Hemodynamic changes in chick embryos precede heart defects after cardiac neural crest ablation.

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.

During normal development, ectomesenchyme from the cardiac neural crest migrates to pharyngeal arches 3, 4, 6 and the developing heart. It participates in the formation of the aorticopulmonary septum and the wall of the great arteries. Removal of the cardiac neural crest resulted in anomalies of the great arteries and in two categories of severe heart defects: (1) outflow septation defects of the persistent truncus arteriosus (PTA) type, (2) alignment defects. It has been hypothesized that PTA occurs if the number of cardiac neural crest cells is reduced below a level critical for complete formation of the aorticopulmonary septum. Alignment defects would be indirect consequences of neural crest defects, possibly caused by altered blood flow in the pharyngeal arch region. We found that these concepts were not in agreement with some experimental facts reported previously, so we considered whether there could be other mechanisms responsible for the heart defects described. To investigate whether mechanical interference with cardiac looping could possibly contribute to the pathogenesis of these anomalies, we removed the entire cardiac neural crest in chick embryos with micro-needles. Postoperative development was checked during cardiac looping and after normal completion of cardiac septation. Our data suggested that abnormal cardiac looping did not contribute to the pathogenesis of the aortic arch artery anomalies and PTA. With respect to the alignment heart defects, we could not elucidate the role of looping anomalies because we did not observe such heart defects. Moreover, PTA occurred only in 28% of survivors. This finding conflicts with previous studies where extensive ablation of the cardiac neural crest has led to a high incidence of PTA (73-100% of survivors). The possible reasons for this discrepancy are discussed. It is shown that the use of different microsurgical techniques (mechanical cutting/microcautery) may be responsible for the different incidence of PTA. We speculate that microcautery hampers a normal complete repair of neural crest defects, possibly by release of abnormally high levels of growth factors.

Ablation of the cardiac neural crest results in abnormal development of the aortic arch arteries leading to altered patterning of the great arteries. The cardiac outflow tract is also affected after neural crest ablation because normally a subset of neural crest cells migrates from the pharyngeal region to form the outflow septum. Using neural crest ablation, it has not been possible to separate the occurrence of aortic arch maldevelopment from cardiac outflow tract dysmorphogenesis. In order to determine whether normal aortic arch artery development is a prerequisite for normal outflow tract development, we have used a combination of antisense treatment with backtransplantation of cardiac neural folds to produce abnormal patterning of the aortic arch arteries. Paralogous groups of Hox messages with their anterior expression domains in pharyngeal arches 3, 4 and 6 were targeted. Antisense targeted to paralogous group 3 Hox message caused aortic arch 3 located within the pharyngeal arch to regress in a manner similar to aortic arch 2, while antisense targeted to paralogous group 5 Hox message caused the appearance of an additional pharyngeal arch containing a novel and completely independent aortic arch artery. Antisense treatment targeting paralogous group 4 Hox message led to no detectable cardiovascular phenotype in the first 6 days of development. While regression of arch artery 3 was associated with abnormal branching patterns of the aorta and pulmonary trunk, this did not involve abnormal separation of the aorta and pulmonary trunks, the semilunar valves or the subvalvular region of the outflow tract. Because none of these changes in pharyngeal or aortic arch artery development was accompanied by abnormal development of the cardiac outflow tract, it appears that normal patterning of the aortic arch arteries is not a prerequisite for normal heart development. Using reverse transcription polymerase chain reaction (RT-PCR) we were unable to detect changes in any of the Hox messages except group 4, thus, using this particular experimental strategy, we are unable to demonstrate or refute that expression of hox genes by cardiac neural crest cells controls aortic arch patterning. Development of the cardiac outflow tract was normal in each instance. This suggests that abnormal aortic arch patterning does not necessarily lead to cardiac malformations.

Cardiac neural crest ablation alters Id2 gene expression in the developing heart

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.

In this study we focused upon whether different levels of postotic neural crest as well as the right and left cardiac neural crest show a segmented or mixed distribution in the extrinsic and intrinsic cardiac nervous system. Different parts of the postotic neural crest were labeled by heterospecific replacement of chick neural tube by its quail counterpart. Quail-chick chimeras (n = 21) were immunohistochemically evaluated at stage HH28+, HH29+, and between HH34-37. In another set of embryos, different regions of cardiac neural crest were tagged with a retrovirus containing the LacZ reporter gene and evaluated between HH35-37 (n = 13). The results show a difference in distribution between the right- and left-sided cardiac neural crest cells at the arterial pole and ventral cardiac plexus. In the dorsal cardiac plexus, the right and left cardiac neural crest cells mix. In general, the extrinsic and intrinsic cardiac nerves receive a lower contribution from the right cardiac neural crest compared with the left cardiac neural crest. The right-sided neural crest from the level of somite 1 seeds only the cranial part of the vagal nerve and the ventral cardiac plexus. Furthermore, the results show a nonsegmented overlapping contribution of neural crest originating from S1 to S3 to the Schwann cells of the cranial and recurrent nerves and the intrinsic cardiac plexus. Also the Schwann cells along the distal intestinal part of the vagal nerve are derived exclusively from the cardiac neural crest region. These findings and the smaller contribution of the more cranially emanating cardiac neural crest to the dorsal cardiac plexus compared with more caudal cardiac neural crest levels, suggests an initial segmented distribution of cardiac neural crest cells in the circumpharyngeal region, followed by longitudinal migration along the vagal nerve during later stages.

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

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.

N,N'-bis(dichloroacetyl)-1,8-octamethylenediamine(bis-diamin e) (100 micrograms) dissolved in dimethylsulfoxide (DMSO) was administered to early developing chick embryos (Hamburger-Hamilton stage 9-21) in order to clarify the teratogenic effects on the cardiovascular system and to determine whether bis-diamine interferes with the migration of neural crest cells. Of 346 cases, 154 (44.5%) survived. The incidence of cardiovascular anomalies was 149 out of 154 cases (96.8%). Infundibular ventricular septal defect, double outlet right ventricle, and persistent truncus arteriosus were the primary cardiac anomalies observed in this study. A high percentage of these anomalies were accompanied by hypoplasia of the right 6th aortic arch artery and persistent left 4th aortic arch artery. Particularly, administration of bis-diamine to chick embryos at stage 13 resulted in a high incidence of persistent truncus arteriosus (64.3%). Bis-diamine has been suspected to inhibiting the migration of neural crest cells. However, neural crest cells were observed in the tunica media of the great arteries and the truncal valves of persistent truncus arteriosus produced by bis-diamine in chimeric embryos at stage 13. Morphological changes such as cell death were not observed.

The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacial clefts, and conotruncal heart defects in the offspring has been shown. Folate shortage results in homocysteine accumulation. Elevated levels of homocysteine have been related to neural tube defects. We studied the behavior of neuroepithelial cells and cranial and cardiac neural crest cells in vitro. Neural tube explants were cultured for 24 and 48 hr in medium after addition of folic acid and/or homocysteine. Folic acid addition increased neuroepithelial cell outgrowth and increased neural crest cell differentiation into nerve and smooth muscle cells. Addition of homocysteine increased neural crest cell outgrowth and migration from the neural tube and inhibited neural crest cell differentiation. Our findings suggest that neural tube defects caused by folate deficiency and hyperhomocysteinemia develop due to increased neuroepithelial to neural crest cell transformation. This increased transformation leads to a shortage of neuroepithelial cells in the neural tube. Defects in orofacial and conotruncal development are explained by abnormal differentiation of neural crest cells in the presence of high homocysteine concentrations. Our findings supports a critical role for folic acid and homocysteine in the development of neural tube defects and neural crest related heart malformations.

FGF8 signaling is chemotactic for cardiac neural crest cells

Retroviral and transgenic lineage-tracing studies have shown that neural crest cells associate with the developing bundles of the ventricular conduction system. Whereas this migration of cells does not provide progenitors for the myocardial cells of the conduction system, the question of whether neural crest affects the differentiation and/or function of cardiac specialized tissues continues to be of interest. Using optical mapping of voltage-sensitive dye, we determined that ventricles from chick embryos in which the cardiac neural crest had been laser ablated did not progress to apex-to-base activation by the expected stage [i.e., Hamburger and Hamilton (HH) 35] but instead maintained basal breakthroughs of epicardial activation consistent with immature function of the conduction system. In direct studies of activation, waves of depolarization originating from the His bundle were found to be uncommon in control hearts from HH34 and HH35 embryos. However, activations propagating from septal base, at or near the His bundle, occurred frequently in hearts from HH34 and HH35 neural crest-ablated embryos. Consistent with His bundle cells maintaining electrical connections with adjacent working myocytes, histological analyses of hearts from neural crest-ablated embryos revealed His bundles that had not differentiated a lamellar organization or undergone a process of compaction and separation from surrounding myocardium observed in controls. Furthermore, measurements on histological sections from optically mapped hearts indicated that, whereas His bundle diameter in control embryos thinned by almost one-half between HH30 and HH34, the His bundle in ablated embryos underwent no such compaction in diameter, maintaining a thickness at HH30, HH32, and HH34 similar to that observed in HH30 controls. We conclude that the cardiac neural crest is required in a novel function involving lamellar compaction and electrical isolation of the basally located His bundle from surrounding myocardium.

Congenital anomalies such as ventricular septal defects and truncus communis have been reported with the prenatal use of antiretroviral therapy. The mechanism of antiretroviral therapy teratogenicity is unclear and is therefore the focus of this study. Some human immunodeficiency virus patients on antiretrovirals are placed on antiepileptic drugs which are also teratogenic. The interactive effects arising from this therapeutic combination may affect their teratogenic propensity through their effects on neural crest cell migration. Appropriately cultured neural crest cells from dissected neural tubes of 32-hr old quail embryos exposed to culture media containing peak plasma levels of Atripla, Topiramate and the combination of both were studied. Distance of migration of neural crest cells was measured using the migration assay and the cells were stained with rhodamine phalloidin to evaluate the cell actin. Also quail neural crest cells were brought into suspension and microinjected into chick hosts to determine the migration of the cells to the interventricular septum. Migration of cultured neural crest cells was extensive in the control cultures, but inhibited in the treated groups. The experimental cultures showed a disarray of actin cytoskeleton contrary to normal distribution of actin filaments in controls. Significantly, few quail neural crest cells migrated to the interventricular septum of chick host embryos compared to the control cultures. The coadministration of topiramate with antiretroviral therapy does not seem to affect the activity of the antiretroviral drug. These results indicate that Atripla and Topiramate cause ventricular septal defects by inhibiting the migration of cardiac neural crest cells.

The cardiac neural crest gene MafB ectopically directs CXCR4 expression in the trunk neural crest

Neural crest cells (NCCs) are multipotent and are attributed to the combination of complex multimodal gene regulatory mechanisms. Cardiac neural crest (CNC) cells, originating from the dorsal neural tube, are pivotal architects of the cardio-neuro-vascular domain, which orchestrates the embryogenesis of critical cardiac and vascular structures. Remarkably, while the scientific community compiled a comprehensive inventory of neural crest derivatives by the early 1980s, our understanding of the CNC's role in various cardiovascular disease processes still needs to be explored. This review delves into the differentiation of NCC, specifically the CNC cells, and explores the diverse facets of non-syndromic cardiovascular neurocristopathies. A systematic review was conducted as per the PRISMA Statement. Three prominent databases, PubMed, Scopus, and Embase, were searched, which yielded 1,840 studies. We excluded 1,796 studies, and the final selection of 44 studies formed the basis of this comprehensive review. Neurocristopathies are a group of genetic disorders that affect the development of cells derived from the NC. Cardiovascular neurocristopathy, i.e., cardiopathy and vasculopathy, associated with the NCC could occur in the form of (1) cardiac septation disorders, mainly the aortico-pulmonary septum; (2) great vessels and vascular disorders; (3) myocardial dysfunction; and (4) a combination of all three phenotypes. This could result from abnormalities in NCC migration, differentiation, or proliferation leading to structural abnormalities and are attributed to genetic, familial, sporadic or acquired causes. Phenotypic characteristics of cardiovascular neurocristopathies, such as bicuspid aortic valve and thoracic aortic aneurysm, share a common embryonic origin and are surprisingly prevalent in the general population, necessitating further research to identify the underlying pathogenic and genetic factors responsible for these cardiac anomalies. Such discoveries are essential for enhancing diagnostic screening and refining therapeutic interventions, ultimately improving the lives of individuals affected by these conditions.