Biomedical subjects

V Lemmon

Publications and source records attributed to V Lemmon.

At least 55 records · Page 3Linked to original sources

PubMed1993

Glial cells of the O-2A lineage bind preferentially to N-cadherin and develop distinct morphologies.

Environmental cues guiding glial cell behavior during development or regeneration of the nervous system are provided by both soluble and nondiffusible factors. We examined the influences of purified extracellular matrix molecules and cell adhesion molecules on the development and proliferation of glial cells from neonatal rat optic nerves. Dissociated optic nerve glia were plated on fibronectin, laminin, collagen type IV, L1, N-cadherin, and N-CAM. Cultures were grown in chemically defined medium to promote formation of oligodendrocytes. Other cultures were grown in 10% serum to support type-1 astrocytes and the differentiation of O-2A progenitor cells to type-2 astrocytes. In short-term adhesion assays to measure cell affinity for the different substrates, cells in the O-2A lineage bound preferentially to N-cadherin while type-1 astrocytes preferentially bound to extracellular matrix components. The cells in the O-2A lineage also developed distinctive morphologies on different substrates after incubation for 4 days. Type-2 astrocytes and oligodendrocytes produced very large membranous expansions on N-cadherin. Measurements of BrdU incorporation indicated that the substrates did not significantly influence cell proliferation rates. Our results showed that O-2A progenitor cells, oligodendrocytes, type-1 astrocytes, and type-2 astrocytes possess different complements of receptors for the adhesion molecules in their environment and that their morphological differentiation can be dramatically altered by these extracellular signals.

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PubMed1992

Modulation of growth cone morphology by substrate-bound adhesion molecules.

The growth cone, a terminal structure on developing and regenerating axons, is specialized for motility and guidance functions. In vivo the growth cone responds to environmental cues to guide the axon to its appropriate target. These cues are thought to be responsible for position-specific morphological changes in the growth cone, but the molecules that control growth cone behavior are poorly characterized. We used scanning electron microscopy to analyze the morphology of retinal ganglion cell growth cones in vitro on different adhesion molecules that axons normally encounter in vivo. L1/8D9, N-cadherin, and laminin each induced distinctive morphological characteristics in growth cones. Growth cones elaborated lamellipodial structures in response to the cell adhesion molecules L1/8D9 and N-cadherin, whereas laminin supported filopodial growth cones with small veils. On L1/8D9, the growth cones were larger and produced more filopodia. Filopodial associations between adjacent growth cones and neurites were frequent on L1/8D9 but were uncommon on laminin or N-cadherin. These results demonstrate that different adhesion molecules have profoundly different effects on growth cone morphology. This is consistent with previous reports suggesting that changes in growth cone morphology in vivo occur in response to changes in substrate composition.

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PubMed1992

Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength.

Growing axons use environmental cues to guide them to their targets. One class of cues is thought to be adhesion molecules on cells and in the extracellular matrix that axons interact with as they grow to their targets. In choosing between two possible pathways, the relative adhesiveness of the two substrates could be an important factor in controlling neurite growth. We conducted experiments in vitro to study how naturally occurring adhesion molecules influence neurite growth. Neurite growth rates, the degree of neurite fasciculation, the choices neurites make between two substrates, and the relative adhesiveness of different substrates were examined. We found that the relative adhesiveness of a substrate was a poor predictor of either axon growth rate or the degree of fasciculation. Furthermore, neurites showed little selectivity between three different naturally occurring substrates, L1, N-cadherin, and laminin. These results suggest that some adhesion molecules may serve as permissive substrates in that they can define axonal pathways but they do not provide information about which path to take at a choice point or about which direction to go along the path. Finally, these results suggest that substrates in vivo may not exert their effects on axon guidance principally via relative adhesiveness.

Adhesiveness↗

PubMed1991

Phosphorylation-dependent regulation of axon fasciculation.

Axons often grow along other axons to produce bundles called fascicles, and a number of cell adhesion molecules (CAMs) found on axon surfaces contribute to this process. The surprising observation that Fab fragments against individual CAMs can completely block fascicle formation suggests that the different axon-associated CAMs are functionally linked. The present studies investigate whether such a linkage might reflect intracellular regulatory mechanisms. Results obtained with chicken retinal explants in culture indicate that fasciculation is highly sensitive to cytoplasmic protein phosphorylation by means of a mechanism that does not alter levels of CAM expression. Moreover, the potent effect of individual Fabs on fasciculation disappears with enhanced phosphorylation. These observations suggest that growing axons possess a general regulatory process for the multiple CAMs that participate in fasciculation.

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PubMed1991

Interactions of Schwann cells with neurites and with other Schwann cells involve the calcium-dependent adhesion molecule, N-cadherin.

During embryogenesis, Schwann cells interact with axons and other Schwann cells, as they migrate, ensheath axons, and participate in organizing peripheral nervous tissues. The experiments reported here indicate that the calcium-dependent molecule, N-cadherin, mediates adhesion of Schwann cells to neurites and to other Schwann cells. Cell cultures from chick dorsal root ganglia and sciatic nerves were maintained in media containing either 2 mM Ca++ or 0.2 mM Ca++, a concentration that inactivates calcium-dependent cadherins. When the leading lamellae of Schwann cells encountered migrating growth cones in medium with 2 mM Ca++, they usually remained extended, and the growth cones often advanced onto the Schwann cell upper surface. In the low Ca++ medium, the frequency of withdrawal of the Schwann cell lamella after contact with a growth cone was much greater, and withdrawal was the most common reaction to growth cone contact in medium with 2 mM Ca++ and anti-N-cadherin. Similarly, when motile leading margins of two Schwann cells touched in normal Ca++ medium, they often formed stable areas of contact. N-cadherin and vinculin were co-concentrated at these contact sites between Schwann cells. However, in low Ca++ medium or in the presence of anti-N-cadherin, interacting Schwann cells usually pulled away from each other in a behavior reminiscent of contact inhibition between fibroblasts. In cultures of dissociated cells in normal media, Schwann cells frequently were aligned along neurites, and ultrastructural examination showed extensive close apposition between plasma membranes of neurites and Schwann cells. When dorsal root ganglia explants were cultured with normal Ca++, Schwann cells migrated away from the explants in close association with extending neurites. All these interactions were disrupted in media with 0.2 mM Ca++. Alignment of Schwann cells along neurites was infrequent, as were extended close apposition between axonal and Schwann cell plasma membranes. Finally, migration of Schwann cells from ganglionic explants was reduced by disruption of adhesive contact with neurites. The addition of antibodies against N-cadherin to medium with normal Ca++ levels had similar effects as lowering the Ca++ concentration, but antibodies against the neuronal adhesive molecule, L1, had no effects on interactions between Schwann cells and neurites.

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PubMed1991

Studies of the developing chick retina using monoclonal antibody 8A2 that recognizes a novel set of gangliosides.

A monoclonal antibody, Mab 8A2, that recognizes a novel set of gangliosides was produced by immunizing a mouse with Embryonic Day 14 chick optic nerve. Immunohistochemical studies of the developing chick retina revealed a complex pattern of Mab 8A2 immunoreactivity. Initially, staining is concentrated in the optic fiber layer in the central retina. Later in development, the most intense staining is seen at the periphery of the retina and 8A2 immunoreactivity appears in other retina layers. In the adult retina, 8A2 immunoreactivity is lost from the optic fiber layer but persists in the inner plexiform layer, inner nuclear layer, and outer plexiform layer. Cell culture experiments showed intense staining of neurites from retinal ganglion cells but no staining of Muller cells. Biochemical characterization of the epitope recognized by Mab 8A2 suggests that it includes a 9-O-acetyl group that is present on five different gangliosides. The 8A2 immunoreactive gangliosides are distinct from and have slower mobilities on thin-layer chromatographs than those recognized by Mab D1.1 which recognizes 9-O-acetyl GD3.

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PubMed1991

Molecular structure and functional testing of human L1CAM: an interspecies comparison.

The rodent, avian, and insect L1-like cell adhesion molecules are members of the immunoglobulin superfamily that have been implicated in axon growth. We have isolated an L1-like molecule from human brain and found that it also supports neurite growth in vitro. We have also cloned and sequenced the entire coding region of human L1CAM and found that it shows a very high degree of homology to mouse L1cam, with 92% identity at the amino acid level. This similarity suggests that L1CAM is an important molecule in normal human nervous system development and nerve regeneration. Overall, there is substantially less homology to chick Ng-CAM; they are 40% identical at the amino acid level but many regions are highly conserved. Comparison of the sequences from human, mouse, chick, and Drosophila indicates that the L1 immunoglobulin domain 2 and fibronectin type III domain 2 are strongly conserved and thus are likely functionally important.

Amino Acid Sequence↗

PubMed1990

The role of cell adhesion molecules in neurite outgrowth on Müller cells.

The roles of neural cell adhesion molecule (NCAM), L1, N-cadherin, and integrin in neurite outgrowth on various substrates were studied. Antibodies against these cell surface molecules were added to explants of chick retina and the neurites from retinal ganglion cells were examined for effects of the antibodies on neurite length and fasciculation. On laminin, an anti-integrin antibody completely inhibited neurite outgrowth. The same antibody did not inhibit neurite outgrowth on polylysine or Müller cells. Antibodies to NCAM, L1, and N-cadherin did not significantly inhibit neurite outgrowth on laminin but produced significant inhibition on Müller cells. The inhibition of neurite outgrowth on glia by anti-L1 antibodies supports the hypothesis that L1 is capable of acting in a heterophilic binding mechanism. On laminin, both anti-N-cadherin and anti-L1 caused defasciculation of neurites from retinal ganglion cells, while anti-NCAM did not. None of these antibodies produced defasciculation on Müller cells. The results indicate that these three cell adhesion molecules may be very important in interactions with glia as axons grow from the retina to the tectum and may be less important in axon-axon interactions along this pathway. No evidence was found supporting the role of integrins in axon growth on Müller cells.

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PubMed1990

Nerve growth cone migration onto Schwann cells involves the calcium-dependent adhesion molecule, N-cadherin.

The role of calcium-dependent adhesion molecules in the migration of nerve growth cones onto the top of Schwann cells was probed by examination of sensory growth cone-Schwann cell interactions in medium containing either 1.0 mM Ca2+ or 0.1 mM Ca2+. In the presence of 1.0 mM Ca2+ growth cones rapidly migrated onto Schwann cells, spread, and remained for extended periods. However, in 0.1 mM Ca2+ growth cones still made frequent contacts with Schwann cells, but migration onto the upper cell surface was much reduced. This contrast in growth cone-Schwann cell interactions could be switched rapidly by changing the Ca2+ concentration of the culture medium. Growth cones of retinal neurons showed similar calcium-dependence in their migration onto Schwann cells. Antibodies to the calcium-dependent adhesion molecule, N-cadherin, also blocked growth cone migration onto Schwann cells, but antibodies to another neuronal adhesion molecule, L1, had no effect on growth cone-Schwann cell interactions. Immunocytochemical staining for N-cadherin and L1 indicated that growth cones and Schwann cells have N-cadherin on their surfaces, while L1 is present only on axons and growth cones. These results provide two kinds of evidence that N-cadherin is important in the initial interactions of growth cones and Schwann cells.

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PubMed1990

Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro.

In vivo studies of the roof plate of the spinal cord and midline optic tectum in rodent and the developing subplate in the telencephalon of the chick showed that two glycosaminoglycans, keratin sulfate and chondroitin sulfate, possibly in the proteoglycan form (KS-PG, CS-PG, or KS/CS-PG), were present at times when axons approach closely but do not invade these territories. To address the question of whether KS/CS-PG actively inhibits growth cone elongation and to determine which component(s) of the proteoglycan may be critical to this phenomenon, we used a technique employing nitrocellulose-coated petri dishes onto which stripes of various purified macromolecules were attached. Isolated E9 chick dorsal root ganglia were grown on lanes of KS/CS-PG in alteration with lanes of the growth-promoting molecule laminin (LN). Neurite outgrowth was abundant along stripes of LN. In contrast, upon encountering a stripe containing KS/CS-PG, neurites either stopped abruptly or turned and traveled along the KS/CS-PG stripe border. The effect was dependent upon the concentration of the proteoglycan with intermediate concentrations producing intermittent patterns of crossing. We mixed LN with the KS/CS-PG, where the LN was in concentrations which alone support outgrowth, and observed that the KS/CS-PG was still inhibitory when such a growth-promoting molecule was present. A 10-fold higher concentration of LN was able to overcome the inhibitory effect of the KS/CS-PG. These results suggest that the interaction of inhibitory and growth-promoting molecules can interact to produce a wide spectrum of neurite patterns ranging from complete inhibition to totally unimpeded outgrowth. Selective enzymatic removal of the KS or CS from the KS/CS-PG permitted various degrees of neurite outgrowth to occur across the previously inhibitory lanes, and digestion of both glycoaminoglycan moieties, leaving only the protein core of the molecule, resulted in a complete lack of inhibition. These assays demonstrated that KS/CS-PG is inhibitory to embryonic dorsal root ganglia neurites in vitro and that complete inhibition requires contributions from both KS and CS moieties.

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PubMed1989

L1-mediated axon outgrowth occurs via a homophilic binding mechanism.

The molecular mechanism by which the L1 cell adhesion molecule mediates neurite outgrowth has been examined. Purified L1 from mouse and L1 from chick brain were attached to nitrocellulose dishes. Both chick and mouse neurons were able to adhere to purified mouse L1 and chick L1. Both molecules promoted neurite extension from chick and mouse neurons. Addition of Fabs specific for chick L1 to the cultures inhibited chick neurite outgrowth on both mouse L1 and chick L1. These findings suggest that L1-like molecules support neurite outgrowth via a "homophilic" binding mechanism.

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PubMed1988

A monoclonal antibody that binds to the surface of photoreceptors.

A monoclonal antibody (MAB), 7H11, that binds to the surface of photoreceptors has been produced. Using immunoaffinity procedures, proteins with molecular weights of 53, 48 and 42 kDa were found to bind a MAB 7H11 column, although the 48- and 42-kDa proteins may be proteolytic breakdown products of the 53 kDa protein. Electron microscopic-immunohistochemical studies show that the antigen is concentrated on the external surface of inner and outer segments of photoreceptors. It is likely that the 7H11 antigen is a component of the interphotoreceptor matrix and, therefore, may contribute to some aspect of photoreceptor-pigment epithelial interactions. However, the expression of the 7H11 antigen in cell culture suggests that it is expressed independently of direct cell-cell interactions with either pigment epithelial cells, Müller cells or other photoreceptors.

Animals↗

PubMed1988

Changes in the vascular extracellular matrix during embryonic vasculogenesis and angiogenesis.

We have previously characterized monoclonal antibodies against chick brain cells. One of them (14-2B2) brightly stained all capillaries in frozen sections of chick brain. Here we show that this antibody is directed against chick fibronectin. Using this antibody and polyclonal antibodies against laminin, we have studied the development of the vascular extracellular matrix. Vasculogenesis, the development of capillaries from in situ differentiating endothelial cells, was studied in yolk sac blood islands and intraembryonic dorsal aorta. Blood islands produced high levels of fibronectin but not laminin. Early intraembryonic capillaries all expressed fibronectin but little if any laminin. The dorsal aorta of a 6-day-old chick embryo has several layers of fibronectin-producing cells, but is devoid of laminin. Laminin expression commenced at Day 8 and by Day 10 an adult-like distribution was found in the aortic vascular wall. Angiogenesis, the formation of capillaries from preexisting vessels, was studied during brain development. Capillary sprouts invading the neuroectoderm at Embryonic Day 4 migrated in a fibronectin-rich matrix devoid of laminin. Ultrastructural immunolocalization demonstrated the presence of fibronectin exclusively on the abluminal site of the endothelial cells. Beginning on Day 6, laminin codistributed with fibronectin in brain capillaries. We conclude that immature capillaries migrate and proliferate in a fibronectin-rich extracellular matrix, which is subsequently remodeled acquiring basement membrane-like characteristics. We suggest that laminin expression is an early indication of vascular maturation.

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PubMed1987

Neurons and glia in purified retinal cultures identified by monoclonal antibodies to intermediate filaments.

Two monoclonal antibodies known to bind intermediate filaments were used to identify neurons and glia from embryonic chick neural retina. Neurofilament specific antibody RT-97-F1, bound neuroepithelial cells, migrating neurons, as well as the photoreceptor layer, plexiform layers and optic fiber layer. The other, 3A7, bound intermediate filaments of Müller cells. In purified neuronal cultures, RT-97-F1 bound 75, 83 and 98% of the cells present at 24, 48 and 72 h respectively, while 3A7 bound 26, 15 and 0.3% for the same times in vitro. In purified glial cultures, RT-97-F1 showed a weak perinuclear binding, while 3A7 strongly bound intermediate filaments of nearly 100% of the cells. These antibodies confirmed and quantitated the high purity of our cultures.

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PubMed1987

An L1-like molecule, the 8D9 antigen, is a potent substrate for neurite extension.

The 8D9 antigen, a cell surface protein isolated from chicken brain that is related to the L1 class of cell adhesion molecules, is shown to contain an activity that promotes the attachment of neurons and the outgrowth of neurites from chicken tecta and mouse cerebellum. When purified 8D9 antigen is attached to a nitrocellulose-coated substrate, neurons rapidly attach and extend unfasciculated neurites. Little or no attachment of astroglia, oligodendroglia, and fibroblast-like cells to the 8D9 antigen is observed. We propose that a function of the 8D9 antigen is that of a neurite extension-promoting substrate in axon fascicles and in regeneration of peripheral nerves.

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PubMed1986

Localization of a filamin-like protein in glia of the chick central nervous system.

Monoclonal antibody 5E10 binds to Muller cells in the chick retina and radial glia in the optic tectum. Biochemical and immunohistochemical experiments indicate that the 5E10 antigen is related to, but may not be identical to, filamin, a high-molecular-weight, actin-binding protein. Developmental studies show that the 5E10 antigen is present in all neuroepithelial cells very early in development, but disappears by about Embryonic Day 10. These results suggest that neurons developmentally regulate not only the type of intermediate filament proteins they express, switching from vimentin to neurofilaments, but also the type of actin-binding proteins.

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PubMed1986

A monoclonal antibody that binds to cones.

A monoclonal antibody that binds to cones has been produced. This antibody, 50-1B11, binds to the outer segments of cones in rhesus monkeys. Immunohistochemical experiments indicate that 50-1B11 binds to a subset of photoreceptors, probably cones, in all vertebrate species tested thus far, including man. In vitro experiments on chicken retina indicate that the antigen is intracellular and associated with the plasma membrane, while electronmicroscopic-immunohistochemical studies demonstrate that the antigen is contained in the lamellae of the outer segments of rhesus cones.

Animals↗

PubMed1986

The appearance of an L1-like molecule in the chick primary visual pathway.

A monoclonal antibody, 8D9, has been obtained that binds to axons in the chick nervous system. Biochemical and immunological experiments indicate that the 8D9 antigen is related to the mouse L1 cell-adhesion molecule. The results of immunohistochemical experiments using monoclonal antibody 8D9 to study the development of the chick visual system are consistent with the 8D9 antigen functioning in axon fasciculation.

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