Fine structure of the organ of attachment of the teleost, Garra gotyla gotyla (Ham)

We describe the morphology of the attachment organ (AO) of the teleost, Garra gotyla gotyla (Cyprinidae). It is located ventrally around the mouth opening and used by the species for attachment to submerged rocks in sub-Himalayan streams and rivers where it lives. The AO consists of three crescentic parts and a central callus part. Scanning electron microscopy (SEM) shows the former to possess numerous tubercles, each of which bears about 23–27 curved spines. Light microscopy shows the epidermis of the tuberculated parts to possess one type of cell arranged into 7–8 rows. Transmission electron microscopy (TEM) reveals these cells to contain abundant tonofilaments (hence called the filament cells). The epidermis of the callus part possesses the filament cells and additionally mucous cells, which are absent in the tuberculated parts. The superficial epidermis is apparently keratinized (thickness: 5–8 μm), and a part of the cells of the outer row is modified into spines. These cells show a thick plasma membrane envelope and possess mucous granules (diameter: 0.1–0.3 μm) and bundles of tonofilaments. The cells of the inner two to four rows possess similar organelles and additionally, prominent Golgi bodies and rough endoplasmic reticulum. Immunohistochemically, the cells of the outer row and the spines stain positively for cytokeratin. The cells of the innermost rows (five to eight) possess few tonofilaments and no mucous granules. It is evident that the filament cells of the mid- to upper epidermis are specialized for the production of mucous granules and tonofilaments, which is unique for the teleost epidermis concerned. It appears that the tuberculated parts with spines assist in anchorage and interlocking with the substratum, while the central callus part probably utilizes both suction and frictional mechanisms, and mucous secretion protects the spines from damage during anchorage and abrasion.     

Structural organization of the toe pads in the amphibian Philautus annandalii (Boulenger, 1906)

We describe the morphology of toe pads in the Himalayan tree frog Philautus annandalii. These are expanded tips of digits and show modifications of their ventral epidermis for adhesion. The outer cells of toe pad epidermis (TPE) bear surface microstructures (0.7 × 0.2 μm), which are keratinized. Their cytoplasm contains no organelles, but pleomorphic nuclei and mucous granules (0.4–0.5 μm) that glue the keratin filaments. In the intermediate cell layer of TPE, similar keratinized microstructures as in the outer cells are present, so that when the outer layer is shed, it is ready with features for adhesion. These cells contain more keratin than the outer cells. The basal cell layer contains thin keratin bundles and usual cell organelles. The dermis contains mucous‐secreting glands, whose ducts open in the outer epidermal cell layer in channels. The dorsal epidermal cells lack surface microstructures and keratin bundles. Ultrastructural features suggest that toe pads utilize the surface microstructures for adhesion aided by mucus, in which the intermediate cell layer seems to bear the shear stress generated during locomotion. Further, TPE can expand and fit into an increased contact area of the substrate. The long, surface microstructures may also help in mechanical interlocking with rough surfaces on plants.     

Keratinization of the outer surface of the avian scutate scale: Interrelationship of alpha and beta keratin filaments in a cornifying tissue

Summary The outer surface of adult Gallus domesticus scutate scale was studied as a model for epidermal cornification involving accumulation of both alpha and beta keratins. Electron-microscopic analysis demonstrated that the basal cells of the adult epidermis contained abundant lipid droplets and that filament bundles and desmosomes were distributed throughout the cell layers. Indirect immunofluorescence microscopy and double-labeling immunogold-electron microscopy confirmed that the stratum germinativum contained alpha keratin but not beta keratin. Beta keratins were first detected in the stratum intermedium and were always found intermingled with filament bundles of alpha keratin. As the differentiating cells moved into the outer regions of the stratum intermedium and the stratum corneum, the large mixed keratin filament bundles labeled increasingly more with beta keratin antiserum and relatively less so with alpha keratin antiserum. Sodium dodecyl sulfate-polyacrylamide gel analysis of vertical layers of the outer surface of the scutate scale confirmed that cells having reached the outermost layers of stratum corneum had preferentially lost alpha keratin. The mixed bundles of alpha and beta keratin filaments were closely associated with desmosomes in the lower stratum intermedium and with electron-dense aggregates in the cytoplasm of cells in the outer stratum intermedium. Using anti-desmosomal serum it was shown that these cytoplasmic plaques were desmosomes.     

Induction of the alpha-type keratinization by hydrocortisone in embryonic chick skins grown in a chemically defined medium: An electron microscopic study

Previous biochemical analyses showed the differential accumulation of the epidermal structural protein, which yielded S-carboxymethylated epidermal protein A (SCMEpA), in the hydrocortisone-induced in vitro keratinization of 13-day embryonic chick tarsometatarsal skin growing in a chemically defined medium (Sugimoto et al., 1974). Fine structural features of such an in vitro keratinization process were studied by electron microscopy in the present work.After 2 days of culture with hydrocortisone (0.02 or 0.2 μM), development of the tonofilament bundles occurred to some extent, but the keratinized layer was not formed. Keratinization was observed after 4 days of culture with hydrocortisone (0.02 or 0.2 μM). Desmosomes and tonofilament bundles were prominent in the cytoplasm of the basal and intermediate cell layers of the epidermis. Keratohyalin granules and lipid droplets appeared in the upper layer. Degradation of cellular organelles such as nuclei and mitochondria then proceeded, leaving only filament bundles and electron-dense amorphous masses in the cytoplasm. Thickened cellular envelopes, which are characteristic of keratinized cells, were also observed. These features are characteristic of alpha-type keratinization which is common for other body surfaces. Beta-type keratinization, typical of normal embryonic scales, was not observed even after 6 days of culture with hydrocortisone. Keratinization of embryonic subperiderm of beta-type did not occur either. These ultrastructural observations clearly showed that hydrocortisone induced the alpha-type keratinization. It was also suggested that SCMEpA was closely related to alpha-type keratinization.     

Sulphur, thiols, and disulphides in the fish epidermis, with remarks on keratinization

Energy dispersive x-ray (EDX) analysis and qualitative and quantitative histochemistry were applied to study the distribution and contents of sulphur, thiols and disulphides in the epidermis of the river lamprey Lampetra fluviatilis , the lesser spotted dogfish Scyliorhinus canicula and the brown trout Salmo trutta fario . Thiols generally reacted weakly throughout the entire epidermis, whereas disulphide reactions were more distinct and differentiated. In the river lamprey, the concentrations of -S-S- groups clearly increased in the developing mucous cells from the stratum basale to the stratum superficiale; skein cells and granular cells reacted negatively to weakly. In the lesser spotted dogfish, amounts of disulphides appeared at moderate concentrations, and only goblet cells displayed a strong reaction. In the brown trout, filament cells showed low concentrations or weak reactions of disulphides, goblet cells and the most outer superficial cells stained strongly. Sulphur distribution and contents generally supported the histochemical observations in normal epidermis cells (absolute sulphur contents: 41–59 mM), only the brown trout showed high amounts of sulphur in the stratum basale (81 mM). The findings corroborate the view that there is an inverse correlation between keratinization and mucous secretion in normal fish epidermis. The sometimes distinct contents of disulphides in the outer mucous layer indicate that this system could endure higher mechanical stress than predictable from its large amounts of neutral glycoproteins.     

Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation

Keratins K14 and K5 have long been considered to be biochemical markers of the stratified squamous epithelia, including epidermis (Moll, R., W. Franke, D. Schiller, B. Geiger, and R. Krepler. 1982. Cell. 31:11-24; Nelson, W., and T.-T. Sun. 1983. J. Cell Biol. 97:244-251). When cells of most stratified squamous epithelia differentiate, they downregulate expression of mRNAs encoding these two keratins and induce expression of new sets of keratins specific for individual programs of epithelial differentiation. Frequently, as in the case of epidermis, the expression of differentiation-specific keratins also leads to a reorganization of the keratin filament network, including denser bundling of the keratin fibers. We report here the use of monospecific antisera and cRNA probes to examine the differential expression of keratin K14 in the complex tissue of human skin. Using in situ hybridizations and immunoelectron microscopy, we find that the patterns of K14 expression and filament organization in the hair follicle are strikingly different from epidermis. Some of the mitotically active outer root sheath (ORS) cells, which give rise to ORS under normal circumstances and to epidermis during wound healing, produce only low levels of K14. These cells have fewer keratin filaments than basal epidermal cells, and the filaments are organized into looser, more delicate bundles than is typical for epidermis. As these cells differentiate, they elevate their expression of K14 and produce denser bundles of keratin filaments more typical of epidermis. In contrast to basal cells of epidermis and ORS, matrix cells, which are relatively undifferentiated and which can give rise to inner root sheath, cuticle and hair shaft, show no evidence of K14, K14 mRNA expression, or keratin filament formation. As matrix cells differentiate, they produce hair-specific keratins and dense bundles of keratin filaments but they do not induce K14 expression. Collectively, the patterns of K14 and K14 mRNA expression and filament organization in mitotically active epithelial cells of the skin correlate with their relative degree of pluripotency, and this suggests a possible basis for the deviation of hair follicle programs of differentiation from those of other stratified squamous epithelia.     

Disruption of the keratin filament network during epithelial cell division

The behaviour of keratin filaments during cell division was examined in a wide range of epithelial lines from several species. Almost half of them show keratin disruption as described previously: by immunofluorescence, filaments are replaced during mitosis by a 'speckled' pattern of discrete cytoplasmic dots. In the electron microscope these ' speckles ' are seen as granules around the cell periphery, just below the actin cortical mesh, with no detectable 10 nm filament structure inside them and no keratin filament bundles in the rest of the cytoplasm. A time course of the filament reorganization was constructed from double immunofluorescence data; filaments are disrupted in prophase, and the filament network is intact again by cytokinesis. The phenomenon is restricted to cells rich in keratin filaments, such as keratinocytes; it is unrelated to the co-existence of vimentin in many of these cells, and vimentin is generally maintained as filaments while the keratin is restructured. Some resistance to the effect may be conferred by an extended cycle time. Filament reorganization takes place within minutes, so that a reversible mechanism seems more likely than one involving de novo protein synthesis, at this metabolically quiet stage of the cell cycle.     

松江鲈鱼皮肤的显微和亚显微结构

采用光学显微镜、扫描电镜和透射电镜,对松江鲈鱼(Trachidermus fasciatus)成体皮肤的显微和亚显微结构进行了观察。结果表明,松江鲈鱼体表不同部位皮肤的厚薄不一,但基本结构相似。皮肤由表皮和真皮层构成。松江鲈鱼的皮肤裸露无鳞,表皮层较薄,由约4～8层细胞构成,主要由复层上皮细胞和黏液细胞及基底细胞组成。表层细胞呈扁平、多边形,细胞之间主要靠桥粒紧密连接,连接处形成增厚的边缘嵴状突起。表皮细胞游离面向内凹陷,表面形成指纹状微嵴。黏液细胞呈圆形或卵圆形,散布在上皮细胞之间。黏液细胞内的黏原颗粒具有椭圆颗粒状、均匀致密的块状和疏松丝状3种不同形态。真皮通过基膜与表皮相连,由稀疏层和致密层构成。真皮结缔组织在腹部较厚而在其他部位较薄。表皮与真皮连接处有色素层,头部、背部、尾柄和体侧皮肤色素细胞分布多,色素层明显,而腹部和颏部皮肤缺少色素。松江鲈鱼黄河群体真皮层中有角质棘状突起,而滦河群体则无。头部、体侧和尾柄处皮肤上还分布有侧线孔和表面神经丘等感觉器官。     

Electron microscopical demonstration of thiols and disulphides in the porcine epidermis

Summary This study describes the electron microscopical distribution of free thiols and disulphides in the epidermis of the domestic pig and the wild boar, as compared to light microscopical histochemistry. With the silver methenamine method, silver labelling of thiols was clearly achieved on the keratohyalin and cytofilament accumulations in the cells of the living epidermis and the plasma membrane of granular cells. To a certain extent, the envelope and cytoplasm of young corneocytes reacted equally intensively. Disulphides were very abundant in the filaments, keratohyalin granules, and cell envelope of granular cells, and, particularly, in the envelope (marginal band) of corneal cells; the latter structure being distinctly delineated from the background. As a specific feature, the viable epidermis of the wild boar stained strongly for disulphides. The results obtained are discussed in view of actual concepts of epidermal keratinization and corneal cell function.     

Larval skin of the flathead sole,Hippoglossoides elassodon

Summary The skin of newly-hatched larval flathead sole, Hippoglossoides elassodon, is described by light and electron microscopy. The epidermis is usually two cells thick and shows differentiation into both squamous and mucous cells. The squamous cells are characterized by numerous cytoplasmic filaments, typical desmosomes, and lack of keratinization; the mucous cells are distended with mucous droplets, which appear to originate in the Golgi apparatus. A basement membrane is present, although thinner and less dense than that of older fish, and the dermis contains loose formations of collagen and pigment cells.This work was supported in part by USPHS research grant CA-08158 from the National Cancer Institute.     

A STUDY OF THE FINE STRUCTURE OF THE EPIDERMIS OF RANA PIPIENS

The epidermis of adult Rana pipiens has been studied by electron microscopy and histological and histochemical methods. It was found that the epidermis is engaged in the production of both keratin and mucus. The basal cells are mainly filled with tonofilaments, whereas the cells located in the mid-portion of the epidermis contain both tonofilaments and mucous granules. Golgi vesicles and endoplasmic reticulum are found in relative abundance in the mucus-producing cells and seem to be involved in the production of mucous granules. The mucus seen was partly retained within the cells and partly secreted into the intercellular spaces. The outermost keratinized cells contain mainly filaments and a few remnants of cell constituents.     

Differentiation of snake epidermis, with emphasis on the shedding layer

Little is known about specific proteins involved in keratinization of the epidermis of snakes. The presence of histidine-rich molecules, sulfur, keratins, loricrin, transglutaminase, and isopeptide-bonds have been studied by ultrastructural autoradiography, X-ray microanalysis, and immunohistochemistry in the epidermis of snakes. Shedding takes place along a shedding complex, which is composed of two layers, the clear and the oberhautchen layers. The remaining epidermis comprises different layers, some of which contain beta-keratins and others alpha-keratins. Weak loricrin, transglutaminase, and sometimes also iso-peptide-bond immunoreactivities are seen in some cells, lacunar cells, of the alpha-layer. Tritiated histidine is mainly incorporated in the shedding complex, especially in dense beta-keratin filaments in cells of the oberhautchen layer and to a small amount in cells of the clear layer. This suggests the presence of histidine-rich, matrix proteins among beta-keratin bundles. The latter contain sulfur and are weakly immunolabeled for beta-keratin at the beginning of differentiation of oberhautchen cells. After merging with beta cells, the dense beta-keratin filaments of oberhautchen cells become immunopositive for beta-keratin. The uptake of histidine decreases in beta cells, where little dense matrix material is present, while pale beta-keratin filaments increase. During maturation, little histidine labeling remains in electron-dense areas of the beta layer and in those of oberhautchen spinulae. Some roundish dense granules of oberhautchen cells rich in sulfur are negative to antibodies for alpha-keratin, beta-keratin, and loricrin. The granules eventually merge with beta-keratin, and probably contribute to the formation of the resistant matrix of oberhautchen cells. In conclusion, beta-keratin, histidine-rich, and sulfur-rich proteins contribute to form snake microornamentations.     

Ultrastructural, histochemical and cytochemical characterization of intestinal epithelial cells in Aplysia depilans (Gastropoda, Opisthobranchia)

To improve the current knowledge about the digestive system in opisthobranchs, light and electron microscopy methods were used to characterize the epithelial cells in the mid‐intestine of Aplysia depilans. This epithelium is mainly formed by columnar cells intermingled with two types of secretory cells, named mucous cells and granular cells. Columnar cells bear microvilli on their apical surface and most of them are ciliated. Mitochondria, multivesicular bodies, lysosomes and lipid droplets are the main components of the cytoplasm in the region above the nucleus of these cells. Peroxisomes are mainly found in middle and basal regions, usually close to mitochondria. Mucous cells are filled with large secretory vesicles containing thin electron‐dense filaments surrounded by electron‐lucent material in which acidic mucopolysaccharides were detected. The basal region includes the nucleus, several Golgi stacks and many dilated rough endoplasmic reticulum cisternae containing tubular structures. The granular cells are characterized by very high amounts of flat rough endoplasmic reticulum cisternae and electron‐dense spherical secretory granules containing glycoproteins. Enteroendocrine cells containing small electron‐dense granules are occasionally present in the basal region of the epithelium. Intraepithelial nerve fibres are abundant and seem to establish contacts with secretory and enteroendocrine cells.     

Characteristics of the structural organization of the cytoskeleton in suspension and monolayer sublines of mouse fibroblasts depending upon the type of growth in culture

In attached cells, the main part of filaments is localized in submembrane area, whereas suspension fibroblasts are situated deeper in the cytoplasm. Another difference involves the form of filament gatherings. In the former these are bundles stretched parallel to the cell surface, whereas in the latter these gatherings are in the form of loose balls near the nucleus. According to their diameter and type of cell localization the latter filaments are similar to intermediate ones. In suspension cells with caps formed by ligand treatment, gatherings of fibrillar elements are observed within the cytoplasm but these filaments are shorter and looser, being located in disorder. In addition, the increase in the number of electron dense granules of unknown nature is observed between these filaments. The above observations suggest that the surface processes may be associated with the inner parts of cytoskeleton.     

Ultrastructural Observations on the Epidermis of Xenoturbella bocki Westblad, 1949; With a Discussion of Epidermal Cytoplasmic Filament Systems of Invertebrates

The highly complex epidermis of Xenoturbella bocki has been studied, mainly employing transmission electron microscopical and histochemical methods. The epidermal organization is described to best advantage in terms of the various cell types present, their intricate interrelationships and their interaction with the highly developed subepidermal membrane complex (SMC). The epidermis is composed mainly of ciliated epidermal supporting cells, at least two types of gland cells (one dominant mucous type with basophilic, alcian blue-positive granules; another possessing acidophilic, PAS-positive granules), several types of nerve cells present in the intraepidermal nerve layer and, finally, some peculiar basally arranged 'pillow cells'. Junctional structures are sparsely developed. The epidermal supporting cell is provided with a distinct cell web, but diagnostic for this cell type is the presence of a very prominent single supporting fibre (SF) composed of supporting filaments. Distally some of these filaments may terminate on the tapering proximal ends of ciliary rootlets, a highly unusual phenomenon. Basally the SF is attached to the SMC in a complex arrangement. The possible relationship of the supporting filaments to cytoskeletal intermediate filaments is discussed. The SF is compared to other cytoplasmic filament systems in mammals and various invertebrates. The ultrastructural organization of Xenoturbella is now fairly well understood, nevertheless, our conclusion must be that it is still not possible to assign an appropriate place for this animal in the systematic-phylogenetic system.     

Variations in the arrangement of tonofilaments in the epidermis of teleost fish

Tonofilaments in epithelial cells of teleost skin can be aligned as bundles or skeins of appreciable bilk, or form a pattern of smaller bundles oriented in various directions, or there may be a condition where individual tonofilaments interlace. If sufficiently close together, interwoven tonofilaments can form a basket-like structure, a “capsule”, proximally in the cell. This arrangement, previously known in epithelial cells of Myxinoids, occurs in localised sites in various teleosts of diverse taxonomic position, for instance in clupeids and gadids. A less intimate interlacing of cortical tonofilaments can accompany a modification of the perinuclear cytoplasm previously described, by light microscopy, as “vesiculated”, as in the middle layers of the epidermis in Periophthalmus. In head epidermis of Sprattus, the outer layers of cells contain proximal capsules, but the middle layers consist of flattened cells with a restricted perinuclear cytoplasm, peripheral tonofilaments, and a second population of filaments of a larger calibre. One implication of these results is that the cytoskeleton can undergo profound modification as cells progress from the basal to the superficial layers of the epidermis.     

Intermediate filament proteins in nonfilamentous structures: Transient disintegration and inclusion of subunit proteins in granular aggregates

The intermediate filament cytoskeleton of cultured bovine kidney epithelial cells and human HeLa cells changes dramatically during mitosis. The bundles of cytokeratin and vimentin filaments progressively unravel into protofilament-like threads of 2–4 nm diameter, and intermediate filament protein is included in numerous, variously sized (2–15 μm) spheroidal aggregates containing densely stained granular particles of 5–16 nm diameter. We describe these mitotic bodies in intact cells and in isolated cytoskeletons. In metaphase to anaphase of normal mitosis and after colcemid arrest of mitotic stages, many cells contain all their detectable cytokeratin and vimentin material in the form of such spheroidal aggregate bodies, whereas in other mitotic cells such bodies occur simultaneously with bundles of residual intermediate filaments. In telophase, the extended normal arrays of intermediate filament bundles are gradually reestablished. We find that vimentin and cytokeratins can be organized in structures other than intermediate filaments. Thus, at least during mitosis of some cell types, factors occur that promote unraveling of intermediate filaments into protofilament-like threads and organization of intermediate filament proteins into distinct granules that form large aggregate bodies. Some cells, at least certain epithelial and carcinoma cells, may contain factors effective in structural modulation and reorganization of intermediate filaments.     

Ultrastructural studies of normal skin and epidermal papillomas of the flathead sole,Hippoglossoides elassodon

Summary Epidermal cells of normal fin web and of the epidermal papilloma of the flathead sole, Hippoglossoides elassodon, were compared by light and electron microscopy. The predominant cells of normal epidermis are squamous and are characterized by complex microvillous interdigitations between adjacent cells, numerous desmosome-like structures, terminal bars, prominent cytoplasmic filaments, and blunt, broad-based microvilli on the outer (free) surfaces of the superficial cells. The less numerous mucous cells of normal epidermis possess abundant ergastoplasm and mucus-filled vacuoles, but lack desmosome-like structures and cytoplasmic filaments.In contrast, in the epidermal papilloma, mucous cells are absent; and the tumor cells are without microvilli, desmosome-like structures, cytoplasmic filaments, and conspicuous ergastoplasm. The papilloma cells are in addition characterized by certain features not seen in normal epidermis, including prominent nuclear pores, very large nucleoli, nuclear dense bodies, vesicular mitochondria with few cristae and dense internal granules, and three types of distinctive cytoplasmic particles of possible viral nature.This investigation was supported by Public Health Service research grant CA 08158 from the National Cancer Institute, and American Cancer Society Institutional Grant No. IN-53 E.We are indebted to Miss Mary Bens for her indispensable technical assistance.     

Keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri (dipnoi)

The differentiation of the epidermis in sarcopterigian fish may reveal some trend of keratinization followed by amphibian ancestors to adapt their epidermis to land. Therefore, the process of keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri was studied by histochemistry, electron microscopy, and keratin immunocytochemistry. The epidermis is tri-stratified in a 2-3-month-old tadpole but becomes 6-8 stratified in young adults. Keratin filaments increase from basal to external cells where loose tonofilament bundles are present. This is shown also by the comparison of positivity to sulfhydryl groups and increasing immunoreactivity to alpha-keratins in more external layers of the epidermis. Two broad-spectrum anti alpha-keratin monoclonal antibodies (AE1 and AE3) stain all epidermal layers as they do in actinopterigian fish. In the adult epidermis, but not in that of the larva, the AE2 antibody (a marker of keratinization in mammalian epidermis) often immunolabels more heavily the external keratinized layers where sulfhydryl groups are more abundant. Mucous granules are numerous and concentrate on the external surface of the epidermis to be discharged and contribute to cuticle formation. Keratin is therefore embedded in a mucus matrix, but neither compact keratin masses nor cell corneous envelope were seen in external cells. It is not known whether specific matrix proteins are associated with mucus. There was no immunolocalization of the keratin-associated proteins, filaggrin and loricrin, which suggests that the epidermis of this species lacks the matrix and cell corneus envelope proteins characteristic of that of amniotes. In conclusion, while specific keratins (AE2 positive) are probably produced in the uppermost layers as in amphibian epidermis, no interkeratin, matrix proteins seem to be present in external keratinocytes of the lungfish other than mucus.