Donna Kumiski

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.

Cardiac neural crest in zebrafish embryos contributes to myocardial cell lineage and early heart function

Myocardial dysfunction is evident within hours after ablation of the cardiac neural crest in chick embryos, suggesting a role for neural crest in myocardial maturation that is separate from its role in outflow septation. This role could be conserved in an animal that does not have a divided systemic and pulmonary circulation, such as zebrafish. To test this hypothesis, we used cell marking to identify the axial level of neural crest that migrates to the heart in zebrafish embryos. Unlike the chick and mouse, the zebrafish cardiac neural crest does not originate from the axial level of the somites. The region of neural crest cranial to somite 1 was found to contribute cells to the heart. Cells from the cardiac neural crest migrated to the myocardial wall of the heart tube, where some of them expressed a myocardial phenotype. Laser ablation of the cardiac premigratory neural crest at the three‐ to four‐somite stage resulted in loss of the neural crest cells migrating to the heart as shown by the absence of AP2‐ and HNK1‐expressing cells and failure of the heart tube to undergo looping. Myocardial function was assessed 24 hr after the cardiac neural crest ablation in a subpopulation of embryos with normal heart rate. Decreased stroke volume, ejection fraction, and cardiac output were observed, indicating a more severe functional deficit in cardiac neural crest‐ablated zebrafish embryos compared with neural crest–ablated chick embryos. These results suggest a new role for cardiac neural crest cells in vertebrate cardiac development and are the first report of a myocardial cell lineage for neural crest derivatives. Developmental Dynamics 226:000–000, 2003.

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.

FGF-8 in the ventral pharynx alters development of myocardial calcium transients after neural crest ablation

Cardiac neural crest ablation results in depressed myocardial calcium transients and elevated proliferation in myocardium at a stage when cardiac neural crest cells are not in contact with the myocardium. To test the hypothesis that cardiac neural crest-derived cells, which migrate into the caudal, ventral pharynx at stage 14, block a signal from the ventral pharynx, we cultured stage 12 chick heart tube or myocardial strips in the presence or absence of ventral pharynx. We found that myocardium cultured with ventral pharynx that had not yet contacted neural crest cells had significantly reduced calcium transients and an increased rate of proliferation. Ventral pharynx from intact embryos at a stage when neural crest-derived cells had reached the pharynx had no effect on myocardial calcium transients. Ventral pharynx from neural crest-ablated embryos continued to suppress myocardial calcium transients at this later stage. Myocardium cultured with FGF-2 also showed a significant reduction in calcium transients. An FGF-2-neutralizing Ab reversed the deleterious effect of the ventral pharynx on myocardial calcium transients and proliferation. We therefore examined the expression of FGF-2 and similar FGFs in the ventral pharynx. Only FGF-8 was expressed in a temporospatial pattern that made it a viable candidate for altering the myocardial calcium transient during stages 14-18. In explant cultures, neutralizing Ab for FGF-8 rescued development of the myocardial calcium transient in neural crest-ablated chick embryos.

Compensatory responses and development of the nodose ganglion following ablation of placodal precursors in the embryonic chick (Gallus domesticus).

The nodose ganglion is the distal cranial ganglion of the vagus nerve which provides sensory innervation to the heart and other viscera. In this study, removal of the neuronal precursors which normally populate the right nodose ganglion was accomplished by ablating the right nodese placode in stage 9 chick embryos. Subsequent histological evaluation showed that in 54% of lesioned embryos surviving to day 6, the right ganglion was absent. Most embryos surviving to day 12, however, had identifiable right ganglia. In day 12 embryos, the right ganglion which developed was abnormal, with ganglion volume and ganglion cell diameter reduced by 50% and 20%, respectively, compared to control ganglia. To investigate the source of the neuron population in the regenerated ganglion, we combined nodose placode ablation with bilateral replacement of chick with quail “cardiac” neural crest (from mid-otic placode to somite 3). These cells normally provide only non-neuronal cells to the nodose ganglion, but produce neurons in other regions. At day 9, quail-derived neurons were identified in the right nodose ganglia of these chimeras, indicating that cardiac neural crest cells can generate neurons in the ganglion when placode-derived neurons are absent or reduced in number. On the other hand, we found that “sympathetic” neural crest (from somites 10 to 20) does not support ganglion development, suggesting that only neural crest cells normally present in the ganglion participate in reconstituting its neuronal population. Our previous work has shown that right nodose placode ablation produces abnormal cardiac function, which mimics a life-threatening human heart condition known as long QT syndrome. The present results suggest that the presence of neural crest-derived neurons in the developing right nodose ganglion may contribute to the functional abnormality in long QT syndrome.