Cardiac neural crest ablation results in early endocardial cushion and hemodynamic flow abnormalities.

Cardiac neural crest cell (CNCC) ablation creates congenital heart defects (CHDs) that resemble those observed in many syndromes with craniofacial and cardiac consequences. The loss of CNCCs causes a variety of great vessel defects, including persistent truncus arteriosus and double-outlet right ventricle. However, because of the lack of quantitative volumetric measurements, less severe defects, such as great vessel size changes and valve defects, have not been assessed. Also poorly understood is the role of abnormal cardiac function in the progression of CNCC-related CHDs. CNCC ablation was previously reported to cause abnormal cardiac function in early cardiogenesis, before the CNCCs arrive in the outflow region of the heart. However, the affected functional parameters and how they correlate with the structural abnormalities were not fully characterized. In this study, using a CNCC-ablated quail model, we contribute quantitative phenotyping of CNCC ablation-related CHDs and investigate abnormal early cardiac function, which potentially contributes to late-stage CHDs. Optical coherence tomography was used to assay early- and late-stage embryos and hearts. In CNCC-ablated embryos at four-chambered heart stages, great vessel diameter and left atrioventricular valve leaflet volumes are reduced. Earlier, at cardiac looping stages, CNCC-ablated embryos exhibit abnormally twisted bodies, abnormal blood flow waveforms, increased retrograde flow percentage, and abnormal cardiac cushions. The phenotypes observed in this CNCC-ablation model were also strikingly similar to those found in an established avian fetal alcohol syndrome model, supporting the contribution of CNCC dysfunction to the development of alcohol-induced CHDs.

Occipital cephalocele with neural crest remnants? Radiological and pathological findings in a newborn boy.

PURPOSE: A cephalocele is a congenital anomaly involving the herniation of intracranial tissue from a skull defect. The sac containing the central nervous system (CNS) with the ventricle system is called the encephalocystocele. An atretic cephalocele is thought to be an abortive form of cephalocele, and the essential nature is still controversial. CASE REPORT: Here, we report the case of a newborn boy with an occipital cephalocele containing a small cystic component which was composed of ependymal cells and the immature CNS tissue. A newborn boy was admitted to our hospital because of an occipital mass, which was about 2.5 cm in diameter, located at the posterior midline, and covered with alopetic skin without CSF leakage. He had a cleft palate. Magnetic resonance imaging (MRI) clearly showed an occipital cephalocele with a tiny cystic component connecting to the subarachnoid space. MRI also showed mild hydrocephalus, hypoplasia of the corpus callosum and tentorium cerebelli, dropping down of the bilateral occipital lobes and vermicular agenesis. We performed the extirpation of the subscalp module under general anesthesia and histologically examined the resected mass. On immunohistopathological examination, most part of the subscalp module was fibrous tissue with numerous vessels and meningeal origin cells. In a small part of the innermost layer, we found a small island consisting of CNS tissue and a tiny cyst lined with a single layer of ependymal cells. CONCLUSION: Based on radiological and immunohistopathological findings, we speculate that the cystic component at the base of the nodule seems to correspond to neural crest remnants but not to true herniation of the brain and cerebral ventricles.

Regulation of pre-otic brain development by the cephalic neural crest.

Emergence of the neural crest (NC) is considered an essential asset in the evolution of the chordate phylum, as specific vertebrate traits such as peripheral nervous system, cephalic skeletal tissues, and head development are linked to the NC and its derivatives. It has been proposed that the emergence of the NC was responsible for the formation of a "new head" characterized by the spectacular development of the forebrain and associated sense organs. It was previously shown that removal of the cephalic NC (CNC) prevents the formation of the facial structures but also results in anencephaly. This article reports on the molecular mechanisms whereby the CNC controls cephalic neurulation and brain morphogenesis. This study demonstrates that molecular variations of Gremlin and Noggin level in CNC account for morphological changes in brain size and development. CNC cells act in these processes through a multi-step control and exert cumulative effects counteracting bone morphogenetic protein signaling produced by the neighboring tissues (e.g., adjacent neuroepithelium, ventro-medial mesoderm, superficial ectoderm). These data provide an explanation for the fact that acquisition of the NC during the protochordate-to-vertebrate transition has coincided with a large increase of brain vesicles.

Calvarial Melanotic Neuroectodermal Tumor of Infancy with Rhabdomyosarcomatous differentiation-A Rare Case.

BACKGROUND: Malignant neuroectodermal tumor of infancy is a rare neural crest cell-derived neoplasm of infants. Histologically, melanotic neuroectodermal tumor of infancy usually consists of 2 types of cells: neuroblast-like and melanocyte-like cells. Here we present a rare case of melanotic neuroectodermal tumor of infancy containing a third type of cell population, that is, rhabdomyoblasts in addition to the above two. CASE DESCRIPTION: We report a case of a 10-month-old female child who was brought to us with complaints of swelling over the right forehead for the last 9 months, which started increasing in size rapidly 3 months before presenting to us. Noncontrast computed tomography scan showed a large well-defined extra-axial lesion in the right frontotemporal region. The child underwent an open biopsy under general anesthesia. Histopathological sections showed a malignant small round cell tumor consisting of hyperchromatic cells lying in sheets and lobules separated by fibrous septae. The patient underwent 7 cycles of neoadjuvant chemotherapy over a period of 2 months. The patient underwent right frontotemporal craniotomy and gross total excision of the lesion as a definitive surgery. Postoperatively, the patient was stable, and there was no new deficit. Histopathology revealed neuroblast-like and melanocyte-like cells with rhabdomyosarcomatous differentiation. The patient received chemotherapy in the postoperative period. The patient had recurrence of the tumor and died 8 months after the surgery. CONCLUSIONS: Calvarial malignant neuroectodermal tumor of infancy with rhabdomyosarcomatous differentiation is a rare entity with no cases being reported before. Neoadjuvant chemotherapy with surgical excision can be a promising modality of treatment.

Shortened outflow tract leads to altered cardiac looping after neural crest ablation.

BACKGROUND: Congenital conotruncal malformations frequently involve dextroposed aorta. The pathogenesis of dextroposed aorta is not known but is thought to be due to abnormal looping and/or wedging of the outflow tract during early heart development. We examined the stage of cardiac looping in an experimental model of dextroposed aorta to determine the embryogenesis of this conotruncal malformation. METHODS AND RESULTS: Hearts were examined from neural crest-ablated embryos by using videocinephotography, scanning electron microscopy, and histological sections. The inflow and outflow limbs of the looped cardiac tube were malpositioned with respect to each other, the inner curvature was diminished, and the outflow limb was straighter and displaced cranially in a manner consistent with diminished length. The altered length could be explained by a significant reduction in the number of cells added to the myocardium of the distal outflow tract from the secondary heart field. CONCLUSIONS: The data are consistent with research showing that normal looping and wedging are essential for normal alignment of the aorta with the left ventricle. These processes are abnormal in neural crest-ablated embryos because of a failure of the outflow tract to lengthen by the addition of myocardial cells from the secondary heart field.

Dissection of Xenopus laevis neural crest for in vitro explant culture or in vivo transplantation.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Cardiac neural crest ablation inhibits compaction and electrical function of conduction system bundles.

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.

L-type Ca2+ channel function in the avian embryonic heart after cardiac neural crest ablation.

In avian and mammalian embryos, surgical ablation or severely reduced migration of the cardiac neural crest leads to a failure of outflow tract septation known as persistent truncus arteriosus (PTA) and leads to embryo lethality due partly to impaired excitation-contraction coupling stemming primarily from a reduction in the L-type Ca(2+) current (I(Ca),(L)). Decreased I(Ca,L) occurs without a corresponding reduction in the alpha(1)-subunit of the Ca(2+) channel. We hypothesize that decreased I(Ca),(L) is due to reduced function at the single channel level. The cell-attached patch clamp with Na(+) as the charge carrier was used to examine single Ca(2+) channel activity in myocytes from normal hearts from sham-operated embryos and from hearts diagnosed with PTA at embryonic days (ED) 11 and 15 after laser ablation of the cardiac neural crest. In normal hearts, the number of single channel events per 200-ms depolarization and the mean open channel probability (P(o)) was 1.89 +/- 0.17 and 0.067 +/- 0.008 for ED11 and 1.14 +/- 0.17 and 0.044 +/- 0.005 for ED15, respectively. These values represent a normal reduction in channel function and I(Ca),(L) observed with development. However, the number of single channel events was significantly reduced in hearts with PTA at both ED11 and ED15 (71% and 47%, respectively) with a corresponding reduction in P(o) (75% and 43%). The open time frequency histograms were best fitted by single exponentials with similar decay constants (tau approximately or equal 4.5 ms) except for the sham operated at ED15 (tau = 3.4 ms). These results indicate that the cardiac neural crest influences the development of myocardial Ca(2+) channels.

The effect of vagal neural crest ablation on the chick embryo cloaca.

The cloaca, the caudal limit of the avian gastrointestinal tract, acts as a collecting chamber into which the gastrointestinal, urinary, and genital tracts discharge. It is intrinsically innervated by the enteric nervous system, which is derived from neural crest emigres that migrate from the vagal and sacral regions of the neural tube. Abnormal cloacal development can cause a number of anorectal anomalies, including persistent cloaca. Ablation of the vagal neural crest has previously been shown to result in an aganglionic hindgut to the extent of the colorectum. The aim of our study was to investigate the effect of vagal neural crest ablation on the cloaca, the limit of the hindgut in the developing chick embryo. Chick embryos were incubated until the 10-12 somite stage. The vagal neural tube corresponding to the level of somites 3-6 was then ablated, and eggs were incubated until harvested on embryonic day 11 (E11). Whole chick embryos were fixed, embedded in paraffin, and sectioned. Immunohistochemistry was then carried out using the HNK-1 monoclonal antibody to label neural crest cells, and results were assessed by light microscopy. Vagal neural crest ablation resulted in a dramatic decrease in the number of neural crest cells colonizing the chick embryo cloaca compared with control embryos. Ablated embryos contained only a small number of HNK-1-positive neural crest cells, which were scattered within the myenteric plexus in a disorganised pattern. Hypoganglionosis was also evident in other regions of the hindgut in ablated embryos. Ablation of the vagal neural crest results in a hypoganglionic cloaca in addition to hypoganglionosis of the hindgut. These results suggest that the cloaca is largely innervated by vagal neural crest emigres. Further studies involving quail-chick chimeras to investigate the exact contribution provided by both vagal and sacral neural crest cells to the cloaca should increase our understanding of the pathophysiology of conditions like persistent cloaca.

Head morphogenesis in embryonic avian chimeras: evidence for a segmental pattern in the ectoderm corresponding to the neuromeres.

Areas of the superficial cephalic ectoderm, including or excluding the neural fold at the same level, were surgically removed from 3-somite chick embryos and replaced by their counterparts excised from a quail embryo at the same developmental stage. Strips of ectoderm corresponding to the presumptive branchial arches were delineated, thus defining anteroposterior 'segments' (designated here as 'ectomeres') that coincided with the spatial distribution of neural crest cells arising from the adjacent levels of the neural fold. This discrete ectodermal metamerisation parallels the segmentation of the hindbrain into rhombomeres. It seems, therefore, that not only is the neural crest patterned according to its rhombomeric origin but that the superficial ectoderm covering the branchial arches may be part of a larger developmental unit that includes the entire neurectoderm, i.e., the neural tube and the neural crest.

Harelip and cleft palate conditions in chick embryos following local destruction of the cephalic neural crest. A preliminary note.

With the aid of micro-laser irradiation, in a total of 156 chick embryos unilaterally (a part of) the cephalic neural crest paralleling the posterior portion of the prosencephalon and mesencephalon, was eliminated. As a result of this treatment, 2 out of 22 embryos, studied 24 hours after irradiation, showed a considerable degree of underdevelopment of the mesenchyme in the homolateral first branchial arch. At the age of 7 days (6 days after irradiation) 11 of the 44 surviving embryos proved to have developed a homolateral harelip condition. In 2 of these embryos, a wide palatal cleft was observed as well. These results demonstrate that facial clefts may develop as a result of a local deficiency of neural crest cells.

Rhombomere rotation reveals that multiple mechanisms contribute to the segmental pattern of hindbrain neural crest migration.

Hindbrain neural crest cells adjacent to rhombomeres 2 (r2), r4 and r6 migrate in a segmental pattern, toward the first, second and third branchial arches, respectively. Although all rhombomeres generate neural crest cells, those arising from r3 and r5 deviate rostrally and caudally (J. Sechrist, G. Serbedzija, T. Scherson, S. Fraser and M. Bronner-Fraser (1993) Development 118, 691-703). We have altered the rostrocaudal positions of the cranial neural tube, adjacent ectoderm/mesoderm or presumptive otic vesicle to examine tissue influences on this segmental migratory pattern. After neural tube rotation, labeled neural crest cells follow pathways generally appropriate for their new position after grafting. For example, when r3 and r4 were transposed, labeled r3 cells migrated laterally to the second branchial arch whereas labeled r4 cells primarily deviated caudally toward the second arch, with some cells moving rostrally toward the first. In contrast to r4 neural crest cells, transposed r3 cells leave the neural tube surface in a polarized manner, near the r3/4 border. Surprisingly, some labeled neural crest cells moved directionally toward small ectopic otic vesicles that often formed in the ectoderm adjacent to grafted r4. Similarly, they moved toward grafted or displaced otic vesicles. In contrast, surgical manipulation of the mesoderm adjacent to r3 and r4 had no apparent effects. Our results offer evidence that neural crest cells migrate directionally toward the otic vesicle, either by selective attraction or pathway-derived cues.

Alteration of early vascular development after ablation of cranial neural crest.

A previous study has shown that, subsequent to ablation of cranial neural crest, heart morphology and pharyngeal arch vessels (aortic arches) are altered before septation of the outflow tract normally occurs. In the present study, we concentrated on very early development of the aortic arch apparatus in the chick (incubation days 3-5). The three-dimensional organization of the arch vessel apparatus was studied by scanning electron microscopy after intravascular injection of Mercox, and by serial sections of embryos embedded in plastic. Alterations in the arch vessel apparatus were already present by day three in embryos with neural crest ablation at stage 9-10. Bilateral symmetry frequently was lost. Arch vessels sometimes were enlarged and occupied most of the arch, with little surrounding mesenchyme. Some arch vessels were small or occluded. Mesenchyme was significantly reduced in quantity in the arches, and was not condensed and symmetrical as in controls. There was a significant increase in the proportion of direct apposition of vessel endothelium with epithelium, without the intervening mesenchyme typical of controls. The surgical manipulation used in this study leads to distinct alterations in the arches of components and relationships which are important in development. Altered blood flow likely affects the development of the heart.

Restriction in cell fates of developing spinal cord cells transplanted to neural crest pathways.

At early neural tube stages, individual stem cells can generate neural crest cells as well as dorsal or ventral spinal cord cells. To determine whether this pluripotency is lost as development proceeds, we back-transplanted quail spinal cells from different developmental stages and different spinal locations into the crest migratory pathways of st 16-20 chicken host embryos. The transplanted spinal cells from st 27 dorsal cord and st 18 ventral cord differentiated within the new crest environment into sensory and sympathetic neurons, satellite and Schwann cells, and melanocytes. St 27 ventral cells still generated several crest derivatives but not sensory or sympathetic neurons. This loss in ability to produce neurons correlates with the end of neurogenesis in ventral cord. The end of neurogenesis in the cord, therefore, results from an intrinsic change in the potential of spinal neuroepithelial cells to generate neurons.

DBHR, a gene with homology to dopamine beta-hydroxylase, is expressed in the neural crest throughout early development.

In a screen for genes involved in neural crest development, we identified DBHR (DBH-Related), a putative monooxygenase with low homology to dopamine beta-hydroxylase (DBH). Here, we describe novel expression patterns for DBHR in the developing embryo and particularly the neural crest. DBHR is an early marker for prospective neural crest, with earliest expression at the neural plate border where neural crest is induced. Furthermore, DBHR expression persists in migrating neural crest and in many, though not all, crest derivatives. DBHR is also expressed in the myotome, from the earliest stages of its formation, and in distinct regions of the neural tube, including even-numbered rhombomeres of the hindbrain. In order to investigate the signals that regulate its segmented pattern in the hindbrain, we microsurgically rotated the rostrocaudal positions of rhombomeres 3/4. Despite their ectopic position, both rhombomeres continued to express DBHR at the level appropriate for their original location, indicating that DBHR is regulated autonomously within rhombomeres. We conclude that DBHR is a divergent member of a growing family of DBH-related genes; thus, DBHR represents a completely new type of neural crest marker, expressed throughout the development of the neural crest, with possible functions in cell-cell signaling.

Excessive microtubules are not responsible for depressed force per cross-bridge in cardiac neural-crest-ablated embryonic chick hearts.

Ablation of the cardiac neural crest (CNCA) in embryonic chicks results in a high incidence of persistent truncus arteriosus, a congenital heart defect associated with decreased myocardial contractility. Using left ventricular trabeculae from chicks at embryonic day (ED) 15, we have previously shown that the twitch force of intact preparations is significantly reduced whereas the maximal calcium-activated force of skinned preparations is not significantly different in CNCA and sham-operated animals. We also previously found that the ventricular content of myosin, as well as of actin and tropomyosin, was nearly doubled in ED 15 hearts after CNCA. Since the number of cross-bridges is proportional to the myosin concentration, these data suggest that the force exerted per cross-bridge is decreased in CNCA hearts. We investigated the possibility that the decrease in force per cross-bridge is caused by inhibition of the contractile apparatus by excessive microtubules. To the contrary, we found that the total beta-tubulin content and the fraction of beta-tubulin polymerized in microtubules measured by Western blotting was the same in ventricular muscle strips from CNCA and sham-operated embryos. Furthermore, exposure to microtubule-destabilizing agents did not improve the force-producing capability of the contractile apparatus in CNCA embryos. We conclude that depression of force per cross-bridge in hearts from CNCA embryos is not due to an excess of microtubules.

Myocardial volume and organization are changed by failure of addition of secondary heart field myocardium to the cardiac outflow tract.

Cardiac neural crest ablation results in primary myocardial dysfunction and failure of the secondary heart field to add the definitive myocardium to the cardiac outflow tract. The current study was undertaken to understand the changes in myocardial characteristics in the heart tube, including volume, proliferation, and cell size when the myocardium from the secondary heart field fails to be added to the primary heart tube. We used magnetic resonance and confocal microscopy to determine that the volume of myocardium in the looped heart was dramatically reduced and the compact layer of myocardium was thinner after neural crest ablation, especially in the outflow tract and ventricular regions. Proliferation measured by 5-bromo-2'-deoxyuridine incorporation was elevated at only one stage during looping, cell death was normal and myocardial cell size was increased. Taken together, these results indicate that there are fewer myocytes in the heart. By incubation day 8 when the heart would have normally completed septation, the anterior (ventral) wall of the right ventricle and right ventricular outflow tract was significantly thinner in the neural crest-ablated embryos than normal, but the thickness of the compact myocardium was normal in all other regions of the heart. The decreased volume and number of myocardial cells in the heart tube after neural crest ablation most likely reflects the amount of myocardium added by the secondary heart field.

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.