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M Chiquet

Publications and source records attributed to M Chiquet.

At least 37 records · Page 2Linked to original sources
PubMed1995

Distinct heparin-binding and neurite-promoting properties of laminin isoforms isolated from chick heart.

Laminin isolated from chick heart is composed of several heterotrimeric variants of 800 and 700 kDa. Here, we used monoclonal antibodies against chick laminin to purify different laminin isoforms from this mixture. Antibody 8D3 specifically removed laminin containing alpha 2 chain from chick heart laminin preparations, leaving behind 700 kDa variants. Using antibody C4 against the laminin beta 2 chain, alpha 2 chain containing variants were further separated into alpha 2 beta 1 gamma 1 and alpha 2 beta 2 gamma 1 laminin, respectively. Laminins containing alpha 2 chain and recognized by antibody 8D3 are cross-shaped molecules. Their expression during embryogenesis is tightly regulated. In 5-day embryos staining with monoclonal antibody 8D3 is restricted to the dermamyotome. Older embryos (8 days) express alpha 2 chain containing variants at myotendinous junction primordia of skeletal muscle, and only late in development these variants are generally expressed in skeletal and heart muscle basement membranes. The 700 kDa laminin variants contain beta 1, beta 2, and gamma 1 subunits affiliated with an immunologically distinct, shorter alpha x chain and appear to be T-shaped in the electron microscope. Whereas laminins with an alpha 2 subunit bind to heparin, variants with the novel alpha x chain do not. Experiments using cultured sympathetic neurons showed that laminins with alpha x chain are less potent than alpha 2 chain containing variants in promoting neurite outgrowth. In contrast, sympathetic neurons cannot discriminate between alpha 2 beta 1 gamma 1 and alpha 2 beta 2 gamma 1 laminin substrates, respectively, and show identical high rates of neurite formation.

Animals↗
PubMed1994

A new nomenclature for the laminins.

The authors have adopted a new nomenclature for the laminins. They are numbered with arabic numerals in the order discovered. The previous A, B1 and B2 chains, and their isoforms, are alpha, beta and gamma, respectively, followed by an arabic numeral to identify the isoform. For example, the first laminin identified from the Engelbreth-Holm-Swarm tumor is laminin-1 with the chain composition alpha 1 beta 1 gamma 1. The genes for these chains are LAMA1, LAMB1 and LAMC1, respectively.

Animals↗
PubMed1994

Tenascin-C expression by fibroblasts is elevated in stressed collagen gels.

Chick embryo fibroblasts cultured on a collagen matrix exert tractional forces leading to the contraction of unrestrained, floating collagen gels and to the development of tension in attached, restrained gels. On a restrained, attached collagen gel the fibroblasts synthesize large quantities of tenascin-C, whereas in a floating, contracting gel tenascin-C synthesis is decreased. This regulation of tenascin-C synthesis can be observed by the secretion of metabolically labeled tenascin-C into the conditioned medium, as well as by the deposition of tenascin-C into the collagen matrix as judged by immunofluorescence. Regulation appears to occur at the transcriptional level, because when cells on attached or floating collagen gels are transfected with promoter constructs of the tenascin-C gene, luciferase expression driven by the tenascin-C promoter parallels the effects measured for endogenous tenascin-C synthesis, whereas luciferase expression under the control of the SV40 promoter does not depend on the state of the collagen gel. The promoter region responsible for tenascin-C induction on attached collagen gels is distinct from the region important for the induction of tenascin-C by serum, and may define a novel kind of response element. By joining this tenascin-C sequence to the SV40 promoter of a reporter plasmid, its activity can be transferred to the heterologous promoter. We propose that the tenascin-C promoter is directly or indirectly activated in fibroblasts generating and experiencing mechanical stress within a restrained collagen matrix. This may be an important aspect of the regulation of tenascin-C expression during embryogenesis as well as during wound healing and other regenerative and morphogenetic processes.

Animals↗
PubMed1994

Tissue-specific expression of the fibril-associated collagens XII and XIV.

Interstitial collagen fibrils form the supporting scaffold of all connective tissues. The synthesis of this framework is subject to a precise spatial and temporal regulation in order to meet the mechanical needs of every tissue type. A subgroup of non-fibrillar collagens termed FACIT seems to play a role in this regulation by providing specific molecular bridges between fibrils and other matrix components. Collagens XII and XIV represent such FACIT molecules and occur preferentially in tissues containing banded type I collagen fibrils. We have used the techniques of indirect immunofluorescence and in situ hybridization to investigate the expression patterns of the two molecules during chicken embryonic development. We detected specific differences in these patterns, which may be related to the respective functions of the two proteins within the connective tissues. Collagen XIV was expressed at very few sites in the 6-day-old embryo, but occurred in virtually every collagen I-containing tissue (skeletal muscle, cardiac muscle, gizzard, tendon, periosteum, nerve) by the end of embryonic development. In contrast, collagen XII was fairly abundant in the 6-day-old embryo but was, at later stages, restricted to only a few dense connective tissue structures (bone, tendon, gizzard). Thus, our results suggest that collagen XII and collagen XIV serve different functions during embryonic development although their structures are highly similar.

Animals↗
PubMed1994

Tenascin-C in peripheral nerve morphogenesis.

The extracellular matrix (ECM) molecule tenascin/cytotactin (TN-C) is expressed at a high level by satellite (glial precursor) cells in developing peripheral nerves of the chick embryo; synthesis of its mRNA peaks at the time period when axonal growth is maximal. When offered as a substrate in vitro, TN-C mediates neurite outgrowth by both motor and sensory neurons. The ability to grow neurites on TN-C is developmentally regulated: sensory neurons from 4-day chick embryos (the stage at which peripheral nerves start to develop) grow immediately and rapidly, whereas neurons from older embryos respond with a long delay. A TN-C domain responsible for this activity is located within the C-terminal (distal) portion of TN-C subunits. Integrin receptors seem to be involved on peripheral neurites because their growth on TN-C is completely blocked by antibodies to beta 1 integrins. In striking contrast to neuronal processes, nerve satellite cells can attach to a TN-C substrate but are completely inhibited in their migratory activity. Artificial substrate borders between tenascin and fibronectin or laminin act as selective barriers that allow neurites to pass while holding up satellite cells. The repulsive action of TN-C on satellite cells is similar to that observed for other cell types and is likely to be mediated by additional TN-C domains. In view of these data, it is surprising that mice seem to develop normally without a functional TN-C gene. TN-C is likely to be redundant, that is, its dual action on cell adhesion is shared by other molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Dual function of tenascin: simultaneous promotion of neurite growth and inhibition of glial migration.

The extracellular matrix molecule tenascin is expressed within the developing peripheral nervous system, first by migrating neural crest cells and later by satellite (Schwann precursor) cells at the growing tips of peripheral nerves. Here we found that the neurite promoting activity of tenascin for sensory neurons is developmentally regulated: very young sensory ganglia of stage 23 (4 days old) embryos grew neurites on tenascin as fast as on laminin and fibronectin. The growth response of older (day 7 and 9) ganglia on laminin and fibronectin was similar to that of 4-day-old ganglia, while on tenascin neurite growth occurred only after a lag phase and at a slower rate. Neurite growth on tenascin was inhibited by antibodies to beta 1 integrin and by heparin. While tenascin promotes neurite outgrowth of peripheral neurons, we found that it does not allow satellite cell migration when it is present on the substratum, and it inhibits migration of satellite cells on fibronectin when added in soluble form. In contrast, soluble tenascin did not significantly alter the rate of neurite growth on tenascin, fibronectin or laminin substrata, although neurites were straighter and less attached. When isolated satellite cells were added to neurites grown on tenascin, they preferentially adhered to and elongated along neurite surfaces. Using patterned substrata of tenascin versus fibronectin or laminin confirmed that tenascin borders allow neurites to pass but act as barriers to migrating satellite cells. We postulate that tenascin or related molecules with dual functions in cell adhesion are important for peripheral nerve morphogenesis. Tenascin allows axonal growth, but may restrict random satellite cell migration into the fibronectin-rich mesenchyme, thereby inducing the compaction of nerve fascicles.

Animals↗
PubMed1992

Tenascin: a modulator of cell growth.

The large, multidomain extracellular matrix protein tenascin displays a markedly restricted tissue distribution during embryogenesis and remains present only in a few adult tissues. The protein is reexpressed, however, during wound healing and in the stroma of malignant tumours. While a variety of studies have dealt with the important role of tenascin in the development of neural and non-neural tissues, there is growing evidence that tenascin expression may be associated with proliferation of cells lining these tissues. The presence of repeating domains in tenascin similar to those in epidermal growth factor prompted us to investigate the ability of tenascin to modulate the growth of different cell types. Tenascin was actually found to be mitogenic for several cell types. This mitogenic activity, however, appears to be associated with a region in the fibronectin type III domains. The mitogenic mechanism is clearly distinct from pathways used by peptide growth factors such as epidermal growth factor and platelet-derived growth factor, which activate the intrinsic tyrosine kinase activity of their cell-surface receptors. However, we show that this large extracellular matrix molecule is efficiently internalised and may be processed by responding cells.

3T3 Cells↗
PubMed1992

A major oligomeric fibroblast proteoglycan identified as a novel large form of type-XII collagen.

Cultured chick embryo skin fibroblasts release a major component with a native molecular mass of about 1 MDa, which resolves into three polypeptide bands of about 300, 350 and 600 kDa upon reduction. We report here the purification of this oligomeric protein and show, by means of polyclonal and monoclonal antibodies, that its three polypeptide constituents are closely related. The 600-kDa polypeptide is likely to be a dimer of two smaller subunits which are cross-linked by non-reducible bonds. By electron microscopy, isolated oligomeric molecules exhibit a novel cruciform structure with a large central globular domain. One arm has the shape of a thin rod about 70 nm in length. The three other arms are thicker, longer (90 nm) and flexible, and carry a prominent double globule at their distal ends. Collagenase treatment of the oligomeric fibroblast protein yields two resistant fragments of about 270 kDa and 320 kDa. The intact 350-kDa and 600-kDa (but not the 300-kDa) polypeptides are chondroitinase sensitive and labeled by metabolic incorporation of [35S]sulfate; collagenase treatment does not remove any [35S] sulfate. Hence, the intact fibroblast protein has glycosaminoglycan chains attached to its non-collagenous domain. Three amino acid sequences obtained from chymotryptic fragments of the fibroblast protein correspond to sequences predicted for chick type-XII collagen from its full-length cDNA [Yamagata, M., Yamada, K. M., Yamada, S. S., Shinomura, T., Tanaka, H., Nishida, Y., Obara, M. & Kimata, K. (1991) J. Cell Biol. 115, 209-221]. However, the novel fibroblast protein described here differs significantly from previously isolated forms of type-XII collagen: its subunits are larger by one third, and it is a proteoglycan.

Amino Acid Sequence↗
PubMed1991

Isolation of chick tenascin variants and fragments. A C-terminal heparin-binding fragment produced by cleavage of the extra domain from the largest subunit splicing variant.

The extracellular-matrix glycoprotein, tenascin, consists of disulfide-linked subunits of 190, 200 and 230 kDa (the three splicing variants reported in chicken) and usually exists as a six-armed structure under the electron microscope. We used monoclonal antibodies to isolate and characterize different splicing variants and proteolytic fragments obtained from the native protein. Purified monomeric tenascin has a native molecular mass of 216 kDa and is structured as single arms. Tenascin fragments obtained by pepsin digestion bind to monoclonal antibody (mAb) TnM1 which is directed against epidermal-growth-factor-like repeats in the N-terminal half of all subunits. These fragments represent the thin proximal part of the tenascin arms and they are still partially linked to dimers and trimers via disulfide bridges. Using mAb Tn68, that reacts with a fibronectin-type-III repeat towards the C-terminus, a tenascin fragment, generated by treatment with pronase, can be isolated. Ultrastructurally, this fragment looks like the thicker distal part of the tenascin arms. Only the 230-kDa variant of tenascin gives rise to this distal fragment after cleavage within the alternatively spliced fibronectin-type-III repeats. Native tenascin and all fragments containing the distal part of its arms bind to heparin-agarose, whereas the proximal fragments do not. Oligomeric and monomeric tenascin inhibit fibronectin-mediated fibroblast spreading with comparable efficiency when added to the culture medium, while the proximal fragment has no effect. The distal fragment as well as reduced and alkylated tenascin are active in this assay, but only at higher molar concentrations when compared to the native protein.

Animals↗
PubMed1991

Purification and partial characterization of Xenopus laevis tenascin from the XTC cell line.

We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.

Animals↗
PubMed1991

Epithelial synthesis of tenascin at tips of growing bronchi and graded accumulation in basement membrane and mesenchyme.

The extracellular matrix protein, tenascin, has been proposed as mediator in epithelial-mesenchymal interactions because of its characteristic distribution during embryogenesis. Here we compared the accumulation of tenascin and laminin in the early chicken lung bud. Laminin is deposited in the basement membrane, starting at the tips and increasing along the shafts of growing primary and secondary bronchi. In contrast, tenascin accumulation is highest in basement membranes and mesenchyme at sites where new bronchial branches are formed. By in situ hybridization, tenascin mRNA was found to be produced exclusively by the epithelium at sites of active growth of bronchial tubes.

Animals↗
PubMed1991

Chick laminin: isolation by monoclonal antibodies and differential distribution of variants in the embryo.

In order to study the expression and function of laminin variants during chick embryonic development, we have generated monoclonal antibodies against chick heart laminin. One monoclonal antibody (mAb), called 9/F-10, could be used to purify chick laminin to homogeneity. By rotary shadowing, cross-shaped and T-shaped laminin particles as well as aggregates of two laminin molecules crosslinked via their short arms could be observed in this preparation. Purified chick laminin was very potent in mediating neurite growth by chick embryonic neurons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced chick heart laminin revealed a complex pattern of polypeptides which are immunologically related to several mammalian laminin chains. The two mAbs, 9/F-10 and 3/E-8, recognize two different types of chick laminin subunits. By immunofluorescence, antibody 3/E-8 labels basement membranes, aortic smooth muscle, and mesenchyme of 6-day-old chick embryos. In contrast, staining by mAb 9/F-10 is confined to basement membranes. Therefore, the two antibodies seem to distinguish between two different chick laminin isoforms.

Animals↗
PubMed1991

Tenascin variants: differential binding to fibronectin and distinct distribution in cell cultures and tissues.

In the chicken, three tenascin variants have been characterized that are generated by alternative splicing of 3 of its 11 fibronectin type III repeats. Using monoclonal antibodies that react with common regions versus extra repeats of tenascin, we could distinguish and separate tenascin variants and investigate their interaction with fibronectin using multiple experimental procedures. Interestingly, in all assays used the smallest tenascin variant bound more strongly to fibronectin than the larger ones. These biochemical data were paralleled by the observation that in chick embryo fibroblast cultures only the smallest form of tenascin could be detected in the fibronectin-rich extracellular matrix network laid down by the cells. Furthermore, each tissue present in adult chicken gizzard contained a distinct set of tenascin variants. Those tissues particularly rich in extracellular matrix, such as the tendon, contained the smallest tenascin only. Intermediate-sized tenascin was present in smooth muscle, whereas the largest form was exclusively detectable underneath the epithelial lining of the villi. Thus it appears that cell type-specific forms of tenascin exist that are appropriate for the functional requirements of the respective extracellular matrices.

Animals↗
PubMed1991

Nerve-dependent and -independent tenascin expression in the developing chick limb bud.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Tenascin accumulates along developing peripheral nerves as they leave the spinal cord and enter the limb mesenchyme (Wehrle and Chiquet, Development 110, 401-415, 1990). Here we found that most but not all tenascin deposited along growing nerves is of glial origin. By in situ hybridization with a tenascin cDNA probe, we determined the site of tenascin mRNA accumulation both in normal and nerve-free limbs. In normal wing buds, tenascin mRNA was first detected within the developing limb nerves. Vinculin-positive glial precursor cells, which comigrate with the axons, are the likely source of this tenascin message. In nerveless wing grafts, tenascin was first expressed in tendon primordia in the absence, and thus independently, from innervation. In contrast to normal limbs, grafted wing buds neither contained vinculin-positive glial precursor cells, nor expressed tenascin in regions proximal to tendon primordia. In normal wing buds, tenascin deposited by tendon primordia transiently parallels and surrounds certain developing nerves. After the major nerve pattern is established, tenascin mRNA disappears from nerves in the upper limb, but is expressed in perichondrium and tendons. We propose that glial tenascin facilitates the penetration of axons into the limb bud and is important for nerve fasciculation. In some places, early tendon primordia might help to guide the migration of axons and glial precursor cells towards their target.

Animals↗
PubMed1990

Exogenous tenascin inhibits mesodermal cell migration during amphibian gastrulation.

We have used amphibian gastrulation as a model system to study the action of the extracellular matrix (ECM) glycoprotein tenascin on mesodermal cell migration. Tenascin function was assayed in vitro during spreading of isolated cells from the dorsal marginal zone (DMZ) and during cell migration from DMZ explants. Plastic coated with bovine fibronectin or gastrula ECM was used as a substratum. In both cases, tenascin added to the medium inhibited spreading and migration of mesodermal cells. In addition, a substratum coated with a mixture of fibronectin and tenascin was found to prevent mesodermal cell migration. Tenascin was also microinjected into the blastocoel cavity of living embryos at the late blastula stage. This led to a complete arrest of gastrulation in more than 80% of the cases. Scanning electron microscopy of fractures from arrested gastrulae showed that mesodermal cell migration was blocked. Similar injection experiments carried out at the middle gastrula stage demonstrated that tenascin is able to inhibit cell migration after cells have already contacted the ECM. Mesodermal cell migration in the presence of tenascin could be restored in vitro and in vivo by the monoclonal antibody mAb Tn68 which is known to mask a cell binding site of the molecule. Finally, tenascin microinjected into the blastocoel of blastula or gastrula stage embryos bound within 15 min to the ECM fibrils at all the stages studied. Our results show that exogenous tenascin can be incorporated into embryonic ECM and interferes in vivo with the interactions of cells with a fibronectin-rich matrix.

Animals↗
PubMed1990

Tenascin is accumulated along developing peripheral nerves and allows neurite outgrowth in vitro.

The extracellular matrix protein, tenascin, appears in a restricted pattern during organ morphogenesis. Here we studied the expression of tenascin along developing peripheral nerves in chick embryos and tested its activity as a substrate for cultured neurons. Motor axons grow out through the tenascin-rich, anterior part of the sclerotome. Shortly after, tenascin surrounds axon fascicles of ventral roots. At the limb levels, outgrowing axons accumulate in the tenascin-containing girdle region forming a plexus. In the limb, tenascin first appears in bracket-like structures that surround the precartilage cell condensations of the femur and humerus, respectively. These regions coincide with the channels along which axons first grow in from the girdle plexus to form the limb nerves. Later, the major tenascin staining is associated with the cartilage and tendon primordia, and not with the limb nerves. We used tenascin as a substrate for cultured neural explants and single cells in order to test for its function in neurite outgrowth. Dissociated embryonic neurons of various types attached to mixed polylysine/tenascin substrates and sprouted rapidly after a lag of several hours. Outgrowth was inhibited and neurites were detached by anti-tenascin antibodies. On substrates coated with tenascin alone, neurite outgrowth was achieved from 3 day spinal cord explants. Whereas growth cones were well spread and rapidly moving, the neurites were poorly attached, straight and rarely branched. We speculate that in vivo tenascin allows axonal outgrowth, but inhibits branching and supports fasciculation of newly formed axons.

Animals↗