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.     

Expression of carbohydrate epitopes L2/HNK-1 and L3 in the larva and imago of Drosophila melanogaster and Calliphora vicina

Summary The carbohydrate epitopes L2/HNK-1 and L3 belong to two overlapping families of adhesion molecules in the vertebrate, and probably the invertebrate nervous systems. To investigate their pattern of expression during the development of insects, cryosections of late third instar larvae and imagoes of Drosophila melanogaster and Calliphora vicina were studied by indirect immunofluorescence using several monoclonal antibodies to the L2/HNK-1 and one monoclonal antibody to the L3 epitope. Each monoclonal antibody to the L2/HNK-1 epitope showed a different immunohistological staining pattern, which differed from that of the L3 monoclonal antibody. In both insect species the immunohistological staining patterns for the two carbohydrate epitopes were similar at the two developmental stages, with immunoreactivity not confined to the nervous system. In larvae, immunoreactivities of the monoclonal antibodies L2.334 and L3.492 were predominantly associated with the extracellular matrix as indicated by co-localization with laminin, particularly in the imaginal discs, while L2.349 revealed a more cell surface-associated distribution. In imagoes, immunoreactivities were detectable in most organs studied.     

Detection of the L2/HNK-1 Carbohydrate Epitope on Glycoproteins and Acidic Glycolipids of the Insect Calliphora vicina

The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid-containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin-layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK-1 (recognizing an epitope on human natural killer cells); and a human IgM M-protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M-protein. After desulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK-1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK-1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.     

Expression and distribution of carbohydrate sequences in chick germ cells: a comparative study with lectins and the NC-1/HNK-1 monoclonal antibody

The expression of end-chain sugar residues and of oligosaccharidic sequences has been investigated in chick germ cells at critical stages during the migration, proliferation and sexual differentiation of these cells. Fluorescent lectins and indirect immunofluorescence studies using the NC-1/HNK-1 monoclonal antibody indicate a remarkable control of glycosylation during germ cell embryonal life. Besides a retained expression of glucose/mannose residues, it was found that alpha- and beta-galactose residues, N-acetyllactosamine and N-N' diacetylchitobiose sequences as well as the sulfated trisaccharidic NC-1 epitope were detectable in a stage-specific pattern. Present at a very high density in the cytoplasm and on the surface of the early germ cells at premigrative and migratory stages, the staining for these carbohydrate sequences gradually disappeared when the germ cells settled and proliferated in the developing gonadal primordia. The disaccharide Gal beta 1----3 Gal NAc was exclusively detected in migrating PGCs. In sexualized gonads, acetyllactosamine and/or diacetylchitobiose were similarly reexpressed in both oogonia and spermatogonia. Spermatogonia displayed beta-galactose residues and a high immunoreactivity with the NC1 Mab, indicating modulations in PGC glycosylations related to the acquisition of sexual phenotypes. In addition NC-1 was found to be expressed in the somatic component of the undifferentiated gonad and in the testis interstitial gland.     

Myelin-Associated Glycoprotein Shares an Antigenic Determinant with a Glycoprotein of Human Melanoma Cells

A sulfated 100K-dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK-1 bind to this glycoprotein. It is shown here that mouse anti-myelin-associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti-MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti-melanoma antibodies (10C5 and 11B5) resemble HNK-1 in binding to MAG and to some 19-28K-dalton glycoproteins and sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti-melanoma antibodies cross-react with neural cell adhesion molecule (N-CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti-melanoma antibodies fall into a class of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, anti-human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N-CAM, and several protein and lipid glycoconjugates of the PNS.     

Sulfated HNK-1 epitope in developing and mature kidney: a new marker for thin ascending loop of Henle and tubular injury in acute tubular necrosis.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.     

The monoclonal antibodies Elec-39, HNK-1 and NC-1 recognize common structures in the nervous system and muscles of vertebrates

The IgM monoclonal antibodies, Elec-39, HNK-1 and NC-1, recognize the same subset of Torpedo electric organ acetylcholinesterase (AChE). We show that they react against a glycosphingolipid (SGPG) containing a sulfated glucuronic acid (SGA). The three antibodies appear essentially identical in their specificity but differ in their affinity for the antigens. We have examined their binding in the CNS, nerves and muscles of several vertebrate species, at the optical and in some cases at the electron microscope level. All three antibodies label the same structures: they show diffuse staining around neuromuscular endplates and label the plasma membrane of the Schwann cells, surrounding the outer layer of myelin sheaths. In the adult rat CNS, the antibodies label certain defined structures, notably extracellular material in the habenula and in the CA2 layer of the hippocampus. In the cortex and cerebellum, they label the surface of neural processes and terminals apposed to large multipolar neurons and Purkinje cells, as well as membranous material contained in inclusions dispersed in the cytoplasm of these neurons. These localizations are consistent with the suggestion that the SGA-antigens may be involved in cellular interactions.     

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.     

HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development.     

An Immunoglobulin M Monoclonal Antibody, Recognizing a Subset of Acetylcholinesterase Molecules from Electric Organs of Electrophorus and Torpedo, Belongs to the HNK-1 Anti-Carbohydrate Family

An immunoglobulin M (IgM) monoclonal antibody (mAb Elec-39), obtained against asymmetric acetylcholinesterase (AChE) from Electrophorus electric organs, also reacts with a fraction of globular AChE (amphiphilic G2 form) from Torpedo electric organs. This antibody does not react with asymmetric AChE from Torpedo electric organs or with the enzyme from other tissues of Electrophorus or Torpedo. The corresponding epitope is removed by endoglycosidase F, showing that it is a carbohydrate. The subsets of Torpedo G2 that react or do not react with Elec-39 (Elec-39+ and Elec-39-) differ in their electrophoretic mobility under nondenaturing conditions; the Elec-39+ component also binds the lectins from Pisum sativum and Lens culinaris. Whereas the Elec-39- component is present at the earliest developmental stages examined, an Elec-39+ component becomes distinguishable only around the 70-mm stage. Its proportion increases progressively, but later than the rapid accumulation of the total G2 form. In immunoblots, mAb Elec-39 recognizes a number of proteins other than AChE from various tissues of several species. The specificity of Elec-39 resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1, NSP-4, as well as IgMs that occur in human neuropathies. Although some human neuropathy IgMs that recognize the myelin-associated glycoprotein did not react with Elec-39+ AChE, mAbs HNK-1, NC-1, and NSP-4 showed the same selectivity as Elec-39 for Torpedo G2 AChE, but differed in the formation of immune complexes.     

Expression and biological functions of sulfoglucuronyl glycolipids (SGGLs) in the nervous system—A review

Sulfoglucuronyl carbohydrate linked to neolactotetraose reacts with HNK-1 antibody. The HNK-1 carbohydrate epitope is found in two major glycolipids, several glycoproteins and in some proteoglycans of the nervous system. Most of the HNK-1 reactive glycoproteins so far identified are neural cell adhesion molecules and/or are involved in cell-cell interactions. HNK-1 carbohydrate is highly immunogenic. Several HNK-1-like antibodies, including IgM of some patients with plasma cell abnormalities and having peripheral neuropathy, have been described. This article summarizes published work mainly on sulfoglucuronyl glycolipids, SGGLs and covers: structural requirements of the carbohydrate epitope for binding to HNK-1 and human antibodies, expression of the lipids in various neural areas, stage and region specific developmental expression in CNS and PNS, immunocytochemical localization, loss of expression in Purkinje cell abnormality murine mutations, biosynthetic regulation of expression by a single enzyme N-acetylglucosaminyl transferase, identification of receptor-like carbohydrate binding neural proteins (lectins), and perceived role of the carbohydrate in physiological functions. The latter includes role in: pathogenesis of certain peripheral neuropathies, in migration of neural crest cells, as a ligand in cell-cell adhesion/interaction and as a promoter of neurite outgrowth for motor neurons. Multiple expression of HNK-1 carbohydrate in several molecules and in various neural cell types at specific stages of nervous system development has puzzled investigators as to its specific biological function, but this may also suggest its importance in multiple systems during cell differentiation and migration processes.Special issue dedicated to Dr. Marjorie B. Lees.     

Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo

We report that a monoclonal antibody, HNK-1, identifies specific regions and cell types during primitive streak formation in the chick blastoderm. Immunohistochemical studies show that the cells of the forming hypoblast are HNK-1 positive from the earliest time at which they can be identified. Some cells of the margin of the blastoderm are also positive. The mesoderm cells of the primitive streak stain strongly with the antibody from the time of their initial appearance. In the epiblast, some cells are positive and some negative at pre-primitive-streak stages, but as the primitive streak develops a gradient of staining intensity is seen within the upper layer, increasing towards the primitive streak. At later stages of development, the notochord and the mesenchyme of the headfold are positive, while the rest of the mesoderm (lateral plate) no longer expresses HNK-1 immunoreactivity. This antibody therefore reveals changes associated with mesodermal induction: before induction, it recognizes the 'inducing' tissue (the hypoblast) and reveals a mosaic pattern in the responding tissue (the epiblast); after primitive streak formation, the mesoderm of the primitive streak that results from the inductive interactions expresses the epitope strongly. Affinity purification of HNK-1-related proteins in various tissues was carried out, followed by SDS-PAGE to identify them. The hypoblast, mesoderm and epiblast of gastrulating chick embryos have some HNK-1-related proteins in common, while others are unique to specific tissues. Attempts have been made to identify these proteins using Western blots and antibodies known to recognize HNK-1-related molecules, but none of the antibodies used identify the bands unique to any of the tissues studied. We conclude that these proteins may be novel members of the HNK-1/L2 family, and that they may have a role in cell interactions during early development.     

Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.     

The spatial and temporal expression of HNK-1 by myogenic and skeletogenic cells in the embryonic rat

The existence of phenotypic differences within a population of cells provides evidence for discrete stages in cellular differentiation and/or identifies subsets of cells with unique functional properties. The monoclonal antibody HNK-1 has been widely shown to identify subpopulations of cells in the developing nervous system. In this paper we focus on the developmental expression of HNK-1 immunoreactivity by derivatives of somitic (paraxial) mesoderm. We show that between embryonic day 12 and 14 (E12–E14) the HNK-1 epitope is transiently expressed by postmitotic myotomal cells. In E14–E17 developing vertebral columns (which are derived from somitic sclerotomal cells), HNK-1 immunolabeling was expressed by subpopulations of skeletogenic cells, including perinotochordal cells associated with the forming annulus fibrosus and cells within or adjacent to the perichondrium. Chondrocytes within forming centra and vertebral arches did not exhibit HNK-1 immunoreactivity. These results, taken together, show that the expression of the HNK-1 epitope can be used to identify subsets of myogenic and skeletogenic cells both spatially and temporally in the developing rat.     

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.     

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° versus 11–12° 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°. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18°. 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. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 659–671, 1998     

Epitope diversity of N-glycans from bovine peripheral myelin glycoprotein P0 revealed by mass spectrometry and nano probe magic angle spinning 1H NMR spectroscopy.

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most abundant constituent of myelin. Using monoclonal antibodies, the homophilic binding of the P0 glycoprotein was shown to be mediated via the human natural keller cell (HNK)-1 epitope (3-O-SO(3)H-GlcUA(beta1-3)Gal(beta1-4)GlcNAc) present on the N-glycans. We recently described the structure of the N-glycan carrying the HNK-1 epitope, present on bovine peripheral myelin P0 (Voshol, H., van Zuylen, C. W. E. M., Orberger, G., Vliegenthart, J. F. G., and Schachner, M. (1996) J. Biol. Chem. 271, 22957-22960). In this study, we report on the structural characterization of the detectable glycoforms, present on the single N-glycosylation site, using state-of-the-art NMR and mass spectrometry techniques. Even though all structures belong to the hybrid- or biantennary complex-type structures, the variety of epitopes is remarkable. In addition to the 3-O-sulfate present on the HNK-1-carrying structures, most of the glycans contain a 6-O-sulfated N-acetylglucosamine residue. This indicates the activity of a 6-O-sulfo-GlcNAc-transferase, which has not been described before in peripheral nervous tissue. The presence of the disialo-, galactosyl-, and 6-O-sulfosialyl-Lewis X epitopes provides evidence for glycosyltransferase activities not detected until now. The finding of such an epitope diversity triggers questions related to their function and whether events, previously attributed merely to the HNK-1 epitope, could be mediated by the structures described here.     

Alternate expression of the HNK-1 epitope in rhombomeres of the chick embryo.

Rhombomeres are regarded as the manifestation of innate segmentation within the vertebrate CNS. To investigate developmental changes occurring in the CNS and PNS, a series of chick embryos were immunostained with several monoclonal antibodies. The HNK-1-immunoreactivity (IR) appeared in rhombomeres (r) 3 and r5 around stage 15, when r2 and r4 were not stained. This alternate pattern is similar to the Krox-20 gene expression in the mouse embryo. At levels of r2 and r4, HNK-1+ neural crest cell masses were attached to the CNS forming cranial sensory ganglia. In these rhombomeres, an accumulation of neuroepithelial cells near the cranial nerve root and early development of neuroblasts in the basal plate were observed. The above observations seem to suggest that the alternate HNK-1-IR in rhombomeres might be related to the expression of cell adhesion molecules, and therefore also to the adhesion of the cranial ganglion precursors to the CNS, which takes place every other rhombomere in the preotic region. Thus, the alternate pattern of the HNK-1-IR seems to be related to the morphogenesis of preotic branchial nerves.     

Immunocytological localization of the HNK-1 carbohydrate in murine cerebellum,hippocampus and spinal cord using monoclonal antibodies with different epitope specificities

The HNK-1 carbohydrate, an unusual 3′-sulfated glucuronic acid epitope characteristic of many neural recognition molecules, serves as a ligand in neural cell interactions and is differentially expressed in the quadriceps and saphenous branches of the femoral nerve in the PNS of adult mice. Based on these observations, we investigated the possibility that the HNK-1 carbohydrate may be differentially distributed in neurons and fiber tracts also in the CNS thereby contributing to different targeting and guidance mechanisms. We have used antibodies with different HNK-1 epitope specificities to probe for subtle differences in expression patterns. In the adult mouse cerebellum the HNK-1 carbohydrate is detectable in stripe-like compartments in the molecular and Purkinje cell layers, whereas N-CAM and its associated α2,8 polysialic acid does not show this compartmentation. In the adult hippocampus, the HNK-1 carbohydrate localizes to perineuronal nets of inhibitory interneurons and marks the inner third of the molecular layer of the dentate gyrus. In the adult spinal cord, HNK-1 labeling is most pronounced in gray matter areas. White matter enriched regions show differential labeling with regard to fiber tracts and antibody specificity. Whereas the different antibodies do not show differences in staining in the cerebellum and the hippocampus, they show differences in staining pattern of fiber tracts and motoneurons in the spinal cord. The HNK-1 expression pattern also differed in the adult spinal cord from that observed at embryonic day 14 and postnatal day 14. Our observations suggest a functional role in the specification of functionally discrete compartments in different areas of the CNS and during development.     

Ascidian neural crest-like cells: phylogenetic distribution, relationship to larval complexity, and pigment cell fate

Migratory neural crest-like cells, which express the cell surface antigen HNK-1 and develop into pigment cells, have recently been identified in the ascidian Ecteinascidia turbinata. Here we use HNK-1 expression as a marker to determine whether neural crest-like cells are responsible for pigment development in diverse ascidian species. We surveyed HNK-1 expression and tyrosinase activity in 12 ascidian species, including those with different adult organizations, developmental modes, and larval sizes and complexities. We observed HNK-1 positive cells in every species, although the timing of HNK-1 expression varied according to the extent of larval complexity. HNK-1 expression was initiated during the late tailbud stage in species in which adult features are formed precociously in large complex larvae. In contrast, HNK-1 positive cells did not appear until the swimming tadpole or juvenile stage in species with small simple larvae in which most adult features appear after metamorphosis. Double labeling experiments indicated that HNK-1 and tyrosinase are expressed in the same subset of pigment-forming mesenchymal cells in species with complex or simple larvae. In addition, the absence of HNK-1 and tyrosinase expression in albino morphs of the colonial ascidian Botryllus schlosseri suggested that the major fate of neural crest-like cells is to become pigment cells. The results suggest that ascidian neural crest-like cells and vertebrate neural crest cells had a common origin during chordate evolution and that their primitive function was to generate body pigmentation.