Purification and characterization of β-galactoside-binding lectin from chick embryonic skin

Lectin activity was found in tarsometatarsal skin of chick embryo. It was specific for β-linked galactosyl residues and required a thiol-reducing agent for hemagglutination activity. The lectin was extracted from dermis and epidermis (skin) with lactose and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. Examination of their biochemical properties showed that although dermis and epidermis develop from different origins, they contain the same lectin. The apparent subunit M_r of lectin was 14 000 and its isoelectric point was 7.0. Under non-dissociating conditions, the lectin exists mainly as a dimer. Radioimmunoassay showed that this skin-type lectin is present in many tissues including skin, muscle, bone, eye, heart, liver and brain at various developmental stages. A wide distribution and a marked change in its content during development strongly suggest that the lectin might have a fundamental role in cellular function, embryonic development and tissue differentiation.     

Changes in expression of two endogenous β-galactoside-binding isolectins in the dermis of chick embryonic skin during development in ovo and in vitro

In order to elucidate the roles of metal-independent animal lectins, we systematically investigated changes in expression of 2 kinds of -galactoside-binding isolectins (MW 14 and 16 kDa) in the dermis of chick embryonic tarsometatarsal skin during the course of development. These lectins were immunohistochemically located at different stages of development both in ovo and in vitro by light and electron microscopy. Light-microscopic observation showed that while positive staining for the 14-kDa lectin was weak at days 8 and 10 it became intense after day 13. In contrast, staining for the 16-kDa lectin was intense at days 8, 10, and 13, but it became weak after day 17 when keratinization of the epidermis was completed. Immuno-electron-microscopic observation revealed that both the 14 and 16-kDa lectins were located on the basement membrane, in the extracellular matrix, and in both the cytoplasm and the nucleus of dermal fibroblasts. Distribution of the 2 isolectins was also examined in cultured skin explants in vitro. The results were almost the same as those obtained in ovo when the skin explant was keratinized in the presence of hydrocortisone. However, in the skin explant where keratinization was prevented and mucous metaplasia was induced by the addition of vitamin A, the distribution of the 14-kDa lectin in the epidermis was significantly affected. These results indicate that (1) the expression of the 2 isolectins is differently regulated in both the dermis and epidermis, (2) the 16-kDa lectin is involved in the early stage of the formation of the dermis and the basement membrane and is replaced by the 14-kDa lectin as keratinization of the epidermis occurs, and (3) the expression of the 2 isolectins in the dermis is not significantly affected by the induction of mucous metaplasia, in contrast to their drastic changes in the epidermis.     

Endogenous beta-galactoside-binding lectin expression is suppressed in retinol-induced mucous metaplasia of chick embryonic epidermis

The fate of endogenous beta-galactoside-binding lectin of chick embryo (14K type) was investigated during the course of skin differentiation. Lectin (14K) was found in keratinized epidermis and was localized mainly in the basal and intermediate cells. However, the protein lectin in the epidermis disappeared when the cultured skin was treated with vitamin A and mucous metaplasia was observed. The synthesis of lectin mRNA was also strongly suppressed by vitamin A in a concentration-dependent manner. On the other hand, in the dermis, in which the lectin was localized in the extracellular matrix, lectin expression was scarcely affected by vitamin A. These results indicated that the lectin was expressed in the keratinized epidermis but that its expression was suppressed in vitamin A-induced mucous-secreting epithelium. The suppression may be a result of a transition of the epidermal regulatory system to one of mucous-secreting epithelium. This is the first finding that 14K lectin expression might be regulated during the course of the epidermal differentiation.     

Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms

Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion.     

Accelerated proliferation and abnormal differentiation of epidermal keratinocytes in endo-beta-galactosidase C transgenic mice

Transgenic (TG) mice that have systemically expressed endo-beta-galactosidase C (EndoGalC) have rough and flaky skin. This skin phenotype is detectable around 5 days postnatal and becomes obscure by 2 weeks after birth. Their epidermis is thickened but the dermis and hair follicles are normal in structure. EndoGalC, which removes the terminal Galalpha1-3Gal disaccharide (alphaGal epitope), was expressed in the epidermis of TG mice. GS-IB4 lectin staining showed that the alphaGal epitope did not exist in the epidermis in TG but existed in wild-type (WT) mice. In TG mice, N-acetylglucosamines were exposed by EndoGalC, which is detected using GS-II lectin. To understand the cause of the epidermal thickening and skin phenotype, we examined the proliferation and differentiation of kerationocytes. BrdU-pulse-labeling revealed that proliferating keratinocytes increased approximately three-fold in TG epidermis compared to WT one. In TG epidermis, the expression domain of cytokeratin 14 increased from 1-2 layers to 4-5 layers and co-expressed with cytokeratin 6 and 10 in the upper layers. The layers expressing involucrin and loricrin also increased but those expressing filaggrin and transglutaminase looked normal. The localization of E-cadherin was similar in both TG and WT mice. Although TG mice showed delayed development of the barrier function around 8 days postnatal, they acquired the function by 12 days after birth. These results suggest that the absence of the alphaGal epitope or the exposed N-acetylglucosamine terminal could play a critical role in the proliferation of basal keratinocytes and differentiation of them into the spinous cells in newborn mice.     

Regional specificity of anuran larval skin during metamorphosis: dermal specificity in development and histolysis of recombined skin grafts

Summary Skins from back and tail were dissected from tadpoles of Rana japonica prior to resorption of the tail and separated into epidermis and dermis by treatment with neutral protease. Homotypically and heterotypically recombined skins were constructed from the separated epidermis and dermis and transplanted into the tail of the original tadpole. Skin grafts using dermis from tail region degenerated simultaneously with resorption of the tail. However, skin grafts containing dermis from back region survived on the posterior part of the juvenile frog beyond metamorphosis. Furthermore, all epidermis underlaid with dermis from back region formed secretory glands and became flattened epithelia characteristic of adult back skin, regardless of region from which the epidermis came. Even when epidermis isolated from tail skin was cultured inside a back skin graft, the tail epidermis survived forming an epithelial cyst and developed secretory glands. These results suggest that regional specificities of anuran larval skin, i.e., development of back skin and even histolysis of tail skin, are determined by regionally specific dermis. The results also suggest that some of epidermal cells of tail skin are able to differentiate into epithelial cells similar to back skin of the adult under the influence of back dermis.     

Stimulation of rat cutaneous fibroblasts and their synthetic activity by implants of powdered nacre (mother of pearl)

The components of the cutaneous envelope, the epidermis and the dermis, change in response to aging or environmental stress factors. The fibroblasts involved in maintaining skin tone are the main targets. Nacre, mother of pearl, from Pinctada maxima, which can stimulate and regulate bone forming cells, was implanted in the dermis of rats to test its action on the skin fibroblasts. This report describes the effect of nacre on the skin fibroblast recruitment and physiological activity. It resulted in enhanced extracellular matrix synthesis and the production of components implicated in cell to cell adhesion and communication (such as decorine) and in tissue regeneration (type I and type III collagens). The nacre implant produced a well vascularized tissue. The physiological conditions in the region around the implant are thus those required for the positive interactions between the dermis and epidermis which are fundamental for the physiological function of the skin.     

Oxytocin-producing and vasopressin-producing eosinophils in the mouse spleen: immunohistochemical,immuno-electron-microscopic and in situ hybridization studies

The localization of an endogenous 14-kDa -galactoside-binding lectin (galectin) and its pattern of gene expression were examined in normal human skin by light- and electron microscopy. Under the light microscope, immunostaining of 14-kDa galectin was observed in the cell membrane of cells in the basal and spinous layers of the epidermis. Galectin was also found in the Langerhans cells, as shown by double labeling using anti-14-kDa galectin and anti-CD1a antibodies. In the dermis, immunostaining for the 14-kDa galectin was positive in the extracellular matrix and fibroblasts. At the electron-microscopic level of resolution, galectin was located primarily along the plasma membrane of keratinocytes, and in both the cytoplasm and nucleus of Langerhans cells in the epidermis, whereas in the dermis it was detected in the extracellular matrix and in both the nucleus and cytoplasm of fibroblasts. The gene expression of 14-kDa galectin was visualized by the HRP-staining method following in situ hybridization techniques. The expression was detected in the cytoplasm of cells in the basal and spinous layers of the epidermis; whereas, in the dermis, it was detected in the cytoplasm of fibroblasts. Moreover, SDS-polyacrylamide gel electrophoresis and lectin-blot analysis revealed that this galectin bound to glycoproteins of approximately 17, 62, and 72 kDa in the epidermis and to those of 29, 54, and 220 kDa in the dermis. The present study indicates that 1) normal human skin produces the -galactoside-binding 14-kDa galectin, and 2) this galectin is located in both the epidermis, particularly in the keratinocytes and Langerhans cells, and in the dermis. These results suggest that galectin is important for cell-cell contact and/or adhesion in the epidermis and for cell-extracellular matrix interaction in the dermis.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

Developmental changes in the heterocellular epidermis of Pelobates syriacus integument.

Changes in characteristic components of the skin epidermis of the large tadpole of Pelobates syriacus were studied throughout its development. The fate of two specific cells in the skin epidermis was followed, from the young tadpole to the adult was studied. It was found that flask-shaped type cells in the tadpole epidermis which are PAS-positive, stain with peanut lectin (PNA). There is no detectable band 3 in the premetamorphosed stages, and mitochondria-rich cells are very rare. This pattern of staining changes completely upon metamorphosis: the PAS-positive cells, specific to the tadpole epidermis disappear, and the mitochondria-rich (MR) cells in the adult skin epithelium react with polyclonal anti-band 3 antibody. Western blot analysis showed the presence of a band 3-like protein of about 95 kDa, only in the adult epithelial extract, corroborating the immunocytochemical observations. The finding of the presence of band 3-like protein in the MR cells of Pelobates, is similar to the observations made in the skin of other amphibian species. On the other hand, the binding of peanut lectin to MR cells is species-specific, since it does not react with the MR cells in the skin epithelium of Pelobates syriacus.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

The skin structure of greater rhea (Rheidae,Palaeognathae)

The aim of this study was to describe the histological structure of the skin of greater rhea (Rhea americana), a ratite bird native to South America. Skin samples were taken from three regions of the trunk (alar, dorsal and pelvic) in 14 specimens which ages ranged from 7 days to adulthood. Serial sections were obtained and subjected to different staining procedures (haematoxylin and eosin, orcein, Masson's trichrome and Gomori), and a morphometric analysis was carried out on stained slides. In general, both epidermis and dermis showed increased thickness of its layers with age. Some differences between regions can be detected both in epidermis and in dermis; for example in adults and 7‐day‐old birds, the stratum corneum of the alar region was thicker than of the dorsal region. In general, the skin of greater rhea was similar to that described in ratites and other birds (a thin epidermis compared to dermis, dermis with scarce elastic fibres, a slender and vascularized stratum superficiale, collagen fibres arranged in three directions). The scarcity of elastic fibres and the general cross‐weaved arrangement of the collagen fibres in the dermis of the adult greater rhea provide strength and flexibility to the dermis, two important features in leather industry.     

花背蟾蜍蝌蚪皮肤的结构与发育

为研究花背蟾蜍(Bufo raddei)蝌蚪在变态发育期皮肤的显微结构特点,选取G19、G22、G26、G36、G41、G43和G46共7个发育期蝌蚪的连续石蜡切片及成体的背部皮肤切片,采用H.E和AB-PAS染色方法,观察了皮肤各层结构的发育时序并进行了相应的测量.结果表明,在G19、G22和G26蝌蚪表皮均为1层细胞;G36蝌蚪皮肤细胞形态和层数在背腹部出现了显著的区别;在G41基本完成了表皮2层细胞的构建;G43期完成完整的真皮构建,其中分布有毛细血管和2种皮肤腺.G46皮肤在厚度、腺体和毛细血管分布等方面表现出了明显的区域性差异,并与成体皮肤结构有明显的差别,显示出蝌蚪在发育过程中皮肤结构的变化与其生存环境之间紧密的关联性.     

Epidermal and hair follicle transglutaminases. Partial characterization of soluble enzymes in newborn mouse skin

Treatment of skins of newborn mice with the neutral protease Dispase in order to separate dermis and epidermis causes pronounced changes in the levels of transglutaminase activity in the epidermis. Two soluble transglutaminases, one anionic enzyme and one cationic enzyme, of Mr approximately 90,000 and approximately 50,000, respectively, are extracted from epidermis; and the activities of both enzymes increase as a function of the time of Dispase treatment of skin. When the anionic Mr approximately 90,000 enzyme is incubated with Dispase after its chromatographic isolation from epidermal extracts, it is converted to a lower molecular weight enzyme. Hair follicles isolated from dermis prepared by a 12-h Dispase treatment of the skin of newborn mice contain two soluble cationic transglutaminases, one of which is indistinguishable from that of epidermis and the other which is not seen in epidermis. Both of these hair follicle enzymes are of Mr approximately 50,000 and appear to exist in monomeric form. They have been partially purified. Based upon these findings, we suggest that transglutaminase processing and control occur during normal differentiation of keratinocytes in epidermis and of hair follicle epidermal cells in dermis and that production of the proper forms of the enzyme may be essential to the formation of mature cornified envelopes and hair shafts, respectively.     

Melanocytes in Human Embryonic and Fetal Skin: A Review and New Findings

Melanocytes account for approximately 5–10% percent of the cells in adult epidermis. Unlike the ectodermally derived keratinocytes, they originate in the neural crest and migrate into the epidermis early in development. There has been an interest in melanocytes in developing human skin since the late 1800s, when concentrated pigmented cells were identified in the sacro-coccygeal skin of Japanese fetuses. This observation led to speculation and subsequent investigation about the racial nature of the melanocytes in this site (the Mongolian spot), the presence of melanocytes in fetuses of other races, the timing of appearance of these cells in both the dermis and epidermis, and their origin. The early investigators relied primarily on histochemical methods that stained either the premelanosome or the pigmented melanosome, or relied upon the activity of tyrosinase within the melanosome to effect the DOPA reaction. Studies by electron microscopy added further documentation to the presence of melanocytes in the skin by resolving the structure of the melanosome regardless of its state of pigmentation. All of these methods recognized, however, only differentiated melanocytes. The thorough investigations of melanocytes in the skin from a large number of black embryos and fetuses by Zimmerman and colleagues between 1948 and 1955 provided insight into the time of appearance of melanocytes in the dermis (10–11 weeks' menstrual age) and the epidermis (11–12 weeks) and revealed the density of these cells in both zones of the skin of several regions of the body. The precise localization of the melanocytes in the developing hair follicles was contributed by the studies of Mishima and Widlan (J Invest Dermatol 1966; 46:263–277). More recently, monoclonal antibodies have been developed that recognize common oncofetal or oncodifferentiation antigens on the surface or in the cytoplasm of melanoma cells and developing melanocytes (but not normal adult melanocytes). These antibodies recognize the cells irrespective of the presence or absence of melanosomes or their activity in the synthesis of pigment and therefore are valuable tools for re-examining the presence, density, and distribution patterns of melanocytes in developing human skin. Using one of these antibodies (HMB-45), it was found that dendritic melanocytes are present in the epidermis between 40 and 50 days estimated gestational age in a density comparable with that of newborn epidermis and are distributed in relatively non-random patterns. A number of questions about the influx of cells into the epidermis, potential reservoirs of melanoblasts retained within the dermis, division of epidermal melanocytes, and the interaction of melanocytes and keratinocytes during development remain unresolved. The tools now appear to be available, however, to begin to explore many of these questions.     

Modulation of catalase in human skin in vivo by acute and chronic UV radiation.

In this study, we demonstrate that catalase is differently regulated either by acute, or chronic UV radiation during the photoaging process. 2MED of UV radiation decreased the activity and expression of catalase gradually in the epidermis and dermis at between 24 and 48 h after the UV exposure. These levels then returned to near normal by 72 h after exposure. The catalase mRNA was also decreased in the skin 24 h after UV irradiation to 50% of the control level, and then started to recover. In contrast, chronic UV irradiation over a lifetime (approximately 50 years) increased the catalase activity in the epidermis and dermis of the human skin in vivo. Our results suggest that catalase might be one of the important enzymes in the skin aging process, and that it plays an important role in the photoprotection of the skin from UV light.     

Expression of Hex during feather bud development

We studied proline-rich divergent homeobox gene Hex/Prh expression in the dorsal skin of chick embryo during feather bud development. Hex mRNA expression was first observed in the dorsolateral ectoderm and mesenchyme at 5 days, then in the epithelium and the dermis of the dorsal skin before placode (primordium of feather bud) formation and then was restricted to the placode and the dermis under the placode. Afterward, Hex expression was seen in the epidermis and the dermis of the posterior region of short bud. In accordance with Hex mRNA expression in the placode, Hex protein was observed in the epidermis as well as in the dermis of the placode. Immunoelectron microscopic study indicated that the protein located both in the nuclei and cytoplasm of the epidermis and the dermis at the short bud stage. The Wnt signaling pathway plays an essential role in the early inductive events in hair (Wnt3a and 7a) and feather (Wnt7a) follicles. The pattern of Hex expression in the epidermis was similar to that of Wnt7a, while little, if any, expression of Wnt7a was detected in the dermis under the placode or the dermis of the short bud compared with that of Hex, suggesting that Hex plays an important role in the initiation of feather morphogenesis.     

Epidermal-dermal tissue interactions between mutant and normal embryonic back skin: site of mutant gene activity determining abnormal feathering is in the epidermis.

The site of the scaleless gene's activity in the development of abnormal feathers was determined by reciprocally recombining epidermis and dermis between normal and scaleless chick embryos and culturing the recombinants for seven days on the chorioallantoic membrane. When recombined with a common dermal source, feather development is enhanced by scaleless high line as compared to scaleless low line epidermis. Against a common responding tissue, 7-day normal back epidermis, significant differences were not found in feather inducing ability between normal, scaleless high line and scaleless low line dermis. It was concluded that, in relation to abnormal feathering, these tissue interactions reveal that the site of the scaleless gene's activity is the epidermis. A model of tissue interaction in the development of normal and abnormal feathers is presented. According to the model, the focus of the scaleless mutation and the genes accumulated by selection for high or low feather numbers is the epidermis, the effect being that the reactivity of the epidermis to dermal stimuli is altered. Subsequently, the epidermis controls the morphogenetic organization of the dermis. The scaleless dermis is presumed to contain normal positional information for the determination of feather structure and pattern.     

Expression of Hex homeobox gene during skin development: Increase in epidermal cell proliferation by transfecting the Hex to the dermis

A number of homeobox genes have been found to be expressed in skin and its appendages, such as scale and feather, and appear to be candidates for the regulation of the development of these tissues. We report that the proline-rich divergent homeobox gene Hex is expressed during development of chick embryonic skin and its appendages (scale and feather). In situ hybridization analysis revealed that, during development of the skin, a transient expression of the Hex gene was observed. While the expression of Hex in the dermis was closely correlated with proliferation activity of epidermal basal cells, that in the epidermis was related to a suppression of epidermal differentiation. When dermal fibroblasts were transfected with Hex, stimulation of both DNA synthesis and proliferation of the epidermal cells followed by two-fold scale ridge elongation and increase in epidermal area was observed during culture of the skin, whereas epidemal keratinization was not affected. This is the first study to demonstrate that Hex is expressed during development of the skin and its appendages and that its expression in the dermal cells regulates epidermal cell proliferation through epithelial mesenchymal interaction.