Localization of fibronectin in the frog skin.

In this review, we present structural and ultrastructural localizations of fibronectin (FN) in the larval and adult skin of the frog (Rana esculenta) either in in vivo or in in vitro conditions. The ventral skin of the tadpole contains membrane-associated FN-plaques disposed around the epidermal and dermal cells during their climactic rearrangement. Moreover, lines of fibrillar FN are detected inside the breaks opened in the derived collagen. The ventral skin of the adult frog reveals FN distributed in the three superimposed tissues forming the skin, i.e. the epidermis, the dermis and the subcutaneous tissue. In vivo, the epidermis is devoid of FN except for the mitochondria-rich cells (MRCs) which contain FN cytoplasmic granules. The dermis reveals two distinct collagenous networks showing FN localizations. A vertically-oriented network formed by thick tracts contains axis of fibrillar FN connecting the upper dermis devoid of FN to the FN-rich subcutaneous tissue. In contiguity with an horizontally-oriented network comprises thin tracts formed by clear spaces separating the superimposed collagen bundles of the dermal stratum compactum. These tracts contain aligned FN-granules. Inside the thick and thin tracts, the dermal and pigment cells present membrane-associated In vitro (in organ culture conditions) MRCs of the epidermis maintain their FN localization and, in addition, the stratum germinativum cells show cytoplasmic FN granules. Epidermal cells, in the vicinity of the cut edges of the cultivated skin fragment, modify their shape and acquire membrane-associated FN-plaques located between desmosomes. The FN localizations in these two collagenous networks of the dermis remain unchanged. In the same way, the FN-rich subcutaneous tissue is unmodified. In summary, the FN distribution in the larval skin is related to the cell rearrangement during the metamorphic climax, and, in the adult skin to the cell migration during the wound healing process and the pigment cell patterning. The cell migration is demonstrated, in organ culture conditions, by antiFN serum used as an experimental tool. FN is an important substrate used in the dermal breaks of the larval skin, and in the dermal tracts of the adult skin, both allowing the dermal and pigment cell migration.

Role of the dermal tracts in the pigment pattern of the frog.

The pigment pattern of the ventral skin of the frog Rana esculenta is compared in skin fragments grown for 24 hr with or without antiserum directed to fibronectin (anti-FN). Melanocyte-stimulating hormone (MSH) was added to the medium during the last hour in culture in order to enhance visibility of melanophores in the ventral region of the frog skin. Comparison of these two treatments provides information regarding the precise localization of melanophores in the dermal tracts and their involvement in the pigment pattern of the ventral frog skin. In this regard, the whitish pigment pattern of skin fragments is compared to the tiny black spots found on anti-FN treated skin fragments and the abundant blotchy spots found on skin cultured alone. The distribution of melanophores in the dermal tracts observed in vertical semithin sections is found to be related to the three different levels of the dermal tracts. This report demonstrates the importance of fibronectin as a substrate for the melanophore migration, the importance of the tract level for the melanophore localization both involved in the pigment pattern of the ventral skin.

Pigment cell localizations in anuran ventral skin at climactic metamorphosis.

In anuran amphibians, the specific color pattern of the skin is expressed after metamorphosis, and its formation involves pigment cell migrations. Pigment cells are differently distributed in the tadpole, larval, and froglet skin. To learn more about their fate during metamorphic climax and in the young froglet, we focused our attention on the different localizations of larval melanophores and iridophores in the ventral skin of Rana esculenta before and during skin homing. Localizations of melanophores and iridophores can be elucidated at the developmental stages suggested by Taylor and Kollros (TK stages). At TK stage II (during early premetamorphosis), large melanophores beneath the larval skin are detected. At TK stage X, dispersed melanophores lie under bundles of muscular striated fibrils near the larval skin; they are also observed at the vascular level. At TK stage XVII (prometamorphosis), melanophores are extended on the inner side of the basement lamellar collagen. At the end of prometamorphosis, iridophores are located with melanophores in the separating space between attached basement collagen and derived basement collagen. At TK stage XX (earlier climax), melanophores and iridophores are detected inside the upper extremities of fractures opened in the derived basement collagen. At TK stage XXIV (later climax), both types of larval pigment cells are observed in the inner extremities of breaks derived from the fractures. During climax, these pigment cells occupy the well-formed breaks. At TK stage XXV in young froglet, the pigment cells remain alone in the breaks formed in the derived basement collagen. Briefly, breaks in the basement lamellar collagen are opened by invading cell processes of mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Dermal breaks: migratory cell pathways opened in anuran tadpole skin at climactic metamorphosis.

In this report, we studied the formation of breaks in the frog dermis during its remodelling at climactic metamorphosis. This remodelling consisted of detachment of the basement lamella collagen from the epidermis. The detached part, called derived collagen, was progressively fractured by breaks. We focused our attention on dermal cell localization during break formation. Firstly, at early climax, dermal cells were localized inside fractures opened in the derived collagen. Secondly, at the later climax, the fractures became breaks, making room for the dermal cells themselves. Thirdly, in derived collagen of the froglet, the well-opened breaks contained elongated dermal cells. At climax, DAB immunoperoxidase staining of fibronectin revealed a granular pattern at the surface of epidermal and dermal cells. Unexpected staining revealed that the dermal breaks contained fibronectin in the form of vertical lines. The foregoing results suggest that the dermal breaks are migratory pathways for dermal cells in derived collagen remodelled at climax.

Fibrillar pattern of self-assembled and cell-assembled collagen: resemblance and analogy.

The geometrical characteristics of fibrillar organizations are studied by electron microscopy in structures obtained in vitro in cell-free assembled collagen gels, and in vivo in dermal tracts of anuran skin. We analyze several characteristics of the fibrils including the diameter, the outline, the curvature and the extrafibrillar space. We analyze also the variation of fibrillar orientation (twist) in longitudinal and transverse thin sections of these structures. The results are compared in the Discussion to determine to what extent these fibrillar patterns are similar to liquid crystalline organizations and to what extent they result from a self-assembly or a cell-assembly process.

Localization of pigment cells in cultured frog skin.

The pigmentation pattern of ventral skin of the frog Rana esculenta consists mainly of melanophores and iridophores, rather than the three pigment cells (xanthophores, iridophores, and melanophores) which form typical dermal chromatophore units in dorsal skin. The present study deals with the precise localization and identification of the types of pigment cells in relation to their position in the dermal tracts of uncultured or cultured frog skins. Iridophores were observed by dark-field microscopy; both melanophores and iridophores were observed by transmission electron microscopy. In uncultured skins, three levels were distinguished in the dermal tracts connecting the subcutaneous tissue to the upper dermis. Melanophores and iridophores were localized in the upper openings of the tracts directed towards the superficial dermis (level 1). The tracts themselves formed level 2 and contained melanophores and a few iridophores. The inner openings of the tracts made up level 3 in which mainly iridophores were present. These latter openings faced the subcutaneous tissue In cultured skins, such pigment-cell distribution remained unchanged, except at level 2 of the tracts, where pigment cells were statistically more numerous; among these, mosaic pigment cells were sometimes observed.

Redistribution of fibronectin and keratin localization patterns in early wounded confluent PtK2 cells.

Single and double-label immunofluorescence were used to study the fibronectin (FN) and keratins (Ks) localization patterns in early wounded confluent PtK2 cells. A time-course study (0 hr, 2 hr, 6 hr and 24 hr) gives the following results: before wounding, the FN localizations of confluent cells are composed of curved and sometimes branched strands or fibrils. The Ks network is formed by radial fluorescent filaments connecting the Ks centers near the nuclei with a linear fluorescence underlying the cell membrane. Two hr after, the FN localizations are redistributed at the cell-cell contact areas. The radial Ks filaments are compacted around the nuclei, some of them delineate the cytoplasmic periphery of the wounded cells. Six hr later, the method shows redistributed FN localizations at the cell-cell contact areas. An alveolar pattern is formed enclosing each of the adjacent cells. The codetected Ks filaments are retracted around the nuclei. The underlying cell-cell contact areas are also well demonstrated. It may be noted that these areas are FN-labelled. Twenty-four hr after wounding, the FN alveolar pattern persists. The redistributed Ks filaments have some similarity to those seen before wounding.

Fibronectin network recovery in confluent PtK2 cells after acrylamide treatment.

Confluent PtK2 cells 4 hr treated with 5 mM acrylamide were FN-detected by indirect immunofluorescence. The initial fibrillar-FN network was replaced by an alveolar-type network located at the cell-cell contacts areas in the form of a thick frame with a lace-like appearance. Afterwards, acrylamide removal was obtained by several washes with fresh FCS-free culture medium. Then, PtK2 cells were returned to the incubator for 20 hr. Cell recovery was indicated by reversion of the initial fibrillar-FN network. These data show that FN reversion was possible without any changes in shape and cytoplasmic organization of non-motile growing cells.

Culture media conditioned by wounded cells modify the fibronectin localization pattern of unwounded confluent PtK2 cells.

A confluent PtK2 cell sheet was incised in a serum-free culture medium, at 15 min, 2 hr and 24 hr after wounding. The culture media were collected in the same way and used as conditioned media. Unwounded confluent cells were cultured in the conditioned medium for 24 hr. They showed a modification of fibronectin localization similar to that which we had previously observed in wounded confluent PtK2 cells: cells lost their normal fibronectin fibrils and were surrounded by fibronectin lace. This finding suggested that during wound healing, the cells released soluble chemical factors which could modify the fibronectin localization pattern of unwounded confluent cells. Subconfluent cells did not respond to conditioned media, showing that confluent cells and subconfluent cells had different susceptibilities.

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.

Skin cultured with or without an NaCl gradient and aldosterone influence epidermal fibronectin localizations.

Fibronectin (FN) localizations in the epidermal cells of the frog Rana esculenta were detected in isolated ventral skin fragments 4 day-cultured with or without an NaCl transepithelial gradient and aldosterone. Without the gradient, few mitochondria-rich cells (MRCs) were FN-detected. Stratum germinativum and spinosum cells also contained fibronectin. With the gradient, numerous MRCs were detected. Below them, in the stratum germinativum, clear spaces were recognized. Aldosterone with or without the gradient modified the above effects: in both cases, many MRC contained fibronectin. It was interesting to note that, for each type of culture, stratum germinativum cells were dramatically FN-detected.

Dermal tracts in frog skin: fibronectin pathways for cell migration.

The dermis of the frog skin (Rana esculenta) displayed a remarkable organization of vertical and horizontal tracts. Vertical thick tracts connected the dermal Stratum spongiosum with the subcutaneous tissue. Horizontal thin tracts were found alongside and contiguous to them. The thick tracts were sheathed by collagen fibrils of the Stratum compactum which were vertically oriented (i.e. parallel to the axes of the tracts) according to the horizontal and orthogonal arrangement of the collagen bundles of the Stratum compactum. The thin tracts devoid of collagenous sheath were formed by clear spaces between superimposed collagen bundles of the dermal Stratum compactum. On vertical sections, the thick tracts were seen to contain fibronectin (FN), detected by indirect immunoperoxidase. Continuous vertical FN lines were centred in these tracts. On horizontal sections, a clear zone around these FN-centred lines was also sheathed by FN. The thick tracts contained flattened pigmentary cells and fibroblasts; these cells were FN-outlined. The thin tracts contained patches of FN and FN-outlined fibroblasts. In culture, in vertical thick tracts, both pigmentary cells and fibroblasts disappeared when antiserum to FN was added to the culture medium. This suggested that thick tracts were pathways allowing pigmentary cells to move upward or downward between their usual upper dermal and lower subcutaneous localizations. Fewer fibroblasts were found in the thin tracts in the presence of antiserum to FN.

Cultured epidermis influences the fibril organization of purified type I collagen gels.

Purified type I collagen gel used as culture substrate was composed of unstriated fibrils. Before culture, gel fragments were coated with culture medium with or without fetal calf serum (FCS+ coated or FCS- coated gels). Each gel fragment was apposed to a fragment of frog skin at the medium/air interface in Trowell culture chamber. After 7 days at 20 degrees C, the coated gels were covered with newly formed epidermis containing fibronectin localized around the keratinocytes, whose morphology was considerably modified. Fibroblast-shaped keratinocytes were localized in the anterior zone of the newly formed epidermis on FCS+ gels. The long axis of the cells was parallel to the gel surface, where numerous unstriated fibrils were located. Polyhedral keratinocytes were located in the posterior zone on FCS+ gels or the anterior and posterior zones on FCS- gels with the long axis perpendicular to the gel surface. Numerous cross-striated fibrils were found under the cultured keratinocytes in the vicinity of the basal filipodia. This model is useful for the study of collagen gel reorganization by keratinocytes.

Fibronectin (FN) localizations in mitochondria-rich cells of frog epidermis.

Indirect immunoperoxidase detection of fibronectin (FN) in the ventral frog epidermis showed that only one type of epidermal cell, the mitochondria-rich cells (MRC), was FN-detected. FN was found in MRC having rounded, flask-like and intermediate shapes, located at different levels of the epidermis between the stratum germinativum and the stratum corneum. These cells contained cytoplasmic FN particularly in the form of small granules. Our results support the view that MRC differ from other epidermal cells in their in vivo FN localizations.

Calcium-modulated desmosome formation and sodium-regulated keratinization in frog skin cultures.

The processes of desmosome formation and keratinization were studied in isolated frog skins cultured in a two-compartment (mucosal and serosal) chamber. Before culture, the skin fragments were trypsinized (stratum corneum together with some parts of stratum granulosum and spinosum were scraped off with forceps) allowing the stratum germinativum to remain on the dermis. When both the mucosal and the serosal culture media contained 1.5 mM calcium and 86 mM sodium concentrations, fully developed desmosomes were differentiated and no keratinization occurred. When the mucosal medium was lowered in two steps to a final calcium concentration of 0.5 mM by dilution with tridistilled sterile water, poorly developed desmosomes were formed, keratinocytes interdigitated and the keratinization was strongly enhanced. The calcium-dependent desmosome formation was affected by the salt gradient established across the skin. These two effects, modulated desmosome formation (calcium) and increased keratinization (sodium), were concomitant with but did not complement one another.

Fibronectin (FN) localizations in early wound healing of cultured frog skins.

The wound healing process of frog skin fragments in epibolic cultures has provided information on FN localizations during the migration of keratinocytes. Mainly two FN localizations were studied by indirect immunodetections: Epidermal localization around keratinocytes which have acquired a fibroblastic shape. Dermal localizations of the sectioned collagen of the stratum spongiosum and stratum compactum detected at the beginning of the culture. Both localizations were observed in this epibolic wound healing process during 6 hr and 24 hr in culture and showed a differential sensitivity to cycloheximide (CHX). It was worth noting that fibronectin was permanently detected in the subcutaneous tissue of non-cultured or cultured skin fragments with or without CHX.

Frog skin epithelium interactions with a bovine purified type I collagen gel in culture.

Isolated epithelium of the ventral frog skin was superimposed to purified type I collagen gels and then cultured. The epithelial anchoring process was dependent on the collagenase type used for epithelial isolation. With type I collagenase, lamina densa remained attached to the basal epithelial pole and epithelial anchorage to the gel was effective. But this anchorage could only take place if gels were precoated with culture media supplemented with fetal calf serum. In these fibrillar gels, 2 types of collagen striated fibers assemblies occurred. In the upper zone, striated fibers assembly was cell-dependent. The basal pole of Malpighian cells generated numerous filopodia inserted into the gel. These filopodia polarized the collagenous assembly around and along them. In the inner zone, aggregates with cholesteric texture were assembled without superimposed epithelium. Their shape and width seemed to be related to the acidosoluble collagen concentration.

Twisted architectures in cell-free assembled collagen gels: study of collagen substrates used for cultures.

Reprecipitated fibrils from collagen solutions assemble into aggregates often showing a remarkable twisted structure. We first observed these aggregates in collagen gels prepared to facilitate culture of epithelial cells. We verified that these structures form in the absence of cells and correspond to a process of self-assembly. Studies on reconstructed fibrils of collagen are generally based on the examination of thin specimens mounted onto coated grids prepared for electron microscopy. We rather applied the classical methods of fixation, embedding and ultramicrotomy, which allowed us to analyze the structure of these aggregates, several microns in diameter. Our gels were prepared from 2.5 mg/ml tropocollagen solutions usually chosen for cell and organ cultures. The time required to obtain twisted architectures, in these aggregates, depends on temperature and the presence of factors such as fetal calf serum proteins. Twist is observed at two different levels of organization. Microfibrils are gathered into twisted bundles which condense into cross-striated fibrils. These fibrils themselves aggregate and show a mutual twist whose orientation is left-handed as is the twist observed within each microfibril bundle. Several models of these architectures are presented. Planar twist, cylindrical twist and toroidal twist are described and their relation to the structure of certain liquid crystals is considered. Examples of orthogonal packing also have been observed. These structures obtained in vitro are very close to patterns already described in vivo in numerous collagen matrices.

Epithelial locomotion and differentiation in frog skin cultures.

Small explants from the medioventral skin of the green frog were maintained in culture for 5 days. During the first hours, a rapid outgrowth of the stratum germinativum was observed at the periphery of the fragment (2 X 3 X 0.75 mm). The Malpighian cells stretched and emitted long lamellipodia following the cut edges of the dermis. These cells acquired a fibroblastic shape and were covered by other flattened cells from the stratum spinosum and even from the stratum granulosum. This progression of cells simulated an epiboly; cell divisions occurred and were revealed by autoradiography. The underlying dermis could be totally covered after 3 days. An increase in the number of cellular layers was observed. The migrating cells redifferentiated, in particular at the lower side of the dermis, which provided a suitable substratum for the newly formed epidermis. A new basal lamina was formed. Some exfoliations of keratinized layers were seen. After 5 or 6 days of culture, epiboly was complete, the epithelium formed a closed system, and degenerative processes occurred, probably by non-penetration of the nutritive medium into the deeper regions of the explant.