Immunocytochemical localization of cysteine‐rich beta‐proteins in the extensible epidermis of the dewlap in the lizard Anolis carolinensis

The dewlap in the lizard Anolis carolinensis is made of scales separated by large interscale regions capable of broad stretching during fan extension. This indicates that the skin contains proteins that allow extension of interscale regions. The immunocytochemical analysis of the epidermis indicates that HgG5, a glycine‐rich hydrophobic beta‐protein poor in cysteine is localized only in the stiff beta‐layer of the outer scale surface, but is completely absent in mesos and alpha‐layers and in hinge regions. HgGC10, a cysteine‐medium‐rich beta‐protein is present in beta‐layers but especially in alpha‐layers of interscale epidermis that presents folds and lacks a beta‐layer. HgGC3 is weakly localized in the alpha‐layer, but is mainly found in hinge regions. HgGC8 and HgG13 are low to absent in the alpha‐ and beta‐layer. The immunolocalization of cysteine‐rich beta‐proteins such as HgGC10/3 in alpha‐layers and interscale epidermis suggests that these small proteins are involved in the formation of a corneous material compatible with dewlap extension. The basement membrane underneath scales is joined to bundles of collagen fibrils in the dermis through anchoring fibrils that likely determine flattening of the epidermis during the extension of the throat fan.     

Microscopic analysis of lizard claw morphogenesis and hypothesis on its evolution

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

Ultrastructural studies of lizard (Anolis carolinensis) myogenesis in vitro

In vitro differentiation of lizard (Anolis carolinensis) skeletal muscle cells was studied by electron microscopy. Myogenesis was studied under conditions in which large numbers of postmitotic prefusion myoblasts accumulate (Growth Medium) and under conditions which are permissive for myotube formation (Fusion Medium). In Growth Medium, myogenic cells proliferate, then assume a characteristic spherical morphology which permits definitive identification of prefusion myoblasts. During the early stages of culture, these round myoblasts resemble myoblasts described in other systems; ultrastructural similarities and differences are discussed. After longer periods of culture in Growth Medium, a continuum of differentiation from isolated myofilaments to assembled myofibrils was seen in these mononucleated cells. These observations confirm the dissociability of contractile protein assembly and myoblast fusion Cultures maintained in Fusion Medium or transferred from Growth Medium to Fusion Medium form multinucleated myotubes on a predictable time scale. Myogenesis was followed in these cultures with particular reference to the early events in myofilament assembly and myofibril formation.     

Immunolocalization of specific beta‐proteins in pad lamellae of the digits in the lizard Anolis carolinensis suggests that cysteine‐rich beta‐proteins provides flexibility

Knowledge of beta‐protein (beta‐keratin) sequences in Anolis carolinensis facilitates the localization of specific sites in the skin of this lizard. The epidermal distribution of two new beta‐proteins (beta‐keratins), HgGC8 and HgG13, has been analyzed by Western blotting, light and ultrastructural immunocytochemistry. HgGC8 includes 16 kDa members of the glycine‐cysteine medium‐rich subfamily and is mainly expressed in the beta‐layer of adhesive setae but not in the setae. HgGC8 is absent in other epidermal layers of the setae and is weakly expressed in the beta‐layer of other scales. HgG13 comprises members of 17‐kDa glycine‐rich proteins and is absent in the setae, diffusely distributed in the beta layer of digital scales and barely present in the beta‐layer of other scales. It appears that the specialized glycine‐cysteine medium rich beta‐proteins such as HgGC8 in the beta‐layer, and of HgGC10 and HgGC3 in both alpha‐ and beta‐layers, are key proteins in the formation of the flexible epidermal layers involved in the function of these modified scales in adaptation to contact and adhesion on surfaces. J. Morphol. 275:504–513, 2014. © 2013 Wiley Periodicals, Inc.     

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.     

Immunolocalization of large corneous beta‐proteins in the green anole lizard (Anolis carolinensis) suggests that they form filaments that associate to the smaller beta‐proteins in the beta‐layer of the epidermis

The distribution of large corneous beta‐proteins of 18–43 kDa (Ac37, 39, and 40) in the epidermis of the lizard Anolis carolinensis is unknown. This study analyses the localization of these beta‐proteins in different body scales during regeneration. Western blot analysis indicates most protein bands at 40–50 kDa suggesting they mix with alpha‐keratin of intermediate filament keratin proteins. Ac37 is present in mature alpha‐layers of most scales and in beta‐cells of the outer scale surface in some scales but is absent in the Oberhäutchen, in the setae and beta‐layer of adhesive pads and in mesos cells. In differentiating beta‐keratinocytes Ac37 is present over 3–4 nm thick filaments located around the amorphous beta‐packets and in alpha‐cells, but is scarce in precorneous and corneous layers of the claw. Ac37 forms long filaments and, therefore, resembles alpha‐keratins to which it probably associates. Ac39 is seen in the beta‐layer of tail and digital scales, in beta‐cells of regenerating scales but not in the Oberhäutchen (and adhesive setae) or in beta‐ and alpha‐layers of the other scales. Ac40 is present in the mature beta‐layer of most scales and dewlap, in differentiating beta‐cells of regenerating scales, but is absent in all the other epidermal layers. The large beta‐proteins are accumulated among forming beta‐packets of beta‐cells and are packed in the beta‐corneous material of mature beta‐layer. Together alpha‐keratins, large beta‐proteins form the denser areas of mature beta‐layer that may have a different consistence that the electron‐paler areas. J. Morphol. 276:1244–1257, 2015. © 2015 Wiley Periodicals, Inc.     

Estrogen localization in the brain of the lizard, Anolis carolinensis (1)

Estrogen induces reproductive behavior in lizards by acting on specific areas of the brain. Neural areas that selectively concentrate radioactivity after 3-h-estradiol administration were mapped using thawmount autoradiography. Major accumulations of hormone-concentrating cells occur in the medial preoptic area, basal hypothalamus, amygdala (n. vetromedialis telencephali) and in and around the torus semicircularis. Numerous smaller accumulations are also described and the topographical distribution is compared with that of avian and mammalian species.     

Structure and site of expression of a murine type II hair keratin

We present here a 1770 bp-long cDNA which encodes a murine type II keratin. Sequence comparisons of the keratin with those of various type II keratins expressed in mouse epidermis and internal stratified epithelia reveal that the new keratin is unrelated to epithelial keratins. Rather the structural organization of its amino- and carboxyterminal domains and the high content of cysteine and proline residues in these regions suggest that the keratin represents a murine type II hair keratin. This assumption was confirmed by in situ hybridization which localized the mRNA of the keratin in upper cells of the hair cortex and in suprabasal cells of the central core unit of filiform papillae of the tongue. Hybrid selection analyses revealed that the keratin has a molecular weight of 58 kD. It remains to be seen whether the keratin corresponds to MHb 3 or MHb 4.     

Ultrastructural changes in skeletal muscle of the tail of the lizard Hemidactylus mabouia immediately following autotomy

Araújo, T.H., Faria, F.P., Katchburian, E. and Freymüller, E. (2009). Ultrastructural changes in skeletal muscle of the tail of the lizard Hemidactylus mabouia immediately following autotomy. —Acta Zoologica (Stockholm) 91 : 440–446. Although autotomy and subsequent regeneration of lizard tails has been extensively studied, there is little information available on ultrastructural changes that occur to the muscle fibers at the site of severance. Thus, in the present study, we examine the ultrastructure of the musculature of the remaining tail stump of the lizard Hemidactylus mabouia immediately after autotomy. Our results show that exposed portions of the skeletal muscle fibers of the stump that are unprotected by connective tissue bulge to produce large mushroom‐like protrusions. These exposed portions show abnormal structure but suffer no leakage of cytoplasmic contents. Many small and large vesicular structures appeared between myofibrils in the interface at this disarranged region (distal) and the other portion of the fibers that remain unchanged (proximal). These vesicles coalesce, creating a gap that leads to the release of the mushroom‐like protrusion. So, our results showed that after the macroscopic act of autotomy the muscular fibers release part of the sarcoplasm as if a second and microscopic set of autotomic events takes place immediately following the macroscopic act of autotomy. Presumably these changes pave the way for the formation of a blastema and the beginning of regeneration.     

Glucosylceramide in the androgen-responsive kidney of the lizard Anolis carolinensis

During the spring breeding season of the American chameleon, Anolis carolinensis, elevated levels of glucosylceramides which contain hydroxy fatty acids are produced in the kidneys of males but not females. Hyperproduction of this glycolipid is also induced by testosterone. The testosterone-induced hypertrophy of epithelial cells in the proximal tubules of the mouse kidney seems an analogous phenomenon and an elevated concentration of specific glycolipids in the male mouse kidney has been previously demonstrated. Thus the formation of renal glycolipids in response to testosterone may be a widespread feature in vertebrates.     

Opsin localization and chromophore retinoids identified within the basal brain of the lizard Anolis carolinensis

Since the beginning of this century evidence has accumulated which demonstrates that non-mammalian vertebrates possess photoreceptors situated deep within the brain. While many attempts have been made to localize these sensory cells, studies have either failed or been inconclusive. In this report we have used several experimental approaches to localize the deep brain photoreceptors of the lizard Anolis carolinensis. Using 3 antibodies that bind vertebrate cone opsins, we have immunolabelled cerebrospinal fluid (CSF)-contacting neurons located at the ventricular border within the nucleus ventromedialis of the septum. Western blot analysis indicates that these antibodies recognized a single 40 kD protein in ocular, anterior brain, and pineal extracts. Immunoblots of rodent brain did not show a similar protein band. We have also identified specific retinoids associated with phototransduction (11-cis and all-trans-3,4-didehydroretinaldehyde) within anterior brain extracts. This combined data provides the most detailed analysis of deep brain photoreceptors in any vertebrate. Consequently, we feel Anolis provides an excellent model to study this unexplored sensory system of the vertebrates.Abbreviations CSF contacting neurons cerebrospinal fluid-contacting neurons - HPLC high performance liquid chromatography - L:D light:dark cycle     

Chromatophores and color change in the lizard,Anolis carolinensis

Summary The skin of the lizard, Anolis carolinensis, changes rapidly from bright green to a dark brown color in response to melanophore stimulating hormone (MSH). Chromatophores responsible for color changes of the skin are xanthophores which lie just beneath the basal lamina containing pterinosomes and carotenoid vesicles. Iridophores lying immediately below the xanthophores contain regularly arranged rows of reflecting platelets. Melanophores containing melanosomes are present immediately below the iridophores. The ultrastructural features of these chromatophores and their pigmentary organelles are described. The color of Anolis skin is determined by the position of the melanosomes within the melanophores which is regulated by MSH and other hormones such as norepinephrine. Skins are green when melanosomes are located in a perinuclear position within melanophores. In response to MSH, they migrate into the terminal processes of the melanophores which overlie the xanthophores above, thus effectively preventing light penetration to the iridophores below, resulting in skins becoming brown. The structural and functional characteristics of Anolis chromatophores are compared to the dermal chromatophore unit of the frog.This study was supported in part by GB-8347 from the National Science Foundation.Contribution No. 244, Department of Biology, Wayne State University.The authors are indebted to Dr. Joseph T. Bagnara for his encouragement during the study and to Dr. Wayne Ferris for his advice and the use of his electron microscope laboratory.     

Thermoregulatory function of the parietal eye in the lizard Anolis carolinensis

Summary Animals were acclimitized to a daily cycle of 15–25°C and a photoperiod of LD 12:12. Parietalectomized animals in an experimental thermal gradient (15°C–40°C, LD 12:12) selected significantly higher temperatures than controls at all hours of the day except from 0800–1200. These results suggest that the lizard parapineal plays a direct role in thermoregulation in addition to the indirect function as an illuminometer.     

Ultrastructural features of skin pigmentation in the lizard Heloderma suspectum with emphasis on xanto‐melanophores

The morphological origin of the dark and pink‐orange areas in the skin of the venomous lizard Heloderma suspectum is not known. Histology and electron microscopy show that dark‐grey areas of the skin derived from dermal chromatophores localized in specific areas present underneath the epidermis. A dynamic chromatophoric unit in the dermis is absent. In the darkest areas of the skin, the accumulation of melanosomes in cells of the beta‐layer contributes to increase the black intensity. In the orange‐pink areas, the superficial dermis contains xantophores storing numerous carotenoid vesicles, rare or absent lamellated pterinosomes and a variable number of melanosomes. These xanto‐melanophores predominate over the remaining chromatophores and form a continuous stratum underneath the epidermis. Beneath this lipoid‐rich stratum, iridophores are infrequent and do not form a continuous layer in the dermis. In the paler areas of the skin, melanophores are sparse in both superficial and deeper part of the dermis where irregularly oriented bundles of collagen fibrils are present. The prevalent xanto‐melanophores localized in the pink‐orange areas of the skin contribute to an effective sunlight protection in desert conditions in addition to the darker regions occupied by melanophores.     

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

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

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The capacity of colour change in intact lizards and in animals with a transected hypophysial stalk was studied during extended periods. It was concluded that, with certain exceptions, the skin colour of the lizards gives information on the circulating levels of MSH, and thereby on the function of the pars intermedia.After transection of the hypophysial stalk, three phases of chromatic behaviour were recognised. During Phase I, which lasted for about 6 days (average), dark skin was retained irrespective of the colour of the background (= high MSH levels), whereas Phase II (19 days) was characterised by the inability of the animals to become brown (= low MSH levels). A gradual recovery toward normal adaptive capacity was seen during Phase III.In the disconnected neuro-intermediate lobe, aldehyde fuchsin (AF)-positive material in the rostral region rapidly clumped and disappeared within a few days. In the caudal portion, the stainability and the varicose arrangement of fibres were retained longer, but disappeared within approximately 14 days after the operation. Proximal to the lesion, the preoptic system exhibited a marked increase in stainability with AF, starting in the median eminence and progressing in a retrograde direction toward the peptidergic nuclei.Revascularisation of the disconnected neuro-intermediate lobe occurred during the first few days. A reinnervation of AF-fibres across the transected area into the neural lobe was observed during Phase III in most animals, but AF-fibres did not reoccupy the entire lobe. In association with an outgrowth of capillaries, the regenerating fibres formed new neural lobules. This regenerative process was accompanied by an increase in blood supply from the primary plexus of the median eminence to the neuro-intermediate lobe.Supported by grants from the Swedish Natural Science Research Council and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Ingrid Hallberg and Mrs. Kirsten Thörneby for valuable technical assistance and skillful attention to the animals and to Miss Inger Norling for photographic aid