HNK-1 Antibody Research Articles

Development of the Ventricular Conduction System of the Crocodilian Heart

Unlike other cold‐blooded vertebrates, crocodilians have a fully septated heart, similar to birds and mammals. It is not known whether they have a ventricular conduction system (VCS) as wellWe studied a group of 8 embryos of the Siamese and Mugger Crocodile between 3 and 84 days of incubation, and serial early embryo sections with HNK‐1/Hematoxylin staining of the Nile Crocodile. We employed optical mapping, ultrasound biomicroscopy, histo‐ and immunohistochemistry, in situ hybridization and 3D reconstructionAt the pre‐septation stage (12 days of incubation), the ventricular activation pattern progressed in a left‐to‐right sweep, similar to that observed at early stages of avian and mammalian cardiogenesis. At two post‐septation stages investigated (45 and 84 days) , epicardial activation patterns showed dual apical breakthroughs on either side of the interventricular septum. Immunohistochemistry using smooth muscle actin (SMA), sarcomeric actin, periostin and HNK‐1 antibodies showed that myocardial SMA staining was present even after septation, while it disappears in comparable chick or mouse hearts. HNK‐1 staining was prominent in the interventricular septum, similar to the situation in the chick and rat where it is considered a marker of the developing ventricular conduction system. In situ hybridization for Cx40 (VCS marker) showed a uniform expression in the entire trabeculated myocardiumWe conclude that this is an evidence of evolutionary correlation between ventricular septation and presence specialized VCS in vertebratesSupported by PRVOUK P35/LF1/5.

Distribution of acetylcholine and catecholamines in fish gills and their potential roles in the hypoxic ventilatory response

Carotid body glomus cells in mammals contain a plethora of different neurochemicals. Several hypotheses exist to explain their roles in oxygen-chemosensing. In the present study we assessed the distribution of serotonin, acetylcholine and catecholamines in the gills of trout (Oncorhynchus mykiss) and goldfish (Carassius auratus) using immunohistochemistry, and an activity-dependent dye, Texas Red hydrazide (TXR). In fish the putative oxygen sensing cells are neuroepithelial cells (NECs) and the focus in recent studies has been on the role of serotonin in oxygen chemoreception. The NECs of trout and goldfish contain serotonin, but, in contrast to the glomus cells of mammals, not acetylcholine or catecholamines. Acetylcholine was expressed in chain and proximal neurons and in extrinsic nerve bundles in the filaments. The serotonergic NECs did not label with the HNK-1 antibody suggesting that if they are derived from the neural crest, they are no longer proliferative or migrating. Furthermore, we predicted that if serotonergic NECs were chemosensory, they would increase their activity during hypoxia (endocytose TXR), but following 30min of hypoxic exposure (45Torr), serotonergic NECs did not take up TXR. Based on these and previous findings we propose several possible models outlining the ways in which serotonin and acetylcholine could participate in oxygen chemoreception in completing the afferent sensory pathway.

Synthesis and Molecular Recognition Studies of the HNK-1 Trisaccharide and Related Oligosaccharides. The Specificity of Monoclonal Anti-HNK-1 Antibodies as Assessed by Surface Plasmon Resonance and STD NMR

The human natural killer cell carbohydrate, HNK-1, plays function-conducive roles in peripheral nerve regeneration and synaptic plasticity. It is also the target of autoantibodies in polyneuropathies. It is thus important to synthesize structurally related HNK-1 carbohydrates for optimizing its function-conducive roles, and for diagnosis and neutralization of autoantibodies in the fatal Guillain-Barré syndrome. As a first step toward these goals, we have synthesized several HNK-1 carbohydrate derivatives to assess the specificity of monoclonal HNK-1 antibodies from rodents: 2-aminoethyl glycosides of selectively O-sulfated trisaccharide corresponding to the HNK-1 antigen, its nonsulfated analogue, and modified structures containing 3-O-fucosyl or 6-O-sulfo substituents in the N-acetylglucosamine residues. These were converted, together with several related oligosaccharides, into biotin-tagged probes to analyze the precise carbohydrate specificity of two anti-HNK-1 antibodies by surface plasmon resonance that revealed a crucial role of the glucuronic acid in antibody binding. The contribution of the different oligosaccharide moieties in the interaction was shown by saturation transfer difference (STD) NMR of the complex consisting of the HNK-1 pentasaccharide and the HNK-1 412 antibody.

The early scaffold of axon tracts in the brain of a primitive vertebrate, the sea lamprey

The development of the early axonal scaffold formed by early-differentiating neurons was studied in a primitive vertebrate (the sea lamprey), by immunohistochemistry against acetylated α-tubulin and a cell surface marker (HNK-1 antibodies), to determine the degree of conservation of this process in vertebrate evolution. The medial and dorsolateral longitudinal fascicles were the first longitudinal axonal bundles observed to develop in the neural tube, followed by the tract of the postoptic commissure and the supraoptic tract. Establishment of the first dorso-ventral tracts occurs after the appearance of the tract of the postoptic commissure and the medial longitudinal fascicle, the basal plate longitudinal axonal system. The dorsolateral longitudinal fascicle appears to be equivalent to the “descending tract” of the mesencephalic nucleus of the trigeminal nerve of mouse and birds; the possible homologies between other early scaffold tracts of the sea lamprey and those of other vertebrates are also discussed. In addition, present results suggest the presence of highly conserved brain regions that would allow for early neuronal differentiation and axonal pathfinding in vertebrates, which were probably defined before the divergence of Agnathans and Gnathostomes.

Vagal neural crest contribution to the chick embryo cloaca

Intrinsic innervation of the developing chick cloaca is provided by the enteric nervous system, a network of neurons and glia that lies within its walls. The enteric nervous system originates from neural crest cells that migrate from the vagal and sacral regions of the neural tube during the early stages of development. Abnormal cloacal development can cause a number of anorectal anomalies including persistent cloaca. Our study aimed to investigate the contribution of vagal neural crest cells to the total population of enteric neurons and glia within the chick embryo cloaca, using quail-chick chimeras. Chicken embryos were incubated until the 10-12 somite stage (ss). The vagal neural tube, corresponding to somites 1-7, was then microsurgically ablated in ovo and isochronic and isotopic quail grafts were performed. The eggs were then reincubated until embryos were harvested at E12. Whole embryos were fixed in Bouin's fluid, embedded in paraffin wax and sectioned. Immunohistochemistry was carried out using the HNK-1 antibody to label all neural crest cells, and the quail-specific antibody, QCPN, to label quail cells. QCPN-immunoreactive cells were seen to make up a large proportion of enteric neurons and glia within the walls of the embryonic cloaca. HNK-1 labelled all neural crest cells in the myenteric and submucosal plexuses as well as the sacral crest-derived nerve of Remak, while QCPN-positive cells were evident in both plexuses but mostly in the submucosal plexus, where they appeared to make up the majority of neurons. Results show that the chick embryo cloaca is primarily innervated by vagal neural crest cells. Further studies to investigate the contribution of sacral neural crest cells to the same region will give further insight into the development of the enteric nervous system within the embryonic cloaca.

The zic1 gene is expressed in chick somites but not in migratory neural crest

Zic transcription factors regulate the expression of neural and neural crest-specific genes and are expressed in the cells of the dorsal neural tube and the premigratory neural crest. Here we characterize zic1 expression in the chick embryo during somite formation and neural crest migration. zic1 is expressed in the dorsomedial portion of epithelial somites and subsequently in the dorsomedial lip of the dermomyotome. Although zic1 is expressed in cells of the nascent myotome, it is absent from differentiated myotome cells that express myosin. As the dorsal root ganglia form, zic1 is expressed at high levels in the dorsal sclerotome and zic1 expression is more pronounced in the caudal regions of the somites. Double-label experiments showed that cells expressing zic1 are not labeled by the HNK-1 antibody specific for migratory neural crest cells. Thus, migrating neural crest cells do not express zic1.

Potential roles for BMP and Pax genes in the development of iris smooth muscle

The embryonic optic cup generates four types of tissue: neural retina, pigmented epithelium, ciliary epithelium, and iris smooth muscle. Remarkably little attention has focused on the development of the iris smooth muscle since Lewis ([1903] J. Am. Anat. 2:405-416) described its origins from the anterior rim of the optic cup neuroepithelium. As an initial step toward understanding iris smooth muscle development, I first determined the spatial and temporal pattern of the development of the iris smooth muscle in the chick by using the HNK1 antibody, which labels developing iris smooth muscle. HNK1 labeling shows that iris smooth muscle development is correlated in time and space with the development of the ciliary epithelial folds. Second, because neural crest is the only other neural tissue that has been shown to generate smooth muscle (Le Lievre and Le Douarin [1975] J. Embryo. Exp. Morphol. 34:125-154), I sought to determine whether iris smooth muscle development shares similarities with neural crest development. Two members of the BMP superfamily, BMP4 and BMP7, which may regulate neural crest development, are highly expressed by cells at the site of iris smooth muscle generation. Third, because humans and mice that are heterozygous for Pax6 mutations have no irides (Hill et al. [1991] Nature 354:522-525; Hanson et al. [1994] Nat. Genet. 6:168-173), I determined the expression of Pax6. I also examined the expression of Pax3 in the developing anterior optic cup. The developing iris smooth muscle coexpresses Pax6 and Pax3. I suggest that some of the eye defects caused by mutations in Pax6, BMP4, and BMP7 may be due to abnormal iris smooth muscle.

Recognition molecule associated carbohydrate inhibits postsynaptic GABA B receptors: a mechanism for homeostatic regulation of GABA release in perisomatic synapses

Extracellular matrix molecules are important cues in the shaping of nervous system structure and function. Here, we describe a novel mechanism by which the HNK-1 carbohydrate carried by recognition molecules regulates perisomatic inhibition in the hippocampus. Neutralization of HNK-1 activity by an HNK-1 antibody results in GABA B receptor-mediated activation of K + currents in CA1 pyramidal cells, which elevates extracellular K + concentration and reduces evoked GABA release in perisomatic inhibitory synapses. This mechanism is supported by pharmacological analysis in hippocampal slices and data showing that the HNK-1 carbohydrate binds to GABA B receptors and inhibits GABA B receptor-activated K + currents in a heterologous expression system. We suggest that the HNK-1 carbohydrate is involved in homeostatic regulation of GABA A receptor-mediated perisomatic inhibition by suppression of postsynaptic GABA B receptor activity.

Regulation of expression of sulfoglucuronyl carbohydrate (HNK-1), Amphoterin and RAGE in retinoic acid-differentiated P19 embryonal carcinoma cells.

HNK-1 antibody reactive sulfoglucuronyl carbohydrate (SGC) and SSEA-1 antibody reactive Lewis X (Lex) epitope are expressed on several glycolipids, glycoproteins, and proteoglycans of the nervous system and have been implicated in cell-cell recognition, neurite outgrowth, and/or neuronal migration during development. Interaction of SGC with its binding protein Amphoterin and interaction of Amphoterin with a cell-signaling molecule, receptor for advance glycation end product (RAGE) have been suggested to regulate neurite outgrowth and neuronal migration. The regulation of expression of SGC, Lex, Amphoterin, and RAGE was studied in embryonal carcinoma P19 cells after treatment with retinoic acid (RA). The untreated proliferating P19 cells strongly expressed the Lex epitope, which was mostly due to Lex-glycoproteins. P19 cells, when differentiated into neuron-like cells by RA, did not express the Lex epitope, but expressed increasing levels of SGC, with time in culture. Quantitative biochemical analyses showed that in the P19 cells after RA treatment, the amount of SGC-glycoproteins increased at a significantly higher level than sulfoglucuronyl glycolipid-1 (SGGL-1). The increase in the levels of SGGL-1 was due to 16-fold upregulation in the activity of lactosylceramide: N-acetylglucosaminyl-transferase (Lc3 synthase), which synthesizes the key intermediate lactotriosylceramide (Lc3Cer), for lacto- and neolacto-glycolipids. The large increase in the activity of Lc3 synthase appeared to regulate the levels of other neolacto glycolipids, such as Lc3Cer, nLc4Cer, nLc6Cer, disialosyl-nLc4Cer (LD1), and Lex-glycolipids. Strong upregulation of glucuronyl-transferase and modest twofold enhancement in the activity of the glucuronyl-sulfotransferase, which catalyze the final steps in the SGC synthesis, also would account for the large increase in the synthesis SGC-glycoproteins. RA also upregulated the synthesis of Amphoterin and RAGE in P19 cells. SGC, RAGE, and Amphoterin were co-localized in the RA-differentiated neurons. The initiation of neurite outgrowth along with co-ordinated upregulation of Amphoterin, RAGE, SGC-glycoproteins, and SGGLs in RA-treated P19 cells support the hypothesis that these molecules are involved in the neuronal process formation.

Developmental changes in the biochemical and immunological characters of the carbohydrate moiety of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan.

Neuroglycan C (NGC), a brain-specific transmembrane proteoglycan, is thought to bear not only chondroitin sulfate but also N- and O-linked oligosaccharides on its core protein. In this study, we isolated and purified NGC from rat brains at various developmental stages by immunoaffinity column chromatography or by immunoprecipitation, and examined the structural characters of its carbohydrate moiety. The chondroitin sulfate disaccharide composition of NGC at postnatal day 10 was significantly different from those of two secreted chondroitin sulfate proteoglycans, neurocan and phosphacan, purified from the brain at the same developmental stage; higher levels of 4-sulfate unit and E unit, a disulfated disaccharide unit, and a lower level of 6-sulfate unit. The levels of both 6-sulfate and E units decreased with a compensatory increase of 4-sulfate unit with postnatal development of the brain. Lectin-blot analysis of the NGC core glycoprotein prepared by chondroitinase digestion confirmed that NGC actually bore both N- and O-linked carbohydrates, and also revealed that lectin-species reactive with NGC did not always recognize other brain-specific proteoglycans, neurocan and phosphacan, and vice versa, even though they were isolated from the brain at the same stage. The reactivity of NGC with lectins and with the HNK-1 antibody markedly changed as the brain matured. These findings indicate that the structure of the carbohydrate moiety of NGC is developmentally regulated, and differs from those of neurocan and phosphacan. The developmentally-regulated structural change of the carbohydrates on NGC may be partly implicated in the modulation of neuronal cell recognition during brain development.

Intrahippocampal Administration of an Antibody against the HNK-1 Carbohydrate Impairs Memory Consolidation in an Inhibitory Learning Task in Mice

Many cell adhesion molecules express the HNK-1 carbohydrate involved in formation and functioning of synapses. To assess its role in learning, we injected the monoclonal HNK-1 antibody or nonimmune IgG into the hippocampus of C57BL/6J mice 1 h after training in a step-down avoidance task. In animals treated with the HNK-1 antibody, latencies of step down in a recall session 48 h after injection did not change compared to training values and were significantly shorter versus IgG-treated controls, which acquired the task normally. Similar differences between the two treatments were also observed after a stronger training protocol in a step-down avoidance paradigm. The HNK-1 antibody was effective only when injected 1 h, but not 48 h after training, thus affecting memory consolidation but not memory recall itself. The HNK-1 antibody impaired memory also in tenascin-R knock-out mice, indicating that extracellular matrix molecule tenascin-R, one of the carriers of the HNK-1epitope in the hippocampus, does not mediate the function of the HNK-1 carbohydrate in this task. Our observations show that the HNK-1 carbohydrate is critically involved in memory consolidation in hippocampus-dependent learning in mammals.

Cell specific-chondroitin sulfate proteoglycan expression during CNS morphogenesis in the chick embryo

There is increasing evidence that proteoglycans, particularly chondroitin sulfate proteoglycans (CSPGs), are integral components in the assembly of the extracellular matrix during early stages of histogenesis. The differential expression of several CSPGs in the developing CNS has raised questions on their origin, phenotype (chemical and structural characteristics), regulation of expression and function. The S103L monoclonal antibody has been an invaluable specific reagent to identify and study a large and abundant CSPG in embryonic chick brain. In the present study we demonstrate that during embryogenesis of the chick CNS, the S103L CSPG (B-aggrecan) is synthesized by neurons of all major neuronal cell types but not by astrocytes, is developmentally regulated, and is associated predominantly with neuronal somata, suggesting that neuronal-specific regulatory mechanisms control the expression of the S103L CSPG in culture. Neurons also exhibit differential expression of glycosaminoglycan type (i.e., KS) and sulfation patterns on different CSPGs when compared to astrocytes, meningial cells or chondrocytes, implying the existence of additional, cell type-specific modes of regulation of the final CSPG phenotype (chemical and structural posttranslational characteristics). A specific temporal pattern of expression of the S103L-CSPG was observed which may contribute to conditions that induce or stabilize specific cell phenotypes during CNS development. In contrast, the other major CSPG in the CNS recognized by the HNK-1 antibody, is synthesized by all cell types of different cell lineages over the entire embryonic period, suggesting a more global cell maintenance function for this CSPG.

The extracellular matrix molecule tenascin-R and its HNK-1 carbohydrate modulate perisomatic inhibition and long-term potentiation in the CA1 region of the hippocampus.

Perisomatic inhibition of pyramidal cells regulates efferent signalling from the hippocampus. The striking presence of HNK-1, a carbohydrate expressed by neural adhesion molecules, on perisomatic interneurons and around somata of CA1 pyramidal neurons led us to apply monoclonal HNK-1 antibodies to acute murine hippocampal slices. Injection of these antibodies decreased GABAA receptor-mediated perisomatic inhibitory postsynaptic currents (pIPSCs) but did not affect dendritic IPSCs or excitatory postsynaptic currents. The decrease in the mean amplitude of evoked pIPSCs by HNK-1 antibodies was accompanied by an increase in the coefficient of variation of pIPSC amplitude, number of failures and changes in frequency but not amplitude of miniature IPSCs, suggesting that HNK-1 antibodies reduced efficacy of evoked GABA release. HNK-1 antibodies did not affect pIPSCs in knock-out mice deficient in the extracellular matrix molecule tenascin-R which carries the HNK-1 carbohydrate as analysed by immunoblotting in synaptosomal fractions prepared from the CA1 region of the hippocampus. For control, HNK-1 antibody was applied to acute sections of mice deficient in the neural cell adhesion molecule NCAM, another potential carrier of HNK-1, and resulted in decrease of pIPSCs as observed in wild-type mice. Reduction in perisomatic inhibition is expected to promote induction of long-term potentiation (LTP) by increasing the level of depolarization during theta-burst stimulation. Indeed, LTP was increased by HNK-1 antibody applied before stimulation. Moreover, LTP was reduced by an HNK-1 peptide mimic, but not control peptide. These results provide first evidence that tenascin-R and its associated HNK-1 carbohydrate modulate perisomatic inhibition and synaptic plasticity in the hippocampus.

Distribution of antigen epitopes shared by nerves and the myocardium of the embryonic chick heart using different neuronal markers.

We examined which neuronal elements and nonneuronal tissues in the embryonic myocardium are stained with antibodies traditionally used for staining nerve tissue. Furthermore, we studied whether nonneuronal myocardial staining was confined to regions determining initial nerve entry points and development of cardiac ganglia. The third focus was whether nerves preferentially distribute in regions of the conduction system. Different neuronal markers were used such as the HNK-1 antibody against neural crest and nerve tissue, Tyrosine Hydroxylase antibody (TH) against putative sympathetic nerve tissue, anti-GFAP against glia cells, antibodies against phosphorylated neurofilaments DO170, RMO270, 3A10, and RT97, and finally the antibody Snap25 against a synaptic protein. Chick embryonic hearts between stage HH25-44 where immunohistochemically evaluated. Transient HNK-1 staining in the basal region of the heart coincided with ingrowing vagal branches and crest-derived neuronal precursor cells seeding the region of the atrioventricular sulcus, suggesting a role for HNK-1 in the homing of the parasympathetic plexus. Transient TH staining was confined to regions of the atrial myocardium coincident with the localization of the few early TH-positive nerve fibers before stage HH40, whereas the second wave of TH-positive nerve fibers at HH42 was mainly localized around myocardial coronary arteries. This transient myocardial TH staining might be involved in early emergence of the catecholaminergic phenotype, while coronary arteries or blood borne factors might be involved in later differentiation. Some myocardial expression, not related with initial nerve ingrowth, using Snap25, TH, HNK-1, DO170, and RMO270 was confined to regions of the ventricular conduction system. HNK-1 is the only marker staining the region of the putative sinoatrial node. Just before hatching nerve fibers, including TH-positive nerve fibers, are uniformly distributed throughout the myocardium, without being specifically confined to regions containing the conduction system or coronary arteries.

Role for cell adhesion and glycosyl (HNK-1 and oligomannoside) recognition in the sharpening of the regenerating retinotectal projection in goldfish.

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7-21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76-86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1-positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26 degrees versus 11-12 degrees in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7 degrees. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18 degrees. Thus, oligomannosidic glycans in particular may play a role in retinotopic sharpening. Blocking glycan-mediated interactions between CAMs and ECM molecules could decrease the extent of exploratory growth of retinal axon collaterals, preventing them from finding their retinotopic sites, or could interfere with L1 or NCAM and laminin binding at the synaptic densities preventing stabilization of retinotopically appropriate synapses. Together, these results support a prominent role for cell adhesion and glycan epitopes in visual synaptic plasticity.

On the Specificity of Anti-Sulfoglucuronosyl Glycolipid Antibodies1

The mouse monoclonal anti-human HNK-1 antibody (also variously known as L2, Leu-7, CD57, VC1.1), monoclonal anti-sulfoglucuronosyl glycosphingolipids (SGGLs) antibody (mAb NGR50), and human sera from patients with demyelinating neuropathy and IgM paraproteinemia, are known to react with not only SGGLs, including sulfoglucuronosyl paragloboside (SGPG) and sulfoglucuronosyl lactosaminyl paragloboside (SGLPG), but also glycoproteins, such as myelin-associated glycoprotein (MAG), PO, PMP22, and certain adhesion molecules. These antigens are known to possess the so-called HNK-1 epitope (3-sulfoglucuronic acid, SGA) moiety. To further define the precise structural requirement of this carbohydrate epitope, we chemically synthesized 14 SGGLs, and their nonsulfated derivatives with defined carbohydrate chain lengths and aglycone structures. The various aglycones include ceramide, 2-(tetradecyl)hexadecyl (B30), and 2-(trimethylsilyl)ethyl (SE). These synthetic SGGLs were tested for their immunoreactivity with the above antibodies by high-performance thin-layer chromatography (HPTLC)-immunoblotting and ELISA. The anti-HNK-1 antibody (VC1.1) reacted with SGGL analogues containing a minimum of two sugars (SGA-Gal-Cer), but not with non-sulfated derivatives of SGGLs nor with SGGLs having a modified ceramide structure. mAb NGR50, on the other hand, reacted with only SGPG and SGLPG. A human patient serum (LT) reacted with all synthetic SGGLs except those with an SE aglycone structure. On the other hand, another human patient serum (YT). like the anti-HNK-1 antibody, VC1.1, reacted with SGPG, SGLPG, and SGGL analogues containing a minimum of two sugars (SGA-Gal-Cer). All antibodies reacted more strongly with synthetic SGGLs with longer carbohydrate chains. These results indicate that anti-SGGL antibodies recognize a minimum of two sugars bearing the following structure (3-sulfoglucuronosyl β 1-3 galactosyl, SGA-Gal-) and that the aglycone (“ceramide”) structure appears to play an important role for antibody-antigen interaction. 1. Dedicated to the memory of Professor Akira Hasegawa.

Disturbed vagal nerve distribution in embryonic chick hearts after treatment with all-trans retinoic acid.

The distribution of the vagal nerve was studied in whole-mount specimens and serial sections of chick embryos after retinoic acid treatment. White Leghorn chick embryos were treated at stage 15 either with 1 microg all-trans retinoic acid (n=11), or with the solvent dimethylsulphoxide (sham-operated embryos, n=8). Eight embryos served as normal controls. At stage 34 all 27 embryos were examined with a dissecting microscope. In order to reveal the vagal patterning, the hearts were removed and whole-mount stained with the HNK-1 antibody. In three hearts of the retinoic acid-treated group a morphologic intracardiac anomaly -- a double outlet right ventricle -- was found. To explore in depth the vagal nerve distribution in the heart, a separate set of hearts of retinoic acid embryos (n=5), sham-operated (n=4) and control embryos (n=5), was devised solely for serial sectioning and staining with the HNK-1 antibody. All hearts of retinoic acid-treated embryos showed a disturbed vagal nerve distribution both over the surface of the heart and within the heart wall. The vagal patterning was not altered in the sham-operated embryos compared to controls. It is concluded that retinoic acid disturbs the development of vagal nerve patterning regardless of the concurrent presence of intracardiac malformations. The mechanism and functional implications remain to be investigated.

Expression of HNK-1 carbohydrate and its binding protein, SBP-1, in apposing cell surfaces in cerebral cortex and cerebellum

Sulfoglucuronyl carbohydrate is the terminal moiety of neolacto-oligosaccharides, expressed on several glycoproteins of the immunoglobulin superfamily involved in cell–cell recognition and on two glycolipids. Sulfoglucuronyl carbohydrate is temporally and spatially regulated in the developing nervous system. It appears to be involved in neural cell recognition and in cell adhesion processes through its interaction with specific proteins on cell surfaces. Previously we have characterized a specific sulfoglucuronyl carbohydrate-binding protein in rat brain. Sulfoglucuronyl carbohydrate binding protein-1 is structurally similar to a 30 000 mol. wt adhesive and neurite outgrowth promoting protein amphoterin [Rauvala and Pihlaskari (1987) J. biol. Chem. 262, p. 16 625]. The pattern of expression of sulfoglucuronyl carbohydrate binding protein-1 in developing rat nervous system was studied to understand the significance of its interaction with sulfoglucoronyl carbohydrate bearing molecules. Biochemical analyses showed that the expression of sulfoglucuronyl carbohydrate binding protein-1 was developmentally regulated similarly to sulfoglucuronyl carbohydrate. Immunocytochemical localization of sulfoglucuronyl carbohydrate binding protein-1 and sulfoglucuronyl carbohydrate was performed by bright-field and fluorescent confocal laser scanning microscopy. In postnatal day 7 rat cerebellum, sulfoglucuronyl carbohydrate binding protein-1 was primarily associated with neurons of the external and internal granule cell layers. The sulfoglucuronyl carbohydrate binding protein-1 immunoreactivity was absent in Purkinje cell bodies and their dendrites in the molecular layer, as well as in Bergmann glial fibres and in white matter. In contrast, sulfoglucuronyl carbohydrate (reactive with HNK-1 antibody) was localized in processes surrounding granule neurons in the internal granule cell layer. Sulfoglucuronyl carbohydrate was also expressed in Purkinje neurons and their dendrites in the molecular layer and their axonal processes in the white matter. To a lesser extent Bergmann glial fibres were also positive for sulfoglucuronyl carbohydrate. In the cerebral cortex, at embryonic day 21, sulfoglucuronyl carbohydrate binding protein-1 was mainly observed in immature neurons of the cortical plate and subplate and dividing cells near the ventricular zone. Whereas, sulfoglucuronyl carbohydrate was strongly expressed in the fibres of the subplate and marginal zone. Sulfoglucuronyl carbohydrate was also found in the processes surrounding the sulfoglucuronyl carbohydrate binding protein-1-expressing neuronal cell bodies in the cortical plate and in ventricular zone. The specific localization of sulfoglucuronyl carbohydrate binding protein-1 in cerebellar granule neurons and neurons of the cerebral cortex was also confirmed by immunocytochemistry of the dissociated tissue cell cultures. The complementary localization of sulfoglucuronyl carbohydrate and sulfoglucuronyl carbohydrate binding protein-1, both in cerebral cortex and cerebellum, in apposing cellular structures indicate possible interaction between the two and signalling during the process of cell migration and arrest of migration.

Local increase level of chondroitin sulfate induces changes in the rhombencephalic neural crest migration.

Numerous studies suggest that chondroitin sulfate proteoglycan (CSPG) inhibits neural crest cells (NCC) migration at the trunk level. However, its action on the cephalic neural crest is not clear. To determine this action, we have microinjected 0.5 nl of different concentrations of chondroitin sulfate (CS) at the anterior rhombencephalon level in 9 stage chick embryos, as well as subgerminally administering beta-D-xyloside to 8 stage chick embryos. Beta-D-xyloside disrupts CSPG synthesis, producing an increase in CS free chains in several embryonal anlages. Chondroitin sulfate microinjected embryos and beta-D xyloside treated embryos were reincubated until attaining 12 stage. Results obtained for both experimental groups were similar. Immunoreactivity with HNK-1 antibody revealed that NCC did not migrate, remaining near the rhombencephalon dorsal wall; in addition, several NCC did not separate from the neural fold, becoming invaginated towards the rhombencephalon cavity. Our findings indicate that an increase in CS free chains in cephalic neural crest migratory routes not only disrupts their migration, but also impedes delamination and detachment of the rhombencephalic neuroepithelium NCC. These data suggest that the inhibitory action upon the neural crest migration attributed to CSPG may rest on its glycosaminoglycan (GAG). We cannot, however, rule out the possibility that increases in other GAGs apart from CS, may produce similar effects on neural crest migration.

Distribution of cells bearing the HNK-1 epitope in the human placenta

HNK-1 (Leu-7 antigen or CD57) is a unique carbohydrate moiety found in certain glycosphingolipids and several cell adhesion glycoproteins on the cell membrane. Previous studies have suggested that HNK-1 carbohydrates act as adhesive ligands in cell-cell interactions. Using a monoclonal antibody reactive to the HNK-1 moiety and an immunoperoxidase method on formalin-fixed paraffin-embedded tissue, the expression of the HNK-1 epitope in human placentae was confined to the intermediate trophoblast (IT) in trophoblastic columns. The number of HNK-1 immunoreactive IT cells increased from the proximal to the midportion of the trophoblastic column, and then disappeared at the junction of the column with the basal plate where IT infiltrates the endomyometrium and becomes extravillous IT. Neither cytotrophoblast nor syncytiotrophoblast reacted with the HNK-1 antibody. In hydatidiform moles, HNK-1 immunoreactivity was localized to areas that structurally resembled trophoblastic columns. In contrast, placental site trophoblastic tumours which do not contain structures analogous to trophoblastic columns did not express HNK-1 epitope. Expression of HNK-1 was only rarely observed in choriocarcinomas, being present in less than 5 per cent of trophoblastic cells in two of 13 cases. The murine placenta, which lacks trophoblastic columns, was negative for HNK-1. Thin-layer chromatography immunostaining demonstrated the HNK-1 reactive glycosphingolipids in placental lipid extracts, whereas Western blot analysis from placental protein extracts failed to reveal detectable glycoproteins that demonstrated HNK-1 immunoreacdvity. In conclusion, the specific localization of HNK-1 reactive glycosphingolipids in trophoblastic columns of the human placenta suggests that the HNK-1 moiety may play an important role in maintaining cohesion between intermediate trophoblastic cells in the trophoblastic columns thereby contributing to the structural integrity of the villi that anchor the placenta to the basal plate.