Premigratory Cardiac Neural Crest Research Articles

FGF8 signaling is chemotactic for cardiac neural crest cells

Cardiac neural crest cells migrate into the pharyngeal arches where they support development of the pharyngeal arch arteries. The pharyngeal endoderm and ectoderm both express high levels of FGF8. We hypothesized that FGF8 is chemotactic for cardiac crest cells. To begin testing this hypothesis, cardiac crest was explanted for migration assays under various conditions. Cardiac neural crest cells migrated more in response to FGF8. Single cell tracing indicated that this was not due to proliferation and subsequent transwell assays showed that the cells migrate toward an FGF8 source. The migratory response was mediated by FGF receptors (FGFR) 1 and 3 and MAPK/ERK intracellular signaling. To test whether FGF8 is chemokinetic and/or chemotactic in vivo, dominant negative FGFR1 was electroporated into the premigratory cardiac neural crest. Cells expressing the dominant negative receptor migrated slower than normal cardiac neural crest cells and were prone to remain in the vicinity of the neural tube and die. Treating with the FGFR1 inhibitor, SU5402 or an FGFR3 function-blocking antibody also slowed neural crest migration. FGF8 over-signaling enhanced neural crest migration. Neural crest cells migrated to an FGF8-soaked bead placed dorsal to the pharynx. Finally, an FGF8 producing plasmid was electroporated into an ectopic site in the ventral pharyngeal endoderm. The FGF8 producing cells attracted a thick layer of mesenchymal cells. DiI labeling of the neural crest as well as quail-to-chick neural crest chimeras showed that neural crest cells migrated to and around the ectopic site of FGF8 expression. These results showing that FGF8 is chemotactic and chemokinetic for cardiac neural crest adds another dimension to understanding the relationship of FGF8 and cardiac neural crest in cardiovascular defects.

Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract

The patterning of the cardiovascular system into systemic and pulmonic circulations is a complex morphogenetic process, the failure of which results in clinically important congenital defects. This process involves extensive vascular remodeling and coordinated division of the cardiac outflow tract (OFT). We demonstrate that the homeodomain transcription factor Pbx1 orchestrates separate transcriptional pathways to control great-artery patterning and cardiac OFT septation in mice. Pbx1-null embryos display anomalous great arteries owing to a failure to establish the initial complement of branchial arch arteries in the caudal pharyngeal region. Pbx1 deficiency also results in the failure of cardiac OFT septation. Pbx1-null embryos lose a transient burst of Pax3 expression in premigratory cardiac neural crest cells (NCCs) that ultimately specifies cardiac NCC function for OFT development, but does not regulate NCC migration to the heart. We show that Pbx1 directly activates Pax3, leading to repression of its target gene Msx2 in NCCs. Compound Msx2/Pbx1-null embryos display significant rescue of cardiac septation, demonstrating that disruption of this Pbx1-Pax3-Msx2 regulatory pathway partially underlies the OFT defects in Pbx1-null mice. Conversely, the great-artery anomalies of compound Msx2/Pbx1-null embryos remain within the same spectrum as those of Pbx1-null embryos. Thus, Pbx1 makes a crucial contribution to distinct regulatory pathways in cardiovascular development.

Cardiac neural crest ablation alters aortic smooth muscle force and voltage-sensitive Ca2+ responses.

Ablation of the premigratory cardiac neural crest (CNC) from the chick embryo results in a malformed outflow tract vasculature termed persistent truncus arteriosus (PTA). In addition, loss of the CNC disrupts myocardial excitation-contraction (EC) coupling, decreases intracellular Ca2+ transients, and depresses force generation. We examined if similar defects occurred in the neural crest-derived smooth muscle of the aortic arch in a test of the hypothesis that loss of elements from the CNC disrupts EC coupling and force production in the smooth muscle of the tunica media of the aortic arch. Aortic arch segments from chicks (embryonic day 15) displaying PTA generated approximately 43% of stress generated by the aortic arch from sham-operated control embryos during potassium depolarization. The depressed force response was associated with a twofold lower Fura-2 transient. In contrast, force and steady-state Fura-2 signals during endothelin-1 stimulation were unchanged. The differences seen in stress generation with potassium depolarization between sham and PTA displaying embryos were not seen in the descending aorta, a tissue not derived from the neural crest. Protein content and immunostaining revealed no differences in the content of actin, myosin, or dihydropyridine receptor from sham or PTA aortic arch. Our results suggest that the CNC is required for normal aortic arch smooth muscle function and support the hypothesis that the loss of CNC impacts the force generating ability, in part by disruption of the EC-coupling processes and altering Ca(2+)-handling.

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.

Lineage and development of the parasympathetic nervous system of the embryonic chick heart.

We were interested in the contribution of the cardiac neural crest to the complete anterior and posterior nerve plexus of the chick heart. This includes the pathways by which these cardiac neural crest-derived neuronal precursors enter the heart. As lineage techniques we used the traditional quail-chick chimera in combination with the newly introduced technique of retroviral reporter gene transfer to premigratory cardiac neural crest cells. Retrovirally infected embryos (n=23) and quail-chick chimeras (n=19) between stages HH27 and 40, were immunohistochemically evaluated, using the lineage markers LacZ (retroviral reporter) and QCPN (anti-quail nuclear marker), respectively and the neuronal differentiation markers HNK-1, RMO-270 and DO-170. Between stages HH27 and 33, quail-derived and LacZ positive cells were situated around the arterial cardiac vagal branches at the arterial pole, and vagal branches along the anterior cardinal veins and the sinal vagal branch at the venous pole. From stage HH35 onward, QCPN/LacZ-positive cardiac ganglia were observed throughout the anterior and posterior plexus and were mainly concentrated in the subepicardium near the distal ends of the arterial cardiac vagal branches and the sinal cardiac vagal branch respectively. From stage HH36 both the anterior and posterior plexus contained a population of large cardiac ganglion cells and a population of smaller cells along nerve branches as well as in the cardiac ganglia, which means that differentiation starts in both plexus at the same time. Furthermore only nerve fiber connections between the anterior and posterior plexus were observed. These results show that the cardiac neural crest contributes to the cardiac ganglion cells from both the entire anterior and posterior plexus. Furthermore these results suggest that these precursor cells enter the arterial pole via the arterial cardiac vagal branches and the venous pole via the sinal cardiac vagal branch without intermixing. Finally we show that in addition to the cardiac ganglia, the cardiac neural crest contributes to small myocardial glia or undifferentiated cells along nerve fibers, and some myocardial nerve fibers as well as nerve tissue in the adventitia of the large veins at the venous pole and in the adventitia of the coronary arteries.

Absence of Neural Crest Cell Regeneration from the Postotic Neural Tube

The preotic neural tube has a variable ability for regeneration of neural crest depending on the neuraxial level. There is robust regeneration of neural crest in the caudal midbrain/rostral hindbrain. In contrast, removal of the cardiac neural crest results in cardiovascular abnormalities suggesting the lack of regeneration in this area, although the regenerative capacity of the cardiac crest region has never been tested directly. Premigratory cardiac neural crest was ablated bilaterally using laser irradiation or extirpation by tungsten needle, and the remaining ventral neural tube was labeled with DiI to examine any neural crest regeneration from the neural tube. The results indicate that there is very little regeneration of crest cells from the cardiac region of the neural tube if the ablation is done prior to the 5-somite stage and no regeneration after the 6-somite stage with either ablation procedure. Furthermore no compensatory response occurs from the adjacent regions of the neural crest. By contrast, we were able to confirm that regeneration of neural crest occurs in the preotic rhombencephalic neural tube even after laser irradiation. An analysis in the trunk region suggests that the trunk neural tube is similar to the cardiac region in that it does not regenerate crest cells in the ventral migratory pathway after ablation. However, melanocytes generated cranial and caudal to the ablated region migrate radially and fill in the ablated region so that there is no interruption of the normal pigment pattern. This study indicates that even though there is a variable capacity for crest regeneration in the preotic neural tube, the postotic neural tube does not have such regenerative ability.

Absence of correlation between transient cranial hemorrhages and congenital malformations following neural crest ablation in chicks.

Ablation of premigratory cardiac neural crest has been used to produce and study extensively a model of abnormal cardiovascular dysmorphology. Previous and continuing research in this laboratory concerns different aspects of the involvement of cranial neural crest in the development of cranial, cervical and cardiac tissues in chick embryos. Recently, we detected the occurrence of transient cranial hemorrhages 24-48 hr after the ablation of selected segments of premigratory cranial neural crest. Since one possible mechanism of action for certain teratogens involves nonreparable damage to a primordial embryonic tissue by an antecedent hemorrhage, the objective of this study was to determine which of three different neural crest ablations is associated with hemorrhages and whether subsequent congenital abnormalities were correlated with the ablation procedure and/or hemorrhage. Premigratory neural crest was ablated from 3 different sites, designated cardiac, mesencephalic and trunk crest, respectively, of stage 8-10 chick embryos. Sham-operated embryos were controls. At 24, 30, and 48 hr after ablation, each embryo was observed for the presence of hemorrhages. On incubation day 11 all the living embryos were killed, fixed, weighed, and analyzed for selected length measurements, developmental stage, and the types and rates of congenital abnormalities. Cardiac and mesencephalic ablation group embryos exhibited significant incidences of cranial hemorrhages and changes in many of the parameters analyzed. It was concluded that the cardiac and mesencephalic, but not the trunk neural crest ablations, produced significant changes in incubation day 11 embryos. However, there was no correlation between the abnormalities and the prior occurrence of the transient cranial hemorrhages.

Proteins Associated with Cardiac Neural Crest in the Pharyngeal Region of Early Chick Embryos

Normal septation and alignment of the outflow tract of the heart depends on ectomesenchymal cells derived from the cardiac neural crest. Removal of the premigratory cardiac neural crest results in a variety of cardiac anomalies. The sequence of events leading to these cardiac anomalies in the absence of the cardiac neural crest cell population is not fully understood. This study is the first in a series directed at understanding the role of the neural crest in cardiovascular development at the molecular level. We have begun by distinguishing proteins uniquely expressed by, or responding to the presence of, cardiac neural crest cells in the pharyngeal apparatus, as well as proteins that are seen in the absence of cardiac neural crest cells. Cardiac neural crest was removed by microcautery from stage 9 to 10 embryos. At stages 14, 18, and 21, the protein patterns in the pharyngeal regions of experimental and sham-operated embryos were compared using isoelectric focusing followed by SDS-polyacrylamide gel electrophoresis. Five proteins were found in sham-operated embryos at stages 14 and 18 that could not be seen in embryos with neural crest ablations. Four proteins were found in embryos with neural crest ablation at stages 14 and 18 that were not seen in sham-operated embryos. By stage 21, the protein patterns of both sham-operated and experimental embryos were identical. These results indicate that there may be a compensatory response to neural crest ablation as development proceeds.

Temporospatial study of the migration and distribution of cardiac neural crest in quail‐chick chimeras

It has been demonstrated that the septation of the outflow tract of the heart is formed by the cardiac neural crest. Ablation of this region of the neural crest prior to its migration from the neural fold results in anomalies of the outflow and inflow tracts of the heart and the aortic arch arteries. The objective of this study was to examine the migration and distribution of these neural crest cells from the pharyngeal arches into the outflow region of the heart during avian embryonic development. Chimeras were constructed in which each region of the premigratory cardiac neural crest from quail embryos was implanted into the corresponding area in chick embryos. The transplantations were done unilaterally on each side and bilaterally. The quail-chick chimeras were sacrificed between Hamburger-Hamilton stages 18 and 25, and the pharyngeal region and outflow tract were examined in serial paraffin sections to determine the distribution pattern of quail cells at each stage. The neural crest cells derived from the presumptive arch 3 and 4 regions of the neuraxis occupied mainly pharyngeal arches 3 and 4 respectively, although minor populations could be seen in pharyngeal arches 2 and 6. The neural crest cells migrating from the presumptive arch 6 region were seen mainly in pharyngeal arch 6, but they also populated pharyngeal arches 3 and 4. Clusters of quail neural crest cells were found in the distal outflow tract at stage 23.

The effects of high phenylalanine concentration on chick embryonic development

Cells from a particular portion of the cranial neural crest (cardiac neural crest) migrate from the neural fold into pharyngeal arches 3, 4 and 6, where they provide the support for the endothelium of the aortic arch arteries, and by migration into the outflow tract become involved in septation of the truncus arteriosus. Ablation of the premigratory cardiac neural crest results in persistent truncus arteriosus and other defects reminiscent of the DiGeorge syndrome in man. Removal of a small area of the cardiac neural crest causes a spectrum of heart defects classified together as dextraposed aorta including changes like that of Fallot's tetralogy in man. Some inflow tract anomalies have also been found. Pilot studies injecting phenylalanine into developing chick embryos at a very early stage had little effect on embryo viability or on the incidence of congenital heart defects. However, sham-treated animals produced predominantly small simple ventricular septal defects but phenylalanine-treated embryos had more serious and complex heart anomalies. It is not possible to say yet that congenital heart disease in the offspring of mothers with untreated phenylketonuria is due to phenylalanine-induced damage to the neural crest, but the pilot studies in chick suggest that this idea is worth pursuing.