Alpha- and beta-keratins of the snake epidermis

Snake scales contain specialized hard keratins (beta-keratins) and alpha- or cyto-keratins in their epidermis. The number, isoelectric point, and the evolution of these proteins in snakes and their similarity with those of other vertebrates are not known. In the present study, alpha- and beta-keratins of snake molts and of the whole epidermis have been studied by using two-dimensional electrophoresis and immunocytochemistry. Specific keratins in snake epidermis have been identified by using antibodies that recognize acidic and basic cytokeratins and avian or lizard scale beta-keratin. Alpha keratins of 40-70 kDa and isoelectric point (pI) at 4.5-7.0 are present in molts. The study suggests that cytokeratins in snakes are acidic or neutral, in contrast to mammals and birds where basic keratins are also present. Beta keratins of 10-15 kDa and a pI of 6.5-8.5 are found in molts. Some beta-keratins appear as basic proteins (pI 8.2) comparable to those present in the epidermis of other reptiles. Some basic "beta-keratins" associate with cytokeratins as matrix proteins and replace cytokeratins forming the corneous material of the mature beta-layer of snake scales, as in other reptiles. The study also suggests that more forms of beta-keratins (more than three different types) are present in the epidermis of snakes.     

Immunological characterization and fine localization of a lizard beta-keratin

Scales of lizards contain beta-keratin of poorly known composition. In the present study, a rat polyclonal serum against a lizard beta-keratin of 14-15 kDa has been produced and the relative protein has been immunolocalized in the epidermis. The observations for the first time show that the isolated protein band derives from the extraction of a protein component of the beta-keratin filaments of lizard epidermis. In immunoblots and immunocytochemistry, the antiserum recognizes most lizard beta-keratins, but produces a variable cross-reactivity with snake beta-keratins, and weak or no reactivity with beta-keratins isolated from tuatara, turtles, alligator and birds. In bidimensional immunoblots of lizard epidermis, three main spots at 15-16 kDa with isoelectric point at 7.0, 7.6 and 8.0, and an unresolved large spot at 29-30 kDa and with pI at 7.5-8.0, are obtained, may be derived from the aggregation of smaller beta-keratin proteins. The ultrastructural immunolocalization with the antibody against lizard beta-keratin shows that only small and large beta-keratin filaments of beta-cells of lizard epidermis are labeled. Keratin bundles in oberhautchen cells are less immunolabeled. Beta-keratin is rapidly polymerized into beta-packets that merge into larger beta-keratin filaments. No labeling is present over other cell organelles or cell layers of lizard epidermis, and is absent in non-epidermal cells. The antiserum recognizes epitope(s) characteristics for lizard beta-keratins, partially recognized in snakes and absent in non-lepidosaurian species. This result indicates that beta-keratins among different reptilian groups posses different immunoreactive regions.     

Distribution and characterization of keratins in the epidermis of the tuatara (Sphenodon punctatus; Lepidosauria, Reptilia)

Reptilian scales are mainly composed of alpha-and beta-keratins. Epidermis and molts from adult individuals of an ancient reptilian species, the tuatara (Sphenodon punctatus), were analysed by immunocytochemistry, mono- and bi-dimensional electrophoresis, and western blotting for alpha- and beta-keratins. The epidermis of this reptilian species with primitive anatomical traits should represent one of the more ancient amniotic epidermises available. Soft keratins (AE1- and AE3-positive) of 40-63 kDa and with isoelectric points (pI) at 4.0-6.8 were found in molts. The AE3 antibody was diffusely localised over the tonofilaments of keratinocytes. The lack of basic cytokeratins may be due to keratin alteration in molts, following corneification or enzymatic degradation of keratins. Hard (beta-) keratins of 16-18 kDa and pI at 6.8, 8.0, and 9.2 were identified using a beta-1 antibody produced against chick scale beta-keratin. The antibody also labeled filaments of beta-cells and of the mature, compact beta-layer. We have shown that beta-keratins in the tuatara resemble those of lizards and snakes, and that they are mainly basic proteins. These proteins replace cytokeratins in the pre-corneoum beta-layers, from which a hard, mechanically resistant corneoum layer is formed over scales. Beta-keratins may have both a fibrous and a matrix role in forming the hard texture of corneoum scales in this ancient species, as well as in more recently evolved reptiles.     

Characterization of keratins and associated proteins involved in the corneification of crocodilian epidermis

Crocodilian keratinocytes accumulate keratin and form a corneous cell envelope of which the composition is poorly known. The present immunological study characterizes the molecular weight, isoelectric point (pI) and the protein pattern of alpha- and beta-keratins in the epidermis of crocodilians. Some acidic alpha-keratins of 47-68 kDa are present. Cross-reactive bands for loricrin (70, 66, 55 kDa), sciellin (66, 55-57 kDa), and filaggrin-AE2-positive keratins (67, 55 kDa) are detected while caveolin is absent. These proteins may participate in the formation of the cornified cell membranes, especially in hinge regions among scales. Beta-keratins of 17-20 kDa and of prevalent basic pI (7.0-8.4) are also present. Acidic beta-keratins of 10-16 kDa are scarce and may represent altered forms of the original basic proteins. Crocodilian beta-keratins are not recognized by a lizard beta-keratin antibody (A68B), and by a turtle beta-keratin antibody (A685). This result indicates that these antibodies recognize specific epitopes in different reptiles. Conversely, crocodilian beta-keratins cross-react with the Beta-universal antibody indicating they share a specific 20 amino acid epitope with avian beta-keratins. Although crocodilian beta-keratins are larger proteins than those present in birds our results indicate presence of shared epitopes between avian and crocodilian beta-keratins which give good indication for the future determination of the sequence of these proteins.     

Characterization of beta-keratins and associated proteins in adult and regenerating epidermis of lizards

Reptilian epidermis contains two types of keratin, soft (alpha) and hard (beta). The biosynthesis and molecular weight of beta-keratin during differentiation of lizard epidermis have been studied by autoradiography, immunocytochemistry and immunoblotting. Tritiated proline is mainly incorporated into differentiating and maturing beta-keratin cells with a pattern similar to that observed after immunostaining with a chicken beta-keratin antibody. While the antibody labels a mature form of beta-keratin incorporated in large filaments, the autoradiographic analysis shows that beta-keratin is produced within the first 30 min in ribosomes, and is later packed into large filaments. Also the dermis incorporates high amount of proline for the synthesis of collagen. The skin was separated into epidermis and dermis, which were analyzed separately by protein extraction and electrophoresis. In the epidermal extract proline-labeled proteic bands at 10, 15, 18-20, 42-45, 52-56, 85-90 and 120 kDa appear at 1, 3 and 5 h post-injection. The comparison with the dermal extract shows only the 85-90 and 120 kDa bands, which correspond to collagen. Probably the glycine-rich sequences of collagen present also in beta-keratins are weakly recognized by the beta-1 antibody. Immunoblotting with the beta-keratin antibody identifies proteic bands according to the isolation method. After-saline or urea-thiol extraction bands at 10-15, 18-20, 40, 55 and 62 kDa appear. After extraction and carboxymethylation, weak bands at 10-15, 18-20 and 30-32 kDa are present in some preparations, while in others also bands at 55 and 62 kDa are present. It appears that the lowermost bands at 10-20 kDa are simple beta-keratins, while those at 42-56 kDa are complex or polymeric forms of beta-keratins. The smallest beta-keratins (10-20 kDa) may be early synthesized proteins that are polymerized into larger beta-keratins which are then packed to form larger filaments. Some proline-labeled bands differ from those produced after injection of tritiated histidine. The latter treatment does not show 10-20 kDa labeled proteins, but tends to show bands at 27, 30-33, 40-42 and 50-62 kDa. Histidine-labeled proteins mainly localize in keratohyalin-like granules and dark keratin bundles of clear-oberhautchen layers of lizard epidermis, and their composition is probably different from that of beta-keratin.     

Soft epidermis of a scaleless snake lacks beta-keratin

Beta-keratins are responsible for the mechanical resistance of scales in reptiles. In a scaleless crotalus snake (Crotalus atrox), large areas of the skin are completely devoid of scales, and the skin appears delicate and wrinkled. The epidermis of this snake has been assessed for the presence of beta-keratin by immunocytochemistry and immunoblotting using an antibody against chicken scale beta-keratin. This antibody recognizes beta-keratins in normal snake scales with molecular weights of 15-18 kDa and isoelectric points at 6.8, 7.5, 8.3 and 9.4. This indicates that beta-keratins of the stratum corneum are mainly basic proteins, so may interact with cytokeratins of the epidermis, most of which appear acidic (isoelectric points 4.5-5.5). A beta-layer and beta-keratin immunoreactivity are completely absent in moults of the scaleless mutant, and the corneous layer comprises a multi-layered alpha-layer covered by a flat oberhautchen. In conclusion, the present study shows that a lack of beta-keratins is correlated with the loss of scales and mechanical protection in the skin of this mutant snake.     

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.     

Immunological characterization of a newly developed antibody for localization of a beta-keratin in turtle epidermis

Turtle scutes are made of hard (beta)-keratins. In order to study size and localization of beta-keratins in turtle shell, we produced a rat polyclonal antiserum against a turtle scute beta-keratin of 13-16 kDa, which allowed the immunolocalization of the protein in the epidermis. In immunoblots the antiserum recognized turtle beta-keratins but showed variable cross-reactivity with lizard, snake, and avian beta-keratins. The turtle antiserum appears less cross-reactive than a chicken scale antiserum (Beta-1). In bidimensional immunoblots, three main protein spots at 15-16 kDa with pI at 7.3, 6.8, 6.4, and an unresolved large spot at 40-45 kDa with pI around 5 were more constantly obtained. The latter may result from the aggregation of the smaller beta-keratin protein. The corneous layer of the carapace and plastron of various species of chelonians appeared immunofluorescent. The ultrastructural immunolocalization showed sparse labeling over beta-keratin filaments of cells of the horny layer of both carapace and plastron. The study for the first time shows that the isolated protein band derived from a component of the beta-keratin filaments of the corneous layer of turtles. This antibody can be used for further studies on beta-keratin expression and sequencing in chelonian shell. No labeling was present over other cell organelles or layers of turtle epidermis and it was absent in non-epidermal cells. The specificity for turtle beta-keratin suggests that the antiserum recognizes some epitope/s specific for chelonians beta-keratins, and that it also variably recognizes other reptilian and avian beta-keratins.     

The epidermis of scales in gecko lizards contains multiple forms of beta-keratins including basic glycine-proline-serine-rich proteins

The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.     

Immunocytochemistry and protein analysis suggest that reptilian claws contain small high cysteine-glycine proteins

The present study analyzes the structure and the main proteins of reptilian claws. Mature claws are formed by two to four layers of keratinocytes, a transitional layer of spindle-shaped cells and a thick corneous layer. Transitional cells elongate and merge into a compact corneous layer that is immunoreactive for beta-keratins, now indicated as sauropsid keratin-associated proteins (sKAPs). Most proteins extracted from claws in representative reptiles have a molecular weight of 13-20 kDa, an acidic to basic isoelectric point, and are identified from the positive immunoreactivity to beta-keratin antibodies. The comparative analysis between lizard and avian claw beta-keratins shows the presence of an internal region of 20 amino acids with the highest identity, indicated as core-box, within an extended 32-amino acid region with a prevalent beta-sheet secondary conformation. This region is structurally equivalent to a 32-amino acid region present in scale beta-keratins of most reptiles. Both reptilian and avian keratins contain glycine-rich regions for stabilization of the beta-keratin polymer. The N- and C-regions contain most cysteine for disulphide-bonds formation. Claw proteins contain higher amount of cysteine and glycine than other scale proteins, suggesting that claw proteins are specialized cysteine-glycine-rich proteins suited to produce a very hard corneous material.     

Distribution and characterization of proteins associated with cornification in the epidermis of gecko lizard

The distribution and molecular weight of epidermal proteins of gecko lizards have been studied by ultrastructural, autoradiographic, and immunological methods. Setae of the climbing digital pads are cross-reactive to antibodies directed against a chick scutate scale beta-keratin but not against feather beta-keratin. Cross-reactivity for mammalian loricrin, sciellin, filaggrin, and transglutaminase are present in alpha-keratogenic layers of gecko epidermis. Alpha-keratins have a molecular weight in the range 40-58 kDa. Loricrin cross-reactive bands have molecular weights of 42, 50, and 58 kDa. Bands for filaggrin-like protein are found at 35 and 42 kDa, bands for sciellin are found at 40-45 and 50-55 kDa, and bands for transglutaminase are seen at 48-50 and 60 kDa. The specific role of these proteins remains to be elucidated. After injection of tritiated histidine, the tracer is incorporated into keratin and in setae. Tritiated proline labels the developing setae of the oberhautchen and beta layers, and proline-labeled proteins (beta-keratins) of 10-14, 16-18, 22-24 and 32-35 kDa are extracted from the epidermis. In whole epidermal extract (that includes the epidermis with corneous layer and the setae of digital pads), beta-keratins of low-molecular weight (10, 14-16, and 18-19 kDa) are prevalent over those at higher molecular weight (34 and 38 kDa). In contrast, in shed epidermis of body scales (made of corneous layer only while setae were not collected), higher molecular weight beta-keratins are present (25-27 and 30-34 kDa). This suggests that a proportion of the small beta-keratins present in the epidermis of geckos derive from the differentiating beta layer of scales and from the setae of digital pads. Neither small nor large beta-keratins of gecko epidermis cross-react with an antibody specifically directed against the feather beta-keratin of 10-12 kDa. This result shows that the 10 and 14-16 kDa beta-keratins of gecko (lepidosaurian) have a different composition than the 10-12 kDa beta-keratin of feather (archosaurian). It is suggested that the smaller beta-keratins in both lineages of sauropsids were selected during evolution in order to build elongated bundles of keratin filaments to make elongated cells. Larger beta-keratins in reptilian scales produce keratin aggregations with no orientation, used for mechanical protection.     

Immunolocalization and characterization of beta-keratins in growing epidermis of chelonians

Beta-keratins constitute most of the corneous material of carapace and plastron of turtles. The production of beta-keratin in the epidermis of a turtle and tortoise (criptodirians) and of a species of pleurodiran turtle was studied after injection of tritiated proline during the growth of carapace, plastron and claws. Growth mainly occurs near hinge regions along the margins of scutes and along most of the claws (growing regions). Proline incorporation occurs mainly in the growing centers, and is more specifically associated with beta-keratin synthesis. Proline-labeled bands of protein at 12-14 kDa and 25-27 kDa, and 37 kDa, in the molecular weight range of beta-keratins, were isolated from the soft epidermis of turtles 3 h after injection of the labeled amino acid. After extraction of epidermal proteins, an antibody directed against a chicken beta-keratin was used for immunoblotting. Bands of beta-keratin at 15-17 kDa, 22-24 kDa, and 36-38 kDa appear in all species. Beta-keratin is present in the growing and compact stratum corneum of the hard (shell) and soft (limbs, neck and tail) epidermis. This was confirmed using a specific antibody against a turtle beta-keratin band of 15-16 kDa. The latter antibody recognized epidermal protein bands in the range of 15-16 kDa and 29-33 kDa, and labels beta-keratin filaments. This result indicates that different forms of beta-keratins are produced from low molecular weight precursors or that larger aggregate form during protein preparation. The present study shows that beta-keratin is abundant in the scaled epidermis of tortoise but also in the soft epidermis of pleurodiran and cryptodiran turtles, indicating that this form of hard keratin is constitutively expressed in the epidermis of chelonians.     

Immunolocalization of keratin-associated beta-proteins (beta-keratins) in scales of the reptiles Sphenodon punctatus indicates that different beta-proteins are present in beta- and alpha-layers

The present ultrastructural immunocytochemical study analyzes the localization of keratin-associated beta-proteins (beta-keratins) in the epidermis of the ancient reptile Sphenodon punctatus, a relict species adapted to mid-cold conditions. The epidermis comprises two main layers, indicated as beta- and alpha-keratin layers. The beta-layer contains small beta-proteins (beta-keratins) identified by using three different antibodies while the alpha-layer is poorly or not labeled for these proteins. Using other two antibodies directed against specific amino acid sequences identified in beta-proteins of lizard it results that a high-glycine beta-protein (HgG5) is specific for the beta-layer. Another antibody that recognizes glycine–cysteine medium-rich beta-proteins (HgGC10) immuno-stains beta- and alpha-layers. This pattern of distribution suggests that both beta- and alpha-layers contain beta-proteins of different types that associate and replace intermediate-filament alpha-keratins during the terminal differentiation of keratinocytes. Therefore the different epidermal layers of the epidermis in S. punctatus, characterized by a specific cytology, material properties and consistency appear to derive from the prevalent type of beta-proteins synthesized in each epidermal layer and not from the alternation between beta- and alpha-keratins. The present observations are discussed in comparison to previous results from lizard epidermis and indicate that beta-keratins correspond to keratin-associated proteins that through their internal beta-pleated region are capable to form filaments in addition to intermediate filaments keratins.     

Hard (Beta-)keratins in the epidermis of reptiles: composition, sequence, and molecular organization

Beta-keratins form the hard corneous material of reptilian scales. In the present review, the distribution and molecular characteristics of beta-keratins in reptiles are presented. In lepidosaurians immunoreactive, protein bands at 12-18 kDa are generally present with less frequent proteins at higher molecular weight. In chelonians, bands at 13-18 and 22-24 kDa are detected. In crocodilians, bands at 14-20 kDa and weaker bands at 30-32 kDa are seen. Protein bands above 25 kDa are probably polymerized beta-keratins or aggregates. Two-dimensional gel electrophoresis shows that beta-keratins are mainly basic and that acidic-neutral keratins may derive from post-translational modifications. Beta-keratins comprise glycine-proline-rich and cystein-proline-rich proteins of 13-19 kDa. Beta-keratin genes may or may not contain introns and are present in multiple copies with a linear organization as in avian beta-keratin genes. Despite amino acid differences toward N- and C-terminals all beta-keratins share high homology in their central, beta-folded region of 20 amino acids, indicated as core-box. This region is implicated in the formation of beta-keratin filaments of scales, claws, and feathers. The homology of the core-box suggests that these proteins evolved from a progenitor sequence present in the stem of reptiles. Beta-keratins have diversified in their amino acid sequences producing secondary (and tertiary) conformations that suited them for their mechanical role in scales. In birds, a small beta-keratin has allowed the formation of feathers. It is suggested that beta-keratins represent the reptilian counterpart of keratin associated or matrix proteins present in mammalian hairs, claws, and horns.     

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

The distribution of three anti-cytokeratin (alpha-keratin) antibodies (AE1, AE2, AE3) in the epidermis of a lizard has been studied by immunocytochemistry at light and electron microscope and by immunoblot analysis. This study shows the expression of different keratins in the resting stage epidermis of the lizard Podarcis sicula. In this stage the epidermis has an external beta-layer, an underlying alpha-layer, some layers of living suprabasal cells and a basal stratum germinativum. The AE1 antibody is localized in the basal and suprabasal cells only in the outer scale surface, but is absent from the inner surface, the hinge region and from the keratinized beta- and alpha-layers. The AE2 antibody is mainly localized at the level of the hinge region and of the alpha-layer and gives a lower reaction in the beta-layer. The AE3 antibody is mainly localized in basal and suprabasal cells, lower in the alpha-layer, and absent from the beta-layer. The electron microscope shows that all the three antibodies immunolabel cytoplasmic fibrillar structures in the deep alpha-layers and that AE2 and AE3 antibodies label small electron-dense areas in the external dense beta-layer within the electron-lucid matrix. Immunoblot analysis of the keratins extracted and separated by gel electrophoresis demonstrates the presence of a band of high molecular weight (67-68 kDa) positive to all three antibodies. In addition AE1 antibody recognizes a 44-45 kDa band and a 57-58 kDa band, AE2 recognizes a 60-61 kDa band, and AE3 recognizes a 47 kDa and a 56-57 kDa band. The localization of the keratins identified by immunoblot analysis in the epithelial layers is discussed taking in account the immunolabeling at light and electron microscope. The present study suggests that also in the normal epidermis of this reptiles, in both the alpha- and the beta-layer, the molecular masses of keratins increase from the basal to the keratinized layers, a phenomenon which is generalized to adult and embryonic amniotes epidermis.     

Beta-keratins of the crocodilian epidermis: composition, structure, and phylogenetic relationships

Nucleotide and deduced amino acid sequences of three beta-keratins of Nile crocodile scales are presented. Using 5'- and 3'-RACE analysis, two cDNA sequences of 1 kb (Cr-gptrp-1) and 1.5 kb (Cr-gptrp-2) were determined, corresponding to 17.4 and 19.3 kDa proteins, respectively, and a pI of 8.0. In genomic DNA amplifications, we determined that the 5'-UTR of Cr-gptrp-2 contains an intron of 621 nucleotides. In addition, we isolated a third gene (Cr-gptrp-3) in genomic DNA amplifications that exhibits seven amino acid differences with Cr-gptrp-2. Genomic organization of the sequenced crocodilian beta-keratin genes is similar to avian beta-keratin genes. Deduced proteins are rich in glycine, proline, serine, and tyrosine, and contain cysteines toward the N- and C-terminal regions, likely for the formation of disulfide bonds. Prediction of the secondary structure suggests that the central core box of 20 amino acids contains two beta-strands and has 75-90% identity with chick beta-keratins. Toward the C-terminus, numerous glycine-glycine-tyrosine and glycine-glycine-leucine repeats are present, which may contribute to making crocodile scales hard. In situ hybridization shows expression of beta-keratin genes in differentiating beta-cells of epidermal transitional layers. Phylogenetic analysis of the available archosaurian and lepidosaurian beta-keratins suggests that feather keratins diversified early from nonfeather keratins, deep in archosaur evolution. However, only the complete knowledge of all crocodilian beta-keratins will confirm whether feather keratins have an origin independent of those in bird scales, which preceded the split between birds and crocodiles.     

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.     

Distribution of specific keratin-associated beta-proteins (beta-keratins) in the epidermis of the lizard Anolis carolinensis helps to clarify the process of cornification in lepidosaurians

The epidermis of different scales in the lizard Anolis carolinensis expresses specific keratin-associated beta-proteins (beta-keratins). In order to localize the sites of accumulation of different beta-proteins, we have utilized antibodies directed against representative members of the main families of beta-proteins, the glycine-rich (HgG5), glycine-cysteine rich (HgGC3), glycine-cysteine medium-rich (HgGC10), and cysteine-rich (HgC1) beta-proteins. Immunoblotting and immunocytochemical controls confirm the specificity of the antibodies made against these proteins. Light and ultrastructural immunocytochemistry shows that the glycine-rich protein HgG5 is present in beta-layers of different body scales but is scarce in the oberhautchen and claws, and is absent in alpha-layers and adhesive setae. The cysteine-glycine-rich protein HgGC3 is low to absent in the oberhautchen, beta-layer, and mesos-layer but increases in alpha-layers. This beta-protein is low in claws where it is likely associated with the hard alpha-keratins previously studied in this lizard. The glycine-cysteine medium-rich HgGC10 protein is low in the beta-layer, higher in alpha-layers, and in the oberhautchen. This protein forms a major component of setal proteins including those of the adhesive spatula that allow this lizard to stick on vertical surfaces. HgC1 is poorly localized in most epidermis analyzed including adhesive setae and claws and appears as a minor component of the alpha-layers. In conclusion, the present study suggests that beta- and alpha-layers of lizard epidermis represent regions with different accumulation of glycine-rich proteins (mainly for mechanical resistance and hydrophobicity in the beta-layer) or cysteine-glycine-rich proteins (for both resistance and elasticity in both alpha- and beta-layers).     

Analysis of morphogenesis and keratinization in transfilter recombinants of feather-forming skin

The relationships between feather morphogenesis, histogenesis, and biochemical differentiation were examined by recombining backskin epidermis and dermis, from chick embryos (Hamburger-Hamilton stages 27-31), with an intervening Nucleopore filter (pore size of 0.4 micron). The filter inhibited normal feather morphogenesis and histogenesis of barb ridges, yet feather-like filaments, which were free of dermal cells, formed from the epidermal cells. Using indirect immunofluorescence, with antiserum against alpha- and beta-keratins, the biochemical differentiation of the feather-like filaments was compared to normal feathers. In the feather-like filaments resulting from tissues of stages 27-29, cells containing beta keratins were occasionally seen at the periphery of the filaments, yet cells containing alpha-keratins were inappropriately located throughout the filaments. In a few feather-like filaments on recombinants resulting from tissues of stages 29.5-31, cells positive for beta-keratins were found in the center of the filament, but again alpha-keratins were also found. Surrounding these cells there were several layers of cells, arranged circumferentially, resembling sheath cells. Some sheath-like cells contained beta-keratins. We conclude that although feather epidermal cells, which are separated from their dermis by a Nuclepore filter, can undergo limited morphogenesis and the production of alpha- and beta-keratins, normal feather morphogenesis, histogenesis, and biochemical differentiation require the intimate associations of epidermis and dermis.