Changes in fibronectin, laminin and type IV collagen distribution relate to basement membrane restructuring during the rat vibrissa follicle hair growth cycle.

Hair growth in adult mammals involves continuous dermal-epidermal interaction across the follicular basement membrane, and repeated reorganisation of lower follicle structure during the hair growth cycle. The immunolocalisation of 3 extracellular matrix components, fibronectin, laminin and type IV collagen was investigated during the course of the rat vibrissa follicle growth cycle, and their distribution correlated with changes in cellular and extracellular ultrastructure, particularly around the basement membrane zone. Laminin and type IV collagen were omnipresent at the follicular dermal-epidermal junction, but were also seen in granular extracellular form within the inner dermal component of the follicle, the dermal papilla. Both the inner papilla-epidermal junction and the thick specialised outer basement membrane (the glassy membrane) revealed labelling by these 2 antibodies around telogen (the period of nonfibre production). By contrast, fibronectin was abundant within the anagen dermal papilla but at telogen stained the dermal-epidermal junction heterogeneously, when it disappeared from the inner papilla-epidermal interface but intensified externally. These changes to extracellular matrix distribution coincided with a modification of basement membrane ultrastructure from a relatively uniform line at anagen, to one which became much broader and multilayered at telogen with a loss of definite structure within the papilla. This shows that the lower part of the vibrissa follicle retains the capacity for very rapid basement membrane modification and remodelling, and implies that it is part of the biological process which enables dermal-epidermal signalling, rather than a secondary product of physical changes to the appendage. The work supports the idea that dermal papilla cells could contribute to basement membrane formation, and also that fibronectin may be involved in regulating cellular activities within the follicle. In the vibrissa follicle, dynamic cellular activity clearly takes place throughout the duration of the hair cycle.

Cellular and extracellular involvement in the regeneration of the rat lower vibrissa follicle.

The sequence of events leading to the reconstruction of a fibre-producing hair follicle, after microsurgical amputation of the lower follicle bulb, has been detailed by immunohistology and electron microscopy. The initial response was essentially found to be a wound reaction, in that hyperproliferative follicle epidermis quickly spread to below the level of amputation--associated with downward movement of mesenchymal (or dermal) sheath cells. Fibronectin was prominent in both dermis and epidermis at this stage and, as in wound repair, preceded laminin and type IV collagen in covering the lower dermal-epidermal junction. Once a new basal line of epidermis and a complete basement membrane were established, laminin and type IV collagen were detected below this junction and within the prospective papilla-forming mesenchyme. This coincided with ultrastructural observations of profuse sub-basement membrane extracellular material in the region of new papilla formation. The glassy membrane displayed extensive ultrastructural modifications at its lower level, and these corresponded with localized variations in staining intensities for all three antibodies over time. The membrane hung below the level of the epidermis, and was crossed by migrating cells from the mesenchymal dermal sheath of the follicle - it acted to segregate the inner group of follicular dermal cells from wound fibroblasts. Extracellular matrix may be a mediator of the dermal-epidermal interactions associated with this hair follicle regeneration phenomenon.

Pattern formation in skin development.

The development of skin and cutaneous appendages in amniote embryos has been submitted to a large number of experimental investigations the results of which have led to a better understanding of the mechanisms whereby this multiform organ arises during embryonic development. In birds, the main appendages are the feathers and the foot scales. Their formation results from a series of inductive events between ectoderm (later epidermis) and subectodermal mesoderm (later individualized dermis). Morphogenetically, the mesodermal (mesenchymal) component of skin is the predominant tissue, insofar as it controls most morphological and physiological features of developing skin and appendages, notably transformation of ectoderm into epidermis, polarization, proliferation and stratification of epidermal cells, initiation, site, size and distribution pattern of epidermal placodes, species-specific architecture of appendages, regional specification of keratin synthesis. The ectodermal (epithelial) component is able to respond to the mesodermal inductive instructions by building feathers and scales in conformity with the specific origin of the dermis. In these epithelial-mesenchymal interactions, extracellular matrix and the microarchitecture of the dermal-epidermal junction appear to play an important role. Indeed extracellular matrix components (primarily collagens, proteoglycans and adhesive glycoproteins) and dermal cell processes close to the epidermal basement membrane become distributed in a microheterogeneous fashion, thus providing a changing substratum for the overlying epidermis. It is assumed that the latter is able to somehow sense the texture and composition of its substratum, and by doing so to appropriately engage in the formation of glabrous, feathered or scaly skin.

Influence of various extracellular matrix components on the behavior of cultured chick embryo dermal cells.

Dermal cells isolated from the back of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III or IV collagen, fibronectin and several glycosaminoglycans: hyaluronate, chondroitin-4, chondroitin-6, dermatan or heparan sulfate). The effect of these substrates on cell behavior was compared with that of culture dish polystyrene. Three parameters of cell behavior were examined: cell proliferation and patterning, spreading (cell surface) and locomotion (velocity and directionality). Data were collected by sequential microphotography and analyzed by computer assisted morphometry. Types I and III collagen, hyaluronate and heparan sulfate had a slowing down effect on cell proliferation and patterning. The inhibitory effect of type I collagen was also detected in mixtures with glycosaminoglycans. The other components had no effect. While the smallest spreading was observed on fibronectin substrate, the largest was recorded on chondroitin-6 sulfate and heparan sulfate. The slowest velocity of locomotion was measured on fibronectin, types I and IV collagen and a mixture of type I collagen and chondroitin-6 sulfate. The fastest speed was recorded on chondroitin-4 sulfate. These effects are discussed in view of our knowledge of the role of the dermis in the development of skin and cutaneous appendages, and in the light of the morphogenetically related microheterogeneous distribution of collagens, fibronectin and various glycosaminoglycans in the developing skin.

Production of fibronectin and collagen types I and III by chick embryo dermal cells cultured on extracellular matrix substrates.

Dermal cells isolated from the back skin of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III, or IV collagen, fibronectin and several glycosaminoglycans (GAGs): hyaluronate, chondroitin-4, chondroitin-6, dermatan and heparan sulfates). The effect of these substrates on the production of fibronectin, of types I, III and IV collagen by cells was compared with that of culture dish polystyrene. Using immunofluorescent labeling of cultured cells, it was observed that, on all substrates, in 1-day and 7-day cultures, 85 to 95% of cells contain type I collagen in the perinuclear cytoplasm; label was absent from cell processes. Type I collagen was also detected in extracellular fibers extending between neighboring cells. By contrast, on all substrates, only 5 to 20% of cells produced type III collagen. Otherwise distribution of type III collagen was similar to that of type I collagen. With anti-type IV collagen antibody no staining of either cell content or extracellular spaces was detected. Staining with anti-fibronectin antibody revealed two types of distribution patterns. On polystyrene and on all but type I collagen substrates, labeling revealed clusters of short thick strands and patches of fibronectin-rich material in extracellular spaces. On type I collagen substrate, however, immunostaining revealed a delicate network of regularly spaced parallel fibrils of fibronectin extending between and along cells. Using quantitative radioimmunoassay of the culture media, it was shown that, after 7 days of culture, cells secreted more type I than type III collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles.

Actin isoform expression may change during development, and in certain physiological, experimental and pathological situations. It is accepted that during sarcomeric (skeletal and cardiac) muscle development, the alpha-skeletal and alpha-cardiac isoforms of actin accumulate rapidly at the onset of muscle fibre formation, while there is a rapid fall in the expression of nonmuscle (beta and gamma) actin isoforms. Here we show that, before birth, both skeletal and myocardial cells express significant amounts of alpha-smooth muscle actin mRNA and protein. This expression is transient and disappears over the 1-7 days following birth. Our findings show that the program regulating actin isoform expression in sarcomeric muscle development is complex and that alpha-smooth muscle actin participates in this process.

Immunolocalization of matricial components during the early stages of chick embryonic liver development.

In the chick embryo, the first liver primordium is observed at the end of the second day of incubation. At 3 and 4 days, ultrastructural analysis of the primitive vascular spaces showed that the endothelial limiting plate was constituted by one or several cell layers. At the vascular pole of the hepatoblasts, mesenchymal cells and connective matrix, present as fibrillar and non fibrillar components, were closely associated. At 5 days, some vascular spaces were limited by a simple endothelial layer. The limiting plate was fenestrated and the connective matrix was reduced to rare collagen fibrils and fibers. Collagen types I, III, IV, procollagen type III, fibronectin and laminin were visualized in the perivascular spaces using immunoperoxidase labeling methods. These components were also detected in the endoplasmic reticulum of hepatoblastic, endothelial and mesenchymal cells. All these appeared to be involved in connective matrix synthesis. Comparing 4 and 5 days, we demonstrated that the number of cells showing intracellular labelling of matricial components dropped dramatically at 5 days, indicating a possible decrease of connective matrix synthesis. Quantification of parenchymal and vascular surfaces was carried out using a semi-automatic image analyzer on consecutive parasagittal sections chosen in the axial part of the embryonic liver. These measurements were performed in order to quantitate the vascular distribution pattern during early development of the liver. These combined immunomorphological studies and morphometrical analyses suggest that during embryogenesis of the liver the synthesis of connective matrix precedes and possibly initiates the vascular differentiation.

Immunofluorescent localization of collagen types I, III and IV, fibronectin, laminin, and basement membrane proteoglycan in developing mouse skin.

The distribution of various extracellular matrix components was studied in frozen sections of embryonic (14-18 days) and early postnatal (birth and 4 days post parturn) dorsal mouse skin using monospecific antibodies and indirect immunofluorescence. Basement membrane zone components - type IV collagen, laminin and heparan sulphate proteoglycan - were found to be uniformly and unchangingly distributed along the dermal-epidermal junction. In contrast, the distribution of interstitial matrix components - types I and III collagen, and fibronectin - was heterogeneous and varied with the stages of hair development. Collagens became sparse and were eventually completely removed from the prospective dermal papilla and from a one-cell-thick sheath of dermal cells around hair buds. They remained absent from the dermal papilla throughout hair organogenesis. Fibronectin was always present around dermal papilla cells and was particularly abundant along the dermal-epidermal junction of hair rudiments, as well as underneath hair buds. In contrast, in interfollicular skin, collagens accumulated in increasing density, while fibronectin became progressively sparser. It thus appears that interstitial collagens and fibronectin are distributed in a manner which is related to hair morphogenesis. In morphogenetically active regions, collagen density is low, while that of fibronectin is high. Conversely, in histologically stabilized zones, collagen is abundant and fibronectin is sparse. This microheterogeneous distribution of interstitial collagens and of fibronectin might thus constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the development of skin and of cutaneous appendages.

Wound healing of human skin transplanted onto the nude mouse. I. An immunohistological study of the reepithelialization process.

Two months after transplantation of human skin onto the nude mouse, excisional wounds were made through the entire thickness of the skin, at the center of the graft, using a 2-mm punch. At various time intervals thereafter, ranging from 2 days to 9 weeks, the graft sites were harvested and processed for an immunohistological study. With a monoclonal antibody directed against HLA-ABC antigens, it was demonstrated that the healing epidermis is of human origin. Moreover, with three different monoclonal antibodies directed against human keratins, named respectively AE1, AE3, and KL1 and with an anti-involucrin antiserum, it is reported that the keratinization and involucrin distribution patterns observed in normal human epidermis are reconstituted, 2 months after transplantation, in the major part of the grafted epidermis, undergo changes during the reepithelialization process, and are restored in the healed epidermis 9 weeks after injury. This study indicates that the nude mouse/human skin model could be a valuable tool to study a major aspect of regeneration such as the reepidermization of human skin without recourse to human volunteers.

Effect of hydrocortisone on skin development in the chick embryo: ultrastructural, immunohistological, and biochemical analysis.

The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.

Influence of collagen and fibronectin substrates on the behaviour of cultured embryonic dermal cells.

The effect of extracellular matrix components on cell patterning was studied in cultures of 7-day chick embryo dorsal dermal cells. A scale of ten stages based on cell density, distribution, and patterning has been defined. Starting from a seeding density of 3.5 X 10(5) cells per dish (diameter 35 mm) in 1.7-1.8 ml of medium supplemented with 5% fetal calf serum, cultures reached stage 8 in 7 days. When cells were cultured on a substrate of native bovine type I collagen, their patterning was retarded by 3 to 4 stages. A substrate of human fibronectin had no effect on the rate of cell patterning, when compared with a plastic substrate. However, when fibronectin was adsorbed on collagen-coated dishes, the retarding effect of collagen was suppressed, and a 'normal' rate of cell patterning was restored. When fibronectin was locally adsorbed on plastic or collagen substrates, so as to offer a heterogeneous substrate to the cells, the border between fibronectin and plastic or between fibronectin and collagen was perceived by the cells as a borderline along which they tended to align.

Reconstitution of the epidermal basement membrane after enzymatic dermal-epidermal separation of embryonic mouse skin.

Pieces of trypsin-isolated 14-day embryonic mouse epidermis were recombined with various living or non-living dermal or non-dermal substrates, in order to analyse the reconstruction of the dermal-epidermal junction. The constitution and ultrastructure of the epidermal basement membrane were characterized by immunolabelling of laminin, type IV collagen and bullous pemphigoid antigen, and by transmission electron microscopy. Trypsin treatment of dorsal skin followed by dermal-epidermal separation does not visibly damage the epidermal basement membrane, which remains attached to the lower face of epidermis. When freshly isolated epidermis is reassociated with dermis, the basement membrane is first degraded during the first 4 h of culture, then reconstituted within 24 h. When epidermis is cultured in isolation the basement membrane disappears within 4 h and is not reconstructed. Epidermis, precultured for 4 h and thus deprived of its basement membrane prior to reassociation, is able to reconstruct an antigenically and ultrastructurally normal basement membrane, when recombined with living or frozen-killed (-20 degrees C) dermis, with muscle tissue, or with a film of fibrous type I collagen. No basement membrane is reconstituted when the epidermis is recombined with heat (100 degrees C) killed dermis. It is concluded that, in the reconstituted epidermal basement membrane, laminin, type IV collagen, bullous pemphigoid antigen, and lamina densa are of exclusive epidermal origin.

Use of retinoic acid for the analysis of dermal-epidermal interactions in the tarsometatarsal skin of the chick embryo.

Feet of chicks are normally covered with scales. Injection of retinoic acid into the amniotic cavity of 10-day chick embryos causes the formation of feathers on the foot scales. To elucidate whether retinoic acid affects primarily the epidermis or the dermis, heterotypic dermal-epidermal recombinants of tarsometatarsal skin were tested as to their morphogenetic capacity, when grafted to the chick chorioallantoic membrane. Recombinants involving treated epidermis and untreated dermis formed feathered scales, while the reverse recombinants of untreated epidermis and treated dermis led to the formation of scales only. Likewise the association of treated tarsometatarsal dermis with untreated epidermis from a non-appendage-forming region (the midventral apterium) resulted in the formation of scales only. These results show that retinoic acid affects primarily the epidermis. Further insight into the mechanism of dermal-epidermal interaction was gained by heterotopic recombinations of early (8.5- and 10-day) untreated tarsometatarsal dermis with epidermis from the midventral apterium. These recombinants formed scales, proving that tarsometatarsal dermis is endowed with scale-forming properties as early as 8.5 days of incubation. Finally, it is concluded that retinoic acid acts on the chick foot epidermal cells by temporarily inhibiting their scale placode-forming properties, allowing their latent feather placode-forming properties to be expressed.

[Distribution of collagen, fibronectin, and laminin during morphogenesis of skin and cutaneous appendages in the chick embryo (author's transl)]. / Répartition du collagène, de la fibronectine et de la laminine au cours de la morphogenèse de la peau et des phanères chez l'embryon de poulet.

In the dermis of inter-appendage and glabrous skin, interstitial collagen types I, III, and V are abundant, while fibronectin is scarce. Conversely, in the morphogenetically active foci of cutaneous appendages, interstitial collagen is scarce or absent, whereas fibronectin is abundant. Type IV collagen and laminin are localized at the dermal-epidermal junction and distributed evenly.

Heterogeneous distribution of bullous pemphigoid antigen during hair development in the mouse.

The appearance and localization of one of the basement membrane constituents, the bullous pemphigoid antigen, were studied during the development of hair follicles in the mouse. The analysis was performed, with the indirect immunofluorescent method, on frozen sections of dorsal skin, between 14 days of gestation and 10 days post partum. Specific labelling was restricted to the epidermal-dermal junction (EDJ). The earliest positive reaction was seen in 15-day embryos as a continuous underlining of the EDJ. At later stages throughout embryonic development and up to 10 days post partum, in hair rudiments labelling of the EDJ was interrupted. In particular, the EDJ along the underside of hair placodes and nodules, the lower half of follicles at the bulb and hair cone stage, as well as around the dermal papilla was not or very faintly labelled. By contrast, in the upper portion of follicles and interplumar skin, the EDJ was brightly labelled. It is concluded that bullous pemphigoid antigen is absent or scarce in zones of morphogenetic activity, where epidermal-dermal interactions are supposedly exerted, whereas it is present in zones of histological stability.

The dermal-epidermal junction during the development of skin and cutaneous appendages in the chick embryo.

The ultrastructure of the junction zone between dermis and epidermis was examined in the chick embryo during the development of feather-forming, scale-forming and glabrous skin. Direct contacts between dermal and epidermal cells were extremely rare and seen sporadically in feather-forming skin only, in connection with anchor filaments. Everywhere else, the basement membrane (BM) comprised an uninterrupted lamina densa. In feather-forming skin, zones of close parallel apposition (CPA) of dermal cell processes against the BM lamina densa were frequent at the margin of feather buds and at the base of feather filaments, and scarce in interplumar skin. In scale-forming skin, the density of CPA was lower, at 10 days, in the interplacode region than within the scale primordium, and, at 11 and 12 days, at the apex of the scale than at its base. At 11 days, dermal cells in scale primordia were equipped with long and thin tubular processes oriented predominantly at right angle with respect to the basal-apical axis of the scale. In the midventral apterium, CPA of dermal cell processes against the BM was very rare at 12 days, and non-existent at later stages, when a complex collagenous matrix was laid down in orthogonal ply-wood fashion underneath the BM lamina densa. Thus, it appeared that the heterogeneity of the distribution of dermal cell processes beneath the basement membrane might represent part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of the appendages.