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L F Reichardt

Publications and source records attributed to L F Reichardt.

At least 73 records · Page 4Linked to original sources
PubMed1995

The integrin receptor alpha 8 beta 1 mediates interactions of embryonic chick motor and sensory neurons with tenascin-C.

This paper identifies a neuronal receptor for tenascin-C (tenascin/cytotactin), an extracellular matrix protein that has previously been detected in developing sensory and motor neuron pathways and has been shown to regulate cell migration in the developing CNS. Antibodies specific for each subunit of the integrin alpha 8 beta 1 are used to demonstrate that alpha 8 beta 1 mediates neurite outgrowth of embryonic sensory and motor neurons on this extracellular matrix protein. In addition, expression of alpha 8 in K562 cells results in surface expression of alpha 8 beta 1 heterodimers that are shown to promote attachment of this cell line to tenascin. The major domain in tenascin that mediates neurite outgrowth is shown to be localized to fibronectin type III repeats 6-8.

Animals↗
PubMed1995

Characterization of beta pat-3 heterodimers, a family of essential integrin receptors in C. elegans.

Members of the integrin family of cell surface receptors have been shown to mediate a diverse range of cellular functions that require cell-cell or cell-extracellular matrix interactions. We have initiated the characterization of integrin receptors from the nematode Caenorhabditis elegans, an organism in which genetics can be used to study integrin function with single cell resolution. Here we report the cloning of an integrin beta subunit from C. elegans which is shown to rescue the embryonic lethal mutation pat-3(rh54) and is thus named beta pat-3. Analysis of the deduced amino acid sequence revealed that beta pat-3 is more similar to Drosophila integrin beta PS and to vertebrate integrin beta 1 than to other integrin beta subunits. Regions of highest homology are in the RGD-binding region and in the cytoplasmic domain. In addition, the 56 cysteines present in the majority of integrin beta subunits are conserved. A major transcript of approximately 3 kilo-base pairs was detected by RNA blot analysis. Immunoblot analysis using a polyclonal antiserum against the cytoplasmic domain showed that beta pat-3 migrates in SDS-PAGE with apparent M(r) of 109 k and 120 k under nonreducing and reducing conditions, respectively. At least nine protein bands with relative molecular weights in the range observed for known integrin alpha subunits coprecipitate with beta pat-3, and at least three of these bands migrate in SDS-PAGE with increased mobility when reduced. This behavior has been observed for a majority of integrin alpha subunits. Immunoprecipitations of beta pat-3 from developmentally staged populations of C. elegans showed that the expression of several of these bands changes during development. The monoclonal antibody MH25, which has been postulated to recognize the transmembrane component of the muscle dense body structure a (Francis, G. R., and R. H. Waterston. 1985. Muscle organization in Caenorhabditis elegans: localization of proteins implicated in thin filament attachment and I-band organization. J. Cell Biol. 101:1532-1549), was shown to recognize beta pat-3. Finally, immunocytochemical analysis revealed that beta pat-3 is expressed in the embryo and in many cell types postembryonically, including muscle, somatic gonad, and coelomocytes, suggesting multiple roles for integrin heterodimers containing this beta subunit in the developing animal.

Amino Acid Sequence↗
PubMed1995

The cadherin-binding specificities of B-cadherin and LCAM.

The cadherin family of calcium-dependent cell adhesion molecules plays an important part in the organization of cell adhesion and tissue segregation during development. The expression pattern and the binding specificity of each cadherin are of principal importance for its role in morphogenesis. B-Cadherin and LCAM, two chicken cadherins, have similar, but not identical, spatial and temporal patterns of expression. To examine the possibility that they might bind to one another in a heterophilic manner, we generated, by cDNA transfection, L-cell lines that express LCAM or B-cadherin. We then examined the abilities of these cells to coaggregate with each other and with other cadherin-expressing cells in short-term aggregation assays. The B-cadherin- and the LCAM-expressing cell lines segregate from P-, N-, or R-cadherin-expressing cells. B-cadherin- and LCAM-expressing cell lines, however, appear to be completely miscible, forming large mixed aggregates. Chick B-cadherin and murine E-cadherin also form mixed aggregates, indistinguishable from homophilic aggregates. Murine E-cadherin and chick LCAM coaggregate less completely, suggesting that the heterophilic interactions of these two cell lines are weak relative to homophilic interactions. These data suggest that heterophilic interactions between B-cadherin and LCAM are important during avian morphogenesis and help identify the amino acids in the binding domain that determine cadherin specificity.

Amino Acid Sequence↗
PubMed1995

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.

Amino Acid Sequence↗
PubMed1995

Expression and in vitro function of beta 1-integrin laminin receptors in the developing avian ciliary ganglion.

In chick development, ciliary ganglion (CG) neurons go through a period of axon extension from approximately embryonic day (E)4 to E8, followed by a period of synaptogenesis and neuronal cell death. By examining the immunohistochemical localization of laminin, in conjunction with Dil labeling of the ciliary nerve projection, we have determined that the pathway taken by these neurons is rich in laminin expression. Therefore, laminins are good candidate molecules for mediating outgrowth of these neurons in vivo. In vitro, the ability of CG neurons to extend neurites on laminin-1 (EHS laminin, alpha 1 beta 1 gamma 1) is maximal up to E8, then declines dramatically. CG neuron outgrowth on laminin-1 requires the activity of beta 1-class integrins. We have used subunit-specific antibodies to determine which of the five beta 1-containing heterodimers known to be laminin receptors (alpha 1 beta 1, alpha 2 beta 1, alpha 6 beta 1, alpha 7 beta 1) are expressed, and which mediate neurite outgrowth. While we could not detect expression of alpha 2 or alpha 7, we have found that alpha 1, alpha 3 beta 1, and alpha 6 beta 1 are expressed on the surface of ciliary ganglion neuron cell bodies and axons, both in vitro and in vivo. Furthermore, antibodies against alpha 3 and alpha 6, but not alpha 1, interfered with CG neurite outgrowth on laminin-1 in vitro. Taken together, these data suggest that interactions of cell surface alpha 3 beta 1 and alpha 6 beta 1 integrins with laminin-1 are likely to mediate growth of CG neurons during pathfinding in vivo.

Animals↗
PubMed1995

TrkA expression in the CNS: evidence for the existence of several novel NGF-responsive CNS neurons.

NGF acts as a neurotrophic factor by binding and activating its receptor on certain neuronal populations in the CNS and PNS. TrkA is a receptor for NGF. Recent findings in vitro indicate that this NGF-activated receptor tyrosine kinase transduces the NGF signal. To further define NGF actions in the CNS, we examined trkA expression in the adult rat brain. We found that trkA mRNA and immunoreactivity (IR) coincided in specific, defined neuronal populations in the forebrain and brainstem. In addition to cholinergic neurons in the basal forebrain and neostriatum, trkA expression was found in noncholinergic neurons in (1) the paraventricular anterior and reuniens thalamic nuclei, (2) the rostral and intermediate subnuclei of the interpeduncular nucleus (IPN), (3) scattered neurons in the ventrolateral and paramedian medulla, (4) the prepositus hypoglossal nucleus, and (5) the area postrema. NGF responsiveness was demonstrated for each of these populations. In contrast to trkA, p75NGFR was found only in a minority of NGF-responsive populations. Our data provide further evidence that expression of trkA marks NGF-responsive CNS neurons and suggests novel roles for NGF in the brain.

Animals↗
PubMed1994

TrkA-immunoreactive profiles in the central nervous system: colocalization with neurons containing p75 nerve growth factor receptor, choline acetyltransferase, and serotonin.

The present investigation used an antibody directed against the extracellular domain of the signal transducing nerve growth factor receptor, trkA, to reveal immunoreactive perikarya or fibers within the olfactory bulb and tubercle, cingulate cortex, nucleus accumbens, striatum, endopiriform nucleus, septal/diagonal band complex, nucleus basalis, hippocampal complex, thalamic paraventricular and reuniens nuclei, periventricular hypothalamus, interpeduncular nucleus, mesencephalic nucleus of the fifth nerve, dorsal nucleus of the lateral lemniscus, prepositus hypoglossal nucleus, ventral cochlear nucleus, ventral lateral tegmentum, medial vestibular nucleus, spinal trigeminal nucleus oralis, nucleus of the solitary tract, raphe nuclei, and spinal cord. Colocalization experiments revealed that virtually all striatal trkA-immunoreactive neurons (> 99%) coexpressed choline acetyltransferase (ChAT) but not p75 nerve growth factor receptor (NGFR). Within the septal/diagonal band complex virtually all trkA neurons (> 95%) coexpressed both ChAT and p75 NGFR. More caudally, dual stained sections revealed numerous trkA/ChAT (> 80%) and trkA/p75 NGFR (> 95%) immunoreactive neurons within the nucleus basalis. In the brainstem, raphe serotonergic neurons (45%) coexpressed trkA. Sections stained with a pan-trk antibody that recognizes primarily trkA, as well as trkB and trkC, labeled neurons within all of these regions as well as within the hypothalamic arcuate, supramammilary, and supraoptic nuclei, hippocampus, inferior and superior colliculus, substantia nigra, ventral tegmental area of T'sai, and cerebellular Purkinje cells. Virtually all of these other regions with the exception of the cerebellum also expressed pan-trk immunoreactivity in the monkey. The widespread expression of trkA throughout the central neural axis suggests that this receptor may play a role in signal transduction mechanisms linked to NGF-related substances in cholinergic basal forebrain and noncholinergic systems. These findings suggest that pharmacological use of ligands for trkA could have beneficial effects on the multiple neuronal systems that are affected in such disorders as Alzheimer's disease.

Animals↗
PubMed1994

An alternatively spliced form of the nerve growth factor receptor TrkA confers an enhanced response to neurotrophin 3.

TrkA, a member of the receptor tyrosine kinase family, binds nerve growth factor (NGF) and subsequently activates intracellular signaling pathways. Previous studies have found variable and weak interaction of the TrkA receptor with neurotrophin 3 (NT-3), another member of the NGF family. TrkA is expressed in two splice forms, differing in the presence of an 18-bp exon in the extracellular domain. The biological responses of each isoform of the TrkA receptor were tested after transfection into the cell line PC12nnr5, a variant of PC12 cells lacking functional TrkA protein. NGF was found to activate each form of the receptor comparably. However, the TrkA isoform containing the variable exon showed significantly higher activation by NT-3, which was detected by stimulation of TrkA autophosphorylation, induction of ZIF268 transcription, and cellular differentiation. Function-perturbing antibodies to the p75 low-affinity NGF receptor potentiated the NT-3 responses of both forms of TrkA in the transfected PC12nnr5 cell lines, suggesting that the low-affinity NGF receptor suppresses the ability of TrkA to respond to NT-3.

Alternative Splicing↗
PubMed1994

Severe sensory and sympathetic deficits in mice lacking neurotrophin-3.

During development, neurotrophins help shape the nervous system by regulating neuronal survival and differentiation. Neurotrophin-3 (refs 1-5) is the most abundant neurotrophin during early development. Neurons responsive to neurotrophin-3 in vitro include primary sensory, sympathetic, motor, enteric, locus coeruleus, hippocampal and cerebellar neurons (ref. 9 for example). Here we report that mice lacking neurotrophin-3 have severe deficits in sensory and sympathetic populations. These mice lack muscle spindles and show abnormal limb positions. In contrast, motor neurons, the enteric nervous system, and the major anatomical regions of the central nervous system seem to develop normally. Comparisons with mutants deficient in other neurotrophins or their receptors indicate that some neurons require more than one neurotrophin during embryogenesis and suggest that neurotrophin-3 functions by binding receptors in addition to its primary receptor trkC (ref. 16). In particular, neurotrophin-3 is essential for survival of sympathetic and sensory neurons that later become dependent on nerve growth factor or brain-derived neurotrophic factor.

Animals↗
PubMed1994

Nerve growth factor and its low-affinity receptor promote Schwann cell migration.

Migrating Schwann cells in developing or regenerating peripheral nerves are known to express dramatically increased levels of nerve growth factor (NGF) and the low-affinity NGF receptor (LNGFR). Schwann cells do not express detectable pp140trk, the NGF-activated receptor tyrosine kinase which is essential for neuronal responses to NGF. The temporal correlation observed in Schwann cells between migration and the enhanced expression of NGF and LNGFR suggests that NGF and LNGFR may promote Schwann cell migration. To test this possibility, we examined the effects of NGF on Schwann cell migration on cryostat sections of biologically relevant NGF-poor and NGF-rich substrates--normal or denervated peripheral (sciatic) nerve, untreated or pretreated with NGF. Results show that Schwann cells migrate more rapidly on denervated than on normal sciatic nerve. Antibodies to NGF or to LNGFR strongly, but incompletely, inhibit enhanced migration on denervated nerves. Pretreatment of denervated nerve sections with NGF increases further the rate of Schwann cell migration. The same antibodies to NGF or to LNGFR abolish this response. These results suggest that one function of the elevated levels of NGF known to be present in embryonic and regenerating peripheral nerves is to promote the migration of Schwann cells. In contrast to neurons, where pp140trk appears to be the functionally critical NGF receptor, NGF responses in Schwann cells depend on LNGFR.

Animals↗
PubMed1994

Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development.

Brain-derived neurotrophic factor (BDNF), a neurotrophin, enhances the survival and differentiation of several classes of neurons in vitro. To determine its essential functions, we have mutated the BDNF gene. Most homozygote mutants die within 2 days after birth, but a fraction live for 2-4 weeks. These develop symptoms of nervous system dysfunction, including ataxia. The BDNF mutant homozygotes have substantially reduced numbers of cranial and spinal sensory neurons. Although their central nervous systems show no gross structural abnormalities, expression of neuropeptide Y and calcium-binding proteins is altered in many neurons, suggesting they do not function normally. In contrast with mice lacking the BDNF receptor TrkB, motor neurons appear normal in the BDNF mutant.

Animals↗
PubMed1994

The expression of B-cadherin during embryonic chick development.

Cadherins compose a family of calcium-dependent cell adhesion molecules that are involved in the segregation of differentiating tissues during development. Each cadherin has a unique spatial and temporal pattern of expression. As has been observed for other cadherins, B-cadherin, when expressed in mouse L929 fibroblasts, confers upon them a calcium-dependent cell aggregation activity. A monoclonal antibody to B-cadherin was isolated and used to determine the pattern of expression of B-cadherin in the developing chick embryo. Antibody staining and in situ hybridization reveal that B-cadherin protein and mRNA are found in diverse epithelia derived from each of the three primary germ layers, where their expression is strikingly regulated during differentiation. In some instances, the regional distribution of B-cadherin within a tissue reflects the distinct functional regions of the tissue. Patterns of staining are similar to, but distinct from, those seen with anti-LCAM antibodies. Although there are many examples where B-cadherin and LCAM are coexpressed, there are also distinctive regional differences.

Animals↗
PubMed1994

NOS induction by NGF in basal forebrain cholinergic neurones: evidence for regulation of brain NOS by a neurotrophin.

Nerve growth factor (NGF) acts through trkA receptors to serve as a trophic factor for cholinergic neurones in the medial septal nucleus (MSN) and vertical limb of the diagonal band (VDB). Herein, we show that brain nitric oxide synthase (NOS), which synthesizes the neuromodulator nitric oxide, is selectively expressed in a large fraction of trkA-containing neurones in the MSN and VDB. Axotomy of these neurones gave evidence that NOS expressing cholinergic neurones innervate the hippocampus. NGF infusion induced a robust, specific increase in NOS expression in basal forebrain cholinergic neurones. These results indicate that brain NOS can be regulated by a neurotrophic factor and suggest that NGF influences forebrain function by regulating production of nitric oxide as well as acetylcholine.

Animals↗
PubMed1994

Embryonic neurons of the developing optic chiasm express L1 and CD44, cell surface molecules with opposing effects on retinal axon growth.

The first retinal ganglion cell axons arriving at the embryonic mouse ventral diencephalon encounter an inverted V-shaped neuronal array defining the midline and posterior boundaries of the future optic chiasm. These neurons express L1, an immunoglobulin superfamily molecule known to promote retinal axon outgrowth, and CD44, a cell surface molecule that we find inhibits embryonic retinal axon growth in vitro. Incoming retinal axons do not penetrate this L1/CD44 neuron array, but turn to establish the characteristic X-shaped optic chiasm along the anterior border of this array. These results suggest that L1/CD44 neurons may serve as an anatomical template for retinal axon pathways at the embryonic mouse ventral diencephalon.

Animals↗
PubMed1994

Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth.

We have studied the role of vinculin in regulating integrin-dependent neurite outgrowth in PC12 cells, a neuronal cell line. Vinculin is a cytoskeletal protein believed to mediate interactions between integrins and the actin cytoskeleton. In differentiated PC12 cells, the cell body, the neurite, and the growth cone contain vinculin. Within the growth cone, both the proximal region of "consolidation" and the more distal region consisting of lamellipodia and filopodia contain vinculin. To study the role of vinculin in neurite outgrowth, we generated vinculin-deficient isolates of PC12 cell lines by transfection with vectors expressing antisense vinculin RNA. In some of these cell lines, vinculin levels were reduced to 18-23% of normal levels. In the vinculin-deficient cell lines, neurite outgrowth on laminin was significantly reduced. In time-lapse analysis, growth cones advanced much more slowly than normal. Further analysis indicated that this deficit could be explained in large part by changes in the behaviors of filopodia and lamellipodia. Filopodia were formed in normal numbers, extended at normal rates, and extended to approximately normal lengths, but were much less stable in the vinculin deficient compared to control PC12 cells. Similarly, lamellipodia formed and grew nearly normally, but were dramatically less stable in the vinculin-deficient cells. This can account for the reduction in rate of growth cone advance. These results indicate that interactions between integrins and the actin-based cytoskeleton are necessary for stability of both filopodia and lamellipodia.

Actins↗
PubMed1994

TrkA cross-linking mimics neuronal responses to nerve growth factor.

TrkA, a tyrosine kinase receptor, is an essential component of the nerve growth factor (NGF) response pathway. The binding of NGF to the receptor induces receptor autophosphorylation and activation of intracellular signaling pathways, resulting in diverse biological effects. We prepared polyclonal antibodies against the entire extracellular domain of rat trkA produced using a baculovirus expression system. These antibodies specifically recognize rat trkA on antigen blots and in immunoprecipitations. Both IgG and Fab fragments block binding of NGF to trkA expressed by the PC12 cell line. In NGF binding studies using anti-trkA and anti-low-affinity NGF receptor (LNGFR) immunoglobulin (Ig) G, essentially all binding of NGF can be inhibited. The results imply that > or = 97% of the NGF binding sites on PC12 cells are accounted for by trkA and the LNGFR. The binding data also argue that all low-affinity NGF binding sites on PC12 cells reflect interactions with the LNGFR, while all high-affinity sites are trkA dependent. A fraction of the high-affinity (or slow) binding sites seem to require both trkA and the LNGFR. Although the monovalent anti-trkA Fab fragments inhibited the biological effects of NGF, such as induction of tyrosine phosphorylation, and survival and neurite outgrowth of sympathetic neurons, the IgG preparation was not effective as an inhibitor. Instead, the IgG fraction by itself was almost as effective as NGF at stimulating receptor activation, cell survival, and neurite outgrowth. Thus, it appears oligomerization of trkA by antibody-induced cross-linking is sufficient to produce the known cellular effects of NGF.

Animals↗
PubMed1994

Specific roles of the alpha V beta 1, alpha V beta 3 and alpha V beta 5 integrins in avian neural crest cell adhesion and migration on vitronectin.

To identify potentially important extracellular matrix adhesive molecules in neural crest cell migration, the possible role of vitronectin and its corresponding integrin receptors was examined in the adhesion and migration of avian neural crest cells in vitro. Adhesion and migration on vitronectin were comparable to those found on fibronectin and could be almost entirely abolished by antibodies against vitronectin and by RGD peptides. Immunoprecipitation and immunocytochemistry analyses revealed that neural crest cells expressed primarily the alpha V beta 1, alpha V beta 3 and alpha V beta 5 integrins as possible vitronectin receptors. Inhibition assays of cellular adhesion and migration with function-perturbing antibodies demonstrated that adhesion of neural crest cells to vitronectin was mediated essentially by one or more of the different alpha V integrins, with a possible preeminence of alpha V beta 1, whereas cell migration involved mostly the alpha V beta 3 and alpha V beta 5 integrins. Immunofluorescence labeling of cultured motile neural crest cells revealed that the alpha V integrins are differentially distributed on the cell surface. The beta 1 and alpha V subunits were both diffuse on the surface of cells and in focal adhesion sites in association with vinculin, talin and alpha-actinin, whereas the alpha V beta 3 and alpha V beta 5 integrins were essentially diffuse on the cell surface. Finally, vitronectin could be detected by immunoblotting and immunohistochemistry in the early embryo during the ontogeny of the neural crest. It was in particular closely associated with the surface of migrating neural crest cells. In conclusion, our study indicates that neural crest cells can adhere to and migrate on vitronectin in vitro by an RGD-dependent mechanism involving at least the alpha V beta 1, alpha V beta 3 and alpha V beta 5 integrins and that these integrins may have specific roles in the control of cell adhesion and migration.

Animals↗