Distribution of different regions of cardiac neural crest in the extrinsic and the intrinsic cardiac nervous system.

In this study we focused upon whether different levels of postotic neural crest as well as the right and left cardiac neural crest show a segmented or mixed distribution in the extrinsic and intrinsic cardiac nervous system. Different parts of the postotic neural crest were labeled by heterospecific replacement of chick neural tube by its quail counterpart. Quail-chick chimeras (n = 21) were immunohistochemically evaluated at stage HH28+, HH29+, and between HH34-37. In another set of embryos, different regions of cardiac neural crest were tagged with a retrovirus containing the LacZ reporter gene and evaluated between HH35-37 (n = 13). The results show a difference in distribution between the right- and left-sided cardiac neural crest cells at the arterial pole and ventral cardiac plexus. In the dorsal cardiac plexus, the right and left cardiac neural crest cells mix. In general, the extrinsic and intrinsic cardiac nerves receive a lower contribution from the right cardiac neural crest compared with the left cardiac neural crest. The right-sided neural crest from the level of somite 1 seeds only the cranial part of the vagal nerve and the ventral cardiac plexus. Furthermore, the results show a nonsegmented overlapping contribution of neural crest originating from S1 to S3 to the Schwann cells of the cranial and recurrent nerves and the intrinsic cardiac plexus. Also the Schwann cells along the distal intestinal part of the vagal nerve are derived exclusively from the cardiac neural crest region. These findings and the smaller contribution of the more cranially emanating cardiac neural crest to the dorsal cardiac plexus compared with more caudal cardiac neural crest levels, suggests an initial segmented distribution of cardiac neural crest cells in the circumpharyngeal region, followed by longitudinal migration along the vagal nerve during later stages.

Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo.

In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10-20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4-14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK-1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro- and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4-14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart.

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.

Expression of the beta 4 integrin subunit in the mouse heart during embryonic development: retinoic acid advances beta 4 expression.

Using immunohistochemical techniques as well as in situ hybridization we were able to elicit the expression pattern of the beta 4 integrin subunit in the murine heart during development. We show that beta 4 is not expressed in the heart before E13 and is afterwards restricted to the endocardium of the atrioventricular canal, the outflow tract, and the venous valves in the right atrium. As these are all sites of high shear stress in the heart, we propose a role for alpha 6 beta 4 in the tight adhesion of the endocardial cells to their basement membranes in these segments. Moreover, mouse embryos were treated with all-trans retinoic acid, which was previously shown to induce congenital malformations, among which malformations of the heart. We show an advanced expression without ectopic localization of cardiac beta 4 after the administration of retinoic acid. This advanced appearance of beta 4 was also shown in extracardiac tissue like migrating neural crest cells. Several hypotheses on the mechanism of beta 4 up-regulation and a possible role for alpha 6 beta 4 in the development of heart malformations after the administration of retinoic acid are discussed.

Differential expression of alpha-6 and other subunits of laminin binding integrins during development of the murine heart.

The development of the heart from a single heart tube to a four chambered organ with two separated unidirectional flows is a highly complex process. Events like looping, septation, tissue remodelling, and development of valves take place in a time period in which the heart already exerts its pump function. Adhesion of cells to each other and to their extracellular matrix as well as the capability to migrate in such a dynamic environment are extremely important. Integrins and extracellular matrix components have already been implicated in this process. In this report, we describe in detail the differential expression of the alpha-6 integrin subunit during late murine heart development, e.g., in the process from looping to the end of septation. We compare mRNA and protein expression patterns with those of beta-1 and other subunits of laminin-binding integrins, alpha-3 and alpha-7. We show a constant and high expression of alpha-6 in the atrial myocardium and a decrease in expression in the ventricular trabecular myocardium. The compact myocardial wall and the ventricular septum do not express alpha-6, except for the myocardium of the distal outflow tract at early stages. Moreover, we describe expression of this integrin subunit in the endocardial cushions that contribute to the development of the atrioventricular and semilunar valves. We propose a role for the alpha-6-beta-1 laminin receptor in the adhesion of cells to their extracellular matrix at sites of high stress due to cardiac contraction or blood flow induced shear stress. Moreover, site specific endothelial expression within the heart and surrounding extracardiac tissue is discussed. This study suggests a distinct role for alpha-6-beta-1 in the heart and provides insight concerning probably important roles of integrins and their extracellular matrix ligands during embryonic development.

The early development of the tunica media in the vascular system of rat embryos.

The use of a monoclonal antibody (HHF-35) against muscle-cell-specific actin has led to new insights into the early development and differentiation of the tunica media of blood vessels. The localization of this substance was studied by light and electron microscopy, in 22 rat embryos ranging between 10 and 15 days post coitum. Actin expression was already seen in the mesoderm around the dorsal aortae at 10 days post coitum, i.e. 1.5 day before circular mesenchymal condensations were detectable by light microscopy. These condensations are usually regarded as the first indication of arterial tunica media formation. The actin expression starts in the dorso-medial mesoderm surrounding the dorsal aortae at the level of the developing pharyngeal arch system, followed at 11.5 days by positive cells in the lateral mesoderm. In 12-day-old embryos most of the mesenchymal cells around the dorsal aortae contain actin, except for those aspects of the dorsal aortae which participate in outgrowing and connecting vessels to specific organs, such as the pharyngeal arch arteries, dorsal intersegmental arteries and aorto-pulmonary plexus, which themselves are still negative. At this stage the vitelline and umbilical arteries, which belong to the early developing ventral segmental arteries, are already surrounded by actin-containing mesenchymal cells. From 13 days onwards the tributary arteries, such as the subclavian and vertebral arteries, start to present the first differentiation of a tunica media in a proximo-distal development. In general the actin-negative areas are involved in vascular remodelling, implying the formation of new vessels, as as well as the disappearance of those previously developed.(ABSTRACT TRUNCATED AT 250 WORDS)