The Sandfish's Skin:Morphology,Chemistry and Reconstruction

The sandfish is a lizard having the remarkable ability to move in desert sand in a swimming-like fashion. The most out-standing adaptations to this mode of life are the low friction behaviour and the extensive abrasion resistance of the sandfish skin against sand, outperforming even steel. We investigated the topography, the composition and the mechanical properties of sandfish scales. These consist of glycosylated keratins with high amount of sulphur but no hard inorganic material, such as silicates or lime. Remarkably, atomic force microscopy shows an almost complete absence of attractive forces between the scale surface and a silicon tip, suggesting that this is responsible for the unusual tribological properties. The unusual glycosylation of the keratins was found to be absolutely necessary for the described phenomenon. The scales were dissolved and reconstituted on a polymer surface resulting in properties similar to the original scale. Thus, we provide a pathway towards exploitation of the reconstituted scale material for future engineering applications.     

Investigating the locomotion of the sandfish in desert sand using NMR-imaging

The sandfish (Scincus scincus) is a lizard having the remarkable ability to move through desert sand for significant distances. It is well adapted to living in loose sand by virtue of a combination of morphological and behavioural specializations. We investigated the bodyform of the sandfish using 3D-laserscanning and explored its locomotion in loose desert sand using fast nuclear magnetic resonance (NMR) imaging. The sandfish exhibits an in-plane meandering motion with a frequency of about 3 Hz and an amplitude of about half its body length accompanied by swimming-like (or trotting) movements of its limbs. No torsion of the body was observed, a movement required for a digging-behaviour. Simple calculations based on the Janssen model for granular material related to our findings on bodyform and locomotor behaviour render a local decompaction of the sand surrounding the moving sandfish very likely. Thus the sand locally behaves as a viscous fluid and not as a solid material. In this fluidised sand the sandfish is able to "swim" using its limbs.     

Avian scale development: XI. Immunoelectron microscopic localization of alpha and beta keratins in the scutate scale

Epithelial-mesenchymal interactions play important roles in morphogenesis, histogenesis, and keratinization of the vertebrate integument. In the anterior metatarsal region of the chicken, morphogenesis results in the formation of distinct overlapping scutate scales. Recent studies have shown that the dermis of scutate scales is involved in the expression of the beta keratin gene products, which characterize terminal differentiation of the epidermis on the outer scale surface (Sawyer et al.: Dev. Biol. 101:8-18, '84; Shames and Sawyer: Dev. Biol. 116:15-22, '86; Shames and Sawyer: In A.A. Moscona and A. Monroy (eds), R.H. Sawyer (Vol. ed): Current Topics in Developmental Biology. Vol. 22: The Molecular and Developmental Biology of Keratins. New York: Academic Press, pp. 235-253, '87). Since alpha and beta keratins are both found in the scutate scale and are members of two different multigene families, it is important to know the precise location of these distinct keratins within the epidermis. In the present study, we have used protein A-gold immunoelectron microscopy with antisera made against avian alpha and beta keratins to specifically localize these keratins during development of the scutate scale to better understand the relationship between dermal cues and terminal differentiation. We find that the bundles of 3-nm filaments, characteristic of tissues known to produce beta keratins, react specifically with antiserum which recognizes beta keratin polypeptides and are found in the embryonic subperiderm that covers the entire scutate scale and in the stratum intermedium and stratum corneum making up the platelike beta stratum of the outer scale surface. Secondly, we find that 8-10-nm tonofilaments react specifically with antiserum that recognizes alpha keratin polypeptides and are located in the germinative basal cells and the lowermost cells of the stratum intermedium of the outer scale surface, as well as in the embryonic alpha stratum, which is lost from the outer surface of the scale at hatching. The alpha keratins are found throughout the epidermis of the inner surface of the scale and the hinge region. Thus, the present study further supports the hypothesis that the tissue interactions responsible for the formation of the beta stratum of scutate scales do not directly activate the synthesis of beta keratins in the germinative cells but influence these cells so that they or their progeny will activate specific beta keratin genes at the appropriate time and place.     

Cytochemical, biochemical and molecular aspects of the process of keratinization in the epidermis of reptilian scales

The characteristics of scaled skin of reptiles is one of their main features that distinguish them from the other amniotes, birds and mammals. The different scale patterns observed in extant reptiles result from a long evolutive history that allowed each species to adapt to its specific environment. The present review deals with comparative aspects of epidermal keratinization in reptiles, chelonians (turtles and tortoises), lepidosaurian (lizards, snakes, sphenodontids), archosaurians (crocodilians). Initially the morphology and cytology of reptilian scales is outlined to show the diversity in the epidermis among different groups. The structural proteins (alpha-keratins and associated proteins), and enzymes utilized to form the corneous layer of the epidermis are presented. Aside cytokeratins (alpha-keratins), used for making the cytoskeleton, reptilian alpha-keratinocytes produce interkeratin (matrix) and corneous cell envelope proteins. Keratin bundles and degraded cell organelles constitute most of the corneous material of alpha-keratinocytes. Matrix, histidine-rich and sulfur-rich proteins are produced in the soft epidermis and accumulated in the cornified cell envelope. Main emphasis is given to the composition and to the evolution of the hard keratins (beta-keratins). Beta-keratins constitute the hard corneous material of scales. These small proteins are synthesized in beta-keratinocytes and are accumulated into small packets that rapidly merge into a compact corneous material and form densely cornified layers. Beta-keratins are smaller proteins (8-20 kDa) in comparison to alpha-keratins (40-70 kDa), and this size may determine their dense packing in corneocytes. Both glycine-sulfur-rich and glycine-proline-rich proteins have been so far sequenced in the corneous material of scales in few reptilian species. The latter keratins possess C- and N-amino terminal amino acid regions with sequence homology with those of mammalian hard keratins. Also, reptilian beta-keratins possess a central core with homology with avian scale/feather keratins. Multiple genes code for these proteins and their discovery and sequentiation is presently an active field of research. These initial findings however suggest that ancient reptiles already possessed some common genes that have later diversified to produce the specific keratin-associated proteins in their descendants: extant reptiles, birds and mammals. The evolution of these small proteins in lepidosaurians, chelonians and archosaurians represent the next step to understand the evolution of cornification in reptiles and derived amniotes (birds and mammals).     

Avian scale development. VI. Ultrastructure of the keratinizing cells of reticulate scales

Reticulate scales develop as radial symmetrical anlagen, in contrast to scuttate scales which appear initially as “epidermal placodes.” Unlike scuttate scales whose outer and inner epidermal surfaces elaborate β-and α-type keratins, respectively, reticulate scales elaborate only one type of epidermal surface which has been reported to give an α-type, X-ray diffraction pattern. We find that, histologically and ultrastructurally, this surface differs from either epidermal surface of scuttate scales. The keratinizing cells become filled with long interweaving bundles of α-filaments which aggregate into rather homogeneous α-fibrils. Keratohyalin granules, which have been shown to be associated with other keratinizing regions in the bird, do not form during the keratinization of reticulate scale epidermis.     

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.     

Material properties of the skin of the Kenyan sand boa Gongylophis colubrinus (Squamata, Boidae)

On the basis of structural data, it has been previously assumed that the integument of snakes consists of a hard, robust, inflexible outer surface (Oberhäutchen and β-layer) and soft, flexible inner layers (α-layers). The aim of this study was to compare material properties of the outer and inner scale layers of the exuvium of Gongylophis colubrinus, to relate the structure of the snake integument to its mechanical properties. The nanoindentation experiments have demonstrated that the outer scale layers are harder, and have a higher effective elastic modulus than the inner scale layers. The results obtained provide strong evidence about the presence of a gradient in the material properties of the snake integument. The possible functional significance of this gradient is discussed here as a feature minimizing damage to the integument during sliding locomotion on an abrasive surface, such as sand.     

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.     

Region-specific expression of scutate scale type beta keratins in the developing chick beak.

This study shows that different patterns of scutate scale type beta keratins are accumulated in the three adjacent structures of the embryonic chick beak: periderm, egg tooth, and cornified beak. The cornified beak accumulates all of the beta keratins of scutate scale except pp2,3. The periderm, which is the outermost, multilayered covering of the whole embryonic beak, accumulates only beta keratins 2,3, and p2,3 of the scutate scale pattern. The egg tooth, which is the rounded elevation on the dorsal surface of the upper beak, and the embryonic claw accumulate greatly reduced levels of 2,3 and p2,3 compared to scutate scale. Like cornified beak, the claw does not accumulate pp2,3, but both tissues express a potentially new beta keratin, beta keratin 8. Neither the histidine rich "fast" proteins (HRPs), which are expressed in embryonic scutate scales and feathers, nor the avian cytokeratin associated proteins (cap-1 and cap-2), which are expressed in scutate and reticulate scales, are expressed in any of the embryonic beak structures or in the claw. The implications of these findings with regard to regulation of terminal differentiation of avian skin are discussed.     

Differences in the histogenesis and keratin expression of avian extraembryonic ectoderm and endoderm recombined with dermis

The responses of the chorionic ectoderm and allantoic endoderm (from 8-day chick embryos) to dermal induction were compared through tissue recombinants grafted onto the chorioallantoic membrane. The chorionic epithelium formed the appropriate epidermis with a fully developed stratum corneum in response to both spur and scutate scale dermises. Analysis of these recombinant epidermal tissues by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that tissue-specific expression of the alpha (alpha) and beta (beta) keratin polypeptides occurred. In addition, indirect immunofluorescence studies with antisera to alpha or beta keratins showed that the beta stratum, which characterizes the epidermis of spurs and scutate scales, was formed, and the alpha keratins were distributed as in the normal epidermal tissues. In contrast, although the allantoic endoderm became stratified in association with either spur or scutate scale dermis, a stratum corneum with a beta stratum did not develop. SDS-PAGE analysis demonstrated that while the characteristic beta keratins of scutate scales and spur were not detected, most of the alpha keratins normally elaborated by these structures were present, suggesting that even without histogenesis of a stratum corneum the expression of alpha keratins of endoderm could be regulated in a tissue-specific manner by dermis. This study also demonstrated that there are differences in the abilities of the chorionic and allantoic epithelia to respond to the same dermal cues, which may reflect earlier restrictions in their developmental potentials.