Correlated TEM, SEM, and histological observations of filiform papillae of the cow tongue

The ultrastructural and histological characteristics of the filiform papillae of the cow tongue are described. These large, conical papillae display a fully keratinized external projection, which demonstrates a dominant hard keratin core partially ensheathed by soft keratin. This biostructure differentiates from a dominant posterior cell line, leading to the hard keratin core, and a thinner anterior cell line which, with contributions from an interpapillary cell line, appears responsible for an attenuating sheath of soft keratin around the hard keratin core. The hard keratin cell line differentiates from cells devoid of keratohyalin granules (KHG), which are rich in thick bundles of tonofilaments. The soft keratin cell line differentiates from cells containing both eosinophil and basophil KHG and dispersed tonofilaments. The bovine filiform papilla also appears similar in biostructure to the mammalian hair shaft and the 'filiform hairs' of the rat penis.     

Degenerative and regenerative changes in epidemrmal organ culture: A morphological study wity reference to membrane-coating granules

Summary Membrane-coating granules (MCG) are poorly understood lamellate organelles unique to keratinized epithelia. This study provides data on a skin model for future in vitro investigations of MCG. Porcine ear epidermal, organ cultures were used under standard cell culture conditions. This system was selected because it is easily established and, following a degenerative period in which MCG are lost, regenerates to form a highly differentiated epidermis. The epidermis appeared healthy during the first 2 din vitro and contained MCG but lost keratohyalin granules (KHG). Overt degenerative changes were evident in the upper epidermis on Day 3, and MCG were now bloated. By Day 4 only one to three layers of viable undifferentiated cells remained. In the overlying necrotic epidermis MCG were rare, presumably due to the bursting of bloated MCG. Epidermal regeneration began around Day 5 and by Day 7 there, were 8 to 13 layers, including a, rudimentary parakeratotic stratum corneum (up to 4 layers). The stratum granulosum (two to three layers) now containe immature KHG and poorly lamellate MCG, but only amorphous material extracellularly. By Day 11 there were three to four layers of granular cells as in vivo, and an orthokeratotic stratum corneum (two to four layers). Improved cornification coincided with an increased number of mature KHG and cross-banded MCG, and lamellate MCG contents extracellularly. This model of epidermal regeneration will factiliate studies into the role played by MCG in keratinization because the epithelium initially lacked MCG but later expressed all the major morphologic features of epidermis. Furthermore the mechanisms by which MCG translaction and extrusion are effected may be probed, by the inclusion of such agents as antimicrotubular drugs and calcium ionophores. This work was supported by a grant from Unilever Research, Port Sunlight, England.     

Formation of the tectorial membrane of the cochlear canal during the embryogenesis of birds

By means of electron microscopy formation of the tectorial membrane of the cochlear canal and differentiation of the cells participating in the process (supporting cells of the basilar papilla and anterior homogeneous cells--AHC) have been studied in chick embryos. The AHC, to which the tectorial membrane is fixed, produce fine fibrillar material, included into the composition of the tectorial membrane. The cells mentioned form a number of cytoskeletal structures connected with the mechanical function of the tectorial membrane. Besides the network of the tonofilaments, gradually filling cytoplasm of the AHC, some peculiar attachings in the form of collagenous fibrillar bundles are revealed, they reach the AHC from the sublying connective tissue and have a direct contact with the basal membrane of the cells. The beginning of the tectorial membrane formation precedes the formation of the cytoskeletal structures. The latter appear only when the mass of the tectorial membrane, and hence, the mechanical loading on the AHC is great enough.     

Fine structure of the epidermis of the mudskipper,Periophthalmus modestus (Gobiidae)

The ultra-structure of the epidermis of the mudskipper,Periophthalmus modestus, was examined by both light and transmission electron microscopies. The epidermis is exceptionally not well endowed with mucous or granular cells. Filament-containing cells occur in three distinct layers of the surface, middle and basal epidermis. The surface layer is further subdivided into two layers, an outermost and less superficial one. Two different cell types were identified in the epidermis. Type I cells are fiat cells in a single stratum. Type II cells are enormous cells, characterized by having a large vacuole in the cytoplasm. The outermost layer is composed of a free surface of Type I cells and numerous microridges covered with a fuzzy, fibrillar substance. The “fuzz” forms a cuticule-like structure, but keratinization as found in terrestrial animals does not occur. The superficial layer contains Type I cells and intraepithelial blood capillaries. When Type I cells become senescent, numerous intercellular spaces are formed in the plasma membranes of adjacent cells, with the senescent cells finally falling off. Just beneath these cells, however, young cells of Type I are always found. The blood capillaries are usually reinforced with young Type I cells. A large volume of oxygen may be absorbed through the skin using the blood capillary network. The middle layer contains several strata of Type II cells. The special corky structure of these cells seems to play an important role in thermal insulation and protection against ultraviolet light in relation to life out of water. However, by comparison with terrestrial animals, the histological design of the epidermis of this goby appears incomplete, so as to reduce desiccation on land, owing to the epidermis lacking a keratinized stratum. The differentiation of the epidermis seems to be an adaptation for a terrestrial habit in this species.     

An electron microscopic study of the human epidermal keratinocyte

Summary 1. The epidermis of the flexor surface of the upper arm of human subjects was studied with the electron microscope. 2. The cytoplasm of the keratinocytes in the basal layer contained many tonofilaments, ribosomes and other cell organelles. The tonofilaments were arranged singly or in loose bundles and many were attached to the inner membrane of the desmosomes. Along the basal border of the cells pinocytotic vesicles could be seen at different stages of development. 3. The keratinocytes in the stratum spinosum differed from those in the basal layer in two main ways: (a) The tonofilaments were grouped together into large compact bundles known as tonofibrils and it was possible to determine a definite beading or cross banding along the length of some of the filaments. (b) The cells were assuming a flattened shape. 4. The keratinocytes in the stratum granulosum possessed large numbers of irregularly shaped keratohyaline granules. The granules were strongly osmiophilic and were always situated on a meshwork of tonofibrils. The keratohyaline granules had no internal structure. The nuclei and mitochondria showed evidence of degeneration. 5. The keratinocytes in the stratum corneum were long and flattened. The cell walls showed increased electron density and were considerably thickened. The cytoplasm was filled with closely packed fibres separated by a small amount of lucent matrix. The fibres were grouped together in bundles running in different directions within the flattened squames. The fibres had along their entire length alternating areas of high and low electron density. The keratohyalin granules had disappeared and nothing remained of the nuclei or the organelles. In the deepest cells of this region the fibres were sometimes loosely packed leaving large irregular open spaces. This area corresponded to the stratum lucidum. In the most superficial layers of the stratum corneum the fibres appeared to be breaking down so that little remained within the keratinocyte except large lucent spaces. The desmosomes showed distinct structural changes. 6. An attempt was made to correlate the structural changes in the different epidermal layers with the process of keratinization. The possible part that keratohyalin may play in the process of thickening of the cell walls was discussed. The relationship between the desmosome and its dynamic environment was considered.I wish to express my sincere thanks to Dr. David Hilding of the Department of Otolaryngology for the use of an R.C.A. electron microscope and other facilities in his laboratory. This research was supported by the United States Public Health Service and American Cancer Society grants. USPHS CA 04679-07, NB 03995.     

Ultrastructure of human amnion and amniotic plaques of normal pregnancy

Summary The fine structure of amniotic and amniotic-plaque epithelia has been studied from normal term pregnancies. The columnar/cuboidal amniotic epithelial cells usually have apical or central nuclei, some free ribosomes, patches of granular endoplasmic reticulum, juxtanuclear Golgi complexes, rod-shaped mitochondria, lipid droplets and some glycogen granules. They have short, blunt microvilli which frequently branch and bathe in the amniotic fluid. The lateral plasma membranes enclose tortuous intercellular spaces which are always interrupted by variously folded processes and desmosomes. The epithelial cells rest on a basal lamina and exhibit highly folded basal processes. The amniotic epithelial cells are neither distinctly Golgi and fibrillar types nor light and dark in appearance.Amnion from near the umbilical cord contains many microscopic and several large plaques. Similar structures are not found on the reflected amnion. The microscopic plaques are whitish and translucent, whereas the large ones are opaque. The large plaques vary between 1–3 mm in diameter, and are over 15 cell layers thick. Each large plaque has a main central region and edges continuous with either the microscopic plaque or the simple amniotic epithelium. The main region shows four zones, namely, stratum basale, stratum spinosum, stratum granulosum and stratum corneum. Such zones are not distinct at the edges. The fine structure of basal cells compares with the amniotic epithelial cells, but the cells of spinosum and granulosum layers possess variable amounts of tonofibrils, keratohyalin granules, free ribosomes and other cytoplasmic organelles and inclusions. The corneum cells are keratinized and are frequently separated by intercellular spaces. They slough into the amniotic cavity singly or as a sheet, and contribute towards the composition of the amniotic fluid. The plaques are of amniotic origin, and are not formed by adhesion of either squamous cells or fetal skin cells (masses of keratinized squames). The present observations suggest that the occurrence of amniotic plaques is normal. The presence of plaques may not be necessarily associated with fetal abnormality. However, increase in numbers of plaques may be caused by conditions of fluid imbalance. The homology and significance of plaques in eutherian mammals have been discussed.This research was supported by USPHS Grant AM-11376 and NIH Grant 69-2136.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Dorsal lingual epithelium of Platemys pallidipectoris (Pleurodira,Chelidae)

Scanning electron microscopy reveals that the flat tongue of Platemys pallidipectoris has shallow grooves and no lingual papillae. The surface of the tongue is covered with dome-shaped bulges, each corresponding to a single cell. Short microvilli are distributed over the cell surface. Light microscopy shows a stratified cuboidal epithelium with an underlying strong connective tissue. Transmission electron microscopy indicates four layers. The basal cells of the epithelium are electron-translucent and have a large central nucleus and a cytoplasm with keratin tonofilaments. Plasma cells with abundant rough endoplasmic reticulum and mitochondria occur in the basal layer. Production of secretory granules begins in the more electron-dense intermediate layers and increases as the cells move toward the surface. The membranes of the cells of the deep intermediate layer form processes that project into relatively wide intercellular spaces. In the superficial intermediate layer, the cytoplasm of the cells contains numerous fine granules; these increase in number but not in size in more distal layers. The cells of the surface layer are electron-translucent with a round nucleus. Contents of their fine granules are secreted into the oral cavity. © 1995 Wiley-Liss, Inc.     

Morphogenesis of rat lingual filiform papillae.

The morphogenesis of filiform papillae on rat tongue was investigated with the electron microscope. Tongue rudiments were first seen on the 12th day of gestation. At 15-17 days, dermal papillae had formed and were arranged in hexagonal array on the dorsal lingual surface. Capping each dermal papilla was a two-layered epithelium that protruded slightly above the lingual surface, thus forming the early filiform papilla. In the next stage of development, at 18-19 days of gestation, the epithelium lining the papilla had differentiated into two cell populations, one producing hard keratin, the other producing soft keratin. Some of the keratinized epithelial cells assumed a position at an acute angle to the tongue surface and extended deep into the epithelium. In the next stage, 20-21 days, a cleft appeared within these angularly oriented cells. This resulted in the division of the epithelium into keraatin-lined individual filiform papillae. Finally, the individual papillae increased in size to the adult form.     

Fine structure of the dorsal lingual epithelium of the little tern, Sterna albifrons Pallas (Aves, Lari).

The dorsal surface of the tongue of the little tern, Sterna albifrons, has a distinctive anterior region for five-sixths of its length and a terminal posterior region. The anterior region observed by scanning electron microscopy is distinguished along its forward half by a median line from which median papillae protrude. The hind half of the anterior region has a median sulcus without papillae. The deciduous epithelium on both sides of the median line and sulcus bears scattered epithelial protrusions. The posterior lingual region has neither median papillae nor deciduous epithelium. So-called giant conical papillae are located in a transverse row between anterior and posterior regions. Delicate microridges adorn the surfaces of all outer epithelial cells in both regions. Examination of the dorsal lingual epithelium by light and electron microscopy provides histologic and cytologic criteria for distinguishing anterior and posterior regions. Basal cells are nearly alike throughout the dorsal epithelium. Intermediate layer cells of the anterior region contain numerous tonofibrils in electron-dense bundles composed of 10 nm tonofilaments. The outer layer is composed of electron-dense, well-keratinized cells, and electron-lucent epithelial protrusions are present on the exposed surface of the outermost cells. Median papillae are composed of typical keratinized cells, which are nearly filled with keratin filaments. Intermediate layer cells in the posterior region of the tongue are nearly filled with unbundled tonofilaments. There is only a very thin outer keratinized layer in this region.     

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.     

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.     

Fine structure of the dorsal lingual epithelium of the lizard, Gekko japonicus (Lacertilia, Gekkonidae)

Three different types of lingual papilla were observed by scanning electron microscopy on the dorsal lingual epithelium of the lizard Gekko japonicus. Dome-shaped lingual papillae were located at the apex. Flat, fan-shaped lingual papillae were seen in the widest area of the lingual body. Long, scale-like lingual papillae were arranged on the latero-posterior dorsal surface. At higher magnification, microvilli and microridges were seen to be widely distributed over the surface of the papillae. By light microscopy, the epithelium of the dome-shaped papillae was composed of single, columnar epithelial cells filled with secretory granules. The tip of the epithelium of the fan-shaped and scale-like papillae was composed of stratified squamous epithelial cells without granules. The major part of the epithelium of these two types of papilla, except the tip area, was also composed of single, columnar epithelial cells with secretory granules. By transmission electron microscopy, a nucleus without a defined shape was seen to be located in the basal part of each of the single, columnar epithelial cells. Rough-surfaced endoplasmic reticulum and Golgi apparatus were well developed around the nucleus. The other, major part of the cytoplasm was filled with the spherical secretory granules, a large number of which had very electron-dense cores and moderately electron-dense peripheral regions. In the stratified squamous epithelium, a nucleus, which tended to be condensed on the free-surface side, was located in the center of each cell. Mitochondria, endoplasmic reticulum, and vesicles were observed in the cytoplasm.     

Fine structure of the horny teeth of the lamprey,Entosphenus japonicus

The fine structure of the horny teeth of the lamprey, Entosphenus japonicus, was examined by light- and electron-microscopy. Most of the horny teeth consisted of two horny and two nonhorny layers. The primary horny layer was well keratinized, and the cells were closely packed and intensely interdigitated, being joined together by many modified desmosomes. The plasma membrane of the horny cell, unlike the membranes of other vertebrates, was not thickened. The intercellular spaces were filled with electron-dense material. Microridges were seen on the free surface. Structures resembling microridges were found on the underside of the primary horny layer. The secondary horny layer displayed various stages of keratinization. The keratinization started at the apex and developed toward the base. In the early stage of keratinization, the superficial cells became cylindrical and were arranged in a row forming a dome-shaped line. Their nuclei were situated in the basal part of the cells. The appearance of the nonhorny layers varied according to the degree of keratinization of the horny layers beneath them. The nonhorny cells were joined together by many desmosomes and possessed many tonofilament bundles. The replacement and keratinization of the horny teeth are discussed in the light of these results.     

Fine structure of the dorsal epithelium of the tongue of the freshwater turtle,Geoclemys reevesii (Chelonia,Emydinae)

Scanning electron microscopy shows that lingual papillae occur all over the dorsal surface of the tongue of the freshwater turtle, Geoclemys reevesii. The surface of each papilla is composed of compactly distributed hemispherical bulges, each composed of a single cell. Microvilli are widely distributed over the surface of cells. Histological examination reveals that the connective tissue penetrates deep into the center of papillae and that the epithelium is stratified columnar. Under the transmission electron microscope, the cells of the basal and the deep intermediate layers of the epithelium appear rounded. A large nucleus lies in the central area of each cell. The cytoplasm contains mitochondria, endoplasmic reticulum and free ribosomes. The cell membrane form numerous processes. The shallow intermediate layer contains two types of cell. The cytoplasm of the first has numerous fine granules, in addition to mitochondria, ribosomes, and endoplasmic reticulum. The other type of cell contains highly electron-dense granules. The surface layer shows two cell types. One type consists of typical mucous cells. The other type of cell contains fine, electron-lucent granules. The latter cells lie on the free-surface side, covering the mucous cells, and have microvilli on their free surfaces.     

Three-dimensional and surface structures of rat filiform papillae

The three-dimensional and surface structures of the simple conical papillae of the rat tongue have been demonstrated with scanning electron microscopy. The papillary projection was organized into the anterior, posterior and central core cell populations, whereas the basal region of the papilla which consisted of circularly arranged cells showed no differentiation into three autonomic cell populations. It is considered that the anterior and posterior cell populations around the central core tend to be mutually attached at the bilateral sides, and that the posterior and core cell contacts are rather close than the anterior one. The anterior papillary cells showed relatively smooth surface with little micropits and without microridges. The reticular microridges on the basal cell surface of the posterior papillary cells appear to later develop the micropits and linear microridges on the tip cell surface. These suggest that the anterior cell surface is more highly keratinized than the posterior one. The microridges or micropits on the outer cell surface and the microprojections on the inner cell surface organizing filiform papilla are considered to be the structures for the purpose of cell adhesion.     

Induction of cAMP receptors by disaggregation of the multicellular complexes of Dictyostelium discoideum

Subcultivated rat lingual epithelial cells when grown on collagen gels at a liquid-gas interface achieve a highly ordered state that closely resembles the parent tissue. Three distinct cell layers are present; basal, spinous, and keratinized. Basal cells are cuboidal in shape and form a complex interface with the underlying collagen fibrils. Spinous cells form a layer 5–10 cells thick and, with the exception of keratohyalin granules, possess an organellar complement identical with native cells, including membrane-coating granules. The keratinized cell layer increases in thickness as a function of time spent in culture. Forty or more plies of terminally differentiated cells are observed following a 30-day culture period. Terminally differentiated cells while retaining pycnotic nuclei and some other organellar debris are principally envelope-enclosed squames filled with tonofilaments. Keratinization is a continuing process which occurs simultaneously across the full expanse of the culture surface. The high degree of tissue organization observed appears to be the result of feeding the cultures from the undersurface.     

Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis

An overview of the morphology and life cycle of Batrachochytrium dendrobatidis, the cause of chytridiomycosis of amphibians, is presented. We used a range of methods to examine stages of the life cycle in culture and in frog skin, and to assess ultrastructural pathology in the skin of 2 frogs. Methods included light microscopy, transmission electron microscopy with conventional methods as well as high pressure freezing and freeze substitution, and scanning electron microscopy with critical point drying as well as examination of bulk-frozen and freeze-fractured material. Although chytridiomycosis is an emerging disease, B. dendrobatidis has adaptations that suggest it has long been evolved to live within cells in the dynamic tissue of the stratified epidermis. Sporangia developed at a rate that coincided with the maturation of the cell, and fungal discharge tubes usually opened onto the distal surface of epidermal cells of the stratum corneum. A zone of condensed, fibrillar, host cytoplasm surrounded some sporangia. Hyperkeratosis may be due to (1) a hyperplastic response that leads to an increased turnover of epidermal cells, and (2) premature keratinization and death of infected cells.     

Culture of endodermal stem/progenitor cells of the mouse tongue

The tongue represents a very accessible source of tissue-specific epithelial stem cells of endodermal origin. However, little is known about the properties of these cells and the mechanisms regulating their proliferation and differentiation. Foxa2, an endodermal marker, is expressed throughout the tongue epithelium during embryonic development but becomes confined to a minority of basal cells and some taste bud sensory cells in the adult tongue. Using a previously described line of transgenic mice in which enhanced green fluorescent protein (eGFP) is expressed under the control of a human keratin 5 promoter region (Krt5-eGFP), we have isolated a subpopulation of cells in the basal epithelial layer of the mouse tongue with a high efficiency of generating holoclones of undifferentiated cells in culture with a feeder layer. Krt5-GFP(hi) cells can both self renew and give rise to differentiated stratified keratinized epithelial cells when cultured on an air-liquid interface.