Contact behaviour exhibited by migrating neural crest cells in confrontation culture with somitic cells

Summary When grown in confrontation culture on a planar substratum, avian neural crest cells and somite cells display both homotypic and heterotypic contact inhibition of movement as judged by analysis of time-lapse video recordings of locomotory and contact behaviour, and by use of a nuclear overlap assay. It is therefore unlikely that migration of neural crest cells within the embryo, and within embryonic tissues, can be explained on the basis of a lack of contact inhibition. The results are discussed in the general context of cell invasiveness.     

Effect of hyaluronic acid on the emergence of neural crest cells from the neural tube of the quail,Coturnix coturnix japonica

Summary Hyaluronic acid (HA) added to the medium of quail neural tubes explanted in vitro influences the number of migratory neural crest cells that emerge, compared with controls. Neural crest cells were counted with an ocular grid after 20 h of migration into 0.1 mm wide areas or bins lying parallel to the neural tube, and the results were analyzed by linear regression. A low concentration of HA (5 g/ml) significantly decreased the total number of neural crest cells in all bins adjacent to the neural tube, whereas several high concentrations of HA (250, 500, and 1000 g/ ml) significantly increased the number of neural crest cells. Intermediate concentrations of HA (50 and 100 g/ml) did not differ from that of controls. Linear regressions of number of cells versus distance from the tube showed no significant differences among the slopes of control, low HA, and high HA treatments, providing evidence that HA does not influence the rate of cell migration. Scanning electron microscopy showed that cells in neuroepithelia exposed to low HA (5 g/ml) appeared in tighter contact, while cells of neuroepithelia in high HA (500 g/ml) appeared more loosely organized, compared with controls. Cells in tight contact could be restrained from leaving the neuroepithelium, whereas cells in loose contact could more readily move out of the neural tube, thus explaining the differences in cell numbers in low HA and high HA, respectively. We conclude that HA can be a factor in the differential adhesivity among neuroepithelial cells and may be important in the initial separation of the neural crest from the neural tube.     

Control of the onset of migration of neural crest cells in avian embryos

Summary To investigate the control of the timing in the epithelio-mesenchymal transformation of the neural crest into a migrating population, neural anlagen (neural tube plus crest) were isolated from 2-day quail embryos by proteases in the presence of Ca^{+ +} and explanted onto substrates favourable for neural crest cell migration. Explants isolated before normal migration had commenced required 3–8 h in vitro before neural crest cells started migration, but explants obtained at migratory stages showed an immediate onset of migration. The schedule was similar to that expected in vivo. When pre-migratory neural anlagen were isolated by protease in Ca^{+ +}- and Mg^{+ +}-free (CMF) medium, or when the protease was followed by a brief (5 min) exposure to CMF medium, neural crest cell migration commenced without delay, and the cohesion of the anlagen was impaired. Ca^{+ +}-free medium duplicated the effects of CMF, but neither Mg^{+ +}-free medium nor CMF treatment before treatment with protease stimulated migration and reduced cohesion. Precocious neural crest cell migration and reduced cohesion also followed when neural anlagen of pre-migratory stages were cultured with membrane. Ca^{+ +}-channel antagonists D600 and Nifedipine, without any exernal Ca^{+ +}-depletion.The decrease of cohesion of these tissues is consistent with results in other systems where protease/Ca^{+ +}-depletion inactivates Ca^{+ +}-dependent cell-cell adhesive mechanisms. Therefore, we suggest that Ca^{+ +}-dependent cell-cell adhesions play a part in preventing neural crest cells from migrating precociously and that the timed inactivation of this adhesion system normally helps trigger the onset of migration. The results with blockers of Ca^{+ +}-channels suggest that Ca^{+ +} levels may be involved in regulating this system.     

The relationship between migration and chondrogenic potential of trunk neural crest cells in Ambystoma mexicanum

Based on results of transplantation experiments, it has long been believed that trunk neural crest cells are incapable of chondrogenesis. When pigmented trunk neural crest cells of Ambystoma mexicanum are transplanted to cranial levels of albino (a/a) embryos, the graft cells ultimately produce ectopic fins, but are incapable of following the chondrogenic cranial neural crest pathways. Therefore, heterotopic transplantation does not expose these cells to the same environment experienced by cranial neural crest cells, and is neither an adequate nor a sufficient test of chondrogenic potential. However, in vitro culture of trunk neural crest cells with pharyngeal endoderm does provide a direct test of chondrogenic ability. That cartilage does not form under these conditions demonstrates conclusively that trunk neural crest cells possess no chondrogenic potential.     

Combined function of HoxA and HoxB clusters in neural crest cells

The evolution of chordates was accompanied by critical anatomical innovations in craniofacial development, along with the emergence of neural crest cells. The potential of these cells to implement a craniofacial program in part depends upon the (non-)expression of Hox genes. For instance, the development of jaws requires the inhibition of Hox genes function in the first pharyngeal arch. In contrast, Hox gene products induce craniofacial structures in more caudal territories. To further investigate which Hox gene clusters are involved in this latter role, we generated HoxA;HoxB cluster double mutant animals in cranial neural crest cells. We observed the appearance of a supernumerary dentary-like bone with an endochondral ossification around a neo-Meckel's cartilage matrix and an attachment of neo-muscle demonstrating that HoxB genes enhance the phenotype induced by the deletion of the HoxA cluster alone. In addition, a cervical and hypertrophic thymus was associated with the supernumerary dentary-like bone, which may reflect its ancestral position near the filtrating system. Altogether these results show that the HoxA and HoxB clusters cooperated during evolution to lead to present craniofacial diversity.     

An assay system to study migratory behavior of cranial neural crest cells in Xenopus

An increasing number of genes are known to show expression in the cranial neural crest area. So far it is very difficult to analyze their effect on neural crest cell migration because of the lack of transplantation techniques. This paper presents a simple method to study the migratory behavior of cranial neural crest cells by homo- and heterotopic transplantations: Green fluorescent protein (GFP) RNA was injected into one blastomere of Xenopus laevis embryos at the 2-cell stage. The cranial neural crest area of stage 14 embryos was transplanted into the head or trunk region of an uninjected host embryo, and the migration was monitored by GFP fluorescence. The transplants were further examined by double immunostaining and confocal microscopy to trace migratory routes inside the embryo, and to exclude contaminations of grafts with foreign tissues. Our results demonstrate that we developed a highly efficient and reproducible technique to study the migratory ability of cranial neural crest cells. It offers the possibility to analyze genes involved in neural crest cell migration by coinjecting their RNA with that of GFP. Received: 28 September 1999 / Accepted: 17 November 1999     

Factors controlling the time of onset of the migration of neural crest cells in the fowl embryo

Summary Transmission electron microscopy of fowl embryos during the 7–10 h preceding migration of trunk-level neural crest (NC) cells revealed extracellular material near the NC-cells. In contrast to the cells of the neural tube, the basal surfaces of NC-cells possessed projections, and were neither contiguous nor covered by a complete basal lamina. The apical zones of NC-cells showed intercellular junctions at the stage of neural-fold fusion, but such junctions were absent in some NC-cells 5 h before migration. The basal laminae of the neural tube and the ectoderm were fused lateral to the NC before migration. In vitro, NC-cell migration commenced immediately when neural anlagen were explanted onto fibronectin-rich matrices, but only when the neural anlagen were from a level where migration had commenced in vivo. Migration was delayed 4–8 h when premigratory-level expiants were used. Short-term cell-adhesion assays showed that NC-cells of both premigratory and migratory levels could adhere to fibronectin-rich matrices and to collagen gels, but only migratory NC-cells could be detached from the neural anlage. The results suggest that the precise schedule of the onset of NC-cell migration correlates with a decrease in the intercellular adhesion of NC-cells.     

PTEN基因对早期胚胎神经嵴细胞迁移的作用研究

目的 初步探讨PTEN基因在早期神经嵴细胞迁移中的作用.方法 首先胚胎整体的原位杂交和免疫荧光方法检测鸡胚胎内源性的PTEN基因及蛋白水平的表达情况;其次,利用鸡胚胎体内半侧神经管转染的方法,使神经管一侧PTEN基因过表达,对侧神经管为正常对照侧;最后,通过Pax7的整体胚胎免疫荧光表达观察PTEN基因对其标记的部分神经嵴细胞迁移的影响.结果 内源性PTEN基因在mRNA和蛋白水平表达显示,其在早期胚胎HH4期的神经板即开始明显的表达;通过半侧过表达PTEN基因后观察到过表达PTEN基因侧的头部神经嵴细胞迁移与对照侧相比明显受到抑制,但对躯干部的影响并不明显.结论 PTEN基因可能抑制早期胚胎头部神经嵴细胞的迁移.     

Ultrastructural and tissue-culture studies on the role of fibronectin,collagen and glycosaminoglycans in the migration of neural crest cells in the fowl embryo

Summary The initial migration of neural crest (NC) cells into cell-free space was studied by transmission electron microscopy at trunk levels of fowl embryos, some of which were fixed in the presence of ruthenium red. Migrating NC cells occurred in zones which contained fewer ruthenium-red stained 15–40 nm diameter granules than other regions. The ruthenium-red stained granules were linked by similarly stained thin ( 3 nm diameter) microfibrils. The granules resemble proteoglycan and the microfibrils may be hyaluronate. NC cells contacted thicker ( 10 nm diameter) fibrils and interstitial bodies, which did not require ruthenium red for visualization. Cytoplasmic microfilaments were sometimes aligned at the point of contact with the extracellular fibrils, which may be fibronectin and collagen.Phase-contrast time-lapse videotaping and scanning electron microscopy showed that NC cells of the fowl embryo in vitro migrated earlier and more extensively on glass coated with fibronectin-rich fibrous material and adsorbed fibronectin molecules than on glass coated with collagen type I (fibres and adsorbed molecules). NC cells became completely enmeshed in fibronectin-rich fibres, but generally remained on the surface of collagen-fibre gels. When given a choice, NC cells strongly preferred fibronectin coatings to plain glass, and plain glass to dried collagen gels. NC cells showed a slight preference for plain glass over glass to which collagen was adsorbed. Addition to the culture medium of hyaluronate (initial conc. 20 mg/ml), chondroitin (5 mg/ml) and fully sulphated chondroitin sulphate and dermatan sulphate (up to 10 mg/ml) did not drastically alter NC cell migration on fibronectin-rich fibrous substrates. However, partially desulphated chondroitin sulphate (5mg/ml) strongly retarded the migration of NC cells.The in vivo and in vitro studies suggest that fibronectin may dictate the pathways of NC cell migration by acting as a highly preferred physical substrate. However, the utilization of these pathways may be reduced by the presence of proteoglycans bearing undersulphated chondroitin sulphate.Abbreviations NC neural crest - ECM extracellular material - GAG glycosaminoglycan - FN fibronectin - CIG cold insoluble globulin - TEM transmission electron microscopy - SEM scanning electron microscopy - DMEM-H HEPES buffered Dulbecco's modified Eagle's medium - FCS foetal calf serum - CEE chick embryo extract - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - PBS phosphate-buffered saline     

The receptor tyrosine kinase RET regulates hindgut colonization by sacral neural crest cells

The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail-chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC.     

How to become neural crest: from segregation to delamination

The development of the neural crest up to the stage where they leave the neural tube can be observed as a series of concatenated but independent events that involve dorsalization of the neural plate/neural tube, neural crest induction, segregation and stabilization, epithelial to mesenchymal transition and delamination. During all these processes, the nascent neural crest cells are subjected to the influence of different signals and have to overcome competition for cell fate and apoptotic signals. In addition, striking rostrocaudal differences unveil how the regulatory cascades are somehow different but still can lead to the production of bona fide neural crest cells.     

A role for chemokine signaling in neural crest cell migration and craniofacial development

Neural crest cells (NCCs) are a unique population of multipotent cells that migrate along defined pathways throughout the embryo and give rise to many diverse cell types including pigment cells, craniofacial cartilage and the peripheral nervous system (PNS). Aberrant migration of NCCs results in a wide variety of congenital birth defects including craniofacial abnormalities. The chemokine Sdf1 and its receptors, Cxcr4 and Cxcr7, have been identified as key components in the regulation of cell migration in a variety of tissues. Here we describe a novel role for the zebrafish chemokine receptor Cxcr4a in the development and migration of cranial NCCs (CNCCs). We find that loss of Cxcr4a, but not Cxcr7b, results in aberrant CNCC migration defects in the neurocranium, as well as cranial ganglia dysmorphogenesis. Moreover, overexpression of either Sdf1b or Cxcr4a causes aberrant CNCC migration and results in ectopic craniofacial cartilages. We propose a model in which Sdf1b signaling from the pharyngeal arch endoderm and optic stalk to Cxcr4a expressing CNCCs is important for both the proper condensation of the CNCCs into pharyngeal arches and the subsequent patterning and morphogenesis of the neural crest derived tissues.     

Morphology and behaviour of neural crest cells of chick embryo in vitro

Summary Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamellae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm or ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.     

Adhesion to extracellular materials by neural crest cells at the stage of initial migration

Summary Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5–2 h incubation, the expiants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.     

Fibronectin in early avian embryos: Synthesis and distribution along the migration pathways of neural crest cells

Summary Immunoperoxidase labelling for fibronectin (FN) in chick embryos showed FN-positive basement membranes surrounding the neural crest cell population prior to crest-cell migration. At cranial levels, crest cells migrated laterally into a large cell-free space. Initially they moved as a tongue of cells contacting the FN-positive basement membrane of the ectoderm, but later the crest cell population expanded into space further from the ectoderm, until eventually the entire cranial cell-free space was occupied by mesenchyme cells. This was accompanied by the appearance of FN among the crest cells. At trunk levels, crest cells entered a relatively small space already containing FN-positive extracellular material. At later stages the migration of trunk crest cells broadly matched the distribution of FN. In vitro, chick and quail embryo ectoderm, endoderm, somites, notochord and neural tube synthesized and organized fibrous FN-matrices, as shown by immunofluorescence. Ectoderm and endoderm deposited this matrix only on the substrate face. The FN content of endoderm and neural tube matrices was transient, the immunofluorescence intensity declining after 1–2 days in culture. Some crest cells of cranial and sacral axial levels synthesized FN. Our data suggests that these were the earliest crest cells to migrate from these levels. This ability may be the first expression of mesenchymal differentiation in these crest cells, and in vivo enable them to occupy a large space. Almost all crest cells from cervico-lumbar axial levels were unable to synthesize FN. In vivo, this inability may magnify the response of these crest cells to FN provided by the neighbouring embryonic tissues.     

Trunk lateral cells are neural crest-like cells in the ascidian Ciona intestinalis: insights into the ancestry and evolution of the neural crest

Neural crest-like cells (NCLC) that express the HNK-1 antigen and form body pigment cells were previously identified in diverse ascidian species. Here we investigate the embryonic origin, migratory activity, and neural crest related gene expression patterns of NCLC in the ascidian Ciona intestinalis. HNK-1 expression first appeared at about the time of larval hatching in dorsal cells of the posterior trunk. In swimming tadpoles, HNK-1 positive cells began to migrate, and after metamorphosis they were localized in the oral and atrial siphons, branchial gill slits, endostyle, and gut. Cleavage arrest experiments showed that NCLC are derived from the A7.6 cells, the precursors of trunk lateral cells (TLC), one of the three types of migratory mesenchymal cells in ascidian embryos. In cleavage arrested embryos, HNK-1 positive TLC were present on the lateral margins of the neural plate and later became localized adjacent to the posterior sensory vesicle, a staging zone for their migration after larval hatching. The Ciona orthologues of seven of sixteen genes that function in the vertebrate neural crest gene regulatory network are expressed in the A7.6/TLC lineage. The vertebrate counterparts of these genes function downstream of neural plate border specification in the regulatory network leading to neural crest development. The results suggest that NCLC and neural crest cells may be homologous cell types originating in the common ancestor of tunicates and vertebrates and support the possibility that a putative regulatory network governing NCLC development was co-opted to produce neural crest cells during vertebrate evolution.     

Immunohistochemical localization of laminin,neural cell adhesion molecule,collagen type IV and T-61 antigen in the embryonic retina of the Japanese quail by in vivo injection of antibodies

Summary Antibodies against laminin (LN), fibronectin (FN), collagen type IV (Col IV), neural cell adhesion molecule (N-CAM), T-61 antigen, actin, tubulin and neurofilament protein were injected into the eyes of quail embryos (Coturnix coturnix japonica) of different ages. Twenty h after injection, the heads of the embryos were fixed and the antibodies visualized in sections with the use of fluorescein-isothiocyanate (FITC) or peroxidase-labeled second antibodies by light- and electron microscopy. Antibodies against cell surface molecules, such as N-CAM, LN, Col IV and T 61, labeled matrix and membrane components of the retinal cells in different antigen-specific patterns. Antibodies against intracellular antigens, such as actin, tubulin and neurofilament protein labeled nonspecifically the vitreous body and the inner basal lamina of the retina, but resulted in only a very weak and diffuse labeling of retinal cells. N-CAM was detected in high concentration in the optic fiber layer on the surface of axons and on the membranes of all retinal cells. Col IV, LN and T 61 antigen were found predominantly in the optic fiber layer. LN and Col IV were located on the surface of axons and the endfeet of ventricular (neuroepithelial) cells in a patchy distribution. The T-61 antigen was found in early stages in the cell-free space of the optic fiber layer, on the surface of ventricular cells and axons, and at later stages also in high-density patches between nerve fibers. The distribution of LN and T-61 antigen together with data from in vitro experiments suggests a crucial role of these proteins in axon extension in the avian retina during early development of the optic fiber layer.     

Abnormalities in neural crest cell migration in laminin alpha5 mutant mice

Although numerous in vitro experiments suggest that extracellular matrix molecules like laminin can influence neural crest migration, little is known about their function in the embryo. Here, we show that laminin alpha5, a gene up-regulated during neural crest induction, is localized in regions of newly formed cranial and trunk neural folds and adjacent neural crest migratory pathways in a manner largely conserved between chick and mouse. In laminin alpha5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin alpha5 in vitro show a reduction by half in the number of migratory neural crest cells. During gangliogenesis, laminin alpha5 mutants exhibit defects in condensing cranial sensory and trunk sympathetic ganglia. However, ganglia apparently recover at later stages. These data suggest that the laminin alpha5 subunit functions as a cue that restricts neural crest cells, focusing their migratory pathways and condensation into ganglia. Thus, it is required for proper migration and timely differentiation of some neural crest populations.