Margaret L. Kirby

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 …

Pathogenesis of persistent truncus arteriosus and dextroposed aorta in the chick embryo after neural crest ablation.

To investigate the contribution of cranial neural crest cells to the developing cardiovascular system in the chick embryo, cauterization of various regions of cranial neural crest was performed. Five regions may be distinguished, each of which contributes mesenchyme to pharyngeal (branchial) arches 1 through 4 and 6. Ablation of arch 3, 4, and 6 regions resulted in a high incidence of persistent truncus arteriosus (PTA) associated with anomalies of the aortic arch. Dextroposed aorta (DPA) or anomalies of the inflow tract were found in all ablation groups. Although anomalies of the aortic arch arteries were induced in all ablation groups and were usually associated with intracardiac anomalies, those of the third and right fourth aortic arch were most frequent in the arch 4 and arch 4 + 6 groups. Anomalies of the sixth aortic arch were most frequent after extensive ablations that included the arch 6 region. We speculate that PTA is a direct result of the decreased population of mesenchymal cells derived from the arch 3 through 6 neural crest regions. DPA or anomalies of the inflow tract may be related to altered hemodynamics due to anomalies induced by neural crest ablation. Anomalies of the aortic arch arteries may be caused by either the direct or indirect process.

Myocardial Lineage Development

The myocardium of the heart is composed of multiple highly specialized myocardial lineages, including those of the ventricular and atrial myocardium, and the specialized conduction system. Specification and maturation of each of these lineages during heart development is a highly ordered, ongoing process involving multiple signaling pathways and their intersection with transcriptional regulatory networks. Here, we attempt to summarize and compare much of what we know about specification and maturation of myocardial lineages from studies in several different vertebrate model systems. To date, most research has focused on early specification, and although there is still more to learn about early specification, less is known about factors that promote subsequent maturation of myocardial lineages required to build the functioning adult heart.

Neural crest origin of cardiac ganglion cells in the chick embryo: Identification and extirpation

Using interspecific grafting of neural crest between quail and chick embryos, it was determined that the cardiac ganglia originate from the cranial region (somites 1-2) of the vagal neural crest (somites 1-7). Neuronal uptake of [3H]choline was used as an index of neuronal development in the chick atrium. Normal uptake was found to be quite high between Days 8 and 14 of incubation. Following extirpation of neural crest over somites 1 to 3 at stages 8 to 10, neuronal uptake in 8-day chick atrium was decreased by 25-60% depending on the stage at which the lesion was performed. It is thought that the residual uptake represents preganglionic terminals from the dorsal motor nucleus of the vagus. Embryos with extirpations of neural crest over somites 1-3 performed at stage 9 showed the greatest decrease of neuronal choline uptake and did not live beyond 11 days of incubation. However, hearts from embryos with partial lesions (performed at stage 10) were treated on incubation Days 12 and 15 for demonstration of acetylcholinesterase in the subepicardial plexus. These hearts showed much less extensive neural plexus with sparse, small cardiac ganglia.

The role of secondary heart field in cardiac development.

Although de la Cruz and colleagues showed as early as 1977 that the outflow tract was added after the heart tube formed, the source of these secondarily added cells was not identified for nearly 25 years. In 2001, three pivotal publications described a secondary or anterior heart field that contributed to the developing outflow tract. This review details the history of the heart field, the discovery and continuing elucidation of the secondarily adding myocardial cells, and how the different populations identified in 2001 are related to the more recent lineage tracing studies that defined the first and second myocardial heart fields/lineages. Much recent work has focused on secondary heart field progenitors that give rise to the myocardium and smooth muscle at the definitive arterial pole. These progenitors are the last to be added to the arterial pole and are particularly susceptible to abnormal development, leading to conotruncal malformations in children. The major signaling pathways (Wnt, BMP, FGF8, Notch, and Shh) that control various aspects of secondary heart field progenitor behavior are discussed.

Cardiac morphogenesis--recent research advances.

ABSTRACT: It has been demonstrated recently that a specific region of neural crest contributes cells to the septa of the outflow tract of the heart. Removal of this region of cardiac neural crest prior to migration from the neural fold results in persistent truncus arteriosus in chick embryos. Removal of other regions of cranial neural crest results in double outlet right ventricle. Since double outlet right ventricle is produced by manipulation of noncardiac neural crest, this malformation is thought to be an indirect rather than direct effect of neural crest ablation. The cranial neural crest forms the walls of all of the aortic arch arteries and it is proposed herein that flow abnormalities are produced in the pharyngeal region by injury to the neural crest. These abnormal hemodynamic characteristics influence heart development. Cardiac neural crest seeds the heart with parasympathetic postganglionic neurons as well as ectomesenchyme. Removal of the cardiac neural crest results in cardiac malformations because of the decreased ectomesenchymal cells. However, the neural population undergoes regeneration and so the innervation of malformed hearts is morphologically normal. The mechanism for this regeneration is not understood.

Optical Coherence Tomography A New High-Resolution Imaging Technology to Study Cardiac Development in Chick Embryos

Background—Optical coherence tomography (OCT) is a depth-resolved, noninvasive, non-destructive imaging modality, the use of which has yet to be fully realized in developmental biology. Methods and Results—We visualized embryonic chick hearts at looping stages using an OCT system with a 22 &mgr;m axial and 27 &mgr;m lateral resolution and an acquisition rate of 4000 A-scans per second. Normal chick embryos from stages 14 to 22 and sham-operated and cardiac neural crest-ablated embryos from stages 15 and 18 were scanned by OCT. Three-dimensional data sets were acquired and processed to create volumetric reconstructions and short video clips. The OCT-scanned embryos (2 in each group) were photographed after histological sectioning in comparable planes to those visualized by OCT. The optical and histological results showing cardiovascular microstructures such as myocardium, the cardiac jelly, and endocardium are presented. Conclusions—OCT is a powerful imaging modality which can provide new insight in assessing and understanding normal and abnormal cardiac development in a variety of animal models.

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 Caudal Proliferating Growth Center Contributes to Both Poles of the Forming Heart Tube

Recent studies have shown that the primary heart tube continues to grow by addition of cells from the coelomic wall. This growth occurs concomitantly with embryonic folding and formation of the coelomic cavity, making early heart formation morphologically complex. A scarcity of data on localized growth parameters further hampers the understanding of cardiac growth. Therefore, we investigated local proliferation during early heart formation. Firstly, we determined the cell cycle length of primary myocardium of the early heart tube to be 5.5 days, showing that this myocardium is nonproliferating and implying that initial heart formation occurs solely by addition of cells. In line with this, we show that the heart tube rapidly lengthens at its inflow by differentiation of recently divided precursor cells. To track the origin of these cells, we made quantitative 3D reconstructions of proliferation in the forming heart tube and the mesoderm of its flanking coelomic walls. These reconstructions show a single, albeit bilateral, center of rapid proliferation in the caudomedial pericardial back wall. This center expresses Islet1. Cell tracing showed that cells from this caudal growth center, besides feeding into the venous pole of the heart, also move cranially via the dorsal pericardial mesoderm and differentiate into myocardium at the arterial pole. Inhibition of caudal proliferation impairs the formation of both the atria and the right ventricle. These data show how a proliferating growth center in the caudal coelomic wall elongates the heart tube at both its venous and arterial pole, providing a morphological mechanism for early heart formation.

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.