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U Rutishauser

Publications and source records attributed to U Rutishauser.

At least 73 records · Page 4Linked to original sources
PubMed1990

Maturation of astrocytes in vitro alters the extent and molecular basis of neurite outgrowth.

In the developing mammalian central nervous system astrocytes have been proposed as an important substrate for axon growth. In the adult central nervous system following injury, astrocytes are a major component of the gliotic response which has been proposed to block axon growth. Experimental transplantation studies using cultured astrocytes have suggested that immature but not mature cultured astrocytes have the capacity to support axon outgrowth when transplanted into the adult rodent CNS. These observations suggest that astrocyte maturation is accompanied by changes in the functional capacity of these cells to support axon outgrowth. To determine whether this functional change reflects an intrisic astrocyte property, the extent and molecular bases of neurite outgrowth from embryonic rat cortical and chick retinal neurons on cultures of purified immature and mature astrocytes have been compared in vitro. The rate and extent of neurite outgrowth from both neuronal populations are consistently greater over the surface of immature than over the surface of mature astrocytes. Furthermore, antibodies to NCAM and G4/L1 significantly reduce neurite outgrowth on immature but not mature astrocytes, while antibodies to the integrin B1 receptor reduced outgrowth on both immature and, to a lesser extent, mature astrocytes. These results suggest that in vitro mature astrocytes have a reduced capacity and different molecular bases for supporting neurite outgrowth compared to immature astrocytes and are consistent with the proposal that functional changes during astrocyte maturation may partially contribute to regulating axon growth in the mammalian CNS.

Animals↗
PubMed1990

Polysialic acid as a regulator of intramuscular nerve branching during embryonic development.

The role of polysialic acid (PSA) during initial innervation of chick muscle was examined. Previously, the adhesion molecules L1 and N-CAM were shown to be important in balancing axon-axon and axon-muscle adhesion during this process. Here we demonstrate developmental changes in the pattern of innervation that are not correlated with levels of L1 or N-CAM expression, but rather with the amount of PSA at the axon surface. Removal of PSA by a specific endoneuraminidase (Endo-N) increased axon fasciculation and reduced nerve branching. In contrast, the nerve trunk defasciculation and increased branching produced by neuromuscular activity blockade were associated with an increase in axonal PSA levels. Furthermore, Endo-N prevented these inactivity-induced effects on branching. Together these results illustrate the potential of PSA as a regulator of cell-cell interactions and provide a direct example of a molecular link between the morphogenic effects of adhesion-mediated and synaptic activity-dependent processes.

Animals↗
PubMed1990

Binding properties of detergent-solubilized NCAM.

An assay has been designed for the identification of NCAM-binding proteins present in an NP-40 detergent extract of brain membranes. This method, which is capable of analyzing both heterophilic and homophilic interactions, uses species-specific antibodies against NCAM in combination with radioiodination, so that after unlabeled chicken and iodinated frog brain membrane proteins were allowed to interact, the chicken NCAM could be specifically isolated by immunoaffinity adsorption. The radiolabeled frog proteins coisolated with chicken NCAM were then characterized by one- and two-dimensional gel electrophoresis in combination with immunoblotting. The only detectable NCAM-binding proteins were identified as the 140- and 180-kD forms of NCAM. The presence and absence of polysialic acid on NCAM did not change the amount or nature of the frog proteins immunopurified under these conditions. As an alternative for detecting heterophilic ligands, a simplified immunoprecipitation method was employed using either iodine or sulfate radiolabels. Again under these conditions only NCAM was detected. These results are consistent with the hypothesis that the major binding protein for NCAM is NCAM itself, and suggest that differences in polysialic acid content do not directly alter the properties of NCAM's homophilic binding site.

Animals↗
PubMed1989

NCAM in the differentiation of embryonic lens tissue.

The role of the neural cell adhesion molecule (NCAM)2 in ocular lens differentiation was investigated in chicken embryos. Changes in expression of NCAM were documented by immunohistology of frozen sections. This analysis revealed that NCAM diminished during lens fiber differentiation, in contrast to the gap junction-associated protein MP26 which became more abundant. The form of NCAM expressed was determined by Western blot analysis of proteins extracted from the different regions of the Embryonic Day 6 lenses. All regions expressed NCAM with an apparent molecular weight of 140 kDa and relatively low levels of polysialylation. The function of NCAM in lens differentiation was investigated using antibodies that inhibit NCAM-mediated adhesion. Two parameters that change during maturation of the lens epithelial cells were monitored: the thickness of the tissue, indicating the length of lens cells, and the particle arrangement of gap junctions, reflecting the state of junctional differentiation. When epithelial cell explants of Embryonic Day 6 lenses were cultured for 5 days, the cells elongated and displayed an increase in the loose, random intramembranous particle arrangements characteristic of maturing lens fiber gap junctions. When the explants were cultured in the presence of anti-NCAM Fabs, the epithelia were thinner than in matched controls and had particle arrangements characteristic of a less mature state. The expression of NCAM during lens differentiation and the effects of attenuating NCAM function suggest that adhesion mediated by NCAM is an essential event in lens cell differentiation.

Animals↗
PubMed1988

The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions.

The neural cell adhesion molecule (NCAM) can influence a number of diverse intercellular events, including junctional communication, the association of axons with pathways and targets, and signals that alter levels of neurotransmitter enzymes. These pleiotropic effects appear to reflect the ability of NCAM to regulate membrane-membrane contact required to initiate specific interactions between other molecules. Such regulation can occur through changes in either NCAM expression or the molecule's content of polysialic acid (PSA). When NCAM with a low PSA content is expressed, adhesion is increased and contact-dependent events are triggered. In contrast, the large excluded volume of NCAM PSA can inhibit cell-cell interactions through hindrance of overall membrane apposition.

Animals↗
PubMed1988

Distinct roles for adhesion molecules during innervation of embryonic chick muscle.

In vitro studies have suggested that the cell adhesion molecules NCAM and G4/L1 contribute to a variety of events during neural development. We have directly tested the role played by these molecules in the process of initial nerve ingrowth and ramification in the embryonic chick iliofibularis muscle by in ovo injections of specific adhesion-blocking antibodies and analysis of the resultant nerve branching pattern in muscle whole mounts. Antibodies against both molecules produced axonal defasciculation, which resulted in an enhanced transverse projection to the fast region of the muscle. In the case of anti-G4/L1, we also observed a large increase in the number of side branches that form from nerve trunks in the slow region and an enhancement of nerve branching in the fast region. Conversely, anti-NCAM produced a striking decrease in both the number and length of side branches in the slow region, and a reduction in nerve branching in the fast region. A similar reduction of nerve branching was obtained following injection of an endosialidase, which removes sialic acid from NCAM, and which was observed to enhance fiber-fiber apposition, presumably by increasing cell adhesion. Based on their biochemical properties in vitro and their in vivo distribution, both NCAM and G4/L1 are in a position to contribute to axon-axon adhesive interactions, whereas NCAM would be expected to also promote axon-myotube interactions. Our observations in fact indicate that these two adhesion molecules play different but complementary roles during muscle innervation and, specifically, that axon-axon fasciculation is influenced by both NCAM and G4/L1 in an anatomically distinct manner to regulate the overall pattern of nerve branching and that NCAM-mediated axon-myotube interactions are necessary for the attainment of the normal stereotyped pattern of nerve branching in both fast and slow regions of this muscle.

Animals↗
PubMed1988

Identification of two structural types of calcium-dependent adhesion molecules in the chicken embryo.

By using an immunological and peptide mapping approach two calcium-dependent cell-cell adhesion molecules (calCAMs) in the embryonic chicken are compared. A third closely related molecule is identified and compared to the two calCAMs. One of the calCAMs appears to be identical to the previously identified adhesion molecule N-cadherin, originally identified in chicken retina and localized to neural tissues. The second is the same as L-CAM, originally identified in chicken liver but localized to a variety of epithelial tissues. The third, also found in liver, is similar to L-CAM but is much closer in structure to N-cadherin. It is, however, immunologically distinct from N-cadherin. We therefore refer to this newly identified molecule as CRM-L for cadherin-related molecule in liver. CRM-L, N-cadherin, and L-CAM are all cell-surface proteins with a similar stability to tryptic digestion in the presence of calcium. CRM-L has the same molecular mass and isoelectric point as N-cadherin but is distinct from L-CAM in these properties. Two-dimensional peptide maps of complete tryptic digests reveal that CRM-L shares 69% of its peptides with N-cadherin and 20% with L-CAM. On the basis of these data, we suggest that there are at least two distinguishable types of calCAMs in the chicken embryo: one represented by the closely related molecules N-cadherin and CRM-L, and another represented by L-CAM.

Animals↗
PubMed1988

Neural cell adhesion molecule regulates cell contact-mediated changes in choline acetyltransferase activity of embryonic chick sympathetic neurons.

E10 chick sympathetic ganglion cells display a cell contact-dependent rise in choline acetyltransferase (ChAT) specific activity over the first several days in culture. This effect can be mimicked by addition of crude membrane fractions prepared from E10 retina and adult chicken brain, but not by those from E10 brain. The effects of both cell-cell and membrane-cell contact are inhibited by the addition of anti-NCAM Fab fragments. The membranes capable of increasing ChAT and those which are ineffective all contain NCAM, however their relative levels of NCAM polysialic acid differ. Whereas membranes with high polysialic acid NCAM are ineffective, selective enzymatic removal of polysialic acid renders them capable of producing an increase in ChAT. The inhibition of NCAM-mediated adhesion produced by Fab fragments can be compensated for by addition of wheat germ agglutinin, but only with membranes whose NCAM has low levels of polysialic acid. Taken together, these data suggest that NCAM can regulate cell contact-mediated increases in ChAT activity. We propose that NCAM-mediated adhesion promotes contact between cell membranes to allow the transmission of an otherwise NCAM-independent signal. In addition, NCAM's polysialic acid moiety appears to influence the ability of cells to transmit this signal, even in the presence of an alternative adhesion mechanism.

Animals↗
PubMed1988

Neural differentiation, NCAM-mediated adhesion, and gap junctional communication in neuroectoderm. A study in vitro.

We studied the development of NCAM and gap junctional communication, and their mutual relationship in chick neuroectoderm in vitro. Expression of NCAM, as detected by monoclonal and polyclonal antibodies, and development of junctional communication, as detected by extensive cell-to-cell transfer of 400-500-D fluorescent tracers, occurred in cultures from stage-2 embryos onward. Both expressions presumably required primary induction. The differentiating cells formed discrete fields of expression on the second to third day in culture, with the NCAM fields coinciding with the junctional communication fields delineated by the tracers. Other neural differentiations developed in the following order: tetanus toxin receptors, neurofilament protein, and neurite outgrowth. Chronic treatment with antibody Fab fragments against NCAM interfered with the development of communication, suggesting that NCAM-mediated adhesion promotes formation of cell-to-cell channels. Temperature-sensitive mutant Rous sarcoma virus blocked (reversibly) communication and the subsequent development of neurofilament protein and neurites, but expression of NCAM continued.

Animals↗
PubMed1988

Patterns of N-CAM expression during myogenesis in Xenopus laevis.

The neural cell adhesion molecule (N-CAM) is seen in the membrane of nerves and muscles from several vertebrate species. Using indirect immunofluorescence, we have examined the expression of this protein during embryonic and postembryonic myogenesis in the African clawed frog, Xenopus laevis. While good staining for N-CAM was seen in neuronal tissues at all stages examined, no staining of embryonic muscle was observed, including both mononucleated and polynucleated myoblasts. In contrast, limb muscles formed at metamorphosis showed strong expression of N-CAM. The developing limb muscles eventually lose their N-CAM, but will reexpress it dramatically when denervated. These observations suggest that myogenesis programs executed at different stages of development can display distinct patterns of N-CAM expression.

Animals↗
PubMed1987

Neural cell adhesion molecule expression in Xenopus embryos.

The spatiotemporal pattern of expression of the neural cell adhesion molecule NCAM was mapped immunohistochemically in embryos of the frog Xenopus, from blastula to early swimming stages, using a polyclonal antibody that recognizes Xenopus NCAM. The neural plate stage was the earliest at which NCAM could be detected. The initial sites of NCAM immunoreactivity were neural ectoderm, somitic mesoderm, and chordamesoderm. During formation of the neural tube, NCAM immunoreactivity became restricted to the neuroectoderm and its derivatives. During closure of the neural tube and for 2-4 hr thereafter, NCAM was expressed in a distinctive radial pattern in coronal sections of the neural tube. NCAM was observed in neural crest cells before migration and after formation of cranial and spinal ganglia. During the period of initial neurite outgrowth, NCAM became concentrated in the developing central nerve fiber pathways. NCAM was seen on peripheral nerves from the time of their initial outgrowth and it was strongly expressed at neuromuscular junctions during the period of their formation. These results show that NCAM is expressed after neural induction and functions during morphogenesis of the neural plate and tube, some neural crest derivatives, development of nerve fiber tracts, and formation of neuromuscular connections.

Animals↗
PubMed1987

Changes in neural cell adhesion molecule (NCAM) structure during vertebrate neural development.

Changes in carbohydrate and polypeptide form of the neural cell adhesion molecule (NCAM) have been documented during the development of central nervous system tissue in both chicken and frog. The carbohydrate variations reflect a high and low content of polysialic acid, and for the two vertebrates examined the expression of these forms is similar. At very early stages of neural development NCAM with a low content of polysialic acid is present, during histogenesis of the central nervous system NCAM with a high content of polysialic acid dominates, and there is a gradual return to NCAM with a low content of polysialic acid as the animals approach maturity. In contrast, the order of expression of the major NCAM polypeptide forms is different in the chicken and frog. These findings suggest that changes in sialic acid are a fundamental aspect of the function of NCAM in development, whereas NCAM polypeptide differences may affect events associated with a particular vertebrate. Studies have demonstrated that a decreased sialic acid content enhances the adhesion properties of NCAM. On this basis, we propose that NCAM with a low content of polysialic acid functions both to maintain integrity of neuroepithelium during morphogenesis of the early embryo and to stabilize differentiated structures in the adult, while the decreased adhesive function of NCAM with a high content of polysialic acid provides more plasticity in cell interactions during cell migration, axon outgrowth, and formation of neural circuits.

Animals↗
PubMed1987

Membrane glycoproteins involved in neurite fasciculation.

Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.

Amino Acid Sequence↗
PubMed1987

Visualization of neural cell adhesion molecule by electron microscopy.

The 130- and 160-kD polypeptide forms of the neural cell adhesion molecule (NCAM) were analyzed by electron microscopy after low angle rotary shadowing and freeze replication. Individual NCAM molecules appeared as uniformly thick rods, with a distinct bend or hinge region near their middle. Aggregates were also present, containing two to six rods in a pinwheel-like configuration without measurable overlap between rods. The 130- and 160-kD NCAM forms had lengths of 38 and 51 nm, respectively, with a difference in arm length distal to the bend, but not toward the center of the pinwheel. Although enzymatic removal of the polysialic acid moiety on NCAM did not alter the appearance of individual molecules, it did increase the average number of arms per aggregate. Monoclonal antibodies that recognize defined regions of the NCAM polypeptide were used to provide landmarks on the observed molecular figures. Two antibodies specific for cytoplasmic epitopes near the COOH terminus were clustered at the distal tip of aggregated arms. Two other antibodies that react with epitopes near the NH2 terminus and the middle of the molecule bound to sites more centrally located on the pinwheel structure. Together, these results suggest that the observed aggregates represent an association of molecules near their NH2-terminal homophilic binding site, and have led to several predictions about the nature of an NCAM-mediated cell-cell bond.

Animals↗
PubMed1987

Axon pathway boundaries in the developing brain. I. Cellular and molecular determinants that separate the optic and olfactory projections.

When optic fibers first approach the chiasmatic region of the diencephalon in the chick embryo on days 3 and 4 (E3-4), they rarely grow rostrally into the olfactory region of the telencephalon. Conversely, olfactory tract axons grow as far as, but never cross the diencephalic/telencephalic (D/T) boundary to enter the optic chiasm. In this study, a region of specialized neuroepithelium, originally named the "knot" in mouse by Silver (1984), has been identified at the D/T border of chick embryos. At pre-axonal stages, the presumptive knot region undergoes a cataclysmic cell death, with concomitant phagocytosis of necrotic debris by the remaining cells. When fibers subsequently appear in the chiasm and olfactory tracts, the knot consists of a very dense, interwoven cluster of non-neuronal cells that lack marginal radial processes, and whose cell bodies directly abut the glial limiting membrane. Thus, the morphology of the knot is in sharp contrast to the cell body-free marginal zone and endfoot regions along which axons tend to grow. In addition, we found that the neural cell adhesion molecule (N-CAM), which is expressed on neuroepithelial cell processes within the central optic and olfactory pathways, is not present on cells in the knot region during periods of axon growth. These results suggest that the knot, through its elimination of the marginal zone processes, absence of large extracellular spaces, and relative absence of adhesion molecules, functions as an axon-refractory barrier that effectively separates the optic and olfactory projections.

Animals↗