The effect of catecholamines and adenosine on the induction of morphological alterations and depigmentation of newt iris epithelial cells in vitro

The J1 glycoproteins have been shown to mediate neuron-astrocyte adhesion and appear in the nervous system as four species of Mr 160,000 (J1-160), 180,000 (J1-180), 200,000 (J1-200), and 220,000 (J1-220), respectively. Tenascin is a disulfide-linked oligomeric, extracellular matrix glycoprotein of subunit Mr 170,000, 190,000, 200,000, and 220,000, which has been proposed to promote epithelial cell proliferation. In view of the structural similarities of the molecules we have used immunohistochemical and immunochemical techniques to compare them. Immunohistochemically, polyclonal J1 and tenascin antibodies yielded identical staining patterns in non-nervous-system tissues, and staining could be completely blocked by preincubating the sera with purified tenascin. In the central nervous system all structures expressing tenascin immunoreactivity were also recognized by J1 antibodies. However, not all J1-positive structures were also tenascin-positive, indicating that J1 antibodies recognized additional epitopes not present on tenascin. Western-blot experiments performed with affinity-purified polyclonal J1 antibodies showed that J1 glycoproteins can be subdivided into two separate pairs, J1-160/180 and J1-200/220, which share a small degree of homology. Western-blot experiments and sequential immunoprecipitations on biosynthetically [35S]methionine- or 125I-radiolabeled J1 glycoproteins carried out with polyclonal J1 and tenascin antibodies demonstrated that J1-200/220 is immunochemically indistinguishable from tenascin. These observations suggest that one set of extracellular glycoproteins is associated with processes as different as neural histogenesis and carcinogenesis of mammary glands.     

J1/tenascin is a repulsive substrate for central nervous system neurons.

J1/tenascin mediates neuron-astrocyte interactions in vitro and is transiently expressed during CNS development in vivo. It is detectable in discrete zones, for example on astrocytes delineating "barrels" in the rodent somatosensory cortex. To investigate the effects of J1/tenascin on neural cell behavior in vitro, we have generated two monoclonal antibodies specific for protein epitopes on J1/tenascin and used them for immunoaffinity isolation of the molecule from postnatal mouse brain. The purified ECM molecule alone did not support attachment and growth of cerebral astrocytes or E14 mesencephalic, E18 hippocampal, and P6 cerebellar neurons. When various ECM constituents were adsorbed to polyornithine-conditioned glass, a favorable substrate for neural cells, the neurons avoided J1/tenascin-, but not laminin- or fibronectin-coated surfaces, while they grew on J1/tenascin-free, polyornithine-containing areas of the coverslip. In contrast, astrocytes formed uniform monolayers on all of these substrates. We conclude that J1/tenascin could serve to define repulsive territories for CNS neurons from different stages of neural development.     

J1/tenascin-related molecules are not responsible for the segmented pattern of neural crest cells or motor axons in the chick embryo

It has been suggested that substrate adhesion molecules of the tenascin family may be responsible for the segmented outgrowth of motor axons and neural crest cells during formation of the peripheral nervous system. We have used two monoclonal antibodies (M1B4 and 578) and an antiserum [KAF9(1)] to study the expression of J1/tenascin-related molecules within the somites of the chick embryo. Neural crest cells were identified with monoclonal antibodies HNK-1 and 20B4. Young somites are surrounded by J1/tenascin immunoreactive material, while old sclerotomes are immunoreactive predominantly in their rostral halves, as described by other authors (Tan et al. 1987--Proc. natn. Acad. Sci. U.S.A. 84, 7977; Mackie et al. 1988--Development 102, 237). At intermediate stages of development, however, immunoreactivity is found mainly in the caudal half of each sclerotome. After ablation of the neural crest, the pattern of immunoreactivity is no longer localised to the rostral halves of the older, neural-crest-free sclerotomes. SDS-polyacrylamide gel electrophoresis of affinity-purified somite tissue, extracted using M1B4 antibody, shows a characteristic set of bands, including one of about 230 x 10(3), as described for cytotactin, J1-200/220 and the monomeric form of tenascin. Affinity-purified somite material obtained from neural-crest-ablated somites reveals some of the bands seen in older control embryos, but the high molecular weight components (120-230 x 10(3] are missing. Young epithelial somites also lack the higher molecular mass components. The neural crest may therefore participate in the expression of J1/tenascin-related molecules in the chick embryo. These results suggest that these molecules are not directly responsible for the segmented outgrowth of precursors of the peripheral nervous system.     

Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

J1/tenascin in substrate-bound and soluble form displays contrary effects on neurite outgrowth

The influence of J1/tenascin adsorbed to polyornithine-conditioned plastic (substrate-bound J1/tenascin) and J1/tenascin present in the culture medium (soluble J1/tenascin) on neurite outgrowth was studied with cultured single cells from hippocampus and mesencephalon of embryonic rats. Neurons at low density grew well on J1/tenascin substrates and extended neurites that were approximately 40% longer than on the polyornithine control substrate after 24 h in vitro. The neurite outgrowth promoting effect of substrate bound J1/tenascin was largely abolished in the presence of mAb J1/tn2, but not by mAb J1/tn1. In contrast to the neurite growth-promoting effects of substrate bound J1/tenascin, neurite outgrowth on polyornithine, laminin, fibronectin, or J1/tenascin as substrates was inhibited by addition of soluble J1/tenascin to the cultures. Neither of the two mAbs neutralized the neurite outgrowth-inhibitory properties of soluble J1/tenascin. In contrast to their opposite effects on neurite outgrowth, both substrate-bound and soluble J1/tenascin reduced spreading of the neuronal cell bodies, suggesting that the neurite outgrowth-promoting and antispreading effects are mediated by two different sites on the molecule. This was further supported by the inability of the mAb J1/tn2 to neutralize the antispreading effect. The J1/tn2 epitope localizes to a fibronectin type III homology domain that is presumably distinct from the putative Tn68 cell-binding domain of chicken tenascin for fibroblasts, as shown by electronmicroscopic localization of antibody binding sites. We infer from these experiments that J1/tenascin contains a neurite outgrowth promoting domain that is distinguishable from the cell-binding site and presumably not involved in the inhibition of neurite outgrowth or cell spreading. Our observations support the notion that J1/tenascin is a multifunctional extracellular matrix molecule.     

Fibroblasts that proliferate near denervated synaptic sites in skeletal muscle synthesize the adhesive molecules tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan

Four adhesive molecules, tenascin(J1), N-CAM, fibronectin, and a heparan sulfate proteoglycan, accumulate in interstitial spaces near synaptic sites after denervation of rat skeletal muscle (Sanes, J. R., M. Schachner, and J. Covault. 1986. J. Cell Biol. 102:420-431). We have now asked which cells synthesize these molecules, and how this synthesis is regulated. Electron microscopy revealed that mononucleated cells selectively accumulate in perisynaptic interstitial spaces beginning 2 d after denervation. These cells were identified as fibroblasts by ultrastructural and immunohistochemical criteria; [3H]thymidine autoradiography revealed that their accumulation results from local proliferation. Electron microscopic immunohistochemistry demonstrated that N-CAM is associated with the surface of the fibroblasts, while tenascin(J1) is associated with collagen fibers that abut fibroblasts. Using immunofluorescence and immunoprecipitation methods, we found that fibroblasts isolated from perisynaptic regions of denervated muscle synthesize N-CAM, tenascin(J1), fibronectin, and a heparan sulfate proteoglycan in vitro. Thus, fibroblasts that selectively proliferate in interstitial spaces near synaptic sites are likely to be the cellular source of the interstitial deposits of adhesive molecules in denervated muscle. To elucidate factors that might regulate the accumulation of these molecules in vivo, we analyzed the expression of tenascin(J1) and fibronectin by cultured fibroblasts. Fibroblasts from synapse-free regions of denervated muscle, as well as skin, lung, and 3T3 fibroblasts accumulate high levels of tenascin(J1) and fibronectin in culture, showing that perisynaptic fibroblasts are not unique in this regard. However, when they are first placed in culture, fibroblasts from denervated muscle bear more tenascin(J1) than fibroblasts from innervated muscle, indicating that expression of this molecule by fibroblasts is regulated by the muscle's state of innervation; this difference is no longer apparent after a few days in culture. In 3T3 cells, accumulation of tenascin(J1) is high in proliferating cultures, depressed in confluent cultures, and reactivated in cells stimulated to proliferate by replating at low density or by wounding a confluent monolayer. Thus, synthesis of tenascin(J1) is regulated in parallel with mitotic activity. In contrast, levels of fibronectin, which increase less dramatically after denervation in vivo, are similar in fibroblasts from innervated and denervated muscle and in proliferating and quiescent 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding.

The localization of the extracellular matrix recognition molecule J1/tenascin was investigated in the crypt-villus unit of the adult mouse ileum by immunoelectron microscopic techniques. In the villus region, J1/tenascin was detected strongly in the extracellular matrix (ECM) between fibroblasts of the lamina propria. It was generally absent in the ECM at the interface between subepithelial fibroblasts and intestinal epithelium, except for some restricted areas along the epithelial basal lamina of villi, but not of crypts. These restricted areas corresponded approximately to the basal part of one epithelial cell. In J1/tenascin-positive areas, epithelial cells contacted the basal lamina with numerous microvillus-like processes, whereas in J1/tenascin-negative areas the basal surface membranes of epithelial cells contacted their basal lamina in a smooth and continuous apposition. In order to characterize the functional role of J1/tenascin in the interaction between epithelial cells and ECM, the intestinal epithelial cell line HT-29 was tested for its ability to adhere to different ECM components. Cells adhered to substratum-immobilized fibronectin, laminin and collagen types I to IV, but not to J1/tenascin. When laminin or collagen types I to IV were mixed with J1/tenascin, cell adhesion was as effective as without J1/tenascin. However, adhesion was completely abolished when cells were offered a mixture of fibronectin and J1/tenascin as substratum. The ability of J1/tenascin to reduce the adhesion of intestinal epithelial cells to their fibronectin-containing basal lamina suggests that J1/tenascin may be involved in the process of physiological cell shedding from the villus.     

Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats

The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.     

Expression of the adhesion molecules L1, N-CAM and J1/tenascin during development of the murine small intestine

We have previously studied the immunohistological localization of the three adhesion molecules L1, N-CAM and J1/tenascin in adult mouse small intestine and shown that L1 expression in epithelial crypt cells underlies the adhesion of these cells to one another [63]. To obtain further insight into the functional roles of L1, N-CAM and J1/tenascin in this organ we studied their expression starting at embryonic day 14 during embryonic and early postnatal morphogenesis and during epithelial cell migration in the adult. Expression of L1 was restricted to neural cells until approximately postnatal day 5, when L1 started to be detectable on crypt but not on villus cells, predominantly on the basolateral membrane infoldings. As in brain, L1-specific mRNA was approximately 6 kb in size. L1 from intestine appears to differ from the brain-derived equivalent in possessing a higher level of glycosylation. N-CAM was detectable from embryonic day 14 onward in neural and also in mesenchymal cells. Expression by smooth muscle cells decreased during development. In the villus core, N-CAM was strongly detectable at contact sites between smooth muscle cells forming the cellular scaffold of the villus. From embryonic day 14 onward, N-CAM appeared in both 180- and 140-kDa forms. J1/tenascin was present in both neural and mesenchymal cells from embryonic day 14 onward. Starting at embryonic day 17, J1/tenascin appeared concentrated at the boundary between mesenchyme and epithelium in an increasing gradient from the crypt base to the villus top. From embryonic day 14 onward J1/tenascin consisted of the 190- and 220-kDa components. J1/tenascin from intestine differed from brain-derived J1 in its carbohydrate composition. These observations show that the three adhesion molecules are expressed by distinct cell populations and may serve as cell-type-specific markers in pathologically altered intestinal tissue.     

Nerve Growth Factor Mediates Monosialoganglioside-Induced Release of Fibronectin and J1/Tenascin from C6 Glioma Cells

C6 rat glioma cells incubated in serum-free medium with D-[14C]glucosamine secrete, on stimulation with nerve growth factor (NGF) or monosialogangliosides (MSGs), several glycoproteins (Gps), the most prominent of which are a 270-, 220-, and 69-kDa Gp. Several growth factors, hormones, phorbol ester, and disialo- and trisialogangliosides did not stimulate secretion. Western blot analysis of the conditioned medium from C6 cells stimulated with NGF or MSG identified one distinct band of approximately 220 kDa for fibronectin and J1/tenascin, which comigrated. Antiserum to NGF prevented NGF-stimulated release and also blocked MSG-evoked release. The 220-kDa band was labeled after pulse labeling with [35S]methionine in the presence of NGF, and by a 15-min chase period radioactively labeled J1/tenascin could be immunoprecipitated. Tunicamycin drastically inhibited almost completely release of the 220-kDa Gp labeled by D-[14C]glucosamine or [35S]methionine. These results extend the range of neurotrophic properties attributed to NGF to cells of glial origin and suggest that NGF regulates secretion of extracellular matrix proteins. MSG stimulation of fibronectin and J1/tenascin secretion may be mediated by NGF or an NGF-like molecule also secreted by the C6 glioma cells.     

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1.

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.     

Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture

The involvement of the adhesion molecules L1, N-CAM, and J1 in adhesion and neurite outgrowth in the peripheral nervous system was investigated. We prepared Schwann cells and fibroblasts (from sciatic nerves) and neurons (from dorsal root ganglia) from 1-d mice. These cells were allowed to interact with each other in a short-term adhesion assay. We also measured outgrowth of dorsal root ganglion neurons on Schwann cell and fibroblast monolayers. Schwann cells (which express L1, N-CAM, and J1) adhered most strongly to dorsal root ganglion neurons by an L1-dependent mechanism and less by N-CAM and J1. Schwann cell-Schwann cell adhesion was mediated by L1 and N-CAM, but not J1. Adhesion of fibroblasts (which express N-CAM, but not L1 or J1) to neurons or Schwann cells was mediated by L1 and N-CAM and not J1. However, inhibition by L1 and N-CAM antibodies was found to be less pronounced with fibroblasts than with Schwann cells. N-CAM was also strongly involved in fibroblast-fibroblast adhesion. Neurite outgrowth was most extensive on Schwann cells and less on fibroblasts. A difference in extent of neurite elongation was seen between small- (10-20 microns) and large- (20-35 microns) diameter neurons, with the larger neurons tending to exhibit longer neurites. Fab fragments of polyclonal L1, N-CAM, and J1 antibodies exerted slightly different inhibitory effects on neurite outgrowth, depending on whether the neurites were derived from small or large neurons. L1 antibodies interfered most strikingly with neurite outgrowth on Schwann cells (inhibition of 88% for small and 76% for large neurons), while no inhibition was detectable on fibroblasts. Similarly, although to a smaller extent than L1, N-CAM appeared to be involved in neurite outgrowth on Schwann cells and not on fibroblasts. Antibodies to J1 only showed a very small effect on neurite outgrowth of large neurons on Schwann cells. These observations show for the first time that identified adhesion molecules are potent mediators of glia-dependent neurite formation and attribute to L1 a predominant role in neurite outgrowth on Schwann cells which may be instrumental in regeneration.     

Boundaries defined by adhesion molecules during development of the cerebral cortex: the J1/tenascin glycoprotein in the mouse somatosensory cortical barrel field

The distribution of the 200/220 KDa J1 glycoprotein (J1-200/220), within the developing vibrissae-related barrel field of the mouse somatosensory cortex, was studied by immunocytochemistry using a monoclonal antibody. J1-200/220, a member of the L2/HNK-1 family of adhesion molecules, also appears to be the mouse homologue of tenascin. J1/tenascin-positive barrel-like structures are visible in the somatosensory cortex between 24 and 48 hr after birth, with the molecule present in prospective barrel boundaries. Immunoelectronmicroscopy reveals labeling that is associated with glial and neuronal plasma membranes, as well as glial end-feet on blood vessels. A possible major source of J1/tenascin expression at this time is astrocyte precursor cells and radial glia. In the putative astrocyte precursor cells, immunolabeling was observed within organelles including the Golgi apparatus. At P6-7 J1/tenascin is most prevalent within prospective interbarrel septae. J1/tenascin-positive barrel boundaries are barely visible on P9 and not observed on P16. The findings indicate that J1/tenascin represents a major component of previously described "hidden" boundaries that we have seen during development using other methodologies. The expression of adhesion molecule-rich boundaries during the critical stages of barrel field formation indicates roles for such molecules during specific cerebral cortical pattern formation events.     

A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain

We have identified a novel carboxyl-terminal splice-variant of the glutamate transporter GLT1, which we denote as GLT1c. Within the rat brain only low levels of protein and message were detected, protein expression being restricted to end feet of astrocytes apposed to blood vessels or some astrocytes adjacent to the ventricles. Conversely, within the retina, this variant was selectively and heavily expressed in the synaptic terminals of both rod- and cone-photoreceptors in both humans and rats. Double-immunolabelling with antibodies to the carboxyl region of GLT1b/GLT1v, which is strongly expressed in apical dendrites of bipolar cells and in cone photoreceptors revealed that in the rat GLT1c was co-localised with GLT1b/GLT1v in cone photoreceptors but not with GLT1b/GLT1v in bipolar cells. GLT1c expression was developmentally regulated, only appearing at around postnatal day 7 in the rat retina, when photoreceptors first exhibit a dark current. Since the glutamate transporter EAAT5 is also expressed in terminals of rod photoreceptor terminals these data indicate that rod photoreceptors express two glutamate transporters with distinct properties. Similarly, cone photoreceptors express two glutamate transporters. We suggest that differential usage of these transporters by rod and cone photoreceptors may influence the kinetics of glutamate transmission by these neurons.     

Monoclonal antibodies (O1 to O4) to oligodendrocyte cell surfaces: an immunocytological study in the central nervous system

Four monoclonal antibodies are characterized that have been obtained from a fusion of mouse myeloma P3-NS1/1-Ag4-1 with spleen cells from BALB/c mice immunized with white matter from bovine corpus callosum. The corresponding antigens (O antigens) are designated O1, O2, O3, and O4. The localization of these antigens was investigated by indirect immunofluorescence in cultures of early postnatal mouse cerebellum, cerebrum, spinal cord, optic nerve, and retina. When tested on live cultures none of the O antibodies reacted with the surface of astrocytes, neurons, or fibroblasts, however, all are positive on the surface of oligodendrocytes. The identity of these cells was determined by double-immunolabeling experiments with indpendent cell-type-specific antigenic markers (glial fibrillary acidic protein, tetanus toxin receptors, fibronectin, and galactocerebroside). Antigen O1 is exclusively expressed on galactocerebroside-positive cells, whereas O2, O3, and O4 are expressed on additional cells that are negative for any of the markers tested. None of the O antigens is expressed on the surface of cultured retinal cells. In fresh-frozen sections of adult mouse cerebellum all O antigens are detectable in white matter tracts and in vesicular structures of the granular layer. O2 and O3 antigens are in addition detectable in GFA protein-positive radial fibers in the molecular layer. In fixed cerebellar cultures, where intracellular antigens are accessible, O1, O2, and O3 antibodies label astrocytes in a GFA protein-like pattern. O antigens are expressed in mouse, rat, chicken, and human central nervous systems. O antibodies belong to the IgM immunoglobulin subclass and have been used in complement-dependent cytotoxic elimination of cerebellar oligodendrocytes in culture. At limiting antibody dilutions all processes of oligodendrocytes are preferably lysed over cell bodies.     

Characterization of the cell adhesion molecules L1, N-CAM and J1 in the mouse intestine.

To gain insight into the cellular and molecular mechanisms underlying epithelial cell surface interactions in the adult mouse intestine, we have characterized the cell adhesion molecules L1, N-CAM and J1 by immunocytological, biochemical and cell biological methods. Whereas N-CAM and J1 expression was found to be confined to the mesenchymal and neuroectodermally-derived parts of the intestine, L1 was localized in the proliferating epithelial progenitor cells of crypts, but not in the more differentiated epithelial cells of villi. L1 was detected in crypt cells by Western blot analysis in the molecular forms characteristic of peripheral neural cells, with apparent mol. wts of 230, 180 and 150 kd. Aggregation of single, enriched crypt, but not villus cells, was strongly inhibited in the presence of Fab fragments of polyclonal L1 antibodies. These observations show that L1 is not confined to the nervous system and that it may play a functional role in the histogenesis of the intestine in the adult animal.     

GLT1, glial glutamate transporter,is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high‐affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin‐like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin‐positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 515–526, 1999     

Glial fibrillary acidic protein, J1-31 antigen and vimentin in adult hamster brain: an immunohistochemical study.

Different regions of the prosencephalon and mesencephalon of the adult hamster brain displayed differences in the immunofluorescence expression of astrocytic proteins, namely glial fibrillary acidic protein and J1-31 antigen (30 kD protein). Neither of these proteins could be detected in layers II-VI of the cerebral cortex. However, varying degrees of immunostaining were detectable in perivascular glia, stria medullaris thalamus, the basal cerebral peduncle and the dentate molecular layer of the hippocampus. Vimentin was conspicuous in neurons, particularly in the cerebral cortex and hippocampus, and in glial fibrillary acidic protein-positive astrocytes in major fibre tracts. These observations are discussed in relation to interspecies differences in the expression of intermediate filament proteins.