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 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.     

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.     

The corneous layer of the claw in the lizard Anolis carolinensis mainly contains the glycine–cysteine-rich beta-protein HgGC3 in addition to hard keratins

The localization of specific claw beta-proteins among the 40 total corneous beta-proteins present in the lizard Anolis carolinensis is not known. The hardness of claws likely depends on glycine–cysteine-rich beta-proteins content, as suggested by previous immunoblot studies. Previous studies have indicated that glycine–cysteine-rich corneous beta-proteins in addition to cysteine-rich alpha-keratins are present in the claw. In order to detect at the ultrastructural level the presence of claw-specific corneous proteins immunofluorescence and electron microscopy immunogold have been utilized. More intense immunoreactivity is obtained for the HgGC3 beta-protein while less intense immunolabeling is seen for HgGC10 and HgG5 beta-proteins and no labeling for the cysteine-rich beta-protein HgC1. The HgGC3 beta-protein appears the prevalent type present in the claw and its numerous cysteines likely form intermolecular disulphide bonds while glycine contributes hydrophobic properties to the corneous material. Other antibodies tagging the core-box and pre-core box regions of beta-proteins label with less intensity the corneous layer. The presence of cysteine-rich alpha-keratins with high homology to some human hair keratins in the dorsal part of the claw suggests that HgGC3-like beta-proteins form numerous disulphide bonds with the larger alpha-keratins giving rise to the hard corneous material of the claw.     

Deleterious Mutations of a Claw Keratin in Multiple Taxa of Reptiles

We have recently shown that homologs of mammalian hair keratins are expressed in the claws of the green anole lizard, Anolis carolinensis. To test whether reptilian hair keratin homologs are functionally associated with claws, we investigated the conservation of the prototypical reptilian hair keratin homolog, hard acidic keratin 1 (HA1), in representative species from all main clades of reptiles. A complete cDNA of HA1 was cloned from the claw-forming epidermis of the lacertid lizard Podarcis sicula, and partial HA1 gene sequences could be amplified from genomic DNA of tuatara, lizards, gekkos, turtles, and crocodiles. In contrast, the HA1 gene of the limbless slow worm, Anguis fragilis, and of two species of turtles contained at least one deleterious mutation. Moreover, an HA1 gene was undetectable in the softshell turtle, snakes, and birds. Mapping the presence and absence of HA1 onto the phylogenetic tree of sauropsids suggested that the HA1 gene has been lost independently in several lineages of reptiles. The species distribution of HA1 is compatible with the hypothesis of a primary function of HA1 in claws but also shows that the formation of reptilian claws does not strictly depend on this keratin.     

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.     

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.     

Microscopic analysis of lizard claw morphogenesis and hypothesis on its evolution

Morphogenesis of claws in the lizard Lampropholis guichenoti has been studied by light and electron microscopy. Claws originate from a thickening of the epidermis covering the tips of digits under which mesenchymal cells aggregate. Mesenchymal cells are in continuity with perichondrial cells of the last phalange, and are connected to the epidermis through numerous cell bridges that cross an incomplete basement membrane. The dense lamella is completed in non‐apical regions of the digit where also collagen fibrils increase. The dorsal side of the developing claw derives from the growth of the outer scale surface of the last scale of the digit. The corneous layer, made of beta‐keratin cells, curves downward by the tip of the growing claw. The epidermis of the ventral side of the claw contains keratohyaline‐like granules and alpha‐keratinocytes like an inner scale surface. The thickness of the horny layer increases in the elongating unguis while a thinner and softer corneous layer remains in the subunguis. These observations show that lizard claws derive from the modification of the last scale or scales of the digit, probably under the influence of the growing terminal phalanx. Some hypotheses on the evolution of claws in reptiles are presented.     

Review: mapping epidermal beta-protein distribution in the lizard <Emphasis Type="Italic">Anolis carolinensis</Emphasis> 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.     

Ultrastructural characteristics of the process of cornification in developing claws of the brushtail possum (Trichosurus vulpecula)

Cornification of developing claws in the brush possum has been analysed by electron microscopy and compared with the process in other tetrapods. Newborns from 3 to 60 days postparturition were studied. After formation of symmetric and round outgrowth in digits the epidermis becomes thicker in the dorsal with respect to the ventral digit tip. The claw elongates forming the unguis and a shorter subunguis. Spinosus keratinocytes in both unguis and subunguis accumulate tonofilaments that fill their cytoplasm. Keratohyaline‐like granules are formed in early stages of differentiation in both unguis and subunguis but they later disappear in highly cornified corneocytes. Tonofilaments become electron‐dense in keratinocytes of the precorneous layer in the large corneocytes of the unguis and in narrow corneocytes of the subunguis. Keratin bundles transform into an amorphous corneous material that embeds or masks the original keratin intermediate filaments. Nucleated corneocytes are accumulated in the unguis while thinner corneocytes are present in the subunguis. The latter contain a dense material, possibly containing high sulphur keratin associated proteins, as occurs during cornifcation of the cortex and cuticle hair cells and in the nail. The process of cornification of mammalian claws is compared with that of reptilian and avian claws.     

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.     

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

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

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.     

Loricrin-like immunoreactivity during keratinization in lizard epidermis

Two modalities of keratinization are present in lizard epidermis: alpha (soft-pliable corneous layers) and beta (hard and inflexible corneous layers). While beta-keratinization is probably due to the synthesis of a new (beta)-keratin gene product, alpha keratinization resembles in part that of mammalian epidermis. The goal of this study was to test whether a sulfur-rich molecule similar to the mammalian corneous cell envelope protein loricrin is also present in lizard epidermis. This was done using X-ray microanalysis and immunocytochemical and ultrastructural methods. In the epidermis of the lizard Podarcis muralis small (0.1-0.3 microm) to large (1-5 microm) keratohyalin-like granules (KHLGs) are produced in alpha-keratinizing cells, especially in the clear layer. Small KHLGs contain sulfur and show weak filaggrin-like and stronger loricrin-like immunoreactivities. The latter is also present in keratinizing alpha-layers but is absent in the beta layers. Large KHLGs in the clear layer derive from the aggregation of the small granules with other components, including lipid material. These large granules show some loricrin-like immunoreactivity and contain sulfur and phosphorous, histidine, but not filaggrin-like immunoreactivity. It is suggested here that phosphorous derives from their phospholipid component. The present study shows that the modality of alpha-keratinization of lizard epidermis resembles that of mammals and suggests that the basic molecular mechanisms of keratin aggregation and formation of the corneous cell envelope were already present in the therapsid line of reptiles from which mammals evolved.     

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.     

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 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.     

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.     

Microscopic and immunohistochemical study on the cornification of the developing beak in the turtle Emydura macquarii

The development and cornification of the ramphoteca (beak) in turtles are not known. The microscopic aspects of beak formation have been analyzed in the pleurodirian turtle Emydura macquarii using histological, immunocytochemical and ultrastructural methods. At embryonic Stage 15 the maxillar beak is originated from discontinuous placodes (one frontal and two oral) formed in the epidermis above and below the mouth that later merge into the epidermis of the beak. The mandibular beak is formed by two lateral placodes. In the placodes, basal keratinocytes in contact with local mesenchymal condensations become columnar, and generate suprabasal cells forming 5–6 layers of embryonic epidermis at Stages 17–20 and a compact shedding alpha‐layer at the base of the embryonic epidermis. These keratinocytes contain irregular or aggregated reticular bodies made of 30–40 nm thick strands of coarse filaments, mixed with tonofilaments and sparse lipid droplets. Beneath the shedding layer are present 3–4 layers of keratinocytes accumulating coarse filaments mixed with beta‐corneous packets, and underneath spindle‐shaped beta‐cells differentiate where beta‐corneous packets completely replace the reticulate bodies. Differently from scales where corneocytes partially merge, beak corneocytes remain separated but they are joined by numerous interlocking spines. The production of beta‐cells in the thick corneous layer of the developing beak, like in claws, occurs before the differentiation of beta‐cells in the body scutes. This indicates that a massive mesenchymal condensation triggers beta‐differentiation before this process is later activated in most of body scutes of the carapace and plastron. J. Morphol. 277:1309–1319, 2016. © 2016 Wiley Periodicals, Inc.     

Fine structure and immunocytochemistry of monotreme hairs, with emphasis on the inner root sheath and trichohyalin-based cornification during hair evolution

The fine structure of hairs in the most ancient extant mammals, the monotremes, is not known. The present study analyzes the ultrastructure and immunocytochemistry for keratins, trichohyalin, and transglutaminase in monotreme hairs and compares their distribution with that present in hairs of the other mammals. The overall ultrastructure of the hair and the distribution of keratins is similar to that of marsupial and placental hairs. Acidic and basic keratins mostly localize in the outer root sheath. The inner root sheath (IRS) comprises 4-8 cell layers in most hairs and forms a tile-like sheath around the hair shaft. No cytological distinction between the Henle and Huxley layers is seen as cells become cornified about at the same time. Externally to the last cornified IRS cells (homologous to the Henle layer), the companion layer contains numerous bundles of keratin. Occasionally, some granules in the companion layer show immunoreactivity for the trichohyalin antibody. This further suggests that the IRS in monotremes is ill-defined, as the companion layer of placental hairs studied so far does not express trichohyalin. A cross-reactivity with an antibody against sheep trichohyalin is present in the IRS of monotremes, suggesting conserved epitopes across mammalian trichohyalin. Trichohyalin granules in the IRS consist of a framework of immunolabeled coarse filaments of 10-12 nm. The latter assume a parallel orientation and lose the immunoreactivity in fully cornified cells. Transglutaminase immunolabeling is diffuse among trichohyalin granules and among the parallel 10-12 nm filaments of maturing inner root cells. Transglutaminase is present where its substrate, trichohyalin, is modified as matrix protein. Cornification of IRS is different from that of hair fiber cuticle and from that of the cornified layer of the epidermis above the follicle. The different consistency among cuticle, IRS, and corneous layer of the epidermis determines separation between hair fiber, IRS, and epidermis. This allows the hair to exit on the epidermal surface after shedding from the IRS and epidermis. Based on comparative studies of reptilian and mammalian skin, a speculative hypothesis on the evolution of the IRS and hairs from the skin of synapsid reptiles is presented.