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

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

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.     

Histidine uptake in the epidermis of lizards and snakes in relation to the formation of the shedding complex

Mammalian epidermis utilizes histidine-rich proteins (filaggrins) to aggregate keratin filaments and form the stratum corneum. Little is known about the involvement of histidine-rich proteins during reptilian keratinization. The formation of the shedding complex in the epidermis of snakes and lizards, made of the clear and the oberhautchen layers, determines the cyclical epidermal sloughing. Differently from snakes, keratohyalin-like granules are present in the clear layer of lizards. The uptake of tritiated histidine into the epidermis of two lizards and one snake has been studied by autoradiography in sections at progressive post-injection periods. At 40 min and 1 hr post-injection keratohyalin-like granules were not or poorly labeled. At 3-22 hr post-injection most of the labeling was present over suprabasal cells destined to form the shedding complex, in keratohyalin-like granules of the clear layer, and in the forming a-layer but was low in the forming b-layer, and in superficial keratinized layers. The analysis of the shedding complex in the pad lamellae (a specialized scale used for climbing) of a gecko showed that the setae and the cytoplasm of clear cells among them are main sites of histidine uptake at 4 hr post-injection. In the snake most of the labeling at 4 hr post-injection was localized in the shedding complex along the boundary between the clear and oberhautchen layers. The present study suggests that, in the epidermis of lepidosaurian reptiles, the synthesis of a histidine-rich protein is involved in the formation of the shedding layer and, as in mammals, in a-keratinization.     

Ultrastructure of the embryonic snake skin and putative role of histidine in the differentiation of the shedding complex.

The morphogenesis and ultrastructure of the epidermis of snake embryos were studied at progressive stages of development through hatching to determine the time and modality of differentiation of the shedding complex. Scales form as symmetric epidermal bumps that become slanted and eventually very overlapped. During the asymmetrization of the bumps, the basal cells of the forming outer surface of the scale become columnar, as in an epidermal placode, and accumulate glycogen. Small dermal condensations are sometimes seen and probably represent primordia of the axial dense dermis of the growing tip of scales. Deep, dense, and superficial loose dermal regions are formed when the epidermis is bilayered (periderm and basal epidermis) and undifferentiated. Glycogen and lipids decrease from basal cells to differentiating suprabasal cells. On the outer scale surface, beneath the peridermis, a layer containing dense granules and sparse 25-30-nm thick coarse filaments is formed. The underlying clear layer does not contain keratohyalin-like granules but has a rich cytoskeleton of intermediate filaments. Small denticles are formed and they interdigitate with the oberhautchen spinulae formed underneath. On the inner scale surface the clear layer contains dense granules, coarse filaments, and does not form denticles with the aspinulated oberhautchen. On the inner side surface the oberhautchen only forms occasional spinulae. The sloughing of the periderm and embryonic epidermis takes place in ovo 5-6 days before hatching. There follow beta-, mesos-, and alpha-layers, not yet mature before hatching. No resting period is present but a new generation is immediately produced so that at 6-10 h posthatching an inner generation and a new shedding complex are forming beneath the outer generation. The first shedding complex differentiates 10-11 days before hatching. In hatchlings 6-10 h old, tritiated histidine is taken up in the epidermis 4 h after injection and is found mainly in the shedding complex, especially in the apposed membranes of the clear layer and oberhautchen cells. This indicates that a histidine-rich protein is produced in preparation for shedding, as previously seen in lizard epidermis. The second shedding (first posthatching) takes place at 7-9 days posthatching. It is suggested that the shedding complex in lepidosaurian reptiles has evolved after the production of a histidine-rich protein and of a beta-keratin layer beneath the former alpha-layer.     

Putative histidin-rich proteins in the epidermis of lizards

In the stratum granulosum of mammalian epidermis, histidin-rich proteins (filaggrins) determine keratin clumping and matrix formation into terminal keratinocytes of the stratum corneum. The nature of matrix, interkeratin proteins in the epidermis of nonmammalian vertebrates, and in particular in that of reptilian, mammalian progenitors are unknown. The present biochemical study is the first to address this problem. During a specific period of the renewal phase of the epidermis of lizards and during epidermal regeneration, keratohyalin-like granules are formed, at which time they take up tritiated histidine. The latter also accumulate in cells of the alpha-keratin layer (soft keratin). This pattern of histidine incorporation resembles that seen in keratohyalin granules of the stratum granulosum of mammalian epidermis. After injection of tritiated histidine, we have analysed the distribution of the radioactivity by histoautoradiography and electrophoretic gel autoradiography of epidermal proteins. Extraction and electrophoretic separation of interfilamentous matrix proteins from regenerating epidermis 3-48 hours post-injection reveals the appearance of protein bands at 65-70, 55-58, 40-43, 30-33, 25-27, and 20-22 kDa. Much weaker bands were seen at 100, 140-160, and 200 kDa. A weak band at 20-22 kDa or no bands at all are seen in the normal epidermis in resting phase and in the dermis. In regenerating epidermis at 22 and 48 hours post-injection, little variation in bands is detectable, but low molecular weight bands tend to increase slightly, suggesting metabolic turnover. Using anti-filaggrin antibodies against rat, human, or mouse filaggrins, some cross-reactivity was seen with more reactive bands at 40-42 and 33 kDa, but it was reduced or absent at 140, 95-100, 65-70, 50-55, and 25 kDa. This suggests that different intermediate degradative proteins of lizard epidermis may share some epitopes with mammalian filaggrins and are different from keratins with molecular weight ranging from 40 to 65-68 kDa. The immunocytochemical observation confirms that a weak filaggrin-like immunoreactivity characterizes differentiating alpha-keratogenic layers in normal and regenerating tail. A weak filaggrin labeling is discernable in small keratohyalin-like granules but is absent from the larger granules and from mature keratinocytes. The present results indicate, for the first time, that histidine-rich proteins are involved in the process of alpha-keratinization in reptilian epidermis. The cationic, interkeratin matrix proteins implicated may be fundamentally similar in both theropsid-derived and sauropsid amniotes.     

Epidermal differentiation during ontogeny and after hatching in the snake Liasis fuscus (Pythonidae,Serpentes, Reptilia), with emphasis on the formation of the shedding complex

Differentiation and localization of keratin in the epidermis during embryonic development and up to 3 months posthatching in the Australian water python, Liasis fuscus, was studied by ultrastructural and immunocytochemical methods. Scales arise from dome-like folds in the skin that produce tightly imbricating scales. The dermis of these scales is completely differentiated before any epidermal differentiation begins, with a loose dermis made of mesenchymal cells beneath the differentiating outer scale surface. At this stage (33) the embryo is still unpigmented and two layers of suprabasal cells contain abundant glycogen. At Stage 34 (beginning of pigmentation) the first layers of cells beneath the bilayered periderm (presumptive clear and oberhautchen layers) have not yet formed a shedding complex, within which prehatching shedding takes place. At Stage 35 the shedding complex, consisting of the clear and oberhautchen layers, is discernible. The clear layer contains a fine fibrous network that faces the underlying oberhautchen, where the spinulae initially contain a core of fibrous material and small beta-keratin packets. Differentiation continues at Stage 36 when the beta-layer forms and beta-keratin packets are deposited both on the fibrous core of the oberhautchen and within beta-cells. Mesos cells are produced from the germinal layer but remain undifferentiated. At Stage 37, before hatching, the beta-layer is compact, the mesos layer contains mesos granules, and cells of the alpha-layer are present but are not yet keratinized. They are still only partially differentiated a few hours after hatching, when a new shedding complex is forming underneath. Using antibodies against chick scale beta-keratin resolved at high magnification with immunofluorescent or immunogold conjugates, we offer the first molecular confirmation that in snakes only the oberhautchen component of the shedding complex and the underlying beta cells contain beta-keratin. Initially, there is little immunoreactivity in the small beta-packets of the oberhautchen, but it increases after fusion with the underlying cells to produce the syncytial beta layer. The beta-keratin packets coalesce with the tonofilaments, including those attached to desmosomes, which rapidly disappear in both oberhautchen and beta-cells as differentiation progresses. The labeling is low to absent in forming mesos-cells beneath the beta-layer. This study further supports the hypothesis that the shedding complex in lepidosaurian reptiles evolved after there was a segregation between alpha-keratogenic cells from beta-keratogenic cells during epidermal renewal.     

Immunocytochemical and autoradiographic studies on the process of keratinization in avian epidermis suggests absence of keratohyalin

The process of keratinization in apteric avian epidermis and in scutate scales of some avian species has been studied by autoradiography for histidine and immunohistochemistry for keratins and other epidermal proteins. Acidic or basic alpha-keratins are present in basal, spinosus, and transitional layers, but are not seen in the corneous layer. Keratinization-specific alpha-keratins (AE2-positive) are observed in the corneous layer of apteric epidermis but not in that of scutate scales, which contain mainly beta-keratin. Alpha-keratin bundles accumulate along the plasma membrane of transitional cells of apteric epidermis. In contrast to the situation in scutate scales, in the transitional layer and in the lowermost part of the corneous layer of apteric epidermis, filaggrin-like, loricrin-like, and transglutaminase immunoreactivities are present. The lack of isopeptide bond immunoreactivity suggests that undetectable isopeptide bonds are present in avian keratinocytes. Using immunogold ultrastructural immunocytochemistry a low but localized loricrin-like and, less, filaggrin-like labeling is seen over round-oval granules or vesicles among keratin bundles of upper spinosus and transitional keratinocytes of apteric epidermis. Filaggrin-and loricrin-labeling are absent in alpha-keratin bundles localized along the plasma membrane and in the corneous layer, formerly considered keratohyalin. Using ultrastructural autoradiography for tritiated histidine, occasional trace grains are seen among these alpha-keratin bundles. A different mechanism of redistribution of matrix and corneous cell envelope proteins probably operates in avian keratinocytes as compared to that of mammals. Keratin bundles are compacted around the lipid-core of apteric epidermis keratinocytes, which do not form complex chemico/mechanical-resistant corneous cell envelopes as in mammalian keratinocytes. These observations suggest that low amounts of matrix proteins are present among keratin bundles of avian keratinocytes and that keratohyalin granules are absent.     

Observations on the histochemistry and ultrastructure of regenerating caudal epidermis of the tuatara Sphenodon punctatus (Sphenodontida,Lepidosauria, Reptilia)

Study of the histology, histochemistry, and fine structure of caudal epidermal regeneration in Sphenodon punctatus through restoration of a scaled form reveals that the processes involved resemble those known in lizards. Following establishment of a wound epithelium (WE), subjacent scale neogenesis involves epidermal downgrowths into the dermis. Although the process is extremely slow, and most new scales do not overlap, their epidermal coverings reestablish epidermal generation (EG) formation. As in lizards, the flat, alpha-keratogenic, WE cells contain lipids as revealed by their affinity for Sudan III. A few mucous cells that store large PAS-positive mucus-like granules also occur in WE. During differentiation of WE cells, among the bundles of 70-nm tonofilaments are many lamellar bodies (LBs) and mucous granules (MGs) that discharge their contents into the cytoplasm and extracellular spaces producing a strongly PAS-positive keratinized tissue. Richness of epidermal lipids coexistent with mucus is a primitive characteristic for amniote vertebrates, probably related to functions as a barrier to cutaneous water loss (CWL). As scale neogenesis begins, beneath the superficial WE appear 3-5 layers of irregularly shaped cells. These contain tonofilament bundles surrounded by small, round keratohyalin-like granules (KHLGs) and a keratinized matrix with beta-keratin packets and a 3-5-nm thick keratin granulation. This mixture of alpha- and beta-keratogenic capacities resembles that seen in the innermost cells of a normal tuatara epidermal generation. As in the latter, but in contrast to both normal and regenerating lizard epidermis, no definable shedding complex with interdigitating clear layer and oberhautchen cells occurs (Alibardi and Maderson, 2003). The tortuous boundaries, and merging beta-keratin packets, identify subjacent keratinizing cells as precursors of the typical stratified, squamous beta-layer seen in long-term regenerated caudal skin wherein the entire vertical sequence of epidermal layers resembles that of normal scales. The sequence of events in caudal epidermal regeneration in S. punctatus resembles that documented for lizards. Observed differences between posttrauma scale neogenesis and scale embryogenesis are responses to functional problems involved in, respectively, restoring, or forming, a barrier to CWL while accommodating rapid somatic growth.     

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.     

Ultrastructural autoradiographic and immunocytochemical analysis of setae formation and keratinization in the digital pads of the gecko Hemidactylus turcicus (Gekkonidae,Reptilia)

The modified subdigital scales of some lizards allow them to climb vertical surfaces. This is due to the action of millions of tiny setae present in the digital pads. Setae are mainly composed of beta-keratin which may have some modality of aggregation similar to that of barbs and barbules of feathers. Keratins and associated proteins are involved in the organization of setae. The formation of setae in the climbing pad lamellae of the gecko Hemidactylus turcicus has been analyzed under the electron microscope after injection of tritiated histidine and immunocytochemistry for a chick scale beta-keratin. Setae are made up of dense and pale filaments, both oriented along the longer axis of setae. Beta-keratin is present in the oberhautchen layer and in the growing setae which are highly modified oberhautchen cells. Most of the immunolabeling concentrated in the central part of setae. This cross-reactivity suggests that some epitopes in chick beta-keratin are also present in gecko setae. Four hours after injection of tritiated histidine, the labeling is localized over setae, in particular in the dense filaments and less in the pale filaments. Some labeling is also seen in the keratinaceous material present in the cytoplasm of clear cells, which are believed to mold setae. The present observations suggest that both beta-keratin and denser matrix proteins, possibly incorporating histidine, are packed into growing setae. These proteins may be mixed to form pale and dense filaments oriented along the longer axis of setae, a pattern resembling that of barb and barbule cells of feathers. The role of matrix material in the orientation of the deposited beta-keratin during setal outgrowth is discussed with the problem of barb and barbule differentiation in avian feathers.     

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.     

Immunocytochemical observations on the cornification of soft and hard epidermis in the turtle Chrysemys picta

The process of cornification in the shell and non-shelled areas of the epidermis of the turtle Chrysemys picta was analyzed by light and ultrastructural immunohistochemistry for keratins, filaggrin and loricrin. Beta-keratin (hard keratin) was only present in the corneus layer of the plastron and carapace. The use of a beta-keratin antibody, developed against a specific chick scale beta-keratin, demonstrated that avian and reptilian hard keratins share common amino acid sequences. In both, shelled and non-shelled epidermis, acidic alpha keratin (AE1 positive) was limited to tonofilament bundles of the basal and suprabasal layer, while basic keratin (AE3 positive) was present in basal, suprabasal, and less intensely, pre-corneus layers, but tended to disappear in the corneus layer. The AE2 antibody, which in mammalian epidermis recognizes specific keratins of cornification, did not stain turtle shell but only the corneus layer of non-shelled (soft) epidermis. Two and four hours after an injection of tritiated histidine, the labelling was evenly distributed over the whole epidermis of both shelled and non-shelled areas, but was absent from the stratum corneum. In the areas of growth at the margin of the scutes of the shell, the labelling increased in precorneus layers. This suggests that histidine uptake is only related to shell growth and not to the production of a histidine-rich protein involved in keratinization. No filaggrin-like and loricrin-like immunoreactivity was seen in the carapace or plastron epidermis. However, in both proteins, some immunoreactivity was found in the transitional layer and in the lower level of the corneus layer of non-shelled areas. Loricrin- and filaggrin-like labelling was seen in small organelles (0.05-0.3 mum) among keratin bundles, identified with mucous-like granules and vesicular bodies. These organelles, present only in non-shelled epidermis, were more frequent along the border with the corneus layer, and labelling was low to absent in mature keratinocytes. This may be due to epitope masking or degradation. The immunolabelling for filaggrin was seen instead in the extracellular space among mature keratinocytes, over a material previously identified as mucus. The possibility that this labelling identified some epitopes derived from degraded portions of a filaggrin-like molecule is discussed. The present study suggests that proteins with some filaggrin- and loricrin-immunoreactivity are present in alpha-keratinocytes but not in beta-keratin cells of the shell.     

Formation of large micro‐ornamentations in developing scales of agamine lizards

The embryonic scales of two Australian agamine lizards (Hypsilurus spinipes and Physignatus lesueuerii) derive from the undulation of the epidermis to form dome‐shaped scale anlage that later become asymmetric and produce keratinized layers. Glycogen is contained in basal and suprabasal cells of the forming outer scale surface that are destined to differentiate into β‐keratin cells. The outer peridermis is very flat, but the second epidermal layer, provisionally identified as an inner peridermis, is composed of large cells that accumulate vesicular bodies and a network of coarse filaments. The sequence of epidermal layers produced beneath the inner peridermis in these agamine lizards corresponds to that of previously studied lizards, but the first subperidermal layer has characteristics of both clear (keratohyalin‐like granules) and oberhautchen (dark β‐keratin packets) cells. This layer is here identified as an oberhautchen since it fuses with the underlying β‐keratinizing cells forming large spinulae as the entire tissue becomes syncytial so that the units appear to increase in size. These spinulae very likely represent sections of honeycomb‐shaped micro‐ornamentations. A mesos layer appears underneath the β‐layer before hatching. J. Morphol. 240:251–266, 1999. © 1999 Wiley‐Liss, Inc.     

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.     

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.     

Observations on the histochemistry and ultrastructure of the epidermis of the tuatara,Sphenodon punctatus (Sphenodontida,Lepidosauria, Reptilia): a contribution to an understanding of the lepidosaurian epidermal generation and the evolutionary origin of the squamate shedding complex

Histochemical and TEM analysis of the epidermis of Sphenodon punctatus confirms previous histological studies showing that skin-shedding in this relic species involves the periodic production and loss of epidermal generations, as has been well documented in the related Squamata. The generations are basically similar to those that have been described in the latter, and their formation involves a cyclic alternation between beta- and alpha-keratogenesis. The six differences from the previously described squamate condition revealed by this study include: 1) the absence of a well-defined shedding complex; 2) the persistence of plasma membranes throughout the mature beta-layer, including the oberhautchen; 3) the concomitant presence of lipogenic lamellar bodies and PAS-positive mucous granules in most presumptive alpha-keratinizing cells; 4) the presence of the secreted contents of these organelles in the intercellular domains of the three derived tissues, the homologues of the squamate mesos, alpha-, and lacunar cells; 5) the paucity of lamellated lipid deposits in such domains; 6) the presence of keratohyalin-like granules (KHLG) in the presumptive lacunar, clear, and oberhautchen cells. In toto, the absence of many of the precisely definable, different pathways of cytogenesis discernible during squamate epidermal generation production might be interpreted as primitive for lepidosaurs. However, when the evolutionary significance of each of the six differences listed is evaluated separately, it becomes clear that the epidermis of S. punctatus possesses primitive amniote, shared and derived lepidosaurian, and some unique characters. This evaluation further elucidates the concept of a lepidosaurian epidermal generation as a derived manifestation of the sauropsid synapomorphy of vertical alternation of keratin synthesis and shows that further study of keratinocyte differentiation in the tuatara may contribute to our understanding of the origin and evolution of beta-keratinization in sauropsid amniotes.     

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

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

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.     

Morphogenesis of the digital pad lamellae in the embryo of the lizard Anolis lineatopus

The present study in the embryo of the lizard Anolis lineatopus describes the modality of cell proliferation responsible for the morphogenesis of the digital pad lamellae and of the epidermal stratification. After tritiated thymidine and 5-bromodeoxy-uridine administration, autoradiographic and immunocytochemical methods have been used. The lamellae originate as long, slightly slanted, undulations of the epidermis of fingers and toes. At an early stage, the epidermis consists of an outer periderm and a basal layer. Cell hypertrophy, and the prevalent cell proliferation in the longer side of the undulation with respect to the shorter side, generate the surface of the outer lamella. Under the peridermis, a shedding complex, composed by clear and oberhautchen layers, is formed and later determines the first intraepidermal shed. The first subperidermal layer derived from the basal layer is a clear layer and the first shed epidermis in the embryo is represented by periderm and clear layer. The heavily granulated clear layer in Anolis lineatopus represents the first epidermal layer produced in the embryonic epidermis, and is connected with the process of shedding. The spinulae of the underlying oberhautchen in the outer scale surface become long setae which grow toward the upper clear layer. Under the shedding complex a β-layer is produced. Autoradiographical study shows that the radioactivity stays in the basal layer for about 4 days before cells move to upper layers. At 6–8 days post-injection labelled cells are visible in the differentiated clear, oberhautchen and β-layers. Under the β-layer differentiating mesos cells are visible before the embryo hatches.     

Keratinization and lipogenesis in epidermal derivatives of the zebrafinch, Taeniopygia guttata castanotis (Aves, Passeriformes, Ploecidae) during embryonic development

Little is known of the lipid content of beta-keratin-producing cells such as those of feathers, scutate scales, and beak. The sequence of epidermal layers in some apteria and in interfollicular epidermis in the zebrafinch embryo (Taeniopygia guttata castanotis) was studied. Also, the production of beta-keratin in natal down feathers and beak was ultrastructurally analyzed in embryos from 3-4 to 17-18 days postdeposition, before hatching. Two layers of periderm initially cover the embryo, but there are eventually 6-8 over the epidermis of the beak. In the beak and sheath cells of feathers, peridermal granules are numerous at 12-14 days postdeposition but they are less frequent in apteria. These granules swell and disappear during sheath or peridermal degeneration at 15-17 days postdeposition. A thin beta-keratin layer forms under the periderm among feather germs of pterylous areas but is discontinuous or disappears in apteria. In differentiating cells of barbs, barbules, and calamus cells of natal down, electron-dense beta-keratin filaments form bundles oriented along the main axis of these cells. Cells of the pulp epidermis and collar, at the base of the follicle, contain lipids and bundles of alpha-keratin filaments. Degenerating pulp cells show vacuolization and nuclear pycnosis. During beta-keratin packing, keratin bundles turn electron-pale, perhaps due to the addition of lipids to produce the final, homogenous beta-keratin matrix. In contrast to the situation in feathers, in the cells of beak beta-keratin packets are irregularly oriented. In both feather and beak epidermal cells the Golgi apparatus and smooth endoplasmic reticulum produce vesicles containing lipid-like material which is also found among forming beta-keratin. The contribution of lipids or lipoprotein to the initial aggregation of beta-keratin molecules is discussed.