Neural Cell Surface Research Articles

Expression and functional roles of neural cell surface molecules and extracellular matrix components during development and regeneration of peripheral nerves

By combining both immunocytochemical and functional investigations, a hypothetical framework will be developed for the molecular mechanisms underlying neuron-glia interactions during development and regeneration of peripheral nerves. In particular, the immunoglobulin-like molecules L1, N-CAM, MAG and P0, the extracellular matrix molecules laminin and tenascin, and the carbohydrates PSA and L2/HNK-1 will be considered. During early stages of limb bud innervation in embryos, L1 and N-CAM are expressed on axons and Schwann cells and are involved in axonal fasciculation, whereas tenascin is thought to be involved in forming a scaffold around the nerve possibly preventing axons and/or Schwann cells from leaving the nerve. PSA has been shown to be involved in pathway selection at initial stages of limb bud innervation. Later on, when motor axons enter muscles, the carbohydrates determine the branching pattern of the nerves. During myelination, L1 appears to play a pivotal role during the formation of the first Schwann cell loops around the prospective myelin-containing axons. MAG and P0 appear also to be functionally involved at initial stages of myelin formation. Additionally, MAG may contribute to the formation and maintenance of non-compacted myelin and axon-Schwann cell apposition whereas P0 is involved in myelin compaction. Under regenerative conditions, L1, N-CAM, laminin, and tenascin are strongly up-regulated by denervated Schwann cells. In vitro observations strongly suggest that these molecules might foster axonal regeneration. The carbohydrate PSA is confined to regrowing axons and is also a candidate to support axonal regrowth. L2/HNK-1, which is found on motor axon-associated Schwann cells, may provide regenerating motor axons with a selective advantage over others resulting in appropriate reinnervation of motor pathways. Since many of the functional studies this review refers to have been performed in vitro, some of the conclusions drawn need reexamination in vivo. Gene manipulations, such as the generation of null mutants followed by a thorough morphological and immunocytochemical investigation may be a powerful tool to resolve this problem.

Immediate Early Gene Expression in Experimental Epilepsy

Neuronal excitation by experimentally induced seizures elicits the rapid induction of a set of genes called immediate early genes (IEGs). The gene products of fos, jun and Krox, multimember gene families that belong to the class of IEGs, participate in a fundamental biological control mechanism, the regulation of gene transcription. IEG encoded proteins act as third messengers in an intracellular signal transduction cascade between neural cell surface receptors, cytoplasmic second messenger systems and specific target genes in the nucleus, a process for which the term 'stimulus transcription coupling' has been given. Almost all types of seizures cause dynamic alterations of IEG expression in neurons of the limbic system, but also in non-limbic areas, such as the cortex, striatum and thalamus. IEG encoded transcription factors are thought to up- or down-regulate effector genes with preferential expression in the central nervous system, including genes for neurotransmitters, growth factors, receptors, synaptic and axonal proteins. If the concept holds true that IEGs act as molecular switches converting epileptic short-term excitation of neurons into alterations of the molecular phenotype, future research may help to explain hitherto unexplained phenomena in epileptogenesis including changes of synaptic efficacy, kindling and sprouting.

Cadherin-mediated adhesion in pancreatic islet cells is modulated by a cell surface N-acetylgalactosaminylphosphotransferase.

Rat pancreatic islet cells and RINm5F cells, an islet derived cell line, have at their cell surface an N-acetylgalactosaminylphosphotransferase (GalNAcPTase) similar to that found at the surface of chick neural retina cells and at the rat neuromuscular junction. On islet cells and RINm5F cells the GalNAcPTase is stably associated with cadherin cell-cell adhesion molecules. The effect of antibodies directed against the GalNAcPTase on homophilic, cadherin mediated adhesion was analyzed by measuring their effect on adhesion of islet and RINm5F cells to an immobilized anti-cadherin antibody. In this experimental paradigm anti-GalNAcPtase antibodies completely inhibit cadherin mediated adhesion. Furthermore, cadherin and GalNAcPTase co-distribute in islet and non-islet tissue. We conclude that pancreatic islet cell-cell adhesion is cadherin mediated and under the control of a tightly associated, cell surface GalNAcPTase.

Borrelia burgdorferi and other related spirochetes bind to galactocerebroside.

Spirochetes are agents of neurologic disease that may utilize specific neural cell surface molecules for adhesion. Borrelia burgdorferi, the etiologic agent of Lyme disease, bound to galactocerebroside (GalCer) in numbers that were two- to threefold greater than to ceramide and glucocerebroside, and four- to fivefold greater than to sphingosine, psychosine, sulfatide, cholesterol, and three membrane phospholipids. The adherence was greater to GalCer and ceramide with a higher content of alpha-hydroxyl fatty acids. Treponema phagedenis Reiter and Borrelia hermsii also bound to GalCer. The binding of B burgdorferi to GalCer was inhibited in a concentration-dependent manner by rabbit polyclonal and murine monoclonal antibodies to this glycosphingolipid component of myelin. The monoclonal antibody to GalCer also inhibited adhesion of the organisms to Schwann cells. Neither free D or L monosaccharides nor the lectin peanut agglutinin inhibited binding. Since B burgdorferi and other spirochetes cause neurologic disease, these results suggest a role for GalCer as a binding site in both the central and peripheral nervous systems.

Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.

We propose that leukocyte-derived cytokines induce the expression of adhesion molecules on the surface of neural cells that facilitates the subsequent attachment of leukocytes. Leukocyte adherence may contribute to some of the neural cell injury seen with various inflammatory diseases of the nervous system. With an in vitro model system, we have shown that mononuclear leukocytes bind to human neuroblastoma and cortical neuron cells only after the neural cells are stimulated with TNF-alpha. TNF-alpha stimulates expression of vascular cell adhesion molecule-1 (VCAM-1) in both of these neural cell lines. VCAM-1 mRNA is increased and VCAM-1 protein can be identified on the neural cell membranes with a new VCAM-1-specific mAb, CL40/2 F8. TNF-alpha also induces ICAM-1 in both of these neural cell lines. Leukocyte beta 1 (CD29) and beta 2 (CD18) integrins and their respective ligands, ICAM-1 and VCAM-1, on neural cells appear to be the dominant ligands mediating MNL:neural cell adhesive interactions. mAb to CD18 block 32 to 57% of the MNL binding to neural cells; similar inhibition is seen with mAb to ICAM-1. mAb to CD29 block 16 to 17% of the MNL binding to the neural cells suggesting that leukocyte beta 1 integrins and neural VCAM-1 may be a second route for MNL:neural cell interactions. Addition of both anti-CD18 and anti-CD29 mAb have an additive blocking effect; both ligand pairs may participate in MNL adhesion to neural cells, reminiscent of the multiplicity of ligands used by MNL when binding to endothelium.

A soluble form of prion protein in human cerebrospinal fluid: Implications for prion-related encephalopathies

The cellular prion protein (PrPc) is a 33-35 kDa sialoglycoprotein anchored to the external surface of neural and non-neural cells by a glycosyl phosphatidylinositol moiety. In addition, a secretory form of PrPc has been found in cell-free translation systems and in cell cultures. On this basis, we investigated human cerebrospinal fluid for the presence of soluble PrP and identified a protein whose molecular weight, antigenic determinants, N-terminal amino acid sequence and sensitivity to protease digestion corresponded to those of PrPc. In prion-related encephalopathies of humans and animals, the secretory form of PrPc might be converted into the abnormal isoform PrPSc and play a role in the dissemination of the disease process and amyloid formation.

The embryonic form of neural cell surface molecule (E-NCAM) in the rat hippocampus and its reexpression on glial cells following kainic acid- induced status epilepticus

The neural cell adhesion molecule (NCAM) changes at the cell surface during development, from highly sialylated forms (embryonic or E-NCAM) to three size classes of less sialylated proteins with apparent molecular mass of 180, 140, and 120 kDa (adult NCAM). In the nervous system, E-NCAM has been localized in developing tissues, where it is thought to play a role in the structuring of neuronal groups and tissue pattern formation. In the present study a monoclonal antibody that specifically detects E-NCAM was used in immunoblot and immunohistochemical procedures. In developing rat hippocampus, E-NCAM cell expression was found to change according to a precise pattern and persisted until 1 month after birth. It was closely associated with the mossy fiber system, an area known for its sprouting propensity. In adult rats, although immunoreactivity considerably decreases and becomes undetectable by immunoblot analysis, E-NCAM was still found to be associated with a few pyramidal-shaped cells in the innermost part of the dentate gyrus. In order to acquire some insight into potential histogenetically plastic functions of E-NCAM, in another series of experiments adult rats were treated with kainic acid, a powerful excitotoxic and convulsant glutamate analog eliciting status epilepticus. When these animals were examined for E-NCAM expression, an intense labeling was found associated with glial-like cells, particularly in the hippocampal formation, and corresponding approximately to the reactive gliosis, as confirmed by staining with anti-glial fibrillary acidic protein antibodies. This expression was detectable from about 3 d following kainic acid administration and persisted for at least 12 weeks; it developed according to an observable spatiotemporal distribution pattern. In animals submitted to amygdala kindling, a nonlesional model of secondarily generalized epilepsy, no such reexpression of E-NCAM was observed. Our observations imply that polysialylation may be a means of identifying neuronal structures capable of plasticity in the CNS. Moreover, intense reexpression of E-NCAM could be a marker of reactive gliosis following brain damage.

Coronavirus induced primary demyelination: indications for the involvement of a humoral immune response.

Coronavirus MHV‐JHM infection of rodents can result in demyelinating encephalomyelitis. We analysed histological changes induced by coronavirus MHV‐JHM infection in Lewis rats. Besides an acute disease (AE), chronic panencephalitis (CPE) and subacute demyelinating encephalomyelitis (SDE) were induced. These disease types were differentiated by the incubation period, the localization of lesions, the type of tissue damage and distribution of virus antigen. In AE and CPE, virus antigen was detected in neurons, astrocytes and oligodendrocytes, whereas in SDE neurons lacked virus antigen. Viral nucleocapsid protein (N) was present in the cytoplasm and the spike protein (S) was displayed on the surface of infected neural cells. However, expression of S protein relative to N protein was severely impaired in SDE lesions. Quantitative analysis of infiltrating inflammatory cells revealed that the number of macrophages and T cells were similar in lesions of AE, CPE and SDE. In contrast to that, SDE lesions contained a significantly higher number of IgG† B cells and plasma cells. In addition active demyelinating SDE lesions displayed an enhanced IgG content and deposits of complement C9. These results indicate that virus induced primary demyelination could be a consequence of antibody mediated cytotoxicity. Furthermore, a reduction in the number of cells producing spike protein in the chronic forms of the disease indicates down‐regulation of this protein, possibly mediated by anti‐S antibodies.

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.

In vitro differentiation of human retinoblastoma cells into neuronal phenotypes

Abstract In order to characterize the cell type(s) of origin of human retinoblastoma cells by immunophenotyping, primary cells from seven retinoblastomas and of the corresponding cell lines (RBL lines), as well as four retinoblastoma (RB) lines established by other groups, were compared with rat and human retina cells, and with the adenovirus E1A-transformed human retinoblast cell line HER-Xhol-CC2. Analyses using monoclonal antibodies (Mabs) RB13-2 and RB21-7, originally raised against prenatal rat brain cells and recognizing neural cell surface antigens expressed in a developmental-stage-dependent manner, and three cell-type-specific Mabs (4211, M501, Mab directed against vimentin) developed by other groups, gave the following results: (i) Retinoblastomas consist of cells expressing differentiated neuronal phenotypes during cultivation in vitro; (ii) All of the newly established RBL lines express neuronal phenotypes; and (iii) Cell lines such as Y79, which have been propagated in vitro for extended periods, do not express antigens specific for the neuronal pathway and cannot, therefore, be considered phenotypically representative of retinoblastoma cells.

Primary structure of the target of calcium vector protein of amphioxus.

CaVPT, a target protein of Ca2(+)-vector from amphioxus muscle, was purified from its complex with CaVP after dissociation by 6 M urea and chromatographies on DEAE-cellulose and calmodulin-Sepharose. The amino acid sequence of CaVPT has been determined. The protein is composed of 243 residues and possesses an unblocked N terminus. Its molecular weight is 26,621, distinctly lower than the apparent molecular weight deduced from electrophoresis on sodium dodecyl sulfate-containing gels. CaVPT contains a potential Asn-linked glycosylation site, four potential protein kinase C phosphorylation sites, and two casein kinase II phosphorylation sites. From the sequence the following three particular domains can be inferred: a collagen-like N-terminal segment, rich in Pro and Ala, that resembles the N-terminal segment of skeletal muscle myosin light chain kinase; next to it (from residues 33 to 50) is located a strongly amphiphilic and basic alpha-helical segment which likely binds the calcium vector protein since a proteolytic cut after Arg50, occurring occasionally during the purification of CaVPT, impairs the binding to immobilized calmodulin. This segment is followed by two immunoglobulin folds. The two immunoglobulin folds typically belong to the C2 subclass and particularly resemble those present in the neural cell surface adhesion molecules NCAM, L1, F11, MAG, TAG-1, fasciclin II, and amalgam. Recently, the presence of immunoglobulin folds of this type has been reported in some intracellular muscular proteins, namely in smooth muscle myosin light chain kinase, striated muscle C protein and titin, as well as in the nematode 600-kDa protein twitchin. From this structural study we can formulate the working hypothesis that CaVPT acts on the structure of the thick filament in muscle or regulates, perhaps via other immunoglobulin fold-containing proteins.

Use of the biotin‐avidin system for labelling, isolation and characterization of neural cell‐surface proteins

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell-surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish-peroxidase-coupled or 35S-labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin-agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.

Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM).

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.

Central nervous system antigen P84 can serve as a substrate for neurite outgrowth

Neurite outgrowth promoting properties of neural cell surface proteins can be assessed by immobilizing isolated membrane proteins on nitrocellulose-coated petri dishes. Using this method, we have identified a unique cell surface antigen, designated P84, as a new neural cell adhesion molecule. Immunoaffinity purified P84 contains three polypeptides with molecular weights of 167, 85, and 66 kDa. When spotted onto nitrocellulose-coated plates, P84 supports adhesion of mouse cerebellar neurons and neurite outgrowth. Glial cell attachment was also observed. Intact monoclonal antibodies directed against P84 inhibit adhesion and outgrowth on a P84 substrate. This antigen is found on the surfaces of neurons in cultures of cerebellar cells. It is also found on a subclass of unidentified flat cells. P84 is not found on oligodendrocytes or GFAP-positive astrocytes. As early as E9, P84 could be detected in the floor plate region of the spinal cord. This pattern persists throughout embryonic development. Postnatally, widespread expression of P84 is observed in a variety of CNS tissues.

The mouse neuronal cell surface protein F3: a phosphatidylinositol-anchored member of the immunoglobulin superfamily related to chicken contactin.

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent-insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development.

Neural cell recognition molecule F11: Membrane interaction by covalently attached phosphatidylinositol

The mode of membrane insertion of F11 130 kDa protein, a neural chick cell surface glycoprotein involved in neurite fasciculation, has been investigated. Up to 41% of total F11 130 kDa is released from adult chick brain plasma membranes by phosphatidylinositol specific phospholipase C (PI-PLC), wheras no release is mediated by lecithin/cephalin specific phospholipase C (PLC). PI-PLC dependent release of F11 is also observed from embryonal chick brain plasma membranes and from the surface of intact retinal cells. Biosynthetic labelling experiments demonstrate that F11 contains ethanolamine. Taken together, these results suggest that F11 interacts with the plasma membrane at least partially through covalently linked glycosyl-phosphatidylinositol (GPI) or a structurally similar lipid.

Interactions between donor and host tissue following cross-species septohippocampal transplants

Interactions between donor and host tissues following xenogeneic transplantation were studied using the neural cell surface antigen, Thy 1.2, as a marker for the donor tissue. Dissociated septal cells from Thy 1.2-positive fetal mice were transplanted to the dentate gyrus of Thy 1.2-negative adult rats. At post-transplantation survival times between 1 and 5 months, an antibody to Thy 1.2 was used to identify donor tissue. The results demonstrate that the donor tissue was capable of migrating and developing within the host following transplantation. Thy 1.2-positive cells and processes were consistently found within the supragranular, infragranular, and molecular layers of the dentate gyrus, and occasionally within the hilus, suggesting that mechanisms existed within the host which influenced the development of the transplanted tissue. Additionally, the survival and growth of the Thy 1.2-positive neurons differed from previous reports describing the growth of acetylcholinesterase (AChE)-positive fibers from xenogeneic transplants. This finding suggested that in addition to growing within the host, xenogeneic transplants may also stimulate a compensatory sprouting response from the host.

Identification and cDNA cloning of a new member of the L2/HNK-1 family of neural surface glycoproteins.

Rabbit antibodies raised against a 135- to 140-kD glycoprotein isolated from the culture medium of mouse forebrain explants were used for the identification and cloning of a complex of mouse neural cell surface glycoproteins. The antibodies recognized a 135-kD surface protein which shared the L2/HNK-1 epitope with several neural cell adhesion molecules. Three homologous complementary deoxyribonucleic acid (cDNA) clones were isolated from a mouse brain cDNA library prepared in the expression vector lambda gt11, one of which was sequenced and found to lack sequence homologies with known proteins. In Northern blots, this clone hybridized with a single 6.3 kb messenger ribonucleic acid (mRNA). In immunoblots of mouse brain extracts, antibodies raised in rabbits against the fusion protein encoded by it stained two glycoproteins of 135 and 90 kD, which we designated F3.135 and F3.90. In the developing mouse cerebellum, F3 antigenic sites were found predominantly on parallel fibers and on postmitotic neurons. In fetal brain cell cultures, F3 antigen was detected at the surface of cells with neuronal morphology, but the antibodies also stained some non-neuronal cells in a pattern characteristic of matrix components. Because all proteins carrying the L2/HNK-1 epitope identified so far have a role in cell adhesion, it can be anticipated that the F3 surface proteins also are involved in cell-interaction phenomena.

Characterization of amalgam: a member of the immunoglobulin superfamily from Drosophila.

The immunoglobulin superfamily is a diverse group of proteins that are involved in various aspects of cell surface recognition. Here, we report the characterization of amalgam (ama), a gene in the Antennapedia complex (ANT-C) of D. melanogaster that exhibits amino acid similarity to vertebrate neural cell adhesion molecules and other members of the immunoglobulin superfamily. The putative 333 amino acid ama protein consists of a signal sequence, three immunoglobulin-like domains, and a short slightly hydrophobic carboxy-terminal region. Antibodies against the ama protein reveal that it accumulates on the surface of various mesodermal and neural cells during embryogenesis. The function of this protein remains elusive, as no mutations have been recovered for ama during saturation EMS mutagenesis of this chromosomal region.

A carbohydrate epitope expressed uniquely on the cell surface of Drosophila neurons is altered in the mutant nac (neurally altered carbohydrate).

Antibodies against horseradish peroxidase (anti-HRP) recognize neural specific cell surface antigens in Drosophila and other insects. The nature of these antigens was investigated in Drosophila and found to include a complex set of developmentally regulated proteins. Their common epitope appears to be a carbohydrate that shares features with the sugar moiety of pineapple stem bromelain, a plant glycoprotein whose carbohydrate structure has been determined. A mutation was identified that eliminates staining by the antibody in imaginal and adult neural tissue. Tissue specific glycoconjugates, although widespread in the animal kingdom, are little understood. This mutation provides a unique opportunity to address the consequences of altering a neural specific carbohydrate moiety in an otherwise intact and behaving animal. The mutation maps to 84F. A second mutation, contained on the third chromosome balancer, TM3, eliminates anti-HRP staining in embryos. These mutations appear to be separate genes.