Peroxidase uptake by photoreceptor terminals of the skate retina.

The photoreceptors of dark-adapted skate retinas bathed in a Ringer solution containing horseradish peroxidase (HRP) incorporate the tracer into membrane-bound compartments within the synaptic terminal of the cell; after 1 or 2 h of incubation, approx. 10-38% of the synaptic vesicles were labeled. The receptors appeared to be functioning normally throughout the incubation period, since electrical potentials of normal amplitude could be elicited in response to dimphotic stimuli. However, it was possible to block the uptake of peroxidase by a regimen of light adaptation that effectively suppressed light-induced activity in the electroretinogram. If, during incubation with peroxidase, retinas were exposed at 10-min intervals to an intense 1-ms flash from a xenon discharge tube, the receptor terminals were almost completely devoid of peroxidase; fewer than 2% of the vesicles were labeled. The suppression of HRP uptake could also be achieved in dark-adapted retinas by adding magnesium to the bathing solution, suggesting that calcium is necessary for transmitter release from vesicles in the receptor terminals. These findings are consistent with the view that vertebrate photoreceptors discharge a neurotransmitter in darkness, and that light decreases the release of this substance. It seems likely that the incorporation of peroxidase into vesicles of physiologically active receptor terminals reflects a mechanism for the retrieval of vesicle membrane after exocytosis.

Elastin-associated microfibrils (10 nm) in a three-dimensional fibroblast culture.

The purpose of this study is to present a three-dimensional dermal fibroblast model. Skin fibroblasts cultured in this system deposit large amounts of collagen and microfibrils. Fibroblasts were seeded onto a nylon filtration mesh and incubated in the presence or absence of ascorbic acid. Collagen fibril formation was found in the presence of ascorbic acid whereas microfibril formation was seen independent of ascorbic acid supplementation. Immunoelectron microscopy revealed that microfibrils were labeled with fibrillin at 67 nm periodicity. Isolated microfibrils studied by rotary shadowing had a beaded appearance consisting of beads linked to each other by a filamentous structure. The spaces between the beads ranged from 10.00-33.33 nm, suggesting that these microfibrils may have an extension-contraction mechanism. Furthermore, the size and spacing of the beads were similar to that seen in microfibrils from tissues (measured after rotary shadowing). Fibroblasts cultured in a three-dimensional mesh represent an effective in vitro model with which to study microfibril formation.

Skin fibroblasts are the only source of nidogen during early basal lamina formation in vitro.

The purpose of this study was to determine whether nidogen, the linkage protein of the basal lamina, is of epidermal or dermal origin. The development of the basal lamina was studied in an in vitro skin model. Preputial fibroblasts seeded onto a nylon mesh attached, proliferated, and developed a rich extracellular matrix (dermal model). Preputial keratinocytes were added to the dermal model to form a keratinocyte dermal model that ultrastructurally resembled in many respects human skin. Ultrastructural analysis revealed early stages of dermal development, including an incomplete basal lamina, aggregates of dermal filamentous material connecting to the lamina densa, bundles of 10-nm microfibrils, formation of premature hemidesmosomes, anchoring filaments, and anchoring fibrils. The cell origin of nidogen was determined in the dermal model and in the epidermal and dermal components of the keratinocyte dermal model. Specific antibodies and a cDNA probe for nidogen were used for immunofluorescence microscopy, Western and Northern blots, and for in situ hybridization studies. Our data show that fibroblasts are the only source of nidogen during early basal lamina formation. Although fibroblasts can synthesize nidogen and deposit it in the dermal matrix, no basal lamina will form unless they are recombined with keratinocytes. This suggests that the epidermis plays a major regulatory role in the production and assembly of nidogen into the basal lamina.

Culturing keratinocytes and fibroblasts in a three-dimensional mesh results in epidermal differentiation and formation of a basal lamina-anchoring zone.

The purpose of this study was to characterize an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 3 and 5 weeks and then processed for electron microscopy. Keratinocytes showed reconstruction of an epidermis consisting of a basal layer with hemidesmosomes, a stratified epithelium with tonofilaments and desmosomes, a granular layer with keratinosomes and keratohyaline granules, and a transitional stratum corneum. Anchoring filaments, lamina densa, anchoring fibrils, bundles of elastin-associated microfibrils (diameters 10 nm) and fine collagen fibrils were formed. Collagen fibrils near the epidermis were much thinner than those in the lower levels. The present study shows that the dermal model containing metabolically active fibroblasts in their natural environment will support epidermal morphogenesis and differentiation including the formation of a basal lamina and anchoring zone.

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Morphology and synaptic connections of HRP-filled, axon-bearing horizontal cells in the Xenopus retina.

Axon-bearing horizontal cells of the Xenopus retina were studied by intracellular injection of HRP following physiological characterization. The profile of the cell viewed in whole mount consisted of a round or oval perikaryon about 50 microns in diameter and an axon about 1 mm long which lacked a prominent terminal expansion. The axonal diameter was 0.5-1.0 microns in its proximal third but 2-4 microns in its distal portion. Along its course the axon emitted 25-40 branchlets each 0.2 micron in diameter, up to 10 micron long and terminating in a cluster of two to six synaptic knobs. Electron microscopic examination revealed that both perikaryal dendrites and axon branchlets ended in both rod and cone synaptic bases; cone contacts outnumbered rod contacts by two- to threefold. We were unable to document synapses of presumed interplexiform cells onto identified horizontal cells. Horizontal cell axons are joined in their distal portions by numerous, small (0.2 micron long) gap junctions. Other gap junctions were noted between horizontal cell processes within the synaptic endings of photoreceptors. An hypothesis is advanced whereby the cluster of axon branchlet synaptic knobs permits dynamic interaction of rod and cone synaptic inputs to the horizontal cell.

Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

There is binding of collagen IV to beta 1 integrin during early skin basement membrane assembly.

This study is concerned with the mechanism of basement membrane assembly in an in vitro 3-dimensional skin-culture system. Dermal fibroblasts alone can synthesize collagen IV, perlecan, and nidogen, but cannot assemble them into a basement membrane. When keratinocytes are added to the culture, however, linear assembly of collagen IV, perlecan, and nidogen is noted at the epidermo-dermal interface. Northern blots and in situ hybridization showed that perlecan and nidogen mRNAs derive exclusively from fibroblasts, while the alpha 2 (IV) collagen chain is expressed by both keratinocytes and fibroblasts, although the major source is in the mesenchyma (80%). Prior to the development of the lamina densa, collagen IV colocalizes with beta 1 integrins, most likely alpha 1 beta 1 and alpha 2 beta 1, which are known receptors for this collagen. Blocking experiments with the AIIB2 mAb (anti-beta 1 integrin subunit) and by peptide inhibition with the CB3(IV) collagen fragment disrupted the assembly of collagen IV. This study suggests that the initiation of basement-membrane formation involves binding of collagen IV molecules to keratinocyte cell-matrix integrins. These complexes act as nucleation sites for further polymerization of collagen IV molecules mostly derived from fibroblasts, by a process of self-assembly.

Local functional models of critical correlations in thin films.

Recent work on local functional theories of critical inhomogeneous fluids and Ising-like magnets has shown them to be a potentially exact, or near exact, description of universal finite-size effects associated with the excess free energy and scaling of one-point functions in critical thin films. This approach is extended to predict the two-point correlation function G in critical thin films with symmetric surface fields in arbitrary dimension d. In d = 2 we show there is exact agreement with the predictions of conformal invariance for the complete spectrum of correlation lengths xi((n)) as well as the detailed position dependence of the asymptotic decay of G. In d = 3 and d>/=4 we present new numerical predictions for the universal finite-size correlation length and scaling functions determining the structure of G across the thin film. Highly accurate analytical closed form expressions for these universal properties are derived in arbitrary dimension.

Dermal collagen fibrils are hybrids of type I and type III collagen molecules.

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.

Decorin interacts with fibrillar collagen of embryonic and adult human skin.

Biglycan (PG-I, BGN) and decorin (PG-II, DCN) are small proteoglycans that have been isolated in cartilage, skin, and bone. Although the function of biglycan is unknown, there is biochemical evidence that decorin interacts with fibrillar collagens (type I, type II). The purpose of this study was to perform immunofluorescence and immunoelectron microscopy and immunoblotting of human embryonic and adult skin with antibodies directed against biglycan and decorin. These antibodies were developed against synthetic peptides of the core proteins of biglycan (amino acid sequence 11-24) and decorin (amino acid sequence 5-17). Immunofluorescence microscopy showed that decorin stained embryonic and adult collagen fibrils. Biglycan did not stain collagen, but it appeared to stain the pericellular matrix of embryonic mesenchymal cells. Immunoelectron microscopy revealed labeling of all collagen fibrils with decorin antibodies regardless of their diameter, often at 60-nm periodicity. Positive stains suggest that most of the labeling was in the gap of the D-period (d and e bands) and also in one of the steps (c band). Decorin was identified by immunoblotting in fetal and adult skin. Also, significant amounts of core protein was identified lacking the dermatan sulfate chain. This study suggests that the core protein of decorin interacts with collagen fibrils although its specific function remains unknown.

Initiation of skin basement membrane formation at the epidermo-dermal interface involves assembly of laminins through binding to cell membrane receptors.

To study the mechanism of basement membrane formation, we determined by immunochemistry temporal and spatial expression of laminin-5 (Ln-5), laminin-1 (Ln-1) and their integrin receptors during early skin morphogenesis. A 3-dimensional skin culture was used that allows the study of the sequential molecular events of basement membrane formation at the epidermodermal interface. During early anchorage of keratinocytes to the extracellular matrix there is expression of Ln-5, BP-230 antigen and alpha3, beta1 integrin subunits. During epidermal stratification and prior to the formation of the lamina densa there is assembly of Ln-5, Ln-1, collagen IV and nidogen accompanied by keratinocyte basal clustering of alpha2, alpha3, alpha6, beta1, and beta4+ integrin subunits. The assembly pattern of Ln-1 and Ln-5 can be disturbed with functional antibodies against the beta1 (AIIB2) and alpha6 (GoH3) integrin subunits. Ln-1 assembly can also be disturbed with antibodies against its E8 domain and by competitive inhibition with a synthetic peptide (AG-73) derived from its G-4 domain. Quantitative RT-PCR showed that the dermis contributes about 80% of the laminin gamma)1 chain mRNA while 20% is produced by the epidermis which emphasizes its dual tissue origin and the major contribution of the mesenchyma in laminin production. The laminin gamma2 chain mRNA, present in Ln-5, was mostly of epidermal origin. This study presents evidence that during the initiation of basement membrane formation, laminins bind to keratinocyte plasma membrane receptors and thus may serve as nucleation sites for further polymerization of these compounds by a self-assembly process.