Secretion of collagen types I and II by epithelial and endothelial cells in the developing chick cornea demonstrated by in situ hybridization and immunohistochemistry

Cells involved in the synthesis of collagen types I and II in the cornea of developing chick embryos have been studied by using in situ hybridization and immunohistochemistry. Corneas processed for in situ hybridization with the type I and II collagen probes demonstrated specific mRNAs in the epithelium of embryos at stage 18 with an increase at stages between 26 and 31, and then gradual decrease to the background level in the next several days. In the endothelium, a small amount of specific mRNA was recognized through these stages. In the stroma, only sections hybridized with the type I probe demonstrated mRNA in fibroblasts. Immunostaining demonstrated specific collagen types in the stroma at sites which were closely associated with cells containing specific mRNAs. Both collagens type I and II were present beneath the epithelium as narrow bands at stage 18; as the thicker primary stroma at stages 20 and 26; and as subepithelial, subendothelial and stromal staining at stage 31. Thereafter, type I collagen was increased in the stroma but it was also noted in the subepithelial and, to a lesser degree, subendothelial regions, whereas type II collagen was gradually confined to the subendothelial matrix. Electron microscopic examination of sections from 5-day-old (stage-27) embryo corneas using antibodies against the carboxyl propeptides of type I and II procollagens revealed the presence of these procollagens within the cisternae of the endoplasmic reticulum and Golgi vesicles in both epithelial and endothelial cells. In the epithelial cells both the periderm and basal cells contained these procollagens within the cytoplasmic organelles. These results indicate that not only the epithelial cells, but also the endothelial cells secrete collagen types I and II during the formation of the primary corneal stroma and for several days after invasion of fibroblasts.     

The behavior of fibroblasts from the developing avian cornea. Morphology and movement in situ and in vitro

The early chick cornea is composed of an acellular collagenous stroma lined with an anterior epithelium and a posterior endothelium. At stage 27-28 of development (5 1/2 days), this stroma swells so that the cornea is 75-120 mum thick. At the same time, fibroblasts that originate from the neural crest begin to invade this stroma. Using Nomarski light microscopy, we have compared the behavior of moving cells in isolated corneas with the migratory activities of the same cells in artificial collagen lattices and on glass. In situ, fibroblasts have cyclindrical bodies from which extend several thick pseudopodia and/or finer filopodia. Movement is accompanied by activity in these cytoplasmic processes. The flat ruffling lamelli-podia that characterize these cells on glass are not seen in situ, but the general mechanism of cell movement seems to be the same as that observed in vitro: either gross contraction or recoil of the cell body (now pear shaped) into the forward cell process, or more subtle "flowing" of cytoplasm into the forward cell process without immediate loss of the trailing cell process. We filmed collisions between cells in situ and in three-dimensional collagen lattices. These fibroblasts show, in their pair-wise collisions, the classical contact inhibition of movement (CIM) exhibited in vitro even though they lack ruffled borders. On glass these cells multi-layer, showing that, while CIM affects cell movement, fibroblasts can use one another as a substratum. Postmitotic cells show CIM in moving away from each other. Interestingly, dividing cells in situ do not exhibit surface blebbing, but do extend filopodia at telophase. The role of CIM in controlling cell movement in vivo and in vitro is stressed in the discussion.     

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.     

Does chondroitin sulfate have a role to play in the morphogenesis of the chick primary corneal stroma?

This paper makes three points about how the chick corneal epithelium lays down the primary stroma, an orthogonally arranged array of well-spaced, 20-nm-diameter collagen fibrils. (1) Isolated corneal epithelia will, when cultured, lay down de novo stromas whose fibril-diameter distribution, fibril spacing, and proteoglycan profile are similar to those laid down in vivo. They differ from embryonic stromas in two ways: first, much of the chondroitin sulfate is released to the medium and, second, there is a relatively small amount of orthogonal organization. Epithelia seem only to lay down such stromas if they are separated from their original stromas with dispase, which leaves an intact basal lamina, and spread out, basal lamina downward, on a Nuclepore filter (poresize, 0.1 micron). (2) Chondroitin sulfate (CS), the predominant proteoglycan (greater than 85%), seems to play no significant role in collagen fibrillogenesis in vitro. Stromas laid down in its absence were indistinguishable from controls as assayed by fibril diameter, organization, and spacing and the amount of collagen synthesized. For these experiments, epithelia were cultured in the presence of hyaluronidase, which degrades CS, and p-nitrophenyl beta-D-xyloside, which inhibits the formation of links between the core protein and glycosaminoglycan side chains in the PG; the absence of intact CS was confirmed by gel filtration. We suggest that, in vivo, CS may facilitate the interfibrillar movement that takes place as the cornea grows. We have also found that keratinase, which degrades the very small amount of keratan sulfate present in the primary stroma, has no effect on stromal deposition. (3) There are substantial amounts of unidentified matrix components in primary stromas laid down both in vivo and in vitro. This conclusion was drawn from SEM observations on both types of stroma after they had been freeze-dried, a process which does not condense hydrated macromolecules. Even after being treated with hyaluronidase to remove the CS, substantial amounts of interfibrillar matrix were still present. Until these components are identified and their interactions with collagen are understood, the mechanisms responsible for stromal morphogenesis are unlikely to be understood.     

Histology and Ultrastructure of the Body Wall in the Phoronid Phoronopsis harmeri

The histology and ultrastructure of the body wall in Phoronopsis harmeriwere studied using light microscopy and TEM. The ectoderm epithelium of tentacles, anterior body region, and ampulla consists of monociliary cells. Gram-negative bacteria were found between microvilli, in the protocuticle of the anterior region, and in the ampulla. The epithelium of the posterior body region lacks both monociliary cells and bacteria. The bundles of nerve fibers run between the layer of epithelial cells and basal membrane. The musculature of the body wall comprises circular and longitudinal muscles. The circular muscle fibers are applied to the basal membrane and constitute a solid layer extending almost throughout the length of the body. This pattern is broken in the posterior body region, where there is no solid layer of circular musculature, and the latter is arranged in isolated muscle bands. In the ampullar (terminal) body region, the inversion of circular and longitudinal muscle layers takes place, so that the latter appears to be pressed against the basal membrane. The apical surfaces of longitudinal muscle cells bear cytoplasmic processes; some of the cells have a flagellum. The basal portion of the longitudinal muscle cells forms a cytoplasmic process containing bundles of tonofilaments. The processes of all cells making up the muscle bands are interwoven and anchored to the basal membrane.     

Scale structure in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Scales of the Australian lungfish, Neoceratodus forsteri, are secreted within the dermis by a capsule of scleroblasts, and enclosed in a pouch made of collagen fibers, in contact with the epidermis over the posterior third of the scale. Each scale grows from a focus, which represents the first formed part of the scale. On the internal surface of the scale is elasmodin, made of collagen fiber bundles arranged in layers. Elasmodin, unmineralized in N. forsteri, contains cells in the living animal, and the number of layers increases as the scales grow. Squamulin, on the thin external part of the scale, is also laid down in layers, and based on a matrix of fine collagen fibrils, mineralized with a poorly crystalline biogenic calcium hydroxylapatite. Squamulin is divided into separate sections called squamulae, and contains long tubules with cells applied to the wall of the tubule. The anterior and lateral surfaces of the squamulin are ornamented with pediculae, and the posterior surface has longitudinal ridges, from which collagen fibers extend to anchor the scale within the pouch. Elasmodin and squamulin are linked by unmineralized collagen fibrils. The layers, formed at irregular intervals, are connected around the margin of the scale, effectively converting the whole scale into a flat structure resembling a pearl, with the first formed tissues deeply embedded inside the scale, and the youngest on the outer surface. Incremental lines in the hard tissue, and the number of layers in the elasmodin, do not reflect the chronological age of the fish. J. Morphol. 276:1137–1145, 2015. © 2015 Wiley Periodicals, Inc.     

Particular structure of the anterior third of the human true vocal cord.

The histological aspects of the true vocal cord mucosa change in the anterior third compared with the posterior two thirds. The anterior third is characterized by an epithelium where the ridges, marked in the posterior two thirds, are very slight or even absent. The underlying basement membrane, which is thin in the posterior two thirds, here appears particularly thick. At the ultrastructural level in this area, beneath a normally thickened basal lamina, a thick layer of finely granulated electron-dense material, interspersed with thin and randomly scattered collagen fibrils and proteoglycan filaments, is detectable. Beneath this thickened basement membrane, a layer of small undulated collagen fibril bundles with very numerous interspersed oxytalan fibres is found. The collagen fibrils, small in diameter (30-40 nm), seem to continue with the collagen fibrils of the basement membrane. In this layer numerous blood vessels with a very thick, delaminated basement membrane are also observed. The underlying area is characterized by the vocal cord ligament, composed by large compact collagen fibril bundles with interspersed elastic fibres. The particular features of the thick basement membrane, the thick-walled and delaminated vessels and the modular distribution of the elastic system together may well form the basic structure enabling the functional integration of the vocal ligament into the overlying mucosa and the underlying vocal muscle.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

Anatomy,histology, and histochemistry of the olfactory organ of the Korean shuttles mudskipper Periophthalmus modestus

The Korean shuttles mudskipper Periophthalmus modestus has paired olfactory organs on its snout, consisting of anterior and posterior nostrils, a single olfactory canal with sensory and nonsensory epithelia, and a single accessory nasal sac. Its sensory epithelium consists of numerous islets forming a pseudostratified layer and contains various cells: olfactory receptor neurons, supporting cells, basal cells, lymphatic cells (LCs), and axon bundles. The sensory epithelium is a stratified squamous layer comprising stratified epithelial cells, mucous cells (MCs) with glycogen, flattened cells (FCs), LCs, and unidentified cells. Specific structures are as follows: (a) a tubular anterior nostril projecting outward, (b) a slit posterior nostril, (c) an elongated olfactory canal, (d) an ethmoidal accessory nasal sac, (e) axon bundles found only in the basal layer of the sensory epithelium, (f) FCs only at the top of the nonsensory epithelium, and (g) glycogen-containing MCs. Such structures seem to be unique in that they have not been observed in most teleost fishes spending their whole life in water.     

Development and fine structure of the bony scutes in Corydoras arcuatus (Siluriformes,callichthyidae)

The development and the structure of the bony scutes have been studied in a growth series of the armored catfish Corydoras arcuatus using light and electron microscopy. Fibroblast-like cell condensations appear in the dermis, in the posterior region of the caudal peduncle, and these will constitute the scute papillae. Collagen bundles of the preexisting dermis colonized by the papilla cells are remodeled and incorporated in the papilla to form, in addition to newly synthesized woven-fibered bony material, the initium of the scute. This process of formation differs from that described for the dermal papilla of an elasmoid scale. During growth, the osteoblasts surrounding the scute constitute the scute sac in which the scute grows. Parallel-fibered bone is deposited on both sides of the initium, and osteoblasts are incorporated within the scute matrix. The remodeling and incorporation of collagen bundles of the preexisting dermis is maintained during growth only in the deep, anterior region of the scute. The posterior region and the upper surface of the scute are close to the epidermal-dermal boundary. When growth slows down in the upper part of the scute, a characteristic, well-mineralized tissue, composed of thin vertical fibrils and granules and devoid of typical striated collagen fibrils, is deposited on the scute surface. A new term, hyaloine, is introduced for this nonosseous, highly mineralized layer constituting the upper part of the scute. Hyaloine shows thin electron-dense lines, which probably correspond to periodic growth arrests. The structure and localization of the hyaloine are compared to other well-mineralized, similar tissues found on the surface of the dermal skeleton in lower vertebrates. © 1993 Wiley-Liss, Inc.     

Morphology of the epithelium in the alimentary tract of the larval lamprey,Petromyzon marinus L.

The alimentary tract of the ammocoete of the lamprey, Petromyzon marinus L., is divisible into three morphologically distinct regions: the oesophagus, the anterior intestine, and the posterior intestine. The epithelium of the oesophagus possesses mucous, ciliated, and columnar cells and appears to be specialized for movement of food particles. The epithelium of the anterior intestine possesses secretory cells with numerous zymogen granules, ciliated cells, and columnar-absorptive cells. Although some absorption occurs in the anterior intestine, the main function of this region seems to be the release of digestive enzymes and the continued movement of food particles. The epithelium of the posterior intestine is entirely comprised of columnar absorptive cells, namely tall (light and dark) columnar and low columnar, and the primary function of this region is one of absorption. The epithelium of the hindgut resembles that of the archinephric duct (Youson and McMillan, '71). The morphology of the alimentary tract of ammocoetes suggests that some differentiation and renewal of cell types may occur in the epithelium of the three regions. Comparison of the alimentary tract of larval lamprey with that of other vertebrates indicates that the gut of the ammocoete represents a less specialized level of vertebrate development.     

Collagen fibril assembly by corneal fibroblasts in three-dimensional collagen gel cultures: small-diameter heterotypic fibrils are deposited in the absence of keratan sulfate proteoglycan.

Extracellular matrix assembly is a multistep process and the various steps in collagen fibrillogenesis are thought to be influenced by a number of factors, including other noncollagenous matrix molecules. The synthesis and deposition of extracellular matrix by corneal fibroblasts grown within three-dimensional collagen gel cultures were examined to elucidate the factors important in the establishment of tissue-specific matrix architecture. Corneal fibroblasts in collagen gel cultures form layers and deposit small-diameter collagen fibrils (approximately 25 nm) typical of the mature corneal stroma. The matrix synthesized contains type VI collagen in a filamentous network and type I and type V collagen assembled as heterotypic fibrils. The amount of type V collagen synthesized is relatively high and comparable to that seen in the corneal stroma. This matrix is deposited between cell layers in a manner reminiscent of the secondary corneal stroma, but is not deposited as densely or as organized as would be found in situ. No keratan sulfate proteoglycan, a proteoglycan found only in the corneal stroma, was synthesized by the fibroblasts in the collagen gel cultures. The assembly and deposition of small-diameter fibrils with a collagen composition and structure identical to that seen in the corneal stroma in the absence of proteoglycans typical of the secondary corneal stroma imply that although proteoglycan-collagen interactions may function in the establishment of interfibrillar spacing and lamellar organization, collagen-collagen interactions are the major parameter in the regulation of fibril diameter.     

Cytochemistry of the skin of patients with mucopolysaccharidoses.

The distribution of complex carbohydrates has been investigated at the light and electron microscope levels in sweat glands of normal subjects and patients with Hurler's or Hunter's disease. Normal sweat glands examined with a battery of light microscopic histochemical methods revealed sulphated complex carbohydrate in secretory granules of the dark cells. These granules lacked affinity for dialysed iron (DI) at the light and electron microscope levels. The DI method demonstrated acid complex carbohydrates ultrastructurally on the surface of the intercellular canaliculi and central lumen in normal sweat glands. DI-reactive acidic material, presumably of mucopolysaccharide nature, surrounded and extended between collagen bundles in the stroma of normal skin, but was absent from the band which ensheathed the sweat gland and consisted of individual rather than bundled collagen fibrils. DI-reactive mucopolysaccharide lined and partially filled vacuoles of dark cells showing a laminar distribution in vacuoles of clear cells in sweat glands of a Hunter patient. The DI method also visualized mucopolysaccharide distributed throughout vacuoles in fibroblasts of this patient. DI-reactive acid material covered the luminal surface of the sweat gland, coated collagen bundles in the stroma and spared the periglandular collagenous sheath in skin from Hurler and Hunter patients as in that from normal controls. Acid phosphatase was localized ultrastructually in vacuoles and nearby cytoplasm and on plasmalemmae of clear cells, dark cells and myoepithelial cells of sweat glands from Hurler and Hunter patients. Vacuoles of dermal fibroblasts and Schwann cells in these specimens also exhibited strong acid phosphatase activity.     

Immunofluorescence study of the extracellular matrix of the human placenta

Distribution of collagen types I, III, IV, V and fibronectin in human placental villi has been studied by indirect immunofluorescence. During 9-12 weeks of pregnancy the extracellular matrix of villi represents a network of filaments organized in bundles and aggregates that contain collagen types I and III and finer filaments of collagen types IV and V. Collagen type IV is regularly detected in basal membrane of capillaries and particularly in villous epithelium, collagen type V and fibronectin are occasionally detected there. Marked immunofluorescent reaction on collagen types IV and V and fibronectin, and weak reaction on collagen type III is observed in cellular islets around cytotrophoblasts. In the fetus born in term placental villi have uniform immunofluorescence in thick basal membranes of fetal capillaries and of chorionic epithelium. The immunofluorescent reaction specific for all collagen types is uniform in villous stroma. Distribution of different collagen types and fibronectin, including the unusual localization of membrane collagen type IV, in villous stroma and cellular islets of early and mature placenta is discussed.     

Acquisition of type IX collagen by the developing avian primary corneal stroma and vitreous

Previous investigations from our laboratory and others have demonstrated that type II collagen, once thought to be a cartilage-specific molecule, is also a component of both the primary corneal stroma and the vitreous of embryonic chickens. In the present immunohistochemical study we have examined the expression in these embryonic matrices of another "cartilage-specific" collagen, type IX, along with type II. In the cornea, type IX collagen is in the primary stroma, but is not detectable in the mature, secondary stroma. Even within the primary stroma this collagen has a brief, transitory existence. It first appears in the peripheral stroma at the time the endothelial cells begin to migrate along its posterior surface, and spreads throughout the stroma during the following 24-36 hr. The epitopes on type IX collagen then suddenly become undetectable just before this matrix swells and becomes populated by the periocular mesenchymal cells (future keratocytes). In comparison, collagen type II (along with type I) is present in the stroma before and long after these events. Deposition of immunodetectable type IX collagen in the developing corneal stroma thus seems to be independent of type II. In the vitreous, we observed type IX collagen along with type II as soon as authentic vitreous could be identified and at all subsequent stages of development. In this tissue, therefore, the expression of collagen types IX and II appears to be coordinate.     

Corneal development. IV. Interruption of collagen excretion into the primary stroma of the cornea with L-azetidine-2-carboxylic acid

The primary stroma of the cornea of the chick embryo contains a cell-free orthogonal ply of collagen fibrils which is delineated clearly by Gomori's silver stain for reticulin and has, in miniature, the same fibrous architecture as the mature stroma. The collagen of this matrix is synthesized by the basal cells of the corneal epithelium and deposited beneath them a layer at a time.     

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

Ultrastructural demonstration of proteoglycans in adult rat cornea

Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl2, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in corneal basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by corneal cells.     

Cell migration in the postembryonic development of the fish lateral line

We examine at the cellular level the postembryonic development of the posterior lateral line in the zebrafish. We show that the first wave of secondary neuromasts is laid down by a migrating primordium, primII. This primordium originates from a cephalic region much like the primordium that formed the primary line during embryogenesis. PrimII contributes to both the lateral and the dorsal branches of the posterior lateral line. Once they are deposited by the primordium, the differentiating neuromasts induce the specialisation of overlying epidermal cells into a pore-forming annulus, and the entire structure begins to migrate ventrally across the epithelium. Thus the final two-dimensional pattern depends on the combination of two orthogonal processes: anteroposterior waves of neuromast formation and dorsoventral migration of individual neuromasts. Finally, we examine how general these migratory processes can be by describing two fish species with very different adult patterns, Astyanax fasciatus (Mexican blind cavefish) and Oryzias latipes (medaka). We show that their primary patterns are nearly identical to that observed in zebrafish embryos, and that their postembryonic growth relies on the same combination of migratory processes that we documented in the case of the zebrafish.     

Radiation-induced changes of fibroblasts in the squamous cell carcinoma of the head and neck

The regrowth of mesenchymal tissue (stroma) surrounding the malignant epithelium is an important step in tissue remodelling during and after irradiation. The radiation-induced fibroblastic changes were studied on tissue samples taken before, during and after the radical irradiation of the squamous cell carcinoma of the head and neck. Elongated fibroblasts with large amount of rough endoplasmic reticulum were seen around the tumor epithelium before radiation. The fibrosis increased during irradiation and at the same time the shape of the fibroblasts changed so that they became more triangular and nuclear structures became more prominent together with hyperchromasia. The amount of cell organelles declined although there was a large amount collagen present. Epithelial cells invaded through the basal lamina. In most samples the basal lamina could not be seen at all and the tumor cells were dispersed between stromal elements. On the other hand there were close contacts between epithelial and mesenchymal cells throughout the study in places where the basal lamina was broken, which might indicate epithelio-mesenchymal interaction. Also the connective tissue formed by fibroblasts and collagen might be part of the radiation induced healing and destruction of the tumor cells.