Carbohydrate histochemistry of the epidermis of Natrix piscator during its sloughing cycle

Carbohydrate histochemistry of the scale and hinge epidermis of the chequered water snake, Nalrix piscator , throughout the sloughing cycle, has been described. The small amount of mucopolysaccharide present in the Oberhautchen, mesos layer, α-layer and β-Mayer (in its initial stage of differentiation) is comparable with that in amphibian epidermis and the epidermis of certain freshwater fish undergoing keratinization. Moderate amounts of mucopolysaccharide in the lacunar tissue and clear layer may protect against environmental pathogens and retain water to protect the epidermis from desiccation. Mucous cells could not be located in the epidermis throughout the sloughing cycle, contrary to some previous observations that they occur in the hinge region. The general absence of glycogen in the epidermis in most stages of the sloughing cycle suggests that the glycogen metabolized in the epidermis is utilized immediately, in view of the high energy requirements of proliferation and differentiation. Accumulation of glycogen granules in the presumptive α-layer in stage 2 and in the clear layer, presumptive Oberhautchen and presumptive β-Mayer in stage 3 is correlated with low energy requirements, indicating a slowing down of the process of keratinization of cells in these layers.     

Enzymes in the epidermis of Natrix piscator during its sloughing cycle

Acid phosphatase, non-specific esterase, alkaline phosphatase, monoamine oxidase and true lipase activities, in the epidermis of Natrix piscator in different stages of the sloughing cycle, have been localized using various histochemical techniques.
Different layers in scale epidermis have staining properties similar to corresponding layers in hinge epidermis.
Acid phosphatase and non-specific esterase activity in cell layers undergoing keratinization, and the lacunar tissue undergoing disintegration are associated with hydrolytic and catabolic wasting processes involving cell death. The activity of these enzymes in the clear layer is associated with the breaking down of the cementing substance resulting in the separation of clear layer from underlying tissue and facilitating the shedding of old slough.
Alkaline phosphatase activity in the stratum germinativum and undifferentiated epidermal cells has been associated with cell proliferation and differentiation. The presence of alkaline phosphatase in the lacunar tissue and clear layer has been correlated with the synthesis of mucopolysaccharides in these layers.
Monoamine oxidase and true lipase activity could not be located in the epidermis at any stage of the sloughing cycle.     

Observations on the epidermis of desert-living iguanids

The epidermis of 146 specimens of Dipsosaurus dorsalis and 182 Uma notata collected throughout the active period of the animals' year has been examined. The morphology of the epidermis is essentially similar to previously described lacertilians but differs in the relatively great degree of development of the mesos layer and the complete keratinization of the lacunar tissue prior to sloughing. Analysis of sloughing frequency throughout the year suggests that species specific patterns may exist, but these do not correlate with any particular known ecologic datum. The patterns do not reflect the reproductive activity of the two species supporting previous experimental conclusions on the lack of effect of gonadial hormones on epidermal activity. There appears to be no evidence of association of femoral gland activity with epidermal activity in D. dorsalis, but the situation is not clearcut in U. notata. These data are discussed in the light of recent studies of the evolutionary origin of epidermal glands in lizards.     

Lipid histochemistry of the epidermis of Natrix piscator during its sloughing cycle

The lipid histochemistry of the scale and hinge epidermis of the chequered water snake, Natrix piscator , throughout the sloughing cycle, has been described. The presence of comparatively high concentrations of phospholipids in the mesos layer and a-layer, in comparison to neutral lipids, has been associated with a permeability barrier to transcutaneous water flux. Free fatty acids, present in almost all epidermal layers and in eosinophilic granular cells, may protect the epidermis from bacterial and fungal attacks. Cholesterol, in addition to phospholipids, in various keratinized layers, is assumed to be derived from membranous structures of epidermal cells and is regarded as a stabilizer of the phospholipids in membranes.     

Keratohyalin-like granules in lizard epidermis: evidence from cytochemical, autoradiographic, and microanalytic studies

Epidermal sloughing in lizards is determined by the formation of an intraepithelial shedding complex in which keratohyalin-like granules are formed. The chemical nature of these granules is unknown, as is their role in keratinization. The goal of this study was to test whether they contain some amino acids similar to those found in mammalian keratohyalin. The embryonic and regenerating epidermis of lizards are useful systems to study the formation of these granules. Histochemically keratohyalin-like granules react to histidine and contain some sulfhydryl groups (cysteine). X-ray microanalysis shows that these granules contain sulfur and often phosphorus, two elements also present in the mature clear, oberhautchen, and beta layer. Instead the mesos, alpha, and lacunar layers contain only sulfur. Most sulfur is probably in a disulfide-bonded form, particularly in mature cells of the shedding complex, in large keratohyalin-like granules, and in the beta-keratin layer. Early differentiating beta-keratin cells have the maximal incorporation of tritiated proline, whereas tritiated arginine is slightly more concentrated in the basal layer of the epidermis. A high uptake of tritiated histidine is observed mainly in keratohyalin-like granules of the clear layer, but also in the oberhautchen layer and forming the alpha-lacunar layer. Immunogold electron microscopy shows that keratohyalin-like granules do not localize keratin but are embedded within a keratin network. These results suggest that keratohyalin-like granules of lizards, like mammalian keratohyalin, contain some sulfur-rich and histidine-rich proteins. These granules participate in the process of hardening of the clear layer that molds the spinulae of the deeper oberhautchen to form the superficial microornamentation.     

Immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) in the regenerating lizard epidermis indicates a new process for the differentiation of the epidermis in lepidosaurians

The process of keratinocyte differentiation was analyzed in the regenerating epidermis of the lizard Anolis carolinensis, where the genes coding for beta‐proteins (beta‐keratins) are known. The regenerating epidermis forms all epidermal layers found in normal scales (Oberhäutchen‐, beta‐, mesos‐, and alpha‐layer). Three specific proteins representing the larger families of beta‐proteins, glycine‐rich (HgG5, 28% glycine, 3.6% cysteine), glycine‐cysteine medium‐rich (HgGC10, 13% glycine, 14.5% cysteine), and glycine‐cysteine rich (HgGC3, 30.4% glycine, 8.7% cysteine) have been immunolocalized at the ultrastructural level. HgG5 is only present in differentiating beta‐cells, a weak or no labeling is observed in Oberhäutchen and is absent in alpha‐cells. The protein is located in the pale corneous material forming the compact beta‐layer but is absent in mature Oberhäutchen cells. HgGC10 is present among beta‐packets in Oberhäutchen and beta‐cells but disappears in more compact and electron‐pale corneous material. The labeling disappears in mesos‐cells and is present with variable intensity in alpha‐cells, whereas lacunar and clear‐cells are low labeled to unlabeled. HgGC3 is sparse or absent in beta‐cells but is lightly present in the darker corneous material of differentiating and mature alpha‐cells, lacunar‐cells, and clear‐cells. The study suggests that while glycine‐rich proteins (electron‐pale) are specifically used for building the resistant and hydrophobic beta‐layer, cysteine–glycine rich proteins (electron‐denser) are used to form the pliable corneous material present in the Oberhäutchen and alpha‐cells. The differential accumulation of beta‐proteins on the alpha‐keratin cytoskeleton scaffold and not the alternance of beta‐ with alpha‐keratins allow the differentiation of different epidermal layers. © 2012 Wiley Periodicals, Inc.     

Ultrastructure of the epidermis of the lizard (Lacerta vivipara) at the resting stage of the sloughing cycle

The ultrastructure of the epidermis of the lizard ( Lacerta vivipara ) one day after sloughing is described. The non-keratinized layers of the epidermis are essentially similar in structure to those of amphibians and mammals. The cells of the basal layer are not however separated from each other by the large spaces described in the amphibian (Farquhar & Palade, 1965). The middle layers of the epidermis at this stage of the sloughing cycle produce neither the characteristic mucous granules found in amphibians nor the keratohyalin granules of mammals. A small number of granules corresponding in size and location to the "Odland bodies" of both mammalian and amphibian epidermis are, however, present. The intermediate layer cells also contain a number of bodies similar in appearance to those described by Farquhar & Palade as lysosomes in amphibian skin. These structures are both osmium iodide and acid phosphatase positive. Unlike the condition in amphibians and mammals, the cytoplasm of cells in the layer immediately beneath the keratinized strata is honeycombed with small vesicles, and contains large irregular vacuoles of uncertain content. Certain nonkeratinizing elements within the epidermis are tentatively interpreted as nerve terminations. Two morphologically distinct keratinized strata can be distinguished, the inner stratum consisting of flattened cells similar to those of the stratum corneum of mammalian epidermis; individual cell outlines cannot be distinguished in the outer stratum, which has a structure similar to that of avian feather keratin. A shallow surface zone of the outer keratinized stratum has been identified as the Oberhautchen. This consists of longitudinally disposed leaflets or laminae which are responsible for the sculptured pattern of the epidermal surface. The observations reported here provide a basis for analysis of changes occurring at other stages of the sloughing cycle.     

Observations on the histochemistry and ultrastructure of the epidermis of the tuatara,Sphenodon punctatus (Sphenodontida,Lepidosauria, Reptilia): a contribution to an understanding of the lepidosaurian epidermal generation and the evolutionary origin of the squamate shedding complex

Histochemical and TEM analysis of the epidermis of Sphenodon punctatus confirms previous histological studies showing that skin-shedding in this relic species involves the periodic production and loss of epidermal generations, as has been well documented in the related Squamata. The generations are basically similar to those that have been described in the latter, and their formation involves a cyclic alternation between beta- and alpha-keratogenesis. The six differences from the previously described squamate condition revealed by this study include: 1) the absence of a well-defined shedding complex; 2) the persistence of plasma membranes throughout the mature beta-layer, including the oberhautchen; 3) the concomitant presence of lipogenic lamellar bodies and PAS-positive mucous granules in most presumptive alpha-keratinizing cells; 4) the presence of the secreted contents of these organelles in the intercellular domains of the three derived tissues, the homologues of the squamate mesos, alpha-, and lacunar cells; 5) the paucity of lamellated lipid deposits in such domains; 6) the presence of keratohyalin-like granules (KHLG) in the presumptive lacunar, clear, and oberhautchen cells. In toto, the absence of many of the precisely definable, different pathways of cytogenesis discernible during squamate epidermal generation production might be interpreted as primitive for lepidosaurs. However, when the evolutionary significance of each of the six differences listed is evaluated separately, it becomes clear that the epidermis of S. punctatus possesses primitive amniote, shared and derived lepidosaurian, and some unique characters. This evaluation further elucidates the concept of a lepidosaurian epidermal generation as a derived manifestation of the sauropsid synapomorphy of vertical alternation of keratin synthesis and shows that further study of keratinocyte differentiation in the tuatara may contribute to our understanding of the origin and evolution of beta-keratinization in sauropsid amniotes.     

Immunogold labeling shows that glycine‐cysteine‐rich beta‐proteins are deposited in the Oberhäutchen layer of snake epidermis in preparation to shedding

Shedding in snakes is cyclical and derives from the differentiation of an intraepidermal shedding complex made of two different layers, termed clear and Oberhäutchen that determine the separation between the outer from the inner epidermal generation that produces a molt. The present comparative immunocytochemical study on the epidermis and molts of different species of snakes shows that a glycine‐cysteine‐rich corneous beta‐protein in a snake is prevalently accumulated in cells of the Oberhäutchen layer and decreases in those of the beta‐layer. The protein is variably distributed in the mature beta‐layer of species representing some snake families when the beta‐layer merges with the Oberhäutchen but disappears in alpha‐layers. Therefore, this protein represents an early marker of the transition between the outer and the inner epidermal generations in the epidermis of snakes in general. It is hypothesized that specific gene activation for glycine‐cysteine‐rich corneous beta‐proteins occurs during the passage from the clear layer of the outer epidermal generation to the Oberhäutchen layer of the replacing inner epidermal generation. It is suggested that in the epidermis of most species glycine‐cysteine‐rich corneous beta‐proteins form part of the dense corneous material that rapidly accumulates in the differentiating Oberhäutchen cells but decreases in the following beta‐layer of the inner epidermal generation destined to be separated from the previous outer generation in the process of shedding. The regulation of the synthesis of these and other proteins is, therefore, crucial in timing the different stages of the shedding cycle in lepidosaurian reptiles. J. Morphol. 276:144–151, 2015. © 2014 Wiley Periodicals, Inc.     

Comparative immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) supports a new explanation for the cyclical process of keratinocyte differentiation in lizard epidermis

Lizard epidermis is made of beta‐ and alpha‐layers. Using Western blot tested antibodies, the ultrastructural immunolocalization of specific keratin‐associated beta‐proteins in the epidermis of different lizard species reveals that glycine‐rich beta‐proteins (HgG5) localize in the beta‐layer, while glycine–cysteine‐medium‐rich beta‐proteins (HgGC10) are present in oberhautchen and alpha‐layers. This suggests a new explanation for the formation of different epidermal layers during the shedding cycle in lepidosaurian epidermis instead of an alternance between beta‐keratins and alpha‐keratins. It is proposed that different sets of genes coding for specific beta‐proteins are activated in keratinocytes during the renewal phase of the shedding cycle. Initially, glycine–cysteine‐medium‐rich beta‐proteins with hydrophilic and elastic properties accumulate over alpha‐keratins in the oberhautchen but are replaced in the next cell layer with glycine‐rich hydrophobic beta‐proteins forming a resistant, stiff, and hydrophobic beta‐layer. The synthesis of glycine‐rich proteins terminates in mesos and alpha‐cells where these proteins are replaced with glycine–cysteine‐rich beta‐proteins. The pattern of beta‐protein deposition onto a scaffold of intermediate filament keratins is typical for keratin‐associated proteins and the association between alpha‐keratins and specific keratin‐associated beta‐proteins during the renewal phase of the shedding cycle gives rise to epidermal layers possessing different structural, mechanical, and texture properties.     

Epidermal differentiation during ontogeny and after hatching in the snake Liasis fuscus (Pythonidae,Serpentes, Reptilia), with emphasis on the formation of the shedding complex

Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water python, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome-like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differentiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of suprabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells beneath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin resolved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen component of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little immunoreactivity in the small beta-packets of the oberhautchen, but it increases after fusion with the underlying cells to produce the syncytial beta layer. The beta-keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differentiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding complex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta-keratogenic cells during epidermal renewal.     

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.     

Immunolocalization of keratin‐associated beta‐proteins in developing epidermis of lizard suggests that adhesive setae contain glycine–cysteine‐rich proteins

The localization of specific keratin‐associated beta‐proteins (formerly referred to as beta‐keratins) in the embryonic epidermis of lizards is not known. Two specific keratin‐associated beta‐proteins of the epidermis, one representing the glycine‐rich subfamily (HgG5) and the other the glycine‐cysteine medium‐rich subfamily (HgGC10), have been immunolocalized at the ultrastructural level in the lizard Anolis lineatopus. The periderm and granulated subperiderm are most immunonegative for these proteins. HgG5 is low to absent in theOberhäutchen layer while is present in the forming beta‐layer, and disappears in mesos‐ and alpha‐layers. Instead, HgGC10 is present in the Oberhäutchen, beta‐, and also in the following alpha‐layers, and specifically accumulates in the developing adhesive setae but not in the surrounding cells of the clear layer. Therefore, setae and their terminal spatulae that adhere to surfaces allowing these lizards to walk vertically contain cysteine–glycine rich proteins. The study suggests that, like in adult and regenerating epidermis, the HgGC10 protein is not only accumulated in cells of the beta‐layer but also in those forming the alpha‐layer. This small protein therefore is implicated in resistance, flexibility, and stretching of the epidermal layers. It is also hypothesized that the charges of these proteins may influence adhesion of the setae of pad lamellae. Conversely, glycine‐rich beta‐proteins like HgG5 give rise to the dense, hydrophobic, and chromophobic corneous material of the resistant beta‐layer. This result suggests that the differential accumulation of keratin‐associated beta‐proteins over the alpha‐keratin network determines differences in properties of the stratified layers of the epidermis of lizards. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Histidine uptake in the epidermis of lizards and snakes in relation to the formation of the shedding complex

Mammalian epidermis utilizes histidine-rich proteins (filaggrins) to aggregate keratin filaments and form the stratum corneum. Little is known about the involvement of histidine-rich proteins during reptilian keratinization. The formation of the shedding complex in the epidermis of snakes and lizards, made of the clear and the oberhautchen layers, determines the cyclical epidermal sloughing. Differently from snakes, keratohyalin-like granules are present in the clear layer of lizards. The uptake of tritiated histidine into the epidermis of two lizards and one snake has been studied by autoradiography in sections at progressive post-injection periods. At 40 min and 1 hr post-injection keratohyalin-like granules were not or poorly labeled. At 3-22 hr post-injection most of the labeling was present over suprabasal cells destined to form the shedding complex, in keratohyalin-like granules of the clear layer, and in the forming a-layer but was low in the forming b-layer, and in superficial keratinized layers. The analysis of the shedding complex in the pad lamellae (a specialized scale used for climbing) of a gecko showed that the setae and the cytoplasm of clear cells among them are main sites of histidine uptake at 4 hr post-injection. In the snake most of the labeling at 4 hr post-injection was localized in the shedding complex along the boundary between the clear and oberhautchen layers. The present study suggests that, in the epidermis of lepidosaurian reptiles, the synthesis of a histidine-rich protein is involved in the formation of the shedding layer and, as in mammals, in a-keratinization.     

Formation of the corneous layer in the epidermis of the tuatara (Sphenodon punctatus, Sphenodontida, Lepidosauria, Reptilia)

The formation of the stratum corneum in the epidermis of the reptile Sphenodon punctatus has been studied by histochemical, immunohistochemical, and ultrastructural methods. Sulfhydryl groups are present in the mesos and pre-alpha-layer but disappear in the keratinized beta-layer and in most of the mature alpha-layer. This suggests a complete cross-linking of keratin filaments. Tyrosine increases in keratinized layers, especially in the beta-layer. Arginine is present in living epidermal layers, in the presumptive alpha-layer, but decreases in keratinized layers. Histidine is present in corneous layers, especially in the intermediate region between the alpha- and a new beta-layer, but disappears in living layers. It is unknown whether histidine-rich proteins are produced in the intermediate region. Small keratohyalin-like granules are incorporated in the intermediate region. The plane of shedding, as confirmed from the study on molts, is located along the basalmost part of the alpha-layer and may involve the degradation of whole cells or cell junctions of the intermediate region. A specific shedding complex, like that of lizards and snakes, is not formed in tuatara epidermis. AE1-, AE2-, or AE3-positive alpha-keratins are present in different epidermal layers with a pattern similar to that previously described in reptiles. The AE1 antibody stains the basal and, less intensely, the first suprabasal layers. Pre-keratinized, alpha- and beta-layers, and the intermediate region remain unlabeled. The AE2 antibody stains suprabasal and forming alpha- and beta-layers, but does not stain the basal and suprabasal layers. In the mature beta-layer the immunostaining disappears. The AE3 antibody stains all epidermal layers but disappears in alpha- and beta-layers. Immunolocalization for chick scale beta-keratins labels the forming and mature beta-layer, but disappears in the mesos and alpha-layer. This suggests the presence of common epitopes in avian and reptilian beta-keratins. Low molecular weight alpha-keratins present in the basal layer are probably replaced by keratins of higher molecular weight in keratinizing layers (AE2-positive). This keratin pattern was probably established since the beginning of land adaptation in amniotes.     

Ultrastructural localization of alpha-keratins in the regenerating epidermis of the lizard Podarcis muralis during formation of the shedding layer

In the epidermis of lizards, alpha- and beta-keratins are sequentially produced during a shedding cycle. Using pre- and post-embedding immunocytochemistry this study shows the ultrastructural distribution of 3 alpha-keratin antibodies (AE1, AE2, AE3) in the renewing epidermis and in the shedding complex of the regenerating tail of the lizard Podarcis muralis. The AE1 antibody that recognizes acidic low MW keratins is confined to tonofilament bundles in basal and suprabasal cells but is not present in keratinizing beta- and alpha-cells. The AE2 antibody that recognises higher MW keratins weakly stains pre-keratinized cells and intensely keratinized alpha-layers. A weak labeling is present in small electrondense areas within the beta-layer. The AE3 antibody, that recognizes low and high MW basic keratins, immunolabels tonofilament bundles in all epidermal layers but intensely the alpha-keratinizing and keratinized layers (mesos, alpha-, lacunar and clear). Keratohyalin-like granules, present in the clear cells of the shedding layer, are negative to these antibodies so that the cornified clear layer contains keratins mixed with non-keratin material. The AE3 antibody shows that the mature beta-layer and the spinulated folds of the oberhautchen are labeled only in small dense areas among the prevalent electron-pale beta-keratin material. Therefore, some alpha-keratin is still present in the beta-layer, and supports the idea that alpha-keratins (basic) function as scaffold for beta-keratin deposition.     

An immunohistochemical study of early embryogenesis in the clawed toad Xenopus laevis by using monoclonal antibodies to intermediate filament proteins

Distribution of cytokeratin epitopes was studied in X. laevis embryos at stages 10-25 using 5 monoclonal antibodies against proteins of the human and rat keratin filaments. Specific staining was observed in chorda, outer layers of ectoderm and presumptive epidermis (late gastrula), and inner layer of presumptive epidermis. The cells of the stained zone (presumptive epidermis) were compressed while the cells of unstained zone (presumptive neuroectoderm) were extended tangentially.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Formation of adherens and communicating junctions coordinate the differentiation of the shedding‐layer and beta‐epidermal generation in regenerating lizard epidermis

In the lizard epidermis, the formation of a stratified alpha‐ and beta‐layer, separated by a shedding complex for molting, suggests that keratinocytes communicate in a coordinated manner after they leave the basal layers during the shedding cycle. I have therefore studied the localization of cell junctional proteins such as beta‐catenin and connexins 43 and 26 during scale regeneration in lizard using immunocytochemistry. Beta‐catenin is also detected in nuclei of basal cells destined to give rise to the Oberhäutchen and beta‐cells suggesting activation of the Wnt‐pathway during beta‐cell differentiation. The observations show that cells of the entire shedding layer (clear and Oberhäutchen) and beta‐layer are connected by beta‐catenin (adherens junctions) and connexins (communicating junctions) during their differentiation. This likely cell coupling determines the formation of a distinct shedding and beta‐layer within the regenerating epidermis. The observed pattern of cell junctional stratification suggests that after departing from the basal layer Oberhäutchen and beta‐cells form a continuous communicating compartment that coordinates the contemporaneous differentiation along the entire scale. While the beta‐layer matures the junctions are lost while other cell junctions are formed in the following mesos‐ and alpha‐cell layers. This process determines the formation of layers with different texture (harder or softer) and the precise localization of the shedding layer within lizard epidermis. J. Morphol. 275:693–702, 2014. © 2014 Wiley Periodicals, Inc.     

A histochemical study of keratinization in the domestic fowl (Gallus gallus)

The microanatomy of the epidermis of the domestic fowl is described and related to the distribution of various histochemical constituents involved in keratinization.
The avian horny layer over the back is composed of a loose network of structurally solid horny cells. This is in contrast to most mammalian epidermal horny cells in which structural keratin is found only in the peripheral cytoplasm, and the interior of the keratinocyte contains soluble products of cytolysis with possibly some free keratin filaments dispersed in the fluid material.
The avian tarsal epidermal horny scales show similarities to both the scales of lizards and snakes and to mammalian tail scales which appear to be homologous structures.
It is suggested that a thin layer of cells containing no detectable disulphide bonds, found in the tarsal scale region of the young chick, is probably mechanically weak and may function as a fission plane for sloughing of the horny layer. A specialized epidermis and thickened horny layer is developed in the fowl on the plantar underside of the toes, but this is quite different in structure from the mammalian plantar epidermis.
The overlapping of zones rich in ribonucleic acid (RNA) and bound cysteine (SH) in the growing feather suggests that protein synthesis and the preparatory stages to keratin disulphide bonding normally occur concurrently in feather formation. This is in contrast to the growing hair which has a region rich in RNA followed immediately before it becomes keratinized by a discrete keratogenous zone weak in RNA but rich in bound cysteine.