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.     

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.     

Quantitative distribution of chick neural crest cells during gangliogenesis

Summary We have quantitated the distribution of chick neural crest cells after they have completed early migration and are aggregating into ganglia. Variables tested for an influence on the distribution of cells include stage, level of somites, position in each of the primary body axes, and individual embryo. The 11th–15th cervical somites of embryos at stages 30, 35, and 40 somites (s) incubated for 2.5, 3.0, and 3.5 days were labeled with antibody to HNK-1 to detect neural crest cells, and doubly labeled with antibody to HNK-1 and to the 150 kD neurofilament subunit to detect neural crest-derived neurons. Significantly more neural crest cells appear at older stages, but cells are uniformly distributed among the 11th–15th somites at any given stage. Significant differences in the total number of neural crest cells among three embryos sampled at the same stage indicate that the number of cells is independent of the staging series used. As early as the 35 s stage about one-third of the neural crest cells throughout the somite exhibit NF staining. At the 40 s stage, doubly labeled NF cells, as well as HNK-1 labeled cells, aggregate in a circumscribed portion of the mediolateral axis to form presumptive sensory ganglia in the dorsal region of the somites. Also at 40 s a wave of cell aggregation into sympathetic ganglia proceeds anteroposteriorly along the ventral border of the somitic mesenchyme. The results show a sequence of phenotypic expression beginning with neurofilament antigen, then ganglionic aggregation, and finally, in the case of sympathetic neurons, catecholamine transmitter.     

Quail neural crest cells cannot read positional values in the dorsal trunk feathers of the chicken embryo

Summary If quail neural crest cells are grafted to the chick, they migrate into the feathers of the host and produce melanin pigment. In one study, the dorsal trunk feathers of the chimaera were found to have quail-like pigment patterns. This was interpreted in terms of a positional information model. By contrast, in another study it was found that pigment patterns in the wing plumage of the chimaera bore little or no resemblance to the quail, showing instead a rather uniform, dark pigmentation. This was interpreted in terms of a prepattern in the ectoderm. This striking difference in results could be because the wing and trunk plumages have their pigment patterns specified in different ways. We have examined this possibility by making detailed maps of the dorsal trunk plumage of the normal quail and the quail-chick chimaera. Using this novel technique, we can accurately record the secondary pigment patterns in the embryonic down plumage. In the quail there are well-defined, longitudinal stripes running down the back, whereas the chimaera shows rather uniform, dark pigment in this area. There is little or no indication of stripes and some chimaerae develop asymmetric, mottled patterns. Grafts to the cephalic region also produce uniform pigmentation with no quail-like patterning. These findings indicate that neural crest cells cannot read positional values in the feathers of another species.     

Morphogenesis of sclerotome and neural crest in avian embryos

Summary The distribution of sclerotome and neural crest cells of avian embryos was studied by light and electron microscopy. Sclerotome cells radiated from the somites towards the notochord, to occupy the perichordal space. Neural crest cells, at least initially, also entered cell-free spaces. At the cranial somitic levels they moved chiefly dorsal to the somites, favouring the rostral part of each somite. These cells did not approach the perichordal space. More caudally (i.e. trunk levels), neural crest cells initially moved ventrally between the somites and neural tube. Adjacent to the caudal half of each somite, these cells penetrated no further than the myosclerotomal border, but opposite the rostral somite half, they were found next to the sclerotome almost as far ventrally as the notochord. However, they did not appear to enter the perichordal space, in contrast to sclerotome cells.When tested in vitro, sclerotome cells migrated towards notochords co-cultured on fibronectin-rich extracellular material, and on collagen gels. In contrast, neural crest cells avoided co-cultured notochords. This avoidance was abolished by inclusion of testicular hyaluronidase and chondroitinase ABC in the culture medium, but not by hyaluronidase from Streptomyces hyalurolyticus. The results suggest that sclerotome and neural crest mesenchyme cells have a different distribution with respect to the notochord, and that differential responses to notochordal extracellular material, possibly chondroitin sulphate proteoglycan, may be responsible for this.     

Wise promotes coalescence of cells of neural crest and placode origins in the trigeminal region during head development

While most cranial ganglia contain neurons of either neural crest or placodal origin, neurons of the trigeminal ganglion derive from both populations. The Wnt signaling pathway is known to be required for the development of neural crest cells and for trigeminal ganglion formation, however, migrating neural crest cells do not express any known Wnt ligands. Here we demonstrate that Wise, a Wnt modulator expressed in the surface ectoderm overlying the trigeminal ganglion, play a role in promoting the assembly of placodal and neural crest cells. When overexpressed in chick, Wise causes delamination of ectodermal cells and attracts migrating neural crest cells. Overexpression of Wise is thus sufficient to ectopically induce ganglion-like structures consisting of both origins. The function of Wise is likely synergized with Wnt6, expressed in an overlapping manner with Wise in the surface ectoderm. Electroporation of morpholino antisense oligonucleotides against Wise and Wnt6 causes decrease in the contact of neural crest cells with the delaminated placode-derived cells. In addition, targeted deletion of Wise in mouse causes phenotypes that can be explained by a decrease in the contribution of neural crest cells to the ophthalmic lobe of the trigeminal ganglion. These data suggest that Wise is able to function cell non-autonomously on neural crest cells and promote trigeminal ganglion formation.     

A role for RhoA in the two-phase migratory pattern of post-otic neural crest cells

Neural crest (NC) cells have been elegantly traced to follow stereotypical migratory pathways throughout the vertebrate embryo, yet we still lack complete information on individual cell migratory behaviors and how molecular mechanisms direct NC cell guidance. Here, we analyze the spatio-temporal migratory pattern of post-otic NC and the in vivo role of the small Rho GTPase, RhoA, using fluorescent cell labeling, molecular perturbation, and intravital 4D (3D+ time) confocal imaging in the intact chick embryo. We find that the post-otic NC cell migratory pattern is established in two phases with distinct cell migratory behaviors. An initial wide front of lateral-directed NC cells, led by NC from rhombomere 7 (r7), move as a distinct subpopulation. This is followed in time by fewer NC cells that migrate collectively from r7 to r8 in a follow-the-leader manner with extensive cellular extensions between cells. We show that post-otic migratory NC cells express RhoA, using RT-PCR on isolated, flow cytometry sorted NC cells and in neural tube culture explants. When RhoA function is altered by expression of a dominant negative or constitutively active form, or injection of C3, there are two major consequences. RhoA constitutively active expressing NC cells are less directional, slower and form fewer follow-the-leader chain assemblies. NC cells expressing RhoA-DN are less affective in retracting filopodia, migrate slower and also form fewer follow-the-leader chain assemblies. Together, these alterations to NC cell intrinsic signaling and cell-cell contact disrupt the precise spatio-temporal post-otic NC cell migratory pattern.     

Redefining the head-trunk interface for the neural crest

The head-trunk interface lies at the occipito-cervical boundary, which corresponds to the somite 5/6 level. Previous studies have demonstrated that neural crest cells also behave differently either side of this boundary and that this may be due to intrinsic differences between cranial and trunk crest. However, it is also possible that some of the observed differences between cranial and trunk crest are assigned by environmental cues. We have therefore scrutinised the behaviour of the neural crest cells generated either side of the occipito-cervical boundary in chick and, interestingly, find that both behave in a truncal fashion by traversing the anterior half of their adjacent somites. Furthermore, although not previously described, we find that transient DRGs form opposite somites 4 and 5. Crest cells produced anterior of the somite 3/4 boundary avoid the somites and behave in a non-truncal fashion; these cells populate the pharyngeal arches, and thus contribute to the developing head. We have further shown, via somite transplantations, that differential behaviour of the posterior versus anterior occipital crest is assigned by the somites. If somites 1 to 3 are replaced by trunk somites, then the anterior occipital crest will behave in a truncal fashion by invading the somites. Correspondingly, if these anterior occipital somites are transplanted in place of trunk somites, they perturb the migration of trunk crest. Thus, for the neural crest, the head-trunk interface does not lie at the occipito-cervical boundary, but rather lies at the somite 3/4 level and is defined by the somites. The fact that this boundary lies at the somite 3/4 level in chick is significant as it reflects the more ancient posterior occipital boundary; in fish, only the first three somites contribute to the occipital bone.     

Neural crest induction at the neural plate border in vertebrates

The neural crest is a transient and multipotent cell population arising at the edge of the neural plate in vertebrates. Recent findings highlight that neural crest patterning is initiated during gastrulation, i.e. earlier than classically described, in a progenitor domain named the neural border. This chapter reviews the dynamic and complex molecular interactions underlying neural border formation and neural crest emergence.     

Neural crest induction by the canonical Wnt pathway can be dissociated from anterior-posterior neural patterning in Xenopus

While Wnt signaling is known to be involved in early steps of neural crest development, the mechanism remains unclear. Because Wnt signaling is able to posteriorize anterior neural tissues, neural crest induction by Wnts has been proposed to be an indirect consequence of posteriorization of neural tissues rather than a direct effect of Wnt signaling. To address the relationship between posteriorization and neural crest induction by Wnt signaling, we have used gain of function and loss of function approaches in Xenopus to modulate the level of Wnt signaling at multiple points in the pathway. We find that modulating the level of Wnt signaling allows separation of neural crest induction from the effects of Wnts on anterior-posterior neural patterning. We also find that activation of Wnt signaling induces ectopic neural crest in the anterior region without posteriorizing anterior neural tissues. In addition, Wnt signaling induces neural crest when its posteriorizing activity is blocked by inhibition of FGF signaling in neuralized explants. Finally, depletion of beta-catenin confirms that the canonical Wnt pathway is required for initial neural crest induction. While these observations do not exclude a role for posteriorizing signals in neural crest induction, our data, together with previous observations, strongly suggest that canonical Wnt signaling plays an essential and direct role in neural crest induction.     

Fate map and morphogenesis of presumptive neural crest and dorsal neural tube

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.     

Homeobox b5 (Hoxb5) regulates the expression of Forkhead box D3 gene (Foxd3) in neural crest

Patterning of neural crest (NC) for the formation of specific structures along the anterio-posterior (A-P) body axis is governed by a combinatorial action of Hox genes, which are expressed in the neuroepithelium at the time of NC induction. Hoxb5 was expressed in NC at both induction and migratory stages, and our previous data suggested that Hoxb5 played a role in the NC development. However, the underlying mechanisms by which Hoxb5 regulates the early NC development are largely unknown. Current study showed that both the human and mouse Foxd3 promoters were bound and trans-activated by Hoxb5 in NC-derived neuroblastoma cells. The binding of Hoxb5 to Foxd3 promoter in vivo was further confirmed in the brain and neural tube of mouse embryos. Moreover, Wnt1-Cre mediated perturbation of Hoxb5 signaling at the dorsal neural tube in mouse embryos resulted in Foxd3 down-regulation. In ovo, Foxd3 alleviated the apoptosis of neural cells induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Foxd3 expression in the chick neural tube. This study demonstrated that Hoxb5 (an A-P patterning gene) regulated the NC development by directly inducing Foxd3 (a NC specifier and survival gene).     

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

Id proteins are negative regulators of basic helix-loop-helix gene products and participate in many developmental processes. We have evaluated the expression of Id2 in the developing chick heart and found expression in the cardiac neural crest, secondary heart field, outflow tract, inflow tract, and anterior parasympathetic plexus. Cardiac neural crest ablation in the chick embryo, which causes structural defects of the cardiac outflow tract, results in a significant loss of Id2 expression in the outflow tract. Id2 is also expressed in Xenopus neural folds, branchial arches, cardiac outflow tract, inflow tract, and splanchnic mesoderm. Ablation of the premigratory neural crest in Xenopus embryos results in abnormal formation of the heart and a loss of Id2 expression in the heart and splanchnic mesoderm. This data suggests that the presence of neural crest is required for normal Id2 expression in both chick and Xenopus heart development and provides evidence that neural crest is involved in heart development in Xenopus embryos.     

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.