Neural crest cell locomotion induced by antibodies to beta 1 integrins. A tool for studying the roles of substratum molecular avidity and density in migration.

Migration of neural crest cells depends on direct, transient interactions between fibronectin molecules and their corresponding Arg-Gly-Asp integrin receptors. We have previously suggested that the moderate-activity interaction between integrin receptors and fibronectin may be critical for the transient association of the cells with their substratum. In order to test this hypothesis, we have examined the in vitro locomotory behavior of neural crest cells on substrata of differing apparent avidities for integrin receptors. As substrata, we used a variety of monoclonal and polyclonal antibodies to the integrin beta 1 subunit that were characterized for their respective relative apparent avidities for the receptor. Neural crest cells were able to migrate on these antibodies and exhibited an organization of substratum-adhesion sites and of cytoskeletal elements virtually identical to that observed on fibronectin, indicating that they can at least partially mimic the migration-promoting activity of fibronectin. However, the number of migrating cells as well as their morphology and their speed of locomotion varied significantly with both the concentration of the antibody substratum and its relative avidity for the receptor. Thus, on high-avidity monoclonal antibodies and on polyclonal divalent antibodies at high concentrations only a limited number of cells escaped from the neural tube, and the rate of their migration was reduced compared to that on fibronectin (23 +/- 5 microns h-1 versus 65 +/- 10 microns h-1). In addition, cells were unusually flattened and cohesive. Time-lapse videomicroscopy revealed that, on high-avidity substrata, neural crest cells were able to extend cell processes that adhered to the substratum, but showed a dramatically reduced capability of breaking pre-existing substratum contacts. In contrast, the same antibodies at low concentrations produced neural crest cell migration at rates very similar to those on fibronectin at the same concentrations. Low-avidity monoclonal antibodies and polyclonal monovalent antibodies at all concentrations tested permitted extensive migration of neural crest cells, which exhibited the same morphology and locomotory behavior as on fibronectin. These results indicate that both the avidity of receptors for the substratum and the number of receptors bound to the substratum are critical in regulating the locomotory behavior of neural crest cells in vitro, and therefore might help to regulate the directionality of migration and final localization pattern of neural crest cells in vivo.

Migration of chick blastoderm under the vitelline membrane: the role of fibronectin.

In the earliest stages of its development the chick blastoderm is a flattened disc at the surface of the yolk. It gradually increases in diameter, partially because the cells are rapidly proliferating, but also because the cells at the periphery (the margin of overgrowth) are migrating in a centrifugal direction. These cells utilize the inner surface of the vitelline membrane as their substratum. In the normal blastoderm, these cells at the edge of the spreading blastoderm are the only cells which are attached to the vitelline membrane. This investigation is concerned with the possible role played by fibronectin in the interaction between these migrating cells and the vitelline membrane. Chick blastoderms, explanted by the New (1955) technique have been treated with synthetic peptides that mimic the adhesive recognition signal of the fibronectin molecule. The pentapeptide GRGDS (containing the specific RGD cell adhesion sequence) caused the edge cells of the blastoderm to detach within minutes, and the expansion of the blastoderm was inhibited for about 4 hr. After this period there was gradual recovery and the cells reattached and spreading resumed. Examination of the margin of the blastoderm by scanning electron microscopy showed that cell processes were lost soon after treatment with GRGDS but concomitant with reattachment and the resumption of spreading, the cell processes reformed. The pentapeptide GRDGS (with the amino acids G and D inverted) produced a brief inhibition of spreading, but after an hour these blastoderms spread at the same rate as controls. Immunocytochemical staining with anti-fibronectin demonstrated that fibronectin was not only present at the interface of the edge cells and the vitelline membrane, but also between the epiblast and the hypoblast. These results indicate that tissue movement during blastoderm spreading is dependent upon fibronectin and that the specific RGD amino acid sequence, and presumably the VLA/integrin family of receptors, is involved in this embryonic morphogenetic movement.

Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF.

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent beta 1 integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-beta 1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin beta 1-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-beta 1 integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the beta 1 integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.