Plasminogen activator activity is associated with neural crest cell motility in tissue culture

We have examined the possibility that proteases such as plasminogen activator (PA) contribute to the extraordinary motile capability of neural crest cells. We show that trunk neural crest cells that migrate from isolated neural tubes in vitro produce PA and that the level of cell-associated PA increases dramatically after 8 days in culture. This increase is not the result of differentiation or time in culture, because neural crest cell clusters that form on top of the neural tube and differentiate into pigment cells but are immotile produce very low levels of PA. If these clusters are removed from the neural tube and replated on a plastic substratum where they migrate, the level of PA associated with the cells increases dramatically, suggesting that PA production is associated with motility. Inhibitors of PA/plasmin activity significantly reduce neural crest cell motility in vitro, further supporting the idea that proteases are important in neural crest cell migration.     

The effect of tenascin and embryonic basal lamina on the behavior and morphology of neural crest cells in vitro

We have investigated the morphology and migratory behavior of quail neural crest cells on isolated embryonic basal laminae or substrata coated with fibronectin or tenascin. Each of these substrata have been implicated in directing neural crest cell migration in situ. We also observed the altered behavior of cells in response to the addition of tenascin to the culture medium independent of its effect as a migratory substratum. On tenascin-coated substrata, the rate of neural crest cell migration from neural tube explants was significantly greater than on uncoated tissue culture plastic, on fibronectin-coated plastic, or on basal lamina isolated from embryonic chick retinae. Neural crest cells on tenascin were rounded and lacked lamellipodia, in contrast to the flattened cells seen on basal lamina and fibronectin-coated plastic. In contrast, when tenascin was added to the culture medium of neural crest cells migrating on isolated basal lamina, a significant reduction in the rate of cell migration was observed. To study the nature of this effect, we used human melanoma cells, which have a number of characteristics in common with quail neural crest cells though they would be expected to have a distinct family of integrin receptors. A dose-dependent reduction in the rate of translocation was observed when tenascin was added to the culture medium of the human melanoma cell line plated on isolated basal laminae, indicating that the inhibitory effect of tenascin bound to the quail neural crest surface is probably not solely the result of competitive inhibition by tenascin for the integrin receptor. Our results show that tenascin can be used as a migratory substratum by avian neural crest cells and that tenascin as a substratum can stimulate neural crest cell migration, probably by permitting rapid detachment. Tenascin in the medium, on the other hand, inhibits both the migration rates and spreading of motile cells on basal lamina because it binds only the cell surface and not the underlying basal lamina. Cell surface-bound tenascin may decrease cell-substratum interactions and thus weaken the tractional forces generated by migrating cells. This is in contrast to the action of fibronectin, which when added to the medium stimulates cell migration by binding both to neural crest cells and the basal lamina, thus providing a bridge between the motile cells and the substratum.     

Transforming growth factor-beta control of cell-substratum adhesion during avian neural crest cell emigration in vitro.

It has been proposed that, in higher vertebrates, the onset of neural crest cell migration from the neural tube involves spatially and temporally coordinated changes in cellular adhesiveness that are under the control of external signals released in the extracellular milieu by neighboring tissues. In the present study, we have analyzed the dynamics of changes in cell-substratum adhesiveness during crest cell emigration and searched for regulatory cues using an in vitro model system. This model is based on the fact that, in vivo, crest cell dispersion occurs gradually along a rostrocaudal wave, allowing us to explant portions of the neural axis, termed migratory and premigratory levels, that differ in the time in culture at which neural crest cells initiate migration and in the locomotory behavior of the cells. We found that neural crest cell emigration is not triggered by the main extracellular matrix molecules present in the migratory pathways, as none of these molecules could abolish the intrinsic difference in the timing of emigration between the different axial levels. Using an in vitro adhesion assay, we found that presumptive neural crest cells from premigratory level explants gradually acquired the ability to respond to extracellular matrix material with time in culture, suggesting that acquisition of appropriate, functional integrin receptors was a necessary step for migration. Finally, we showed that members of the transforming growth factor-beta family reduced in a dose-dependent manner the delay of neural crest cell emigration from premigratory level explants and were able to increase significantly the substratum-adhesion properties of crest cells. Our results suggest that acquisition of substratum adhesion by presumptive neural crest cells is a key event during their dispersion from the neural tube in vitro, and that members of the transforming growth factor-beta family may act as potent inducers of crest cell emigration, possibly by increasing the substratum adhesion of the cells.     

Dynamic alterations in gene expression after Wnt-mediated induction of avian neural crest

The Wnt signaling pathway is important in the formation of neural crest cells in many vertebrates, but the downstream targets of neural crest induction by Wnt are largely unknown. Here, we examined quantitative changes in gene expression regulated by Wnt-mediated neural crest induction using quantitative PCR (QPCR). Induction was recapitulated in vitro by adding soluble Wnt to intermediate neural plate tissue cultured in collagen, and induced versus control tissue were assayed using gene-specific primers at times corresponding to premigratory (18 and 24 h) or early (36 h) stages of crest migration. The results show that Wnt signaling up-regulates in a distinct temporal pattern the expression of several genes normally expressed in the dorsal neural tube (slug, Pax3, Msx1, FoxD3, cadherin 6B) at "premigratory" stages. While slug is maintained in early migrating crest cells, Pax3, FoxD3, Msx1 and cadherin 6B all are down-regulated by the start of migration. These results differ from the temporal profile of these genes in response to the addition of recombinant BMP4, where gene expression seems to be maintained. Interestingly, expression of rhoB is unchanged or even decreased in response to Wnt-mediated induction at all times examined, though it is up-regulated by BMP signals. The temporal QPCR profiles in our culture paradigm approximate in vivo expression patterns of these genes before neural crest migration, and are consistent with Wnt being an initial neural crest inducer with additional signals like BMP and other factors maintaining expression of these genes in vivo. Our results are the first to quantitatively describe changes in gene expression in response to a Wnt or BMP signal during transformation of a neural tube cell into a migratory neural crest cell.     

Perturbation of cranial neural crest migration by the HNK-1 antibody

The HNK-1 antibody recognizes a carbohydrate moiety that is shared by a family of cell adhesion molecules and is also present on the surface of migrating neural crest cells. Here, the effects of the HNK-1 antibody on neural crest cells were examined in vitro and in vivo. When the HNK-1 antibody was added to neural tube explants in tissue culture, neural crest cells detached from laminin substrates but were unaffected on fibronectin substrates. In order to examine the effects of the HNK-1 antibody in vivo, antibody was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. The injected antibody persisted for approximately 16 hr on the injected side of the embryo and appeared to be most prevalent on the surface of neural crest cells. Embryos fixed within the first 24 hr after injection of HNK-1 antibodies (either whole IgMs or small IgM fragments) showed one or more of the following abnormalities: (1) ectopic neural crest cells external to the neural tube, (2) an accumulation of neural crest cell volume on the lumen of the neural tube, (3) some neural tube anomalies, or (4) a reduction in the neural crest cell volume on the injected side. The ectopic cells and neural tube anomalies persisted in embryos fixed 2 days postinjection. Only embryos having 10 or less somites at the time of injection were affected, suggesting a limited period of sensitivity to the HNK-1 antibody. Control embryos injected with a nonspecific antibody or with a nonblocking antibody against the neural cell adhesion molecule (N-CAM) were unaffected. Previous experiments from this laboratory have demonstrated than an antibody against integrin, a fibronectin and laminin receptor caused defects qualitatively similar to those resulting from HNK-1 antibody injection (M. Bronner-Fraser, J. Cell Biol., 101, 610, 1985). Coinjection of the HNK-1 and integrin antibodies resulted in a greater percentage of affected embryos than with either antibody alone. The additive nature of the effects of the two antibodies suggests that they act at different sites. These results demonstrate that the HNK-1 antibody causes abnormalities in cranial neural crest migration, perhaps by perturbing interactions between neural crest cells and laminin substrates.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.     

Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion

The possible role of a 140-kD cell surface complex in neural crest adhesion and migration was examined using a monoclonal antibody JG22, first described by Greve and Gottlieb (1982, J. Cell. Biochem. 18:221-229). The addition of JG22 to neural crest cells in vitro caused a rapid change in morphology of cells plated on either fibronectin or laminin substrates. The cells became round and phase bright, often detaching from the dish or forming aggregates of rounded cells. Other tissues such as somites, notochords, and neural tubes were unaffected by the antibody in vitro even though the JG22 antigen is detectable in embryonic tissue sections on the surface of the myotome, neural tube, and notochord. The effects of the JG22 on neural crest migration in vivo were examined by a new perturbation approach in which both the antibody and the hybridoma cells were microinjected onto neural crest pathways. Hybridoma cells were labeled with a fluorescent cell marker that is nondeleterious and that is preserved after fixation and tissue sectioning. The JG22 antibody and hybridoma cells caused a marked reduction in cranial neural crest migration, a build-up of neural crest cells within the lumen of the neural tube, and some migration along aberrant pathways. Neural crest migration in the trunk was affected to a much lesser extent. In both cranial and trunk regions, a cell free zone of one or more cell diameters was generally observed between neural crest cells and the JG22 hybridoma cells. Two other monoclonal antibodies, 1-B and 1-N, were used as controls. Both 1-B and 1-N bind to bands of the 140-kD complex precipitated by JG22. Neither control antibody affected neural crest adhesion in vitro or neural crest migration in situ. This suggests that the observed alterations in neural crest migration are due to a functional block of the 140-kD complex.     

Distribution of laminin and collagens during avian neural crest development

The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscle. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues. The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and lamin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development. These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny. (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration. (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis. (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.     

The Delayed Entry of Thoracic Neural Crest Cells into the Dorsolateral Path Is a Consequence of the Late Emigration of Melanogenic Neural Crest Cells from the Neural Tube

Neural crest cells migrate along two pathways in the trunk: the ventral path, between the neural tube and somite, and the dorsolateral path, between the somite and overlying ectoderm. In avian embryos, ventral migration precedes dorsolateral migration by nearly 24 h, and the onset of dorsolateral migration coincides with the cessation of ventral migration. Neural crest cells in the ventral path differentiate predominantly as neurons and glial cells of the peripheral nervous system, whereas those in the dorsolateral path give rise to the melanocytes of the skin. Thus, early- and late-migrating neural crest cells exhibit unique morphogenetic behaviors and give rise to different subsets of neural crest derivatives. Here we present evidence that these differences reflect the appearance of specified melanocyte precursors, or melanoblasts, from late- but not early-migrating neural crest cells. We demonstrate that serum from Smyth line (SL) chickens specifically immunolabels melanocyte precursors, or melanoblasts. Using SL serum as a marker, we first detect melanoblasts immediately dorsal and lateral to the neural tube beginning at stage 18, which is prior to the onset of dorsolateral migration. At later stages every neural crest cell in the dorsolateral path is SL-positive, demonstrating that only melanoblasts migrate dorsolaterally. Thus, melanoblast specification precedes dorsolateral migration, and only melanoblasts migrate dorsolaterally at the thoracic level. Together with previous work (Erickson, C. A., and Goins, T. L.,Development121, 915–924, 1995), these data argue that specification as a melanoblast is a prerequisite for dorsolateral migration. This conclusion suggested that the delay in dorsolateral migration (relative to ventral migration) may reflect a delay in the emigration of melanogenic neural crest cells from the neural tube. Several experiments support this hypothesis. There are no melanoblasts in the ventral path, as revealed by the absence of SL-positive cells in the ventral path, and neural crest cells isolated from the ventral path do not give rise to melanocytes when explanted in culture, suggesting that early, ventrally migrating neural crest cells are limited in their ability to differentiate as melanocytes. Similarly, neural crest cells that emigrate from the neural tubein vitroduring the first 6 h fail to give rise to any melanocytes or SL-positive melanoblasts, whereas neural crest cells that emigrate at progressively later times show a dramatic increase in melanogenesis under identical culture conditions. Thus, the timing of dorsolateral migration at the thoracic level is ultimately controlled by the late emigration of melanogenic neural crest cells from the neural tube.     

Immunohistochemical localisation of chondroitin sulphate proteoglycans and the effects of chondroitinase ABC in 9- to 11-day rat embryos

Studies on cell behaviour in vitro have indicated that the chondroitin sulphate proteoglycan (CSPG) family of molecules can participate in the control of cell proliferation, differentiation and adhesion, but its morphogenetic functions had not been investigated in intact embryos. Chondroitin/chondroitin sulphates have been identified in rat embryos at low levels at the start of neurulation (day 9) and at much higher levels on day 10. In this study we have sought evidence for the morphogenetic functions of CSPGs in rat embryos during the period of neurulation and neural crest cell migration by a combination of two approaches: immunocytochemical localization of CSPG by means of an antibody, CS-56, to the chondroitin sulphate component of CSPG, and exposure of embryos to the enzyme chondroitinase ABC. Staining of the CS-56 epitope was poor at the beginning of cranial neurulation; bright staining was at first confined to the primary mesenchyme under the convex neural folds late on day 9. In day 10 embryos, all mesenchyme cells were stained, but at different levels of intensity, so that primary mesenchyme, neural crest and sclerotomal cells could be distinguished from each other. Basement membranes were also stained, particularly bright staining being present where two epithelial were basally apposed, e.g., neural/surface ectoderms, dorsal aorta/neural tube, prior to migration of a population of cells between them. Staining within the neural epithelium was first confined to the dorsolateral edge region, and associated with the onset of neural crest cell emigration; after neural tube closure, neuroepithelial staining was more general. Neural crest cells were stained during migration, but the reaction was absent in areas associated with migration end-points (trigeminal ganglion anlagen, frontonasal mesenchyme). Embryos exposed to chondroitinase ABC in culture showed no abnormalities until early day 10, when cranial neural crest cell emigration from the neural epithelium was inhibited and neural tube closure was retarded. Sclerotomal cells failed to take their normal pathway between the dorsal aorta and neural tube. Correlation of the results of these two methods suggests: (1) that by decreasing adhesiveness within the neural epithelium at specific stages, CSPG facilitates the emigration of neural crest cells and the migratory movement of neuroblasts, and may also provide increased flexibility during the generation of epithelial curvatures; (2) that by decreasing the adhesiveness of fibronectin-containing extracellular matrices, CSPG facilitates the migration of neural crest and sclerotomal cells. This second function is particularly important when migrating cells take pathways between previously apposed tissues.     

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.     

Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and "synergy" sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin alpha5beta1.     

Neural crest cell migration: requirements for exogenous fibronectin and high cell density

Cells of the neural crest participate in a major class of cell migratory events during embryonic development. From indirect evidence, it has been suggested that fibronectin (FN) might be involved in these events. We have directly tested the role of FN in neural crest cell adhesion and migration using several in vitro model systems. Avian trunk neural crest cells adhered readily to purified plasma FN substrates and to extracellular matrices containing cellular FN. Their adhesion was inhibited by antibodies to a cell-binding fragment of FN. In contrast, these cells did not adhere to glass, type I collagen, or to bovine serum albumin in the absence of FN. Neural crest cell adhesion to laminin (LN) was significantly less than to FN; however, culturing of crest cells under conditions producing an epithelioid phenotype resulted in cells that could bind equally as well to LN as to FN. The migration of neural crest cells appeared to depend on both the substrate and the extent of cell interactions. Cells migrated substantially more rapidly on FN than on LN or type I collagen substrates; if provided a choice between stripes of FN and glass or LN, cells migrated preferentially on the FN. Migration was inhibited by antibodies against the cell-binding region of FN, and the inhibition could be reversed by a subsequent addition of exogenous FN. However, the migration on FN was random and displayed little persistence of direction unless cells were at high densities that permitted frequent contacts. The in vitro rate of migration of cells on FN-containing matrices was 50 microns/h, similar to their migration rates along the narrow regions of FN-containing extracellular matrix in migratory pathways in vivo. These results indicate that FN is important for neural crest cell adhesion and migration and that the high cell densities of neural crest cells in the transient, narrow migratory pathways found in the embryo are necessary for effective directional migration.     

Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)     

Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways.

Cranial neural crest cells are a pluripotent population of cells derived from the neural tube that migrate into the branchial arches to generate the distinctive bone, connective tissue and peripheral nervous system components characteristic of the vertebrate head. The highly conserved segmental organisation of the vertebrate hindbrain plays an important role in patterning the pathways of neural crest cell migration and in generating the distinct or separate streams of crest cells that form unique structures in each arch. We have used focal injections of DiI into the developing mouse hindbrain in combination with in vitro whole embryo culture to map the patterns of cranial neural crest cell migration into the developing branchial arches. Our results show that mouse hindbrain-derived neural crest cells migrate in three segregated streams adjacent to the even-numbered rhombomeres into the branchial arches, and each stream contains contributions of cells from three rhombomeres in a pattern very similar to that observed in the chick embryo. There are clear neural crest-free zones adjacent to r3 and r5. Furthermore, using grafting and lineage-tracing techniques in cultured mouse embryos to investigate the differential ability of odd and even-numbered segments to generate neural crest cells, we find that odd and even segments have an intrinsic ability to produce equivalent numbers of neural crest cells. This implies that inter-rhombomeric signalling is less important than combinatorial interactions between the hindbrain and the adjacent arch environment in specific regions, in the process of restricting the generation and migration of neural crest cells. This creates crest-free territories and suggests that tissue interactions established during development and patterning of the branchial arches may set up signals that the neural plate is primed to interpret during the progressive events leading to the delamination and migration of neural crest cells. Using interspecies grafting experiments between mouse and chick embryos, we have shown that this process forms part of a conserved mechanism for generating neural crest-free zones and contributing to the separation of migrating crest populations with distinct Hox expression during vertebrate head development.     

Combined intrinsic and extrinsic influences pattern cranial neural crest migration and pharyngeal arch morphogenesis in axolotl

Cranial neural crest cells migrate in a precisely segmented manner to form cranial ganglia, facial skeleton and other derivatives. Here, we investigate the mechanisms underlying this patterning in the axolotl embryo using a combination of tissue culture, molecular markers, scanning electron microscopy and vital dye analysis. In vitro experiments reveal an intrinsic component to segmental migration; neural crest cells from the hindbrain segregate into distinct streams even in the absence of neighboring tissue. In vivo, separation between neural crest streams is further reinforced by tight juxtapositions that arise during early migration between epidermis and neural tube, mesoderm and endoderm. The neural crest streams are dense and compact, with the cells migrating under the epidermis and outside the paraxial and branchial arch mesoderm with which they do not mix. After entering the branchial arches, neural crest cells conduct an "outside-in" movement, which subsequently brings them medially around the arch core such that they gradually ensheath the arch mesoderm in a manner that has been hypothesized but not proven in zebrafish. This study, which represents the most comprehensive analysis of cranial neural crest migratory pathways in any vertebrate, suggests a dual process for patterning the cranial neural crest. Together with an intrinsic tendency to form separate streams, neural crest cells are further constrained into channels by close tissue apposition and sorting out from neighboring tissues.     

Molecular regulation of neural crest development

The neural crest is a transient embryonic structure that gives rise to a multitude of different cell types in the vertebrate. As such, it is an iideal model to study the processes of vertebrate differentiation and development. This review focuses on two major questionsrelated to neural crest development. The first question concerns the degree and time of commitment of the neural crest cellsto differntt cell lineages and the emerging role of the homebox containing genes in regulating this process. Evidence from the cephalic crest suggests that the commitment process does start before the neural crest cells migrate away from the neural tube and gene ablation experiments suggest that different homeobox genes are required for the development of neural and mesenchymal tissue derivatives. However, clonal analysis of neural crest cell before migration suggests that many of the cells remain multi-potential indicating that the final determinative steps occur progressively during migration and in association with environmental influences. The second question concerns the nature of the environmental factors that determine the differentiation of neural crest cells into discrete lineages. Evidence is provided, mainly from in vitro experiments, that purified growth factors selectively promote the differentiation of neural crest cells down either sympathetic, adrenal, sensory, or melanocytic cell lineages.     

An antibody to a receptor for fibronectin and laminin perturbs cranial neural crest development in vivo

Previous studies from this laboratory (M. Bronner-Fraser (1985). J. Cell Biol. 101, 610) have demonstrated that an antibody to a cell surface receptor complex caused alterations in avian neural crest cell migration. Here, these observations are extended to examine the distribution and persistency of injected antibody, the dose dependency of the effect, and the long-term influences of antibody injection. The CSAT antibody, which recognizes a cell surface receptor for fibronectin and laminin, was injected lateral to the mesencephalic neural tube at the onset of cranial neural crest migration. Injected antibody molecules did not cross the midline, but appeared to diffuse throughout the injected half of the mesencephalon, where they remained detectable by immunocytochemistry for about 22 hr. Embryos were examined either during neural crest migration (up to 24 hr after injection) or after formation of neural crest-derived structures (36-48 hr after injection). In those embryo fixed within the first 24 hr, the major defects were a reduction in the neural crest cell number on the injected side, a buildup of neural crest cells within the lumen of the neural tube, and ectopically localized neural crest cells. In embryos allowed to survive for 36 to 48 hr after injection, the neural crest derivatives appeared normal on both the injected and control side, suggesting that the embryos compensated for the reduction in neural crest cell number on the injected side. However, the embryos often had severely deformed neural tubes and ectopic aggregates of neural crest cells. In contrast, several control antibodies had no effect. These findings suggest that the CSAT receptor complex is important in the normal development of the neural crest and neural tube.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively, and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: 1) what establishes the pathways of migration and 2) what controls the final destination and differentiation of various neural crest subpopulations. These questions will be addressed in this review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube, and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted, or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk. The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: 1) the vagal-level neural crest cells exhibit modest developmental bias; 2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively; 3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.