Keratohyalin-like granules in lizard epidermis: evidence from cytochemical, autoradiographic, and microanalytic studies

Epidermal sloughing in lizards is determined by the formation of an intraepithelial shedding complex in which keratohyalin-like granules are formed. The chemical nature of these granules is unknown, as is their role in keratinization. The goal of this study was to test whether they contain some amino acids similar to those found in mammalian keratohyalin. The embryonic and regenerating epidermis of lizards are useful systems to study the formation of these granules. Histochemically keratohyalin-like granules react to histidine and contain some sulfhydryl groups (cysteine). X-ray microanalysis shows that these granules contain sulfur and often phosphorus, two elements also present in the mature clear, oberhautchen, and beta layer. Instead the mesos, alpha, and lacunar layers contain only sulfur. Most sulfur is probably in a disulfide-bonded form, particularly in mature cells of the shedding complex, in large keratohyalin-like granules, and in the beta-keratin layer. Early differentiating beta-keratin cells have the maximal incorporation of tritiated proline, whereas tritiated arginine is slightly more concentrated in the basal layer of the epidermis. A high uptake of tritiated histidine is observed mainly in keratohyalin-like granules of the clear layer, but also in the oberhautchen layer and forming the alpha-lacunar layer. Immunogold electron microscopy shows that keratohyalin-like granules do not localize keratin but are embedded within a keratin network. These results suggest that keratohyalin-like granules of lizards, like mammalian keratohyalin, contain some sulfur-rich and histidine-rich proteins. These granules participate in the process of hardening of the clear layer that molds the spinulae of the deeper oberhautchen to form the superficial microornamentation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Formation of the corneous layer in the epidermis of the tuatara (Sphenodon punctatus, Sphenodontida, Lepidosauria, Reptilia)

The formation of the stratum corneum in the epidermis of the reptile Sphenodon punctatus has been studied by histochemical, immunohistochemical, and ultrastructural methods. Sulfhydryl groups are present in the mesos and pre-alpha-layer but disappear in the keratinized beta-layer and in most of the mature alpha-layer. This suggests a complete cross-linking of keratin filaments. Tyrosine increases in keratinized layers, especially in the beta-layer. Arginine is present in living epidermal layers, in the presumptive alpha-layer, but decreases in keratinized layers. Histidine is present in corneous layers, especially in the intermediate region between the alpha- and a new beta-layer, but disappears in living layers. It is unknown whether histidine-rich proteins are produced in the intermediate region. Small keratohyalin-like granules are incorporated in the intermediate region. The plane of shedding, as confirmed from the study on molts, is located along the basalmost part of the alpha-layer and may involve the degradation of whole cells or cell junctions of the intermediate region. A specific shedding complex, like that of lizards and snakes, is not formed in tuatara epidermis. AE1-, AE2-, or AE3-positive alpha-keratins are present in different epidermal layers with a pattern similar to that previously described in reptiles. The AE1 antibody stains the basal and, less intensely, the first suprabasal layers. Pre-keratinized, alpha- and beta-layers, and the intermediate region remain unlabeled. The AE2 antibody stains suprabasal and forming alpha- and beta-layers, but does not stain the basal and suprabasal layers. In the mature beta-layer the immunostaining disappears. The AE3 antibody stains all epidermal layers but disappears in alpha- and beta-layers. Immunolocalization for chick scale beta-keratins labels the forming and mature beta-layer, but disappears in the mesos and alpha-layer. This suggests the presence of common epitopes in avian and reptilian beta-keratins. Low molecular weight alpha-keratins present in the basal layer are probably replaced by keratins of higher molecular weight in keratinizing layers (AE2-positive). This keratin pattern was probably established since the beginning of land adaptation in amniotes.     

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.     

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.     

Scanning electron microscopy of changes in epidermal structure occurring during the shedding cycle in squamate reptiles

Previous studies of squamate epidermal structure have focused on either histology and ultrastructure or oberhautchen surface texture as revealed by scanning electron microscopy (SEM). Using SEM data drawn from a variety of lizard taxa (primarily iguanids, but also agamids, chamaeleonids, and scincids), as well as amphisbaenians and colubrid snakes, we relate the surfaces encountered in gross dissection of squamate skin to histologically identifiable layers, and characterize their surface structure. Only the oberhautchen bears the repeating pattern of ornamentation noted by previous authors. Because the clear layer is a perfect template of the oberhautchen surface, it is the only layer with which the oberhautchen might be confused. However, the clear layer can be identified by its tendency to curl and crack during preparation. All other surfaces encountered were relatively featureless, except for impressions left by dermal “papillae” associated with mechanoreceptors. Using a method for examining preserved specimens to determine the stage in the shedding cycle, we assess two sources of variation in epidermal surface structure: stage in the shedding cycle and wear. Examination of immature renewal-phase epidermis suggests that the oberhautchen does not mature synchronously across a single scale or across body regions. Comparing inner- and outer-generation oberhautchen in sheddingphase epidermis, we conclude that changes in surface appearance caused by natural wear fall into two categories: discrete scratches and accumulation of debris. We see no evidence of overall “buffing” on a microscopic level, though surface structure may be obscured by scratches and gouges. Many squamate taxa show a gradient from low relief surface structure on elevated regions such as keels to high relief patterns at scale edges. This gradient is not due to wear; its significance is unknown.     

Immunogold labeling shows that glycine‐cysteine‐rich beta‐proteins are deposited in the Oberhäutchen layer of snake epidermis in preparation to shedding

Shedding in snakes is cyclical and derives from the differentiation of an intraepidermal shedding complex made of two different layers, termed clear and Oberhäutchen that determine the separation between the outer from the inner epidermal generation that produces a molt. The present comparative immunocytochemical study on the epidermis and molts of different species of snakes shows that a glycine‐cysteine‐rich corneous beta‐protein in a snake is prevalently accumulated in cells of the Oberhäutchen layer and decreases in those of the beta‐layer. The protein is variably distributed in the mature beta‐layer of species representing some snake families when the beta‐layer merges with the Oberhäutchen but disappears in alpha‐layers. Therefore, this protein represents an early marker of the transition between the outer and the inner epidermal generations in the epidermis of snakes in general. It is hypothesized that specific gene activation for glycine‐cysteine‐rich corneous beta‐proteins occurs during the passage from the clear layer of the outer epidermal generation to the Oberhäutchen layer of the replacing inner epidermal generation. It is suggested that in the epidermis of most species glycine‐cysteine‐rich corneous beta‐proteins form part of the dense corneous material that rapidly accumulates in the differentiating Oberhäutchen cells but decreases in the following beta‐layer of the inner epidermal generation destined to be separated from the previous outer generation in the process of shedding. The regulation of the synthesis of these and other proteins is, therefore, crucial in timing the different stages of the shedding cycle in lepidosaurian reptiles. J. Morphol. 276:144–151, 2015. © 2014 Wiley Periodicals, Inc.     

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.     

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.     

Cell adhesion and junctional proteins in the developing skin of snakes indicate they coordinate the differentiation of the epidermis

The development of scales and the sequence of epidermal layers during snake embryogenesis has been studied by immunofluorescence for the localization of cell adhesion, adherens, and communicating cell junctional proteins. At about 2nd/3rd of embryonic development in snakes the epidermis forms symmetric bumps at the beginning of scale formation, and they rapidly become asymmetric and elongate forming outer and inner surfaces of the very overlapped scales seen at hatching. The dermis separates a superficial loose from a deeper dense part; the latter is joined to segmental muscles and nerves, likely acting on scale orientation during snake movements. N-cam is present in the differentiating epidermis and mesenchyme of forming scales while L-cam is only/mainly detected in the periderm and epidermis. Mesenchymal N-cam is associated with the epidermis of the elongating dorsal scale surface and with the beta-differentiation that occurs in the overlapping outer surface of scales. Beta-catenin and Connexin-43 show a similar distribution, and they are mainly present in the periderm and differentiating suprabasal keratinocytes likely forming an intense connectivity during epidermal differentiation. Beta-catenin also shows nuclear localization in differentiating cells of the shedding and beta-layers at late stages of scale morphogenesis, before hatching. The study suggests that intensification of adhesion and gap junctions allows synchronization of the differentiation of suprabasal cells to produce the ordered sequence of epidermal layers of snake scales, starting from the shedding complex and the beta-layer.

     

Keratinization in the epidermis of amphibians and the lungfish: comparison with amniote keratinization

Keratinization in the epidermis of amphibians and the lungfish has been studied by electron microscopy, autoradiography and immunocytochemistry to determine whether histidine-rich proteins, filaggrin and loricrin are present. In the lungfish and amphibian tadpoles, anti-keratin antibodies (AE1 and AE3) stain the whole epidermis but not the AE2 antibody, a marker for keratinization. In adult epidermis, the AE2 antibody mainly stains keratinized layers, AE1 mainly stained basal cells, less suprabasal cells and no pre-keratinized and keratinized layers, and AE3 stains all epidermal layers. This staining pattern resembles that of amniote epidermis. Little tritiated histidine is taken up in toad epidermis at 4-6 h post-injection but 24 h after injection the radioactivity is most concentrated in the replacement layer beneath the corneus. This indicates that protein synthesis takes place in the epidermis but, due to the metabolic conversion that takes place in 24 h, it is unlikely that histidine-rich proteins are formed. Neither filaggrin-like nor loricrine-like immunoreactivities are present in amphibian and lungfish epidermis. This indicates absence of histidine-rich matrix proteins and corneous cell envelope proteins and only mucus is present among keratin filaments. Filaggrine-like and loricrin-like proteins are characteristic of amniotes epidermis and might have originated in basic amniotes (cotylosaurs).     

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.     

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.     

Presence of a glycine-cysteine-rich beta-protein in the oberhautchen layer of snake epidermis marks the formation of the shedding layer

The complex differentiation of snake epidermis largely depends on the variation in the production of glycine-cysteine-rich versus glycine-rich beta-proteins (beta-keratins) that are deposited on a framework of alpha-keratins. The knowledge of the amino acid sequences of beta-proteins in the snake Pantherophis guttatus has allowed the localization of a glycine-cysteine-rich beta-protein in the spinulated oberhautchen layer of the differentiating shedding complex before molting takes place. This protein decreases in the beta-layer and disappears in mesos and alpha-layers. Conversely, while the mRNA for a glycine-rich beta-protein is highly expressed in differentiating beta-cells, the immunolocalization for this protein is low in these cells. This discrepancy between expression and localization suggests that the epitope in glycine-rich beta-proteins is cleaved or modified by posttranslational processes that take place during the differentiation and maturation of the beta-layer. The present study suggests that among the numerous beta-proteins coded in the snake genome to produce epidermal layers with different textures, the glycine-cysteine-rich beta-protein marks the shedding complex formed between alpha- and beta-layers that allows for molting while its disappearance between the beta- and alpha-layers (mesos region for scale growth) is connected to the formation of the alpha-layers.