Neural Crest Cell Adhesion Research Articles

The novel ECM protein SNED1 mediates cell adhesion via α5β1 integrin.

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays critical roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by α5β1integrin. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis.

Aberrant neural crest development causes craniofacial and other malformations in an animal model of Musculocontractural Ehlers-Danlos syndrome

Musculocontractural Ehlers-Danlos syndrome (MC-EDS) is a rare recessive disorder that is characterized by connective tissue fragility, distinct craniofacial features and congenital malformations. MC-EDS patients have defects in the enzymes dermatan sulfate epimerase-1 and dermatan 4-O-sulfotransferase-1, which are involved in the biosynthesis of iduronic acid in the chondroitin sulfate/dermatan sulfate (CS/DS) chains of proteoglycans (PGs). While the connective tissue defect is a result of disturbed collagen fibril assembly based on a decreased iduronic acid content of interacting CS/DS-PGs, the cause of the developmental malformations in MC-EDS is not well understood. This review focuses on a new role of CS/DS-PGs in the development of multipotent and highly migratory neural crest (NC) cells in the Xenopus embryo model of MC-EDS. Single iduronic acid residues in CS/DS-PGs are involved in the formation of NC-derived craniofacial structures by facilitating the migration and adhesion of NC cells to fibronectin. Our results suggest a defect in NC development as cause of the craniofacial and other congenital anomalies in MC-EDS patients, which might contribute to an improved diagnosis and etiology-based therapy. (Less)

PINCH‐1 expression during early avian embryogenesis: Implications for neural crest and heart development

The invasion of the cardiac neural crest (CNC) into the outflow tract (OFT) and subsequent OFT septation are critical events during vertebrate heart development. We previously had performed four modified differential display (DD) screens in the chick embryo to identify genes that may be involved in CNC and heart development. Full-length sequence of one of the DD clones has been obtained and identified as chick PINCH-1. This particularly interesting new cysteine-histidine-rich protein contains five protein-binding LIM domains (five double zinc fingers), a nuclear localization signal, and a nuclear export signal, allowing it to participate in integrin and growth factor signaling and possibly act as a transcription factor. We show here for the first time that chick PINCH-1 is expressed in neural crest cells, both in the neural fold and cardiac OFT, and is also expressed in mesoderm derived-structures, including the myocardium, during avian embryogenesis. The normal expression pattern and overexpression in neural crest cell explants suggest that PINCH-1 may be a regulator of neural crest cell adhesion and migration.

Central role of the alpha4beta1 integrin in the coordination of avian truncal neural crest cell adhesion, migration, and survival.

Based on functional and histological studies, the fibronectin receptor of the integrin family alpha4beta1 has been ascribed a critical role during neural crest cell migration in the vertebrate embryo. In the present study, because integrins have been shown to participate in multiple basic cellular processes, including cell adhesion, migration, survival, proliferation, and differentiation, we have reexamined in detail the role of alpha4beta1 during avian truncal neural crest cell migration. RT-PCR and immunocytochemical studies revealed that migrating neural crest cells but not premigratory cells explanted in vitro expressed detectable levels of alpha4 messengers and proteins suggesting that alpha4beta1 expression was induced at the time of the initiation of the migration phase. In agreement with this observation, antibody inhibition of alpha4beta1 activity in vitro resulted in a strong, immediate and sustained reduction of neural crest cell motion on fibronectin, as judged on videomicroscopy analyses, but apparently did not prevent their delamination from the neural tube. However, alpha4beta1 appeared to exhibit a broader role in the control of cell migration on a variety of extracellular matrix molecules, presumably by regulating cellular events downstream from integrins. Moreover, blocking alpha4beta1 function caused a severe increase in apoptotic cell death among the neural crest population without influencing notably cell proliferation. Collectively, these results indicate that, notwithstanding its critical implication in cell motion, alpha4beta1 integrin could play a central role in neural crest cell development by coordinating multiple cellular events, such as cell adhesion, locomotion, and survival.

F-spondin is a contact-repellent molecule for embryonic motor neurons.

The floor plate plays a key role in patterning axonal trajectory in the embryonic spinal cord by providing both long-range and local guidance cues that promote or inhibit axonal growth toward and across the ventral midline of the spinal cord, thus acting as an intermediate target for a number of crossing (commissural) and noncrossing (motor) axons. F-spondin, a secreted adhesion molecule expressed in the embryonic floor plate and the caudal somite of birds, plays a dual role in patterning the nervous system. It promotes adhesion and outgrowth of commissural axons and inhibits adhesion of neural crest cells. In the current study, we demonstrate that outgrowth of embryonic motor axons also is inhibited by F-spondin protein in a contact-repulsion fashion. Three independent lines of evidence support our hypothesis: substrate-attached F-spondin inhibits outgrowth of dissociated motor neurons in an outgrowth assay; F-spondin elicits acute growth cone collapse when applied to cultured motor neurons; and challenging ventral spinal cord explants with aggregates of HEK 293 cells expressing F-spondin, causes contact-repulsion of motor neurites. Structural-functional studies demonstrate that the processed carboxyl-half protein that contains the thrombospondin type 1 repeats is more prominent in inhibiting outgrowth, suggesting that the processing of F-spondin is important for enhancing its inhibitory activity.

Phosphotyrosine signalling as a regulator of neural crest cell adhesion and motility.

We demonstrate that neural crest cell-cell adhesion, cell-substrate adhesion, and ultimately cell motility, are highly dependent on the balanced action of tyrosine kinases and tyrosine phosphatases. Neural crest cell migration on fibronectin is diminished in the presence of the tyrosine phosphatase inhibitor vanadate or tyrosine kinase inhibitor herbimycin A, while cadherin-rich cell-cell adhesions are significantly increased. In contrast, cells treated with the kinase inhibitor genistein have decreased motility, rearrange rapidly and reversibly into a pavement-like monolayer, but have no increase in cadherin interactions. Genistein-sensitive tyrosine kinases may therefore abrogate a latent sensitivity of neural crest cells to contact-mediated inhibition of movement. Furthermore, we show that the activity of herbimycin A-sensitive kinases is necessary for focal adhesion formation in these cells. Moreover, the size and distribution of these adhesions are acutely sensitive to the actions of tyrosine phosphatases and genistein-sensitive kinases. We propose that in migrating neural crest cells there is a balance in phosphotyrosine signalling which minimises both cell-cell adhesion and contact inhibition of movement, while enhancing dynamic cell-substrate interactions and thus the conditions for motility.

ADAM 13: A Novel ADAM Expressed in Somitic Mesoderm and Neural Crest Cells duringXenopus laevisDevelopment

Embryonic development involves a series of cell adhesive interactions that provide mechanical and instructive information required for morphogenesis. The ADAMs family of membrane-anchored proteins, containinga disintegrinandmetalloprotease domain, is well suited for participating in such developmental events. They encode not only a potential adhesive function, through an integrin-binding disintegrin domain, but also a potential antiadhesive function, through a zinc-dependent metalloprotease domain. In order to investigate the role of ADAMs in early development we cloned a cDNA encoding a novel member of the ADAM family from aXenopus laevisneurula stage library. We call this cDNA, and the 915-amino-acid protein it encodes, ADAM 13. X-ADAM 13 RNA is expressed during embryogenesis from the midblastula stage through tadpole stage 45. X-ADAM 13 is localized to somitic mesoderm and cranial neural crest cells during gastrulation, neurulation, and in tail bud stages. Sequence analyses of the X-ADAM 13 metalloprotease and disintegrin domains indicate that the protein is likely to be involved in both proteolytic and cell-adhesive functions. The X-ADAM 13 sequence is most closely related to that of mouse meltrin α, which is implicated in myoblast fusion. Our data suggest that X-ADAM 13 may be involved in neural crest cell adhesion and migration as well as myoblast differentiation.

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.

Repertoire and function of the fibronectin-binding integrins in avian neural crest cell adhesion and migration

Repertoire and function of the fibronectin-binding integrins in avian neural crest cell adhesion and migration

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

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

Neural Crest Cell Interactions with Laminin: Structural Requirements and Localization of the Binding Site for α1β1 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 α-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 α1 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 α1, whereas later attachment and migration were mediated by α1 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.

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 Mr 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-β1 integrin antibodies, there is evidence that this class of integrins is essential for the neural crest cell-Col VI interaction.

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.

Effects of HNK-1 monoclonal antibody on the substratum attachment and survival of neural crest and sclerotome cells in culture.

The sclerotome portion of the differentiating embryonic chick somite becomes infiltrated by neural crest cells prior to its dispersal towards the embryonic axis. This means that sclerotome cells explanted into culture for the purpose of examining their interactions in vitro are contaminated with a proportion of neural crest cells. The purpose of this study was to explore the neural crest cell adhesion epitope recognized by the HNK-1 monoclonal antibody and ways in which this antibody can be used to eliminate neural crest cells from mixed culture by selective cytotoxicity. Immunofluorescence technique, under the conditions used here, indicated that the antibody appeared to stain all the mesenchymal (i.e. neural crest) cells emigrating from pieces of embryonic neural tube in culture. Examination of the effects of HNK-1 suggests that the antibody interacts with substratum-binding sites on the neural crest cell surface. On fibronectin-coated substrata the antibody tended to inhibit neurite outgrowth but left the cells relatively well spread, while on laminin substrata the effect was to discourage both neurite extension and cell spreading, causing cell retraction. These results suggest that the cell surface epitope recognized by HNK-1 influences neurite outgrowth, neurite adhesiveness or both. Failure of cell spreading on laminin suggests interaction with the laminin binding sites on the cell body. Elimination of the crest cells from mixed culture with sclerotome was achieved by culturing the cells in the presence of HNK-1 antibody and complement during the period required for complete cell outgrowth from the sclerotome explant.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell adhesion and migration in the early vertebrate embryo: location and possible role of the putative fibronectin receptor complex.

Using a combined in vivo and in vitro approach, we have analyzed the immunofluorescent localization and function of a 140,000-mol-wt glycoprotein complex implicated in cell adhesion to fibronectin (FN), with particular emphasis on neural crest cell adhesion and migration. This putative fibronectin receptor complex (FN-receptor) was detectable in almost all tissues derived from each of the three primary germ layers. It was present in both mesenchymal and epithelial cells, and was particularly enriched at sites close to concentrations of FN, e.g., at the basal surfaces of epithelial cells. It was also present on neural crest cells. The distribution and function of this putative receptor was then analyzed on individual cells in vitro. It was diffusely organized on highly locomotory neural crest cells and somitic fibroblasts. Both motile cell types also displayed relatively low numbers of focal contacts and microfilament bundles and limited amounts of localized vinculin, alpha-actinin, and endogenous FN. In contrast, the FN-receptor in stationary embryonic cells, i.e., somitic cells after long-term culture or ectodermal cells, existed in characteristic linear patterns generally co-distributed with alpha-actinin and fibers of endogenous FN. Anti-FN-receptor antibodies inhibited the adhesion to FN of motile embryonic cells, but not of stationary fibroblasts. However, these same antibodies adsorbed to substrata readily mediated adhesion and spreading of cells, but were much less effective for cell migration. Our results demonstrate a widespread occurrence in vivo of the putative FN-receptor, with high concentrations near FN. Embryonic cell migration was associated with a diffuse organization of this putative receptor on the cell surface in presumably labile adhesions, whereas stationary cells were anchored to the substratum at specific sites linked to the cytoskeleton near local concentrations of FN-receptor.

Effects of alterations of cell surface carbohydrates on adhesion of neural crest cells of [formula omitted]: [formula omitted], Dept. of Anatomy, University of Alberta, Edmonton, Alberta, Canada. T6G 2H7

Effects of alterations of cell surface carbohydrates on adhesion of neural crest cells of [formula omitted]: [formula omitted], Dept. of Anatomy, University of Alberta, Edmonton, Alberta, Canada. T6G 2H7

Neural crest cell migration: requirements for exogenous fibronectin and high cell density.

Cells of the neural crest participate in a major class of cell migratory events during embryonic development. From indirect evidence, it has been suggested that fibronectin (FN) might be involved in these events. We have directly tested the role of FN in neural crest cell adhesion and migration using several in vitro model systems. Avian trunk neural crest cells adhered readily to purified plasma FN substrates and to extracellular matrices containing cellular FN. Their adhesion was inhibited by antibodies to a cell-binding fragment of FN. In contrast, these cells did not adhere to glass, type I collagen, or to bovine serum albumin in the absence of FN. Neural crest cell adhesion to laminin (LN) was significantly less than to FN; however, culturing of crest cells under conditions producing an epithelioid phenotype resulted in cells that could bind equally as well to LN as to FN. The migration of neural crest cells appeared to depend on both the substrate and the extent of cell interactions. Cells migrated substantially more rapidly on FN than on LN or type I collagen substrates; if provided a choice between stripes of FN and glass or LN, cells migrated preferentially on the FN. Migration was inhibited by antibodies against the cell-binding region of FN, and the inhibition could be reversed by a subsequent addition of exogenous FN. However, the migration on FN was random and displayed little persistence of direction unless cells were at high densities that permitted frequent contacts. The in vitro rate of migration of cells on FN-containing matrices was 50 microns/h, similar to their migration rates along the narrow regions of FN-containing extracellular matrix in migratory pathways in vivo. These results indicate that FN is important for neural crest cell adhesion and migration and that the high cell densities of neural crest cells in the transient, narrow migratory pathways found in the embryo are necessary for effective directional migration.

Role of collagen and fibronectin in neural crest cell adhesion and migration

Cultured neural crest cells which are freshly trypsinized require serum or purified fibronectin to attach to collagen substrates of types I–V. Furthermore, neural crest cells migrate in a Boyden chamber in response to fibronectin, and a “checkerboard” analysis demonstrates that fibronectin is both chemotactic and chemokinetic for these cells. It is proposed that collagen serves as a substrate for neural crest cells as they migrate in the early embryo. By mediating the cells' attachment to collagen, fibronectin may influence the movement of the cells. Local differences in fibronectin concentration or availability in the matrix could affect the degree of attachment of the cells to the collagen substrate and could also direct their migration by serving as a chemoattractant.