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.     

Correlation between the expression of the HNK-1 epitope and cellular invasiveness in prestreak epiblast cells of chick embryos

During avian gastrulation, certain cells present in the epiblast layer ingress through the basement membrane sealing the basal surface of themselves. Previously we reported that chick prestreak epiblast cells show two different behavioral phenotypes upon reconstituted basement membrane and laminin gel in vitro. Half of the dissociated epiblast cells invade the gel substratum after one-day of culture, whereas the others attach to the gel but do not invade. It is expected that such heterogeneity in the behavior of the epiblast cells reflects some mechanism that sorts the cells into those that will ingress into the blastocoelic cavity and those that will remain in the epiblast layer. To test this hypothesis, we dissociated chick prestreak epiblast cells into single cells, cultured them on the laminin gel, and then stained them with anti-HNK-1 antibody. This antibody binds to an epitope present on half of the prestreak epiblast cells which are thought to differentiate into presumptive mesoendodermal cells. We found that 80% of the invasive epiblast cells were HNK-1-positive whereas 77% of the non-invasive cells were HNK-1 negative. In the case of invasive cells, the edges of the proteolytic holes made by the invasive cells were often stained. These results suggest that the cells expressing the HNK-1 carbohydrate chain are preferentially invasive, and this induces selective ingression of the carrier cells for mesoendodermal differentiation in vivo.     

Molecular cloning and expression of a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope

A cDNA encoding a novel glucuronyltransferase was cloned from a rat brain cDNA library. The cDNA sequence contained an open reading frame encoding 324 amino acids, with type II transmembrane topology. The amino acid sequence revealed 49% homology to rat GlcAT-P, a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope of glycoproteins, [Terayama et al. (1997) Proc. Natl. Acad. Sci. USA 94, 6093-6098] and the highest sequence homology was found in the catalytic region. Northern blot analysis indicated that this newly cloned glucuronyltransferase is expressed in the nervous system, consistent with the selective localization of the HNK-1 carbohydrate epitope in the nervous system. Transfection of this cDNA into COS-1 cells induced the expression of the HNK-1 carbohydrate epitope on cell surfaces, and induced the morphological changes in these cells. These results indicated that this newly cloned cDNA is a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope.     

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.     

Differential expression of two glucuronyltransferases synthesizing HNK-1 carbohydrate epitope in the sublineages of the rat myogenic progenitors

HNK-1 epitope is a cell-surface carbohydrate mediating various cell-cell or cell-substrate interactions. We found HNK-1 epitope in longitudinally arrayed fibers in the subpopulation of the epaxial myotome, and hypaxial myoblasts migrating into the limb bud in the rat embryo. We next investigated the expression patterns of genes encoding two glucuronyltransferases (GlcAT-P, GlcAT-D) and sulfotransferase (Sul-T), which are required for biosynthesis of HNK-1 epitope. GlcAT-P gene was expressed in the non-migrating longitudinal fibers, whereas GlcAT-D gene was expressed in the migrating myoblasts in the limb bud. Sul-T gene expression was ubiquitously observed in all these myogenic populations. Thus, differential expression of GlcAT genes may relate to the epaxial/hypaxial or migrating/non-migrating myoblast lineages.     

Immunolocalization of the HNK-1 epitope in the autonomic innervation to the liver and upper digestive tract of the developing rat embryo

The immunohistochemical analysis of the HNK-1 epitope presence in the liver and upper digestive tract nerves was carried out in 12- to 18-day-old rat embryos embedded in acrylamide–agarose and observed with laser scanning confocal microscopy. The vagus and sympathetic trunk were intensely immunostained at all ages; branches of both structures were also HNK-1 positive, and ramified ventrocaudally following the course of the thoracic and abdominal aorta, caval vein, portal vein and ductus venosus. As early as day 12, some immunostained cells were seen in the mesentery that formed the enteric nervous system. Clearly immunostained HNK-1-immunoreactive fibres were detected innervating the digestive wall after day 14, forming both myenteric and submucosal plexuses. After day 16, the Glisson sheath showed streams of HNK-1-positive fibres coming from dorsal areas, lining the peritoneal surface of the diaphragm, invading the capsule, and ramifying superficially around the lobes of the liver. We saw no immunoreactive structures pervading the hepatic lobes at all ages studied, with the exception of occasional HNK-1-positive cells in the superficial parenchyma, which were visualized after 16 days of gestation. Our findings can help to understand the development of the gastrointestinal and liver innervation in the rat.     

beta-Hexosaminidase expression in chick embryo fibroblasts in vitro.

1. Two forms of beta-hexosaminidase, similar to hexosaminidase A and hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in vitro. 2. beta-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro. 3. Concanavalin-A treatment determines the increase of the neutral form, hexosaminidase C, during development. 4. Concanavalin-A reduces the specific activity of beta-hexosaminidase during development.     

Asymmetric expression of antivin/lefty1 in the early chick embryo

Mammalian lefty and zebrafish antivin, highly related to lefty, are shown to be expressed asymmetrically and involved in the specification of the left body side of early embryos. We isolated a chick homologue of the antivin/lefty1 cDNA and studied its expression pattern during early chick development. We found that antivin/lefty1 is expressed asymmetrically on the left side of the prospective floorplate, notochord and lateral plate mesoderm of the chick embryo.     

Stage-specific gene expression in early chick embryo

Inhibition of DNA replication by aphidicolin in the chick morula interferes with its progression to a normal blastula and prevents induction of the first morphogenetic cell movements of primitive streak formation. Embryos in aphidicolin synthesize some polypeptides typical of blastula but do not display all the characteristic features of morula to blastula transition. Inhibition of DNA replication interferes with the sequential synthesis of maternally coded polypeptides and with the activation of the embryonic genome in the chick embryo.     

Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney

The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N-linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina.     

Identification of a peptide mimic of the L2/HNK-1 carbohydrate epitope

The L2/HNK-1 carbohydrate is carried by many neural recognition molecules and is involved in neural cell interactions during development, regeneration in the peripheral nervous system, synaptic plasticity, and autoimmune-based neuropathies. Its key structure consists of a sulfated glucuronic acid linked to lactosaminyl residues. Because of its biological importance but limited availability, the phage display method was used to isolate a collection of peptide mimics that bind specifically to an L2/HNK-1 antibody. The phages isolated from a 15-mer peptide library by adsorption to this antibody share a consensus sequence of amino acids. The peptide mimicked several important functions of the L2/HNK-1 carbohydrate, such as binding to motor neurons in vitro, and preferential promotion of in vitro neurite outgrowth from motor axons compared with sensory neurons. A scrambled version of the peptide had no activity. The combined observations indicate that we have isolated a mimic of the L2/HNK-1 carbohydrate that is able to act as its functional substitute.     

Collagen type IX and HNK-1 epitope in tears of patients with pseudoexfoliation syndrome

Pseudoexfoliation syndrome (PEX) is an age-related condition, which may cause open-angle glaucoma and has increasing interest since it seems to affect additional human tissues, i.e., cardiovascular tissue, skin, and still lacks elucidated pathogenesis. Collagen type IX and HNK-1 epitope have been considered characteristic constituents of the aqueous humour of PEX patients, since their amounts were increased in PEX aqueous humour compared to normal eyes. Since it has been proposed that the initial manifestations of PEX syndrome occur in conjunctiva, the present study was undertaken to investigate the presence of the same antigens in tears of PEX patients and their possible use as the biochemical markers for early diagnosis. Tears of PEX patients and healthy individuals were subjected to western blotting analysis for various basement membrane components identified in aqueous humour. It was found that collagen type IX and HNK-1 epitope were present in tears, the amount of the former being increased 2.7 times compared to normal (P<0.05), surprisingly high as compared with total protein or lysozyme activity in tears, which were found to be increased in PEX patients about 25% with no statistical differences (P approximately 0.4). The results suggest the possible use of tears' collagen type IX for the diagnosis of PEX syndrome.     

Claudin-5 expression in the vasculature of the developing chick embryo

The claudin family of proteins are integral components of tight junctions and are responsible for determining the ion specificity and permeability of paracellular transport within epithelial and endothelial cell layers. Studies in human, mouse, Xenopus, and zebrafish have shown that only a limited number of claudins are expressed in endothelial cells. Here, we report the expression pattern of Claudin-5 during chick development. Between HH stage 4 and 6 Claudin-5 expression was observed exclusively in extraembryonic tissue. Claudin-5 expression was not observed in the embryo until HH stage 8, coincident with the onset of embryonic vascularization. Claudin-5 expression was maintained in the developing vasculature in the embryonic and extraembryonic tissue throughout organogenesis (HH stage 19–35), including the vasculature of the ectoderm and of organs derived from the mesoderm and endoderm lineages. These data describe a conserved expression pattern for Claudin-5 in the endothelial tight junction barrier and is the first report of the onset of Claudin-5 expression in a vertebrate embryo.     

A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.