Biomedical subjects

R Perris

Publications and source records attributed to R Perris.

At least 37 records · Page 2Linked to original sources

PubMed1997

Differential cation regulation of the alpha 5 beta 1 integrin-mediated adhesion of leukemic cells to the central cell-binding domain of fibronectin.

Normal and neoplastic leukocytes interact with the central cell-binding and carboxyl-terminal regions of fibronectin (FN) primarily via the alpha(4) beta(1) and alpha(5) beta(1) integrins. By using a unique centrifugation-based cell adhesion assay and affinity chromatography of the cell surface-labeled integrins, we show in this study that the constitutive alpha(5) beta(1)-dependent attachment of three leukemic cell lines, BV-173, K562, and Nalm-6, to FN or to a 110-kDa central cell-binding fragment of FN was totally inhibited at 37 degrees C by preincubation of the substrate with either antibodies to the arginine-glycine-aspartic acid-containing region (3Fn-9 module) or to the synergistic region (3Fn-9 module) of FN. Similar results were obtained when assays were carried out at 4 degrees C, suggesting that energy-dependent events were not involved. On the other hand, only the antibody against the 3Fn-10 module was able to detach most firmly adherent cells. Constitutive cell attachment to the 110-kDa fragment was cation dependent, with the order of efficacy of the cation being Mn2+ > Mg2+ > Ca2+, and Ca2+ was only effective for BV-173 cells. Antibodies against the alpha(5) beta(1) integrin or the alpha(5) subunit completely impaired cell attachment of all cell lines, whereas several blocking anti-beta(1) subunit antibodies, including 4B4, P4C10, and AIIB2, differentially perturbed cell adhesion of BV-173, depending on which cation was present. These anti-beta(1) blocking antibodies, whose epitopes map to a region distant from the one expected to contain the beta(1) putative cation binding sites, seemed to lock the higher activation state of this integrin attained in the presence of Mg2+ and to preserve it during the subsequent adhesion events irrespectively of the presence of the low avidity state-inducing Ca2+ ion. Because the higher binding avidity displayed by BV-173 could not be explained by a higher degree of preclustering of alpha(5) beta(1) integrin in this cell line compared to K562, these findings suggest that cations might act as allosteric activators of integrin function. Whether this novel cation-dependent parameter of alpha(5) beta(1) integrin-FN interaction might contribute to the growth control and/or tissue dissemination of leukemic cells remains to be determined.

Antibodies, Blocking↗

PubMed1996

Differential neural crest cell attachment and migration on avian laminin isoforms.

A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian alpha 2, beta 2 and gamma 1 laminin chains. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin-2 (alpha 2 beta 1 gamma 1), Laminin-4 (alpha 2 beta 2 gamma 1), and laminins comprising the beta 1, beta 2 and gamma 1 chains associated with a shorter alpha chain which, in SDS-PAGE, co-migrate with the beta/gamma complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin-alpha x (heart tissue) and Laminin-G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as "T"-shaped particles similar to Laminin-6 and Laminin-7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin-2 and Laminin-4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin-G. Similarly to the EHS Laminin-1, neural crest cells recognized all avian isoforms through their alpha 1 beta 1 integrin, shown previously to be the primary laminin-binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the alpha-helical coiled-coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the alpha 1 beta 1 integrin.

Animals↗

PubMed1996

Inhibitory effects of PG-H/aggrecan and PG-M/versican on avian neural crest cell migration.

Aggrecans and PG-M/versicans represent two newly defined families of hyaluronan-binding proteoglycans for which the function is still poorly understood. Using the avian neural crest as a model system, we have examined the molecular mechanisms entailed in the cell-proteoglycan interaction during embryonic cell motility. Both the primary cartilage aggrecan of the avian embryo (PG-H/aggrecan) and the largest variant of the avian mesenchymal versican (PG-M/versican VO) failed to support neural crest cell adhesion and migration when topographically immobilized onto the substrate. Conversely, solely the PG-H/aggrecan, and similar aggrecans from other species, counteracted the migration-promoting effect of a number of matrix molecules lacking proteoglycan affinity. This inhibitory effect was not reproduced by the isolated glycosaminoglycan chains, the isolated core protein, the reduced and alkylated macromolecule, or the aggrecan in which the G1 hyaluronan-binding domain had been inactivated with hyaluronan fragments or antibodies. Limited depolymerization of the side chains and preincubation of the PG-H/aggrecan with anti-glycosaminoglycan antibodies differentially reduced the inhibitory activity of the proteoglycan on cell motility. The results demonstrate a diverse inhibitory effect of aggrecans and PG-M/versicans on embryonic cell movement and show that the inhibitory action of aggrecans is independent of substrate binding, is dependent on a G1 domain-mediated association of the intact proteoglycan with cell surface-bound hyaluronan, and is differentially mediated by its glycosaminoglycan side chains.

Aggrecans↗

PubMed1996

Distribution of keratan sulphate and chondroitin sulphate in wild type and white mutant axolotl embryos during neural crest cell migration.

In embryos of the white mutant axolotl, prospective pigment cells are unable to migrate from the neural crest (NC) due to a deficiency in the subepidermal extracellular matrix (ECM). This raises the question of the molecular nature of this functional defect. Some PGs can inhibit cell migration on ECM molecules in vitro, and an excess of this class of molecules in the migratory pathways of neural crest cells might cause the restricted migration of prospective pigment cells seen in the white mutant embryo. In the present study, we use several monoclonal antibodies against epitopes on keratan sulphate (KS) and chondroitin sulphate (CS) and LM immunofluorescence to examine the distribution of these glycosaminoglycans at initial (stage 30) and advanced (stage 35) stages of neural crest cell migration. Most KS epitopes are more widely distributed in the white mutant than in the wild type embryo, whereas CS epitopes show very similar distributions in mutant and wild type embryos. This is confirmed quantitatively by immunoblotting: certain KS epitopes are more abundant in the white mutant. TEM immunogold staining reveals that KS as well as CS are present both in the basal lamina and in the interstitial ECM in both types of embryos. It remains to be investigated whether the abundance of certain KS epitopes in the white mutant embryo might contribute to the deficiency in supporting pigment cell migration shown by its ECM.

Ambystoma↗

PubMed1996

Effects of extracellular matrix molecules on subepidermal neural crest cell migration in wild type and white mutant (dd) axolotl embryos.

Migration of neural crest (NC) derived pigment cells is restricted in the white mutant (dd) axolotl embryo (Ambystoma mexicanum). Transplantations between mutant and wild type embryos show that the extracellular matrix (ECM) of the white mutant is unable to support the migration of prospective pigment cells in wild type embryos (Löfberg et al., 1989, Dev. Biol. 131:168-181). In the present study, we test the effects of various purified ECM molecules on NC cell migration in the subepidermal migratory pathway of wild type (D/-) and white mutant (dd) axolotl embryos. We adsorbed the ECM molecules onto membrane microcarriers, which were then implanted under the epidermis. Fibronectin (FN), tenascin (TN), collagens I and VI, and a chick aggrecan stimulated migration in both types of embryos. Laminin-nidogen, rat chondrosarcoma aggrecan, and shark aggrecan stimulated migration in dd embryos but did not affect migration in D/- embryos. Collagen III, fibromodulin and bovine aggrecan had no effect on migration in either type of embryo. NC cells did not migrate on control microcarriers, which lacked ECM molecules. Some cells observed contacting, and presumably migrating on, coated microcarriers could be identified as pigment cells by their ultrastructure. Enzymatic digestion in vivo with chondroitinase ABC had no effect on NC cell migration. The neutral or stimulatory effect of the aggrecans is surprising; when tested in vitro they inhibited NC cell migration. The effect of three-dimensionality and other molecules present either in the embryonic ECM or in solution may overcome the inhibitory effect of aggrecans.

Ambystoma↗

PubMed1994

Differential attachment of human neoplastic B cells to purified extracellular matrix molecules.

Recirculation of normal and neoplastic lymphocytes occurs via binding to the endothelial luminar surface, followed by extravasation and the subsequent interaction of the cells with components of the underlying basement membrane and stromal extracellular matrix (ECM). To identify matrix constituents that could be involved in the tissue dissemination of neoplastic B cells, we have examined the ability of three lymphoma B-cell lines and one Philadelphia chromosome (Ph1)-positive cell line established from the lymphoid transformation of a chronic myeloid leukemia (CML) to adhere to a range of purified ECM molecules. Immunophenotyping with a panel of markers suggested that the lines derived from cells that had undergone transformation at distinct stages of B-cell maturation. The four cell lines displayed a differential ability to adhere to the ECM molecules tested. BV-173, Ci-1, and Sc-1 cells attached to various degrees to fibronectin (FN). Ri-1, Ci-1, and Sc-1 cells attached to human laminin (LN) variants, whereas only Ci-1 and Sc-1 cells showed some affinity for collagen (Col) type VI. All four cell lines interacted with fibrillar Col I, but only BV-173 and Ri-1 cells attached to fibrillar Col III. The subendothelial Col VIII only was active as a substratum for BV-173 cells. In all cases, cells bound to fibrillar collagens when they were assembled into polymeric fibrils, and were incapable of adhering to monomeric and denatured collagen. In contrast to cell adhesion to FN and LN, which showed a plateau at high substrate concentrations, adhesion to fibrillar Col I reached a peak at intermediary concentrations and decreased thereafter, suggesting that cells respond to a definite macromolecular arrangement of collagenous fibrils. Adhesion to individual ECM molecules was not directly correlated with the apparent maturation state of the cells, nor with the relative density of known ECM receptors. Taken together, these results suggest that interaction of neoplastic B cells with selected matrix components may influence their dispersion throughout tissues. We further suggest that the use of quantitative cell adhesion assays in vitro may provide means of defining the behavioral traits of neoplastic B cells in vivo.

Burkitt Lymphoma↗

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.

Animals↗

PubMed1993

Collagen type VI in neural crest development: distribution in situ and interaction with cells in vitro.

We have examined the spatio-temporal distribution of collagen type VI (Col VI) during neural crest development in vivo and its ability to promote neural crest cell attachment and migration in vitro. An affinity purified antiserum and chain-specific monoclonal antibodies against chicken Col VI were employed to immunolocalize the collagen in tissue sections and by immunoblotting. At stages of initial neural crest cell migration, the alpha 1(VI) and alpha 2(VI) chains were immunolocalized in apposition with basement membranes of the neural tube, somites, notochord and ectoderm, whereas no immunoreactivity was seen for the alpha 3(VI) chain. Immunoblotting analysis confirmed the expression of alpha 1(VI) and alpha 2(VI) chains and the lack of detectable immunoreactivity for the alpha 3(VI) chain at these early phases of neural crest development. Conversely, at advanced phases of migration and following gangliogenesis, expression of alpha 3(VI) chain coincided with that of alpha 1(VI) and alpha 2(VI) chains in apposition with basement membranes, around the dorsal root ganglia, and in fibrillar arrangements within the developing dermis and ventral sclerotome. The ability of Col VI to promote neural crest cell attachment and migration was tested in vitro using quantitative assays for these processes. Both native microfilaments and isolated tetramers of Col VI strongly promoted neural crest cell attachment and migration. Optimal stimulation of neural crest cell adhesion and migration was dependent upon structural integrity of Col VI since unfolded and disassembled alpha chains only weakly promoted cell attachment and were virtually inactive in supporting cell movement. The importance of a native macromolecular organization of Col VI further was analyzed in experiments in which dissociated tetramers were reassociated by Ca(2+)- and temperature-dependent self-aggregation. In contrast to native microfilaments, these oligomeric complexes were less effective in promoting neural crest cell movement, but still retained the ability to stimulate maximal cell attachment. The results indicate that Col VI is a primary component of the extracellular matrix deposited along neural crest migratory pathways, where it may participate in the regulation of cell movement by functioning as a migratory substrate. The ability of Col VI to promote neural crest cell adhesion and motility is highly dependent upon maintainance of a native macromolecular arrangement.

Actin Cytoskeleton↗

PubMed1993

The role of cell-cell and cell-matrix interactions in the morphogenesis of the neural crest.

The neural crest is an excellent model system with which to study the molecular controls of morphogenesis. Neural crest cells initiate their migration from the dorsal portion of the neural epithelium owing to an epithelial-mesenchymal transformation. They then followed stereotyped pathways for long distances before reaching their multiple destinations, and they cease migration in defined regions where they undergo terminal differentiation. Recent experimental evidence, which is summarized in this review, suggests that these various steps in neural crest morphogenesis are influenced by cell-cell and cell-matrix adhesions. Future studies employing genetic mutants and molecular techniques should provide more direct evidence for the role of cell-cell and cell-matrix interactions in neural crest morphogenesis.

Animals↗

PubMed1993

Neural crest cell interaction with type VI collagen is mediated by multiple cooperative binding sites within triple-helix and globular domains.

Collagen type VI (Col VI) is a primary constituent of the extracellular matrix encountered by migrating avian neural crest cells in situ and is effective in promoting attachment and motility of these cells in vitro. In this study, we have explored the molecular mechanisms of neural crest-Col VI interaction by using quantitative assays for cell attachment and migration in vitro, proteolytic fragments of the collagen, and a panel of domain-specific monoclonal antibodies. Removal of the predominant portion of the amino-terminal globular domains of Col VI tetramers by pepsin digestion (P6 fragment) resulted in a > fivefold decrease in their cell adhesion and motility-promoting activity. Further digestion of P6 with bacterial collagenase, which causes a complete loss of the amino-terminal domains plus an adjacent triple-helical segment, did not affect adhesion but reduced migration down to 40% of that seen on undigested P6. Untreated and pepsin-digested Col VI monomers were significantly less effective than their tetrameric counterparts and a M(r) 200,000 fragment, generated from pepsin-digested monomers by a second pepsin treatment, only retained 40% of the motility-promoting activity while preserving the adhesive capacity. A mixture of amino- and carboxyl-terminal globular domains supported both cell attachment and migration. While neural crest cells adhered equally well to the individual intact alpha 1 (VI)/alpha 2(VI) and alpha 3(VI) chains, they migrated most extensively on the alpha 3(VI) chain. Conversely, pepsin-digested individual alpha chains were significantly less effective in promoting cell adhesion and locomotion. Selective preincubation of Col VI microfilaments and isolated tetramers with a panel of monoclonal antibodies against triple helix, carboxyl-terminal, and amino-terminal epitopes of the different constituent chains differentially perturbed neural crest cell attachment and migration. Sites differentially involved in neural crest cell attachment and migration seemed to be present at the carboxyl termini of the alpha 1(VI) and alpha 2(VI) chains and at the amino-terminus of the alpha 3(VI) chain. The results suggest that neural crest cells interact with Col VI through multiple and cooperative binding sites present within its triple-helical and globular domains. The differential involvement and efficiency of these sites in stimulating neural crest cell adhesion and migration is strongly determined by the supramolecular organization of the collagen and requires inter- and intramolecular structural integrity. Since neural crest cell attachment and migration on Col VI was completely inhibited by anti-beta 1 integrin antibodies, there is evidence that this class of integrins is essential for the neural crest cell--Col VI interaction.

Animals↗

PubMed1993

Molecular mechanisms of neural crest cell attachment and migration on types I and IV collagen.

We have examined the mechanisms involved in the interaction of avian neural crest cells with collagen types I and IV (Col I and IV) during their adhesion and migration in vitro. For this purpose native Col IV was purified from chicken tissues, characterized biochemically and ultrastructurally. Purified chicken Col I and Col IV, and various proteolytic fragments of the collagens, were used in quantitative cell attachment and migration assays in conjunction with domain-specific collagen antibodies and antibodies to avian integrin subunits. Neural crest cells do not distinguish between different macromolecular arrangements of Col I during their initial attachment, but do so during their migration, showing a clear preference for polymeric Col I. Interaction with Col I is mediated by the alpha 1 beta 1 integrin, through binding to a segment of the alpha 1(I) chain composed of fragment CNBr3. Neural crest cell attachment and migration on Col IV involves recognition of conformation-dependent sites within the triple-helical region and the noncollagenous, carboxyl-terminal NC1 domain. This recognition requires integrity of inter- and intrachain disulfide linkages and correct folding of the molecule. Moreover, there also is evidence that interaction sites within the NC1 domain may be cryptic, being exposed during migration of the cells in the intact collagen as a result of the prolonged cell-substratum contact. In contrast to Col I, neural crest cell interaction with Col IV is mediated by beta 1-class integrins other than alpha 1 beta 1.

Animals↗

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.

Animals↗

PubMed1991

Collagens in avian neural crest development: distribution in vivo and migration-promoting ability in vitro.

This study examines the spatiotemporal distribution of collagen (Col) types I-V and IX during neural crest development in vivo and their ability to support neural crest cell movement in vitro. Col I, III and IV were widespread throughout the embryo, including the neural crest migratory pathways, whereas Col II, V and IX preferentially localized to regions from which migrating neural crest cells were absent. Col I-IV and IX occurred both in association with basement membranes and within interstitial matrices, whereas Col V only was detected in juxtaposition to basement membranes. Although initially distributed throughout the rostrocaudal extent of the somitic sclerotome, Col I and III rearranged to the caudal portion with progressive neural crest cell migration through the rostral portion of the sclerotome. This rearrangement does not occur in neural crest-ablated embryos, suggesting that it is a direct consequence of neural crest cell migration. The perinotochordal matrix, avoided by neural crest cells, contained a metameric Col II/IX immunoreactivity along the rostrocaudal axis which alternated with that of Col I and III. In contrast, Col IV and V were not observed in this matrix, but lined the basement membranes of the notochord and ventrolateral neural tube. To determine their functional significance for neural crest cell migration in vivo, purified collagens were tested for their ability to promote neural crest cell motility in vitro. Neural crest cell migration on isolated collagens was most pronounced on Col I and IV, whereas Col II, V and the triple-helical fragment of Col VII were unable to support cell motility. Substrata created by copolymerization of Col I and fibronectin, or Col I and laminin-nidogen, supported cell motility better than Col I alone, whereas both Col V and a cartilage-type chondroitin sulfate proteoglycan reduced cell movement on Col I. Fibronectin bound to pre-immobilized monomeric Col I, II or V had a reduced ability to support neural crest cell movement when compared to fibronectin alone. A similar reduction was seen for Col IV bound to the low density heparan sulfate proteoglycan from the EHS mouse tumor. The results demonstrate that Col I-IX are differentially distributed in the early avian embryo. During neural crest development several of these collagens undergo dynamic reorganizations that correlate with the migration of neural crest cells. Furthermore, various collagens possess distinct abilities to support neural crest cell migration in vitro, and their migration-promoting activity can be modulated by their conformation and/or association with other matrix components.

Animals↗

PubMed1990

Inhibition of neural crest cell migration by aggregating chondroitin sulfate proteoglycans is mediated by their hyaluronan-binding region.

We have recently shown that the large hyaluronan-aggregating chondroitin sulfate proteoglycan from cartilage (PG-LA) is unfavorable as a substrate for neural crest cell migration in vitro and that this macromolecule inhibits cell dispersion on fibronectin substrates when included in the medium (R. Perris and S. Johansson, 1987, J. Cell Biol. 105, 2511-2521). In this study we present data on the specificity of the migration-repressing activity of PG-LA and data on the molecular mechanisms by which the proteoglycan might impair neural crest cell motility. Soluble PG-LA potently impaired cell migration on substrates of laminin/laminin-nidogen, vitronectin, and collagen types I, III, IV, and VI. When tested in solid-phase binding assays, PG-LA bound avidly to substrates of collagen types I-III and V. Conversely, minimal amounts of the proteoglycan bound to substrates of laminin-nidogen, vitronectin, collagen types IV and VI, and fibronectin or to a proteolytic fragment encompassing its cell-binding domain (105 kDa). Preincubation of these substrates with soluble PG-LA prior to plating of the cells had no effect on their locomotory behavior. These results indicate that PG-LA affects neural crest cell movement primarily through an interaction with the cell surface, rather than by association with the cell motility-promoting substrate molecules. The molecular interaction of soluble PG-LA with neural crest cells was further examined by analyzing the effects of isolated domains of the proteoglycan on cell migration on fibronectin. Addition of chondroitin sulfate chains, the core protein free of glycosaminoglycans, the isolated hyaluronan-binding region (HABr), or a proteolytic fragment corresponding to the keratan sulfate-enriched domain of the PG-LA to neural crest cells migrating on fibronectin or the 105-kDa fibronectin fragment had no significant effect on their motility. After reduction and alkylation, PG-LA was considerably less efficient in perturbing cell movement on fibronectin substrates and virtually ineffective in altering migration on the 105-kDa fragment. In the presence of hyaluronan fragments of 16-30 monosaccharides in length, or an antiserum against the HABr, the migration repressing activity of PG-LA was reduced in a dose-dependent fashion. Furthermore, the inhibitory action of PG-LA was significantly reduced by treatment of the cells with Streptomyces hyaluronidase.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗

PubMed1990

Structural and compositional divergencies in the extracellular matrix encountered by neural crest cells in the white mutant axolotl embryo.

The skin of the white mutant axolotl larva is pigmented differently from that of the normal dark due to a local inability of the extracellular matrix (ECM) to support subepidermal migration of neural crest-derived pigment cell precursors. In the present study, we have compared the ECM of neural crest migratory pathways of normal dark and white mutant embryos ultrastructurally, immunohistochemically and biochemically to disclose differences in their structure/composition that could be responsible for the restriction of subepidermal neural crest cell migration in the white mutant axolotl. When examined by electron microscopy, in conjunction with computerized image analysis, the structural assembly of interstitial and basement membrane ECMs of the two embryos was found to be largely comparable. At stages of initial neural crest cell migration, however, fixation of the subepidermal ECM in situ with either Karnovsky-ruthenium red or with periodate-lysine-paraformaldehyde followed by ruthenium red-containing fixatives, revealed that fibrils of the dark matrix were significantly more abundant in associated electron-dense granules. This ultrastructural discrepancy of the white axolotl ECM was specific for the subepidermal region and suggested an abnormal proteoglycan distribution. Dark and white matrices of the medioventral migratory route of neural crest cells had a comparable appearance but differed from the corresponding subepidermal ECMs. Immunohistochemistry revealed only minor differences in the distribution of fibronectin, laminin, collagen types I, and IV, whereas collagen type III appeared differentially distributed in the two embryos. Chondroitin- and chondroitin-6-sulfate-rich proteoglycans were more prevalent in the white mutant embryo than in the dark, especially in the subepidermal space. Membrane microcarriers were utilized to explant site-specifically native ECM for biochemical analysis. Two-dimensional gel electrophoresis of these regional matrices revealed a number of differences in their protein content, principally in constituents of apparent molecular masses of 30-90,000. Taken together our observations suggest that local divergences in the concentration/assembly of low and high molecular mass proteins and proteoglycans of the ECM encountered by the moving neural crest cells account for their disparate migratory behavior in the white mutant axolotl.

Ambystoma↗

PubMed1989

Molecular mechanisms of avian neural crest cell migration on fibronectin and laminin.

We have examined the molecular interactions of avian neural crest cells with fibronectin and laminin in vitro during their initial migration from the neural tube. A 105-kDa proteolytic fragment of fibronectin encompassing the defined cell-binding domain (65 kDa) promoted migration of neural crest cells to the same extent as the intact molecule. Neural crest cell migration on both intact fibronectin and the 105-kDa fragment was reversibly inhibited by RGD-containing peptides. The 11.5-kDa fragment containing the RGDS cell attachment site was also able to support migration, whereas a 50-kDa fragment corresponding to the adjacent N-terminal portion of the defined cell-binding domain was unfavorable for neural crest cell movement. In addition to the putative "cell-binding domain," neural crest cells were able to migrate on a 31-kDa fragment corresponding to the C-terminal heparin-binding (II) region of fibronectin, and were inhibited in their migration by exogenous heparin, but not by RGDS peptides. Heparin potentiated the inhibitory effect of RGDS peptides on intact fibronectin, but not on the 105-kDa fragment. On substrates of purified laminin, the extent of avian neural crest cell migration was maximal at relatively low substrate concentrations and was reduced at higher concentrations. The efficiency of laminin as a migratory substrate was enhanced when the glycoprotein occurred complexed with nidogen. Moreover, coupling of the laminin-nidogen complex to collagen type IV or the low density heparan sulfate proteoglycan further increased cell dispersion, whereas isolated nidogen or the proteoglycan alone were unable to stimulate migration and collagen type IV was a significantly less efficient migratory substrate than laminin-nidogen. Neural crest cell migration on laminin-nidogen was not affected by RGDS nor by YIGSR-containing peptides, but was reduced by 35% after addition of heparin. The predominant motility-promoting activity of laminin was localized to the E8 domain, possessing heparin-binding activity distinct from that of the N-terminal E3 domain. Migration on the E8 fragment was reduced by greater than 70% after addition of heparin. The E1' fragment supported a minimal degree of migration that was RGD-sensitive and heparin-insensitive, whereas the primary heparin-binding E3 fragment and the cell-adhesive P1 fragment were entirely nonpermissive for cell movement.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Timing in the regulation of neural crest cell migration: retarded "maturation" of regional extracellular matrix inhibits pigment cell migration in embryos of the white axolotl mutant.

In larvae of the white axolotl mutant (Ambystoma mexicanum), contrary to normal dark ones, trunk pigmentation is restricted because the epidermis is unable to support subepidermal migration of pigment cells from the neural crest (NC). This study examines whether the subepidermal extracellular matrix (ECM) is the defective component which prevents pigment cell migration in the white embryo. We transplanted subepidermal ECM, adsorbed in vivo on membrane microcarriers, from and to white and dark embryos in various combinations. White embryos have demonstrated normal NC cell migration along the medioventral pathway, and in order to test the effects of medial ECM on subepidermal migration, this ECM was similarly transplanted. Carriers with ECM attached were inserted subepidermally in host embryos at a premigratory NC stage. Control carriers without ECM and carriers with subepidermal ECM from white donors did not affect NC cell migration in white or dark embryos. In contrast, subepidermal ECM from dark donors triggered NC cell migration in the subepidermal space of both white and dark hosts. Remarkably, subepidermal ECM from white donors which were older than those normally used also stimulated migration in embryos of both strains. Likewise, medial ECM from white donors elicited migration in white as well as dark hosts. Pigment cells occurred among those NC cells that were stimulated to migrate in response to contact with ECM on carriers. These results indicate that the subepidermal ECM of the white embryo is transiently defective as a substrate for pigment cell migration, implying that "maturation" of the ECM is retarded beyond the times during which pigment cells are able to respond. In contrast, the medial ECM of the white embryo appears to mature normally. These findings suggest that the effect of the d gene is expressed regionally through the subepidermal ECM during a limited period of development. Hence, the action of the d gene seems to retard ECM maturation, bringing it out of phase with the migratory capability of the pigment cells. We propose that such a shift in relative timing of the developmental phenomena involved inhibits pigment cell migration in embryos of the white axolotl mutant and, accordingly, that the restricted pigmentation of the mutant larva is generated through heterochrony.

Ambystoma mexicanum↗

PubMed1988

Local embryonic matrices determine region-specific phenotypes in neural crest cells.

Membrane microcarriers were used to determine the ability of regional extracellular matrices to direct neural crest cell differentiation in culture. Neural crest cells from the axolotl embryo responded to extracellular matrix material explanted from the subepidermal migratory pathway by dispersing and by differentiating into pigment cells. In contrast, matrix material from the presumptive site of dorsal root ganglia stimulated pronounced cell-cell association and neurotypic expression. Cell line segregation during ontogeny of the neural crest that leads to diversification into pigment cells of the skin or into elements of the peripheral nervous system appears to be controlled in part by local cell-matrix interactions.

Ambystoma mexicanum↗