Substratum effects on cell dispersal, morphology, and differentiation in cultures of avian neural crest cells

Adhesive extracellular matrix (ECM) molecules appear to play roles in the migration of neural crest cells, and may also provide cues for differentiation of these cells into a variety of phenotypes. We are studying the influences of specific ECM components on crest differentiation at the levels of both individual cells and cell populations. We report here that the glycoproteins fibronectin and laminin differentially affect melanogenesis in cultures of avian neural crest-derived cells. Clusters of neural crest cells were allowed to form on explanted neural tubes for 24 and 48 hr, and then subcultured on uncoated glass coverslips or coverslips coated with fibronectin or laminin. The morphology of cells varied on the three substrata, as did patterns of cell dispersal. Crest cells dispersed most rapidly and extensively on fibronectin. In contrast, cells on laminin dispersed initially, but then assumed a stellate morphology and rapidly formed small aggregates. Cell dispersal was minimal on glass substrata, resulting in a uniformly dense distribution. These patterns of dispersal were similar in subcultures of both 24- and 48-hr clusters, although dispersal of cells from older clusters was less extensive. The rate and extent of melanogenesis correlated with patterns of cell dispersal. Cell from 24-hr clusters underwent melanogenesis significantly more slowly on fibronectin than on the other two substrata. Pigment cells began to differentiate by 2 days of subculture in the cell aggregates on laminin and in the dense centers of cultures on untreated glass. By 5 days, there was significantly more melanogenesis in cultures on laminin and glass than on fibronectin substrata. Melanogenesis in cultures of 48-hr clusters was more rapid and extensive on control (glass) substrata than on fibronectin or laminin, correlating with reduced cell dispersal. We conclude that fibronectin and laminin, which are found along neural crest migratory pathways in vivo, can affect melanogenesis in vitro by regulating patterns of cell dispersal.     

Laminin induces the stable expression of surface galactosyltransferase on lamellipodia of migrating cells

We have previously shown that cell surface galactosyltransferase (GalTase) mediates cell spreading and migration on basal lamina matrices by binding N-linked oligosaccharide substrates within laminin. In this study we have examined the distribution and expression of cell surface GalTase during mesenchymal cell migration on various extracellular matrices. Antisera raised against affinity-purified beta 1,4 GalTase, as well as anti-GalTase Fab fragments, inhibited cell migration on laminin-containing matrices, whereas under identical conditions, anti-GalTase IgG had no effect on the rate of cell migration on fibronectin substrates. Cells migrating on laminin had three times the level of surface GalTase, assayed by 125I-antibody binding and by direct enzyme assay, than similar cells migrating on fibronectin. On the other hand, total cellular GalTase, assayed either enzymatically or by Northern blot analysis, was similar when cells were grown on laminin or fibronectin. The laminin-dependent increase in surface GalTase was due to its expression onto the leading and trailing edges of migrating cells in association with actin-containing microfilaments assayed by double-label indirect immunofluorescence. On stationary cells, surface GalTase levels were low, but as cells began to migrate on laminin GalTase became polarized to the growing lamellipodia. GalTase was not detectable on lamellipodia or filopodia when cells migrated on fibronectin substrates. These results show that laminin-containing matrices induce the stable expression of GalTase onto cell lamellipodia and filopodia where it mediates subsequent cell spreading and migration. Since fibronectin was unable to induce GalTase expression onto lamellipodia, these studies also suggest that the extracellular matrix can selectively influence which intracellular components are maintained on the cell surface.     

In vitro studies on the control of nerve fiber growth by the extracellular matrix of the nervous system

1. Cultured neurons from embryonic chick sympathetic ganglia or dorsal root ganglia grow nerve fibers extensively on simple substrata containing fibronectin, collagens (types I, III, IV), and especially laminin. 2. The same neurons cultured on substrata containing glycosaminoglycans grow poorly. Glycosaminoglycans (heparin) inhibit nerve fiber growth on fibronectin substrata. 3. Proteolytic fragments of fibronectin support nerve fiber growth only when the cell attachment region is intact. For example, a 105 kD fragment, encompassing the cell attachment region, supports growth when immobilized in a substratum, but a 93 kD subfragment, lacking the cell attachment region, is unable to support fiber growth. When it is added to the culture medium, the 105 kD fragment inhibits fiber growth on substrata containing native fibronectin. 4. In culture medium lacking NGF, DRG neurons extend nerve fibers only on laminin and not on fibronectin, collagen or polylysine. Studies with radioiodinated laminin indicate that laminin binds with a relatively high affinity (kd approximately equal to 10(-9) M) to DRG neurons, and to a variety of other neural cells (NG108 cells, PC12 cells, rat astrocytes, chick optic lobe cells). We have isolated a membrane protein (67 kD) by affinity chromatography on laminin columns and are characterizing this putative laminin receptor. 5. Dissociated DRG neurons or ganglionic explants cultured on complex substrata consisting of tissue sections of CNS or PNS tissues extend nerve fibers onto the PNS (adult rat sciatic nerve) but not CNS (adult rat optic nerve) substrata. Other tissue substrata which support fiber growth in vivo (embryonic rat spinal cord, goldfish optic nerve) support growth in culture. While substrata from adult CNS, which support meager regeneration in vivo (adult rat spinal cord) support little fiber growth in culture. 6. Ganglionic explants cultured in a narrow space between a section of rat sciatic nerve and optic nerve grow preferentially onto the sciatic nerve suggesting that diffusible growth factors are not responsible for the differential growth on the two types of tissues. 7. Dissociated neurons adhere better to sections of sciatic nerve than optic nerve. Laminin, rather than fibronectin or heparan sulfate proteoglycan, is most consistently identifiable by immunocytochemistry in tissues (sciatic nerve, embryonic spinal cord, goldfish optic nerve) which support nerve fiber growth. Taken together, these data suggest that ECM adhesive proteins are important determinants of nerve regeneration.     

Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti- adhesive

Regulation by the extracellular matrix (ECM) of migration, motility, and adhesion of olfactory neurons and their precursors was studied in vitro. Neuronal cells of the embryonic olfactory epithelium (OE), which undergo extensive migration in the central nervous system during normal development, were shown to be highly migratory in culture as well. Migration of OE neuronal cells was strongly dependent on substratum- bound ECM molecules, being specifically stimulated and guided by laminin (or the laminin-related molecule merosin) in preference to fibronectin, type I collagen, or type IV collagen. Motility of OE neuronal cells, examined by time-lapse video microscopy, was high on laminin-containing substrata, but negligible on fibronectin substrata. Quantitative assays of adhesion of OE neuronal cells to substrata treated with different ECM molecules demonstrated no correlation, either positive or negative, between the migratory preferences of cells and the strength of cell-substratum adhesion. Moreover, measurements of cell adhesion to substrata containing combinations of ECM proteins revealed that laminin and merosin are anti-adhesive for OE neuronal cells, i.e., cause these cells to adhere poorly to substrata that would otherwise be strongly adhesive. The evidence suggests that the anti- adhesive effect of laminin is not the result of interactions between laminin and other ECM molecules, but rather an effect of laminin on cells, which alters the way in which cells adhere. Consistent with this view, laminin was found to interfere strongly with the formation of focal contacts by OE neuronal cells.     

Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF): I. Effects on short-term adhesiveness and on long-term organization of the culture

A growth factor (EDGF) derived from the retina controls the proliferation and shape of adult bovine epithelial lens (BEL) cells in vitro as well as extracellular matrix (ECM) assembly. In order to analyse this mechanism and the specificity of the interactions between BEL cells and the extracellular matrix we have investigated the adhesion and growth of BEL cells on various substrata (fibronectin, laminin, ECM). BEL cells treated with EDGF adhered more slowly to plastic Petri dishes than untreated cells, in part due to EDGF inhibition of fibronectin deposition. The untreated BEL cells spread less well on ECM or laminin than on fibronectin-coated plastic. The preferential adhesiveness of BEL cells on fibronectin vs laminin was confirmed by attachment experiments performed on replicas of SDS-PAGE of these proteins. However, in long-term cultures, 8 days after seeding, BEL cells were very differently arranged on plastic or on ECM. ECM by itself did not increase the proliferation rate but helped to restore an organized cell monolayer. BEL cells stimulated to grow on ECM by treatment with EDGF exhibited at least transiently contact inhibition producing a perfectly organized epithelium similar to the one observed in vivo. These results suggest specific interactions between ECM or ECM components with BEL cell that restrain excessive cell spreading and restore an original polarized phenotype of the cells seen in vivo.     

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

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

Interactions of rat hepatocytes with type IV collagen, fibronectin and laminin matrices. Distinct matrix-controlled modes of attachment and spreading

We have examined the interaction of adult rat hepatocytes in primary culture, to type IV collagen, fibronectin, and laminin, the major basement membrane proteins of normal rat liver. Culture substrata consisted of glass coverslips, which were covalently derivatized with individual purified basement membrane constituents at varying densities of protein. The attachment of freshly prepared hepatocytes was examined after incubation at 37 degrees C for 30 min as a function of the amount of protein on the coverslips. For each of the three types of substratum under study, distinct modes of cell attachment were observed, with the apparent affinity of hepatocytes for type IV collagen being three-fold greater than for fibronectin and ten-fold greater than for laminin. Cell attachment exhibited saturation on all substrata. Hepatocyte spreading was measured by scanning electron microscopy of cells incubated at 37 degrees for 2 h on similarly prepared coverslips. A five-fold greater surface density of type IV collagen was required for maximal spreading compared with attachment. For cells on fibronectin or laminin the maximal cell spreading reached on type IV collagen did not occur even at coverslip protein densities 10 to 20 times those providing for maximal cell attachment. A very similar qualitative pattern of cell proteins was secreted within a few hours of plating on the various substrata and further studies failed to reveal any evidence that attachment and spreading was mediated by endogenously produced matrix molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of basal epithelial cell surface and Cytoskeleton to solubilized extracellular matrix molecules

Corneal epithelium removed from underlying extracellular matrix (ECM) extends numerous cytoplasmic processes (blebs) from the formerly smooth basal surface. If blebbing epithelia are grown on collagen gels or lens capsules in vitro, the basal surface flattens and takes on the smooth contour typical of epithelium in contact with basal lamina in situ. This study examines the effect of soluble extracellular matrix components on the basal surface. Corneal epithelia from 9- to 11-d-old chick embryos were isolated with trypsin-collagenase or ethylenediamine tetraacetic acid, then placed on Millipore filters (Millipore Corp., Bedford, Mass.), and cultured at the medium-air interface. Media were prepared with no serum, with 10% of calf serum, or with serum from which plasma fibronectin was removed. Epithelia grown on filters in this medium continue to bleb for the duration of the experiments (12-14 h). If soluble collagen, laminin, or fibronectin is added to the medium, however, blebs are withdrawn and by 2-6 h the basal surface is flat. Epithelia grown on filters in the presence of albumin, IgG, or glycosaminoglycans continue to bleb. Epithelia cultured on solid substrata, such as glass, also continue to bleb if ECM is absent from the medium. The basal cell cortex in situ contains a compact cortical mat of filaments that decorate with S-1 myosin subfragments; some, if not all, of these filaments point away from the plasmalemma. The actin filaments disperse into the cytoplasmic processes during blebbing and now many appear to point toward the plasmalemma. In isolated epithelia that flatten in response to soluble collagens, laminin, and fibronectin, the actin filaments reform the basal cortical mat typical or epithelial in situ. Thus, extracellular macromolecules influence and organize not only the basal cell surface but also the actin-rich basal cell cortex of epithelial cells.     

Laminin alters cell shape and stimulates motility and proliferation of murine skeletal myoblasts

Proliferating skeletal myoblasts show multiple specific responses to laminin, one of the major glycoprotein components of basement membranes. Using MM14Dy myoblasts, a myogenic cell strain derived from a normal adult mouse skeletal muscle, we show in this study that substrate-bound laminin but not other matrix proteins such as collagens or fibronectin specifically and rapidly induces the outgrowth of cell processes, resulting in bipolar, spindle-shaped cells. This effect is independent from the presence of collagens or serum, and was also observed in primary cultures of fetal mouse skeletal myoblasts. The outgrowth of cell processes on laminin is associated with a dramatic stimulation of cell motility: MM14 myoblasts migrate about five times faster on laminin than on fibronectin. In another series of experiments the effect of laminin and fibronectin on thymidine uptake and proliferation of myoblasts was tested. On top of a type I collagen substrate which was provided to ensure complete adhesion even at low doses of laminin or fibronectin, laminin stimulated myoblast proliferation and incorporation of [3H]thymidine in a dose-dependent manner. The stimulation is two- to threefold higher than on dishes coated with equivalent amounts of fibronectin and is observed both in the presence and in the absence of serum. These results suggest that laminin, a major component of the muscle basal lamina, may be actively involved in the development and regeneration of skeletal muscle.     

Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata

We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line. In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil. Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin. With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin. With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+. Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata. The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin. Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA. Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation. These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.     

Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding.

The localization of the extracellular matrix recognition molecule J1/tenascin was investigated in the crypt-villus unit of the adult mouse ileum by immunoelectron microscopic techniques. In the villus region, J1/tenascin was detected strongly in the extracellular matrix (ECM) between fibroblasts of the lamina propria. It was generally absent in the ECM at the interface between subepithelial fibroblasts and intestinal epithelium, except for some restricted areas along the epithelial basal lamina of villi, but not of crypts. These restricted areas corresponded approximately to the basal part of one epithelial cell. In J1/tenascin-positive areas, epithelial cells contacted the basal lamina with numerous microvillus-like processes, whereas in J1/tenascin-negative areas the basal surface membranes of epithelial cells contacted their basal lamina in a smooth and continuous apposition. In order to characterize the functional role of J1/tenascin in the interaction between epithelial cells and ECM, the intestinal epithelial cell line HT-29 was tested for its ability to adhere to different ECM components. Cells adhered to substratum-immobilized fibronectin, laminin and collagen types I to IV, but not to J1/tenascin. When laminin or collagen types I to IV were mixed with J1/tenascin, cell adhesion was as effective as without J1/tenascin. However, adhesion was completely abolished when cells were offered a mixture of fibronectin and J1/tenascin as substratum. The ability of J1/tenascin to reduce the adhesion of intestinal epithelial cells to their fibronectin-containing basal lamina suggests that J1/tenascin may be involved in the process of physiological cell shedding from the villus.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

Neural crest migration in 3D extracellular matrix utilizes laminin, fibronectin, or collagen

The trunk neural crest originates by transformation of dorsal neuroepithelial cells into mesenchymal cells that migrate into embryonic interstices. Fibronectin (FN) is thought to be essential for the process, although other extracellular matrix (ECM) molecules are potentially important. We have examined the ability of three dimensional (3D) ECM to promote crest formation in vitro. Neural tubes from stage 12 chick embryos were suspended within gelling solutions of either basement membrane (BM) components or rat tail collagen, and the extent of crest outgrowth was measured after 22 hr. Fetal calf serum inhibits outgrowth in both gels and was not used unless specified. Neither BM gel nor collagen gel contains fibronectin. Extensive crest migration occurs into the BM gel, whereas outgrowth is less in rat tail collagen. Addition of fibronectin or embryo extract (EE), which is rich in fibronectin, does not increase the extent of neural crest outgrowth in BM, which is already maximal, but does stimulate migration into collagen gel. Removal of FN from EE with gelatin-Sepharose does not remove the ability of EE to stimulate migration. Endogenous FN is localized by immunofluorescence to the basal surface of cultured neural tubes, but is not seen in the proximity of migrating neural crest cells. Addition of the FN cell-binding hexapeptide GRGDSP does not affect migration into either the BM gel or the collagen gel with EE, although it does block spreading on FN-coated plastic. Thus, although crest cells appear to use exogenous fibronectin to migrate on planar substrata in vitro, they can interact with 3D collagenous matrices in the absence of exogenous or endogenous fibronectin. In BM gels, the laminin cell-binding peptide, YIGSR, completely inhibits migration of crest away from the neural tube, suggesting that laminin is the migratory substratum. Indeed, laminin as well as collagen and fibronectin is present in the embryonic ECM. Thus, it is possible that ECM molecules in addition to or instead of fibronectin may serve as migratory substrata for neural crest in vivo.     

Integrin-mediated migration of murine B82L fibroblasts is dependent on the expression of an intact epidermal growth factor receptor

To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell rounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lines. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors. Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF. The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration.     

Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cell model

Myogenesis is a complex process in which committed myogenic cells differentiate and fuse into myotubes that mature into the muscle fibres of adult organisms. This process is initiated by a cascade of myogenic regulatory factors expressed upon entry of the cells into the myogenic differentiation programme. However, external signals such as those provided by the extracellular matrix (ECM) are also important in regulating muscle differentiation and morphogenesis. In the present work, we have addressed the role of various ECM substrata on C2C12 myoblast behaviour in vitro. Cells grown on fibronectin align and fuse earlier than cells on laminin or gelatine. Live imaging of C2C12 myoblasts on fibronectin versus gelatine has revealed that fibronectin promotes a directional collective migratory behaviour favouring cell-cell alignment and fusion. We further demonstrate that this effect of fibronectin is mediated by RGD-binding integrins expressed on myoblasts, that N-cadherin contributes to this behaviour, and that it does not involve enhanced myogenic differentiation. Therefore, we suggest that the collective migration and alignment of cells seen on fibronectin leads to a more predictable movement and a positioning that facilitates subsequent fusion of myoblasts. This study highlights the importance of addressing the role of fibronectin, an abundant component of the interstitial ECM during embryogenesis and tissue repair, in the context of myogenesis and muscle regeneration.     

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.     

Avian vasculogenesis and the distribution of collagens I, IV, laminin, and fibronectin in the heart primordia

The heart-forming regions of the early embryo are composed of splanchnic mesoderm, endoderm, and the associated ECM. The ECM of the heart-forming regions in stage 7-9 chicken embryos was examined using immunofluorescence. Affinity purified antibodies to chicken collagens type I and IV, chicken fibronectin, and mouse laminin were used as probes. We report that (1) the basement membrane of the endoderm contains immunoreactive laminin and collagen IV; (2) the nascent basement membrane of the heart splanchnic mesoderm contains immunoreactive laminin, but not type IV collagen, and (3) the prominent ECM between the splanchnic mesoderm and the endoderm (the primitive-heart ECM) contains collagen IV, collagen I, fibronectin, but not laminin. In addition, we describe microscopic observations on the spatial relationship of cardiogenic cells to the primitive-heart ECM and the endodermal basement membrane.     

Interaction of embryonic corneal epithelium with exogenous collagen,laminin, and fibronectin: Role of endogenous protein synthesis

Previously, we have shown that the embryonic corneal epithelium is capable of interacting with exogenous collagen, laminin, and fibronectin in soluble form, each of which causes isolated epithelium cultured on Millipore filter to stop blebbing, reorganize the basal cytoskeleton, and flatten. Here we examine the involvement of endogenously derived extracellular matrix (ECM) molecules in the interaction of the basal epithelial cell surface with the added ECM molecules. We demonstrate here that the isolated avian corneal epithelium cultured on Millipore filter is capable of synthesizing collagens and laminin, but not fibronectin. To examine whether the epithelium is capable of interacting directly with exogenous ECM components or if there is the necessity for production of a linker molecule, epithelial protein synthesis was inhibited with cycloheximide (CHX). The blebbing epithelium in the presence of CHX was then confronted with soluble ECM molecules added to the medium under the filter; such epithelia are able to interact with, and flatten in response to, both collagen and laminin. However, such inhibited epithelia continue to bled in the presence of fibronectin. We next used l-azetidine-4-carboxylic acid (LACA) to interfere with collagen secretion. Epithelia exposed to LACA are still capable of interacting with collagen and laminin, but not fibronectin, indicating a dependence on collagen secretion. These results suggest that fibronectin requires a linker protein, probably collagen, to interact with the basal epithelial surface, whereas both collagen and laminin may interact directly with the cell surface to transform the basal cytoskeleton into the cortical mat typical of differentiating corneal epithelium in situ.     

Differential response of arterial and venous endothelial cells to extracellular matrix is modulated by oxygen

Binding of endothelial cell (EC) integrins to extracellular-matrix (ECM) components is one of the key events to trigger intracellular signaling that will ultimately result in proper vascular development. Even within one tissue, the endothelial phenotype differs between arteries and veins. Here, we tested the hypothesis that anchorage-dependent processes, such as proliferation, viability, survival and actin organization of venous (VEC) and arterial EC (AEC) differently depend on ECM proteins. Moreover, because of different oxygen tension in AEC and VEC, we tested oxygen as a co-modulator of ECM effects. Primary human placental VEC and AEC were grown in collagens I and IV, fibronectin, laminin, gelatin and uncoated plates and exposed to 12 and 21% oxygen. Our main findings revealed that VEC are more sensitive than AEC to changes in the ECM composition. Proliferation and survival of VEC, in contrast to AEC, were profoundly increased by the presence of collagen I and fibronectin when compared with gelatin or uncoated plates. These effects were reversed by inhibition of focal adhesion kinase (Fak) and modulated by oxygen. VEC were more susceptible to the oxygen-dependent ECM effects than AEC. However, no differential ECM effect on actin organization was observed between the two cell types. These data provide first evidence that AEC and VEC from the same vascular loop respond differently to ECM and oxygen in a Fak-dependent manner.     

Bronchial branching correlates with specific glycosidase activity, extracellular glycosaminoglycan accumulation, TGF beta(2), and IL-1 localization during chick embryo lung development.

During organ differentiation, cell-extracellular matrix (ECM) interactions are required. The components of the ECM, such as glycosaminoglycans, fibronectin, laminin, and collagens, change in relation to cytokine and enzyme activity. Moreover, glycosaminoglycans (GAGs) are components of the ECM that play an important role in both cytokine regulation and cell activities. In this work we studied the accumulation of hyaluronic acid and chondroitin sulfate and heparan sulfate proteoglycans (PGs), beta-N-acetyl-D-glucosaminidase activity, the presence of transforming growth factor beta(2) (TGF beta(2)), and interleukin-1 (IL-1), and the localization of fibronectin, laminin, and collagen I and IV during the early stages of chick embryo lung development. We also determined the levels of hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate GAGs and the activity of beta-N-acetyl-D-glucosaminidase with biochemical methods. Our data show that beta-N-acetyl-D-glucosaminidase activity increases in each cell, especially in the epithelial growth front at the emergence of each bronchial bud, where hyaluronic acid and IL-1 are located in the surrounding mesenchymal areas. Chondroitin sulfate and heparan sulfate PGs, fibronectin, laminin, and collagen I and IV are evident in the area near the basal membrane along the sides where the forming structures are stabilized. Biochemical data show that beta-N-acetyl-D-glucosaminidase activity increases in cells during lung development and is related to GAG decrease and to modifications of the nonsulfated/sulfated GAG ratio. These modifications could change cytokine activity and play an important role in bronchial branching development.