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Recent advances in defining the role of the extracellular Matrix in neural crest development
No full textDifferential neural crest cell migration on isolated collagens: role of RGD as a potential recognition site
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Molecular mechanisms of neural crest cell attachment and migration on types I and IV collagen
No full textWe 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
Collagens in avian neural crest development: distribution in vivo and migration-promoting ability in vitro
Full text linkThis 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
Molecular Mechanisms of Avian Neural Crest Cell Migration on Fibronectin and Laminin
No full textWe 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 >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. Preincubation of laminin-nidogen substrates with antisera against the E8 fragment, but not against the E1′ or the E4 fragment, potently reduced migration on the complex, further suggesting that the E8 domain is the predominant motility-promoting region of laminin. We conclude that initial neural crest cell migration on fibronectin occurs primarily through an interaction with the RGDS site within the cell-binding domain, whereas other potential attachment/motility-promoting sites may act to stabilize cell-fibronectin linkages. Neural crest cell migration on laminin is primarily mediated by the E8 domain. The efficiency of this domain as well as the ability of other potential motility-promoting domains to stimulate cell movement may be influenced by the association of laminin with other extracellular matrix molecules
Collagen type VI in neural crest development: distribution in situ and interaction with cells in vitro
No full textWe 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
An autocrine TGF-beta/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition
Full text linkData source: Supplementary material, http://www.molbiolcell.org/content/22/10/1686Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and tumor metastasis. A double-negative feedback loop involving the miR-200 family and ZEB (zinc finger E-box-binding homeobox) transcription factors has been postulated to control the balance between epithelial and mesenchymal states. Here we demonstrate using the epithelial Madin Darby canine kidney cell line model that, although manipulation of the ZEB/miR-200 balance is able to repeatedly switch cells between epithelial and mesenchymal states, the induction and maintenance of a stable mesenchymal phenotype requires the establishment of autocrine transforming growth factor-β (TGF-β) signaling to drive sustained ZEB expression. Furthermore, we show that prolonged autocrine TGF-β signaling induced reversible DNA methylation of the miR-200 loci with corresponding changes in miR-200 levels. Collectively, these findings demonstrate the existence of an autocrine TGF-β/ZEB/miR-200 signaling network that regulates plasticity between epithelial and mesenchymal states. We find a strong correlation between ZEBs and TGF-β and negative correlations between miR-200 and TGF-β and between miR-200 and ZEBs, in invasive ductal carcinomas, consistent with an autocrine TGF-β/ZEB/miR-200 signaling network being active in breast cancers.Philip A. Gregory, Cameron P. Bracken, Eric Smith, Andrew G. Bert, Josephine A. Wright, Suraya Roslan, Melanie Morris, Leila Wyatt, Gelareh Farshid, Yat-Yuen Lim, Geoffrey J. Lindeman, M. Frances Shannon, Paul A. Drew, Yeesim Khew-Goodall and Gregory J. Goodal
Neural Crest Cell Interaction with Type VI Collagen Is Mediated by Multiple Cooperative Binding Sites within Triple-Helix and Globular Domains
No full textCollagen 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 motilitypromoting 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 α1(VI)/α2(VI) and α3(VI) chains, they migrated most extensively on the α3(VI) chain. Conversely, pepsin-digested individual a 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, carboxylterminal, 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 α1(VI) and α2(VI) chains and at the amino-terminus of the α3(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-β_1 integrin antibodies, there is evidence that this class of integrins is essential for the neural crest cell-Col VI interaction
Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development
Full text linkIn 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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