Lizard Epidermis Research Articles

Comparative immunohistochemical analysis suggests a conserved role of EPS8L1 in epidermal and hair follicle barriers of mammals.

The mammalian skin and its appendages depend on tightly coordinated differentiation of epithelial cells. Epidermal growth factor receptor (EGFR) pathway substrate 8 (EPS8) like 1 (EPS8L1) is enriched in the epidermis among human tissues and has also been detected in the epidermis of lizards. Here, we show by the analysis of single-cell RNA-sequencing data that EPS8L1 mRNA is co-expressed with filaggrin and loricrin in terminally differentiated human epidermal keratinocytes. Comparative genomics indicated that EPS8L1 is conserved in all main clades of mammals, whereas the orthologous gene has been lost in birds. Using a polyclonal antibody against EPS8L1, we performed an immunohistochemical screening of skin from diverse mammalian species and immuno-electron microscopy of human skin. EPS8L1 was detected predominantly in the granular layer of the epidermis in monotremes, marsupial, and placental mammals. The labeling was partly associated with cell membranes, and it was evident along the perimeter of keratinocytes at the transition with the cornified layer of the epidermis, similar to involucrin distribution. Basal, spinous, and the fully mature cornified layers lacked immunolabeling of EPS8L1. In addition to the epidermis, the hair follicle inner root sheath (IRS) was immunolabeled. Both epidermal granular layer and IRS contribute to the barrier function of the skin, suggesting that EPS8L1 is involved in the regulation of these barriers.

Immunolocalization of Pglyrp3 and Eps8l1 proteins in the regenerating lizard epidermis indicates they contribute to epidermal barrier formation

During tail regeneration in lizards the new corneous layer formed in the regenerating epidermis includes antimicrobial peptides, cystatin and serpins, likely forming an anti-microbial barrier. The present study aims to reveal other proteins potentially contributing to this protective barrier of the epidermis. Using immunohistochemistry we have detected a peptidoglycan-like recognition protein-3 (pglyrp3), an antimicrobial molecule, and an epidermal growth factor receptor kinase 8 l (eps8l), a receptor of EGF (Epidermal Growth Factor) that stimulates epidermal formation. The study shows that the two proteins are mostly accumulated in the forming wound epidermis and in the shedding layer of the regenerating scales. The shedding layer is the intra-epidermal layer that allows the separation of the initial corneous layer from the regenerating epidermis. While presence of pglyrp3 is likely related to the formation of the anti-microbial barrier, the function of the eps8l protein in epidermal regeneration remains unknown. Whether the latter protein is involved in keratinocyte movement within the regenerating epidermis has to be specifically determined in future studies. Together with the antimicrobial peptides cystatin and serpins, previously detected in the wound epidermis and shedding layer, the present study indicates that pglyp3, and potentially eps8l, contribute to protect the new skin and underlying regenerated tissues from the potential microbe invasion.

Immunolocalization of the EDWM-Protein Indicates a Matrix Role in Cornification of Lizard Epidermis

During epidermal differentiation in the scales of lizards and snakes, from the basal layer beta- and later alpha-keratinocytes are generated to form beta-and alpha-corneous layers. In the lizard Anolis carolinensis, minor proteins derived from the EDC (Epidermal Differentiation Complex) are added to the main constituent proteins, IFKs (Intermediate Filament Keratins) and CBPs (Corneous Beta Proteins, formerly indicated as beta keratins). One of these proteins that previous studies showed to be exclusively expressed in the skin, EDWM (EDC protein containing high GSRC amino acids) is rich in cysteine and arginine, amino acids that form numerous –S–S– and electro-static chemical bonds in the corneous material. Light and electron microscopy immunolbeling for EDWM show a diffuse localization in differentiating beta-cells and in some alpha-cells, in particular those of the clear-layer, involved in epidermal shedding. The study suggests that EDWM may function as a matrix protein that binds to IFKs and CBPs, contributing to the formation of the specific corneous material present in beta- and alpha-corneous layers. In particular, its higher immunolocalization in the maturing clear layer indicates that this protein is important for its differentiation and epidermal shedding in A. carolinensis and likely also in other lepidosaurian reptiles.

Immunolocalization of Cell Adhesion Molecules during tail regeneration in the lizard Podarcis muralis indicates coordinated control of epithelial differentiation

AbstractImmunolocalization of cell adhesion molecules during tail regeneration in the lizard Podarcis muralis indicates coordinated control of epithelial differentiation. Acta Zoologica (Stockolm). Lizard epidermis is formed by alternating layers of hard corneous beta‐layers and softer and pliable alpha‐layers. After tail amputation in the lizard Podarcis muralis, the tail regenerates within a month forming neogenic scales. The differentiation of beta‐ and alpha‐layers in the epidermis has been studied by immunofluorescence detection of cell adhesion molecules. Initially, the thick wound epidermis shows high content in L‐CAM and N‐CAM in comparison with normal epidermis, including pre‐corneous layers where labelling is intense in the cytoplasm and along the keratinocyte perimeter. L‐ and N‐CAMs are also observed in the regenerating ependyma and N‐CAM in mesenchymal cells of blastema. The apical regenerating (wound) epidermis often shows nuclear localization in basal layers, especially for E‐cadherin, perhaps indicating epithelial–mesenchymal transition. Sections of regenerating scales show the typical renewal phase of the epidermis, with an intensification of L‐CAM, N‐CAM and general cadherin immunofluorescence in the forming clear‐Oberhautchen layers, and in the following beta‐ and alpha‐layers as they start to differentiate from the basal layer. A higher immunolabelling for cell adhesion cadherins suggests that these proteins contribute to form an integrated epidermal unit along the regenerating scales that allows the ordered stratification and shedding of these layers in lizard epidermis.

Ontogeny of the Moorish gecko Tarentola mauritanica with emphasis on morphogenesis of the skin and its derivatives.

Studying reptilian embryonic development provides answers to many questions related to the development of tetrapods. Reptilian skin has been recently considered in studies at the evo-devo level. The lizard epidermis has to be shed periodically. At the embryonic level, contention exists regarding the first layers to appear, whether the oberhautchen or the clear layer, and whether the shedding complex develops before hatching. Geckos exhibit diverse morphologies independently evolved multiple times within the clade, such as subdigital pad lamellae. Here we investigate the embryonic development of Tarentola mauritanica and establishing its embryonic table. Primarily we follow the development of the integument. This is a closely related species to Tarentola annularis and it is crucial to investigate whether it has the same derived digital condition of claw regression. Eleven embryonic stages are described according to the external morphological characteristics of the embryos. Interestingly, the oviposition stage appears earlier than its close relative T. annularis, and the total incubation time is less. We also describe skin development, adding clear evidence to the debate on the development of the shedding complex, which we found is developed before hatching. We describe one layer of periderm and the clear layer as the first embryonic epidermal layers. Generally, our results show the genus Tarentola to have the advantage of being a unique taxon, easily breed at the laboratory, with multiple clutches per year, and with an earlier stage at oviposition. That could be a model animal for embryonic development and experimental embryology studies.

Disulfide-bond-mediated cross-linking of corneous beta-proteins in lepidosaurian epidermis

Corneous beta-proteins (CBPs), formerly referred to as beta-keratins, are major protein components of the epidermis in lepidosaurian reptiles and are largely responsible for their material properties. These proteins have been suggested to form filaments of 3.4nm in thickness and to interact with themselves or with other proteins, including intermediate filament (IF) keratins. Here, we performed immunocytochemical labeling of CBPs in the epidermis of different lizards and snakes and investigated by immunoblotting analysis whether the reduction of disulfide bonds or protein oxidation affects the solubility and mobility of these CBPs. Immunogold labeling suggested that CBPs are partly co-localized with IF-keratins in differentiating and mature beta-cells. The chemical reduction of epidermal proteins from lizard and snake epidermis increased the abundance of CBP-immunoreactive bands in the size range of CBP monomers on Western blots. Conversely, in vitro oxidation of epidermal proteins reduced the abundance of putative CBP monomers. Some modifications in the IF-keratin range were also noted. These results strongly indicate that CBPs associate with IF-keratins and other proteins via disulfide bonds in the epidermis of lizards and snakes, which likely contributes to the resilience of the cornified beta- and alpha-layers of the lepidosaurian epidermis in live animals and after shedding.

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.

Crack/Fold Hybrid Structure-Based Fluidic Networks Inspired by the Epidermis of Desert Lizards

A bioinspired fluidic system with cracks and folds was introduced to emulate the structures and functions of desert lizards' integuments, which show marked ability of water management. Because there was a structural analogy between scales and interscalar channels of lizard's skin and cracks and folds of a bilayer elastic material, we can mimic lizard's skin by controlling the stress distribution on patterned elastomers. Our system showed not only capillary-driven water retention within confined fluidic network, but also stretching-driven biaxial water transport. Observed features of our system may enhance understanding of water management in relation to morphogenetic aspects of lizards.

Review: mapping epidermal beta-protein distribution in the lizard Anolis carolinensis shows a specific localization for the formation of scales, pads, and claws.

The epidermis of lizards is made of multiple alpha- and beta-layers with different characteristics comprising alpha-keratins and corneous beta-proteins (formerly beta-keratins). Three main modifications of body scales are present in the lizard Anolis carolinensis: gular scales, adhesive pad lamellae, and claws. The 40 corneous beta-proteins present in this specie comprise glycine-rich and glycine-cysteine-rich subfamilies, while the 41 alpha-keratins comprise cysteine-poor and cysteine-rich subfamilies, the latter showing homology to hair keratins. Other genes for corneous proteins are present in the epidermal differentiation complex, the locus where corneous protein genes are located. The review summarizes the main sites of immunolocalization of beta-proteins in different scales and their derivatives producing a unique map of body distribution for these structural proteins. Small glycine-rich beta-proteins participate in the formation of the mechanically resistant beta-layer of most scales. Small glycine-cysteine beta-proteins have a more varied localization in different scales and are also present in the pliable alpha-layer. In claws, cysteine-rich alpha-keratins prevail over cysteine-poor alpha-keratins and mix to glycine-cysteine-rich beta-proteins. The larger beta-proteins with a molecular mass similar to that of alpha-keratins participate in the formation of the fibrous meshwork present in differentiating beta-cells and likely interact with alpha-keratins. The diverse localization of alpha-keratins, beta-proteins, and other proteins of the epidermal differentiation complex gives rise to variably pliable, elastic, or hard corneous layers in different body scales. The corneous layers formed in the softer or harder scales, in the elastic pad lamellae, or in the resistant claws possess peculiar properties depending on the ratio of specific corneous proteins.

Immunolocalization of sulfhydryl oxidase in reptilian epidermis indicates that the enzyme participates mainly to the hardening process of the beta-corneous layer.

Reptilian skin is tough and scaled representing an evolutionary adaptation to the terrestrial environment. The presence of sulfhydryl oxidase during the process of hardening of the corneous layer in reptilian epidermis has been analyzed by immunocytochemistry and immunoblotting. Sulfhydryl oxidase-like immunoreactivity of proteins in the 50-65kDa range of molecular weight is mainly observed in the transitional and pre-corneous layers of crocodilians, chelonian, and in the forming beta-layer of lepidosaurians. The ultrastructural localization of the enzyme by immunogold in lizard epidermis during renewal and resting stages shows that the labeling is mainly distributed in the cytoplasm and along the accumulating beta-packets of differentiating beta-cells while it appears very low to undetectable in differentiating alpha-cells of the lacunar, clear, mesos, and alpha-layers. The labeling however becomes absent or undetectable also in the fully mature beta-layer. The study shows that an oxidative enzyme is likely responsible of the cross-linking of the numerous cysteines present in the main proteins accumulated in corneocytes of reptilian epidermis, known as corneous beta-proteins (beta-keratins). This process of disulphide bond formation is probably largely responsible for the formation of hard beta-corneous layers in reptilian scales, a difference with alpha-corneous layers where substrate proteins of transglutaminase appear predominant.

Immunolocalization of loricrin in the maturing α-layer of normal and regenerating epidermis of the lizard Anolis carolinensis.

Numerous corneous proteins are produced during the differentiation of the complex lizard epidermis, comprising hard β-layers and softer α-layers. In the present ultrastructural and immunocytochemical study, we have localized a homolog of the mammalian skin barrier protein loricrin in the skin of the green anole lizard (Anolis carolinensis). We used an antibody specific to the carboxyterminus of loricrin 1, a gene of the epidermal differentiation complex (EDC) of A. carolinensis. Lizard loricrin is present in the maturing α-layer (lacunar cells) of normal scale epidermis and in the accumulating corneocytes of the wound epidermis (lacunar cells) of the regenerating epidermis. The protein appears as a component of the α-layer but not of the β-layer. Lizard loricrin is diffused in the cytoplasm of pre-corneous α-keratinocytes but eventually concentrates in the packing corneous material of the maturing corneocytes of the α-layer (lacunar) in normal epidermis or in the wound epidermis of regenerating epidermis. The protein likely contributes to the composition and pliability of the corneous material but is not specifically accumulated on the corneous cell envelope (marginal layer) that is scarcely differentiated in these cells. The study contributes to the knowledge on the distribution of specific corneous proteins that give rise to the different material properties of α-layers versus β-layers in lizard epidermis.

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.

Transition from embryonic to adult epidermis in reptiles occurs by the production of corneous beta-proteins.

The adaptation of the epidermis in amniote vertebrates to life on land took place by a drastic change from an embryonic epidermis made of two-four periderm layers to a terrestrial-proof epidermis. This transition occurred by the increase in types and number of specialized corneous proteins coded by genes of the Epidermal Differentiation Complex. The prevalent types of corneous proteins produced in the reptilian epidermis contain a beta-sheet region of high amino acid homology which allows their polymerization into a meshwork of filaments forming the hard corneous material of scales and claws. The present immunogold ultrastructural study shows that this transition occurs with the synthesis of glycine-rich corneous beta-proteins (formerly indicated as beta-keratins) that are added to the initial framework of acidic intermediate filaments produced in the embryonic epidermis of lizards, snake, alligator and turtle. These corneous beta-proteins are accumulated in the transitional and definitive layers of reptilian epidermis formed underneath the transitory two-four layered embryonic epidermis. In the more specialized reptiles capable of shedding the epidermis as a single unit, such as lizards and snakes, special glycine-cysteine rich beta-proteins are initially produced in a single layer immediately formed beneath the embryonic epidermis, the oberhautchen. The latter layer allows the in ovo shedding of the embryonic epidermis in preparation for hatching, and in the following shedding cycles of the adult epidermis. The production of specialized corneous-specific beta-proteins in addition to intermediate filament keratins was probably an essential addition for terrestrial life during the evolution of reptiles into different lineages, including birds. The increase of glycine and cysteine in epidermal proteins enhanced the hydrophobicity, insolubility and mechanical strength of the stratum corneum in these amniotes.

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.

Ultrastructural immunolocalization of beta-defensin-27 in granulocytes of the dermis and wound epidermis of lizard suggests they contribute to the anti-microbial skin barrier

The high resistance to infections in lizard wounds suggests that these reptiles possess effective antimicrobial peptides in their tissues. The present immunocytochemical study shows the cellular localization of beta-defensin 27 in tail tissues and in the blood, a defensin previously identified in the lizard Anolis carolinensis through biomolecular methods. Beta-defensin-27 immunoreactivity is only observed in some large granules mainly contained in heterophilic granulocytes that are sparse within the dermis of the skin or in the isolated blood. This peptide is absent in other cell types of the skin, in keratinocytes and in subdermal muscle tissue of the tail in normal conditions. Pre-corneous keratinocytes of the regenerating tail epidermis are unlabeled or show a weak labeling for the peptide only in sparse cytoplasmic areas or in the extracellular spaces among corneocytes of the wound and regenerating epidermis. The study suggests that beta-defensin 27 is normally stored in granulocytes present in the blood or in connective tissues while in the epidermis keratinocytes do not show the presence of this peptide unless these cells are stimulated from injury to produce and likely release beta-defensins.

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.

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

AbstractLizard 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.

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.

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.

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).