Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

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.     

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.     

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.     

Low‐cysteine alpha‐keratins and corneous beta‐proteins are initially formed in the regenerating tail epidermis of lizard

During tail regeneration in lizards, the stratified regenerating epidermis progressively gives rise to neogenic scales that form a new epidermal generation. Initially, a soft, un‐scaled, pliable, and extensible epidermis is formed that is progressively replaced by a resistant but non‐extensible scaled epidermis. This suggests that the initial corneous proteins are later replaced with harder corneous proteins. Using PCR and immunocytochemistry, the present study shows an upregulation in the synthesis of low‐cysteine type I and II alpha‐keratins and of corneous beta‐proteins with a medium cysteine content and a low content in glycine (formerly termed beta‐keratins) produced at the beginning of epidermal regeneration. Quantitative PCR indicates upregulation in the production of alpha‐keratin mRNAs, particularly of type I, between normal and the thicker regenerating epidermis. PCR‐data also indicate a higher upregulation for cysteine‐rich corneous beta‐proteins and a high but less intense upregulation of low glycine corneous protein mRNAs at the beginning of scale regeneration. Immunolabeling confirms the localization of these proteins, and in particular of beta‐proteins with a medium content in cysteine initially formed in the wound epidermis and later in the differentiating corneous layers of regenerating scales. It is concluded that the wound epidermis initially contains alpha‐keratins and corneous beta‐proteins with a lower cysteine content than more specialized beta‐proteins later formed in the mature scales. These initial corneous proteins are likely related to the pliability of the wound epidermis while more specialized alpha‐keratins and beta‐proteins richer in glycine and cysteine are synthesized later in the mature and inflexible scales. J. Morphol. 278:119–130, 2017. ©© 2016 Wiley Periodicals,Inc.     

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.     

Immunolocalization of specific beta‐proteins in pad lamellae of the digits in the lizard Anolis carolinensis suggests that cysteine‐rich beta‐proteins provides flexibility

Knowledge of beta‐protein (beta‐keratin) sequences in Anolis carolinensis facilitates the localization of specific sites in the skin of this lizard. The epidermal distribution of two new beta‐proteins (beta‐keratins), HgGC8 and HgG13, has been analyzed by Western blotting, light and ultrastructural immunocytochemistry. HgGC8 includes 16 kDa members of the glycine‐cysteine medium‐rich subfamily and is mainly expressed in the beta‐layer of adhesive setae but not in the setae. HgGC8 is absent in other epidermal layers of the setae and is weakly expressed in the beta‐layer of other scales. HgG13 comprises members of 17‐kDa glycine‐rich proteins and is absent in the setae, diffusely distributed in the beta layer of digital scales and barely present in the beta‐layer of other scales. It appears that the specialized glycine‐cysteine medium rich beta‐proteins such as HgGC8 in the beta‐layer, and of HgGC10 and HgGC3 in both alpha‐ and beta‐layers, are key proteins in the formation of the flexible epidermal layers involved in the function of these modified scales in adaptation to contact and adhesion on surfaces. J. Morphol. 275:504–513, 2014. © 2013 Wiley Periodicals, Inc.     

Immunolocalization of beta‐proteins and alpha‐keratin in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material

Immunolocalization of beta‐proteins in the epidermis of the soft‐shelled turtle explains the lack of formation of hard corneous material, Acta Zoologica, Stockholm. The corneous layer of soft‐shelled turtles derives from the accumulation of higher ratio of alpha‐keratins versus beta‐proteins as indicated by gene expression, microscopic, immunocytochemical and Western blotting analysis. Type I and II beta‐proteins of 14–16 kDa, indicated as Tu2 and Tu17, accumulate in the thick and hard corneous layer of the hard‐shelled turtle, but only type II is present in the thinner corneous layer of the soft‐shelled turtle. The presence of proline–proline and proline–cysteine–hinge dipeptides in the beta‐sheet region of all type II beta‐proteins so far isolated from the epidermis of soft‐shelled turtles might impede the formation of beta‐filaments and of the hard corneous material. Western blot analysis suggests that beta‐proteins are low to absent in the corneous layer. The ultrastructural immunolocalization of Tu2 and Tu17 beta‐proteins shows indeed that a diffuse labelling is seen among the numerous alpha‐keratin filaments present in the precorneous and corneous layers of the soft epidermis and that no dense corneous material is formed. Double‐labelling experiments confirm that alpha‐keratin prevails on beta‐proteins. The present observations support the hypothesis that the soft material detected in soft‐shelled turtles derives from the prevalent activation of genes producing type II beta‐proteins and high levels of alpha‐keratins.     

Immunolocalization of large corneous beta‐proteins in the green anole lizard (Anolis carolinensis) suggests that they form filaments that associate to the smaller beta‐proteins in the beta‐layer of the epidermis

The distribution of large corneous beta‐proteins of 18–43 kDa (Ac37, 39, and 40) in the epidermis of the lizard Anolis carolinensis is unknown. This study analyses the localization of these beta‐proteins in different body scales during regeneration. Western blot analysis indicates most protein bands at 40–50 kDa suggesting they mix with alpha‐keratin of intermediate filament keratin proteins. Ac37 is present in mature alpha‐layers of most scales and in beta‐cells of the outer scale surface in some scales but is absent in the Oberhäutchen, in the setae and beta‐layer of adhesive pads and in mesos cells. In differentiating beta‐keratinocytes Ac37 is present over 3–4 nm thick filaments located around the amorphous beta‐packets and in alpha‐cells, but is scarce in precorneous and corneous layers of the claw. Ac37 forms long filaments and, therefore, resembles alpha‐keratins to which it probably associates. Ac39 is seen in the beta‐layer of tail and digital scales, in beta‐cells of regenerating scales but not in the Oberhäutchen (and adhesive setae) or in beta‐ and alpha‐layers of the other scales. Ac40 is present in the mature beta‐layer of most scales and dewlap, in differentiating beta‐cells of regenerating scales, but is absent in all the other epidermal layers. The large beta‐proteins are accumulated among forming beta‐packets of beta‐cells and are packed in the beta‐corneous material of mature beta‐layer. Together alpha‐keratins, large beta‐proteins form the denser areas of mature beta‐layer that may have a different consistence that the electron‐paler areas. J. Morphol. 276:1244–1257, 2015. © 2015 Wiley Periodicals, Inc.     

Immunocytochemical localization of cysteine‐rich beta‐proteins in the extensible epidermis of the dewlap in the lizard Anolis carolinensis

The dewlap in the lizard Anolis carolinensis is made of scales separated by large interscale regions capable of broad stretching during fan extension. This indicates that the skin contains proteins that allow extension of interscale regions. The immunocytochemical analysis of the epidermis indicates that HgG5, a glycine‐rich hydrophobic beta‐protein poor in cysteine is localized only in the stiff beta‐layer of the outer scale surface, but is completely absent in mesos and alpha‐layers and in hinge regions. HgGC10, a cysteine‐medium‐rich beta‐protein is present in beta‐layers but especially in alpha‐layers of interscale epidermis that presents folds and lacks a beta‐layer. HgGC3 is weakly localized in the alpha‐layer, but is mainly found in hinge regions. HgGC8 and HgG13 are low to absent in the alpha‐ and beta‐layer. The immunolocalization of cysteine‐rich beta‐proteins such as HgGC10/3 in alpha‐layers and interscale epidermis suggests that these small proteins are involved in the formation of a corneous material compatible with dewlap extension. The basement membrane underneath scales is joined to bundles of collagen fibrils in the dermis through anchoring fibrils that likely determine flattening of the epidermis during the extension of the throat fan.     

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.     

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 alpha-keratins and associated beta-proteins in lizard epidermis shows that acidic keratins mix with basic keratin-associated beta-proteins

The differentiation of the corneous layers of lizard epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against alpha-keratins and keratin associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins). Both beta-cells and alpha-cells of the corneous layer derive from the same germinal layer. An acidic type I alpha-keratin is present in basal and suprabasal layers, early differentiating clear, oberhautchen, and beta-cells. Type I keratin apparently disappears in differentiated beta- and alpha-layers of the mature corneous layers. Conversely, a basic type II alpha-keratin rich in glycine is absent or very scarce in basal and suprabasal layers and this keratin likely does not pair with type I keratin to form intermediate filaments but is weakly detected in the pre-corneous and corneous alpha-layer. Single and double labeling experiments show that in differentiating beta-cells, basic KAbetaPs are added and replace type-I keratin to form the hard beta-layer. Epidermal alpha-keratins contain scarce cysteine (0.2–1.4 %) that instead represents 4–19 % of amino acids present in KAbetaPs. Possible chemical bonds formed between alpha-keratins and KAbetaPs may derive from electrostatic interactions in addition to cross-linking through disulphide bonds. Both the high content in glycine of keratins and KAbetaPs may also contribute to increase the hydrophobicy of the beta- and alpha-layers and the resistance of the corneous layer. The increase of gly-rich KAbetaPs amount and the bonds to the framework of alpha-keratins give rise to the inflexible beta-layer while the cys-rich KAbetaPs produce a pliable alpha-layer.     

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.     

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.     

Synthesis of interkeratin matrix in differentiating lizard epidermis: an ultrastructural autoradiographic study after injection of tritiated proline and histidine

During epidermal differentiation in mammals, keratins and keratin-associated matrix proteins rich in histidine are synthesized to produce a corneous layer. Little is known about interkeratin proteins in nonmammalian vertebrates, especially in reptiles. Using ultrastructural autoradiography after injection of tritiated proline or histidine, the cytological process of synthesis of beta-keratin and interkeratin material was studied during differentiation of the epidermis of lizards. Proline is mainly incorporated in newly synthesized beta-keratin in beta-cells, and less in oberhautchen cells. Labeling is mainly seen among ribosomes within 30 min postinjection and appears in beta-keratin packets or long filaments 1-3 h later. Beta-keratin appears as an electron-pale matrix material that completely replaces alpha-keratin filaments in cells of the beta-layer. Tritiated histidine is mainly incorporated into keratohyalin-like granules of the clear layer, in dense keratin bundles of the oberhautchen layer, and also in dense keratin filaments of the alpha and lacunar layer. The detailed ultrastructural study shows that histidine-labeling is localized over a dense amorphous material associated with keratin filaments or in keratohyalin-like granules. Large keratohyalin-like granules take up labeled material at 5-22 h postinjection of tritiated histidine. This suggests that histidine is utilized for the synthesis of keratins and keratin-associated matrix material in alpha-keratinizing cells and in oberhautchen cells. As oberhautchen cells fuse with subjacent beta-cells to form a syncytium, two changes occur : incorporation of tritiated histidine, but uptake of proline increases. The incorporation of tritiated histidine in oberhautchen cells lowers after merging with cells of the beta-layer, whereas instead proline uptake increases. In beta-cells histidine-labeling is lower and randomly distributed over the cytoplasm and beta-keratin filaments. Thus, change in histidine uptake somehow indicates the transition from alpha- to beta-keratogenesis. This study indicates that a functional stratum corneum in the epidermis of amniotes originates only after the association of matrix and corneous cell envelope proteins with the original keratin scaffold of keratinocytes.     

Embryonic keratinization in vertebrates in relation to land colonization

The embryogenesis and cytology of the epidermis in different vertebrates is variable in relation to the formation of a stratum corneum of different complexity. The latter process was essential for land colonization during vertebrate evolution and produced an efficient barrier in amniotes. Keratinocytes are made of cross‐linked keratins associated with specific proteins and lipids that are produced at advanced stages of embryogenesis when the epidermis becomes stratified. In these stages the epidermis changes from an aquatic to a terrestrial type, preadapted in preparation for the impact with the dry terrestrial environment that occurs at hatching or parturition. The epidermal barrier against water‐loss, mechanical and chemical stress, and microbe penetration is completely formed shortly before birth. Beneath the outer periderm, variably stratified embryonic layers containing glycine‐rich alpha‐keratins are formed in preparation for adult life. The following layers of the epidermis produce proteins for the formation of the cornified cell membrane and of the cornified material present in keratinocytes of the adult epidermis in reptiles, birds and mammals. The general features of the process of soft cornification in the embryonic epidermis of vertebrates are presented. Cornification in developing scales in reptiles, avian feathers and mammalian hairs is mainly related to the evolution of keratin‐associated proteins. The latter proteins form the resistant matrix of hard skin derivatives such as claws, beaks, nails and horns.     

Differentiation of the epidermis of scutes in embryos and juveniles of the tortoise Testudo hermanni with emphasis on beta-keratinization

The sequence of differentiation of the epidermis of scutes during embryogenesis in the tortoise Testudo hermanni was studied using autoradiography, electron microscopy and immunocytochemistry. The study was mainly conducted on the epidermis of the carapace, plastron and nail. Epidermal differentiation resembles that described for other reptiles, and the embryonic epidermis is composed of numerous cell layers. In the early stages of differentiation of the carapacial ridge, cytoplasmic blebs of epidermal cells are in direct contact with the extracellular matrix and mesenchymal cells. The influence of the dermis on the formation of the beta‐layer is discussed. The dermis becomes rich in collagen bundles at later stages of development. The embryonic epidermis is formed by a flat periderm and four to six layers of subperidermal cells, storing 40–70‐nm‐thick coarse filaments that may represent interkeratin or matrix material. Beta‐keratin is associated with the coarse filaments, suggesting that the protein may be polymerized on their surface. The presence of beta‐keratin in embryonic epidermis suggests that this keratin might have been produced at the beginning of chelonian evolution. The embryonic epidermis of the scutes is lost around hatching and leaves underneath the definitive corneous beta‐layer. Beneath the embryonic epidermis, cells that accumulate typical large bundles of beta‐keratin appear at stage 23 and at hatching a compact beta‐layer is present. The differentiation of these cells shows the progressive replacement of alpha‐keratin bundles with bundles immunolabelled for beta‐keratin. The nucleus is degraded and electron‐dense nuclear material mixes with beta‐keratin. In general, changes in tortoise skin when approaching terrestrial life resemble those of other reptiles. Lepidosaurian reptiles form an embryonic shedding layer and crocodilians have a thin embryonic epidermis that is rapidly lost near hacthing. Chelonians have a thicker embryonic epidermis that accumulates beta‐keratin, a protein later used to make a thick corneous layer.     

Epidermal differentiation in the developing scales of embryos of the Australian scincid lizard Lampropholis guichenoti

Formation of the first epidermal layers in the embryonic scales of the lizard Lampropholis guichenoti was studied by optical and electron microscopy. Morphogenesis of embryonic scales is similar to the general process in lizards, with well‐developed overlapping scales being differentiated before hatching. The narrow outer peridermis is torn and partially lost during scale morphogenesis. A second layer, probably homologous to the inner peridermis of other lizard species, but specialized to produce lipid‐like material, develops beneath the outer peridermis. Two or three lipogenic layers of this type develop in the forming outer surface of scales near to the hinge region. These layers form a structure here termed “sebaceous‐like secretory cells.” These cells secrete lipid‐like material into the interscale space so that the whole epidermis is eventually coated with it. This lipid‐like material may help to reduce friction and to reduce accumulation of dirt between adjacent extremely overlapping scales. At the end of their differentiation, the modified inner periderm turns into extremely thin cornified cells. The layer beneath the inner peridermis is granulated due to the accumulation of keratohyalin‐like granules, and forms a shedding complex with the oberhautchen, which develops beneath. Typically tilted spinulae of the oberhautchen are formed by the aggregation of tonofilaments into characteristically pointed cytoplasmic outgrowths. Initially, there is little accumulation of β‐keratin packets in these cells. During differentiation, the oberhautchen layer merges with cells of the β‐keratin layer produced underneath, so that a typical syncytial β‐keratin layer is eventually formed before hatching. Between one‐fourth distal and the scale tip, the dermis under epidermal cells is scarce or absent so that the mature scale tip is made of a solid rod of β‐keratinized cells. At the time of hatching, differentiation of a mesos layer is well advanced, and the epidermal histology of scales corresponds to Stage 5 of an adult shedding cycle. The present study confirms that the embryonic sequence of epidermal stratification observed in other species is basically maintained in L. guichenoti. J. Morphol. 241:139–152, 1999. © 1999 Wiley‐Liss, Inc.     

Immunocytochemical analysis of beta keratins in the epidermis of chelonians,lepidosaurians, and archosaurians

Beta (beta) keratins are present only in the avian and reptilian epidermises. Although much is known about the biochemistry and molecular biology of the beta keratins in birds, little is known for reptiles. In this study we have examined the distribution of beta keratins in the adult epidermis of turtle, lizard, snake, tuatara, and alligator using light and electron immunocytochemistry with a well-characterized antiserum (anti-beta(1) antiserum) made against a known avian scale type beta keratin. In lizard, snake, and tuatara epidermis this antiserum reacts strongly with the beta-layer, more weakly with the oberhautchen before it merges with the beta-layer, and least intensely with the mesos layer. In addition, the anti-beta(1) antiserum reacts specifically with the setae of climbing pads in gekos, the plastron and carapace of turtles, and the stratum corneum of alligator epidermis. Electron microscopic studies confirm that the reaction of the anti-beta(1) antiserum is exclusively with characteristic bundles of the 3-nm beta keratin filaments in the cells of the forming beta-layer, and with the densely packed electron-lucent areas of beta keratin in the mature bet- layer. These immunocytochemical results suggest that the 3-nm beta keratin filaments of the reptilian integument are phylogenetically related to those found in avian epidermal appendages.