Karen L. Waldo

Neural Crest and Cardiovascular Patterning

Ablation of the cardiac neural crest has provided a powerful model of cardiovascular dysmorphogenesis because removal or manipulation of neural crest cells can be done before migration in sites distant from central cardiovascular development in chick embryos. Because the heart begins to function so early in its own morphogenetic history, the cardiovascular system is exquisitely sensitive to the embryonic environment, and virtually any manipulation of the embryo has cardiovascular consequences. Thus, although neural crest manipulation is not devoid of experimental artifact, it has resulted in significant advances in our understanding of certain of the factors involved in normal heart development in a way that direct manipulation of heart development does not. We have now identified two major roles for the neural crest in cardiovascular patterning. These are (1) participation in the patterning of the pharyngeal arches and their derivatives, including the aortic arch arteries, which will become the great arteries of the thorax, and (2) migration of a discrete population of neural crest cells into the cardiac outflow tract and participation in formation of the outflow septum. With the advent of genetically based animal models of cardiovascular dysmorphogenesis, some of which are neural crest related, it becomes important to understand what we know definitively from the ablation model along with the questions that remain. This review will focus on the roles of the neural crest in cardiovascular patterning as seen in the ablation model. An important part of the emerging neural crest–ablation phenotype is an early change in the functional competency of the developing myocardium. We believe that the neural crest–ablation phenotype provides a prototype for comparison with newer models of neural crest–related cardiovascular dysmorphogenesis and that the changes in myocardial functional development provide a means of understanding the early mortality associated with neural crest–type cardiac dysmorphogenesis. The four-chambered heart …

Cardiac neural crest is essential for the persistence rather than the formation of an arch artery

Double‐label immunohistochemistry was used to compare early aortic arch artery development in cardiac neural crest‐ablated and sham‐operated quail embryos ranging from stage 13 to stage 18. The monoclonal antibody QH‐1 labeled endothelial cells and their precursors, and HNK‐1 labeled migrating neural crest cells. In the sham‐operated embryos, the third aortic arch artery developed from a lumenizing strand of endothelial precursors that became separated from the pharyngeal endoderm by migrating cardiac neural crest cells as they ensheathed the artery. The arch artery of the neural crest‐ablated embryos lumenized but failed to become separated from the pharyngeal endoderm, indicating that neural crest is unnecessary for the early formation of the aortic arch artery. However, once blood flow was initiated through the third arch artery of crest‐ablated embryos at stage 16, the artery became misshapen and sinusoidal. By embryonic day 3, abnormal connections to the dorsal aorta occurred and bilateral symmetry was lost, suggesting that the loss of neural crest‐derived ectomesenchyme destabilizes the nascent artery. Although here we show no loss of the third arch artery, past studies have reported hypoplasia or missing carotids in older neural crest‐ablated embryos (Bockman et al. [1987] Am. J. Anat. 180:332–341; Bockman et al. [1989] Anat. Rec. 225:209–217; Nishibatake et al. [1987] Circulation 75:255–264; Tomita et al. [1991] Circulation 84:1289–1295). We suggest that the cardiac neural crest is essential for the persistence of an arch artery, but not its formation. Furthermore, since changes in the development of the arch artery are seen prior to the formation of the tunica media, it is suggested that a critical period is reached in the development of the arch artery, after lumenization, but prior to the formation of the tunica media, which necessitates the presence of the cardiac neural crest.

A NOVEL ROLE FOR CARDIAC NEURAL CREST IN HEART DEVELOPMENT

Ablation of premigratory cardiac neural crest results in defective development of the cardiac outflow tract. The purpose of the present study was to correlate the earliest functional and morphological changes in heart development after cardiac neural crest ablation. Within 24 hours after neural crest ablation, the external morphology of the hearts showed straight outflow limbs, tighter heart loops, and variable dilations. Incorporation of bromodeoxyuridine in myocytes, an indication of proliferation, was doubled after cardiac neural crest ablation. The myocardial calcium transients, which are a measure of excitation-contraction coupling, were depressed by 50% in both the inflow and outflow portions of the looped heart tube. The myocardial transients could be rescued by replacing the cardiac neural crest. The cardiac jelly produced by the myocardium was distributed in an uneven, rather than uniform, pattern. An extreme variability in external morphology could be attributed to the uneven distribution of cardiac jelly. In the absence of cardiac neural crest, the myocardium was characterized by somewhat disorganized myofibrils that may be a result of abnormally elevated proliferation. In contrast, endocardial development appeared normal, as evidenced by normal expression of fibrillin-2 protein (JB3 antigen) and normal formation of cushion mesenchyme and trabeculae. The signs of abnormal myocardial development coincident with normal endocardium suggest that the presence of cardiac neural crest cells is necessary for normal differentiation and function of the myocardium during early heart development. These results indicate a novel role for neural crest cells in myocardial maturation.

Gap Junction Communication and the Modulation of Cardiac Neural Crest Cells

The analyses of transgenic and knockout mice with perturbations in alpha 1 connexin (Cx43) function have revealed an important role for gap junctions in cardiac development. This likely involves the modulation of cardiac crest migration and function. Studies carried out with these mouse models suggest that clinically there may be a novel category of cardiac defects involving crest perturbations that do not include outflow septation defects, but rather involve more subtle defects in the pulmonary outflow tract.

Hensen’s node gives rise to the ventral midline of the foregut: implications for organizing head and heart development

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensens node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

A Novel Role for Cardiac Neural Crest in Heart Development

It is well known that cardiac neural crest participates in development of the cardiac outflow septation and patterning of the great arteries. Less well known is that ablation of the cardiac neural crest leads to a primary myocardial dysfunction. Recent data suggests that the myocardial dysfunction occurs because of the absence of an interaction of neural crest and pharyngeal endoderm to alter signaling from the endoderm. Continuation of an FGF-like signal from the endoderm past a precise time in development appears to be detrimental to myocardial maturation.

Myocardial volume and organization are changed by failure of addition of secondary heart field myocardium to the cardiac outflow tract

Cardiac neural crest ablation results in primary myocardial dysfunction and failure of the secondary heart field to add the definitive myocardium to the cardiac outflow tract. The current study was undertaken to understand the changes in myocardial characteristics in the heart tube, including volume, proliferation, and cell size when the myocardium from the secondary heart field fails to be added to the primary heart tube. We used magnetic resonance and confocal microscopy to determine that the volume of myocardium in the looped heart was dramatically reduced and the compact layer of myocardium was thinner after neural crest ablation, especially in the outflow tract and ventricular regions. Proliferation measured by 5‐bromo‐2′‐deoxyuridine incorporation was elevated at only one stage during looping, cell death was normal and myocardial cell size was increased. Taken together, these results indicate that there are fewer myocytes in the heart. By incubation day 8 when the heart would have normally completed septation, the anterior (ventral) wall of the right ventricle and right ventricular outflow tract was significantly thinner in the neural crest‐ablated embryos than normal, but the thickness of the compact myocardium was normal in all other regions of the heart. The decreased volume and number of myocardial cells in the heart tube after neural crest ablation most likely reflects the amount of myocardium added by the secondary heart field. Development Dynamics 228:152–160, 2003.

Development of the Great Arteries

Most information about the embryology of the great arteries in the human comes from elaborate descriptions by researchers such as Congdon (1922) and Padget (1948) earlier in this century. A broader understanding of the mechanisms behind vascular development has been gained more recently from animal models other than humans. Because avian embryos such as chick and quail are used extensively in developmental cardiovascular research, some of the major differences between human and chick will be mentioned as we discuss the development of the great arteries in humans. To avoid confusion in the ensuing discussion, “aortic arch” will refer to the definitive arch whereas “aortic arch artery” refers to one of the transient pharyngeal arch arteries that are precursors of all the adult great arteries.