Developmental Expression of HNK-1-Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei

Monoclonal antibody HNK-1-reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK-1-reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto-N-neotetraosylceramide (SGGL-1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto-N-norhexaosyl ceramide (SGGL-2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK-1-reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK-1-reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK-1-reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N-CAM-180. A diffuse HNK-1-reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK-1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK-1-reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental Expression of HNK-1-Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.     

Analysis of the development of cellular subsets present in the neural crest using cell sorting and cell culture

We have tested the hypothesis that developmentally significant cellular subsets are present in the early stages of neural crest ontogenesis. Cultured quail trunk neural crest cells probed with the monoclonal antibodies HNK-1 and R24 exhibited heterogeneous staining patterns. Fluorescence-activated cell sorting was used to isolate the HNK-1+ and HNK-1- cell populations at 2 days in vitro. When these cell populations were cultured, the HNK-1+ sorted cells differentiated into melanocytes, unpigmented cells, and numerous catecholamine-positive (CA+) cells. In contrast, the HNK-1- sorted cells gave rise to melanocytes and unpigmented cells, but few, if any, CA+ cells. When neural crest cells at 2 days in vitro were labeled with R24 and sorted, both the R24+ the R24- sorted cell populations produced numerous CA+ cell, melanocytes, and unpigmented cells. These results provide evidence for the existence of developmental preferences in some subsets of neural crest cells early in embryogenesis.     

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.     

Characterization of HNK-1 antigens during the formation of the avian enteric nervous system.

During vertebrate embryogenesis, interaction between neural crest cells and the enteric mesenchyme gives rise to the development of the enteric nervous system. In birds, monoclonal antibody HNK-1 is a marker for neural crest cells from the entire rostrocaudal axis. In this study, we aimed to characterize the HNK-1 carrying cells and antigen(s) during the formation of the enteric nervous system in the hindgut. Immunohistological findings showed that HNK-1-positive mesenchymal cells are present in the gut prior to neural crest cell colonization. After neural crest cell colonization this cell type cannot be visualized anymore with the HNK-1 antibody. We characterized the HNK-1 antigens that are present before and after neural crest cell colonization of the hindgut. Immunoblot analysis of plasma membranes from embryonic hindgut revealed a wide array of HNK-1-carrying glycoproteins. We found that two HNK-1 antigens are present in E4 hindgut prior to neural crest cell colonization and that the expression of these antigens disappears after neural crest colonization. These two membrane glycoproteins, G-42 and G-44, have relative molecular masses of 42,000 and 44,000, respectively, and they both have isoelectric points of 5.5 under reducing conditions. We suggest that these HNK-1 antigens and the HNK-1-positive mesenchymal cells have some role in the formation of the enteric nervous system.     

Sulfoglucuronyl Glycolipids Bind Laminin

Previous studies have shown that HNK-1 antibody reactive glycoconjugates, including the glycolipids 3-sulfoglucuronylneolactotetraosylceramide (SGGL-1) and 3-sulfoglucuronylneolactohexaosylceramide (SGGL-2), are temporally and spatially regulated antigens in the developing mammalian cortex. Extracellular matrix glycoprotein laminin is involved in cell adhesion by interacting with cell surface components and also promotes neurite outgrowth. Laminin has been shown to bind sulfatide. The interaction of sulfated glycolipids SGGL-1 and SGGL-2 with laminin was studied by employing a solid-phase radioimmunoassay and by HPTLC-immunoblotting. Laminin binding was detected with anti-laminin antibodies followed by 125I-labelled Protein A and autoradiography. Laminin binds SGGL-1 and SGGL-2, besides sulfatide, but does not bind significantly gangliosides and neutral glycolipids. The binding of SGGLs to laminin was two to three times less compared to sulfatide when compared on a molar basis. Desulfation of SGGLs and sulfatide by mild acid treatment resulted in abolition of laminin binding. On the other hand, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect. This showed that the sulfate group was essential for laminin binding. Of the various glycosaminoglycans tested, only heparin inhibited the binding of laminin to SGGLs and sulfatide in a dose-dependent manner. This indicated that SGGLs and sulfatide bind to the heparin binding site present in the laminin molecule. The availability of HNK-1 reactive glycolipids and glycoproteins such as SGGLs and several neural cell adhesion molecules to bind laminin at critical stages of neural development may serve as important physiological signals.     

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis

Summary A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.     

Spectrum of in vitro differentiation of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody and analysis of the adrenergic development of HNK-1+ sorted subpopulations.

Previous work has demonstrated that catecholamine-containing cells differentiate preferentially from populations of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody (Maxwell, Forbes, and Christie, 1988). In the present work, we examine several additional features of the differentiation of these sorted cell populations. As one part of this study, the development of subpopulations of the HNK-(1+)-sorted neural crest cells has been investigated. Twice as many catecholamine-positive and total cells developed from the brightest third of the HNK-1+ cells compared to the remaining HNK-1+ cells, but the proportion of catecholamine-containing cells was similar in both populations. When either of these HNK-1+ subpopulations were grown together with HNK-1- cells, no reduction in the number of adrenergic cells was observed. These results indicate that subpopulations of HNK-1+ cells are qualitatively similar and that their adrenergic development is not affected by HNK-1- cells. In the second part of this study, we investigate the specificity of differentiation of HNK-(1+)- and HNK-(1-)-sorted cells by examining several additional phenotypic markers of development. We found that tyrosine hydroxylase and somatostatin immunoreactive cells developed from the HNK-(1+)-sorted population, while few, if any, cells bearing these phenotypic markers appeared in the HNK-(1-)-sorted population. In marked contrast, substantial numbers of cells immunoreactive for A2B5, E/C8, and NF-160 differentiated from both the HNK-(1+)- and the HNK-(1-)-sorted cell populations. The A2B5, E/C8, and NF-160 immunoreactive cells exhibited a variety of morphologies ranging from nonneuronal to neuronal in both sorted populations. Taken together, these results indicate that the presence of the HNK-1 antigen(s) on the trunk neural crest cell surface at 2 days in vitro is rather tightly correlated with the differentiation of adrenergic and some peptidergic cells, but much less so with other classes of neural cells including A2B5, E/C8, and NF-160 immunoreactive cells. Thus, these findings support the view that cell surface differences are correlated with and may contribute to the generation of the phenotypic diversity of neural crest cell derivatives.     

Loss of Sulfoglucuronyl and Other Neolactoglycolipids in Purkinje Cell Abnormality Murine Mutants

A significant reduction in the content of two members of the sulfoglucuronyl-neolacto series of glycolipids (SGGLs), 3-sulfoglucuronyl-lacto-N-neotetraosylceramide (SGGL-1) and 3-sulfoglucuronyl lacto-N-norhexaosylceramide (SGGL-2), in the cerebellum of the Purkinje cell abnormality mutants, Purkinje cell degeneration (pcd/pcd), lurcher (Lc/+), and staggerer (sg/sg), was also confirmed in the mildly affected nervous (nr/nr) mutant. The expression of SGGLs was studied during development of the pcd/pcd mutant cerebellum, and it was shown that the rate of decline in the level of SGGLs practically coincided with the loss of Purkinje cell perikarya. This indicated that SGGLs are primarily localized in Purkinje cells and that initially, at least, there is no genetic defect in the biosynthesis of SGGLs in the mutant. The precursors of SGGLs, viz., lacto-N-neotetraosylceramide (paragloboside) and lacto-N-norhexaosylceramide, as well as other glycolipids derived from these precursors, such as X-determinant fucoglycolipids and disialosyllacto-N-neotetraosylceramide, were also present in normal cerebellum. Levels of paragloboside and its other derivatives, similar to SGGLs, were also significantly reduced in the Purkinje cell abnormality mutants pcd/pcd, sg/sg, Lc/+, and nr/nr but were normal in other cerebellar mutants, such as quaking (qk/qk), weaver (wv/wv), and reeler (rl/rl), where Purkinje cells are not involved. Thus, the entire paragloboside family of glycolipids is primarily associated with Purkinje cells in the cerebellum. Although levels of monoclonal antibody HNK-1-reactive glycolipids were reduced in the Purkinje cell abnormality mutants, HNK-1-reactive glycoproteins were not affected in these mutants.     

Characterization of glycoconjugate antigens in mouse embryonic neural precursor cells

Neuronal and glial cells organizing the central nervous system (CNS) are generated from common neural precursor cells (NPCs) during neural development. However, the expression of cell-surface glycoconjugates that are crucial for determining the properties and biological function of these cells at different stages of development has not been clearly defined. In this study, we investigated the expression of several stage-specific glycoconjugate antigens, including several b-series gangliosides GD3, 9-O-acetyl GD3, GT1b and GQ1b, stage-specific embryonic antigen-1 (SSEA-1) and HNK-1, in mouse embryonic NPCs employing immunocytochemistry and flow cytometry. In addition, several of these antigens were positively identified by chemical means for the first time. We further showed that the SSEA-1 immunoreactivity was contributed by both glycoprotein and glycolipid antigens, and that of HNK-1 was contributed only by glycoproteins. Functionally, SSEA-1 may participate in migration of the cells from neurospheres in an NPC cell culture system, and the effect could be repressed by anti-SSEA-1 antibody. Based on this observation, we identified beta1 integrin as one of the SSEA-1 carrier glycoproteins. Our data thus provide insights into the functional role of certain glycoconjugate antigens in NPCs during neural development.     

Phenotypic characterization of Ewing sarcoma cell lines with monoclonal antibodies

The histogenesis of Ewing sarcoma, the second most frequent bone tumor in humans, remains controversial. Four Ewing cell lines were analyzed by immunological methods. A panel of antibodies directed to T, B, and myelomonocytic markers gave negative results. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. Ganglioside GD2, a marker of neuroectodermal tissues and tumors, was present on all lines. These were also stained by the mouse monoclonal antibody HNK-1, which detects a carbohydrate epitope present on several glycoconjugates of the nervous system, including two glycoproteins, the myelin-associated glycoprotein and the neural cell-adhesion molecule (N-CAM), and an acidic glycolipid of the peripheral nervous system. The P61 monoclonal antibody, which reacts with a peptide moiety of N-CAM, and a rabbit antiserum, raised to purified mouse N-CAM and not recognizing the HNK-1-defined epitope, were also reactive. By contrast, all antibodies specific for hematopoietic cell surface antigens were totally negative. Besides these antigenic features, Ewing sarcoma cells are characterized by a specific t(11;22)(q24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor, suggesting a possible evolutionary related origin. The recent finding that the human N-CAM gene is located at the vicinity of the breakpoint on chromosome 11 indicates that it might be involved in genetic rearrangements occurring in this region.     

Effect of different fixatives on immunocytochemical localization of HNK-1-reactive antigens in cerebellum: a method for differentiating the localization of the same carbohydrate epitope on proteins vs lipids.

Monoclonal antibody (MAb) HNK-1 recognizes a carbohydrate epitope present in certain glycolipids, glycoproteins, and proteoglycans. Five different fixation methods, together with biochemical analyses of the antigens, were evaluated to study immunocytochemical localization of this epitope in layers of adult rat cerebellum; 4% paraformaldehyde/0.5% cetylpyridinium chloride was found to be optimal for overall immunoreactivity, and the antigens were apparent in all cerebellar layers. To differentially localize HNK-1-reactive carbohydrate epitope on proteins vs lipids in cerebellar layers, we tested the effect of 0.2%, 2%, or 4% glutaraldehyde combined with 2% paraformaldehyde (GT/PF) on HNK-1 and other MAb-reactive protein and lipid antigens; 2% or 4% GT/PF significantly reduced or abolished immunoreactivity of MAb HNK-1 and 5F9 (reacting with microtubule-associated protein 2) with cerebellar proteins analyzed on Western blots, but did not decrease HNK-1 reactivity to lipid antigens on HPTLC blots. In cerebellar tissue sections, HNK-1 and 5F9 immunoreactivity was reduced after 2% or 4% GT/PF fixation. However, significant amounts of HNK-1 immunoreactivity remained in molecular layer and deep cerebellar nuclei. GT/PF fixation did not cause significant changes in immunoreactivity patterns of other carbohydrate lipid antigens, such as those that react with MAb A2B5, 7A, and WCC4. Therefore, carbohydrate epitope on lipids, as opposed to that on proteins, may be preferentially detectable by immunocytochemistry after fixation with 2% or 4% GT/PF. The selective localization of HNK-1-reactive carbohydrate in the molecular layer and deep cerebellar nuclei with 2% or 4% GT/PF fixation correlates well with the observed presence of HNK-1-reactive lipids in these areas but not in the granular layer and white matter, as determined by microdissection of the individual layers and biochemical analysis. The application of 2% or 4% GT/PF fixation as a general method for differentiating the same carbohydrate epitope on proteins vs lipids in immunocytochemistry for other tissues and other antibodies remains to be further evaluated.     

Explanted Fish Neural Tubes Give Rise to Differentiating Neural Crest Cells

Neural tubes were explanted from the trunk of various embryonic stages of three teleost fish, Xiphophorus maculatus (platyfish), X. helleri (swordtail), and Oryzias latipes (Japanese medaka) with the aim to obtain in vitro differentiating neural crest cells. Outgrowth of cells was observed immediately after attachment of the explants on dishes coated with fibronectin. The outgrowing cells stained with the HNK-1 monoclonal antibody indicating that they were neural crest cells. Maximum cell outgrowth was obtained from explants of stage 9 of Xiphophorus and 19 of medaka, i.e., from stages characteristic of maximal neural crest cell segregation, and by the use of Leibovitz's (L-15) medium supplemented with 20% FBS. In this medium cells survived for more than two weeks; M199 also gave satisfactory results but DMEM allowed only poor cell growth and survival. Neuronal cells could be observed in all cultures after 48 hr, in some medaka cultures these cells were mixed with pigment cells but homogeneous pigment cell cultures were also observed. This in vitro system will be invaluable for the study of the developmental potential of fish neural crest cells and the contributions of extrinsic factors in neural crest cell fate.     

Spectrum of in vitro differentiation of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody and analysis of the adrenergic development of HNK-1+ sorted subpopulations

Previous work has demonstrated that catecholamine-containing cells differentiate preferentially from populations of quail trunk neural crest cells isolated by cell sorting using the HNK-1 antibody (Maxwell, Forbes, and Christie, 1988). In the present work, we examine several additional features of the differentiation of these sorted cell populations. As one part of this study, the development of subpopulations of the HNK-1⁺-sorted neural crest cells has been investigated. Twice as many catecholamine-positive and total cells developed from the brightest third of the HNK-1⁺ cells compared to the remaining HNK-1⁺ cells, but the proportion of catecholamine-containing cells was similar in both populations. When either of these HNK-1⁺ subpopulations were grown together with HNK-1^? cells, no reduction in the number of adrenergic cells was observed. These results indicate that subpopulations of HNK-1⁺ cells are qualitatively similar and that their adrenergic development is not affected by HNK-1^? cells. In the second part of this study, we investigate the specificity of differentiation of HNK-1⁺- and HNK-1^?-sorted cells by examining several additional phenotypic markers of development. We found that tyrosine hydroxylase and somatostatin immunoreactive cells developed from the HNK-1⁺-sorted population, while few, if any, cells bearing these phenotypic markers appeared in the HNK-1^?-sorted population. In marked contrast, substantial numbers of cells immunoreactive for A2B5, E/C8, and NF-160 differentiated from both the HNK-1⁺- and the HNK-1^?-sorted cell populations. The A2B5, E/C8, and NF-160 immunoreactive cells exhibited a variety of morphologies ranging from nonneuronal to neuronal in both sorted populations. Taken together, these results indicate that the presence of the HNK-1 antigen(s) on the trunk neural crest cell surface at 2 days in vitro is rather tightly correlated with the differentiation of adrenergic and some peptidergic cells, but much less so with other classes of neural cells including A2B5, E/C8, and NF-160 immunoreactive cells. Thus, these findings support the view that cell surface differences are correlated with and may contribute to the generation of the phenotypic diversity of neural crest cell derivatives.     

Expression of Schwann cell markers by mammalian neural crest cells in vitro

During embryonic development, neural crest cells differentiate into a wide variety of cell types including Schwann cells of the peripheral nervous system. In order to establish when neural crest cells first start to express a Schwann cell phenotype immunocytochemical techniques were used to examine rat premigratory neural crest cell cultures for the presence of Schwann cell markers. Cultures were fixed for immunocytochemistry after culture periods ranging from 1 to 24 days. Neural crest cells were identified by their morphology and any neural tube cells remaining in the cultures were identified by their epithelial morphology and immunocytochemically. As early as 1 to 2 days in culture, approximately one third of the neural crest cells stained with m217c, a monoclonal antibody that appears to recognize the same antigen as rat neural antigen-1 (RAN-1). A similar proportion of cells were immunoreactive in cultures stained with 192-IgG, a monoclonal antibody that recognizes the rat nerve growth factor receptor. The number of immunoreactive cells increased with time in culture. After 16 days in culture, nests of cells, many of which had a bipolar morphology, were present in the area previously occupied by neural crest cells. The cells in the nests were often associated with neurons and were immunoreactive for m217c, 192-IgG and antibody to S-100 protein and laminin, indicating that the cells were Schwann cells. At all culture periods examined, neural crest cells did not express glial fibrillary acidic protein. These results demonstrate that cultured premigratory neural crest cells express early Schwann cell markers and that some of these cells differentiate into Schwann cells. These observations suggest that some neural crest cells in vivo may be committed to forming Schwann cells and will do so provided that they then proceed to encounter the correct environmental cues during embryonic development.     

Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins.

The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick beta 1 subunit of integrin, suggesting that beta 1-integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (greater than 1 microgram ml-1; Low-LM), but not at high coating concentration of laminin (10 micrograms ml-1; High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required greater than 0.1 mM Ca2+ or Mn2+. Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165,000 Mr which is also found in immunoprecipitates using antibodies against the beta 1 subunit of integrin and is likely to be an integrin alpha subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of beta 1-integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin.     

Ganglioside composition of growth cone-deficient nerve cell cultures

The ganglioside concentration and composition in growth cone-deficient nerve cells, induced by inclusion of cytochalasin B (CB) are compared with those of 2-day-old control cells from primary cultures of embryonic rat cerebral cortex. Ganglioside GM1 and GD1a are the major gangliosides in the growth cone. Ganglioside GM1 may be one of the membrane components of growth cones that function in neural recognition during development.     

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

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