Cephalic Neural Crest Research Articles

Immunohistochemical demonstration of cytokeratin in human embryonic neurons arising from placodes

Sensory neurons of the olfactory, trigeminal, facial, vestibulo-cochlear, glossopharyngeal and vagal nerves, and neurons migrating along the olfactory nerve to the brain have special anlagen, made up of placodes located in the epithelial layer. To investigate the characteristic phenotype of placode-derived neurons, immunohistochemical analysis of intermediate filaments was conducted on formalin-fixed human embryonic tissues. Neurons arising from placodes including luteinizing-hormone releasing hormone (LHRH) neurons migrating from the olfactory placode to the brain had immunoreactivity to antibodies specific to cytokeratin, AE1 and CAM5.2 during the embryonic stage. However, this immunoreactivity disappeared during the late embryonic to the post-embryonic stage and was not observed in the roots of these nerves in the post-natal stage. Immunoreactivity was detected in both the somata and processes, and the distribution differed from that described in rodent brain neurons. With this exception, no other human peripheral neurons, including spinal dorsal root ganglia, had immunoreactivity with anti-cytokeratin antibodies throughout the entire developmental stage. Although the cephalic neural crest also directly generates neurons to most of the cranial sensory ganglia, we could not find any evidence that it contributed to the genesis of cytokeratin-positive embryonic neurons. We concluded that cytokeratin is an intermediate filament common to human embryonic neurons of cephalic placodal origin and that this immunohistochemical marker may be useful in analyzing the developmental sequence of several congenital diseases involving the cranial nerves, such as Moebius syndrome and Goldenhar syndrome.

Msx2 Is a Transcriptional Regulator in the BMP4-Mediated Programmed Cell Death Pathway

Homeobox-containing genes play an important role in patterning processes that occur during embryogenesis. Programmed cell death is a key process during pattern formation. The mechanisms by which programmed cell death is spatially regulated are not well characterized. Msx1 and Msx2 are two closely related homeobox-containing genes that are expressed at sites where cellular proliferation and programmed cell death occur, including the developing limb and the cephalic neural crest. Tissue interactions are necessary for the maintenance of Msx1 and Msx2 expression and programmed cell death. It has been demonstrated that BMP4 can regulate cell death at these same sites as well as induce Msx expression. These observations lead to the hypothesis that Msx2 is a key regulator of cell death in the BMP-mediated pathway. Embryonic stem cell lines will undergo processes typical of early embryogenesis upon aggregation and have recently been shown to provide a model system for programmed cell death. In contrast to ES cells, we see that P19 cells do not undergo pronounced cell death upon aggregation; however, constitutive ectopic Msx2 expression in P19 cells results in a marked increase in apoptosis induced upon aggregation but has no effect when cells are grown as a monolayer. If aggregates are allowed to interact with a substrate, the process of programmed cell death is completely inhibited. Addition of BMP4 to aggregated P19 cells also results in cell death; however, BMP4 does not increase levels of cell death in Msx2-expressing cells. Addition of BMP4 to P19 cells results in an induction of Msx2 transcription consistent with its proposed role in cell death in the embryo. Our data support a model by which BMP4 induces programmed cell death via an Msx2-mediated pathway and provide direct functional evidence that Msx2 expression is a regulator of this process.

Taste buds develop autonomously from endoderm without induction by cephalic neural crest or paraxial mesoderm.

Although it had long been believed that embryonic taste buds in vertebrates were induced to differentiate by ingrowing nerve fibers, we and others have recently shown that embryonic taste buds can develop normally in the complete absence of innervation. This leads to the question of which tissues, if any, induce the formation of taste buds in oropharyngeal endoderm. We proposed that taste buds, like many specialized epithelial cells, might arise via an inductive interaction between the endodermal epithelial cells that line the oropharynx and the adjacent mesenchyme that is derived from both cephalic neural crest and paraxial mesoderm. Using complementary grafting and explant culture techniques, however, we have now found that well-differentiated taste buds will develop in tissue completely devoid of neural crest and paraxial mesoderm derivatives. When the presumptive oropharyngeal region was removed from salamander embryos prior to the onset of cephalic neural crest migration, taste buds developed in grafts and explants coincident with their appearance in intact control embryos. Similarly, explants from neurulae in which movement of paraxial mesoderm had not yet begun also developed taste buds after 9-12 days in vitro. We conclude that neither cranial neural crest nor paraxial mesoderm is responsible for the induction of embryonic taste buds. Surprisingly, the ability to develop taste buds late in embryonic development seems to be an intrinsic feature of the oropharyngeal endoderm that is determined by the completion of gastrulation.

The regeneration of the cephalic neural crest, a problem revisited: the regenerating cells originate from the contralateral or from the anterior and posterior neural fold.

The mesencephalic and rhombencephalic levels of origin of the hypobranchial skeleton (lower jaw and hyoid bone) within the neural fold have been determined at the 5-somite stage with a resolution corresponding to each single rhombomere, by means of the quail-chick chimera technique. Expression of certain Hox genes (Hoxa-2, Hoxa-3 and Hoxb-4) was recorded in the branchial arches of chick and quail embryos at embryonic days 3 (E3) and E4. This was a prerequisite for studying the regeneration capacities of the neural crest, after the dorsal neural tube was resected at the mesencephalic and rhombencephalic level. We found first that excisions at the 5-somite stage extending from the midmesencephalon down to r8 are followed by the regeneration of neural crest cells able to compensate for the deficiencies so produced. This confirmed the results of previous authors who made similar excisions at comparable (or older) developmental stages. When a bilateral excision was followed by the unilateral homotopic graft of the dorsal neural tube from a quail embryo, thus mimicking the situation created by a unilateral excision, we found that the migration of the grafted unilateral neural crest (quail-labelled) is bilateral and compensates massively for the missing crest derivatives. The capacity of the intermediate and ventral neural tube to yield neural crest cells was tested by removing the chick rhombencephalic neural tube and replacing it either uni- or bilaterally with a ventral tube coming from a stage-matched quail. No neural crest cells exited from the ventral neural tube but no deficiency in neural crest derivatives was recorded. Crest cells were found to regenerate from the ends of the operated region. This was demonstrated by grafting fragments of quail neural fold at the extremities of the excised territory. Quail neural crest cells were seen migrating longitudinally from both the rostral and caudal ends of the operated region and filling the branchial arches located inbetween. Comparison of the behaviour of neural crest cells in this experimental situation with that showed by their normal fate map revealed that crest cells increase their proliferation rate and change their migratory behaviour without modifying their Hox code.

Dysgenesis of cephalic neural crest derivatives in Pax7−/− mutant mice

Pax7 is a member of the paired box containing gene family. Its expression pattern suggests a function in cephalic neural crest derivatives, skeletal muscle and central nervous system development. To understand the role of Pax7 during mouse embryogenesis, we used the homologous recombination technique in embryonic stem cells and generated Pax7-/- mice. Homozygous animals are born but die shortly afer weaning. They exhibit malformations in facial structures involving the maxilla and nose. Our analysis suggests that the observed phenotype is due to a cephalic neural crest defect. No obvious phenotype could be detected in the central nervous system and skeletal muscle. Functional redundancy between Pax7 and Pax3 is discussed.

Choanal Atresia

Choanal atresia is an abnormality of canalization during development of the nasal passages. It involves bone and/or soft tissue and may result in either partial (choanal stenosis) or complete obstruction of the posterior nasal airway. The most widely accepted mechanism for the development of choanal atresia is the persistence of the oronasal membrane beyond the sixth week of gestation, but abnormal migration of cephalic neural crest cells following neural tube closure also has been implicated. The incidence of choanal atresia is 1 in 7000 to 8000 live births. It is more common in females (2:1), more likely to be bony or cartilaginous than membranous (9:1), and more commonly unilateral and right-sided (2:1).

Embryonic Origin of Amphibian Taste Buds

Despite numerous descriptive studies, the embryonic origin of vertebrate taste buds has never been experimentally determined. A number of different alternatives have been suggested for taste bud origins, including epibranchial placodes, the neural crest, and the local epithelium of the oropharyngeal cavity. The role of a series of epibranchial placodes and the cephalic neural crest, which together give rise to the cranial nerves innervating taste buds, was examined with regard to the development of oropharyngeal taste buds in an ambystomatid salamander, the axolotl. When pigmented placodal ectoderm or neural folds were grafted isotopically and isochronically into nonpigmented host embryos, known derivatives of each tissue contained pigmented cells, but labeled taste buds were never encountered. Thus, neither epibranchial placodes nor neural crest contribute cells to taste buds during embryogenesis. The majority of the oropharyngeal cavity of ambystomatid salamanders is lined by an endodermal epithelium. In order to demonstrate conclusively that taste buds arise from this local epithelium, the presumptive cephalic endoderm of early axolotl gastrulae was microinjected with the lipophilic dye, DiI. In the oropharyngeal epithelium of all larvae examined, both taste buds and general epithelial cells were labeled with DiI, indicating their common endodermal origin. Our findings are novel in that this is the first experimental demonstration of the endodermal origin of a vertebrate sensory receptor cell class.

Restoration of Normal Hox Code and Branchial Arch Morphogenesis after Extensive Deletion of Hindbrain Neural Crest

Among the derivatives of the cephalic neural crest is the ectomesenchyme which subsequently constitutes most of the craniofacial skeleton. There is evidence to suggest that the skeletogenic fate of the hindbrain neural crest is specified before emigration from the neural tube and that Antennapedia class Hox genes are involved in that process. To explore the putative causal link between Hox expression and craniofacial morphology, we produced a specific series of bilateral crest deletions in chick embryos and assessed branchial arch morphology, Hox gene expression, and patterning of skeletal structures in the postoperative embryo. Surprisingly, we found that deletion of the bulk of the rhombencephalic crest and substantial portions of the dorsal rhombencephalon did not prevent normal branchial arch morphogenesis and normal patterns of Hox gene (-A3 and -B4) expression 48 h after operation. Neural crest-like cells have been identified on crest migration pathways at the level of the original ablation, further confirming that ablated cephalic neural crest is replaced by regeneration from the cut edge of the neuroepithelium. Furthermore, in such embryos ectomesenchyme from regenerated crest is able to form a facial skeleton in which the mandible and hyoid apparatus are normal in size and organization. These findings demonstrate that the cranial neuroepithelium has more extensive regenerative capacities than was previously thought, which has important implications for investigations of craniofacial development.

Retinoic Acid Promotes the Differentiation of Adrenergic Cells and Melanocytes in Quail Neural Crest Cultures

The neural crest (NC) generates very diverse neuronal and nonneuronal cell types in the vertebrate embryo. Pluripotency and plasticity of NC cells have been demonstrated, but the mechanisms whereby extrinsic factors control NC cell diversification are still unknown. Here we have investigated in vitro the influence of the morphogen retinoic acid (RA) in cultures of quail NC cells explanted at different developmental stages, when they start to migrate from the neural primordium, and when they reach their target organs. We found that addition of RA to mass cultures of early-migrating trunk NC promotes the differentiation of melanocytes and adrenergic cells. These responses are dose-dependent and vary according to the time of RA exposure. Testing RA effect on crest-derived cells located in Embryonic Day 3 (E3) somites and in E4 skin showed that the adrenergic-promoting action of RA is restricted to early-migratory cells, while melanogenic precursors colonizing the skin remain sensitive to RA for a longer period. Furthermore, analysis of cephalic and trunk NC clonal cultures revealed that RA influences the differentiation of cells when they are in a pluripotent state: among the progeny of isolated crest cells, RA specifically increases the number of the adrenergic cells, very likely by influencing their commitment; moreover, RA stimulates pigment synthesis in the melanoblasts. In conclusion, these results demonstrate that RA enhances in vitro the differentiation of NC cells and support a role for endogenous retinoids in the ontogeny of NC derivatives.

Diabetic embryopathy: Possible pathogenesis

Despite recent emphasis upon improved metabolic control during early diabetic pregnancy, the offspring of insulin-dependent diabetic women continue to have a 2- to 4-fold increased risk of congenital malformations. We recently evaluated the affected offspring of 4 insulin-dependent diabetic women. All had abnormal ears in association with vertebral defects. Our analysis of the structural defects of these infants and a review of the literature suggest that the pathogenesis of some cases of the diabetic embryopathy may involve a primary insult to developing somite mesoderm and associated cephalic neural crest cells.

Localization of PDGF A and PDGFRα mRNA in Xenopus embryos suggests signalling from neural ectoderm and pharyngeal endoderm to neural crest cells

In situ hybridization analysis of Xenopus laevis embryos reveals that mRNA encoding the platelet-derived growth factor α receptor (PDGFRα) is expressed in cephalic neural crest masses prior to migration from the future neural tube and during their migration into the visceral arches. The analysis of fluorescently labeled neural crest tissue transplanted to unlabeled host embryos demonstrates that neural crest cells are the only detectable source of PDGFRα mRNA within visceral arches. Transcripts encoding PDGF A are present in neural ectoderm, otic vesicle and pharyngeal endoderm. Their location suggests that PDGF A provides a signal, first from the neural epithelium and later from the otic vesicle and pharyngeal endoderm, to cephalic neural crest cells during their migration in the arch region.

Chondrogenesis of neural crest cells: Effect of poly-l-lysine and bone extract

The mechanisms of chondrogenic differentiation are generally studied in vitro by analyzing the action of agents that promote or affect chondrogenesis in embryonic mesenchyme, such as cells of the embryonic limb bud. However, it is not known whether progenitor cells of the craniofacial skeleton, which are of a different embryonic origin and derived from the neural crest, are similarly responsive to such agents. To gain insight into the regulation of chondrogenic differentiation in cells derived from neural crest, we have treated chick embryonic neural crest explants in vitro with poly-L-lysine (PL, M(r) 380 kDa) or bovine bone extract (BBE), two agents known to enhance chondrogenesis of limb mesenchymal cells. Both cephalic (normally chondrogenic) and trunk (normally nonchondrogenic) neural crest cells were analyzed. Chondrogenic differentiation was determined by histological, immunohistochemical and autoradiographic methods. Our results indicate that both PL (380 kDa) and BBE significantly enhance chondrogenesis of cephalic neural crest cells, suggesting that the mechanism of chondrogenesis of these ectodermally derived cells is similar to that of mesodermally derived limb mesenchymal cells. However, trunk neural crest cells did not undergo chondrogenesis in response to PL or BBE. These data show that chondrogenesis can be enhanced in cranial ectodermal neural crest cells in a manner similar to that in the limb mesenchyme. However, since nonchondrogenic trunk neural crest cells are not responsive, an inherent potential for cartilaginous differentiation is necessary for exogenous stimulation of chondrogenesis.

Analysis of the embryonic lineage of vertebrate taste buds.

In all vertebrates, taste buds are the last sensory receptors to appear late in embryonic development. They are thought to arise locally from the oropharyngeal epithelium, although this hypothesis has not been tested experimentally. Alternatively, taste buds have been proposed to arise from neuroectodermal cells that migrate from peripheral neurogenic sources to the oropharyngeal epithelium and give rise to taste bud precursor cells. In order to determine the exact embryonic lineage of the cells of vertebrate taste buds, we have employed a combination of endogenous and exogenous cell marking techniques to follow neuroectodermal and endodermal cells through development. We find, in the ambystomatid salamander used in our studies, taste buds arise locally within the endodermally-derived epithelium lining the oropharyngeal cavity, and do not receive a contribution from neuroectodermal sources, i.e. ectodermal placodes or cephalic neural crest.

Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.

Mesoderm formation is critical for the establishment of the animal body plan and in Drosophila requires the snail gene. This report concerns the cloning and expression pattern of the structurally similar gene snail1 from zebrafish. In situ hybridization shows that the quantity of snail1 RNA increases at the margin of the blastoderm in cells that involute during gastrulation. As gastrulation begins, snail1 RNA disappears from the dorsal axial mesoderm and becomes restricted to the paraxial mesoderm and the tail bud. snail1 RNA increases in cells that define the posterior border of each somite and then disappears when somitic cells differentiate. Later in development, expression appears in cephalic neural crest derivatives. Many snail1-expressing cells were missing from mutant spadetail embryos and the quantity of snail1 RNA was greatly reduced in mutant no tail embryos. The work presented here suggests that snail1 is involved in morphogenetic events during gastrulation, somitogenesis and development of the cephalic neural crest, and that no tail may act as a positive regulator of snail1.

Heterotopic neuroglial tissue of the face: Report of six cases and review of the literature

We report six cases of heterotopic neuroglial tissue of the face. All cases appeared at birth with a laterofacial mass and, in most cases, a respiratory disorder. The initial diagnosis has frequently been lymphangioma. But the rapidly growing character of the mass with resistance to high dose corticoid therapy, the fluid aspect at computerized tomography imaging with no connection to the central nervous system, and the presence of cerebrospinal fluid at the needle puncture were the most helpful features in narrowing the differential diagnoses. Patients were treated by surgical removal of the mass. The pathologic study showed the presence of neuroglial tissue. The management of the patients is discussed. The thirteen previous cases reported in the literature and our six cases lead to a discussion on the embryologic origin of this rare entity—the cephalic neural crest.

Maturational dysautonomia and facial anomalies associated with esophageal atresia: Support for neural crest involvement

Patients with esophageal atresia (EA) or choanal atresia/stenosis (CA) present with many clinical features of maturational dysautonomia (DY). Since CA and DY are considered manifestations of cephalic neurocristopathy, we tested the hypothesis that EA may also be related to faulty development of cephalic neural crest. Forty-eight patients with EA and 53 with CA were followed up to study the frequency of the facial anomalies which are regarded as the phenotypic expression of an abnormal cephalic neural crest contribution to facial embryogenesis. Forty-eight patients with EA and 51 with CA had clinical manifestations of DY. Forty-four patients with EA (91%) and 49 with CA (92%) had one or more facial anomalies. Comparing the groups, patients with EA had an increased frequency of unilateral facial anomalies of branchial arch derivatives ( P < .01); those with CA had an increased frequency of anomalies of frontonasal process derivatives ( P < .01). These findings support the hypothesis that EA may be related to an abnormal contribution from the cephalic neural crest. The presence of facial anomalies may facilitate the diagnosis of subclinical DY.

Thyroid C cells in the DiGeorge anomaly: a quantitative study.

The developmental field defect in the DiGeorge anomaly (DGA) principally affects derivatives of the third and fourth branchial pouches (areas containing a large population of cephalic neural crest cells), including the ultimobranchial body (UB), the reputed source of thyroid calcitonin-producing cells (C cells) in humans. To evaluate the content of C cells in children with DGA, sections of the thyroid in 16 cases of DGA (group I) (8 incomplete and 8 complete forms) and 16 age-matched controls (group II) were stained by immunoperoxidase for calcitonin and chromogranin. Eleven of 16 (69%) cases in group I [4 of 8 (50%) of the complete and 7 of 8 (88%) of of the incomplete cases] and 14 of 16 (88%) of group II exhibited positive-staining cells for both markers, either individually or in small clusters within the follicular epithelial basement membrane. The average number of C cells per high-power field (HPF) (400x) for group I was 1.6 +/- 0.9 and for group II 4.9 +/- 1.7 (P < .005). Although the percentage of positive incomplete DGA cases was the same as that of the control cases, the average number of C cells/HPF was 2.0 +/- 1.1 and similar to that of complete DGA cases (1.2 +/- 0.6). These results demonstrated that C cells are present in the thyroid of patients with DGA more frequently than expected, although in deficient numbers when compared quantitatively to age-matched controls showing a normal infantile pattern of thyroid C cell distribution. Although this observation confirms that there is deficiency of thyroid C cell development in DGA and is in keeping with the assumption that the cells are of neural crest origin, our data raise the possibility of an additional source of C cells, perhaps from thyroid endoderm, in a manner analogous to the endocrine cells of the gut and respiratory tract.

Isolation of Sna, a mouse gene homologous to the Drosophila genes snail and escargot: its expression pattern suggests multiple roles during postimplantation development.

The Drosophila gene snail encodes a zinc-finger protein that is required zygotically for mesoderm formation. Snail acts as a transcriptional repressor during the period of mesoderm formation by preventing expression of mesectodermal and ectodermal genes in the mesoderm anlage. A Xenopus homolog (xsnail) of snail has been cloned and it too is expressed early in the mesodermal germ layer. We have isolated cDNA clones of a mouse gene (termed Sna) closely related to snail and xsnail and another Drosophila gene termed escargot that also encodes a zinc-finger protein. Sna encodes a 264 amino acid protein that contains four zinc fingers. Developmental RNA blot analysis showed that Sna transcripts are expressed throughout postimplantation development. Analysis of the spatial and temporal localization of Sna transcripts by in situ hybridization to both whole-mount and sectioned embryos revealed that, in the gastrulating embryo, Sna is expressed throughout the primitive streak and in the entire mesodermal germ layer. By 9.5 days post coitum (dpc) Sna is expressed at high levels in cephalic neural crest and limb bud mesenchyme. In fact, by 10.5 dpc Sna expression is observed in most mesenchymal cells, whether of neural crest or mesodermal origin. Later in gestation, high levels of Sna expression are observed in condensing cartilage and in the mesenchymal component of several tissues (lung, kidney, teeth and vibrissae) that undergo epithelial-mesenchymal inductive interactions during development. These results suggest multiple roles for the Sna gene in gastrulation and organogenesis during murine development.

Amniotic band sequence: Streeter's hypothesis reexamined.

Recently published reports of 54 subjects with the amniotic band syndrome (ABS) were reviewed, paying particular attention to internal anomalies. Evidence from the internal anomalies suggests that in most cases reviewed, damage occurred in a definable time period, probably prior to 26 days postconception and before the establishment of effective embryonic circulation. Most defects are explicable in terms of interference with neuropore closure, malmigration of cephalic neural crest tissue, and damage to the mesonephros consistent with local interference of the graded expression of organizational genes resulting in a local defect in the organization of the embryo.

Statistical evidence for a random commitment of pluripotent cephalic neural crest cells.

The neural crest (NC) of vertebrate embryos yields cell types belonging to the neural, melanocytic and mesectodermal lineages. To test the possibility that the precursors of these lineages segregate from pluripotent cells by a process involving stochastic determinants, we have analyzed with statistical methods the associations between six differentiated cell types in 201 clones obtained in vitro from migrating cephalic NC cells. Our analysis suggests that neuronal, adrenergic and Schwann cells are not randomly associated, whereas these neural cell types differentiate in the clones independently of both melanocytes and cartilage. These results raise the possibility that pluripotent NC progenitors give rise to the precursors of the major NC-derived lineages (neural, melanocytic and mesectodermal) by a process involving stochastic restrictions of their developmental potentialities.