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T Lallier

Publications and source records attributed to T Lallier.

10 recordsLinked to original sources
PubMed1996

Inhibition of cranial neural crest adhesion in vitro and migration in vivo using integrin antisense oligonucleotides.

Although it is well-established that beta1 integrins play a functional role in the migration of cranial neural crest cells, little is known about the number or importance of their associated alpha subunits. Here, we have utilized antisense oligonucleotides (aONs) against various mammalian integrin alpha subunits to functionally "knock out" integrins in vitro and in vivo. First, we examined the attachment in vitro of cranial neural crest cells to fibronectin and laminin in the presence of antisense or reversed-sense oligonucleotides using a quantitative adhesion assay. We found three alpha integrin aONs that blocked attachment to fibronectin substrates only, one that blocked attachment to laminin substrates only, and one that blocked attachment to both fibronectin and laminin. As expected, an aON to chick beta1 integrin reduced attachment to both fibronectin and laminin substrates. These results suggest that there are three or more functionally distinct integrin heterodimers on avian cranial neural crest cells. Second, we examined the ability of aONs against various alpha integrin subunits to perturb cranial neural crest migration in vivo by injecting the oligonucleotides into the cranial mesenchyme through which neural crest cells migrate. Those alpha aONs that inhibited cell attachment in vitro also caused neural crest and/or neural tube abnormalities after injection in vivo. In addition, two aONs that had no effect in vitro did affect emigration of neural crest cells in vivo. Immunoprecipitations revealed that some integrin subunits were depleted after treatment with antisense but not reversed-sense oligonucleotides both in vivo and in vitro. The results suggest that integrin alpha subunits are required for cranial neural crest cell attachment and emigration.

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PubMed1994

Neural crest cell interactions with laminin: structural requirements and localization of the binding site for alpha 1 beta 1 integrin.

We have identified the sites of neural crest cell interaction with laminin in vitro by examining their ability to attach to and migrate on proteolytic fragments of the molecule and the ability of fragment-specific antibodies to inhibit these interactions. The binding site on laminin was localized to the E8 domain on the long arm of laminin, as well as the T8' fragment within this domain, but not the E1', E3, or E4 fragments. Only subfragments containing the carboxy-terminal rod-like portion of the A chain plus the corresponding B1 and B2 chains retained the attachment-promoting activity of the parent E8 fragment. In addition, interactions required maintenance of the triple-stranded and alpha-helical coiled-coil structure of this domain. Reduction and alkylation of laminin and the E8 and T8 fragments significantly reduced neural crest cell attachment and migration. An antiserum against chick alpha 1 integrin reduced migration and adhesion of neural crest cells on an intact laminin-nidogen complex, the E8 fragment, and all its active subfragments. Furthermore, we observed that neural crest cells modified laminin substrata prepared in the absence of divalent cations. Early stable attachment to these substrata was mediated by an integrin other than alpha 1, whereas later attachment and migration were mediated by alpha 1 integrins. Our results suggest that neural crest cells selectively bind to the B1-A-B2 mid-portion (T8') of the E8 domain of laminin, requiring structural integrity of this region and that they modify laminin substrata as a result of prolonged cell-matrix interactions.

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PubMed1993

Inhibition of neural crest cell attachment by integrin antisense oligonucleotides.

Neural crest cell interactions with extracellular matrix molecules were analyzed with the use of antisense oligonucleotides to block synthesis of integrin subunits. When added to the culture medium of quail neural crest cells, selected antisense phosphorothiol oligonucleotides reduced the amounts of cell surface alpha 1 or beta 1 integrin subunits by up to 95 percent and inhibited neural crest cell attachment to laminin or fibronectin substrata. Differential effects on specific alpha integrins were noted after treatment with alpha-specific oligonucleotides. Cells recovered the ability to bind to substrata 8 to 16 hours after treatment with inhibitory oligonucleotides. The operation of at least three distinct alpha integrin subunits is indicated by substratum-selective inhibition of cell attachment.

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PubMed1992

Alpha 1 beta 1 integrin on neural crest cells recognizes some laminin substrata in a Ca(2+)-independent manner.

Neural crest cells migrate along pathways containing laminin and other extracellular matrix molecules. In the present study, we functionally and biochemically identify an alpha 1 beta 1 integrin heterodimer which bears the HNK-1 epitope on neural crest cells. Using a quantitative cell adhesion assay, we find that this heterodimer mediates attachment to laminin substrata prepared in the presence of Ca2+. Interestingly, neural crest cells bind to laminin-Ca2+ substrata in the presence or absence of divalent cations in the cell attachment medium. In contrast, the attachment of neural crest cells to laminin substrata prepared in the presence of EDTA, heparin, Mg2+, or Mn2+ requires divalent cations. Interactions with these laminin substrata are mediated by a different integrin heterodimer, since antibodies against beta 1 but not alpha 1 integrins inhibit neural crest cell attachment. Thus, the type of laminin substratum appears to dictate the choice of laminin receptor used by neural crest cells. The laminin conformation is determined by the ratio of laminin to Ca2+, though incorporation of heparin during substratum polymerization alters the conformation even in the presence of Ca2+. Once polymerized, the substratum appears stable, not being altered by soaking in either EDTA or divalent cations. Our findings demonstrate: (a) that the alpha 1 beta 1 integrin can bind to some forms of laminin in the absence of soluble divalent cations; (b) that substratum preparation conditions alter the conformation of laminin such that plating laminin in the presence of Ca2+ and/or heparin modulates its configuration; and (c) that neural crest cells utilize different integrins to recognize different laminin conformations.

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PubMed1992

Cranial and trunk neural crest cells use different mechanisms for attachment to extracellular matrices.

We have used a quantitative cell attachment assay to compare the interactions of cranial and trunk neural crest cells with the extracellular matrix (ECM) molecules fibronectin, laminin and collagen types I and IV. Antibodies to the beta 1 subunit of integrin inhibited attachment under all conditions tested, suggesting that integrins mediate neural crest cell interactions with these ECM molecules. The HNK-1 antibody against a surface carbohydrate epitope under certain conditions inhibited both cranial and trunk neural crest cell attachment to laminin, but not to fibronectin. An antiserum to alpha 1 intergrin inhibited attachment of trunk, but not cranial, neural crest cells to laminin and collagen type I, though interactions with fibronectin or collagen type IV were unaffected. The surface properties of trunk and cranial neural crest cells differed in several ways. First, trunk neural crest cells attached to collagen types I and IV, but cranial neural crest cells did not. Second, their divalent cation requirements for attachment to ECM molecules differed. For fibronectin substrata, trunk neural crest cells required divalent cations for attachment, whereas cranial neural crest cells bound in the absence of divalent cations. However, cranial neural crest cells lost this cation-independent attachment after a few days of culture. For laminin substrata, trunk cells used two integrins, one divalent cation-dependent and the other divalent cation-independent (Lallier, T. E. and Bronner-Fraser, M. (1991) Development 113, 1069-1081). In contrast, cranial neural crest cells attached to laminin using a single, divalent cation-dependent receptor system. Immunoprecipitations and immunoblots of surface labelled neural crest cells with HNK-1, alpha 1 integrin and beta 1 integrin antibodies suggest that cranial and trunk neural crest cells possess biochemically distinct integrins. Our results demonstrate that cranial and trunk cells differ in their mechanisms of adhesion to selected ECM components, suggesting that they are non-overlapping populations of cells with regard to their adhesive properties.

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PubMed1991

Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development.

In this study, we describe the distribution of various classes of proteoglycans and their potential matrix ligand, hyaluronan, during neural crest development in the trunk region of the chicken embryo. Different types of chondroitin and keratan sulfate proteoglycans were recognized using a panel of monoclonal antibodies produced against specific epitopes on their glycosaminoglycan chains. A heparan sulfate proteoglycan was identified by an antibody against its core protein. The distribution of hyaluronan was mapped using a biotinylated fragment that corresponds to the hyaluronan-binding region of cartilage proteoglycans. Four major patterns of proteoglycan immunoreactivity were observed. (1) Chondroitin-6-sulfate-rich proteoglycans and certain keratin sulfate proteoglycans were absent from regions containing migrating neural crest cells, but were present in interstitial matrices and basement membranes along prospective migratory pathways such as the ventral portion of the sclerotome. Although initially distributed uniformly along the rostrocaudal extent of the sclerotome, these proteoglycans became rearranged to the caudal portion of the sclerotome with progressive migration of neural crest cells through the rostral sclerotome and their aggregation into peripheral ganglia. (2) A subset of chondroitin/keratan sulfate proteoglycans bearing primarily unsulfated chondroitin chains was observed exclusively in regions where neural crest cells were absent or delayed from entering, such as the perinotochordal and subepidermal spaces. (3) A subset of chondroitin/keratan sulfate proteoglycans was restricted to the perinotochordal region and, following gangliogenesis, was arranged in a metameric pattern corresponding to the sites where presumptive vertebral arches form. (4) Certain keratan sulfate proteoglycans and a heparan sulfate proteoglycan were observed in basement membranes and in an interstitial matrix uniformly distributed along the rostrocaudal extent of the sclerotome. After gangliogenesis, the neural crest-derived dorsal root and sympathetic ganglia contained both these proteoglycan types, but were essentially free of other chondroitin/keratan-proteoglycan subsets. Hyaluronan generally colocalized with the first set of proteoglycans, but also was concentrated around migrating neural crest cells and was reduced in neural crest-derived ganglia. These observations demonstrate that proteoglycans have diverse and dynamic distributions during times of neural crest development and chondrogenesis of the presumptive vertebrae. In general, chondroitin/keratan sulfate proteoglycans are abundant in regions where neural crest cells are absent, and their segmental distribution inversely correlates with that of neural crest-derived ganglia.

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PubMed1991

Avian neural crest cell attachment to laminin: involvement of divalent cation dependent and independent integrins.

The mechanisms of neural crest cell interaction with laminin were explored using a quantitative cell attachment assay. With increasing substratum concentrations, an increasing percentage of neural crest cells adhere to laminin. Cell adhesion at all substratum concentrations was inhibited by the CSAT antibody, which recognizes the chick beta 1 subunit of integrin, suggesting that beta 1-integrins mediate neural crest cell interactions with laminin. The HNK-1 antibody, which recognizes a carbohydrate epitope, inhibited neural crest cell attachment to laminin at low coating concentrations (greater than 1 microgram ml-1; Low-LM), but not at high coating concentration of laminin (10 micrograms ml-1; High-LM). Attachment to Low-LM occurred in the absence of divalent cations, whereas attachment to High-LM required greater than 0.1 mM Ca2+ or Mn2+. Neural crest cell adherence to the E8 fragment of laminin, derived from its long arm, was similar to that on intact laminin at high and low coating concentrations, suggesting that this fragment contains the neural crest cell binding site(s). The HNK-1 antibody recognizes a protein of 165,000 Mr which is also found in immunoprecipitates using antibodies against the beta 1 subunit of integrin and is likely to be an integrin alpha subunit or an integrin-associated protein. Our results suggest that the HNK-1 epitope on neural crest cells is present on or associated with a novel or differentially glycosylated form of beta 1-integrin, which recognizes laminin in the apparent absence of divalent cations. We conclude that neural crest cells have at least two functionally independent means of attachment to laminin which are revealed at different substratum concentrations and/or conformations of laminin.

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PubMed1990

Distribution and biochemical characterization of the INO antigen during chick neural crest cell migration.

The INO (inhibitor of neurite outgrowth) antibody recognizes a laminin-heparan sulfate proteoglycan complex and was isolated for its ability to functionally inhibit axonal outgrowth of peripheral neurons. Here, we examine the distribution and biochemical characteristics of INO in the early chick embryo. Because the INO antigen is sensitive to most classical fixation procedures and fixation leads to abundant nuclear staining, the antibody was directly injected into 1.5-2.5-day-old embryos prior to fixation. The distribution of the injected antibody was then observed in cryostat sections by indirect immunofluorescence. Particular attention was focussed upon regions of ongoing neural crest cell migration. The INO antigen was observed along both cranial and trunk neural crest cell migratory pathways. The antigen was seen around the basement membrane surrounding the neural tube and notochord, and underneath the ectoderm and endoderm. In addition, fibrillar staining was observed in the cranial mesenchyme and in both rostral and caudal halves of the somitic sclerotome in the trunk. The distribution pattern was identical to that previously observed for laminin or heparan sulfate proteoglycan. To confirm the nature of the INO antigen, we performed immunoprecipitations of chick embryos ranging from 1.5 to 9 days of incubation. Half of each sample was digested with heparinase prior to SDS-PAGE and silver staining. In material from young embryos, bands of 200 and 180 kD (probably corresponding to the B-chains of laminin) plus two broad smears of bands at 180-150 kD and 130-85 kD were observed without heparinase digestion. Following enzymatic digestion, the 200-kD and 180-kD bands remained, while the smears disappeared and were replaced by numerous low-molecular-weight bands. In contrast to preparations from young embryos, samples taken from embryos at day 3 or beyond did not enter the 8% gel without heparinase digestion, though the banding pattern appeared identical to younger samples after heparinase digestion in the presence or absence of Ca2+. This change in the INO antigen with age could result from an increase in the heparin-side-chains attached to similar core proteins, or from an increase in the stability of the laminin-heparan sulfate proteoglycan containing complex with time.

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PubMed1988

A monoclonal antibody against a laminin-heparan sulfate proteoglycan complex perturbs cranial neural crest migration in vivo.

INO (inhibitor of neurite outgrowth) is a monoclonal antibody that blocks axon outgrowth, presumably by functionally blocking a laminin-heparan sulfate proteoglycan complex (Chiu, A. Y., W. D. Matthew, and P. H. Patterson. 1986. J. Cell Biol. 103: 1382-1398). Here the effect of this antibody on avian neural crest cells was examined by microinjecting INO onto the pathways of cranial neural crest migration. After injection lateral to the mesencephalic neural tube, the antibody had a primarily unilateral distribution. INO binding was observed in the basal laminae surrounding the neural tube, ectoderm, and endoderm, as well as within the cranial mesenchyme on the injected side of the embryo. This staining pattern was indistinguishable from those observed with antibodies against laminin or heparan sulfate proteoglycan. The injected antibody remained detectable for 18 h after injection, with the intensity of immuno-reactivity decreasing with time. Embryos ranging from the neural fold stage to the 9-somite stage were injected with INO and subsequently allowed to survive for up to 1 d after injection. These embryos demonstrated severe abnormalities in cranial neural crest migration. The predominant defects were ectopic neural crest cells external to the neural tube, neural crest cells within the lumen of the neural tube, and neural tube deformities. In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. When embryos with ten or more somites were injected with INO, no effects were noted, suggesting that embryos are sensitive for only a limited time during their development. Immunoprecipitation of the INO antigen from 2-d chicken embryos revealed a 200-kD band characteristic of laminin and two broad smears between 180 and 85 kD, which were resolved into several bands at lower molecular mass after heparinase digestion. These results indicate that INO precipitates both laminin and proteoglycans bearing heparan sulfate residues. Thus, microinjection of INO causes functional blockage of a laminin-heparan sulfate proteoglycan complex, resulting in abnormal cranial neural crest migration. This is the first evidence that a laminin-heparan sulfate proteoglycan complex is involved in aspects of neural crest migration in vivo.

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