Anti-fibronectin serum inhibits the disorganization of the dermal-epidermal junction in cultured wounded skin

During wound-healing in cultured frog skin fragments, fibronectin (FN) was detected in the dermal-epidermal junction. Intracellular fibronectin was stained using permeabilization and DAB immunoperoxidase. With electron microscopy intracytoplasmic FN granules were localized in the epidermal processes of the stratum germinativum cells protruding towards the dermis and in their marginal regions (membrane-associated plaques). Faint staining was visible at the level of the lamina densa and inside some parts of the lamina lucida. In comparison, contrasted ultrathin sections revealed classical disorganization of the dermal-epidermal junction. In the presence of anti-fibronectin serum during the whole time of culture, fibronectin-antifibronectin binding was visualized in the form of sparse cytoplasmic granules in the epidermal processes of the stratum germinativum cells. Contrasted ultrathin sections emphasized the continuity between the tonofilaments, the anchoring filaments and the anchoring fibrils. Briefly, anti-fibronectin serum inhibits the disorganization of the dermal-epidermal junction in cultured wounded skin.     

Basal lamina formation by porcine thyroid cells grown in collagen- and laminin-deficient medium

Summary Porcine thyroid cells isolated by dispase treatment were cultured in either (a) Matrigel, (b) agarose with the addition of different combinations of basic fibroblast growth factor and laminin, or (c) on agarose-coated dishes. The formation of follicles and the presence of a basal lamina was investigated by routine electron microscopy of Araldite-embedded material and by light and electron microscopical immunocytochemical detection of the basal lamina components, laminin and collagen type IV. After 10 days of culture in Matrigel or agarose, a basal lamina-like structure surrounded most follicles. Follicles of cells growing in agarose and overlaid with a medium containing thyrotropin and fibroblast growth factor showed a fluorescent band at the basal side of the follicles after immunocytochemical staining with anti-laminin and anti-collagen antibodies. Routine electron microscopy showed that a basal lamina-like structure lined the outside of the follicle. This structure could be subdivided into a lamina lucida and a lamina densa. Electron microscopical immunogold labelling revealed that immunologically detectable laminin was confined to the lamina densa. These findings suggest that even in the absence of basal lamina components in the culture medium, thyroid cells are able to form follicles with a regular basal lamina when they are cultured in a three-dimensional environment.     

Ultrastructure of the basal lamina and its relationship to extracellular matrix of embryos of the starfish Pisaster ochraceus as revealed by anionic dyes

The ultrastructure of the 51/2–6-day-old embryonic asteroid basal lamina (BL) was studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and after treatment with anionic dyes. Conventional fixation in glutaraldehyde and osmium reveals a BL consisting of a lamina densa separated from the basal cell surface by a lamina lucida. Little or no reticular lamina is present. Material similar in appearance to the basal lamina extends into the blastocoel, forming an extracellular matrix (ECM). Following fixation in the presence of the dye ruthenium red, proteoglycan (PG) granules are visible in the lamina lucida and immediately beneath the lamina densa. The ECM consists of granules of a similar appearance, which are associated with fibers of an intermediate electron density resembling invertebrate collagen. After fixation in the presence of alcian blue under polyanionic conditions, all aspects of the basal lamina and the ECM stain very densely. The use of alcian blue in 0.3 M MgCl₂ (monoanionic condition) or in low concentrations reveals a lamina densa consisting of a fine feltwork and tubule-like structures. A meshwork composed of thick, densely stained and thinner, intermediately stained strands is embedded in the inner aspect (that adjacent to the blastocoel) of the ectodermal lamina densa. Similar elements are present in the endodermal BL, but the dense material is represented by short regions that do not form a meshwork. The dense and intermediate strands of both basal laminae also extend into the blastocoel as ECM. The tubule-like structures extend from the dense material of the inner meshwork into the lamina densa. They also cross both the lamina densa and lucida to associatee with the basal cell membranes. The fact that the basal cell surfaces are often puckered outward at the points of contact suggests that this configuration might be providing a means whereby forces can be transferred from the ECM through the basal lamina to the cells.     

Light-microscopic immunocytochemical localization of fibronectin in the developing rat lung

Summary The development of the rat lung is a process of continuing morphological change. Indications from work in other mammalian systems suggest that fibronectin may be important in the control of this process. The present study has examined embryonic, neonatal, and adult lung tissue of the rat by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate fibronectin at the light-microscopic level. Positive reaction was observed with anti-fibronectin serum in all stages examined. Control sections treated with pre-immune serum or no primary serum gave negative results in each case. Fibronectin in adult tissue was localized to the alveolar surface and alveolar basal lamina. Neonatal tissue showed fibronectin on pulmonary tubule walls and in basal lamina while embryonic tissue revealed localization of the protein in the basal lamina and in association with small groups of cells at the base of septal buds. These findings suggest a role for fibronectin in the control of rat lung development. The results are discussed in terms of the known functions of fibronectin as a preliminary matrix for the subsequent deposition of collagenous connective tissue, as a cellular adhesion protein, and as surface-bound material for cellular migration.     

Ultrastructural immunocytochemical localization of fibronectin in the developing rat lung

In a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra- and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.     

Immunoelectron microscopic localization of fibronectin in the smooth muscle layer of mouse small intestine

We studied the ultrastructural distribution of fibronectin in the smooth muscle layer of mouse small intestine with affinity-purified antibodies using the immunogold technique. Fibronectin was present over the pericellular area extending from the cell membrane to the extracellular matrix beyond the basal lamina. Distribution of the glycoprotein over the pericellular area was heterogeneous, i.e., it was localized more abundantly in the narrow space between smooth muscle cells, the gaps having a width of 60-80 nm where the two dense bands in adjacent cells matched each other. Such localization suggests that fibronectin contributes to cell adhesion. Within the basement membrane, gold label was localized both in lamina lucida and lamina densa, more densely in the latter than in the former. Fibronectin was also co-distributed with collagen fibers in the extracellular matrix. Within smooth muscle cells, gold particles were observed on rough endoplasmic reticulum and secretory vesicle-like structures. These results suggest that smooth muscle cells synthesize fibronectin and secrete it as a component of the basal lamina and extracellular matrix.     

Development of the basal lamina and extracellular materials in the early chick embryo

Summary Chick embryos at developmental stages up to primitive streak formation were fixed in a mixture of tannic acid and glutaraldehyde. A basal lamina was present in the unincubated embryo and consisted of a lucent lamina interna and a lamina densa. At the primitive streak stage the lamina densa showed a periodicity of stained elements. Densely stained materials were present on the cell surfaces lining the cavity between the epiblast and endoblast, and on the mesoderm cells within this cavity. Considerable amounts of extracellular material were observed in the cavity. Hyaluronidase treatment removed the cell surface and extracellular material, indicating that hyaluronic acid is a major component. This enzyme disrupted the basal lamina, leaving a fibrillar remnant with no periodic structure. It is therefore suggested that the dense periodicities consist of glycosaminoglycan built on an enzyme-resistant framework which is probably collagen. Enzyme-resistant fibrils, presumably collagen precursors, are present elsewhere within the tissue spaces.     

Extracellular Matrix in Platyhelminths,with Special Reference to the Presence of Fibronectin

The extracellular matrix (ECM) was studied in the turbellarians Polycelis nigra and Microstomum lineare, the cestode Diphyllobothrium dendriticum and the trematode Dicrocoelium dendriticum. Routine LM staining methods for connective tissue gave positive results only in P. nigra. Positive staining reactions were observed in the subepithelial basal lamina, around the pharynx and as strings in the tissues. Peroxidase-anti-peroxidase and immunofluorescence methods were used to identify fibronectin. Positive results were obtained in all species. Positive reactivity to anti-fibronectin was observed in the subepithelial basal lamina and as a thin network between the cells of the tissues. Some intracellular reactivity occurred, but the cell type was not identified. In M. lineare a strong positive reactivity was observed in the regenerating area.     

Masking of antigenic sites of fibronectin by glycosaminoglycans in ethanol-fixed embryonic tissue

We studied the interaction between glycosaminoglycans (GAGs) and fibronectin in the basement membrane of the epiblast in the chicken blastoderm using testicular-hyaluronidase digestion of GAGs either on fixed tissue sections or in vivo after microinjection of the enzyme preparation prior to immunostaining for fibronectin. In the choice of fixatives, special attention was paid to their preservation of GAGs. The controls included alcian-blue staining of serial sections to test the efficiency of the digestion, and incubations in the presence of protease inhibitors to abolish contaminating proteolytic activity in the commercial hyaluronidase preparations. The results indicate that fixation in solutions which preserve GAGs, i.e. ethanolic solutions or aqueous solutions containing cetylpyridinium chloride, allows the immunocytochemical demonstration of fibronectin in the basement membrane of the epiblast at the level of the endophyllic crescent, but masks this glycoprotein at the epithelial-mesenchymal interface. As shown by both approaches, this masking of immunoreactivity is reversible. Moreover, the in vivo clearance of GAGs before fixation shows that the masking at the epithelial-mesenchymal interface is not an experimental peculiarity due to the use of a particular technique, but is the consequence of an interaction between GAGs and fibronectin in that particular area of the basement membrane that is used by mesoblast cells as a substrate for migration. The observation that fibronectin may be masked by GAGs in ethanol-fixed tissue--a commonly used fixation method--may require the re-evaluation of some negative results mentioned in the literature.     

Masking of antigenic sites of fibronectin by glycosaminoglycans in ethanol-fixed embryonic tissue

Summary We studied the interaction between glycosaminoglycans (GAGs) and fibronectin in the basement membrane of the epiblast in the chicken blastoderm using testicular-hyaluronidase digestion of GAGs either on fixed tissue sections or in vivo after microinjection of the enzyme preparation prior to immunostaining for fibronectin. In the choice of fixatives, special attention was paid to their preservation of GAGs. The controls included alcian-blue staining of serial sections to test the efficiency of the digestion, and incubations in the presence of protease inhibitors to abolish contaminating proteolytic activity in the commercial hyaluronidase preparations. The results indicate that fixation in solutions which preserve GAGs, i.e. ethanolic solutions or aqueous solutions containing cetylpyridinium chloride, allows the immunocytochemical demonstration of fibronectin in the basement membrane of the epiblast at the level of the endophyllic crescent, but masks this glycoprotein at the epithelial-mesenchymal interface. As shown by both approaches, this masking of immunoreactivity is reversible. Moreover, the in vivo clearance of GAGs before fixation shows that the masking at the epithelial-mesenchymal interface is not an experimental peculiarity due to the use of a particular technique, but is the consequence of an interaction between GAGs and fibronectin in that particular area of the basement membrane that is used by mesoblast cells as a substrate for migration. The observation that fibronectin may be masked by GAGs in ethanol-fixed tissue — a commonly used fixation method—may require the re-evaluation of some negative results mentioned in the literature.     

Fine structure of the glomerular basement membrane and immunolocalization of five basement membrane components to the lamina densa (basal lamina) and its extensions in both glomeruli and tubules of the rat kidney

Electron microscopic immunostaining was used to examine the localization of type IV collagen, laminin, entactin , heparan sulfate proteoglycan, and fibronectin within the basement membranes of the rat kidney. In preliminary experiments, various methods of processing formaldehyde-fixed kidney were compared using antilaminin antiserum and the indirect immunoperoxidase method. Little or no laminin immunostaining of the glomerular basement membrane was present in sections unless they had been frozen-thawed; and even in this case, the immunostaining was light in comparison to that of basement membranes in adjacent tubules. However, when frozen-thawed sections were treated with 0.5% sodium borohydride, immunostaining was then as strong in glomerular as in tubular basement membranes. Accordingly, this treatment was applied to frozen-thawed sections before immunostaining for any of the substances under study. Immunostaining of the glomerular basement membrane for each of the five substances was fairly uniform throughout the lamina densa (also called basal lamina), but uneven in the lamina lucida interna and externa (also called lamina rara interna and externa) in which stained bands extended from the lamina densa. Similarly in the basement membranes of tubules, immunostaining for the five substances was localized to the lamina densa and bands extending into the lamina lucida. When the ultrastructure of the glomerular basement membrane was examined, three structures were found: (1) a network of 4-nm-thick "cords," which seems to be the main component; the cords are closely packed in the lamina densa and more loosely arranged in the lamina lucida interna and externa; (2) straight, hollow 7-10-nm-thick structures referred to as " basotubules "; and (3) 3.5-nm elements composed of minute paired rods, referred to as "double pegs." The distribution of the cords, but not that of the other two structures, was related to the immunostaining pattern. It is concluded that (1) to fully reveal the antigenicity of the glomerular basement membrane, frozen-thawed sections must be treated with sodium borohydride prior to immunostaining, possibly because this basement membrane is more compact than the others; and (2) in both glomerular and tubular basement membranes, type IV collagen, laminin, entactin , heparan sulfate proteoglycan and fibronectin are colocalized in the lamina densa and its extensions to the laminae lucidae . Since the distribution of the cords corresponds to that of immunostaining, it is likely that the five substances are present within the cords.     

Regional Ultrastructural Differences in Basal Laminae Isolated from the Starfish Pisaster ochraceus

The ultrastructure of different regions of the basal laminae isolated from 5-1/2-6 day-old embryos of the starfish, Pisaster ochraceus , has been described after fixation in the presence of anionic dyes. Isolated basal laminae from all regions of the embryo exhibit a lamina lucida and lamina densa. No lamina fibroreticularis is present. Instead, a coarse meshwork of thick densely stained and thinner intermediately stained fibers is embedded in the lamina densa and extends into the blastocoel forming the extracellular matrix. The coarse meshwork associated with the ectodermal basal lamina consists primarily of thick densely stained fibers with a small number of intermediate ones while that associated with the endodermal one contains much less densely stained material. These structures were morphologically identical to those found in control embryos. Examination of different regions of the endodermal basal lamina shows that the amount of dense material varies from region to region. These differences in dense material may reflect biochemical differences, particularly of proteoglycans, which could provide positional information to migrating mesenchyme cells.     

Ultrastructural localization of laminin in rat sensory ganglia

We adapted immunocytochemical methods for localization of laminin to examine its disposition in neural tissue at the ultrastructural level. In dorsal root ganglia, laminin was found in basal laminae of the satellite and Schwann cells ensheathing neuronal perikarya and nerve fibers, respectively, and around blood vessels. Within the basal lamina, the immunostain was found in the lamina lucida and lamina densa. Occasional immunostained coated pits were identified in satellite and Schwann cells, but virtually no intracellular label was seen even in freeze-thawed/detergent-permeabilized specimens. In the perineurium, only the basal lamina of the inward-facing surface of the inner-most cell layer was usually stained.     

Deposition of extracellular matrix along the pathways of migrating fibroblasts

Summary Fibroblasts from rat, mouse and chick embryos cultured on poly-lysine/fibronectin- or poly-lysine/laminin-coated dishes were stained with antibodies directed to extracellular matrix molecules. The staining showed that cells had migrated during culture and deposited extracellular matrix components along their migration trails. Depending on the antigen, the staining of the matrix revealed fibrils, spots or a diffuse smear along the migration pathways. The major matrix components were fibronectin and heparan sulfate proteoglycan; however, laminin nidogen, tenascin, glia-derived nexin (GDN) and chondroitin-4-sulfate proteoglycan were also found. The migration trails were also detectable by scanning electron microscopy. Here, the fibrils were the prominent structures. The deposition of matrix was independent from the substratum: fibronectin was deposited on laminin, plain poly-lysine, basal lamina and even on fibronectin. Functional assays using anti-fibronectin or an antiserum to embryonic pigment epithelium basement membrane disturbed the formation of matrix fibrils, but did not inhibit cell attachment and translocation. Likewise, heparin in the culture medium only partially inhibited cell migration, despite the fact that it disturbed the formation of proper matrix fibrils. Our results suggest that the deposition of extracellular matrix by cells may not be mandatory for attachment and translocation. However, the deposition of matrix along defined trails might be important for the pathfinding of cells or nerve fibers that appear later in development.     

Rat endometrial stromal-epithelial response to estrogen infusion

Morphologic changes at the interface of rat endometrial luminal epithelial cells and the stromal cells immediately adjacent were examined and correlated with hypertrophy of the epithelial cells during estradiol (E2) infusion (1 microgram E2/24 h). While the lamina densa in castrate endometrium was thread-like, it became thicker and apparently more granular in some areas below the luminal epithelium during E2 infusion. However, no changes were seen in the intensity of laminin-like immunoreactivity at various time points up to 96 hours after beginning infusion, suggesting that these alterations were due to changes in nonlaminin components. The stromal cells adjacent to the basal lamina in the castrate state had cell processes extending toward the epithelium that terminated on the basal lamina. Under estrogen infusion, stromal cell bodies migrated close to and became oriented along the basal lamina. No interruptions were seen in the lamina densa or in the laminin-like immunoreactivity in the basal lamina. Thus, there were no direct morphologic interactions between epithelial and stromal cells induced by estrogen. Some of the stromal cells developed a dilated rough endoplasmic reticulum and some developed multiple elaborate processes within 41 hours after minipump implantation. Within 28 hours, nuclear hypertrophy had occurred in 15% of the epithelial cell layer. If interactions occur between stromal and epithelial cells, and morphologic evidence presented here suggests they do, then all such interactions are through an intact lamina densa-laminin layer, and any chemical mediators affecting cells on opposite sides of the lamina densa must migrate through it.     

Localization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin to the basal lamina of basement membranes

Electron microscopic immunostaining of rat duodenum and incisor tooth was used to examine the location of four known components of the basement-membrane region: type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. Antibodies or antisera against these substances were localized by direct or indirect peroxidase methods on 60-microns thick slices of formaldehyde-fixed tissues. In the basement- membrane region of the duodenal epithelium, enamel-organ epithelium, and blood-vessel endothelium, immunostaining for all four components was observed in the basal lamina (also called lamina densa). The bulk of the lamina lucida (rara) was unstained, but it was traversed by narrow projections of the basal lamina that were immunostained for all four components. In the subbasement-membrane fibrous elements or reticular lamina, immunostaining was confined to occasional "bridges" extending from the epithelial basal-lamina to that of adjacent capillaries. The joint presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basal lamina indicates that these substances do not occur in separate layers but are integrated into a common structure.     

Behaviour of dissociated hypoblast cells on the basal lamina and on extracellular fibrils in the gastrulating chicken embryo.

The spreading behaviour of dissociated hypoblast cells on and besides a band of aligned fibrils associated with the basal lamina of the epiblast was investigated by the use of scanning electron microscopy. A horse-shaped band of aligned fibrils, first demonstrated by Wakely and England (1979), is present during the gastrulation stages of chicken embryos on the ventral side of the epiblast at the cranial and lateral borders of the area pellucida. The basal lamina of the area pellucida situated inside the fibrillar band enables the spreading and probably the locomotion of dissociated cells, which appeared as polarized cells. Numerous cells were also found on the fibrillar band, and these cells lacked distinct lamellae and a polarized shape. Extensions of the cells contacted the extracellular fibrils and, at these sites of contact, the pattern of the fibrils was frequently deformed. From these observations and from previous results emerged the concept that spreading and locomotion of dissociated hypoblast cells, as well as single mesoblast cells and healing hypoblast epithelium, are inhibited by the band of extracellular fibrils, which acts as a physical barrier. The cell biological basis of the mechanism by which extracellular fibrils associated with the basal lamina arrest the migration of hypoblast and mesoblast cells, but guide the migration of primordial germ cells, is discussed.     

Extracellular matrix of cultured glial cells: selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.     

Fibronectin in basement membrane of Hertwig's epithelial sheath. Light and electron immunohistochemical localization

The distribution of fibronectin throughout the basement membrane of Hertwig's epithelial sheath was studied using specific antibodies with the immunoperoxidase technique in both light and electron microscopy. Our results demonstrate that, after collagenase digestion in situ, the basement membrane was strongly labelled by antifibronectin antibodies on the lamina lucida, the lamina densa and the lamina (pars) fibroreticularis which contained aperiodic fibrils of 5-10 nm in diameter.     

Expression of different regional patterns of fibronectin immunoreactivity during mesoblast formation in the chick blastoderm

The appearance and distribution of the extracellular material glycoprotein, fibronectin, was investigated in gastrulating chick embryos using affinity-purified anti-human plasma fibronectin antibodies. Preservation of tissue structure and immunoreactivity was carried out by ethanol/acetic acid fixation or by formaldehyde/glutaraldehyde fixation. Using the former fixation method, fibronectin immunoreactivity was detected (1) at the ventral surface of the upper layer or epiblast, mainly anterior and lateral to Hensen's node, in regions where middle-layer or mesoblast cells are not yet present, and (2) sparsely in extracellular spaces of the deep layer. Using the latter fixation method, fibronectin immunoreactivity was, moreover, found at the entire ventral surface of the upper layer, i.e., also at the epithelial-mesenchymal interface, where a basement membrane was previously described. At the light microscope level, we could not detect significant immunoreactivity in the middle layer. Treatment of sections of ethanol-fixed blastoderms with testicular hyaluronidase before immunostaining for fibronectin partially demasked the antigenic sites of this glycoprotein at the epithelial-mesenchymal interface. The present report indicates that the different regional patterns of fibronectin immunoreactivity in the basement membrane of the upper layer are spatially and temporally correlated with migration and positioning of mesoblast cells. These regional patterns are probably due to differences in the composition of fibronectin-associated material such as chondroitin sulfate A and/or C proteoglycans, and/or hyaluronate, before and after mesoblast expansion, rather than to differences in the distribution of fibronectin itself. In this respect, it is noteworthy that the chemical composition of the basement membrane of an epithelium changes as mesenchyme cells migrate over it. The results also favor the idea that fibronectin is a structural component of the whole basement membrane which is used as a substrate for migration of mesenchymal cells.