protein, smoothelin, have been described. In the adult chick, the 55-kDa smoothelin-A is expressed in visceral SMC, whereas the 120-kDa smoothelin-B is the major product in vascular SMC. Chicken was chosen to study smoothelin expression during embryogenesis and neonatally. Smoothelin-B was found in vascular SMC from stage 20 onward. In visceral SMC, smoothelin-B was present from stage 29 until hatching. Perinatally, a strong up-regulation of smoothelin synthesis was observed in visceral tissues, coinciding with a switch to the A-isoform. Transient smoothelin synthesis was observed in the somites and the developing heart. Western blotting revealed in these tissues a 62-kDa smoothelin isoform, designated smoothelin-C. Expression of the smoothelin isoforms seems to be strictly controlled with respect to cell type and developmental stage and may be related to the mode of contraction of the different cells.

Epicardial outgrowth inhibition leads to compensatory mesothelial outflow tract collar and abnormal cardiac septation and coronary formation.

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.

Loss of function of the Prx1 and Prx2 homeobox genes alters architecture of the great elastic arteries and ductus arteriosus.

Prx1 (MHox) and Prx2 (S8) are non-clustered homeobox genes that are expressed in a complex, mostly mesenchyme-specific pattern throughout embryogenesis. The expression pattern and gene-targeted mice previously revealed a major role for Prx1 in skeletogenesis. In addition, specific and high expression of both Prx genes was reported in the developing cardiovascular system, predominantly in prospective connective tissues of the heart and in the great arteries and veins. We examined embryos of previously generated gene-targeted mice. Prx2-/- mutants were viable and did not show cardiovascular malformations. Intracardiac morphology of Prxl-/- and Prx1/Prx2-combined null mutants also appeared normal throughout development. However, the Prx1-/- and Prx1/Prx2 double-null mutants showed a vascular abnormality with an abnormal positioning and awkward curvature of the aortic arch in addition to a misdirected and elongated ductus arteriosus, and in two of seven combined mutants, an anomalous retro-oesophageal right subclavian artery. Generally, all great arteries appeared to run somewhat tortuously through the surrounding mesenchyme. The vascular histology and vessel wall thickness were normal in all mutants. Prx1-/- and Prx double-gene-targeted mice revealed similar spectra of vascular anomalies, but double mutants appeared to be more seriously affected. The current findings suggest that other genes may compensate for the loss of Prx in the heart, but, in contrast, our data support a role for Prx in the development of vascular and perivascular matrix.

Comparative anatomy and ontogeny of the ductus arteriosus, a vascular outsider.

In its function of separating pulmonary and systemic arterial blood flow, the ductus arteriosus, which connects both circuits, either closes permanently at a certain stage in development or attains a capacity to close and reopen depending on the physiological needs in certain species. In air-breathing vertebrates varying from lungfish to mammals, the ductus arteriosus derives from the sixth pharyngeal arch artery, and in preparation for its specific task, undergoes its own unique differentiation programme, starting early in development. To date, the mechanisms involved in defining this unique status, as compared to the other great arteries, are unclear. This review clarifies some of the elusiveness of the ductus arteriosus. It includes a comparative description of this artery in species exemplifying the different classes of air-breathing vertebrates, and illustrates similarities and differences in morphogenesis and closure mechanisms among the species. It also deals with possible influences of vascular innervation and with congenital anomalies in which the ductus arteriosus is involved. New data suggest that HOXB5 expression in the neural crest along the dorsal half of the sixth arch artery may be involved in the instigation of ductus arteriosus differentiation.

Unique vascular morphology of the fourth aortic arches: possible implications for pathogenesis of type-B aortic arch interruption and anomalous right subclavian artery.

OBJECTIVE: Neural crest-derived cells were previously shown to participate in vessel wall formation of the great thoracic arteries, and their contribution was proposed to affect morphology and physiology of these vessels in the chick. The present investigation was undertaken to examine vascular differentiation and morphogenesis of the neural crest-derived aortic arches in mammals. METHODS: Using immunohistochemical markers for smooth muscle cell differentiation and a neurofilament marker, we examined morphogenesis of the great arteries in mice, ranging from embryonic day 11.5 to the adult. RESULTS: We observed that in the 4th aortic arch arteries early media formation differed from the other arteries, in that they almost completely lacked (or showed decreased) actin expression in certain areas. This discontinuity in actin expression persisted throughout much of foetal development, in the form of circular segments of cells displaying decreased staining for smooth muscle markers, both at the left and right side of the arterial tree. In adult mice, the 4th arch artery derivatives, segment B of the aortic arch and the proximal right subclavian artery, were observed to differ from adjoining vessels in their smooth muscle and elastic composition. Staining for neurofilaments revealed close association of the developing segments with apparent sensory afferent vascular innervation. CONCLUSION: The unique areas of the 4th arch artery identified here reflect the basic segmental patterning of the early embryonic pharyngeal arches. These segments correlate with sites that are predisposed to interruption or severe hypoplasia, and may thus reveal part of the aetiology of type-B aortic arch interruptions and arteria lusoria.

Patterns of paired-related homeobox genes PRX1 and PRX2 suggest involvement in matrix modulation in the developing chick vascular system.

PRX1 (MHox) and PRX2 (S8) were previously shown to be expressed throughout embryogenesis in complex, mostly mesenchyme-specific patterns. In the developing cardiovascular system both genes were highly expressed in prospective connective tissues, that is, endocardial cushions and valves, the epicardium, and the wall of the great arteries and veins. We further scrutinised expression of PRX1 and PRX2 in the developing vascular system of the chicken embryo and compared patterns with those of established vascular differentiation markers (muscle-actin, procollagen I, and fibrillin-2). PRX1 and PRX2 expression were associated with the primary vessel wall from early stages onward and became increasingly restricted to the adventitial and outer medial cell layers. PRX1 eventually colocalised strikingly with procollagen I and fibrillin-2 expression and generally excluded high smooth muscle actin expression. Furthermore, PRX1 expression preceded the segregation of very distinct nonmuscular cells and smooth muscle cells in the media of the great arteries. PRX2 patterns deviated at later stages from those of PRX1 and showed specific and high transcript levels in the ductus arteriosus from embryonic day 6 onward. Results suggest that PRX genes are not essential in smooth muscle contractile differentiation, but may be involved in matrix modulation in the vascular system and possibly in defining the noncontractile cellular phenotype and in media-adventitia definition.

Neural crest cell contribution to the developing circulatory system: implications for vascular morphology?

In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular morphogenesis. For this purpose, we performed long-term lineage tracing using quail-chick chimera techniques and premigratory NCC infection with a replication-incompetent retrovirus containing the LacZ reporter gene in combination with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the anterior cardinal veins was also determined to be NC-derived. NC-derived elastic arteries differed from non-NC elastic vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating a muscular or elastic artery. Boundaries between NC-infested areas and mesodermal vessel structures were mostly very sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in the aortic and pulmonary trunks.

Onset of elastogenesis and downregulation of smooth muscle actin as distinguishing phenomena in artery differentiation in the chick embryo.

During development, the arterial system is grossly divided into elastic and muscular vessel types. Apart from local environmental factors, it has been suggested that vascular smooth muscle cell origin (mesoderm or neural crest) is involved in this, as yet poorly understood, arterial differentiation. We describe differentiation of the thoracic arterial system in the chick embryo, using immunohistochemical techniques staining for muscle-specific actin, vinculin and desmin and histological staining to visualise elastin. The initial developmental stages of the vessel wall in all arteries appeared to be highly similar, with all arteries showing peri-endothelial actin and vinculin staining. Major alterations did not occur until the start of elastogenesis, which coincided with complete loss of actin staining from the proximal part of the great arteries. Later in development, however, actin was re-expressed in a subpopulation of medial cells, which also expressed vinculin and desmin. Concomitantly another, nonmuscular, cell type became evident in the great arteries. Transient loss of actin expression and segregation of very distinct cell populations occurred only in vessels prone to elastic development and known to receive a neural crest contribution. In contrast, arteries that developed a muscular phenotype never lost the initially acquired peri-endothelial actin expression. We also show a significant difference in the organisation of elastic fibres between elastic vessels that contain neural crest derivatives and those that do not. The ductus arteriosus still presents as an enigma in the sense that it is the only part of the pharyngeal arch complex that develops a muscular phenotype.