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.     

A monoclonal antibody against a laminin-heparan sulfate proteoglycan complex perturbs cranial neural crest migration in vivo

INO (inhibitor of neurite outgrowth) is a monoclonal antibody that blocks axon outgrowth, presumably by functionally blocking a laminin-heparan sulfate proteoglycan complex (Chiu, A. Y., W. D. Matthew, and P. H. Patterson. 1986. J. Cell Biol. 103: 1382-1398). Here the effect of this antibody on avian neural crest cells was examined by microinjecting INO onto the pathways of cranial neural crest migration. After injection lateral to the mesencephalic neural tube, the antibody had a primarily unilateral distribution. INO binding was observed in the basal laminae surrounding the neural tube, ectoderm, and endoderm, as well as within the cranial mesenchyme on the injected side of the embryo. This staining pattern was indistinguishable from those observed with antibodies against laminin or heparan sulfate proteoglycan. The injected antibody remained detectable for 18 h after injection, with the intensity of immuno-reactivity decreasing with time. Embryos ranging from the neural fold stage to the 9-somite stage were injected with INO and subsequently allowed to survive for up to 1 d after injection. These embryos demonstrated severe abnormalities in cranial neural crest migration. The predominant defects were ectopic neural crest cells external to the neural tube, neural crest cells within the lumen of the neural tube, and neural tube deformities. In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. When embryos with ten or more somites were injected with INO, no effects were noted, suggesting that embryos are sensitive for only a limited time during their development. Immunoprecipitation of the INO antigen from 2-d chicken embryos revealed a 200-kD band characteristic of laminin and two broad smears between 180 and 85 kD, which were resolved into several bands at lower molecular mass after heparinase digestion. These results indicate that INO precipitates both laminin and proteoglycans bearing heparan sulfate residues. Thus, microinjection of INO causes functional blockage of a laminin-heparan sulfate proteoglycan complex, resulting in abnormal cranial neural crest migration. This is the first evidence that a laminin-heparan sulfate proteoglycan complex is involved in aspects of neural crest migration in vivo.     

Distribution of a putative cell surface receptor for fibronectin and laminin in the avian embryo

The cell substratum attachment (CSAT) antibody recognizes a 140-kD cell surface receptor complex involved in adhesion to fibronectin (FN) and laminin (LM) (Horwitz, A., K. Duggan, R. Greggs, C. Decker, and C. Buck, 1985, J. Cell Biol., 101:2134-2144). Here, we describe the distribution of the CSAT antigen along with FN and LM in the early avian embryo. At the light microscopic level, the staining patterns for the CSAT receptor and the extracellular matrix molecules to which it binds were largely codistributed. The CSAT antigen was observed on numerous tissues during gastrulation, neurulation, and neural crest migration: for example, the surface of neural crest cells and the basal surface of epithelial tissues such as the ectoderm, neural tube, notochord, and dermomyotome. FN and LM immunoreactivity was observed in the basement membranes surrounding many of these epithelial tissues, as well as around the otic and optic vesicles. In addition, the pathways followed by cranial neural crest cells were lined with FN and LM. In the trunk region, FN and LM were observed surrounding a subpopulation of neural crest cells. However, neither molecule exhibited the selective distribution pattern necessary for a guiding role in trunk neural crest migration. The levels of CSAT, FN, and LM are dynamic in the embryo, perhaps reflecting that the balance of surface-substratum adhesions contributes to initiation, migration, and localization of some neural crest cell populations.     

The Timing of Appearance and Pathway of Migration of Cranial Neural Crest-derived Precursors of Melanocytes in Chick Embryos

The timing of appearance and pathway of migration of precursors of melanocytes in cranial regions of chick embryos were examined by the monoclonal antibody MEBL-1, which can identify precursors of melanocytes soon after their emigration from the neural tube (7). Precursors of melanocytes were first detected on the dorsal side of the mesencephalic neural tube at stage 16, when other neural crest cells had already left the dorsal side of the neural tube. Then precursors of melanocytes at more rostral and caudal levels appeared. After the first appearance on the neural tube, precursors of melanocytes migrated along a dorsolateral pathway under the superficial ectoderm, which followed other neural crest cells. These results indicate that precursors of melanocytes migrate along spatially the same pathway as other neural crest cells, but temporally the different time as considered previously.     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.     

Translocation of latex beads after laser ablation of the avian neural crest

Previous studies from this laboratory (M. E. Bronner-Fraser, 1982, Dev. Biol.91, 50–63) have demonstrated that latex beads translocate ventrally after injection into avian embryos during the phase of neural crest migration, to settle in the vicinity of neural-crest-derived structures. In order to examine the role of host neural crest cells in the ventral translocation of implanted beads, latex beads have been injected into regions of embryos from which the neural crest cells have been ablated using a laser microbeam. Prior to their migratory phase, neural crest cells reside in the dorsal portion of the neural tube. Laser irradiation of the dorsal neural tube was used to reproducibly achieve either partial or complete ablation of neural crest cells from the irradiated regions. The effectiveness of the ablation was assessed by the degree of reduction in dorsal root ganglia, a neural crest derivative. Because of the rapidity and precision of this technique, it was possible to selectively remove neural crest cells without significantly altering other embryonic structures. The results indicate that, after injection of latex beads into the somites of embryos whose neural crest cells were removed by laser irradiation, the beads translocate ventrally in the absence of the endogenous neural crest.     

Perturbation of beta 1 integrin-mediated adhesions results in altered somite cell shape and behavior.

Cell contact and adhesion between somites and the axial extracellular matrix (ECM) is likely to play a fundamental role in vertebrate development. In a preliminary report we showed that injection of the monoclonal antibody CSAT, which recognizes the avian beta 1 integrins, causes a lateral separation of both somites and segmental plate tissue from the embryonic axis (Drake and Little, 1991). In this study we addressed the cell biological response to CSAT injection, particularly the cell-ECM interactions involved in maintaining normal somite-axial relationships. A total of 150 stage 7-10 quail embryos have been injected with CSAT and then cultured for varying periods (1-30 hr). CSAT caused somitic cells to behave abnormally. Changes include, rounding-up, extensive blebbing, and formation of retraction fibers. A majority of separated somites were able to assume normal axial position with further time culture. Whether a somite subsequently aligned at the axis was dependent on the amount of CSAT injected and the postinjection culture period. Embryos in which somites remained separated from the axis after relatively long culture intervals (18-24 hr) displayed abnormal sclerotomal cell migrations. In no case did control injected embryos exhibit cellular alterations. Similarly, the injection of RGD-containing peptides had no detectable effect on somitogenesis or somite/segmental plate adhesion to the axis. On the basis of these data, we conclude that beta 1 integrins are necessary for normal somitic cell adhesions to the axis, but not somite segmentation and differentiation.     

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.     

Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube.

We have examined the distribution and function of the defined cell adhesion molecules, N-cadherin and N-CAM, in the emigration of cranial neural crest cells from the neural tube in vivo. By immunocytochemical analysis, both N-cadherin and N-CAM were detected on the cranial neural folds prior to neural tube closure. After closure of the neural tube, presumptive cranial neural crest cells within the dorsal aspect of the neural tube had bright N-CAM and weak N-cadherin immunoreactivity. By the 10- to 11-somite stage, N-cadherin was prominent on all neural tube cells with the exception of the dorsal-most cells, which had little or no detectable immunoreactivity. N-CAM, but not N-cadherin, was observed on some migrating neural crest cells after their departure from the cranial neural tube. To examine the functional significance of these molecules, perturbation experiments were performed by injecting antibodies against N-CAM or N-cadherin into the cranial mesenchyme adjacent to the midbrain. Fab' fragments or whole IgGs of monoclonal and polyclonal antibodies against N-CAM caused abnormalities in the cranial neural tube and neural crest. Predominantly observed defects included neural crest cells in ectopic locations, both within and external to the neural tube, and mildly deformed neural tubes containing some dissociating cells. A monoclonal antibody against N-cadherin also disrupted cranial development, with the major defect being grossly distorted neural tubes and some ectopic neural crest cells outside of the neural tube. In contrast, nonblocking N-CAM antibodies and control IgGs had few effects. Embryos appeared to be sensitive to the N-CAM and N-cadherin antibodies for a limited developmental period from the neural fold to the 9-somite stage, with older embryos no longer displaying defects after antibody injection. These results suggest that the cell adhesion molecules N-CAM and N-cadherin are important for the normal integrity of the cranial neural tube and for the emigration of neural crest cells. Because cell-matrix interactions also are required for proper emigration of cranial neural crest cells, the results suggest that the balance between cell-cell and cell-matrix adhesion may be critical for this process.     

Partial restriction in the developmental potential of late emigrating avian neural crest cells.

Trunk neural crest cells migrate along two major pathways: a ventral pathway through the somites whose cells form neuronal derivatives and dorsolateral pathway underneath the ectoderm whose cells become pigmented. In avian embryos, the latest emigrating neural crest cells move only along the dorsolateral pathway. To test whether late emigrating neural crest cells are more restricted in developmental potential than early migrating cells, cultures were prepared from the neural tubes of embryos at various stages of neural crest cell migration. "Early" and "middle" aged neural crest cells differentiated into many derivatives including pigmented cells, neurofilament-immunoreactive cells, and adrenergic cells. In contrast, "late" neural crest cells differentiated into pigment cells and neurofilament-immunoreactive cells, but not into adrenergic cells even after 10-14 days. To further challenge the developmental potential of early and late emigrating neural crest cells, they were transplanted into embryos during the early phases of neural crest cell migration, known to be permissive for adrenergic neuronal differentiation. The cells were labeled with the vital dye, DiI, and injected onto the ventral pathway at stages 14-17. Two and three days after injection, some early neural crest cells were found to express catecholamines, suggesting they were adrenergic neuroblasts. In contrast, DiI-labeled late neural crest cells never became catecholamine-positive. These results suggest that the late emigrating neural crest cell population has a more restricted developmental potential than the early migrating neural crest cell population.     

Effect of hyaluronidase treatment on the distribution of cranial neural crest cells in the chick embryo

The cranial paraxial mesoblast is patterned into segmental units termed somitomeres. Recently we demonstrated the morphological relationship between the migratory pathways of cranial neural crest cells and the patterned primary mesenchyme of chick embryos (Anderson and Meier, '81). Since extracellular matrix, particularly hyaluronate, is also distributed in cranial crest pathways, embryos were given sub-blastodisc injections of hyaluronidase just prior to neural tube fusion and neural crest migration to remove matrix. Histological sections of enzyme-treated embryos showed that Alcian blue staining of hyaluronate was significantly reduced. Surface ectoderm appeared collapsed on the subjacent mesoderm as well. Examination of embryos with the scanning electron microscope (SEM) revealed that paraxial mesoderm remained segmentally patterned even though it appeared more condensed because of a reduction in intercellular space between mesenchymal cells. In enzyme-treated embryos, the rostral crest cells spread over the dorsal surfaces of the first four somitomeres, as they would do normally. This distribution of neural crest cells occurs even when enzyme treatment interferes with neural tube fusion at that level. We conclude that 1) neural tube fusion is not a prerequisite for the timely release of cranial crest in the chick embryo and 2) that much of the organized hyaluronate-rich matrix that lies in the path of cranial crest is not essential for crest emigration or patterned distribution.     

Pathways of trunk neural crest cell migration in the mouse embryo as revealed by vital dye labelling

Analysis of neural crest cell migration in the mouse has been difficult due to the lack of reliable cell markers. Recently, we found that injection of DiI into the chick neural tube marks premigratory neural crest cells whose endfeet are in contact with the lumen of the neural tube (Serbedzija et al. Development 106, 809-819 (1989)). In the present study, this technique was applied to study neural crest cell migratory pathways in the trunk of the mouse embryo. Embryos were removed from the mother between the 8th and the 10th days of development and DiI was injected into the lumen of the neural tube. The embryos were then cultured for 12 to 24 h, and analyzed at the level of the forelimb. We observed two predominant pathways of neural crest cell migration: (1) a ventral pathway through the rostral portion of the somite and (2) a dorsolateral pathway between the dermamyotome and the epidermis. Neural crest cells were observed along the dorsolateral pathway throughout the period of migration. The distribution of labelled cells along the ventral pathway suggested that there were two overlapping phases of migration. An early ventrolateral phase began before E9 and ended by E9.5; this pathway consisted of a stream of cells within the rostral sclerotome, adjacent to the dermamyotome, that extended ventrally to the region of the sympathetic ganglia and the dorsal aorta.(ABSTRACT TRUNCATED AT 250 WORDS)     

In ovo time-lapse analysis after dorsal neural tube ablation shows rerouting of chick hindbrain neural crest

Previous analyses of single neural crest cell trajectories have suggested important roles for interactions between neural crest cells and the environment, and amongst neural crest cells. To test the relative contribution of intrinsic versus extrinsic information in guiding cells to their appropriate sites, we ablated subpopulations of premigratory chick hindbrain neural crest and followed the remaining neural crest cells over time using a new in ovo imaging technique. Neural crest cell migratory behaviors are dramatically different in ablated compared with unoperated embryos. Deviations from normal migration appear either shortly after cells emerge from the neural tube or en route to the branchial arches, areas where cell-cell interactions typically occur between neural crest cells in normal embryos. Unlike the persistent, directed trajectories in normal embryos, neural crest cells frequently change direction and move somewhat chaotically after ablation. In addition, the migration of neural crest cells in collective chains, commonly observed in normal embryos, was severely disrupted. Hindbrain neural crest cells have the capacity to reroute their migratory pathways and thus compensate for missing neural crest cells after ablation of neighboring populations. Because the alterations in neural crest cell migration are most dramatic in regions that would normally foster cell-cell interactions, the trajectories reported here argue that cell-cell interactions have a key role in the shaping of the neural crest migration.     

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.     

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

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 fibronectin in the early phase of avian cephalic neural crest cell migration

Immunofluorescence and immunoperoxidase labeling for fibronectin was used to study the early events of cephalic neural crest cell migration in avian embryos. Prior to crest cell appearance, fibronectin was associated with the basement membranes of all tissues. The loose mesenchymal cells were also surrounded by this glycoprotein. The crest cell individualization phase included a transient rounding up and a rapid increase in cell number in a very limited space. Whereas the neural tube basement membrane was not formed dorsally at the site of emergence of crest cells, it was partially fused laterally with the ectoderm basement membrane apparently preventing immediate crest cell emigration. Further increase in cell number occurred concomitantly with their penetration between the two developing basement membranes of the neural tube and the ectoderm. The localization of migrating crest cells is apparently greatly influenced by local interactions between the ectoderm and the neural tube, whose morphogenesis differs considerably at each axial level: at the mesencephalic-rhombencephalic levels, crest cells rapidly reached a cell-free space that was mostly devoid of fibronectin. Further migration occurred laterally in that space while pioneer crest cells became surrounded by fibronectin in their environment. Crest cells progressed as a confluent multicellular layer with an apparent velocity of 70 μm/hr. At the prosencephalic and median rhombencephalic levels, crest cells accumulated between the fibronectin-rich basement membranes of the ectoderm and the neural tube. Pioneer crest cells were arrested at the site of attachment of the ectoderm and the neural tube basement membranes (i.e., optic vesicles and otic placodes). Crest cells resumed their migration when more space became available during the constriction of the optic vesicles and the invagination of the otic placodes.