Reptilian Epidermis Research Articles

Widespread Growth Factor Expression in the Reptilian Epidermis Indicates Roles During Homeostasis and Wound Healing

The skin is the primary interface between an organism and its external environment. As such, it participates in the maintenance of homeostasis, as well as functions as a physical barrier, protecting against physical trauma, solar radiation, and pathogens. These functions are mediated, at least in part, by various growth factors, including members of the vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and transforming growth factor β (TGFβ) families. Although growth factors have long been documented in the dermis, particularly during wound healing, less is known about their expression in the overlying epidermis. Here we performed a spatio‐temporal investigation of growth factor expression within the epidermis of a representative reptile, the leopard gecko (Eublepharis macularius). Recent work has demonstrated that leopard gecko skin has an intrinsic capacity to heal scar‐free. Therefore, we investigated the pattern of growth factor expression before, during, and after scar‐free wound healing. We demonstrate that the gecko epidermis expresses a diverse panel of growth factor ligands and receptors, including VEGF, VEGF receptors 1 (VEGFR1) and 2 (VEGFR2), FGF‐2 and FGFR1, and TGFβ1 and activin βA. Curiously, only VEGFR1 and FGFR1 were dynamically expressed, and only during the earliest phases of re‐epithelization; otherwise all the proteins of interest were constitutively present. Using double‐immunofluorescence, we observed co‐localization and/or overlapping expression patterns of VEGF/VEGFR2/VEGFR1 and FGF‐2/FGFR1. These findings indicate that homeostatic and injury‐mediated functions of the epidermis may involve both paracrine and autocrine signalling. Furthermore, they provide compelling evidence that keratinocytes expressing these growth factors may participate in non‐angiogenic roles during homeostasis. We propose that constitutive growth factor expression by keratinocytes is associated with functions specific to reptilian biology, including body‐wide skin shedding, protection against ultraviolet photodamage, and photobiosynthesis of vitamin D.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants 400358 (to MV) and 401013 (to JP), and Ontario Veterinary College Scholarship (to NS)

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

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

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

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

Immunoreactivity to the pre-core box antibody shows that most glycine-rich beta-proteins accumulate in lepidosaurian beta-layer and in the corneous layer of crocodilian and turtle epidermis

The differentiation of the corneous layers of reptilian epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against the conserved pre-core box region of their keratin-associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins) and silver-intensification. The epitope analysis in the sequences of different reptilian KAbetaPs indicates that this antibody recognizes mainly glycine-rich beta-proteins in lizards and snakes. The immunoreactivity of the beta-layer of the tuatara to this antibody also suggests that a similar epitope is present in beta-proteins of this relict species. In crocodilians the antibody recognizes glycine-rich beta-proteins, so far representing all the known crocodilian KAbetaPs. In hard-shelled turtle the antibody labels mainly type 1 KAbetaPs that represent most types found in this turtle. The antibody does not label the corneous layer of the soft-shelled turtle that contains exclusively type 2 KAbetaPs, with a low identity to the epitope recognized by the antibody. The prevalent labeling of the beta-layers in lepidosaurian epidermis and of the corneous layer in turtle and crocodilian epidermis suggest that this antibody is mainly directed toward KAbetaPs rich in glycine. The latter are main constituents of the corneous layer in turtles and crocodilians and of the beta-layer in lizards, snakes and the tuatara. These proteins are largely responsible for the inflexibility, mechanical resistance, chromophobicity and relative hydrophobicity of the reptilian corneous layer.

Ultrastructural immunocytochemistry for the central region of keratin associated-beta-proteins (beta-keratins) shows the epitope is constantly expressed in reptilian epidermis

The presence of beta-proteins containing a core-box region in specific regions of reptilian epidermis has been studied by immunological methods. Alpha-keratins are detected by the antibody AK2 that recognizes a sequence toward the C-terminal of acidic alpha-keratins of 48–52kDa. Beta-proteins are recognized by an antibody directed to the core-box region specific for these proteins of 18–37kDa. The AK2 antibody labels with variable intensity alpha-keratin bundles in basal and suprabasal keratinocytes in the epidermis of representative species of reptiles but immunolabeling decreases or disappears in pre-corneous and corneous cells. As opposite, the core-box antibody only labels with variable intensity the dense beta-corneous material formed in pre-corneous and corneous layers of crocodilian and turtle epidermis. In lepidosaurian epidermis the core-box antibody labels the beta-layer while the mesos and alpha-layers are poorly or not labeled. The immunological evidence indicates that beta-proteins are synthesized in the upper spinosus and pre-corneous layers of the epidermis and replace or mask the initial alpha-keratin framework present in keratinocytes as they differentiate into cells of the beta-layer. In the specialized pad lamellae of gecko and anoline lizards charged beta-proteins accumulate in the adhesive setae and may affect the mechanism of adhesion that allows these lizards to walk vertical surfaces. The addition of beta-proteins to the alpha-keratins in upper cell layers of the epidermis recalls the process of cornification of mammalian epidermis where specific keratin-associated proteins (involucrin, loricrin and filaggrin) associate with the keratin framework in terminally differentiating keratinocytes of the stratum corneum.

Immunocytochemistry indicates that glycine-rich beta-proteins are present in the beta-layer, while cysteine-rich beta-proteins are present in beta- and alpha-layers of snake epidermis

Immunolocalization of glycine-rich and cysteine–glycine-medium-rich beta-proteins (Beta-keratins) in snake epidermis indicates a different distribution between beta- and alpha-layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta-keratin layers alternated with softer and pliable alpha-keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta-proteins (formerly beta-keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine-rich beta-proteins of 12–15 kDa, is poorly or not reactive with the beta-layer of snake epidermis. This suggests that glycine-rich proteins similar to those present in lizards are altered during maturation of the beta-layer. Conversely, a glycine–cysteine-medium-rich beta-protein (HgGC10) of 10–12 kDa is present in beta- and alpha-layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta- and alpha-cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta-proteins rich in glycine mainly accumulate on a scaffold of alpha-keratin producing a resistant and hydrophobic beta-layer. Conversely, beta-proteins lower in glycine but higher in cysteine accumulate on alpha-keratin filaments present in beta- and alpha-layers producing resistant but more pliable layers.

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha-keratins and keratin-associated beta-proteins (formerly beta-keratins) has allowed the production of epitope-specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha-keratin range (40-70 kDa) or beta-protein range (10-30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha-proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin-associated beta-proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin-associated beta-proteins is typical for keratin-associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine-rich or sulfur-rich proteins). In turtles and crocodilians epidermis, keratin-associated beta-proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine-rich versus cysteine-glycine-rich keratin-associated beta-proteins in cells sequentially produced from the basal layer and not from the alternation of beta- with alpha-keratins. The process gives rise to Oberhäutchen, beta-, mesos-, and alpha-layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin-associated proteins (KAPs) of the epidermis, the keratin-associated beta-proteins of sauropsids are capable to form filaments of 3-4 nm which give rise to an X-ray beta-pattern as a consequence of the presence of a beta-pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates.

Hard cornification in reptilian epidermis in comparison to cornification in mammalian epidermis

The structure of reptilian hard (beta)-keratins, their nucleotide and amino acid sequence, and the organization of their genes are presented. These 13-19 kDa proteins are basic, rich in glycine, proline and serine, and different from cytokeratins. Their mRNAs are expressed in beta-cells. The central part of beta-keratins (this region has been previously termed 'core-box' and is peculiar of all sauropsid proteins) is composed of two beta-folded regions and shows a high identity with avian beta-keratins. This central part present in all beta-keratins, including feather keratins, is the site of polymerization to build the framework of beta-keratin filaments. Beta-keratins appear cytokeratin-associated proteins. Their central region might have originated in an ancestral glycine-rich protein present in stem reptiles from which beta-keratins evolved and diversified into reptiles and birds. Stem reptiles of the Carboniferous period might have possessed glycine-rich proteins derived from exons/domains corresponding to the variable, glycine-rich region of cytokeratins. Beta-keratins might have derived from a gene coding for small glycine-rich keratin-associated proteins. The glycine-rich regions evolved differently in the lineage leading to modern reptiles and birds versus that leading to mammals. In the reptilian lineage some amino acid regions produced by point mutations and amino acid changes might have given rise to originate the central beta-pleated region. The latter allowed the formation of filamentous proteins (beta-keratins) associated with intermediate filament keratins and replaced them in beta-keratin cells. In the mammalian lineage no beta-pleated region was generated in their matrix proteins, the glycine-rich keratin-associated proteins. The latter evolved as glycine-tyrosine-rich, sulphur-rich, and ultra-sulphur-rich proteins that are used for building hairs, horns and nails.

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.

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 langerhans cells in the terrestrial turtle, Kinosternum integrum

In mammalian epidermis, Langerhans cells (LC) are the only antigen-presenting dendritic cells that possess the ectoenzyme adenosine triphosphase (ATPase) and constitutively express class II molecules encoded by the major histocompatibility complex. Recently, we demonstrated the presence of LC in chicken epidermis. The aim of the present study is to demonstrate the presence of LC-like cells in turtle Kinosternum integrum, epidermis by light and ultrastructural ATPase histochemistry. ATPase-positive dendritic cells were observed in epidermal sheets whose maximum mean number was 192 cells/mm 2. Electron microscopy for ATPase stained sections showed an electrondense precipitate in the plasma membrane of dendritic clear cells located among basal and suprabasal keratinocytes, ultrastructurally similar to LC. In serial sections, some dendritic cells showed LC (Birbeck) granules. The present study demonstrates for the first time ATPase-positive dendritic cells, morphologically similar to LC, in reptilian epidermis.

Keratin diversity in the reptilian epidermis

AbstractThe ϕ keratins extracted from individual turtles and snakes were compared. The extent and nature of the molecular differentiation associated with the production of structurally distinct epidermal tissues was determined. The electrophoretic comparisons, molecular weight, and chemical fractionation indicate that these tissues contain unique proportions of the constituent keratin monomers specific to each species. This pattern of differentiation is similar to that previously observed for avian scale, claw, and beak, and for mammalian horn and hoof. This suggests that several “scale‐like” structures with distinctive chemical properties may be produced by a single individual without the synthesis of wholly unique proteins. The implications of these observations for the evolution of mammalian hair and avian feathers are discussed.

Lamellar granules in mammalian, avian, and reptilian epidermis

Evidence is presented that mammalian membrane-coating granules, avian multigranular bodies, and reptilian mesos granules are homologous structures. All three types which will here be called lamellar granules have the following items in common: (1) A lamellar pattern produced by disks stacked coin-like above each other. (2) Major dense lines separating adjacent disks, each of which contains a minor dense line. (3) Spacing between and thickness of major dense lines. (4) Positive reaction with various lipid stains, PAS, and the Thiery silver method. (5) Disappearance of the lamellar pattern after treatment with lipid solvents or phospholipase C. (6) Extrusion of contents into the intercellular space and its rearrangement into broad sheets running parallel to the surface of the horny cells. Based on these observations and biochemical results (Elias et al., 1979, J. Invest. Dermatol. 73, 339–348) the lamellar contents (disks) are interpreted as “flattened” liposomes.

Formation of cutaneous appendages in dermo-epidermal recombinations between reptiles, birds and mammals.

1. Previous experiments on dermo-epidermal recombinations between birds and mammals have shown that the class-specific quality of the cutaneous appendages depends on intrinsic properties of the epidermis but that several steps of their morphogenesis are controlled by the dermis. This morphogenetic interplay has been tested further in new experiments with reptilian skin. 2. Reconstituted homo- and heterospecific skin explants, involving epidermis and dermis of lizard, chick and mouse, were cultured for 8 days on the chorioallantoic membrane of the chick embryo. 3. Homospecific recombinations of dorsal, caudal or ventral lizard epidermis and dorsal lizard dermis gave rise to small dorsal-type scales. Recombinants of dorsal, caudal or ventral lizard epidermis and ventral lizard dermis gave rise to large ventral-type scales. 4. Heterospecific recombinations of dorsal, caudal or ventral lizard epidermis and chick dermis from the glabrous comb region did not differentiate any scale structures. 5. Heterospecific recombinations of dorsal or caudal lizard epidermis and tarsometatarsal chick dermis formed large chick-type scales. 6. Heterospecific recombinations of dorsal, caudal or ventral lizard epidermis and chick feather-forming, or mouse hair-forming or whisker-forming dermis gave rise to tubercular scale primordia. The diameter and distribution of these primordia were in conformity with the feather, pelage hair and vibrissal patterns respectively. 7. Heterospecific association of lizard dermis and chick or mouse epidermis led to the formation of few epidermal placode-like pegs; those differentiated by the mouse epidermis were interpreted as hair bud structures. 8. The differentiation of reptilian scales is the result of dermo-epidermal interactions. Reptilian epidermis, when confronted with either reptilian, avian, or mammalian dermis, always responds to the dermal messages by forming scale buds. For final scale morphogenesis, however, reptilian dermis or avian scale-forming dermis is required. Reptilian dermis appears to be unable to induce extensive appendage formation in avian or mammalian epidermis. 9. A remarkable similarity exists in the mechanisms of skin differentiation in the three classes of amniotes. Indeed scales, feathers and hairs require two kinds of dermal messages for their complete morphogenesis: early ones, which can be transmitted from one class to another, and which are responsible for the initiation, site, size and distribution pattern of appendage primordia, whose class-specific quality (scale, feather or hair buds) is determined by the epidermis; and later specific ones which can only be understood within the class and which are necessary for the completion of the specific architecture of the cutaneous appendage.

The structural protein of reptilian scales.

AbstractPrevious studies of reptilian epidermis have shown that scales contain both a and feather fibrous proteins. The outer stratum (feather) and inner stratum (a) of snake (Boa constrictor) scales were separated and the tissue extracted with 6 M urea in 0.1 M Tris, pH 9.0, with 0.1 M mercaptoethanol. Following alkylation to the S‐carboxymethyl derivatives (SCM), the proteins were separated on the basis of their solubility at pH 4.5. X‐ray diffraction analysis indicated that pH 4.5 insoluble proteins represented the fibrous proteins while the soluble ones had the characteristics of matrix material. Disc electrophoretic patterns and amino acid analyses of the two types of fibrous proteins were different while those of the matrix proteins from both strata appeared to be very similar. The epidermal proteins of shell (feather) and extremity (a) skin from turtle (Pseudemys) were similarly isolated and shown to have different amino acid compositions. Although separation of the various components by differences in their solubility at pH 4.5 was not possible, a comparison of electrophoretic patterns suggested that the proteins extracted from the epidermis of the shell and extremity skin had different fibrous but similar matrix proteins. These studies indicated a strong similarity between reptilian scales and hair, where fibrous and matrix proteins are present and are separable on the basis of the solubility of their SCM derivatives. Although the a and feather fibrous proteins form filaments of very different size, it appears that they are associated with similar matrix proteins.

Keratinization of the reptilian epidermis: An ultrastructural study of the turtle skin

The turtle epidermis was studied in an effort to obtain information about differentiation products and sequential events leading to the formation of the horny layer. Reproductive basal cells contain many ribosomes, and both granular and agranular endoplasmic reticulum. Filaments are relatively few; lipid droplets and glycogen particles appear in a moderate amount. Differentiating cells are filled with synthetic organelles, lipid droplets, mucous granules, and vesicular bodies. Mucous granules reveal a variable internal structure, and they are displaced toward the cell periphery. Ultimately, mucus is discharged into the intercellular space. Vesicular bodies contain several small vesicles and a peculiar disklike component. When the cells enter the transformation stage, the plasma membrane thickens and the synthetic organelles disintegrate. The horny cells are filled with loosely packed filaments, a relatively translucent matrix, and lipid. The keratinization process is characterized by comparing these features to those seen in amphibian, avian, and mammalian epidermis.

An autoradiographic study of the sites of protein synthesis in the epidermis of the Indigo snake (Drymarchon corais couperi).

AbstractIn order to delineate more clearly the sites of protein synthesis during the synchronous proliferation of reptilian epidermis, H3‐leucine was injected intradermally into the Indigo snake (Drymarchon corais couperi) at various times in the sloughing cycle. Autoradiograms were made of biopsies, taken during the early and late proliferative stages of the molting cycle. These confirmed morphologic observations (Roth and Jones, '66) that moderate numbers of spinous cells are added to the α‐layer during the resting phase. The number of cells added, and the amount of protein synthesized, reaches a maximum in the midresting phase and then decreases. No synthesis of upper α‐layer, μϵσºζ‐layer or β‐layer proteins occurs during the resting phase. Synthesis of the specialized proteins of the β‐layer, the μϵσºζ‐layer and the upper stratum of the α‐layer occurs during the proliferative phase, after the cells have left the basal layer, (while they represent inner epidermal generation), in contrast to mammalian epidermis in which the fibrous proteins are largely synthesized in the basal layer and the lower‐most spinous cells.