Cardiac Neural Crest Migration Research Articles

Network-based analysis reveals the potential involvement of proteasome subunit alpha-2 in tetralogy of Fallot

Introduction: Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects. However, there is no effective therapeutic approach and current therapies have limited curative efficacy. Moreover, the exact etiology of TOF has remained largely unknown. Improved understanding of molecular mechanisms can give an insight into TOF pathogenesis and development of therapeutic approaches. Methods: Here, we conducted a systematic study on the right ventricular myocardium of 24 infants (16 ToF/8 control) using weighted gene co-expression network analysis (WGCNA) to identify meaningful modules or candidate biomarkers. Results: Co-expression network analysis by WGCNA suggested that a highly preserved turquoise module with 2,493 genes and a P-value of 3×10-11 was significantly correlated to TOF. The top 5 hub genes of this module were PSMA2, MYL12A, C11ORF71, COMMD6, and CREG1. The result of turquoise module enrichment showed that the most correlation topic in biological processes and KEGG pathways were positive regulation of cardiac neural crest migration involved in outflow tract morphogenesis and positive regulation of neural crest cell differentiation. Also, we recognized 4 FDA-approved drug candidates for other indications could potentially use for the treatment of TOF patients through regulation of two hub genes of the co-expression network (PSMA2 and NDUFA4). Our findings also showed that the 13 experimentally validated microRNAs regulated the co-expression network through 5 hub genes. Conclusion: We systematically recognized co-expressed gene modules and hub genes associated with TOF progression, which offered insights into the mechanisms underlying TOF progression and some potential drugs for the treatment of TOF.

The relationship between gene GATA6 defect and congenital heart disease

Recent studies have showed GATA6 may be a candidate gene of congenital heart disease (CHD).GATA6 is a member of the GATA transcription factors,and GATA6 is expressed within the precardiac mesoderm from early stages in its specification.Some studies about outflow tract (OFT) development show that GATA6 can directly regulate the migration of cardiac neural crest cells.Researchers find some missense mutation on GATA6 (p.A178V,p.L198V,p.D404Y,p.E460X) by directly sequencing.These mutations may influence transcriptional activity of GATA6 and are closely associated with CHD.Appropriate level of GATA6 in embryos is critical to cardiogenesis,and over-expression GATA6 can disrupt this process.Cardiogenesis needs a cascade of transcription factors includes GATA6 and other factors under fight temporal and spatial control.This paper reviews the latest studies about the relationship between gene GATA6 defect and CHD. Key words: GATA6 ; Congenital heart disease

Notch pathway regulation of neural crest cell development in vivo

The function of Notch signaling in murine neural crest-derived cell lineages in vivo was examined. Conditional gain (Wnt1Cre;Rosa(Notch)) or loss (Wnt1Cre;RBP-J(f/f)) of Notch signaling in neural crest cells (NCCs) in vivo results in craniofacial, cardiac, and trunk abnormalities. Severe craniofacial malformations are apparent in Wnt1Cre;Rosa(Notch) embryos, while less severe skull abnormalities are evident in Wnt1Cre;RBP-J(f/f) mice. Deficient cardiac neural crest migration, resulting in cardiac outflow tract malformations, occurs with increased or decreased Notch signaling in NCCs. Smooth muscle cell differentiation also is impaired in pharyngeal NCC derivatives in both Wnt1Cre;Rosa(Notch) and Wnt1Cre;RBP-J(f/f) embryos. Neurogenesis is absent and gliogenesis is increased in the dorsal root ganglia of Wnt1Cre;Rosa(Notch) embryos, while neurogenesis is increased and gliogenesis is decreased in Wnt1Cre;RBP-J(f/f) embryos. Together, these studies demonstrate essential cell-autonomous roles for appropriate levels of Notch signaling during NCC migration, proliferation, and differentiation with critical implications in craniofacial, cardiac, and neurogenic development and disease.

High‐affinity folate receptor in cardiac neural crest migration: A gene knockdown model using siRNA

Folate supplementation reduces the incidence of congenital heart defects, but the nature of this protective mechanism remains unclear. Immunolabeling demonstrated that the neural tube and neural crest (NC) cells were rich in the high-affinity folate receptor FOLR1and during the early stages of development FOLR1 was found principally in these cells. Suppression of Folr1 expression in the nascent cardiac NC by site-directed short-interfering RNA (siRNA) altered cardiac NC cell mitosis and subsequent migration patterns leading to abnormal development of the pharyngeal arch arteries (PAA) and outflow tract. qPCR analysis demonstrated that the siRNA treatment significantly reduced Folr1 24 hr after treatment. These treatments also significantly reduced mitosis in the neural tube, but adjacent, nontreated areas were unaffected. In summary, a brief reduction in the expression of Folr1 during a critical stage of NC development had long-term consequences for the development of the PAA and outflow tract.

Cardiac outflow tract septation failure in Pax3-deficient embryos is due to p53-dependent regulation of migrating cardiac neural crest

During neural tube closure, Pax3 is required to inhibit p53-dependent apoptosis. Pax3 is also required for migration of cardiac neural crest (CNC) from the neural tube to the heart and septation of the primitive single cardiac outflow tract into the aorta and pulmonary arteries. Whether Pax3 is required for CNC migration and outflow tract septation by inhibiting p53-dependent apoptosis is not known. In this study, mouse strains carrying reporters linked to Pax3 alleles were used to map the fate of CNC cells in embryos which were either Pax3-sufficient (expressing one or two functional Pax3 alleles) or Pax3-deficient (expressing two null Pax3 alleles), and in which p53 had been inactivated or not. Migrating CNC cells were observed in both Pax3-sufficient and -deficient embryos, but CNC cells were sparse and disorganized in Pax3-deficient embryos as migration progressed. The defective migration was associated with increased cell death. Suppression of p53, either by null mutation of the p53 gene, or administration of a p53 inhibitor, pifithrin-α, prevented the defective CNC migration and apoptosis in Pax3-deficient embryos, and also restored proper development of cardiac outflow tracts. These results indicate that Pax3 is required for cardiac outflow tract septation because it blocks p53-dependent processes during CNC migration.

Oxidative stress during diabetic pregnancy disrupts cardiac neural crest migration and causes outflow tract defects

Maternal diabetes increases risk for congenital malformations, particularly cardiac outflow tract defects. Maternal diabetes inhibits expression of Pax3 in neuroepithelium through hyperglycemia-induced oxidative stress. The neuroepithelium gives rise to the neural crest, and Pax3 expression in cardiac neural crest (CNC) is required for CNC migration to the heart and for outflow tract septation. Here we tested whether maternal diabetes, through hyperglycemia-induced oxidative stress, before the onset of CNC delamination, impairs CNC migration and cardiac outflow tract septation. CNC migration was mapped in mouse embryos whose mothers were diabetic, or transiently hyperglycemic, or in which oxidative stress was transiently induced, using reporters linked to Pax3 expression. CNC apoptosis was examined by TUNEL assay. Outflow tract septation was examined histologically and by gross inspection. Few, if any, migrating CNC cells were observed in embryos of diabetic mice, and this was associated with increased apoptosis along the path of CNC migration. Outflow tract defects were significantly increased in fetuses of diabetic mice. Notably, induction of hyperglycemia or oxidative stress on the day prior to the onset of Pax3 expression and CNC migration also impaired CNC migration, increased apoptosis, and caused outflow tract defects. However, antioxidants administered on the day prior to the onset of Pax3 expression and CNC migration prevented these effects of hyperglycemia or oxidative stress. In diabetic pregnancy, oxidative stress, which inhibits expression of genes required for CNC viability, causes subsequent CNC depletion by apoptosis during migration, which leads to outflow tract defects.

Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect.

A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by 'cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. We used focal cell labelling and orthotopic grafting, followed by whole embryo culture, to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonisation of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resembled wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotch cardiac neural crest defect, we performed reciprocal grafts of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrated normally in the +/+ environment, and +/+ cells migrated normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurred when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2H environment. We conclude that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment.

The Role of Neural Crest during Cardiac Development in a Mouse Model of DiGeorge Syndrome

The velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a genetic disorder characterized by phenotypic abnormalities of the derivatives of the pharyngeal arches, including cardiac outflow tract defects. Neural crest cells play a major role in the development of the pharyngeal arches, and defects in these cells are likely responsible for the syndrome. Most patients are hemizygous for a 1.5- to 3.0-Mb region of 22q11, that is suspected to be critical for normal pharyngeal arch development. Mice hemizygous for a 1.5-Mb homologous region of chromosome 16 (Lgdel/+) exhibit conotruncal cardiac defects similar to those seen in affected VCFS/DGS patients. To investigate the role of Lgdel genes in neural crest development, we fate mapped neural crest cells in Lgdel/+ mice and we performed hemizygous neural crest-specific inactivation of Lgdel. Hemizygosity of the Lgdel region does not eliminate cardiac neural crest migration to the forming aortic arches. However, neural crest cells do not differentiate appropriately into smooth muscle in both fourth and sixth aortic arches and the affected aortic arch segments develop abnormally. Tissue-specific hemizygous inactivation of Lgdel genes in neural crest results in normal cardiovascular development. Based on our studies, we propose that Lgdel genes are required for the expression of soluble signals that regulate neural crest cell differentiation.

Synthetic matrix metalloproteinase inhibitor decreases early cardiac neural crest migration in chicken embryos.

During early embryonic development, cardiac neural crest (NC) cells emerge from the forming neural tube, migrate beneath the ectoderm, enter the pharyngeal arches, and subsequently participate in the septation of the heart. Like tumor cells, NC cells penetrate through basement membranes and invade extracellular matrix during their emigration and migration and, therefore, are liable to use similar invasive mechanisms. Matrix metalloproteinases (MMPs) are a family of zinc proteolytic enzymes known to be important in cell migration and invasion of normal and metastatic cells. In an earlier study, we found that the spatial and temporal distribution pattern of MMP-2 positively correlates with cardiac NC migration, suggesting MMP enzymatic activity may be important in mediating cardiac cell NC migration. To test this hypothesis, a synthetic MMP inhibitor, KB8301, was used to block MMP enzymatic activity during in vitro and in vivo cardiac NC cell migration in chick embryos. Injection of KB8301 into the cell-free space adjacent to the neural tube at the level of the second somite before the NC cells emigrated caused major morphologic anomalies in embryos and disrupted cardiac NC morphogenesis. Unilateral injection of KB8301 at lower concentrations, significantly decreased cardiac NC migration on the injected side compared with the noninjected side and compared with that of the injected controls. This decrease correlated with a decrease in MMP activity in the embryos and was not attributable to differences in embryo size or rate of embryonic development after injection. KB8301 also significantly decreased the rate of NC cell motility and distance NC cells migrated from explanted neural tubes and increased cell area and perimeter. These data suggest that MMP enzymatic activity is an important mediator of early cardiac NC migration and that perturbation of endogenous MMP activity may lead to NC-related congenital defects.

Effect of elevated homocysteine on cardiac neural crest migration in vitro.

A positive correlation between elevated maternal homocysteine (Hcys) and an increased risk of neural tube, craniofacial, and cardiac defects is well known. Studies suggest Hcys perturbs neural crest (NC) development and may involve N-methyl-D-aspartate (NMDA) receptors (Rosenquist et al., 1999). However, there is no direct evidence that Hcys alters NC cell behavior. Here, we evaluated the effect of Hcys on cardiac NC cell migratory behavior in vitro. Neural tube segments from chick embryos treated in ovo with or without Hcys were placed in culture and the migratory behavior of emigrating NC cells was monitored. Hcys significantly increased in vitro NC cell motility at all embryonic stages examined. NC cell surface area and perimeter were also increased. However, the relative distance NC cells migrated from their original starting point only increased in NC cells treated in ovo at stage 6 or at the time neural tube segments were cultured. Cysteine had no effect. NMDA mimicked Hcys' effect on NC motility and migration distance but had no effect on cell area or perimeter. The noncompetitive inhibitor of NMDA receptors, MK801+, significantly inhibited NC cell motility, reduced migration distance, and also blocked the effects of NMDA and Hcys on NC motility and migratory distance in vitro. A monoclonal antibody directed against the NMDA receptor immunostained NC cells in vitro and, in western blots, bound a single protein with the appropriate molecular weight for the NMDA receptor in NC cell lysates. These data are consistent with the hypothesis that a Hcys-sensitive NMDA-like receptor is expressed by early emigrating NC cells or their precursors, which is important in mediating their migratory behavior. Perturbation of this receptor may be related to some of the teratogenic effects observed with elevated Hcys.

Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest.

The neural crest plays a crucial part in cardiac development. Cells of the cardiac subpopulation of cranial neural crest migrate from the hindbrain into the outflow tract of the heart where they contribute to the septum that divides the pulmonary and aortic channels. In Splotch mutant mice, which lack a functional Pax3 gene, migration of cardiac neural crest is deficient and aorticopulmonary septation does not occur. Downstream genes through which Pax3 regulates cardiac neural crest development are unknown. Here, using a combination of genetic and molecular approaches, we show that the deficiency of cardiac neural crest development in the Splotch mutant is caused by upregulation of Msx2, a homeobox gene with a well-documented role as a regulator of BMP signaling. We provide evidence, moreover, that Pax3 represses Msx2 expression via a direct effect on a conserved Pax3 binding site in the Msx2 promoter. These results establish Msx2 as an effector of Pax3 in cardiac neural crest development.

PlexinA2 and semaphorin signaling during cardiac neural crest development.

Classic studies using avian model systems have demonstrated that cardiac neural crest cells are required for proper development of the cardiovascular system. Environmental influences that perturb neural crest development cause congenital heart defects in laboratory animals and in man. However, little progress has been made in determining molecular programs specifically regulating cardiac neural crest migration and function. Only recently have complex transgenic tools become available that confirm the presence of cardiac neural crest cells in the mammalian heart. These studies have relied upon the use of transgenic mouse lines and fate-mapping studies using Cre recombinase and neural crest-specific promoters. In this study, we use these techniques to demonstrate that PlexinA2 is expressed by migrating and postmigratory cardiac neural crest cells in the mouse. Plexins function as co-receptors for semaphorin signaling molecules and mediate axon pathfinding in the central nervous system. We demonstrate that PlexinA2-expressing cardiac neural crest cells are patterned abnormally in several mutant mouse lines with congenital heart disease including those lacking the secreted signaling molecule Semaphorin 3C. These data suggest a parallel between the function of semaphorin signaling in the central nervous system and in the patterning of cardiac neural crest in the periphery.

Recovery from arterial growth delay reduces penetrance of cardiovascular defects in mice deleted for the DiGeorge syndrome region.

Chromosome 22q11.2 heterozygous deletions cause the most common deletion syndrome, including the DiGeorge syndrome phenotype. Using a mouse model of this deletion (named Df1) we show that the aortic arch patterning defects that occur in heterozygously deleted mice (Df1/+) are associated with a differentiation impairment of vascular smooth muscle in the 4th pharyngeal arch arteries (PAAs) during early embryogenesis. Using molecular markers for neural crest, endothelial cells and vascular smooth muscle, we show that cardiac neural crest migration into the 4th arch and initial formation of the 4th PAAs are apparently normal in Df1/+ embryos, but affected vessels are growth-impaired and do not acquire vascular smooth muscle. As in humans, not all deleted mice present with cardiovascular defects at birth. However, we found, unexpectedly, that all Df1/+ embryos have abnormally small 4th PAAs during early embryogenesis. Many embryos later overcome this early defect, coincident with the appearance of vascular smooth muscle differentiation, and develop normally. Embryos born with aortic arch patterning defects probably represent a more severely affected group that fails to attain sufficient 4th PAA growth for normal remodelling of the PAA system. Our data indicate that Df1/+ embryos are able to overcome a localized arterial growth impairment and thereby reduce the penetrance of birth defects.

Retinoic Acid Inhibits Cardiac Neural Crest Migration by Blocking c-Jun N-Terminal Kinase Activation

Retinoic acid (RA), a potent teratogen, produces a characteristic set of embryonic cardiovascular malformations similar to those observed in neural crest ablated avians. While the effects of RA on neural crest are well described, the molecular mechanism(s) of RA action on these cells is less clear. The present study examines the relationship between RA and mitogen-activated protein kinase signaling in neural crest cells and demonstrates that c-Jun N-terminal kinase (JNK) activation is severely repressed by RA. RA suppressed migration and proliferation of primary cultures of mouse neural crest cells treated in vitro as well as from animals treated in vivo. On Western blots, JNK activation/phosphorylation in neural crest cultures was reduced, while neither extracellular signal-regulated kinase (ERK) nor p38 pathways were affected. Both the dose-dependent stimulation of neural crest outgrowth and JNK phosphorylation by platelet-derived growth factor AA, which promotes outgrowth but not proliferation of neural crest cultures, were completely abrogated by RA. To establish the relevance of the JNK signaling pathway to cardiac neural crest migration, dominant negative adenoviral constructs were used to inhibit upstream activation of JNK or c-Jun downstream responses. Both adenoviral constructs markedly reduced neural crest cell outgrowth, while a dominant negative inhibitor of the p38 pathway had no effect. These data demonstrate that the JNK signaling pathway and c-Jun activation are critical for cardiac neural crest outgrowth and are potential targets for the action of RA.

Migration of cardiac neural crest cells in Splotch embryos.

Pax3 encodes a transcription factor expressed during mid-gestation in the region of the dorsal neural tube that gives rise to migrating neural crest populations. In the absence of Pax3, both humans and mice develop with neural crest defects. Homozygous Splotch embryos that lack Pax3 die by embryonic day 13.5 with cardiac defects that resemble those induced by neural crest ablation in chick models. This has led to the hypothesis that Pax3 is required for cardiac neural crest migration. However, cardiac derivatives of Pax3-expressing precursor cells have not been previously defined, and Pax3-expressing cells within the heart have not been well demonstrated. Hence, the precise role of Pax3 during cardiac development remains unclear. Here, we use a Cre-lox method to fate map Pax3-expressing neural crest precursors to the cardiac outflow tract. We show that although Pax3 itself is extinguished prior to neural crest populating the heart, derivatives of these precursors contribute to the aorticopulmonary septum. We further show that neural crest cells are found in the outflow tract of Splotch embryos, albeit in reduced numbers. This indicates that contrary to prior reports, Pax3 is not required for cardiac neural crest migration. Using a neural tube explant culture assay, we demonstrate that neural crest cells from Splotch embryos show normal rates of proliferation but altered migratory characteristics. These studies suggest that Pax3 is required for fine tuning the migratory behavior of the cardiac neural crest cells while it is not essential for neural crest migration.

Gap junction-mediated cell-cell communication modulates mouse neural crest migration.

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in alpha1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for alpha1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing alpha1 connexins, and from alpha1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative alpha1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and alpha1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous alpha1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and alpha1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the alpha1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium- an elevation in the CMV43 mice vs. a reduction in the alpha1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non-neural crest- derived tissues. Overall, these findings suggest that gap junction communication mediated by alpha1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of alpha1 connexin function.

Identification of Genes involved in Cardiac Development • 120

Objective: Cardiac morphogenesis in the mouse embryo occurs over a short period of time. More specifically, cardiac outflow tract and aortopulmonary septum formation occur between E9.5 and E13.5, concurrent with cardiac neural crest migration, which is required for conotruncal septation. The purpose of this study is to identify known and novel genes that participate in cardiac development either directly or indirectly through regulation of cardiac neural crest migration.

Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp2H) mutant.

Neural crest cells originating in the occipital region of the avian embryo are known to play a vital role in formation of the septum of the cardiac outflow tract and to contribute cells to the aortic arches, thymus, thyroid and parathyroids. This 'cardiac' neural crest sub-population is assumed to exist in mammals, but without direct evidence. In this paper we demonstrate, using RT-PCR and in situ hybridisation, that Pax3 expression can serve as a marker of cardiac neural crest cells in the mouse embryo. Cells of this lineage were traced from the occipital neural tube, via branchial arches 3, 4 and 6, into the aortic sac and aorto-pulmonary outflow tract. Confirmation that these Pax3-positive cells are indeed cardiac neural crest is provided by experiments in which hearts were deprived of a source of colonising neural crest, by organ culture in vitro, with consequent lack of up-regulation of Pax3. Occipital neural crest cell outgrowths in vitro were also shown to express Pax3. Mutation of Pax3, as occurs in the splotch (Sp2H) mouse, results in development of conotruncal heart defects including persistent truncus arteriosus. Homozygotes also exhibit defects of the aortic arches, thymus, thyroid and parathyroids. Pax3-positive neural crest cells were found to emigrate from the occipital neural tube of Sp2H/Sp2H embryos in a relatively normal fashion, but there was a marked deficiency or absence of neural crest cells traversing branchial arches 3, 4 and 6, and entering the cardiac outflow tract. This decreased expression of Pax3 in Sp2H/Sp2H embryos was not due to down-regulation of Pax3 in neural crest cells, as use of independent neural crest markers, Hoxa-3, CrabpI, Prx1, Prx2 and c-met also revealed a deficiency of migrating cardiac neural crest cells in homozygous embryos. This work demonstrates the essential role of the cardiac neural crest in formation of the heart and great vessels in the mouse and, furthermore, shows that Pax3 function is required for the cardiac neural crest to complete its migration to the developing heart.

Cardiac neural crest contribution to the pulmonary artery and sixth aortic arch artery complex in chick embryos aged 6 to 18 days.

Previous studies of cardiac neural crest (CNC) migration in early chick embryos demonstrated CNC cells in the media of pharyngeal arch arteries three, four, and six, and in the most proximal part of the developing pulmonary arteries. The objectives of this study were to learn 1) to what extent the CNC is involved in the later development of the pulmonary arteries, 2) how the CNC cells are distributed in the sixth aortic arch artery including the wall of the ductus arteriosus in the older embryo, and 3) what happens to the CNC as the pulmonary artery/sixth arch complex grows into its adult configuration. Quail-to-chick chimeras were used to study CNC distribution in embryos aged 6 to 18 days. Controls (undisturbed chick embryos) were collected with chimeras. Each was fixed, processed, sectioned, stained with Feulgen-Rossenbeck stain, and analyzed. The results demonstrated that CNC disappeared from the proximal pulmonary arteries by embryonic day 9 and played no further role in pulmonary artery development. With the exception of the endothelium, CNC completely filled the wall of the sixth aortic arch artery as far distally as its junction with the dorsal aorta in younger embryos and with the aorta in older embryos, thus suggesting the possibility of proximodistal migration of CNC along the sixth aortic arch. The ductus wall, filled with CNC, was intimately associated with the recurrent laryngeal nerve, also filled with CNC, thereby strongly suggesting a role for CNC in ductal closure.