Localization and characterization of specific cornification proteins in avian epidermis

Little is known about proteins involved in the formation of the stratum corneum in the avian apteric epidermis. The present immunocytochemical, autoradiographic and electrophoretic study shows that antibodies against characteristic proteins of mammalian cornification (alpha-keratins, loricrin, sciellin, filaggrin, transglutaminase) recognize avian epidermal proteins. This suggests the presence of avian protein with epitopes common to related mammalian proteins. These proteins may also be involved in the formation of the cornified core and cell envelope of mature avian corneocytes. The immunoblotting study suggests that protein bands, cross-reactive for antibodies against loricrin (45, 52-57 kDa), sciellin (54, 84 kDa), filaggrin (32, 38, 45-48 kDa), and transglutaminase (40, 50, 58 kDa), are present in the avian epidermis. Immunocytochemistry shows that immunoreactivity for the above proteins is localized in the transitional and lowermost corneous layer of apteric epidermis. Their epitopes are rapidly masked/altered in cornifying cells and are no longer detectable in mature corneocytes. In scaled epidermis a thick layer made of beta-keratins of 14-18, 20-22, and 33 kDa is formed. Only in feathered epidermis (not in scale epidermis), an antifeather chicken beta-keratin antibody recognized a protein band at 8-12 kDa. This small beta-keratin is probably suitable for the formation of long, axial filaments in elongated barb, barbule and calamus cells. Conversely, the larger beta-keratins in scales are irregularly deposited forming flat plates. Tritiated histidine coupled to autoradiography show an absence of both keratohyalin and histidine-rich proteins in adult feathered and scaled epidermis. Most of the labeling appears in proteins within the range of beta- and alpha-keratins. These data on apteric epidermis support the hypothesis of an evolution of the apteric and interfollicular epidermis from the expansion of hinge regions of protoavian archosaurians.     

Vertebrate keratinization evolved into cornification mainly due to transglutaminase and sulfhydryl oxidase activities on epidermal proteins: An immunohistochemical survey

The epidermis of vertebrates forms an extended organ to protect and exchange gas, water, and organic molecules with aquatic and terrestrial environments. Herein, the processes of keratinization and cornification in aquatic and terrestrial vertebrates were compared using immunohistochemistry. Keratins with low cysteine and glycine contents form the main bulk of proteins in the anamniote epidermis, which undergoes keratinization. In contrast, specialized keratins rich in cysteine-glycine and keratin associated corneous proteins rich in cysteine, glycine, and tyrosine form the bulk of proteins of amniote soft cornification in the epidermis and hard cornification in scales, claws, beak, feathers, hairs, and horns. Transglutaminase (TGase) and sulfhydryl oxidase (SOXase) are the main enzymes involved in cornification. Their evolution was fundamental for the terrestrial adaptation of vertebrates. Immunohistochemistry results revealed that TGase and SOXase were low to absent in fish and amphibian epidermis, while they increased in the epidermis of amniotes with the evolution of the stratum corneum and skin appendages. TGase aids the formation of isopeptide bonds, while SOXase forms disulfide bonds that generate numerous cross-links between keratins and associated corneous proteins, likely increasing the mechanical resistance and durability of the amniote epidermis and its appendages. TGase is low to absent in the beta-corneous layers of sauropsids but is detected in the softer but pliable alpha-layers of sauropsids, mammalian epidermis, medulla, and inner root sheath of hairs. SOXase is present in hard and soft corneous appendages of reptiles, birds, and mammals, and determines cross-linking among corneous proteins of scales, claws, beaks, hairs, and feathers.     

Immunolocalization and characterization of cornification proteins in snake epidermis

Little is known about specific proteins involved in keratinization of the epidermis of snakes, which is composed of alternating beta- and alpha-keratin layers. Using immunological techniques (immunocytochemistry and immunoblotting), the present study reports the presence in snake epidermis of proteins with epitopes that cross-react with certain mammalian cornification proteins (loricrin, filaggrin, sciellin, transglutaminase) and chick beta-keratin. alpha-keratins were found in all epidermal layers except in the hard beta- and alpha-layers. beta-keratins were exclusively present in the oberhautchen and beta-layer. After extraction and electrophoresis, alpha-keratins of 40-67 kDa in molecular weights were found. Loricrin-like proteins recorded molecular weights of 33, 50, and 58 kDa; sciellin, 55 and 62 kDa; filaggrin-like, 52 and 65 kDa; and transglutaminase, 45, 50, and 56 kDa. These results suggest that alpha-layers of snake epidermis utilize proteins with common epitopes to those present during cornification of mammalian epidermis. The beta-keratin antibody on extracts from whole snake epidermis showed a strong cross-reactive band at 13-16 kDa. No cross-reactivity was seen using an antibody against feather beta-keratin, indicating absence of a common epitope between snake and feather keratins.     

Immunolocalization of Scaffoldin,a Trichohyalin‐Like Protein,in the Epidermis of the Chicken Embryo

Recent comparative genomic studies have identified a chicken gene that codes for a trichohyalin‐like protein rich in arginine and glutamic acid termed scaffoldin. Immunocytochemistry and immunoelectron microscopy show that this protein is predominantly localized in periderm granules, subcellular structures present in the periderm of the embryonic epidermis of chick scales, beak, claw, and in the sheath of developing and regenerating feathers. This suggests that scaffoldin contributes to the formation of periderm granules and to the soft cornification of the embryonic epidermis before the definitive epidermis is formed. Scaffoldin is absent from the definitive and adult epidermis generated underneath the periderm in scales and in inter‐follicular regions. Scaffoldin mixes with corneous beta‐proteins (beta‐keratins) synthesized in keratinocytes of the transitional layers formed beneath the periderm in the subunguis of the developing claws. Immunoreactivity for scaffoldin is absent in keratinocytes that accumulate corneous beta‐proteins such as those of scales, claws, and barbule‐barb cells of feathers. Corneous beta‐proteins represent the prevalent type of proteins present in adult epidermis of claws, scales, and feathers. These observations indicate that scaffoldin is a protein of transitional epidermal cells of the avian integument and might represent an important component of periderm granules. Anat Rec, 298:479–487, 2015. © 2014 Wiley Periodicals, Inc.     

Ultrastructural and immunohistochemical observations on the process of horny growth in chelonian shells

The process of growth of horny scutes of the carapace and plastron in chelonians is poorly understood. In order to address this problem, the shell of the terrestrial tortoise Testudo hermanni, the freshwater turtle Chrysemys picta, and the soft shelled turtle Trionix spiniferus were studied. The study was carried out using immunohistochemistry, electron microscopy and autoradiography following injection of tritiated histidine. The species used in the present study illustrate three different types of shell growth that occur in chelonians. In scutes of Testudo and Chrysemys, growth mainly occurs in the hinge regions by the production of cells that accumulate beta-keratin and incorporate tritiated histidine. Newly produced bundles of alpha- and beta-keratin incorporate most of the histidine. No keratohyalin is observed in the epidermis of any of the species studied here. In Testudo, newly generated corneocytes containing beta-keratin form a corneous layer to form the growing rings of scutes. In Chrysemys, newly generated corneocytes containing beta-keratin form the new, expanded corneous layer. In the latter species, at the end of the growing season (autumn/fall), thin corneocytes containing little beta-keratin are produced underneath the corneous layer, and gradually form a scission layer. In the following growing season (spring-summer) the shedding layer matures and determines the loss of the outer corneous layer. In this way, scutes expand their surface at any new molt. In Trionix, no distinct scutes and hinge regions are present and during the growing season, new corneocytes are mainly produced along the perimeter of the shell. Corneocytes of Trionix contain little beta-keratin and form a thick corneous layer in which cells resemble the alpha-layer of the softer epidermis of the limbs, tail and neck. Neither keratohyalin nor specific histidine incorporation was observed in these cells. Corneocytes are gradually lost from the epidermal surface. Dermal scutes are absent in Trionix, but the dermis is organized in 6-10 layers of plywood-patterned collagen bundles. The stratified layers gradually disappear toward the growing border of the shell. The mode of growth of horny scutes in these different species of chelonians is discussed.     

Isolation of a new class of cysteine�Cglycine�Cproline-rich beta-proteins (beta-keratins) and their expression in snake epidermis

Scales of snakes contain hard proteins (beta‐keratins), now referred to as keratin‐associated beta‐proteins. In the present study we report the isolation, sequencing, and expression of a new group of these proteins from snake epidermis, designated cysteine–glycine–proline‐rich proteins. One deduced protein from expressed mRNAs contains 128 amino acids (12.5 kDa) with a theoretical pI at 7.95, containing 10.2% cysteine and 15.6% glycine. The sequences of two more snake cysteine–proline‐rich proteins have been identified from genomic DNA. In situ hybridization shows that the messengers for these proteins are present in the suprabasal and early differentiating beta‐cells of the renewing scale epidermis. The present study shows that snake scales, as previously seen in scales of lizards, contain cysteine‐rich beta‐proteins in addition to glycine‐rich beta‐proteins. These keratin‐associated beta‐proteins mix with intermediate filament keratins (alpha‐keratins) to produce the resistant corneous layer of snake scales. The specific proportion of these two subfamilies of proteins in different scales can determine various degrees of hardness in scales.     

Cornification of the pulp epithelium and formation of pulp cups in downfeathers and regenerating feathers

During most of feather growth (anagen), the dermal papilla stimulates the collar epithelium to give rise to feather keratins accumulating cells that form most of the corneous material of barbs and the rachis. Aside from the induction of differentiated cells of the feather, the distal part of the papilla forms a loose connective tissue that nourishes the growing feather, termed the pulp. In the last stages of feather growth, the pulp undergoes a process of re-absorption and leaves empty cavities indicated as pulp cups surrounded by keratinized cells inside the calamus. The process of cornification of pulp cups in different species of birds has been described here by using electron microscopy and immunocytochemistry for keratins. Pulp cells accumulate bundles of soft (alpha)-keratin, but do not synthesise feather keratins as in the surrounding calamus cells. Cells of the pulp epithelium accumulate large amounts of lipids and form a softer keratinized epithelium surrounding the pulp. This type of keratinization resembles the formation of soft epidermis in apteric and interfollicular regions. The role of the cornified pulp epithelium is to limit water loss and to form a microbe barrier before the mature feather is moulted.     

Immunocytochemical localization of keratins,associated proteins and uptake of histidine in the epidermis of fish and amphibians

Keratinization and the role of histidine in some species of fish and amphibians have been analyzed by immunocytochemistry and autoradiography. In cartilaginous and bony fishes, staining of acidic (AE1-positive) and basic (AE3-positive) keratins was strong and their distribution patterns were uniform in all epidermal layers. The AE2 antibody (for keratins K1 and K10 that are typical for keratinization) did not produce any positivity. This was also observed in lungfish epidermis but the AE2 antibody often produced some positivity in the more keratinized layers. In the axolotl (urodele), that is adapted to aquatic conditions, as well as in other species of urodele (newts) that are more adapted to terrestrial conditions, the same pattern was present as in fish. In the latter, the AE2 antibody non-specifically stained all epidermal layers. In more terrestrially-adapted anurans (frog and toad) AE1 immunopositivity was mainly found in basal layers, the AE3 antibody stained the entire epidermis, and AE2 immunopositivity was often localized in the external layers of the epidermis. This pattern resembled that in the epidermis of amniotes. Administration of tritiated histidine to goldfish epidermis showed that at 1, 4 and 24 h after injection, labelling was low and uniformely distributed in all epidermal layers. In newt and toad epidermis, histidine labelling increased from 1 to 4 h after injection but tended to remain evenly distributed throughout the epidermis. However, from 4 up to 24 h after injection, labelling became concentrated in the upper intermediate and replacement layers, suggesting that turnover proteins were produced. Histidine was probably converted into other metabolites at 4-24 h after injection. Whether the newly synthetized proteins were a form of keratin or a specific histidine-rich protein remains to be determined biochemically. Uptake of tritiated thymidine in newt epidermis indicated that keratinocytes move into the uppermost stratum intermedium within 4 days, and reach the replacement layer in approximately 6 days. Taken together, the data obtained with tritiated histidine and thymidine suggest that most histidine is taken up in the upper intermedium and replacement layer at 4-24 h after injection. Neither a granular layer nor crossreaction with filaggrin and loricrin were observed in fish and amphibian epidermis. Although the cell membrane of superficial corneous cells of amphibian epidermis became thicker, the absence of loricrine immunolabelling suggests that a cell corneous envelope containing this protein is not present or undetectable.     

Development,structure, and protein composition of the corneous beak in turtles

The beak or rhamphotheca in turtles is a horny lamina that replaces the teeth. Its origin, development, structure, and protein composition are here presented. At mid-development stages, the epidermis of the maxilla and mandible gives rise to placodes that enlarge and merge into laminae through an intense cell proliferation. In these expanding laminae, the epidermis gives rise to 5–8 layers of embryonic epidermis where coarse filaments accumulate for the initial keratinization of cells destined to be sloughed before hatching. Underneath the embryonic epidermis of the beak numerous layers of spindle-shaped beta-cells are produced while they are absent in other skin regions. Beta-cells contain hard corneous material and give rise to the corneous layer of the beak whose external layers desquamate due to wearing and mechanical abrasion. Beta-catenin is present in nuclei of proliferating keratinocytes of the germinal layer likely responding to a wnt signal, but also is part of the adhesive junctions located among beak keratinocytes. The thick corneous layer is made of mature corneocytes connected one to another along their irregular perimeter by an unknown cementing material and junctional remnants. Immunolabeling shows that the main components of the horny beak are Corneous Beta Proteins (CBPs) of 10–15 kDa which genes are located in the Epidermal Differentiation Complex (EDC) of the turtle genome. Specific CBPs, in addition to a lower amount of Intermediate Filament Keratins, accumulate in the horny beak. Compaction of the main proteins with other unknown, minor proteins give rise to the hard corneous material of the beak.     

Protein synthesis studied by autoradiography in the epidermis of different species

This paper reports autoradiographic studies of protein synthesis related to epidermal cornification in several different species. A high concentration of injected histidine appeared in granular cells of man, the monkey, guinea pig, hairless mouse, and newborn rat, indicating that synthesis of relatively histidine-rich protein is involved in formation of keratohyalin granules. Two steps in the cornification process: synthesis of this histidine-rich protein, in addition to protein synthesized in the lower layers of the epidermis are postulated in epidermis containing keratohyalin granules. In the epidermis of the turtle, which does not contain keratohyalin granules, concentration of histidine is not observed, suggesting that protein of the cornified layer in this species seems to be synthesized as a 1-stage process.     

Ultrastructural immunolocalization of involucrin in the medulla and inner root sheath of the human hair

The participation of involucrin in the cornification of the human hair has been studied by light and electron microscopy immunohistochemistry. Immunoreactivity for involucrin is absent in keratinized cuticle and cortical cells although some immunolabeling is observed in the corneous membrane of internal cortical cells surrounding the hair medulla. Conversely, immunolabeling for involucrin is present in the cytoplasm of keratinizing cells of the medulla and inner root sheath. During the maturation and final cornification of medullary and inner root sheath cells the immunolabeling for involucrin tends to concentrate in the peripheral cytoplasm and along the cornified cell plasma membrane in both medullary and inner root sheath cells, a pattern similar to that known for corneocytes of the epidermis. This observation suggests that in the hair involucrin mainly participates in the formation of the corneous material of the medulla and inner root sheath in conjunction with trichohyalin, probably by the formation of isopeptide-bonds. Therefore, together with trichohyalin, the cross-linking due to involucrin is also responsible for the mechanical resistance of the corneous trabeculae present among the empty spaces of the medulla of the human hair.     

Body plan of turtles: an anatomical, developmental and evolutionary perspective

The evolution of the turtle shell has long been one of the central debates in comparative anatomy. The turtle shell consists of dorsal and ventral parts: the carapace and plastron, respectively. The basic structure of the carapace comprises vertebrae and ribs. The pectoral girdle of turtles sits inside the carapace or the rib cage, in striking contrast to the body plan of other tetrapods. Due to this topological change in the arrangement of skeletal elements, the carapace has been regarded as an example of evolutionary novelty that violates the ancestral body plan of tetrapods. Comparing the spatial relationships of anatomical structures in the embryos of turtles and other amniotes, we have shown that the topology of the musculoskeletal system is largely conserved even in turtles. The positional changes seen in the ribs and pectoral girdle can be ascribed to turtle-specific folding of the lateral body wall in the late developmental stages. Whereas the ribs of other amniotes grow from the axial domain to the lateral body wall, turtle ribs remain arrested axially. Marginal growth of the axial domain in turtle embryos brings the morphologically short ribs in to cover the scapula dorsocaudally. This concentric growth appears to be induced by the margin of the carapace, which involves an ancestral gene expression cascade in a new location. These comparative developmental data allow us to hypothesize the gradual evolution of turtles, which is consistent with the recent finding of a transitional fossil animal, Odontochelys, which did not have the carapace but already possessed the plastron.     

Ultrastructural localization of desmoglein and plakophilin in the human hair suggests that the cell membrane complex is a long desmosomal remnant

Unlike the superficial part of the corneous layer of the epidermis (Stratum corneum) where desmosomes are degraded and corneocytes flake away, the trichocytes in the hair remain attached to each other after cornification. The permanence and fine localization of cell junctions, in particular of desmosomal proteins in the cornifying and mature human hair, is not known. The present electron microscope immunolocalization study indicates that two protein markers for desmosomes such as desmoglein 4 and plakophilins 1 and 3 are still present in mature cortical and cuticle cells. These proteins remain mainly localized in the cornified cytoplasmic side of desmosomal remnants of cortical cells, but also in the delta layer of the extracellular region of the membrane complex. This suggests that the delta layer represents an extensive desmosomal remnant formed between mature cortical cells and in cuticle cells. The endocuticle appears to be the site of accumulation of desmosomal proteins and degraded nuclear material. The cornification of desmosomal junctions in both cortical and cuticle cells likely contributes to stabilize the integrity of the hair shaft.     

Structure and functions of keratin proteins in simple, stratified, keratinized and cornified epithelia

Historically, the term ‘keratin’ stood for all of the proteins extracted from skin modifications, such as horns, claws and hooves. Subsequently, it was realized that this keratin is actually a mixture of keratins, keratin filament‐associated proteins and other proteins, such as enzymes. Keratins were then defined as certain filament‐forming proteins with specific physicochemical properties and extracted from the cornified layer of the epidermis, whereas those filament‐forming proteins that were extracted from the living layers of the epidermis were grouped as ‘prekeratins’ or ‘cytokeratins’. Currently, the term ‘keratin’ covers all intermediate filament‐forming proteins with specific physicochemical properties and produced in any vertebrate epithelia. Similarly, the nomenclature of epithelia as cornified, keratinized or non‐keratinized is based historically on the notion that only the epidermis of skin modifications such as horns, claws and hooves is cornified, that the non‐modified epidermis is a keratinized stratified epithelium, and that all other stratified and non‐stratified epithelia are non‐keratinized epithelia. At this point in time, the concepts of keratins and of keratinized or cornified epithelia need clarification and revision concerning the structure and function of keratin and keratin filaments in various epithelia of different species, as well as of keratin genes and their modifications, in view of recent research, such as the sequencing of keratin proteins and their genes, cell culture, transfection of epithelial cells, immunohistochemistry and immunoblotting. Recently, new functions of keratins and keratin filaments in cell signaling and intracellular vesicle transport have been discovered. It is currently understood that all stratified epithelia are keratinized and that some of these keratinized stratified epithelia cornify by forming a Stratum corneum. The processes of keratinization and cornification in skin modifications are different especially with respect to the keratins that are produced. Future research in keratins will provide a better understanding of the processes of keratinization and cornification of stratified epithelia, including those of skin modifications, of the adaptability of epithelia in general, of skin diseases, and of the changes in structure and function of epithelia in the course of evolution. This review focuses on keratins and keratin filaments in mammalian tissue but keratins in the tissues of some other vertebrates are also considered.     

Ultrastructural localization of histidine-labelled interkeratin matrix during keratinization of amphibian epidermis

Interkeratin histidine-rich proteins (filaggrins) play a functional role in aggregation of keratin filaments into dense bundles during terminal differentiation of mammalian keratinocytes when forming the dense matrix of the stratum corneum. The origin of the stratum corneum during adaptation to land in amphibious vertebrate progenitors was probably linked with the synthesis of matrix proteins. However, whether similar proteins are present in living amphibians is unknown. The possible involvement of interkeratin matrix molecules rich in basic amino acids such as histidine during keratinization of amphibian epidermis has been evaluated in the present study by ultrastructural autoradiography after administration of tritiated histidine. At 4 and 8 h post-injection, labelling was mainly localized over electron-dense amorphous material or irregular granules in between keratin filaments in cells of the upper intermedium, replacement, and immature corneous layers. Nuclear material incorporating tritiated histidine was also present in the maturing corneous layer. Small mucous-like granules did not take up tritiated histidine and X-ray microanalysis indicated that the latter granules contained sulphur. The present study suggests that small amounts of histidine-rich molecules which were not sufficient to form microscopically-visible keratohyalin granules were present in ancestral amphibian epidermis. However, this material was sufficient to promote aggregation of keratin filaments in the cytoplasm of amphibian differentiating keratinocytes, especially near the external corneous cell envelope. Electron-dense material associated with the corneous cell envelope also contained sulphur as indicated by X-ray microanalysis. It is unknown whether sulphur is derived from either sulphated mucins, or disulphide bonds in aggregated keratins, or specific sulphur-rich proteins.     

Claw development and cornification in the passeraceous bird zebrafinch (<Emphasis Type="Italic">Taeniatopygia guttata castanotis</Emphasis>)

The histogenesis and cornification of claws in zebrafinch embryos has been analyzed. At 10–12 days post-deposition, the epidermis at the tip of the toes forms placode-like anlage associated with a mesenchymal condensation and with a terminal phalange. Claws seem to be modified scales, the dorsal side of which becomes the unguis whereas a ventral scale is the origin of the sub-unguis. At 14–15 days, numerous keratinocytes form the unguis, the corneous layer of which becomes thicker than in the sub-unguis and accumulates beta-keratin and lipids. Keratin bundles are mainly directed toward the tip of the claw and have a prevalent parallel orientation. Unguis corneocytes are thicker and accumulate more beta-keratin than corneocytes of the sub-unguis. Mature corneocytes become partially fused in a compact corneous layer at 17–18 days, near hatching. During growth of the unguis, the embryonic epidermis and beta-keratin cells curve over the tip of the claw and localize in the ventral part of the claw, forming the claw pad. The latter is shed at hatching leaving the pointed claw made of harder corneous layers in the unguis side of the claw.     

The characterization of two monoclonal anti-keratin antibodies and their use in the study of epithelial disorders

The present paper describes the production and characterization of two monoclonal antibodies, K20 and K92. Immunohistological staining showed these two antibodies to be specific for keratinizing epithelium. However, whereas K20 stained all layers of the epidermis K92 reacted with only the suprabasal epidermal layers. Immunoblotting studies with preparations of keratins from both the non-cornified (i.e. the basal, spinous and granular layers) and cornified (stratum corneum) layers of epidermis showed that K20 recognized the 46, 48, 50, 55, 56, 56.5, 59, 61, 62, 64, 65, 66 and 67 kd bands, of which the 50 and 46 kd bands appeared to be masked in tissue sections. In contrast, antibody K92 was more restricted in its activity, recognizing only the 55 and 56 kd bands strongly. These antibodies were used in the study of various epithelial disorders and revealed alterations in the epithelial intermediate filament expression in both benign and malignant disease processes.