Segmental origin and migration of neural crest cells in the hindbrain region of the chick embryo.

A vital dye analysis of cranial neural crest migration in the chick embryo has provided a positional fate map of greater resolution than has been possible using labelled graft techniques. Focal injections of the fluorescent membrane probe DiI were made into the cranial neural folds at stages between 3 and 16 somites. Groups of neuroepithelial cells, including the premigratory neural crest, were labelled by the vital dye. Analysis of whole-mount embryos after 1-2 days further development, using conventional and intensified video fluorescence microscopy, revealed the pathways of crest cells migrating from mesencephalic and rhombencephalic levels of the neuraxis into the subjacent branchial region. The patterns of crest emergence and emigration correlate with the segmented disposition of the rhombencephalon. Branchial arches 1, 2 and 3 are filled by crest cells migrating from rhombomeres 2, 4 and 6 respectively, in register with the cranial nerve entry/exit points in these segments. The three streams of ventrally migrating cells are separated by alternating regions, rhombomeres 3 and 5, which release no crest cells. Rostrally, rhombomere 1 and the caudal mesencephalon also contribute crest to the first arch, primarily to its upper (maxillary) component. Both r3 and r5 are associated with enhanced levels of cell death amongst cells of the dorsal midline, suggesting that crest may form at these levels but is then eliminated. Organisation of the branchial region is thus related by the dynamic process of neural crest immigration to the intrinsic mechanisms that segment the neuraxis.     

Effects of mesodermal tissues on avian neural crest cell migration

We have used microsurgical techniques to investigate the effects of embryonic mesodermal tissues on the pattern of chick neural crest cell migration in the trunk. Segmental plate or lateral plate mesenchyme was transplanted into regions encountered by neural crest cells. We found that neural crest cells are able to migrate through lateral plate mesenchyme but not through segmental plate tissue until this tissue differentiates into a sclerotome. After this stage, segmental migration is controlled by the subdivision of the sclerotome into a rostral and a caudal half; when the rostrocaudal orientation of the sclerotomes is reversed by rotating the segmental plate 180 degrees about its rostrocaudal axis, neural crest cells migrate through the portion of the sclerotome that was originally rostral.     

Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.

Cranial neural crest (CNC) cells migrate extensively, typically in a pattern of cell streams. In Xenopus, these cells express the adhesion molecule Xcadherin-11 (Xcad-11) as they begin to emigrate from the neural fold. In order to study the function of this molecule, we have overexpressed wild-type Xcad-11 as well as Xcad-11 mutants with cytoplasmic (deltacXcad-11) or extracellular (deltaeXcad-11) deletions. Green fluorescent protein (GFP) was used to mark injected cells. We then transplanted parts of the fluorescent CNC at the premigratory stage into non-injected host embryos. This altered not only migration, but also the expression of neural crest markers. Migration of transplanted cranial neural crest cells was blocked when full-length Xcad-11 or its mutant lacking the beta-catenin-binding site (deltacXcad-11) was overexpressed. In addition, the expression of neural crest markers (AP-2, Snail and twist) diminished within the first four hours after grafting, and disappeared completely after 18 hours. Instead, these grafts expressed neural markers (2G9, nrp-I and N-Tubulin). Beta-catenin co-expression, heterotopic transplantation of CNC cells into the pharyngeal pouch area or both in combination failed to prevent neural differentiation of the grafts. By contrast, deltaeXcad-11 overexpression resulted in premature emigration of cells from the transplants. The AP-2 and Snail patterns remained unaffected in these migrating grafts, while twist expression was strongly reduced. Co-expression of deltaeXcad-11 and beta-catenin was able to rescue the loss of twist expression, indicating that Wnt/beta-catenin signalling is required to maintain twist expression during migration. These results show that migration is a prerequisite for neural crest differentiation. Endogenous Xcad-11 delays CNC migration. Xcad-11 expression must, however, be balanced, as overexpression prevents migration and leads to neural marker expression. Although Wnt/beta-catenin signalling is required to sustain twist expression during migration, it is not sufficient to block neural differentiation in non-migrating grafts.     

In ovo time-lapse analysis of chick hindbrain neural crest cell migration shows cell interactions during migration to the branchial arches

Hindbrain neural crest cells were labeled with DiI and followed in ovo using a new approach for long-term time-lapse confocal microscopy. In ovo imaging allowed us to visualize neural crest cell migration 2-3 times longer than in whole embryo explant cultures, providing a more complete picture of the dynamics of cell migration from emergence at the dorsal midline to entry into the branchial arches. There were aspects of the in ovo neural crest cell migration patterning which were new and different. Surprisingly, there was contact between neural crest cell migration streams bound for different branchial arches. This cell-cell contact occurred in the region lateral to the otic vesicle, where neural crest cells within the distinct streams diverted from their migration pathways into the branchial arches and instead migrated around the otic vesicle to establish a contact between streams. Some individual neural crest cells did appear to cross between the streams, but there was no widespread mixing. Analysis of individual cell trajectories showed that neural crest cells emerge from all rhombomeres (r) and sort into distinct exiting streams adjacent to the even-numbered rhombomeres. Neural crest cell migration behaviors resembled the wide diversity seen in whole embryo chick explants, including chain-like cell arrangements; however, average in ovo cell speeds are as much as 70% faster. To test to what extent neural crest cells from adjoining rhombomeres mix along migration routes and within the branchial arches, separate groups of premigratory neural crest cells were labeled with DiI or DiD. Results showed that r6 and r7 neural crest cells migrated to the same spatial location within the fourth branchial arch. The diversity of migration behaviors suggests that no single mechanism guides in ovo hindbrain neural crest cell migration into the branchial arches. The cell-cell contact between migration streams and the co-localization of neural crest cells from adjoining rhombomeres within a single branchial arch support the notion that the pattern of hindbrain neural crest cell migration emerges dynamically with cell-cell communication playing an important guidance role.     

Evidence for collapsin-1 functioning in the control of neural crest migration in both trunk and hindbrain regions

Collapsin-1 belongs to the Semaphorin family of molecules, several members of which have been implicated in the co-ordination of axon growth and guidance. Collapsin-1 can function as a selective chemorepellent for sensory neurons, however, its early expression within the somites and the cranial neural tube (Shepherd, I., Luo, Y. , Raper, J. A. and Chang, S. (1996) Dev. Biol. 173, 185-199) suggest that it might contribute to the control of additional developmental processes in the chick. We now report a detailed study on the expression of collapsin-1 as well as on the distribution of collapsin-1-binding sites in regions where neural crest cell migration occurs. collapsin-1 expression is detected in regions bordering neural crest migration pathways in both the trunk and hindbrain regions and a receptor for collapsin-1, neuropilin-1, is expressed by migrating crest cells derived from both regions. When added to crest cells in vitro, a collapsin-1-Fc chimeric protein induces morphological changes similar to those seen in neuronal growth cones. In order to test the function of collapsin-1 on the migration of neural crest cells, an in vitro assay was used in which collapsin-1-Fc was immobilised in alternating stripes consisting of collapsin-Fc/fibronectin versus fibronectin alone. Explanted neural crest cells derived from both trunk and hindbrain regions avoided the collapsin-Fc-containing substratum. These results suggest that collapsin-1 signalling can contribute to the patterning of neural crest cell migration in the developing chick.     

The distribution of tenascin coincides with pathways of neural crest cell migration

The distribution of the extracellular matrix (ECM) glycoprotein, tenascin, has been compared with that of fibronectin in neural crest migration pathways of Xenopus laevis, quail and rat embryos. In all species studied, the distribution of tenascin, examined by immunohistochemistry, was more closely correlated with pathways of migration than that of fibronectin, which is known to be important for neural crest migration. In Xenopus laevis embryos, anti-tenascin stained the dorsal fin matrix and ECM along the ventral route of migration, but not the ECM found laterally between the ectoderma and somites where neural crest cells do not migrate. In quail embryos, the appearance of tenascin in neural crest pathways was well correlated with the anterior-to-posterior wave of migration. The distribution of tenascin within somites was compared with that of the neural crest marker, HNK-1, in quail embryos. In the dorsal halves of quail somites which contained migrating neural crest cells, the predominant tenascin staining was in the anterior halves of the somites, codistributed with the migrating cells. In rat embryos, tenascin was detectable in the somites only in the anterior halves. Tenascin was not detectable in the matrix of cultured quail neural crest cells, but was in the matrix surrounding somite and notochord cells in vitro. Neural crest cells cultured on a substratum of tenascin did not spread and were rounded. We propose that tenascin is an important factor controlling neural crest morphogenesis, perhaps by modifying the interaction of neural crest cells with fibronectin.     

Distribution and migration pathways of HNK-1-immunoreactive neural crest cells in teleost fish embryos.

Whole mounts and cross-sections of embryos from three species of teleost fish were immunostained with the HNK-1 monoclonal antibody, which recognizes an epitope on migrating neural crest cells. A similar distribution and migration was found in all three species. The crest cells in the head express the HNK-1 epitope after they have segregated from the neural keel. The truncal neural crest cells begin to express the epitope while they still reside in the dorsal region of the neural keel; this has not been observed in other vertebrates. The cephalic and anterior truncal neural crest cells migrate under the ectoderm; the cephalic cells then enter into the gill arches and the anterior truncal cells into the mesentery of the digestive tract where they cease migration. These cephalic and anterior trunk pathways are similar to those described in Xenopus and chick. The neural crest cells of the trunk, after segregation, accumulate in the dorsal wedges between the somites, however, unlike in chick and rat, they do not migrate in the anterior halves of the somites but predominantly between the neural tube and the somites, the major pathway observed in carp and amphibians; some cells migrate over the somites. The HNK-1 staining of whole-mount embryos revealed a structure resembling the Rohon-Beard and extramedullary cells, the primary sensory system in amphibians. Such a system has not been described in fish.     

Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

Cooperation between the PDGF receptors in cardiac neural crest cell migration

Neural crest cells (NCCs) are essential components of the sympathetic nervous system, skin, craniofacial skeleton, and aortic arch. It has been known for many years that perturbation of migration, proliferation, and/or differentiation of these cells leads to birth defects such as cleft palate and persistent truncus arteriosus (PTA). Previously, we had shown that disruption of the platelet-derived growth factor receptor (PDGFR) alpha in NCCs resulted in defects in craniofacial and aortic arch development, the latter with variable penetrance. Because we observed ventricular septal defects in embryos that are null for the PDGFRbeta, we hypothesized that both PDGF receptors are involved in NCC formation. Here, we show that both receptors are expressed in cardiac NCCs and that the combined loss of the PDGFRalpha and PDGFRbeta in NCCs resulted in NCC-related heart abnormalities, including PTA and a ventricular septal defect (VSD). Using NCC lineage tracing, we observed that loss of PDGF receptor signaling resulted in reduced NCCs in the conotruncus region, leading to defects in aortic arch septation. These results indicate that while PDGFRalpha plays a predominant role in NCC development, the PDGFRbeta is expressed by and functions in cardiac NCCs. Combined PDGF receptor signaling is required for sufficient recruitment of cardiac NCCs into the conotruncal region and for formation of the aortico-pulmonary and ventricular septum.     

Molecular mechanisms of segmental patterning in the vertebrate hindbrain and neural crest

Recent work has shown that segmentation underlies the patterning of the vertebrate hindbrain and its neural crest derivatives. Several genes have been identified with segment-restricted expression, and evidence is now emerging regarding their function and regulatory relationships. The expression patterns of Hox genes and the phenotype of null mutants indicate roles in specifying segment identity. A zinc finger gene Krox-20 is a segment-specific regulator of Hox expression, and it seems probable that retinoic acid receptors also regulate Hox genes in the hindbrain. The receptor tyrosine kinase gene Sek may mediate cell-cell interactions that lead to segmentation. These studies provide a starting point for understanding the molecular basis of segmental patterning in the hindbrain.     

Plasticity in zebrafish hox expression in the hindbrain and cranial neural crest

The anterior-posterior identities of cells in the hindbrain and cranial neural crest are thought to be determined by their Hox gene expression status, but how and when cells become committed to these identities remain unclear. Here we address this in zebrafish by cell transplantation, to test plasticity in hox expression in single cells. We transplanted cells alone, or in small groups, between hindbrain rhombomeres or between the neural crest primordia of pharyngeal arches. We found that transplanted cells regulated hox expression according to their new environments. The degree of plasticity, however, depended on both the timing and the size of the transplant. At later stages transplanted cells were more likely to be irreversibly committed and maintain their hox expression, demonstrating a progressive loss of responsiveness to the environmental signals that specify segmental identities. Individual transplanted cells also showed greater plasticity than those lying within the center of larger groups, suggesting that a community effect normally maintains hox expression within segments. We also raised experimental embryos to larval stages to analyze transplanted cells after differentiation and found that neural crest cells contributed to pharyngeal cartilages appropriate to the anterior-posterior level of the new cellular environment. Thus, consistent with models implicating hox expression in control of segmental identity, plasticity in hox expression correlates with plasticity in final cell fate.     

Patterning the cranial neural crest: hindbrain segmentation and Hox gene plasticity

Understanding the patterning mechanisms that control head development--particularly the neural crest and its contribution to bones, nerves and connective tissue--is an important problem, as craniofacial anomalies account for one-third of all human congenital defects. Classical models for craniofacial patterning argue that the morphogenic program and Hox gene identity of the neural crest is pre-patterned, carrying positional information acquired in the hindbrain to the peripheral nervous system and the branchial arches. Recently, however, plasticity of Hox gene expression has been observed in the hindbrain and cranial neural crest of chick, mouse and zebrafish embryos. Hence, craniofacial development is not dependent on neural crest prepatterning, but is regulated by a more complex integration of cell and tissue interactions.     

Mechanisms of neural crest cell migration

Neural crest cells are remarkable in their extensive and stereotypic patterns of migration. The pathways of neural crest migration have been documented by cell marking techniques, including interspecific neural tube grafts, immunocytochemistry and Dil-labelling. In the trunk, neural crest cells migrate dorsally under the skin or ventrally through the somites, where they move in a segmental fashion through the rostral half of each sclerotome. The segmental migration of neural crest cells appears to be prescribed by the somites, perhaps by an inhibitory cue from the caudal half. Within the rostral sclerotome, neural crest cells fill the available space except for a region around the notochord, suggesting the notochord may inhibit neural crest cells in its vicinity. In the cranial region, antibody perturbation experiments suggest that multiple cell-matrix interactions are required for proper in vivo migration of neural crest cells. Neural crest cells utilize integrin receptors to bind to a number of extracellular matrix molecules. Substrate selective inhibition of neural crest cell attachment in vitro by integrin antibodies and antisense oligonucleotides has demonstrated that they possess at least three integrins, one being an α₁β₁ integrin which functions in the absence of divalent cations. Thus, neural crest cells utilize complex sets of interactions which may differ at different axial levels.     

Molecular analysis of neural crest migration

The neural crest (NC) cells have been called the 'explorers of the embryos' because they migrate all over the embryo where they differentiate into a variety of diverse kinds of cells. In this work, we analyse the role of different molecules controlling the migration of NC cells. First, we describe the strong similarity between the process of NC migration and metastasis in tumour cells. The epithelial-mesenchymal transition process that both kinds of cells undergo is controlled by the same molecular machinery, including cadherins, connexins, Snail and Twist genes and matrix metalloproteases. Second, we analysed the molecular signals that control the patterned migration of the cephalic and trunk NC cells. Most of the factors described so far, such as Eph/ephrins, semaphorins/neuropilins and Slit/Robo, are negative signals that prohibit the migration of NC cells into target areas of the embryo. Finally, we analyse how the direction of migration is controlled by regulation of cell polarity and how the planar cell polarity or non-canonical Wnt signalling is involved in this process.     

Ultrastructure of neural crest formation in the midbrain/rostral hindbrain and preotic hindbrain regions of the mouse embryo

In the mouse embryo, neural crest mesenchyme associated with the first and second pharyngeal arches escapes from the epithelium that forms the tips of the midbrain/rostral hindbrain and preotic hindbrain neural folds. To investigate the ultrastructure of crest formation, embryos with four to eight pairs of somites were processed for transmission electron microscopy. In the earliest event related to crest formation, crest precursors in the midbrain/rostral hindbrain elongated and moved all or most of their contents to the basal region of the epithelium. Elongation was probably mediated by apical bands of microfilaments and longitudinally oriented microtubules. Elongated cells then relinquished apical associations while nonelongated cells maintained those associations and withdrew from the basal lamina. This resulted in an epithelium stratified into apical and basal (crest precursor) layers. The coalescence of enlarging extra-cellular spaces opened a delaminate gap between the two layers. Additional crest precursors entered this gap from the apical layer. From the time crest precursors began moving basally, some formed microfilament- and/or microtubule-containing processes, which penetrated the basal lamina. Some of these cells moved their contents into the larger, microtubule-containing processes, perhaps thereby escaping from the epithelium. Soon after elongating cells appeared, the basal lamina beneath the epithelium began to degrade in a pattern unrelated to process formation. This ultimately resulted in disruption of the lamina, dispersal of the basal layer of the epithelium, and release of the crest precursors in the delaminate gap. Once crest formation was complete, the apical layer reformed a basal lamina on a patch-by-patch, cell-by-cell basis. In the preotic hindbrain, elongating crest precursors apparently forced their basal faces through the basal lamina and then relinquished apical association to escape. As a result, the lamina was disrupted before the epithelium could stratify, and enlarged extracellular spaces appeared among mesenchymal cells rather than creating a delaminate gap. The failure of elongation to disrupt the basal lamina in the midbrain/rostral hindbrain and its success in the preotic hindbrain might be due to less-vigorous, less-concerted elongation in the midbrain/rostral hindbrain or to earlier, more rapid degradation of the lamina in the preotic hindbrain.     

A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration

To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway.     

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.     

Environmental influences on neural crest cell migration

Neural crest cells migrate extensively and interact with numerous tissues and extracellular matrix components during their movement. Cell marking techniques have shown that neural crest cells in the trunk of the avian embryo migrate through the anterior, but not posterior, half of each sclerotome and avoid the region around the notochord. A possible mechanism to account for this migratory pattern is that neural crest cells may be inhibited from entering the posterior sclerotome and the perinotochordal space. Thus, interactions with other tissue may prescribe the pattern of neural crest cell migration in the trunk. In contrast, interactions between neural crest cells and the extracellular matrix may mediate the primary interactions controlling neural crest cells migration in the head region. © 1993 John Wiley & Sons, Inc.     

Control of neural crest cell behavior and migration

Neural crest cells (NCCs) are a remarkable, dynamic group of cells that travel long distances in the embryo to reach their target sites. They are responsible for the formation of craniofacial bones and cartilage, neurons and glia in the peripheral nervous system, and pigment cells. Live imaging of NCCs as they traverse the embryo has been critical to increasing our knowledge of their biology. NCCs exhibit multiple behaviors and communicate with each other and their environment along each step of their journey. Imaging combined with molecular manipulations has led to insights into the mechanisms controlling these behaviors. In this review, we highlight studies that have used live imaging to provide novel insight into NCC migration and discuss how continued use of such techniques can advance our understanding of NCC biology.