Reduced dosage of ß-catenin provides significant rescue of cardiac outflow tract anomalies in a Tbx1 conditional null mouse model of 22q11.2 deletion syndrome.

The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of TBX1, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of Tbx1 in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical Wnt/ß-catenin has major roles in cardiac OFT development that may act upstream of Tbx1. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in ß-catenin loss of function embryos compared to Tbx1 loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of Tbx1 and one allele of ß-catenin were inactivated in the Mef2c-AHF-Cre domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, ß-catenin was increased in expression when Tbx1 was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and Tbx1 in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.

Local and regional variations in myofibrillar patterns in looping rat hearts.

BACKGROUND: In chickens, cytodifferentiation, right side dominance in myofibril development, and variations in myofibrillar patterns in different areas and layers of the myocardial wall exist which have been implicated in the process of heart looping. Little comparable information is available for developing myofibrillar patterns in the early development of mammalian hearts. METHODS: We have used transmission electron microscopy (TEM), confocal scanning laser microscopy (CSLM), and 3-D reconstruction techniques also present in the looping hearts of embryonic day (ED) 9.5 to 11.5 rat hearts. RESULTS: Local and regional variations and right side dominance in myofibrillar patterns were shown during looping in 9.5 through 11.5 days of development in embryonic rat heart. At 9.5 days of development, myofibrils near the lumen of the myocardial wall were primarily in circumferential bands while near the pericardial surface they were primarily in longitudinal bands. In older embryos, regional variations in myofibrillar organization was found in areas associated with the cardiac cushions, trabeculae, and myocardial wall of the developing heart chambers. Based on sarcomeric structure, myofibrils in the ventricle and outflow tract were more advanced than those found in the atrial wall. CONCLUSIONS: The local and regional patterns of myofibrils in looping rat hearts are similar to those which have been found in developing chicken hearts. This study and others indicate cytodifferentiation and development of the contractile apparatus has a crucial role in the process of heart looping.

Development of the musculoelastic septation complex in the avian truncus arteriosus.

It is now well established that cells from the cardiac neural crest (CNC) are essential for normal conotruncal septation. The truncal septation complex consists of the aorticopulmonary (AP) septum and the myocardial sheath of the truncus. The principal role of the CNC cells during septation appears to be their differentiation into the elastogenic smooth muscle that forms the AP septum proper. The objective of this study was to integrate serial reconstruction and specific histochemical markers in order to provide a unified analysis of the relationships between the CNC and the other components of the truncal septation complex. The development of the septation complex was compared normal embryos vs. embryos from which the CNC had been surgically ablated. Embryos from each group were harvested after incubation periods of 4-8 days (Hamburger-Hamilton stages 23-34). Histochemical procedures were performed for positive identification of the elastic matrix and smooth muscle alpha-actin; the presence of these proteins was used as the criterion for "septal cells" and to define the boundaries of the septum. The results indicate that the shape, components, boundaries, and degree of organization of the septation complex may be different from previous descriptions. Furthermore, all of the components of the truncal septation complex are dysgenic in the absence of the CNC. Of special significance in the absence of CNC. Of special significance in the absence of CNC are: 1) the failure of the myocardial sheath to retract; 2) the apparently random distribution of surrogate ectomesenchyme; and 3) the impairment of truncal elastogenesis. These results indicate that the cells of neural crest origin interact with the surrounding mesenchyme during septation and that the entire septation complex depends upon the presence of the neural crest cells for normal development.

Distribution of vitronectin in the embryonic chick heart during endocardial cell migration.

In the early phase of heart development, the endocardial cells migrate into the truncal swellings and atrioventricular (AV) cushions, and become mesenchymal cells. Vitronectin is a glycoprotein which is thought to mediate cell migration. The present study demonstrates by immunohistochemistry the distribution of vitronectin in order to elucidate its contribution to endocardial cell migration in the developing chick heart. At Hamburger and Mamilton's stage 23, the network of fibrillar material filled the extracellular space of both truncal swellings and AV cushions. The fibrillar network has been thought to be a matrix for endocardial cell migration. The network was stained with the anti-vitronectin antibody. At stage 29, the swellings and cushions were packed with mesenchymal cells, though immunoreactivity to the antibody was still observed in the extracellular matrix. The myocardium facing the AV cushions reacted to the antibody, but the myocardium surrounding the truncus arteriosus did not. The intensity of the immunohistochemical staining of the myocardium facing the AV cushions increased and reached a peak at stages 24 to 26, and then became weak by stage 29. The endocardial sheet, aortico-pulmonary septum and developing tunica media of the great arteries were not stained by the antibody at any stage. These results strongly suggest that vitronectin is involved in the migration of endocardial cells, and that the myocardium facing the AV cushions produces vitronectin.

Distribution of the neural crest cells in the heart of birds: a three dimensional analysis.

The distribution of neural crest derived cells (NC) in the heart of quail-chick chimeric embryos was analyzed three-dimensionally after computer reconstruction. During the division of the truncus arteriosus into the aorta and the pulmonary trunk, ventral and dorsal columns of NC-derived cells were found in the truncal swellings. These columns were elongations from the aorticopulmonary (AP) septum. The dorsal column extended more proximally than did the ventral column. Around hatching, NC-derived cells located between the proximal aorta and the pulmonary trunk, differentiated into cartilage and connective tissue. They formed a part of the cardiac skeleton. A small number of NC-derived cells were scattered in the cusps of the arterial valves. Cells derived from the right NC were located around the aorta and the right arch arteries but not around the distal pulmonary trunk and the left arch arteries. At the proximal level, cells derived from the right NC were located in both the dorsal and ventral columns. These results suggest that the AP septum is mainly formed by NC-derived cells, right and left NC cells migrating into assigned areas in the heart. Location of two columns of NC-derived cells may support a translocation hypothesis for the AP septum during truncal division.

The distribution and ultrastructure of sensory elements in the baroreceptor region of the truncus arteriosus of the lizard Trachydosaurus rugosus.

The proximal truncus arteriosus of the lizard Trachydosaurus rugosus was studied with light-, fluorescence- and electron-microscopical techniques. Three vessels comprised the truncus; the pulmonary, left aortic, and carotico-aortic arteries. Right and left truncal nerves, each derived from the ipsilateral vagus nerve, innervated the truncus, particularly its proximal 3 mm. Ultrastructurally, the nerves had a variety of appearances: some were clearly adrenergic, c-type or p-type. A number of profiles contained large numbers of mitochondria and were classified as sensory. Some profiles defied exact classification, having characteristics common to two different types of profile. Within the outer medial layers, profiles up to 7 micrometers in diameter were found. These contained large numbers of mitochondria, myelin bodies and structures intermediate between the two. In addition, the profiles contained large amounts of glycogen and small numbers of vesicles. These nerve fibres were classified as baroreceptors, since they closely resemble carotid sinus and aortic arch baroreceptors in mammals. Large numbers of chromaffin cells were found, particularly in the common wall of the pulmonary and left aortic arteries. Many of these cells emitted a long tapering process, which sometimes entered a nearby nerve bundle. Sensory, p-type and c-type profiles, but not adrenergic profiles, made extensive close contacts with chromaffin cells.

Morphogenesis of the truncus arteriosus of the chick embryo heart: tissue reorganization during septation.

Septation of the truncus arteriosus of the normal chick embryo heart was surveyed systematically with the light microscope. Tissue from replicate samples at successive periods of development was sectioned within an arbitrary coordinate system based on positional reference points along the external surface of the heart. Correlation of several aspects of tissue morphology within this spatial and temporal reference-frame yielded a new description of tissue associations and kinetics during septation. A stable complex of tissue structures appeared in the downstream, distal truncus at Stage 25 and persisted throughout the septation process. This complex consisted of (1) the cephalic margin of the myocardial sheath, and (2) the adjacent bifurcation of the vascular lumen, linked together by (3) the newly condensed Y-shaped strap of cells forming the aorticopulmonary septum. The apparent motion of this septation-complex toward the ventricle(s), the appearance within the thoracic cavity of the adjacent segments of the aortic arches, and measures of tissue length and width suggested that septation was accompanied, and perhaps initiated, by increased tension along the truncus. The truncal ridges remained upstream from the complex, with mesenchymal condensations beneath the endocardium differentiating into the definitive semilunar valves. Downstream from the bifurcation, mesenchyme in the aortic arch region condensed around the separate lumens to form the smooth muscular tunica media of the great arteries. The epicardium developed in a caudocephalic direction along the heart tube. Vagal innervation approached the heart cephalocaudally. Capillaries formed along the dividing truncus in both directions. Autoradiography following 3H-thymidine labelling demonstrated reduced DNA synthetic activity in the cephalic margin of the myocardium and aorticopulmonary septum, compared with the associated loose mesenchyme.

Morphogenesis of the truncus arteriosus of the chick embryo heart: the formation and migration of mesenchymal tissue.

The appearance and migration of mesenchymal cushion tissue within the truncus arteriosus of the normal 2.5 to 6-day chick embryo heart was surveyed systemically with the light microscope. Series of cross-sections taken from replicate hearts at successive developmental stages allowed comparison of the following qualitative and quantitative aspects of early truncal morphogenesis. Mesenchyme within the truncus was derived from two distinct sources. The first mesenchyme appeared to migrate caudally into the cardiac jelly of the distal truncus from the nearby aortic arch region, coincident with slowing of the anterior elongation of the heart tube (Hamburger-Hamilton Stage 17-18). A second, separate mesenchymal population, derived from endocardium, began to fill the conus and proximal truncus in a radial direction, coicident with expansion of the bulbs cordis (Stage 12-19). The measured kinetics of relative cell numbers, distributions, and mitotic indices suggest substantial contributions from both sources. By Stage 26, the conotruncal region was filled with mesenchyme, which then condensed to form the anlagen of three future structures: the semilunar valves, the aorticopulmonary septum, and the tunica media of the great arteries.